Improved growth and physioloies in white clover
Under drought stress, our results showed that exogenous 1 μM IAA pretreatment on root mitigated plant wilt whereas L-AOPP worsened it (Fig. 1A). Meanwhile, IAA pretreatment significantly improved stem dry weight, relative water content and total chlorophyll content in leaves, however, L-AOPP decreased all of them (Fig. 1B-1D). Studies have shown that IAA is closely related to drought tolerance in plants, and wild type Arabidopsis plants pre-treated with IAA exhibited enhanced drought resistance [35]. Under water deficiency, IAA effectively maintained relative water content and enhanced photosynthetic efficiency and growth of barley. Application of IAA could alleviate the adverse effects brought by dehydration and succeed in enhancing barley growth [36]. Apparently, IAA made morphological and physiological state of white clover in IAA+D set much better than in PEG-6000 set (Figure1). In contrast to appearance of plants in L-AOPP+D, it could be concluded that IAA pretreatment had a positive effect in improving drought tolerance in white clover.
Content Variations of Endogenous Phytohormones
It was showed that drought stress induced an increase in ABA and JA content, but a decrease in CTK, GA3 and SA content (Figure 2A-2E). IAA significantly increased ABA, GA3 and JA content (Figure 3A, 3C, 3D). These results implied that IAA played an important role in synthesizing or accumulating ABA, GA3 and JA in white clover.
Transcriptome data revealed that increase of ABA content induced and activated the expression of a large number of drought resistant genes [37]. ABA regulated downstream response of RD29B (dehydration stress gene) by regulating bZIP gene [38]. In our studies, we also found that there was a consistent correlation between content of ABA and expression of RD22 under stress (Figure 3A, Figure 5B), suggesting that ABA also probably regulated expression of RD22 gene in white clover.
In Arabidopsis and rice, the accumulation of ABA regulated the polar transport of auxin [39, 40]. It has been found that the interaction between IAA and ABA promoted the development of lateral roots in plants, and this pattern of root growth regulation was important for plants to respond to severe drought stress [41]. Besides, exogenous ABA enhanced the recovery of photosynthetic rate in upland rice under PEG stress [42]. Based on these experimental results and combined with Figure 1 and Figure 3 A, we also could speculate that an increase in ABA content enhanced drought resistance through multiple ways, such as improved RD22 gene expression, more content of total chlorophyll and more stem dry weight. However, L-AOPP had opposite effects on them, further confirming that these changes were caused by IAA.
As far as IAA and GAs were concerned, some researchers proved that normal level of bioactive GA1 required normal level of IAA in elongating pea stems [43] and IAA promoted GA1 synthesis [44]. It was found that GA induced the formation of porosity [45]. Here, our results showed that content of exgenous IAA significantly increased the content of GA3 (Figure 3C), and IAA content and GA3 content had consistency in change. And this consistency probably was a result of IAA’s activation in enzemes ralated to GA3 synthesis, like IAA’s promotion on GA1 synthesis [44].
Transgenic creeping bentgrass over-expressing prenyltransferase had a higher endogenous CTK content and improved photosynthesis and water use efficiency and further enhanced drought resistance [46]. In the interaction between IAA and CTK, it was found that IAA down-regulated biosynthesis level of CTK [47]. Our results showed that the increase in endogenous IAA content significantly decreased content of CTK under water sufficiency, but had no significant effect on the level of CTK under drought stress. It was found that CTK inhibited auxin transport protein PIN and reduced the accumulation of IAA, but inhibited lateral root growth [48]. However, the application of CTK resulted in a rapid increase in IAA in young parts [49]. According to these results, interaction between CTK and IAA possibly could be complicated and dependent on different tissues.
JA content increased rapidly under drought stress, and a strong interaction between JA and ABA signaling pathway was observed [50]. Our results indicated that exogenous IAA also increased content of JA (Figure 3D). Some studies have shown that JA is in the upstream of ABA biosynthesis, and the accumulation of JA at early stage led to accumulation of jasmonic acid isoleucine, which is one necessary condition for ABA synthesis under drought stress [51]. Like these results, improved content of JA could spur content of ABA to an improved level.
