Shouhong Zhu, Ynjun Li, Xinyu Zhng, Feng Liu, Fei Xue, Yongshn Zhng,Zhosheng Kong, Qin-Ho Zhu, Jie Sun,

a College of Agriculture/The Key Laboratory of Oasis Eco-agriculture, Shihezi University, Shihezi 832000, Xinjiang, China

b State Key Laboratory of Cotton Biology, Institute of Cotton Research, Academy of Agricultural Sciences, Anyang 455000, Henan, China

c State Key Laboratory of Plant Genomics, Institute of Microbiology Academy of Seed Design, Chinese Academy of Sciences, Beijing 100101,China

d CSIRO Agriculture and Food, GPO Box 1700, Canberra 2601, Australia

Keywords:

ABSTRACT Fiber length is one of the most important quality parameters of cotton fibers.Transcriptomic analyses of developing cotton fibers have identified genes preferentially expressing in fiber elongation stage, but few have been functionally characterized.Here, on the basis of confirmation of the preferential expression profile of GhAlaRP (Gh_A09G1166 and Gh_D09G1172), an alanine rich protein gene,in the rapid elongating fibers, we investigated the role of GhAlaRP in fiber development by generating transgenic cottons with an increased or decreased expression level of GhAlaRP.Our results showed that the fiber length was consistently significantly shorter in both the GhAlaRP-RNAi lines and the alarp mutant generated by genome editing than in the control YZ-1.GhAlaRP was localized on plasma membrane,nucleus and endoplasmic reticulum.The yeast two-hybrid assay and bimolecular fluorescence complementation assay showed that GhAlaRP co-expresses and interacts with GhAnnexin(Gh_D11G2184)and GhEXPA(Gh_A10G2323)that are involved in fiber elongation.Down-regulation of GhAlaRP co-suppressed the expression levels of GhAnnexin and GhEXPA.These results suggest a role of GhAlaRP in regulation of cotton fiber elongation,which could be achieved by regulating the functions of GhAnnexin and GhEXPA.

1.Introduction

Cotton is the most important source of natural fiber and textile industry materials in the world.Cotton fiber is a single cell that differentiates and develops from the ovule epidermal cells [1,2].Its development process includes four distinct and overlapping stages: initiation, elongation, secondary cell wall thickening and maturation [1,3,4].There is no clear distinction between each of the two consecutive stages.During the process of fiber cell development, many genes are involved in regulating the growth and development of cotton fiber cells by affecting important physiological and biochemical activities of fiber cells [1,4], which changes the morphological structure of fiber cells, and affects the formation of cotton fiber yield and quality [1,3-6].

The duration of each stage of cotton fiber cell development varies by genotype and environment.The elongation stage of cotton fiber cells lasts for a long time.The elongation rate and time of cotton fiber cells determine the final length of fiber.The elongation of cotton fiber cells is a complex physiological and biochemical process involving vacuolar swell, cell wall relaxation, biosynthesis and transportation of membrane lipids and cell wall components.In this process, genes involved in osmotic pressure regulation, cytoskeletal assembly, cell wall relaxation, glucose and fatty acid metabolism have important functions [6].Annexin, sucrose synthase,actin, tubulin, and saturated long-chain fatty acids all have been shown to be essential for proper fiber elongation [6-11].TCP14 plays a role in auxin-mediated differentiation and elongation of fiber epidermal cells [12].GhHOX3, a class IV homeodomain-leucine zipper (HD-ZIP) transcription factor,is highly expressed during the early fiber elongation stage.Silencing of GhHOX3 inhibited fiber elongation, while overexpression of GhHOX3 promoted fiber length increase, indicating that GhHOX3 is involved in the regulation of cotton fiber cell elongation [13].WLIM1a is preferentially expressed in the elongation and secondary wall synthesis stages of fibers[5].Fibers of cotton plants overexpressing WLIM1a were longer and had a thinner and more compact secondary cell wall, which contributed to improved fiber strength and fineness,indicating that WLIM1a plays a dual role in fiber,elongation and secondary cell wall formation [5].

The fiber elongation phase involves synthesis of primary cell wall [14,15], in which newly synthesized cell wall polypeptides are deposited in the lumen of endoplasmic reticulum (ER).The membrane systems, including ER, Golgi and plasma membrane, play roles in processing and transporting cell wall components.Glycosylation of structural proteins and their polymerization with hemicellulose and pectin are carried out in the Golgi apparatus, and then transported to outside the plasma membrane through transport of vesicles [16].GhEXP, a gene encoding α-expansin protein that is associated with cell wall expansion, is highly expressed during the time of fiber elongation and regulates cell wall loosening, which leads to cotton fiber cell elongation [17].Fiber elongation involves both unidirectional fast elongation via polar or tip growth and expand via diffuse growth and is probably driven by a unique tip-biased diffuse growth [18].

