Modulation of chlorophyll biosynthesis by water stress in rice seedlings during chloroplast
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biogenesis1
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Vijay K. Dalal and Baishnab C. Tripathy*
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School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
Accepted Article
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*corresponding author
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Supported by a grant from the Department of Biotechnology, Government of India grant
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(BT/PR14827/BCE/08/841/2010), University Grants Commission capacity build up funds, and
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Department of Science and Technology purse grant from Jawaharlal Nehru University, New
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Delhi.
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Running title: Modulation of chlorophyll biosynthesis by drought
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Address correspondence to:
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Baishnab C Tripathy
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School of Life Sciences
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Jawaharlal Nehru University
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New Delhi 110067
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India
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Phone: +91-11-26704524
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FAX: +91-11-26742558
This article has been accepted for publication and undergone full scientific peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as an ‘Accepted
Article’, doi: 10.1111/j.1365-3040.2012.02520.x
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© 2012 Blackwell Publishing Ltd
Email: [email protected]
Accepted Article
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World count: 12985
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Abstract
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To understand the impact of water stress on the greening process, water stress was applied to six-
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day old etiolated seedlings of a drought-sensitive cultivar of rice (Oryza sativa), Pusa Basmati-1
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by immersing their roots in 40 mM PEG 6000 (-0.69 MPa) or 50 mM PEG 6000 (-1.03 MPa)
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dissolved in half-strength MS-nutrient-solution, 16 h prior to transfer to cool-white-fluorescent +
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incandescent light. Chlorophyll accumulation substantially declined in developing water-stressed
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seedlings.
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biosynthetic
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protoporphyrin IX monomethylester and protochlorophyllide. Although, 5-aminolevulinic acid
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synthesis decreased, the gene expression and protein abundance of the enzyme responsible for its
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synthesis, glutamate-1-semialdehyde aminotransferase increased, suggesting its crucial role in
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the greening process in stressful environment. The biochemical activities of Chl biosynthetic
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enzymes
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Coproporphyrinogen
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Protochlorophyllide oxidoreductase were down-regulated due to their reduced protein
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abundance/gene expression in water-stressed seedlings. Down regulation of Protochlorophyllide
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oxidoreductase resulted in impaired Shibata shift. Our results demonstrate that reduced synthesis
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of early intermediates i.e. glutamate-1-semialdehyde and 5-aminolevulinic acid could modulate
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the gene expression of later enzymes of Chl biosynthesis pathway.
Reduced Chl synthesis was due to decreased accumulation of chlorophyll
intermediates
i.e.
i.e.
glutamate-1-semialdehyde,
5-aminolevulinic
III
oxidase,
acid
dehydratase,
Porphyrinogen
IX
5-aminolevulinic
Porphobilinogen
oxidase,
acid,
Mg-
deaminase,
Mg-chelatase
and
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Key words: Chlorophyll biosynthesis, Chloroplast biogenesis, Photosynthesis, Rice, Water-
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stress.
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© 2012 Blackwell Publishing Ltd
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Introduction:
Accepted Article
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Plant growth, development, photosynthesis and plant productivity are severely affected
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due to environmental stresses, particularly during early seedling growth. When seeds germinate
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beneath the soil, their seedlings remain in near-darkness for a while. In angiosperms, the
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differentiation of etioplast to chloroplast does not take place in dark as protochlorophyllide
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oxidoreductase (POR) enzyme (EC 1.3.33.1) requires light as a substrate to photo-transform
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Pchlide via trans addition of hydrogen across the C17–C18 double bond of the D-ring to form
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Chlide (Apel et al. 1980; Griffiths 1978; Santel & Apel 1981). Therefore, etiolated rice seedlings
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do not synthesize chlorophyll (Chl) and contain a special form of plastids called etioplasts or
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etiochloroplasts. As seedlings come out of soil, they are exposed to light and light-mediated
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chlorophyll biosynthesis and other associated greening processes are initiated resulting in
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transformation of etioplasts to chloroplasts (Waters & Pyke 2005). Chloroplast development
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involves the biosynthesis of components of photosynthetic apparatus involving synthesis of Chl,
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carotenoids, lipids and proteins which involves a coordinated function of chloroplast and nuclear
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genomes (Gray et al. 2003; Leon et al. 1998; Nott et al. 2006). Most steps of Chl biosynthesis
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are well understood (Bollivar 2006; Goslings et al. 2004; Meskauskiene et al. 2001; Tanaka &
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Tanaka 2007; Tripathy & Rebeiz 1986, 1987, 1988; Manohara & Tripathy 2000; Mohapatra &
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Tripathy 2002, 2007; Pattanayak et al. 2002, 2005; Pattanayak & Tripathy 2011; Wang et al.
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2010; Wu et al. 2007; Shalygo et al. 2009; Peter et al. 2010; Hedtke et al. 2007).
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Chloroplast development is influenced by external and internal factors such as light
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quality, salt, temperature, nutrition, leaf age and leaf water potential etc. (Dutta, Mohanty &
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Tripathy 2009; Bhardwaj & Singhal 1981; Bengston et al. 1978; Mohanty & Tripathy 2011;
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Virgin 1965). Chill-, heat- or salt-stressed seedlings have impaired Chl biosynthesis due to
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down-regulation of gene expression and protein abundance or due to post-translational
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modification of several enzymes involved in tetrapyrrole metabolism (Eskins et al. 1986; Sood,
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Tyagi & Tripathy 2004; Sood, Gupta & Tripathy 2005; Tewari & Tripathy 1998, 1999;
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Mohanty, Grimm & Tripathy 2006; Lay et al. 2000, 2001; Satpal & Tripathy unpublished).
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Water stress is another very important stress frequently encountered by plants due to
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scanty rainfall. It is also an important factor influencing the chloroplast development. Drought
© 2012 Blackwell Publishing Ltd
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substantially alters plant metabolism i.e. plant growth, photosynthesis and yield (Boyer 1982;
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Gimenez et al. 1992; Munns 2002; Loggini et al. 1999; Lawlor & Cornic 2002; Lawlor & Tezara
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2009; Chaves et al. 2009). Many studies have been already performed to know the impact of
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water stress on photosynthesis in developed leaves (Loggini et al. 1999; Giardi et al. 1996;
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Galmes et al. 2007; Massacci et al. 2008). Two principal mechanisms are invoked for decreased
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photosynthesis, i) restricted diffusion of CO2 into the intercellular spaces of leaves, caused by
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stomatal closure (Cornic 2000; Cornic & Briantais 1991), and ii) metabolic inhibition (Tezara et
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al. 1999; Gimenez et al. 1992). Metabolic down-regulation in developing green leaves could
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result from the reduced and improper assembly of photosynthetic complexes in thylakoid
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membranes due to reduction in Chl biosynthesis under water stress (Bhardwaj & Singhal 1981;
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Bengston et al. 1978). Photosynthetic proteins require Chl and carotenoids for their correct
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folding, assembly and insertion into thylakoid membranes (Kim et al. 1994; Horn & Paulsen
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2002). Because of reduced photosynthetic CO2 fixation in water-stressed plants, most of the
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NADPH is not utilized resulting in a highly reduced electron transport chain (ETC) prone to
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oxygen attack (Noctor et al. 2002; Chaves et al. 2009; Cornic & Briantais 1991; Lawlor &
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Tezara 2009). After extracting electrons from over-reduced ETC, O2 is converted to O2- that
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could potentially damage the photosynthetic apparatus and other cellular components (Chaves &
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Oliveira 2004). Singlet oxygen is also generated under such conditions. ROS could down-
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regulate the synthesis of tetrapyrroles intermediate Pchlide probably due to impaired MPE
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cyclase activity (Aarti et al. 2006; Stenbaek et al. 2008), that results in reduced Chl synthesis in
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plants.
Accepted Article
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Most of the pigment biosynthesis and their assembly to pigment-protein complexes occur
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in de-etiolating seedlings and stress severely affects early seedling growth. Therefore, the impact
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of water-stress on early seedling development and Chl biosynthesis was monitored to understand
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the mechanism of regulation of the greening process and Chl biosynthesis during light-induced
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chloroplast development. In the present study, we investigated the effect of water stress on
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pigment synthesis, gene and protein expression and activities of enzymes involved in Chl
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biosynthesis in drought-sensitive rice cultivar, PB-1 during light-induced chloroplast
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development. Our results demonstrate that the Shibata shift, observed during very early light-
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induced chloroplast development is impaired and Chl biosynthesis is substantially down-
© 2012 Blackwell Publishing Ltd
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regulated due to water stress. It further reveals that Chl biosynthesis is down-regulated to
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prevent the accumulation of harmful singlet oxygen generating tetrapyrroles at a very early step
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i.e. ALA synthesis, due to reduced gene expression of early enzymes of Chl biosynthesis
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pathway.
Accepted Article
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Materials and Methods
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Plant Material
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Drought-sensitive rice (Oryza sativa L.) cultivar Pusa Basmati-1 (PB-1) was used as
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experimental material. Seeds of PB-1 were obtained from Indian Agricultural Research Institute
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(I.A.R.I.), Pusa, New Delhi.
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Plant Growth Conditions and application of water-stress.
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Seeds were washed with tap water and soaked in water for 24 h. Seeds were grown on
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germination paper using half-strength Murashige and Skoog (MS) liquid media having no agar
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or vitamins. Seeds were grown first in complete darkness for six days at 28 0C before giving
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water stress. To study the regulation of chlorophyll biosynthesis by water stress, different
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concentrations of polyethylene glycol 6000 (PEG 6000) dissolved in half strength MS growth
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medium [40 mM PEG (osmotic potential, -0.69 MPa) or 50 mM PEG 6000 (osmotic potential, -
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1.03 MPa)] (Michel & Kaufmann 1973) were added in dark to the roots of seedlings maintained
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at 28 0C, 16 h prior to transfer to cool-white-fluorescent + incandescent light (100 µmoles
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photons m-2 s-1). After every 12 h, water was added to compensate for the evaporation from
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growth medium.