Exogenous application of SA could improve photosynthetic activity, leaf water content and membrane permeability, thus enhanced tolerance of tomato to drought [52]. Our studies found that content of SA was decreased by drought stress but not regularly influenced by exogenous IAA. Additionally, Alonso-Ramírez A found that GA positively regulated SA and exogenous application of GA could increase content of SA and alleviated environmental stress [53]. Here, GA3 probably also alleviated drought stress through some certain regulatory ways except its influence on variation of SA content.
Taken together, we speculated that the altered phytohormones tend to reach to a new homeostasis after application of exogenous IAA under drought stress. These variations of major phytohormones could contribute to drought resistance for plants through certain signal transduction and gene regulation pathways.
Changes of expression of genes responsing to IAA and TF genes
Transcriptome data showed that rice AUX/IAA genes were induced by exogenous IAA and drought [54]. AUX/IAA1 in Sorghum was also up-regulated by drought [55]. IAA8 and IAA27 gene relative expression got a homeostasis during all time when water was sufficient. Drought stress enorrmously prompted their expressions on 7 d and 14 d. We found that exogenous IAA up-regulated expression of IAA8 and down -regulated expression of IAA27. In zinnia, transcript level of IAA8 was particularly induced by auxin and was expressed in plant vascular development [56]. Tomato transgenic plants with under-expression of Sl-IAA27 gene showed multiple phenotypes interrelated to vegetative growth. Silencing of it resulted in higher auxin sensitivity, with change of root development and diminished Chl content in leaves [57]. Here, down-regulation of IAA27 also probably had multiple effects on growth and root development in white clover.
High content ARF gene expression nearly corresponded to high content of endogenous IAA, meaning that ARF genes expression were largely modulated by endogenous IAA. One study also found that auxin treatment could affect transcript abundance of seveal OsARF genes, and these ARF genes might play crucial roles in varied metabolic pathways and some cellular processes in rice [58]. At present, there are few reports on the functional verification of ARF gene in other plants. It is necessary to further study the specific role of ARF gene in plants responsing to IAA and drought stress.
GH3 family genes were also involved in plant responsing to biotic and abiotic stresses. Our studies showed that expressions of GH3.1, GH3.3, GH3.6 and GH3.9 were induced by drought stress (Figure 3C, 3D, 3F and 3G), denoting that these GH family genes could respond to drought stress. Besides, exogenous IAA also prompted expressions of GH3.1 and GH3.9 genes (Figure 3C and 3G), indicating that these two genes may have relation to endogenous IAA content. Arabidopsis thaliana seedlings pretreated with IAA showed strong drought tolerance, and other studies showed that exogenous IAA regulated a variety of gene expression related to stress [59]. It was found that decreased endogenous IAA content in rice mutants accompanied with deficiency in carotenoid and transgenic plants overexpressing OsGH3.2 showed the sensitivity to drought [11]. Activation of OsGH3.13 enhanced drought resistance in Rice [60]. Moreover, exogenous IAA activated responsive gene GH3.9 and resulted in the strong drought resistance in plant [61]. These results indicated that exogenous IAA could enhance drought resistance in white clover and GH3.1 and GH3.9 gene was involved in drought tolerance indeed.
Transcription factors (TFs) play important regulatory roles in growth, development, morphogenesis and response to external environments. At present, hundreds of transcription factors regulating plant resistance to drought, low temperature, high salt and disease have been found in higher plants. They were divided into bZIP, DREB, MYB and WRKY family groups etc, according to differences of DNA binding domains. Due to their abilities to regulate a number of genes associated with stress, TFs activations by special phytohormone are important to improve stress resistance of plants.
