OsPEX1,a leucine-rich repeat extensin protein,functions in the regulation of caryopsis development and quality in rice

Xin Lun,Shnwen Ke,Shuchun Liu,Guojin Tng,Dhui Hung,Minyi Wei,Yuexiong Zhng,Gng Qin,*,Xing-Qin Zhng,c,*

a Guangdong Engineering Research Center of Grassland Science,College of Forestry and Landscape Architecture,South China Agricultural University,Guangzhou 510642,Guangdong,China

b Rice Research Institute,Guangxi Academy of Agricultural Sciences/Guangxi Key Laboratory of Rice Genetics and Breeding,Nanning 530007,Guangxi,China

c Guangdong Laboratory of Lingnan Modern Agricultural Science and Technology,Guangzhou 510642,Guangdong,China

Keywords:Rice Caryopsis Grain size Glume Extensin-like protein

ABSTRACT Rice caryopses are enclosed by outer glumes.The size and dimension of the outer glume are the main determinants of caryopsis size.However,it is unclear whether caryopsis development is completely dependent on the size of the glume,or whether it can grow and expand autonomously despite the constraint of glume enclosure.We report the identification of a mutant line that produces normal-sized glumes with smaller mature caryopses that do not fill the entire glume cavity.The caryopsis phenotype in the pex1 mutant is caused by a reduction in cell size.OsPEX1,a leucine-rich repeat extensin gene,was highly expressed in the developing caryopsis.Overexpression of OsPEX1 driven by a constitutive promoter recapitulated the mutant phenotype,showing that the small-caryopsis phenotype is caused by overexpression of the OsPEX1 gene.Free amino acids,including several essential amino acids,and crude protein were increased in pex1 relative to the wild type,endowing pex1 with improved nutritional quality.Our results suggest that caryopsis development can be genetically uncoupled from maternally controlled glume development and that OsPEX1 might be a new resource for improving nutritional quality of rice cultivars.

1.Introduction

Rice,a staple food crop worldwide,is consumed by humans primarily as polished white or unpolished brown rice.Grain size,usually measured by grain length,width,thickness,and weight,affects the yield and quality of rice and is a major target of research and breeding.The rice grain is derived from a floret with caryopsis that is encased by the glume(lemma and palea).The caryopsis consists of diploid maternal tissues (pericarp,testa,and nucellus),triploid endosperm,and a diploid embryo.During flower development,the glume expands in volume and grows to maximum size prior to anthesis.Inside the mature glume,the caryopsis starts to grow and increase in size after fertilization,and reaches its final size around 20 days after fertilization [1].Although the glume and caryopsis grow and develop at different time points,the mature caryopses perfectly fill the inner chamber enclosed by the glumes.It has long been accepted that the size and dimension of the outer glume are the main determinants of caryopsis size.

Recent studies have begun to shed light on the regulatory mechanism of glume development via identification and functional characterization of genes associated with grain size and yield.Nearly 100 genes associated with grain shape and weight have been isolated in rice [2,3].However,almost all of them affect glume size.For example,natural mutants of grain width(GW)produce wider glumes and widened caryopses.Other genes influence glume size by affecting either cell number and size in lemmas and palea,or cell cycle regulation [4–11].

With respect to the regulatory mechanism of glume development,phytohormone and G-protein signaling have been shown to act in regulating grain size and yield in rice.Several dwarf mutants in brassinosteroid (BR) hormone biosynthesis and signaling pathways affect grain and panicle size,including d61,d2,d11,sg1,and RAV6 [11–16].Mutations in Dwart1 (D1),also known as the rice heterotrimeric G protein alpha subunit (RGA1),affect multiple signaling pathways such as BR and GA and exert pleiotropic effects on organ growth and reduction in glume and seed size [17–19].GW6,encoding a GAST family protein,controls grain size via the gibberellin pathway [20].A recent study [21]indicated that the ubiquitin–proteasome pathway and mitogenactivated protein kinase (MAPK) signaling are also involved in the regulation of glume size.However,little is known about how caryopsis size is controlled and regulated.It is unclear whether the development of the caryopsis is completely dependent on glume size or whether it can grow and expand autonomously,independent of the constraint of glume enclosure.

