To identify genes of potential importance to cold, salt, and drought tolerance, global expression profiling was performed on Arabidopsis plants subjected to stress treatments of 4°C, 100 mm NaCl, or 200 mm mannitol, respectively. RNA samples were collected separately from leaves and roots after 3-and 27-h stress treatments. Profiling was conducted with a GeneChip microarray with probe sets for approximately 8,100 genes. Combined results from all three stresses identified 2,409 genes with a greater than 2-fold change over control. This suggests that about 30% of the transcriptome is sensitive to regulation by common stress conditions. The majority of changes were stimulus specific. At the 3-h time point, less than 5% (118 genes) of the changes were observed as shared by all three stress responses. By 27 h, the number of shared responses was reduced more than 10-fold (Ͻ 0.5%), consistent with a progression toward more stimulus-specific responses. Roots and leaves displayed very different changes. For example, less than 14% of the cold-specific changes were shared between root and leaves at both 3 and 27 h. The gene with the largest induction under all three stress treatments was At5g52310 (LTI/COR78), with induction levels in roots greater than 250-fold for cold, 40-fold for mannitol, and 57-fold for NaCl. A stress response was observed for 306 (68%) of the known circadian controlled genes, supporting the hypothesis that an important function of the circadian clock is to "anticipate" predictable stresses such as cold nights. Although these results identify hundreds of potentially important transcriptome changes, the biochemical functions of many stress-regulated genes remain unknown.Plants have a remarkable ability to cope with highly variable environmental stresses, including cold, drought, and soils with changing salt and nutrient concentrations (i.e. abiotic stress). Nevertheless, these stresses together represent the primary cause of crop loss worldwide (Boyer, 1982), reducing average yields for most major crop plants by more than 50% (Bray et al., 2000). In contrast, the estimated yield loss caused by pathogens is typically around 10% to 20%.Significant progress has been made to understand and manipulate abiotic stress responses (for reviews, see Shinozaki and Yamaguchi-Shinozaki, 1996;Bohnert and Sheveleva, 1998;Smirnoff, 1998;Blumwald, 2000;Bray et al., 2000;Cushman and Bohnert, 2000;Hasegawa et al., 2000;Knight, 2000;Schroeder et al., 2001;Serrano and Rodriguez-Navarro, 2001;Thomashow, 2001;Zhu, 2001bZhu, , 2001a. Three important themes have emerged.First, the initiation of most stress treatments correlates with a cytosolic calcium release, in some cases with stimulus-specific patterns of oscillation (Allen et al., 2000;Knight, 2000;Posas et al., 2000). Second, stimulus-specific changes in gene expression are often observed alongside a set of shared stress responses. For example, in a survey of 1,300 Arabidopsis genes, the majority of cold and drought stressregulated genes were observed as a shared stress ...

Previous work on the adaptation of maize (Zea mays) primary roots to water deficit showed that cell elongation is maintained preferentially toward the apex, and that this response involves modification of cell wall extension properties. To gain a comprehensive understanding of how cell wall protein (CWP) composition changes in association with the differential growth responses to water deficit in different regions of the elongation zone, a proteomics approach was used to examine water soluble and loosely ionically bound CWPs. The results revealed major and predominantly region-specific changes in protein profiles between well-watered and water-stressed roots. In total, 152 water deficit-responsive proteins were identified and categorized into five groups based on their potential function in the cell wall: reactive oxygen species (ROS) metabolism, defense and detoxification, hydrolases, carbohydrate metabolism, and other/unknown. The results indicate that stress-induced changes in CWPs involve multiple processes that are likely to regulate the response of cell elongation. In particular, the changes in protein abundance related to ROS metabolism predicted an increase in apoplastic ROS production in the apical region of the elongation zone of water-stressed roots. This was verified by quantification of hydrogen peroxide content in extracted apoplastic fluid and by in situ imaging of apoplastic ROS levels. This response could contribute directly to the enhancement of wall loosening in this region. This large-scale proteomic analysis provides novel insights into the complexity of mechanisms that regulate root growth under water deficit conditions and highlights the spatial differences in CWP composition in the root elongation zone.Roots often continue to grow under water deficits that completely inhibit shoot and leaf elongation (Sharp and Davies, 1979;Westgate and Boyer, 1985), and this is considered an important mechanism of plant adaptation to water-limited conditions (Sharp and Davies, 1989). Investigation of the mechanisms of root growth adaptation to water deficit is important for improving plant performance under drought, because water resources for agriculture are becoming increasingly limited.The physiology of maize (Zea mays) primary root elongation at low water potentials has been studied extensively (for review, see Sharp et al., 2004), which has provided the foundation for an understanding of the complex network of responses involved. Analysis of the relative elongation rate profile within the root elongation zone showed that under severe water deficit, elongation rates are fully maintained in the apical few millimeters but progressively inhibited as cells are displaced further from the root apex (Sharp et al., 1988;Liang et al., 1997). To help understand the maintenance of elongation in the apical region of roots growing under water deficit conditions, Spollen and Sharp (1991) measured the spatial distribution of turgor pressure and found that values were uniformly decreased by over 50% throughout the e...

It is common for the root/shoot ratio of plants to increase when water availability is limiting. This ratio increases because roots are less sensitive than shoots to growth inhibition by low water potentials. The physiological and molecular mechanisms that assist root growth under drought conditions are reviewed, with a focus on changes in cell walls. Maize seedlings adapt to low water potential by making the walls in the apical part of the root more extensible. In part, this is accomplished by increases in expansin activity and in part by other, more complex changes in the wall. The role of xyloglucan endotransglycosylase, peroxidase and other wall enzymes in root adaptation to low water potential is evaluated and some of the complications in the field of study are listed.

