Full-length cDNAs are essential for functional analysis of plant genes. Using the biotinylated CAP trapper method, we constructed full-length Arabidopsis cDNA libraries from plants in different conditions, such as drought-treated, cold-treated, or unstressed plants, and at various developmental stages from germination to mature seed. We prepared a cDNA microarray using ∼1300 full-length Arabidopsis cDNAs to identify drought- and cold-inducible genes and target genes of DREB1A/CBF3, a transcription factor that controls stress-inducible gene expression. In total, 44 and 19 cDNAs for drought- and cold-inducible genes, respectively, were isolated, 30 and 10 of which were novel stress-inducible genes that have not been reported as drought- or cold-inducible genes previously. Twelve stress-inducible genes were identified as target stress-inducible genes of DREB1A, and six of them were novel. On the basis of RNA gel blot and microarray analyses, the six genes were identified as novel drought- and cold-inducible genes that are controlled by DREB1A. Eleven DREB1A target genes whose genomic sequences have been registered in the GenBank database contained the dehydration-responsive element (DRE) or DRE-related CCGAC core motif in their promoter regions. These results show that our full-length cDNA microarray is a useful material with which to analyze the expression pattern of Arabidopsis genes under drought and cold stresses, to identify target genes of stress-related transcription factors, and to identify potential cis-acting DNA elements by combining the expression data with the genomic sequence data.

Monitoring the Expression Pattern of 1300 Arabidopsis Genes under Drought and Cold Stresses by Using a Full-Length cDNA Microarray

Glucose modulates many vital processes in photosynthetic plants. Analyses of Arabidopsis glucose insensitive2 (gin2) mutants define the physiological functions of a specific hexokinase (HXK1) in the plant glucose-signaling network. HXK1 coordinates intrinsic signals with extrinsic light intensity. HXK1 mutants lacking catalytic activity still support various signaling functions in gene expression, cell proliferation, root and inflorescence growth, and leaf expansion and senescence, thus demonstrating the uncoupling of glucose signaling from glucose metabolism. The gin2 mutants are also insensitive to auxin and hypersensitive to cytokinin. Plants use HXK as a glucose sensor to interrelate nutrient, light, and hormone signaling networks for controlling growth and development in response to the changing environment.

http://science.sciencemag.org/content/sci/300/5617/332.full.pdf

Role of the Arabidopsis Glucose Sensor HXK1 in Nutrient, Light, and Hormonal Signaling

https://www.cell.com/cell/pdf/S0092-8674(08)00214-6.pdf

Rapid synthesis of auxin via a new tryptophan-dependent pathway is required for shade avoidance in plants.

Nitrogen assimilation is a vital process controlling plant growth and development. Inorganic nitrogen is assimilated into the amino acids glutamine, glutamate, asparagine, and aspartate, which serve as important nitrogen carriers in plants. The enzymes glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), aspartate aminotransferase (AspAT), and asparagine synthetase (AS) are responsible for the biosynthesis of these nitrogen-carrying amino acids. Biochemical studies have revealed the existence of multiple isoenzymes for each of these enzymes. Recent molecular analyses demonstrate that each enzyme is encoded by a gene family wherein individual members encode distinct isoenzymes that are differentially regulated by environmental stimuli, metabolic control, developmental control, and tissue/cell-type specificity. We review the recent progress in using molecular-genetic approaches to delineate the regulatory mechanisms controlling nitrogen assimilation into amino acids and to define the physiological role of each isoenzyme involved in this metabolic pathway.

The molecular-genetics of nitrogen assimilation into amino acids in higher plants

We identified Arabidopsis thaliana sterility mutants by screening T-DNA and EMS-mutagenized lines and characterized several male-sterile mutants with defects specific for different anther processes. Approximately 44 and 855 sterile mutants were uncovered from the T-DNA and EMS screens, respectively. Several mutants were studied in detail with defects that included the establishment of anther morphology, microspore production, pollen differentiation, and anther dehiscence. Both non-dehiscencing and late-dehiscencing mutants were identified. In addition, pollenless mutants were observed with either apparent meiotic defects and/or abnormalities in cell layers surrounding the locules. Two mutant alleles were identified for the POLLENLESS3 locus which have defects in functional microspore production that lead to the degeneration of cells within the anther locules. pollenless3–1 contains a T-DNA insertion that co-segregates with the mutant phenotype and pollenless3–2 has a large deletion in the POLLENLESS3 gene. The POLLENLESS3 gene has no known counterparts in the GenBank, but encodes a protein containing putative nuclear localization and protein-protein interaction motifs. The POLLENLESS3 gene was shown recently to be the same as MS5, a previously described Arabidopsis
thaliana male-sterility mutant. Three genes were identified in the POLLENLESS3 genomic region: GENEY, POLLENLESS3, and β9-TUBULIN. The segment of the Arabidopsis
thaliana genome containing the POLLENLESS3 and β9-TUBULIN genes is duplicated and present on a different chromosome. Analysis of the POLLENLESS3 expression pattern determined that the 1.3-kb POLLENLESS3 mRNA is localized specifically within meiotic cells in the anther locules and that POLLENLESS3 mRNA is present only during late meiosis.