Using the mRNA fluorescence differential display-PCR(mRNA FDD-PCR)technology,we identified GhF1,a gene preferentially expressed in elongating cotton fibers [19].Spatial and temporal expression patterns of GhF1 suggest a potential role in cotton fiber elongation [19].GhF1 encodes a short protein containing 66 amino acids, of which, 14 (21.2%) are alanine.We, therefore, in this study, renamed it as cotton alanine-rich-protein gene,or GhAlaRP and investigated its function in cotton fiber development by overexpressing,RNAi and genome editing.We showed that down-regulation of GhAlaRP by RNAi or genome editing had a negative effect on fiber length although overexpressing the gene did not increase fiber length.GhAlaRP interacts with GhAnnexin and GhEXPA,which were co-suppressed with GhAlaRP in both the RNAi and genome editing lines.The function of GhAlaRP in fiber elongation could be achieved by forming a regulatory network through interaction with GhAnnexin and GhEXPA.

2.Materials and methods

2.1.Plant materials and growth conditions

Gossypium hirsutum Xinluzao 33 and YZ-1 were used as materials in these experiments.Cotton YZ-1 was used for transforming.Cotton Xinluzao 33 was grown in the experimental field at Shihezi University (Shihezi, Xinjiang, China).On the day of flowering, cotton boll age were tagged and marked as 0 day post anthesis (DPA).Fibers from 6, 12, 18, and 24 DPA bolls were removed from the ovules.Some tissues (roots,stems,and leaves)were harvested from 14-day-old seedlings.All collected tissues were immediately frozen in liquid nitrogen and stored at -70 °C.The control cotton and the transgenic lines were grown in the experiment field at Shihezi University (Shihezi, Xinjiang, China) under standard farming conditions.The control cotton and the alarp mutant lines were grown in the experiment field at the plant breeding base of Southern (Sanya, Hainan, China) under standard farming conditions.

The tobacco (Nicotiana benthamiana) plants were grown in an environmental growth chambers (16 h light/8h dark, 22 °C) and used in infiltration and observation of subcellular localization of GhAlaRP.

2.2.Gene cloning, sequence and phylogenetic analyses

The full-length GhAlaRP cDNA was isolated from Xinluzao 33 fiber cDNA library.The conserved protein domains were searched using the online software InterProScan (http://www.ebi.ac.uk/interpro/search/sequence-search) [20].The DNAMAN software was used to perform alignment of AlaRP sequences from the three cotton species (G.hirsutum, G.arboreum, G.raimondii) and other plants.The phylogenetic tree was constructed by using the Neighbor-joining (NJ) method in the MEGA 7.0 software [21].

2.3.Quantitative RT-PCR analysis

Total RNA was extracted from various cotton tissues (roots,stems, leaves, whole flowers at 0 DPA and fibers at 6, 12, 18,and 24 DPA) of the non-transgenic cotton (Xinluzao 33 and YZ-1), the different transgenic cotton lines (GhAlaRPoverexpressing and GhAlaRP-RNAi) and the genome edited cotton lines (alarp), and reverse transcribed to cDNA using the method described by Zhu et al.[22].The qRT-PCR was performed using a SYBR Green I Master mixture (Roche, Basel,Switzerland) in a Light Cycler 480II system (Roche, Switzerland).All fiber samples were collected from the same boll positions at the same time.The same method was used to detect the expression level of GhAlaRP,GhAnnexin and GhEXPA in the control YZ-1,the transgenic lines and the alarp mutant lines.The qRT-PCR primers for GhAlaRP, GhAnnexin and GhEXPA are shown in Table S1.GhUBQ7 was selected as an internal reference gene.In each case three independent biological replicates were used for expression analysis.

