Indole -3-acetic acid improves drought tolerance of white clover associated with activating auxin-related genes, abscisic acid and jasmonic acid-induced stress responsive transcription factors, and inhibiting senescence genes

Auxin plays an important role in regulating plant development. To aquire feasible regulation effects and mechanism of IAA on drought tolerance in white clover, relative water content, chlorophyll content, several major phytohormone level, the expression of gene-related to IAA signal perception and synthesis, as well as drought-resistance related transcription factor genes and senescence genes were investigated in white clover plants under 15% polyethylene glycol-6000 (PEG-6000) after 1µM exogenous IAA pre-treatment and 100µM L-AOPP (inhibitor of IAA synthesis) pre-treatment. Compared to control, drought stress signicantly diminished stem dry weight (g/10 plants), relative water content (RWC, %) and total chlorophyll content (Chl, mg/g). Exogenous IAA signicantly increased RWC and Chl, however, L-AOPP, drastically decreased stem dry weight (g/10 plants), RWC and Chl compared with direct drought treatment. Besides that, compared with direct drought treatment, exogenous IAA signicantly improved ABA content and JA content, up-regulated expression level of auxin-responsing genes (GH3.1, GH3.9, IAA8), drought-resistance related transcription factor genes (bZIP11, DREB2, MYB14, MYB48, WRKY2, WRKY56, WRKY108715) and drought resistance gene (RD22), meanwhile, down-regulated expression level of auxin-responsing genes (GH3.3, GH3.6, IAA27) and leaf senescence genes (SAG101 and SAG102). L-AOPP, contrarily, compared with direct drought treatment, signicantly reduced ABA, GA3 and JA content, down-regulated expression level of GH3.1, GH3.9, IAA8, bZIP11, DREB2, MYB14, MYB48, WRKY2, WRKY56, WRKY108715, ERD and RD22, meanwhile, up-regulated SAG101 and SAG102. Collectively, these data suggested a positive role of exogenous IAA in alleviating drought stress damage in white clover.


Introduction
Drought continuously attracted researchers' concern partly because of its in uence in restricting plant growth and distribution worldwide [1,2,3]. Adaptability, capability of being or becoming adapted, would give rise to some biochemical and physiological changes to confer plants with improved survival rate in arid environments [4,5,6,7]. In essence, these changes are results of expression of direct or indirect related drought responsive genes, including early-response to dehydration (ERD) stress genes and resoponse to dehydration (RD) stress genes which are closely related with plant senescence [8,9] and senescence-associated genes (SAG) which regulate expression of some genes related to chlorophyll degradation and cytoplasm damage [10]. Also, it was reported that transcriptional activation of transcriptional factors (TFs), such as basic region/leucine-zipper motif (bZIP), dehydration responsive element binding (DREB), myeloblastosis (MYB) and WRKY, could be in uenced by drought and regulate plant physiological state.
White clover (Trifolium repens), one legume plant, is one of important forages for many domesticated animals in the world due to its high yield, good quality and rich nutritional value, and thus is planted in large quantities. However, white clover is susceptible to drought stress and exhibits signi cant changes in leaf senescence when confronting to dehydration stimulation [30]. However, there are few detailed studies about IAA's effects on dehydration in white clover, from phytohormones to TFs, from IAA responsive genes to senescence genes. In order to explore biochemical and physiological changes of white clover in drought stress and changes caused by IAA addition, we investigated contents of six major phytohormones (including IAA itself) expression of ARF gene, GH3 family genes (belonging to auxinresponsive gene), IAA repsonse gene, TFs genes, ERD gene and RD gene, SAG genes in white clover under drought stress, with the purpose of provding some possible perspectives of the varied adaptations of white clover in dehydration. And we formulate hypotheses that exogenous IAA improved drouhgt resistance in white clover through several biochemical and physiological changes, ranging from contents of several phytohormones, to expressions of auxin-responsive genes, some TFs genes and senescence genes. These changes confer a better appreance of white clover and reinforce adaptability of it in drought.

Plant material and growth condition
The seeds of 'Pixie' (Trifolium repens cv.) were purchased from Beijing Mammoth Seed Industry Company in China. The seeds were sterilized with 1% (W/V) sodium hypochlorite solution for 3 min and then rinsed with sterile water for 3 times, ~0.8 g of seeds were evenly spread in culture pot (9 cm depth, 18 cm width, 24 cm length) containing sterilized quartz sand, then placed in the growth chamber (23℃/16 h day and 19℃/ 8 h night cycle, irradiance of about 300μmol quanta·m -2 ·s -1 , 75% relative humidity). 25 mL of deionized water was added to each pot in every day. After germination, instead of deionized water, Hoagland's nutrition solution was used. In order to minimize the environmental effects, the positions of the pots in the culture chamber were also daily replaced.

