Functions Of Wheat F-box Gene TaFBA1 In ABA-mediated Plant Drought Tolerance

Drought stress is one of the most common environmental stresses,and affects the growth and developmentment of plants.It had great significance to strengthen the research on the mechanism of plant drought tolerance to improve the quality and yield of crops.ABA level in plants increased under drought stress,which further improved plant stress resistance.The F-box protein is a key subunit in the SCF E3 ligase that is involved in responses to abiotic stresses.However,the relationship between F-box protein and ABA-mediated stress tolerance in wheat remains unclear.A gene encoding the wheat F-box protein,TaFBA1,was previously obtained in our lab by homologous cloning.We found that overexpression of TaFBA1 improved the drought stress tolerance in transgenic tobacco plants.It was also found that the stomatal opening of tobacco with overexpression of TaFBA1 was larger,suggesting that TaFBA1 played a negative regulatory role in ABA-regulated stomatal closure.This paper aims to analyze the physiological mechanism of TaFBA1 in ABA-mediated plants drought tolerance and stomatal movement.The main results were as follows:?1?The genomic PCR and qRT-PCR was use to detect the TaFBA1 overexpression in Arabidopsis lines,Arabidopsis mutants lines and the recovery lines of TaFBA1 Arabidopsis mutants.We selected TaFBA1 overexpression lines OE4,OE5,OE6,mutants lines atfbw2-4,atfbw2-5,atfbw2-6 and recovery lines R-4,R-5,and R-6 lines were used in subsequent experiments.?2?We tested the seed germination rate,cotyledon greening rates and root length of Arabidopsis under mannitol?artificial drought?and mannitol+Na₆WO₄?ABA synthesis inhibitor?conditions.The results indicated that drought stress inhibited seed germination,cotyledon greening and root growth of all lines,but the inhibition to overexpressing lines was less than that of WT,but the inhibition of mutants was greater than that of WT;the drought tolerance of mutants was significantly improved after recovery TaFBA1.After inhibiting ABA synthesis by Na₆WO₄,the inhibition effect of drought on all plants was alleviated,but the difference among the different lines was not changed,which suggested that TaFBA1 may not be involved in ABA synthesis.ABA levels in different lines also proved this conciution.?3?The growth and leaf photosynthetic capacity of grown Arabidopsis plants were detected under natural drought treatment.The results showed that the effect of drought treatment on grown Arabidopsis were similar to that of seedling.Under normal conditions,there was no significant difference in stomatal conductance?Gs?,transpiration rate?E?,net photosynthetic rate?Pn?and intercellular CO₂ concentration?Ci?between overexpressed and mutant lines.After drought treatment,Gs,E and Pn of TaFBA1 overexpressed lines were higher than WT,while the mutant was reversed;there was no significant difference in Ci.The results indicated that TaFBA1 overexpression enhanced the photosynthetic performance of Arabidopsis.?4?When ABA was used to treat every kind of lines,TaFBA1 overexpressed Arabidopsis had higher seed germination rate and cotyledon greening,faster elongation of roots and better growth,while mutant plants were opposite;recovery lines can reduce the sensitivity of mutant to ABA.The photosynthetic capacity of TaFBA1 overexpressed lines was better than that of WT and mutants after ABA treatment.These results suggested that TaFBA1overexpression alleviates the inhibitory effect of ABA treatment on Arabidopsis.?5?Especially,the TaFBA1 overexpression plants had higher Gs under ABA and drought conditions than WT.Stomatal closure of overexpressed plants responded slowly to ABA,and stomatal closure of mutant responded faster.This indicated that TaFBA1 overexpression decreased the sensitivity of stomatal closure to ABA in transgenic plants.?6?To further explored the molecular mechanism of TaFBA1 regulating ABA signaling,we analyzed the effect of TaFBA1 on the expressions of ABA-related genes,including ABA response genes and ABA-synthesis associated genes.The results showed that TaFBA1 could regulate the expressions of ABA signal response gene,but had little effect on ABA synthesis genes.Stomatal movement-related genes were downregulated in TaFBA1 overexpression plants.These suggested that TaFBA1 may participate in the regulation of stomatal closure by regulating the expression of ABA response and stomatal movement related genes.?7?We performed yeast two-hybrid screening with TaFBA1 as the bait,and were lucky to obtained two interaction proteins:RCAR1?an ABA receptor?and ABI5?ABA response protein?.We further verified the interaction between TaFBA1 and RCAR1 through BiFC assays in vivo,and the results showed that they might interact on the cell membrane.?8?The antioxidant capacity of each line was analyzed by detecting the physiological and biochemical activities.Under drought and ABA conditions,the contents of H₂O₂,O₂^·?production rate and MDA content in TaFBA1 overexpressed lines were lower than that in WT,while mutant was on the contrary,and recovery lines was lower than that in mutant.These results indicated that overexpression of TaFBA1 enhanced the antioxidant capacity of Arabidopsis under ABA and drought conditions.This may be the most important mechanisms underiying TaFBA1 improved drought tolerance of transgenic Arabidopsis.In conclusion,wheat TaFBA1 positively regulates plant drought tolerance,which is independent of ABA biosynthesis.But TaFBA1 negatively regulates sensitivity of Arabidopsis plants to ABA.TaFBA1-regulated ABA-insensitivity may be achieved by regulating ABA-mediated gene expressions through interactions with RCAR1 and ABI5.Increased antioxidant competence and decreased ROS accumulation may be the most important mechanism underlying the improved drought tolerance of TaFBA1-overexpressing plants.