A Cation-Chloride Cotransporter Gene Is Required for Cell Elongation and Osmoregulation in Rice^,

OsCCC1 functions as a K⁺, Na⁺, and Cl⁻ cotransporter in rice to maintain osmotic potential for cell elongation through increasing internal solute concentrations.

Abstract

Rice (Oryza sativa) is characterized by having fibrous root systems; however, the molecular mechanisms underlying the root development are not fully understood. Here, we isolated a rice mutant with short roots and found that the mutant had a decreased cell size of the roots and shoots compared with wild-type rice. Map-based cloning combined with whole-genome sequencing revealed that a single nucleotide mutation occurred in a gene, which encodes a putative cation-chloride cotransporter (OsCCC1). Introduction of OsCCC1 cDNA into the mutant rescued the mutant growth, indicating that growth defects of both the roots and shoots are caused by loss of function of OsCCC1. Physiological analysis showed that the mutant had a lower concentration of Cl⁻ and K⁺ and lower osmolality in the root cell sap than the wild type at all KCl supply conditions tested; however, the mutant only showed a lower Na⁺ concentration at high external Na⁺. Expression of OsCCC1 in yeast increased accumulation of K⁺, Na⁺, and Cl⁻. The expression of OsCCC1 was found in both the roots and shoots, although higher expression was found in the root tips. Furthermore, the expression in the roots did not respond to different Na⁺, K⁺, and Cl⁻ supply. OsCCC1 was expressed in all cells of the roots, leaf, and basal node. Immunoblot analysis revealed that OsCCC1 was mainly localized to the plasma membrane. These results suggest that OsCCC1 is involved in the cell elongation by regulating ion (Cl⁻, K⁺, and Na⁺) homeostasis to maintain cellular osmotic potential.

Root architecture is a very important trait for plant growth and development because roots are essential for the uptake of water and mineral nutrients from soils. In addition, roots also play an important role in detoxification of harmful minerals in soils, structural support of aboveground parts, and environmental sensing (Marschner, 2012; Jung and McCouch, 2013). An ideotype of root system is determined by many factors, such as root length, number, diameter, and root configuration in the soil profile (de Dorlodot et al., 2007; Petricka et al., 2012). These factors differ with plant species and environments; therefore, understanding of molecular mechanisms underlying root development in different species and response to environmental changes is very important for crop productivity.

Rice (Oryza sativa) is characterized by having a fibrous root system, which is composed of a seminal root, crown roots, lateral roots, and root hairs (Rebouillat et al., 2009; Coudert et al., 2010). Anatomically, rice roots have two Casparian strips at the exodermis and endodermis cells and aerenchyma due to destruction of cortical cells in the root mature zones (Kawai et al., 1997; Coudert et al., 2010). A number of genes involved in root development in rice have been identified by different approaches. These genes are involved in various biological processes controlling the development of primary root (Zhuang et al., 2006; Qi et al., 2012; Qin et al., 2013; Xia et al., 2014), crown root (Wang et al., 2011; Woo et al., 2007; Inukai et al., 2005), lateral root (Nakamura et al., 2006; Zhu et al., 2012; Kitomi et al., 2012), and root hair (Yuo et al., 2009; Kim et al., 2007; Won et al., 2010).

Osmotic pressure is an important component to drive cell elongation. Potassium (K) is the most abundant cation in the cytosol and K⁺ with its accompanying anions contributing greatly to the osmotic potential of plant cells and tissues (Marschner, 2012). Potassium transporters and channels in plant have been extensively studied in gene families such as Shaker, KUP/HAK/KT, HKT, NHX, and CHX (Ashley et al., 2006; Shabala and Cuin, 2008; Wang and Wu, 2013). Some of these family members also have the transport activity of sodium (Na) due to the similar physico-chemical properties between sodium and potassium (Hamamoto et al., 2015). For plants, sodium usually is not essential, but in some cases, sodium could replace the role of potassium to maintain the cell osmotic potential (Blumwald, 2000; Horie et al., 2007). Nonselective cation channels are proposed to be the dominant pathways of Na⁺ influx in many plant species (Kronzucker and Britto, 2011; Hasegawa, 2013; Yamaguchi et al., 2013), but the molecular identity of many Na⁺ uptake mechanisms is still unknown. On the other hand, chloride (Cl), together with potassium, has a particular function to stabilize the osmotic potential and turgor pressure (White and Broadley, 2001; Marschner, 2012). Recently, a plasma membrane-localized Nitrate/Peptide Transporter, NPF2.4, was reported to be involved in the long-distance transport of Cl⁻ in Arabidopsis (Arabidopsis thaliana; Li et al., 2016). However, the molecular mechanism for Cl⁻ transport in plants is still poorly understood.

In animals, it has been reported that a cation-chloride cotransporter (CCC) family (also called SLC12) is involved in transport of K⁺, Na⁺, and Cl⁻ (Russell, 2000; Hebert et al., 2004; Gamba, 2005). It has been divided into three groups: K⁺-Cl⁻ cotransporters, Na⁺-Cl⁻ cotransporters, and Na⁺-K⁺-Cl⁻ cotransporters. These transporters have a variety of functions, including transepithelial salt transport, hearing, neuronal development, and cell volume regulation (Hoffmann et al., 2009; Lindinger et al., 2011; Moes et al., 2014). CCC family genes were also found in the plant genome, but only a few of them have been functionally characterized. AtCCC in Arabidopsis has been suggested to be involved in long-distance Cl⁻ transport (Colmenero-Flores et al., 2007). AtCCC catalyzed the coordinated symport of K⁺, Na⁺, and Cl⁻ in Xenopus laevis oocytes. It showed preferential expression in the root and shoot vasculature at the xylem/symplast boundary, root tips, trichomes, leaf hydathodes, leaf stipules, and anthers. Knockout of this gene resulted in shorter organs, including inflorescence stems, roots, leaves, and siliques (Colmenero-Flores et al., 2007), indicating that AtCCC is involved in development processes and Cl homeostasis. More recently, Henderson et al. (2015) characterized a CCC gene (VviCCC) from grapevine (Vitis vinifera). They found that VviCCC was able to complement the atccc mutant, indicating their similar role in plants. Furthermore, both VviCCC and AtCCC were observed to be localized at the Golgi and trans-Golgi network (Henderson et al., 2015). On the other hand, OsCCC1 in rice was partially characterized in terms of salt stress (Kong et al., 2011). Knockdown of this gene resulted in increased sensitivity to salt stress, especially to high KCl (Kong et al., 2011). The concentration of K⁺ and Cl⁻ was decreased in both the roots and shoots of knockdown lines compared with the wild type, whereas that of Na⁺ was hardly affected by suppression of this gene. In contrast to AtCCC and VviCCC, OsCCC1 was localized to the plasma membrane examined by transient expression of OsCCC1-GFP in onion epidermal cells.

