Plant Physiology Preview. Published on January 26, 2016, as DOI:10.1104/pp.15.01624
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Suggested running title: Rice leaf photosynthetic development
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Corresponding author: Andrew J. Fleming
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Department of Animal and Plant Sciences,
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University of Sheffield,
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Sheffield S10 2TN,
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UK
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Tel: +44 (0)114 222 4830
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Fax: +44 (0)114 222 0002
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Email: [email protected]
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Research Area: Genes, Development and Evolution
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Copyright 2016 by the American Society of Plant Biologists
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Title: Combined Chlorophyll Fluorescence and Transcriptomic Analysis
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Identifies the P3/P4 Transition as a Key stage in Rice Leaf Photosynthetic
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Development
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Julia C. van Campen1, Muhammad N. Yaapar1, Supatthra Narawatthana1,a,
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Christoph Lehmeier1,b, Samart Wanchana2,c, Vivek Thakur2, Caspar Chater1,d, Steve
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Kelly3, Stephen A. Rolfe1, W. Paul Quick2, Andrew J. Fleming1
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1
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2TN, UK
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International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines
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Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, UK
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One sentence summary: A combined transcriptomic and physiological analysis of
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rice leaf development identifies the stage (P3/P4 transition) when photosynthetic
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competence is first established.
Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10
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Present addresses of authors:
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a
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Bangkok 10900, Thailand
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b
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66047 KS, USA
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c
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Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand
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d
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Universidad Nacional Autónoma de México, Mexico
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Corresponding author: Andrew J Fleming- [email protected]
Present address: Rice Department, Ministry of Agriculture and Cooperatives,
Present address: Kansas Biological Survey, The University of Kansas, Lawrence,
National Center for Genetic Engineering and Biotechnology, 113 Phahonyothin Rd.,
Departamento de Biologia Molecular de Plantas, Instituto de Biotecnologia,
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AUTHOR CONTRIBUTIONS
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AJF and WPQ designed the research; JvC, MNY, and SN performed research; SAR
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contributed new analytic tools and SK contributed new computational tools; SK, VT,
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SW, JvC, CC and MNY analyzed data; AJF and JvC wrote the paper, with all authors
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contributing to the final version.
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ABSTRACT
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Leaves are derived from heterotrophic meristem tissue which at some point must
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make the transition to autotrophy via the initiation of photosynthesis. However, the
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timing and spatial co-ordination of the molecular and cellular processes underpinning
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this switch are poorly characterised. In this paper we report on the identification of a
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specific stage in rice (Oryza sativa) leaf development (P3/P4 transition) when
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photosynthetic competence is first established. Using a combined physiological and
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molecular approach, we show that elements of stomatal and vascular differentiation
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are co-ordinated with the onset of measurable light absorption for photosynthesis.
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Moreover, by exploring the response of the system to environmental perturbation we
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show that the earliest stages of rice leaf development have significant plasticity with
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respect to elements of cellular differentiation of relevance for mature leaf
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photosynthetic performance. Finally, by performing an RNAseq analysis targeted at
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the early stages of rice leaf development, we uncover a palette of genes whose
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expression likely underpins the acquisition of photosynthetic capability. Our results
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identify the P3/P4 transition as a highly dynamic stage in rice leaf development
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where several processes for the initiation of photosynthetic competence are co-
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ordinated. As well as identifying gene targets for future manipulation of rice leaf
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structure/function, our data highlight a developmental window during which such
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manipulations are likely to be most effective.
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INTRODUCTION
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Rice is a C3 grass whose seeds are the single most important staple food, providing
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a fifth of the world’s dietary energy supply (Elert, 2014). The source of all carbon in
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these seeds is photosynthesis and there is significant interest in improving this
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fundamental process through alteration of several morphological, biochemical and
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physiological traits in leaves (Long et al., 2006; Hibberd et al., 2008). However there
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are still large gaps in our knowledge of leaf development and the ontogeny of
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photosynthesis, both in rice and other species. For example, although we have a
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deep and detailed understanding of the mechanism of photosynthesis, it is still
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unclear at exactly which stage in development a rice leaf first gains the capacity to
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capture light energy for carbon fixation. Linked to this, although it is well established
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that leaves in many species (including rice) can undergo morphological and
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photosynthetic acclimation to the environment (Hikosaka and Terashima, 1995;
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Murchie and Horton, 1997; Oguchi et al., 2003), and it is known that the cellular
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processes underpinning this acclimation occur during relatively early stages of leaf
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development (Jurik et al., 1979; Sims and Pearcy, 1992; Murchie et al., 2005), the
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point when developmental plasticity is lost has not been defined precisely.
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Rice leaves are initiated by a co-ordinated process of cell growth and division which
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lead to the formation of a protrusion on the shoot apical meristem (Itoh et al., 2005).
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The primordium (enclosed within the encircling sheaths of older leaves) then
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undergoes further development to form a mature leaf which consists of a flattened
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blade and a sheath encompassing the younger leaves, which develop sequentially
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from the shoot apical meristem. The young plant thus consists of a series of
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concentric leaves all of which originate from the meristem and are produced in a
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series separated by a time period, termed the plastochron (P). The definition of
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leaves by their sequential order of formation (L1, L2, L3…) and plastochron age (P1,
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P2, P3…) allows the comparison of leaves from different plants at equivalent
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developmental stages. P1 stage is characterised by a small protrusion forming on
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the flank of the shoot apical meristem. This protrusion then forms a hood shaped
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structure around the shoot apical meristem (P2 stage) before completely enclosing
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the shoot apical meristem and taking on an elongated conical shape (P3 stage) (Itoh
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et al., 2005). The subsequent P4 stage is characterised by a phase of rapid
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elongation of the leaf blade and visible greening of the tissue. Due to the degree of
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elongation that occurs during P4 the stage can be sub-divided into stages (1-12)
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defined by particular blade lengths (Kusumi et al., 2010). At P5 stage the distal tip of
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the leaf starts to protrude from the sheaths of older leaves, being pushed up by rapid
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elongation of the more proximal sheath, before growth stops as the leaf reaches
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maturity (P6 stage).
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In addition to distinct developmental stages over time, individual grass leaves (such
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as rice) show a clear gradient of development along the longitudinal axis (Li et al.,
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2010). This has been successfully exploited in a number of investigations to allow
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comparisons of transcriptomes, proteomes and metabolomes in cells undergoing
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different stages of differentiation. These analyses have revealed key pathways and
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fundamental regulators of photosynthesis in rice and also in maize (Li et al., 2010;
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Majeran et al., 2010; Pick et al., 2011; Wang et al., 2014). These patterning events
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at the base of mature leaves reflect a template set by the more mature differentiated
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tissue, distinct from the processes that must occur within a new leaf primordium
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where the pattern arises de novo. Thus, studying the morphology, physiology and
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gene expression dynamics of rice leaf primordia has the potential to provide novel
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insights that may not be evident in studies of mature leaf gradients.
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With respect to the analysis of the very earliest stages of grass leaf development,
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previous work has successfully used laser micro-dissected portions of maize shoot
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apical meristem domains to identify transcripts associated with leaf initiation (Ohtsu
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et al., 2007). This work was restricted to the extremely early processes of leaf
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determination and initiation (up to P1) and did not encompass subsequent stages of
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leaf development. This was achieved by Wang et al. (2013a) who recently provided
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a detailed transcriptomic analysis of early leaf development in maize using dissected
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primordia. These data provided the first analysis of a monocot leaf at this
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developmental resolution. To date a similar analysis has not been reported for rice.
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Most of the work on early leaf development (both in monocots and dicots) has
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focussed on changes in the transcriptome, yet relating these data to the
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physiological function of the leaf is often problematic. Gene expression at the
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transcript level does not necessarily infer biochemical or physiological function
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(Amiour et al., 2012; Fernie and Stitt, 2012; Lan et al., 2012). In the case of
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photosynthesis, chlorophyll fluorescence has developed as a routine technique for
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the measurement of photosynthetic function (Krause and Weis, 1991; Maxwell and
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Johnson, 2000; Meng et al., 2001; Baker, 2008), however most equipment is
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designed for measurements in leaves which can be clamped within a chamber,
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greatly restricting the minimum size of the material that can be analysed.
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Microfluorescence techniques have been developed which provide the appropriate
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resolution for the analysis of small leaf primordia, however these have generally
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been applied to the analysis of plant/microbe interactions (reviewed in Berger et al.,
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2007) and plant stress response (reviewed in Baker and Rosenqvist, 2004) rather
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than used in the context of leaf development.
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A significant body of work has shown that many plants have the ability to acclimate
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their leaves to the light environment, leading to the formation of sun or shade leaves
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(Kubinova, 1991; Murchie and Horton, 1997; Oguchi et al., 2003; Kim et al., 2005;
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Terashima et al., 2006). The ambient environment is sensed by mature leaves,
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leading to the formation of an as yet uncharacterised signal that influences the
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morphogenesis of developing leaves at a distance from the mature leaves. It is clear
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that this systemic signalling system exists in rice and that responding leaves are
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young (Murchie et al., 2005), but the precise developmental stage during which this
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developmental plasticity exists is not known, nor whether this developmental window
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of sensitivity to acclimation correlates with the expression of particular sets of genes
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associated with specific elements of leaf differentiation.
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In this paper we report on a combined physiological and transcriptomic analysis of
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the early stages of rice leaf development. These data provide a description of the
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gene expression changes occurring during the earliest phase of acquisition of
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photosynthetic competence. In addition, by exploiting a series of transfer
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experiments between high and low irradiance regimes at distinct leaf stages, we
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identify the P3/P4 transition as a key stage in rice leaf development where a
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coordinated differentiation of photosynthetic tissue, vasculature and stomata occurs.
