Dawid Perlikowski

Changes in the chloroplast proteome following water deficit and subsequent watering in a high- and a low-drought-tolerant genotype of Festuca arundinacea

Festuca arundinacea is one of the most drought-tolerant species within the Lolium-Festuca complex and was used as a model for research aimed at identifying the chloroplast components involved in the proteomic response for drought stress in forage grasses. Individual F. arundinacea genotypes with contrasting levels of drought tolerance, the high-drought-tolerant (HDT) and the low-drought-tolerant (LDT) genotypes, were selected for comparative physiological and proteomic work. Measurements of water uptake, chlorophyll fluorescence, relative water content, electrolyte leakage, and gas exchange during drought and rewatering periods were followed by investigations on accumulation levels of chloroplast proteins before drought conditions, on d 3 and 11 of drought treatment, and after 10 d of subsequent watering, using two-dimensional gel electrophoresis. The proteins that were accumulated differentially between the selected plants were then identified by mass spectrometry. The LDT genotype revealed lower levels of water uptake and relative water content as drought progressed, and this was accompanied by lower levels of transpiration and net photosynthesis, and a higher level of electrolyte leakage observed in this genotype. Eighty-two protein accumulation profiles were compared between the HDT and LDT genotypes and ten proteins were shown to be differentially accumulated between them. The functions of the selected proteins in plant cells and their probable influence on the process of recovery after drought treatment in F. arundinacea are discussed.

Influence of short-term drought conditions and subsequent re-watering on the physiology and proteome of Lolium multiflorum/Festuca arundinacea introgression forms, with contrasting levels of tolerance to long-term drought.

Festuca arundinacea is a drought tolerant species. Lolium multiflorum has better forage quality but lower tolerance to abiotic stresses. Their hybrids offer an opportunity to perform research on the molecular basis of tolerance to drought. The aim of this work was to recognise the mechanisms of response to short-term drought (11 days) in a glasshouse in two L. multiflorum/F. arundinacea introgression forms with distinct levels of tolerance to long-term drought (14 weeks) in the field. Measurements of physiological parameters, analyses of protein accumulation profiles using two-dimensional gel electrophoresis, and mass spectrometry identification of proteins, which were accumulated differentially between the selected genotypes during short-term drought, were performed. Genotype 7/6, with lower yield potential during 14 weeks of drought, and lower ability to re-grow after watering, had a higher capacity for photosynthesis during 11 days of drought. Genotype 4/10, more tolerant to long-term drought, was able to repair damaged cell membranes after watering and was also characterised by lower transpiration during short-term drought. A total of 455 proteins were analysed, and the 17 that were differentially accumulated between the two genotypes were identified. The results of physiological and proteomic research led to a hypothesis that the higher photosynthetic capacity of genotype 7/6 could be due to a more efficient Calvin cycle, supported by higher accumulation of crucial proteins involving chloroplast aldolase.

Garlic (Allium sativum L.) fertility: transcriptome and proteome analyses provide insight into flower and pollen development

Commercial cultivars of garlic, a popular condiment, are sterile, making genetic studies and breeding of this plant challenging. However, recent fertility restoration has enabled advanced physiological and genetic research and hybridization in this important crop. Morphophysiological studies, combined with transcriptome and proteome analyses and quantitative PCR validation, enabled the identification of genes and specific processes involved in gametogenesis in fertile and male-sterile garlic genotypes. Both genotypes exhibit normal meiosis at early stages of anther development, but in the male-sterile plants, tapetal hypertrophy after microspore release leads to pollen degeneration. Transcriptome analysis and global gene-expression profiling showed that >16,000 genes are differentially expressed in the fertile vs. male-sterile developing flowers. Proteome analysis and quantitative comparison of 2D-gel protein maps revealed 36 significantly different protein spots, 9 of which were present only in the male-sterile genotype. Bioinformatic and quantitative PCR validation of 10 candidate genes exhibited significant expression differences between male-sterile and fertile flowers. A comparison of morphophysiological and molecular traits of fertile and male-sterile garlic flowers suggests that respiratory restrictions and/or non-regulated programmed cell death of the tapetum can lead to energy deficiency and consequent pollen abortion. Potential molecular markers for male fertility and sterility in garlic are proposed.

Remodeling of Leaf Cellular Glycerolipid Composition under Drought and Re-hydration Conditions in Grasses from the Lolium-Festuca Complex.

