【作者】 于延冲；

【导师】 向凤宁；

【作者基本信息】 山东大学 ， 细胞生物学， 2011， 博士

【摘要】 在长期的进化过程中,植物形成了一套完整的机制,用于调节自身的生长发育以适应或抵御外界生物和非生物胁迫,在这些进程中转录因子(WRKY、DREB、NAC、MYB等)发挥了重要的调控作用。WRKY转录因子家族是近十几年来发现的一类植物特有的转录因子,它们在植物体内是非组成型表达的,受生物及非生物胁迫(水杨酸、病原诱导子、高盐、干旱、低温等)诱导,主要参与生物与非生物胁迫应答和植物衰老,有关对植物发育的调控研究报道很少,仅见参与调控了种子和表皮毛的发育。迄今为止,尚未见它们参与调控植物开花和分枝发育的报道。植物开花是植物从营养生长到生殖生长转折的关键,具有很强的可塑性。在各种外界环境和内部因子的影响下,植物会选择在适当的时机开花进而获得生殖的成功。植物开花是一个涉及多因子相互作用的复杂系统。目前,在拟南芥中主要存在4条调控植物开花的途径：光周期途径,春化途径,自主途径和赤霉素途径。研究表明,其它一些因子也参与了开花调控,比如蔗糖和非生物胁迫因子。一些外界胁迫因子(干旱、紫外线和病原菌)能够促进植物开花,这是植物逃避逆境的一种适应机制。通常情况下,高盐胁迫会抑制拟南芥开花,其调控机制已有-些研究。但尚未见有关高盐胁迫促进植物开花的报道。植物的分枝是从茎顶端分生组织衍生出的腋生分生组织中分化出来的,包含两个关键的发育过程：腋生分生组织在叶腋部位的形成和腋生分生组织的生长发育(腋/侧芽的生长)。它决定着植物地上部分的株型,与作物的产量密切相关。植物的分枝发育受到各种外界环境条件、遗传因素和植物激素的影响。目前,多个调控植物分枝发育的基因已被发掘。迄今,有关WRKY转录因子调控植物分枝发育的研究尚未见报道。本研究从拟南芥激活标签突变体库中筛选得到了一株较Co1-0开花早、花器官大、植株矮小、分枝多的突变体D27。Tail-PCR和RT-PCR分析推测,该表型可能由WRKY转录因子基因WRKY71的过表达引起。遗传分析发现,该突变体的表型稳定。在此基础上,利用Col-0、D27 (WRKY71-1D)及其敲除系wrky71-1研究了WRKY71在正常条件和高盐胁迫下对拟南芥开花的作用,揭示了WRKY71介导高盐诱导拟南芥开花提前的分子机制,分析了WRKY71对拟南芥分枝发育的贡献及其可能机制,为进一步阐明拟南芥开花和分枝发育的分子调控机制奠定了基础。主要研究过程及实验结果包括：1.WRKY71转录因子的分离鉴定及其功能初探1.1突变体的筛选和基因分离鉴定从拟南芥激活标签突变体库中,筛选得到了一株突变体D27,与野生型Col-0相比,该突变体植株矮小、开花早、花器官增大、分枝多。Tail-PCR和RT-PCR分析推测,该突变体的表型可能是由WRKY71的过表达引起。购买并筛选获得了wrky71-1敲除系。1.2 WRKY71的表达模式时空表达模式：Real time-PCR分析发现,WRKY71在拟南芥Col-0生长的前三周内表达逐步上调,随后逐步下降；其在各个组织器官中均有表达,其中在角果中表达量最高。进一步的GUS染色分析发现,WRKY71亦在各组织器官中表达,在根、表皮毛及托叶中表达显著。亚细胞定位发现,WRKY71定位于洋葱表皮细胞和拟南芥原生质体的细胞核中。转录激活活性实验证实了该基因具有转录激活活性。对非生物胁迫和激素的应答模式：Real time-PCR分析发现,WKRY71受高盐和ABA的诱导而高表达,其由ABA依赖的途径受高盐诱导。1.3 WRKY71介导盐胁迫下拟南芥的早开花以200mM NaCl处理在土壤中生长的Col-0、WRKY71-1D和wrky71-1植株(7天和10天各浇灌1L 200mM NaCl),发现WRKY71-1D在第23天开花,仅比正常条件下延迟1天,而Col-0和wrky71-1在第35天开花,比正常条件下延迟1周,说明WRKY71-1D的开花进程对高盐不敏感。2. WRKY71调控拟南芥花发育的分子机制研究2.1 WRKY71促进拟南芥开花的确定WRKY71过表达植株35S：：WRKY71的表型与WRKY71-1D一致,佐证了WRKY71-1D的表型是由WRKY71过表达引起的。2.2 WRKY71在花序分生组织、花原基及花器官原基中表达RNA原位杂交发现WRKY71在15天的花序分生组织和花原基中表达,在17天的花序分生组织和花器官原基中表达。表明WRKY71可能参与了顶端花序分生组织的形成以及花的发育。2.3 WRKY71促进开花转折组织学纵向切片观察发现花原基出现(开花转折)的时间分别为：100%的WRKY71-1D在第11天、90.4±5.7%的Col-0在第15天、而仅有37.6±3.3%的wrky71-1在第15天。扫描电镜结果同该结果一致,表明WRKY71具有促进拟南芥开花转折的作用。2.4高盐胁迫下WRKY71亦促进开花转折组织学纵向切片分析发现高盐胁迫下花原基出现的时间分别为：100%的WRKY71-1D在第12天,32.7±3.3%的Col-0在第17天,而仅有21.6±2.9%的wrky71-1在17天,表明WRKY71-1D的开花起始几乎不受到高盐的抑制。可见,WRKY71在高盐胁迫下亦促进开花转折。综上所述,WRKY71受高盐的诱导,WRKY71的过表达促进拟南芥的开花,因此,推断WRKY71介导了高盐诱导拟南芥开花提前。2.5 WRKY71不参与四条主要开花途径Col-0、WRKY71-1D和wrky71-1均能正常应答日照长度(长日照和短日照)、低温和GA信号,在处理条件下,WRKY71-1D的开花时间依然早于Col-0和wrky71-1。四条途径中的一些相关基因在三系中表达水平相近,而WRKY71在一些开花突变体(gi-2、co、ft、fve-4、Id-1、fld-1、fca-9)中的表达变化亦不明显,表明WRKY71可能不参与上述4条途径。2.6 WRKY71通过影响蔗糖运输而促进拟南芥开花RT-PCR分析发现蔗糖合成代谢和信号转导的相关基因在三系中的表达差异不明显,表明WRKY71可能不影响拟南芥蔗糖的合成代谢和信号转导。蔗糖转运子基因SUC8和SUC9在WRKY71-1D中表达下调,EMSA实验证明WRKY71与SUC8和SUC9启动子区的W-box结合,表明WRKY71通过直接抑制SUC8和SUC9的表达,促进胞外蔗糖运输到茎顶端以促进拟南芥的开花。2.7 WRKY71促进花分生组织决定基因表达Real time-PCR分析发现在非盐胁迫和盐胁迫条件下,花分生组织决定基因LFY、API、CAL和FUL在WRKY71-1D中均上调表达。这与WRKY71-1D在非盐胁迫和盐胁迫条件下开花早的表型一致。2.8 WRKY71结合LFY启动子的W-boxEMSA实验证明WRKY71能强烈地结合在LFY启动子的W-box上,微弱地结合在CAL启动子的W-box上,不能与AP1启动子的W-box结合；ChIP实验亦证明了WRKY71能结合在LFY启动子的W-box上,而不能与CAL结合,表明WRKY71通过直接上调LFY表达而促进拟南芥开花。2.9杂交互补验证对(?)WRKY71-1D×LFY::GUS杂交种和LFY::GUS进行GUS染色分析发现,前者的GUS活性高于后者,表明WRKY71可提高LFY的翻译水平。WRKY71-1D×lfy植株与WRKY71-1D表型相似,但是不能开花结实,表明LFY的缺失阻断了WRKY71-1D的开花,从而证实了WRKY71是通过调控上调LFY表达来促进拟南芥的开花。2.10 WRKY71促进拟南芥花器官的发育WRKY71-1D的萼片、花瓣、雄蕊和心皮均比Col-0和wrky71-1的大,其花器官发育快速。