Proteomic analysis of papaya (Carica papaya L.) displaying typical sticky disease symptoms

Papaya (Carica papaya L.) hosts the only described laticifer-infecting virus (Papaya meleira virus, PMeV), which is the causal agent of papaya sticky disease. To understand the systemic effects of PMeV in papaya, we conducted a comprehensive proteomic analysis of leaf samples from healthy and diseased plants grown under field conditions. First, a reference 2-DE map was established for proteins from healthy samples. A total of 486 reproducible spots were identified, and MALDI-TOF-MS/MS data identified 275 proteins accounting for 159 distinct proteins from 231 spots that were annotated. Second, the differential expression of proteins from healthy and diseased leaves was determined through parallel experiments, using 2-DE and DIGE followed by MALDI-TOF-MS/MS and LC-IonTrap-MS/MS, respectively. Conventional 2-DE analysis revealed 75 differentially expressed proteins. Of those, 48 proteins were identified, with 26 being upregulated (U) and 22 downregulated (D). In general, metabolism-related proteins were downregulated, and stress-responsive proteins were upregulated. This expression pattern was corroborated by the results of the DIGE analysis, which identified 79 differentially expressed proteins, with 23 identified (17 U and 6 D). Calreticulin and the proteasome subunits 20S and RPT5a were shown to be upregulated during infection by both 2-DE and DIGE analyses. These data may help shed light on plant responses against stresses and viral infections.     

Photoprotection mutants of Arabidopsis thaliana acclimate to high light by increasing photosynthesis and specific antioxidants

Biochemical and physiological acclimation to different light environments is crucial for plant growth and survival. In high light (HL), feedback de-excitation (qE) is a well-known photoprotective mechanism that dissipates excess excitation energy in the light-harvesting antenna of photosystem II (PSII) and relieves excitation pressure in the photosynthetic electron transport chain. The xanthophylls zeaxanthin (Z) and lutein (L) function in qE, but also have roles as antioxidants. Although several studies have shown that qE is important during short-term fluctuations in light intensity, here we show that it is not required for the growth of Arabidopsis thaliana in prolonged HL conditions in the laboratory. Mutants that are deficient in qE alone, qE and Z synthesis, or in qE, Z synthesis and also L synthesis were able to grow at 1800 micromol photons m(-2) s(-1) and exhibited no major symptoms of photooxidative stress. The mutants (and wild type) acclimated to HL by increasing photosynthetic capacity and decreasing light harvesting, which together rendered qE less important for photoprotection. At a metabolite level, the HL-grown mutants appeared to compensate for their remaining qE deficit with increased alpha-tocopherol and ascorbate levels compared to the wild type. The specificity of this response provides insight into the relationship between qE and the antioxidant network in plants.     

Photoinhibition induced alterations in energy transfer process in phycobilisomes of PS II in the cyanobacterium, Spirulina platensis

Exposure of algae or plants to irradiance from above the light saturation point of photosynthesis is known as high light stress. This high light stress induces various responses including photoinhibition of the photosynthetic apparatus. The degree of photoinhibition could be clearly determined by measuring the parameters such as absorption and fluorescence of chromoproteins. In cyanobacteria and red algae, most of the photosystem (PS) II associated light harvesting is performed by a membrane attached complex called the phycobilisome (PBS). The effects of high intensity light (1000-4000 micromol photons m(-2) s(-1)) on excitation energy transfer from PBSs to PS II in a cyanobacterium Spirulina platensis were studied by measuring room temperature PC fluorescence emission spectra. High light (3000 micromol photons m(-2) s(-1)) stress had a significant effect on PC fluorescence emission spectra. On the other hand, light stress induced an increase in the ratio of PC fluorescence intensity of PBS indicating that light stress inhibits excitation energy transfer from PBS to PS II. The high light treatment to 3000 micromol photons m(-2) s(-1) caused disappearance of 31.5 kDa linker polypeptide which is known to link PC discs together. In addition we observed the similar decrease in the other polypeptide contents. Our data concludes that the Spirulina cells upon light treatment causes alterations in the phycobiliproteins (PBPs) and affects the energy transfer process within the PBSs.     

Proteomic identification of toxic volatile organic compound-responsive proteins in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The proteins that are responsive to toxic volatile organic compounds (VOC) such as formaldehyde and toluene were analyzed with proteome analysis using two-dimensional difference image gel electrophoresis (DIGE) technology. Twenty-one days after germination (DAG) seedlings of Arabidopsis thaliana were exposed either to the gaseous formaldehyde or toluene in an airtight box installed in a plant growth chamber maintained at 24°C under the long day condition with relatively low light condition. Comparative expression analysis revealed 14 and 22 protein spots as proteins displaying at least 1.5-fold differences in expression upon formaldehyde and toluene treatment, respectively, compared to those of untreated control. Most of the isolated spots were successfully identified by peptide analysis using LC-MS-MS. The VOC-responsive proteins contain ATP synthase CF1, ribulose-1,5-bisphosphate carboxylase/oxygenase, photosystem II light harvesting complex, and enolase, which are components of photosynthesis and carbohydrate metabolism. Despite the relatively low light intensity was applied, many identified VOC-induced proteins were previously known to be up-regulated upon high light stimulus. In addition, proteins involved in the toxin catabolic process and stress hormone-related proteins were identified as toluene-induced proteins. Although the exact function of most of the VOC-responsive proteins identified in these experiments had not been characterized, the protein expression analysis using DIGE was clearly demonstrated that plants are capable of responding actively to VOCs at translational level, and identified proteins may provide valuable tools to account for the effects of abiotic stress caused by air pollutants such as VOCs in plant.     