In this study, we isolated a rice mutant showing a distinct short-root phenotype. Map-based cloning combined with whole-genome sequencing revealed that the phenotype was caused by a point mutation of the gene OsCCC1 belonging to CCC family. A detailed functional analysis showed that OsCCC1, encoding a plasma membrane-localized transporter for K⁺, Na⁺, and Cl⁻, is required for cell elongation of both the roots and shoots through maintaining cellular osmotic potential.

RESULTS

Isolation and Phenotypic Characterization of a Short-Root Rice Mutant

A rice mutant showing short-root phenotype was obtained from a Tos-17 transposon insertion line (NG2024). NG2024 has two Tos-17 insertion sites that are located at chromosomes 3 and 7 (https://tos.nias.affrc.go.jp/), respectively; however, neither of them was associated with the short-root phenotype by PCR identification, indicating that the short-root phenotype was caused by other mutation site. The mutant showed a much shorter length of seminal, lateral, and crown roots than the wild type (cv Nipponbare) at both the seedling stage and reproductive stage (Fig. 1, A–D; Supplemental Fig. S1, A and B). A time-dependent root elongation measurement showed that the root elongation rate was much slower in the mutant than in the wild type (Fig. 1E). However, the number of crown roots and the density of the lateral roots were similar between the wild type and the mutant (Fig. 1, F and G).

Figure 1.

Phenotypic comparison of the wild-type rice and short-root mutant. A to C, Phenotypes of the wild type (cv Nipponbare; left) and the mutant (right) grown hydroponically for 5 d (A), 10 d (B), and 30 d (C). Bars = 1 cm (A), 5 cm (B), and 10 cm (C). D, Phenotypes of the wild type (left) and the mutant (right) grown in the field at harvest. Bar = 30 cm. E, Time-dependent root growth. Germinated seedlings of both the wild type and the mutant were exposed to a 0.5 mm CaCl₂ solution, and the seminal root length was measured at different days indicated. Error bars represent ± sd (n = 15). F, Lateral root density. The lateral root numbers on primary root of 7-d-old seedlings were counted, and the lateral root density was calculated by dividing the number of lateral roots by the primary root length for each plant. Data are means ± sd (n = 20). G, Crown root number of the wild type and the mutant grown hydroponically for 20 d.

The mutant also showed a shorter shoot height compared with the wild type (Fig. 1, B–D). The width of both leaf blade and basal stem was smaller in the mutant than in the wild type (Supplemental Fig. S1, C–F).

When cultivated in a field, the mutant showed much smaller size of the whole plants (Supplemental Fig. S2A). The plant height of the mutant was significantly lower than that of the wild type at harvest (Supplemental Fig. S2B). All yield components, including panicle number, 1000-grain weight, spikelet number per panicle, and percentage of filled spikelets, were greatly decreased in the mutant (Supplemental Fig. S2, C–H), resulting in a significant reduction of grain yield (Supplemental Fig. S2I).

Observation of longitudinal sections of root tip region showed that the length of root apical meristem (from the quiescent center to start of the elongation zone) was significantly shorter in the mutant than that in the wild type (0.59 ± 0.04 µm versus 0.85 ± 0.07 µm; Fig. 2A). Both wild-type and mutant roots had similar radial structure, including the epidermis, exodermis, sclerenchyma, cortex, endodermis, pericycle, and stele, at both elongation zone and mature zone (Fig. 2, B–E). Furthermore, both the wild type and the mutant had the same number of root cortical cell layer (Supplemental Fig. S3A). However, the diameter of the roots was significantly smaller in the mutant than in the wild type (Fig. 2, B–E). The length and width of the root cells of the mutant was 43.9% and 71.9%, respectively, of the wild type (Fig. 2, F and G).

Figure 2.

Morphological comparison of the wild-type rice and short-root mutant. A, Longitudinal sections of the root tip in the wild type (cv Nipponbare; left) and the mutant (right). The length of root apical meristem is evaluated by the distance between the quiescent center (QC) and the start point of the elongation zone. Bar = 1 mm. B to E, Root transverse sections at 1 mm (B and C) and 10 mm (D and E) from the root apex of the wild type (B and D) and the mutant (C and E). Three-week-old seedlings were used for observation of longitudinal and cross sections of crown root. Bars = 200 μm. F, Longitudinal cell length of cortical cells in the mature region (at 10 mm from the apex) of the root (n = 90). G, Transverse cell width of cortical cells in the mature region (at 10 mm from the apex) of the root (n = 80). H to K, Shoot transverse sections at 5 and 30 mm from the root-shoot junction of the wild type (H and J) and the mutant (I and K). Bar = 1 mm. These photos showed a rolled-up young leaf blade (YB) enclosed by an old leaf sheath (OS). L, Transverse cell width of adaxial epidermal cells in leaf sheath (n = 140). The asterisk in A, C, E to G, and L shows a significant difference between the wild type and mutant (P < 0.05 by Tukey’s test).

Shoot cell size was also compared between the mutant and the wild type at the shoot basal region. Observation of transverse cross sections showed that the cell size of leaf sheath in mutant was smaller than that in the wild type in both elongating and elongated zones (Fig. 2, H–K). The cell width of the leaf epidermal cells was 20.0 µm in the wild type, in contrast to 14.7 µm in the mutant (Fig. 2L). However, there was no difference in the cell numbers of adaxial epidermis of leaf sheath between the wild type and the mutant (Supplemental Fig. S3B). These results indicate that the shorter roots and shoots of the mutant are derived from decreased cell size but not from the cell numbers and tissue structure.

Cloning of the Responsible Gene for the Short-Root Phenotype

We first performed a genetic analysis by using a heterogeneous population derived from a Tos-17 insertion line. Among 200 seedlings tested, 54 seedlings showed short-root phenotype, while 146 seedlings showed normal root phenotype. This segregation ratio fits to 1:3, indicating that the short-root phenotype is controlled by a recessive gene.

