Dong-Hui Wang

Genome-Wide ORFeome Cloning and Analysis of Arabidopsis Transcription Factor Genes

Here, we report our effort in generating an ORFeome collection for the Arabidopsis transcription factor (TF) genes. In total, ORFeome clones representing 1,282 Arabidopsis TF genes have been obtained in the Gateway high throughput cloning pENTR vector, including 411 genes whose annotation lack cDNA support. All the ORFeome inserts have also been mobilized into a yeast expression destination vector, with an estimated 85% rate of expressing the respective proteins. Sequence analysis of these clones revealed that 34 of them did not match with either the reported cDNAs or current predicted open-reading-frame sequences. Among those, novel alternative splicing of TF gene transcripts is responsible for the observed differences in at least five genes. However, those alternative splicing events do not appear to be differentially regulated among distinct Arabidopsis tissues examined. Lastly, expression of those TF genes in 17 distinct Arabidopsis organ types and the cultured cells was profiled using a 70-mer oligo microarray.

DNA damage in the early primordial anther is closely correlated with stamen arrest in the female flower of cucumber (Cucumis sativus L.)

To investigate the regulatory mechanisms of sex expression in cucumber, morphological observations and biochemical analyses were carried out on inappropriate stamen development of female flowers of cucumber. It was found that developmental arrest of the inappropriate stamen mainly occurs at the anther primordium. This arrest is closely correlated with DNA damage, as detected by TUNEL assay, and might result from anther-specific DNase activation. It was also found that the DNA damage does not lead to cell degeneration, although chromatin condensation is observed in the anther primordia.

Stamen development in Arabidopsis is arrested by organ-specific overexpression of a cucumber ethylene synthesis gene CsACO2

Cucumber (Cucumis sativus L.) has served as a model to understand hormone regulation in unisexual flower development since the 1950s and the role of ethylene in promoting female flower development has been well documented. Recent studies cloned the F-locus in gynoecious lines as an additional copy of the ACC synthase (ACS) gene, which further confirmed the role of ethylene in the promotion of female cucumber flowers. However, no direct evidence was generated to demonstrate that increases in endogenous ethylene production could induce female flowers by arresting stamen development. To clarify the relationship between ethylene production and stamen development, we overexpressed the ethylene synthesis cucumber gene CsACO2 to generate transgenic Arabidopsis, driven by the organ-specific promoter PAP3. We found that organ-specific overexpression of CsACO2 significantly affected stamen but not carpel development, similar to that in the female flowers of cucumber. Our results suggested that increases in ethylene production directly disturb stamen development. Additionally, our study revealed that among all floral organs, stamens respond most sensitively to exogenous ethylene.

Characterization of an ethylene‐inducible, calcium‐dependent nuclease that is differentially expressed in cucumber flower development

• Production of unisexual flowers is an important mechanism that promotes cross-pollination in angiosperms. We previously identified primordial anther-specific DNA damage and organ-specific ethylene perception responsible for the arrest of stamen development in female flowers, but little is known about how the two processes are linked. • To identify potential links between the two processes, we performed suppression subtractive hybridization (SSH) on cucumber (Cucumis sativus L.) stamens of male and female flowers at stage 6, with stamens at stage 5 of bisexual flowers as a control. • Among the differentially expressed genes, we identified an expressed sequence tag (EST) encoding a cucumber homolog to an Arabidopsis calcium-dependent nuclease (CAN), designated CsCaN. Full-length CsCaN cDNA and the respective genomic DNA sequence were cloned and characterized. The CsCaN protein exhibited calcium-dependent nuclease activity. CsCaN showed ubiquitous expression; however, increased gene expression was detected in the stamens of stage 6 female flowers compared with male flowers. As expected, CsCaN expression was ethylene inducible. It was of great interest that CsCaN was post-translationally modified. • This study demonstrated that CsCaN is a novel cucumber nuclease gene, whose DNase activity is regulated at multiple levels, and which could be involved in the primordial anther-specific DNA damage of developing female cucumber flowers.

The B-box family gene STO (BBX24) in Arabidopsis thaliana regulates flowering time in different pathways.