Mutation in OsKinesin-13A (sar1) causes small glumes and rounded grains due to defects in cell elongation.Although glumes and caryopses are both reduced in length,sar1 affects specifically glume elongation and length and the reduction in caryopsis size is an indirect effect of the reduced space of the shortened glumes,as sar1 caryopses can grow to the wild-type (WT) length after removal of the glume-height constraint[22].This observation suggests a model in which cell length in the glume and caryopsis are regulated independently.However,little genetic evidence has been uncovered to support such model.

Leucine-rich repeat extensins (LRXs) are a class of cell walllocalized chimeric proteins with a leucine-rich repeat (LRR)domain and an extensin domain containing Ser-Pro(3–5)repeated modules near the C terminus[23].Several LRXs in Arabidopsis have been studied in detail[24–26].The rice genome encodes 8 LRX proteins [23],but little is known about their functions.In this study,we identified and characterized a rice small-caryopsis mutant from an Ac/Ds transposon-tagging population.Our work revealed that rice LRX gene OsPEX1 plays an important role in the regulation of caryopsis development and quality in rice.

2.Materials and methods

2.1.Plant materials and growth conditions

The japonica rice cultivar Zhonghua 11 was used as the wild type.The pex1 mutant was derived from an Ac/Ds transposontagging population of Zhonghua 11 [27,28].Rice seeds were propagated in a paddy field in Guangzhou,China.

2.2.Plasmid construction and rice transformation

To produce PEX1-overexpressing transgenic plants,the fulllength PEX1 coding sequence was amplified from cDNA derived from Zhonghua 11 using primer pairs listed in Table S1.After confirmation by DNA sequencing,the amplified sequence was cloned into the binary vector pCUbi1390.The final constructs were electroporated into Agrobacterium tumefaciens strain EHA105.

For promoter analysis,a 1572-bp DNA fragment immediately upstream of the OsPEX1 start codon was amplified using primers LRXPF and LRXPF (Table S1) and inserted into the EcoRI and NcoI sites of the pCAMBIA1305.1 vector.The resulting plasmid was introduced into a wild-type plant by A.tumefaciens-mediated transformation.Transgenic plants were selected on hygromycin medium and T2transgenic plants were used to test βglucuronidase (GUS) activity.

2.3.Quantitative RT-PCR

Total RNA was extracted from frozen samples with TRIzol reagent (Invitrogen Corporation,Carlsbad,CA,USA) according to the manufacturer’s instructions.The RNA was pre-treated with DNase I,and first-strand cDNA was generated using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific,Waltham,MA,USA).Quantitative RT-PCRs were performed using a SYBR Premix Ex Taq RT-PCR kit (Takara Biotechnology (Dalian)Co.,Ltd.,Dalian,Liaoning,China) following the manufacturer’s instructions.Investigated genes and their specific primer sets are shown in Table S1.The relative expression level of a target gene was normalized to that of rice 25sRNA.

2.4.Determination of crude protein content and free amino acids

The crude protein content was estimated by multiplying the organic nitrogen content by 6.25.Nitrogen concentration was determined by the Kjeldahl method as previously described [29].Oven-dried brown rice flour samples (0.5 g) were placed into a 50-mL Kjeldahl digestion flask.To each flask,8 mL of concentrated H2SO4was added and the flask was gently heated.When water evaporation had ceased,the heat was increased to 400 °C and 30% (v/v) H2O2was added to the flask intermittently until the digest cleared.After complete digestion,the flask was allowed to cool and water was slowly added to make up the volume to 100 mL.A 10-mL sample was used to determine N content.Free amino acids were determined by reverse-phase HPLC [30].

2.5.Physicochemical properties analysis of seed starch

Apparent amylose content(AAC)was tested as described previously[31].Total starch content of brown rice was estimated by the HCl hydrolysis-DNS method,as described [32].Alkali spreading value (ASV) was evaluated as previously described [33].Adhesive strength (or named gel consistency) was determined as described[34].