Previous work on the growth biophysics of maize (Zea mays L.)primary roots suggested that cell walls in the apical 5 mm of the elongation zone increased their yielding ability as an adaptive response to low turgor and water potential (+,.,). To test this hypothesis more directly, we measured the acid-induced extension of isolated walls from roots grown at high (-0.03 MPa) or low (-1.6 MPa) +, using an extensometer. Acid-induced extension was greatly increased in the apical 5 mm and was largely eliminated in the 5-to 10-mm region of roots grown at low JI,. This pattern is consistent with the maintenance of elongation toward the apex and the shortening of the elongation zone in these roots. Wail proteins extracted from the elongation zone possessed expansin activity, which increased substantially in roots grown at low qW. Western blots likewise indicated higher expansin abundance in the roots at low +, . , . Additionally, the susceptibility of walls to expansin action was higher in the apical 5 mm of roots at low +, . , than in roots at high +, . , . The basal region of the elongation zone (5-10 mm) did not extend in response to expansins, indicating that loss of susceptibility to expansins was associated with growth cessation in this region.Our results indicate that both the increase in expansin activity and the increase in cell-wall susceptibility to expansins play a role in enhancing cell-wall yielding and, therefore, in maintaining elongation in the apical region of maize primary roots at low +, . , .

Laccase, EC 1.10.3.2 or p-diphenol:dioxygen oxidoreductase, has been proposed to be involved in lignin synthesis in plants based on its in vitro enzymatic activity and a close correlation with the lignification process in plants. Despite many years of research, genetic evidence for the role of laccase in lignin synthesis is still missing. By screening mutants available for the annotated laccase gene family in Arabidopsis, we identified two mutants for a single laccase gene, AtLAC15 (At5g48100) with a pale brown or yellow seed coat which resembled the transparent testa (tt) mutant phenotype. A chemical component analysis revealed that the mutant seeds had nearly a 30% decrease in extractable lignin content and a 59% increase in soluble proanthocyanidin or condensed tannin compared with wild-type seeds. In an in vitro enzyme assay, the developing mutant seeds showed a significant reduction in polymerization activity of coniferyl alcohol in the absence of H(2)O(2). Among the dimers formed in the in vitro assay using developing wild-type seeds, 23% of the linkages were beta-O-4 which resembles the major linkages formed in native lignin. The evidence strongly supports that AtLAC15 is involved in lignin synthesis in plants. To our knowledge, this is the first genetic evidence for the role of laccase in lignin synthesis. Changes in seed coat permeability, seed germination and root elongation were also observed in the mutant.

Laccases, EC 1.10.3.2 or p-diphenol:dioxygen oxidoreductases, are multi-copper containing glycoproteins. Despite many years of research, genetic evidence for the roles of laccases in plants is mostly lacking. In this study, a reverse genetics approach was taken to identify T-DNA insertional mutants (the SALK collection) available for genes in the Arabidopsis laccase family. Twenty true null mutants were confirmed for 12 laccase genes of the 17 total laccase genes (AtLAC1 to AtLAC17) in the family. By examining the mutants identified, it was found that four mutants, representing mutations in three laccase genes, showed altered phenotypes. Mutants for AtLAC2, lac2, showed compromised root elongation under PEG-induced dehydration conditions; lac8 flowered earlier than wild-type plants, and lac15 showed an altered seed colour. The diverse phenotypes suggest that laccases perform different functions in plants and are not as genetically redundant as previously thought. These mutants will prove to be valuable resources for understanding laccase functions in vivo.

Cell wall proteins (CWPs) play important roles in various processes, including cell elongation. However, relatively little is known about the composition of CWPs in growing regions. We are using a proteomics approach to gain a comprehensive understanding of the identity of CWPs in the maize (Zea mays) primary root elongation zone. As the first step, we examined the effectiveness of a vacuum infiltration-centrifugation technique for extracting water-soluble and loosely ionically bound (fraction 1) CWPs from the root elongation zone. The purity of the CWP extract was evaluated by comparing with total soluble proteins extracted from homogenized tissue. Several lines of evidence indicated that the vacuum infiltration-centrifugation technique effectively enriched for CWPs. Protein identification revealed that 84% of the CWPs were different from the total soluble proteins. About 40% of the fraction 1 CWPs had traditional signal peptides and 33% were predicted to be nonclassical secretory proteins, whereas only 3% and 11%, respectively, of the total soluble proteins were in these categories. Many of the CWPs have previously been shown to be involved in cell wall metabolism and cell elongation. In addition, maize has type II cell walls, and several of the CWPs identified in this study have not been identified in previous cell wall proteomics studies that have focused only on type I walls. These proteins include endo-1,3;1,4-beta-D-glucanase and alpha-L-arabinofuranosidase, which act on the major polysaccharides only or mainly present in type II cell walls.

Flowering at the appropriate time of year is essential for successful reproduction in plants. We found that HAP3b in Arabidopsis (Arabidopsis thaliana), a putative CCAAT-binding transcription factor gene, is involved in controlling flowering time. Overexpression of HAP3b promotes early flowering while hap3b, a null mutant of HAP3b, is delayed in flowering under a long-day photoperiod. Under short-day conditions, however, hap3b did not show a delayed flowering compared to wild type based on the leaf number, suggesting that HAP3b may normally be involved in the photoperiod-regulated flowering pathway. Mutant hap3b plants showed earlier flowering upon gibberellic acid or vernalization treatment, which means that HAP3b is not involved in flowering promoted by gibberellin or vernalization. Further transcript profiling and gene expression analysis suggests that HAP3b can promote flowering by enhancing expression of key flowering time genes such as FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1. Our results provide strong evidence supporting a role of HAP3b in regulating flowering in plants grown under long-day conditions.