| Wheat is one of the most important crops worldwide, but its growth and productivity are adversely affected by abiotic stresses such as drought and salt. Therefore, to enhance wheat stress tolerance is critical for coping with these adverse stimuli and improving its productivity. Stress tolerance is a multigenes-controlled phenotype, which may include stress response, ion transport, secondary metabolism and energy flow. Thus, cell engineering technology is an available strategy for breeding new wheat cultivars with salt and drought tolerance. In our previous work, a new somatic hybrid introgression line Shanrong No.3 (SR3) was generated using common wheat Jinan 177 (JN177) and Thinopyum politician, a salt and drought tolerant grass. Former results showed that some chromatin fragments of T. ponticum intergrated into SR3’s genome, and a high frequency of allelic variation and a vast transcriptomic and proteomic change in SR3 happened, indicating that SR3 is an excellent material for investigating genetic variation, isolating stress tolerance associated genes, and dissecting the mechanisms underlying stress response.Excess ROS production is a common response when plants are exposed to abiotic and biotic stresses, which brings about serious oxidative damage. Plants possess diverse adaptive mechanisms to reduce such oxidative damage by synthesizing large amount of enzymatic and non-enzymatic ROS scavengers, and the latter cover flavonoids. Flavonoids are secondary metabolites with low molecular weight, plays important roles in various bio-events, including UV protection, defense against pathogens and pests, signaling with microorganisms, auxin transport regulation, and pigmentation, especially including apparent roles in stress protection.Previous omics-study showed that the high salt and drought tolerance of SR3 is in part attributed to its superior anti-oxidation capacity, and that a set of genes that are involved in flavonoid synthesis display differential expression patterns between SR3 and JN177, suggesting the relationship between flavonoids and the high salt and drought tolerance of SR3. Based on this information, here, we proposed to measure the flavonoid contents between SR3 and JN177. and compare the transcriptional patterns of genes in flavonoid synthesis; clone a wheat flavonol synthase gene TaFLS1, and a wheat anthocyanin synthase gene TaANS1, which play key roles in flavonol and anthocyanin synthesis branches, respectively; detect their in vitro and in vivo enzymatic activities, and the effects of their overexpressions on development and abiotic stress tolerance in Arabidopsis, with aim to assume the relationship between flavonoids and the high salt and drought tolerance of SR3, and to uncover the mechanisms of these metabolites governing development and abiotic stress response in plants. The main research contents and results achieved in this work were summarized as follows.1. Flavonoids contents and expression patterns of flavonoid biosynthetic genes in SR3 and JN177To know the putative role of flavonoids in SR3’s superior ROS scavenging capacity and high salt/drought tolerance, we measured the contents of flavonoids in SR3 and JN177 seedlings at three-leaf stage. In root, the contents of total flavonoids of SR3 were higher than those of JN177 under both the control and salt/H2O2-stressful condition; oppositely, the contents of anthocyanins were lower; In leaf, the amounts of total flavonoids and anthocyanins of SR3 were both lower than that of JN177 under the control and salt treatment. These results confirm that flavonoids exactly participate in the response to salt/ H2O2 stress, and suggest that flavonoid synthesis may be different between SR3 and JN177.Previous transcriptomic study showed that the genes involved in flavonoid (especially flavonols and anthocyanidins) synthesis had transcriptional difference between SR3 and JN177. Here, semi-quantitative RT-PCR and real-time quantitative PCR were conducted to confirm their expression patterns. The results indicated that flavonols synthesis pathway was induced by salt stress, whereas anthocyanidins pathway was reduced. Several genes exhibited differential salt-response profiles between two cultivars, of which TaFLSl was induced during the whole course in SR3, and was slightly induced only in short terms in JN177, while the response of TaDFR and TaANS1 was opposite. These results confirm that flavonoids exactly participate in the response to salt, and the different in response between SR3 and JN177 mirror the association between flavonoids with the high salt tolerance of SR3.2. The Function of TaFLS1 in abiotic stress tolerance and