2.4.Vector plasmid construction and cotton transformation

The coding region of GhAlaRP was amplified and cloned into pGWB17 and pA35HK vector by the Gateway technology [23,24]to construct the GhAlaRP-overexpressing (pGWB17-GhAlaRP) and GhAlaRP-RNAi (35HK-GhAlaRP) plasmid, respectively (Table S2).For genome editing, the sgRNA targets were predicted using the online software (http://cbi.hzau.edu.cn/cgi-bin/CRISPR) based on the G.hirsutum genome [25-28].Among the predicted target sites, the one (5′-GTTTGTTGTGT CCGGGACCA-3′) located at exon and simultaneously targeting the two homoeologs of GhAlaRP, GhAlaRP-A (Gh_A09G1166) and GhAlaRP-D (Gh_D09G1172) [29], was selected for constructing the CRISPR/Cas9 vector as previously described [30].Agrobacterium (strain LB4404) harboring the pGWB17-GhAlaRP, pA35HK-GhAlaRP or 35S-Cas9-AtU6-GhAlaRP-sgRNA vector was used to infect cotton hypocotyls.Callus induction and plant regeneration were performed according to the published protocol [31].

2.5.Subcellular localization and co-localization analysis

To investigate the localization of the GhAlaRP protein, the pGWB5-GhAlaRP-GFP vector was generated by inserting the coding region of GhAlaRP into the pGWB5 vector using the Gateway technology [23,24](Table S2).The pGWB5-GhAlaRPGFP plasmid was then transformed into Agrobacterium (strain GV3101).Three-week-old tobacco leaves were infiltrated with Agrobacterium.Subcellular localization was examined with a confocal microscope(Leica TCS SP5)at 2-4 days after infiltration.To further verify colocalization of GhAlaRP with ERs,the RFP-HDEL vector with a RFP tag and the pGWB5-GhAlaRP-GFP vector with a GFP tag were separately transformed into Agrobacterium (strain GV3101).Tobacco leaves were agroinfiltrated with a mixture of the two vectors (the volume ratio of pGWB5-GhAlaRP-GFP and RFP-HDEL was 1:1, OD600= 0.5).Subcellular localization was examined with a confocal microscope (Leica TCS SP5) after 2-4 days infiltration.

2.6.Yeast two hybrid assay and bimolecular fluorescence complementation (BiFC) assay

The coding sequence of GhAlaRP was cloned into the pGBKT7 vector(Clontech)for constructing the bait vector(Table S2).A prey library of cotton fiber was constructed by fusing cDNAs in the pGADT-7 vector (Clontech) (Table S2).The yeast twohybrid assay was performed according to the manufacturer’s protocol (Clontech) and the proteins encoded by the cDNA library of cotton fiber were screened for their interactions with GhAlaRP.Briefly, the plasmids of positive clones were screened and extracted; the positive plasmid containing cDNA sequence was sequenced.Sequences were confirmed using Blastn search in the upland cotton genome.The full length of the interacting genes was fused in the pGADT7 or pGBKT7 vector (Clontech), and co-transformed into Saccharomyces cerevisiae Y2HGold yeast cells to test their interaction.

The cDNAs encoding GhAlaRP and interacting proteins(GhAnnexin and GhEXPA) were cloned into BiFC constructs using the ClonExpress Multis One Step Cloning Kit.The CDS sequence of GhAlaRP was amplified and cloned into the BamH I sites of the pSAT1-nEYFP vector to form GhAlaRP-nEYFP(Table S2).The coding sequences of GhAnnexin and GhEXPA were amplified and cloned into the BamH I sites of the pSAT1-cEYFP vector to generate GhAnnexin-cEYFP and GhEXPA-cEYFP, respectively.Subsequently, the BiFC constructs were transferred into Agrobacterium tumefaciens strain GV3101 and transiently expressed in tobacco leaves via Agrobacterium injection [32].The fluorescence of YFP was assayed using a confocal laser-scanning microscope (Leica TCS SP5).

2.7.Determination of fiber quality

The plants(both transgenics and controls)used in phenotyping were grown in the experimental fields at Shihezi University (Shihezi, Xinjiang, China) and Sanya (Hainan, China).Standard cotton field management was applied to all plants.To minimize the effect of flowering time and boll position on fiber quality, we sampled mature bolls at the same time from the 1st and 2nd nodes of the middle fruit branches of cotton plants.After ginning, sample 20 g fiber was prepared for each replicate and analyzed for fiber quality using the High Volume Instrument (HVI) systems by the Center of Cotton Fiber Quality Inspection and Testing, Chinese Ministry of Agriculture (Anyang, Henan, China).Three biological replicates were assayed for each line.

2.8.Analysis of transcriptome data and accession numbers

The transcriptome data of different tissues of the G.hirsutum cultivar TM-1 were downloaded from the NCBI Sequence Read Archive (SRA: PRJNA248163, http://www.ncbi.nlm.nih.-gov/sra/?term=PRJNA248163) [29].Fragments per kilobase of transcript per million mapped reads(FPKM)were used in estimation of gene expression level[33].The heatmap was generated using the HemI 1.0 software [34].