Experimental design
When the second leaf of white clover fully unfolded, seedlings were subjected to four sets: (1) Control, Hoagland's nutrition solution was used as previously; (2) PEG-6000 treatment (labeled with D), Hoagland's nutrition solution with 15% PEG-6000 (W/V, -0.3Mpa); (3) IAA pre-treatment and PEG-6000 treatment (labeled with IAA+D), Hoagland's nutrition solution with 1 μM IAA for the rst 7-day, and then Hoagland's nutrition solution with 15% PEG-6000 for the subsequent days; (4) L-AOPP pre-treatment and PEG-6000 treatment (labeled with L-AOPP+D), Hoagland's nutrition solution with 100 μM L-AOPP for the rst 7-day, and then Hoagland's nutrition solution with 15% PEG-6000 for the last days. All plants were simultaneously treated with PEG-6000. Each treatment had 4 biological replicates. Samples were collected on 0 d, 7 d and 14 d after adding PEG-6000.

Physiological measurements
Relative water content (RWC), stem dry weight and Total Chlorophyll content (Chl) For RWC, approximately 0.2 g of leaves (FW) were wrapped with clean gauze and then dipped into distilled water. After 24 hours at 4 ℃, weighed them as saturated weight (SW). Samples were placed in kraft paper bags, kept in an oven at 105 ℃ for 30 min, then at 75 ℃ for 48 h, and weigh them as dry weight (DW). RWC was calculated through formula [31]: RWC(%)=(FW-DW)/( SW-DW)×100% For total chlorophyll content, approximately 0.1 g of leaves (FW) were extracted with 10 mL of mixed solution (80% acetone/95% methanol, 1:1 V/V) in glass tubes in dark till leaves became colorless.The extract solution was instantly assayed spectrophotometrically. Speci c absorption of Chl a and Chl b was recorded at 645 nm and 663 nm, respectively. Total chlorophyll content was calculated according to the following formula [32]: Chl (a+b)(mg/g) =(20.2×OD 645 +8.02×OD 663 )/(DW×1000) Quanti cation of IAA, ABA, CTK (iPAs and ZRs), GA3, JA and SA HPLC-ESI-MS method [33] with minor modi cation was used to quantify IAA, ABA, CTK (iPAs and ZRs), GA3, JA and SA. Leaves of white clover were weighted (~0.2g) and transferred to a 5 ml screw-cap tube with one steel ball whose diameter was less than 5mm. Kept tubes in liquid nitrogen for 10 min to frezze plant samples and ground them into powder through a Plant Tissue Breaker. Add 200 µl of the working solution of internal standards [33] to each tube. Add 2 ml extraction solvent [33]. Shake tubes at a speed of 100 rpm for 30 min at 4 °C. Add 2 dichloromethane to each tube and shake for 30 min in a 4 °C room.
Centrifuge at a speed of 12,000 rpm for 5 min at 4 °C. Transfer ~1.8 ml of the solvent from the lower phase into a screw-cap vial and concentrate it by using a nitrogen evaporator. The samples were redissolved in 0.2 ml methanol. Inject 20 µl of the last methanol solution into the C18 (reverse-phase) HPLC column for analysis. The HPLC-ESI-MS (HPLC system, LC-10AD series, Shimadzu, Japan; ESI-MS system, ABI 4000 QTRAP) conditions and settings were used. Applied Biosystems Analyst software version 1.5.1 was used to control MS system and to perform data analysis and data management.
Expression of drought-induced transcription factor (TF) genes, response to drought (ERD/RD22) and  [34]. Primer sequences of drought-induced transcription factors and drought-induced genes and their corresponding GeneBank accession numbers are listed in Table 2 in Appendixes.

Statistics and mapping
In this paper, Origin 8.5.1 was used to generate the histogram, SPSS 19.0 to analysis of variance (ANOVA) at 0.05 probability level. Data were transformed to meet normality and homogeneity of variance. Fisher's LSD was used to determine differences among sets.