2.6.Histochemical analysis

Mature caryopses were soaked in FAA solution(50%ethanol,5%glacial acetic acid,5% formaldehyde) overnight at 4 °C for cell fixation and dehydrated in a graded alcohol series.They were then sectioned transversely into 2-mm slices with a razor blade and embedded in paraffin.Semi-thin sections were cut using a glass knife in a Leica ultra-microtome and stained with 0.05%(w/v)Fast Green and 0.1% (w/v) safranin.The histochemical slices were observed under a light microscope.The cell size was calculated with Image J.

2.7.Pasting viscosity

Rice pasting properties were determined on a Rapid Visco Analyzer (RVA,model TCW3,Newport Scientific,Warriewood,Australia).Three grams of rice flour were mixed with 25 mL of distilled water.A programmed heating–cooling cycle was used,in which samples were held at 50 °C for 1 min,heated to 95 °C in 3.8 min,held at 95 °C for 2.5 min before cooling to 50 °C in 3.8 min,and held at 50°C for 1.4 min.The parameters used to characterize the pasting curves were peak viscosity(PV),hot paste viscosity (HPV),cool paste viscosity (CPV),and their derivative parameter,breakdown (BD=PV -HPV).

3.Results

3.1.pex1 produced normal size of glumes but smaller caryopsis

The pex1 mutant had glumes of almost the same size as wild type (WT) plants,but markedly smaller caryopses (Fig.1A).Compared with the WT,caryopsis width and length in the heterozygous pex1 mutant (pex1/+) were reduced by 18.5% and 4.0%,respectively,while the homozygous pex1 mutant (pex1/pex1) showed a 25.3% reduction in caryopses width and a 7.2% reduction in caryopses length(Fig.1B).As a result,the 1000-grain weights of pex1/+and pex1/pex1 were reduced by 25.0% and 33.8%,respectively(Fig.1B).

Fig.1.Phenotypic characterization of the pex1 mutant.(A)pex1 exhibits normal glume size but smaller caryopses.(B)Comparisons of glume length,glume width,caryopsis length,caryopsis width,1000-caryopsis weight,and 1000-glume weight of WT and the pex1 mutant.All values are means±SD(n=30).**,P ＜0.01,determined by Student’s ttest.

3.2.Developmental characteristics of pex1 caryopsis

Morphological analysis was performed for the grain and caryopses of the WT and the pex1 mutant grown in a paddy field.The anthesis date of each floret was marked on the surface of the lemma,and samples were collected.The glumes of pex1 plants were almost indistinguishable from those of WT plants (Fig.2A).However,the pex1 mutant produced significantly smaller caryopses from 7 days after pollination (DAP) compared with the WT(Fig.2 B,C).The major extension of the rice caryopsis along the long axis occurred from 1 to 7 DAP,while major expansion along the transversal axis occurred between 1 and 15 DAP (Fig.2 B,C).The results suggested that the growth pattern for caryopsis width differed from that of caryopsis length.

3.3.OsPEX1 affects caryopsis size by influencing cell expansion

To test whether cell size is affected in caryopses,aleurone cells of the caryopses from the WT and pex1 plants were examined and smaller aleurone cells were observed in the caryopses of pex1 plants (Fig.3A–C).In order to examine the phenotype by molecular analyses,expression of several representative genes involved in cell expansion was investigated by quantitative RTPCR (Fig.3D).Cell expansion-associated genes such as OsEXP6(LOC_Os03g21820) and xyloglucan endo-transglycosylase/hydrolase (XTH) genes including XTH10 (LOC_Os06g48200) and XTH17 (LOC_Os08g13920) were significantly downregulated in the caryopses of pex1 mutants compared with the WT control.These results suggested that the pex1 mutation affected caryopsis size by reducing cell size.

Fig.2.Dynamic characteristics of the pex1caryopsis development.(A)Dynamics of grain development in WT and pex1 mutant.(B)Caryopsis development from 1 to 25 days after pollination (DAP).The rapid elongation of the caryopsis occurred in the first 5 DAP;the major period of expansion occurred in the first 15 DAP.Scale bar,5 mm.(C)Comparison of caryopsis length and caryopsis width of WT and pex1 mutant.**, P ＜0.01,determined by Student’s t-test.