The sequence data from this article can be found in the Cotton Functional Genomics Database or GenBank under the following accession numbers: GhAlaRP-A (Gh_A09G1166),GhAlaRP-D (Gh_D09G1172), GrAlaRP (Gorai.006G138900), GaAlaRP (Cotton_A_07799), TcAlaRP (XP_007028878), PtAlaRP(XP_006373772.1), GsAlaRP (KHN26345.1), MtAlaRP(XP_013467503.1),GhAnnexin(NM_001327088.1(Gh_D11G2184))and GhEXPA (MG000819.1 (Gh_A10G2323)).

3.Results

3.1.GhAlaRP is highly expressed in rapid elongating cotton fibers

GhAlaRP was predominantly expressed in developing fibers and very lowly expressed in root,stem,leaf and whole flower(Fig.1).In the 6-24 DPA fibers, GhAlaRP showed the highest expression level at 6 DPA, and then gradually decreased with the development of cotton fibers (Fig.1).This expression pattern of GhAlaRP in developing fibers was consistent with the previous Northern blot result [19].The high expression level of GhAlaRP in the early stages of fiber development, particularly in the fiber rapid elongation stage, suggested a role of GhAlaRP in fiber cell elongation.

Fig.1–Expression patterns of GhAlaRP in different tissues of cotton.R,root;S,stem;L,leaf;F,whole flower;6–24,fibers of 6, 12, 18, 24 days post anthesis.Error bars represent the standard deviation of triplicate experiments.GhUBQ7 was used as an internal control.

3.2.Phylogenetic analysis of AlaRP from different plant species

The G.hirsutum genome contains a pair of homoeologous GhAlaRP, i.e.GhAlaRP-A (Gh_A09G1166) and GhAlaRP-D (Gh_D09G1172), with identical amino acid sequences.Using GhAlaRP as a query, we searched its homologs in the two diploid cotton species, G.arboreum and G.raimondii, and other plant species.As expected, a single copy of AlaRP (GaAlaRP and GrAlaRP) was found in each diploid cotton species.The amino acid sequences of GaAlaRP and GrAlaRP are identical to that of GhAlaRP (Fig.2A).We also found a single copy of AlaRP in four other plant species (Populus trichocarpa, Glycine soja, Medicago truncatula, and Theobroma cacao) (Fig.2A).Based on phylogenetic analysis, the cotton AlaRP proteins were closely related to TcAlaRP, the AlaRP from Theobroma cacao, and were divergent from the AlaRP from other three species (Fig.2B).

3.3.Suppression of GhAlaRP inhibits fiber elongation

To explore the role of GhAlaRP in cotton fiber elongation, we generated transgenic GhAlaRP-RNAi and GhAlaRPoverexpression cottons.In total, we obtained thirteen independent GhAlaRP-RNAi lines and fifteen independent GhAlaRP-overexpression lines.The three independent GhAlaRP-RNAi and GhAlaRP-overexpression lines with the lowest and the highest expression level of GhAlaRP, respectively, were selected and preceded to the T3generation for further characterization and phenotyping (Figs.S1, S2).Compared with the control YZ-1, the three independent GhAlaRP-overexpression lines had no significant difference in the length of mature fibers (Fig.3A, B).However, the three independent GhAlaRP-RNAi lines had a significantly decreased fiber length (reduced by 2.66-4.45%) (Fig.3A, B; Table S3).These results suggest that inhibiting the expression of GhAlaRP affected fiber elongation and shortened the length of cotton fibers.

To further confirm the repressing function of GhAlaRP in cotton fiber elongation, we generated a transgenic cotton plant (named n1) with mutated GhAlaRP-A and GhAlaRP-D using the genome editing technology.Four types of editing were found in n1(T0), a single base insertion (+1 bp) and three different length of deletion (3, 13, and 14 bp) (Fig.S3).In the T1generation, all 17 plants analyzed were found to be edited in the target sites (Fig.S4; Table S4).Among them, n1-1, n1-6, and n1-16 contained the same editing types, n1-7 and n1-11 contained the same editing types, n1-10 and n1-17 contained the same editing types.Three plants (n1-7, n1-11, and n1-12) did not contain the exogenous T-DNA insertion (based on the absence of both NPTII and GFP genes) (Fig.S4; Table S4) and were selected for further analysis and phenotyping because their mutations in GhAlaRP would be stable.The expression level of GhAlaRP was significantly downregulated in these three T1plants compared with the control YZ-1 (Fig.S5).The fiber length of these three T1plants was significantly shorter than that of YZ-1 (reduced by 6.94-9.84%) (Fig.3C, D; Table S5).This result was consistent with the fiber phenotype observed in the GhAlaRP-RNAi lines, pro-viding strong evidence for the involvement of GhAlaRP in reg-ulation of cotton fiber elongation.

