Plant Physiology Preview. Published on March 16, 2016, as DOI:10.1104/pp.16.00017
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Running title
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Involvement of a cation-chloride cotransporter in rice cell elongation
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Corresponding Author
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Jian Feng MA
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Institute of Plant Science and Resources, Okayama University,
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Chuo 2-20-1, Kurashiki 710-0046, Japan
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Tel: +81-86-434-1209
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Fax: +81-86-434-1209
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E-mail: [email protected]
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Research area
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Membranes, Transport, and Biogenetics
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A cation-chloride cotransporter gene is required for cell elongation and
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osmoregulation in rice
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Zhi Chang Chen1,2, Naoki Yamaji2, Miho Fujii-Kashino2 and Jian Feng Ma2*
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and Forestry University, Fujian, Fuzhou 350002, China
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Japan
Root Biology Center, Haixia Institute of Science and Technology, Fujian Agriculture
Institute of Plant Science and Resources, Okayama University, Chuo 2-20-1, Kurashiki,
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One Sentence Summary:
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OsCCC1 functions as a K＋, Na＋ and Cl－ cotransporter in rice to maintain osmotic
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potential for cell elongation through increasing internal solute concentrations.
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* Address correspondence to [email protected]
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ABSTRACT
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Rice (Oryza sativa) is characterized by having fibrous root systems; however, the
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molecular mechanisms underlying the root development are not fully understood.
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Here, we isolated a rice mutant with short roots and found that the mutant had a
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decreased cell size of the roots and shoots compared with the wild-type rice (WT). A
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map-based cloning combined with whole genome sequencing revealed that a single
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nucleotide mutation occurred in a gene, which encodes a putative cation-chloride
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cotransporter (OsCCC1). Introduction of OsCCC1 cDNA into the mutant rescued the
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mutant growth, indicating that growth defect of both the roots and shoots are caused by
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loss of function of OsCCC1. Physiological analysis showed that the mutant had a lower
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concentration of Cl－ and K＋, and lower osmolality in the root cell sap than WT at all
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KCl supply conditions tested; however, the mutant only showed a lower Na ＋
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concentration at high external Na ＋ . Expression of OsCCC1 in yeast increased
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accumulation of
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roots and shoots although higher expression was found in the root tips. Furthermore, the
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expression in the roots did not respond to different Na ＋ , K ＋ and Cl － supply.
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OsCCC1 was expressed in all cells of the roots, leaf and basal node. Immunoblot
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analysis revealed that OsCCC1 was mainly localized to the plasma membrane. These
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results suggest that OsCCC1 is involved in the cell elongation by regulating ion (Cl－, K
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＋
K＋, Na＋ and Cl－. The expression of OsCCC1 was found in both the
and Na＋) homeostasis to maintain cellular osmotic potential.
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INTRODUCTION
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Root architecture is a very important trait for plant growth and development because
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roots are essential for the uptake of water and mineral nutrients from soils.
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roots also play an important role in detoxification of harmful minerals in soils, structural
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support of above-ground parts and environmental sensing (Marschner, 2012; Jung and
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McCouch, 2013). An ideotype of root system is determined by many factors such as
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root length, number, diameter and root configuration in the soil profile (De Dorlodot et
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al., 2007; Petricka et al., 2012).
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environments, and therefore understanding of molecular mechanisms underlying root
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development in different species and response to environmental changes is very
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important for crop productivity.
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In addition,
These factors differ with plant species and
Rice (Oryza sativa L.) is characterized by having a fibrous root system, which is
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composed of a seminal root, crown roots, lateral roots and root hairs (Rebouillat et al.,
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2009; Coudert et al., 2010). Anatomically, rice roots have two Casparian strips at the
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exodermis and endodermis cells and aerenchyma due to destruction of cortical cells in
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the root mature zones (Kawai et al., 1998; Coudert et al., 2010).
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involved in root development in rice have been identified by different approaches.
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These genes are involved in various biological processes controlling the development of
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primary root (Qi et al., 2012; Zhuang et al., 2005; Qin et al., 2013; Xia et al., 2014),
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crown root (Wang et al., 2011; Woo et al., 2007; Inukai et al., 2005), lateral root
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(Nakamura et al., 2006; Zhu et al., 2011; Kitomi et al., 2012) and root hair (Yuo et al.,
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2009; Kim et al., 2007; Won et al., 2010).
A number of genes
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Osmotic pressure is an important component to drive cell elongation. Potassium (K)
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is the most abundant cation in the cytosol and K＋ with its accompanying anions
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contribute greatly to the osmotic potential of plant cells and tissues (Marschner, 2012).
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Potassium transporters and channels in plant have been extensively studied such as the
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gene families of Shaker, KUP/HAK/KT, HKT, NHX, and CHX (Ashley et al., 2006;
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Shabala and Cuin, 2007; Wang and Wu, 2013). Some of these family members also
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have the transport activity of sodium (Na), due to the similar physico-chemical
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properties between sodium and potassium (Hamamoto et al., 2015). For plants, sodium
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usually is not essential, but in some cases, sodium could replace the role of potassium to
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maintain the cell osmotic potential (Blumwald, 2000; Horie et al., 2007). Non-selective
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cation channels (NSCCs) are proposed to be the dominant pathways of Na＋ influx in
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many plant species (Kronzucker and Britto, 2011; Hasegawa, 2013; Yamaguchi et al.,
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2013), but the molecular identity of many Na+ uptake mechanisms is still unknown. On
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the other hand, chloride (Cl), together with potassium, has a particular function to
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stabilize the osmotic potential and turgor pressure (White and Broadley, 2001;
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Marschner, 2012).
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NPF2.4 was reported to be involved in the long-distance transport of Cl － in
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Arabidopsis (Li et al., 2015). However, the molecular mechanism for Cl－ transport in
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plants is still poorly understood.
Recently, a plasma membrane-localized Nitrate/Peptide Transporter
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In animals, it has been reported that a cation-chloride cotransporter (CCC) family
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(also called SLC12) is involved in transport of K＋, Na＋ and Cl－ (Russell, 2000;
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Hebert et al., 2004; Gamba, 2005).
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cotransporters (KCC), Na＋-Cl－ cotransporters (NCC) and Na＋-K－-Cl－ cotransporters
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(NKCC). These transporters have a variety of functions including transepithelial salt
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transport, hearing, and neuronal development and cell volume regulation (Hoffmann et
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al., 2009; Lindinger et al., 2011; Moes et al., 2014). CCC family genes were also found
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in the plant genome, but only few of them has been functionally characterized.
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AtCCC in Arabidopsis has been suggested to be involved in long-distance Cl －
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transport (Colmenero-Flores et al., 2007). AtCCC catalyzed the coordinated symport
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of K＋, Na＋ and Cl－ in Xenopus laevis oocytes. It showed preferential expression in
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the root and shoot vasculature at the xylem/symplast boundary, root tips, trichomes, leaf
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hydathodes, leaf stipules and anthers. Knockout of this gene resulted in shorter organs
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including inflorescence stems, roots, leaves and siliques (Colmenero-Flores et al., 2007),
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indicating that AtCCC is involved in development processes and Cl homeostasis.
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More recently, Henderson et al. (2015) characterized a CCC gene (VviCCC) from
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grapevine (Vitis vinifera L.). They found that VviCCC was able to complement atccc
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mutant, indicating their similar role in plants.
