Functions Of Wheat F-box Gene TaFBA1 In Plant Heat And Drought Stress Tolerance

As an important food crop,wheat(Triticum aestivum L.)has been widely cultivated in most parts of northern China.However,wheat often suffers from a variety of abiotic stresses,such as drought,salinity,high temperature,low temperature during their whole growth process.These extreme environment stresses may directly affect the wheat yield and seed quality.Therefore,improving the abiotic stress tolerance of wheat is of great significance to ensure food supply.E3 ligase is an important component of ubiquitin/26S proteasome system.It was found that E3 is closely related to the stress adaptability of plants.As the key subunit of SCF-type E3 ligase,F-box also plays an important role in plant stress tolerance.An F-box protein gene TaFBA1 was cloned from wheat'Shannong 16'in the previous research in our laboratory,and we found that it played a positive role in drought and saline-alkali stresses in transgenic tobacco.In order to explore the function of TaFBA1 in response to abiotic stress in wheat,the transgenic wheat with overexpression or knockdown of TaFBA1 were obtained by homologous transformation.Combined with transgenic tobacco and wheat lines,the functions of TaFBA1 in plant abiotic stress tolerance were analyzed,and the mechanisms underlying were explored.The main results are as follows:(1)TaSkp1 gene was cloned from wheat,and then TaSkp1-AD and TaFBA1-BD vectors were constructed.Yeast two-hybrid assay was carried out to examine the interaction between TaSkp1 and TaFBA1.The results suggested that TaFBA1 was a F-box protein.(2)The transgenic tobacco with overexpression of TaFBA1 at seedling and adult stages were exposed to 45°C heat stress.The results showed that the transgenic lines had better growth phenotype,higher chlorophyll content and stronger photosynthetic capacity than that in WT,which indicated that the overexpression of TaFBA1 improved the heat tolerance of tobacco plants.(3)The mechanism underlying the heat tolerance of transgenic tobacco was analyzed from the aspect of physiological level and gene regulation.The stronger antioxidant capacity was discovered in transgenic lines than that in WT.Compared with WT,transgenic tobacco showed less ROS accumulation,lighter protein carbonylation,and higher antioxidant enzyme(CAT,SOD,APX,POD)activities under heat stress.Moreover,the expression of abiotic stress-related genes and HSP70 protein abundance were also higher in transgenic lines.(4)The homozygous transgenic wheat lines were identified by quantitative real-time PCR(q RT-PCR),and then two overexpressing lines TaFBA1-OE3(FO3),TaFBA1-OE5(FO5)and two knockdown lines TaFBA1-RNAi2(FR2),TaFBA1-RNAi(FR8)were chosen for further analysis.We examined the activity of E3 ligase of transgenic wheat lines.The results showed that the E3 ligase activities in OE lines were higher than that in WT before and after drought stress,which was in contrast to the RNAi lines,indicating that TaFBA1 was successfully overexpressed or silenced in wheat.And the transgenic plants could be used in the following research works.(5)We analysed the growth state of transgenic plants when exposed to drought stress at seedling and adult stages.Compared with WT,OE lines exhibited a lower leaf wilting rate,higher plant height and stronger photosynthetic capacity under drought stress,while RNAi lines had the opposite phenotype.The results suggested that TaFBA1 played a positive role in regulating the drought tolerance in wheat.According to the statistics of yield traits at grain maturity stage,we found that OE lines had fuller grains and higher grain weight under normal growth conditions.These differences were more obvious after drought stress.However,the number of grains per panicle was comparable among all lines.Therefore,we speculated that OE lines had stronger grain filling ability,which was helpful to increase wheat yield under drought stress.(6)From the aspect of antioxidation,we analyzed the drought tolerance of the transgenic wheat.The results showed the contents of O₂^·(?)and H₂O₂in OE lines were lower than that in WT after drought treatment,indicating that OE lines accumulated less ROS.Meanwhile,the protein oxidation in OE lines was also more lighter.MDA content and REC were lower than those of WT,suggesting that the membrane damage of OE lines was less serious.Also,the activities of some antioxidant enzymes were markedly increased in OE lines than that of WT under drought stress.We hypothesized that the overexpression of TaFBA1 improved the drought tolerance of OE lines by enhancing the antioxidant capacity.(7)In terms of water maintenance,the stomatal aperture of wheat leaves was observed after drought treatment.We discovered that the proportion of completely open and partially open stomas in OE lines was more than that in WT,and the leaf water loss rate was also higher in OE lines,which implied that the more water was lost in OE lines leaves.Further,The sensitivity of OE lines to ABA was decreased,which may be the important factor for their slower stomatal closure under drought stress.But the higher Gs was helpful for OE plants to absorb more CO₂for photosynthesis,which was contributed to higher photosynthetic rate under drought stress.We found that there was no significant difference in root growth phenotype among all lines after 20%PEG treatment,while the root vitality and AQP activity of OE lines were higher,which implied that OE lines had stronger water absorption capacity.At the same time,the higher soluble sugar contents were found in OE lines than that in WT.The higher RWC of OE lines under drought stress may be related to the stronger water absorption and osmotic adjustment capacity.This may be another important reason for the stronger drought tolerance of OE lines.(8)By yeast two-hybrid library assay,we found that a wheat stress response protein,TaASRP1,interacted with TaFBA1.The interaction between TaFBA1 and TaASRP1 was further verified by BiFC and LUC assays.q RT-PCR was used to detect the expression of TaFBA1 and TaASRP1 under various abiotic stress.We found that the responses of TaFBA1and TaASRP1 to different stresses(20%PEG6000,200 m M Na Cl,10?M ABA,and 4°C)were synchronous.We transformed TaFBA1 and TaASRP1 into Arabidopsis and observed the growth of transgenic Arabidopsis under drought stress.The results showed that the root length of atfbw2-4,atfbw2-5 was shorter than that of WT,while the root length of TaASRP1-OE lines was longer than that of WT.When overexpressing TaASRP1 in the mutant,the root length phenotype of Arabidopsis was restored.With the increase of mannitol concentration,the phenotypic difference became more and more obvious.We speculated that TaFBA1 and TaASRP1 played a synergistic role in regulating the drought tolerance in wheat.