| As an important protein quality control system,endoplasmic reticulum associated degradation(ERAD)plays important roles in plant growth,development and stress response via eliminates misfolded or unassembled proteins from ER.At present,the components of maize ERAD system are poorly understood.Discovering the key components of maize ERAD and identifying their biological functions are expected to provide new ideas for maize genetic improvement.Previously,we screened and characterized a plant weakened mutant nlsl,which have narrow leaves.We isolated the causal gene ZmNLS1 by map-based cloning,which encodes two transcripts,ZmNLSl-T01 and ZmNLS1-T02.In the study,the molecular mechanism of ZmNLS1 regulating maize plant growth and development was carried out.1.Morphological and histocytological observation of nls1 mutantMutant nls1 and wild-type(WT)plants grow to the four-leaf stage in the nutrient solution.Morphological observations showed that the plant height,leaf width and length,number of lateral root and seminal roots,average length of seminal roots and biomass of nls1 were significantly reduced compare to WT.The histological observations showed that nls1 has obvious histocytological variation compared with the WT,including the number of kranz anatomy that is significantly reduced between adjacent leaf veins,the midrib is enlarged,the size of stem apex growth point is significantly reduced,and the number of epidermal cells between adjacent leaf veins is significantly reduced,stems and roots become thinner,and the number of cells in the root tip meristem is significantly reduced,etc.The above results indicated that the decrease in cell number may be the main cytological reason for the shortness of nis1 plants and narrow leaves caused by the loss of function of ZmNLS1.2.Expression pattern analysis and subcellular localizationWe use the promoter of ZmNLS1 to drive GUS expression in maize.Histochemical staining showed that ZmNLS1 was expressed in various tissues at different developmental stages,whereas obvious differences were detected in different types of cells in the same tissue.In the leaves,it is mainly expressed in the kranz anatomy.In roots,it is mainly expressed in the pericycle,primary phloem,lateral root primordium,root cap and outer cortex cells.In tassel and ear,it is mainly expressed in vascular bundle cells.These results suggested that ZmNLS1 may be involved in cell differentiation and morphogenesis of specific tissues and organs.We constructed the pZmNLS1::ZmNLS1-T01:EGFP and pZmNLS1::ZmNLSl-T02:EGFP plasmids and introduced them together with a ER marker plasmid with mCherry fluorescent protein into Nicotiana benthamiana epidermal cells.Laser confocal microscopy revealed that the GFP signals displayed overlapping pattern with the mCherry signals.These results showed that ZmNLS1-T01 and ZmNLS1-T02 is located in the endoplasmic reticulum.3.Functional identification of ZmNLS1 through genetic transformationBoth nls1 and allelic nls2 are point mutations that affect the alternative splicing of ZmNLS1,resulting in a series of transcript variants.Whether these variants still have partial functions is unknown.In this study,the ZmNLS1 gene was knockout in maize by Crispr/Cas9 technology.Their homozygous progeny all showed a weakened plant phenotype similar to nls1 and nls2.Therefore,the loss of function of ZmNLSl results in severe plant weakening,but not lethal.To clarify the ability of ZmNLS1-T01 and ZmNLS1-T02 transcripts to complement the nls1 phenotype,we used the maize ubiquitioh1 promoter to drive their expression and transferred them into nls1.Compared with ZmNLS1-T01,ZmNLS1-T02 can complement the phenotype of nls1 to a greater extent.With leaf width as a detection index,ZmNLSl-T02 can restore the phenotype of nls1 to more than 85%of WT.4.Interaction identification of ZmNLSl and several ERAD componentsPrevious studies and bioinformatics analysis suggested that ZmNLSl-T02 may be a component of the ERAD system.We cloned the ORF sequences of multiple ERAD system components from maize.Bimolecular fluorescence complementation(BiFC)assay showed that ZmNLSl-T02 may interact with ZmDerlin1,ZmErlin2,ZmRNF5 and ZmRNF185,which will be further verified by CoIP analysis in the future.5.Effect of ZmNLS1 mutation on the protein degradation ability of ERAD pathwayThe homeostasis maintenance of sterol synthase HMG-CoA reductase(HMGR)depends on the ERAD pathway in in yeast,animals and plants.To determine whether ZmNLS1 mutation causes the weakening of the ability to maintain the protein homeostasis by ERAD pathway,we cloned the ORF sequences of ZmHMGR1(Zm00001d006040)and AtHMGR1(AT1G76490),and constructed ZmHMGR1:MYC and AtHMGR1:EGFP plasmids.The above vectors were transformed into nls1 and WT,respectively.At present,T0-generation transgenic plants have been obtained.After harvesting,the offspring will be used to detect the differences in protein content of ZmHMGR1 and AtHMGR1 in nls1 and WT,to clarify the effect of ZmNLS1 mutation on the ERAD pathway.6.ZmNLS1 mutation may induce ER-stress responseThe unfolded protein response(UPR)induced by ER-stress in maize mainly involves two pathways:IRE1-ZmbZIP60 and ATF6-ZmbZIP17.The results of qPCR and semi-quantitative PCR showed that the mRNA splicing variant of ZmbZIP60 in nls1 leaves was 5 times that of WT,which suggested that the ZmNLS1 mutation might induce the ER-stress response.We constructed the EGFP:ZmbZIP60 and EGFP:ZmbZIP17 reporter plasmids,which were transformed into nls1 and WT,respectively.At present,we have obtained the positive T0-generation transgenic plants,which were self-pollinated to harvest offspring.Their offspring will be used to detect the content differences of the nucleuslocalized ZmbZIP60 and ZmbZIP17 splice variants in nls1 and WT by Western blotting,to further determine whether the ZmNLS1 mutation activates ER-stress response.7.The ZmNLS1 mutation causes severe DNA fragmentation and weakened cell proliferation ability in nls1Persistent ER-stress will cause cells to turn from surviving to death.In plants,ERstress-induced programmed cell death(PCD)has obvious characteristics of DNA fragmentation.TUNEL analysis is an effective method to detect DNA fragmentation caused by apoptosis signals.In the study,TUNEL assay showed that the leaf,root,stem tip and root tip of nls1 were more severely DNA fragmented than WT,indicating that the ZmNLSl mutation may have caused severe PCD in nls1.DNA damage usually block cell cycle process.In the study,we found that the growth rate of nls1 callus was only about 50%of that of WT,indicating that the ZmNLS1 mutation seriously affected the proliferation ability of nls1 cells.Based on all the above results,we believe that ZmNLS1 may be involved in maintaining protein homeostasis as an ERAD component.The loss-of-function of ZmNLS1 may lead to the weakening of ERAD patnway,causing misfolded or new unfolded proteins to accumulate in the ER,which in turn induces ER-stress and persistent UPR response,and contribute to cell death and weakened cell proliferation,ultimately lead to the defects in the growth and development of nls1 plants. |
