Overexpression of Oscasp1 Improves Calcium Tolerance in Rice

International Journal of Molecular Sciences

Article Overexpression of OsCASP1 Improves Calcium Tolerance in Rice

Zhigang Wang †, Zhiwei Chen †, Xiang Zhang †, Qiuxing Wei, Yafeng Xin, Baolei Zhang, Fuhang Liu and Jixing Xia *

State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Science and Technology, Guangxi University, Nanning 530004, China; [email protected] (Z.W.); [email protected] (Z.C.); [email protected] (X.Z.); [email protected] (Q.W.); [email protected] (Y.X.); [email protected] (B.Z.); [email protected] (F.L.) * Correspondence: [email protected]; Tel.: +86-0771-3236616 † These authors contributed equally to this work.

Abstract: The Casparian strip domain protein 1 (OsCASP1) is necessary for the formation of the Casparian strip (CS) in the rice endodermis. It also controls Ca2+ transport to the stele. Here, we demonstrated that OsCASP1 overexpression enhanced Ca tolerance in rice. Under normal conditions, OsCASP1-overexpressed lines showed similar concentrations of essential metals in the roots and shoots compared to the wild type, while under high Ca conditions, Ca in the roots, shoots, and xylem sap of the OsCASP1-overexpressed lines was signiﬁcantly decreased. This did not apply to other essential metals. Ca-inhibited growth was signiﬁcantly alleviated in the OsCASP1-overexpressed lines. Furthermore, OsCASP1 overexpression resulted in earlier formation of both the CS and functional apoplastic barrier in the endodermis but did not induce ectopic CS formation in non-

endodermal cell layers and affect suberin accumulation in the endodermis. These results indicate that the overexpression of OsCASP1 promotes CS formation in endodermal cells and inhibits Ca2+

Citation: Wang, Z.; Chen, Z.; Zhang, transport by the apoplastic pathway, restricting Ca accumulation in the roots and shoots under high X.; Wei, Q.; Xin, Y.; Zhang, B.; Liu, F.; Ca conditions. Taken together, the results suggest that OsCASP1 overexpression is an effective way Xia, J. Overexpression of OsCASP1 to improve rice adaptation to high Ca environments. Improves Calcium Tolerance in Rice. Int. J. Mol. Sci. 2021, 22, 6002. Keywords: Casparian strip; suberin lamellae; apoplastic barrier; calcium; lignin https://doi.org/10.3390/ijms22116002

Academic Editor: Marcello Iriti 1. Introduction Received: 13 April 2021 Calcium (Ca) is one of the most abundant metal elements in soil, which normally Accepted: 30 May 2021 contains large amounts of exchangeable Ca (300–5000 ppm) [1]. It is an essential macronu- Published: 1 June 2021 trient for plant growth and is involved in many biological processes, including cell division and expansion, structural integrity, and cellular signaling [2–6]. However, high cytosolic Publisher’s Note: MDPI stays neutral Ca2+ concentrations are cytotoxic [7,8]. Excessive Ca2+ restricts plant growth in calcare- with regard to jurisdictional claims in ous soils [7]. Therefore, optimal Ca concentrations are required for normal plant growth published maps and institutional afﬁl- and development. iations. Divalent cation Ca2+ in the soil solution are taken up by the root system and radially delivered to the xylem by apoplastic and symplastic pathways [9–11]. In roots, two endodermal barriers, Casparian strip (CS) and suberin lamellae, are considered to limit the apoplastic ﬂow of Ca across the endodermis into the xylem. These two barriers develop Copyright: © 2021 by the authors. gradually during the root differentiation [12,13]. At the primary developmental stage of Licensee MDPI, Basel, Switzerland. endodermis, CS formation is initiated at a few millimeters from the root apex. After the CS This article is an open access article formation and maturation in the root differentiation zone, suberin lamellae are deposited distributed under the terms and between the primary cell wall and the plasma membrane surrounding endodermal cells. conditions of the Creative Commons Attribution (CC BY) license (https:// When the CS located in the anticlinal cell wall between endodermal cells is completely 2+ creativecommons.org/licenses/by/ formed, transport of Ca to the xylem must use a symplastic pathway. In this case, the 2+ 4.0/). apoplastic Ca diffuses into the cytoplasm of the endodermal cells through Ca channels