To isolate the gene responsible for the short-root phenotype, we constructed an F2 population by crossing the mutant with Kasalath, an indica cultivar. Using 3460 F2 seedlings showing short-root phenotype, the candidate gene was mapped to a 440-kb region near the centromere of chromosome 8 by map-based cloning using markers shown in Supplemental Table S1 (Supplemental Fig. S4A). There are 56 predicted genes within this region based on the Rice Annotation Project Database (http://rapdb.dna.affrc.go.jp/). To further clone the responsible gene, we performed MutMap (Abe et al., 2012; Supplemental Fig. S5) by sequencing the whole genome of bulked DNA from normal-root and short-root pools. Sequence alignment revealed one point mutation occurred in the 440-kb region. In the genome of short-root pool, all showed adenine (A) in this locus, whereas in the genome of normal-root pool, it contained adenine (A) and cytosine (C) (Supplemental Fig. S6A). To confirm this result, we resequenced this locus by using a PCR product. The results showed that the wild type presented C, the mutants presented A, and the heterozygote presented both A and C in this locus (Supplemental Fig. S6B). This was consistent with the whole-genome sequencing results.

The point mutation is located at the 12th exon of a gene encoding a putative cation-chloride cotransporter (OsCCC1; Supplemental Fig. S4B). This mutation resulted in one amino acid change from Cys (C) in the wild type to Phe (F) in the mutant. OsCCC1 (LOC_Os08g23440) contains 14 exons and 13 introns (Supplemental Fig. S4B), encoding a peptide of 989 amino acids according to RGAP (http://rice.plantbiology.msu.edu/). We confirmed the sequence of entire open reading frame (ORF) from cDNA of rice (cv Nipponbare).

In the rice genome, there are two CCC genes: OsCCC1 and OsCCC2. They share 82% identity with each other. OsCCC1 shares 79% identity with AtCCC in Arabidopsis. A BLAST search on NCBI revealed OsCCC1 homologs in other plant species, including maize (Zea mays), sorghum (Sorghum bicolor), soybean (Glycine max), tobacco (Nicotiana tabacum), rape (Brassica napus), and Medicago truncatula (Supplemental Fig. S7A). Using the SOSUI program (http://bp.nuap.nagoya-u.ac.jp/sosui/) and TMHMM server (http://www.cbs.dtu.dk/services/TMHMM-2.0/), OsCCC1 was predicted to be a membrane protein with 11 transmembrane domains (Supplemental Fig. S7B).

Complementation Test

To confirm whether the mutation in OsCCC1 is responsible for the short-root and -shoot phenotypes, we performed a complementation test by introducing 2.5-kb promoter sequence of OsCCC1 fused with OsCCC1 cDNA into the mutant. Analysis with two independent transgenic lines showed that their root and shoot growth recovered the same as the wild type (Fig. 3, A and B), indicating that these phenotypes are caused by mutation of OsCCC1.

Figure 3.

Complementation test and mineral analysis. A and B, Complementation of the short-root phenotype. Germinated seedlings of wild-type rice, short-root mutant, and two independent complementation lines (T1) transformed with an ORF of OsCCC1 driven by 2.5-kb promoter of OsCCC1 were exposed to a solution containing Ca(NO₃)₂ for 3 d. The root was photographed (A), and the root length was measured by a ruler (B). Data are means ± sd (n = 10). Bar = 5 cm. C and D, Macro (C) and micro (D) mineral concentrations in the root cell sap. The root tips (0–15 mm) were excised for root cell sap collection. The metal concentrations were determined by inductively coupled plasma mass spectrometer. Data are means ± sd (n = 3). The asterisk in B to D indicates significant differences compared with the wild type (*P < 0.05 by Tukey’s test).

Mineral Profile Analysis of Short-Root Mutant

Since CCC was reported to be a cation-chloride cotransporter in animals and Arabidopsis (Russell, 2000; Hebert et al., 2004; Colmenero-Flores et al., 2007), we compared the cation profile of the root cell sap among the wild type, mutant, and two complementation lines. Among cations tested, K⁺ was the dominant one, being 40 mm in the wild type (Fig. 3C). The wild type and two complementation lines showed similar cation profiles (Fig. 3, C and D). By contrast, the mutant showed a 64% reduction in K⁺ concentration, while the concentration of other cations, including Na, Mn, Cu, and Zn, increased, but that of Mg, Ca, and Fe remained unchanged in the mutant (Fig. 3, C and D).

To further investigate whether the short-root phenotype in the mutant is caused by low K⁺ or high Na⁺ concentration, we exposed the plants to different KCl and NaCl supply conditions. The results showed that the concentration of K⁺ and Cl⁻ was significantly lower in root of mutant than the wild type and two complementation lines irrespective of KCl or NaCl supply (Fig. 4, A, C, E, and G). Na⁺ concentration was much higher in the mutant at low NaCl condition (Fig. 4, B and F) but remarkably decreased at high NaCl supply condition (50 mm; Fig. 4F). The Na⁺ concentration in the root cell sap was significantly decreased with increasing external K⁺ concentration in both the wild type and mutant (Fig. 4B). In contrast, the effect of external Na⁺ on K⁺ concentration in the root cell sap was not large (Fig. 4E). The root growth of mutant was not completely restored by any condition of KCl or NaCl supply (Supplemental Fig. S8).

Figure 4.

Concentration of ions (K⁺, Na⁺, and Cl⁻) and osmolality in root cell sap in response to KCl and NaCl. Germinated seedlings of wild-type rice, short-root mutant, and two independent complementation lines were exposed to a solution containing different concentration of KCl (A–D) or NaCl (E–H) for 3 d. The root tips (0–15 mm) were excised for root cell sap collection. The concentration of K⁺ (A and E), Na⁺ (B and F), and Cl⁻ (C and G) and osmolality (D and H) were determined in the wild type, mutant, and two complementation lines. Data are means ± sd (n = 3). The asterisk indicates significant differences compared with the wild type (*P < 0.05 by Tukey’s test).

Distribution of K⁺, Na⁺, and Cl⁻ was also examined in the roots of the wild type and mutant using scanning electron microscopy with energy dispersive x-ray spectroscopy. At 50 mm NaCl supply condition, a much stronger signal of K⁺, Na⁺, and Cl⁻ was observed in root cells of the wild type than that of the mutant (Supplemental Fig. S9, A–F). Moreover, quantitative analysis showed the K⁺, Na⁺, and Cl⁻ were highly accumulated in the root cortical cells rather than the stele and exodermis in both the wild type and mutant (Supplemental Fig. S9, G–L). The concentration of Na⁺, K⁺, and Cl⁻ in the shoot was also compared between the wild type and mutant. Similar to the roots, concentration of K⁺ and Cl⁻ was lower in the shoots of the mutant compared with the wild type and two complementation lines (Supplemental Fig. S10, A and B). The Na⁺ concentration was also decreased in both the shoots and roots under the condition of high Na⁺ supply (Supplemental Fig. S10C).

Osmolality in the Root and Shoot Cell Sap

The osmolality in the root and shoot cell sap was compared among the mutant, the wild type, and two complementation lines using vapor pressure osmometer. The results showed that the mutant had a decreased root and shoot osmolality compared with the wild type and two complementation lines, irrespective of KCl or NaCl supply conditions (Fig. 4, D and H; Supplemental Fig. S11).