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were observed to be localized at the Golgi and trans-Golgi network (Henderson et al.,
It has been divided into three groups: K＋-Cl－
Furthermore, both VviCCC and AtCCC
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2015). On the other hand, OsCCC1 in rice was partially characterized in terms of salt
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stress (Kong et al., 2011). Knockdown of this gene resulted in increased sensitivity to
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salt stress, especially to high KCl (Kong et al., 2011). The concentration of K＋ and Cl
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－
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WT, whereas that of Na＋ was hardly affected by suppression of this gene. In contrast to
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AtCCC and VviCCC, OsCCC1 was localized to the plasma membrane examined by
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transient expression of OsCCC1-GFP in onion epidermal cells.
was decreased in both the roots and shoots of knockdown lines compared with the
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In the present study, we isolated a rice mutant showing a distinct short-root phenotype.
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A map-based cloning combined with whole genome sequencing revealed that the
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phenotype was caused by a point mutation of the gene (OsCCC1) belonging to CCC
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family.
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membrane-localized transporter for K＋, Na＋ and Cl－ is required for cell elongation of
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both the roots and shoots through maintaining cellular osmotic potential.
A detailed functional analysis showed that OsCCC1 encoding a plasma
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RESULTS
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Isolation and Phenotypic Characterization of a Short-root Rice Mutant
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A rice mutant showing short-root phenotype was obtained from a Tos-17 transposon
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insertion line (NG2024). NG2024 has two Tos-17 insertion sites which are located at
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chromosome 3 and 7 (https://tos.nias.affrc.go.jp/), respectively; however, neither of
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them was associated with short-root phenotype by PCR identification, indicating that
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the short-root phenotype was caused by other mutation site. The mutant showed a
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much shorter length of seminal, lateral and crown roots than the WT (cv. Nipponbare) at
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both the seedling stage and reproductive stage (Fig. 1A-D, Supplemental Fig. S1A-B).
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A time-dependent root elongation measurement showed that the root elongation rate
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was much slower in the mutant than in the WT (Fig. 1E). However, the number of
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crown roots and the density of the lateral roots were similar between WT and the mutant
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(Fig. 1F-G).
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The mutant also showed a shorter shoot height compared with WT (Fig. 1B-D). The
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width of both leaf blade and basal stem was smaller in the mutant than in the WT
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(Supplemental Fig. S1C-F).
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When cultivated in a field, the mutant showed much smaller size of the whole plants
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(Supplemental Fig. S2A). The plant height of the mutant was significantly lower than
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that of WT at harvest (Supplemental Fig. S2B).
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panicle number, 1000-grain weight, spikelet number per panicle and percentage of filled
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spikelets were greatly decreased in the mutant (Supplemental Fig. S2C-H), resulting in
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a significant reduction of grain yield (Supplemental Fig. S2I).
All yield components including
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Observation of longitudinal sections of root tip region showed that the length of root
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apical meristem (LRAM, from the quiescent center (QC) to start of the elongation zone)
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was significantly shorter in the mutant than that in WT (0.59±0.04 µm vs 0.85±0.07 µm,
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Fig. 2A). Both WT and the mutant roots had similar radial structure including the
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epidermis, exodermis, sclerenchyma, cortex, endodermis, pericycle and stele at both
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elongation zone and mature zone (Fig. 2B-E). Furthermore, both WT and the mutant
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had the same number of root cortical cell layer (Supplemental Fig. S3A). However, the
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diameter of the roots were significantly smaller in the mutant than in WT (Fig. 2B-E).
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The length and width of the root cells of the mutant was 43.9% and 71.9%, respectively,
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of the WT (Fig. 2F, G).
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Shoot cell size was also compared between the mutant and WT at the shoot basal
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region. Observation of transverse cross sections showed that the cell size of leaf sheath
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in mutant was smaller than that in WT in both elongating and elongated zones (Fig.
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2H-K). The cell width of the leaf epidermal cells was 20.0 µm in the WT, in contrast to
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14.7 µm in the mutant (Fig. 2L). However, there was no difference in the cell numbers
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of adaxial epidermis of leaf sheath between WT and the mutant (Supplemental Fig.
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S3B). These results indicate that the shorter roots and shoots of the mutant is derived
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from decreased cell size but not from the cell numbers and tissue structure.
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Cloning of the Responsible Gene for the Short-root Phenotype
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We first performed a genetic analysis by using a heterogeneous population derived from
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a Tos-17 insertion line.
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phenotype, while 146 seedlings showed normal root phenotype.
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fits to 1:3, indicating that the short-root phenotype is controlled by a recessive gene.
Among 200 seedlings tested, 54 seedlings showed short-root
This segregation ratio
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To isolate the gene responsible for the short-root phenotype, we constructed an F2
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population by crossing the mutant with Kasalath, an indica cultivar. Using 3460 F2
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seedlings showing short-root phenotype, the candidate gene was mapped to a 440-kb
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region near the centromere of chromosome 8 by map-based cloning using markers
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shown in Supplemental Table S1 (Supplemental Fig. S4A). There are 56 predicted
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genes within this region based on the Rice Annotation Project Database (RAP-DB,
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http://rapdb.dna.affrc.go.jp/). To further clone the responsible gene, we performed
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MutMap (Abe et al., 2012, Supplemental Fig. S5) by sequencing the whole genome of
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bulked DNA from normal-root and short-root pools. Sequence alignment revealed one
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point mutation occurred in the 440 kb region.
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showed adenine (A) in this locus, whereas in the genome of normal-root pool, it
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contained adenine (A) and cytosine (C) (Supplemental Fig. S6A). To confirm this result,
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we re-sequenced this locus by using a PCR product. The results showed that WT
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presented C, the mutants presented A, and the heterozygote presented both A and C in
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this locus (Supplemental Fig. S6B).
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sequencing results.
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In the genome of short-root pool, all
This was consistent with the whole genome
The point mutation is located at the 12th exon of a gene encoding a putative
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cation-chloride cotransporter (OsCCC1, Supplemental Fig. S4B).
This mutation
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resulted in one amino acid change from cysteine (C) in WT to phenylalanine (F) in the
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mutant.
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(Supplemental Fig. S4B), encoding a peptide of 989 amino acids according to RGAP
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(http://rice.plantbiology.msu.edu/). We confirmed the sequence of entire ORF from
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cDNA of rice (cv. Nipponbare).
OsCCC1 (LOC_Os08g23440) contains 14 exons and 13 introns
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In the rice genome, there are two CCC genes; OsCCC1 and OsCCC2. They share
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82% identity with each other. OsCCC1 shares 79% identity with AtCCC in Arabidopsis.
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A Blast search on NCBI revealed OsCCC1 homologs in other plant species, including
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maize, sorghum, soybean, tobacco, rape and Medicago truncatula (Supplemental Fig.
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S7A). Using the SOSUI program (http://bp.nuap.nagoya-u.ac.jp/sosui/) and TMHMM
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sever (http://www.cbs.dtu.dk/services/TMHMM-2.0/), OsCCC1 was predicted to be a
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membrane protein with 11 transmembrane domains (Supplemental Fig. S7B).
