First insights into the biochemical and molecular response to cold stress in <Emphasis Type="Italic">Cicer microphyllum</Emphasis>, a crop wild relative of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis>)

Identifying a potential crop wild relative (CWR) of legumes, especially one with high abiotic stress tolerance, has been a priority of plant breeders for many decades. Traditionally CWRs have been selected based on biometrical traits observed in the field, however this methodology is insufficient for research into nonmorphological traits such as stress tolerance. Biochemical and molecular analysis of potential CWRs allows for more informed selection. Specifically, we focus on Cicer microphyllum Benth, a CWR of cultivated chickpea Cicer arietinum L., which is distributed in Trans Himalayan ranges adjacent to glaciers of India and Pakistan at the alpine altitude gradient between 2700 to 6000 m. The objective of this study is to begin characterization of the biochemical and molecular bases of adaptation of C. microphyllum to cold stress and compare it to its cultivated relative (Cold susceptible genotype ILC533). Significant differences were recorded in terms of malondialdehyde (MDA) concentration, electrolyte leakage and proline accumulation in C. microphyllum, as compared to C. arietinum, upon cold exposure (4°C/24h). C. microphyllum exhibits more membrane stability under cold stress. Furthermore, proline overaccumulation and an increase in the enzymatic activities of antioxidants including superoxide dismutase, catalase, and ascorbate peroxidase were also observed in C. microphyllum under cold stress treatment. Expression of pyrroline-5-carboxylate synthetase, chalcone reductase, flavonoid 3',5'-hydroxylase and flavonoid 3'-monooxygenase are all upregulated under cold treatment in C. microphyllum. The characteristics recommend C. microphyllum both as a model for plant response to cold stress and as a potential source for abiotic stress resistant germplasm for chickpea breeding programs.     

Methyl jasmonate induces ATP biosynthesis deficiency and accumulation of proteins related to secondary metabolism in Catharanthus roseus (L.) G. hairy roots

Jasmonates are specific signal molecules in plants that are involved in a diverse set of physiological and developmental processes. However, methyl jasmonate (MeJA) has been shown to have a negative effect on root growth and, so far, the biochemical mechanism for this is unknown. Using Catharanthus roseus hairy roots, we were able to observe the effect of MeJA on growth inhibition, cell disorganization and cell death of the root cap. Hairy roots treated with MeJA induced the perturbation of mitochondrial membrane integrity and a diminution in ATP biosynthesis. Furthermore, several proteins were identified that were involved in energy and secondary metabolism; the changes in accumulation of these proteins were observed with 100 μM MeJA. In conclusion, our results suggest that a switch of the metabolic fate of hairy roots in response to MeJA could cause an increase in the accumulation of secondary metabolites. This is likely to have important consequences in the production of specific alkaloids important for the pharmaceutical industry.     

Differential Secretion and Accumulation of Terpene Indole Alkaloids in Hairy Roots of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> Treated with Methyl Jasmonate

The induction of several secondary metabolites in plants is one of the most commonly observed effects after the external addition of methyl jasmonate (MeJA). After the elicitation of Catharanthus roseus hairy roots with different concentrations of MeJA, changes in the accumulation of alkaloids such as ajmalicine, serpentine, ajmaline and catharanthine were observed. In addition to the increased accumulation of alkaloids in the tissues, the root exudation of phytochemicals increased compared to that of the non-treated control hairy roots. Moreover, MeJA induced differential secretion of several C. roseus hairy root metabolites.     

An efficient protocol for in vitro propagation of the wild legume Cicer microphyllum Benth., a crop wild relative of chickpea (Cicer arietinum L.)

In Vitro Cellular & Developmental Biology - Plant - Cicer microphyllum Benth. is a wild legume that is adapted to the extremely adverse climatic conditions of the cold Himalayan deserts. This...     

A Comparative Study of Lectin Affinity Based Plant N-Glycoproteome Profiling Using Tomato Fruit as a Model