Yeast Complementation Test of OsCCC1

To test whether OsCCC1 is permeable to K⁺, we first introduced OsCCC1 into yeast strain CY162 using a Gal-inducible promoter. CY162 is a mutant sensitive to K⁺ deficiency due to lack of K⁺ transporters TRK1 and TRK2 (Anderson et al., 1992). OsKAT1, a known K⁺ transporter in rice (Obata et al., 2007), was used as a positive control. In the presence of Glc, when the gene expression was not induced, the yeast carrying empty vector pYES2 (negative control), OsCCC1, mutated OsCCC1 (C568F), and OsKAT1 showed the same growth (Fig. 5A). However, when the gene expression was induced by Gal, the growth of yeast carrying OsCCC1 and OsKAT1 was much better than that of empty vector and mutated OsCCC1 (Fig. 5B). These results suggest that OsCCC1 is involved in uptake of K⁺ in yeast.

Figure 5.

Yeast complementation test of OsCCC1. A to D, OsCCC1-mediated tolerance to K⁺ deficiency (A and B) and Na⁺ toxicity (C and D). OsCCC1, mutated OsCCC1 (C568F), empty vector (pYES2, negative control), OsKAT1 (positive control), or OsHKT2;1 (positive control) was introduced into yeast mutant strain CY162 sensitive to K⁺ deficiency (A and B) or G19 sensitive to Na⁺ stress (C and D). The yeast was cultured on the synthetic complete medium (SC-uracil) containing 20 mm KCl (A and B) or 300 mm NaCl (C and D) in the presence of Glc (A and C) or Gal (B and D) at 30°C for 3 d. Four serial 1:10 dilutions (from left to right) of yeast cell suspensions starting from OD₆₀₀ = 0.5 were spotted on plates. E to H, Dose-dependent uptake of K⁺ and Cl⁻ in yeast CY162 (E and F), and Na⁺ and Cl⁻ in yeast G19 (G and H). Yeast strain CY162 carrying OsCCC1, OsKAT1, or empty vector (pYES2) was exposed to a SC-uracil solution containing KCl (25, 50, 75, and 100 mm) in the presence of Gal. Yeast strain G19 carrying OsCCC1, OsHKT2;1, or empty vector (pYES2) was exposed to a SC-uracil solution containing NaCl (0, 100, 300, and 500 mm) in the presence of Gal. Yeast strains were sampled at the exponential phase for elemental analysis. Data are means ± sd (n = 3). The asterisk shows a significant difference compared with empty vector (P < 0.05 by Tukey’s test).

To determine whether OsCCC1 is also involved in Na⁺ uptake, we then introduced OsCCC1 into the yeast mutant strain G19, which lacks major Na⁺ pumps and shows high sensitivity to salt stress (Quintero et al., 1996). The Na⁺ transporter gene OsHKT2;1 in rice (also named OsHKT1; Horie et al., 2001) was used as a positive control. In the presence of Glc, there was no difference in the growth among the yeast cells carrying pYES2 (negative control), OsCCC1, mutated OsCCC1, and OsHKT2;1 (Fig. 5C). Since the medium used contained 10 mm KCl rather than 1 mm used previously (Amtmann et al., 2001), the vector control yeast was able to grow in the presence of 300 mm NaCl. However, in the presence of Gal, expression of OsHKT2;1 and OsCCC1 resulted in a higher sensitivity to salt stress compared with the empty vector and mutated OsCCC1 (Fig. 5D). Compared with OsHKT2;1, OsCCC1 showed a relatively lower affinity to Na⁺ (Fig. 5D).

Furthermore, we quantified the uptake of K⁺ and Cl⁻ by OsCCC1 in CY162, Na⁺, and Cl⁻ in G19 using liquid culture. A dose-dependent experiment showed that K uptake by the yeast CY162 expressing OsCCC1 and OsKAT1 was significantly higher than that by vector control at each KCl concentration in the presence of Gal (Fig. 5E). The Cl⁻ uptake was also increased in the yeast carrying OsCCC1 compared within the vector control but not in yeast carrying OsKAT1 (Fig. 5F). Similarly, a dose-dependent experiment showed that Na⁺ uptake by the yeast G19 expressing OsCCC1 and OsHKT2;1 was significantly higher than that by vector control (Fig. 5G), but the Cl⁻ uptake was increased only in the yeast carrying OsCCC1, but not in yeast carrying OsHKT2;1 (Fig. 5H). These results showed that different from OsKAT1 and OsHKT2;1, OsCCC1 likely functions as a Na⁺, K⁺-Cl⁻ cotransporter in yeast.

Expression Pattern of OsCCC1

OsCCC1 was expressed in both the roots and shoots, but much higher expression was found in the roots (Fig. 6A). In the roots, the expression was higher in the root tip (0–1 cm) than in the mature region (1–2 cm; Fig. 6B). Furthermore, the expression level of OsCCC1 was similar between central cylinder and outer tissues in root mature region (Fig. 6C). The expression of OsCCC1 in the roots showed no response to external K⁺ and Na⁺ concentrations added up to 5 mm and Cl⁻ up to 10 mm (Fig. 6D). The expression of OsCCC1 in the roots was also hardly affected by high NaCl or KCl (50 mm; Fig. 6E).

Figure 6.

Expression pattern of OsCCC1 in rice. A, Expression of OsCCC1 in the roots and shoots. The roots and shoots of rice seedlings grown hydroponically for 5 d were sampled for RNA extraction. B, Root spatial expression. Root segments (0–1 and 1–2 cm) of rice seedlings (5 d old) were excised for RNA extraction. C, Tissue specificity of OsCCC1 expression. The root segments at 1.75 to 2.25 cm from the root tips were collected for the tissue sections. The central cylinder (pericycle and inner tissues) and outer tissues (cortex and epidermis) were separated by laser microdissection. D and E, Expression of OsCCC1 in response to Na⁺, K⁺, and Cl⁻. Rice seedlings were pretreated with K deficiency for 1 week and then exposed to a solution containing different NaCl + KCl (1:1 ratio) concentrations for 6 h (D). Rice seedlings were exposed to nutrient solution without or with 50 mm KCl or 50 mm NaCl supply for 12 h (E). The root part was excised for RNA extraction. The expression level was determined by real-time RT-PCR. Histone H3 was used as an internal standard. The expression relative to root (A), root tip (B), central cylinder (C), 0 μm Cl⁻ (D), or 0 mm NaCl/KCl (E) is shown. Data are means ± sd (n = 3). The asterisk in A and B indicates a significant difference compared with root and root tip, respectively (P < 0.05 by Tukey’s test).

