Functions Of Wheat E3 Ligase Gene Ta PUB1 In Plant Abiotic Stress Tolerance

Wheat?Triticum aestivum L.?is one of the main food crops of humans,and abiotic stress seriously affects wheat growth and yield.Therefore,to excavate abiotic related gene resources is significance for the selection of resistant,high-yield and stable-yielding wheat varieties,increasing wheat yield and ensuring world food security.E3s ligases are the key component of the ubiquitin system.Recent studys have shown that the E3 ligases are involved in plant growth and development regulation and stress response processes.In our previous study,a U-box gene TaPUB1 was isolated from wheat.The results showed that TaPUB1 could be induced by abiotic stress such as drought,salt,and plant hormones such as ABA.In this study,we further investigated the functions of the TaPUB1gene in drought,heavy metal Cd stress and ABA response.The main results are as follows:1.Wheat TaPUB1 modulated plant drought stress resistance by improving antioxidant capability and osmotic adjustment?1?Three transgenic lines?OE9,OE10,and OE11?and wild type?WT?were used as experimental materials.The ELISA results showed that TaPUB1 had E3 ligase activity.?2?Seed germination experiment was preformed by PEG6000,the results showed that overexpression of TaPUB1 increased seed germination rates.Under drought conditions,the seedling survival rate and adult growth of the transgenic lines were significantly increased than those of WT plants.These results indicated that overexpression of TaPUB1 improved drought stress tolerance of plants.?3?We analyzed the mechanisms of TaPUB1 that in drought tolerance through physiological assays.The results showed that overexpression of TaPUB1 increased the chlorophyll content and net photosynthetic rate?Pn?of transgenic lines under drought stress.Compared with WT,the relative content of transgenic lines was higher,and the osmotic type was lower than that of WT under drought stress.And the content of osmolytes,such as,proline and soluble sugar of transgenic lines were higher than WT.Furthermore,the electrolyte leakage,MDA content,and protein carbonylation in the TaPUB1-overexpression plants were lower than that of in WT.Moreover,less accumulation of reactive oxygen species?ROS?and stronger antioxidant capacity were observed in the TaPUB1-overexpression plants than that of WT plants.The qRT-PCR results showed that overexpression of TaPUB1 increased expression levels of some drought-related and antioxidant-related genes.?4?Methyl viologen?MV?was used to induce oxidative stress.Compared with WT,the higher seed germination rates,low root length inhibition,the higher chlorophyll content and lower ROS were detected in transgenic plants.These results indicated that TaPUB1 can improve the antioxidant tolerance of plants2.The wheat TaPUB1 mediated ABA response and seed development by facilitating TaPYL4 and TaABI5 degradation?1?Transgenic lines of TaPUB1-overexpressed or RNAi were treated with ABA.The results showed that TaPUB1-OE lines had higher germination rate,longer root length,and larger stomata aperture after ABA treatment compared with WT.But the RNAi lines showed the opposite phenotype.TaPUB1 decreased the sensitivity of wheat to ABA.Our results further showed that TaPUB1 influenced the expression of several ABA-responsive genes.?2?Furthermore,we found that TaPUB1 interacted with TaPYL4?wheat pyrabactin resistance 1-like 4?and TaABI5?wheat ABA-insensitive 5?,two members in ABA signal transduction by Y2H,LCI,BiFC and Pull down assays.Ubiquitination and protein degradation experiments showed that TaPUB1 promoted ubiquitination degradation of TaPYL4 and TaABI5.Those results suggested that TaPYL4 and TaABI5 were ubiquitinated substrates of TaPUB1.?3?In addition,we observed the agronomic traits of transgenic wheat and found that overexpression of TaPUB1 resulted in the less single-split grain numbers,but larger grain size and higher thousand kernel weight than those of the WT lines,but TaPUB1-RNAi showed a significant opposite phenotype.Genetic experiments showed that TaABI5 can restore the large size phenotype of abi5-1 Arabidopsis mutant.3.Wheat TaPUB1 regulated Cd uptake and tolerance via promoting the degradation of TaIRT1 and TaIAA17?1?The qRT-PCR results showed that TaPUB1 could be induced by Cd stress.Furthermore,after Cd stress treatment,TaPUB1-OE lines had higher germination rate,better wheat growth at seedling and adult stage,and higher photosynthetic rates compared with WT.But TaPUB1-RNAi showed a significant opposite phenotype.?2?The Cd²⁺content assays by ICP-MS were performed,and the results showed that the Cd²⁺content in the TaPUB1-OE lines was lower than that in the WT,and the Cd²⁺content in TaPUB1-RNAi lines was higher than that in the WT after Cd stress treatment.The NMT results suggested that overexpression of TaPUB1 reduced Cd uptake,whereas RNAi plants exhibited a significant increase in Cd accumulation after Cd treatment.TaIRT1,a wheat iron-responsive transporter 1?IRT1?,is a ZIP transporter responsible for the high-affinity metal uptake under iron deficiency,transporting Cd²⁺in plants.We found that TaPUB1interacted with and ubiquitinates TaIRT1.?3?TaPUB1 also interacted with and ubiquitinated TaIAA17?INDOLE-3-ACETIC ACID17?,which played a key role in auxin signal activation and root growth modulation,and resulted in alleviating root growth inhibition of Cd.?4?Lower reactive oxygen species accumulation,higher antioxidant enzyme activity and expression of antioxidant genes in TaPUB1-OE lines than WT were detected.