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Int. J. Mol. Sci. 2021, 22, 6002 2 of 11 the apoplastic Ca2+ diffuses into the cytoplasm of the endodermal cells through Ca channels in the plasma membrane [10]. Subsequently, Ca2+ is exported from the symplast of in the plasmathe membrane endodermal [10]. cells Subsequently, into the stele Ca 2+by isCa exported2+ transporters from thesuch symplast as Ca2+-ATPases of the [10]. When endodermal cellssuberin into lamellae the stele cover by Ca the2+ endodermaltransporters cells, such direct as Ca Ca2+-ATPases2+ uptake from [10]. the When apoplast into the suberin lamellaeendodermis cover the endodermalis prevented, cells, and the direct Ca Ca2+ must2+ uptake be taken from up the into apoplast the symplast into the from the outer endodermis iscell prevented, layers [10]. and However, the Ca2+ must the precise be taken roles up of into these the two symplast endodermal from the barriers outer in regulating cell layers [10].Ca However,2+ transport the into precise the xylem roles of for these transfer two endodermalto the shoot are barriers poorly in understood. regulating Ca2+ transport intoInterestingly, the xylem for in transfer Arabidopsis to the, several shoot areCS poorlymutants understood. such as casp1, casp3, esb1, myb36, and Interestingly,lotr1 indisplayedArabidopsis similar, several shoot CS ionomic mutants alterations, such as casp1, including casp3, esb1 low, myb36Ca accumulation, and [14–16]. lotr1 displayedIt similarwas proposed shoot ionomic that an alterations, increase inincluding endodermal low suberization Ca accumulation in these [14 –mutants16]. inhibited It was proposedCa2+ that transport an increase into the in endodermal xylem by the suberization symplastic pathway, in these mutants leading inhibitedto the reduced Ca con- Ca2+ transportcentration into the in xylem the shoots by the [15,16]. symplastic Rice differs pathway, from leading Arabidopsis to the in reducedhaving two Ca CSs in the ex- concentrationodermis in the shoots and endodermis, [15,16]. Rice differsrespectively from Arabidopsis[17]. Apoplasticin having Ca2+ twouptake CSs may in the occur largely in exodermis andthe endodermis, root tip where respectively the CS is [ 17formed]. Apoplastic in the endodermis Ca2+ uptake but may not occur the exodermis largely in [18]. A recent the root tip wherereport the shows CS is formedthat OsCASP1 in the endodermis is only involved but not in theCS formation exodermis in [18 the]. A root recent endodermis [18]. report shows thatIn oscasp1OsCASP1, defectiveis only endodermal involved in CSCS formationformation inresulted the root in endodermis uncontrolled [18 apoplastic]. Ca2+ In oscasp1, defectivetransport endodermal to the stele, CS which formation is prevented resulted by in th uncontrollede CS in the wild apoplastic type, resulting Ca2+ in the over- transport to theaccumulation stele, which of isCa prevented in the shoot by [18]. the CSTherefore, in the wildthe role type, of resultingthe CS in controlling in the Ca accu- over-accumulationmulation of Ca in inshoots the shoot differs [18 between]. Therefore, Arabidopsis the role and of therice. CS in controlling Ca accumulation in shootsTo further differs investigate between Arabidopsis the role ofand OsCASP1 rice. in rice CS formation and Ca uptake, in To furtherthis investigate study, we the generated role of OsCASP1 OsCASP1in overexpression rice CS formation transgenic and Ca uptake,lines and in characterized this study, wethem. generated We foundOsCASP1 thatoverexpression OsCASP1 overexpression transgenic lines induced andcharacterized early formation them. of the CS in the We found thatrootOsCASP1 tip andoverexpression selectively reduced induced Ca early accumula formationtion in of the CSroot in and the rootshoot tip under high Ca and selectivelyconditions. reduced Ca Furthermore, accumulation the in overexpre the root andssion shoot lines under enhanced high rice Ca conditions. tolerance to excess Ca. Furthermore,This the overexpressionwork enriches our lines understanding enhanced rice of tolerancethe interrelationships to excess Ca. between This work CS formation, Ca enriches our understandinguptake, and Ca oftolerance the interrelationships in rice. between CS formation, Ca uptake, and Ca tolerance in rice. 2. Results 2. Results 2.1. Expression2.1. of OsCASP1 Expression in of Transgenic OsCASP1 Lines in Transgenic Lines OsCASP1 wasOsCASP1 overexpressed was overexpressed in rice plants underin rice theplants control under of the maizecontrol ubiquitin of the maize ubiquitin promoter. Twopromoter. independent Two transgenic independent lines transgenic (pOX-1 and lines pOX-2) (pOX-1 were and produced pOX-2) were for subse- produced for sub- quent experiments.sequent RT-qPCR experiments. analysis RT-qPCR showed anal signiﬁcantlyysis showed higher significantlyOsCASP1 higherexpression OsCASP1 expression in both the rootsin both and the shoots roots of and the twoshoots transgenic of the two lines transg thanenic in thelines wild-type than in the rice wild-type (WT) rice (WT) (Figure1A), ranging(Figure from 1A), a ranging 10 to 35-fold from increase.a 10 to 35-fold Furthermore, increase. a Furthermore, spatial analysis a spatial revealed analysis revealed higher OsCASP1higherexpression OsCASP1 in the expression different rootin the segments different of root the transgenic segments linesof the compared transgenic lines com- with the WT (Figurepared 1withB). the WT (Figure 1B).

