The nuclear export receptor OsXPO1 is required for rice development and involved in abiotic stress responses

Qiufei Peng,Jieyu Qiu,Xintong Li,Xuezhong Xu,Xinxing Peng,Guohui Zhu,b,*

a Guangdong Laboratory for Lingnan Modern Agriculture,College of Life Sciences,South China Agricultural University,Guangzhou 510640,Guangdong,China

b Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms,Guangzhou 510640,Guangdong,China

Keywords:Exportin 1 Nucleocytoplasmic transport Plant development Abiotic stress

ABSTRACT The transport of proteins to and from the nucleus is necessary for many cellular processes and is one of the ways plants respond to developmental signals and environmental stresses.Nucleocytoplasmic trafficking of proteins is mediated by the nuclear transport receptor(NTR).Although NTR has been extensively studied in humans and Arabidopsis,it has rarely been identified and functionally characterized in rice.In this study,we identified exportin 1 in rice(OsXPO1)as a nuclear export receptor.OsXPO1 shares high protein identity with its functional homologs in Arabidopsis and other organisms.OsXPO1 localized to both the nucleus and the cytoplasm,directly interacted with the small GTPases OsRAN1 and OsRAN2 in the nucleus,and mediated their nuclear export.Loss-of-function osxpo1 mutations were lethal at the seedling stage.Suppression of OsXPO1 expression in RNA interference lines produced multifaceted developmental defects,including arrested growth,premature senescence,abnormal inflorescence,and brown and mouth-opened spikelets.Overexpression of OsXPO1 in rice reduced plant height and seed-setting rate,but increased plant tolerance in response to PEG-mimicked drought stress and salt stress.These results indicate that OsXPO1 is a nuclear export receptor and acts in regulating plant development and abiotic stress responses.

1.Introduction

In eukaryotic cells,the nuclear envelope is a selective barrier separating the nucleoplasm from the cytoplasm.Nuclear pore complexes(NPCs)provide gateways for the exchange of proteins and macromolecular complexes between the nucleus and the cytoplasmic compartment[1,2].The nucleocytoplasmic trafficking of proteins is mediated by the nuclear transport receptor(NTR).NTR belongs to the importin β family proteins and is classified as importin(for nuclear import)and exportin(for nuclear export).More than 14 importin β-like NTRs have been identified in the yeast genome,20 members in vertebrates,and 18 members in Arabidopsis[2,3].

Chromosome region maintenance 1(CRM1)is one of the most important and best characterized exportins[4].CRM1 is present in all eukaryotic cells.It was originally identified as a chromosomal mutation in the fission yeast Schizosaccharomyces pombe,and was later renamed exportin 1(XPO1)for its role in protein nuclear export[5,6].During nuclear export,CRM1/XPO1 binds with RanGTP(a GTP-bound Ran protein)and its export cargo to form a ternary complex in the nucleus.After translocation from the nucleus,the complex is dissociated in the cytoplasm to release cargo proteins[2,4].More than 1050 proteins in humans and 700 proteins in yeast cells have been identified as CRM1 cargoes,including proteins associated with translation,ribosome biogenesis,mRNA degradation,vesicular transport,autophagy,and cellular regulatory circuits[7].In addition to its role in nuclear export,CRM1 plays an essential role in mitosis by recruiting partner proteins in the complex connected to the kinetochore at centromeres,thereby promoting proper chromosomal segregation[8].

Arabidopsis AtXPO1 is a homolog of CRM1 in humans and yeast and mediates the nuclear export of proteins containing leucinerich nuclear export signal(NES)[9].The Arabidopsis genome encodes two homologous proteins,XPO1A and XPO1B,which regulate plant development and stress responses.A single xpo1a or xpo1b mutant appears phenotypically normal,whereas a double mutation is lethal to gametophytes[10].XPO1A and XPO1B both mediate the nucleocytoplasmic trafficking of XIW1,an XPO1-interacting WD40 protein 1,and thereby regulate plant responses to abscisic acid(ABA)and salt stress[11,12].Interestingly,only xpo1a/hit2(heat intolerant 2)mutant,but not xpo1b,showed defects in basal thermotolerance,indicating that XPO1A could have its own cargoes and specific functions that are important for plant survival under heat stress[13].

