Yanchong Yu

WRKY71 accelerates flowering via the direct activation of FLOWERING LOCUS T and LEAFY in Arabidopsis thaliana

Flowering is crucial for achieving reproductive success. A large number of well-delineated factors affecting flowering are involved in complex genetic networks in Arabidopsis thaliana. However, the underlying part played by the WRKY transcription factors in this process is not yet clear. Here, we report that WRKY71 is able to accelerate flowering in Arabidopsis. An activation-tagged mutant WRKY71-1D and a constitutive over-expresser of WRKY71 both flowered earlier than the wild type (WT). In contrast, both the RNA interference-based multiple WRKY knock-out mutant (w71w8 + 28RNAi) and the dominant repression line (W71-SRDX) flowered later. Gene expression analysis showed that the transcript abundance of the flowering time integrator gene FLOWERING LOCUS T (FT) and the floral meristem identity genes LEAFY (LFY), APETALA1 (AP1) and FRUITFULL (FUL) were greater in WRKY71-1D than in the WT, but lower in w71w8 + 28RNAi and W71-SRDX. Further, WRKY71 was shown to bind to the W-boxes in the FT and LFY promoters in vitro and in vivo. The suggestion is that WRKY71 activity hastens flowering via the direct activation of FT and LFY.

Repression of callus initiation by the miRNA-directed interaction of auxin–cytokinin in Arabidopsis thaliana

In tissue culture systems plant cells can be induced to regenerate to whole plants. A particularly striking example of cellular reprogramming is seen in this regeneration process, which typically begins with the induction of an intermediate cell mass referred to as callus. The identity of the key genetic cues associated with callus formation is still largely unknown. Here a microRNA-directed phytohormonal interaction is described which represses callus initiation and formation in Arabidopsis thaliana. miR160 and ARF10 (At2g28350), a gene encoding an auxin response factor, were shown to exhibit a contrasting pattern of transcription during callus initiation from pericycle-like cells. The callus initiation is faster and more prolific in a miR160-resistant form of ARF10 (mARF10), but slower and less prolific in the transgenic line over-expressing miR160c (At5g46845), arf10 and arf10 arf16 mutants than that in the wild type. ARF10 repressed the expression of Arabidopsis Response Regulator15 (ARR15, At1g74890) via its direct binding to the genes promoter. The loss of function of ARR15 enhanced callus initiation and partly rescued the phenotype induced by the transgene Pro35S:miR160c. Overexpression of ARR15 partly rescues the callus initiation defect of mARF10 plants. Our findings define miR160 as a key repressor of callus formation and reveal that the initiation of callus is repressed by miR160-directed interaction between auxin and cytokinin.

The effect of the heterologous expression of Phragmites australis γ-glutamylcysteine synthetase on the Cd2+ accumulation of Agrostis palustris

Heavy metal pollution has become one of the most serious environmental problems today. To develop a more efficient plant to clean up heavy metal contaminated soils, a gamma-glutamylcysteine synthetase (GCS) cDNA, named PaGCS, was isolated by PCR from Phragmites australis. The PaGCS sequence was transformed via agroinfection into the heavy metal intolerant grass Agrostis palustris. Five confirmed transgenic A. palustris plants expressing PaGCS were compared with the wild-type line for growth and Cd(2+) accumulation, as well as for the expression of a number of phytochelatin synthesis and stress-responsive enzymes when challenged with Cd(2+) stress. GCS and phytochelatin synthase (PCS) were up-regulated in the transgenic lines. All the transgenic lines accumulated more Cd(2+) and phytochelatins (PCs) than the wild-type line, and three of the five lines grew more effectively than the wild-type after either five or 21 d of Cd(2+) stress. Variation among the transgenics was observed for the distribution of Cd(2+) in the root, shoot and leaf. The malondialdehyde content of all the transgenic lines was lower than that of the wild type under Cd(2+) treatment, while the activity of both superoxide dismutase and peroxidase present in the transgenic lines increased markedly 24 h after Cd(2+) stress, and then rapidly declined.

Genome-wide identification of soybean WRKY transcription factors in response to salt stress

Members of the large family of WRKY transcription factors are involved in a wide range of developmental and physiological processes, most particularly in the plant response to biotic and abiotic stress. Here, an analysis of the soybean genome sequence allowed the identification of the full complement of 188 soybean WRKY genes. Phylogenetic analysis revealed that soybean WRKY genes were classified into three major groups (I, II, III), with the second group further categorized into five subgroups (IIa–IIe). The soybean WRKYs from each group shared similar gene structures and motif compositions. The location of the GmWRKYs was dispersed over all 20 soybean chromosomes. The whole genome duplication appeared to have contributed significantly to the expansion of the family. Expression analysis by RNA-seq indicated that in soybean root, 66 of the genes responded rapidly and transiently to the imposition of salt stress, all but one being up-regulated. While in aerial part, 49 GmWRKYs responded, all but two being down-regulated. RT-qPCR analysis showed that in the whole soybean plant, 66 GmWRKYs exhibited distinct expression patterns in response to salt stress, of which 12 showed no significant change, 35 were decreased, while 19 were induced. The data present here provide critical clues for further functional studies of WRKY gene in soybean salt tolerance.

