ZmGOLS2, a target of transcription factor ZmDREB2A, offers similar protection against abiotic stress as ZmDREB2A.

GALACTINOL SYNTHASE is the first committed enzyme in the raffinose biosynthetic pathway. We have previously characterized the maize (Zea mays) GALACTINOL SYNTHASE2 gene (ZmGOLS2) as abiotic stress induced. To further investigate the regulation of ZmGOLS2 gene expression, individual luciferase expression vectors,in which the luciferase gene was controlled by different lengths of the ZmGOLS2 promoter, were co-transfected into maize protoplasts with either a ZmDREB2A- or a GFP-expression vector. Over-expression of ZmDREB2A up-regulated both the expression of the luciferase gene controlled by the ZmGOLS2 promoter and the endogenous ZmGOLS2 gene in protoplasts. Only one of the two DRE elements in the ZmGOLS2 promoter was identified as necessary for this up-regulation. Expression vectors of GFP, ZmGOLS2 or ZmDREB2A were stably transformed into Arabidopsis. Expression of ZmDREB2A up-regulated the AtGOLS3 gene but only over-expression of ZmGOLS2 resulted in hyper-accumulation of galactinol and raffinose. Regardless, under drought-, heat shock-, high osmotic- or salinity-stress conditions, both the ZmGOLS2- and the ZmDREB2A- expressing plants had greater germination percentages, greater percentages of seedlings becoming autotropic, and/or greater survival percentages during/after stress than the control plants. Under normal growing conditions, transgenic Arabidopsis plants expressing the ZmGOLS2 gene had similar growth to that of untransformed wild type or GFP-expressing control plants, whereas ZmDREB2A over-expressing plants exhibited retarded growth relative to either of the controls. These data suggest that over-expression of ZmGOLS2, rather than the transcription factor ZmDREB2A, is a more practical target for generation of abiotic-stress tolerant crops.

Functional analysis of the 5' regulatory region of the maize GALACTINOL SYNTHASE2 gene.

Maize (Zea mays) GALACTINOL SYNTHASE (GolS) is a key enzyme in the raffinose biosynthetic pathway. We have previously characterized the maize GolS2 (ZmGolS2) gene as heat shock induced in maize germinating seeds and cultured cells. Here we report the identification, isolation and characterization of the 1.5Kb, 5' regulatory region of the ZmGolS2 gene. The 1.5kb fragment and its deletions were tested for promoter activity by their regulation of the Renilla (Renilla reniformis) luciferase reporter gene expression in maize protoplasts cultured at either 25°C or 42°C for 24h. The expression of a constitutively expressed firefly (Photinus ssp.) luciferase gene in the same vector was used as a reference. One heat shock element (HSE) was identified by comparing the promoter activity of each fragment under normal and heat shock conditions. Deletion or triplication of this HSE motif, abolished or enhanced the heat shock response of the ZmGolS2 promoter, respectively. This HSE motif is specifically bound by proteins in the nuclear extracts of heat shock stressed, but not unstressed maize cells as confirmed by DNA-EMSA. This work helps to understand the regulatory mechanism of the ZmGolS2 gene under stress conditions.

Genome-wide landscape of polyadenylation in Arabidopsis provides evidence for extensive alternative polyadenylation.

Alternative polyadenylation (APA) has been shown to play an important role in gene expression regulation in animals and plants. However, the extent of sense and antisense APA at the genome level is not known. We developed a deep-sequencing protocol that queries the junctions of 3'UTR and poly(A) tails and confidently maps the poly(A) tags to the annotated genome. The results of this mapping show that 70% of Arabidopsis genes use more than one poly(A) site, excluding microheterogeneity. Analysis of the poly(A) tags reveal extensive APA in introns and coding sequences, results of which can significantly alter transcript sequences and their encoding proteins. Although the interplay of intron splicing and polyadenylation potentially defines poly(A) site uses in introns, the polyadenylation signals leading to the use of CDS protein-coding region poly(A) sites are distinct from the rest of the genome. Interestingly, a large number of poly(A) sites correspond to putative antisense transcripts that overlap with the promoter of the associated sense transcript, a mode previously demonstrated to regulate sense gene expression. Our results suggest that APA plays a far greater role in gene expression in plants than previously expected.

Light-induced phosphorylation and degradation of the negative regulator PHYTOCHROME-INTERACTING FACTOR1 from Arabidopsis depend upon its direct physical interactions with photoactivated phytochromes.

