A Cation-Chloride Cotransporter Gene Is Required forCell Elongation and Osmoregulation in Rice1[OPEN]

Zhi Chang Chen, Naoki Yamaji, Miho Fujii-Kashino, and Jian Feng Ma*

Root Biology Center, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University,Fujian, Fuzhou 350002, China (Z.C.C.); and Institute of Plant Science and Resources, Okayama University,Chuo 2-20-1, Kurashiki, Japan (Z.C.C., N.Y., M.F.-K., J.F.M.)

ORCID ID: 0000-0003-3411-827X (J.F.M.).

Rice (Oryza sativa) is characterized by having fibrous root systems; however, the molecular mechanisms underlying the rootdevelopment are not fully understood. Here, we isolated a rice mutant with short roots and found that the mutant had adecreased cell size of the roots and shoots compared with wild-type rice. Map-based cloning combined with whole-genomesequencing revealed that a single nucleotide mutation occurred in a gene, which encodes a putative cation-chloridecotransporter (OsCCC1). Introduction of OsCCC1 cDNA into the mutant rescued the mutant growth, indicating that growthdefects of both the roots and shoots are caused by loss of function of OsCCC1. Physiological analysis showed that the mutanthad a lower concentration of Cl2 and K+ and lower osmolality in the root cell sap than the wild type at all KCl supply conditionstested; however, the mutant only showed a lower Na+ concentration at high external Na+. Expression of OsCCC1 in yeastincreased accumulation of K+, Na+, and Cl2. The expression of OsCCC1 was found in both the roots and shoots, although higherexpression was found in the root tips. Furthermore, the expression in the roots did not respond to different Na+, K+, and Cl2

supply. OsCCC1 was expressed in all cells of the roots, leaf, and basal node. Immunoblot analysis revealed that OsCCC1 wasmainly localized to the plasma membrane. These results suggest that OsCCC1 is involved in the cell elongation by regulating ion(Cl2, K+, and Na+) homeostasis to maintain cellular osmotic potential.

Root architecture is a very important trait for plantgrowth and development because roots are essentialfor the uptake ofwater andmineral nutrients from soils.In addition, roots also play an important role in de-toxification of harmful minerals in soils, structuralsupport of aboveground parts, and environmentalsensing (Marschner, 2012; Jung and McCouch, 2013).An ideotype of root system is determined by manyfactors, such as root length, number, diameter, and rootconfiguration in the soil profile (de Dorlodot et al., 2007;Petricka et al., 2012). These factors differ with plantspecies and environments; therefore, understandingof molecular mechanisms underlying root develop-ment in different species and response to environmen-tal changes is very important for crop productivity.

Rice (Oryza sativa) is characterized by having a fi-brous root system, which is composed of a seminal root,crown roots, lateral roots, and root hairs (Rebouillatet al., 2009; Coudert et al., 2010). Anatomically, riceroots have two Casparian strips at the exodermis andendodermis cells and aerenchyma due to destruction ofcortical cells in the root mature zones (Kawai et al.,1997; Coudert et al., 2010). A number of genes involvedin root development in rice have been identified bydifferent approaches. These genes are involved in var-ious biological processes controlling the developmentof primary root (Zhuang et al., 2006; Qi et al., 2012; Qinet 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 drivecell elongation. Potassium (K) is the most abundantcation in the cytosol and K+ with its accompanyinganions contributing greatly to the osmotic potential ofplant cells and tissues (Marschner, 2012). Potassiumtransporters and channels in plant have been extensivelystudied in gene families such as Shaker, KUP/HAK/KT,HKT, NHX, and CHX (Ashley et al., 2006; Shabala andCuin, 2008; Wang and Wu, 2013). Some of these familymembers also have the transport activity of sodium (Na)due to the similar physico-chemical properties betweensodium and potassium (Hamamoto et al., 2015). Forplants, sodiumusually is not essential, but in some cases,sodium could replace the role of potassium to maintain

1 This work was supported by a Grant-in-Aid for Scientific Re-search on Innovative Areas from the Ministry of Education, Culture,Sports, Science, and Technology of Japan (22119002 and 24248014 toJ.F.M.).

* Address correspondence to maj@rib.okayama-u.ac.jp.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Jian Feng Ma (maj@rib.okayama-u.ac.jp).

Z.C.C., N.Y., and J.F.M. conceived and designed the experiments;Z.C.C. performedmost of the experiments; M.F.-K. prepared the lasermicrodissection sample for RT-PCR; Z.C.C. and J.F.M. analyzed data;Z.C.C., N.Y., and J.F.M. wrote the article.

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the cell osmotic potential (Blumwald, 2000; Horie et al.,2007). Nonselective cation channels are proposed to bethe dominant pathways of Na+ influx in many plantspecies (Kronzucker and Britto, 2011; Hasegawa, 2013;Yamaguchi et al., 2013), but the molecular identity ofmany Na+ uptake mechanisms is still unknown. On theother hand, chloride (Cl), together with potassium, has aparticular function to stabilize the osmotic potential andturgor pressure (White and Broadley, 2001; Marschner,2012). Recently, a plasma membrane-localized Nitrate/Peptide Transporter, NPF2.4, was reported to be in-volved in the long-distance transport of Cl2 in Arabi-dopsis (Arabidopsis thaliana; Li et al., 2016). However,the molecular mechanism for Cl2 transport in plants isstill poorly understood.In animals, it has been reported that a cation-chloride

cotransporter (CCC) family (also called SLC12) is in-volved in transport of K+, Na+, and Cl2 (Russell, 2000;Hebert et al., 2004; Gamba, 2005). It has been dividedinto three groups: K+-Cl2 cotransporters, Na+-Cl2

cotransporters, and Na+-K+-Cl2 cotransporters. Thesetransporters have a variety of functions, includingtransepithelial salt transport, hearing, neuronal devel-opment, and cell volume regulation (Hoffmann et al.,2009; Lindinger et al., 2011; Moes et al., 2014). CCCfamily genes were also found in the plant genome, butonly a few of them have been functionally character-ized. AtCCC in Arabidopsis has been suggested to beinvolved in long-distance Cl2 transport (Colmenero-Flores et al., 2007). AtCCC catalyzed the coordinatedsymport of K+, Na+, and Cl2 inXenopus laevis oocytes. Itshowed preferential expression in the root and shootvasculature at the xylem/symplast boundary, root tips,trichomes, leaf hydathodes, leaf stipules, and anthers.Knockout of this gene resulted in shorter organs, in-cluding inflorescence stems, roots, leaves, and siliques(Colmenero-Flores et al., 2007), indicating that AtCCCis involved in development processes and Cl homeo-stasis. More recently, Henderson et al. (2015) charac-terized a CCC gene (VviCCC) from grapevine (Vitisvinifera). They found that VviCCC was able to comple-ment the atccc mutant, indicating their similar role inplants. Furthermore, both VviCCC and AtCCC wereobserved to be localized at the Golgi and trans-Golginetwork (Henderson et al., 2015). On the other hand,OsCCC1 in rice was partially characterized in terms ofsalt stress (Kong et al., 2011). Knockdown of this generesulted in increased sensitivity to salt stress, especiallyto high KCl (Kong et al., 2011). The concentration of K+

and Cl2 was decreased in both the roots and shootsof knockdown lines compared with the wild type,whereas that of Na+was hardly affected by suppressionof this gene. In contrast to AtCCC and VviCCC,OsCCC1 was localized to the plasma membrane exam-ined by transient expression of OsCCC1-GFP in onionepidermal cells.In this study, we isolated a rice mutant showing

a distinct short-root phenotype. Map-based cloningcombined with whole-genome sequencing revealedthat the phenotype was caused by a point mutation of

the gene OsCCC1 belonging to CCC family. A detailedfunctional analysis showed that OsCCC1, encoding aplasma membrane-localized transporter for K+, Na+,and Cl2, is required for cell elongation of both the rootsand shoots through maintaining cellular osmotic po-tential.

