Mechanical wounding not only damages plant tissues, but also provides pathways for pathogen invasion. To understand plant responses to wounding at a genomic level, we have surveyed the transcriptional response of 8,200 genes in Arabidopsis plants. Approximately 8% of these genes were altered by wounding at steady-state mRNA levels. Studies of expression patterns of these genes provide new information on the interactions between wounding and other signals, including pathogen attack, abiotic stress factors, and plant hormones. For example, a number of wound-responsive genes encode proteins involved in pathogen response. These include signaling molecules for the pathogen resistance pathway and enzymes required for cell wall modification and secondary metabolism. Many osmotic stress- and heat shock-regulated genes were highly responsive to wounding. Although a number of genes involved in ethylene, jasmonic acid, and abscisic acid pathways were activated, many in auxin responses were suppressed by wounding. These results further dissected the nature of mechanical wounding as a stress signal and identified new genes that may play a role in wounding and other signal transduction pathways.

Although calcium is a critical component in the signal transduction pathways that lead to stress gene expression in higher plants, little is known about the molecular mechanism underlying calcium function. It is believed that cellular calcium changes are perceived by sensor molecules, including calcium binding proteins. The calcineurin B-like (CBL) protein family represents a unique group of calcium sensors in plants. A member of the family, CBL1, is highly inducible by multiple stress signals, implicating CBL1 in stress response pathways. When the CBL1 protein level was increased in transgenic Arabidopsis plants, it altered the stress response pathways in these plants. Although drought-induced gene expression was enhanced, gene induction by cold was inhibited. In addition, CBL1-overexpressing plants showed enhanced tolerance to salt and drought but reduced tolerance to freezing. By contrast, cbl1 null mutant plants showed enhanced cold induction and reduced drought induction of stress genes. The mutant plants displayed less tolerance to salt and drought but enhanced tolerance to freezing. These studies suggest that CBL1 functions as a positive regulator of salt and drought responses and a negative regulator of cold response in plants.

Members of the Arabidopsis calcineurin B–like Ca²⁺ binding protein (AtCBL) family are differentially regulated by stress conditions. One AtCBL plays a role in salt stress; another is implicated in response to other stress signals, including drought, cold, and wounding. In this study, we identified a group of novel protein kinases specifically associated with AtCBL-type Ca²⁺ sensors. In addition to a typical protein kinase domain, they all contain a unique C-terminal region that is both required and sufficient for interaction with the AtCBL-type but not calmodulin-type Ca²⁺ binding proteins from plants. Interactions between the kinases and AtCBLs require micromolar concentrations of Ca²⁺, suggesting that increases in cellular Ca²⁺ concentrations may trigger the formation of AtCBL–kinase complexes in vivo. Unlike most serine/threonine kinases, the AtCBL-interacting kinase efficiently uses Mn²⁺ to Mg²⁺ as a cofactor and may function as a Mn²⁺ binding protein in the cell. These findings link a new type of Ca²⁺ sensors to a group of novel protein kinases, providing the molecular basis for a unique Ca²⁺ signaling machinery in plant cells.

Calcium is a critical component in a number of plant signal transduction pathways. A new family of calcium sensors called calcineurin B-like proteins (AtCBLs) have been recently identified from Arabidopsis. These calcium sensors have been shown to interact with a family of protein kinases (CIPKs). Here we report that each individual member of AtCBL family specifically interacts with a subset of CIPKs and present structural basis for the interaction and for the specificity underlying these interactions. Although the C-terminal region of CIPKs is responsible for interaction with AtCBLs, the N-terminal region of CIPKs is also involved in determining the specificity of such interaction. We have also shown that all three EF-hand motifs in AtCBL members are required for the interaction with CIPKs. Several AtCBL members failed to interact with any of the CIPKs presented in this study, suggesting that these AtCBL members either have other CIPKs as targets or they target distinct proteins other than CIPKs. These results may provide structural basis for the functional specificity of CBL family of calcium sensors and their targets.

Although Ser/Thr and Tyr kinases/phosphatases play a critical role in cell growth and development in animals, none of the typical Tyr kinases has been characterized from higher plants. The existence of Tyr phosphatases in plants has also been controversial until recently when several members of protein Tyr phosphatase (PTP) family were characterized from Arabidopsis (Gupta et al., 1998; Xu et al., 1998; Fordham-Skelton et al., 1999, 2002; Ulm et al., 2001, 2002; Gupta et al., 2002). These studies suggest that protein Tyr (de) phosphorylation performs critical functions in plants. In support of this hypothesis, biochemical and genetic analyses confirm that Tyr phosphorylation, like that in animals and fungi, is involved in the regulation of MAP kinase activities in plant cells (for review, see Tena et al., 2001; Zhang and Klessig, 2001; Luan, 2002; Ulm et al., 2002).

Protein tyrosine kinases and phosphatases play a vital role in the regulation of cell growth and differentiation in animal systems. However, none of these enzymes has been characterized from higher plants. In this study, we isolated a cDNA encoding a putative protein tyrosine phosphatase (PTPase) from Arabidopsis (referred to as AtPTP1). The expression level of AtPTP1 is highly sensitive to environmental stresses. High-salt conditions increased AtPTP1 mRNA levels, whereas cold treatment rapidly eliminated the AtPTP1 transcript. The recombinant AtPTP1 protein specifically hydrolyzed phosphotyrosine, but not phosphoserine/threonine, in protein substrates. Site-directed mutagenesis defined two highly conserved amino acids, cysteine-265 and aspartate-234, as being essential for the phosphatase activity of the AtPTP1 protein, suggesting a common catalytic mechanism for PTPases from all eukaryotic systems. In summary, we have identified AtPTP1 as a tyrosine-specific protein phosphatase that may function in stress responses of higher plants.