In addition to XPO1,many importin β-like NTRs have been identified and functionally characterized in Arabidopsis.The Arabidopsis exportin 5/HASTY(HST)acts in miRNA biogenesis[14,15]and exportin-T/PAUSED(PSD)is associated with the export of tRNA[16,17].However,there is only limited information available for rice.In this study,we identified an Arabidopsis XPO1 homolog in rice.The rice genome encodes only one copy of XPO1,OsXPO1,which has conserved importin β-like domains and directly interacts with the OsRAN protein.Our results suggest that OsXPO1 is necessary for plant development and abiotic stress response.

2.Materials and methods

2.1.Plant materials and growth conditions

Zhonghua 11 rice(Oryza sativa L.,as wild type,WT)was used to generate OsXPO1-knockout,RNA interference(RNAi),and overexpression plants.Germinated seeds were cultivated in Kimura B complete nutrient solution and grown in a greenhouse with a day/night temperature of 30–35 °C/23–26 °C and a photoperiod of 12 h/12 h.Pot planting employed the same greenhouse conditions,with each pot containing 3–4 plants.Field planting was under natural conditions in spring 2021(April-July)in the field of the experimental farm of South China Agricultural University in Guangzhou,China.For a stress experiment,four-leaf-stage seedlings were grown in an environmental chamber at temperatures of 30/25°C(day/night)and treated with 20%PEG6000 and 200 mmol L-1NaCl for various lengths of time.

2.2.Gene identification and sequence analysis

Rice XPO1 gene was identified by a BLAST search in the NCBI database(https://blast.ncbi.nlm.nih.gov/Blast.cgi)using the previously reported amino acid sequences of Arabidopsis XPO1[9].Amino acid multiple sequence alignments were constructed with Clustal X[18].A phylogenetic tree of XPO1 proteins was constructed using the neighbor-joining method in MEGA(version 6.06)[19].The Pfam domain program(https://pfam.xfam.org)was used to perform sequence alignments.

2.3.Vector construction and plant transformation

OsXPO1 knockout plants were constructed using the CRISPR/Cas9 system.The sgRNA targeting OsXPO1 was designed with CRISPR-GE[20].CRISPR/Cas9 binary construct preparation followed Ma et al.[21].To generate the OsXPO1 RNAi construct,a 909-bp coding sequence of OsXPO1(from 1142 to 2050 bp)was forward-and reverse-amplified and then inserted into the pYLRNAi vector.For overexpression of OsXPO1,the full-length OsXPO1 coding sequence was amplified and introduced into the pYL-OX vector driven by the ubiquitin promoter(pYL-RNAi and pYL-OX vectors were provided by Yao-Guang Liu,South China Agricultural University).All the constructs were introduced into Agrobacterium tumefaciens strain EHA105 and transformed into Zhonghua 11 through Agrobacterium-mediated transformation.The primers used for vector construction are listed in Table S1.

2.4.Quantitative real-time PCR(qRT-PCR)

Total RNA was isolated with a MiniBEST Plant RNA Extraction Kit(TaKaRa,Tokyo,Japan).The extracted RNA was then treated with DNase I and used as templates for first-strand cDNA synthesis with a PrimeScript RT reagent Kit(TaKaRa).qRT-PCR was performed with a SYBR Green PCR Master Mix(Accurate Biology,Changsha,Hunan,China),with three biological repeats.Rice Actin1(LOC_Os03g50885)was used as an internal reference gene to quantify cDNA abundance.Relative expression levels were calculated by the 2-ΔΔCTmethod[22].The primers used for qRT-PCR are listed in Table S1.