3.4.Physicochemical properties of pex1 caryopsis

To identify the effects of OsPEX1 on grain quality,we compared the starch physicochemical properties of caryopses in the WT and pex1 mutant.Compared with the WT,the apparent amylose content (AAC) of brown rice was decreased significantly (～2.5 folds)in the pex1 mutant,but with no clear decrease in total starch content,whereas alkali spreading value and adhesive strength were significantly increased in the pex1 mutant (Fig.4A).The pex1 mutant differed significantly from the WT in almost all pasting property parameters,including peak viscosity,hot paste viscosity,cool paste viscosity,and breakdown value (Fig.4B).These results suggest that OsPEX1 may mediate AAC content,alkali spreading value,adhesive strength,and starch pasting properties.

We next investigated the effect of OsPEX1 on the crude protein and free amino acid (FAA) content in brown rice of WT and pex1.The crude protein content in the pex1 was increased by 23.6%compared to the WT(Fig.4C).Free amino acids were also increased significantly by 31.1% to 312.5%,leading to an increase of 114.4% in the total FAA content in pex1 relative to the WT (Fig.4C;Table S1).In particular,several essential amino acids (e.g.,Val,Lys,Met,Thr,Leu,Ile,and His) were much higher in the pex1 mutant,especially Val,which increased more than threefold,than in the WT (Fig.4C;Table S1).These results suggested a role of OsPEX1 in FAA biosynthesis and protein content.Overall,the physicochemical properties of pex1 starch were markedly altered in comparison with the WT.

Fig.3.The pex1 mutation affected caryopsis size by reducing cell size.(A) Cross sections of mature caryopses at the ventral positions of WT and pex1 endosperms.Dotted lines indicate sites of cross sections in the WT and pex1caryopses.(B)Magnified images of the boxed areas in(A).(C)Comparison of average length of each cell in the aleurone layer.All values are means±SD(n=10).**,P ＜0.01,determined by Student’s t-test.(D)Transcript levels of cell expansion-associated genes in caryopses of pex1/pex1 at 7 DAP,relative to WT.The rice 25sRNA gene was used as an internal control.Values are means ± SD (n=3),**, P ＜0.01,determined by Student’s t-test.

3.5.OsPEX1 affects the expression levels of starch,protein and amino acid biosynthesis-associated genes in developing caryopsis

To determine whether changes in the accumulation of grain proteins and starch were reflected in altered mRNA levels,we investigated the expression of key genes involved in the accumulation of storage materials in filling rice grain.The transcript levels of four genes involved in grain protein biosynthesis and three genes involved in amino acid biosynthesis in developing grain at 7 DAP were upregulated in the pex1 mutant relative to the WT (Fig.5).Transcript levels of the Wx gene,involved in amylose biosynthesis,were markedly downregulated in pex1 relative to wild-type seeds.These results strongly suggest that OsPEX1 influences the expression of genes participating in starch,protein and amino acid biosynthesis in developing rice grains.

3.6.Overexpression of PEX1 leads to a pex1-like phenotype

To confirm that overexpression of OsPEX1 (LOC_Os11g43640) is responsible for the small caryopsis in pex1,we overexpressed LOC_Os11g43640 in the WT plant under the control of the maize(Zea mays) Ubiquitin promoter.Compared to WT control,all positive transformants displayed glumes of normal size but smaller caryopsis,a phenotype similar to that of the pex1 mutants(Fig.6).These results indicate that the abnormal phenotypes of the pex1 mutant result from overexpression of OsPEX1(LOC_Os11g43640).

Fig.4.The pex1 mutation affected rice quality.(A) Comparisons of apparent amylose,total starch,alkali spreading value,and adhesive strength in mature seeds of WT and pex1.(B)Pasting properties of WT and pex1 grains analyzed with a Rapid Visco Analyzer.(C) Contents of free amino acids (FAA)and crude protein in brown rice of WT and pex1.All values are means ± SD (n=3).**, P ＜0.01,determined by Student’s t-test.

