Genome-Wide Identification and Preliminary Functional Analysis of BAM (ß-Amylase) Gene Family in Upland Cotton.

The ß-amylase (BAM) gene family encodes important enzymes that catalyze the conversion of starch to maltose in various biological processes of plants and play essential roles in regulating the growth and development of multiple plants. So far, BAMs have been extensively studied in Arabidopsis thaliana (A. thaliana). However, the characteristics of the BAM gene family in the crucial economic crop, cotton, have not been reported. In this study, 27 GhBAM genes in the genome of Gossypium hirsutum L (G. hirsutum) were identified by genome-wide identification, and they were divided into three groups according to sequence similarity and phylogenetic relationship. The gene structure, chromosome distribution, and collinearity of all GhBAM genes identified in the genome of G. hirsutum were analyzed. Further sequence alignment of the core domain of glucosyl hydrolase showed that all GhBAM family genes had the glycosyl hydrolase family 14 domain. We identified the BAM gene GhBAM7 and preliminarily investigated its function by transcriptional sequencing analysis, qRT-PCR, and subcellular localization. These results suggested that the GhBAM7 gene may influence fiber strength during fiber development. This systematic analysis provides new insight into the transcriptional characteristics of BAM genes in G. hirsutum. It may lay the foundation for further study of the function of these genes.

Recruitment of AtWHY1 and AtWHY3 by a distal element upstream of the kinesin gene AtKP1 to mediate transcriptional repression.

A 43-bp distal element, the AtKP1-related element (KPRE), was previously shown to repress the promoter activity of the kinesin gene AtKP1 in Arabidopsis thaliana. In order to identify KPRE-binding factor 1 (KBF1), a combination of ion-exchange chromatography, gel-filtration chromatography and DNA-affinity chromatography was used to purify KBF1 from whole cell extracts of Arabidopsis seedlings. Mass spectrometric identification showed that KBF1 contains two members of the whirly family of transcription factors, AtWHY1 and AtWHY3. KBF1 is a single and double-stranded DNA-binding factor. A ChIP assay showed that AtWHY1 and AtWHY3 bind to the upstream region of AtKP1 gene in vivo. Over-expression of AtWHY1 and AtWHY3 led to an obvious decrease of AtKP1 transcripts, based on quantitative real-time PCR analysis. Interestingly, salicylic acid treatment resulted in an increase of AtWHY1 and AtWHY3 transcripts, and a decrease of AtKP1 transcripts. Thus, AtWHY1 and AtWHY3, as two components of KBF1, can be recruited at the KPRE site to mediate the transcriptional repression of AtKP1. Our results prove that AtKP1 is a new downstream target of the whirly family of transcription factors.

A 43-bp A/T-rich element upstream of the kinesin gene AtKP1 promoter functions as a silencer in Arabidopsis.

The expression of the Arabidopsis thaliana kinesin-like protein 1 (AtKP1) gene is restricted to tender tissues. We used a 5'-deletion assay to identify and characterize the regulatory regions controlling tissue-specific AtKP1 expression. Multiple enhancer regions, located 470- and 2,808-bp upstream of the translational start codon, were critical for activation, while a silencer region located at -2,987 to -2,808 (A + T = 71%) was required for repression. Within this 180-bp fragment, a 43-bp element (termed KPRE, A + T = 58%) mediated repression of the CaMV35S promoter by using a gain-of-function approach that was orientation-dependent in leaves and orientation-independent in roots. Electrophoretic mobility shift assay (EMSA) showed that the GAGAAATT octamer (corresponding to neucleotides -2,908 - -2,900) in KPRE was the core negative regulatory motif for interacting with DNA-binding proteins in leaves and roots. However, using a second gain-of-function experiment with KPRE fused to CaMV35S, we found that the mutant negatively affected transcription in transgenic leaves and positively affected transcription in transgenic roots. This indicated that these two modes mediate repressive regulation in leaves and roots, respectively. The EMSA experiment using different mutant KPRE as probes confirmed that two distinct sets of proteins bound to KPRE at an overlapping site AGAAAT in the leaf. Taken together, these data suggest that two different modes control the negatively transcriptional regulation of KPRE in leaves and roots, and provide new insight into the mechanism of transcriptional repression of A/T-rich sequences in higher plants.