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Complementation Test
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To confirm whether the mutation in OsCCC1 is responsible for the short-root and -shoot
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phenotypes, we performed a complementation test by introducing 2.5-kb promoter
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sequence of OsCCC1 fused with OsCCC1 cDNA into the mutant. Analysis with two
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independent transgenic lines showed that their root and shoot growth recovered to the
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same as WT (Fig. 3A-B), indicating that these phenotypes are caused by mutation of
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OsCCC1.
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Mineral Profile Analysis of Short-root Mutant
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Since CCC was reported to be a cation-chloride cotransporter in animals and
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Arabidopsis (Russell, 2000; Hebert et al., 2004; Colmenero-Flores et al., 2007), we
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compared cation profile of the root cell sap among WT, mutant and two
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complementation lines.
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mM in the WT (Fig. 3C). WT and two complementation lines showed similar cation
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profiles (Fig. 3C-D).
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concentration, while the concentration of other cations including Na＋, Mn2＋, Cu2＋ and
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Zn2＋ increased, but that of Mg2＋, Ca2＋ and Fe3＋ remained unchanged in the mutant
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(Fig. 3C-D).
Among cations tested, K＋ was the dominant one, being 40
By contrast, the mutant showed a 64% reduction in K ＋
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To further investigate whether the short-root phenotype in the mutant is caused by
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low K＋ or high Na＋ concentration, we exposed the plants to different KCl and NaCl
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supply conditions. The results showed that the concentration of K＋ and Cl－ was
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significantly lower in root of mutant than WT and two complementation lines
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irrespective of KCl or NaCl supply (Fig. 4A, C, E, G). Na＋ concentration was much
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higher in mutant at low NaCl condition (Fig. 4B, F), but remarkably decreased at high
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NaCl supply condition (50 mM, Fig. 4F). The Na＋ concentration in the root cell sap
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was significantly decreased with increasing external K＋ concentration in both the WT
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and mutant (Fig. 4B). In contrast, the effect of external Na＋ on K＋ concentration in
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the root cell sap was not large (Fig. 4E).
The root growth of mutant was not
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completely restored by any condition of KCl or NaCl supply (Supplemental Fig. S8).
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Distribution of K＋, Na＋ and Cl－ was also examined in the roots of WT and mutant
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by using the Scanning Electron Microscopy (SEM) with Energy Dispersive X-ray
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Spectroscopy (EDX). At 50 mM NaCl supply condition, a much stronger signal of K＋,
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Na＋ and Cl－ was observed in root cells of WT than that of mutant (Supplemental Fig.
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S9A-F). Moreover, quantitative analysis showed K ＋ , Na ＋ and Cl － were highly
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accumulated in the root cortical cells rather than the stele and exodermis in both the WT
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and mutant (Supplemental Fig. S9G-L). The concentration of Na＋, K＋ and Cl－ in the
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shoot was also compared between the WT and mutant. Similar to the roots,
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concentration of K＋ and Cl－ was lower in the shoots of the mutant compared with WT
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and two complementation lines (Supplemental Fig. S10A, B). The Na＋ concentration
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was also decreased in both the shoots and roots under the condition of high Na＋ supply
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(Supplemental Fig. S10C).
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Osmolality in the Root and Shoot Cell Sap
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The osmolality in the root and shoot cell sap was compared among the mutant, WT and
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two complementation lines using vapor pressure osmometer. The results showed that the
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mutant had a decreased root and shoot osmolality compared with WT and two
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complementation lines, irrespectively of KCl or NaCl supply conditions (Fig. 4D, 4H,
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and Supplemental Fig. S11). .
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Yeast Complementation Test of OsCCC1
To test whether OsCCC1 is permeable to K＋, we firstly introduced OsCCC1 into
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yeast strain CY162 using a galactose-inducible promoter.
CY162 is a mutant sensitive
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to K＋ deficiency due to lack of K＋ transporters TRK1 and TRK2 (Anderson et al.,
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1992). OsKAT1, a known K＋ transporter in rice (Obata et al., 2007), was used as a
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positive control.
In the presence of glucose, when the gene expression was not
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induced, the yeast carrying empty vector pYES2 (negative control), OsCCC1, mutated
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OsCCC1 and OsKAT1 showed the same growth (Fig. 5A). However, when the gene
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expression was induced by galactose, the growth of yeast carrying OsCCC1 and
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OsKAT1 was much better than that of empty vector and mutated OsCCC1 (Fig. 5B).
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These results suggest that OsCCC1 is involved in uptake of K＋ in yeast.
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To determine whether OsCCC1 is also involved in Na＋uptake, we then introduced
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OsCCC1 into the yeast mutant strain G19, which lacks major Na+ pumps and shows
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high sensitivity to salt stress (Quintero et al., 1996).
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OsHKT2;1 in rice (also named OsHKT1, Horie et al., 2001) was used as a positive
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control. In the presence of glucose, there was no difference in the growth among the
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yeast cells carrying pYES2 (negative control), OsCCC1, mutated OsCCC1 and
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OsHKT2;1 (Fig. 5C). Since the medium used contained 10 mM KCl rather than 1 mM
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used previously (Amtmann et al. 2001), the vector control yeast was able to grow in the
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presence of 300 mM NaCl. However, in the presence of galactose, expression of
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OsHKT2;1 and OsCCC1 resulted in a higher sensitivity to salt stress compared with the
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empty vector and mutated OsCCC1 (Fig. 5D). Compared with OsHKT2;1, OsCCC1
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showed a relatively lower affinity to Na＋ (Fig. 5D).
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A Na ＋ transporter gene
Furthermore, we quantified the uptake of K＋ and Cl－ by OsCCC1 in CY162, Na＋
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and Cl－ in G19 using liquid culture.
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uptake by the yeast CY162 expressing OsCCC1 and OsKAT1 were significantly higher
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than that by vector control at each KCl concentration in the presence of galactose (Fig.
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5E). The Cl－ uptake was also increased in the yeast carrying OsCCC1 compared within
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the vector control, but not in yeast carrying OsKAT1 (Fig. 5F). Similarly, a
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dose-dependent experiment showed that Na＋ uptake by the yeast G19 expressing
A dose-dependent experiment showed that K
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OsCCC1 and OsHKT2;1 was significantly higher than that by vector control (Fig. 5G),
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but the Cl－ uptake was increased only in the yeast carrying OsCCC1, but not in yeast
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carrying OsHKT2;1 (Fig. 5H). These results showed that different from OsKAT1 and
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OsHKT2;1, OsCCC1 likely functions as a Na＋, K＋-Cl－ cotransporter in yeast.
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Expression Pattern of OsCCC1
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OsCCC1 was expressed in both the roots and shoots, but much higher expression was
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found in the roots (Fig. 6A). In the roots, the expression was higher in the root tip (0-1
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cm) than in the mature region (1- 2 cm) (Fig. 6B). Furthermore, the expression level of
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OsCCC1 was similar between central cylinder and outer tissues in root mature region
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(Fig. 6C). The expression of OsCCC1 in the roots showed no response to external K＋
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and Na＋ concentrations added up to 5 mM, and Cl－ up to 10 mM (Fig. 6D). The
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expression of OsCCC1 in the roots was also hardly affected by high NaCl or KCl (50
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mM) (Fig. 6E).