2.5.Transient expression and green fluorescent protein(GFP)assays

For subcellular localization,the coding sequence of OsXPO1 was amplified and cloned into pCAMBIA1300-GFP driven by the 35S promoter,and the plasmid was transformed into Nicotiana benthamiana epidermal cells.For assays of the nucleocytoplasmic trafficking of proteins,the coding sequences of OsRAN1 and OsRAN2,the cytosolic fructose-1,6-bisphosphatase(OsFBP)gene(LOC_Os01g64660),and the NLS-OsFBP-NES fusion protein,where the nuclear localization signal(NLS)sequence is from SV40 large T antigen and the NES sequence is from the HIV-1 protein Rev[9],were cloned into pCAMBIA1300-GFP driven by the 35S promoter.The plasmids were transformed into rice protoplast or epidermal cells of N.benthamiana.The transformed rice protoplasts or N.benthamiana cells were treated with or without 40 nm leptomycin B(LMB)for 4 h.GFP signal was visualized under a confocal microscope(Leica sp5,Heidelberg,Germany)one day after transformation into protoplast or two days after infiltration into N.benthamiana cells.DAPI staining was used as a nuclear marker.The primers used for vector construction are listed in Table S1.

2.6.Yeast two-hybrid assays

The coding sequence of OsXPO1 was cloned into a pGAD-T7 vector to generate the AD-OsXPO1 plasmid,and the coding sequences of OsRAN1 and OsRAN2 were cloned into pGBK-T7 to generate BDOsRAN1 and BD-OsRAN2 plasmids,respectively.The AD and BD constructs were co-transformed into the yeast strain Y2HGold(Clontech,Mountain View,CA,USA).A series of diluted cultures were spotted on SD plates lacking Trp and Leu or lacking Trp,Leu,His,and Ade.The plates were incubated at 30 °C for 3 days to observe yeast growth.The primers used for vector construction are listed in Table S1.

2.7.Bimolecular fluorescence complementation(BiFC)assays

The coding sequence of OsXPO1 was cloned into the binary BiFC vector pSPYNE173 to produce the OsXPO1-YFPNplasmid.The coding sequences of OsRAN1 and OsRAN2 were cloned into pSPYCE(M)to produce RAN1-YFPCand RAN2-YFPCplasmids,respectively.The constructs were introduced into Agrobacterium strain GV3101 and then infiltrated into N.benthamiana leaves.YFP signal was observed by confocal microscopy-two days after infiltration.The primers used for vector construction are listed in Table S1.

3.Results

3.1.Identification of XPO1 homologs in rice

Fig.1.Protein identity and phylogenetic analysis of XPO1 in various species.(A)Gene and protein similarity of OsXPO1 with its Arabidopsis homologs.(B)Phylogenetic tree of XPO1 in five species.The sequences from rice(OsXPO1,XP_015632087.1),Arabidopsis thaliana(XPO1A,NP_197204.1 and XPO1B,NP_566193.2),humans(hCRM1,NP_003391.1),Caenorhabditis elegans(XPO-1,NP_741567.1),and Schizosaccharomyces pombe(SpCrm1p,NP_001342911.1)were aligned.Bootstrap values were estimated(with 1000 replicates)to assess the relative support for each branch,and bootstrap values were labeled with cutoff=20.Protein identities(%)to rice OsXPO1 sequence are listed.(C)Predicted domains in OsXPO1.IBN_N,importin-β N-terminal;Xpo1,exportin 1-like protein;CRM1_C,CRM1 C-terminal.Asterisk represents LMB-sensitive cysteine.

To identify XPO1 candidate genes in the rice genome,we performed a BLAST search using Arabidopsis XPO1 protein sequences[9].Only one candidate,OsXPO1(Os03g0858100),was identified in the NCBI database.DNA sequencing confirmed that the OsXPO1 gene had a 3213-bp-long coding sequence,corresponding to a protein of 1070 amino acid residues.OsXPO1 showed 82.7%and 80.5%protein identities with Arabidopsis XPO1A and XPO1B,respectively(Fig.1A).OsXPO1 also showed high(43.2%–48.9%)protein identity with its functional homologs in human,C.elegans,and S.pombe[6,23,24].A phylogenetic tree was constructed using MEGA based on XPO1 protein sequence homologs and is displayed in Fig.1B.Phylogenetic analysis also showed that XPO1 is evolutionarily conserved from lower to higher plants.XPO1 proteins were classified into two clusters in higher plants(Fig.S1A),suggesting divergence between monocotyledons and dicotyledons.