RESULTS

Isolation and Phenotypic Characterization of a Short-RootRice Mutant

A rice mutant showing short-root phenotype wasobtained from a Tos-17 transposon insertion line(NG2024). NG2024 has two Tos-17 insertion sites thatare located at chromosomes 3 and 7 (https://tos.nias.affrc.go.jp/), respectively; however, neither of themwas associated with the short-root phenotype by PCRidentification, indicating that the short-root phenotypewas caused by other mutation site. The mutant showeda much shorter length of seminal, lateral, and crownroots than the wild type (cv Nipponbare) at both theseedling stage and reproductive stage (Fig. 1, A–D;Supplemental Fig. S1, A and B). A time-dependent rootelongation measurement showed that the root elonga-tion rate was much slower in the mutant than in thewild type (Fig. 1E). However, the number of crownroots and the density of the lateral roots were similarbetween the wild type and the mutant (Fig. 1, F and G).

The mutant also showed a shorter shoot heightcomparedwith the wild type (Fig. 1, B–D). Thewidth ofboth leaf blade and basal stem was smaller in the mu-tant than in the wild type (Supplemental Fig. S1, C–F).

When cultivated in a field, the mutant showed muchsmaller size of thewhole plants (Supplemental Fig. S2A).The plant height of the mutant was significantly lowerthan that of the wild type at harvest (Supplemental Fig.S2B). All yield components, including panicle number,1000-grain weight, spikelet number per panicle, andpercentage of filled spikelets, were greatly decreasedin the mutant (Supplemental Fig. S2, C–H), resultingin a significant reduction of grain yield (SupplementalFig. S2I).

Observation of longitudinal sections of root tip re-gion showed that the length of root apical meristem(from the quiescent center to start of the elongationzone) was significantly shorter in the mutant than thatin the wild type (0.596 0.04 mm versus 0.856 0.07 mm;Fig. 2A). Both wild-type and mutant roots had similarradial 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 thesame number of root cortical cell layer (Supplemental Fig.S3A). However, the diameter of the roots was signifi-cantly smaller in the mutant than in the wild type (Fig. 2,B–E). The length and width of the root cells of the mutantwas 43.9% and 71.9%, respectively, of the wild type (Fig.2, F and G).

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Shoot cell size was also compared between the mu-tant and the wild type at the shoot basal region. Ob-servation of transverse cross sections showed that thecell size of leaf sheath inmutant was smaller than that inthe wild type in both elongating and elongated zones(Fig. 2, H–K). The cell width of the leaf epidermal cellswas 20.0 mm in the wild type, in contrast to 14.7 mm inthe mutant (Fig. 2L). However, there was no differencein the cell numbers of adaxial epidermis of leaf sheathbetween the wild type and the mutant (SupplementalFig. S3B). These results indicate that the shorter rootsand shoots of the mutant are derived from decreasedcell size but not from the cell numbers and tissuestructure.

Cloning of the Responsible Gene for the Short-Root Phenotype

We first performed a genetic analysis by using aheterogeneous population derived from a Tos-17 in-sertion line. Among 200 seedlings tested, 54 seedlingsshowed short-root phenotype, while 146 seedlingsshowed normal root phenotype. This segregation ratiofits to 1:3, indicating that the short-root phenotype iscontrolled by a recessive gene.

To isolate the gene responsible for the short-rootphenotype, we constructed an F2 population by cross-ing the mutant with Kasalath, an indica cultivar. Using3460 F2 seedlings showing short-root phenotype, thecandidate gene was mapped to a 440-kb region near thecentromere of chromosome 8 by map-based cloningusing markers shown in Supplemental Table S1(Supplemental Fig. S4A). There are 56 predicted geneswithin this region based on the Rice Annotation ProjectDatabase (http://rapdb.dna.affrc.go.jp/). To furtherclone the responsible gene, we performed MutMap(Abe et al., 2012; Supplemental Fig. S5) by sequencingthe whole genome of bulked DNA from normal-rootand short-root pools. Sequence alignment revealedone point mutation occurred in the 440-kb region. In thegenome of short-root pool, all showed adenine (A)in this locus, whereas in the genome of normal-rootpool, it contained adenine (A) and cytosine (C)(Supplemental Fig. S6A). To confirm this result, weresequenced this locus by using a PCR product. Theresults showed that the wild type presented C, themutants presented A, and the heterozygote presentedboth A and C in this locus (Supplemental Fig. S6B).This was consistent with the whole-genome sequencingresults.

Figure 1. Phenotypic comparison of the wild-type rice and short-root mutant. A to C, Pheno-types of thewild type (cvNipponbare; left) and themutant (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 atharvest. Bar = 30 cm. E, Time-dependent rootgrowth. Germinated seedlings of both the wildtype and the mutant were exposed to a 0.5 mM

CaCl2 solution, and the seminal root length wasmeasured at different days indicated. Error barsrepresent6 SD (n = 15). F, Lateral root density. Thelateral root numbers on primary root of 7-d-oldseedlings were counted, and the lateral root den-sity was calculated by dividing the number oflateral roots by the primary root length for eachplant. Data are means 6 SD (n = 20). G, Crownroot number of the wild type and the mutantgrown hydroponically for 20 d.

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

and OsCCC2. They share 82% identity with each other.OsCCC1 shares 79% identity with AtCCC in Arabi-dopsis. A BLAST search on NCBI revealed OsCCC1homologs in other plant species, including maize(Zea mays), sorghum (Sorghum bicolor), soybean (Glycinemax), tobacco (Nicotiana tabacum), rape (Brassica napus),andMedicago truncatula (Supplemental Fig. S7A). Usingthe SOSUI program (http://bp.nuap.nagoya-u.ac.jp/sosui/) and TMHMM server (http://www.cbs.dtu.dk/services/TMHMM-2.0/), OsCCC1 was predictedto be a membrane protein with 11 transmembrane do-mains (Supplemental Fig. S7B).

Complementation Test

To confirm whether the mutation in OsCCC1 is re-sponsible for the short-root and -shoot phenotypes, weperformed a complementation test by introducing2.5-kb promoter sequence of OsCCC1 fused withOsCCC1 cDNA into the mutant. Analysis with two in-dependent transgenic lines showed that their root andshoot growth recovered the same as the wild type (Fig.3, A and B), indicating that these phenotypes are causedby mutation of OsCCC1.

Mineral Profile Analysis of Short-Root Mutant

Since CCC was reported to be a cation-chloridecotransporter in animals and Arabidopsis (Russell,2000; Hebert et al., 2004; Colmenero-Flores et al., 2007),we compared the cation profile of the root cell sapamong thewild type,mutant, and two complementationlines. Among cations tested, K+ was the dominant one,being 40mM in thewild type (Fig. 3C). Thewild type andtwo 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

Figure 2. Morphological comparison of the wild-type rice and short-root mutant. A, Longitudinalsections of the root tip in the wild type (cv Nip-ponbare; left) and the mutant (right). The length ofroot apical meristem is evaluated by the distancebetween the quiescent center (QC) and the startpoint of the elongation zone. Bar = 1 mm. B to E,Root transverse sections at 1 mm (B and C) and10 mm (D and E) from the root apex of the wildtype (B and D) and the mutant (C and E). Three-week-old seedlings were used for observation oflongitudinal and cross sections of crown root.Bars = 200 mm. F, Longitudinal cell length ofcortical cells in the mature region (at 10 mm fromthe apex) of the root (n = 90). G, Transverse cellwidth of cortical cells in the mature region (at10 mm from the apex) of the root (n = 80). H to K,Shoot transverse sections at 5 and 30mm from theroot-shoot junction of the wild type (H and J) andthe mutant (I and K). Bar = 1 mm. These photosshowed a rolled-up young leaf blade (YB)enclosed by an old leaf sheath (OS). L, Transversecell width of adaxial epidermal cells in leaf sheath(n = 140). The asterisk in A, C, E to G, and L showsa significant difference between the wild type andmutant (P , 0.05 by Tukey’s test).