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Tissue-specific Expression of OsCCC1
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To investigate tissue-specific expression of OsCCC1, we generated a transgenic rice
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carrying 2.5 kb promoter sequence of OsCCC1 fused with GFP. Immunostaining of the
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transgenic rice with a GFP antibody showed that the root tips showed stronger signal
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than other parts (Fig. 7A). The signal was observed in all root cells at both the
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elongation zone and mature zone of the roots (Fig. 7B, D). No signal was observed in
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the WT (Fig. 7C, E), indicating the specificity of the antibody. The signal was also
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observed in the leaf blade (Fig. 7F), leaf sheath (Fig. 7H) and basal node (Fig. 7J) of the
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transgenic lines, but not in the WT (Fig. 7G, I, K).
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Cellular and subcellular localization of OsCCC1
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To investigate the localization of OsCCC1 protein in different tissues, we performed
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immunostaining using an antibody against C-terminal peptide of OsCCC1. Western blot
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analysis showed that there was only one band observed in both the shoots and roots
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(Supplemental Fig. S11). The size of this band was about 100 kDa, which corresponds
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to the predicted size of OsCCC1 protein (108 kDa), indicating that this antibody is
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highly specific to OsCCC1. In addition, the protein abundance in roots was much higher
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than that in shoots (Supplemental Fig. S12), which is consistent with the expression
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level as shown in Fig. 6A. The fluorescence signal was strongly detected in all cells of
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the roots (Fig. 8A). In the leaf blade, the signal was stronger in the vascular bundle
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compared to the other tissues (Fig. 8B) and in the basal region, the signal could be
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detected in both phloem and xylem region (Fig. 8C).
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To examine the in situ subcellular localization of OsCCC1, we performed a double
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staining with 4’,6-diamidino-2-phenylindole (DAPI) for nuclei showed that the
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fluorescence signal from OsCCC1 antibody (red color) was localized mainly at the
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peripheral region of the cells, which was circumscribed but did not envelope the nuclei
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(blue color) (Figure 8D, E, F). This result indicate that OsCCC1 is localized to the
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plasma membrane although additional proof (e.g. analysis of membrane fractions)
333
would be required to establish the exact subcellular localization pattern.
334
335
23
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
336
DISCUSSION
337
338
Isolation of rice mutants with altered root morphology is a good approach for studying
339
molecular mechanisms underlying root development. In this study, we obtained a rice
340
root mutant (osccc1) with distinct morphology from a Tos-17 insertion line. This
341
mutant showed extremely shorter seminal, lateral and crown roots (Fig. 1A-D,
342
Supplemental Fig. S1A, B). There was no difference in the number of lateral roots,
343
crown roots, and cortical cell layer (Fig. 1F-G, 2A-E, Supplemental Fig. S3A); however,
344
the cell size of the mutant roots was significantly smaller than that of WT (Fig. 2F, G).
345
Furthermore, the mutant also presented a shorter shoot height and smaller leaf cell size
346
(Fig. 1B-D, 2H-L). Therefore, the short-root and -shoot phenotypes of the mutant result
347
from the decreased cell size of both the roots and shoots.
348
Positional cloning combined with whole-genome sequencing led to isolation of a
349
gene responsible for the short-root phenotype (Supplemental Fig. S4-6).
This was
350
confirmed by the complementation test (Fig. 3A-B).
351
belongs to a cation-chloride cotransporter gene family (CCC). A single amino acid
352
substitution (C568F) of OsCCC1 occurred in the last transmembrane domain in the
353
mutant (Supplemental Fig. S4B). This mutation resulted in loss of function of this gene
354
as shown in yeast assay experiment (Fig. 5).
The gene (OsCCC1) cloned
355
OsCCC1 was partially characterized previously and suggested to be involved in salt
356
stress tolerance (Kong et al., 2011); however, the exact role of this gene is unknown.
357
In this study, we further characterized this gene in terms of transport activity, tissue and
358
subcellular localization, expression pattern and detailed analysis of knockout mutants.
359
Our immunostaining result suggests that OsCCC1 is located to the plasma membrane in
24
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
360
rice cells although additional experiments would be required to determine the exact
361
location (Fig. 8). This is consistent with the result of transient expression in onion
362
epidermal cells (Kong et al., 2011).
363
from that of VviCCC in grapevine and AtCCC in Arabidopsis, which are localized at the
364
Golgi (Henderson et al., 2015). This difference may determine their different role in
365
plants. In fact, AtCCC has been proposed to be involved in the long-distance ion
366
transport (Henderson et al., 2015), but OsCCC1 is required for cell enlargement,
367
indicating diverse functions of plant CCC in different plant species.
368
needs investigation whether AtCCC is also involved in cell enlargement because atccc
369
mutant showed a reduced size, a similar phenotype to osccc1.
However, this subcellular localization is different
However, it still
370
Our results also show that OsCCC1 is likely a Na＋, K＋-Cl－ co-transporter in rice.
371
This is supported by yeast assay experiment and phenotypic analysis of the knockout
372
line.
373
defect as OsKAT1 (Fig. 5A, B). However, different from OsKAT1, expression of
374
OsCCC1 also increased Cl－ uptake in the yeast (Fig. 5E, F), indicating that OsCCC1
375
functions as a co-transporter for K ＋ and Cl － .
376
concentration in the yeast, the Cl－ concentration was much lower (Fig. 5E, F). The
377
reason for this phenomenon remains to be examined in future, but one possibility is that
378
Cl－ taken up by OsCCC1 is effluxed since yeast is not able to sequester Cl－ (Coury et
379
al., 1999). By contrast, K＋ is sequestered into the vacuoles, resulting in difference
380
concentration of K＋ and Cl－ in the cells. In rice root cell sap, the concentration of K
381
＋
382
decreased concentration of K＋ and Cl－ similarly at different external K concentrations
383
(Fig. 4A, C).
In yeast mutant detective in K＋ uptake, OsCCC1 complemented its growth
However, compared with K ＋
and Cl － was relatively comparable (Fig. 4). Knockout of OsCCC1 resulted in
This result further indicates that OsCCC1 is a K ＋ andCl －
25
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
384
co-transporter.
385
Although Na＋ is not an essential element for plant growth and the mutant phenotype
386
was also observed in the absence of Na＋, OsCCC1 shows its permeability to Na＋ in
387
yeast mutant defective in Na＋ uptake (Fig. 5C, D). Compared with OsHKT2;1, it
388
seems that the affinity for Na＋ by OsCCC1 is weak (Fig. 5D). However, expression of
389
OsCCC1 in the yeast increased both Na ＋ and Cl － uptake, while expression of
390
OsHKT2;1 only increased Na＋ uptake (Fig. 5G, H). In rice root cell sap, the Na
391
concentration in the mutant differed with external Na concentrations.
392
accumulated less Na＋ than WT at a high Na＋ concentration (50 mM) but not at low
393
Na＋ concentrations (Fig. 4F). These results suggest that OsCCC1 in rice could mediate
394
Na＋ transport with low affinity. On the other hand, it was observed that the mutant
395
presented a higher Na＋ concentration than WT at low Na＋ concentrations (Fig. 4F).
396
This difference might be attributed to the competition between K＋ and Na＋. K＋ and
397
Na＋ uptake by the roots are also mediated by other channels and transporters (Sauer et
398
al. 2013). Besides, at lower Na＋ supply in the presence of low K＋, the Na＋ uptake
399
may be enhanced in the mutant due to K＋-deficiency induced up-regulation of other
400
potassium/sodium transporters such as OsHKT2;1 (Horie et al., 2007; Fig. 4B, F).