3.7.Expression pattern of OsPEX1

Given that pex1 exhibits glumes of normal size but smaller caryopses than WT,we compared the transcriptional levels of OsPEX1 in glumes and caryopses between WT and the pex1 mutant.In the caryopsis,the OsPEX1 gene in pex1 mutants was expressed approximately fourfold more abundantly than in WT,whereas in the glumes OsPEX1 was not differentially expressed between the WT and pex1 mutants(Fig.7A).These findings indicate that OsPEX1 negatively modulates caryopsis size in rice.To elucidate the role of OsPEX1 in caryopsis development,we further investigated the expression pattern of OsPEX1 during caryopsis development.As shown in Fig.7B,transcripts of OsPEX1 rapidly increased after flowering,reaching their peak (approximately seven folds) at 7 DAP,and then decreasing but remaining high at 10 DAP.

Fig.5.Expression levels of genes involved in starch,protein,and amino acid biosynthesis in developing rice grains in the pex1 mutant and WT plants.Values are based on three biological replications.**, P ＜0.01.Error bars show SD.The annotated names of the genes are shown in Table S1.

Fig.6.Overexpression of OsPEX1 phenocopies the pex1 mutant phenotype.The grain (A) and caryopsis (B) morphologies of the wild-type (WT) and transgenic plants overexpressing OsPEX1.OE indicated transgenic plants overexpressing rice PEX1.(C)Characterization of glume length,glume width,caryopsis length,and caryopsis width in WT and transgenic plants (OE)overexpressing rice OsPEX1.Values are means ±SD.In each plot,statistically significant differences are indicated by asterisks(*,P ＜0.05;**,P ＜0.01).

Fig.7.Expression pattern of OsPEX1.(A) Comparisons of the transcriptional level of OsPEX1 in glumes and caryopsis between WT and pex1 mutant plants.(B) Change over time in the OsPEX1 transcription levels of rice caryopsis from 0 to 10 day after flowering.(C) OsPEX1p::Gus expression pattern in developing caryopses.

Promoter-GUS fusion analysis revealed that OsPEX1 was expressed in the outer surface of the endosperm,including the aleurone layer of developing caryopses(Fig.7C),especially the dorsal vascular bundle of developing caryopses with high levels of GUS expression (Fig.S1).Interestingly,GUS expression was restricted to the embryo of mature seeds.No GUS staining was detected at the aleurone layer of mature seed (Fig.7C).

We also conducted expression analysis of several genes involved in glume regulation in WT and pex1 mutant.As expected,RT-qPCR (quantitative reverse-transcription PCR) results showed that the transcriptional levels of eight genes tested in the pex1 mutant were similar to those in WT (Fig.S2),suggesting that the overexpression of OsPEX1 exerted lower effects on genes associated with glume size,consistent with the pex1 phenotype with normal glume compared to WT plants.

Fig.8.Identification and expression analysis of putative rice PEX genes.(A)Phylogenic analysis of the LRXs in Arabidopsis and rice.The phylogenetic trees were constructed by the maximum likelihood method using Mega 7.0;bootstrap analysis was performed with 1000 replicates,excluding positions with gaps.Based on the analysis,OsLRXs were divided into two subgroups.(B) Gene structures and conserved domains of the LRXs.Thin lines represent introns,dark bars refer to putative protein-coding sequence(CDS),untranslated regions are shown in gray rectangles.LRX proteins contain a signal peptide for protein export,an LRR domain,a cysteine-rich linker domain(CRD)and an extensin domain.(C)RT-PCR analysis of OsPEX1-3 gene transcripts in the WT and pex1 mutant.Total RNAs were extracted from the WT and pex1 mutant roots at 3 days after germination (DAG),leaves at 15 DAG,and caryopses at 7 days after pollination (DAP. 25S rRNA is shown at the bottom as an internal control.

3.8.Expression of rice PEX genes is not limited to reproductive tissue

We investigated the phylogenetic relationship between rice and Arabidopsis LRXs genes by comparing the putative full-length protein sequences,which can be divided into two subgroups:LRX and PEX(Fig.8A),following Baumberger et al.[35].All of the putative LRX proteins contain conserved leucine-rich repeat (LRR)motifs for protein–protein interactions and extensin domain probably involved in crosslinking to cell wall components (Fig.8B).A cysteine-rich domain (CRD) might act to stabilize the structure of the LRX protein via formation of disulfide bonds with cysteine residues of the LRR domain [36,37].None of the LRX genes except for OsPEX2 has introns (Fig.8B).OsPEX1 has the longest coding sequence in the LRX members of rice (Fig.8B).