Drought tolerant plant genotypes are able to maintain stability and integrity of cellular membranes in unfavorable conditions, and to regenerate damaged membranes after stress cessation. The profiling of cellular glycerolipids during drought stress performed on model species such as Arabidopsis thaliana does not fully cover the picture of lipidome in monocots, including grasses. Herein, two closely related introgression genotypes of Lolium multiflorum (Italian ryegrass) × Festuca arundinacea (tall fescue) were used as a model for other grass species to describe lipid rearrangements during drought and re-hydration. The genotypes differed in their level of photosynthetic capacity during drought, and in their capacity for membrane regeneration after stress cessation. A total of 120 lipids, comprising the classes of monogalactosyldiacyloglycerol, digalactosyldiacyloglycerol, sulfoquinovosyldiacylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, diacylglicerol, and triacylglicerol, were analyzed. The results clearly showed that water deficit had a significant impact on lipid metabolism in studied forage grasses. It was revealed that structural and metabolic lipid species changed their abundance during drought and re-watering periods and some crucial genotype-dependent differences were also observed. The introgression genotype characterized by an ability to regenerate membranes after re-hydration demonstrated a higher accumulation level of most chloroplast and numerous extra-chloroplast membrane lipid species at the beginning of drought. Furthermore, this genotype also revealed a significant reduction in the accumulation of most chloroplast lipids after re-hydration, compared with the other introgression genotype without the capacity for membrane regeneration. The potential influence of observed lipidomic alterations on a cellular membrane stability and photosynthetic capacity, are discussed. HIGHLIGHTS A higher drought tolerance of grasses could be associated with an earlier lipidome response to a stress signal and with a membrane regeneration after stress cessation accompanied by a turnover of chloroplast lipids

Water Deficit Affects Primary Metabolism Differently in Two Lolium multiflorum/Festuca arundinacea Introgression Forms with a Distinct Capacity for Photosynthesis and Membrane Regeneration

Understanding how plants respond to drought at different levels of cell metabolism is an important aspect of research on the mechanisms involved in stress tolerance. Furthermore, a dissection of drought tolerance into its crucial components by the use of plant introgression forms facilitates to analyze this trait more deeply. The important components of plant drought tolerance are the capacity for photosynthesis under drought conditions, and the ability of cellular membrane regeneration after stress cessation. Two closely related introgression forms of Lolium multiflorum/Festuca arundinacea, differing in the level of photosynthetic capacity during stress, and in the ability to regenerate their cellular membranes after stress cessation, were used as forage grass models in a primary metabolome profiling and in an evaluation of chloroplast 1,6-bisphosphate aldolase accumulation level and activity, during 11 days of water deficit, followed by 10 days of rehydration. It was revealed here that the introgression form, characterized by the ability to regenerate membranes after rehydration, contained higher amounts of proline, melibiose, galactaric acid, myo-inositol and myo-inositol-1-phosphate involved in osmoprotection and stress signaling under drought. Moreover, during the rehydration period, this form also maintained elevated accumulation levels of most the primary metabolites, analyzed here. The other introgression form, characterized by the higher capacity for photosynthesis, revealed a higher accumulation level and activity of chloroplast aldolase under drought conditions, and higher accumulation levels of most photosynthetic products during control and drought periods. The potential impact of the observed metabolic alterations on cellular membrane recovery after stress cessation, and on a photosynthetic capacity under drought conditions in grasses, are discussed.

Proteomic analysis of developing rye grain with contrasting resistance to preharvest sprouting

Significant differences in the two-dimensional electrophoresis patterns of proteins from developing rye grain were found to be associated with resistance and susceptibility to preharvest sprouting (PHS). Mass spectrometry of individual spots showing different abundance in PHS-resistant and PHS-susceptible lines identified proteins involved in: reaction to biotic and abiotic stresses, including oxidative stress, energy metabolism and regulation of gene expression. Highly differentiated abundance of proteins found in developing grain suggest that the diversification of processes leading to developing PHS resistance or PHS susceptibility starts from an early stage of grain development. A part of the identified proteins in rye grain were also reported to be associated with PHS in wheat and rice, which suggests that some mechanisms affecting precocious germination might be common for different cereal species.

Identification of Kernel Proteins Associated with the Resistance to Fusarium Head Blight in Winter Wheat (Triticum aestivum L.)

Numerous potential components involved in the resistance to Fusarium head blight (FHB) in cereals have been indicated, however, our knowledge regarding this process is still limited and further work is required. Two winter wheat (Triticum aestivum L.) lines differing in their levels of resistance to FHB were analyzed to identify the most crucial proteins associated with resistance in this species. The presented work involved analysis of protein abundance in the kernel bulks of more resistant and more susceptible wheat lines using two-dimensional gel electrophoresis and mass spectrometry identification of proteins, which were differentially accumulated between the analyzed lines, after inoculation with F. culmorum under field conditions. All the obtained two-dimensional patterns were demonstrated to be well-resolved protein maps of kernel proteomes. Although, 11 proteins were shown to have significantly different abundance between these two groups of plants, only two are likely to be crucial and have a potential role in resistance to FHB. Monomeric alpha-amylase and dimeric alpha-amylase inhibitors, both highly accumulated in the more resistant line, after inoculation and in the control conditions. Fusarium pathogens can use hydrolytic enzymes, including amylases to colonize kernels and acquire nitrogen and carbon from the endosperm and we suggest that the inhibition of pathogen amylase activity could be one of the most crucial mechanisms to prevent infection progress in the analyzed wheat line with a higher resistance. Alpha-amylase activity assays confirmed this suggestion as it revealed the highest level of enzyme activity, after F. culmorum infection, in the line more susceptible to FHB.

