Effects of IAA on White clover under no PEG stress
In the absence of PEG stress, IAA significantly increased stem dry weight of white clover, indicating that IAA pre-treatment caused white clover to accumulate more organic matter during the same growth time. At this time, the chlorophyll content of white clover leaves also increased significantly. Subsequently, increased content of chlorophyll contributed to an increase in photosynthetic rate.
As far as IAA and GA were concerned, some researchers proved that an average level of bioactive GA1 required normal level of IAA in elongating pea stems , and IAA promoted GA1 synthesis . Here, our results showed that exogenous IAA increased GA3 (Figure 2C).This probably because of IAA's activation in enzymes related to GA3 syntheses, like IAA's promotion on GA1 synthesis .
In zinnia, the transcript level of IAA8 was induced by auxin . Here, we found that exogenous IAA up-regulated the expression of IAA8 in white clover. Therefore, it may be true that IAA is essential for the expression of IAA8 gene. IAA8 was found to play a crucial role in floral organ development and abnormal formation of floral in IAA8-mutation Arabidopsis could be changed through JA application, meaning that a strong link between IAA8 and JA . Fortunately, exogenous IAA significantly increased the expression of IAA8 and JA levels in our findings. For the ARF gene, IAA significantly increased the expression of it in the present study. One study also found that auxin treatment could affect the transcript abundance of several OsARF genes, and these ARF genes might play crucial roles in varied metabolic pathways and some cellular processes in rice .
Several studies also showed that the high expression level of ERD and RD22 subserved plant resistance to drought [42, 43]. ERD genes are a group of genes that are rapidly induced (in 1 h) under stress . Our results showed that IAA significantly reduced the expression of ERD gene. It may be inferred that the ERD gene was not necessary for plants suffering no PEG stress, and IAA also inhibited the expression of it.
Improved growth and physiologies in white clover under PEG stress
Under PEG stress, the results showed that exogenous IAA mitigated plant dehydration, and L-AOPP worsened it (Figure 1A). IAA improved stem dry weight, relative water content, and total chlorophyll content in leaves, however, L-AOPP decreased all of them (Figure 1B-1D). Studies have shown that IAA is related to drought tolerance in plants, and wild Arabidopsis plants pre-treated with IAA exhibited enhanced drought resistance . The application of IAA could ease the adverse effects brought by PEG stress and enhanced barley growth . IAA conferred white clover with the better morphological and physiological state in the IAA+D group than that in the D group (Figure1), suggesting that IAA had a positive effect in improving drought tolerance of white clover.
Changes in Phytohormones and subsequent effects in white clover under PEG stress
Transcriptome data revealed that an increase of ABA content activated expression of many drought-resistant genes . ABA regulated downstream response of RD29B (dehydration stress gene) by regulating the bZIP gene . In our studies, we also found that there was a consistent correlation between ABA and expression of RD22 under PEG stress (Figure 2A, Figure 5B), suggesting that ABA also probably regulated expression of RD22 gene in white clover and increased drought resistance.
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 necessary for plants to respond to severe drought stress . Besides, exogenous ABA enhanced the recovery of photosynthetic rate in upland rice under PEG stress . Based on these experimental results and Figure 1 and Figure 2 A, we could speculate that an increase in ABA content might enhance drought resistance through multiple ways, such as improved RD22 expression, higher total chlorophyll, and more stem dry weight. Moreover, the opposite effects L-AOPP on these indicators further confirmed that these changes arose from IAA.
GA3 application reduced cell permeability and electrolyte leakage under drought stress . Therefore, increased content of GA3 could enhance drought resistance in plants. In the present study, the results showed that IAA significantly increased the content of GA3 in white clover under PEG stress, and improved its resistance to PEG stress.
And the strong interaction between JA and ABA was observed . Some researchers have shown that JA was upstream of ABA biosynthesis, and the accumulation of JA at an early stage led to an accumulation of jasmonic acid isoleucine, which was one necessary condition for ABA synthesis under drought stress . Here, our results showed that exogenous IAA increased the content of both JA (Figure 2D) and ABA (Figure 2A). And we could conclude that IAA might regulate ABA synthesis via JA in white clover.
In summary, we believed that plant hormones had reached a new homeostasis after applying exogenous IAA under PEG stress. Changes in these plant hormones may promote plant drought resistance through specific signal transduction and gene regulatory pathways.
Expressions of genes responding to IAA and TF genes under PEG stress
Transcriptome data showed that rice AUX/IAA genes were induced by exogenous IAA and drought . AUX/IAA1 in Sorghum was also up-regulated by drought . In this experiment, PEG stress significantly increased the expression of IAA8 and IAA27. Interestingly, IAA significantly increased the expression of IAA8 rather than IAA27. It was found that IAA8 was involved in lateral root formation in Arabidopsis . White clover under PEG stress would instinctively improve the expression of IAA8 to form more lateral root to get more water, and IAA may enhance its expression. Some researchers revealed that the Sl-IAA27 gene was down-regulated by auxin . Our result of IAA27 was in line with this finding. Tomato transgenic plants with under-expression of the Sl-IAA27 gene showed multiple phenotypes interrelated to vegetative growth. Here, the down-regulation of IAA27 may have multiple effects on growth and root development in white clover.