Figure 1. OsCASP1Figure expression 1. OsCASP1 in differentexpression tissues in of different wild-type tissues plants of (WT) wild-type and OsCASP1 plants-overexpressing (WT) and OsCASP1 rice lines- (pOX- 1 and pOX-2).overexpressing (A) Expression rice of lines OsCASP1 (pOX-1 in androots pOX-2). and shoots. (A) ExpressionExpression ofrelativeOsCASP1 to thein wild-type roots and root shoots. expression is shown. (B) SpatialExpression OsCASP1 relative expression to the wild-type in roots. rootExpression expression relative is shown. to the wild-type (B) Spatial rootOsCASP1 expressionexpression (0–3 mm) in is shown. RNA was extractedroots. Expression from different relative root to parts the wild-type(0 to 3, 3 to root 6, 6 expressionto 9, and 9 to (0–3 12 mm)mm from is shown. the root RNA tip) was of 5-day-old extracted seedlings. Histone H3 wasfrom used different as an root internal parts standard. (0 to 3, 3 to Data 6, 6 represent to 9, and 9 means to 12 mm ± SD from (n = the 3). root Different tip) of letters 5-day-old indicate seedlings. significant differences (Tukey test, p < 0.05). Histone H3 was used as an internal standard. Data represent means ± SD (n = 3). Different letters indicate signiﬁcant differences (Tukey test, p < 0.05).

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2.2. The Role of OsCASP1 in Ca Tolerance 2.2. TheTwo Role pOX of OsCASP1lines, the inknockout Ca Tolerance line (oscasp1-1) and the WT Nipponbare, were used to measureTwo phenotypic pOX lines, thedifferences knockout under line ( oscasp1-1normal (0.18) and mM the WTCaCl Nipponbare,2) and high wereCa2+ (20 used mM to 2+ measureCaCl2) conditions. phenotypic Under differences normal under conditions, normal similar (0.18 mMgrowth CaCl was2) and observed high Ca between(20 mM the CaCloverexpressed2) conditions. lines Under and the normal WT, while conditions, shoot similargrowth growthin the oscasp1-1 was observed knockout between line was the overexpressedslightly retarded lines (Figure and the 2A). WT, However, while shoot under growth high inCa the2+ conditions,oscasp1-1 knockout significant line differ- was slightlyences in retarded plant growth (Figure were2A). observed However, among under highthe WT, Ca 2+oscasp1-1conditions,, and signiﬁcantthe pOX lines differences (Figure in2B). plant The growth leaves wereof both observed WT and among the mutant the WT, showedoscasp1-1 evidence, and the of pOX necrosis. lines (Figure Moreover,2B). Thegrowth leaves in the of bothmutant WT line and was the more mutant seriously showed retarded evidence than of necrosis.in the WT, Moreover, with most growth of the inleaves the mutantdry and line wilted was (Figure more seriously 2B). In contrast, retarded the than leaves in the in WT,the two with pOX most lines of the appeared leaves drylargely and normal wilted and (Figure unaffected2B). In contrast, by the high the Ca leaves concentration in the two (Figure pOX lines 2B). appearedCorrespondingly, largely normalthe dry andweights unaffected of theby shoots the high and Ca roots concentration from these (Figure lines were2B). Correspondingly,significantly higher the than dry weightsthose of ofthe the WT shoots and knockout and roots lines from under these high lines Ca were2+ conditions signiﬁcantly (Figure higher 2C,D). than Taken those to- of thegether, WT andthe phenotypic knockout lines data under indica highted Cathat2+ theconditions overexpression (Figure2 C,D).of OsCASP1 Taken together, can increase the phenotypicCa tolerance data in rice. indicated that the overexpression of OsCASP1 can increase Ca tolerance in rice.

Figure 2. Growth of OsCASP1-overexpressing and wild-type rice seedlings. (A) Growth under the controlFigure 2. condition Growth (0.18of OsCASP1 mM CaCl-overexpressing2). (B) Growth and with wild high-type Ca rice (20 seedlings. mM CaCl (A2).) Growth (C) Dry under weights the ofcontrol shoots. condition (D) Dry (0.18 weights mM ofCaCl roots.2). ( Two-week-oldB) Growth with seedlings high Ca of(20 the mM wild CaCl type2). (WT),(C) Dryoscasp1 weights, and of twoshoots.OsCASP1 (D) Dry-overexpressing weights of roots. lines Two-week-old (pOX-1 and seedlings pOX-2) were of the grown wild type in half-strength (WT), oscasp1 Kimura, and two B

solutionOsCASP1 with-overexpressing 20 mM CaCl 2lines. After (pOX-1 12 d, and plants pOX-2) were photographedwere grown in andhalf-strength sampled. Kimura Bar = 10 B cm. solution Data arewith mean 20 mM± SD; CaCln =2. After 3. Different 12 d, plants letters were indicate photographed signiﬁcant differencesand sampled. (Tukey Bar = test, 10 cm.p < Data 0.05). are mean ± SD; n = 3. Different letters indicate significant differences (Tukey test, p < 0.05). 2.3. Mineral Analysis of OsCASP1 Overexpression Lines 2.3. MineralNo differences Analysis were of OsCASP1 observed Overexpression in tissue mineral Lines concentrations between the pOX and WT linesNo differences under normal were conditions observed (Figure in tissue3A,B) mi (Supplementaryneral concentrations Figures between S2A,C the and pOX S3A,C). and UnderWT lines conditions under ofnormal high Caconditions2+, the Ca (Figur levels ine 3A,B) both shoots (Supplementary and roots of Figures the pOX S2A,C lines were and signiﬁcantlyS3A,C). Under lower conditions than in of the high WT Ca shoots2+, the (FigureCa levels3A,B). in both No shoots differences and roots were of observed the pOX inlines the were concentrations significantly of lower other metals,than in includingthe WT shoots K, Mg, (Figure Cu, and 3A,B). Zn, No between differences the plants were (Supplementaryobserved in the concentrations Figures S2B,D of and other S3B,D). meta Thesels, including results K, indicated Mg, Cu, thatand Zn, overexpression between the

