Proteomic analysis of defense response of wildtype Arabidopsis thaliana and plants with impaired NO- homeostasis

In recent years, nitric oxide (NO) has been recognized as a signalling molecule of plants, being involved in diverse processes like germination, root growth, stomatal closing, and responses to various stresses. A mechanism of how NO can regulate physiological processes is the modulation of cysteine residues of proteins (S-nitrosylation) by S-nitrosoglutathione (GSNO), a physiological NO donor. The concentration of GSNO and the level of S-nitrosylated proteins are regulated by GSNO reductase, which seems to play a major role in NO signalling. To investigate the importance of NO in plant defense response, we performed a proteomic analysis of Arabidopsis wildtype and GSNO-reductase knock-out plants infected with both the avirulent and virulent pathogen strains of Pseudomonas syringae. Using 2-D DIGE technology in combination with MS, we identified proteins, which are differentially accumulated during the infection process. We observed that both lines were more resistant to avirulent infections than to virulent infections mainly due to the accumulation of stress-, redox-, and defense-related proteins. Interestingly, after virulent infections, we also observed accumulation of defense-related proteins, but no or low accumulation of stress- and redox-related proteins, respectively. In summary, we present here the first detailed proteomic analysis of plant defense response.     

Adaptation and growth response of Arabidopsis thaliana to deuterium

Summary Arabidopsis seeds were sown aseptically on mineral media containing between 0 and 90% of heavy water (D₂O). Initially, a D₂O level of over 50% was lethal for the plants. However, after culture for six successive generations on 50% D₂O, plants were capable of growing marginally on media containing up to 70% D₂O, but not higher. With increasing concentration, deuterium progressively delays germination, slows growth, reduces survival, results in bleaching of the leaves and delays flowering. Pollen fertility is not affected measurably but seed set is reduced with increasing levels of deuteration so that at 70% D₂O few seeds were obtained. The viability of the seeds harvested from plants grown on deuterated media is low. No chlorophyll or morphological mutants were observed among a large number of plant progenies. Seeds from plants cultured on D₂O media for several generations grow normally on proteated media in the very first generation.     

An analysis of learned kin recognition in hymenoptera

A model is developed to explain data on the probability with which a strange conspecific hymenopteran female will be accepted into a group of sisters. The analysis is based on a genetic labeling system (primarily odors) of m loci and n_i equally frequent alleles at the i-th locus (i = 1,…, m). Three recognition mechanisms are considered (viz: genotype recognition; foreign-label rejection; and habituated-label acceptance) where all three mechanisms depend on individuals learning the labels represented in their group. The probability with which non-kin will be accepted into large sibling group is calculated for a number of different labeling systems. These different labeling systems are compared and a comparision is also made between the three recognition mechanisms mentioned above. A general expression is then derived, in terms of the number of loci and alleles in the labeling system and the size of the sibling group, for the probability with which “strang” sisters are accepted into a group of sisters with whom they have had no prior contact. These results are applied to existing data on the primitively eusocial sweat bee Lasioglossum zephyrum and the present indication is that recognition in L. zephyrum can be modeled by foreign-label rejection with a genetic labeling system of four or five loci with eight to ten alleles in total (i.e. each locus will have two or at most three alleles).     

Proteome analysis of cold stress response in Arabidopsis thaliana using DIGE-technology

A proteome study based on 2-D gel electrophoresis was performed in order to analyse the cold-stress response of Arabidopsis plants. The emphasis was to monitor the overall changes in the protein complement after prolonged exposure rather than short-term responses. Two different temperature regimes were used (6 degrees C and 10 degrees C) and plants were exposed to cold-stress exposure for 1 week. Protein patterns were also monitored after re-shifting plants to control conditions for a further week. To monitor gradual changes in the response to the two cold-stress conditions, the analysis was performed with DIGE technology with the inclusion of an internal standard. In the experiments using 6 degrees C, 22 spots with at least 2-fold altered expression were found; among them 18 were increased and four were decreased. When plants were exposed to 10 degrees C, 18 of these 22 spots still showed a 2-fold change; however, the alterations were, in general, more moderate than observed under 6 degrees C. Spot identification was performed by MALDI-TOF and ESI-MS/MS. Many of the proteins identified have previously been described in the context of cold-stress responses, indicating the validity of this proteome approach for further in-depth studies.     

Proteomic analysis of Arabidopsis thaliana ecotypes with contrasted root architecture in response to phosphate deficiency

Owing to a weak availability in soil, plants have developed numerous morphological, physiological and biochemical adaptations to acquire phosphate (Pi). Identification and characterisation of key genes involved in the initial steps of Pi-signalling might provide clues about the regulation of the complex Pi deficiency adaptation mechanism. A two-dimensional gel electrophoresis approach was performed to investigate proteome responses to Pi starvation in Arabidopsis. Two ecotypes were selected according to contrasting responses of their root system architecture to low availability of Pi. Thirty protein spots were shown to be affected by Pi deficiency. Fourteen proteins appeared to be up-regulated and ten down-regulated with ecotype Be-0, wheras only thirteen proteins were observed as down-regulated for ecotype Ll-0. Furthermore, systematic and opposite responses to Pi deficiency were observed between the two ecotypes. The sequences of these 30 differentially expressed protein spots were identified using mass spectrometry, and most of the proteins were involved in oxidative stress, carbohydrate and proteins metabolism. The results suggested that the modulation of alcohol dehydrogenase, malic enzyme and aconitate hydratase may contribute to the contrasted adaptation strategy to Pi deficiency of Be-0 and Ll-0 ecotypes. A focus on aconitate hydratase highlighted a complex reverse response of the pattern of corresponding spots between the two ecotypes. This protein, also potentially involved in iron homeostasis, was speculated to contribute, at least indirectly, to the root architecture response of these ecotypes.     