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RESULTS
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Photosynthetic function in rice leaves is established at the P3/P4 transition
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The first three leaves of a rice plant (P1 – P3) are already initiated in the embryo
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prior to germination. Following germination additional leaves are produced at regular
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intervals by the shoot apical meristem (Itoh et al., 2005). Leaf 5 showed a typical and
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reproducible growth pattern (Fig. 1A) and was chosen for use in all subsequent
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analyses. Observing developing leaves in rice is complicated by the fact that they
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are hidden from view by the sheaths of older leaves. We therefore characterised a
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plastochron index (Erickson and Michelini, 1957; Hill and Lord, 1990; Fournier et al.,
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2005) (Fig. 1C), which allowed us to use the length of leaf 3 to robustly assess the
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developmental stage of leaf 5 without destruction of the plant. Plants could thus be
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selected and treated at specific plastochron stages of leaf 5 without invasive
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manipulations. Fig. 1B and D show the P1-P6 (mature) stages of leaf 5 growth and
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Fig. S1 provides an overview of the histology of the different stages of rice leaf
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development.
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To investigate at exactly which developmental stage and where within the leaf the
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ability to generate electron flow via the absorption of light energy occurred, we used
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chlorophyll fluorescence microscopy. Figure 2 shows a series of example images
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from leaves at P3, P4 and P5 stages, as well as mature leaf blades. Due to their
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relative size, only portions of the P5 and mature leaf blades are shown. For each
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sample the raw fluorescence output is shown adjacent to the calculated values and
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distribution of φPSII, which provides a measure of the quantum efficiency of electron
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transport. P3 stage primordia occasionally displayed measurable electron transport
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(Fig. 2A) but this was very weak and tightly localised to the distal tip of the primordia.
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Four out of nine P3 primordia analysed did not display any measurable signal. Early
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P4 stage leaves showed a much more robust ability to generate electron transport,
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with all (10 out of 10) samples analysed showing detectable signal. The signal was
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again restricted to the distal tip of the leaves but was higher than that observed in P3
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samples (Fig. 2B). For comparison, all P5 stage leaves analysed (which were all
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visibly green) and all mature leaves analysed showed a more uniform and very high
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signal (Fig. 2C,D).
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These physiological data suggested that the ability of rice leaf tissue to perform a
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basic function of photosynthesis (absorption of light energy to generate an electron
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flow) develops at the very end of the P3 stage at the distal tip but that there is a then
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a rapid transition to photosynthetic competence during early P4 stage. Analysis of
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the ultrastructure of plastids during leaf 5 development supported this interpretation.
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Plastids in P3 primordia lacked any obvious granal structure (Fig. 3A) whereas by P4
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stage plastids with occasional grana were observed (Fig. 3B). In contrast, P5
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plastids had distinct grana (Fig 3C) which looked very similar to those in mature
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leaves (Fig. 3D). It was noticeable that even in the P3 stage plastids (i.e., in leaves in
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which we infer photosynthesis was not occurring or was very limited) distinct starch
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grains were observed (Fig. 3A). We also investigated the developmental phase of
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acquisition of potential photosynthetic capability by examining the pattern of
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chlorophyll autofluorescence using confocal microscopy. In P4 stage primordia a
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clear maximum of signal intensity was observed near the tip of the leaf, with more
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proximal regions displaying a striated pattern of chlorophyll fluorescence (Figure 3E).
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We did not extract and measure the chlorophyll content of P3 and P4 stage leaves
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due to technical difficulties and the oversimplification of the spatial differences in
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chlorophyll content in P3 and P4 stage leaves this would represent. However,
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chlorophyll content was around 593 mg m-2 in mature leaf blades (n=5; standard
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deviation= 177mg m-2) and 335 mg m-2 in P5 stage leaves (n=5; standard deviation=
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54.9 mg m-2). The average raw fluorescence signal in regions of P3 and P4 stage
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leaves where it was detectable was around 10-20% (P3 stage) or 10-60% (P4 stage)
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of the raw fluorescence signal observed in mature leaves. Thus, we estimate the
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average chlorophyll content of these regions of P3 and P4 stage leaves to be around
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60-120mg m-2 (P3 stage leaves) or 60-360 mg m-2 (P4 stage leaves), depending on
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the sampling location.
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To further investigate the nature of the biochemical and physiological events
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underpinning the very early stages of the acquisition of photosynthetic potential, we
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analysed the induction kinetics of φPSII (the quantum efficiency of photosystem II in
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the light; φPSII=(Fm’-Fs)/Fm’; Maxwell and Johnson, 2000; Figure 4A-D). Data were
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extracted from up to 7 regions of interest (ROI) along the length of three of the five
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P3 stage primordia in which φPSII was measurable (Fig. 4A), as well as three P4
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stage primordia (Fig. 4B). The results indicated a high degree of variation both along
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individual primordia (from base to tip) and between primordia, both at the P3 and P4
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stage, consistent with the idea that rapid changes were occurring in the capacity for
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electron transport around this transition. Very fast induction rates were observed in
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the P3 samples, consistent with P3 leaves being developing sink tissues with high
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levels of reduced metabolites (Turgeon, 1989; Meng et al., 2001). Slightly slower
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rates of φPSII induction were observed in tip regions of P4 stage primordia,
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consistent with our proposal that the generation of Calvin cycle intermediates, the
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activation of Calvin cycle enzymes and possibly stomatal opening require more time
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in this tissue at this stage of leaf development. These data can be compared with
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those observed in P5 stage and mature leaves which showed slower induction
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kinetics but much higher values of φPSII and overall more robust patterns of
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induction despite the higher irradiance used (Fig. 4C,D). These patterns are
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characteristic of source leaves in which photosynthetic function has been fully
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established (Makino et al., 2002).
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After leaf primordia at different developmental stages had undergone induction and
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reached a steady state of electron transport, they were exposed to a series of
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increasing irradiances (Figure 4E-H). At each irradiance steady state φPSII images
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were collected and data was extracted from regions along the length of leaf
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primordia, as described for φPSII induction. In P3 stage leaf primordia a light
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response of φPSII was only measurable in the regions nearest the distal tip, and
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φPSII rapidly dropped to very low levels as the irradiance increased (Fig. 4E). In P4
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stage leaf primordia, a light response of φPSII was measurable in regions further
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away from the tip than in P3 primordia and all 10 primordia analysed still showed a
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φPSII of 0.2-0.1 in regions nearest the tip at the highest irradiance used (Fig. 4F).
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Generally, the heterogeneity seen in steady state φPSII values after induction was
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also seen in the light response of φPSII in P3 stage leaves (compare Fig. 4A and
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4E). This can be compared with the pattern of light induction seen in P5 and mature
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leaf samples (Fig. 4G,H) which showed relatively consistent induction kinetics along
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the length of the leaves analysed and high consistency between different samples.
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Non-photosynthetic quenching (NPQ) was also measured during induction and at a
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range of irradiances, but slight movement of the samples in between recording of the
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Fm and Fm’ in P3 and P4 stage leaves limited the interpretation of these data
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(Supplemental Figure S3).
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A loss of leaf developmental plasticity occurs after the P3 stage
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The above data highlighted the P3/P4 transition as a key stage in the transition to
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photosynthetic competence during rice leaf development. To investigate to what
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extent this physiological transition correlated with specific elements of leaf
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differentiation, we performed a histological analysis of leaf 5 from P2 through to P6.
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At P2 stage there was little overt cellular differentiation to indicate the future position
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of vascular tissue across the leaf blade (Fig. 5A and Fig. S1). By P3 stage, co-
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ordinated patterns of cell division indicated regions of vascular formation which
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became more pronounced during P4, so that by P5 stage and the mature P6 stage a
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classical pattern of vascular differentiation into xylem and phloem was apparent (Fig
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5A).
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It has long been established that rice leaves grown under different irradiances
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undergo a process of acclimation to yield distinct leaf morphologies (Murchie et al.,
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2005). To investigate the degree of developmental plasticity in this system and to
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define when this plasticity is lost, we performed a series of experiments in which
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plants containing leaf 5 at particular developmental stages (P1, P3, P5) were
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transferred from a high light (700 µmol m-2s-1) to a low light (250 µmol m-2s-1)
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environment and the structure and photosynthetic function of the mature leaves were
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measured (Fig. 5B-F). As shown in Figure 5C, leaf 5 grown continuously under a HL
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environment had a mean thickness of 0.63 mm whereas leaf five grown continuously
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under a LL environment was significantly thinner (0.41 mm; p<0.05, Tukey HSD).
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When plants were transferred from HL to LL when leaf 5 was at P1 or P3 stage, the
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mature leaves attained an appropriate thickness for the new LL environment (0.40
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mm when transferred at P1 stage; 0.43 mm when transferred at P3 stage). However,
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when the transfer occurred at the P5 stage the mature leaf was of intermediate
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thickness (0.50 mm) indicating that at the P5 stage leaves were no longer able to
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morphologically acclimate fully to the new light environment, consistent with previous
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work (Murchie et al., 2005). Measurement of leaf blade width (Fig. 5D) and area (Fig.
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5E) revealed that growth in the surface plane of the blade showed a similar pattern
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of determination by the P5 stage with respect to light environment as leaf thickness.
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Combined gas exchange/chlorophyll fluorescence analysis indicated that leaf 5 of
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plants grown under continual HL conditions had a higher assimilation rate at ambient
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CO2 than the equivalent leaf grown under LL conditions (Fig. 5F). When leaf 5 was
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transferred from HL to LL at the P1, P3 or P5 stage it was able to physiologically
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acclimate at all stages so that the measured leaf assimilation rate in the mature leaf
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was similar to a leaf maintained continuously under LL conditions. These data
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indicated that photosynthetic capacity could acclimate post-P5 via altered
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biochemistry, consistent with previous observations (Murchie et al., 2002; Murchie et
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al., 2005).
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Having established that rice leaves were showing an appropriate acclimation
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response at the gross morphological level, we set out to investigate the light
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response at the cellular level, with particular emphasis on stomatal patterning since
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one might expect this to relate to the acquisition of photosynthetic function. Stomata
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in rice leaves form in specific epidermal cell files (Fig. 6A; Luo et al., 2012).
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Epidermal cell files are formed in a series of interveinal gaps (IVGs) defined by
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parallel vascular strands along the long axis of the leaf. Stomatal density (SD) within
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each IVG at the mid-point along mature leaf 5 was measured in plants grown under
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HL and LL (Fig. 6B). Under HL leaves had a maximum of 16 IVGs whereas under LL
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up to 18 IVGs formed. Values of SD were found to be reasonably constant across all
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IVGs under both treatments, with the exception of the IVGs at the leaf margin which
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tended to have a relatively high SD. With respect to interveinal distance (IVD) these
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were generally smaller in LL across all regions of the leaf (Fig. 6C). Again the IVD
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values at the leaf margin for both HL and LL leaves tended to be different from the
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rest, with the margin IVD being relatively small, mirroring the relatively high SD
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values observed in this IVG (Fig. 6B).