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plants (Supplementary Figures S2B,D and S3B,D). These results indicated that overexpres- sionof ofOsCASP1 OsCASP1resulted resulted in in reduced reduced Ca Ca accumulation accumulation in in both both roots roots and and shoots shoots under under high highCa Ca conditions. conditions. To determineTo determine the the reason reason for for the the low low Ca Ca accumulation accumulation in thethe pOXpOX shoots,shoots, we we compared com- paredCa andCa and Sr concentrations Sr concentrations in the in xylem the xylem sap of sap the of plants the plants under differentunder different Ca or Sr Ca treatments. or Sr treatments.Sr has similar Sr has chemicalsimilar chemical properties properties to Ca and to Ca was and used was as used a tracer as a of tracer apoplastic of apoplastic transport transportfrom root from to shoot.root to Under shoot. normal Under conditions,normal conditions, the Ca and the Sr Ca concentrations and Sr concentrations in the xylem in sap the werexylem similar sap were between similar the pOXbetween and WTthe plantspOX and (Figure WT3 C,D).plants However, (Figure 3C,D). under highHowever, Ca or Sr undertreatment, high Ca the or xylemSr treatment, sap in thethe pOXxylem lines sap showed in the pOX signiﬁcantly lines showed lower significantly Ca/Sr concentrations lower Ca/Srthan concentrations in the WT rice than (Figure in 3theC,D), WT suggesting rice (Figure that 3C,D), the overexpression suggesting that of theOsCASP1 overexpres-inhibited sionapoplastic of OsCASP1 Ca2+ inhibitedtransport apoplastic to the shoot, Ca2+ resulting transport in to low the Ca shoot, accumulation resulting in in the low pOX Ca shootsac- cumulationunder high in the Ca pOX conditions. shoots under high Ca conditions.

FigureFigure 3. Ca 3. concentrationsCa concentrations in OsCASP1 in OsCASP1-overexpressing-overexpressing lines lines (pOX-1, (pOX-1, pOX-2) pOX-2) and and wild-type wild-type rice rice plantsplants (WT). (WT). (A) (CaA) concentration Ca concentration in shoots. in shoots. (B) Ca (B) concentration Ca concentration in roots. in roots. Two-week-old Two-week-old seedlings seedlings werewere grown grown in nutrient in nutrient solution solutionss containing containing 0.18 0.18 or 20 or mM 20 mM CaCl CaCl2 for2 12for d. 12 (C d.) (CaC) concentration Ca concentration in in xylemxylem sap. sap. Xylem Xylem sap sapwas was collected collected from from plants plants exposed exposed to different to different Ca2+ Ca concentrations2+ concentrations (0.18 (0.18 and and 10 mM)10 mM) for 6 for h. 6(D h.) (SrD )concentration Sr concentration in xylem in xylem sap. sap. Xylem Xylem sap sapwas was collected collected from from plants plants exposed exposed to to 2+ differentdifferent Sr Srconcentrations2+ concentrations (0.18 (0.18 and and20 mM) 20 mM) for 6 for h.6 Data h. Data are aremeans means ± SD± (SDn = (3).n = Different 3). Different letters letters indicateindicate significant signiﬁcant differences differences (Tukey (Tukey test, test, p < p0.05).< 0.05).

2.4.2.4. Effect Effect of OsCASP1 of OsCASP1 Overexpression Overexpression on CS on CSFormation Formation To Toinvestigate investigate the theeffect effect of OsCASP1 of OsCASP1 overexpressionoverexpression on CS on CSformation, formation, we weinvesti- investi- gatedgated lignification ligniﬁcation in the in theroots roots as lignin as lignin is a is main a main component component of the of theCS. CS. Histochemical Histochemical stainingstaining of sections of sections at 1 at and 1 and 3 mm 3 mm from from the theroot root tip tipshowed showed no nolignin lignin deposition deposition in WT in WT endodermalendodermal cells cells (Figure (Figure 4A,D)4A,D) (Supplementary (Supplementary FigureFigure S4A,D), S4A,D which), which is consistent is consistent with with previousprevious results results [18]. [18 In]. contrast, In contrast, in the in thetwo two OsCASP1OsCASP1-overexpressed-overexpressed lines, lines, the theendoder- endoder- malmal cells cells exhibited exhibited restricted, restricted, dot-like dot-like ligni ligninn staining staining (Figure (Figure 4B,C,E,F)4B,C,E,F) (Supplementary (Supplementary FigureFigure S4B,C,E,F). S4B,C,E,F However,). However, further further from from th thee tip tip (5 (5 mm), mm), the lignin depositiondeposition pattern pattern was wassimilar similar to to that that of of the the WT WT (Figure (Figure4G–I) 4G–I) (Supplementary (Supplementary Figure Figure S4G–I). S4G–I). This This indicates indicates that thatOsCASP1 OsCASP1overexpression overexpression induced induced early early CS CS formation formation in in the the root root tip tip region. region.

