Sajad Majeed Zargar

Quantitative proteomics of Arabidopsis shoot microsomal proteins reveals a cross-talk between excess zinc and iron deficiency.

Iron (Fe) deficiency significantly effects plant growth and development. Plant symptoms under excess zinc (Zn) resemble symptoms of Fe‐deficient plants. To understand cross‐talk between excess Zn and Fe deficiency, we investigated physiological parameters of Arabidopsis plants and applied iTRAQ‐OFFGEL quantitative proteomic approach to examine protein expression changes in microsomal fraction from Arabidopsis shoots under those physiological conditions. Arabidopsis plants manifested shoot inhibition and chlorosis symptoms when grown on Fe‐deficient media compared to basal MGRL solid medium. iTRAQ‐OFFGEL approach identified 909 differentially expressed proteins common to all three biological replicates; the majority were transporters or proteins involved in photosynthesis, and ribosomal proteins. Interestingly, protein expression changes between excess Zn and Fe deficiency showed similar pattern. Further, the changes due to excess Zn were dramatically restored by the addition of Fe. To obtain biological insight into Zn and Fe cross‐talk, we focused on transporters, where STP4 and STP13 sugar transporters were predominantly expressed and responsive to Fe‐deficient conditions. Plants grown on Fe‐deficient conditions showed significantly increased level of sugars. These results suggest that Fe deficiency might lead to the disruption of sugar synthesis and utilization.

Unraveling the iron deficiency responsive proteome in Arabidopsis shoot by iTRAQ-OFFGEL approach

Iron (Fe) is required by plants for basic redox reactions in photosynthesis and respiration, and for many other key enzymatic reactions in biological processes. Fe homeostatic mechanisms have evolved in plants to enable the uptake and sequestration of Fe in cells. To elucidate the network of proteins that regulate Fe homeostasis and transport, we optimized the iTRAQ-OFFGEL method to identify and quantify the number of proteins that respond to Fe deficiency in the model plant Arabidopsis. In this study, Fe deficiency was created using Fe-deficient growth conditions, excess zinc (Zn), and use of the irt1-1 mutant in which the IRT1 Fe transporter is disrupted. Using the iTRAQ-OFFGEL approach, we identified 1139 proteins, including novel Fe deficiency-responsive proteins, in microsomal fractions isolated from 3 different types of Fe-deficient shoots compared with just 233 proteins identified using conventional iTRAQ-CEX. Further analysis showed that greater numbers of low-abundance proteins could be identified using the iTRAQ-OFFGEL method and that proteins could be identified from numerous cellular compartments. The improved iTRAQ-OFFGEL method used in this study provided an efficient means for identifying greater numbers of proteins from microsomal fractions of Arabidopsis shoots. The proteome identified in this study provides new insight into the regulatory cross talk between Fe-deficient and excess Zn conditions.

Quantitative Proteomic Analysis of the Response to Zinc, Magnesium, and Calcium Deficiency in Specific Cell Types of Arabidopsis Roots

The proteome profiles of specific cell types have recently been investigated using techniques such as fluorescence activated cell sorting and laser capture microdissection. However, quantitative proteomic analysis of specific cell types has not yet been performed. In this study, to investigate the response of the proteome to zinc, magnesium, and calcium deficiency in specific cell types of Arabidopsis thaliana roots, we performed isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomics using GFP-expressing protoplasts collected by fluorescence-activated cell sorting. Protoplasts were collected from the pGL2-GFPer and pMGP-GFPer marker lines for epidermis or inner cell lines (pericycle, endodermis, and cortex), respectively. To increase the number of proteins identified, iTRAQ-labeled peptides were separated into 24 fractions by OFFGFEL electrophoresis prior to high-performance liquid chromatography coupled with mass spectrometry analysis. Overall, 1039 and 737 proteins were identified and quantified in the epidermal and inner cell lines, respectively. Interestingly, the expression of many proteins was decreased in the epidermis by mineral deficiency, although a weaker effect was observed in inner cell lines such as the pericycle, endodermis, and cortex. Here, we report for the first time the quantitative proteomics of specific cell types in Arabidopsis roots.

Unraveling the efficiency of RAPD and SSR markers in diversity analysis and population structure estimation in common bean

Increase in food production viz-a-viz quality of food is important to feed the growing human population to attain food as well as nutritional security. The availability of diverse germplasm of any crop is an important genetic resource to mine the genes that may assist in attaining food as well as nutritional security. Here we used 15 RAPD and 23 SSR markers to elucidate diversity among 51 common bean genotypes mostly landraces collected from the Himalayan region of Jammu and Kashmir, India. We observed that both the markers are highly polymorphic. The discriminatory power of these markers was determined using various parameters like; percent polymorphism, PIC, resolving power and marker index. 15 RAPDs produced 171 polymorphic bands, while 23 SSRs produced 268 polymorphic bands. SSRs showed a higher PIC value (0.300) compared to RAPDs (0.243). Further the resolving power of SSRs was 5.241 compared to 3.86 for RAPDs. However, RAPDs showed a higher marker index (2.69) compared to SSRs (1.279) that may be attributed to their higher multiplex ratio. The dendrograms generated with hierarchical UPGMA cluster analysis grouped genotypes into two main clusters with various degrees of sub clustering within the cluster. Here we observed that both the marker systems showed comparable accuracy in grouping genotypes of common bean according to their area of cultivation. The model based STRUCTURE analysis using 15 RAPD and 23 SSR markers identified a population with 3 sub-populations which corresponds to distance based groupings. High level of genetic diversity was observed within the population. These findings have further implications in common bean breeding as well as conservation programs.

