ANNEXIN1 mediates calcium-dependent systemic defense in Arabidopsis plants upon herbivory and wounding.

Funder: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior; Id: http://dx.doi.org/10.13039/501100002322Cellular calcium (Ca) transients are endogenous signals involved in local and systemic signaling and defense activation upon environmental stress, including wounding and herbivory. Still, not all Ca2+ channels contributing to the signaling have been identified, nor are their modes of action fully known. Plant annexins are proteins capable of binding to anionic phospholipids and can exhibit Ca channel-like activity. Arabidopsis ANNEXIN1 (ANN1) is suggested to contribute to Ca transport. Here, we report that wounding and simulated-herbivory-induced cytosolic free Ca elevation was impaired in systemic leaves in ann1 loss-of-function plants. We provide evidence for a role of ANN1 in local and systemic defense of plants attacked by herbivorous Spodoptera littoralis larvae. Bioassays identified ANN1 as a positive defense regulator. Spodoptera littoralis feeding on ann1 gained significantly more weight than larvae feeding on wild-type, whereas those feeding on ANN1-overexpressing lines gained less weight. Herbivory and wounding both induced defense-related responses on treated leaves, such as jasmonate accumulation and defense gene expression. These responses remained local and were strongly reduced in systemic leaves in ann1 plants. Our results indicate that ANN1 plays an important role in activation of systemic rather than local defense in plants attacked by herbivorous insects

Annexins as Overlooked Regulators of Membrane Trafficking in Plant Cells

Annexins are an evolutionary conserved superfamily of proteins able to bind membrane phospholipids in a calcium-dependent manner. Their physiological roles are still being intensively examined and it seems that, despite their general structural similarity, individual proteins are specialized toward specific functions. However, due to their general ability to coordinate membranes in a calcium-sensitive fashion they are thought to participate in membrane flow. In this review, we present a summary of the current understanding of cellular transport in plant cells and consider the possible roles of annexins in different stages of vesicular transport

Structure, function and membrane interactions of plant annexins: an update

Knowledge accumulated over the past 15 years on plant annexins clearly indicates that this disparate group of proteins builds on the common annexin function of membrane association, but possesses divergent molecular mechanisms. Functionally, the current literature agrees on a key role of plant annexins in stress response processes such as wound healing and drought tolerance. This is contrasted by only few established details of the molecular level mechanisms that are driving these activities. In this review, we appraise the current knowledge of plant annexin molecular, functional and structural properties with a special emphasis on topics of less coverage in recent past overviews. In particular, plant annexin post-translational modification, roles in polar growth and membrane stabilisation processes are discussed.Full Tex

Is annexin 1 a multifunctional protein during stress responses?

Accumulating evidence suggest that certain annexins can play a role in abiotic stress responses in plants. We found that for one member of the Arabidopsis thaliana annexin gene family, annexin 1 (AnnAt1), loss-of-function mutants are more sensitive to drought stress and gain-of-function mutants are more tolerant.1 We also found that AnnAt1 is able to regulate accumulation of H2O2 in vivo in Arabidopsis cells based on the observation that the level of ROS accumulation following induction by ABA correlates with the level of AnnAt1 protein in transgenic Arabidopsis plants. Here we provide more commentary on the antioxidant activity of AnnAt1, critically assess the evidence that AnnAt1 and other annexins possess peroxidase activity, emphasize a redox-induced posttranslational modification which occurs to AnnAt1 during ABA signaling, and discuss ways this annexin’s membrane associations could mediate stress signaling while addressing the potential that AnnAt1 is a multifunctional protein in plants

Collagenase as a useful tool for the analysis of plant cellular peripheries

A technique for the selective loosening of the cell wall structure and the isolation of proteins permanently knotted in the cell walls was elaborated. Following treatment with collagenase, some proteins, such as calreticulin (CRT) and auxin binding protein 1 (ABP1) were released from purified cell walls, most probably through destruction of respective interacting proteins. The results were confirmed by the immunolocalization of the ABP1 and CRT with confocal and electron microscopy. On the other hand, potential substrates of collagenase, among them annexin 1 have been recognized. Mass spectra of annexin 1 obtained after collagenase digestion and results from analysis of potential cleavage sites suggested that the mechanism of enzyme cleavage might not depend on the amino acid sequence. Summarizing, collagenase was found to be a very useful tool for exploring molecules involved in the functioning of cellular peripheries