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Then, we investigated diffusion of the apoplastic tracer PI into the stele as a measure of theThen, functionality we investigated of the endodermal diffusion of apoplastic the apoplastic barrier tracer [15]. PI Less into PI the penetrated stele as a the measure stele ofof the the overexpressing functionality of thelines endodermal at 5 mm from apoplastic the root barrier apex [15 than]. Less in PIthe penetrated WT (Figure the 5A–F). stele of However,the overexpressing at the start lines of the at 5root mm elongation from the root zone apex (1 thancm from in the the WT root (Figure apex),5A–F). no PI However,penetra- tionat the was start observed of the rootin any elongation of the lines zone (Figure (1 cm 5G–L). from theThese root results apex), suggested no PI penetration that the dif- was fusionalobserved barrier in any formation of the lines in (Figurethe overexpr5G–L).essing These lines results was suggested developed that earlier the diffusionalcompared withbarrier the formation WT. in the overexpressing lines was developed earlier compared with the WT.

FigureFigure 4. 4. CasparianCasparian strip formationformation ininOsCASP1 OsCASP1-overexpressing-overexpressing lines lines (pOX-1, (pOX-1, pOX-2) pOX-2) and and wild-type wild- type rice plants (WT). (A) to (I) Casparian strip formation in the wild type and two OsCASP1-over- rice plants (WT). (A–I) Casparian strip formation in the wild type and two OsCASP1-overexpressing expressing lines. Lignin staining of root sections (1, 3, and 5 mm from the apex) of wild-type lines. Lignin staining of root sections (1, 3, and 5 mm from the apex) of wild-type (A,D,G), pOX- (A,D,G), pOX-1 (B,E,H), and pOX-2 (C,F,I). Magenta color represents lignin and blue represents cellulose.1 (B,E,H ),The and sections pOX-2 were (C,F, Istained). Magenta with color0.2% representsBasic Fuchsin lignin and and 0.1% blue Calc representsofluor White cellulose. for 30 min, The respectively.sections were Five stained roots withof each 0.2% line Basic (WT, Fuchsin pOX-1, andand 0.1%pOX-2) Calcoﬂuor are examined White and for show 30 min, the respectively. same phenotype.Five roots The of Casparian each line strip (WT, is pOX-1, indicated and by pOX-2) arrowh areeads. examined en, endodermis; and show x, the xylem same vessel. phenotype. Scale bars, The 20Casparian μm. strip is indicated by arrowheads. en, endodermis; x, xylem vessel. Scale bars, 20 µm.

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FigureFigure 5. Casparian 5. Casparian strip strip functionality functionality of OsCASP1 of OsCASP1-overexpressing-overexpressing lines lines (pOX-1, (pOX-1, pOX-2) pOX-2) and and wild- wild- type rice plants (WT) evaluated by PI penetration. PI penetration in the roots of wild-type (A,D,G,J), type rice plants (WT) evaluated by PI penetration. PI penetration in the roots of wild-type (A,D,G,J), pOX-1 (B,E,H,K), and pOX-2 (C,F,I,L) plants at 5 mm (A–F) and 10 mm (G–L) from the root apex. pOX-1 (B,E,H,K), and pOX-2 (C,F,I,L) plants at 5 mm (A–F) and 10 mm (G–L) from the root apex. Five-day-old seedlings were exposed to PI in the dark for 40 min. (D–F,J–L) are magnified images of theFive-day-old yellow boxed seedlings area in were(A–C exposed,G–I) respectively. to PI in the Five dark roots for 40of min.each (lineD– F(WT,,J–L) pOX-1, are magniﬁed and pOX-2) images are ofexamined the yellow and boxed show area the insame (A– Cphenotype.,G–I) respectively. The Casparian Five roots strip of is each indicated line (WT, by pOX-1,arrowheads. and pOX-2) en, endodermis;are examined x, xylem and showvessel. the Scale same bars, phenotype. 100 μm. The Casparian strip is indicated by arrowheads. en, endodermis; x, xylem vessel. Scale bars, 100 µm. 2.5. Effect of OsCASP1 Overexpression on Suberin Accumulation 2.5. Effect of OsCASP1 Overexpression on Suberin Accumulation To test whether overexpression of OsCASP1 affected suberin accumulation in the en- To test whether overexpression of OsCASP1 affected suberin accumulation in the endodermal cells, we investigated suberin deposition in different parts of the roots. Minimal dodermal cells, we investigated suberin deposition in different parts of the roots. Minimal suberin accumulation was observed at 15 mm from the root tip in both the WT and pOX suberin accumulation was observed at 15 mm from the root tip in both the WT and pOX lines (Figure 6D,F), while at 30 mm from the tip, suberin was visible in several endodermal lines (Figure6D,F), while at 30 mm from the tip, suberin was visible in several endodermal cells (Figure 6A–C). However, there was no difference in suberin accumulation in the en- cells (Figure6A–C). However, there was no difference in suberin accumulation in the dodermis between the WT and two pOX lines (Figure 6A–C). These results indicated that endodermis between the WT and two pOX lines (Figure6A–C). These results indicated that the overexpression of OsCASP1 did not affect suberin accumulation in the endodermis. the overexpression of OsCASP1 did not affect suberin accumulation in the endodermis.