The phytochrome (phy) family of photoreceptors regulates changes in gene expression in response to red/far-red light signals in part by physically interacting with constitutively nucleus-localized phy-interacting basic helix-loop-helix transcription factors (PIFs). Here, we show that PIF1, the member with the highest affinity for phys, is strongly sensitive to the quality and quantity of light. phyA plays a dominant role in regulating the degradation of PIF1 following initial light exposure, while phyB and phyD and possibly other phys also influence PIF1 degradation after prolonged illumination. PIF1 is rapidly phosphorylated and ubiquitinated under red and far-red light before being degraded with a half-life of approximately 1 to 2 min under red light. Although PIF1 interacts with phyB through a conserved active phyB binding motif, it interacts with phyA through a novel active phyA binding motif. phy interaction is necessary but not sufficient for the light-induced phosphorylation and degradation of PIF1. Domain-mapping studies reveal that the phy interaction, light-induced degradation, and transcriptional activation domains are located at the N-terminal 150-amino acid region of PIF1. Unlike PIF3, PIF1 does not interact with the two halves of either phyA or phyB separately. Moreover, overexpression of a light-stable truncated form of PIF1 causes constitutively photomorphogenic phenotypes in the dark. Taken together, these data suggest that removal of the negative regulators (e.g., PIFs) by light-induced proteolytic degradation might be sufficient to promote photomorphogenesis.

Glycerol-3-phosphate levels are associated with basal resistance to the hemibiotrophic fungus Colletotrichum higginsianum in Arabidopsis.

Glycerol-3-phosphate (G3P) is an important component of carbohydrate and lipid metabolic processes. In this article, we provide evidence that G3P levels in plants are associated with defense to a hemibiotrophic fungal pathogen Colletotrichum higginsianum. Inoculation of Arabidopsis (Arabidopsis thaliana) with C. higginsianum was correlated with an increase in G3P levels and a concomitant decrease in glycerol levels in the host. Plants impaired in utilization of plastidial G3P (act1) accumulated elevated levels of pathogen-induced G3P and displayed enhanced resistance. Furthermore, overexpression of the host GLY1 gene, which encodes a G3P dehydrogenase (G3Pdh), conferred enhanced resistance. In contrast, the gly1 mutant accumulated reduced levels of G3P after pathogen inoculation and showed enhanced susceptibility to C. higginsianum. Unlike gly1, a mutation in a cytosolic isoform of G3Pdh did not alter basal resistance to C. higginsianum. Furthermore, act1 gly1 double-mutant plants were as susceptible as the gly1 plants. Increased resistance or susceptibility of act1 and gly1 plants to C. higginsianum, respectively, was not due to effects of these mutations on salicylic acid- or ethylene-mediated defense pathways. The act1 mutation restored a wild-type-like response in camalexin-deficient pad3 plants, which were hypersusceptible to C. higginsianum. These data suggest that G3P-associated resistance to C. higginsianum occurs independently or downstream of the camalexin pathway. Together, these results suggest a novel and specific link between G3P metabolism and plant defense.

Rapid and efficient subcloning of DNA without dephosphorylation or gel electrophoresis.

Conventional subcloning into plasmid vectors often involves dephosphorylation, gel electrophoresis, DNA extraction, and purification to isolate the target insert and the cleaved plasmid. This is not only time-consuming but very often problematic. We have developed strategies that can circumvent these steps by mixing digested donor and recipient plasmids together for ligation. These strategies capitalizes on: (1) the ability to enhance the ligation efficiency of desired DNA fragments into the target vector by the generation and removal of small (< 50 bp) fragments from nontarget DNA using peripheral restriction sites and spin column technology and (2) the elimination of undesired ligation products after ligation by using the Lac Z gene, differences in antibiotic resistance among plasmid vectors, and unique restriction sites situated in nontarget DNA fragments.

Expression of a GALACTINOL SYNTHASE gene in tomato seeds is up-regulated before maturation desiccation and again after imbibition whenever radicle protrusion is prevented.

Raffinose family oligosaccharides (RFOs) have been implicated in mitigating the effects of environmental stresses on plants. In seeds, proposed roles for RFOs include protecting cellular integrity during desiccation and/or imbibition, extending longevity in the dehydrated state, and providing substrates for energy generation during germination. A gene encoding galactinol synthase (GOLS), the first committed enzyme in the biosynthesis of RFOs, was cloned from tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds, and its expression was characterized in tomato seeds and seedlings. GOLS (LeGOLS-1) mRNA accumulated in developing tomato seeds concomitant with maximum dry weight deposition and the acquisition of desiccation tolerance. LeGOLS-1 mRNA was present in mature, desiccated seeds but declined within 8 h of imbibition in wild-type seeds. However, LeGOLS-1 mRNA accumulated again in imbibed seeds prevented from completing germination by dormancy or water deficit. Gibberellin-deficient (gib-1) seeds maintained LeGOLS-1 mRNA amounts after imbibition unless supplied with gibberellin, whereas abscisic acid (ABA) did not prevent the loss of LeGOLS-1 mRNA from wild-type seeds. The presence of LeGOLS-1 mRNA in ABA-deficient (sitiens) tomato seeds indicated that wild-type amounts of ABA are not necessary for its accumulation during seed development. In all cases, LeGOLS-1 mRNA was most prevalent in the radicle tip. LeGOLS-1 mRNA accumulation was induced by dehydration but not by cold in germinating seeds, whereas both stresses induced LeGOLS-1 mRNA accumulation in seedling leaves. The physiological implications of LeGOLS-1 expression patterns in seeds and leaves are discussed in light of the hypothesized role of RFOs in plant stress tolerance.