Real time-PCR分析发现,花器官发育相关基因AP1、AP3和AG,花器官大小的基因UFO和干细胞维持和分化相关基因WUS和CLV3在WRKY71-1D中表达上调。ChIP和EMSA实验证明WRKY71仅能与AP3启动子的W-box结合,而不能与UFO和WUS结合,表明WRKY71通过直接或间接地上调上述基因引起花器官发育提前和花器官增大。总之,拟南芥WRKY71通过直接上调LFY,间接上调AP1、CAL和FUL来促进开花起始；通过直接上调AP3,间接上调AP1和AG而促进花器官发育；间接上调UFO而促进花器官增大；间接上调WUS和CLV3而提高顶端分生组织活性而加快茎顶端分生组织干细胞的增殖和分化。3.WRKY71调控拟南芥分枝发育的功能研究3.1 WRKY71-1D突变体分枝数目增多对长日照下生长7周的Col-0、WRKY71-1D和wrky71-1进行了株高、角果数、分枝数目和附着枝数目统计,发现：三者的平均株高分别为34.2±3.4、9.1±0.6、34.7±2.8cm；平均结荚率分别为128.7±30.2、62.5±18.8、129.6±32.2个；分枝总数分别为55.1±6.1、27.3±2.3、25.3±3.6个；附着枝数分别为22.6±5.3、1.1±0.8、1.2±0.4个。3.2 WRKY71-1D的腋芽不休眠组织学纵向切片分析发现,WRKY71-1D植株腋芽出现在13天,15天时已膨大,而Col-0和wrky71-1在15天还未见腋芽发生。扫描电镜观察发现,WRKY71-1D莲座叶腋芽在21天已见花原基,而Col-0仅分化出叶子,wrky71-1仅见叶原基。生长至32天时,WRKY71-1D腋芽生长发育成侧枝,而Col-0和wrky71-1侧芽仍处于休眠状态,表明WRKY71参与调控了拟南芥侧枝的形成和发育过程。3.3 WRKY71在叶腋处表达通过RNA原位杂交分析发现,WRKY71在Col-0的叶腋处表达,进一步表明WRKY71参与了拟南芥侧枝的形成进程。3.4 WRKY71促进分枝基因的表达调控分枝发育相关基因的表达分析发现,WRKY71-1D中RAX2表达明显的上调,LAS表达微弱的上调。EMSA实验证明,WRKY71与RAX2启动子的W-box结合,表明WRKY71通过直接上调RAX2而引起拟南芥的多分枝。3.5 WRKY71-1D维管束发育缺陷对Col-0、WRKY71-1D和wrky71-1茎的横切面结构的观察发现,WRKY71-1D茎很细,仅有6个维管束,比Col-0少两个；其束间形成层细胞层数比Col-0多1-2层,表明WRKY71-1D的茎维管束系统发育有缺陷。已知拟南芥维管束发育与生长素的功能密切相关,尤其是生长素运输能力对维管束发育影响大,因此,推测WRKY71可能参与了生长素运输的调控。3.6 WRKY71促进生长素的运输离体节点实验分别将带有腋芽的Col-0、WRKY71-1D和wrky71-1茎段上端插入含有1μM 2,4-D的1/2MS培养基中,下端插入不含2,4-D的1/2MS培养基中,腋芽朝上培养7天,发现Col-0和wrky71-1腋芽有一定程度的伸长生长,而WRKY71-1D腋芽未见生长,推测在WRKY71-1D中侧芽生长的抑制可能与其生长素运输能力的增强有关。生长素运输基因表达生长素运输相关基因的表达分析发现,PIN1在WRKY71-1D中上调表达,表明WRKY71可能通过提高生长素运输能力对分枝发育起作用。3.7 WRKY71不影响生长素的生物合成分析生长素合成相关基因表达水平发现,NIT4和CYP79B3在WRKY71-1D中下调表达。但HPLC分析发现,Col-0、WRKY71-1D和wrky71-1中自由IAA含量相近,表明WRKY71不影响拟南芥体内生长素的合成。3.8 WRKY71不影响生长素的信号转导将生长4天的Col-0、WRKY71-1D和wrky71-1转入含10nM和1μM的IAA的1/2MS培养基上生长至14天,发现三系的主根、侧根数目及其长度变化不大,表明WRKY71-1D能正常的响应生长素信号。RT-PCR分析发现,生长素信号转导相关基因在三系中表达差异不明显,表明WRKY71可能不参与生长素的信号转导。结论,WRKY71通过直接上调RAX2引起了拟南芥分枝增多,通过提高生长素的运输能力对分枝发育起作用。更多还原

【Abstract】 In the long process of evolution, plants have formed a set of mechanisms for regulating their own growth and development to adapt to and resist external biotic and abiotic stresses, in which transcription factors (WRKY, DREB, NAC, MYB, etc.) played important roles.WRKY transcription factor family has been identified specifically in plants within the last decade. Their expressions are non-constitutive and induced by biotic and abiotic stress (salicylic acid, pathogen elicitors, high salinity, drought, low temperature etc.) They are mainly involved in biotic and abiotic stress responses and senescence, while barely involved in the regulation of plant development except seed and trichome development. However, their roles in regulating plant flower and branch development have not been demonstrated.Flowering, which is plastic, is the key point for plant to transit from vegetative to reproductive phase. Affected by various external and internal cues, plants will choose to flower at an appropriate time to achieve reproductive success. Flowering is a complex process and multiple factors are involved in this system. So far, there are four major pathways controlling flowering time in Arabidopsis:the photoperiod, vernalization, autonomous and GA pathways. Studies have shown that other factors are also involved in flowering regulation, such as sucrose and abiotic stress factors. Some external stress factors (drought, UV and pathogens) are able to promote