Sequence analysis using the Pfam domain program showed that OsXPO1 has an importin-β N-terminal domain(IBN_N),an exportin 1-like protein domain(Xpo1),and a CRM1 C-terminal domain(CRM_C;Figs.1C,S1B),which is similar to that of the functional XPO1 proteins.A characteristic cysteine residue was found at position 528 of OsXPO1,which determines sensitivity to leptomycin B(LMB)and is conserved in importin β family proteins(Figs.1C,S1C)[9].

3.2.Subcellular localization and expression of OsXPO1

To investigate the subcellular localization of OsXPO1,35S:OsXPO1-GFP was transiently expressed in N.benthamiana cells.OsXPO1-GFP fluorescence was detected in both the cytoplasm and the nucleus(Fig.2A),indicating the nucleocytoplasmic localization of the OsXPO1 protein.The pattern of subcellular localization of OsXPO1 is consistent with that of Arabidopsis XPO1A protein[25].

Quantitative RT-PCR analysis showed that OsXPO1 was ubiquitously expressed in leaves,roots,stems,leaf sheaths,and panicles.The transcription levels of OsXPO1 in reproductive tissues were higher than those in seedling tissues,with the highest expression in panicles(Fig.2B),suggesting the potential role of OsXPO1 in regulating plant development.The expression of OsXPO1 was induced by stress and ABA treatments.The mRNA levels of XPO1 in the leaves increased by respectively 6.1,2.5,and 5.4 times after four hours of PEG,NaCl,or ABA treatment(Fig.2C).

3.3.OsXPO1 interacts with and facilitates nuclear export of rice OsRAN proteins

In humans and yeast,XPO1 has been shown[2,4]to interact with RAN protein(a small GTPase)to mediate the nuclear export of specific cargoes.To investigate the possibility of rice OsXPO1 as a nuclear export receptor,we tested the interaction between OsXPO1 and OsRAN proteins(OsRAN1 and OsRAN2).To this end,OsXPO1A-YFPN,along with OsRAN1-YFPCor OsRAN2-YFPC,were co-transformed into N.benthamiana leaves for BiFC assays.Fluorescence detection showed that the YFP signal was distributed primarily in the nucleus,with slight fluorescence in the cytosol(Fig.3A).Yeast two-hybrid assays showed that OsXPO1 physically interacts with rice OsRAN1 and OsRAN2(Fig.3B).These results suggested that OsXPO1 directly interacts with rice OsRAN protein

in vivo and in vitro.

To test whether OsXPO1 is essential for nuclear export of OsRAN proteins,35S:GFP-OsRAN1 and 35S:GFP-OsRAN2 were transiently expressed in either rice protoplasts or N.benthamiana leaves.GFP fluorescence indicated that OsRAN1 and OsRAN2 were located in both the nucleus and the cytosol,predominantly in the nucleus(Fig.4A,B).These results are consistent with previous reports[26,27].Given that osxpo1 mutations are lethal under normal growth conditions(Fig.5B),the specific inhibitor of XPO1/CRM1,leptomycin B(LMB)[28],was used to test the relevance of the OsXPO1 function for the nuclear export of OsRAN proteins.After LMB treatment,GFP fluorescence apparently accumulated in the nucleus(Fig.4A,B).These results suggested that nuclear export of OsRAN1 and OsRAN2 is OsXPO1-dependent.

We used rice cytosolic fructose-1,6-bisphosphatase(OsFBP)to verify the general function of OsXPO1 in protein nuclear export.To this end,the plasmids of GFP-OsFBP and GFP-OsFBP fused with NLS and NES sequences were constructed(Fig.S2),and then transiently expressed in rice protoplasts or N.benthamiana leaves.GFP fluorescence was detected only in the cytosol of GFP-OsFBP(Fig.S2).For the GFP-NLS-OsFBP-NES fusion protein,it was distributed in both the nucleus and the cytosol,and almost in the nucleus after LMB treatment(Fig.S2).These results suggest that OsXPO1 is essential for the nuclear export of proteins containing NES sequences.