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other cations, including Na, Mn, Cu, and Zn, increased,but that of Mg, Ca, and Fe remained unchanged in themutant (Fig. 3, C and D).

To further investigate whether the short-root phe-notype in the mutant is caused by low K+ or high Na+

concentration, we exposed the plants to different KCland NaCl supply conditions. The results showed thatthe concentration of K+ and Cl2 was significantly lowerin root of mutant than the wild type and two comple-mentation lines irrespective of KCl or NaCl supply (Fig.4, A, C, E, and G). Na+ concentration was much higherin themutant at lowNaCl condition (Fig. 4, B and F) butremarkably decreased at high NaCl supply condition(50 mM; Fig. 4F). The Na+ concentration in the rootcell sap was significantly decreased with increasingexternal K+ concentration in both the wild type andmutant (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 com-pletely restored by any condition of KCl orNaCl supply(Supplemental Fig. S8).

Distribution of K+, Na+, and Cl2 was also examinedin the roots of the wild type and mutant using scanningelectron microscopy with energy dispersive x-rayspectroscopy. At 50 mM NaCl supply condition, amuch stronger signal of K+, Na+, and Cl2 was observedin root cells of the wild type than that of the mutant(Supplemental Fig. S9, A–F). Moreover, quantitativeanalysis showed the K+, Na+, and Cl2 were highly ac-cumulated in the root cortical cells rather than the steleand exodermis in both the wild type and mutant(Supplemental Fig. S9, G–L). The concentration of Na+,

K+, and Cl2 in the shoot was also compared betweenthe wild type and mutant. Similar to the roots, con-centration of K+ and Cl2 was lower in the shoots of themutant compared with the wild type and two com-plementation lines (Supplemental Fig. S10, A and B).The Na+ concentration was also decreased in both theshoots and roots under the condition of high Na+ sup-ply (Supplemental Fig. S10C).

Osmolality in the Root and Shoot Cell Sap

The osmolality in the root and shoot cell sap wascompared among the mutant, the wild type, and twocomplementation lines using vapor pressure osmome-ter. The results showed that the mutant had a decreasedroot and shoot osmolality compared with the wild typeand two complementation lines, irrespective of KCl orNaCl supply conditions (Fig. 4, D and H; SupplementalFig. S11).

Yeast Complementation Test of OsCCC1

To test whether OsCCC1 is permeable to K+, we firstintroduced OsCCC1 into yeast strain CY162 using aGal-inducible promoter. CY162 is a mutant sensitive toK+ deficiency due to lack of K+ transporters TRK1 andTRK2 (Anderson et al., 1992). OsKAT1, a known K+

transporter in rice (Obata et al., 2007), was used as apositive control. In the presence of Glc, when the geneexpression was not induced, the yeast carrying emptyvector pYES2 (negative control), OsCCC1, mutated

Figure 3. Complementation test and mineralanalysis. A and B, Complementation of the short-root phenotype. Germinated seedlings of wild-type rice, short-root mutant, and two independentcomplementation lines (T1) transformed with anORF of OsCCC1 driven by 2.5-kb promoter ofOsCCC1 were exposed to a solution containingCa(NO3)2 for 3 d. The root was photographed (A),and the root length was measured by a ruler (B).Data are means 6 SD (n = 10). Bar = 5 cm. C andD, Macro (C) and micro (D) mineral concentra-tions in the root cell sap. The root tips (0–15 mm)were excised for root cell sap collection. Themetal concentrations were determined by induc-tively coupled plasma mass spectrometer. Dataare means 6 SD (n = 3). The asterisk in B to D in-dicates significant differences compared with thewild type (*P , 0.05 by Tukey’s test).

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OsCCC1 (C568F), and OsKAT1 showed the samegrowth (Fig. 5A). However, when the gene expressionwas induced by Gal, the growth of yeast carryingOsCCC1 and OsKAT1 was much better than that ofempty vector and mutated OsCCC1 (Fig. 5B). Theseresults suggest that OsCCC1 is involved in uptake of K+

in yeast.To determine whether OsCCC1 is also involved in

Na+ uptake, we then introduced OsCCC1 into the yeastmutant strain G19, which lacks major Na+ pumps andshows high sensitivity to salt stress (Quintero et al.,1996). The Na+ transporter gene OsHKT2;1 in rice (alsonamed OsHKT1; Horie et al., 2001) was used as apositive control. In the presence of Glc, there was nodifference in the growth among the yeast cells carryingpYES2 (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 wasable to grow in the presence of 300 mM NaCl. However,in the presence of Gal, expression of OsHKT2;1 andOsCCC1 resulted in a higher sensitivity to salt stresscompared with the empty vector and mutated OsCCC1(Fig. 5D). Compared with OsHKT2;1, OsCCC1 showeda relatively lower affinity to Na+ (Fig. 5D).

Furthermore, we quantified the uptake of K+ and Cl2

by OsCCC1 in CY162, Na+, and Cl2 in G19 using liquidculture. A dose-dependent experiment showed that Kuptake by the yeast CY162 expressing OsCCC1 andOsKAT1 was significantly higher than that by vectorcontrol at each KCl concentration in the presence of Gal(Fig. 5E). The Cl2 uptake was also increased in the yeastcarrying OsCCC1 compared within the vector controlbut not in yeast carrying OsKAT1 (Fig. 5F). Similarly, a

Figure 4. Concentration of ions (K+, Na+, and Cl2)and osmolality in root cell sap in response to KCland NaCl. Germinated seedlings of wild-type rice,short-root mutant, and two independent comple-mentation lines were exposed to a solution con-taining different concentration of KCl (A–D) orNaCl (E–H) for 3 d. The root tips (0–15 mm) wereexcised for root cell sap collection. The concen-tration of K+ (A and E), Na+ (B and F), and Cl2 (Cand G) and osmolality (D and H) were determinedin the wild type, mutant, and two complementa-tion lines. Data aremeans6 SD (n = 3). The asteriskindicates significant differences comparedwith thewild type (*P , 0.05 by Tukey’s test).

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dose-dependent experiment showed that Na+ uptakeby the yeast G19 expressing OsCCC1 and OsHKT2;1was significantly higher than that by vector control (Fig.5G), but the Cl2 uptake was increased only in the yeastcarrying OsCCC1, but not in yeast carrying OsHKT2;1(Fig. 5H). These results showed that different fromOsKAT1 and OsHKT2;1, OsCCC1 likely functions as aNa+, K+-Cl2 cotransporter in yeast.

Expression Pattern of OsCCC1

OsCCC1 was expressed in both the roots and shoots,but much higher expressionwas found in the roots (Fig.6A). In the roots, the expression was higher in the roottip (0–1 cm) than in the mature region (1–2 cm; Fig. 6B).Furthermore, the expression level of OsCCC1 wassimilar between central cylinder and outer tissues inroot mature region (Fig. 6C). The expression ofOsCCC1

in the roots showed no response to external K+ and Na+

concentrations added up to 5 mM and Cl2 up to10 mM (Fig. 6D). The expression of OsCCC1 in theroots was also hardly affected by high NaCl or KCl(50 mM; Fig. 6E).