flowering, which is a kind of adaptive mechanisms to avoid stress for plants. Typically, high salinity stress will inhibit flowering, and the related mechanism has been reported. However, there is no report about high salinity stress promoting flowering.Shoot branching is from the axillary meristem (AM) which derived from shoot apical meristem, and has two key processes:formation of AM in the axils of leaves and the outgrowth of AM (Growth of lateral bud). Branching determines the aerial architecture of plant and is closely related with crop yields. Branch development is affected by various external environmental conditions, genetic factors and plant hormones. Currently, a number of genes regulating plant branch development have been identified. So far, there is no report about the WRKY transcription factors regulating plant branch development.In this study, from a collection of several thousand independent activation tagging Arabidopsis lines, we screened one line, D27, which was early flowering and dwarf, and had bigger floral organs and more branches compared with Col-0. Using RT-PCR and Tail-PCR, we indicated that the phenotype of D27 was caused by the activation of WRKY71. Genetic analysis showed that the mutant phenotype was stable. On this basis, using Col-0, D27 (WRKY71-1D) and the knockout mutant wrky71-1, we studied the function of WRKY71 on Arabidopsis flowering under normal and high salinity stress conditions, revealed the molecular mechanisms that WRKY71 mediated salinity-inducible early flowering in Arabidopsis, analyzed the contribution of WRKY71 to the branch development of Arabidopsis and its possible mechanism, and provided the basis for further clarification of the molecular mechanism of flower and branch development.The main process and results of this research were as follows:1. Identity of WRKY71 transcription factor and preliminary analysis of its function1.1 Mutant selection and gene identityFrom a collection of several thousand independent activation tagging Arabidopsis lines, we screened one line, D27, which was dwarf and early flowering, and had bigger floral organs and more branches compared with wild type Col-0. Using RT-PCR and Tail-PCR, we indicated that the phenotype of D27 was caused by the activation of WRKY71. In addition, we obtained a homozygous wrky71-1 knockout mutant.1.2 Expression pattern of WRKY71Temporal and special expression pattern:Real time-PCR analysis indicated that WRKY71 was gradually up-regulated within the first three weeks in Col-0 and then down-regulated. WRKY71 was expressed in all tissues, especially in silique. GUS staining showed that WRKY71 was also expressed in all tissues, especially in root, trichome and stipule. The sub-cellular localization of WRKY71 gene product was limited to the nucleus of the onion epidermal cells and Arabidopsis protoplasts. WRKY71 possessed transcriptional activation activity.Response to abiotic stress and hormone:Real time-PCR analysis indicated that WRKY71 was induced by high salinity and ABA. The high salinity induction of WRKY71 appeared to operate via an ABA-dependent pathway.1.3 WRKY71 mediates salt-inducible early flowering in ArabidopsisOne-week old Col-0, WRKY71-1D and wrky71-1 plants grown in soil were watered twice with 1L 200mM NaCl (at 