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As in other grass leaves, stomata in rice are formed via an intricate but stereotypical
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series of cell divisions leading to the development of a stomatal complex consisting
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of a pair of guard cells and supporting cells (Fig. 6E; described in detail in Luo et al.,
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2012). When stomatal complex area (SCA) was measured in mature HL grown
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leaves a relatively consistent value was observed in all IVGs (Fig. 6D). When leaves
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were grown under continual LL a much smaller SCA was measured in all IVGs (Fig.
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6D). To further investigate the nature of this difference in SCA and to identify the
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developmental window during which SCA was set, we analysed stomata in a series
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of plants which had been transferred from HL to LL at specific stages of development
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of leaf 5 (as described in Fig. 5B). These data displayed a startling significant
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difference in variance between samples transferred at different developmental
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stages (Brown-Forsythe test, p<0.001), with leaves transferred at P1 and P3 stages
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showing very high variances (Fig. 6F). A non-parametric analysis of the data
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(Kruskal-Wallis with a post hoc Dunn’s multiple comparison) indicated that the
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median SCA values for P1-transfered and LL samples were significantly different
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from the other treatments (p< 0.05), suggesting a fluidity in the setting of stomatal
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size in early rice leaf development which is later lost.
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To investigate the cause of this variation in SCA we measured stomatal complex
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length (SCL) and stomatal complex width (SCW). A similar pattern was observed to
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that recorded for SCA, suggesting that the variation observed in SCA reflected
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changes in both SCL and SCW (Fig. 6G,H). Comparison of the different SCL
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samples revealed a significant difference in variance (Brown-Forsythe p< 0.01) with
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P1 and P3 transferred samples showing the highest variances but a non-parametric
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test did not distinguish any of the samples for SCL. The SCW data also showed a
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significant variation in variance (Brown-Forsythe p< 0.05) and the median values
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obtained from leaves transferred at P1 and P3 stages could be distinguished from
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the other treatments (Kruskal-Wallis, Dunns multiple comparison p < 0.05).
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Interestingly, although comparison of the mean values of SD in the transferred
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leaves indicated no difference between the different treatments, the data indicated a
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higher variance in SD in the P1 and P3 transferred leaves compared to the other
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samples (Fig. 6I) with some transferred leaves showing very low (< 150 stomata per
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mm2) or very high values (>600 stomata per mm2) (Supplemental Figure S4). Thus a
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plasticity in the setting of various stomatal parameters was observed during very
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early leaf development which seemed to be lost later.
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An analysis of stomatal differentiation by scanning electron microscopy (SEM)
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supported the interpretation that early stage primordia (P1, P3) show greatest
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flexibility in the light response of SCA. At P3 stage epidermal cell files were clearly
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visible along the surface of the primordium but no overt signs of stomatal
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differentiation were apparent (Fig. 6N,O). By late P4 stage differentiated stomata
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were clearly visible (Fig. 6P), consistent with the P4 stage being a phase of rapid
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stomatal differentiation, subsequent to patterning events during P3.
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Since stomata in rice form along epidermal cell files, we also investigated the extent
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to which the parameters of stomatal size and density might be set by the overall
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parameters of epidermal cell file number (CFN) and width (CFW) within IVGs, and
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the relationship of these parameters to whole leaf values of vein number and
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interveinal distance (IVD). The overall pattern of CFW was similar to that observed
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for SCA (Fig. 6F,J). The differences in variance for CFW were less than for SCA and
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the P1 and P3-transferred samples had a significantly lower CFW than the other
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treatments (ANOVA with post hoc Tukey test, p < 0.05).
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The differences in CFW were accompanied by differences in IVD (Fig. 6K). Most
362
notably, the P1 transferred leaves had a significantly smaller IVD compared to HL
363
leaves (p < 0.005) and LL leaves also had smaller average IVD (p< 0.01). A
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364
decrease in IVD is predicted to lead to an increase in the frequency of veins across
365
the leaf blade (since veins set the boundaries for IVD). This was observed for the P1
366
transferred leaves which showed a significantly higher vein frequency than the other
367
treatments (p< 0.05) (Fig. 5L). It is interesting to note that although under HL IVD
368
was higher than under LL and CFN was essentially unchanged (Fig. 6M), vein
369
frequency across the blade of HL and LL leaves was very similar (Fig. 6L). This
370
apparent discrepancy was resolved by the observation that under HL conditions the
371
width of the vascular bundles was smaller than under LL. There thus appeared to be
372
a compensation process between IVD and vascular bundle width which acted to
373
maintain vein frequency and a relatively constant number of intervening epidermal
374
cell files.
375
In addition to investigating the outcome on leaf development of transitions from HL to
376
LL, we also analysed the reverse situation when leaf primordia at specific
377
plastochron stages from plants grown under LL were transferred to HL. For this
378
analysis we focussed on those traits which showed the most marked differences in
379
the high to low irradiance experiments described above. As shown in Supplemental
380
Figure S5A, leaves grown under LL then transferred to HL at P5 stage did not attain
381
a typical HL thickness at maturity, whereas leaves transferred at the P3 stage
382
achieved a thickness which could not be discriminated from leaves continually kept
383
under HL. Interestingly, P1 stage leaves transferred to HL conditions remained
384
relatively thin at maturity, suggesting that under some conditions very early stage
385
primordia can remain fixed with respect to final leaf thickness. When stomatal
386
complex area (SCA) was analysed a similar pattern was observed, with the P5
387
transferred leaves displaying a mean SCA which was similar to that observed under
388
LL conditions and distinct from that observed under HL (Supplemental Figure S5B),
389
with P1 and P3 transferred leaves showing a more intermediate phenotype. There
390
was no difference in mean stomatal density (SD) between the different treatments
391
(Supplemental Figure S5C) but there was trend for higher variance in the leaves
392
transferred at P3 and P5 stages, with densities of over 500 stomata per mm2 being
393
observed. Similarly, the various treatments did not lead to a significant difference in
16
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
394
final cell file width (CFW) although there was a trend again for higher variance in the
395
transferred at P3 and P5 stages (Supplemental Figure S5D).
396
Gene expression patterns underpinning the P3/P4 transition
397
Our analysis of both physiology and structure identified the P3/P4 transition as an
398
important stage in rice leaf development with respect to the acquisition of
399
photosynthetic capability and associated cellular differentiation, as well as the ability
400
to respond to environmental signals by altering aspects of cellular differentiation. To
401
investigate the gene expression changes underpinning this transition we performed
402
RNA sequencing analysis on leaf 5 at P3 stage, P4 stage, and the blade at P5 stage.
403
Overall, 14502 genes were identified that were differentially expressed during early
404
leaf development (out of a total of 25768 for which expression was detected). Having
405
validated the RNAseq data (Supplemental Figure S6), we interrogated the datasets
406
to identify patterns of gene expression during early leaf development which might
407
underpin the key processes identified by our morphological and physiological
408
analysis. These results are summarised in Figure 7 and described below.
409
Since our analysis indicated that photosynthetic electron transport first develops at
410
the P3/P4 stage transition, we first investigated the pattern of transcript accumulation
411
for those genes annotated as ‘photosynthetic’ (MapMan bin ‘PS’).
412
Our results indicated that 40 genes involved in photosynthesis (out of 351 rice genes
413
annotated with MapMan bin ‘PS’ (photosynthesis)) were already at 50% or more of
414
their mature leaf expression level at the earliest developmental stage studied, P3
415
(Supplemental Table S2), suggesting that a small number of genes involved in the
416
photosynthetic process are already transcriptionally expressed in tissues that are not
417
yet photosynthetically active.
418
annotated as involved in photosynthesis was an increase in expression from both P3
419
to P4 and from P4 to P5 stages (159 genes; Supplemental Table S3A). Broadly,
420
functional groups of genes up regulated from P3 to P5 stage were found to include
421
those involved in the Calvin-Benson cycle, photosynthetic light reactions, and
422
subunits of both photosystem I and photosystem II. These expression changes were
The most common expression pattern of genes
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423
accompanied by upregulation of photoprotective mechanisms such as carotenoid
424
biosynthesis (18/21 annotated genes; Supplemental Table S3B) and around half of
425
genes involved in ascorbate and glutathione metabolism (49/106 annotated genes;
426
Supplemental Table S3C).
427
A range of expression patterns was seen in genes previously identified through rice
428
mutant studies as playing a role in photosynthetic development (Table 1). A small
429
number of these were most highly expressed at P3 stage. However, many other
430
genes with known rice photosynthetic mutant phenotypes showed a peak in
431
expression at P4 stage, as well as several genes with Arabidopsis thaliana
432
photosynthetic mutant phenotypes. A further group of genes with rice photosynthetic
433
mutant phenotypes were maximally expressed at P5 stage. Genes showing this
434
expression pattern also included orthologs of those known to play a role in
435
photosystem assembly in Arabidopsis. Only a small number of genes that have
436
known mutant phenotypes to do with photosynthesis in rice showed a neutral
437
expression pattern in these early stages of leaf development.
438
To investigate the control of expression of these photosynthesis-associated genes at
439
the P3/P4 stages we also analysed transcription factors showing similar patterns to
440
these known photosynthetic genes during rice leaf development, which led to the
441
identification of 30 genes with increasing expression patterns during early leaf
442
development and 21 genes whose transcripts peaked at P4 stage. (See methods for
443
full selection criteria; and see Supplemental Table S4A). This list included genes
444
previously shown to play a role in regulating chloroplast development (e.g. GLK1,
445
GLK2; (Fitter et al., 2002; Wang et al., 2013b), supporting the notion that the other
446
TF genes in this list may also play a role in facilitating or regulating the onset of
447
photosynthesis. Notably, there are also several CCT family transcription factors in
448
our list of potential regulators of photosynthetic development.