Tissue-Specific Expression of OsCCC1

To investigate tissue-specific expression of OsCCC1, we generated transgenic rice carrying 2.5-kb promoter sequence of OsCCC1 fused with GFP. Immunostaining of the transgenic rice with a GFP antibody showed that the root tips showed stronger signal than other parts (Fig. 7A). The signal was observed in all root cells at both the elongation zone and mature zone of the roots (Fig. 7, B and D). No signal was observed in the wild type (Fig. 7, C and E), indicating the specificity of the antibody. The signal was also observed in the leaf blade (Fig. 7F), leaf sheath (Fig. 7H), and basal node (Fig. 7J) of the transgenic lines, but not in the wild type (Fig. 7, G, I, and K).

Figure 7.

Tissue specificity of OsCCC1 expression. Immunostaining with an anti-GFP antibody was performed in different tissues of pOsCCC1-GFP transgenic rice (A, B, D, F, H, and J) and wild-type rice (C, E, G, I, and K), including longitudinal section of root tip (A), cross sections at 1 mm (B and C) and 10 mm (D and E) from root apex, leaf blade (F and G), leaf sheath (H and I), and basal node (J and K). Red color shows signal from GFP antibody detected with a secondary antibody. Cyan color shows cell wall autofluorescence (F and G). Yellow-dotted area is magnified in the insets in J. EN, Endodermis; EX, exodermis; P, phloem region; X, xylem region. Five independent transgenic lines were investigated, and the representative results are shown. Bars = 200 μm.

Cellular and Subcellular Localization of OsCCC1

To investigate the localization of OsCCC1 protein in different tissues, we performed immunostaining using an antibody against C-terminal peptide of OsCCC1. Western-blot analysis showed that there was only one band observed in both the shoots and roots (Supplemental Fig. S11). The size of this band was about 100 kD, which corresponds to the predicted size of OsCCC1 protein (108 kD), indicating that this antibody is highly specific to OsCCC1. In addition, the protein abundance in roots was much higher than that in shoots (Supplemental Fig. S12), which is consistent with the expression level as shown in Figure 6A. The fluorescence signal was strongly detected in all cells of the roots (Fig. 8A). In the leaf blade, the signal was stronger in the vascular bundle compared to the other tissues (Fig. 8B), and in the basal region, the signal could be detected in both phloem and xylem region (Fig. 8C).

Figure 8.

Cellular and subcellular localization of OsCCC1 in rice. Immunostaining with a polyclonal antibody against OsCCC1 in different organs of rice was performed, including root (A), leaf blade (B), and basal node (C–F). Red color indicates the OsCCC1 antibody-specific signal. Blue color indicates cell wall and nucleus stained by DAPI (yellow arrowheads). Yellow-dotted areas in A to C were magnified and inserted in A, B, and D to F. D and E are signal from OsCCC1 antibody and DAPI/cell wall, respectively. F is a merged image of D and E. Bar = 50 μm.

To examine the in situ subcellular localization of OsCCC1, we performed a double staining with 4′,6-diamino-phenylindole (DAPI) for nuclei, showing that the fluorescence signal from OsCCC1 antibody (red color) was localized mainly at the peripheral region of the cells, which was circumscribed but did not envelope the nuclei (blue color; Fig. 8, D–F). This result indicates that OsCCC1 is localized to the plasma membrane, although additional proof (e.g. analysis of membrane fractions) would be required to establish the exact subcellular localization pattern.

DISCUSSION

Isolation of rice mutants with altered root morphology is a good approach for studying molecular mechanisms underlying root development. In this study, we obtained a rice root mutant (osccc1) with distinct morphology from a Tos-17 insertion line. This mutant showed extremely shorter seminal, lateral, and crown roots (Fig. 1, A–D; Supplemental Fig. S1, A and B). There was no difference in the number of lateral roots, crown roots, and cortical cell layer (Figs. 1, F and G, and 2, A–E; Supplemental Fig. S3A); however, the cell size of the mutant roots was significantly smaller than that of the wild type (Fig. 2, F and G). Furthermore, the mutant also presented a shorter shoot height and smaller leaf cell size (Figs. 1, B–D, and 2, H–L). Therefore, the short-root and -shoot phenotypes of the mutant result from the decreased cell size of both the roots and shoots.

Positional cloning combined with whole-genome sequencing led to isolation of a gene responsible for the short-root phenotype (Supplemental Figs. S4–S6). This was confirmed by the complementation test (Fig. 3, A and B). The gene (OsCCC1) cloned belongs to a cation-chloride cotransporter gene family (CCC). A single amino acid substitution (C568F) of OsCCC1 occurred in the last transmembrane domain in the mutant (Supplemental Fig. S4B). This mutation resulted in loss of function of this gene as shown in yeast assay experiment (Fig. 5).

OsCCC1 was partially characterized previously and suggested to be involved in salt stress tolerance (Kong et al., 2011); however, the exact role of this gene is unknown. In this study, we further characterized this gene in terms of transport activity, tissue and subcellular localization, expression pattern, and detailed analysis of knockout mutants. Our immunostaining result suggests that OsCCC1 is located in the plasma membrane in rice cells, although additional experiments would be required to determine the exact location (Fig. 8). This is consistent with the result of transient expression in onion epidermal cells (Kong et al., 2011). However, this subcellular localization is different from that of VviCCC in grapevine and AtCCC in Arabidopsis, which are localized at the Golgi (Henderson et al., 2015). This difference may determine their different role in plants. In fact, AtCCC has been proposed to be involved in the long-distance ion transport (Henderson et al., 2015), but OsCCC1 is required for cell enlargement, indicating diverse functions of plant CCC in different plant species. However, it still needs investigation whether AtCCC is also involved in cell enlargement because atccc mutant showed a reduced size, a similar phenotype to osccc1.

Our results also show that OsCCC1 is likely a Na⁺, K⁺-Cl⁻ cotransporter in rice. This is supported by yeast assay experiment and phenotypic analysis of the knockout line. In yeast mutant defective in K⁺ uptake, OsCCC1 complemented its growth defect as OsKAT1 (Fig. 5, A and B). However, different from OsKAT1, expression of OsCCC1 also increased Cl⁻ uptake in the yeast (Fig. 5, E and F), indicating that OsCCC1 functions as a cotransporter for K⁺ and Cl⁻. However, compared with K⁺ concentration in yeast, the Cl⁻ concentration was much lower (Fig. 5, E and F). The reason for this phenomenon remains to be examined in future, but one possibility is that Cl⁻ taken up by OsCCC1 is effluxed since yeast is not able to sequester Cl⁻ (Coury et al., 1999). By contrast, K⁺ is sequestered into the vacuoles, resulting in different concentration of K⁺ and Cl⁻ in the cells. In rice root cell sap, the concentration of K⁺ and Cl⁻ was relatively comparable (Fig. 4). Knockout of OsCCC1 resulted in decreased concentration of K⁺ and Cl⁻ similarly at different external K concentrations (Fig. 4, A and C). This result further indicates that OsCCC1 is a K⁺ and Cl⁻ cotransporter.