7th and 10th day). Under this conditions, WRKY71-1D bolted at 23rd day, which was only delayed by one day compared to under non-stressed conditions, while both Col-0 and wrky71-1 bolted at 35th day, which was delayed by one week compared to under non-stressed conditions, indicating that the flowering of WRKY71-1D was insensitive to high salinity.2. The molecular mechanism of WRKY71 in regulating flower development2.1 WRKY71 promotes flowering in ArabidopsisThe WRKY71-overexpression lines showed similar phenotype with WRKY71-1D, which confirmed that the phenotype of WRKY71-1D was caused by the activation of WRKY71.2.2 WRKY71 is expressed in inflorescence meristem, flower and flower organ primordiaIn situ hybridization showed that WRKY71 was expressed in inflorescence meristem and flower primordia of 15-day-old Col-0 and in inflorescence meristem and flower organ primordia of 17-day-old Col-0, indicating that WRKY71 may be involved in the formation of inflorescence meristem and flower development.2.3 WRKY71 promotes floral transitionHistology section showed that the floral transition of Col-0, WRKY71-1D and wrky71-1 was as follows:all WRKY71-1D initiated at 11th day,90.4±5.7% of Col-0 initiated at 15th day, while only 37.6±3.3% of the wrky71-1 mutant initiated at 15th day. Observation by using SEM confirmed this result, suggesting that WRKY71 promoted floral transition of Arabidopsis.2.4 WRKY71 promotes floral transition under high salinity stress conditionHistology section showed that the floral transition of Col-0, WRKY71-1D and wrky71-1 under high salinity stress condition was as follows:all WRKY71-1D initiated at 12nd day,32.7±3.3% of Col-0 initiated at 17th day, while only 21.6±2.9% of the wrky71-1 mutant initiated at 17th day, indicating that the floral transition of WRKY71-1D was insensitive to high salinity. Thus WRKY71 also promoted floral transition of Arabidopsis under high salinity stress condition.In conclusion, WRKY71 is induced by high salinity, the overexpression of WRKY71 promotes floral transition in Arabidopsis. Therefore, WRKY71 mediates salt-inducible early flowering in Arabidopsis.2.5 WRKY71 may be not involved in four major flowering pathwaysCol-0, WRKY71-1D and wrky71-1 were all responded normally to day-length changes, prolonged vernalization treatment and GA application. Under these conditions, the flowering time of WRKY71-1D was still earlier than that of Col-0 and wrky71-1. The expression of genes involved in the four major pathways was unaltered in WRKY71-1D, as was the expression of WRKY71 in any of flowering mutants (gi2, co,ft,fve-4, ld-1, fld-1,fca-9). Thus, WRKY71 may be not involved in four major flowering pathways.2.6 WRKY71 promotes flowering of Arabidopsis via affecting sucrose transportRT-PCR analysis indicated that the expression of genes related with sucrose biosynthesis and signaling transduction was similar in Col-0, WRKY71-1D and wrky71-1, indicating that WRKY71 may be not involved in sucrose biosynthesis and signaling transduction. Sucrose transporter genes SUC8 and SUC9 were down-regulated in WRKY71-1D, EMSA experiment showed that WRKY71 was able to interact with the W-boxes of SUC8 and SUC9 promoter, suggesting that WRKY71 repressed SUC8 and SUC9 directly, and increased the extracellular