| 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. |
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