Although Na⁺ is not an essential element for plant growth and the mutant phenotype was also observed in the absence of Na⁺, OsCCC1 shows its permeability to Na⁺ in yeast mutant defective in Na⁺ uptake (Fig. 5, C and D). Compared with OsHKT2;1, it seems that the affinity for Na⁺ by OsCCC1 is weak (Fig. 5D). However, expression of OsCCC1 in the yeast increased both Na⁺ and Cl⁻ uptake, while expression of OsHKT2;1 only increased Na⁺ uptake (Fig. 5, G and H). In rice root cell sap, the Na concentration in the mutant differed with external Na concentrations. The mutant accumulated less Na⁺ than the wild type at a high Na⁺ concentration (50 mm) but not at low Na⁺ concentrations (Fig. 4F). These results suggest that OsCCC1 in rice could mediate Na⁺ transport with low affinity. On the other hand, it was observed that the mutant presented a higher Na⁺ concentration than the wild type at low Na⁺ concentrations (Fig. 4F). This difference might be attributed to the competition between K⁺ and Na⁺. K⁺ and Na⁺ uptake by the roots are also mediated by other channels and transporters (Sauer et al., 2013). Besides, at lower Na⁺ supply in the presence of low K⁺, the Na⁺ uptake may be enhanced in the mutant due to K⁺ deficiency-induced up-regulation of other potassium/sodium transporters such as OsHKT2;1 (Horie et al., 2007; Fig. 4, B and F). However, at higher Na⁺ supply, the contribution of OsCCC1 to the whole uptake became larger, resulting in decreased Na⁺ uptake in the mutant (Fig. 4, B and F).

OsCCC1 was expressed in almost all cells of rice plant (Fig. 7), and its expression was unaffected by external K⁺ and Na⁺ concentration up to 50 mm (Fig. 6, D and E). This result is different from a previous study by Kong et al. (2011), who found that the expression of OsCCC1 was induced by high concentration of KCl (150 mm). This discrepancy could be attributed to the concentrations of salts used for treatment. Since rice is a relatively salt-sensitive species, high salt (150 mm) will cause the growth inhibition. Therefore, the up-regulation of OsCCC1 by 150 mm KCl could be an indirect result due to reduced growth. In fact, we found the expression of internal standards (Actin and HistoneH3) was also changed at higher salt concentration (150 mm). Our results show that the constitutive expression of OsCCC1 is required for K⁺, Na⁺, and Cl⁻ homeostasis in cells for maintaining appropriate osmotic pressure for cell elongation. This is supported by that knockout of this gene significantly decreased the osmolality (Fig. 4, D and H).

K⁺ is the most abundant cation in the cytosol and cell extension depends on K⁺ accumulation in the cells for increasing the osmotic potential (Dolan and Davies, 2004). In this study, we also found high K⁺ concentration (40–150 mm) in the root cell sap depending on external K⁺ concentrations (Fig. 4A). This high K⁺ concentration is maintained through different transporters involved in the uptake. For example, AtHAK5 mainly expressed at the epidermis and stele of roots, was reported to be involved in K⁺ uptake in Arabidopsis (Gierth et al., 2005). The expression of AtHAK5 is rapidly up-regulated by potassium starvation. Different from other transporter genes, OsCCC1 shows no response to K⁺ and is highly expressed in all cells of the root tip region (Fig. 6, B and D), where cell elongation occurs. High KCl supply did not completely complement the root growth in the short-root mutant (Supplemental Fig. S8), although the KCl concentration and osmolality in mutant were increased with increasing external KCl supply, but not to the level of the wild type (Fig. 4D). These findings suggest that the KCl uptake mediated by OsCCC1 represents a basic component for maintaining the K⁺ concentrations required for cell enlargement. High K⁺ supply also cannot restore the root hair growth in the K⁺ transporter mutant trh1 in Arabidopsis (Rigas et al., 2001).

In conclusion, OsCCC1 functions as a K⁺, Na⁺-Cl⁻ cotransporter in rice. It is important for maintaining osmotic potential by transporting K⁺, Na⁺, and Cl⁻ into the cells for cell elongation.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

The short-root mutant was isolated from a Tos-17 transposon insertion line (NG2024), which was regenerated from callus of a japonica cultivar Nipponbare of rice (Oryza sativa; Miyao et al., 2003). Seeds of wild-type rice (cv Nipponbare) and the mutant were soaked in deionized water at 30°C in the dark for 2 d and then transferred to a net floating on 0.5 mm CaCl₂ solution in a 1.5-liter plastic container for 3 to 7 d before being used for various experiments. The seedlings were then transferred to a 3.5-liter plastic pot containing one-half-strength Kimura B solution (pH 5.6; Yamaji and Ma, 2007). The nutrient solution was changed once every 2 d.

Morphological Analysis

For measuring the root length, germinated seedlings were exposed to a 0.5 mm CaCl₂ solution and the root length was measured by a ruler at different days. The lateral root numbers in primary root of 7-d-old seedlings were counted and the lateral root density was calculated by dividing the number of lateral roots by the primary root length for each plant. Three-week-old seedlings were used for observation of longitudinal and cross sections of crown root (at 1 and 10 mm from the root apex) and cross section of leaf sheath (at 5 and 30 mm from the root-to-shoot junction). The samples were imbedded into 5% agar and then were sectioned by a microslicer (100 µm thickness, LinearSlicer PRO10; Dosaka EM). These sections were observed under a confocal laser scanning microscope (LSM 700; Carl Zeiss). The length of root apical meristem (the distance between the quiescent center and the start point of the elongation zone) was also determined. For the measurement of cell size, longitudinal and cross sections of the root elongation zone were used. The length of the cortical cells was determined for nine roots each with 10 cells (n = 90). The width of the cortical cells was determined for 10 roots each with eight cells (n = 80). The width of epidermal cells in leaf sheath was determined for 14 samples each with 10 cells (n = 140).

