SUMMARYPlant roots respond to environmental stresses or the exogenous plant hormone abscisic acid (ABA) by undergoing marked physiological and morphological changes. We show here that PERK4, a gene that encodes a member of the Arabidopsis thaliana proline-rich extensin-like receptor kinase family, plays an important role in ABA responses. Mutation of PERK4 by T-DNA insertion decreased sensitivity to ABA with respect to seed germination, seedling growth and primary root tip growth. The effect on root growth was due to enhanced cell elongation rather than cell division. The cytosolic free calcium concentration and Ca 2+ channel currents were lower in perk4 root cells than in wild-type cells in the presence of ABA. Root growth was similar in wild-type and perk4 plants after the application of a Ca 2+ channel blocker. PERK4 localised to the plasma membrane, and was shown to be an ABA-and Ca 2+ -activated protein kinase. Our data suggest that the receptor-like kinase encoded by PERK4 functions at an early stage of ABA signalling to inhibit root cell elongation by perturbing Ca 2+ homeostasis.

Plants can grow in soils containing highly variable amounts of mineral nutrients, like Ca2+ and Mn2+, though the mechanisms of adaptation are poorly understood. Here, we report the first genetic study to determine in vivo functions of a Ca2+ pump in plants. Homozygous mutants of Arabidopsis harboring a T-DNA disruption in ECA1 showed a 4-fold reduction in endoplasmic reticulum-type calcium pump activity. Surprisingly, the phenotype of mutant plants was indistinguishable from wild type when grown on standard nutrient medium containing 1.5 mmCa2+ and 50 μm Mn2+. However, mutants grew poorly on medium with low Ca2+ (0.2 mm) or high Mn2+ (0.5 mm). On high Mn2+, the mutants failed to elongate their root hairs, suggesting impairment in tip growth processes. Expression of the wild-type gene (CAMV35S::ECA1) reversed these conditional phenotypes. The activity of ECA1 was examined by expression in a yeast (Saccharomyces cerevisiae) mutant, K616, which harbors a deletion of its endogenous calcium pumps. In vitro assays demonstrated that Ca2+, Mn2+, and Zn2+stimulated formation of a phosphoenzyme intermediate, consistent with the translocation of these ions by the pump. ECA1 provided increased tolerance of yeast mutant to toxic levels of Mn2+ (1 mm) and Zn2+(3 mm), consistent with removal of these ions from the cytoplasm. These results show that despite the potential redundancy of multiple Ca2+ pumps and Ca2+/H+ antiporters in Arabidopsis, pumping of Ca2+ and Mn2+ by ECA1 into the endoplasmic reticulum is required to support plant growth under conditions of Ca2+ deficiency or Mn2+ toxicity.

Ca21 is required for protein processing, sorting, and secretion in eukaryotic cells, although the particular roles of the transporters involved in the secretory system of plants are obscure. One endomembrane-type Ca-ATPase from Arabidopsis (Arabidopsis thaliana), AtECA3, diverges from AtECA1, AtECA2, and AtECA4 in protein sequence; yet, AtECA3 appears similar in transport activity to the endoplasmic reticulum (ER)-bound AtECA1. Expression of AtECA3 in a yeast (Saccharomyces cerevisiae) mutant defective in its endogenous Ca 21 pumps conferred the ability to grow on Ca 21 -depleted medium and tolerance to toxic levels of Mn 21. A green fluorescent protein-tagged AtECA3 was functionally competent and localized to intracellular membranes of yeast, suggesting that Ca 21 and Mn 21 loading into internal compartment(s) enhanced yeast proliferation. In mesophyll protoplasts, AtECA3-green fluorescent protein associated with a subpopulation of endosome/prevacuolar compartments based on partial colocalization with the Ara7 marker. Interestingly, three independent eca3 T-DNA disruption mutants showed severe reduction in root growth normally stimulated by 3 mM Ca 21 , indicating that AtECA3 function cannot be replaced by an ER-associated AtECA1. Furthermore, root growth of mutants is sensitive to 50 mM Mn 21 , indicating that AtECA3 is also important for the detoxification of excess Mn 21 . Curiously, Ateca3 mutant roots produced 65% more apoplastic protein than wild-type roots, as monitored by peroxidase activity, suggesting that the secretory process was altered. Together, these results demonstrate that the role of AtECA3 is distinct from that of the more abundant ER AtECA1. AtECA3 supports Ca 21 -stimulated root growth and the detoxification of high Mn 21 , possibly through activities mediated by post-Golgi compartments that coordinate membrane traffic and sorting of materials to the vacuole and the cell wall.

TIFY, previously known as ZIM, comprises a plant-specific family annotated as transcription factors that might play important roles in stress response. Despite TIFY proteins have been reported in Arabidopsis and rice, a comprehensive and systematic survey of ZmTIFY genes has not yet been conducted. To investigate the functions of ZmTIFY genes in this family, we isolated and characterized 30 ZmTIFY (1 TIFY, 3 ZML, and 26 JAZ) genes in an analysis of the maize (Zea mays L.) genome in this study. The 30 ZmTIFY genes were distributed over eight chromosomes. Multiple alignment and motif display results indicated that all ZmTIFY proteins share two conserved TIFY and Jas domains. Phylogenetic analysis revealed that the ZmTIFY family could be divided into two groups. Putative cis-elements, involved in abiotic stress response, phytohormones, pollen grain, and seed development, were detected in the promoters of maize TIFY genes. Microarray data showed that the ZmTIFY genes had tissue-specific expression patterns in various maize developmental stages and in response to biotic and abiotic stresses. The results indicated that ZmTIFY4, 5, 8, 26, and 28 were induced, while ZmTIFY16, 13, 24, 27, 18, and 30 were suppressed, by drought stress in the maize inbred lines Han21 and Ye478. ZmTIFY1, 19, and 28 were upregulated after infection by three pathogens, whereas ZmTIFY4, 13, 21, 23, 24, and 26 were suppressed. These results indicate that the ZmTIFY family may have vital roles in response to abiotic and biotic stresses. The data presented in this work provide vital clues for further investigating the functions of the genes in the ZmTIFY family.