Chloroplast proteins involved in drought stress response in selected cultivars of common bean (Phaseolus vulgaris L.).

One of the major cell organelles, whose functions are affected during drought stress are chloroplasts. In this study, chloroplast proteome under drought was studied in two cultivars of common bean (Phaseolus vulgaris L), Tiber and more sensitive to drought, Starozagorski cern, which were subjected to drought for 6 and 13 days. A comparative proteomic analysis with 2D-DIGE was performed on the isolated chloroplast proteins from leaves. Together, 44 proteins with changed abundance between control and stressed plants were identified with LC-MS/MS from both cultivars. The majority of the identified proteins were involved in photosynthetic processes. The results showed a decrease in abundance in different structure components of photosystem I and II, and ATP synthase, which may indicate a suppression of light-dependent reactions by drought stress. Similar proteomic response for both cultivars after 6 and 13 days of drought was observed. Proteins with contrasting abundance patterns between the cultivars or proteins specific for only one cultivar, such as ferredoxin-NADP reductase, photosystem II stability/assembly factor HCF136, curvature thylakoid protein 1B, and plastidial membrane protein porin were pointed out as major identified proteins revealing differential abundance between the cultivars. Taken together, our results provide insight into the molecular response of chloroplasts in common bean under drought stress, whereas conclusions about the tolerance mechanisms require further studies.

Towards a better understanding of protein changes in common bean under drought: A case study of N-glycoproteins.

Drought is one of the major abiotic stress conditions limiting crop growth and productivity. Glycosylation of proteins is very important post-translational modification that is involved in many physiological functions and biological pathways. To understand the involvement of N-glycoproteins in the mechanism of drought response in leaves of common bean, a proteomic approach using lectin affinity chromatography, SDS-PAGE and LC-MS/MS was applied. Quantification of N-glycoproteins was performed using MaxQuant with a label free quantification approach. Thirty five glycoproteins were changed in abundance in leaves of common bean under drought. The majority of these proteins were classified into functional groups that include cell wall processes, defence/stress related proteins and proteins related to proteolysis. Beta-glucosidase showed the highest increase in abundance among proteins involved in cell wall metabolism, suggesting its role in cell wall modification under drought stress. These results fit with the general concept of the stress response in plants and suggest that drought stress might affect biochemical metabolism in the cell wall. The structures of N-glycans were determined manually from spectra, where structures of high mannose, complex and hybrid types of N-glycans were found. The present study provided an insight into the glycoproteins related to drought stress in common bean at the proteome level, which is important for further understanding of molecular mechanisms of drought response in this important legume.

Proteomic analysis of common bean stem under drought stress using in-gel stable isotope labeling.

Drought is an abiotic stress that strongly influences plant growth, development and productivity. Proteome changes in the stem of the drought-tolerant common bean (Phaseolus vulgaris L.) cultivar Tiber have were when the plants were exposed to drought. Five-week-old plants were subjected to water deficit by withholding irrigation for 7, 12 and 17days, whereas control plants were regularly irrigated. Relative water content (RWC) of leaves, as an indicator of the degree of cell and tissue hydration, showed the highest statistically significant differences between control and drought-stressed plants after 17days of treatment, where RWC remained at 90% for control and declined to 45% for stressed plants. Plants exposed to drought for 17days and control plants at the same developmental stage were included in quantitative proteomic analysis using in-gel stable isotope labeling of proteins in combination with mass spectrometry. The quantified proteins were grouped into several functional groups, mainly into energy metabolism, photosynthesis, proteolysis, protein synthesis and proteins related to defense and stress. 70kDa heat shock protein showed the greatest increase in abundance under drought of all the proteins, suggesting its role in protecting plants against stress by re-establishing normal protein conformations and thus cellular homeostasis. The abundance of proteins involved in protein synthesis also increased under drought stress, important for recovery of damaged proteins involved in the plant cell's metabolic activities. Other important proteins in this study were related to proteolysis and folding, which are necessary for maintaining proper cellular protein homeostasis. Taken together, these results reveal the complexity of pathways involved in the drought stress response in common bean stems and enable comparison with the results of proteomic analysis of leaves, thus providing important information to further understand the biochemical and molecular mechanisms of drought response in this important legume.

Differential proteomic analysis of drought stress response in leaves of common bean (Phaseolus vulgaris L.).