Alterations in Kernel Proteome after Infection with Fusarium culmorum in Two Triticale Cultivars with Contrasting Resistance to Fusarium Head Blight

Highlight: The level of pathogen alpha-amylase and plant beta-amylase activities could be components of plant-pathogen interaction associated with the resistance of triticale to Fusarium head blight. Triticale was used here as a model to recognize new components of molecular mechanism of resistance to Fusarium head blight (FHB) in cereals. Fusarium-damaged kernels (FDK) of two lines distinct in levels of resistance to FHB were applied into a proteome profiling using two-dimensional gel electrophoresis (2-DE) to create protein maps and mass spectrometry (MS) to identify the proteins differentially accumulated between the analyzed lines. This proteomic research was supported by a measurement of alpha- and beta-amylase activities, mycotoxin content, and fungal biomass in the analyzed kernels. The 2-DE analysis indicated a total of 23 spots with clear differences in a protein content between the more resistant and more susceptible triticale lines after infection with Fusarium culmorum. A majority of the proteins were involved in a cell carbohydrate metabolism, stressing the importance of this protein group in a plant response to Fusarium infection. The increased accumulation levels of different isoforms of plant beta-amylase were observed for a more susceptible triticale line after inoculation but these were not supported by a total level of beta-amylase activity, showing the highest value in the control conditions. The more resistant line was characterized by a higher abundance of alpha-amylase inhibitor CM2 subunit and simultaneously a lower activity of alpha-amylase after inoculation. We suggest that the level of pathogen alpha-amylase and plant beta-amylase activities could be components of plant-pathogen interaction associated with the resistance of triticale to FHB.

Insight into cellular proteome of Lolium multiflorum/Festuca arundinacea introgression forms to decipher crucial mechanisms of cold acclimation in forage grasses

Frost tolerance is the main component of winter-hardiness. To express this trait, plants sense low temperature, and respond by activating the process of cold acclimation. The molecular mechanisms of this acclimation have not been fully understood in the agronomically important group of forage grasses, including Lolium-Festuca species. Herein, the introgression forms of L. multiflorum/F. arundinacea distinct with respect to their frost tolerance, were used as models for the comprehensive, proteomic and physiological, research to recognize the crucial components of cold acclimation in forage grasses. The obtained results stressed the importance of photosynthetic performance under acclimation to low temperature. The stable level of photochemical processes after three weeks of cold acclimation in the introgression form with a higher level of frost tolerance, combined simultaneously with only slightly (but not significantly) decreased level of CO2 assimilation after that period, despite significantly lower stomatal conductance, indicated the capacity for that form to acclimate its photosynthesis to low temperature. This phenomenon was driven by the Calvin cycle efficiency, associated with revealed here accumulation profiles and activities of chloroplastic aldolase. The capacity to acclimate the photosynthetic machinery to cold could be one of the most crucial components of forage grass metabolism to improve frost tolerance.

Host extracts induce changes in the proteome of plant pathogen Fusarium proliferatum

Fusarium proliferatum is a polyphagous pathogenic fungus able to infect many crop plants worldwide. Differences in proteins accumulated were observed when maize- and asparagus-derived F. proliferatum strains were exposed to host extracts prepared from asparagus, maize, garlic, and pineapple tissues. Seventy-three unique proteins were up-regulated in extract-supplemented cultures compared to the controls. They were all identified using mass spectrometry and their putative functions were assigned. A major part of identified proteins was involved in sugar metabolism and basic metabolic processes. Increased accumulation of proteins typically associated with stress response (heat shock proteins, superoxide dismutases, and glutaredoxins) as well as others, putatively involved in signal transduction, suggests that some metabolites present in plant extracts may act as elicitors inducing similar reaction as the abiotic stress factors. As a case study, thirteen genes encoding the proteins induced by the extracts were identified in the genomes of diverse F. proliferatum strains using gene-specific DNA markers. Extract-induced changes in the pathogens metabolism are putatively a result of differential gene expression regulation. Our findings suggest that host plant metabolites present in the extracts can cause biotic stress resulting in elevated accumulation of diverse set of proteins, including those associated with pathogens stress response.