Ascorbate deficiency can limit violaxanthin de-epoxidase activity in vivo

As a response to high light, plants have evolved non-photochemical quenching (NPQ), mechanisms that lead to the dissipation of excess absorbed light energy as heat, thereby minimizing the formation of dangerous oxygen radicals. One component of NPQ is pH dependent and involves the formation of zeaxanthin from violaxanthin. The enzyme responsible for the conversion of violaxanthin to zeaxanthin is violaxanthin de-epoxidase, which is located in the thylakoid lumen, is activated by low pH, and has been shown to use ascorbate (vitamin C) as its reductant in vitro. To investigate the effect of low ascorbate levels on NPQ in vivo, we measured the induction of NPQ in a vitamin C-deficient mutant of Arabidopsis, vtc2-2. During exposure to high light (1,500 micromol photons m(-2) s(-1)), vtc2-2 plants initially grown in low light (150 micromol photons m(-2) s(-1)) showed lower NPQ than the wild type, but the same quantum efficiency of photosystem II. Crosses between vtc2-2 and Arabidopsis ecotype Columbia established that the ascorbate deficiency cosegregated with the NPQ phenotype. The conversion of violaxanthin to zeaxanthin induced by high light was slower in vtc2-2, and this conversion showed saturation below the wild-type level. Both the NPQ and the pigment phenotype of the mutant could be rescued by feeding ascorbate to leaves, establishing a direct link between ascorbate, zeaxanthin, and NPQ. These experiments suggest that ascorbate availability can limit violaxanthin de-epoxidase activity in vivo, leading to a lower NPQ. The results also demonstrate the interconnectedness of NPQ and antioxidants, both important protection mechanisms in plants.     

Over-expression of a pepper plastid lipid-associated protein in tobacco leads to changes in plastid ultrastructure and plant development upon stress

Proteins homologous to fibrillin, a pepper plastid lipid-associated protein involved in carotenoid storage in fruit chromoplasts, have been recently identified in leaf chloroplasts from several species and shown to be induced upon environmental stress. To further investigate the role of the protein, transgenic Nicotiana tabacum plants over-expressing fibrillin using a constitutive promoter were generated. Transgenics grown under standard light intensities (300 micromol photons m-2 sec-1) were found to contain substantial amounts of fibrillin in flowers and leaves. In leaves, the protein was immunolocalized within chloroplasts in both stromal and thylakoid subfractions. No change was noticed in thylakoid structures from transgenics, but chloroplasts contained an increased number of plastoglobules organized in clusters. In petals, leucoplasts were also found to contain more agglutinated plastoglobules. The effects of environmental factors on fibrillin gene expression and protein localization were studied in tobacco leaves. Less fibrillin was present in plants grown under low light intensities, which can be explained by the involvement of a light-dependent splicing step in the control of fibrillin gene expression in leaves. Analysis of protein subfractions from plants subjected to drought or high light showed that both stresses resulted in fibrillin association with thylakoids. Whereas no growth difference between wild-type (WT) and transgenic plants was noticed under low light conditions, transgenics exhibit a longer main stem, enhanced development of lateral stems and accelerated floral development under higher light intensities. These data suggest that fibrillin-related proteins fulfil an important function in plant development in relation to environmental constraints.     

IMMUTANS does not act as a stress-induced safety valve in the protection of the photosynthetic apparatus of Arabidopsis during steady-state photosynthesis

IMMUTANS (IM) encodes a thylakoid membrane protein that has been hypothesized to act as a terminal oxidase that couples the reduction of O(2) to the oxidation of the plastoquinone (PQ) pool of the photosynthetic electron transport chain. Because IM shares sequence similarity to the stress-induced mitochondrial alternative oxidase (AOX), it has been suggested that the protein encoded by IM acts as a safety valve during the generation of excess photosynthetically generated electrons. We combined in vivo chlorophyll fluorescence quenching analyses with measurements of the redox state of P(700) to assess the capacity of IM to compete with photosystem I for intersystem electrons during steady-state photosynthesis in Arabidopsis (Arabidopsis thaliana). Comparisons were made between wild-type plants, im mutant plants, as well as transgenics in which IM protein levels had been overexpressed six (OE-6 x) and 16 (OE-16 x) times. Immunoblots indicated that IM abundance was the only major variant that we could detect between these genotypes. Overexpression of IM did not result in increased capacity to keep the PQ pool oxidized compared to either the wild type or im grown under control conditions (25 degrees C and photosynthetic photon flux density of 150 micromol photons m(-2) s(-1)). Similar results were observed either after 3-d cold stress at 5 degrees C or after full-leaf expansion at 5 degrees C and photosynthetic photon flux density of 150 micromol photons m(-2) s(-1). Furthermore, IM abundance did not enhance protection of either photosystem II or photosystem I from photoinhibition at either 25 degrees C or 5 degrees C. Our in vivo data indicate that modulation of IM expression and polypeptide accumulation does not alter the flux of intersystem electrons to P(700)(+) during steady-state photosynthesis and does not provide any significant photoprotection. In contrast to AOX1a, meta-analyses of published Arabidopsis microarray data indicated that IM expression exhibited minimal modulation in response to myriad abiotic stresses, which is consistent with our functional data. However, IM exhibited significant modulation in response to development in concert with changes in AOX1a expression. Thus, neither our functional analyses of the IM knockout and overexpression lines nor meta-analyses of gene expression support the model that IM acts as a safety valve to regulate the redox state of the PQ pool during stress and acclimation. Rather, IM appears to be strongly regulated by developmental stage of Arabidopsis.