3.4.Impaired OsXPO1 expression leads to developmental defects in rice

The Arabidopsis XPO1 gene is essential for plant development.The xpo1a xpo1b double mutant exhibits a lethal gametophytic phenotype[10].To identify the potential role of OsXPO1 in plant development,we used the CRISPR/Cas9 system to generate OsXPO1 knockout mutants.No homozygous lines of OsXPO1 mutation were obtained in the T0generation.A homozygous mutant osxpo1-1 was obtained from the T1heterozygous lines that lacked 6 bp in the coding sequence of OsXPO1,leading to deficiency of respectively Met and Trp at positions 49 and 50 of OsXPO1(Fig.5A).However,the osxpo1-1 homozygous line did not grow normally,showing pale-green leaves and severe dehydration phenotypes,and eventually died at the two-leaf growth stage(Fig.5B).

Fig.2.Subcellular localization and expression of OsXPO1.(A)Subcellular localization of OsXPO1.35S:OsXPO1-GFP plasmids were transiently expressed in N.benthamiana for 2 d.The GFP signals were observed by confocal microscopy.Scale bars,50 μm.(B)Transcriptional expression of OsXPO1 in various organs.Four-leaf stage seedlings and flowering-stage reproductive plants were sampled for OsXPO1 expression analysis.(C)Expression of OsXPO1 in response to stresses and ABA treatment.Four-leaf stage seedlings were treated with 20% PEG6000,200 mmol L-1 NaCl,or 50 μmol L-1 ABA.The leaves were sampled 1 and 4 h after treatments.

Fig.3.Interaction between OsXPO1 and OsRAN proteins.(A)BiFC assays of OsXPO1 and OsRAN.OsXPO1-YFPN,along with OsRAN1-YFPC or OsRAN2-YFPC fusion proteins,were co-expressed in N.benthamiana,and YFP was imaged with a confocal microscope 2 d post-infiltration.Scale bars,50 μm.(B)Yeast two-hybrid assay of the interactions between OsXPO1 and OsRAN.The OsXPO1 protein is fused to the GAL4 activation domain(AD),and the OsRAN1 and OsRAN2 are fused to the GAL4 binding domain(BD).

We accordingly pivoted to generating OsXPO1 RNA interference(RNAi)plants.A 909-bp coding sequence of OsXPO1 was used as the targeted interference DNA and three independent OsXPO1-RNAi lines(T0generation)were obtained.In comparison with wild-type plants,the expression levels of OsXPO1 in the OsXPO1-RNAi lines decreased by approximately 49%,41%,and 97%(Fig.5C).OsXPO1-RNAi plants displayed multiple developmental defects,including severely impaired growth,premature senescence of the whole plant,abnormal inflorescence development,and brown and mouth-open spikelets(Fig.5D,E).The seed-setting rate of OsXPO1-RNAi was nearly zero,and no subsequent T1seeds were obtained.These results strongly indicate that OsXPO1 is essential for rice development and growth.

Fig.4.OsXPO1 facilitates nuclear export of OsRAN1 and OsRAN2 protein.35S:GFP-OsRAN1 and 35S:GFP-OsRAN2 plasmids were transiently expressed either in rice protoplasts for one day(A)or in the N.benthamiana leaves for 2 d(B).The transformed protoplasts and N.benthamiana leaves were incubated with or without 40 nm LMB for 4 h.The GFP signals were observed by confocal microscopy.DAPI staining was used as a nuclear marker.Scale bars,5 μm for(A)and 50 μm for(B).

Fig.5.Impairment of OsXPO1 expression results in developmental defects.(A)Generation of osxpo1 mutant by CRISPR/Cas9 system.Deleted bases in osxpo1 are indicated by dashed lines.The underlined CCA is the protospacer adjacent motif(PAM)sequence in sgRNA.(B)Phenotypes of the osxpo1 plants are described in(A).The 20-d-old heterozygous(Heter)and homozygous(Homo)lines of osxpo1 are shown.(C)Expression of OsXPO1 in OsXPO1-RNAi lines.(D,E)Developmental phenotypes of OsXPO1-RNAi lines.RNAi plants(left)displayed arrested growth and premature senescence(D),as well as brown and mouth-open spikelets(E).ZH11 plants(wild type,right)were used as controls.All plants are grown under greenhouse conditions in pots containing paddy soil.