Transcripts of AtPEX1-4 were detected almost exclusively in flowers including mature anthers,pollen,and pollinated carpels,suggesting that Arabidopsis PEX genes were expressed specifically in reproductive organs [35].To determine whether rice PEX genes acquired their tissue-specific expression in reproductive organs,we conducted the expression analysis of three PEX genes in roots,leaves,and developing seeds.In contrast to the tissue specificity of Arabidopsis PEX genes in reproductive organs,OsPEX1 transcripts were detected in roots as well as seeds at 7 DAF,OsPEX3 was expressed in roots,leaves,and seeds,whereas OsPEX2 was not expressed in these tissues (Fig.8C).Thus,rice PEX genes were expressed in vegetative and reproductive organs,an expression pattern differing from that of Arabidopsis PEX genes.

In contrast to the higher expression levels of OsPEX1 in the pex1 mutant,the transcript levels of OsPEX3 by RT-qPCR were not significantly different between the WT and pex1 mutant,suggesting that overexpression of OsPEX1 had no effect on the transcript levels of OsPEX3 in the pex1 mutant (Fig.S3).

4.Discussion

Deng and colleagues[22]reported that OsKinesin-13A,a microtubule depolymerase,acts in regulation of glume length by affecting cell elongation.The mutant of OsKinesin-13A,sar1,displayed length reduction in the glume.However,WT and sar1 caryopses maturing under glume-cutting conditions were similar in length.These observations indicated that the grain phenotype was caused by reduction in glume length,which indirectly restricted caryopsis size.Although some studies of the relationship between glume and caryopsis development were discussed based on notched grains in rice [38,39],these observations suggested that the size and length of floral glume and caryopsis can be regulated independently.

The length reduction in sar1 caryopses was not a direct consequence of mutation in OsKinesin-13A,but resulted from the space restrictions due to shortened glumes [22],indicating that OsKinesin-13A controls glume size but not caryopsis size.By contrast,mutation in OsPEX1 directly affected caryopsis size but showed little effect on glume size,indicating that OsPEX1 is directly involved in regulating caryopsis size.Thus,our findings strongly support a model in which caryopsis development can be separated from maternally controlled glume development.

The OsPEX1 gene is a member of the LRX (LRR/EXTENSIN) gene family.The LRX proteins are characterized by a domain with leucine-rich repeats in addition to the extension domain.LRRs are frequently implicated in protein–protein interactions and signal transduction during development or in pathogen recognition and defense [40–42].One proposed function of extensins is to lock-in cell shape upon cell expansion[43,44].In view of the properties of LRR and extensins,LRXs could potentially be involved in the regulation of cell wall expansion in response to signals.Such a function in cell morphogenesis is supported by the finding that the Arabidopsis lrx1 mutant developed aberrant root hairs [25].The idea is also supported by the observation in this study that pex1 had smaller aleurone cells than WT (Fig.3),consistent with a role of OsPEX1 in cell wall expansion.Together with the observations that overexpression of OsPEX1 resulted in reduction of caryopsis size,our results support a negative regulatory role for OsPEX1 in caryopsis development.

According to their expression pattern,LRX genes of higher plants can be classified as vegetatively expressed or expressed predominantly in reproductive tissue,two categories that coincide almost completely with their phylogenetic clades[35,36].Interestingly,even though OsPEX1 belongs to the PEX (pollen expression LRX) subfamily,it is highly expressed in root and stem as well as reproductive tissue such as developing caryopsis,but is expressed at much lower level in leaf and glume,in accord with the pleiotropic phenotypes of the pex1 plants with dwarfism [28] and small caryopsis(Fig.1),but with less effect on leaf[28]and glume development (Fig.1).

Proper lignin deposition is essential for appropriate plant development.Cell expansion cannot occur smoothly unless the cell wall is sufficiently soft to allow cell expansion but rigid enough to resist turgor pressure.Lignin is a major component of the secondary cell wall and the biosynthesis and deposition of lignin is closely associated with cell expansion.Our previous results [28] indicate that OsPEX1 functions in lignin biosynthesis and deposition.Not surprisingly,overexpression of OsPEX1 led to reduced cell size in stem[28]and caryopsis(present study).In fact,previous studies in Arabidopsis[45]and rice[46,47]have revealed that the lignin pathway is involved in cell expansion and regulates seed size.

