Annexins act as targets of calcium signals in eukaryotic cells, and recent results suggest that they play an important role in plant stress responses. We found that in Arabidopsis (Arabidopsis thaliana), AnnAt1 (for annexin 1) mRNA levels were upregulated in leaves by most of the stress treatments applied. Plants overexpressing AnnAt1 protein were more drought tolerant and knockout plants were more drought sensitive than ecotype Columbia plants. We also observed that hydrogen peroxide accumulation in guard cells was reduced in overexpressing plants and increased in knockout plants both before and after treatment with abscisic acid. Oxidative protection resulting from AnnAt1 overexpression could be due to the low level of intrinsic peroxidase activity exhibited by this protein in vitro, previously linked to a conserved histidine residue found in a peroxidase-like motif. However, analyses of a mutant H40A AnnAt1 protein in a bacterial complementation test and in peroxidase activity assays indicate that this residue is not critical to the ability of AnnAt1 to confer oxidative protection. To further examine the mechanism(s) linking AnnAt1 expression to stress resistance, we analyzed the reactive S3 cluster to determine if it plays a role in AnnAt1 oligomerization and/or is the site for posttranslational modification. We found that the two cysteine residues in this cluster do not form intramolecular or intermolecular bonds but are highly susceptible to oxidation-driven S-glutathionylation, which decreases the Ca 2+ affinity of AnnAt1 in vitro. Moreover, S-glutathionylation of AnnAt1 occurs in planta after abscisic acid treatment, which suggests that this modification could be important in regulating the cellular function of AnnAt1 during stress responses.

A BSTR ACTA 12-residue peptide AcDKDGDGY-ISAAENH 2 analogous to the third calcium-binding loop of calmodulin strongly coordinates lanthanide ions (K ‫؍‬ 10 5 M ؊1 ). When metal saturated, the peptide adopts a very rigid structure, the same as in the native protein, with three last residues AAE fixed in the ␣-helical conformation. Therefore, the peptide provides an ideal helix nucleation site for peptide segments attached to its C terminus. NMR and CD investigations of peptide AcDKDGDGYISAAEAAAQNH 2 presented in this paper show that residues A13-Q16 form an ␣-helix of very high stability when the La 3؉ ion is bound to the D1-E12 loop. In fact, the lowest estimates of the helix content in this segment give values of at least 80% at 1°C and 70% at 25°C. This finding is not compatible with existing helix-coil transition theories and helix propagation parameters, s, reported in the literature. We conclude, therefore, that the initial steps of helix propagation are characterized by much larger s values, whereas helix nucleation is even more unfavorable than is believed. In light of our findings, thermodynamics of the nascent ␣-helices is discussed. The problem of CD spectra of very short ␣-helices is also addressed.

SNF1-related protein kinases 2 (SnRK2s) are plant-specific enzymes involved in environmental stress signaling and abscisic acid-regulated plant development. Here, we report that SnRK2s interact with and are regulated by a plant-specific calcium-binding protein. We screened a Nicotiana plumbaginifolia Matchmaker cDNA library for proteins interacting with Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK), a member of the SnRK2 family. A putative EF-hand calcium-binding protein was identified as a molecular partner of NtOSAK. To determine whether the identified protein interacts only with NtOSAK or with other SnRK2s as well, we studied the interaction of an Arabidopsis thaliana orthologue of the calcium-binding protein with selected Arabidopsis SnRK2s using a two-hybrid system. All kinases studied interacted with the protein. The interactions were confirmed by bimolecular fluorescence complementation assay, indicating that the binding occurs in planta, exclusively in the cytoplasm. Calcium binding properties of the protein were analyzed by fluorescence spectroscopy using Tb 3؉ as a spectroscopic probe. The calcium binding constant, determined by the protein fluorescence titration, was 2.5 ؎ 0.9 ؋ 10 5 M ؊1 . The CD spectrum indicated that the secondary structure of the protein changes significantly in the presence of calcium, suggesting its possible function as a calcium sensor in plant cells. In vitro studies revealed that the activity of SnRK2 kinases analyzed is inhibited in a calcium-dependent manner by the identified calcium sensor, which we named SCS (SnRK2-interacting calcium sensor). Our results suggest that SCS is involved in response to abscisic acid during seed germination most probably by negative regulation of SnRK2s activity.Plants respond to environmental stresses by induction of various defense mechanisms. Stress signals are recognized and transmitted to different cellular compartments by specialized signaling pathways in which protein kinases and phosphatases are key components. The SnRK2 family members are plant-specific kinases considered as important regulators of plant response to abiotic stresses. Ten members of the SnRK2 family have been identified in both Arabidopsis thaliana and Oryza sativa (1, 2). All of them, except SnRK2.9 from A. thaliana, were shown by transient expression in protoplasts to be rapidly activated by treatment with different osmolytes, such as sucrose, mannitol, sorbitol, or NaCl and some of them also by abscisic acid (ABA), 3 suggesting that these kinases are involved in a general response to osmotic stress (1-3). It was also reported that in tobacco BY-2 cells Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK), a member of the SnRK2 subfamily, is activated rapidly in response to hyperosmotic stress (4 -6).Ample data indicate that SnRK2s are positive regulators of plant response to drought. ABA-activated protein kinase (AAPK) is activated by ABA in guard cells of fava bean (Vicia faba) in response to drought and is involved in the regula...

S100A1 is a typical representative of a group of EF‐hand calcium‐binding proteins known as the S100 family. The protein is composed of two α subunits, each containing two calcium‐binding loops (N and C). At physiological pH (7.2) and NaCl concentration (100 mm), we determined the microscopic binding constants of calcium to S100A1 by analysing the Ca2+‐titration curves of Trp90 fluorescence for both the native protein and its Glu32 → Gln mutant with an inactive N‐loop. Using a chelator method, we also determined the calcium‐binding constant for the S100A1 Glu73 → Gln mutant with an inactive C‐loop. The protein binds four calcium ions in a noncooperative way with binding constants of K1 =4 ± 2 × 103 m−1 (C‐loops) and K2≈ 102 m−1 (N‐loops). Only when both loops are saturated with calcium does the protein change its global conformation, exposing to the solvent hydrophobic patches, which can be detected by 2‐p‐toluidinylnaphthalene‐6‐sulfonic acid – a fluorescent probe of protein‐surface hydrophobicity. S‐Glutathionylation of the single cysteine residue (85) of the α subunits leads to a 10‐fold increase in the affinity of the protein C‐loops for calcium and an enormous – four orders of magnitude – increase in the calcium‐binding constants of its N‐loops, owing to a cooperativity effect corresponding to ΔΔG = −6 ± 1 kcal·mol−1. A similar effect is observed upon formation of the mixed disulfide with cysteine and 2‐mercaptoethanol. The glutathionylated protein binds TRTK‐12 peptide in a calcium‐dependent manner. S100A1 protein can act, therefore, as a linker between the calcium and redox signalling pathways.