3.4.Subcellular localization of GhAlaRP

In order to understand the subcellular localization of GhAlaRP, a pGWB5-GhAlaRP fusion expression vector was constructed and transformed into Agrobacterium tumefaciens GV3101.After transient expression of the vector in tobacco leaves, the fluorescence of GFP was observed under confocal laser microscopy, which showed distribution in cell membrane and nucleus as well as in endoplasmic reticulum (ER)(Fig.4A).

To verify co-localization of GhAlaRP with ER, Agrobacterium tumefaciens (GV3101) containing an ER-marker RFP-HDEL and a GFP reporter pGWB5-GhAlaRP-GFP were co-injected into tobacco leaves that were then observed under laser confocal microscopy after 2-4 days infiltration.Both RFP and GFP were observed in ER, confirming localization of GhAlaRP in ER (Fig.4B, C).

3.5.GhAlaRP interacts with GhAnnexin and GhEXPA

To know whether GhAlaRP interacts with other proteins and to identify the proteins interacting with GhAlaRP in fiber cells, we used GhAlaRP protein as a bait to screen a yeast twohybrid library transformed with a cDNA library prepared from developing fibers.Based on selection and sequencing of positive clones, we identified 30 candidate proteins interacting with GhAlaRP (Table S6).

Fig.2–Sequence alignment and phylogenic relationship of AlaRP proteins.(A)Alignment of amino acid sequences of AlaRP from cotton and other plants.The black underlines indicate the two transmembrane domains(the 7th–28th amino acids,the 43rd–65th amino acids) of AlaRP.The black asterisks indicate the alanine residues.(B) Phylogenic relationship between GhAlaRP and other AlaRP proteins.The AlaRP proteins used are GhAlaRP-A (G.hirsutum, Gh_A09G1166), GhAlaRP-D (G.hirsutum, Gh_D09G1172), GrAlaRP (G.raimondii, Gorai.006G138900), GaAlaRP (G.arboretum, Cotton_A_07799), TcAlaRP(Theobroma cacao,XP_007028878),PtAlaRP(Populus trichocarpa,XP_006373772),GsAlaRP(Glycine soja,KHN26345)and MtAlaRP(Medicago truncatula,XP_013467503).The numbers on the tree branches represent bootstrap confidence values as Bootstrap is 1000; the scale bar represents genetic distance.

Among these 30 candidates, two proteins (Annexin and expansin (EXPA)), whose homologous genes have been reported to be involved in the development of cotton fibers were selected for further investigation [11,18,35,36].GhAnnexin (Gh_D11G2184), an annexin involved in calcium signal transduction, and GhEXPA (Gh_A10G2323), an expansin protein associated with cell wall, were used in the yeast twohybrid assay and BIFC verification.Yeast cells cotransformed with BD-GhAlaRP and AD-GhAnnexin or BDGhAlaRP and AD-GhEXPA could grow on the selective medium SD-Leu-Ade-Trp-His (X-α-gal + ABA) (Fig.5A), suggesting that GhAlaRP could bind to GhAnenxin and GhEXPA.

To confirm their interactions in vivo, we used BiFC to identify the interaction localization in tobacco leaf cells.The BiFC assay is based on the formation of a fluorescent complex comprising of two fragments of YFP, which are brought together by the association of two interacting proteins fused to the YFP N/C-terminals [32].When GhAlaRP-nEYFP was co-infiltrated with GhAnnexin-cEYFP, fluorescence was observed in the leaf cells, and when GhAlaRP-nEYFP was coinfiltrated with GhEXPA-cEYFP, the yellow fluorescence was also observed in tobacco leaf cells (Fig.5B).The results of yeast interaction and BIFC indicated that GhAlaRP could interact with GhAnnexin and GhEXPA, suggesting that GhA-laRP might regulate the elongation of cotton fiber cells by regulating membrane-associated proteins and cell wall relaxation-related proteins.