449
The setting of aspects of CFW, SCA and vein frequency, as well as the overall
450
parameters of leaf width and thickness, prior to the P5 stage suggested that genes
451
involved in cell growth and division at the P3/P4 transition play an important role in
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452
determining morphological parameters linked to photosynthesis and acclimation. In
453
line with this, functional groups of genes down regulated from P3 to P5 stage
454
included those involved in the cell cycle (24 genes including ARF75, TANGLED1 and
455
many cyclins), regulation of transcription (270 genes including TFs and chromatin
456
remodelling factors), DNA synthesis (97 genes including histones and DNA
457
polymerase subunits) and protein synthesis (both plastid and non-plastid; 108 genes
458
mostly involved in ribosome biogenesis). Notably, the P3/P4 transition was
459
associated with an increase in the expression of genes associated with inhibition of
460
progress through the cell cycle (KRP1, KRP6) and an increased relative expression
461
of genes associated with cell wall differentiation and re-modelling.
462
The P3/P4 transition is associated with vascular differentiation and patterning (Figure
463
5A) and of the 36 rice genes identified as vascular development regulators or
464
orthologs of vascular development regulators in Arabidopsis (Scarpella and Meijer,
465
2004) (Supplemental Table S5), 22 reached their highest level of expression by P3
466
stage, nine peaked at P4 stage and only five were expressed most highly at P5
467
stage (Figure 7B). Thus, most of these putative vascular development regulators
468
showed a decrease in expression from P3 to P5. Of the few genes known to have a
469
vascular mutant phenotype in rice, several were most highly expressed at P3 stage,
470
including OsKn3, OsAGO7, and NARROW AND ROLLED LEAF1 (Postma-Haarsma
471
et al., 1999; Shi et al., 2007; Hu et al., 2010). However, a neutral expression pattern
472
across the three stages studied was observed for the NARROW LEAF1, NARROW
473
LEAF2 and NARROW LEAF3 genes (Qi et al., 2008; Cho et al., 2013). As auxin has
474
long been known to be involved in vascular development, we also studied the
475
expression patterns of OsPIN genes previously found to be expressed in the
476
vasculature (Wang et al., 2009). These genes showed a mixture of expression
477
patterns, with OsPIN1a most highly expressed at P3 stage, OsPIN5b showing a
478
neutral expression pattern, OsPIN10a peaking at P4 stage and OsPIN5a and
479
OsPIN1b reaching maximal expression at P5 stage. Expression of OsPIN2 was not
480
detected.
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481
Finally, since our results indicated that the P3 stage was characterized by a plastic
482
response in terms of stomatal size in response to altered light environment (Figure 6)
483
but that this plasticity was lost by P5 stage, we interrogated our data to identify which
484
rice genes and orthologs of maize (Zea mays) and Arabidopsis genes linked with
485
stomatal differentiation and patterning were expressed during P3 and P4 stage. Of
486
the 17 genes identified as putative stomatal development regulators in rice, 11 were
487
most highly expressed at P3 stage (Figure 7B; Supplemental Table S6). This
488
included orthologs of SPEECHLESS, TOO MANY MOUTHS, EPF2, EPFL7 and
489
YODA (Bergmann et al., 2004; Liu et al., 2009; Rychel et al., 2010). A further five
490
reached their highest expression level at P4 stage, including rice orthologs of
491
SCREAM1, EPFL9 and MUTE (Hunt et al., 2010; Liu et al., 2009). The expression of
492
all stomatal differentiation genes was less in P5 than in earlier stages.
493
DISCUSSION
494
Despite the importance of leaves as the basic engine for photosynthesis-derived
495
nutrition, aspects of our understanding of how these organs develop in monocots are
496
incomplete. In this paper we set out to address this knowledge gap by addressing
497
the specific questions: When does a rice leaf gain capacity for light capture to
498
generate reducing power for carbon fixation? How well does this acquisition of
499
physiological capability correlate with morphological aspects of leaf differentiation
500
and with underlying patterns of gene expression? How flexible are these
501
developmental processes and when is developmental plasticity lost?
502
The P3/P4 transition is when photosynthetic competence is initiated
503
By creating a plastochron index we were able to stage rice leaf development in a
504
robust fashion, enabling comparison of equivalent leaves. Although the main
505
structural and anatomical changes that occur during rice leaf development have
506
been characterised (Itoh et al., 2005), our results provide an analysis of how this
507
structure relates to function, i.e., when a leaf actually gains the capacity to perform
508
an essential element of photosynthesis (absorption of light energy to generate an
509
electron flow).
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510
Although the micro-fluorescence technique used for this analysis places some
511
constraints on data interpretation (for example, no absolute measures of electron
512
transport rate or photosynthetic capacity were calculated since absorbance was
513
found to be variable (Supplemental Figure S2) and other assumptions made in the
514
comparison of these very different tissues may not be met), we are confident that
515
true quenching of chlorophyll fluorescence was occurring in our primordia and our
516
φPSII measurements are valid. We cannot rule out the presence of some cyclic
517
electron flow, reduction of the plastoquinone pool in the dark (Corneille et al., 1998),
518
or the presence of a small proportion of light harvesting complexes not connected to
519
reaction centres. However, we do not observe the very high raw fluorescence signal
520
that might be observed if a large proportion of light harvesting complexes were
521
uncoupled, and the reasonable Fv/Fm values observed indicate non-zero amounts of
522
quenching. Furthermore, the light response of φPSII observed in primordia shows
523
the expected pattern of decreasing φPSII at increasing irradiances, consistent with a
524
functioning electron transport chain.
525
Our results indicate that the ability to absorb light energy to generate an electron flow
526
is gained at the very end of the P3 stage of development at the distal tip of the
527
primordium, followed by a very rapid transition to photosynthetic competence during
528
early P4. Electron transport efficiency does not reach the level of that of a mature
529
leaf until the P5 stage, while little additional photosynthetic electron transport
530
efficiency is gained between P5 stage and the mature leaf. The high variability of the
531
light response of φPSII between samples and between regions is consistent with P3
532
and P4 stage primordia undergoing rapid photosynthetic development, with different
533
leaves having widely differing abilities to use increasing amounts of light to drive
534
electron transport. This interpretation was supported by a structural analysis of
535
plastid differentiation. Plastids prior to P3 stage lacked obvious grana whereas by P4
536
stage plastids had internal structure consistent with capacity for photosynthetic
537
activity. P3 plastids frequently contained starch granules, suggesting that the plastids
538
were importing carbon from source leaves and that starch grains were being used as
539
a temporary carbon store, potentially enabling rapid growth during the fundamental
21
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540
biochemical switch from sink to source metabolism. Recently, Kusumi et al. (2010)
541
used a similar structural analysis of plastid differentiation alongside chlorophyll
542
fluorescence imaging to show that in rice, leaves at the P4-2 stage (around 2 cm
543
long; before emergence) have measureable φPSII. Other than Kusumi et al. (2010),
544
there are few studies reporting photosynthetic measurements on very young leaves.
545
Meng et al. (2001) showed that in the developmental gradient present in a
546
developing tobacco leaf (the youngest used was 3.9cm long), even regions at the
547
base have a measurable rate of electron transport. Peterson et al. (2014) reported
548
changes in the relative abundance of PSI and PSII along maize leaves (the third leaf
549
of 12-day old seedlings was used), and conclude that redox mediation of chlorophyll
550
biosynthesis may regulate PS assembly and thus ensure the development of a
551
suitable PSI/PSII excitation balance. In the most immature (basal) leaf segment
552
studied, Peterson et al. (2014) also report a larger non-variable component of
553
fluorescence, which they attribute to a moderate accumulation of incompletely
554
assembled, non-functional PSII complexes. However, no detailed measurements of
555
φPSII kinetics in at different developmental stages of the leaf are reported in these
556
studies. Our data agree with that of Kusumi et al. (2010), but we also show that the
557
first detectable electron transport occurs before this in the tip of P3 stage leaves, and
558
that development of photosynthetic function occurs basipetally thereafter. Thus, the
559
large longitudinal developmental gradient in the acquisition of φPSII along P3 and P4
560
stage primordia can be visualized to a high spatial resolution using our approach.
561
These physiological data can be compared with our mRNA analysis. In particular we
562
addressed the questions: which photosynthetic genes become transcriptionally
563
active at the onset of the physiological process? Is there a ‘core’ set of early
564
photosynthetic genes in rice?
565
Analysis of gene expression in the very earliest stage of leaf initiation in maize
566
indicated that there is no significant difference in the expression of photosynthesis-
567
related genes between the SAM proper and leaves at the P0/P1 stages (Brooks et
568
al., 2009), whereas in tomato (a dicot) a gene encoding the small subunit of ribulose-
569
1,5-bisphosphate carboxylase/oxygenase was already expressed at P1 stage but not
22
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570
expressed in the SAM (Fleming et al., 1993). In another study, it was found that in
571
older intact maize seedlings, 39 photosynthesis-related genes were upregulated
572
compared to SAMs (Ohtsu et al., 2007). However, both studies in maize used
573
microarrays enriched in probes reflecting specific subsets of tissues, limiting the
574
extent to which the results can be compared to our RNAseq-based analysis (Ohtsu
575
et al., 2007; Brooks et al., 2009). In rice, we found that by the P3 stage 40 genes
576
involved in photosynthesis (out of 351 rice genes annotated as photosynthetic) were
577
already at 50% or more of their mature leaf expression level. These data suggest
578
that over a tenth of genes involved in photosynthesis are already highly expressed
579
before a leaf can actually utilise the encoded enzymatic machinery. Since light is
580
required for the final step in chlorophyll biosynthesis and our analysis indicated a
581
wave of acquisition of chlorophyll fluorescence from primordium tip towards the base
582
at the P3/P4 transition, our data suggest that the basic machinery for photosynthetic
583
electron transport is developmentally set prior to the late P3 stage by a core set of
584
genes, with a light-related trigger initiating actual capability for photosynthetic
585
electron flow. Analysis of P2 stage primordia would allow further testing of this
586
hypothesis, however we found the dissection of such young primordia in rice to be
587
technically very challenging.