For bZIPs, only a small part of them were identified to play roles in plant growth and development, abiotic stress and hormone signal transduction, however their potential molecular mechanisms are still unknown, and need further exploration [62]. Previous study has shown that OsbZIP23 in maize is involved in ABA signalings and actively regulates drought and salt stress [63]. Other researchers found that bZIP11 in Arabidopsis interacted with one adapter proteins via an amino-terminal activation domain to recruit histone acetylation system to specific auxin-responsive genes [29]. bZIP37 expressed highly in the salt-stressed plant, which might effectively activated downstream of ABA-inducible gene expression [64]. We found that expression of bZIP11 was also induced by exogenous IAA (Figure 4A), and that of bZIP37 was induced by PEG-6000 stress (Figure 4B). So, we speculated that bZIP11 gene responded to IAA and bZIP37 responded to drought stimulus.
DREBs (dehydration-responsive element-binding proteins) play important roles in plant response to drought stress and were found to be activated in ways dependent on ABA or not [65]. It was showed that exogenous IAA positively enhanced expression of DREB2 and DREB4, and L-AOPP negatively regulated expression of DREB2 and DREB4 (Figure 4D, 4E) in our studies. Other study has shown that DREBs regulate expression of many downstream genes of drought resistance and over-expression of DREB gene can enhance drought resistance in plants [66]. Our results showed that the improved drought resistance of white clover by exogenous IAA could be associated with the role of DREB2 and DREB4 expression.
MYBs also are important in regulating plant growth, development, metabolism and stress response, and almost all eukaryotes have MYB transcription factors. The response mechanisms of MYBs in stress environment are not very clear. Our studies found that both exogenous IAA and drought stress positively regulated expression of MYB14 and MYB48 and L-AOPP decreased their expression (Figure 4G, 4H). Xiong found that over-expression of MYB48-1 promoted biosynthesis of ABA and improved drought resistance of transgenic rice [67]. AtMYB60 regulated stomatal movement and promoted Arabidopsis thaliana to respond to drought stress [27]. Different MYBs showed varied functions in progress of responding to drought and improved drought resistance directly or indirectly.
At present, the research progress of WRKY transcription factors in abiotic stress has been gradually developed. It was found that cold, heat, salt, drought and hormone induced WRKY gene expression quickly. WRKYs also played important roles in plant drought stress and regulated plant response to abiotic stress through interaction with hormones and protein kinases [68], However the molecular mechanism of its regulation were still limited. It was found that WRKYs were involved in plant stress regulatory networks and WRKY proteins expressions were induced by drought stress [69]. WRKY transcription factor ABO3 induced expression of drought resistance genes, such as RD29A and COR47, and positively regulated drought resistance [70]. In terms of WRKY2, WRKY56, and WRKY108715 genes, we found that drought also induced their expressions. Moreover, exogenous IAA also significantly up-regulated expression levels of WRKY family genes, comparing with direct drought treatment. It seemed that these WRKYs played considerably important role in white clover response to drought and IAA improved capability of this response.
Expression of stress gene and senescence -associated gene
In term of ERD and RD22 genes, our results suggested that drought up-regulated expression levels of them. Furthermore, exogenous IAA also prompted their expression levels. L-AOPP decreased their expression levels. Several studies also showed that high expression level of ERD and RD22 subserved plant resistance to drought [71, 72]. ERD11 and ERD13 genes could encode some polypeptides which were homologous to glutathione S-transferases in tobacco and maize. Besides, expressions of ERD11 and ERD13 genes were induced by dehydration, but not influenced by GA, ABA, 6-BA and 2,4-D [73]. Other studies also found that RD22 gene was double improved by both ABA and MYB proteins [74].
As far as SAG101 and SAG102 genes were concerned, our studies found that drought extremely improved their expression levels, IAA significantly decreased expression levels of them and L-AOPP enhanced expression levels of them. SAG101 in Arabidopsis encoded an Acyl Hydrolase involved in leaf senescence [75]. It was found that exogenous IAA inhibited transcription level of SAG12 [76] and retarded senescence of leaves. The plant with over-expression of YUCCA6 gene improved content of endogenous IAA and hindered senescence of plant through down-regulated expression of SAG12 [77]. Similarily, the depressed expression of SAG101 and SAG102 by IAA could play a part in delaying senescence resulted from drought stress in white clover.