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Chlorophyll, carotenoid and protein estimation
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Chl and carotenoid contents were estimated as described by Porra, Thompson &
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Kriedemann (1989) and Welburn & Lichtenthaler (1984). Protein was estimated according to
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Bradford (1976).
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© 2012 Blackwell Publishing Ltd
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Accepted Article
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Calculation of correction factors
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Several parameters, i.e. Chl content, protein contents etc. were expressed on per g fresh
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weight basis. However, due to water stress, there was severe loss of moisture that resulted in
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reduction in relative water contents of plants. To correct for the loss of moisture, the pigment or
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protein determined on fresh weight basis was multiplied by simultaneously measured correction
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factor, determined as DW of 100 mg of control samples/DW of 100 mg of stressed samples.
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GSA Content
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GSA content was measured as described by Kannangara & Schouboe (1985) and Sood et
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al. (2005). Leaf samples (200 mg) were taken, weighed and one set was processed immediately
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for GSA estimation (0 h); another set was incubated in 500 µM gabaculine in 0.1 M MES (pH
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7.0) for 4 h in light. The tissues were hand homogenized in pre-chilled mortar and pestle in 5.0
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mL of 0.1 N HCl and centrifuged at 10000 rpm for 10 min at 4 0C. Supernatant was taken for
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GSA estimation. Reaction mixture contained: 400 µL of supernatant, 360 µL of 0.1 N HCl, 80
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µL of 2% 3-methyl-2-benzothiazolinonehydrazone (MBTH). This was then incubated in boiling
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water bath for two minutes and cooled rapidly. Boiling was done in capped tubes. Reference
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cuvette contained 400 µL of 0.1 N HCl and no supernatant was added to it. After cooling 760 µL
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of distilled water and 40 µL of 20% FeCl3 was added to it, mixed by vortexing and read at 620
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nm. Extinction coefficient used was 16.9 mM-1. Net GSA synthesized during 4 h period was
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measured by subtracting the 0 h GSA from 4 h GSA content.
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ALA Content
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ALA content was measured according to Harel & Klein (1972); Tewari & Tripathy
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(1998). Leaf samples (200 mg) were taken at different time points. One set was kept in 60 mM
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levulinic acid (LA) for 4 h in 100 µmoles photons m-2 s-1 light and another set was processed
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immediately for ALA estimation (0 h). Tissues were hand homogenized in a pre-chilled mortar
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and pestle in 5 mL of 1 M sodium acetate buffer (pH 4.6). The homogenate was centrifuged at
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10000 rpm for 10 min and supernatant was taken for assay. The assay mixture consisted of 1 mL
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of supernatant, 4 mL of distilled water and 250 µL of acetylacetone. The assay medium was
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mixed properly and heated in a boiling water bath for 10 min. Then the extract was cooled at
© 2012 Blackwell Publishing Ltd
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room temperature and an equal volume of modified Ehrlich’s reagent was added to it and
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vortexed for 2 min. After 10 min of incubation, absorbance of the extract was measured at 555
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nm. ALA content was determined from the standard curve prepared from known concentration
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of ALA (Sood et al. 2005). Net ALA synthesized during 4 h period was measured by subtracting
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the 0 h ALA from 4 h ALA content.
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Pchlide Content
Accepted Article
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Pchlide contents were estimated according to Tewari & Tripathy (1998) and Hukmani &
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Tripathy (1992). Briefly, leaf tissues were homogenized in 90% chilled ammoniacal acetone (10
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mL) in a pre-chilled mortar and pestle. The homogenate was centrifuged at 10000 rpm for 10
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min at 4 0C. Supernatant was taken and hexane extracted acetone residue solvent mixture
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(HEAR) was prepared according to Chakraborty & Tripathy (1992). Emission spectra E440,
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E400 of HEAR were recorded from 580 nm to 700 nm and corrected for photomultiplier tube
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response.
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ALA Dehydratase (ALAD, EC 5.4.3.8)
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Preparation of Ehlrich reagent: Ehlrich reagent was prepared as described by
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Mauzerall & Granick (1956). For preparing Ehlrich reagent 2 g of dimethyl amino benzaldehyde
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(DMAB) was dissolved in 30 mL of glacial acetic acid and 16 mL of 70% perchloric acid (4 N)
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was added to it. Final volume was made upto 50 mL with glacial acetic acid.
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ALAD assay was performed according to Hukmani & Tripathy 1994; Tewari & Tripathy
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1998. Three replicates of 200 mg leaves were taken from seedlings grown in white light. Leaves
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were homogenized at 4 0C in 5 mL of 0.1 M Tris (pH 7.6), 10 mM 2-mercaptoethanol in a pre-
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chilled mortar and pestle. The homogenate was centrifuged 10000 rpm for 10 min at 4 0C.
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Supernatant was taken for enzyme assay, performed as described by Shemin (1962) and Tewari
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& Tripathy (1998). PBG formed was calculated using the absorption coefficient of 6.2 x 104 M-1
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cm-1.
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Porphobilinogen Deaminase (PBGD, EC 2.5.1.61)
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Three replicates of 200 mg seedlings were hand homogenized in mortar and pestle in 5.0
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mL of phosphate buffer (pH 8.0) and 0.6 mM EDTA at 4 °C and passed through 4 layers of
© 2012 Blackwell Publishing Ltd
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cheese cloth. Homogenate was centrifuged at 4 °C. Supernatant was taken for assay. Enzyme
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assay was performed according to Hukmani & Tripathy (1994) and Tewari & Tripathy (1998).
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Enzyme activity was assayed as amount of porphyrin synthesized in 3.0 mL of reaction mixture
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having following composition as final concentrations: freshly prepared 0.6 mL PBG (550 µM),
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90 µL EDTA solution (0.6 mM), 428 µL phosphate buffer (pH 8.0), 1.8 mL of enzyme extract
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and 82 µL distilled water. In blank distilled water was added instead of enzyme. The reaction
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mixture was incubated at 37 °C for 1 h. 750 µL of this reaction mixture was taken to which 1.7
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mL of TCA (7.1%) was added to stop the reaction. Centrifuged at 10000 rpm for 10 min in
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sorvall SS-34 tubes at 4 °C. Then 10 µL of iodine solution (1%) was added and incubated for 5
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min at 37 oC. Finally 20 µL of freshly prepared sodium thiosulphate (2%) was added to reduce
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iodine.
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porphyrin was 5.48 x105 M-1 cm-1. Absorbance of all samples was taken from 400-700 nm to
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check for a peak at 405.5 or 406 nm.
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Preparation of Coprogen III, and Protogen IX
Accepted Article
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Absorbance was measured at 406 nm. Extinction coefficient used at 406 nm for
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Sodium amalgam was prepared according to Jacobs & Jacobs (1982). Reduction of
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porphyrins was carried out according to the method of Poulson & Polglosse (1974) with some
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modifications. Porphyrinogens were freshly prepared by reducing porphyrins, namely
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coproporphyrin III, or Proto IX. Porphyrins were dissolved in 20% ethanol containing 0.1 N
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KOH and centrifuged at room temperature. Supernatant was taken for performing reduction.
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Oxygen present in the solution was removed by flushing the solution with nitrogen gas. To this
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solution 5 g of 3% sodium amalgam was added with continuous flushing of solution with
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nitrogen gas. The reduction was carried out for 2 to 3 min; the solution becomes colorless during
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this time. Reduction was continued for 2 to 3 min more to ensure complete reduction of the
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porphyrin. Immediately, the contents were filtered. The pH of the porphyrinogen solution was
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carefully adjusted to 7.5-7.8 with 40% orthophosphoric acid.
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Coproporphyrinogen oxidase (CPO, EC 1.3.3.3)
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Leaves were taken from seedlings after 24 h of greening and hand homogenized in pre-
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chilled mortar and pestle in isolation buffer containing 0.5 M sucrose, 20 mM Hepes (pH 7.7), 1
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mM MgCl2, 1 mM Na2EDTA, 0.2% BSA (w/v). Homogenate was passed through 8 layers of
© 2012 Blackwell Publishing Ltd
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cheese cloth, one layer of Mira cloth and centrifuged at 4 0C. Pellet containing plastids was
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suspended in lysis buffer containing 0.01 M Tris-HCl (pH 7.7), 20 mM MgCl2, and 2.5 mM
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EDTA and kept on ice for 10 min. This was then centrifuged at 4 0C. Supernatant was taken for
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assay, done as described by Tewari & Tripathy (1998). Synthesis of protoporphyrin IX from
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reduced coporphyrinogen was measured by spectrofluorimetry (Hukmani & Tripathy 1992;
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Tewari & Tripathy 1998).
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Protoporphyrinogen oxidase (protox, EC 1.3.3.4)
Accepted Article
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Protox assay was performed as described by Tewari & Tripathy (1998). Leaves were
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taken from seedlings after 24 h of greening and hand homogenized in prechilled mortar and
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pestle in isolation buffer containing 0.5 M sucrose, 20 mM Hepes (pH 7.7), 1 mM MgCl2, 1 mM
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Na2EDTA, 0.2% BSA (w/v). Homogenate was passed through 8 layers of cheese cloth, one layer
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of mira cloth and centrifuged at 4 0C. Pellet containing plastids was suspended in lysis buffer
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containing 0.01 M Tris-HCl (pH 7.7), 20 mM MgCl2, and 2.5 mM EDTA and kept on ice for 10
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min. This was then centrifuged at 4 0C. Supernatant was taken for assay and synthesis of
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protoporphyrin IX from reduced protoporphyrinogen was measured by spectrofluorimetry
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(Hukmani & Tripathy 1992; Tewari & Tripathy 1998.