401
However, at higher Na＋ supply, the contribution of OsCCC1 to the whole uptake
402
became larger, resulting in decreased Na＋ uptake in the mutant (Fig. 4B, F).
The mutant
403
OsCCC1 was expressed in almost all cells of rice plant (Fig. 7) and its expression
404
was unaffected by external K＋ and Na＋ concentration up to 50 mM (Fig. 6D, E).
405
This result is different from a previous study by Kong et al. (2011), who found that the
406
expression of OsCCC1 was induced by high concentration of KCl (150 mM). This
407
discrepancy could be attributed to the concentrations of salts used for treatment. Since
26
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
408
rice is a relatively salt-sensitive species, high salt (150 mM) will cause the growth
409
inhibition.
410
indirect result due to reduced growth. In fact, we found the expression of internal
411
standards (Actin and HistoneH3) was also changed at higher salt concentration (150
412
mM). Our results show that the constitutive expression of OsCCC1 is required for K＋,
413
Na＋ and Cl－ homeostasis in cells for maintaining appropriate osmotic pressure for cell
414
elongation. This is supported by that knockout of this gene significantly decreased the
415
osmolality (Fig. 4D, H).
Therefore, the up-regulation of OsCCC1 by 150 mM KCl could be an
416
K＋ is the most abundant cation in the cytosol and cell extension depends on K＋
417
accumulation in the cells for increasing the osmotic potential (Dolan and Davies, 2004).
418
In the present study, we also found high K＋ concentration (40-150 mM) in the root cell
419
sap depending on external K＋ concentrations (Fig. 4A). This high K＋ concentration
420
is maintained through different transporters involved in the uptake.
421
AtHAK5 mainly expressed at the epidermis and stele of roots, was reported to be
422
involved in K＋ uptake in Arabidopsis (Gierth et al., 2005). The expression of AtHAK5
423
is rapidly up-regulated by potassium starvation.
424
OsCCC1 shows no response to K＋ and is highly expressed in all cells of the root tip
425
region (Fig. 6B, D), where cell elongation occurs. High KCl supply did not completely
426
complement the root growth in the short-root mutant (Supplemental Fig. S8), although
427
the KCl concentration and osmolality in mutant were increased with increasing external
428
KCl supply, but not to the level of the WT (Fig. 4D). These findings suggest that the
429
KCl uptake mediated by OsCCC1 represents a basic components for maintaining the K
430
＋
431
the root hair growth in a K＋ transporter mutant trh1 in Arabidopsis (Rigas et al., 2001).
concentrations required for cell enlargement.
For example,
Different from other transporter genes,
High K＋ supply also cannot restore
27
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
432
In conclusion, OsCCC1 functions as K ＋ , Na ＋ -Cl ＋ cotransporter in rice. It is
433
important for maintaining osmotic potential by transporting K＋, Na＋ and Cl＋ into the
434
cells for cell elongation.
435
436
MATERIALS AND METHODS
437
438
Plant Materials and Growth Conditions
439
The short-root mutant was isolated from a Tos-17 transposon insertion line (NG2024),
440
which was regenerated from callus of a japonica cultivar Nipponbare of rice (Oryza
441
sativa L., Miyao et al., 2003). Seeds of wild type rice (cv. Nipponbare) and mutant were
442
soaked in deionized water at 30°C in the dark for two days, and then transferred to a net
443
floating on a 0.5 mM CaCl2 solution in a 1.5-L plastic container for 3 to 7 days before
444
being used for various experiments. The seedlings were then transferred to a 3.5-L
445
plastic pot containing half-strength Kimura B solution (pH 5.6) (Yamaji and Ma, 2007).
446
The nutrient solution was changed once two days.
447
448
Morphological Analysis
449
For measuring the root length, germinated seedlings were exposed to a 0.5 mM CaCl2
450
solution and the root length was measured by a ruler at different days. The lateral root
451
numbers in primary root of 7-d-old seedlings were counted and the lateral root density
452
was calculated by dividing the number of lateral roots by the primary root length for
453
each plant. Three-week-old seedlings were used for observation of longitudinal and
454
cross sections of crown root (at 1 mm and 10 mm from the root apex), and cross section
455
of leaf sheath (at 5 mm and 30 mm from the root-to-shoot junction). The samples were
28
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
456
imbedded into 5% agar and then were sectioned by a microslicer (100 µm in thickness,
457
LinearSlicer PRO10; Dosaka EM, http://www.dosaka-em.jp/). These sections were
458
observed under a confocal laser scanning microscope (LSM 700; Carl Zeiss,
459
http://www.zeiss.com). The length of root apical meristem (LRAM, the distance between
460
the quiescent center (QC) and the start point of the elongation zone) was also
461
determined.
462
root elongation zone were used. The length of the cortical cells was determined for 9
463
roots each with 10 cells (n = 90). The width of the cortical cells was determined for 10
464
roots each with eight cells (n = 80). The width of epidermal cells in leaf sheath was
465
determined for 14 samples each with 10 cells (n = 140).
For the measurement of cell size, longitudinal and cross sections of the
466
467
Positional Cloning of OsCCC1 and Whole Genome Sequencing
468
For mapping the responsible gene, the short-root mutant was crossed with Kasalath to
469
obtain F2 population.
470
(short-root vs normal-root) was firstly performed to identify the molecular markers
471
linked to OsCCC1 (Michelmore et al., 1991). To further mapping this gene,
472
polymorphic molecular markers were designed based on the sequence comparison in the
473
corresponding genomic region between Nipponbare and 93-11 (Supplemental Table S1).
474
Using 3460 F2 seedlings showing short-root phenotype, OsCCC1 finally was mapped to
475
440-kb region near the centromere of chromosome 8 according to the RAP-DB
476
(http://rapdb.dna.affrc.go.jp/).
A bulked segregant analysis using two bulked DNA samples
477
For whole genome sequencing, the short-root mutant plant was crossed with the
478
wild-type rice (cv. Nipponbare). The F1 plant was self-pollinated to obtain F2 progeny.
479
For genetic analysis, 200 seeds were used for phenotypic analysis. The genomic DNA
29
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
480
of 50 F2 plants each showing the short-root or normal-root phenotype were bulked in an
481
equal ratio and subjected to whole genome sequencing for 50 cycles by the sequencer.
482
The SNP analysis was conducted by MutMap (Abe et al., 2012).
483
484
Complementation Test
485
The native 2.5 kb promoter sequence of OsCCC1 was amplified by PCR using the
486
primer
487
TCGATCTCCCCGTTCTCCATCCCTCACTCTAGCAACTACA-3’). The ORF with
488
3’UTR of OsCCC1 was amplified by PCR using the primer pairs (5’-
489
TGTAGTTGCTAGAGTGAGGGATGGAGAACGGGGAGATCGA-3’
490
CGGACTAGTACCAATAATTTCAGCTGA-3’).
491
ProOsCCC1-OsCCC1-3’UTR was acquired by overlap PCR and inserted into
492
pPZP2H-lac (with NOS terminator) using ApaI and SpeI. The construct was introduced
493
into the calluses of rice (cv. Nipponbare) via Agrobacterium-mediated transformation
494
(Hiei et al., 1994).
pairs
(5’-
AATGGGCCCTTGTTGAGGTATAAGGTCA-3’
The
and
and
fragment
5’-
5’-
containing
495
496
RNA Isolation, Gene Cloning and Expression Analysis
497
Total RNA from rice roots was extracted using the RNeasy Mini Kit (Qiagen). One
498
microgram of total RNA was used for first strand cDNA synthesis using a ReverTra
499
Ace qPCR RT Master Mix kit (TOYOBO) following the manufacturer’s instructions.