588
Many of the genes that are maximally expressed at P4 stage are known to have
589
chloroplast biogenesis mutant phenotypes, emphasizing the importance of this stage
590
for the development of photosynthetic capability. Transcripts for most genes
591
encoding the core photosynthetic machinery (including those for most electron
592
transport chain components and Calvin-Benson cycle enzymes) increase from both
593
P3 to P4 and P4 to P5, showing that bulk production of these vital components of
594
autotrophy occurs over a relatively longer developmental time. Kusumi et al. (2010)
595
split photosynthetic development in rice into three stages: DNA replication and
596
plastid division (late P3/ early P4 stage); activation of the chloroplast genetic system
597
(particularly the transcription/translation machinery; early P4 stage); and activation of
598
the photosynthetic machinery (late P4 stage to P5 stage and beyond). Our results
599
are largely consistent with their findings. However, we also pinpoint a varied set of
23
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600
photosynthesis-related genes that are already expressed at the very early P3 stage,
601
we add more genes with known mutant phenotypes into the expression picture, and
602
we identify a novel set of transcription factors that show expression changes
603
accompanying those of these known genes. This diverse list of transcription factors
604
contains known regulators of photosynthetic development as well as many CCT
605
family genes. In Arabidopsis, the CCT family gene CIA2 has been found to regulate
606
chloroplast protein synthesis and import (Sun et al., 2009). Thus, this list of
607
transcription factors may provide a useful toolbox for future modification of
608
photosynthetic function in rice.
609
The developmental stages of which we have studied the transcriptomic dynamics are
610
directly comparable to those previously studied in maize (Wang et al., 2013a). In
611
these maize primordia, key photosynthetic genes such as those encoding Calvin
612
cycle enzymes and photosystem II reaction centre subunits are already expressed at
613
low levels and are upregulated in later developmental stages. In maize primordia, the
614
chlorophyll biosynthetic enzyme PORB seems not to be expressed until later
615
developmental stages (equivalent to rice P4/P5), which is also when tetrapyrrole
616
biosynthesis shows a peak of activity. Other available datasets on monocot
617
developmental
618
developmental gradient (Li et al., 2010; Tausta et al. 2014; the third leaf of 9-day old
619
seedlings was used). Although it is more difficult to directly compare these studies of
620
leaf segments to our entire (but much younger) primordia, the general pattern of
621
photosynthetic genes already being expressed in the youngest segments at the base
622
of the leaf (before the sink-source transition) is consistent with our findings.
623
Major patterning events linked to photosynthesis occur in the leaf at the P3/P4
624
transition
625
In addition to the appropriate biochemical apparatus for light capture and carbon
626
fixation, photosynthesis requires the integrated supply and export of material,
627
including CO2, water, carbon and nitrogen. Since our physiological analysis indicated
628
the P3/P4 transition is key to the acquisition of photosynthetic competence, we
transcriptomics
are
from
segments
along
the
24
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
maize
leaf
629
investigated whether associated elements of leaf differentiation including vasculature
630
and stomata were co-ordinated with this phase. Our data indicate that this is the
631
case and identify a series of genes whose expression is likely to be involved in the
632
appropriate spatial and temporal differentiation of these elements. Undoubtedly, the
633
development of morphological characteristics is affected by the longitudinal
634
developmental gradient existing within rice leaf primordia, as visualised in our
635
chlorophyll fluorescence imaging analysis. We capture precisely positioned
636
snapshots along this gradient which demonstrate major changes in morphological
637
development at the P3, P4 and P5 developmental stages. This provides guidance for
638
interpretation of our stage-specific gene expression data.
639
Stomatal differentiation occurs during P4 stage and our analysis revealed the
640
expression during this phase of a number of genes associated in other systems with
641
processes of stomatal patterning and differentiation. Previous attempts to detect the
642
expression of some of these genes in rice (e.g., OsSPCH1 and OsSPCH2) have
643
failed (Liu et al., 2009), suggesting that the highly targeted and staged RNAseq
644
approach taken here was essential for the detection of these transcripts. The overall
645
pattern of stomatal associated gene expression was consistent with the observed
646
limitation of stomatal differentiation to the P3/P4 stage. Expression of most of these
647
genes was minimal in P5 stage leaves and the others tended to show a gradual
648
decline in expression from P3 to P5 with some showing a peak in expression at P4
649
(e.g., OsMUTE, OsSCREAM1, OsEPFL9). In light of the role of these genes in
650
controlling stomatal patterning and differentiation in other systems, these rice genes
651
likely play a vital role in setting parameters of stomatal pattern and number.
652
Since there is significant interest in modifying aspects of rice leaf structure for crop
653
improvement, we thought it would be informative to explore the limits of flexibility of
654
the normal process of leaf differentiation, since this would inform on both the
655
boundaries of the system and identify whether particular stages of development
656
should be targeted for modification. To do this we exploited the knowledge that
657
transfer of rice plants from high to low irradiance leads to an acclimation process in
658
developing leaves so that morphology appropriate to the new light environment is
25
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
659
generated. Focussing on stomata, these experiments supported and extended
660
previous observations that this morphological acclimation process occurs prior to the
661
P5 stage, whereas photosynthetic capacity can acclimate post-P5 stage through
662
altered biochemistry (Murchie et al., 2005). Our data indicate that stomata in high
663
irradiance grown rice are larger than those grown under low irradiance but with no
664
significant shift in mean stomatal density. Most strikingly, they indicate that at the P1
665
and P3 stages the process of stomatal patterning is highly plastic and can respond to
666
a shift in irradiance to generate mature leaves with high variation in stomatal density
667
and size. Thus, rice leaves do possess an inherent plasticity in the stomatal
668
patterning mechanism which is subject to environmental regulation, but this is limited
669
to the P1-P3 stages. This is clearest in leaves transferred from HL to LL but is also
670
apparent in leaves transferred in a reciprocal fashion from LL to HL. The situation
671
here is slightly more complex but again it is the P3 stage which is appears to be the
672
key developmental phase in terms of plasticity of response to external environment.
673
Although well documented in eudicots, the mechanism of systemic control of leaf
674
development by environmental factors remains unclear (Lake et al., 2001; Lake et
675
al., 2002; Murchie et al., 2005; Coupe et al., 2006). Our results suggest that
676
manipulation of this system could allow the generation of rice leaves with very
677
distinct stomatal properties and, thus, water relations. We suggest that targeting the
678
P3/P4 stage of development would be the most appropriate for such manipulations.
679
Due to the anatomy of rice leaves, the width of a stomatal complex will be influenced
680
by the width of the epidermal cell file within which it arises. Coupled to the number of
681
cell files within an interveinal gap and the number of interveinal gaps in a leaf (which
682
will be set by the number of veins and the overall width of a leaf), a complex
683
interaction of cellular and leaf-scale patterning and differentiation events will
684
influence the final number and size of stomata. Our data show that transfer of a rice
685
leaf from a HL to LL environment at a very early stage of development (P1) leads to
686
narrower leaves with an increased number of (but smaller) IVGs. The absolute
687
number of cell files within these narrower IVGs remains relatively constant, but the
688
width of the cell files tends to be smaller, i.e., the leaf acclimates to the new
26
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
689
environment at the cellular level. At the level of gene expression, genes involved in
690
cell division processes tend to decrease in transcript level from P3 through to P5 but
691
it is interesting to note that some members of the KRP gene family (associated with
692
the termination of cell division; De Veylder et al., 2001; Barroco et al., 2006) show
693
peaks of relative transcript level at the P3 and P5 stages. Modulation of expression
694
of these genes may play a role in determining the number and size of cells as leaves
695
enter the P5 stage of rice leaf maturation.
696
Our data indicate that changes in epidermal cell file number and size were linked
697
with changes in vein number and size. As in maize, the midvein is initiated early (by
698
rice P2 stage, maize P1/2 stage), with development of lateral veins occurring at the
699
next developmental stage (rice P3, maize P3/4 stages) (Wang et al., 2013b).
700
However, in maize, foliar leaves develop the spacing (by P4 stage) and
701
characteristic Kranz anatomy (by P5 stage) of C4 plants, which does not occur in our
702
C3 rice leaves. The venation pattern that develops in rice is instead broadly similar to
703
that of many other C3 grasses (Sage and Sage, 2009; Sakaguchi and Fukuda, 2008;
704
reviewed in Nelson and Dengler, 1997).
705
To investigate the regulation of vascular development in rice further, we studied the
706
developmental timing of expression in rice of a number of genes previously linked
707
with the control of vascular development in rice, Arabidopsis and maize (Scarpella
708
and Meijer, 2004). Most of these genes reached maximal expression at the P3 stage
709
followed by a general decrease by P5. The general decline in the number of highly
710
expressed vascular-linked genes from P3 to P5 coincides with the decrease in rate
711
of differentiation seen at these later stages, suggesting that this loss of gene
712
expression may limit the ability of older stages to initiate new vascular tissue.
713
Mutation in some of these genes has been shown to lead to altered vascular
714
differentiation in rice (Nishimura et al., 2002; Yamaguchi et al., 2004), suggesting
715
that they represent a panel of relevance for the future investigation of rice leaf
716
vascular development. However, the number of rice vein patterning mutants
717
described at a molecular level is very limited and many of those mentioned here
718
show pleiotropic developmental defects including a strong link between vein
27
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
719
patterning and leaf width (Qi et al., 2008). Thus, suitable targets for manipulation of
720
rice vascular patterning remain elusive.
721
How differentiation of vascular and surrounding photosynthetic tissue is organised
722
has been much debated (Berleth et al., 2000; Scarpella et al., 2003). Chlorophyll
723
fluorescence arose in stripes along the developing leaf, consistent with the idea that
724
already by the early P4 stage, leaf tissue was set on a developmental trajectory
725
setting apart non-photosynthetic vascular and photosynthetic intervening mesophyll
726
tissue. The differentiation of photosynthetic tissue may be organised in reference to
727
the network of developing vascular tissue, which provides a ‘scaffold’ from which
728
positional signals inform the differentiation of the mesophyll. Conversely, Andriankaja
729
et al. (2012) showed that the exit from proliferation of Arabidopsis leaf cells may be
730
regulated by their photosynthetic differentiation, and Scarpella et al. (2004)
731
demonstrate in Arabidopsis that mesophyll differentiation terminates the iterative
732
process of vascular initiation. Which of these processes are at play in monocots, and
733
rice specifically, is unclear. However, we provide evidence that vascular
734
development and the initiation of photosynthetic differentation are temporally and
735
spatially coordinated in rice, with both occurring around the P3/P4 stages. In
736
addition, the striated pattern of chlorophyll autofluorescence observed in P4 stage
737
primordia (Figure 3) is reminiscent of vascular patterning. Direct evidence for
738
positional signals from the vasculature informing photosynthetic development in rice
739
is not available, however in plants with very clear compartmentalisation of
740
photosynthetic and non-photosynthetic function, such as the C4 plant maize, such
741
positional information is known to be key (Nelson and Langdale, 1989). The
742
molecular nature of positional information derived from the vasculature has begun to
743
be pinned down in root development (Sabatini et al., 1999; Nakajima et al., 2001; Wu
744
et al., 2014), suggesting this is a tractable problem.