3.6.Downregulation of GhAlaRP suppressed the expression levels of GhAnnexin and GhEXPA in cotton fibers

GhAnnexin belongs to the Annexin protein family [37,38].The upland cotton genome contains 25 Annexin genes, which could be divided into three subfamilies (Fig.6A).GhAnnexin interacting with GhAlaRP belongs to subfamily I.The coding sequence of this gene is 951 bp in length and encodes 316 amino acids.The protein contains four Annexin repeat domains (IPR018502) (Fig.6B).RNA-seq data analysis found that GhAnnexin has the highest transcriptional level at the rapid fiber elongation stage (5 and 10 DPA) (Fig.6C), indicat-ing that this gene is involved in the elongation process of cot-ton fibers.Compared with the control YZ-1, the GhAlaRP-RNAi lines had a significantly decreased expression level of GhAnnexin in 6 DPA fibers (Fig.6D).Similar results were observed in all three alarp mutant T1plants (Fig.6E).These results sug-gest that GhAnnexin is co-suppressed with GhAlaRP in elonga-tion fibers.

Previous studies have shown that the elongation of cotton fiber cells was closely related to the expansin of cell wall, in which EXP proteins play an important role [18,36].Based on BlastP search against the upland cotton genome [29], we identified 77 EXPs that could be clustered into five subfamilies.GhEXPA was located in subfamily II (Fig.7A).The coding sequence of GhEXPA is 777 bp in length and encodes 258 amino acids.As typical expansins, GhEXPA contains an Expansin/Pollen allergen_DPBB domain (IPR007112) and an Expansin/Cellulose binding like domain (IPR007117) (Fig.7B).Base on RNA-seq data, among the 12 GhEXPAs of the subfamily II, GhEXPA had the highest level of transcription in 5 and 10 DPA fibers (Fig.7C), suggesting a role of GhEXPA in the elongation process of cotton fiber cells.Similar to GhAn-nexin, the expression level of GhEXPA in the 6 DPA cotton fibers was significantly lower in both the GhAlaRP-RNAi lines and the three alarp mutant T1plants than in the control YZ-1 (Fig.7D, E).These results suggest a co-expression relation-ship between GhAnnexin, GhEXPA and GhAlaRP.

Fig.3–Fiber phenotype of the transgenic cotton lines and alarp lines.(A) Phenotype of mature fibers from the GhAlaRPoverexpressing lines(O3,O6,and O14),the GhAlaRP-RNAi lines(R7,R16,and R20)and the control YZ-1.(B)Mature fiber length of the GhAlaRP-overexpressing lines (O3, O6, and O14), the GhAlaRP-RNAi lines (R7, R16, and R20) and the control YZ-1.The mature fiber length is measured using the HVI system.(C)Comparison of mature fibers from the alarp mutant lines(n1-7,n1-11, and n1-12) generated by genome editing and the control YZ-1.(D) Mature fiber length of the control YZ-1 and the alarp mutant lines(n1-7,n1-11,and n1-12).The mature fiber length is measured using the HVI system.Data are means±SD,and three replications were performed in each analysis.The letters above the columns indicate a statistically significant difference based on the Tukey’s multiple comparison test (P＜0.05) for the transgenic lines, the alarp mutant lines data compared to the control lines (YZ-1) under the normal conditions, respectively.

4.Discussion

Cotton fibers are seed trichomes developed from single epidermal cells.The development process of cotton fibers is not only regulated by complex regulatory networks involving many genes but also affected by environmental conditions [1,2].Apart from being reported to participate in the cold stress in cabbage [39], the function of AlaRP has not been reported in other plants.GhAlaRP is preferentially expressed in developing fibers with the highest expression level observed in the rapid elongating fibers (Fig.1).Mature fibers of both the GhAlaRP-RNAi lines and the alarp mutant plants generated by genome editing were shorter than those of the control YZ-1 (Fig.3), indicating that inhibiting the expression of GhAlaRP had a negative effect on the elongation of cotton fibers.