Tissue-Specific Expression of OsCCC1

To investigate tissue-specific expression of OsCCC1,we generated transgenic rice carrying 2.5-kb promotersequence of OsCCC1 fused with GFP. Immunostainingof the transgenic rice with a GFP antibody showed thatthe root tips showed stronger signal than other parts(Fig. 7A). The signal was observed in all root cells atboth the elongation zone and mature zone of the roots(Fig. 7, B and D). No signal was observed in the wildtype (Fig. 7, C and E), indicating the specificity of theantibody. The signal was also observed in the leaf blade

Figure 5. Yeast complementation test ofOsCCC1. A to D, OsCCC1-mediated toleranceto K+ deficiency (A and B) and Na+ toxicity (Cand D). OsCCC1, mutated OsCCC1 (C568F),empty vector (pYES2, negative control),OsKAT1(positive control), or OsHKT2;1 (positive con-trol) was introduced into yeast mutant strainCY162 sensitive to K+ deficiency (A and B) orG19 sensitive to Na+ stress (C and D). The yeastwas cultured on the synthetic complete medium(SC-uracil) containing 20 mM KCl (A and B) or300mMNaCl (C andD) in the presence of Glc (Aand C) or Gal (B and D) at 30˚C for 3 d. Fourserial 1:10 dilutions (from left to right) of yeastcell suspensions starting from OD600 = 0.5 werespotted on plates. E to H, Dose-dependent up-take of K+ and Cl2 in yeast CY162 (E and F), andNa+ and Cl2 in yeast G19 (G and H). Yeast strainCY162 carrying OsCCC1, OsKAT1, or emptyvector (pYES2) was exposed to a SC-uracil so-lution containing KCl (25, 50, 75, and 100 mM)in the presence of Gal. Yeast strain G19 carryingOsCCC1, OsHKT2;1, or empty vector (pYES2)was exposed to a SC-uracil solution containingNaCl (0, 100, 300, and 500 mM) in the presenceof Gal. Yeast strains were sampled at the expo-nential phase for elemental analysis. Data aremeans 6 SD (n = 3). The asterisk shows a signif-icant difference compared with empty vector(P , 0.05 by Tukey’s test).

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(Fig. 7F), leaf sheath (Fig. 7H), and basal node (Fig. 7J) ofthe transgenic lines, but not in the wild type (Fig. 7, G, I,and K).

Cellular and Subcellular Localization of OsCCC1

To investigate the localization of OsCCC1 protein indifferent tissues, we performed immunostaining usingan antibody against C-terminal peptide of OsCCC1.Western-blot analysis showed that there was only onebandobserved in both the shoots and roots (SupplementalFig. S11). The size of this band was about 100 kD,which corresponds to the predicted size of OsCCC1protein (108 kD), indicating that this antibody ishighly specific to OsCCC1. In addition, the proteinabundance in roots was much higher than that inshoots (Supplemental Fig. S12), which is consistentwith the expression level as shown in Figure 6A. Thefluorescence signal was strongly detected in all cells ofthe roots (Fig. 8A). In the leaf blade, the signal was

stronger in the vascular bundle compared to the othertissues (Fig. 8B), and in the basal region, the signalcould be detected in both phloem and xylem region(Fig. 8C).

To examine the in situ subcellular localization ofOsCCC1, we performed a double staining with49,6-diamino-phenylindole (DAPI) for nuclei, showingthat the fluorescence signal from OsCCC1 antibody(red color) was localized mainly at the peripheral re-gion of the cells, which was circumscribed but did notenvelope the nuclei (blue color; Fig. 8, D–F). This resultindicates that OsCCC1 is localized to the plasmamembrane, although additional proof (e.g. analysis ofmembrane fractions) would be required to establish theexact subcellular localization pattern.

DISCUSSION

Isolation of rice mutants with altered root morphol-ogy is a good approach for studying molecular

Figure 6. Expression pattern of OsCCC1 in rice. A, Expression of OsCCC1 in the roots and shoots. The roots and shoots of riceseedlings grown hydroponically for 5 dwere sampled for RNA extraction. B, Root spatial expression. Root segments (0–1 and 1–2cm) of rice seedlings (5 d old) were excised for RNA extraction. C, Tissue specificity ofOsCCC1 expression. The root segments at1.75 to 2.25 cm from the root tipswere collected for the tissue sections. The central cylinder (pericycle and inner tissues) and outertissues (cortex and epidermis) were separated by laser microdissection. D and E, Expression of OsCCC1 in response to Na+, K+,and Cl2. 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 H3was used as an internal standard. The expression relative to root (A), root tip (B), central cylinder (C), 0 mM Cl2 (D), or0 mMNaCl/KCl (E) is shown. Data are means6 SD (n = 3). The asterisk in A and B indicates a significant difference compared withroot and root tip, respectively (P , 0.05 by Tukey’s test).

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mechanisms underlying root development. In thisstudy, we obtained a rice root mutant (osccc1) withdistinct morphology from a Tos-17 insertion line. Thismutant showed extremely shorter seminal, lateral, andcrown roots (Fig. 1, A–D; Supplemental Fig. S1, A and

B). There was no difference in the number of lateralroots, crown roots, and cortical cell layer (Figs. 1, F andG, and 2, A–E; Supplemental Fig. S3A); however, thecell size of the mutant roots was significantly smallerthan that of thewild type (Fig. 2, F andG). Furthermore,

Figure 7. Tissue specificity of OsCCC1 ex-pression. Immunostaining with an anti-GFPantibody was performed in different tissues ofpOsCCC1-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 antibodydetected with a secondary antibody. Cyancolor shows cell wall autofluorescence (F andG). Yellow-dotted area is magnified in theinsets in J. EN, Endodermis; EX, exodermis; P,phloem region; X, xylem region. Five inde-pendent transgenic lines were investigated,and the representative results are shown.Bars = 200 mm.

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the mutant also presented a shorter shoot height andsmaller leaf cell size (Figs. 1, B–D, and 2, H–L). There-fore, the short-root and -shoot phenotypes of the mu-tant result from the decreased cell size of both the rootsand shoots.Positional cloning combined with whole-genome

sequencing led to isolation of a gene responsible forthe 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 acation-chloride cotransporter gene family (CCC). Asingle amino acid substitution (C568F) of OsCCC1 oc-curred in the last transmembrane domain in the mutant(Supplemental Fig. S4B). This mutation resulted in lossof function of this gene as shown in yeast assay exper-iment (Fig. 5).OsCCC1 was partially characterized previously and

suggested to be involved in salt stress tolerance (Konget al., 2011); however, the exact role of this gene is un-known. In this study, we further characterized this genein terms of transport activity, tissue and subcellularlocalization, expression pattern, and detailed analysisof knockout mutants. Our immunostaining result sug-gests that OsCCC1 is located in the plasma membranein rice cells, although additional experiments would berequired to determine the exact location (Fig. 8). This isconsistent with the result of transient expression inonion epidermal cells (Kong et al., 2011). However, thissubcellular localization is different from that of VviCCCin grapevine and AtCCC in Arabidopsis, which arelocalized at the Golgi (Henderson et al., 2015). Thisdifference may determine their different role in plants.In fact, AtCCC has been proposed to be involved in thelong-distance ion transport (Henderson et al., 2015), butOsCCC1 is required for cell enlargement, indicatingdiverse functions of plant CCC in different plant spe-cies. However, it still needs investigation whether

AtCCC is also involved in cell enlargement becauseatccc mutant showed a reduced size, a similar pheno-type to osccc1.

