The Functional Study Of Lamin-like Protein OsNMCP1 In Regulating Drought Resistance And Root Growth In Rice

Rice(Oryza sativa L.)is one of the major food crops in the world.The extreme climates such as high temperature,cold and drought pose a serious of threats to food security.Due to severe shortage of fresh water resources and the low used efficiency of agricultural irrigation water,drought stress have developed to be one of the biggest problems for the agriculture.The advantageous root system architecture plays a key role in the drought avoidance mechanism by increasing water absorption and utilization.In our previous rice microarray analysis,Os NMCP1 was identified as drought-inducible gene.With the drought stress treatment and root traits measurement of Os NMCP1 overexpression(OE)plants and osnmcp1 mutant plants,we found that Os NMCP1 was involved in the regulation of root growth and drought resistance.The function of Os NMCP1 was further studied in this work.The lamin proteins are mainly localized at the nuclear periphery in human cells.We found that Os NMCP1 was localized in the nucleus by subcellular localization.Further more,immunofluorescence staining was performed in the roots of GFP-Os NMCP1 transgenic plants.Close observation of single cells under high magnification showed that Os NMCP1 was predominantly localized at the nuclear periphery.The results indicate that the lamin-like protein Os NMCP1 is localized at the nuclear periphery.The Os NMCP1 transcript level was increased both in the leaf and root tissues after ABA and drought stress treatments.The Os NMCP1-OE lines showed increased drought resistance at the seedling and the reproductive stages.Contrary to the overexpression results,the osnmcp1 mutant lines were hypersensitive to drought stress treatment at the seedling stage and the reproductive stage.These results suggest that Os NMCP1 has a positive effect on drought resistance.We further investigated the root traits at the seed maturation stage and noticed that the Os NMCP1-OE plants had longer root,while the osnmcp1 mutant plants exhibited shorter root than the corresponding WT plants under normal growth conditions,and the difference was more significant under drought stress conditions.The Os NMCP1 transcript level was analyzed by q RT-PCR in the root cap,meristem zone,elongation zone,and mature zone,the highest expression was detected in the elongation zone.Os NMCP1 may modulate root growth by regulating cell division in the root meristem and elongation zone.These results together suggest that Os NMCP1plays a positive role in regulating drought resistance,which may be partially due to the increased root growth.The lamin proteins have a wide range of nuclear functions and are involved in chromatin interaction,signal transduction,gene regulation,genomic stability and cell differentiation.To examine whether Os NMCP1 participates in the regulation of gene expression associated with chromatin status,we conducted integrative analyses of Assay for Transposase-Accessible Chromatin Sequencing(ATAC-seq)and RNA Sequencing(RNA-seq)using the root tissues of Os NMCP1-OE and wild type rice ZH11 plants under normal and drought stress conditions.The results showed that the number of increased differentially accessible regions(DARs)were significantly greater than that of decreased DARs under both normal(8791 vs 1524)and drought stress(12742 vs 2511)conditions in the Os NMCP1-OE line.Moreover,the distribution of intensity increased peaks was mainly enriched around the transcription start site(TSS)of the adjacent genes,while the intensity decreased peaks distributed almost evenly across the genome regions in relation to the TSS,with no obvious pattern,under both normal and drought stress conditions.We conducted RNA-seq analysis and discovered 728 and 3042 up-regulated differentially expressed genes(DEGs)and 434 and 2696 down-regulated DEGs under normal and drought stress conditions,respectively.The genes with DARs in the promoter region were defined as differentially accessible genes(DAGs).We integrated DAGs and up-regulated DEGs and found 362 and 619 common genes in both gene sets,accounting for 49.93% and 20.35% of total DEGs under normal and drought conditions,respectively.We identified the common genes as promoter-accessibility and expression increased genes(PEIGs)under normal and drought conditions(NPEIGs and DPEIGs)by Os NMCP1.PEIGs and DEGs gene ontology(GO)enrichment analysis showed that the GO terms closely related to "stress response".We found that 17.4% of NPEIGs and 7.9% of DPEIGs encode transcription factors(TFs),which was the largest category of the PEIGs composition,and the drought resistance related genes Os ERF48,Os NAC10,SNAC1,and Osb ZIP23 were included.We collected and re-analyzed the public RNA-seq data on the root architecture regulatory TFs Os NAC10 and Os ERF48.The Os NAC10 and Os ERF48can induce 298(40.93%)and 103(14.15%)DEGs expression,which were up-regulated in the Os NMCP1-OE plants,respectively.These results indicate that Os NMCP1 regulates drought response and root growth by changing the relevant genes chromatin accessibility.Furthermore,we found an Os NMCP1-interacting protein Os SWI3 C,a subunit of chromatin remodeling complexes,and further confirmed the interaction by bimolecular flurescence complementation(Bi FC)and Co-Immunoprecipitation(Co-IP)assays.Os SWI3 C overexpression plants showed drought sensitivity while Os SWI3 C RNA interference lines showed increased drought resistance compared with the wild type plants,respectively.This result indicates that Os SWI3 C is also involved in drought resistance pathway.The ATAC-seq analysis of Os SWI3C-RNAi plants under drought stress showed that 15199 DARs were increased in accessibility among the total 17925 DARs,which was similar to the result in Os NMCP1-OE,indicating Os SWI3 C plays a negative role in chromatin accessibility under drought stress conditions.Compared the Os SWI3C-RNAi DAGs with Os NMCP1-OE DPEIGs,it's revealed 544 common genes(Co PEIGs)that accounting for 87.88% of DPEIGs,indicating that Os NMCP1 co-regulatory with Os SWI3 C.Co PEIGs GO enrichment analysis showed that the term of 'transcription regulation' including 45 TFs.Besides,884 and 726 co-expression relations were found between the TF genes and DEGs,involving 459 and 546 DEGs up and down-regulated,respectively.These results suggest that Os NMCP1 may act together with Os SWI3 C in regulating the expression of downstream genes with diverse functions under drought conditions.Furthermore,we detected some genes that positively regulates root growth and drought resistance including Os NAC10,Os ERF48,Os SGL,Os MSR15,Os CML15,Os CML16,Os MSR2,Os CML25,Os ASR1,Os ASR2 and Os ASR3,which were up-regulated by Os NMCP1 and down-regulated by Os SWI3 C.This result indicates that Os SWI3 C plays a negative regulatory role in response to drought resistance.Based on the above results,we summarized the drought resistance mechanism of Os NMCP1 in rice.Os NMCP1 could influence chromatin accessibility by interacting with the Os SWI3C-containing chromatin remodeler complex thus to reduce the suppression effect of the complex under drought stress conditions.The Os NMCP1 expression was induced by drought stress,and the accumulated Os NMCP1 protein may lead to more nonperiphery Os NMCP1 'meeting' with Os SWI3 C.Os SWI3 C interacted with Os NMCP1 and released from the SWI/SNF complex that might partially relax the condensed chromatin,and thus directly or indirectly modulate many root growth and drought response related genes.The Os NMCP1 over-expression plants with developed root system architecture enhanced the drought resistance of rice.The function of this lamin-like gene in regulating drought resistance may be explored for the engineering and breeding of drought-resistant crops.