Annexins are calcium-dependent or independent membrane phospholipids and cytoskeleton binding proteins,which exist widely in most eukaryotic cells [37,38].Because of its ability to bind to calcium and lipid membranes,Annexin can be involved in signaling and membrane trafficking[40,41],including secretion, signal transduction, ion channel formation, and cytoskeletal interactions [42,43].Plant Annexins are concentrated in the dilated tip regions of polar growing cells,such as pollen tubes and root hairs,whose location corresponds to the directionality of secretion [44-46].Cell polarity amplification is regulated by the expression level and precise localization of Annexin [47].In cotton, AnnGh3 and AnnGh5 were highly expressed in cotton fibers during rapid elongation, and their expression levels gradually decreased with the development of cotton fibers, suggesting that they play an important role during the rapid elongation stage of cotton fiber cells [11].Inhibiting the expression of GhAnn2 resulted in shorter and thinner cotton fibers [35].AnxGb6 may interact with GbACT1 to regulate cotton fiber development [48].In this study, we found that GhAnnexin(Gh_D11G2184),which contains four Annexin repeat domains(IPR018502), was able to interact with GhAlaRP (Fig.6).Annexin repeat domains are thought to bind to Ca2+and play an important role in cell throughput, endocrine and signal transduction [49].RNA-seq data showed that GhAnnexin has a higher expression level in 5 and 10 DPA fibers than in other tissues(Fig.6).Importantly,GhAnnexin and GhAlaRP seem to be co-expressed as the expression level of GhAnnexin was significantly lower in the GhAlaRP-RNAi lines and the alarp mutant plants than in the control YZ-1 (Fig.6).These results suggested that GhAlaRP may interact with GhAnnexin to regulate the elongation of cotton fibers.In rice,the histidine-and alanine-rich protein (OsHARP) interacts with calreticulin, a major calcium-sequestering protein in ERs and with a variety of cellular functions.Transgenic rice plants with a downregulated level of OsHARP are consistently shorter than the control plants [50].

Fig.4–Subcellular localization of GhAlaRP.(A)Transiently expressed GFP-GhAlaRP was localized in plasma membrane(Scale bars,20 μm).The figures on the left,middle,and right are GFP fluorescence image,bright field image and merged image of the same leaf cell,respectively.(B and C)GFP-GhAlaRP was co-expressed with an ER marker(RFP-HDEL),in the different surfaces of tobacco leaf epidermal cells (Scale bars, 25 μm).

During cotton fiber elongation, the cell wall of fibers must be relaxed and expanded to allow cell growth, in which expansins play an essential role.Expansins contain two domains, Expansin/Pollen allergen_DPBB domain (IPR007112)and an Expansin/Cellulose binding like domain (IPR007117)(Fig.7).They function on cell wall to promote its extensibility by destroying the hydrogen bonds between cellulose microfilaments [51,52].Cotton genome contains many expansinencoding genes; some of them have been demonstrated to function in fiber development.For instance, GhEXP1 and GhEXP2 are specifically expressed during fiber development [18].Overexpressing GbEXPATR, an expansin gene unique to G.barbadense, promotes fiber elongation [36].In this study, we identified an expansin protein GhEXPA from cotton fiber cDNA library by yeast two-hybrid assay.RNA-seq data showed that GhEXPA and GhAlaRP had similar expression patterns.Both had the highest expression levels observed in the rapid elongating fiber cells (5 and 10 DPA) (Fig.7C).Similar to GhAnnexin, GhEXPA had a decreased expression level in the GhAlaRP-RNAi lines (Fig.7D).The direct interaction and coexpression relationship between GhAlaRP and GhEXPA indicate that both genes positively regulate fiber cell development.

Cotton fiber development is a complex and orderly process, and is accomplished through coordinated expression and interaction of numerous genes.Fiber elongation requires biosynthesis of cell membrane lipids, cell wall components and other related proteins, as well as proper trafficking of the newly synthesized substances to their final cellular destinations [6-11].Annexin localized on the membrane is involved in regulating the elongation of cotton fibers[10,35,48].EXPA proteins are synthesized in ERs, processed,modified and sheared to produce the mature forms of EXPA,which are then further processed, transported or secreted to the cell wall to perform its function [18,36,51].Interestingly, GhAlaRP contains two transmembrane domains(Fig.2).GhAlaRP was found to be localized not only on plasma membrane,but also in nucleus and ERs(Fig.4),speculate that GhAlaRP might be involved in transport of substances not only intercellularly but also intracellularly.Together with the observations of direct interaction and co-suppression between GhAlaRP and both GhAnnexin and GhEXPA, we speculate that GhAlaRP might act as a small protein partner,not only interacting with Annexin on the membrane to regulate fiber elongation, but also involving in the modification process of EXPA in ERs, from which the modified EXPA proteins, are further transported or secreted to the cell wall to regulate the elongation of cotton fiber cells.GhAlaRP is thus an essential component of the regulatory network including GhAnnexin and GhEXPA that contributes to cotton fiber elongation.

