Many G2/M phase-specific genes in plants contain mitosis-specific activator (MSA) elements, which act as G2/M phase-specific enhancers and bind with R1R2R3-Myb transcription factors. Here, we examined the genome-wide effects of NtmybA2 overexpression, one of the R1R2R3-Myb transcription factors in tobacco (Nicotiana tabacum). We used a custom-made 16K cDNA microarray for comparative transcriptome analysis of transgenic tobacco BY2 cell lines that overexpress NtmybA2 or its truncated hyperactive form. The microarray was also used to determine the transcript profile during the cell cycle in synchronized cultures of BY2 cells. Combined microarray data from transgenic lines and synchronized cells revealed that overexpression of the truncated hyperactive form of NtmybA2, but not its full-length form, preferentially up-regulated many G2/M phase-specific genes in BY2 cells. We determined promoter sequences of several such up-regulated genes and showed that all contain MSA-like motifs in the proximal regions of their promoters. One of the up-regulated genes, NtE2C, encoding for cyclin-specific ubiquitin carrier proteins contained a single functional MSA-like motif, which specifically controlled the expression of a reporter gene in the G2/M phase in BY2 cells. Furthermore, a genomic footprint experiment showed that the MSA element in the NtE2C promoter interacted with nuclear proteins in vivo. Therefore, we propose that the transcription of many G2/M phase-specific genes in tobacco is positively regulated by NtmybA2, in most cases through direct binding to the MSA elements.

NIMA-related kinases (Neks) are a family of serine/threonine kinases that have been linked to cell-cycle regulation in fungi and mammals. Information regarding the function of Neks in plants is very limited. We screened the three plant species that have had their genomes sequenced in an attempt to improve our understanding of their role in plants. We retrieved seven members in Arabidopsis thaliana, nine in Populus trichocarpa and six in Oryza sativa. Phylogenetic analysis showed that plant Neks are closely related to each other and contain paralogous genes. Moreover, their chromosome distribution and their exon-intron structure revealed that the actual plant Nek family was derived from a single representative followed by large segmental duplication events. Functional expression analyses in the three species relied on RTqPCR in poplar and publicly available microarray data for Arabidopsis and rice. Although plant Neks are present in every organ analyzed, their expression profiles suggest their involvement in plant development processes. Furthermore, we showed that PNek1, a member of the poplar family, is expressed at sites of free auxin synthesis and is specifically involved during the vascularization process.

* The transcription factors C-repeat binding factors/dehydration-responsive element binding proteins (CBFs/DREBs) control the expression of many stress-inducible genes in Arabidopsis.* A cDNA clone, designated GhDREB1, was isolated from cotton (Gossypium hirsutum) by cDNA library screening.* Northern blot analysis indicated that mRNA accumulation of GhDREB1 was induced by low temperatures and salt stress, but was not induced by abscisic acid (ABA) or drought stress in cotton seedlings. Transgenic tobacco (Nicotiana tabacum) plants overexpressing GhDREB1 displayed stronger chilling tolerance than wild-type plants. Their leaf chlorophyll fluorescence, net photosynthetic rate and proline concentrations were higher than those of control plants during low-temperature treatment. However, under normal growth conditions, the transgenic tobacco plants exhibited retarded growth and delayed flowering. Interestingly, GhDREB1 transcripts in cotton seedlings were negatively regulated by gibberellic acid (GA(3)) treatment. Analysis of the promoter of the GhDREB1 gene revealed the presence of one low-temperature and four gibberellin-responsive elements. Green fluorescent protein (GFP) signal intensity or beta-glucuronidase (GUS) activity driven by the GhDREB1 promoter was clearly enhanced by low temperature but repressed by GA(3).* These results suggest that GhDREB1 functions as a transcription factor and plays an important role in improving cold tolerance, and also affects plant growth and development via GA(3).

Calmodulins (CaMs) are the most ubiquitous calcium sensors in eukaryotes. A number of CaM-binding proteins have been identified through classical methods, and many proteins have been predicted to bind CaMs based on their structural homology with known targets. However, multicellular organisms typically contain many CaM-like (CML) proteins, and a global identification of their targets and specificity of interaction is lacking. In an effort to develop a platform for large-scale analysis of proteins in plants we have developed a protein microarray and used it to study the global analysis of CaM/CML interactions. An Arabidopsis thaliana expression collection containing 1,133 ORFs was generated and used to produce proteins with an optimized medium-throughput plant-based expression system. Protein microarrays were prepared and screened with several CaMs/CMLs. A large number of previously known and novel CaM/CML targets were identified, including transcription factors, receptor and intracellular protein kinases, F-box proteins, RNA-binding proteins, and proteins of unknown function. Multiple CaM/CML proteins bound many binding partners, but the majority of targets were specific to one or a few CaMs/CMLs indicating that different CaM family members function through different targets. Based on our analyses, the emergent CaM/CML interactome is more extensive than previously predicted. Our results suggest that calcium functions through distinct CaM/CML proteins to regulate a wide range of targets and cellular activities.

The eukaryotic cell cycle is controlled by cyclin-dependent kinases (CDKs). Plants possess six types of CDK, among which the B-type CDK (CDKB) is expressed specifically from the late S- to the M-phase. We demonstrate that the expression of Arabidopsis CDKB2 is under the control of protein degradation machinery. The beta-glucuronidase fused to a putative PEST motif of CDKB2 was unstable in tobacco Bright Yellow-2 cells and Arabidopsis plants, and its degradation was arrested by the proteasome inhibitor MG132. We propose that the abundance of CDKB2 protein is regulated not only at the transcriptional level, but also through proteasome-mediated protein degradation.

Progression through the cell cycle is regulated by cyclin-dependent kinases (CDKs). Plants possess a unique class of CDKs, designated B-type CDKs, but seem to lack a functional CDC25 phosphatase, which is a crucial activator of the onset of mitosis in non-plant species. Based on a striking number of functional parallels between the Arabidopsis thaliana CDKB1;1 and the Drosophila melanogaster CDC25 (string), we hypothesize that the acquisition of B-type CDKs and the disappearance of CDC25 in plants might have been associated; in these coupled events, the CDC25-controlled onset of mitosis might have been evolutionarily replaced by a B-type CDK-dominated pathway, eventually resulting in the loss of the CDC25 gene.