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Mg-chelatase (EC 6.6.1.1)
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Assay was done as described by Tewari & Tripathy (1998). Leaves were taken from
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seedlings after 24 h of greening and hand homogenized in pre-chilled mortar and pestle in
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isolation buffer containing 0.5 M sucrose, 20 mM Hepes (pH 7.7), 1 mM MgCl2, 1 mM
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Na2EDTA, 0.2% BSA. Homogenate was passed through 8 layers of cheese cloth, one layer of
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Mira cloth and centrifuged at 4 0C. The pellet was gently suspended in suspension buffer
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containing 0.5 M Sucrose, 0.2 M Tris-HCl, 20 mM MgCl2, 2.5 mM Na2EDTA, and 20 mM ATP,
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at room-temperature (pH 7.7). The synthesis of MP(E) from protoporphyrin IX was monitored as
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described before (Tewari & Tripathy 1998; Hukmani & Tripathy 1992).
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Protochlorophyllide oxidoreductase (POR, EC 1.3.33.1)
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POR activity was measured as described elsewhere (Tewari & Tripathy 1998). Water-stress was
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applied to etiolated rice seedlings 16 h prior to transfer to light for 72 h. Five replicates of 50 mg
© 2012 Blackwell Publishing Ltd
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leaves were harvested from the seedlings under illumination and were transferred to 6 h dark and
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subsequently transferred to light (100 µmoles photons m-2 s-1) for 1 h. Five batches of 50 mg
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leaves were excised from the seedlings before the end of 6 h dark period and immediately after 1
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h photo-period. Leaves were immediately homogenized in 90% ammoniacal acetone, acetone:
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0.1 N NH4OH=9:1), in dark under safe green light and their Pchlide contents were estimated by
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spectrofluorimetry (Hukmani & Tripathy 1992). The difference between the net amount of
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Pchlide synthesized in dark and the same remaining after 1 h of light treatment is the amount of
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Pchlide phototransformed from Pchlide to Chlide.
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percent phototransformation of Pchlide, accumulated during 6 h dark period, to Chlide as
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follows: [(Net amount of Pchlide synthesized in 6 h of dark period - Pchlide content remaining
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after 1 h of light exposure)/Net amount of Pchlide synthesized in 6 h of dark period] x 100.
Accepted Article
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POR activity was measured as relative
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RNA Isolation, RT-PCR Analysis
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RNA was extracted from fresh, control and water stressed rice seedlings using Tri®
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reagent (Sigma, USA) according to the manufacturer’s instructions. RNA quantification was
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done using Nanodrop spectrophotometer (ND 1000, USA) and confirmed by applying equal
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amounts of total RNA to an agarose gel using Ethidium bromide staining. First-strand cDNA
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was synthesized using 2 µg of total RNA using RevertAidTM H Minus M-MuLV Reverse
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Transcriptase (Fermentas, USA) in a 20 µL reaction according to manufacturer’s protocol.
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Semiquantitative RT-PCR was performed as described by Burch-Smith et al. (2006). After
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synthesis, the cDNA was diluted 1:10, and 2 µL of cDNA was used as a template for PCR
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amplification in a 20 µL reaction mixture.
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PCR was performed for 26 to 29 cycles within a linear range of amplification for all
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genes. The number of cycles and annealing temperature were optimized for each specific primer
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pairs. Gene specific primers used in the study are provided in Supplimentary Information Table
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S1. Ten µL of the PCR products were loaded and separated on 0.8-1% agarose Tris-acetate
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EDTA gel. Ethidium bromide-stained PCR products were quantified using an Alpha Imager
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3400. RT-PCR for each gene was performed in triplicates. The average values were determined
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using Alpha Ease FC software.
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© 2012 Blackwell Publishing Ltd
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Plastids Isolation and Western Blotting
Accepted Article
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Plastids were isolated as described by Chakraborty & Tripathy 1992. Transfer of proteins
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from polyacrylamide gels to nitrocellulose (NC) membranes was carried out in a semi-dry
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Transblot apparatus (ATTO Corp., Tokyo, Japan), as recommended by the manufacturer. Blots
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were stained with alkaline phosphatase using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and
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nitroblue tetrazolium (NBT) (Jilani et al. 1996). The Western-blot analysis was performed twice.
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Polyclonal and heterologous antibodies were used (Supplimentary Information Table S2) for
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some major enzymes of Chl biosynthetic pathway.
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Fluorescence spectra of leaves for Shibata shift
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Water stress was given in dark to six-day old etiolated rice seedlings. After 16 h of water-
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stress-treatment etiolated leaves were excised and were given a red light flash. Subsequently,
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they were frozen in liquid N2 after 0, 1 or 15 min of dark incubation and their fluorescence
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emission spectra (77K) were recorded at excitation wavelength of 440 nm and emission
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wavelength of 600-740 nm. Spectra were not corrected for photomultiplier tube response.
16
17
Results
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Plants growth:
19
A drought-sensitive and high yielding cultivar of rice, [Oryza sativa L. cv Pusa Basmati-
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1, (PB-1)] was taken for study. Rice seeds were germinated and grown in polyvinyl chloride
21
boxes on moist germination paper at 28 0C in dark. Six-day old etiolated rice seedlings were
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treated with 40 mM or 50 mM PEG 6000 dissolved in half-strength MS salt solution for 16 h and
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subsequently transferred to cool-white-fluorescent + incandescent light (100 µmoles photons m-2
24
s-1) at 28 0C for 24-72 h. After water-stress was applied, seedlings progressively rolled their
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leaves. As the greening progressed, water-stressed seedlings showed reduced greening and
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stunted growth (Fig. 1).
© 2012 Blackwell Publishing Ltd
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Shoot/Root length
Accepted Article
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As compared to their respective controls, the shoot and root length of water-stressed
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seedlings (50 mM PEG-treated) declined by 35% and 10% respectively after 72 h of stress
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treatment (Fig. 1E).
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Shoot Dry Weight
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The shoot dry weight (DW) of control seedlings increased by 19% and 32% after 48 h
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and 72 h of light exposure (Fig. 2) compared to 24 h DW. However, in water-stressed seedlings,
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shoot dry weight declined as the greening of leaves was substantially down-regulated. As
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compared control, after 72 h of stress, their dry weight was reduced by 22% and 28% due to 40
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mM or 50 mM PEG-treatment respectively.
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Protein content
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The protein content of stressed seedlings determined after 24-72 h of greening was
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multiplied by simultaneously measured correction factor to take into account the loss of moisture
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due to water stress. Protein content was 21.05 mg gFW-1 in control seedlings. It declined to14.94
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mg gFW-1 (-29%) and 11.31 mg gFW-1 (-46%) in 40 mM or 50 mM PEG treated seedlings
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respectively (Fig. 2).
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Chlorophyll
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Chl content of control and water-stressed seedlings is shown in Fig. 3. Shoots of control
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seedlings had maximum Chl biosynthesis after 72 h of greening. The Chl content of stressed
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seedlings determined after 24-72 h of greening was multiplied by simultaneously measured
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correction factor to take into account the loss of moisture due to water stress. Chl content,
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corrected for the loss of moisture in stressed-seedlings, decreased from 1.054 mg gFW-1 in
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control to 0.931 mg gFW-1 (-12%) or 0.607 mg gFW-1 (-42%) in 40mM or 50 mM PEG-treated
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rice seedlings respectively after 72 h of continuous de-etiolation (Fig. 3).
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Carotenoids
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Carotenoids contents, corrected for the loss of moisture as described for Chl, of rice
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seedlings increased with increase of greening period and decreased in response to water stress
© 2012 Blackwell Publishing Ltd
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(Fig. 3). Rice seedlings treated with 50 mM PEG, had reduced (27%) carotenoids abundance
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after 72 h of greening (Fig. 3).
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Chl Biosynthesis intermediates
Accepted Article
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Chloroplast biogenesis and Chl accumulation was down-regulated in water-stressed
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seedlings. To understand the mechanism of down-regulation of Chl accumulation, the steady
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state levels of Chl biosynthetic intermediates were measured.
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Glutamate 1-semialdehyde (GSA): GSA is the precursor of ALA in chlorophyll biosynthesis.
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To prevent the conversion of accumulated GSA to ALA, gabaculine, an inhibitor of GSA-AT,
9
was added to the incubation medium. GSA accumulation, corrected for the loss of moisture as
10
described for Chl, decreased in response to water stress. After 72 h of greening in the presence of
11
50 mM PEG, GSA accumulation declined from 8.84 µmols gFW-1 in control seedlings to 3.98
12
µmols gFW-1 in stressed seedlings i.e. by 55% (Fig. 4A).
13
δ-Amino levulinic acid (ALA): After GSA, next early intermediate of chlorophyll biosynthesis
14
is ALA. Net accumulation of ALA was measured in the presence of levulinic acid (LA), an
15
inhibitor of ALA dehydratase.
16
described for Chl, decreased drastically in response water stress. In control seedlings, after 72 h
17
of greening, ALA content was 251.33 nmols gFW-1, however in stressed samples it was reduced
18
by 70% to only 75.46 nmols gFW-1 (Fig. 4B).