500
The cDNA fragment containing OsCCC1 open reading frame was amplified by PCR
501
using
502
CGGACTAGTACCAATAATTTCAGCTGA-3’. The fragment was cloned into the
503
pGEM-T easy vector (Promega) for sequence confirmation using the ABI PRISM 310
the
primers
5’-AATGTCGACATGGAGAACGGGGAGATC-3’
30
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
and
5’-
504
Genetic Analyzer and the BigDye Terminators v3.1 cycle sequencing kit (Applied
505
Biosystems).
506
For the spatial expression, RNA was extracted from root tips (0-1 cm) and basal
507
region (1-2 cm) of 5-d-old seedlings.
For root tissue-specific expression, the segments
508
at 1.75 to 2.25 cm from the root tip of 4-d-old seedlings were collected for the tissue
509
sections. The central cylinder (pericycle and inner tissues) and outer tissues (cortex and
510
epidermis) were separated using a Veritas Laser Microdissection System LCC1704
511
(Molecular Devices) followed by total RNA extraction. To investigate the response of
512
OsCCC1 expression to KCl and NaCl, 10-d-old seedlings were pretreated with
513
potassium deficiency for one week, and then exposed to a half strength Kimura B
514
solution (removal of K) containing different NaCl + KCl (1:1 ratio) concentrations for 6
515
hours. For high salt condition, rice seedlings (14-d-old) were exposed to a nutrient
516
solution without or with 50 mM KCl or 50 mM NaCl supply for 12 h.
517
The gene expression level was determined by real-time RT-PCR using Thunderbird
518
SYBR qPCR Mix (TOYOBO) on Mastercycler ep realplex (Eppendorf). The primers
519
used
520
5′-CTTGAGAATCGTCCTGTGGA-3′ for OsCCC1. Histone H3 (forward primer,
521
5′-AGTTTGGTCGCTCTCGATTTCG-3′;
522
5′-TCAACAAGTTGACCACGTCACG-3′) was used as an internal control. The
523
expression was normalized by the ΔΔCt method.
were
5′-AAGCCGTTGTCATTGTGAAG-3′
reverse
and
primer,
524
525
Tissue-specificity of Expression
526
The native 2.5 kb promoter sequence of OsCCC1 was amplified by PCR. The primer
527
sequences
(5’-AATGGGCCCTTGTTGAGGTATAAGGTCA-3’
31
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
and
528
5’-CGGACTAGTGCCGCTTTACTTGTACAG-3’) were used for amplification and
529
introduction of the ApaI and SpeI restriction sites. The fragment was linked to sGFP
530
gene by overlap PCR. The amplified PCR product was inserted into pPZP2H-lac (with
531
NOS terminator) using ApaI and SpeI to create the OsCCC1 promoter–GFP construct.
532
The construct was introduced into the calluses of rice (cv. Nipponbare) via
533
Agrobacterium-mediated transformation (Hiei et al., 1994). The root, leaf sheath and
534
leaf blade of one-month-old seedlings of WT (cv. Nipponbare) and five independent
535
transgenic rice (T0) were used for immunostaining with a rabbit GFP antibody as
536
described below.
537
538
Tissue and Subcellular Localization of OsCCC1
539
The synthetic peptide SGAPQDDSQEAYTSAQRR (positions 870 to 887 of OsCCC1)
540
was used to immunize rabbits to obtain antibodies against OsCCC1. The obtained
541
antiserum was purified through a peptide affinity column before use. The root, leaf
542
blade and basal node of one-month-old rice seedlings (cv. Nipponbare) were used for
543
immunostaining as described below. Double staining with DAPI for nuclei was also
544
performed to investigate the subcellular localization.
545
546
Immunohistological Analysis
547
For western blot, microsomal proteins were extracted from the roots and shoots of
548
wild-type rice (cv. Nipponbare) according to Mitani et al. (2009). After determining the
549
protein concentrations by the Bradford assay (Biorad), the same amount of each sample
550
was loaded onto SDS/PAGE using 5-20% gradient polyacrylamide gels (ATTO). Rabbit
551
Anti-OsCCC1 polyclonal antibody (1:500) was used as the primary antibodies.
32
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
552
Anti-rabbit IgG (H+L) conjugated to horseradish peroxidase (1:10000; Promega) was
553
used as a secondary antibody, and an ECL Plus Western Blotting Detection System (GE
554
Healthcare) was used for chemiluminescence detection.
555
Immunostaining was performed according to the method modified from Yamaji and
556
Ma (2007). Rice roots, leaf blade, leaf sheath and node were fixed in 4% (w/v)
557
paraformaldehyde and 60 mM sucrose buffered with 50 mM cacodylic acid (pH 7.4) for
558
two h at room temperature. After three times washed by phosphate-buffered saline (PBS;
559
10 mM PBS, pH 7.4, 138 mM NaCl, 2.7 mM KCl), the samples were embedded in 5%
560
agar and sectioned 100-μm thick with LinearSlicer PRO 10 (Dosaka EM). Sections
561
were placed on microscope slides, incubated with PBS containing 0.1% (w/v)
562
pectolyase and 0.3% (v/v) Triton X-100 for 2 h at room temperature. After washed three
563
times with PBS, samples were blocked with PBS containing 5% (w/v) bovine serum
564
albumin (BSA) and add anti-GFP (1:1000 dilution) or anti-OsCCC1 (1: 500 dilution)
565
primary antibody. Slides were incubated at room temperature overnight, and washed
566
four times with PBS. The slides were exposed to secondary antibodies (Alexa Fluor 555
567
goat anti-rabbit IgG; Molecular Probes) in PBS with BSA for 2 h at room temperature,
568
washed five times in PBS, and mounted with 50% (v/v) glycerol in PBS. Samples were
569
examined with a laser-scanning confocal microscope (LSM700; Carl Zeiss).
570
571
Elemental and Osmotic Pressure Analysis
572
Germinated seedlings of WT, short-root mutant and two complementation lines were
573
exposed to 0.1 mM Ca(NO3)2 solution with different concentrations of KCl or NaCl.
574
After one day, the shoots and roots of half plants were harvested and dried at 70°C for 2
575
d and then boiled at 100°C for 2 hours. After 3 days, the root length of another half
33
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
576
plants was measured by a ruler for root recovery test. At end of the experiment, the
577
root tips (0-15 mm) and the shoot were excised for cell sap collection according to Chen
578
et al. (2012). Metal concentration in the roots and shoots and the cell sap was
579
determined
580
(http://www.agilent.com). Cl concentration was determined by ion chromatograph
581
(ICS-900, Dionex) with the column IonPac AS12A. The osmolality in the roots and
582
shoots was measured using 10 μl cell sap of each sample by vapor pressure osmometer
583
5520 (WESCOR, USA).
by
ICP-MS
using
an
Agilent
7700
mass
spectrometer
584
585
OsCCC1 Expression in Yeast
586
The entire ORF for OsCCC1, mutated OsCCC1, OsHKT2;1 and OsKAT1 were
587
amplified by PCR and were cloned into the pYES2 vector (Invitrogen), respectively.