Our results also show that OsCCC1 is likely a Na+,K+-Cl2 cotransporter in rice. This is supported by yeastassay experiment and phenotypic analysis of theknockout 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 Cl2 uptake in theyeast (Fig. 5, E and F), indicating that OsCCC1 func-tions as a cotransporter for K+ and Cl2. However,compared with K+ concentration in yeast, the Cl2 con-centration was much lower (Fig. 5, E and F). The reasonfor this phenomenon remains to be examined in future,but one possibility is that Cl2 taken up by OsCCC1 iseffluxed since yeast is not able to sequester Cl2 (Couryet al., 1999). By contrast, K+ is sequestered into thevacuoles, resulting in different concentration of K+ andCl2 in the cells. In rice root cell sap, the concentration ofK+ and Cl2 was relatively comparable (Fig. 4). Knock-out of OsCCC1 resulted in decreased concentration ofK+ and Cl2 similarly at different external K concen-trations (Fig. 4, A and C). This result further indicatesthat OsCCC1 is a K+ and Cl2 cotransporter.

Although Na+ is not an essential element for plantgrowth and themutant phenotypewas also observed inthe absence of Na+, OsCCC1 shows its permeability toNa+ in yeast mutant defective in Na+ uptake (Fig. 5, Cand D). Compared with OsHKT2;1, it seems that theaffinity for Na+ byOsCCC1 is weak (Fig. 5D). However,expression of OsCCC1 in the yeast increased both Na+

and Cl2 uptake, while expression of OsHKT2;1 onlyincreased Na+ uptake (Fig. 5, G and H). In rice root cellsap, the Na concentration in the mutant differed withexternal Na concentrations. The mutant accumulatedless Na+ than the wild type at a high Na+ concentration

Figure 8. Cellular and subcellular lo-calization of OsCCC1 in rice. Immu-nostaining with a polyclonal antibodyagainst OsCCC1 in different organs ofrice was performed, including root (A),leaf blade (B), and basal node (C–F). Redcolor indicates the OsCCC1 antibody-specific signal. Blue color indicates cellwall and nucleus stained by DAPI (yel-low arrowheads). Yellow-dotted areas inA to Cwere magnified and inserted in A,B, and D to F. D and E are signal fromOsCCC1 antibody and DAPI/cell wall,respectively. F is a merged image of Dand E. Bar = 50 mm.

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(50 mM) but not at low Na+ concentrations (Fig. 4F).These results suggest that OsCCC1 in rice could me-diate Na+ transport with low affinity. On the otherhand, it was observed that the mutant presented ahigher Na+ concentration than the wild type at low Na+

concentrations (Fig. 4F). This difference might be at-tributed to the competition between K+ andNa+. K+ andNa+ uptake by the roots are also mediated by otherchannels 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 be-came larger, resulting in decreased Na+ uptake in themutant (Fig. 4, B and F).

OsCCC1was expressed in almost all cells of rice plant(Fig. 7), and its expression was unaffected by externalK+ and Na+ concentration up to 50 mM (Fig. 6, D and E).This result is different from a previous study by Konget al. (2011), who found that the expression of OsCCC1was induced by high concentration of KCl (150 mM).This discrepancy could be attributed to the concentra-tions of salts used for treatment. Since rice is a relativelysalt-sensitive species, high salt (150 mM) will cause thegrowth inhibition. Therefore, the up-regulation ofOsCCC1 by 150 mM KCl could be an indirect result dueto reduced growth. In fact, we found the expression ofinternal standards (Actin and HistoneH3) was alsochanged at higher salt concentration (150 mM). Our re-sults show that the constitutive expression ofOsCCC1 isrequired for K+, Na+, and Cl2 homeostasis in cells formaintaining appropriate osmotic pressure for cellelongation. This is supported by that knockout of thisgene significantly decreased the osmolality (Fig. 4, Dand H).

K+ is the most abundant cation in the cytosol and cellextension depends on K+ accumulation in the cells forincreasing the osmotic potential (Dolan and Davies,2004). In this study, we also found high K+ concentra-tion (40–150 mM) in the root cell sap depending onexternal K+ concentrations (Fig. 4A). This high K+ con-centration is maintained through different transportersinvolved in the uptake. For example, AtHAK5 mainlyexpressed at the epidermis and stele of roots, wasreported to be involved in K+ uptake in Arabidopsis(Gierth et al., 2005). The expression of AtHAK5 is rap-idly up-regulated by potassium starvation. Differentfrom other transporter genes, OsCCC1 shows no re-sponse to K+ and is highly expressed in all cells of theroot tip region (Fig. 6, B and D), where cell elongationoccurs. High KCl supply did not completely comple-ment the root growth in the short-root mutant(Supplemental Fig. S8), although the KCl concentrationand osmolality in mutant were increased with in-creasing external KCl supply, but not to the level of thewild type (Fig. 4D). These findings suggest that the KCluptake mediated by OsCCC1 represents a basic com-ponent for maintaining the K+ concentrations required

for cell enlargement. HighK+ supply also cannot restorethe root hair growth in the K+ transportermutant trh1 inArabidopsis (Rigas et al., 2001).

In conclusion, OsCCC1 functions as a K+, Na+-Cl2

cotransporter in rice. It is important for maintainingosmotic potential by transporting K+, Na+, and Cl2 intothe 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 Nipponbareof 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 andthen transferred to a net floating on 0.5 mM CaCl2 solution in a 1.5-liter plasticcontainer for 3 to 7 d before being used for various experiments. The seedlingswere then transferred to a 3.5-liter plastic pot containing one-half-strengthKimura B solution (pH 5.6; Yamaji and Ma, 2007). The nutrient solution waschanged once every 2 d.

Morphological Analysis

For measuring the root length, germinated seedlings were exposed to a0.5 mM CaCl2 solution and the root length was measured by a ruler at differentdays. The lateral root numbers in primary root of 7-d-old seedlings werecounted and the lateral root density was calculated by dividing the number oflateral roots by the primary root length for each plant. Three-week-old seed-lings 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 and30 mm from the root-to-shoot junction). The samples were imbedded into 5%agar and then were sectioned by a microslicer (100 mm thickness, LinearSlicerPRO10; Dosaka EM). These sections were observed under a confocal laserscanning microscope (LSM 700; Carl Zeiss). The length of root apical meristem(the distance between the quiescent center and the start point of the elongationzone) was also determined. For the measurement of cell size, longitudinal andcross sections of the root elongation zone were used. The length of the corticalcells was determined for nine roots each with 10 cells (n = 90). The width of thecortical cells was determined for 10 roots each with eight cells (n = 80). Thewidth of epidermal cells in leaf sheath was determined for 14 samples eachwith10 cells (n = 140).

Positional Cloning of OsCCC1 and Whole-Genome Sequencing

For mapping the responsible gene, the short-root mutant was crossed withKasalath to obtain an F2 population. A bulked segregant analysis using twobulked DNA samples (short-root versus normal-root) was first performed toidentify the molecular markers linked to OsCCC1 (Michelmore et al., 1991). Tofurther mapping this gene, polymorphic molecular markers were designedbased on the sequence comparison in the corresponding genomic region be-tween Nipponbare and 93-11 (Supplemental Table S1). Using 3460 F2 seedlingsshowing short-root phenotype, OsCCC1 finally was mapped to 440-kb regionnear the centromere of chromosome 8 according to the Rice Annotation ProjectDatabase (http://rapdb.dna.affrc.go.jp/).

