José A. Casaretto

A Rice WRKY Gene Encodes a Transcriptional Repressor of the Gibberellin Signaling Pathway in Aleurone Cells

The molecular mechanism by which GA regulates plant growth and development has been a subject of active research. Analyses of the rice (Oryza sativa) genomic sequences identified 77 WRKY genes, among which OsWRKY71 is highly expressed in aleurone cells. Transient expression of OsWRKY71 by particle bombardment specifically represses GA-induced Amy32b α-amylase promoter but not abscisic acid-induced HVA22 or HVA1 promoter activity in aleurone cells. Moreover, OsWRKY71 blocks the activation of the Amy32b promoter by the GA-inducible transcriptional activator OsGAMYB. Consistent with its role as a transcriptional repressor, OsWRKY71 is localized to nuclei of aleurone cells and binds specifically to functionally defined TGAC-containing W boxes of the Amy32b promoter in vitro. Mutation of the two W boxes prevents the binding of OsWRKY71 to the mutated promoter, and releases the suppression of the OsGAMYB-activated Amy32b expression by OsWRKY71, suggesting that OsWRKY71 blocks GA signaling by functionally interfering with OsGAMYB. Exogenous GA treatment decreases the steady-state mRNA level of OsWRKY71 and destabilizes the GFP:OsWRKY71 fusion protein. These findings suggest that OsWRKY71 encodes a transcriptional repressor of GA signaling in aleurone cells.

The Transcription Factors HvABI5 and HvVP1 Are Required for the Abscisic Acid Induction of Gene Expression in Barley Aleurone Cells

The abscisic acid (ABA) response promoter complexes (ABRCs) of the HVA1 and HVA22 genes have been shown to confer ABA-induced gene expression in cereals. A barley basic domain/Leu zipper (bZIP) transcription factor, HvABI5, is able to recognize ABRCs in vitro in a sequence-specific manner and to transactivate ABRC–β-glucuronidase reporter genes when introduced to barley aleurone cells via particle bombardment. This transactivation is dependent on the presence of another transcription factor, HvVP1, and cannot be blocked by the negative regulator abi1-1. Using the double-stranded RNA interference technique, we show that HvABI5 and HvVP1 are necessary for the ABA induction of gene expression but have no effect on another hormone-regulated process, the gibberellin-induced and ABA-suppressed expression of α-amylase. Our work indicates that although other typical plant bZIP transcription factors may bind ABRCs in vitro, HvABI5 is related to a subfamily of bZIPs responsible for the ABA induction of gene expression. Furthermore, HvABI5 and HvVP1 are not involved in the ABA suppression of gene expression.

The transcription factor SlAREB1 confers drought, salt stress tolerance and regulates biotic and abiotic stress-related genes in tomato

Members of the abscisic acid-responsive element binding protein (AREB)/abscisic acid-responsive element binding factor (ABF) subfamily of basic leucine zipper (bZIP) transcription factors have been implicated in abscisic acid (ABA) and abiotic stress responses in plants. Here we describe two members identified in cultivated tomato (Solanum lycopersicum), named SlAREB1 and SlAREB2. Expression of SlAREB1 and SlAREB2 is induced by drought and salinity in both leaves and root tissues, although that of SlAREB1 was more affected. In stress assays, SlAREB1-overexpressing transgenic tomato plants showed increased tolerance to salt and water stress compared to wild-type and SlAREB1-down-regulating transgenic plants, as assessed by physiological parameters such as relative water content (RWC), chlorophyll fluorescence and damage by lipoperoxidation. In order to identify SlAREB1 target genes responsible for the enhanced tolerance, microarray and cDNA-amplified fragment length polymorphism (AFLP) analyses were performed. Genes encoding oxidative stress-related proteins, lipid transfer proteins (LTPs), transcription regulators and late embryogenesis abundant proteins were found among the up-regulated genes in SlAREB1-overexpressing lines, especially in aerial tissue. Notably, several genes encoding defence proteins associated with responses to biotic stress (e.g. pathogenesis-related proteins, protease inhibitors, and catabolic enzymes) were also up-regulated by SlAREB1 overexpression, suggesting that this bZIP transcription factor is involved in ABA signals that participate in abiotic stress and possibly in response to pathogens.