Positional Cloning of OsCCC1 and Whole-Genome Sequencing

For mapping the responsible gene, the short-root mutant was crossed with Kasalath to obtain an F2 population. A bulked segregant analysis using two bulked DNA samples (short-root versus normal-root) was first performed to identify the molecular markers linked to OsCCC1 (Michelmore et al., 1991). To further mapping this gene, polymorphic molecular markers were designed based on the sequence comparison in the corresponding genomic region between Nipponbare and 93-11 (Supplemental Table S1). Using 3460 F2 seedlings showing short-root phenotype, OsCCC1 finally was mapped to 440-kb region near the centromere of chromosome 8 according to the Rice Annotation Project Database (http://rapdb.dna.affrc.go.jp/).

For whole-genome sequencing, the short-root mutant plant was crossed with wild-type rice (cv Nipponbare). The F1 plant was self-pollinated to obtain F2 progeny. For genetic analysis, 200 seeds were used for phenotypic analysis. The genomic DNA of 50 F2 plants each showing the short-root or normal-root phenotype was bulked in an equal ratio and subjected to whole-genome sequencing for 50 cycles by the sequencer. The single nucleotide polymorphism analysis was conducted by MutMap (Abe et al., 2012).

Complementation Test

The native 2.5-kb promoter sequence of OsCCC1 was amplified by PCR using the primer pairs 5′-AATGGGCCCTTGTTGAGGTATAAGGTCA-3′ and 5′-TCGATCTCCCCGTTCTCCATCCCTCACTCTAGCAACTACA-3′. The ORF with 3′ untranslated region of OsCCC1 was amplified by PCR using the primer pairs 5′-TGTAGTTGCTAGAGTGAGGGATGGAGAACGGGGAGATCGA-3′ and 5′-CGGACTAGTACCAATAATTTCAGCTGA-3′. The fragment containing ProOsCCC1-OsCCC1-3′UTR was acquired by overlap PCR and inserted into pPZP2H-lac (with NOS terminator) using ApaI and SpeI. The construct was introduced into the calluses of rice (cv Nipponbare) via Agrobacterium tumefaciens-mediated transformation (Hiei et al., 1994).

RNA Isolation, Gene Cloning, and Expression Analysis

Total RNA from rice roots was extracted using the RNeasy Mini Kit (Qiagen). One microgram of total RNA was used for first-strand cDNA synthesis using a ReverTra Ace qPCR RT Master Mix kit (TOYOBO) following the manufacturer’s instructions. The cDNA fragment containing OsCCC1 ORF was amplified by PCR using the primers 5′-AATGTCGACATGGAGAACGGGGAGATC-3′ and 5′-CGGACTAGTACCAATAATTTCAGCTGA-3′. The fragment was cloned into the pGEM-T easy vector (Promega) for sequence confirmation using the ABI PRISM 310 genetic analyzer and the BigDye Terminators v3.1 cycle sequencing kit (Applied Biosystems).

For the spatial expression, RNA was extracted from root tips (0–1 cm) and basal region (1–2 cm) of 5-d-old seedlings. For root tissue-specific expression, the segments at 1.75 to 2.25 cm from the root tip of 4-d-old seedlings were collected for the tissue sections. The central cylinder (pericycle and inner tissues) and outer tissues (cortex and epidermis) were separated using the LCC1704 Veritas Laser Microdissection System (Molecular Devices) followed by total RNA extraction. To investigate the response of OsCCC1 expression to KCl and NaCl, 10-d-old seedlings were pretreated with potassium deficiency for 1 week and then exposed to a one-half-strength Kimura B solution (removal of K) containing different NaCl + KCl (1:1 ratio) concentrations for 6 h. For high-salt condition, rice seedlings (14 d old) were exposed to a nutrient solution without or with 50 mm KCl or 50 mm NaCl supply for 12 h.

The gene expression level was determined by real-time RT-PCR using Thunderbird SYBR qPCR Mix (TOYOBO) on Mastercycler ep realplex (Eppendorf). The primers used were 5′-AAGCCGTTGTCATTGTGAAG-3′ and 5′-CTTGAGAATCGTCCTGTGGA-3′ for OsCCC1. Histone H3 (forward primer, 5′-AGTTTGGTCGCTCTCGATTTCG-3′; reverse primer, 5′-TCAACAAGTTGACCACGTCACG-3′) was used as an internal control. The expression was normalized by the ΔΔCt method.

Tissue Specificity of Expression

The native 2.5-kb promoter sequence of OsCCC1 was amplified by PCR. The primer sequences (5′-AATGGGCCCTTGTTGAGGTATAAGGTCA-3′ and 5′-CGGACTAGTGCCGCTTTACTTGTACAG-3′) were used for amplification and introduction of the ApaI and SpeI restriction sites. The fragment was linked to sGFP gene by overlap PCR. The amplified PCR product was inserted into pPZP2H-lac (with NOS terminator) using ApaI and SpeI to create the OsCCC1 promoter-GFP construct. The construct was introduced into the calluses of rice (cv Nipponbare) via A. tumefaciens-mediated transformation (Hiei et al., 1994). The root, leaf sheath, and leaf blade of 1-month-old seedlings of the wild type (cv Nipponbare) and five independent transgenic rice (T0) were used for immunostaining with a rabbit GFP antibody as described below.

Tissue and Subcellular Localization of OsCCC1

The synthetic peptide SGAPQDDSQEAYTSAQRR (positions 870 to 887 of OsCCC1) was used to immunize rabbits to obtain antibodies against OsCCC1. The obtained antiserum was purified through a peptide affinity column before use. The root, leaf blade, and basal node of 1-month-old rice seedlings (cv Nipponbare) were used for immunostaining as described below. Double staining with DAPI for nuclei was also performed to investigate the subcellular localization.

Immunohistological Analysis

For western-blot analysis, microsomal proteins were extracted from the roots and shoots of wild-type rice (cv Nipponbare) according to Mitani et al. (2009). After determining the protein concentrations by the Bradford assay (Bio-Rad), the same amount of each sample was loaded onto SDS/PAGE using 5 to 20% gradient polyacrylamide gels (ATTO). Rabbit anti-OsCCC1 polyclonal antibody (1:500) was used as the primary antibody. Anti-rabbit IgG (H+L) conjugated to horseradish peroxidase (1:10,000; Promega) was used as a secondary antibody, and an ECL Plus western blotting detection system (GE Healthcare) was used for chemiluminescence detection.