Forwhole-genome sequencing, the short-rootmutant plantwas crossedwithwild-type rice (cv Nipponbare). The F1 plant was self-pollinated to obtain F2progeny. For genetic analysis, 200 seeds were used for phenotypic analysis. Thegenomic DNA of 50 F2 plants each showing the short-root or normal-rootphenotype was bulked in an equal ratio and subjected to whole-genome se-quencing for 50 cycles by the sequencer. The single nucleotide polymorphismanalysis was conducted by MutMap (Abe et al., 2012).

Complementation Test

The native 2.5-kb promoter sequence of OsCCC1 was amplified by PCRusing the primer pairs 59-AATGGGCCCTTGTTGAGGTATAAGGTCA-39 and59-TCGATCTCCCCGTTCTCCATCCCTCACTCTAGCAACTACA-39. The ORF

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with 39 untranslated region ofOsCCC1was amplified by PCR using the primerpairs 59-TGTAGTTGCTAGAGTGAGGGATGGAGAACGGGGAGATCGA-39and 59-CGGACTAGTACCAATAATTTCAGCTGA-39. The fragment contain-ing ProOsCCC1-OsCCC1-39UTRwas acquired by overlap PCR and inserted intopPZP2H-lac (with NOS terminator) using ApaI and SpeI. The construct wasintroduced into the calluses of rice (cv Nipponbare) via Agrobacterium tumefa-ciens-mediated transformation (Hiei et al., 1994).

RNA Isolation, Gene Cloning, and Expression Analysis

Total RNA from rice rootswas extracted using theRNeasyMini Kit (Qiagen).One microgram of total RNA was used for first-strand cDNA synthesis using aReverTraAce qPCRRTMasterMix kit (TOYOBO) following themanufacturer’sinstructions. The cDNA fragment containing OsCCC1 ORF was amplified byPCR using the primers 59-AATGTCGACATGGAGAACGGGGAGATC-39 and59-CGGACTAGTACCAATAATTTCAGCTGA-39. The fragment was clonedinto the pGEM-T easy vector (Promega) for sequence confirmation using theABI PRISM 310 genetic analyzer and the BigDye Terminators v3.1 cycle se-quencing kit (Applied Biosystems).

For the spatial expression, RNA was extracted from root tips (0–1 cm) andbasal 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 werecollected for the tissue sections. The central cylinder (pericycle and inner tis-sues) and outer tissues (cortex and epidermis) were separated using theLCC1704 Veritas Laser Microdissection System (Molecular Devices) followedby total RNA extraction. To investigate the response of OsCCC1 expression toKCl and NaCl, 10-d-old seedlings were pretreated with potassium deficiencyfor 1 week and then exposed to a one-half-strength Kimura B solution (removalof 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 solutionwithout or with 50 mM KCl or 50 mM NaCl supply for 12 h.

The gene expression level was determined by real-time RT-PCR usingThunderbird SYBR qPCR Mix (TOYOBO) on Mastercycler ep realplex(Eppendorf). The primers used were 59-AAGCCGTTGTCATTGTGAAG-39and 59-CTTGAGAATCGTCCTGTGGA-39 for OsCCC1. Histone H3 (forwardprimer, 59-AGTTTGGTCGCTCTCGATTTCG-39; reverse primer, 59-TCAA-CAAGTTGACCACGTCACG-39) was used as an internal control. The ex-pression was normalized by the DDCt method.

Tissue Specificity of Expression

The native 2.5-kb promoter sequence of OsCCC1 was amplified by PCR.The primer sequences (59-AATGGGCCCTTGTTGAGGTATAAGGTCA-39 and59-CGGACTAGTGCCGCTTTACTTGTACAG-39) were used for amplificationand introduction of the ApaI and SpeI restriction sites. The fragment was linkedto sGFP gene by overlap PCR. The amplified PCR product was inserted intopPZP2H-lac (with NOS terminator) using ApaI and SpeI to create the OsCCC1promoter-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 forimmunostaining with a rabbit GFP antibody as described below.

Tissue and Subcellular Localization of OsCCC1

The synthetic peptide SGAPQDDSQEAYTSAQRR (positions 870 to 887 ofOsCCC1) was used to immunize rabbits to obtain antibodies against OsCCC1.The obtained antiserum was purified through a peptide affinity column beforeuse. The root, leaf blade, and basal node of 1-month-old rice seedlings (cvNipponbare) were used for immunostaining as described below. Doublestaining with DAPI for nuclei was also performed to investigate the subcellularlocalization.

Immunohistological Analysis

Forwestern-blot analysis,microsomal proteinswere extracted from the rootsand 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 anti-body (1:500) was used as the primary antibody. Anti-rabbit IgG (H+L) conju-gated 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 fromYamaji andMa (2007). Rice roots, leaf blade, leaf sheath, and node were fixed in4% (w/v) paraformaldehyde and 60mM Suc bufferedwith 50mM 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 mm thick with a LinearSlicer PRO 10 (Dosaka EM).Sections were placed on microscope slides and incubated with PBS containing0.1% (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 containing5% (w/v) bovine serum albumin and anti-GFP (1:1,000 dilution) or anti-OsCCC1 (1: 500 dilution) primary antibody. Slides were incubated at roomtemperature overnight and washed four times with PBS. The slides were ex-posed to secondary antibodies (Alexa Fluor 555 goat anti-rabbit IgG; MolecularProbes) in PBSwith bovine serum albumin for 2 h at room temperature, washedfive times in PBS, and mounted with 50% (v/v) glycerol in PBS. Samples wereexamined 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 com-plementation lines were exposed to 0.1 mM Ca(NO3)2 solution with differentconcentrations of KCl orNaCl. After 1 d, the shoots and roots of half plants wereharvested 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 recoverytest. At end of the experiment, the root tips (0–15 mm) and the shoot were ex-cised for cell sap collection according to Chen et al. (2012). Metal concentrationin the roots, shoots, and cell sapwas determined by inductively coupled plasmamass spectrometer using an Agilent 7700 mass spectrometer. Cl concentrationwas determined by ion chromatograph (ICS-900; Dionex) with the IonPacAS12A column. The osmolality in the roots and shoots was measured using10 mL 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 OsKAT1were amplified by PCR and cloned into the pYES2 vector (Invitrogen). Aftersequence confirmation, the OsCCC1, mutated OsCCC1 (C568F), OsKAT1,OsHKT2;1, or the empty vector were introduced into yeast according to themanufacturer’s protocols (S.c.easy comp transformation kit; Invitrogen). TheK+-sensitive mutant strain CY162 (MATa ura3 his3 his4 trk1D trk2D1::pCK64)andNa-sensitive mutant strain G19 (MATa ade2 ura3 leu2 his3 trp1 ena1D::HIS3::ena4D) were used for study. Primer pairs used for amplification and introduc-tion of restriction sites were 59-ATGAGCTCAAAATGGAGAACGGGGA-GATC-39 and 59-CCCTCGAGTCATGTGAAGAATGTGAC-39 forOsCCC1 andmutatedOsCCC1, 59-ACGAGCTCAAAATGCCACGTTCTTCTCGT-39 and59-CCCTCGAGTTATACGTTCACTTGCTG-39 for OsKAT1, and 59-ATGAG-CTCAAAATGACGAGCATTTACCATGA-39 and 59-CCCTCGAGTTACCA-TAGCCTCCAATATT-39 for OsHKT2;1.

Yeast transformants were selected on uracil-deficient medium and grown insynthetic complete (SC-uracil) yeast medium containing 2% Glc, 0.67% yeastnitrogen base without amino acids (Difco), 0.2% appropriate amino acids, and2% agar at pH 6.0. One colony was selected in each transformation strain andgrown in liquid SC-Glc-uracil medium. For the plate experiment, four serialdilutions of yeast cell suspensionswere spotted onplates containingGal andGlcand cultured at 30°C for 3 d. The SC-uracil plate was added with 10 mM KCl toachieve the total K+ to 20 mM or added with 300 mM NaCl to achieve the totalNa+ to 300 mM.

