Hong Zhai

Positional cloning and characterization reveal the molecular basis for soybean maturity locus E1 that regulates photoperiodic flowering

The complex and coordinated regulation of flowering has high ecological and agricultural significance. The maturity locus E1 has a large impact on flowering time in soybean, but the molecular basis for the E1 locus is largely unknown. Through positional cloning, we delimited the E1 locus to a 17.4-kb region containing an intron-free gene (E1). The E1 protein contains a putative bipartite nuclear localization signal and a region distantly related to B3 domain. In the recessive allele, a nonsynonymous substitution occurred in the putative nuclear localization signal, leading to the loss of localization specificity of the E1 protein and earlier flowering. The early-flowering phenotype was consistently observed in three ethylmethanesulfonate-induced mutants and two natural mutations that harbored a premature stop codon or a deletion of the entire E1 gene. E1 expression was significantly suppressed under short-day conditions and showed a bimodal diurnal pattern under long-day conditions, suggesting its response to photoperiod and its dominant effect induced by long day length. When a functional E1 gene was transformed into the early-flowering cultivar Kariyutaka with low E1 expression, transgenic plants carrying exogenous E1 displayed late flowering. Furthermore, the transcript abundance of E1 was negatively correlated with that of GmFT2a and GmFT5a, homologues of FLOWERING LOCUS T that promote flowering. These findings demonstrated the key role of E1 in repressing flowering and delaying maturity in soybean. The molecular identification of the maturity locus E1 will contribute to our understanding of the molecular mechanisms by which a short-day plant regulates flowering time and maturity.

GmFT4, a Homolog of FLOWERING LOCUS T, Is Positively Regulated by E1 and Functions as a Flowering Repressor in Soybean

The major maturity gene E1 has the most prominent effect on flowering time and photoperiod sensitivity of soybean, but the pathway mediated by E1 is largely unknown. Here, we found the expression of GmFT4, a homolog of Flowering Locus T, was strongly up-regulated in transgenic soybean overexpressing E1, whereas expression of flowering activators, GmFT2a and GmFT5a, was suppressed. GmFT4 expression was strongly up-regulated by long days exhibiting a diurnal rhythm, but down-regulated by short days. Notably, the basal expression level of GmFT4 was elevated when transferred to continous light, whereas repressed when transferred to continuous dark. GmFT4 was primarily expressed in fully expanded leaves. Transcript abundance of GmFT4 was significantly correlated with that of functional E1, as well as flowering time phenotype in different cultivars. Overexpression of GmFT4 delayed the flowering time in transgenic Arabidopsis. Taken together, we propose that GmFT4 acts downstream of E1 and functions as a flowering repressor, and the balance of two antagonistic factors (GmFT4 vs GmFT2a/5a) determines the flowering time of soybean.

Molecular identification of genes controlling flowering time, maturity, and photoperiod response in soybean

Most plants activate the developmental transition from the vegetative to the reproductive phase in response to photoperiod length, temperature, and other environmental stimuli. Successful identification of major genes underlying flowering time and maturity in soybean is a prerequisite for understanding of the regulation of flowering time. Recent progress has been made toward molecular bases of soybean maturity loci by using both candidate gene and positional cloning approaches. In particular, successful identification of the molecular identity of the soybean maturity locus E1 is a remarkable achievement, because this gene is essential for understanding the regulation of flowering time and maturity in soybean. The E1 gene has a putative bipartite nuclear localization signal, and a domain distantly related to B3. Transcriptional profiling showed the E1 gene is under photoperiodic regulation. The E2 gene in soybean encodes GmGIa, a homolog of Arabidopsis GIGANTEA that has multiple functions involved in the circadian clock and flowering. Both of the E3 and E4 genes encode copies of PHYTOCHROME A proteins, and both genes response differentially to light with different red to far-red quantum (R:FR) ratios. In addition, two homologs (GmFT2a and GmFT5a) of FLOWERING LOCUS T coordinately promote photoperiodic flowering in soybean. Public availability of the soybean genome sequence to the research community will greatly facilitate fine mapping and cloning of more genes underlying flowering time and photoperiodic response. Further research on identified genes will help us to understand the exquisite regulatory network of parallel and intertwining pathways controlling flowering time and photoperiodic response in soybean.