sucrose level to promote flowering.2.7 WRKY71 promotes floral identity genesReal time-PCR analysis indicated that under both control and salinity-stressed conditions, floral identity genes LFY, API, CAL and FUL were up-regulated in WRKY71-1D, consistent with the early flowering phenotype under both control and salinity-stressed conditions.2.8 WRKY71 binds to LFY promoter W-boxEMSA experiment showed that WRKY71 was able to strongly interact with the LFY promoter W-box, weakly interact with the CAL promoter W-box, not able to interact with the API promoter W-box. ChIP experiment also showed that WRKY71 was able to strongly interact with the LFY promoter W-box, while not able to interact with the CAL promoter W-box, indicating that WRKY71 accelerated flowering of Arabidopsis via up-regulating LFY directly.2.9 Cross complementaryGUS staining of WRKY71-1D×LFY::GUS and LFY::GUS showed that the GUS activity of the former was higher than that of the latter, suggesting that WRKY71 was able to promote the translation level of LFY.WRKY71-1D×lfy showed similar phenotype with WRKY71-1D, but did not flower and produce fruits. These results suggested that the loss of LFY arrested the flowering of WRKY71-1D, which confirmed that WRKY71 promoted flowering via up-regulating LFY directly.2.10 WRKY71 promotes floral organ developmentThe sepal, petal, stamen and carpel of WRKY71-1D were all bigger than that of Col-0 and wrky71-1, and developed rapidly. Real time-PCR analysis showed that floral organ development related genes AP1, AP3 and AG, floral organ size related gene UFO and stem cell maintenance and differentiation related genes WUS and CLV3 were up-regulated in WRKY71-1D. ChIP and EMSA experiments also showed that WRKY71 was only able to interact with the AP3 promoter W-box, but not UFO and WUS, suggesting that WRKY71 accelerated floral organ development and increases floral organ size via directly or indirectly up-regulating the above genes.In all, WRKY71 promotes flowering via directly up-regulating LFY, indirectly up-regulating API, CAL and FUL, and directly down-regulating SUC8 and SUC9; promotes floral organ development via directly up-regulating AP3, indirectly up-regulating API and AG; increases floral organ size via indirectly up-regulating UFO; activates shoot apical meristem to accelerate the proliferation and differentiation of the stem cells via indirectly up-regulating WUS and CLV3.3. The roles of WRKY71 in regulating branch development of Arabidopsis3.1 WRKY71-1D has more branchesPlant height and the number of silique, branch and accessory paraclade of 7-week-old Col-0, WRKY71-1D and wrky71-1 were as follows:their average height was 34.2±3.4,9.1±0.6 and 34.7±2.8 cm respectively; the number of their average silique was 128.7±30.2,62.5±18.8 and 129.6±32.2 respectively; the number of their average branch was 55.1±6.1,27.3±2.3 and 25.3±3.6 respectively; the number of their average branch was respectively 22.6±5.3, 1.1±0.8 and 1.2±0.4.3.2 Axillary buds of WRKY71-1D are not dormantHistology section showed that the axillary buds of WRKY71-1D formed at 13rd day and expanded at 15th day, while that of Col-0 and wrky71-1 had not yet formed. Observation by using SEM showed that at 21st day, axillary buds of WRKY71-1D developed floral primordia, that