Date Published: August 26, 2013
Publisher: Public Library of Science
Author(s): Xiao Luo, Xiaoli Sun, Baohui Liu, Dan Zhu, Xi Bai, Hua Cai, Wei Ji, Lei Cao, Jing Wu, Mingchao Wang, Xiaodong Ding, Yanming Zhu, Ji-Hong Liu.
Flowering is a critical event in the life cycle of plants; the WRKY-type transcription factors are reported to be involved in many developmental processes sunch as trichome development and epicuticular wax loading, but whether they are involved in flowering time regulation is still unknown. Within this study, we provide clear evidence that GsWRKY20, a member of WRKY gene family from wild soybean, is involved in controlling plant flowering time. Expression of GsWRKY20 was abundant in the shoot tips and inflorescence meristems of wild soybean. Phenotypic analysis showed that GsWRKY20 over-expression lines flowered earlier than the wild-type plants under all conditions: long-day and short-day photoperiods, vernalization, or exogenous GA3 application, indicating that GsWRKY20 may mainly be involved in an autonomous flowering pathway. Further analyses by qRT-PCR and microarray suggests that GsWRKY20 accelerating plant flowering might primarily be through the regulation of flowering-related genes (i.e., FLC, FT, SOC1 and CO) and floral meristem identity genes (i.e., AP1, SEP3, AP3, PI and AG). Our results provide the evidence demonstrating the effectiveness of manipulating GsWRKY20 for altering plant flowering time.
In higher plants, a phase transition from vegetative to reproductive development is one of the most important events in their life history [1,2]. This transition is tightly coordinated through a diverse array of signaling networks that integrate various endogenous and exogenous signals . Flowering time is a key trait in adaptation, as it is vital for reproductive success. Arabidopsis thaliana contains at least four flowering pathways that are responsive to these cues: the photoperiod pathway monitors changes in day length; the gibberellin pathway plays a promotive role in flowering under non-inductive photoperiods; the vernalization pathway senses the prolonged exposure to low temperature; and the autonomous pathway mediates flowering by perceiving plant developmental status [3–5]. Most recently, an endogenous pathway that adds plant age to the control of flowering time has been described . Several genes, such as CONSTANS (CO), FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), and FLOWERING LOCUS C (FLC) have been identified as key components in these flowering signal pathways . CO, which encodes a zinc-finger transcriptional activator, controls the timing of flowering by positively regulating two floral integrators, FT  and SOC1 ; FLC, a flowering repressor gene, also acts as an upstream regulator gene of FT and SOC1 . Moreover, these flowering integrators have been shown to exhibit both overlapping and independent functions in the determination of flowering time and they integrate signals from multiple flowering pathways and their expression levels eventually determine the exact flowering time [3,10].
WRKY TFs have been reported to regulate plant various developments, but no data is available about whether WRKY TFs are involved in flowering time regulation. In the present study, our data provide clear genetic evidence for the function of GsWRKY20 in controlling floral initiation. We found that GsWRKY20 acts as a positive regulator of flowering, since the transgenic plants over-expressing GsWRKY20 demonstrated early flowering compared to WT. Flowering time is known to be coordinated by at least four pathways, namely autonomous, photoperiod, vernalization, and GA pathways . GsWRKY20ox plants flowered earlier than the WT under both LD and SD conditions. Flower development of Arabidopsis is promoted by LD condition and delayed by SD condition . In our case, GsWRKY20ox plants grown under SD condition flowered significantly earlier than WT plants; however, flower production of both WT and transgenic plants was delayed compared to the plants grown under LD condition. Thus, over-expression of GsWRKY20 can accelerate flower formation, but cannot overcome the photoperiodic effect, suggesting that GsWRKY20ox plants were still sensitive to photoperiod and GsWRKY20 should be independent of photoperiod flowering pathway. Vernalization flowering pathway indicates that low temperature treatment of germinating seed can induce early flowering , to judge whether GsWRKY20 was involved in vernalization flowering pathway mainly based on whether vernalization will suppress the early flowering phenotype of the GsWRKY20ox plants, although the transgenic acceptor Col-0 is capable to flower without vernalization, we demonstrated that GsWRKY20ox plants and the WT plants responded normally to vernalization, vernalization treatment promoted flowering of GsWRKY20ox plants and the WT plants compared to the normal condition, but GsWRKY20ox plants still flowered much more earlier than WT plants both in LD and SD conditions, so vernalization cannot suppress the early flowering phenotype of GsWRKY20 transgenic lines. Since the GsWRKY20ox plants showed a normal response to vernalization and exhibited earlier flowering, a role of GsWRKY20 in vernalization flowering pathway was excluded. With this approach, many flowering-related Arabidopsis lines of Columbia (Col-0) background were examined whether they were involved in vernalization flowering pathway. For example, with this approach, the Col-0 background mutants sr45-1  and syp22-1  were demonstrated that they were not involved in vernalization flowering pathway. On the other hand, all GsWRKY20ox plants flowered earlier than WT plant after sprayed with exogenous GA3 or watered with the GA biosynthesis inhibitor PAC, suggesting that GsWRKY20 is excluded from GA3 pathway. From these data, we can conclude that GsWRKY20 was involved in the regulation of flowering time through regulatory pathways other than the above three pathways.