An abiotic stress-responsive bZIP transcription factor from wild and cultivated tomatoes regulates stress-related genes

Wild relatives of cultivated tomato (Solanum lycopersicum) are resistant to a wide range of abiotic and biotic stress conditions. In an effort to understand the molecular mechanisms of salt stress resistance in the wild and cultivated Solanum species, a basic leucine zipper (bZIP) transcription factor was identified in S. chilense, S. peruvianum and S. lycopersicum and named ScAREB1, SpAREB1 and SlAREB1, respectively. Deduced amino acid sequences of the three proteins are 97% identical among them and present high homology with the ABF/AREB subfamily of transcription factors described in different plant species, including Arabidopsis (ABF2, 54% identical) and tobacco (PHI-2, 50% identical). Expression of these orthologous genes is upregulated similarly in the three species by salt stress. The expression of SlAREB1 was further investigated in S. lycopersicum and found to be induced by drought, cold and abscisic acid. To investigate the possible role of this transcription factor in response to abiotic stress, a simple transient expression assay was used for rapid analysis of genes regulated by SlAREB1 in tomato and tobacco by means of Agrobacterium-mediated transformation. Tobacco leaves expressing SlAREB1 showed upregulation of stress-responsive genes such as RD29B, the LEA genes ERD10B and TAS14, the transcription factor PHI-2 and a trehalose-6-phosphate phosphatase gene. These results suggest that this class of bZIP plays a role in abiotic stress response in the Solanum genus.

Functional definition of ABA-response complexes: the promoter units necessary and sufficient for ABA induction of gene expression in barley (Hordeum vulgare L.)

Abscisic acid (ABA)-response promoter complexes (ABRCs), consisting of an ACGT core-containing element (ACGT box) and a coupling element (CE), have been shown to be necessary and sufficient for ABA induction of gene expression in cereal plants. In this work, the component elements of two ABRCs are defined in terms of base sequence, orientation, and distance from each other. The ACGT element requires the sequence 5′-ACGTGGC-3′ and the elements CE1 and CE3 require the sequences CCACC and GCGTGTC, respectively. The ACGT element and CE3 are next to each other in the barley ABA-inducible gene HVA1, and lengthening the distance between them gradually decreases their activity in conferring ABA response. On the other hand, the ACGT element and CE1 are separated by about 20 bp in the promoter of another ABA-inducible gene, HVA22, and need to be separated by multiples of 10 bp in order to confer high ABA induction, suggesting that these two elements have to be located in the same side of the DNA double helix. Although the coupling between an ACGT box and a CE is sufficient for ABA induction, two copies of the ACGT element are equally active. However, two copies of CE3 appear to be less active. Specific interactions between ABRC and nuclear proteins have been detected. In vitro binding activities of nuclear proteins to an ABRC and to its mutant forms appear to be proportional to the biological activities of these sequences in vivo. Our data suggest that the specific response to ABA is determined by the presence of two ACGT boxes or an ACGT box plus a CE as well as by the flanking sequences of the ACGT boxes and the CEs.

VvCO and VvCOL1, two CONSTANS homologous genes, are regulated during flower induction and dormancy in grapevine buds

Two previously uncharacterized Vitis viniferaCONSTANS-like genes (VvCO, VvCOL1), which are predicted to encode proteins with homology to members of the Arabidopis CONSTANS family, were identified. Under controlled conditions, both genes show a diurnal expression pattern with peak at dawn. During grapevine bud development, VvCOL1 is mainly expressed in dormancy, suggesting a participation in the transcriptional photoperiod control of bud dormancy induction and maintenance in this species. On the other hand, VvCO expression in latent buds is in agreement with a function during flowering induction. A spatial and temporal relationship in the expression of VvCO, VFY and VvMADS8 (the ArabidopsisLEAFY and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 orthologues) in latent buds is observed, suggesting that these genes are involved in the seasonal periodicity of flowering in grapevines. Furthermore, our results provide a new molecular insight into tendril development showing that grapevine CO homologues are also expressed in this distinctive organ.

Modulation of organic acids and sugar content in tomato fruits by an abscisic acid-regulated transcription factor

Growing evidence suggests that the phytohormone abscisic acid (ABA) plays a role in fruit development. ABA signaling components of developmental programs and responses to stress conditions include the group of basic leucine zipper transcriptional activators known as ABA-response element binding factors (AREBs/ABFs). AREB transcription factors mediate ABA-regulated gene expression involved in desiccation tolerance and are expressed mainly in seeds and in vegetative tissues under stress; however, they are also expressed in some fruits such as tomato. In order to get an insight into the role of ABA signaling in fruit development, the expression of two AREB-like factors were investigated during different developmental stages. In addition, tomato transgenic lines that overexpress and downregulate one AREB-like transcription factor, SlAREB1, were used to determine its effect on the levels of some metabolites determining fruit quality. Higher levels of citric acid, malic acid, glutamic acid, glucose and fructose were observed in SlAREB1-overexpressing lines compared with those in antisense suppression lines in red mature fruit pericarp. The higher hexose concentration correlated with increased expression of genes encoding a vacuolar invertase (EC 3.2.1.26) and a sucrose synthase (EC 2.4.1.13). No significant changes were found in ethylene content which agrees with the normal ripening phenotype observed in transgenic fruits. These results suggest that an AREB-mediated ABA signal affects the metabolism of these compounds during the fruit developmental program.