Fig.5–Yeast two-hybrid assays and BIFC of the interactions of GhAlaRP with GhAnnexin and GhEXPA.(A)Yeast two-hybrid assays of the interactions of GhAlaRP with GhAnnexin and GhEXPA.From top to bottom, the growth of the positive control Y2HGold (pGBKT7-53 + pGADT7-T), the negative control Y2HGold (pGBKT7-Lam + pGADT7-T, BD (pGBKT7)-GhAlaRP + AD(pGADT7), BD + AD-GhAnnexin (Gh_D11G2184), BD + AD-GhEXPA (Gh_A10G2323), Y2HGold (BD-GhAlaRP + AD-GhAnnexin(Gh_D11G2184)), and Y2HGold (BD-GhAlaRP + AD-GhEXPA (Gh_A10G2323)).Three dilutions (10-1, 10-2, and 10-3) of yeast liquid were spread on SD/-Leu-Trp-His-Ade (+X-α-gal-ABA) plate.(B) BiFC assay of GhAlaRP and two proteins (GhAnnexin(Gh_D11G2184) and GhEXPA (Gh_A10G2323)) in tobacco.Detection of the YFP fluorescence signal indicates protein–protein interaction.Scale bars, 20 μm.

Fig.6–Expression of GhAnnexin(Gh_D11G2184)is suppressed in the GhAlaRP-RNAi lines and the alarp lines.(A)Phylogenetic analysis of Annexin-encoding gene in G.hirsutum.A total of 25 Annexin genes from G.hirsutum were used to construct the phylogenetic tree using the MEGA 5.0 software.The Annexin gene interacting with GhAlaRP is underlined.(B) Schematic representation of the structure of GhAnnexin (Gh_D11G2184), which contains four Annexin repeat domains (IPR018502).(C)RNA-seq analysis of GhAnnexin genes in different tissues and organs of G.hirsutum.Yellow and blue indicate up- and downregulated genes, respectively.DPA, day post anthesis.(D) The expression level of GhAnnexin (Gh_D11G2184) in 6 DPA fibers of GhAlaRP-RNAi (T3) lines (R7, R16, and R20) and the control YZ-1.Data are means ± SD, and three replications were performed in each analysis.(E) The transcript levels of GhAnnexin (Gh_D11G2184) in fibers of the alarp(T1) lines(n1-7,n1-11,and n1-12) and the control YZ-1.Data are means ± SD, and three replications were performed in each analysis.

Fig.7–GhEXPA (Gh_A10G2323) expression is suppressed in the GhAlaRP-RNAi lines and the alarp lines.(A) Phylogenetic analysis of EXPs from G.hirsutum.A total of 77 EXPA-encoding genes from G.hirsutum were used to construct the phylogenetic tree using the MEGA 7.0 software.The EXPA interacting with GhAlaRP is underlined.(B) Schematic representation of the GhEXPA (Gh_A10G2323) protein structure, which contains an Expansin/Pollen allergen_DPBB domain(IPR007112) and an Expansin/Cellulose-binding-like domain (IPR007117).(C) RNA-seq analysis of GhEXPA genes in different tissues and organs of G.hirsutum.Yellow and blue indicate up-and down-regulation,respectively.DPA,day post anthesis.(D)Transcript levels of GhEXPA(Gh_A10G2323)in 6 DPA fibers of GhAlaRP-RNAi(T3)lines(R7,R16,and R20)and the control YZ-1.Data are means±SD,and three replications were performed in each analysis.(E)Transcript levels of GhEXPA(Gh_A10G2323)in fibers of the alarp (T1) lines (n1-7, n1-11, and n1-12) and the control YZ-1.Data are means ± SD, and three replications were performed in each analysis.

Declaration of competing interest

Authors declare that there are no conflicts of interest.

Author contributions

Jie Sun and Qian-Hao Zhu conceived and designed the experiments.Shouhong Zhu performed the experiments and analyzed the data.Yanjun Li, Xinyu Zhang, Feng Liu, Fei Xue,Yongshan Zhang, and Zhaosheng Kong contributed reagents/materials/analysis tools.Shouhong Zhu, Jie Sun,and Qian-Hao Zhu drafted and wrote the manuscript.

Acknowledgments

We are thankful to Professor Guixian Xia (Chinese Academy of Sciences, China) for insightful comments on the manuscript and helpful discussions.This work was supported by the National Key Research and Development Program of China (2016YFD0101900, 2017YFD0100200), the Genetically Modified Organisms Breeding Major Project of China(2016ZX08005-005).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2020.08.007.