745
CONCLUSIONS
746
The results reported here identify the P3/P4 transition as a highly dynamic stage in
747
rice leaf development where initial competence for photosynthesis is achieved. This
28
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
748
is co-ordinated with processes of vascular and stomatal differentiation and we
749
identify sets of genes involved in the setting of each of these processes. As well as
750
identifying targets for future manipulation, our results suggest that such
751
manipulations should target stages prior to P4 to exploit the endogenous plasticity of
752
differentiation exhibited by rice leaves during this phase of development.
753
754
755
29
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
756
MATERIALS AND METHODS
757
Plant material and growth conditions
758
Rice plants (Oryza sativa L. var. indica, IR64) were grown in hydroponics as
759
described (Makino et al., 1997). Plants were grown in a growth chamber (Conviron,
760
www.conviron.com) at high light (700 µmol m-2s-1) or low light (250 µmol m-2s-1) with
761
a 12h/12h light/dark cycle, 50% humidity, ambient CO2 and a temperature of 28 °C.
762
Histology
763
For leaf thickness measurements, 1 cm sections were cut from mature 5th leaf
764
blades, fixed in 4:1 ethanol: acetic acid for 24 hours, and embedded in Technovit
765
7100 (TAAB, www.taab.co.uk). Samples were sectioned at 2 µm thickness using a
766
Leica RM2145 microtome (Leica, www.leica-microsystems.com), stained with
767
Toluidine Blue (Sigma-Aldrich, www.sigmaaldrich.com) and imaged using an
768
Olympus
769
drawings of vascular development were rendered manually in Adobe Photoshop
770
CS5. Thickness was measured at the bulliform cells using Adobe Photoshop version
771
12.0. For transmission electron microscopy, primordia at different developmental
772
stages were dissected into 3% glutaraldehyde (Sigma-Aldrich) in 0.1 M phosphate
773
buffer. Further fixation and processing were as described (Wallace et al., 2015). For
774
confocal microscopy, P4 stage leaf primordia were dissected, mounted in water, and
775
imaged
776
www.zeiss.co.uk). Excitation was with a 488 nm argon laser. Emitted light was
777
detected at 650-710 nm. Noise in the image background only was removed using
778
Adobe Photoshop.
779
Scanning electron microscopy
780
Rice leaf primordia were fixed for six hours on a 2RPM orbital shaker in glass vials
781
containing 4% (w/v) paraformaldehyde (Thermo Fisher Scientific Inc.), 2.5% (w/v)
782
glutaraldehyde (Agar Scientific Ltd), 0.5% (v/v) Tween-20 (Sigma-Aldrich Co.) and
783
0.2M PBS (pH 7.4) (Sigma-Aldrich Co.), all prepared using ultra high purified water.
DP71
using
microscope
an
inverted
(Olympus,
LSM510
www.olympus-lifescience.com).
Meta
confocal
microscope
30
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Line
(Zeiss,
784
Fixative was replaced with two changes of PBS for 30 minutes each. Samples were
785
dehydrated in acetone (Fisher Scientific UK Ltd.) series of 5%, 10%, 25%, 40%,
786
55%, 70%, 85%, 95% and 2 times 100% for 1 hour each. Samples were dried with
787
liquid CO2 using a Polaron critical point dryer (Agar Scientific, U.K). Samples were
788
then mounted onto aluminium stubs using black carbon stickers (Agar Scientific,
789
U.K) and sputter coated with gold (Edwards S150B gold sputter coater) in an argon
790
gas atmosphere. Images were obtained using SEM (Philips XL-20) and processed
791
with embedded XL-20 software.
792
Light response curves
793
Light response curves were recorded on mature 5th leaves in which blade elongation
794
was no longer occurring. Gas exchange was recorded using a LICOR LI-6400
795
portable photosynthesis system (LICOR Biosciences, www.licor.com) at irradiances
796
of 50, 100, 150, 200, 250, 300, 400, 500, 600, 800, 1000, 1250, 1500 and 2000 µmol
797
m-2 s-1 using a constant air flow rate of 200 µmol m-2 s-1, a sample CO2 concentration
798
of 400 µmol m-2 s-1, and a block temperature of 28 °C with a relative humidity of 50%.
799
Plants were allowed to acclimate to each subsequent irradiance level for 3 minutes.
800
The average of up to 54 photosynthetic assimilation rate measurements recorded at
801
5 second intervals was taken at each irradiance level.
802
Chlorophyll Fluorescence microscopy
803
P3 and P4 stage leaf primordia were dissected, mounted on cooled set agarose in a
804
humid environment and imaged using a modified Olympus BX50WI microscope
805
(Rolfe and Scholes, 2002). After exposing leaf primordia to an initial dark period of
806
five minutes, the minimal level of fluorescence was recorded (Fo) and a saturating
807
pulse was applied (3000 μmol m-2 s-1) to measure the initial maximum fluorescence
808
(Fm). Subsequently, an actinic light was switched on, and Fs/Fm' was recorded every
809
30 seconds over a period of 20 minutes as primordia went through induction.
810
Optimal induction irradiances to avoid photodamage of 50 μmol m-2 s-1 for P3 and P4
811
stage leaves and 200 µmol m-2 s-1 for P5 stage and mature leaves were determined
812
through pilot experiments. For light response curves, primordia were exposed to
31
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
813
irradiances of 30, 50, 100, 150, 200, 300, 400 and 600 μmol m-2 s-1after undergoing
814
induction. Plants were allowed to acclimate to each subsequent irradiance level for
815
five minutes, after which four Fs/Fm’ measurements were taken at 30 second
816
intervals. For induction and light response curves, data were extracted from several
817
circular regions of interest (ROIs), each with a diameter of 40 µm. ROIs were evenly
818
spaced along the entire length of the primordium (P3 and P4 stage leaves; up to
819
seven ROIs) or positioned at the base, midpoint, and tip of P5 or mature leaf blades
820
(three ROIs per leaf blade), with positioning over the midvein or major veins avoided
821
(Supplemental Figure 1C). Where no signal was recorded in multiple ROIs
822
throughout the course of the induction or light response experiment, plotted traces
823
may overlap. Absorbance was imaged using the red/far red method (Rolfe and
824
Scholes, 2010).
825
Chlorophyll content analysis
826
Chlorophyll was extracted and chlorophyll content quantified using the method
827
described in Lichtenthaler and Wellburn (1983). Briefly, two leaf disks (each 4 mm in
828
diameter) were collected from the middle of the P5 or mature leaf blade (n=5 per
829
stage) and ground up in 10 ml cold 80% acetone using a pestle and mortar.
830
Chlorophyll a and b concentration were measured using a spectrophotometer
831
(Perkin Elmer), with the following calculations used: chlorophyll a (µg ml-1) = 12.21
832
(A663) - 2.81 (A646); chlorophyll b (µg ml-1) = 20.13 (A646) - 5.03 (A663).
833
Analysis of stomatal and vein patterning
834
For each rice leaf a 1 cm segment at the midpoint was cut and fixed in ethanol:
835
acetic acid (7:1 v/v) for 2-3 days at room temperature, then bleached (25% economic
836
bleach; Ottimo Supplies Ltd.) for 1 day. Samples were cleared using 2-3 drops of
837
chloral hydrate solution (10 g chloral hydrate in 2.5 ml water and 1 g glycerol) for one
838
hour at room temperature. 1 to 2 drops of modified Hoyer’s Solution (10 g chloral
839
hydrate from Riedel-de Haen®, 1 g glycerol from Sigma-Aldrich Co. LLC and 3 g of
840
20% Arabic gum solution from Minerals-Water Ltd) was employed as mountant and
841
samples placed on glass slides. Samples were observed under an Olympus BX51
32
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
842
light
microscope
using
843
Photomicrographs were taken with an Olympus camera DP71 (12.5 megapixels)
844
using Olympus a U-TVO.63XC camera adapter, both mounted on the microscope.
845
Images were captured and processed using CELL-B Version 2.7 and ImageJ®
846
software for stomata, interveinal gaps and veins counting as well as measuring. The
847
areas of stomatal complexes, aperture length and guard cells (GC) width were
848
measured using a Bamboo® Pen Tablet from Wacom Co., Ltd. Statistical analysis
849
was done using Minitab16 and Graphpad Prism6.
850
RNA extraction and Sequencing
851
For P3 stage leaves, 240 primordia were used per RNA sample; for P4 stage leaves,
852
five primordia per sample (P4 stage leaves around 1 cm in length were used); and
853
for P5 stage leaves, three leaves per sample (blade tissue down to the collar only).
854
Samples were harvested between three to five hours into the photoperiod to
855
minimise the potential influence of circadian factors and the analysis was performed
856
with three biological replicates. RNA was extracted using TriZol (Invitrogen) and
857
cleaned up and DNAse1 treated using the Sigma Plant Total RNA kit (Sigma
858
Aldrich). The quality of the resulting RNA was assessed using the Agilent 2100
859
BioAnalyzer (www.genomics.agilent.com), and all RIN values were found to be
860
above 8. RNASeq was carried out at the Liverpool Centre for Genomic Research
861
(www.liv.ac.uk/genomic-research)
862
construction
863
(www.illumina.com). Sequencing (Illumina HiSeq 2000) produced paired end reads
864
with a read length of 100 bp.
865
Transcript quantification and differential gene expression analysis
866
Paired end reads were subject to quality trimming and adaptor filtering using
867
Trimmomatic (Bolger et al., 2014) using the settings “LEADING:10 TRAILING:10
868
SLIDINGWINDOW:5:15 MINLEN:50”. The quality filtered paired-end reads were
869
then mapped to the complete set of CDS from version 7.0 of the Oryza sativa L. var.