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FigureFigure 6. 6. SuberinSuberin staining staining of of the the OsCASP1OsCASP1-overexpressing-overexpressing lines lines (pOX-1, (pOX-1, pOX-2) pOX-2) an andd wild-type wild-type rice rice plants plants (WT). (WT). Suberin Suberin staining of roots (15 and 30 mm from the apex) from WT (A,D), pOX-1 (B,E), and pOX-2 (C,F). Root cross sections were staining of roots (15 and 30 mm from the apex) from WT (A,D), pOX-1 (B,E), and pOX-2 (C,F). Root cross sections were stained with 0.01% (w/v) Fluorol Yellow 088 in lactic acid at 70 °C for 30 min. Five roots of each line (WT, pOX-1, and pOX- stained with 0.01% (w/v) Fluorol Yellow 088 in lactic acid at 70 ◦C for 30 min. Five roots of each line (WT, pOX-1, and 2) are examined and show the same phenotype. Yellow color shows suberin signal. Bar = 100 μm. pOX-2) are examined and show the same phenotype. Yellow color shows suberin signal. Bar = 100 µm. 3. Discussion 3. Discussion The CS functions as a physical barrier that prevents unfavorable inflow and backflow The CS functions as a physical barrier that prevents unfavorable inflow and backflow of nutrients between the soil and the stele and is considered to play an important role in of nutrients between the soil and the stele and is considered to play an important role environmental adaptation in plants [12,13]. For example, the Arabidopsis CS mutant in environmental adaptation in plants [12,13]. For example, the Arabidopsis CS mutant gso1/sgn3 displayed a severe potassium deficiency phenotype under low potassium con- gso1/sgn3 displayed a severe potassium deficiency phenotype under low potassium conditions [19]. Another Arabidopsis CS mutant lotr1 showed hypersensitivity to low Ca2+ conditions [19]. Another Arabidopsis CS mutant lotr1 showed hypersensitivity to low Ca2+ ditions [16]. Recently, the mutation of both CS integrity factor1 (CIF1) and CIF2 in Ara- conditions [16]. Recently, the mutation of both CS integrity factor1 (CIF1) and CIF2 in bidopsis produced noticeable growth defects under excess iron conditions [20,21]. More Arabidopsis produced noticeable growth defects under excess iron conditions [20,21]. More recently,recently, the the oscasp1oscasp1 mutantmutant with with defective defective CS CS formation formation showed showed hypersensitivity hypersensitivity to to high high CaCa conditions conditions in in rice rice [18]. [18]. However, However, the the role role of of the the CS CS in in plant plant adaptation adaptation to to the the growth growth environmentenvironment remains remains unclear. unclear. Rice Rice is is usua usuallylly cultivated cultivated under under flooded flooded conditions, conditions, where where 2+ CaCa2+ concentrationconcentration in in soil soil solution solution is is high high due due to to the the dissolution dissolution of of calcium calcium carbonate carbonate in in 2+ acidacid soils. soils. Especially, Especially, thethe soilsoil ofof karst karst areas areas contains contains a highera higher Ca 2+Caconcentration concentration [22 [22].]. In theIn thepresent present study, study, we reportedwe reported that thethat overexpression the overexpression of OsCASP1 of OsCASP1can improve can improve rice adaptation rice ad- aptationto high Cato high stress. Ca The stress. expression The expression of OsCASP1 of OsCASP1in the pOX in linesthe pOX was lines significantly was significantly enhanced enhancedin all tissues, in all including tissues, including the root tip, the compared root tip, compared with the WT. with Under the WT. normal Under conditions, normal con- the ditions,concentrations the concentrations of various essentialof various metals essential in the metals roots in and the shoots roots wereand shoots found towere be similarfound tobetween be similar the between WT and the pOX WT lines. and Under pOX lines. high CaUnder conditions, high Ca theconditions, Ca2+ concentration the Ca2+ concen- in the trationroots, shoots,in the roots, and xylemshoots, sap and of xylem the pOX sap linesof the was pOX significantly lines was significantly decreased indecreased contrast in to contrastother essential to other metals. essential Furthermore, metals. Furthermore, Ca-inhibited Ca-inhibited growth was growth significantly was significantly alleviated al- in leviatedthe pOX in lines. the pOX In addition, lines. InOsCASP1 addition,overexpression OsCASP1 overexpression resulted in earlierresulted CS in formation earlier CS in for- the mationendodermis in the but endodermis did not affect but suberindid not accumulation.affect suberin accumulation. This early CS formationThis early inhibited CS for- mationapoplastic inhibited Ca transport, apoplastic resulting Ca transport, in less resulting Ca2+ in thein less shoot. Ca2+ These in the results shoot. indicatedThese results that indicatedthe overexpression that the overexpression of OsCASP1 can of reduceOsCASP1 Ca accumulationcan reduce Ca in accumulation the shoots and in enhancethe shoots Ca andtolerance enhance in rice.Ca tolerance in rice.