of Col-0 developed leaf, and that of wrky71-1 only developed leaf primordia. At 32nd day, axillary buds of WRKY71-1D outgrew and developed into branches, while that of Col-0 and wrky71-1 were still dormant. These results suggesting that WRKY71 may be involved in the formation and development of Arabidopsis branches.3.3 WRKY71 is expressed in the axilsIn situ hybridization showed that WRKY71 was expressed in the axils of Col-0, further indicating that WRKY71 may be involved in the formation of Arabidopsis branches.3.4 WRKY71 promotes genes related with branch developmentAnalysis the expression of genes related with branch development showed that RAX2 was significantly up-regulated in WRKY71-1D, LAS was slightly up-regulated in WRKY71-1D. EMS A experiment showed that WRKY71 was able to strongly interact with the RAX2 promoter W-box, suggesting that WRKY71 increased Arabidopsis branches via up-regulating RAX2 directly.3.5 The vascular bundle of WRKY71-1D is deficientHistology section showed that the stem of WRKY71-1D was thin, and had only 6 vascular bundles which was two less than that of Col-0; its cell layers of interfascicular cambium was one or two less than that of Col-0, indicating that the vascular bundle of WRKY71-1D was deficient. According to the present knowledge, the function of auxin is closely related with vascular bundle development, especially the auxin transport. Thus, WRKY71 may be involved in regulating the auxin transport.3.6 WRKY71 promotes auxin transportExciting node assay:The the upper part of Col-0, WRKY71-1D and wrky71-1 stems with axillary buds were inserted into the 1/2MS medium containing 1μM 2,4-D and the bottom part into the 1/2MS medium, The stems were cultured for 7 days with the buds upward. The axillary bud of Col-0 and wrky71-1 outgrew to some extent, while that of WRKY71-1D did not outgrow, suggesting that the inhibition of the axillary bud in WRKY71-1D was probably related with the enhance of auxin transport.3.7 WRKY71 does not affect the biosynthesis of auxinAnalysis the expression of genes related with auxin biosynthesis showed that N1T4 and CYP79B3 were down-regulated in WRKY71-1D. However, HPLC analysis indicated that the content of free IAA in Col-0, WRKY71-1D and wrky71-1 was similar, suggesting that WRKY71 did not affect the biosynthesis of auxin. 3.8 WRKY71 does not affect the transduction of auxin signaling Four-day-old Col-0, WRKY71-1D and wrky71-1 were transferred to the 1/2MS medium containing 10nM or 1μM IAA and continued growing to 14th day, the primary root and the number and length of lateral root were similar in three lines, indicating that WRKY71-1D responded normally to auxin signaling. RT-PCR analysis showed that the expression of genes related with transduction of auxin signaling was similar in in three lines, suggesting that WRKY71 may be not involved in the transduction of auxin signaling.Expression of genes related with auxin transport:Analysis of the expression of genes related with auxin transport showed that PIN1 was up-regulated in WRKY71-1D, suggesting that WRKY71 may promote auxin transport to act on branch development.In conclusion, WRKY71 is able to increase Arabidopsis branches via directly up-regulating RAX2, promote auxin transport to act on branch development.更多还原