Receptor kinases of Catharanthus roseus RLK1-like (CrRLK1L)regulate cell expansion throughout the plant [48].RAPID ALKALINIZATION FACTOR (RALF) peptides as a ligand of CrRLK1L are perceived by heterocomplexes of CrRLK1L and GPI-anchored proteins[49].Recent studies [36] have shown that RALF-CrRLK1L pathway is highly conserved in plants and could regulate multiple processes including fertility,growth,and stress response via interaction with various factors.There is increasing evidence that the RALF-CrRLK1L module serves as a node of energy metabolism,balancing starch and protein biosynthesis in plants.For example,FERONIA (FER),a CrRLK1L family member,can regulate carbon and nitrogen utilization by interacting with the E3 ubiquitin ligase ATL6 [50].The RALF1-FERONIA complex phosphorylates eIF4E1,a eukaryotic translation initiation factor,to promote protein synthesis in Arabidopsis roots [51].FER serves as a regulator of starch metabolism by interacting with glyceraldehyde-3-phosphate dehydrogenase[52].Recently,it was shown[53]that the rice FERONIA-like receptor(FLR1)negatively controls grain size by interaction with OsRac1(a ROP GTPase) and also affects the accumulation of starch and protein in seeds.The grain quality,including chalkiness,of the OsRac1 mutant and overexpression lines did not differ from that of the wild type [53].These studies show that the RALF-CrRLK1L pathway could regulate grain size and quality,possibly via different mechanisms.

Our results indicate that OsPEX1 functions in caryopsis development.The mechanisms regulating caryopsis size and quality by OsPEX1 remain to be elucidated.LRX proteins are highly conserved in higher plants and can directly interact with RALF peptide hormone and CrRLK1L transmembrane receptors [36].These findings prompt the hypothesis that OsPEX1 controls caryopsis size and quality via direct interaction with RALF and CrRLK1L members.Rice RALF and CrRLK1L family contain 46 and 16 members,respectively (http://rice.plantbiology.msu.edu/).Which RALF or CrRLK1L members influence the functions of OsPEX1 and whether they can physically interact with the OsPEX1 protein await further study.

It is generally believed that LRX family proteins are located in the cell wall and function as a structural protein.Emerging evidence suggests that LRXs are involved in the regulation of cell wall expansion in response to signals [54,55].However,how cell wall proteins are involved in signaling is a mystery.It is becoming clear that any disturbance in the plant cell wall has a direct impact on cell membrane systems,and likewise that the disruption of any intracellular component also may influence the plant cell wall[55,56].Arabidopsis LRX11,an ortholog of OsPEX1,can bind small RALF peptides,which function as unique signal molecules [57].An attractive hypothesis would thus be that OsPEX1 has signal or regulatory functions beyond those of a structural protein of the cell wall.This point should be considered in future studies aimed at dissecting the contributions of OsPEX1 to caryopsis development and grain quality via the RALF-mediated signaling cascade in rice.

5.Conclusions

We propose that OsPEX1 regulates caryopsis size and simultaneously improves nutrition quality in rice.Our results suggest that caryopsis development can be separated from maternally controlled glume development.

CRediT authorship contribution statement

Xin Luan:Investigation,Methodology,Visualization.Shanwen Ke:Investigation.Shuchun Liu:Investigation.Guojian Tang:Investigation.Dahui Huang:Investigation.Minyi Wei:Investigation.Yuexiong Zhang:Formal analysis,Validation.Gang Qin:Conceptualization,Resources.Xiang-Qian Zhang:Conceptualization,Supervision,Visualization,Writing– original draft,Writing–review &editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank Dr.Qingjun Xie(South China Agricultural University)for his help with plasmid construction.This work was supported by the National Natural Science Foundation of China (31671594 and 30900884 to Xiangqian Zhang),Guangxi Key Laboratory of Rice Genetics and Breeding Open Foundation (2018-05-Z06-KF02 and 2018-15-Z06-KF15),and Guangdong Basic and Applied Basic Research Foundation (2020A1515110067).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2021.10.001.