Allelic Variations at Four Major Maturity E Genes and Transcriptional Abundance of the E1 Gene Are Associated with Flowering Time and Maturity of Soybean Cultivars

The time to flowering and maturity are ecologically and agronomically important traits for soybean landrace and cultivar adaptation. As a typical short-day crop, long day conditions in the high-latitude regions require soybean cultivars with photoperiod insensitivity that can mature before frost. Although the molecular basis of four major E loci (E1 to E4) have been deciphered, it is not quite clear whether, or to what degree, genetic variation and the expression level of the four E genes are associated with the time to flowering and maturity of soybean cultivars. In this study, we genotyped 180 cultivars at E1 to E4 genes, meanwhile, the time to flowering and maturity of those cultivars were investigated at six geographic locations in China from 2011 to 2012 and further confirmed in 2013. The percentages of recessive alleles at E1, E2, E3 and E4 loci were 38.34%, 84.45%, 36.33%, and 7.20%, respectively. Statistical analysis showed that allelic variations at each of four loci had a significant effect on flowering time as well as maturity. We classified the 180 cultivars into eight genotypic groups based on allelic variations of the four major E loci. The genetic group of e1-nf representing dysfunctional alleles at the E1 locus flowered earliest in all the geographic locations. In contrast, cultivars in the E1E2E3E4 group originated from the southern areas flowered very late or did not flower before frost at high latitude locations. The transcriptional abundance of functional E1 gene was significantly associated with flowering time. However, the ranges of time to flowering and maturity were quite large within some genotypic groups, implying the presence of some other unknown genetic factors that are involved in control of flowering time or maturity. Known genes (e.g. E3 and E4) and other unknown factors may function, at least partially, through regulation of the expression of the E1 gene.

GmCOL1a and GmCOL1b Function as Flowering Repressors in Soybean Under Long-Day Conditions

CONSTANS (CO) has a central role in the photoperiod response mechanism in Arabidopsis. However, the functions of legume CO genes in controlling flowering remain unknown. Here, we analyze the expression patterns of E1, E2 and GmCOL1a/1b using near-isogenic lines (NILs), and we further analyze flowering-related genes in gmcol1b mutants and GmCOL1a-overexpressing plants. Our data showed that both E3 and E4 up-regulate E1 expression, with the effect of E3 on E1 being greater than the effect of E4 on E1. E2 was up-regulated by E3 and E4 but down-regulated by E1. GmCOL1a/1b were up-regulated by E1, E2, E3 and E4. Although the spatial and temporal patterns of GmCOL1a/1b expression were more similar to those of AtCOL2 than to those of AtCO, gmcol1b mutants flowered earlier than wild-type plants under long-day (LD) conditions, and the overexpression of GmCOL1a caused late flowering under LD or natural conditions. In addition, GmFT2a/5a, E1 and E2 were down-regulated in GmCOL1a-overexpressing plants under LD conditions. Because E1/2 influences the expression of GmCOL1a, and vice versa, we conclude that these genes may function as part of a negative feedback loop, and GmCOL1a/b genes may serve as suppressors in photoperiodic flowering in soybean under LD conditions.

Recent Achievement in Gene Cloning and Functional Genomics in Soybean

Soybean is a model plant for photoperiodism as well as for symbiotic nitrogen fixation. However, a rather low efficiency in soybean transformation hampers functional analysis of genes isolated from soybean. In comparison, rapid development and progress in flowering time and photoperiodic response have been achieved in Arabidopsis and rice. As the soybean genomic information has been released since 2008, gene cloning and functional genomic studies have been revived as indicated by successfully characterizing genes involved in maturity and nematode resistance. Here, we review some major achievements in the cloning of some important genes and some specific features at genetic or genomic levels revealed by the analysis of functional genomics of soybean.

Functional conservation and diversification of the soybean maturity gene E1 and its homologs in legumes

Gene regulatory networks involved in flowering time and photoperiodic responses in legumes remain unknown. Although the major maturity gene E1 has been successfully deciphered in soybean, knowledge on the functional conservation of this gene is limited to a certain extent to E1 homologs in legumes. The ectopic expression of Phvul.009G204600 (PvE1L), an E1 homolog from common bean, delayed the onset of flowering in soybean. By contrast, the ectopic expression of Medtr2g058520 (MtE1L) from Medicago truncatula did not affect the flowering of soybean. Characterization of the late-flowering mte1l mutant indicated that MtE1L promoted flowering in Medicago truncatula. Moreover, all transgenic E1, PvE1L and MtE1L soybean lines exhibited phenotypic changes in terms of plant height. Transgenic E1 or PvE1L plants were taller than the wild-type, whereas transgenic MtE1L plants produced dwarf phenotype with few nodes and short internode. Thus, functional conservation and diversification of E1 family genes from legumes in the regulation of flowering and plant growth may be associated with lineage specification and genomic duplication.

Diurnal Expression Pattern, Allelic Variation, and Association Analysis Reveal Functional Features of the E1 Gene in Control of Photoperiodic Flowering in Soybean.