Immunostaining was performed according to the method modified from Yamaji and Ma (2007). Rice roots, leaf blade, leaf sheath, and node were fixed in 4% (w/v) paraformaldehyde and 60 mm Suc buffered with 50 mm cacodylic acid (pH 7.4) for 2 h at room temperature. After washing three times in PBS (10 mm PBS, pH 7.4, 138 mm NaCl, and 2.7 mm KCl), the samples were embedded in 5% agar and sectioned 100 μm thick with a LinearSlicer PRO 10 (Dosaka EM). Sections were placed on microscope slides and incubated with PBS containing 0.1% (w/v) pectolyase and 0.3% (v/v) Triton X-100 for 2 h at room temperature. After washing three times in PBS, samples were blocked with PBS containing 5% (w/v) bovine serum albumin and anti-GFP (1:1,000 dilution) or anti-OsCCC1 (1: 500 dilution) primary antibody. Slides were incubated at room temperature overnight and washed four times with PBS. The slides were exposed to secondary antibodies (Alexa Fluor 555 goat anti-rabbit IgG; Molecular Probes) in PBS with bovine serum albumin for 2 h at room temperature, washed five times in PBS, and mounted with 50% (v/v) glycerol in PBS. Samples were examined with a confocal laser scanning microscope (LSM700; Carl Zeiss).

Elemental and Osmotic Pressure Analysis

Germinated seedlings of the wild type, short-root mutant, and two complementation lines were exposed to 0.1 mm Ca(NO₃)₂ solution with different concentrations of KCl or NaCl. After 1 d, the shoots and roots of half plants were harvested and dried at 70°C for 2 d and then boiled at 100°C for 2 h. After 3 d, the root length of another half plants was measured by a ruler for root recovery test. At end of the experiment, the root tips (0–15 mm) and the shoot were excised for cell sap collection according to Chen et al. (2012). Metal concentration in the roots, shoots, and cell sap was determined by inductively coupled plasma mass spectrometer using an Agilent 7700 mass spectrometer. Cl concentration was determined by ion chromatograph (ICS-900; Dionex) with the IonPac AS12A column. The osmolality in the roots and shoots was measured using 10 μL cell sap of each sample by vapor pressure osmometer (5520; Wescor).

OsCCC1 Expression in Yeast

The entire ORFs for OsCCC1, mutated OsCCC1, OsHKT2;1, and OsKAT1 were amplified by PCR and cloned into the pYES2 vector (Invitrogen). After sequence confirmation, the OsCCC1, mutated OsCCC1 (C568F), OsKAT1, OsHKT2;1, or the empty vector were introduced into yeast according to the manufacturer’s protocols (S.c.easy comp transformation kit; Invitrogen). The K⁺-sensitive mutant strain CY162 (MATa ura3 his3 his4 trk1Δ trk2Δ1::pCK64) and Na-sensitive mutant strain G19 (MATa ade2 ura3 leu2 his3 trp1 ena1Δ::HIS3::ena4Δ) were used for study. Primer pairs used for amplification and introduction of restriction sites were 5′-ATGAGCTCAAAATGGAGAACGGGGAGATC-3′ and 5′-CCCTCGAGTCATGTGAAGAATGTGAC-3′ for OsCCC1 and mutated OsCCC1, 5′-ACGAGCTCAAAATGCCACGTTCTTCTCGT-3′ and 5′-CCCTCGAGTTATACGTTCACTTGCTG-3′ for OsKAT1, and 5′-ATGAGCTCAAAATGACGAGCATTTACCATGA-3′ and 5′-CCCTCGAGTTACCATAGCCTCCAATATT-3′ for OsHKT2;1.

Yeast transformants were selected on uracil-deficient medium and grown in synthetic complete (SC-uracil) yeast medium containing 2% Glc, 0.67% yeast nitrogen base without amino acids (Difco), 0.2% appropriate amino acids, and 2% agar at pH 6.0. One colony was selected in each transformation strain and grown in liquid SC-Glc-uracil medium. For the plate experiment, four serial dilutions of yeast cell suspensions were spotted on plates containing Gal and Glc and cultured at 30°C for 3 d. The SC-uracil plate was added with 10 mm KCl to achieve the total K⁺ to 20 mm or added with 300 mm NaCl to achieve the total Na⁺ to 300 mM.

For the liquid culture uptake experiment, yeast transformants were first grown to linear phase. After washing three times with Mill-Q water, the yeast was adjusted to an OD₆₀₀ value of 0.15 and cultured in a SC-uracil (+Gal) medium with KCl (25, 50, 75, or 100 mm) or NaCl (0, 100, 300, or 500 mm) until OD₆₀₀ = 1.5 to 2.0. Cells were collected and washed by 20 mm Ca(NO₃)₂ solution on ice three times. The samples were boiled for 1 h before elemental analysis.

Root Elemental Distribution Analysis

Both wild-type rice and the short-root mutant (21 d old) were cultivated in the nutrient solution containing 50 mm NaCl. After 24 h, the roots were washed three times in 5 mm Ca(NO₃)₂ solution on ice, excised, and fixed by 5% agar powder. The transverse section at about 10 mm from the root apex was sectioned by a microslicer (LinearSlicer PRO10; Dosaka EM) and immediately used for analysis by scanning electron microscope (TM3000; Hitachi) in vacuum condition at −20°C. The elemental distribution photos were generated by energy dispersive x-ray spectrometer (SwiftED 3000; Oxford Instruments).

Accession Numbers

The accession number of OsCCC1 is registered as LC085614 in the GenBank/EMBL databases.

Supplemental Data

The following supplemental materials are available.

• Supplemental Figure S1. Phenotypic comparison of lateral roots, leaf blade, and basal stem between the wild-type rice and short-root mutant.

• Supplemental Figure S2. Growth and grain yield of wild-type rice (cv Nipponbare) and short-root mutant grown in a field.

• Supplemental Figure S3. Comparison of cell number in root and shoot between the wild-type rice and short-root mutant.

• Supplemental Figure S4. Map-based cloning of the gene responsible for the short-root phenotype.

• Supplemental Figure S5. Scheme for MutMap using whole-genome sequencing.

• Supplemental Figure S6. Alignment of mutation region between the bulked DNA from normal-root and short-root pools by MutMap and confirmation of mutation by PCR.

• Supplemental Figure S7. Phylogenetic tree of OsCCC1 in plants and predicated topology of OsCCC1.

• Supplemental Figure S8. Partial recovery of the root growth in short-root mutant by addition of NaCl/KCl.

• Supplemental Figure S9. Elemental distribution in short-root mutant using SEM and EDX.

• Supplemental Figure S10. Concentration of K⁺, Na⁺, and Cl⁻ in roots and shoots in response to KCl and NaCl.

• Supplemental Figure S11. Osmolality in shoot cell sap in response to KCl and NaCl.

• Supplemental Figure S12. Western-blot analysis of OsCCC1.

• Supplemental Table S1. Primers for InDel markers used for mapping of OsCCC1.

Glossary

ORF

open reading frame

DAPI

4′,6-diamino-phenylindole