Evolutionary and Functional Analysis of Membrane-Bound NAC Transcription Factor Genes in Soybean

Multiple levels of functional divergence contributed to gene retention after whole-genome duplication in the soybean membrane-bound NAC transcription factors gene family. Functional divergence is thought to be an important evolutionary driving force for the retention of duplicate genes. We reconstructed the evolutionary history of soybean (Glycine max) membrane-bound NAC transcription factor (NTL) genes. NTLs are thought to be components of stress signaling and unique in their requirement for proteolytic cleavage to free them from the membrane. Most of the 15 GmNTL genes appear to have evolved under strong purifying selection. By analyzing the phylogenetic tree and gene synteny, we identified seven duplicate gene pairs generated by the latest whole-genome duplication. The members of each pair were shown to have variously diverged at the transcriptional (organ specificity and responsiveness to stress), posttranscriptional (alternative splicing), and protein (proteolysis-mediated membrane release and transactivation activity) levels. The dormant (full-length protein) and active (protein without a transmembrane motif) forms of one pair of duplicated gene products (GmNTL1/GmNLT11) were each separately constitutively expressed in Arabidopsis (Arabidopsis thaliana). The heteroexpression of active but not dormant forms of these proteins caused improved tolerance to abiotic stresses, suggesting that membrane release was required for their functionality. Arabidopsis carrying the dormant form of GmNTL1 was more tolerant to hydrogen peroxide, which induces its membrane release. Tolerance was not increased in the line carrying dormant GmNTL11, which was not released by hydrogen peroxide treatment. Thus, NTL-release pattern changes may cause phenotypic divergence. It was concluded that a variety of functional divergences contributed to the retention of these GmNTL duplicates.

Genetic and biochemical characterization of somatic hybrids between Bupleurum scorzonerifolium and Gentianopsis paludosa

Ultraviolet-irradiated protoplasts of Gentianopsis paludosa were fused with those of Bupleurum scorzonerifolium and 28 independent hybrid calli were identified, five of which later differentiated into plants. A genetic analysis of these calli and regenerated plants based on chromosome number, esterase, random amplified polymorphic DNA, and 5S rDNA spacer profiling showed that the majority of their nuclear genomes were represented by the recipient biparent B. scorzonerifolium. A restriction fragment length polymorphism analysis of the plastidial genomes confirmed that DNA from both biparents was present in some of the hybrids. The secondary metabolite composition of the hybrids was analyzed by a combination of high-performance liquid chromatography and gas chromatography–mass spectrometry analysis. The content of oleanolic acid in two of the hybrid lines was substantially higher than in the donor G. paludosa, while that of swertiamarin was equal to that in G. paludosa in two of the six hybrids analyzed. A number of both G. paludosa and B. scorzonerifolium specific compounds were detected in the three hybrids analyzed by GC–MS as were several not present in either of the biparents.

Membrane-bound NAC transcription factors in maize and their contribution to the oxidative stress response

NAC membrane-bound transcription factors (NTM1-like, NTL proteins) participate in the regulation of plant development and the abiotic stress response. While their function has been thoroughly explored in Arabidopsis thaliana, this is not the case in maize. Seven ZmNTL genes were identified by an in silico scan of relevant genome sequence. All seven included a NAC domain at their N terminus, and an α-helical membrane-bound structure domain in their C terminal region. Based on their gene structure and content of conserved motifs, the seven sequences were distributed into four clades. Six of the seven ZmNTLs were associated with the plasma membrane, and the remaining one with the endoplasmic reticulum. ZmNTL2-7 were more strongly transcribed in the stem than in either the leaf or root, while ZmNTL1 transcript abundance was highest in the leaf. When the plants were exposed to either abscisic acid or hydrogen peroxide treatment, all seven genes were up-regulated in the root and stem and down-regulated in the leaf. The heterologous expression of ZmNTL1-ΔTM, 2-ΔTM and 5-ΔTM in A. thaliana reduced the level of sensitivity of the plant to hydrogen peroxide.

WRKY71 Acts Antagonistically Against Salt-Delayed Flowering in Arabidopsis thaliana

Soil salinity affects various aspects of plant growth and development including flowering. Usually, plants show a delayed flowering phenotype under high salinity conditions, whereas some plants will risk their life to continue to grow, thereby escaping serious salt stress to achieve reproductive success. However, the molecular mechanisms of the escape strategies are not clear yet. In this work, we report that the transcription factor WRKY71 helps escape salt stress in Arabidopsis. The expression of the WRKY71 wild-type (WT) allele was salinity inducible. Compared with Col-0, high salt stress caused only a marginal delay in the flowering time of the activation-tagged mutant WRKY71-1D. However, flowering in the RNA interference (RNAi)-based multiple WRKY knock-out mutant (w71w8 + 28RNAi) was dramatically later than in the WT under high salinity conditions. Meanwhile, expression of FLOWERING LOCUS T (FT) and LEAFY (LFY) was greater in WRKY71-1D than in the WT, and lower in w71w8 + 28RNAi under salinity-stressed conditions. The suggestion is that WRKY71 activity hastens flowering, thereby providing a means for the plant to complete its life cycle in the presence of salt stress.