The responses of wild-type and ABA mutant Arabidopsis thaliana plants to phosphorus starvation

The growth patterns of plants subjected to phosphorus starvation resemble those caused by treatment with ABA, suggesting that ABA could mediate the response of the plant to phosphorus starvation. We examined the role of ABA in phosphorus stress by comparing growth and biochemical responses of Arabidopsis thaliana ABA mutants aba-1 and abi2-1 to those of wild-type plants. We first characterized acid phosphatase production of wild-type Arabidopsis in response to phosphorus starvation. We found that several acid phosphatase isozymes are present in roots and shoots, but only a subset of these isozymes are induced by phosphorus stress, and they are induced in both organs. Production of acid phosphatase in response to phosphorus stress was not affected by the aba-1 or abi2-1 mutations. Low phosphorus also resulted in decreased growth of both wild-type and ABA mutant plants, and the root-to-shoot ratio was increased in both wild type and mutants. Anthocyanins accumulated in response to phosphorus stress in both wild-type and mutant plants, but the increase was reduced in the aba-1 mutant. Thus, two different ABA mutants responded normally in most respects to phosphorus stress. Our data do not support a major role for ABA in coordinating the phosphorus-stress response.     

Identification of genes required for pollen-stigma recognition in Arabidopsis thaliana

In higher plants, cell-cell recognition reactions taking place following pollination allow the selective restriction of self-pollination and/or interspecific pollination. Many of these systems function by regulating the process of water transfer from the cells found at the stigmatic surface to the individual pollen grain. Interspecific pollination studies on the cruciferous weed Arabidopsis thaliana revealed only a broad specificity of pollen recognition such that pollen from all tested members of the crucifer family were recognized, whereas pollen from almost all other species failed to hydrate. Genetic analysis of A. thaliana has identified three genes that are essential for this recognition process. Recessive mutations in any of these genes result in male sterility due to the production of pollen grains that fail to hydrate when placed on the stigma, but that are capable of hydrating and growing a pollen tube in vitro. Results from mixed pollination experiments suggest that the mutant pollen grains specifically lack a functional pollen-stigma recognition system. All three mutations described also result in a defect in the wax layer normally found on stems and leaves, similar to previously described eceriferum (cer) mutations. Genetic complementation and mapping experiments demonstrated that the newly identified mutants are allelic to the previously identified genes cer1, cer3 and cer6. TEM analysis of the ultrastructure of the pollen coating revealed that all of the mutant pollen grains bear coatings of normal thickness and that tryphine lipid droplets are missing in cer1-147, are reduced in size in cer6-2654 and appear normal in cer3-2186. Temperature shift experiments revealed that the block in the recognition step of the mutant pollen grains can be suppressed by pollination at lower temperatures but not by reduced temperatures during pollen development. These results suggest that the lipids which are altered in the cer mutations may be important in regulating some biophysical property of the pollen coating.     

Simulations of virtual plants reveal a role for SERRATE in the response of leaf development to light in Arabidopsis thaliana

The SERRATE gene (SE) was shown to determine leaf organogenesis and morphogenesis patterning in Arabidopsis thaliana. The se-1 mutant was used here to investigate the role of SE in leaf development in response to incident light. Virtual plants were modelled to analyse the phenotypes induced by this mutation. Plants were grown under various levels of incident light. The amount of light absorbed by the plant was estimated by combining detailed characterizations of the radiative environment and virtual plant simulations. Four major changes in leaf development were induced by the se-1 mutation. Two constitutive leaf growth variables were modified, with a lower initial expansion rate and a higher duration of expansion. Two original responses to a reduced incident light were identified, concerning the leaf-initiation rate and the duration of leaf expansion. The se-1 mutation dramatically affects both changes in the leaf development pattern and the response to reduced incident light. Virtual plants helped to reveal the combined effects of the multiple changes induced by this mutation.     

Hypersensitive response in cucumber mosaic virus-inoculated Arabidopsis thaliana

The responses of 12 Arabidopsis ecotypes to cucumber mosaic virus strain Y (CMV(Y)) or strain O (CMV(O)) were characterized. Except for ecotype C24, all ecotypes were susceptible to both strains of CMV. In C24, CMV(O) multiplied systemically, but CMV(Y) did not spread systemically and induced only local necrotic spots in virus-inoculated leaves 21–27 h after inoculation. In CMV(Y)-inoculated C24 leaves, virus was confined to the inoculated leaves, and the amount of the pathogenesis-related-1 protein increased during the progress of local necrotic spot formation. These results indicate that C24 mounts a hypersensitive response (HR) to CMV(Y). By genetic analysis of crosses between C24 and ecotype Columbia or Landsberg (erecta) which are susceptible to CMV(Y) infection, the HR to CMV(Y) in C24 was found to be determined by a single major dominant gene whose function was influenced by a modifier gene from the Landsberg ecotype. Comparison of the responses between C24 leaves inoculated with pseudorecombinants of both strains of CMV suggested that the HR was controlled by CMV RNA3. The molecular interaction between the single major gene and CMV(Y) RNA3 is likely to induce the HR in CMV(Y)-inoculated C24.