The phytohormone abscisic acid (ABA) mediates the adaptation of plants to environmental stresses such as drought and regulates developmental signals such as seed maturation. Within plants, the PYR/PYL/RCAR family of START proteins receives ABA to inhibit the phosphatase activity of the group-A protein phosphatases 2C (PP2Cs), which are major negative regulators in ABA signalling. Here we present the crystal structures of the ABA receptor PYL1 bound with (+)-ABA, and the complex formed by the further binding of (+)-ABA-bound PYL1 with the PP2C protein ABI1. PYL1 binds (+)-ABA using the START-protein-specific ligand-binding site, thereby forming a hydrophobic pocket on the surface of the closed lid. (+)-ABA-bound PYL1 tightly interacts with a PP2C domain of ABI1 by using the hydrophobic pocket to cover the active site of ABI1 like a plug. Our results reveal the structural basis of the mechanism of (+)-ABA-dependent inhibition of ABI1 by PYL1 in ABA signalling.

The DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN2A (DREB2A) transcription factor controls water deficitinducible gene expression and requires posttranslational modification for its activation. The activation mechanism is not well understood; however, the stability of this protein in the nucleus was recently found to be important for its activation. Here, we report the isolation of Arabidopsis thaliana DREB2A-INTERACTING PROTEIN1 (DRIP1) and DRIP2, C3HC4 RING domain-containing proteins that interact with the DREB2A protein in the nucleus. An in vitro ubiquitination assay showed that they function as E3 ubiquitin ligases and are capable of mediating DREB2A ubiquitination. Overexpression of DRIP1 in Arabidopsis delayed the expression of DREB2A-regulated drought-responsive genes. Drought-inducible gene expression was slightly enhanced in the single T-DNA mutants of drip1-1 and drip2-1. By contrast, significantly enhanced gene expression was revealed in the drip1 drip2 double mutant under dehydration stress. Collectively, these data imply that DRIP1 and DRIP2 function negatively in the response of plants to drought stress. Moreover, overexpression of full-length DREB2A protein was more stable in drip1-1 than in the wild-type background. These results suggest that DRIP1 and DRIP2 act as novel negative regulators in drought-responsive gene expression by targeting DREB2A to 26S proteasome proteolysis.

Plants have a variety of mechanisms for defending against plant pathogens and tolerating environmental stresses such as drought and high salinity. Ginkbilobin2 (Gnk2) is a seed storage protein in gymnosperm that possesses antifungal activity and a plant-specific cysteine-rich motif (domain of unknown function26 [DUF26]). The Gnk2-homologous sequence is also observed in an extracellular region of cysteine-rich repeat receptor-like kinases that function in response to biotic and abiotic stresses. Here, we report the lectin-like molecular function of Gnk2 and the structural basis of its monosaccharide recognition. Nuclear magnetic resonance experiments showed that mannan was the only yeast (Saccharomyces cerevisiae) cell wall polysaccharide that interacted with Gnk2. Gnk2 also interacted with mannose, a building block of mannan, with a specificity that was similar to those of mannose-binding legume lectins, by strictly recognizing the configuration of the hydroxy group at the C4 position of the monosaccharide. The crystal structure of Gnk2 in complex with mannose revealed that three residues (asparagine-11, arginine-93, and glutamate-104) recognized mannose by hydrogen bonds, which defined the carbohydrate-binding specificity. These interactions were directly related to the ability of Gnk2 to inhibit the growth of fungi, including the plant pathogenic Fusarium spp., which were disrupted by mutation of arginine-93 or the presence of yeast mannan in the assay system. In addition, Gnk2 did not inhibit the growth of a yeast mutant strain lacking the a1,2-linked mannose moiety. These results provide insights into the molecular basis of the DUF26 protein family.

An angiotensin I-converting enzyme (ACE) inhibitory peptide Ile-Gln-Pro with an IC(50) value of 5.77 +/- 0.09 microM was purified from the alcalase digests of Spirulina platensis by gel filtration chromatography and two steps of reverse-phase high-performance liquid chromatography (RP-HPLC). The peptide was synthesized and showed resistance to in vitro digestion by gastrointestinal proteases. Kinetics studies indicated that the peptide was a noncompetitive inhibitor and that the K(i) value was 7.61 +/- 0.16 microM. Oral administration of Ile-Gln-Pro at a dosage of 10 mg/kg showed significant decreases of the weighted systolic blood pressure (SBP) and diastolic blood pressure (DBP) in spontaneously hypertensive rats (SHR) at 4, 6, and 8 h after treatment. The results showed that the ACE inhibitory peptide from Spirulina platensis may have potential for use in the prevention and treatment of hypertension.

A novel basic protein with antifungal activity was isolated from the seeds of Ginkgo biloba and purified to homogeneity. The protein inhibited the growth of some fungi (Fusarium oxysporum, Trichoderma reesei, and Candida albicans) but did not exhibit antibacterial action against Escherichia coli. Furthermore, this protein showed weak inhibitory activity against the aspartic protease pepsin. To design primers for gene amplification, the NH(2)-terminal and partial internal amino acid sequences were determined using peptides obtained from a tryptic digest of the oxidized protein. The full-length cDNA of the antifungal protein was cloned and sequenced by RT-PCR and rapid amplification of cDNA ends (RACE). The cDNA contained a 402-bp open reading frame encoding a 134-aa protein with a potential signal peptide (26 residues), suggesting that this protein is synthesized as a preprotein and secreted outside the cells. The antifungal protein shows approximately 85% identity with embryo-abundant proteins from Picea abies and Picea glauca at the amino acid level; however, there is no homology between this protein and other plant antifungal proteins, such as defensin, and cyclophilin-, miraculin- and thaumatin-like proteins.