Recent advances in molecular marker techniques: Insight into QTL mapping, GWAS and genomic selection in plants

Recent advances in sequencing technology have brought several novel platforms for marker development and subsequent genotyping. The high-throughput and cost effective marker techniques have changed the entire scenario of marker applications. The huge genotypic data obtained with next generation sequencing (NGS) also demands analytical tools, statistical advances, and comprehensive understanding to cope with breeding applications. In the present review, we discussed different available marker techniques, their strengths, and limitations. Emphasis was given on software tools, analytical pipelines, workbenches, and online resources available for marker development. Comparison of SNP genotyping involving complexity reduction techniques like GBS, RRL, RAD, and array-based platforms were presented in a view to describe suitability for specific purposes. We found that genotyping by whole genome re-sequencing has great potential, and could be a routine application in the near future with continuously decreasing cost of sequencing. Microsatellites, still a valuable option for breeders, have also advanced with NGS. Here a catalogue of tools for microsatellite evaluation in short sequence reads was provided. The most common applications of molecular marker like QTL mapping, genome-wide association mapping (GWAS), and genomic selection were highlighted. The present review will be helpful for the effective utilization of available resources and for the planning of crop improvement programs employing molecular marker techniques.

Quantitative proteomics reveals role of sugar in decreasing photosynthetic activity due to Fe deficiency

1 Centre for Plant Biotechnology, Division of Biotechnology, S K University of Agricultural Sciences and Technology of Kashmir, Srinagar, India, 2 Research Laboratory for Biotechnology and Biochemistry, Kathmandu, Nepal, 3 GRADE Academy Private Limited, Birgunj, Nepal, 4 Faculty of Health and Sport Sciences and Tsukuba International Academy for Sport Studies, University of Tsukuba, Tsukuba, Japan, 5 Department of Bioinformatics, Ritsumeikan University, Shiga, Japan

Towards a common bean proteome atlas: looking at the current state of research and the need for a comprehensive proteome

1 School of Biotechnology, SK University of Agricultural Sciences and Technology of Jammu, Jammu, India, Department of Botany, Jamia Hamdard University, New Delhi, India, National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi, India, 4 Reproduction and Developmental Biology, Institute of Plant Genetics and Biotechnology, Slovak Academy of Science, Nitra, Slovakia, 5 Research Laboratory for Biotechnology and Biochemistry, Kathmandu, Nepal, Organization for Educational Initiatives, University of Tsukuba, Tsukuba, Japan

Population Structure Analysis and Selection of Core Set among Common Bean Genotypes from Jammu and Kashmir, India

Understanding the genetic diversity of a crop is useful for its effective utilization in breeding programmes. For better understanding of the genetic variability in common bean, the first and foremost step is to study its genetic diversity. In the present investigation, 138 genotypes of common bean collected from various regions of Jammu and Kashmir, India, representing major common bean growing areas of this region, were evaluated using 23 SSRs. These SSRs were found highly polymorphic and possess high values for various parameters indicating their high discriminatory power. The average PIC value observed was 0.692, with 0.730 as average gene diversity value, and 0.267 as heterozygosity. Twenty-three SSRs produced a total of 251 alleles. The dendrogram generated with un-weighted neighbour joining cluster analysis grouped genotypes into three main clusters with various degrees of sub-clustering within the clusters. The model-based STRUCTURE analysis using 23 SSR markers identified a population with 3 sub-populations which corresponds to distance-based groupings with average FST value and expected heterozygosity of 0.1497 and 0.6696, respectively, within the sub-population, as such high level of genetic diversity was observed within the population. Further, Core Hunter II was used to identify a core set of 96 diverse genotypes. This core set of diverse 96 genotypes is a potential resource for association mapping studies and can be used by breeders as a material to make desirable genetic crosses to generate elite varieties for the fulfilling global market needs. These findings have further implications in common bean breeding as well as conservation programs.

Shift from Gel Based to Gel Free Proteomics to Unlock Unknown Regulatory Network in Plants: A Comprehensive Review

Sajad Majeed Zargar1*, Nancy Gupta2, Rakeeb A Mir3, Vandna Rai4 1Centre for Plant Biotechnology, Division of Biotechnology, Sher-e-Kashmir University of Agricultural Sciences & Technology of Kashmir, Shalimar, Srinagar, J&K, India 2School Biotechnology, Sher-e-Kashmir University of Agricultural Sciences & Technology of Jammu, Chatha, Jammu, J&K, India 3School of Bio resources & Biotechnology, BGSB University, Rajouri, J&K, India 4NRCPB, New Delhi, India Journal of Advanced Research in Biotechnology Open Access Review Article

Peptide Separation Methodologies for In-depth Proteomics

The integration of proteomics to other omics technologies and generation of proteome maps of a particular cell/tissue requires the identification and quantification of a maximum number of proteins. Traditional 2-D gel-based approach though provides a clear proteome map has its limitations, such as time consuming, requiring high skill, and most importantly, inability to identify low-abundance proteins. The most common drawback of 2-D gel electrophoresis is the masking of low amount proteins by the highly expressed (high abundance) proteins. Therefore, the elucidation of complete regulatory networks of a cell/tissue demands identification of low-abundance proteins. Low-abundance protein identification requires the use of usually gel-free mass spectrometry (MS)-based approaches. Using Arabidopsis thaliana as a model system, in this chapter, we describe all the steps followed for the extraction of microsomal proteins to MS analysis of separated peptides with a major focus on three different methods, namely, OFFGEL fractionation, 2D-LC, and long-column method for the identification of low-abundance proteins. Separation of such peptides will lead to in-depth proteomics-based investigations to answer biological questions.