Flowering at the appropriate time is crucial for reproductive success and is strongly influenced by various pathways such as photoperiod, circadian clock, FRIGIDA and vernalization. Although each separate pathway has been extensively studied, much less is known about the interactions between them. In this study we have investigated the relationship between the photoperiod/circadian clock gene and FRIGIDA/FLC by characterizing the function of the B-box STO gene family. STO has two B-box Zn-finger domains but lacks the CCT domain. Its expression is controlled by circadian rhythm and is affected by environmental factors and phytohormones. Loss and gain of function mutants show diversiform phenotypes from seed germination to flowering. The sto-1 mutant flowers later than the wild type (WT) under short day growth conditions, while over-expression of STO causes early flowering both in long and short days. STO over-expression not only reduces FLC expression level but it also activates FT and SOC1 expression. It also does not rely on the other B-box gene CO or change the circadian clock system to activate FT and SOC1. Furthermore, the STO activation of FT and SOC1 expression is independent of the repression of FLC; rather STO and FLC compete with each other to regulate downstream genes. Our results indicate that photoperiod and the circadian clock pathway gene STO can affect the key flowering time genes FLC and FT/SOC1 separately, and reveals a novel perspective to the mechanism of flowering regulation.

Why is ethylene involved in selective promotion of female flower development in cucumber

Our recent work by Wang et al (2010), together with previous studies published in the last decade, have provided evidence suggesting a link between ethylene signaling and primordial anther specific DNA damage in female cucumber flowers. These findings explained ethylene promotion of female flower by ethylene inhibition of stamen development. However, it is not determined if ethylene promotes carpel development. In addition, an explanation of why the naturally occurring gas ethylene was selected to be involved in such developmental events remains elusive. In this study, we carried out a phylogenetic analysis of cucumber ACS genes, and analyzed the expression levels of some pre-miRNAs in male, female and hermaphrodite flowers. We found the M gene might have evolved prior to, or “co-opted” into unisexual flower development before the F gene, and miRNA might be involved in carpel development regulation. Based on these observations, we propose a new hypothesis to explain why ethylene was selected to be involved in the evolution of the unisexual cucumber flower.

A simple treatment to significantly increase signal specificity in immunohistochemistry

It is increasingly important to understand a gene’s function at the protein level. Immunohistochemistry is used to identify new functions of phytohormones by finding their precise localization patterns. To facilitate protein immunolocalization, we developed a protocol that can significantly reduce background and give highly specific signals of antibody to traget proteins. The key improvement is to treat secondary antibodies by mixing with homogenized fresh and fixed plant tissue. Using this protocol, we have successfully carried out immunolocalization of proteins encoded by genesAP3 andHoMADS2, as well as plant hormones ABA and IAA.

CsAP3: A Cucumber Homolog to Arabidopsis APETALA3 with Novel Characteristics

In our previous efforts to understand the regulatory mechanisms of cucumber unisexual flower development, we observed a stamen-specific down-regulation of the ethylene receptor CsETR1 in stage 6 female flowers of cucumber (Cucumis sativus L.). This down-regulation is correlated with the primordial anther-specific DNA damage that characterizes inappropriate stamen development in cucumber female flowers. To understand how CsETR1 is down regulated in the stamen, we characterized a cucumber MADS box gene homologous to Arabidopsis AP3, CsAP3. We demonstrated that CsAP3 is functionally equivalent to the Arabidopsis B-class MADS gene AP3. However, three novel characteristics of CsAP3 were found. These include firstly, binding and activating CsETR1 promoter in vitro and in vivo; secondly, containing a GV repeat in its C-terminus, which is conserved in cucurbits and required for the transcription activation; and thirdly, decreased expression as the node number increases, which is similar to that found for CsETR1. These findings revealed not only the conserved function of CsAP3 as a B-class floral identity gene, but also its unique functions in regulation of female flower development in cucumber.

Transcription Factor OsTGA10 Is a Target of the MADS Protein OsMADS8 and Is Required for Tapetum Development

The transcription factor OsTGA10, a target of OsMADS8, regulates tapetum development via direct binding to other tapetum-regulatory factors and to the regulatory regions of genes involved in tapetum development. This study aimed at elucidating regulatory components behind floral organ identity determination and tissue development. It remains unclear how organ identity proteins facilitate development of organ primordia into tissues with a determined identity, even though it has long been accepted that floral organ identity is genetically determined by interaction of identity genes according to the ABC model. Using the chromatin immunoprecipitation sequencing technique, we identified OsTGA10, encoding a bZIP transcription factor, as a target of the MADS box protein OsMADS8, which is annotated as an E-class organ identity protein. We characterized the function of OsTGA10 using genetic and molecular analyses. OsTGA10 was preferentially expressed during stamen development, and mutation of OsTGA10 resulted in male sterility. OsTGA10 was required for tapetum development and functioned by interacting with known tapetum genes. In addition, in ostga10 stamens, the hallmark cell wall thickening of the endothecium was defective. Our findings suggest that OsTGA10 plays a mediator role between organ identity determination and tapetum development in rice stamen development, between tapetum development and microspore development, and between various regulatory components required for tapetum development. Furthermore, the defective endothecium in ostga10 implies that cell wall thickening of endothecium is dependent on tapetum development.