588
After sequence confirmation, the OsCCC1, mutated OsCCC1, OsKAT1, OsHKT2;1 or
589
the empty vector were introduced into yeast according to the manufacturer’s protocols
590
(S.c.easy comp transformation kit, Invitrogen). The K＋-sensitive mutant strain CY162
591
(MATa ura3 his3 his4 trk1Δ trk2Δ1::pCK64) and Na-sensitive mutant strain G19 (MATa
592
ade2 ura3 leu2 his3 trp1 ena1Δ::HIS3::ena4Δ) were used for study. Primer pairs used
593
for
594
5’-ATGAGCTCAAAATGGAGAACGGGGAGATC-3’
595
5’-CCCTCGAGTCATGTGAAGAATGTGAC-3’ for OsCCC1 and mutated OsCCC1,
596
and
597
CCCTCGAGTTATACGTTCACTTGCTG-3’
598
ATGAGCTCAAAATGACGAGCATTTACCATGA-3’
599
CCCTCGAGTTACCATAGCCTCCAATATT-3’ for OsHKT2;1.
amplification
and
introduction
of
restriction
were
and
5’-ACGAGCTCAAAATGCCACGTTCTTCTCGT-3’
for
sites
and
OsKAT1,
and
and
5’5’5’-
600
Yeast transformants were selected on uracil-deficient medium and grown in synthetic
601
complete (SC-uracil) yeast medium containing 2% glucose, 0.67% yeast nitrogen base
34
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
602
without amino acids (Difco), 0.2% appropriate amino acids, and 2% agar at pH 6.0. One
603
colony was selected in each transformation strain and grown in the liquid
604
SC-glucose-uracil medium. For the plate experiment, four serial dilutions of yeast cell
605
suspensions were spotted on plates containing galactose and glucose, and cultured at
606
30°C for 3 days. The SC-uracil plate was added with 10 mM KCl to achieve the total K
607
＋
to 20 mM or added with 300 mM NaCl to achieve the total Na＋ to 300 mM.
608
For the liquid culture uptake experiment, yeast transformants were firstly grown to
609
linear phase. After washed three times with Mill-Q water, the yeast was adjusted to an
610
OD600 value of 0.15 and cultured in a SC-uracil (+galactose) medium with KCl (25, 50,
611
75, or 100 mM) or NaCl (0, 100, 300, or 500 mM) till OD600=1.5-2.0. Cells were
612
collected and washed by 20 mM Ca(NO3)2 solution on ice for three times. The samples
613
were boiled for one hour before elemental analysis.
614
615
Root Elemental Distribution Analysis
616
Both wild type rice (WT) and the short-root mutant (21-d-old) were cultivated in the
617
nutrient solution containing 50 mM NaCl. After 24 hours, the roots were washed three
618
times by 5 mM Ca(NO3)2 solution on ice, the root was excised and fixed by 5% agar
619
powder. The transverse section at about 10 mm from the root apex was sectioned by a
620
microslicer (LinearSlicer PRO10; Dosaka EM, http://www.dosaka-em.jp/) and
621
immediately used for analysis by scanning electron microscope (TM3000, HITACHI) in
622
vacuum condition at -20°C. The elemental distribution photos were generated by energy
623
dispersive X-ray spectrometer (SwiftED 3000, Oxford Instruments).
624
625
Accession Number
35
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
626
Accession number of OsCCC1 is registered as LC085614 in the GenBank/EMBL
627
databases.
628
629
List of Author Contribution
630
Z.C.C., N.Y. and J.F.M. conceived and designed the experiments; Z.C.C. performed
631
most of the experiments; M. K. prepared the laser microdissection sample for RT-PCR;
632
Z.C.C. and J.F.M. analyzed data; Z.C.C., N.Y. and J.F.M. wrote the article.
633
634
SUPPLEMENTAL DATA
635
The following materials are available in the online version of this article.
636
Supplemental Figure S1. Phenotypic comparison of lateral roots, leaf blade and basal
637
stem between the wild-type rice and short-root mutant.
638
Supplemental Figure S2. Growth and grain yield of wild-type rice (cv. Nipponbare)
639
and short-root mutant grown in a field.
640
Supplemental Figure S3. Comparison of cell number in root and shoot between the
641
wild-type rice (WT) and short-root mutant.
642
Supplemental Figure S4. Map-based cloning of the gene responsible for the short-root
643
phenotype.
644
Supplemental Figure S5. Scheme for MutMap using whole genome sequencing.
645
Supplemental Figure S6. Alignment of mutation region between the bulked DNA from
646
normal-root and short-root pools by MutMap (A) and confirmation of mutation by PCR
647
(B).
648
Supplemental Figure S7. Phylogenetic tree of OsCCC1 in plant (A) and predicated
649
topology of OsCCC1 (B).
36
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2016 American Society of Plant Biologists. All rights reserved.
650
Supplemental Figure S8. Partial recovery of the root growth in short-root mutant by
651
addition of NaCl/KCl.
652
Supplemental Figure S9. Elemental distribution in short-root mutant using SEM &
653
EDX.
654
Supplemental Figure S10. Concentration of K＋, Na＋ and Cl－ in roots and shoots in
655
response to KCl and NaCl.
656
Supplemental Figure S11. Osmolality in shoot cell sap in response to KCl and NaCl.
657
Supplemental Figure S12. Western blot analysis of OsCCC1.
658
Supplemental Table S1. Primers for InDel markers used for mapping of OsCCC1.
659
660
ACKNOWLEDGMENTS
661
We thank Rice Genome Resource Center in Tsukuba for providing the Tos-17 insertion
662
line. We thank Dr. T. Horie for kindly providing the yeast strains CY162 and G19 and
663
for critical discussion. We also thank Nao Komiyama for helping in generating
664
transgenic rice. This research was supported by a Grant-in-Aid for Scientific Research
665
on Innovative Areas from the Ministry of Education, Culture, Sports, Science and
666
Technology of Japan (22119002 and 24248014 to J.F.M.).
667
668
FIGURE LEGENDS
669
Figure 1. Phenotypic comparison of the wild-type rice (WT) and short-root mutant.
670
A-C, Phenotypes of WT (cv. Nipponbare, left) and the mutant (right) grown
671
hydroponically for 5 d (A), 10 d (B) and 30 d (C). Scale bar =1 cm (A), 5 cm (B), and
672
10 cm (C), respectively. D, Phenotypes of WT (left) and the mutant (right) grown in
673
field at harvest. Scale bar = 30 cm. E, Time-dependent root growth. Germinated
37
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
674
seedlings of both WT and the mutant were exposed to a 0.5 mM CaCl2 solution and the
675
seminal root length was measured at different days indicated. Error bars represent ± SD
676
(n=15). F, Lateral root density. The lateral root numbers on primary root of 7-d-old
677
seedlings were counted and the lateral root density was calculated by dividing the
678
number of lateral roots by the primary root length for each plant. Data are means ± SD
679
(n=20). G, Crown root number of WT and the mutant grown hydroponically for 20
680
days.
681
682
Figure 2. Morphological comparison of the wild-type rice (WT) and short-root mutant.