870
japonica MSU Release 7 using bowtie2 (Langmead and Salzberg, 2012) and
following
Nomarski
the
differential
using
Illumina
interference
RiboZero
treated
TruSeq
stranded
contrast
RNA
with
mRNA
33
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
(DIC).
library
protocol
871
transcript abundances were estimated using RSEM (Li and Dewey, 2011). All
872
pairwise comparisons between developmental stages were made using DESeq
873
(Anders and Huber, 2010), using the default normalization method and identifying
874
differentially expressed genes as those with a Benjamini-Hochberg corrected p-value
875
≤ 0.05 (Benjamini and Hochberg, 1995). A PCA plot of all count data by replicate
876
was generated using SIMCA-P+ (version 12).
877
Validation of RNASeq data by quantitative RT-PCR
878
After RNA analysis by Illumina sequencing both in silico quality control and qPCR
879
validation of the expression patterns of selected genes were used to assess the
880
quality of the data (Supplemental Figure S6; Supplemental Table S1). These
881
indicated a low variance between biological replicates and verified that the patterns
882
of expression identified by bioinformatic analysis were reflected at the level of qPCR
883
quantification. Validation of RNASeq data was carried out using RNA retained from
884
the original samples submitted for RNASeq. M-MLV reverse transcriptase
885
(Invitrogen, www.lifetechnologies.com) was used for cDNA synthesis following the
886
manufacturer’s protocol. cDNA from each of three biological replicates for each
887
developmental stage was used, except for P3 stage, were two biological replicates
888
were used. Primers were designed using QuantPrime (Arvidsson et al., 2008)
889
(details in Supplemental Table S1B). PCR amplification was carried out in an ABI
890
StepOne Plus qPCR system using SYBR Green Master Mix (Invitrogen). Two
891
technical replicates from two separate cDNA synthesis reactions were averaged for
892
each biological replicate and relative fold changes in expression between
893
developmental stages were calculated using the
894
Livak, 2008).
895
Functional term enrichment analysis and identification of genes of interest
896
The direction of the log2(FoldChange) in expression between stages and its
897
Benjamini-Hochberg corrected significance (p<0.05) were used to cluster all loci into
898
11 expression clusters (Supplemental Figure S7). Custom Perl scripts were used to
899
group genes into clusters. Loci for which there were less than 10 fragment
ΔΔ
CT method (Schmittgen and
34
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
900
alignments were not clustered. The 11 clusters were used to identify the stage at
901
which individual genes reached their maximum expression level.
902
For MapMan (version 3.5.1R2; Usadel et al., 2005) term enrichment analysis testing,
903
significantly enriched gene groups were identified as those with a Benjamini-
904
Hochberg corrected p-value of less than 0.05 following Wallenius approximation and
905
length normalisation of uncorrected p-values using GOseq (Young et al., 2010). ‘Up
906
regulated’ functional terms were those over represented in clusters of genes that
907
were up regulated between at least two developmental stages and not down
908
regulated between any two developmental stages. ‘Down regulated’ functional terms
909
were those over represented in clusters of genes that were down regulated between
910
at least two developmental stages and not up regulated between any two
911
developmental stages.
912
Potential novel regulators of photosynthetic development (Supplemental table S4A)
913
were identified by applying the following rules: genes must be transcription factors;
914
they must be in clusters ‘up 2’ (significantly up regulated from P3 to P4, no significant
915
change P4 to P5), ‘up 3’ (significantly up regulated from P3 to P4 and P4 to P5), or
916
‘peak’ (significantly up regulated from P3 to P4, significantly down regulated P4 to
917
P5); the log2(FoldChange)P3-P4 ≥2; the level of expression in P4 stage ≥5; the level
918
of expression in mature leaves ≥5 (not for genes in the ‘peak’ cluster; data from
919
Davidson et al., 2012); the level of expression in rice endosperm ≤5 (Davidson et al.,
920
2012).
921
For genes putatively involved in vascular and stomatal development, genes of
922
interest from Arabidopsis thaliana and maize (Zea mays) were identified from the
923
literature (Scarpella and Meijer, 2004; Liu et al., 2009) (Supplemental Tables S5 and
924
S6). Rice orthologs of these genes were identified using conditional orthology
925
assignment as described by Aubry et al. (2014).
926
Accession numbers
927
Sequence data from this article can be found in the EMBL/GenBank data libraries
928
under accession number SRP062323.
35
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
929
930
Table 1 Expression patterns of rice genes known to be involved in photosynthetic
931
development.
Locus
Gene name(s)
Reference
Cluster
LOC_Os09g38980 TCD9
Jiang et al., 2014a
down 1
LOC_Os05g37160 OsFtsz2
Vitha et al., 2001
down 1
LOC_Os06g07210 RNRL1
Yoo et al., 2009
down 3
LOC_Os06g14620 RNRS1
Yoo et al., 2009
down 3
LOC_Os02g56100 RNRL2
Yoo et al., 2009
down 3
LOC_Os06g03720 RNRS2
Yoo et al., 2009
down 3
LOC_Os08g07840 OsPOLP1
Takeuchi et al., 2007
down 3
LOC_Os03g20460 OsGKpm, VIRESCENT2
Sugimoto et al., 2007
neutral
LOC_Os03g16430 SIG2B
Kasai et al., 2004
neutral
LOC_Os02g39340 OsDG2
Jiang et al., 2014b
neutral
LOC_Os03g45400 NUS1, VIRESCENT1
Kusumi et al., 1997; peak
Kusumi et al., 2011
LOC_Osp1g00240 rpoB
Hanaoka et al., 2005
peak
LOC_Osp1g00250 rpoC1
Hanaoka et al., 2005
peak
LOC_Osp1g00260 rpoC2
Hanaoka et al., 2005
peak
LOC_Os11g26160 SIG2A
Kasai et al., 2004
peak
LOC_Os06g44230 OsRpoTp
Kusumi et al., 2004
peak
LOC_Os09g21250 YLC1
Zhou et al., 2013
peak
LOC_Os05g23740 OsHsp70CP1
Kim and An, 2013
peak
LOC_Os03g29810 VYL, OsClp6
Dong et al., 2013
peak
LOC_Os05g32680 PAC
Meurer et al., 1998
peak
LOC_Os04g56970 OsFtsz1
Vitha et al., 2001
peak
LOC_Os04g42030 SNOW-WHITE LEAF1
Hayashi-Tsugane
et peak
al., 2014
LOC_Os08g06630 SIG1
Kasai et al., 2004
36
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
up 1
LOC_Osp1g00660 rpoA
Hanaoka et al., 2005
up 2
LOC_Os05g51150 SIG3
Kasai et al., 2004
up 2
LOC_Os04g39970 OsV4
Gong et al., 2014
up 2
LOC_Os05g50930 SIG5
Kasai et al., 2004
up 3
LOC_Os08g14450 SIG6
Kasai et al., 2004
up 3
LOC_Os06g40080 YGL2
Chen et al., 2013
up 3
LOC_Os10g35370 PORB
Kang et al., 2015
up 3
not found in rice
Kasai et al., 2004
N/A
SIG4
932
37
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
933
Supplemental Data Files
934
Supplemental Figure S1. Anatomy of the rice shoot apical meristem and developing
935
leaves.
936
Supplemental Figure S2. Absorbance of developing leaves.
937
Supplemental Figure S3. Induction and light response of NPQ.
938
Supplemental Figure S4. Stomatal density extremes.
939
Supplemental Figure S5 Leaf and stomatal characteristics after transfer from LL to
940
HL conditions.
941
Supplemental Figure S6. RNASeq quality control.
942
Supplemental Figure S7. Enrichment analysis of gene expression clusters.
943
Supplemental Table S1. A. Verification of RNASeq results by qPCR. B. qPCR primer
944
details.
945
Supplemental Table S2. Genes annotated with MapMan bin ‘PS’ (photosynthesis)
946
that reach ≥50% of maximal expression by P3 stage.
947
Supplemental Table S3. Genes annotated with MapMan bins A. 'Photosynthesis', B.
948
'Carotenoids' or C. 'Ascorbate and glutathione' and their expression patterns during
949
rice early leaf development.
950
Supplemental Table S4. Transcription factors that are potential regulators of
951
photosynthetic development.
952
Supplemental Table S5. A. Arabidopsis and maize genes known to regulate vascular
953
development (Scarpella and Meijer 2004) and their putative rice orthologs. B.
954
Expression patterns of other rice genes known to be involved in vascular
955
development.
956
Supplemental Table S6. Putative regulators of stomatal development in rice and their
957
expression patterns during early leaf development.
958
Supplemental Data 1. All rice RNASeq count data, median normalised.
38
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
959
ACKNOWLEDGEMENTS
960
The authors are grateful to Dr. Nicola Green at the Kroto Research Institute for
961
assistance with confocal imaging, Chris Hill for assistance with TEM and Marion
962
Bauch and staff at the NERC Biomolecular Analysis Facility in Sheffield for technical
963
assistance. JvC was supported by BBSRC CASE studentship BB/J012610/1 in
964
collaboration with the International Rice Research Institute (IRRI); MNY by a Malay
965
Government (JPA) Studentship and SN by a Thai Government Studentship; and CL,
966
SAR and AF by BBSRC grant BB/J004065. SK is a Royal Society University
967
Research Fellow.
968
AUTHOR CONTRIBUTIONS
969
AJF and WPQ designed the research; JvC, MNY, and SN performed research; SAR
970
contributed new analytic tools and SK contributed new computational tools; SK, VT,
971
SW, JvC, CC and MNY analyzed data; AJF and JvC wrote the paper, with all authors
972
contributing to the final version.
973
39
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
974
FIGURE LEGENDS
975
Figure 1 Analysis of leaf growth. (A) Leaf blade elongation over time for the first 5
976
leaves of IR64 rice. Error bars show standard deviation (n=10). (B) Dissected apices
977
to reveal leaf primordia at P1 (greater magnification in inset), P2, P3 and P4-1 (<1
978
cm) stages. (C) Plastochron index showing the relationship between the length of
979
leaf 3 and developmental stage (P1, P2, P3, P4) of leaf 5. (n=7). (D) Leaf primordia
980
at stages P3, P4-2 (<2 cm), P4-12 (<12 cm), P5 and P6 stages. Scale bars in B=
981
0.25 mm (P1, P2, P3) or 1 mm (P4-1); D = 1 cm.