19
Mg-protoporphyrin IX monomethyl ester [MP(E)]: Later metabolic products i.e. Mg-
20
protoporphyrin IX and its monomethyl ester [MP(E)], corrected for the loss of moisture as
21
described for Chl, were measured after 72 h of light exposure in control and 50 mM PEG-treated
22
seedlings. MP(E) contents were reduced to 47% of control (3.28 nmols gFW-1) in stressed
23
seedlings (1.53 nmols gFW-1) (Fig. 4C).
24
Protochlorophyllide (Pchlide):
25
process. After 72 h of greening, the steady state Pchlide content corrected for the loss of moisture
26
as described for Chl, declined by 66% (Fig. 4D) from 14.48 nmols gFW-1 in control seedlings to
27
5.40 nmols gFW-1 in water-stressed seedlings.
ALA accumulation, corrected for the loss of moisture as
MP(E) is further metabolized to Pchlide during greening
28
© 2012 Blackwell Publishing Ltd
13
Chlorophyll biosynthetic enzymes:
Accepted Article
1
2
To understand the mechanism of impairment of Chl biosynthesis and that of Chl
3
biosynthetic intermediates due to water stress, different enzymes involved in Chl biosynthesis
4
were studied.
5
ALA dehydratase (ALAD):
6
Enzyme activity of ALAD that converts two molecules of ALA to PBG was determined
7
in 50 mM PEG-induced water-stressed seedlings after 72 h of greening. In stressed seedlings,
8
ALAD activity decreased by 33% (Fig. 5A). ALAD activity was 31.93 nmols mg protein-1 h-1 in
9
control seedlings and 21.26 nmols mg protein-1 h-1 in treated seedlings.
10
Porphobilinogen deaminase (PBGD):
11
The next step in the Chl biosynthesis is the conversion of four molecules of
12
porphobilinogen to uroporphyrinogen, which is catalyzed by PBGD. The enzyme activity was
13
estimated by measuring the amount of porphyrin synthesis from porphobilinogen (PBG). After
14
72 h of greening the PBGD activity declined by 32% in stressed seedlings (Fig. 5B). PBGD
15
activity was 5.03 nmols mg protein-1 h-1 in control seedlings and 3.44 nmols mg protein-1 h-1 in
16
water-stressed seedlings.
17
Coproporphyrinogen III oxidase (CPO):
18
This enzyme catalyzes the conversion of coproporphyrinogen III to protoporphyrinogen
19
IX. Coupled activity of CPO was estimated by measuring the amount of proto IX formed from
20
coproporphyrinogen III. Its activity is high during early plastid development and chlorophyll
21
biosynthesis. The CPO activity, measured at 24 h of greening, was reduced by 33% in response
22
to water stress (Fig. 5C). Its enzymatic activity reduced from 53.93 nmols 100 mg protein-1 h-1 in
23
control seedlings to 35.66 nmols 100 mg protein-1 h-1 in treated seedlings.
24
Protoporphyrinogen IX oxidase (protox):
25
Protox catalyzes the conversion of protoporphyrinogen IX to protoporphyrin IX. Its
26
activity was estimated by measuring the amount of protoporphyrin IX formed from
27
protoporphyrinogen IX after 24 h of greening. As compared to control, the protox activity was
28
reduced by 38% in water-stressed seedlings (Fig. 5D) to 1.38 µmols 100 mg protein-1 h -1.
© 2012 Blackwell Publishing Ltd
14
Mg- chelatase (EC 6.6.1.1):
Accepted Article
1
2
Mg-chelatase is the first enzyme in the Mg branch of the Chl biosynthetic pathway that
3
inserts Mg into Proto IX to form Mg-protoporphyrin IX (Mg-Proto IX). Proto IX was used as a
4
substrate and Mg-chelatase activity was measured as the amount of Mg-Proto IX and its
5
monomethylester [MP(E)] synthesized in control and stressed samples. MP(E) synthesizing
6
capacity of Mg-chelatase was reduced by half from 2.46 nmols 100 mg protein-1 h-1 in control
7
seedlings to 1.22 nmols 100 mg protein-1 h-1 in water-stressed seedlings (Fig. 5E) after 24 h of
8
stress.
9
Protochlorophyllide oxidoreductase (POR):
10
Protochlorophyllide (Pchlide) accumulates in dark and conversion of Pchlide to
11
chlorophyllide (Chlide) is catalyzed in angiosperms by a light-dependent enzyme
12
protochlorophyllide oxidoreductase (POR). The POR activity was estimated by measuring the
13
photo-transformation of Pchlide to Chlide. After 72 h of greening seedlings were transferred to
14
dark for 6 h, a few leaves were harvested and homogenized in dark. Subsequently, seedlings
15
were transferred to light (100 µmoles photons m-2 s-1) for 1 h and their leaves were harvested and
16
homogenized. Pchlide contents of dark incubated and 1 h light-exposed seedlings were estimated
17
as described in materials and methods. POR activity, calculated as relative percent photo-
18
transformation of Pchlide, accumulated during 6 h dark period to Chlide, was 38% of control in
19
stressed seedlings (Fig. 5F).
20
21
Shibata shift:
22
To understand the impact of water stress during very early seedling development the
23
Shibata shift was monitored in 50 mM PEG-treated etiolated seedlings. Six-day old etiolated
24
control or water-stressed seedlings were illuminated by a pulse of red light (1500 µmoles
25
photons m-2 s-1) of 0.2 seconds and were frozen in liquid nitrogen either immediately after red
26
flash or after 1 min or 15 min of dark incubation subsequent to flash illumination. Fluorescence
27
emission spectra (E440) of leaves were recorded before as well as after the red flash, at low
28
temperature (77K). Before flash illumination fluorescence spectra showed a characteristic peak
29
at 632 nm due to non-phototransformable Pchlide and a peak at 657 nm due to
© 2012 Blackwell Publishing Ltd
15
phototransformable Pchlide. The non-phototransformable to phototransformable Pchlide ratio i.e.
2
E440F632:E440F657 increased from 0.1 in control etiolated leaves to 0.15 in water-stressed
3
etiolated leaves. In control samples, following flash illumination, the 657 nm peak declined due
4
to photo-transformation of Pchlide with simultaneous appearance of peak at 691 nm (Fig. 6A).
5
After 1 min of dark incubation, peak shifted to a longer wavelength at 694 nm. After 15 min of
6
dark incubation of flash-illuminated samples, the 694 nm-peak shifted to a lower wavelength i.e.
7
680 nm with a concomitant increase of peak at 657 nm due to regeneration of
8
phototransformable Pchlide (Fig. 6A).
Accepted Article
1
9
Leaves from stressed seedlings exhibited a peak at 692 nm with a concomitant decrease
10
of phototransformable peak at 657 nm, immediately after flash illumination (Fig. 6B). After 1
11
min of flash, the peak shifted to 694 nm. After 15 min of dark incubation following red flash, the
12
peak at 694 nm shifted to 690 nm and only a hump appeared around 680 nm (Fig. 6B). The peak
13
at 657 nm due to regeneration of phototransformable Pchlide was substantially reduced in
14
stressed samples.
15
16
17
18
Gene expression of chlorophyll biosynthetic enzymes:
To correlate the enzymatic activities with the gene expression, RT-PCR analysis was
performed after 24 h and 72 h of greening in response to water stress (Fig. 7).
19
HemA1 encodes Glutamyl-t-RNA reductase 1 that catalyses the conversion of Glutamyl-
20
t-RNA into Glutamate-1-semialdehyde (GSA) in photosynthetic tissues. In stressed seedlings,
21
HemA1 expression was reduced by 26-30% after 24-72 h of greening (Figs. 7 A & B). In the
22
same vein, the GSA synthesis declined in stressed seedlings.
23
24
25
26
27
28
Although, the ALA synthesis decreased, the gene expression of gsa was highly up-
regulated i.e. by 230% at 24 h and 90% at 72 h of stress treatment (Figs. 7 A & B).
The message abundance of Alad declined by 34% and 65% after 24 h and 72 h of stress-
treatment respectively (Figs. 7 A & B).
In water-stressed seedlings, transcript levels of pbgd decreased by 50% after 24-72 h of
greening (Figs. 7 A & B).
© 2012 Blackwell Publishing Ltd
16
The message abundance of UroD declined in response to water stress by 50-60% after
Accepted Article
1
2
3
4
24-72 h of greening (Figs. 7 A & B).
Coprox/CPO transcript abundance decreased in stressed samples by 20% after 24-72 h of
greening (Figs. 7 A & B).
5
The gene expression of PPX-1 that codes for protporphyrinogen oxidase, declined in
6
water-stressed seedlings by 17% and 28% after 24 h and 72 h of water stress treatment
7
respectively (Figs. 7 A & B).
8
9
10
11
The Chlorina 1 (ChlD) that encodes for subunit D of Mg-chelatase was down-regulated
by 40% after 24 h and by 59% after 72 h of stress treatment (Fig. 7 A & B).
The transcript levels of Chlorina 9 (ChlI) decreased in stressed seedlings after 24-72 h of
stress application by 26-28% as compared to that of control (Figs. 7 A & B).
12
The expression of HemH2 that encodes for ferrochelatase 2, the first enzyme of heme
13
biosynyhetic pathway, shares substrate (Protoporphyrin IX) with Mg-chelatase and is mostly
14
expressed in green tissue, decreased after 24 h of stress by 35% (Figs. 7 A & B).
15
The transcripts abundance of rice homolog of Xantha-l (Chl27) that encodes for the
16
membrane-bound subunit of MPE cyclase decreased by 36% after 24-72 h of stress treatment
17
(Figs. 7 A & B).
18
19
In stressed seedling, Por B expression was lesser than control at both time points. Por B
transcript abundance decreased by 45-55% after 24-72 h of water stress (Figs. 7 A & B).
