A Study On The Molecular Mechanism Of ZmBET5L1 On Regulating Osmotic Stress Tolerance In Maize

Osmotic stress,one of the most important stress factors,is frequently caused by drought and saline alkali,and then results in the loss of crop yield.Therefore,improving osmotic stress tolerance is critical for crop production.However,osmotic stress tolerance of crop plants is a quantitative trait controlled by multiple minor effect loci.The genetic improvement to minor effect genes is difficult and the genetic gain is small.Mining the key genes and creating new germplasm of crop osmotic stress tolerance are meaningful to improve crop osmotic stress tolerance.The cargo proteins across and between the membranes of eukaryotic cells are mainly transported by vesicle trafficking.Transport protein particles(TRAPPs)serve as tethering factors in vesicle trafficking.Vesicle transport is considered to be particular in the regulation of cell response to abiotic stresses,including osmotic stress by protein transport and signal transduction.Thus,modifying vesicle transport in cells is expected to improve the response and tolerance of plant cells to osmotic stress.Based on this theoretical cognition,we studied the function and mechanism of vesicle transport-related genes in maize response and tolerance to osmotic stress.The main results are as follows:1.Knowledge-driven QTL analysis showed ZmBET5L1(BET5-like1)may be a candidate gene related to drought tolerance in maize.In order to study the role of tethering factors in drought osmotic stress,we collected all tethering factors in maize,and mined the genes with the most QTL coverage in the published database on drought osmotic stress tolerance,and then screened the genes that highly expressed in seedling roots and respond to polyethylene glycol(PEG)-simulated osmotic stress.The result showed that ZmBET5L1 is considered as a candidate gene for drought osmotic stress tolerance.Phylogenetic analysis showed BET5 protein is conserved across animals,plants and microorganisms,and is highly conserved in monocotyledon species.Additionally,we demonstrated that ZmBET5L1 interacts with other subunits of maize TRAPP complex by firefly luciferase complementation imaging(LCI)assay and yeast two hybrid(Y2H).2.ZmBET5L1 negatively regulates the early development of primary roots.To explore the effect of ZmBET5L1 in the development of primary roots,we created two knock-out lines(zmbet5l1-1 and-2)by CRISPR/Cas9 and two overexpression lines(ZmBET5L1-OE1 and-OE2)using maize Ubiquitin promoter-driven ZmBET5L1 constructs.We found the primary roots of zmbet5l1 lines were significantly longer than that of wild-type(WT)siblings from 5 to 9 days after germination(DAG).For ZmBET5L1-OE lines,primary roots were shorter than that of non-transgenic(NT)siblings from 3 to 11 DAG in ZmBET5L1-OE1 and 7 to 11 DAG in ZmBET5L1-OE2.The results indicate that ZmBET5L1 negatively regulates the early development of primary roots.Furthermore,loss of ZmBET5L1 function led to reduced pollen fertility but had no effect on important agronomically traits.3.ZmBET5L1 negatively regulates the maize tolerance to drought osmotic stress.After drought treatment with rewatering,we found a significant improvement in survival rate for zmbet5l1 compared to WT siblings.The 11 DAG-siblings were treated with or without 20% PEG for 4 days,for control treatments,zmbet5l1 lines showed a similar primary root elongation rate to WT siblings;whereas with 20% PEG-simulated osmotic treatment,zmbet5l1 knockout lines showed a higher primary root elongation rate than WT siblings.Therefore,we suggest that ZmBET5L1 negatively regulates primary root development and resistance to drought osmotic stress in maize.4.ZmBET5L1 acts as a tethering factor to participate in vesicle trafficking.We observed a wide distribution of ZmBET5L1-GFP signal in Golgi and ER,mostly in cisGolgi,by a co-localization with the endoplasmic reticulum(ER),cis-Golgi and TGN/EE(trans-Golgi Network/Early Endosome)markers.We also found that Golgitargeting marker proteins displayed a punctate signal pattern in WT cells but extra ER signals in zmbet5l1-1 cells.In addition,we used TGN/EE marker FM4-64 treated with Brefeldin A(BFA)to evaluate the vesicle transport rate by BFA bodies.The BFA bodies aggregation rate in zmbet5l1-1 root cells are significantly lower than in WT under normal condition;PEG treatment could accelerate the induction of BFA bodies in WT root cells but didn’t show such a trend in the zmbet5l1-1.These results suggest that ZmBET5L1 involves in vesicle trafficking between ER to Golgi,and is facilitated to TGN aggregation.5.ZmBET5L1 regulates auxin content,polar localization and distribution..We performed anti-GFP immunoprecipitation and quantitative mass spectrometry(IP-MS)using transgenic maize seedling roots expressing ZmBET5L1-GFP in vivo.The results showed that ZmBET5L1 interacts four PP2 As,which are considered to involve in PIN1 polar localization and signal transduction pathways in abiotic stresses.Furthermore,we found that the auxin content increased and the PIN1 polar localization signal and distribution changed,and the application of auxin could make maize plants tolerant to drought stress in zmbet5l1 mutants.Therefore,we found that ZmBET5L1 moderates PIN1 polar localization and auxin flow to maintain normal root growth and tolerance to drought stress.In summary,through knowledge-driven data mining,we found a vesicle trafficking-related tethering factor,ZmBET5L1.ZmBET5L1 protein participates in compose of TRAPP,ER-to-Golgi vesicle trafficking,and vesicle aggregation of TGN,and then negatively regulates the early development of primary roots and the drought osmotic stress tolerance by regulating PIN1 polar localization and auxin flow.