Wan-Chen Li

Expression Profile of Maize MicroRNAs Corresponding to Their Target Genes Under Drought Stress

Microarray assay of four inbred lines was used to identify 303 microRNAs differentially expressed under drought stress. The microRNAs were used for bioinformatics prediction of their target genes. The majority of the differentially expressed microRNA families showed different expression profiles at different time points of the stress process among the four inbred lines. Digital gene expression profiling revealed 54 genes targeted by 128 of the microRNAs differentially expressed under the same stress conditions. The differential expression of miR159 and miR168 was further validated by locked nucleic acid northern hybridization. These results indicated that miR159 and miR168, as well as numerous other microRNAs, play critical roles in signaling pathways of maize response to drought stress. However, the level of the post-transcriptional regulation mediated by microRNAs had different responses among genotypes, and the gene expression related to signaling pathways under drought stress is also regulated, possibly by multiple mechanisms.

Interaction between abscisic acid receptor PYL3 and protein phosphatase type 2C in response to ABA signaling in maize

Abscisic acid (ABA) is a ubiquitous hormone that regulates plant growth, development and responses to environmental stresses. In recent researches, pyrabactin resistance 1-like protein (PYL) and protein phosphatase type 2C (PP2C) were identified as the direct receptor and the second component of ABA signaling pathway, respectively. However, a lot of PYL and PP2C members were found in Arabidopsis and several other plants. Some of them were found not to be involved in ABA signaling. Because of the complex diversity of the genome, few documents have been available on the molecular details of the ABA signal perception system in maize. In the present study, we conducted bioinformatics analysis to find out the candidates (ZmPYL3 and ZmPP2C16) of the PYL and PP2C members most probably involved in ABA signaling in maize, cloned their encoding genes (ZmPYL3 and ZmPP2C16), verified the interaction between these two proteins in response to exogenous ABA induction by yeast two-hybrid assay and bimolecular fluorescence complementation, and investigated the expression patterns of these two genes under the induction of exogenous ABA by real-time fluorescence quantitative PCR. The results indicated that the ZmPYL3 and ZmPP2C16 proteins interacted in vitro and in vivo in response to the induction of exogenous ABA. The downregulated expression of the ZmPYL3 gene and the upregulated expression of the ZmPP2C16 gene are responsive to the induction of exogenous ABA. The ZmPYL3 and ZmPP2C16 proteins are the most probable members of the receptors and the second components of ABA signaling pathway, respectively.

Heterologous expression of antifreeze protein gene AnAFP from Ammopiptanthus nanus enhances cold tolerance in Escherichia coli and tobacco.

Antifreeze proteins are a class of polypeptides produced by certain animals, plants, fungi and bacteria that permit their survival under the subzero environments. Ammopiptanthus nanus is the unique evergreen broadleaf bush endemic to the Mid-Asia deserts. It survives at the west edge of the Tarim Basin from the disappearance of the ancient Mediterranean in the Tertiary Period. Its distribution region is characterized by the arid climate and extreme temperatures, where the extreme temperatures range from -30 °C to 40 °C. In the present study, the antifreeze protein gene AnAFP of A. nanus was used to transform Escherichia coli and tobacco, after bioinformatics analysis for its possible function. The transformed E. coli strain expressed the heterologous AnAFP gene under the induction of isopropyl β-D-thiogalactopyranoside, and demonstrated significant enhancement of cold tolerance. The transformed tobacco lines expressed the heterologous AnAFP gene in response to cold stress, and showed a less change of relative electrical conductivity under cold stress, and a less wilting phenotype after 16 h of -3 °C cold stress and thawing for 1h than the untransformed wild-type plants. All these results imply the potential value of the AnAFP gene to be used in genetic modification of commercially important crops for improvement of cold tolerance.

A betaine aldehyde dehydrogenase gene from Ammopiptanthus nanus enhances tolerance of Arabidopsis to high salt and drought stresses

Glycine betaine functions as an osmotic protectant in response of plant to abiotic stresses. In higher plants, the betaine aldehyde dehydrogenase (BADH) catalyzes the key step of glycine betaine biosynthesis. A lot of effort has been paid to cloning and heterologous expression of the BADH genes from different plants. However, different phenotypes were observed from the transgenic plants. Diversity of subcellular location and functions was found among the members of the BADH family. Our previous report described the cloning of an AnBADH gene from xerophyte Ammopiptanthus nanus and preliminary functional validation in Escherichia coli. In the present study, the function of the AnBADH gene for abiotic tolerance was further characterized by quantitative real time PCR and heterologous expression in Arabidopsis mutant. The results showed that the endogenous expression of the AnBADH gene was strongly induced by the treatments of high salt, dehydration, abscisic acid, heat, and cold. The heterologous expression of the AnBADH gene significantly enhanced tolerance of the Arabidopsis mutant to high salt and drought stresses. Under the stress conditions, the transgenic lines exhibited a more robust root system and a larger fresh weight, higher content of glycine betaine and proline, higher relative water content, lower relative electrolyte leakage and malondialdehyde content, compared to the untransformed mutant. These results suggest that the AnBADH gene encodes a functional betaine aldehyde dehydrogenase, and plays a critical role in the adaptation of A. nanus to its harsh habitat. It provides one more choice for genetic improvement of commercial crops for abiotic tolerance.

Cloning and characterization of BES1/BZR1 transcription factor genes in maize

BES1/BZR1 transcription factors regulate the expression of brassinosteroid-responsive genes and play vital roles in plant growth and response to environmental stimuli. Their regulation mechanism has been well elucidated in genetic model plants. The complexity of the maize genome might lead to evolutional and functional diversification among the members of the ZmBES1/BZR1 gene family. In the present study, eleven members of the ZmBES1/BZR1 gene family were identified by genome-wide analysis, and ten of their open reading frames were successfully amplified. Bioinformatics analysis showed that these genes unevenly distributed on seven of the ten maize chromosomes, with three pairs of segmental duplication genes, and their encoding proteins shared similar motif composition and conserved domains. The expression of the ZmBES1/BZR1 genes displayed much differential in different organs and developmental stages, as well as in response to abscisic acid and light signal. Subcellular localization confirmed that most of them localized in nucleus. More attention should be paid to ZmBES1/BZR1-4 and -5, which were clustered into a distinguished phylogenetic clade, and ZmBES1/BZR1-2 and -7, which localized in chloroplast. The results indicated their similar but not identical functions in brassinosteroid-mediated signaling pathway and would be helpful in further functional study of the ZmBES1/BZR1s in maize.