20
21
Immunoblot analysis of chlorophyll biosynthesis pathway enzymes:
22
To correlate the enzymatic activities and gene expression with that of protein abundance,
23
western blot analysis of certain enzymes involved in Chl biosynthesis was performed in control
24
and stressed seedlings with plastidic protein (30 µg). Equal amount of protein was loaded in each
25
lane as shown in Fig. 8A.
26
Gluamate-1-semialdehyde aminotransferase (GSA-AT, EC 5.4.3.8):
© 2012 Blackwell Publishing Ltd
17
In agreement with that of gene expression, the protein abundance increased in control
2
seedlings during greening. Due to stress treatment, its protein abundance was up-regulated by
3
35% after 72 h of greening (Figs. 8 B & C).
4
Uroporphyrinogen III decarboxylase (UroD EC 4.1.1.37):
Accepted Article
1
5
Protein abundance of UROD which catalyses the conversion of uroporphyrinogen III to
6
coproporphyrinogen III deceased greatly in water-stressed seedlings at 24 h (by 35%) and 72 h
7
(by 38%) of greening (Figs. 8 B & C).
8
Coproporphyrinogen III oxidase (CPO):
9
Abundance of CPO which catalyses the conversion of coproporphyrinogen III to
10
protoporphyrinogen IX, deceased by 45-50% after 24-72 h of greening in water-stressed
11
seedlings (Figs. 8 B & C).
12
Protoporphyrinogen oxidase:
13
The protein abundance of plastidic protoporphyrinogen oxidase I (PPX1), decreased
14
drastically in water-stressed seedlings by 65-70% after 24-72 h of stress treatment (Figs 8. B &
15
C).
16
Magnesium chelatase subunit I (Chl I):
17
ChlI protein abundance was only partially reduced in water-stressed seedlings after 24 h
18
and 72 h of greening (Figs. 8 B & C).
19
Protochlorophyllide oxidoreductase (POR):
20
POR protein abundance declined in stressed seedlings by 87-89% after 24-72 h of stress
21
treatment (Figs. 8 B & C).
22
Geranyl-geranyl reductase (Chl P, EC 1.3.1.83):
23
24
In water-stressed seedlings Chl P declined severely after 24 h and 72 h of stress (Figs. 8
B & C) by 62-68%.
25
26
Discussion:
© 2012 Blackwell Publishing Ltd
18
Results demonstrate that during the de-etiolation of control rice seedlings, Chl contents
2
per gram fresh weight increased with period of light exposure and saturated at 72 h of
3
illumination (Fig. 3). In response to water-stress, Chl biosynthesis was down-regulated (42%).
4
Almost similar response during de-etiolation was seen in cucumber, wheat and other seedlings in
5
response to temperature and salt stress (Mohanty et al. 2006; Tewari & Tripathy 1998, Satpal &
6
Tripathy unpublished).
Accepted Article
1
7
The reduced Chl synthesis in stressed seedlings was mostly due to down-regulation of
8
early intermediates of Chl biosynthesis i.e. GSA and ALA (Figs. 4A & 4B). In the same vein,
9
the down-regulation of GSA/ALA contents was observed in other stresses (Table 1) during early
10
seedling development in salt- (Satpal & Tripathy unpublished), chill- and heat-stressed
11
rice/maize/cucumber/Pinus seedlings (Hodgins and Van Huystee 1986 Tewari & Tripathy 1998;
12
Hodgins and Oquist 2006).
13
Reduced GSA synthesis in water-stressed rice seedlings was due to down-regulation of
14
HemA1 transcript abundance (Fig. 7). Similarly, its expression was down-regulated in chill- and
15
heat-stressed cucumber and wheat seedlings. In contrast, in response to salt-stress, the gene
16
expression of HemA1 was partially up-regulated in salt-tolerant and was not affected in salt-
17
sensitive rice genotype. HemA1 is mainly expressed in photosynthetic tissue. HemA2 is
18
predominantly expressed in roots and non-photosynthetic tissue of plants (Tanaka et al. 1996;
19
Nagai et al. 2007). Glutamyl t-RNA reductase is known to be post-translationally regulated by
20
phytochrome, heme, Pchlide and FLU (Vothknecht et al. 1998; Pontoppidan & Kannangara
21
1994; Goslings et al. 2004; Meskauskiene et al. 2001; McCormac et al. 2001) and also could be
22
similarly regulated by environmental stresses.
23
In water-stressed seedlings, although the protein/transcript abundance of the next enzyme
24
involved in ALA biosynthesis i.e. GSA-AT increased, the ALA contents declined. This suggests
25
that GSA-AT enzyme involved in ALA biosynthesis may be inactivated by post-translational
26
modification during stress condition. Similar increase in gsa transcript/protein abundance and
27
reduction in ALA contents were reported in response to salt stress and heat stress in rice or wheat
28
(Mohanty et al. 2006; Satpal & Tripathy unpublished; Table 1). The reduced ALA synthesis was
29
most likely partly due to limiting amounts of the substrate GSA. These results demonstrate that
© 2012 Blackwell Publishing Ltd
19
Chl biosynthesis pathway is down-regulated at the early steps under stress conditions in order to
2
prevent the accumulation of harmful singlet oxygen generating tetrapyrroles.
Accepted Article
1
3
Similar to decline in ALAD activity and its gene expression observed in salt-stressed rice,
4
chill- and heat-stressed cucumber or wheat seedlings (Mohanty et al. 2006; Satpal & Tripathy
5
unpublished; Table 1), the ALAD activity was reduced in water-stressed rice seedlings due to the
6
down-regulation of its transcript abundance. Limitation of ALA, a substrate for ALAD probably
7
reduced its gene expression in water stress and other stress conditions. These results suggest that
8
the increased or decreased availability of the substrate of the enzyme could positively or
9
negatively regulate the gene expression of the enzyme. The enzymatic activity of PBGD that
10
deaminates PBG to form Urogen was reduced due to down-regulation of its transcript abundance
11
in water-stressed rice seedlings (Figs. 5B & 7B). Similarly, PBGD enzyme activity and its
12
transcript abundance were down-regulated in rice/cucumber by salt or temperature stress (Satpal
13
& Tripathy unpublished, Tewari & Tripathy 1998, 1999; Table 1).
14
The subsequent enzyme in the porphyrin biosynthesis pathway is UROD, responsible for
15
copropoporphyrinogen synthesis. UROD protein abundance decreased in water-stressed
16
seedlings, which well correlates with the declined message abundance of UroD in response to
17
water stress. The UROD protein and transcript abundance also declined in salt-stressed and chill-
18
stressed rice/wheat seedlings (Satpal & Tripathy unpublished; Mohanty et al. 2006). This is in
19
contrast with our previous observations in cucumber and wheat where UROD activity and its
20
transcript/protein abundance increased in response to heat-stress (Mohanty et al. 2006; Tewari &
21
Tripathy 1998).
22
coproporphyrinogen oxidase and protoporphyrinogen oxidase. Similar to salt-, chill-stressed
23
rice/cucumber seedlings (Satpal & Tripathy unpublished, Tewari & Tripathy 1998; Table 1), the
24
enzyme activity of CPO and protox decreased in water-stressed seedlings (Figs. 5C & 5D) due to
25
down-regulation of their gene/protein abundance (Fig. 7 and Fig. 8).
Next two enzymes involved in protoporphyrin IX biosynthesis are
26
All the steps in the biosynthesis of Mg-porphyrins and Fe-porphyrins are shared up to
27
protoporphyrin IX. Mg chelatase performs the Mg insertion step in porphyrin ring. It has three
28
subunits, Chl D, Chl H and Chl I (Gibson et al. 1995, 1999; Willows et al. 1998; Jensen et al.
29
1996). In the present study, Mg-chelatase activity decreased by 50% in water-stressed seedlings
30
(Fig. 5E). In the same vein, the gene/protein expression of ChlI/Chlorina9 and ChlD/Chlorina1
© 2012 Blackwell Publishing Ltd
20
subunits of Mg-chelatase (Zhang et al. 2006) partially declined in water-stressed seedlings (Figs.
2
7 & 8).
3
(Ikegami et al. 2007) and therefore could have impaired function in altered redox environment in
4
water-stressed seedlings. Stoichiometric imbalance among the subunits of Mg-chelatase has
5
previously been shown to decrease the Mg-chelatase activity as seen in Chl I over-expressing or
6
under-expressing transgenic Arabidopsis plants (Papenbrock et al. 2000). Inadequate proportion
7
of all subunits is known to hamper the correct assembly of active Mg-chelatase (Guo et al 1998;
8
Hansson et al. 1999; Jensen et al. 1999). We have also previously shown that in salt- and chill-
9
stressed rice/cucumber seedlings, the Mg-chelatase activity and gene/protein expression were
10
down-regulated (Satpal & Tripathy unpublished; Tewari & Tripathy 1998; Table 1). ChlH was
11
proposed as a receptor of ABA, although there are reports to the contrary (Muller and Hansson
12
2009; Shen et al. 2006; Wu et al. 2009). ABA synthesis increases due to water stress. Increased
13
ABA could bind to ChlH and might down-regulate Chl biosynthesis. However, this needs
14
experimental verification.
Accepted Article
1
Chl I1 subunit is also post-translationally regulated by chloroplastic thioredoxin
15
16
MPE cyclase performs a very complex reaction of fifth ring formation in tertrapyrrole
17
biosynthesis. It is proposed to have two soluble subunits and a membrane subunit i.e. Xantha-l in
18
barley and Chl27 in Arabidopsis (Rzeznicka et al. 2005; Tottey et al. 2003). Transcripts level of
19
membrane subunit of MPE cyclase, encoded by AtChl27/Xantha-l homolog in rice, showed a
20
marginal decline in rice seedlings under water stress (Fig. 7). Its expression is also down-
21
regulated by salt-stress (Satpal & Tripathy unpublished; Table 1). MPE cyclase activity was
22
found to be protected by NADPH-dependent thioredoxin and 2-Cys peroxiredoxins system that
23
removes the H2O2 from the vicinity of MPE cyclase to protect its activity (Stenbaek et al. 2008).