GH3 family genes were also involved in plants responding to biotic and abiotic stress. Our studies showed that expression 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), showing that these two genes may have a relation to endogenous IAA content. Arabidopsis thaliana seedlings pre-treated with IAA showed an improved drought tolerance, and a variety of expressions of GH genes related to stress were regulated by exogenous IAA . It was found that decreased endogenous IAA content in rice mutants accompanied by a deficiency in carotenoid and transgenic plants over-expressing OsGH3.2 showed the sensitivity to drought . Activation of OsGH3.13 enhanced drought resistance in Rice . Exogenous IAA activated responsive gene GH3.9 and resulted in the strong drought resistance in plants . These results showed that exogenous IAA could enhance drought resistance in white clover, and GH3.1 and GH3.9 gene was involved in drought tolerance.
For bZIPs, only a few members were identified to play roles in plant growth and development, abiotic stress, and hormone signal transduction, but their potential molecular mechanisms were still unknown and need further exploration . An earlier study has shown that OsbZIP23 in maize is involved in ABA signaling and regulates drought stress . Other researchers found that bZIP11 in Arabidopsis interacted with one adapted proteins via an amino-terminal activation domain to recruit the histone acetylation system to specific auxin-responsive genes . bZIP37 expressed in the salt-stressed plant activating downstream of ABA-induced gene expression . We also found that expression of bZIP11 was also induced by exogenous IAA (Figure 4A), and that of bZIP37 was induced by PEG-6000 (Figure 4B).
Also, DREBs (dehydration-responsive element-binding proteins) play essential roles in plant response to drought stress and were found to be activated in ways dependent on ABA . It was shown that exogenous IAA enhanced expression of DREB2 and DREB4, and L-AOPP negatively regulated expression of DREB2 and DREB4 (Figure 4D, 4E) in our studies. Another study has shown that DREBs regulating the expression of many downstream genes of drought resistance and over-expression of the DREB gene can enhance drought resistance in plants . Our results showed that improved drought resistance of white clover by exogenous IAA could be associated with the expression of the DREB2 and DREB4.
MYBs are also essential in regulating plant growth, development, metabolism, and stress response, and almost all eukaryotes have MYB transcription factors. The response mechanisms of MYBs in the stress environment are not very clear. Our studies found that both exogenous IAA and drought stress-regulated expression of MYB14 and MYB48 and L-AOPP decreased their expressions (Figure 4G, 4H). Xiong found that the over-expression of MYB48-1 promoted biosynthesis of ABA and improved drought resistance of transgenic rice . AtMYB60 regulated stomatal movement and promoted Arabidopsis thaliana to respond to drought stress . Different MYBs showed varied functions in the progress of responding to drought and improved drought resistance.
At present, research of WRKY transcription factors in abiotic stress has been progressed. It was found that WRKYs were involved in plant stress regulatory networks, and WRKY proteins were induced by drought stress . WRKYs also played essential roles in plant drought stress and regulated plant response to abiotic stress through interaction with hormones and protein kinases ; however, the molecular mechanisms of their regulations were still limited. WRKY transcription factor ABO3 induced expression of drought resistance genes, such as RD29A and COR47, and improved drought resistance . In terms of WRKY2, WRKY56, and WRKY108715 gene, we found that drought induced their expressions, and exogenous IAA also up-regulated the expression of WRKY family genes in white clover.
Expression of stress gene and senescence-associated gene under PEG stress
ERD and RD22 subserved plant resistance to drought [42, 43], and our results suggested that both PEG stress and exogenous IAA up-regulated the expression of them in white clover, but L-AOPP decreased them. Besides, the expression of ERD11 and ERD13 gene was induced by dehydration, but not influenced by GA, ABA, 6-BA, and 2,4-D . Other studies also found that the RD22 gene was doubled by both ABA and MYB proteins .
As far as SAG101 and SAG102 gene was concerned, our study found that PEG stress and L-AOPP increased their expressions, but IAA decreased them. SAG101 in Arabidopsis encoded an Acyl hydrolase involved in leaf senescence . It was found that exogenous IAA inhibited the transcription level of SAG12  and retarded the senescence of leaves. The plant with over-expression of the YUCCA6 gene improved the content of endogenous IAA and hindered senescence of plant by down-regulating expression of SAG12 . Similarly, the decreased expression of SAG101 and SAG102 by IAA could play a role in delaying senescence resulted from PEG stress in white clover.