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In Arabidopsis, localization of AtCASPs at the CS membrane domain (CSD) initiates CS formation [23]. Subsequently, other proteins such as enhanced suberin 1 (ESB1) and peroxidase 64 (PER64) are recruited to the CSD for lignin biosynthesis and deposition by the AtCASPs [24,25]. OsCASP1 functions in a similar manner to the AtCASPs, forming a transmembrane scaffold that mediates CS formation in the endodermis [18]. In the WT, we observed an initiation of CS formation at 5 mm from the root tip in conjunction with high OsCASP1 expression (Figure4A,D). In the pOX lines, OsCASP1 was highly expressed throughout the root. As a result, the start site of lignin deposition in the endodermis in the pOX lines was closer to the root apex relative to the WT (Figure4B,C,E,F). However, ectopic CS formation in non-endodermal cells was not observed (Figure4B,C,E,F). These results suggested that the initiation of CS formation in rice endodermal cells is likely to be related to high levels of OsCASP1 expression. The underlying mechanism remains to be determined. Previous studies have reported that Ca2+ enters the xylem radially via the apoplast in the root tip regions where CS is not completely formed or via the cytoplasm of non- suberized endodermal cells where the CS is formed [10]. In Arabidopsis, several CS mutants such as esb1 and lotr1 exhibited an enhanced suberization of endodermal cells, restricted Ca uptake, and showed low Ca accumulation in shoots [14,16]. Another CS mutant sgn3-3 with no ectopic suberin accumulation also exhibited the lower Ca concentration in shoots, suggesting that CS is also important for Ca2+ transport into the xylem for transfer to the shoot [19]. In the present study, we found that the endodermal apoplastic barrier was developed early in the overexpressing lines (Figure5A–F), which could be attributed to the early CS formation. However, the overexpression of OsCASP1 did not affect suberin deposition in root endodermal cells (Figure6A–F). Therefore, the decreased Ca accumulation in the roots and shoots of the OsCASP1-overexpressing lines was mainly caused by early formation of the endodermal apoplastic barrier. Early endodermal CS formation inhibited the apoplastic flow of Ca2+ across the endodermis into the xylem, resulting in less Ca uptake in the roots and less movement of Ca2+ to the shoots of the pOX lines. However, this inhibitory effect is not especially large because the effects of OsCASP1 overexpression on Ca accumulation were observed only at high Ca concentrations. This is supported by the results from the short-term Sr uptake that OsCASP1 overexpression does not affect apoplastic transport from root to shoot under normal conditions while reducing apoplastic transport with high Sr2+ treatment. Additionally, both apoplastic and symplastic pathways contribute to Ca accumulation in roots and shoots. There is one possibility that the contribution of the apoplastic pathway to Ca accumulation is higher in shoots than in roots. This may explain the smaller difference in Ca2+ concentrations between the WT and the pOX lines in roots compared to those in the shoots. In conclusion, our results demonstrated that the overexpression of OsCASP1 enhanced tolerance to high Ca stress in rice by decreasing the apoplastic flow of Ca2+ into the xylem and subsequently reducing Ca accumulation in the shoots. Our findings also provide the potential application of this strategy for improving the adaptation of other crops to high Ca environments, especially to a karst calcium-rich environment.

4. Materials and Methods 4.1. Generation of OsCASP1-Overexpressed Lines For the overexpression of OsCASP1 in rice, the OsCASP1 coding region (Os04g0684300) was ampliﬁed by RT-PCR from rice total RNA using the primers 5’-AAAACTGCAGATGA GCTCCGGCGAGCCTGCCG-3’ (PstI site underlined) and 5’-CCGACGCGTCTAGCGCTTG CGGATAGAGCAG-3’ (MluI site underlined). Using PstI and MluI, the ampliﬁed frag- ment was cloned into the pCAMBIA1300-Ubi vector containing the maize ubiquitin promoter and the nopaline synthase gene terminator to form the Pubi-OsCASP1 construct (Supplementary Figure S1). The resulting vector was introduced into Agrobacterium tumefa- ciens (strain EHA101) and then into rice (Nipponbare) by Agrobacterium-mediated transfor- Int. J. Mol. Sci. 2021, 22, 6002 9 of 11

mation. Hygromycin was used to select putative transgenic plants. OsCASP1 knockout line oscasp1-1 was obtained by CRISPR/Cas9 technology and prepared before [18].