24
Decreased gene expression of xantha-l homolog and its post-translational regulation due to the
25
oxidizing environment in stressed seedlings could have resulted in the down-regulation of MPE
26
cyclase activity and Pchlide accumulation.
27
Protochlorophyllide
(Pchlide)
is
converted
to
chlorophyllide
(Chlide)
by
28
protochlorophyllide oxidoreductase (POR). In Arabidopsis, three POR iso-enzymes have been
29
reported namely POR A, POR B and POR C encoded by POR A, POR B and POR C genes
30
respectively (Armstrong et al. 1995; Pattanayak et al. 2002). However, several plants including
© 2012 Blackwell Publishing Ltd
21
rice have only two iso-enzymes i.e. POR A and POR B. POR activity, measured as relative
2
percent phototransformation of Pchlide to Chlide, decreased by 60% in response to water stress
3
(Fig. 5F) after 72 h of greening. In the same vein, the POR protein abundance (Fig. 8) and PorB
4
expression (Fig. 7) were substantially down-regulated in stressed seedlings. A decrease in POR
5
activity/protein abundance and transcript expression of PorB was also reported in chill- and salt-
6
stressed cucumber/rice seedlings (Satpal & Tripathy unpublished; Table 1). Although plants
7
down-regulate the POR upon transfer from dark to light, the POR abundance is not affected by
8
light in seedlings exposed to low temperature likely due to the down-regulation of POR
9
degrading enzymes (Mohanty et al. 2006; Table 1).
Accepted Article
1
10
The ChlP (geranyl-geranyl reductase) is responsible for phytol synthesis. Its protein
11
levels were highly down-regulated in seedlings due to water-stress treatment (Fig. 8). Similar
12
results were reported in salt-stressed rice seedlings (Satpal & Tripathy unpublished).
13
Table 1 examines the responses of Chl biosynthetic enzymes to various environmental
14
stresses.
These stresses broadly down-regulate most of the enzymes of Chl biosynthesis
15
pathway. However, gene/protein expression of a certain enzyme i.e. GSA-AT is up-regulated in
16
most stresses, i.e. heat, water, salt etc.
17
temperature. As GSA-AT is a crucial enzyme involved in the last step of synthesis of ALA,
18
plants most likely up-regulate its expression to compensate for the reduced expression of earlier
19
enzymes of the ALA biosynthesis.
20
Shibata shift:
The expression of UroD is up-regulated in high-
21
When etiolated leaves are subjected to a flash of light, the large aggregates of POR–
22
Phlide–NADPH ternary complexes are converted to POR–Chlide–NADPH complexes. Such
23
ternery complexes have higher emission and are slowly dissociated into smaller complexes
24
accompanied by the progressive release of Chlide from POR catalytic site. This leads to a large
25
blue shift in absorption and emission maxima of Chlide, and is called Shibata shift. The process
26
ends with the formation of Chlide absorbing at 672 nm and emitting at 682 nm (Chlide682).
27
Crosslinking experiments have shown that C672-682 is partly composed of Chlide still bound to
28
POR complexes and partly by Chlide bound to other proteins (Ryberg et al. 1992; Wiktorsson et
© 2012 Blackwell Publishing Ltd
22
al. 1993). Shibata shift is followed by formation of photoactive photosystem II (PSII) units
2
(Franck 1993).
Accepted Article
1
3
During plant development in dark, both the photo- and non-photo active pools of Pchlide
4
accumulate at different proportions, which is reflected in the modifications of fluorescence
5
spectra. The Photoreduction of Pchlide to Chlide is mediated by several short lifetime
6
intermediates, e.g. semireduced Pchlide radical species formed by hydrogen transfer from
7
NADPH (Belyaeva et al. 1988; Lebedev & Timko 1999) and characterized by their very low
8
fluorescence yield (Schoefs 2001). In contrary to the formation of first intermediate, which
9
requires light, the transfer of the second hydrogen ion would not need light and spontaneously
10
occurs at temperatures higher than 193K. In vitro experiments have shown that Chlide absorbing
11
at 676 nm and emitting at 690 nm has same organization as that of photoactive Pchlides i.e.
12
Chlide-LPOR-NADP complex (Oliver & Griffiths 1982; Wiktorsson et al. 1993) indicating that
13
this form of Chlide originated from photoactive Pchlides. Chlide 676-690 undergoes molecular
14
modifications at room temperature depending on the release of Chlide from LPOR active sites
15
along with formation of large aggregates of POR-NADP complexes (Chlide 670-675) (Sirnoval
16
et al. 1968).
17
Since the POR gene expression and protein abundance declined in water-stressed rice
18
seedlings, it was imperative to assess any changes in Shibata shift that is mediated by this photo-
19
enzyme. The etiolated control seedlings had a smaller emission fluorescence peak (77K) at 637
20
nm due to non-phototransformable Pchlide and a larger peak at 657 nm due to
21
phototransformable Pchlide (Fig. 6A).
22
monomeric Pchlide complex or esterified Pchlide i.e. protochlorophyll (Lindsten et al. 1988),
23
which spontaneously dimerizes to form (POR-Pchlide-NADPH)2.
24
monomeric Pchlide is not flash-photoactive; instead it regenerates the long wavelength Pchlide
25
forms (Schoefs & Franck 1993; He et al. 1994; Schoefs et al. 1994, 2000a, 2000b). The dimer
26
has the absorption maximum at 638 nm and emission maximum at 645 nm (Ouazzani Chahdi et
27
al. 1998; Lebedev & Timko 1999). The dimeric POR-Pchlide-NADPH complex further
28
polymerizes to form 16-mer or larger aggregates of POR-Pchlide-NADPH complex i.e. (POR-
29
Pchlide-NADPH)n having absorption maximum at 650 nm and emission maximum at 657-658
30
nm (Böddi et al. 1989; Wiktorsson et al. 1993) and is flash photoactive (Böddi et al. 1991).
© 2012 Blackwell Publishing Ltd
The non-phototransformable Pchlide peak is due to
23
The short-wavelength,
Upon 16 h of stress treatment, the etiolated seedlings displayed an emission fluorescence peak
2
(77K) at 632 nm due to non-phototransformable Pchlide and a peak at 657 nm due to
3
phototransformable Pchlide (Fig. 6B). However, as compared to control, the ratio of non-
4
phototransformable/phototransformable Pchlide (F657/F632) increased from 0.10 to 0.15 in
5
stressed seedlings suggesting an impairment of aggregation of monomeric POR-Pchlide-NADPH
6
to 16-mer or larger aggregates of POR-Pchlide-NADPH complex i.e. (POR-Pchlide-NADPH)n.
7
This may be due to reduced availability of NADPH in water-stressed etiolated seedlings or due
8
to degradation of polymeric complexes in the stressed environment.
Accepted Article
1
9
The flash-induced phototransformation and Shibata shift leading to chloroplast
10
biogenesis was substantially affected in 16 h water-stressed samples. Upon red light flash
11
illumination (0.2 sec) of control leaves the phototransformable Pchlide peak at 657 nm
12
emanating from large aggregates of polymeric (POR-Pchlide-NADPH)n complexes almost
13
disappeared due to photo-reduction of Pchlide to Chlide and a new peak appeared at 691 nm due
14
to formation of Chlide-LPOR-NADP+ complexes (Oliver & Griffiths 1982; El Hamouri 1981;
15
Franck 1993; Franck et al. 1999; Wiktorsson et al. 1993). Transformation of Pchlide658 into
16
Chlide692 was previously observed by exposing the leaf primordia of common ash (Fraxinus
17
excelsior L.) and Hungarian ash, Fraxinus angustifolia Vahl. (Solymosi et al. 2006), wheat
18
(Franck et al., 1999) and that of Horse chestnut (Aesculus hippocastanum) (Solymosi et al. 2006)
19
to light flash. One min after flash, 691 nm-peak shifted to 694 nm (Fig. 6A) in control leaves due
20
to the formation of Chlide-LPOR-NADPH complexes (Oliver & Griffiths 1982; El Hamouri
21
1981; Franck et al. 1999). Subsequently, this peak blue-shifted to 680 nm after 15 min post-
22
flash incubation of control leaves (Fig. 6A) due to the release of Chlide from the active site of
23
LPOR and disaggregation of multimeric complexes a process called Shibata shift (Böddi et al.
24
1990; Shibata 1957; Franck 1993).