For the liquid culture uptake experiment, yeast transformants were firstgrown to linear phase. After washing three times with Mill-Q water, the yeastwas adjusted to an OD600 value of 0.15 and cultured in a SC-uracil (+Gal) me-dium with KCl (25, 50, 75, or 100 mM) or NaCl (0, 100, 300, or 500 mM) untilOD600 = 1.5 to 2.0. Cells were collected andwashed by 20mMCa(NO3)2 solutionon ice three times. The samples were boiled for 1 h before elemental analysis.

Root Elemental Distribution Analysis

Bothwild-type rice and the short-rootmutant (21dold)were cultivated in thenutrient solution containing 50 mM NaCl. After 24 h, the roots were washedthree times in 5 mM Ca(NO3)2 solution on ice, excised, and fixed by 5% agarpowder. The transverse section at about 10 mm from the root apex was

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sectioned by a microslicer (LinearSlicer PRO10; Dosaka EM) and immediatelyused for analysis by scanning electronmicroscope (TM3000; Hitachi) in vacuumcondition at 220°C. The elemental distribution photos were generated by en-ergy dispersive x-ray spectrometer (SwiftED 3000; Oxford Instruments).

Accession Numbers

The accession number ofOsCCC1 is registered as LC085614 in theGenBank/EMBL databases.

Supplemental Data

The following supplemental materials are available.

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

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

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

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

Supplemental Figure S5. Scheme for MutMap using whole-genome se-quencing.

Supplemental Figure S6. Alignment of mutation region between thebulked DNA from normal-root and short-root pools by MutMap andconfirmation of mutation by PCR.

Supplemental Figure S7. Phylogenetic tree of OsCCC1 in plants and pred-icated topology of OsCCC1.

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

Supplemental Figure S9. Elemental distribution in short-root mutant us-ing SEM and EDX.

Supplemental Figure S10. Concentration of K+, Na+, and Cl2 in roots andshoots in response to KCl and NaCl.

Supplemental Figure S11. Osmolality in shoot cell sap in response to KCland NaCl.

Supplemental Figure S12. Western-blot analysis of OsCCC1.

Supplemental Table S1. Primers for InDel markers used for mapping ofOsCCC1.

ACKNOWLEDGMENTS

We thank the Rice Genome Resource Center in Tsukuba for providing theTos-17 insertion line. We thank Dr. T. Horie for kindly providing the yeaststrains CY162 and G19 and for critical discussion. We also thank NaoKomiyama for helping in generating transgenic rice.

Received January 7, 2016; accepted March 15, 2016; published March 16, 2016.

LITERATURE CITED

Abe A, Kosugi S, Yoshida K, Natsume S, Takagi H, Kanzaki H, Matsumura H,Yoshida K, Mitsuoka C, Tamiru M, et al (2012) Genome sequencing revealsagronomically important loci in rice using MutMap. Nat Biotechnol 30:174–178

Amtmann A, Fischer M, Marsh EL, Stefanovic A, Sanders D, SchachtmanDP (2001) The wheat cDNA LCT1 generates hypersensitivity to sodiumin a salt-sensitive yeast strain. Plant Physiol 126: 1061–1071

Anderson JA, Huprikar SS, Kochian LV, Lucas WJ, Gaber RF (1992)Functional expression of a probable Arabidopsis thaliana potassium channel inSaccharomyces cerevisiae. Proc Natl Acad Sci USA 89: 3736–3740

Ashley MK, Grant M, Grabov A (2006) Plant responses to potassium de-ficiencies: a role for potassium transport proteins. J Exp Bot 57: 425–436

Blumwald E (2000) Sodium transport and salt tolerance in plants. CurrOpin Cell Biol 12: 431–434

Chen ZC, Yamaji N, Motoyama R, Nagamura Y, Ma JF (2012) Up-regulation of a magnesium transporter gene OsMGT1 is required forconferring aluminum tolerance in rice. Plant Physiol 159: 1624–1633

Colmenero-Flores JM, Martínez G, Gamba G, Vázquez N, Iglesias DJ,Brumós J, Talón M (2007) Identification and functional characterizationof cation-chloride cotransporters in plants. Plant J 50: 278–292

Coudert Y, Périn C, Courtois B, Khong NG, Gantet P (2010) Geneticcontrol of root development in rice, the model cereal. Trends Plant Sci15: 219–226

Coury LA, McGeoch JEM, Guidotti G, Brodsky JL (1999) The yeastSaccharomyces cerevisiae does not sequester chloride but can express afunctional mammalian chloride channel. FEMS Microbiol Lett 179: 327–332

de Dorlodot S, Forster B, Pagès L, Price A, Tuberosa R, Draye X (2007)Root system architecture: opportunities and constraints for genetic im-provement of crops. Trends Plant Sci 12: 474–481

Dolan L, Davies J (2004) Cell expansion in roots. Curr Opin Plant Biol 7:33–39

Gamba G (2005) Molecular physiology and pathophysiology of electro-neutral cation-chloride cotransporters. Physiol Rev 85: 423–493

Gierth M, Mäser P, Schroeder JI (2005) The potassium transporterAtHAK5 functions in K(+) deprivation-induced high-affinity K(+) uptakeand AKT1 K(+) channel contribution to K(+) uptake kinetics in Arabi-dopsis roots. Plant Physiol 137: 1105–1114

Hamamoto S, Horie T, Hauser F, Deinlein U, Schroeder JI, Uozumi N(2015) HKT transporters mediate salt stress resistance in plants: fromstructure and function to the field. Curr Opin Biotechnol 32: 113–120

Hasegawa PM (2013) Sodium (Na+) homeostasis and salt tolerance ofplants. Environ Exp Bot 92: 19–31

Hebert SC, Mount DB, Gamba G (2004) Molecular physiology of cation-coupled Cl- cotransport: the SLC12 family. Pflugers Arch 447: 580–593

Henderson SW, Wege S, Qiu J, Blackmore DH, Walker AR, Tyerman SD,Walker RR, Gilliham M (2015) Grapevine and Arabidopsis cation-chloride cotransporters localise to the Golgi and trans-Golgi networkand indirectly influence long-distance ion transport and plant salt tol-erance. Plant Physiol 169: 2215–2229

Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation ofrice (Oryza sativa L.) mediated by Agrobacterium and sequence analysisof the boundaries of the T-DNA. Plant J 6: 271–282

Hoffmann EK, Lambert IH, Pedersen SF (2009) Physiology of cell volumeregulation in vertebrates. Physiol Rev 89: 193–277

Horie T, Costa A, Kim TH, Han MJ, Horie R, Leung HY, Miyao A,Hirochika H, An G, Schroeder JI (2007) Rice OsHKT2;1 transportermediates large Na+ influx component into K+-starved roots for growth.EMBO J 26: 3003–3014

Horie T, Yoshida K, Nakayama H, Yamada K, Oiki S, Shinmyo A (2001)Two types of HKT transporters with different properties of Na+ and K+

transport in Oryza sativa. Plant J 27: 129–138Inukai Y, Sakamoto T, Ueguchi-Tanaka M, Shibata Y, Gomi K, Umemura

I, Hasegawa Y, Ashikari M, Kitano H, Matsuoka M (2005) Crownrootless1, which is essential for crown root formation in rice, is a targetof an AUXIN RESPONSE FACTOR in auxin signaling. Plant Cell 17:1387–1396