The majority of common bean plants are cultivated under drought conditions. Maintaining crop yields under drought stress is thus one of the biggest challenges facing bean production. In order to improve our understanding of the complex mechanisms involved in the response of common bean (Phaseolus vulgaris) to drought stress, a proteomic approach was used to identify drought-responsive proteins in leaves of two cultivars differing in their response to drought, Tiber and more sensitive Starozagorski cern. 2D-DIGE was used to compare differences in protein abundance between control and stressed plants. Fifty-eight proteins whose abundance changed significantly were identified by LC-MS/MS in Tiber and 64 in Starozagorski cern. The majority of identified proteins were classified into functional categories that include energy metabolism, photosynthesis, ATP interconversion, protein synthesis and proteolysis, stress and defence related proteins. Details of the function of the identified proteins and their abundance profiles in Tiber and Starozagorski are discussed. Interactions between identified proteins were demonstrated by bioinformatics analysis, enabling a more complete insight into biological pathways and molecular functions affected by drought stress. The results form the basis for a further understanding of the biochemical mechanisms of drought response in common bean.

Development of sampling approaches for the determination of the presence of genetically modified organisms at the field level.

In order to comply with the European Union regulatory threshold for the adventitious presence of genetically modified organisms (GMOs) in food and feed, it is important to trace GMOs from the field. Appropriate sampling methods are needed to accurately predict the presence of GMOs at the field level. A 2-year field experiment with two maize varieties differing in kernel colour was conducted in Slovenia. Based on the results of data mining analyses and modelling, it was concluded that spatial relations between the donor and receptor field were the most important factors influencing the distribution of outcrossing rate (OCR) in the field. The approach for estimation fitting function parameters in the receptor (non-GM) field at two distances from the donor (GM) field (10 and 25 m) for estimation of the OCR (GMO content) in the whole receptor field was developed. Different sampling schemes were tested; a systematic random scheme in rows was proposed to be applied for sampling at the two distances for the estimation of fitting function parameters for determination of OCR. The sampling approach had already been validated with some other OCR data and was practically applied in the 2009 harvest in Poland. The developed approach can be used for determination of the GMO presence at the field level and for making appropriate labelling decisions. The importance of this approach lies in its possibility to also address other threshold levels beside the currently prescribed labelling threshold of 0.9% for food and feed.

Different classes of proteases are involved in the response to drought of Phaseolus vulgaris L. cultivars differing in sensitivity.

Protein breakdown and recycling, which depend on the levels of proteolytic enzymes, are an essential part of the plant response to environmental stress. In order to study changes in the activity of proteases in Phaseolus vulgaris L. subjected to water deficit, three cultivars of European origin that exhibit different degrees of sensitivity to drought were chosen on the basis of changes in water potential, psiw, water and protein contents of leaves during progressive water deficit, and loss of membrane integrity after osmotic stress. Twenty-day-old plants were subjected to water deficit by withholding irrigation. Specific enzyme activities in leaf extracts were determined for plants under different degrees of drought stress using different substrates and protease inhibitors. Proteolytic activities were partially characterized by gel exclusion chromatography. Activities of two of the three identified serine proteinases changed under water deficit. The activity of the one with apparent molecular mass of approximately 65 kDa was observed to increase progressively with increasing withdrawal of water in the more sensitive cultivars, but to decrease in the more resistant cultivar. The same activity was elevated in senescent leaves. Under conditions of severe water deficit, the most sensitive cultivar exhibited a marked increase in the activity of two different aminopeptidases, while the more resistant cultivar showed a significant decrease in the activity of these aminopeptidases. These results point to complex and probably specific roles for different proteases in the plant response to drought.

Determination of aneuploids in hop (Humulus lupulus L.) using flow cytometry.

In order to study the possibility that high-resolution flow cytometry can be used for determination of aneuploids, different genotypes of Humulus lupulus were analyzed. Triploid cultivars are bred by hybridization between diploid and tetraploid lines, and as the result of this process, some aneuploids are occasionally also formed. We analyzed eight triploid cultivars and seven putative aneuploids. Triploid cultivars Cerera, Cicero, Celeia, Cekin, Blisk, Mt. Hood, Huller Bit. and Willamette (3x = 30) were measured for nuclear DNA content using Trifolium repens as reference. No significant differences among peak positions of triploid cultivars (having an average CV value per peak of 1.94%) were found. Measurement of nuclear DNA content was also performed for seven lines: 175/75, 89/113, 89/154, 91/215, 175/17, 89/87 and 91/74 previously determined by chromosome counting to be aneuploids (CV per peak was 1.41%). A statistically lower DNA content was found for line 175/75 and higher values were measured for lines 89/154, 89/113 and 91/215. Repeated chromosome counting revealed that the number of chromosomes in line 175/75 was 29, while lines 89/154, 89/113 and 91/215 possessed 31 chromosomes. The other lines were identified as triploids. We conclude that flow cytometry can be efficiently used for determination of aneuploidy in Humulus lupulus.