982
Figure 2 Chlorophyll fluorescence and photosynthetic efficiency during early
983
leaf development. Raw chlorophyll fluorescence (left image) and φPSII (right image)
984
at 50 µmol m-2 s-1 in (A) P3, (B) P4, (C) P5 and (D) mature leaves. Images are
985
shown for 3 biological replicates for each leaf stage. Scale bars 0.25 mm. φPSII value
986
is indicated by the scale adjacent to D.
987
Figure
988
Transmission electron micrographs of plastids in leaves at (A) P3 stage; (B) P4
989
stage; (C) P5 stage; (D) mature leaves. (E) Chlorophyll autofluorescence in a P4
990
stage leaf. Scale bars in A-C= 0.5 µm, D=1 µm, E= 100 µm. G: stacked grana, *:
991
starch grain. Arrows indicate plastids. Locations sections were taken from are shown
992
in Supplemental Figure S1C.
993
Figure 4 Induction kinetics of photosynthesis during early leaf development.
994
Induction of φPSII at 50 µmol m-2 s-1 in different regions of (A) P3 stage and (B) P4
995
stage leaves and at 200 µmol m-2 s-1 in (C) P5 stage and (D) mature leaves. Light
996
response of φPSII in different regions of (E) P3 stage, (F) P4 stage, (G) P5 stage and
997
(H) mature leaves. Images are shown for 3 biological replicates for each leaf stage.
998
Regions of measurement, from tip to base, are indicated by the colour legend
999
adjacent to E.
3
Plastid differentiation during early leaf development. (A-D)
1000
Figure 5 Leaf morphogenesis in response to altered irradiance during early
1001
leaf development. (A) Vein differentiation in primordia at P2, P3, P4, P5 stages and
1002
at maturity. Locations sections were taken from are shown in Supplemental Figure
40
Downloaded from on June 14, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
1003
S1C. (B) Schematic of irradiance transfer experiments. Plants grown under high
1004
irradiance (HL) were either maintained under HL or transferred to low irradiance (LL)
1005
when leaf 5 had achieved specific developmental stages (P1, P3, P5), then
1006
maintained under LL until analysis. (C-E) Measurements of thickness (C), width (D)
1007
and area (E) in leaves grown under high irradiance (HL) and transferred to low
1008
irradiance (LL) at P1, P3 or P5 stages of leaf 5 development. Error bars show
1009
standard error (n=7). Different letters indicate statistically significant difference
1010
(Tukey HSD, P<0.05). (F) Light response curves for assimilation rates in leaf 5 of
1011
plants as described in B. Error bars show standard deviation, (n≥4). Scale bars in
1012
A= 0.05 mm.
1013
Figure 6 Stomatal size and density after leaf transfer between different
1014
irradiances. (A) Epidermis showing an interveinal gap with cell files containing
1015
stomatal complexes. (B-D) Measurements of stomatal density (B), interveinal
1016
distance (C), and stomatal complex area (D) in interveinal gaps (IG) across mature
1017
rice leaves grown under high irradiance, HL (solid line) and low irradiance, LL
1018
(broken line). Under LL conditions more IGs were generated, leading to the
1019
observation of IG17 and IG18 in a few LL leaves (number indicated by value n).
1020
Error bars indicate standard error, n = 5 except where indicated. (E) Individual
1021
stomatal complex indicating the supporting cells (SC) and the dimensions taken for
1022
stomatal complex area (SCA), stomatal complex length (SCL) and stomatal complex
1023
width (SCW). (F-M) Measurements of SCA (F), SCL (G), SCW (H), stomatal density
1024
(SD) (I), cell file width (CFW) (J), interveinal distance (IVD) (K), vein frequency (L)
1025
and cell file number (CFN) (M) in leaves either grown continually under high
1026
irradiance (HL), low irradiance (LL) or transferred from HL to LL at P1, P3 or P5
1027
stage. Where appropriate, identical letters in a graph indicate samples that could not
1028
be distinguished from each other by statistical analysis (see text). (N-P) Scanning
1029
Electron Microscope images of a P3 primordium (N), a higher magnification of the
1030
surface of a P3 primordium (no stomatal complexes visible) (O) and a mature
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stomatal complex in a P4 primordium (P). Scale bar in A = 30 µm. Scale bar in E = 5
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µm. Scale bars in N and O = 30 µm. Scale bar in P = 10 µm.
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Figure 7 Key changes in gene expression during early rice leaf development.
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(A) Leaf developmental stages used for RNA sequencing analysis and their position
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within the plant (not to scale; colours added for clarity). (B) Key functional terms up
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regulated and down regulated during development and selected genes reaching their
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maximum expression level at P3, P4 or P5 stage. Where a gene name is shown
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twice, rice has two orthologs of the Arabidopsis gene of interest. See also
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Supplemental Tables S2, S3, S4, S5, S6 and S7 and Supplemental Figure S7.
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Table 1 Expression patterns of rice genes known to be involved in photosynthetic
development.
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B
Figure 1 Analysis of leaf growth. (A) Leaf blade elongation over time for the first 5
leaves of IR64 rice. Error bars show standard deviation (n=10). (B) Dissected apices
to reveal leaf primordia at P1 (greater magnification in inset), P2, P3 and P4-1 (<1 cm)
stages. (C) Plastochron index showing the relationship between the length of leaf 3
and developmental stage (P1, P2, P3, P4) of leaf 5. (n=7). (D) Leaf primordia at stages
P3, P4-2 (<2 cm), P4-12 (<12 cm), P5 and P6 stages. Scale bars, B: 0.25 mm (P1
inset) or 1 mm (other images); D: 1 cm.
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Figure 2 Chlorophyll fluorescence and photosynthetic efficiency during early
leaf development. Raw chlorophyll fluorescence (left image) and φPSII (right image)
at 50 µmol m-2 s-1 in (A) P3, (B) P4, (C) P5 and (D) mature leaves. Images are shown
for 3 biological replicates for each leaf stage. Scale bars 0.25 mm. φPSII value is
indicated by the scale adjacent to D.
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Figure 3 Plastid differentiation during early leaf development. (A-D) Transmission
electron micrographs of plastids in leaves at (A) P3 stage; (B) P4 stage; (C) P5 stage;
(D) mature leaves. (E) Chlorophyll autofluorescence in a P4 stage leaf. Scale bars in
A-C= 0.5 µm, D=1 µm, E= 100 µm. G: stacked grana, *: starch grain. Arrows indicate
plastids. Locations sections were taken from are shown in Supplementary Figure S1C.
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Figure 4 Induction kinetics of photosynthesis during early leaf development.
Induction of φPSII at 50 µmol m-2 s-1 in different regions of (A) P3 stage and (B) P4
stage leaves and at 200 µmol m-2 s-1 in (C) P5 stage and (D) mature leaves. Light
response of φPSII in different regions of (E) P3 stage, (F) P4 stage, (G) P5 stage and
(H) mature leaves. Images are shown for 3 biological replicates for each leaf stage.
Regions of measurement, from tip to base, are indicated by the colour legend adjacent
to E.
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Figure 5 Leaf morphogenesis in response to altered irradiance during early leaf
development. (A) Vein differentiation in primordia at P2, P3, P4, P5 stages and at
maturity. Locations sections were taken from are shown in Supplementary Figure S1C.
(B) Schematic of irradiance transfer experiments. Plants grown under high irradiance
(HL) were either maintained under HL or transferred to low irradiance (LL) when leaf
5 had achieved specific developmental stages (P1, P3, P5), then maintained under LL
until analysis. (C-E) Measurements of thickness (C), width (D) and area (E) in leaves
grown under high irradiance (HL) and transferred to low irradiance (LL) at P1, P3 or
P5 stages of leaf 5 development. Error bars show standard error (n=7). Different letters
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indicate statistically significant difference (Tukey HSD, P<0.05). (F) Light response
curves for assimilation rates in leaf 5 of plants as described in B. Error bars show
standard deviation, (n≥4). Scale bars in A= 0.05 mm.
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Figure 6 Stomatal size and density after leaf transfer between different
irradiances. (A) Epidermis showing an interveinal gap with cell files containing
stomatal complexes. (B-D) Measurements of stomatal density (B), interveinal distance
(C), and stomatal complex area (D) in interveinal gaps (IG) across mature rice leaves
grown under high irradiance, HL (solid line) and low irradiance, LL (broken line). Under
LL conditions more IGs were generated, leading to the observation of IG17 and IG18
in a few LL leaves (number indicated by value n). Error bars indicate standard error, n
= 5 except where indicated. (E) Individual stomatal complex indicating the supporting
cells (SC) and the dimensions taken for stomatal complex area (SCA), stomatal
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complex length (SCL) and stomatal complex width (SCW). (F-M) Measurements of
SCA (F), SCL (G), SCW (H), stomatal density (SD) (I), cell file width (CFW) (J),
interveinal distance (IVD) (K), vein frequency (L) and cell file number (CFN) (M) in
leaves either grown continually under high irradiance (HL), low irradiance (LL) or
transferred from HL to LL at P1, P3 or P5 stage. Where appropriate, identical letters
in a graph indicate samples that could not be distinguished from each other by
statistical analysis (see text). (N-P) Scanning Electron Microscope images of a P3
primordium (N), a higher magnification of the surface of a P3 primordium (no stomatal
complexes visible) (O) and a mature stomatal complex in a P4 primordium (P). Scale
bar in A = 30 µm. Scale bar in E = 5 µm. Scale bars in N and O = 30 µm. Scale bar in
P = 10 µm.
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Figure 7 Key changes in gene expression during early rice leaf development. (A)
Leaf developmental stages used for RNA sequencing analysis and their position within
the plant (not to scale; colours added for clarity). (B) Key functional terms up regulated
and down regulated during development and selected genes reaching their maximum
expression level at P3, P4 or P5 stage. Where a gene name is shown twice, rice has
two orthologs of the Arabidopsis gene of interest. See also Supplementary Tables S2,
S3, S4, S5, S6 and S7 and Supplementary Figure S7.
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