Anther developmental defects in Arabidopsis thaliana male-sterile mutants

The goal of our investigations is to define the genetic control of microtubule-based processes in a higher plant. The available evidence suggests that we have achieved our first objective: the characterization of the complete alpha-tubulin and beta-tubulin gene families of Arabidopsis. Four additional alpha-tubulin genes (TUA2, TUA4, TUA5, and TUA6) of Arabidopsis have been cloned and sequenced to complete the analysis of the gene structure for all six alpha-tubulin genes detectable on DNA gel blots of Arabidopsis genomic DNA hybridized with alpha-tubulin coding sequences. TUA1 and TUA3 were characterized earlier in our laboratory. Noncoding gene-specific hybridization probes have been constructed for all six alpha-tubulin genes and used in RNA gel blot analyses to demonstrate that all six genes are transcribed. The six genes encode four different alpha-tubulin isoforms; TUA2 and TUA4 encode a single isoform, as do TUA3 and TUA5. Two-dimensional protein gel immunoblot analyses have resolved at least four alpha-tubulin isoforms from plant tissues, suggesting that all of the predicted TUA gene products are synthesized in vivo.

The small genome of Arabidopsis contains at least six expressed alpha-tubulin genes.

Like the skeleton of any vertebrate animal, the cytoskeleton plays a major role in detennining the three-dimensional form of a cell. However, in plants the cytoskeleton is probably more analogous to a scaffold, being directly involved in the establishment of cell morphology and less involved in the maintenance of cell shape once the relatively rigid cell walls are in place. The term \"skeleton\" unfortunately tends to connote a rigid, somewhat permanent structure, but it is clear that much of the cytoskeleton is dynamic and polymorphous. Microtubules in particular are continually being disassembled, assembled, and rearranged into new configurations or arrays as a cell progresses through division and differentiation. The cellulosic wall of a typical plant cell largely precludes cell migration, which plays a central role in animal development. Thus, morphogenesis in plants is govemed primarily by the processes that establish the planes of cell divisions and the axes of cell expansions, and components of the cytoskeleton play important roles in both processes. In addition, the plant cytoskeleton is directly involved in the intracellular transport of a wide variety of macromolecules, vesicles, organelles, and other macromolecular structures essentia1 to plant growth and development. A11 three major classes of cytoskeletal filaments-microtubules, intermediate filaments, and microfilaments-are present in plant cells. However, our discussion will be restricted to the microtubule-based components of the plant cytoskeleton. First, we will describe the major microtubule arrays present in plant cells and discuss their known and/or predicted functions. Then we will focus on (a) recent information about the large tubulin gene families in plant cells and their possible significance, (b) questions about MTOCs in plants and the probable nucleation of microtubule assembly by y-tubulin, and (c) the roles of MAPs in plant cells. Finally, we emphasize that a clear picture of plant cell growth and differentiation will become available only when we understand the coordinated interactions between the cytoskeleton, the cell nucleus, the nuclear envelope, the ER, the plasma membrane, and the cell wall. At present there is too little definitive information about most of these important interactions to warrant further discussion here.

http://www.plantphysiol.org/content/plantphysiol/104/1/1.full.pdf

Microtubule Components of the Plant Cell Cytoskeleton

The pool of tubulin protein in tissues of Arabidopsis is provided by the expression of multiple alpha-tubulin (TUA) and beta-tubulin genes. Whereas most tubulin genes are expressed in many tissues, previous evidence suggested that the TUA1 gene might be expressed primarily in pollen. We now report a detailed analysis of TUA1 expression during Arabidopsis development. In RNA from tissues of dissected flowers, TUA1 transcripts were detected only in stamens and mature pollen. Chimeric genes containing TUA1 5' flanking DNA fused to the beta-glucuronidase (GUS) coding region were used to create transgenic Arabidopsis plants. Plants containing a chimeric gene with 533 bp of 5' flanking sequence were analyzed by histochemical assay to localize GUS expression within the plant. The blue product of GUS enzyme activity accumulated very rapidly in postmitotic pollen grains. Much lower levels of GUS activity were detected in anthers with uninucleate pollen grains, in flower receptacles, and in a few vegetative tissues. Analysis of 5' deletions of the TUA1 promoter suggested that 97 bp of 5' flanking DNA is sufficient to drive GUS expression in pollen and young anthers, whereas at least 380 bp is required to detect GUS expression in the receptacle. Examination of the TUA1 promoter sequence revealed several motifs that are repeated within the TUA1 promoter and are similar to sequences in other pollen-specific promoters.

Preferential expression of an alpha-tubulin gene of Arabidopsis in pollen.

The genome of Arabidopsis thaliana (Linnaeus) Heynhold was shown to contain an alpha-tubulin gene family consisting of at least four genes and/or pseudogenes. The primary structure of a transcribed alpha-tubulin gene was determined. A comparison of the predicted amino acid sequence of the A. thaliana alpha-tubulin with the predicted amino acid sequences of alpha-tubulins of Chlamydomonas reinhardtii, Stylonychia lemnae, and Homo spaiens reveals a high degree of homology; 90%, 87%, and 83% identity, respectively. Thus, a plant alpha-tubulin exhibits a high degree of homology to the alpha-tubulins of protists and animals. The coding sequence of the A. thaliana alpha-tubulin gene is interrupted by four introns, which occur at positions different from those of the less numerous introns of C. reinhardtii and rat alpha-tubulin genes. S1 nuclease mapping data showed that transcription is initiated 99 +/- 1 base pairs upstream from the translation initiation codon. Both 5' and 3' noncoding gene-specific probes were used to examine the expression of the alpha-tubulin gene in leaves, roots, and flowers by hybridization to total RNA isolated from these tissues. The results showed that the alpha-tubulin gene was transcribed in all three tissues.

https://www.pnas.org/content/pnas/84/16/5833.full.pdf

D. P. Snustad

Papers

The small genome of Arabidopsis contains at least six expressed alpha-tubulin genes.

Microtubule Components of the Plant Cell Cytoskeleton

Preferential expression of an alpha-tubulin gene of Arabidopsis in pollen.

Characterization of the alpha-tubulin gene family of Arabidopsis thaliana

The β-tubulin gene family of Arabidopsis thaliana: preferential accumulation of the β1 transcript in roots