683
A, Longitudinal sections of the root tip in the WT (cv. Nipponbare; left) and the mutant
684
(right). The length of root apical meristem is evaluated by the distance between the
685
quiescent center (QC) and the start point of the elongation zone. Scale bar =1 mm. B-E,
686
Root transverse sections at 1 mm (B, C) and 10 mm (D, E) from the root apex of the
687
WT (B, D) and the mutant (C, E). Three-week-old seedlings were used for observation
688
of longitudinal and cross sections of crown root. Scale bars = 200 μm. F, Longitudinal
689
cell length of cortical cells in the mature region (at 10 mm from the apex) of the root
690
(n=90). G, Transverse cell width of cortical cells in the mature region (at 10 mm from
691
the apex) of the root (n=80). H-K, Shoot transverse sections at 5 mm and 30 mm from
692
the root-shoot junction of the WT (H, J) and the mutant (I, K). Scale bar = 1 mm. These
693
photos showed a rolled-up young leaf blade (YB) enclosed by an old leaf sheath (OS). L,
694
Transverse cell width of adaxial epidermal cells in leaf sheath (n=140). The asterisk in
695
(A), (C), (E), (F), (G) and (L) shows a significant difference between WT and mutant
696
(P<0.05 by Tukey’s test).
697
38
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
698
Figure 3. Complementation test and mineral analysis.
699
A-B, Complementation of the short-root phenotype. Germinated seedlings of wild-type
700
rice (WT), short-root mutant and two independent complementation lines (T1)
701
transformed with an ORF of OsCCC1 driven by 2.5 kb promoter of OsCCC1, were
702
exposed to a solution containing Ca(NO3)2 for 3 days. The root was photographed (A)
703
and the root length was measured by a ruler (B). Data are means ± SD (n=10). Scale bar
704
=5 cm. C-D, Macro (C) and micro (D) mineral concentrations in the root cell sap. The
705
root tips (0-15 mm) were excised for root cell sap collection. The metal concentrations
706
were determined by ICP-MS. Data are means ± SD (n=3). The asterisk in (B), (C) and
707
(D) indicates significant differences compared with WT (*P<0.05 by Tukey’s test).
708
709
Figure 4. Concentration of ions (K＋, Na＋ and Cl－) and osmolality in root cell sap in
710
response to KCl and NaCl.
711
Germinated seedlings of wild-type rice (WT), short-root mutant and two independent
712
complementation lines were exposed to a solution containing different concentration of
713
KCl (A-D) or NaCl (E-H) for three days. The root tips (0-15 mm) were excised for root
714
cell sap collection. The concentration of K ＋ (A, E), Na ＋ (B, F), Cl － (C, G),
715
osmolality (D, H) were determined in the WT, mutant and two complementation lines.
716
Data are means ± SD (n=3). The asterisk indicates significant differences compared
717
with WT (*P<0.05 by Tukey’s test).
718
719
Figure 5. Yeast complementation test of OsCCC1.
720
A-D, OsCCC1-mediated tolerance to K＋ deficiency (A, B) and Na＋ toxicity (C, D).
721
OsCCC1, mutated OsCCC1, empty vector (pYES2, negative control), OsKAT1 (positive
39
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
722
control) or OsHKT2;1 (positive control) was introduced into yeast mutant strain CY162
723
sensitive to K＋ deficiency (A, B) or G19 sensitive to Na＋ stress (C, D). The yeast was
724
cultured on the synthetic complete medium (SC-uracil) containing 20 mM KCl (A, B)
725
or 300 mM NaCl (C, D) in the presence of glucose (A, C) or galactose (B, D) at 30ºC
726
for 3 days. Four serial 1:10 dilutions (from left to right) of yeast cell suspensions
727
starting from OD600=0.5 were spotted on plates. E-H, Dose-dependent uptake of K＋
728
and Cl－ in yeast CY162 (E, F), Na＋ and Cl－ in yeast G19 (G, H). Yeast strain CY162
729
carrying OsCCC1, OsKAT1, or empty vector (pYES2) was exposed to a SC-uracil
730
solution containing KCl (25, 50, 75, 100 mM) in the presence of galactose.
731
strain G19 carrying OsCCC1, OsHKT2;1, or empty vector (pYES2) was exposed to a
732
SC-uracil solution containing NaCl (0, 100, 300, 500 mM) in the presence of galactose.
733
Yeast strains were sampled at the exponential phase for elemental analysis. Data are
734
means ± SD (n=3). The asterisk shows a significant difference compared with empty
735
vector (P<0.05 by Tukey’s test).
Yeast
736
737
Figure 6. Expression pattern of OsCCC1 in rice.
738
A, Expression of OsCCC1 in the roots and shoots. The roots and shoots of rice
739
seedlings grown hydroponically for 5 days were sampled for RNA extraction. B, Root
740
spatial expression. Root segments (0-1 cm and 1-2 cm) of rice seedlings (5-d-old) were
741
excised for RNA extraction. C, Tissue specificity of OsCCC1 expression. The root
742
segments at 1.75 to 2.25 cm from the root tips were collected for the tissue sections. The
743
central cylinder (pericycle and inner tissues) and outer tissues (cortex and epidermis)
744
were separated by laser microdissection. D-E, Expression of OsCCC1 in response to Na
745
＋
, K＋ and Cl－. Rice seedlings were pretreated with K deficiency for one week, and
40
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
746
then exposed to a solution containing different NaCl + KCl (1:1 ratio) concentrations
747
for six hours (D). Rice seedlings were exposed to nutrient solution without or with 50
748
mM KCl or 50 mM NaCl supply for 12 h (E). The root part was excised for RNA
749
extraction. The expression level was determined by real-time RT-PCR. Histone H3 was
750
used as an internal standard. The expression relative to root (A), root tip (B), central
751
cylinder (C), 0 μM Cl－ (D) or 0 mM NaCl/KCl (E) is shown. Data are means ± SD
752
(n=3). The asterisk in (A) and (B) indicates a significant difference compared with root
753
and root tip, respectively (P<0.05 by Tukey’s test).
754
755
Figure 7. Tissue specificity of OsCCC1 expression.
756
Immunostaining with an anti-GFP antibody was performed in different tissues of
757
pOsCCC1-GFP transgenic rice (A, B, D, F, H, J) and wild-type rice (C, E, G, I, K),
758
including longitudinal section of root tip (A), cross sections at 1 mm (B, C) and 10 mm
759
(D, E) from root apex, leaf blade (F, G), leaf sheath (H, I) and basal node (J, K). Red
760
color shows signal from GFP antibody detected with a secondary antibody. Cyan color
761
shows cell wall autofluorescence (F, G). Yellow-dotted area is magnified and insets in
762
(J). EN, endodermis; EX, exodermis; P, phloem region; X, xylem region. Five
763
independent transgenic lines were investigated and the representative results are shown.
764
Bars = 200 μm.
765
766
Figure 8. Cellular and subcellular localization of OsCCC1 in rice.
767
Immunostaining with a polyclonal antibody against OsCCC1 in different organs of rice
768
was performed, including root (A), leaf blade (B) and basal node (C-F). Red color
769
indicates the OsCCC1 antibody-specific signal. Blue color indicates cell wall and
41
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
770
nucleus stained by DAPI (yellow arrowheads). Yellow-dotted areas in (A), (B) and (C)
771
were magnified and inserted in (A), (B) and (D-F). (D) and (E) are signal from
772
OsCCC1 antibody and DAPI/Cell wall, respectively. (F) is a merged image of (D) and
773
(E).
Bar = 50 μm
774
775
42
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
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