Jung JK, McCouch S (2013) Getting to the roots of it: Genetic and hormonalcontrol of root architecture. Front Plant Sci 4: 186

Kawai M, Samarajeewa PK, Barrero RA, Nishiguchi M, Uchimiya H(1997) Cellular dissection of the degradation pattern of cortical cell deathduring aerenchyma formation of rice roots. Planta 204: 277–287

Kim CM, Park SH, Je BI, Park SH, Park SJ, Piao HL, Eun MY, Dolan L,Han CD (2007) OsCSLD1, a cellulose synthase-like D1 gene, is requiredfor root hair morphogenesis in rice. Plant Physiol 143: 1220–1230

Kitomi Y, Inahashi H, Takehisa H, Sato Y, Inukai Y (2012) OsIAA13-mediated auxin signaling is involved in lateral root initiation in rice.Plant Sci 190: 116–122

Kong XQ, Gao XH, Sun W, An J, Zhao YX, Zhang H (2011) Cloning andfunctional characterization of a cation-chloride cotransporter geneOsCCC1. Plant Mol Biol 75: 567–578

Kronzucker HJ, Britto DT (2011) Sodium transport in plants: a criticalreview. New Phytol 189: 54–81

Li B, Byrt C, Qiu J, Baumann U, HrmovaM, Evrard A, Johnson AA, BirnbaumKD, Mayo GM, Jha D, et al (2016) Identification of a stelar-localized

506 Plant Physiol. Vol. 171, 2016

Chen et al.

www.plantphysiol.orgon June 23, 2019 - Published by Downloaded from Copyright © 2016 American Society of Plant Biologists. All rights reserved.

transport protein that facilitates root-to-shoot transfer of chloride inArabidopsis. Plant Physiol 170: 1014–1029

Lindinger MI, Leung M, Trajcevski KE, Hawke TJ (2011) Volumeregulation in mammalian skeletal muscle: the role of sodium-potassium-chloride cotransporters during exposure to hypertonic solutions. J Physiol589: 2887–2899

Marschner H (2012) Mineral Nutrition of Higher Plants, Ed 3. AcademicPress, London

Michelmore RW, Paran I, Kesseli RV (1991) Identification of markerslinked to disease-resistance genes by bulked segregant analysis: a rapidmethod to detect markers in specific genomic regions by using segre-gating populations. Proc Natl Acad Sci USA 88: 9828–9832

Mitani N, Yamaji N, Ma JF (2009) Identification of maize silicon influxtransporters. Plant Cell Physiol 50: 5–12

Miyao A, Tanaka K, Murata K, Sawaki H, Takeda S, Abe K, Shinozuka Y,Onosato K, Hirochika H (2003) Target site specificity of the Tos17 ret-rotransposon shows a preference for insertion within genes and againstinsertion in retrotransposon-rich regions of the genome. Plant Cell 15:1771–1780

Moes AD, van der Lubbe N, Zietse R, Loffing J, Hoorn EJ (2014) Thesodium chloride cotransporter SLC12A3: new roles in sodium, potas-sium, and blood pressure regulation. Pflugers Arch 466: 107–118

Nakamura A, Umemura I, Gomi K, Hasegawa Y, Kitano H, Sazuka T,Matsuoka M (2006) Production and characterization of auxin-insensitive rice by overexpression of a mutagenized rice IAA protein.Plant J 46: 297–306

Obata T, Kitamoto HK, Nakamura A, Fukuda A, Tanaka Y (2007) Riceshaker potassium channel OsKAT1 confers tolerance to salinity stress onyeast and rice cells. Plant Physiol 144: 1978–1985

Petricka JJ, Winter CM, Benfey PN (2012) Control of Arabidopsis rootdevelopment. Annu Rev Plant Biol 63: 563–590

Qi Y, Wang S, Shen C, Zhang S, Chen Y, Xu Y, Liu Y, Wu Y, Jiang D (2012)OsARF12, a transcription activator on auxin response gene, regulatesroot elongation and affects iron accumulation in rice (Oryza sativa). NewPhytol 193: 109–120

Qin C, Li Y, Gan J, Wang W, Zhang H, Liu Y, Wu P (2013) OsDGL1, ahomolog of an oligosaccharyltransferase complex subunit, is involved inN-glycosylation and root development in rice. Plant Cell Physiol 54:129–137

Quintero FJ, Garciadeblás B, Rodríguez-Navarro A (1996) The SAL1 geneof Arabidopsis, encoding an enzyme with 39(29), 59-bisphosphate nu-cleotidase and inositol polyphosphate 1-phosphate activities, increasessalt tolerance in yeast. Plant Cell 8: 529–537

Rebouillat J, Dievart A, Verdeil JL, Escoute J, Giese G, Breitler JC, GantetP, Espeout S, Guiderdoni E, Périn C (2009) Molecular genetics of riceroot development. Rice (NY) 2: 15–34

Rigas S, Debrosses G, Haralampidis K, Vicente-Agullo F, Feldmann KA,Grabov A, Dolan L, Hatzopoulos P (2001) TRH1 encodes a potassiumtransporter required for tip growth in Arabidopsis root hairs. Plant Cell13: 139–151

Russell JM (2000) Sodium-potassium-chloride cotransport. Physiol Rev 80:211–276

Sauer DB, Zeng W, Canty J, Lam Y, Jiang Y (2013) Sodium and potassiumcompetition in potassium-selective and non-selective channels. NatCommun 4: 2721

Shabala S, Cuin TA (2008) Potassium transport and plant salt tolerance.Physiol Plant 133: 651–669

Wang XF, He FF, Ma XX, Mao CZ, Hodgman C, Lu CG, Wu P (2011)OsCAND1 is required for crown root emergence in rice. Mol Plant 4:289–299

Wang Y, Wu WH (2013) Potassium transport and signaling in higherplants. Annu Rev Plant Biol 64: 451–476

White PJ, Broadley MR (2001) Chloride in soils and its uptake andmovement within the plant: A review. Ann Bot (Lond) 88: 967–988

Won SK, Choi SB, Kumari S, Cho M, Lee SH, Cho HT (2010) Root hair-specific EXPANSIN B genes have been selected for Graminaceae roothairs. Mol Cells 30: 369–376

Woo YM, Park HJ, Su’udi M, Yang JI, Park JJ, Back K, Park YM, An G(2007) Constitutively wilted 1, a member of the rice YUCCA gene family,is required for maintaining water homeostasis and an appropriate rootto shoot ratio. Plant Mol Biol 65: 125–136

Xia J, Yamaji N, Che J, Shen RF, Ma JF (2014) Normal root elongationrequires arginine produced by argininosuccinate lyase in rice. Plant J 78:215–226

Yamaguchi T, Hamamoto S, Uozumi N (2013) Sodium transport system inplant cells. Front Plant Sci 4: 410

Yamaji N, Ma JF (2007) Spatial distribution and temporal variation of therice silicon transporter Lsi1. Plant Physiol 143: 1306–1313

Yuo T, Toyota M, Ichii M, Taketa S (2009) Molecular cloning of a roothairless gene rth1 in rice. Breed Sci 59: 13–20

Zhu ZX, Liu Y, Liu SJ, Mao CZ, Wu YR, Wu P (2012) A gain-of-functionmutation in OsIAA11 affects lateral root development in rice. Mol Plant5: 154–161

Zhuang X, Jiang J, Li J, Ma Q, Xu Y, Xue Y, Xu Z, Chong K (2006) Over-expression of OsAGAP, an ARF-GAP, interferes with auxin influx,vesicle trafficking and root development. Plant J 48: 581–591

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