4.2. RNA Isolation and Gene Expression Analysis To compare the expression of OsCASP1 in the WT and transgenic plants, we extracted total RNA from the roots and shoots of both (5 days old) using the TRIzol reagent kit (Life Technologies, Carlsbad, CA, USA). First-strand cDNA was synthesized from 1 µg total RNA using the PrimeScript II 1st Strand cDNA Synthesis kit (Takara, Toyko, Japan) according to the manufacturer’s instructions. The SYBR Premix ExTaq II (Takara) was used for expression analysis by qTOWER 2.0 (Analytik Jena, Jena, Germany). The primer sequences for qRT-PCR were 50-TCCCGGCCTTCCTGTTCTTC-30 and 50- CATGCATATGGCCACCCAGT- 30 for OsCASP1, 50-GGTCAACTTGTTGATTCCCCTCT-30 and 50-AACCGCAAAATCCAAA GAACG-30 for Histone H3. Histone H3 was used as an internal standard. Three biological replicates were analyzed for each sample.

4.3. Determination of Ca Tolerance and Element Concentration The WT and OsCASP1-overexpressing plants (15 days old) were grown in half-strength Kimura B solution containing different Ca2+ concentrations (0.18 and 20 mM) replacing the solution every two days. After 14 d, both control and experimental plants were washed three times in deionized water and photographed. The roots and shoots were ◦ ◦ collected separately and dried at 70 C for 3 d before digestion with HNO3 at 140 C. After appropriate dilution with 2% HNO3, the concentrations of zinc, manganese, iron, copper, potassium, magnesium, calcium, and strontium were measured by ICP-MS (Plasma Quant MS Elite; Analytik).

4.4. Xylem Sap Collection Thirty-day-old seedlings were cultured for 6 h in half-strength Kimura B solution containing different Ca (0.18 and 10 mM) or Sr (0.18 and 20 mM) concentrations. The shoots (2 cm above the roots) were excised, and the xylem sap was collected using a 2+ micropipette for 1 h. After dilution with 2% HNO3, the Ca concentration in the xylem sap was measured as described above.

4.5. Lignin/Cellulose Staining To investigate the lignin deposition in the endodermis of the WT and OsCASP1- overexpressing plant roots, root cross-sections (100 µm thickness) were cut at several posi- tions (1, 3, and 5 mm from the root apex) using a microslicer (VT1000 S; Leica, Germany). The sections were stained with 0.2% Basic Fuchsin for lignin and 0.1% Calcofluor White for cellulose as described previously [26]. Fluorescence was measured with a confocal laser scanning microscope (TCS SP8MP, Leica Microsystems). Z-stack magnified images were taken (20 optical sections with 0.3–0.4 µm intervals for each sample) and 3D images were constructed by LAS X 3D software (Leica Microsystems). The excitation and emission settings were 580 nm/600–615 nm for Basic Fuchsin and 405 nm/415–440 nm for Calcofluor White.

4.6. PI Penetration Assay Propidium iodide (PI) staining was performed according to the method of Alassimone et al. [27]. Five-day-old seedling roots were treated with 15 µg/mL PI (P4170; Sigma Aldrick, St. Louis, MO, USA) in a dark incubator at 28 ◦C for 40 min. After three washes with water, the roots were sliced into 100 µm sections 15 and 30 mm from the root tip and evaluated by confocal microscopy using 488 nm and 600–650 nm as the excitation and emission wavelengths, respectively. Int. J. Mol. Sci. 2021, 22, 6002 10 of 11

4.7. Suberin Staining Suberin staining was performed as previously described [28]. Root cross-sections, prepared as described in 2.5, were stained with 0.01% (w/v) Fluorol Yellow 088 (Sigma) in lactic acid at 70 ◦C for 30 min. After washing three times at 70 ◦C, the specimens were observed under confocal microscopy. The following excitation and emission settings were used: 488 nm/510–525 nm.

4.8. Statistical Analysis GraphPad Prism 8 software was used for statistical analysis. Data were analyzed using one-way ANOVA followed by Tukey’s test. Signiﬁcance of differences at p < 0.05 is indicated by different letters.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms22116002/s1. Author Contributions: J.X. conceived and designed the experiment. Z.W., Z.C. and X.Z. performed the experiments. Q.W., Y.X., B.Z. and F.L. participated in the research. Z.W. and J.X. analyzed the data. J.X. wrote the paper. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by National Natural Science Foundation of China (32070275), Guangxi Natural Science Foundation (2016GXNSFFA380013), Innovation Project of Guangxi Grad- uate Education (YCBZ2020022), Guangxi innovation-driven development special funding project (Grant no. Guike-AA17204070), and Hundred-Talent Program of Guangxi (2014). Data Availability Statement: All the data supporting the conclusions of this article are provided within the article and in its additional files. All data and materials are available upon reasonable request from the corresponding author. Conflicts of Interest: The authors declare no conflict of interest.

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