Crosstalk Between Gibberellin and Abscisic Acid in Cereal Aleurone

The antagonism between gibberellins (GA) and abscisic acid (ABA) is an important factor regulating the developmental transition from embryogenesis to seed germination. In cereal aleurone layers, the expression of genes encoding hydrolytic enzymes needed for seedling growth, such as α-amylases and proteases, is induced by GA but suppressed by ABA. In addition, ABA induces the expression of genes that may play a role in the establishment of stress tolerance. Because of these well-defined biochemical and molecular markers, the cereal aleurone layers have been used as a convenient system for studying GA/ABA actions. Both gain- and loss-of-function approaches have been followed by the constitutive or the RNAi-mediated knockdown expression of specific regulatory molecules. The GA signaling pathway is anchored by the transcription factor GAMyb, which interacts with a specific region in the promoter of GA up-regulated genes, and an upstream regulatory molecule SLN1 (SLR1) that appears to be a functional homolog of the Arabidopsis GAI/RGA regulatory proteins. It is established that the ABA induction and suppression of gene expression follow two distinct signaling pathways, with the former requiring a transcription factor ABI5, but inhibited by a protein phosphatase 2C, and the latter mediated by a protein kinase PKABA1 and an unknown factor. The ABA suppression action has been pinpointed to be upstream from the formation of functional GAMyb but downstream from the site of action of SLN1 (SLR1).

VvBOR1, the Grapevine Ortholog of AtBOR1, Encodes an Efflux Boron Transporter That is Differentially Expressed Throughout Reproductive Development of Vitis vinifera L.

Boron (B) is an essential micronutrient for normal development of roots, shoots and reproductive tissues in plants. Due to its role in the structure of rhamnogalacturonan II, a polysaccharide required for pollen tube growth, B deficiency has been associated with the occurrence of parthenocarpic seedless grapes in some varieties of Vitis vinifera L. Despite that, it is unclear how B is mobilized and accumulated in reproductive tissues. Here we describe the characterization of an efflux B transporter, VvBOR1, homolog to AtBOR1, which is involved in B xylem loading in Arabidopsis thaliana roots. VvBOR1-green fluorescent protein (GFP) fusion protein expressed in A. thaliana localizes in the proximal plasma membrane domain in root pericycle cells, and VvBOR1 overexpression restores the wild-type phenotype in A. thaliana bor1-3 mutant plants exposed to B deficiency. Complementation of a mutant yeast strain indicates that VvBOR1 corresponds to a B efflux transporter. Transcriptional analyses during grapevine reproductive development show that the VvBOR1 gene is preferentially expressed in flowers at anthesis and a direct correlation between the expression pattern and B content in grapes was established, suggesting the involvement of this transporter in B accumulation in grapevine berries.

Transcriptional regulation by abscisic acid in barley (Hordeum vulgare L.) seeds involves autoregulation of the transcription factor HvABI5

The barley bZIP transcription factor HvABI5 mediates abscisic acid (ABA)-upregulated gene expression in barley (Hordeum vulgare L.) seeds. HvABI5 specifically recognizes cis-elements of the ABA response complexes present in the promoters of the ABA-induced genes HVA1 and HVA22. HvABI5 together with another transcription factor, HvVP1, are required for the transactivation of these promoters, and this transactivation process is insensitive to the negative regulator abi1–1. The expression of HvABI5 itself appeared to be induced by ABA and can be suppressed by abi1-1. Gain- and loss-of-function studies in barley aleurone cells show that HvABI5 expression is positively regulated by a feed-forward circuit that involves HvABI5 itself and HvVP1. Mutation of the Ser residue in HvABI5, which has been shown to be phosphorylated in an ABA-dependent manner in the rice orthologue of HvABI5, reduces the transactivation activity of the factor by 50%. Although levels of HvABI5 and its transcript are enhanced by ABA treatment, the nuclear localization of HvABI5 is not affected by ABA. A model based on these observations is presented to explain the ABA upregulation of gene expression.