Although four maturity genes, E1 to E4, in soybean have been successfully cloned, their functional mechanisms and the regulatory network of photoperiodic flowering remain to be elucidated. In this study, we investigated how the diurnal expression pattern of the E1 gene is related to photoperiodic length; and to what extent allelic variation in the B3-like domain of the E1 gene is associated with flowering time phenotype. The bimodal expression of the E1 gene peaked first at around 2 hours after dawn in long-day condition. The basal expression level of E1 was enhanced by the long light phase, and decreased by duration of dark. We identified a 5bp (3 SNP and 2-bp deletion) mutation, referred to an e1-b3a, which occurs in the middle of B3 domain of the E1 gene in the early flowering cultivar Yanhuang 3. Subcellular localization analysis showed that the putative truncated e1-b3a protein was predominately distributed in nuclei, indicating the distribution pattern of e1-b3a was similar to that of E1, but not to that of e1-as. Furthermore, genetic analysis demonstrated allelic variations at the E1 locus significantly underlay flowering time in three F2 populations. Taken together, we can conclude the legume specific E1 gene confers some special features in photoperiodic control of flowering in soybean. Further characterization of the E1 gene will extend our understanding of the soybean flowering pathway in soybean.

Overexpression of Glycine soja WRKY20 enhances drought tolerance and improves plant yields under drought stress in transgenic soybean

Drought stress is a major constraint to the production and yield stability of soybean (Glycine max L. Merrill). Transgenic breeding offers new opportunities for developing drought-resistant varieties. However, soybean is much more difficult to transform than other species; so in our previous studies, several drought-related genes, which were identified from the transcriptome profiles of Glycine soja, were first heterologous expressed in Arabidopsis or alfalfa (Medicago sativa L.) for function characterization. Among these genes, GsWRKY20 shows effective roles in drought tolerance. In this study, to breed high drought-tolerant soybean cultivar, GsWRKY20 was overexpressed in soybean under the control of the cauliflower mosaic virus 35S promoter. We found that transgenic soybean overexpressing GsWRKY20 showed greatly enhanced tolerance to drought stress compared with the non-transformed plants. Under drought stress conditions, lower relative membrane permeability and malondialdehyde (MDA) content were observed in transgenic soybean, indicating a less degree of membrane injury of transgenic plants. Higher antioxidant enzyme activity and more free proline content were observed in transgenic soybean, which help plants to resist drought stress. GsWRKY20 overexpressing soybean plants have lower stomatal density, faster stomatal closure and so exhibited lower stomatal conductance, which reduced water loss under drought stress conditions. GsWRKY20 overexpressing soybean plants exhibited higher yields under drought stress conditions, with higher plant height, longer root, and higher seed yield at the adult developmental stage. In conclusion, the transgenic soybean generated in this study could be used for farming in arid and semi-arid areas that are prone to extremely severe drought stress.

DNA-binding protein phosphatase AtDBP1 acts as a promoter of flowering in Arabidopsis

Main conclusionWe provide evidence thatAtDBP1promotes flowering by regulating the transcript levels of several important integrators and floral meristem identity genes, includingFLC, CO, SOC1, LFY, FTandFD.DNA-binding protein phosphatases (DBP) which exhibit both sequence specific DNA-binding and protein phosphatase 2C activities are important regulators that are involved in both the transcriptional and post-translational regulations. DBP factors are known to mediate susceptibility to potyviruses; however, whether they are involved in other processes is still unclear. In this study, under both long day (LD) and short day conditions, AtDBP1 overexpressing plants displayed early flowering, while the knock out mutants, atdbp1, exhibited a delay in flowering relative to the wild-type plants; both the overexpressing lines and atdbp1 mutants remained photoperiodic sensitive, indicating that AtDBP1 was involved in the autonomous pathway. AtDBP1 does not respond to vernalization at transcript level, and both AtDBP1 overexpressing plants and atdbp1 mutants remain responsive to vernalization, indicating that AtDBP1 may not be directly involved in vernalization. Real-time PCR analysis showed that AtDBP1 can suppress FLOWERING LOCUC C (FLC) expression, a key integrator of the autonomous and vernalization pathways, and enhance the expression levels of CONSTANS and FLOWERING LOCUC T, key regulators of the LD pathway. Furthermore, expression of floral meristem identity genes including SUPPRESSOR OF OVEREXPRESSION OF CO 1, LEAFY and FD was also promoted in AtDBP1overexpressing plants. AtDBP1 transcription can be detected in root, leaf, stem, flower and silique. AtDBP1–GFP and YFP–AtDBP1 fusion protein were localized in the cytosol and nucleus. Our results provide the evidence demonstrating the effective role of AtDBP1 for flowering time regulation and report a novel function of DBP factors in planta besides in plant defense.