25
In stressed leaves, upon red light flash illumination of etiolated leaves the
26
phototransformable Pchlide peak at 657 nm disappeared and a new peak appeared at 692 nm
27
(Fig. 6B) due to formation of Chlide-LPOR-NADP+ complexes (Oliver & Griffiths 1982; El
28
Hamouri 1981) demonstrating that phototransformation of Pchlide to Chlide could still take
29
place in 16 h water-stressed samples. After 1 min post-flash incubation this peak shifted to 694
30
nm due to the formation of Chlide-LPOR-NADPH complexes (Fig. 6B). In water-stressed
© 2012 Blackwell Publishing Ltd
24
leaves shift to lower wavelengths was substantially delayed. A shoulder appeared at 680 nm
2
after 15 min of dark incubation, in contrast to complete shift to 680 nm in control seedlings,
3
suggesting a slow release of Chlide from the active site of LPOR (Böddi et al. 1990; Shibata
4
1957). In a non-physiological environment i.e. after dessication of detached barley leaves a
5
slowdown of Shibata was earlier reported (Lay et al. 2000, 2001). In the same vein, arrest of
6
Shibata shift was demonstrated in heat-stressed (Mohanty & Tripathy 2011) and salt-stressed
7
(Abdelkader et al. 2007) wheat seedlings. On the contrary, in chill-stress the Shibata shift was
8
partially affected (Mohanty & Tripathy 2011). Arrest of Shibata shift in heat-stressed wheat
9
seedlings was due to disaggregation of polymeric Pchlide-POR-NADPH molecules which
10
delayed the conversion of non-phototransformable Pchlide to its phototransformable form
11
resulting in belated development of the core antenna protein complex CP47 of Photosystem II
12
(PS II) (Mohanty & Tripathy 2011). Upon 15 min of dark incubation after flash illumination, a
13
good amount of phototransformable Pchlide (F657) was regenerated in control seedlings (Fig.
14
6A) and substantially less in stressed seedlings (Fig. 6B) demonstrating the down-regulation of
15
synthesis of Pchlide and its conversion to phototransformable form.
Accepted Article
1
16
The extent of down-regulation of ALA biosynthesis matches with that of Chl
17
biosynthesis in water-stressed seedlings implying that reduced gene expression and activity of
18
later enzymes of Chl biosynthesis pathway i.e. UROD, CPO, protox (PPX1), Mg-chelatase
19
(ChlI), POR etc. could be regulated by the abundance of early intermediates GSA or ALA. In the
20
same vein reduced expression and activity of later enzymes of Chl biosynthesis pathway i.e. Chl
21
synthase, Chl27 and Chl M in antisense plants caused a feedback-inactivation of the initial step
22
of the pathway leading to down-regulation of the metabolic flow to Chl (Shalygo et al. 2009;
23
Alawady & Grimm 2005; Peter et al. 2010; Bang et al. 2008; Rzeznicka et al. 2005; Pontier et
24
al. 2007). Observations from our laboratory in relation to PORC over-expression (Pattanayak &
25
Tripathy 2011), those of GUN4 (Peter & Grimm 2009) and ChlM (Alawady & Grimm 2005) led
26
to a general activation of the enzymes of Chl biosynthesis and consequent increase in Chl
27
contents. These studies along with our present observation of down-regulation of later enzymes
28
of Chl biosynthesis by reduced ALA synthesis in water-stressed seedlings demonstrates a
29
regulatory network of genes involved in tetrapyrrole biosynthesis.
© 2012 Blackwell Publishing Ltd
25
In conclusion the Chl biosynthesis is substantially down-regulated due to water stress
2
during seedling development. Chl biosynthesis is down-regulated at a very early step i.e. ALA
3
synthesis, due to reduced gene expression of early enzymes of Chl biosynthesis pathway under
4
stress conditions that prevents the accumulation of harmful singlet oxygen generating
5
tetrapyrroles. Down-regulation of Chl content could act as a regulatory mechanism in plants to
6
resist drought. Minimization of light absorption by reduced amounts of Chl would down-regulate
7
the electron transport to reduce the ROS production. The increased gene expression and protein
8
abundance of GSA-AT coupled with our previous observation of up-regulation of gsa gene
9
expression in heat-stressed and salt-stressed seedlings suggest that it may play a crucial role in
10
tolerance to abiotic stresses. Sense and antisense expression of GSA-AT will shed light on the
11
role played by GSA-AT under stress conditions.
Accepted Article
1
12
13
Acknowledgements: We thank C.G. Kannangara (Washington State University, USA) for the
14
gift of barley antibodies for glutamate-1-semialdehyde aminotransferase, W.T. Griffiths
15
(University of Bristol, UK) for wheat antibody for POR and B. Grimm, Humboldt University,
16
Berlin for UROD, CPO, PPOX1 and ChlP tobacco antibodies used for Western-blot analysis.
17
18
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© 2012 Blackwell Publishing Ltd
38
Table 1: Impact of various stress-induced alterations in activity, protein and transcript levels of
Accepted Article
1
2
enzymes involved in Chl biosynthesis.
GluTR
Protein
Transcript
GSA-AT
Protein
Transcript
ALAD
Enzyme Activity
Transcript
PBGD
Enzyme Activity
Transcript
UROD
Enzyme Activity
Protein
Transcript
CPO
Enzyme Activity
Protein
Transcript
Protox
Enzyme Activity
Protein
Transcript
Mg-chelatase
Enzyme Activity
Protein
Transcript
MPE cyclase
Enzyme Activity
Transcript
POR
Enzyme Activity
Protein
Transcript
CAO
Transcript
ChlP
Protein
Transcript
Salt-stress
Chill-stress
Heat-stress
−
+
−
−
−
−
+
+
+
+
+
−
+
+
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
0
−
−
−
−
−
−
−
+
+
+
−
−
−
−
−
0
0
0
0
0
−
−
−
−
−
−
−
0
−
0
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
0
0
−
0
−
−
Water-stress
−
−
−
© 2012 Blackwell Publishing Ltd
−
39
Data compiled from the present study, Tewari & Tripathy 1998; Mohanty et al. 2006; Mohanty
2
& Tripathy 2011; Satpal & Tripathy unpublished. The 0, + and  denote no change, up-
3
regulation and down-regulation, respectively.
Accepted Article
1
© 2012 Blackwell Publishing Ltd
40
Figures Legends:
2
Fig 1. Control and water-stressed (50 mM PEG) rice seedlings after 24 h (A and B) and 72 h (C
3
and D) of greening respectively. (E) Shoot and root length of control (black) and water-stressed
4
(grey, patterned) seedlings after 72 h of greening. Six-day old etiolated seedlings were treated
5
with 50 mM PEG 6000 dissolved in half strength MS nutrient soln., 16 h prior to the transfer to
6
cool white fluorescent + incandescent light (100 µmoles photons m-2 s-1), at 28 0C. To see the
7
difference in greening, of control and stressed samples, rolled leaves from stressed seedlings
8
were spread open and put between two slides to take pictures (B & D).
9
Fig 2. Dry weight, DW (closed symbols) and protein content (open symbols) of control (circle)
10
and water-stressed seedlings. Six-day old etiolated seedlings were treated with 40 mM (triangle)
11
or 50 mM PEG 6000 (square) dissolved in half strength MS nutrient soln., 16 h prior to the
12
transfer to cool white fluorescent + incandescent light (100 µmoles photons m-2 s-1), at 28 0C.
13
Seedlings were harvested at 24 h, 48 h and 72 h of greening and their DW and protein contents
14
were measured. Each data point is the average of three replicates. The error bars represent SD.
15
Fig 3. Total Chl (closed symbols) and carotenoids (open symbols) content of control (circle) and
16
water-stressed seedlings. Six-day old etiolated seedlings were treated with 40 mM (triangle) or
17
50 mM (square) PEG 6000 dissolved in half strength MS nutrient soln., 16 h prior to the transfer
18
to cool white fluorescent + incandescent light (100 µmoles photons m-2 s-1), at 28 0C. Seedlings
19
were harvested at desired time points (24 h, 48 h and 72 h) and their Chl and carotenoids
20
contents were measured. Each data point is the average of three replicates. The error bars
21
represent SD.
22
Fig 4. Chl biosynthesis intermediates after 72 h of greening. Net synthesis of Glutamate 1-
23
semialdehyde (GSA), δ-amino levulinic acid (ALA), MP(E) and Pchlide in control (black) and
24
water-stressed (grey, patterned) seedlings. Six-day old etiolated seedlings were treated with 50
25
mM PEG 6000 dissolved in half strength MS nutrient soln., 16 h prior to the transfer to cool
26
white fluorescent + incandescent light (100 µmoles photons m-2 s-1), at 28 0C. The GSA and
27
ALA content was estimated after 4 h of illumination in presence of gabaculine or levulinic acid
28
respectively. Each data point is the average of three replicates and the error bars represent SD.
Accepted Article
1
© 2012 Blackwell Publishing Ltd
41
Fig 5. Activities of enzymes involved in Chl biosynthesis, control (black) vs 50 mM PEG (grey,
2
patterned) treated seedlings. Six-day old etiolated seedlings were treated with 50 mM PEG 6000
3
dissolved in half strength MS nutrient soln., 16 h prior to the transfer to cool white fluorescent +
4
incandescent light (100 µmoles photons m-2 s-1), at 28
5
Porphobilinogen deaminase (C) Coproporphyrinogen oxidase, (D) Protoporphyrinogen oxidase
6
(E) Mg-chelatase and (F) Protochlorophyllide oxidoreductase activities were measured after 24 h
7
or 72 h of greening as described in materials and methods. Each data point is the average of three
8
replicates. The error bars represent SD.
9
Fig 6. Low temperature (77K) fluorescence emission spectrum (E440) of (A) control and (B)
Accepted Article
1
0
C. (A) ALA-dehydratase, (B)
10
water-stressed (16 h) etiolated seedlings showing Shibata-shift.
11
Fig 7. Gene expression of chlorophyll biosynthetic enzymes. Six-day old etiolated seedlings
12
were treated with 50 mM PEG 6000 dissolved in half strength MS nutrient soln., 16 h prior to the
13
transfer to cool white fluorescent + incandescent light (100 µmoles photons m-2 s-1) at 28 0C for
14
24 h or 72 h. Total RNA was isolated from control and water-stressed leaves of rice seedlings
15
after 24 h and 72 h of greening and (A) semiquantitative RT-PCR was performed with cDNA
16
made from 2 µg of RNA. (B) Percent intensity of gene expression. Black bars represent control
17
samples after 24 h of greening; white, coarse patterned bars represent water-stressed samples
18
after 24 h of greening; grey, fine patterned and white, crossed bars represent control and water-
19
stressed samples respectively after 72 h of greening.