3.5.Overexpression of OsXPO1 inhibits plant growth but increases stress tolerance

To further evaluate the effects of OsXPO1 on plant development and stress response,we generated OsXPO1 overexpression plants by constructing the full-length coding sequence of OsXPO1 under the control of the ubiquitin promoter.Several homozygous transgenic lines were obtained.Unexpectedly,the transcriptional expression of OsXPO1 was significantly increased in only one transgenic line(OsXPO1-OE-5)(Fig.6A).In comparison with wild-type plants,the plant height and seed-setting rate of OsXPO1-OE-5 were significantly reduced,and the number of effective tillers and 1000-kernel weight remained unchanged(Figs.6B–G,S3).Transgenic lines with slightly increased OsXPO1 expression(OsXPO1-OE-8 and OE-1)did not show statistical changes in agronomic traits in comparison with the wild type(Figs.6B–G,S3).These results suggested that maintaining the expression of OsXPO1 within a certain range is important for plant growth and development and that its overexpression may harm plants.

We also tested stress response using the OsXPO1-OE lines,given that OsXPO1 expression can be induced by abiotic stresses.Wildtype and OsXPO1 transgenic plants were treated with salt stress and PEG-mimicked drought stress and then maintained under normal culture conditions.Compared with wild-type plants,OsXPO1-OE-5 showed significantly increased tolerance to salt stress and drought stress.Almost all wild-type plants were lethal under salt stress,whereas most OsXPO1-OE-5 plants maintained green leaves(Fig.7).PEG-mimicked drought stress severely affected the leaf-wilting phenotypes of the wild type,whereas OsXPO1-OE-5 plants recovered vigorous growth after removal of stress treatment(Fig.7).

4.Discussion

4.1.OsXPO1 is an exportin protein in rice

NTR is essential for nucleocytoplasmic trafficking in eukaryotic cells,which determines the subcellular localization of macromolecules and participates in various cellular biological processes[2,3].However,few NTRs in rice have been identified and functionally characterized.OsImpβ1 is an importin β protein that can mediate nuclear import in rice and is specifically required for pollen tube elongation[29,30].CRD1,the rice ortholog of Arabidopsis HST,is essential for maintaining normal miRNA levels in plant cells[31].In this study,we identified OsXPO1 as an exportin in rice based on the following observations.First,OsXPO1 shares high(43.2%–82.7%)protein identity with XPO1/CRM1 proteins that have been characterized,including human CRM1,C.elegans XPO-1,S.pombe SpCrm1p,and Arabidopsis XPO1A and XPO1B[6,10,23,24].OsXPO1 harbors conserved domains similar to those of these proteins(Fig.1).Second,during nuclear export,XPO1 binds cooperatively with RanGTP in the nucleus to power the translocation of the XPO1-cargo-RanGTP complex through an NPC.Once it reaches the cytoplasm,the complex is dissociated to facilitate cargo release[2].In this study,OsXPO1 was localized to both the nucleus and the cytoplasm(Fig.2).Protein interaction assays showed that OsXPO1 interacted directly with OsRAN proteins,primarily in the nucleus with slight signals in the cytoplasm(Fig.3).Third,using a specific inhibitor of XPO1,we verified that OsXPO1 was essential for the nuclear export not only of OsRAN proteins,but of the protein containing NES sequence(Figs.4,S2).Taken together,these results are consistent with patterns of functional XPO1 proteins,indicating that the machinery of nuclear export of proteins is evolutionarily conserved in eukaryotes.

Fig.6.Overexpression of OsXPO1 affects rice development and yield.(A)Relative expression of OsXPO1 in OsXPO1-OE transgenic plants.(B)Phenotypes of OsXPO1-OE plants at reproductive stages.(C)Phenotypes of spikelets of OsXPO1-OE plants.(D–G)Effective tiller number(D),plant height(E),seed setting rate(F),and 1000-kernel weight(G)of OsXPO1-OE plants grown in the field.Rice plants were grown in a growth chamber in Kimura B nutrient solution at seedling stage and transferred to pots(B)or field(C–G)conditions at the four-leaf stage.Values are means±SD(n=3 for A and G;n=30 for D,E,and F).Asterisks indicate a significant difference compared with ZH11(Student’s ttest,P＜0.01).

Fig.7.Overexpression of OsXPO1 increased plant stress tolerance.Four-leaf stage of wild-type and OsXPO1 transgenic plants grown in the growth chamber were treated with 200 mmol L-1 NaCl for 3 d and allowed to recover for 3 d,or treated with 20% PEG for 20 d and allowed to recover for 3 d.

