Role of AP2/EREBP transcription factors in gene regulation during abiotic stress.

Crop plants are exposed to many types of abiotic stress during their life cycle. Water deficit derived from drought, low temperature or high salt concentration in the soil, is one of the most common environmental stresses that affects growth and development of plants through alterations in metabolism and gene expression. Adaptation to these conditions may involve passive tolerance or active homeostatic mechanisms for maintaining water balance. Active responses occur at different levels in the plant and may represent a concomitant protection against other types of stress such as pathogen attack. Many morphological and physiological adaptations to water stress are under the control of the plant hormone abscisic acid and involve specific activation of target genes that in one way or another protect cells against water deficit or participate in the regulation of the drought response. Here, we discuss recent advances in our understanding of drought adaptation mediated by specific changes in gene expression and the role of AP2/EREBP nuclear factors in these processes.

Domain fusion between SNF1-related kinase subunits during plant evolution.

Members of the conserved SNF1/AMP-activated protein kinase (AMPK) family regulate cellular responses to environmental and nutritional stress in eukaryotes. Yeast SNF1 and animal AMPKs form a complex with regulatory SNF4/AMPKgamma and SIP1/SIP2/GAL83/AMPKbeta subunits. The beta-subunits function as target selective adaptors that anchor the catalytic kinase and regulator SNF4/gamma-subunits to their kinase association (KIS) and association with the SNF1 complex (ASC) domains. Here we demonstrate that plant SNF1-related protein kinases (SnRKs) interact with an adaptor-regulator protein, AKINbetagamma, in which an N-terminal KIS domain characteristic of beta-subunits is fused with a C-terminal region related to the SNF4/AMPKgamma proteins. AKINbetagamma is constitutively expressed in plants, suppresses the yeast delta snf4 mutation, and shows glucose-regulated interaction with the Arabidopsis SnRK, AKIN11. Our results suggest that evolution of AKINbetagamma reflects a unique function of SNF1-related protein kinases in plant glucose and stress signalling.

Constitutive protein-DNA interactions on the abscisic acid-responsive element before and after developmental activation of the rab28 gene.

Transcription of the rab28 gene from maize is induced in late embryo development and in response to abscisic acid. We have studied the regulation of the activity of the rab28 promoter in embryos. Two abscisic acid-responsive elements (ABREs) were necessary for expression in embryos of transgenic Arabidopsis and in transient transformation in maize embryos. In vivo footprinting showed that there was protein binding to the ABREs and to other cis elements in the promoter in young embryos before expression of rab28. This shows that the rab28 promoter is in an open chromatin structure before developmental activation. The ABREs are important for the induction and have protein binding in young embryos. Nuclear proteins extracted from embryos before activation of rab28 bound to the ABREs in band shift assays. A complex with different mobility was formed between nuclear proteins and the ABREs after induction of rab28 suggesting a modification of the ABRE-binding factor or an exchange of proteins. The footprints on the ABREs were unaltered by induction with abscisic acid or during developmental activation of rab28. These results indicate that constitutive binding of transcription factor(s) on the ABRE is central in embryonic regulation of the rab28 gene.

The use of an alternative promoter in the Arabidopsis thaliana HMG1 gene generates an mRNA that encodes a novel 3-hydroxy-3-methylglutaryl coenzyme A reductase isoform with an extended N-terminal region.

The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR, EC 1.1.1.34) catalyses the synthesis of mevalonate, the committed precursor of the great variety of isoprenoid compounds and derivatives synthesized in higher plants. It has previously been reported that Arabidopsis thaliana contains two differentially expressed genes, HMG1 and HMG2, that encode two HMGR isoforms (HMGR1 and HMGR2, respectively). This paper reports the characterization of a novel HMGR mRNA (HMGR1L mRNA) derived from the HMG1 gene. This mRNA is initiated 121 bp upstream from the transcription start site previously characterized. In contrast with the previously reported HMGR1 mRNA (HMGR1S mRNA), which is detected at high levels in all tissues of the plant, HMGR1L mRNA is present at relatively low levels and its expression is restricted mostly to seedlings, roots and inflorescences. HMGR1L and HMGR1S mRNAs are transcribed from alternative promoters. HMGR1L mRNA contains an in-phase AUG start codon which allows the synthesis of a novel HMGR isoform (HMGR1L) having 50 additional amino acid residues at its N-terminal end. Using an in vitro transcription-translation system we have shown that HMGR1L is inserted into ER-derived microsomes. It is thus unlikely that the extended N-terminal region of HMGR1L might have a role in targeting the enzyme to plastids or mitochondria. These results support the previous proposal that the endoplasmic reticulum is the only cell compartment for the primary targeting of HMGR in Arabidopsis and reinforce the view that plant HMGR is under the control of complex mechanisms operating at both transcriptional and post-transcriptional levels.

Expression of the Arabidopsis HMG2 gene, encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase, is restricted to meristematic and floral tissues.

The synthesis of mevalonate, which is considered the first rate-limiting step in isoprenoid biosynthesis, is catalyzed by the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR; EC 1.1.1.34). In Arabidopsis, HMGR is encoded by two differentially expressed genes (HMG1 and HMG2). The transcriptional activity of the HMG2 gene was studied after fusing different regions of its 5' flanking region to the beta-glucuronidase (GUS) reporter gene and transforming the resulting constructs into tobacco plants. The spatial and temporal expression directed by the HMG2 promoter in the transgenic plants is consistent with the expression pattern previously established by RNA analysis using an HMG2-specific probe. HMG2 expression is restricted to meristematic (root tip and shoot apex) and floral (secretory zone of the stigma, mature pollen grains, gynoecium vascular tissue, and fertilized ovules) tissues. Deletion analysis of the HMG2 5' flanking region was conducted in transgenic plants and transfected protoplasts. The region containing nucleotides -857 to +64 of the HMG2 gene was sufficient to confer high levels of expression in both floral and meristematic tissues, although deletion to nucleotide -503 resulted in almost complete loss of expression. Sequences contained within the 5' transcribed, untranslated region are also important for gene expression. The biological significance of the restricted pattern of expression of HMG2 is also discussed.