20
Fig 8. Immnoblot analysis of Chl biosynthetic enzymes. Six-day old etiolated seedlings were
21
treated with 50 mM PEG 6000 dissolved in half strength MS nutrient soln., 16 h prior to the
22
transfer to cool white fluorescent + incandescent light (100 µmoles photons m-2 s-1) at 28 0C.
23
Plastids were isolated from control and water-stressed seedlings after 24 and 72 h of greening.
24
(A) Equal amount of (30 µg) of thylakoid proteins were separated by SDS-PAGE (12.5%). (B)
25
Western blot was performed with different antibodies as described in material and methods. (C)
26
Percent intensity of protein expression. Black bars represent control samples after 24 h of
27
greening; white, coarse patterned bars represent water-stressed samples after 24 h of greening;
28
grey, fine patterned and white, crossed bars represent control and water-stressed samples
29
respectively after 72 h of greening.
© 2012 Blackwell Publishing Ltd
42
Accepted Article
1
2
3
4
Supporting Information
Additional Supporting Information may be found in the online version of this article:
5
6
Table S1. Gene specific primers used in this study
7
Table S2. Antibodies used in this study
8
9
10
11
© 2012 Blackwell Publishing Ltd
43
Control
A
C 50 mM PEG
50 mM PEG
Control
B
After 72 h of greening
Control
50 mM PEG Control
D
Sh
hoot /Root len
ngth (cm) afterr 72h of green
ning
Accepted Article
50 mM PEG
After 24 h of greening
E
14
12
10
8
6
4
2
0
Shoot
Root
Fig 1. Control and water-stressed (50 mM PEG) rice seedlings after 24 h (A and B) and 72 h (C and D) of greening
respectively E,
respectively.
E Shoot and root length of control (black) and water-stressed (grey,
(grey patterned) seedlings after 72 h of
greening. Six-day old etiolated seedlings were treated with 50 mM PEG 6000 dissolved in half strength MS nutrient soln.,
16 h prior to the transfer to cool white fluorescent + incandescent light (100 μmoles photons m-2 s-1), at 280C. To see the
difference in greening, of control and stressed samples, rolled leaves from stressed seedlings were spread open and put
between two slides to take pictures (B & D).
6
30
5
20
4
Prote
ein, mg gFW -1
7
DW
W, mg shoot -1
Accepted Article
40
3
10
2
24 h
48 h
72 h
Greening period
Fig 2. Dry weight, DW (closed symbols) and protein content (open symbols) of control (circle) and waterstressed seedlings. Six-day old etiolated seedlings were treated with 40 mM (triangle) or 50 mM PEG 6000
(square) dissolved in half strength MS nutrient soln., 16 h prior to the transfer to cool white fluorescent +
incandescent light (100 μmoles photons m-2 s-1), at 280C. Seedlings were harvested at 24 h, 48 h and 72 h of
greening and their DW and protein contents were measured. Each data point is the average of three replicates.
The error bars represent SD.
0.20
-1
0.6
0.15
0.4
0.2
0.10
0.0
-0.2
Carotenoids
s, mg gFW
-1
08
0.8
Chl , mg
g gFW
Accepted Article
1.0
0.05
-0.4
24 h
48 h
72 h
Greening period
Fig 3. Total Chl (closed symbols) and carotenoids (open symbols) content of control (circle) and water-stressed
seedlings Six-day old etiolated seedlings were treated with 40 mM (triangle) or 50 mM (square) PEG 6000 dissolved in
seedlings.
half strength MS nutrient soln., 16 h prior to the transfer to cool white fluorescent + incandescent light (100 μmoles
photons m-2 s-1), at 280C. Seedlings were harvested at desired time points (24 h, 48 h and 72 h) and their Chl and
carotenoids contents were measured. Each data point is the average of three replicates. The error bars represent SD.
A
10
18
C
D
150
100
50
GSA, μm
mols g FW -1
200
8
6
4
2
3
2
1
Pchlide, nm
mols g FW -1
16
250
ALA, nm
mols g FW -1
4
B
MP(E), nm
mols g FW -1
Accepted Article
300
14
12
10
8
6
4
2
0
0
0
0
Fig 4. Chl biosynthesis intermediates after 72 h of greening. Net synthesis of Glutamate 1-semialdehyde (GSA), δ-amino
levulinic acid (ALA), MP(E) and Pchlide in control (black) and water-stressed (grey, patterned) seedlings. Six-day old
etiolated seedlings were treated with 50 mM PEG 6000 dissolved in half strength MS nutrient soln., 16 h prior to the
transfer to cool white fluorescent + incandescent light (100 μmoles photons m-2 s-1), at 280C. The GSA and ALA content
was estimated after 4 h of illumination in presence of gabaculine or levulinic acid respectively. Each data point is the
average of three replicates and the error bars represent SD.
D
10
0
72 h of greening
2.5
B
2.0
15
1.5
1.0
0.5
0.0
24 h of greening
E
Proto IX, nmols 100 mg prrotein-1 h-1
20
MP(E
E) nmols 100 mg protein-1 h-1
PBG, nmols mg protein
n-1 h-1
Proto
o IX, µmols 100 m
mg protein-1 h-1
A
30
6
5
4
3
2
1
0
72 h of greening
C
70
60
50
40
30
20
10
0
24 h of greening
120
3.0
PO
OR activity, relatiive percent
phototransform
mation
Porphyrin
ns, nmols mg pro
otein-1 h-1
Accepted Article
40
2.5
2.0
1.5
1.0
0.5
100
80
60
40
20
0
0.0
24 h of greening
F
72 h of greening
Fig 5. Activities of enzymes involved in Chl biosynthesis, control (black) vs 50 mM PEG (grey, patterned) treated seedlings.
Six-day old etiolated seedlings were treated with 50 mM PEG 6000 dissolved in half strength MS nutrient soln.,
soln 16 h prior to
-2
-1
0
the transfer to cool white fluorescent + incandescent light (100 μmoles photons m s ), at 28 C. (A) ALA-dehydratase, (B)
Porphobilinogen deaminase (C) Coproporphyrinogen oxidase, (D) Protoporphyrinogen oxidase (E) Mg-Chelatase and (F)
Protochlorophyllide oxidoreductase activities were measured after 24 h or 72 h of greening as described in materials and
methods. Each data point is the average of three replicates. The error bars represent SD.
Accepted Article
A
B
Fig 6. Low temperature (77K)
fluorescence emission spectra
(E440) of (A) control and (B)
water-stressed (16 h) etiolated
seedlings
dli
showing
h i Shibata-shift.
Shib
hif
Accepted Article
Con, 24h
WS, 24h
Con, 72h
WS, 72h
A
Arbitrarry Units
B
250
200
150
Fig 7. Gene expression of chlorophyll
biosynthetic enzymes. Six-day old etiolated
HemA1
seedlings were treated with 50 mM PEG 6000
dissolved in half strength MS nutrient soln., 16
gsa
h prior to the transfer to cool white fluorescent +
Alad
incandescent light (100 μmoles photons m-2 s-1),
at 280C for 24 h or 72 h. Total RNA was
pbgd
isolated from control and water-stressed leaves
of rice seedlings after 24 h and 72 h of greening
UroD
and (A) semiquantitative RT-PCR was
CPO
performed with cDNA made from 2µg of RNA.
(B) Percent intensity of gene expression. Black
PPX‐I
bars represent control samples after 24 h of
Chlorina 1(ChlD) greening; white, coarse patterned bars represent
water-stressed samples after 24 h of greening;
Chlorina 9(Chl I) grey, fine patterned and white crossed bars
represent control and water-stressed samples
HemH2
respectively after 72 h of greening.
Xantha‐l homolog (Chl27)
Por B
Rac2
C o n tr o l, 2 4 h o f g r e e n in g
W a te r s tr e s s , 2 4 h o f g r e e n in g
C o n tr o l, 7 2 h o f g r e e n in g
W a te r s tr e s s , 7 2 h o f g r e e n in g
100
50
0
Hem A1
gsa
A la d
Pbgd
U ro D
CPO
P P X -1
C h l o r i n a 1 C h l o r in a 9 H e m H 2
X a n th a -l
Por B
WS, 24h Con, 72h WS, 72h
Accepted Article
Con, 24h
B
A
97kD
66kD
GSA-AT
UroD
CPO
PPOX I
Chl I
43kD
29kD
20kD
POR
ChlP
14kD
C
Control, 24 h of greening
Water stress, 24 h of greening
Control, 72 h of greening
Water stress, 72 h of greening
Arbitrary Units
200
150
100
50
0
GSA-AT
UroD
CPO
PPOX-1
Chl l
POR
Chl P
Fig 8. Immnoblot analysis of Chl biosynthetic enzymes. Six-day old etiolated seedlings were treated with 50 mM PEG
6000 dissolved in half strength MS nutrient soln., 16 h prior to the transfer to cool white fluorescent + incandescent light
(100 μmoles photons m-2 s-1), at 280C. Plastids were isolated from control and water-stressed seedlings after 24 and 72 h of
greening. (A) Equal amount of (30µg) of plastid proteins were separated by SDS-PAGE (12.5%). (B) Western blot was
performed with different antibodies as described in material and methods. (C) Percent intensity of protein expression.
Black bars represent control samples after 24 h of greening; white, coarse patterned bars represent water-stressed samples
after 24 h of greening; grey, fine patterned and white crossed bars represent control and water-stressed samples respectively
after 72 h of greening.