its putative acting mechanismsThe transcriptional response to various abiotic stresses was measured by RT-PCR and real-time PCR. Under the control conditions, TaFLSl had a higher expression level than JN177. TaFLS1 was slightly induced only at 6 h in JN177, while its expression was gradually increased in SR3 over the first 24 h when treated with 200 mM NaCl. TaFLSl showed similar expression profiles between SR3 and JN177 under 18% PEG6000,100μM ABA and 10 mM H2O2 treatments, with higher induction strength in SR3:the expression was dramatically induced and peaked at 6 h by PEG and ABA, and was continuously improved by H2O2. Its response sensitivity to 200 mM NaCl,18% PEG6000,100μM ABA and 10 mM H2O2 was different from that of JN177, with a higher transcription extent in SR3. Such difference possibly owes to lower methylation frequency of its TaFLSl promoter sequence in SR3 than in JN177 according to our experimental result.TaFLSl can in vitro convert dihydroquercetin and dihydrokaemferol to quercetin and kaemferol, respectively. Arabidopsis TaFLSl overexpression (OE) lines accumulated more flavonols, had lower abundance of endogenous AtFLSl transcripts than vector control (VC) line, indicated that the flavonols accumulation owed to TaFLS1 overexpression, that is, TaFLSl had in vivo enzymatic activity. By contrast, OE lines had less anthocyanins, and the color of their cotyledon petiole, hypocotyl and seed became lighter; the expression of genes involved in anthocyanin synthesis branch was restricted, indicating that this branch was inhibited, and the flavonoid synthesis was redirected from anthocyanins to flavonols. OE lines showed more vigorous growth ability than VC line in medium plate containing NaCl; in soils, OE lines survived from the drought treatment (a 14-day water withholding followed by a 3-day rewatering), but VC lines were dead. The detached leaves of OE lines had lower water lose rate, and their stomatal aperture was also smaller under ABA and drought treatments. The activities of ROS scavenging enzymes such as SOD and CAT as well as the transcriptional levels of their encoding genes were increased in OE lines, while ROS and MDA contents was decreased; OE lines had higher tolerance capacity to H2O2. These results indicated that TaFLSl enhanced salt and drought tolerance partially through improving ROS removal and promoting stomatal closure under stressful conditions.Real-time PCR results showed that in OE lines, ABA synthesis and signaling genes ABA1, AA03, AB11 and ABI2 as well as ABA-dependent response pathway genes RD22 and RD29B were induced, indicating that the salt and drought tolerance.Inconsistent with the effect under ABA and drought treatments, TaFLSl overexpression promoted stomatal opening under the normal condition, demonstrating that TaFLS1 plays dual roles in modulating stomatal movement, and the promotion of stomata opening may be relative with the decrease in ROS production by inhibiting PI3K activity, the increase in ROS removal by enhancing ROS scavenging enzymes, and the block of auxin transporting, which are the consequence of flavonols accumulation.3. The Function of TaANSl in abiotic stress tolerance and its putative acting mechanismsTo know the similarity and difference between flaovnols and anthocyanins in abiotic tolerance, we also carried out a functional analysis of TaANSl. Real-time PCR analysis showed that TaANSl was reduced in SR3 when exposure to salinity/drought stress; it was also reduced under 100μM ABA and 10 mM H2O2 treatments.However, TaANS1 OE lines were more sensitive to drought tolerance than VC line. Real-time PCR analysis showed that alike TaFLSl, TaANS1 OE lines had higher transcriptional levels of ABA synthesis genes AA03 and NCED3, while had lower transcript abundances of ABA signaling and ABA-dependent response pathway genes ABF3, RAB18, RD22 and RD29B. This indicated that TaANS1 ovcrexprcssion reduced drought tolerance via weakening ABA-dcpcndent response pathway.Taken together, we found that overexpressions of TaFLS1 and TaANS1 redirected two synthesis branches, and enhanced contents of flavonols and anthocyanins, respectively; the overexpressions of TaFLSl and TaANS1 performed opposite roles in drought tolerance via positively and negatively regulating ABA-dependent response pathway; flavonols played dual roles in stomatal movement-promoting stomatal opening under the normal conditions and stomatal closure under the stressful conditions, and the promotion of stomatal opening is possibly achieved via inhibiting ROS production, enhancing ROS removal and blocking auxin transporting. |
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