4.2.OsXPO1 is essential for rice development and growth

Regulation of nucleocytoplasmic trafficking is involved in many plant signaling pathways,including hormone response,abiotic and biotic stresses,and environmental changes[3,32,33].Owing to the involvement of NTR in nucleocytoplasmic trafficking,many NTR mutants exhibit a pleiotropic developmental phenotype.Arabidopsis HST/exportin 5 and PSD/exportin-T are indispensable for normal plant development.The hst and psd mutants showed multiple developmental defects[15,34].Our experiment showed that loss of function in osxpo1 plants is lethal at the seedling stage(Fig.5).The suppression of OsXPO1 expression in OsXPO1-RNAi plants led to defective pleiotropic phenotypes during the reproductive stage,including reduced growth,abnormal inflorescence,premature senescence,and decreased fertility.These phenotypes are very similar to those described for the hst and psd mutants of Arabidopsis.Although OsXPO1-RNAi produced seeds in the T0generation,the seed coat was brown and mouth-opened and the seeds were aborted(Fig.5).Interestingly,the phenotype of brown and mouth-opened spikelets has also been found in OsRAN2-RNAi plants[26].This finding is not surprising,given that OsXPO1 and OsRAN2 form a complex and function synergistically in the nuclear export of proteins[2].

Although our results indicate that OsXPO1 is essential for rice development and growth,overexpression of OsXPO1 in rice impaired plant development by reducing plant height and seed-setting rate(Fig.6).It has also been reported[27,35]that overexpression of wheat TaRAN1 and rice OsRAN1 in transgenic Arabidopsis and rice resulted in abnormal plant morphology,owing to the impairment of cellular mitosis and the auxin signaling pathway Together,these results suggest that NTR-dependent nucleocytoplasmic trafficking is important for plant development,acting possibly by regulating the timing and position of molecular components within the cell[32].

4.3.OsXPO1 is involved in stress response

In addition to affecting plant development,some NTRs also participate in stress responses by regulating the nucleocytoplasmic trafficking of specific proteins[3,33].Arabidopsis SAD2(sensitive to ABA and drought 2)encodes an importin β-domain family protein and mediates the nuclear import of MYB4,an R2R3-type transcription repressor.Loss-of-SAD2 mutations led to a decrease in MYB4 accumulation in the nucleus,increasing tolerance to UV-B radiation[36].Arabidopsis XPO1 mediates the nuclear export of XIW1,a positive regulator of ABA signaling,and thereby affects plant response to ABA and drought stress[11].In the present study,overexpression of OsXPO1 in rice increased plant tolerance to salt and drought stress(Fig.7).The results are similar to the description of OsRAN-overexpression plants.Overexpression of OsRAN1 and OsRAN2 affects plant development but increases plant abiotic tolerance,such as to cold stress[27,37].

Although our results indicate that OsXPO1 is important for plant development and stress response,the mechanism remains unknown.In humans and yeast,CRM1/XPO1 is reported[4,8]to be involved in two biological processes:nuclear export of target proteins and RNA,and relocation of target proteins in the nucleus.If OsXPO1 is involved in similar processes in rice,impaired OsXPO1 function could affect the transport of factors essential for cell division,plant development,and stress response.The specific cargoes of OsXPO1 remain to be identified and functionally characterized.

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.

CRediT authorship contribution statement

Qiufei Peng:Visualization,Methodology,Data curation,Writing–original draft.Jieyu Qiu:Visualization,Methodology,Writing–review & editing.Xintong Li:Visualization,Methodology.Xuezhong Xu:Conceptualization,Visualization,Methodology.Xinxiang Peng:Funding acquisition,Supervision.Guohui Zhu:Conceptualization,Writing–original draft,Funding acquisition,Supervision.

Acknowledgments

This work was supported by the National Key Research and Development Program(2020YFA0907600),the Laboratory of Lingnan Modern Agriculture Project(NZ2021004),the Natural Science Foundation of Guangdong Province(2020A1515010157),and the Science and Technology Program of Guangzhou(202102080499).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2022.06.008.