Results
Effects of exogenous IAA on drought tolerance of white clover Morphological appearance of white clover plants in all experiment sets were shown as Figre 1A. IAA pretreatment had an obvious facilitation to white clover under normal condition, followed by a signi cant higher stem dry weight as compared to other treatments ( Figure 1B). However, L-AOPP (inhibitor of auxin synthesis) pretreatment remarkably reduced total chlorophyll content Chl Figure 1D . In control, stem dry weight increased ( Figure 1B) by days, RWC and Chl remained stable throughout the experiment (Figure1C-1D). When drought stress went on, stem dry weight, RWC and Chl content gradually decreased in white clover in all sets (Figure1C-1D). However, stem dry weight, RWC and Chl were signi cantly higher in IAA pretreatment than those without IAA pretreatment. Furthermore, L-AOPP signi cantly diminished them when compared to drought sets ( Figure 1B-1D).
Content of ABA, CTK (iPAs and ZRs), GA3, JA and SA On 0 day, IAA reduced CTK content by 90.2% and improved GA3 and JA content by 45.2% and 18.4% ( Figure 2B-2D), respectively. And, it had no in uence on ABA and SA content (Figure 2A-2E). However, L-AOPP increased CTK content by 61.4% and SA content by 130% ( Figure 2B, 2E), decreased GA3 and JA content by 28.8% and 13.8% ( Figure 2C, 2D), respectively. And it had no impact on ABA content ( Figure   2A). In PEG-6000 treatment, ABA and JA content gradually got rose, accompanied by the gradual decline in CTK, GA3 and SA content during experiment period. On 7 day and 14 day, ABA, GA3 and JA contents were signi cantly higher in IAA set than those without IAA pretreatment (Figure 2A, 2C, 2D), while SA content was signi cantly lower in IAA set than it in others. L-AOPP nearly had the opposite appearances when compared with IAA (Figure 2A, 2C, 2D).
Endogenous IAA content and relative expression of auxin response genes IAA set showed a signi cant increase in endogenous IAA content while L-AOPP set exhibited a signi cant decrease of endogenous IAA content in white clover under normal condition. The content of endogenous IAA remained at a stable level in control but decreased much in PEG-6000 treatment from start to end ( Figure 3A). However, compared with PEG-6000 treatment, exogenous IAA pretreatment maintained a considerably higher endogenous IAA content in white clover exposed to stress, inversely, the L-AOPP pretreatment showed a remarkable decreased trendancy in endogenous IAA content ( Figure 3A).
Under normal condition, exogenous IAA improved the expression of ARF (auxin response factors), GH3.9 and IAA8, lowered the expression of GH3.5 and IAA27, and had no effects on the expression of GH3.1, GH3.3 and GH3.6. L-AOPP mainly restrained the expression of GH3.1 and GH3.3 and had no prominent in uence on other genes. PEG-6000 inhibited the expression of ARF, up-regulated the expression of other genes. Under drought stress, signi cantly higher expression level of ARF, GH3.1, GH3.5, GH3.9 and IAA8 and lower expression level of GH3.3 , GH3.6 and IAA27 were detected in IAA set compared to IAA-absence set. However, L-AOPP down-regulated all genes expression except GH3.5 ( Figure 3B-3J) .

Relative expression of transcription factor (TF) genes responsed to drought stress in white clover
In this experiment, we chose 3 genes in bZIP, DREB, MYB and WRKY transcription factor family, respectively. Under no stress, IAA signi cantly up-regulated expression of bZIP107, MYB48, WRKY2 and WRKY56, down-regulated expression of DREB5 and MYB48, and had no remarkable in uence on expression of bZIP11, bZIP37, DREB2, DREB4, MYB14 and WRKY108715. L-AOPP signi cantly downregulated bZIP107 and up-regulated DREB5. All concerned transcription factor genes were induced by PEG-6000 except that expression of bZIP107 was depressed in present experiment. Under drought stress, addition of IAA further up-regulated the expression level of all the transcription factor genes, L-AOPP lowered the expression of these genes, exception for bZIP37 and bZIP107compared with PEG-6000 set ( Figure 4B, 4C).
Expression of drought-response genes and senescence-associated genes Under no stress, exogenous IAA showed a conspicuous inhibition to the relative expression of ERD. However, L-AOPP signi cantly induced expression of RD22 gene. Drought enhanced expression of ERD and RD22 in large. IAA also improved expression of ERD and RD22 and L-AOPP lowered their expressions ( Figure 5). In addition, drought stress extremely activated expression of SAG101 and SAG102, L-AOPP upregulated expression of SAG101, while IAA displayed a prominent suppression to expression of SAG101 and SAG102 in white clover.

Discussion
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 signi cantly 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 de ciency, IAA effectively maintained relative water content and enhanced photosynthetic e ciency 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 signi cantly 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 con rming 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 signi cantly 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 e ciency 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 signi cantly decreased content of CTK under water su ciency, but had no signi cant 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 in uenced 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 in uence 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 su cient. Drought stress enorrmously prompted their expressions on 7 d and 14 d. We found that exogenous IAA up-regulated expression of IAA8 and downregulated 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 veri cation of ARF gene in other plants. It is necessary to further study the speci c 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 de ciency 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 identi ed 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 speci c 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 signi cantly upregulated 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 in uenced 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 signi cantly 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.

Conclusion
Under normal condition, endogenous IAA content of leaves in IAA+D was the highest ( Figure 3A). Under PEG-6000 treatment, IAA pretreament on roots of white clover improved endogenous IAA level while L-AOPP pretreatment decreased IAA content of leaves ( Figure 3A