Shu-Nong Bai

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.

Developmental analyses reveal early arrests of the spore-bearing parts of reproductive organs in unisexual flowers of cucumber (Cucumis sativus L.)

To understand the regulatory mechanisms governing unisexual flower development in cucumber, we conducted a systematic morphogenetic analysis of male and female flower development, examined the dynamic changes in expression of the C-class floral organ identity gene CUM1, and assessed the extent of DNA damage in inappropriate carpels of male flowers. Accordingly, based on the occurrence of distinct morphological events, we divided the floral development into 12 stages ranging from floral meristem initiation to anthesis. As a result of our investigation we found that the arrest of stamen development in female flowers, which occurs just after the differentiation between the anther and filament, is mainly restricted to the primordial anther, and that it is coincident with down-regulation of CUM1 gene expression. In contrast, the arrest of carpel development in the male flowers occurs prior to the differentiation between the stigma and ovary, given that no indication of ovary differentiation was observed even though CUM1 gene expression remained detectable throughout the development of the stigma-like structures. Although the male and female reproductive organs have distinctive characteristics in terms of organ differentiation, there are two common features regarding organ arrest. The first is that the arrest of the inappropriate organ does not affect the entirety of the organ uniformly but occurs only in portions of the organs. The second feature is that all the arrested portions in both reproductive organs are spore-bearing parts.

Duplication and Functional Diversification of HAP3 Genes Leading to the Origin of the Seed-Developmental Regulatory Gene, LEAFY COTYLEDON1 (LEC1), in Nonseed Plant Genomes

The HAP3 gene encodes a subunit of the CCAAT-box-binding factor (CBF), a highly conserved trimeric activator that recognizes and binds the ubiquitous CCAAT promoter element with high affinity. Two types of HAP3 gene have been identified in plant genomes. The LEAFY COTYLEDON1 (LEC1)-type HAP3 genes encode a functionally specialized subunit of CBF, which is expressed specifically in developing seeds. In contrast, most non-LEC1-type HAP3 genes are expressed in various tissues. It has been proposed that the LEC1-type HAP3 genes originated from the duplication and functional divergence of non-LEC1-type HAP3 genes. However, it is not yet known when this duplication event took place or whether the LEC1-type HAP3 genes appeared at the same time as the origin of seed plants. Here we describe a comprehensive comparison of the duplication patterns of HAP3 genes in different plant genomes. We recognize a major expansion of the HAP3 gene family accompanying the origin and early diversification of land plants and postulate that retrotransposition and other mechanisms of gene duplication have been involved in the expansion of the plant HAP3 gene family. We provide evidence that the LEC1-type HAP3 genes originated in nonseed vascular plant genomes and demonstrate that they are inductively expressed under drought stress in nonseed plants. These genes, however, were recruited to a novel regulatory network in the early stages of seed plant evolution and steadily expressed during seed development and maturation.

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.

HDA18 Affects Cell Fate in Arabidopsis Root Epidermis via Histone Acetylation at Four Kinase Genes

HDA18 has deacetylase activity and affects epidermal cell patterning of Arabidopsis roots by regulating the transcription of a set of kinase genes. This set of kinase genes functions in a positional information relay system. Both down- and upregulation of HDA18 expression result in the same phenotype and increase the expression the kinase genes. The differentiation of hair (H) and non-hair (N) cells in the Arabidopsis thaliana root epidermis is dependent on positional relationships with underlying cortical cells. We previously found that histone acetylation relays positional information and that a mutant altered in the histone deacetylase gene family member HISTONE DEACETYLASE 18 (HDA18) exhibits altered H and N epidermal cell patterning. Here, we report that HDA18 has in vitro histone deacetylase activity and that both mutation and overexpression of HDA18 led to cells at the N position having H fate. The HDA18 protein physically interacted with histones related to a specific group of kinase genes, which are demonstrated in this study to be components of a positional information relay system. Both down- and upregulation of HDA18 increased transcription of the targeted kinase genes. Interestingly, the acetylation levels of histone 3 lysine 9 (H3K9), histone 3 lysine 14 (H3K14) and histone 3 lysine 18 (H3K18) at the kinase genes were differentially affected by down- or upregulation of HDA18, which explains why the transcription levels of the four HDA18-target kinase genes increased in all lines with altered HDA18 expression. Our results reveal the surprisingly complex mechanism by which HDA18 affects cellular patterning in Arabidopsis root epidermis.

Molecular analysis of early rice stamen development using organ-specific gene expression profiling

Elucidating the regulatory mechanisms of plant organ formation is an important component of plant developmental biology and will be useful for crop improvement applications. Plant organ formation, or organogenesis, occurs when a group of primordial cells differentiates into an organ, through a well-orchestrated series of events, with a given shape, structure and function. Research over the past two decades has elucidated the molecular mechanisms of organ identity and dorsalventral axis determinations. However, little is known about the molecular mechanisms underlying the successive processes. To develop an effective approach for studying organ formation at the molecular level, we generated organ-specific gene expression profiles (GEPs) reflecting early development in rice stamen. In this study, we demonstrated that the GEPs are highly correlated with early stamen development, suggesting that this analysis is useful for dissecting stamen development regulation. Based on the molecular and morphological correlation, we found that over 26 genes, that were preferentially up-regulated during early stamen development, may participate in stamen development regulation. In addition, we found that differentially expressed genes during early stamen development are clustered into two clades, suggesting that stamen development may comprise of two distinct phases of pattern formation and cellular differentiation. Moreover, the organ-specific quantitative changes in gene expression levels may play a critical role for regulating plant organ formation.

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.

Nectar production and transportation in the nectaries of the female Cucumis sativus L. flower during anthesis

Summary.In an effort to gain a greater understanding of nectar production, we studied the dynamic mechanisms of starch accumulation and transformation and nectar transportation in the Cucumis sativus L. female flower. Starch, which is the main precursor of nectar, accumulates in the epidermis and underlying parenchyma, with the most active accumulation occurring in the parenchyma cells within 3 days prior to anthesis. Thereafter, the starch was successively hydrolyzed and the hydrolyte was transported from the amyloplasts to vacuoles, suggesting that amyloplasts and vacuoles are the centers of nectar production. In addition, we observed few plasmodesmata and the presence of invaginated plasmalemma and electron-dense material in the intercellular spaces, suggesting that the apoplast system is involved in nectar transportation in an ATPase-dependent fashion.

The concept of the sexual reproduction cycle and its evolutionary significance

The concept of a “sexual reproduction cycle (SRC)” was first proposed by Bai and Xu (2013) to describe the integration of meiosis, sex differentiation, and fertilization. This review discusses the evolutionary and scientific implications of considering these three events as part of a single process. Viewed in this way, the SRC is revealed to be a mechanism for efficiently increasing genetic variation, facilitating adaptation to environmental challenges. It also becomes clear that, in terms of cell proliferation, it is appropriate to contrast mitosis with the entire SRC, rather than with meiosis alone. Evolutionarily, it appears that the SRC was first established in unicellular eukaryotes and that all multicellular organisms evolved within that framework. This concept provides a new perspective into how sexual reproduction evolved, how generations should be defined, and how developmental processes of various multicellular organisms should properly be compared.

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.