Down-regulation of CBP80 gene expression as a strategy to engineer a drought-tolerant potato

Developing new strategies for crop plants to respond to drought is crucial for their innovative breeding. The down-regulation of nuclear cap-binding proteins in Arabidopsis renders plants drought tolerant. The CBP80 gene in the potato cultivar Desiree was silenced using artificial microRNAs. Transgenic plants displayed a higher tolerance to drought, ABA-hypersensitive stomatal closing, an increase in leaf stomata and trichome density, and compact cuticle structures with a lower number of microchannels. These findings were correlated with a higher tolerance to water stress. The level of miR159 was decreased, and the levels of its target mRNAs MYB33 and MYB101 increased in the transgenic plants subjected to drought. Similar trends were observed in an Arabidopsis cbp80 mutant. The evolutionary conservation of CBP80, a gene that plays a role in the response to drought, suggests that it is a candidate for genetic manipulations that aim to obtain improved water-deficit tolerance of crop plants

The Role of Annexin 1 in Drought Stress in Arabidopsis1[W]

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 up-regulated 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 Ca2+ 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

NPQ assayed in leaf of well-watered potato plants.

<p>Potato WT (dashed line) and transgenic S-7 (solid line) grew in the walk-in growth chamber under controlled conditions and were watered to maintained FC at 65%. Performance of gross non-photochemical quenching (NPQ) were assayed on the first fully developed composite leaf from the top of plant at 4 hours after turning the light with Dual PAM-100. For measurement plants were adapted to dark for 20 minutes and then stimulated with repeated light pulses of actinic light (94 PPFD) for 5 minutes and once again subjected to dark for 6 minutes. Each point represents the mean ±SD (<i>n</i> = 3–4). Experiment was repeated three times and gave comparable results.</p

Profiling of annexin expression in WT potato leaves during drought.

<p>Potato WT plants grew in the walk-in growth chamber under controlled conditions. After 8–10 weeks irrigation was gradually reduced to decrease the field capacity (FC) to 25% (which took approximately 10 days) and then maintained at this level till 14^th day. Samples were collected from the first fully developed composite leaf from the top at indicated time points (D0 – beginning of drought, D6 – sixth day of drought, and D14 – fourteenth day of drought). RNA was isolated with Trizol and sq-RT-PCR was performed with primer sets specific for certain annexins. The level of expression was normalized against <i>EF1a</i> mRNA. Results are means ±SE (n≤4). Homogenic groups are determined by Tukey HSD (Honestly Significant Differences) test. The same letters designate values belong to the same homogenic group (p<0.05). Experiment was repeated twice.</p

Accumulation of ROS (hydrogen peroxide and superoxide anion) and lipid peroxidation.

<p>Potato WT (white bars) and transgenic line S-7 (black bars) grew in walk-in growth chamber under controlled conditions. Leaf discs were expunged from the third, fourth and fifth upper fully expanded leaves and immediately vacuum infiltrated with methyl viologen (50 μM). After 1 hour incubation in dark discs were exposed to high light irradiance (850 PPFD) for indicated times (0.5–24 hours). Superoxide anion was determined colorimetrically with nitro blue tetrazolium chloride 9NBT). Hydrogen peroxide was stained in tissue with diaminobenzidine tetrahydrochloride (DAB) and quantified using the ImageJ. Lipid peroxidation was estimated spectrophotometrically with thiobarbituric acid (TBA). Results are means ±SE (n = 5). Homogenic groups are determined by Tukey HSD (Honestly Significant Differences) test. The same letters designate values belong to the same homogenic group (p<0.05). Experiment was repeated twice.</p