Lectin affinity chromatography (LAC) can provide a valuable front-end enrichment strategy for the study of N-glycoproteins and has been used to characterize a broad range eukaryotic N-glycoproteomes. Moreover, studies with mammalian systems have suggested that the use of multiple lectins with different affinities can be particularly effective. A multi-lectin approach has also been reported to provide a significant benefit for the analysis of plant N-glycoproteins; however, it has yet to be determined whether certain lectins, or combinations of lectins are optimal for plant N-glycoproteome profiling; or whether specific lectins show preferential association with particular N-glycosylation sites or N-glycan structures. We describe here a comparative study of three mannose-binding lectins, concanavalin A, snowdrop lectin, and lentil lectin, to profile the N-glycoproteome of mature green stage tomato (Solanum lycopersicum) fruit pericarp. Through coupling lectin affinity chromatography with a shotgun proteomics strategy, we identified 448 putative N-glycoproteins, whereas a parallel lectin affinity chromatography plus hydrophilic interaction chromatography analysis revealed 318 putative N-glycosylation sites on 230 N-glycoproteins, of which 100 overlapped with the shotgun analysis, as well as 17 N-glycan structures. The use of multiple lectins substantially increased N-glycoproteome coverage and although there were no discernible differences in the structures of N-glycans, or the charge, isoelectric point (pI) or hydrophobicity of the glycopeptides that differentially bound to each lectin, differences were observed in the amino acid frequency at the −1 and +1 subsites of the N-glycosylation sites. We also demonstrated an alternative and complementary in planta recombinant expression strategy, followed by affinity MS analysis, to identify the putative N-glycan structures of glycoproteins whose abundance is too low to be readily determined by a shotgun approach, and/or combined with deglycosylation for predicted deamidated sites, using a xyloglucan-specific endoglucanase inhibitor protein as an example.N-glycosylation is one of the most heterogeneous and common post-translational modifications of eukaryotic proteins and one that affects many aspects of protein targeting, enzymatic properties, stability and intermolecular interactions (1–3). There is therefore considerable interest in developing robust and sensitive high throughput analytical pipelines to isolate and structurally characterize N-glycoprotein populations (2, 4–8), allowing glycoprotein identification and analysis of the glycosylation site occupancy and N-glycan structure. To this end, lectin affinity chromatography (LAC)¹ is increasingly popular: specifically, various lectins are known to have different binding affinities for N-glycans and so the selective binding of N-glycoproteins in complex protein extracts to these lectins and their subsequent release allows a critical enrichment step before sequencing and glycan analysis by MS (9).As a refinement of this approach, the use of multiple lectin affinity chromatography (MLAC) in yeast and animal studies (4, 6, 10), using different proteomic platforms, has been shown to increase the numbers of isolated N-glycoproteins or N-glycopeptides. Collectively, these studies of taxonomically diverse eukaryotic N-glycoproteomes suggest a general conservation of the glycosylation site (N-X-S/T, where X can be any amino acid except proline), as well as conserved features of three-dimensional protein structure (4, 6, 10). Although there have been several studies to determine the structural basis of the binding specificity of specific lectins to yeast and animal N-glycoproteins (11), larger scale N-glycoproteomic analyses have typically not attempted to determine whether a particular combination of lectins provides optimal enrichment, or whether specific features, such as N-glycan structure or amino acid sequence at and around the N-glycosylation site, are associated with different lectins. Therefore, systematic comparative studies are essential to determine whether particular lectins can be optimal for specific tissues, organs, and organisms.LAC has also been used in plant N-glycoprotein analyses to enrich for populations of cell wall localized proteins (8, 12, 13) and a recent report (13) described the application of MLAC to map substantial numbers of N-glycosylation sites in a range of key experimental model organisms, included the plant Arabidopsis thaliana. Using a LTQ-Orbitrap Velos mass spectrometer, the authors identified 2186 unique N-glycosylation sites in proteins extracted from five different arabidopsis organs (13), which represents a substantial increase in the number of identified N-glycosylation sites that have resulted from previous plant N-glycoprotein studies. However, the particular analytical platform that was used did not allow the structural characterization of the N-glycans or N-glycopeptides (13). Indeed, certain features of N-glycopeptides, such as poor fragmentation, heterogeneity and a large dynamic range in most complex mixtures often limits the structural analysis of N-glycans in high throughput systematic analyses (2). It is also important to note that the structures of plant N-glycans differ from those of animals and yeast, as exemplified by the presence of β-1, 2-xylose and α-1, 3-fucose and the complete absence of multiantennary N-glycans and sialic acid in plant N-glycoproteins (1, 14). Therefore, assumptions that are made with regard to the lectin binding of animal and yeast proteins do not necessarily apply to those from plants. Consequently, there is a need to investigate the structural basis of lectin binding to plant N-glycoproteins. Moreover, the limitations of typical shotgun based profiling approaches in identifying and characterizing low abundance N-glycoproteins in complex protein extracts need to be addressed to allow more comprehensive plant N-glycoproteome profiling.In the present study we address both these issues using mature green stage tomato (Solanum lycopersicum) fruit pericarp as an experimental model to carry out a comparative analysis of N-glycoproteins associated with each of three mannose-binding lectins: concanavalin A (ConA), snowdrop lectin (GNA), and lentil lectin (LCH). Fruit development is associated with substantial cell wall metabolism and the expression of many wall localized N-glycoproteins (8) and tomato in particular represents an excellent model for studies of fleshy fruits and cell wall N-glycoproteins (15). We established an MLAC analytical pipeline that included shotgun proteomic profiling and deglycosylation and deamidation analysis, to allow the determination of N-glycoprotein protein identity, N-glycosylation site and N-glycan structures. This information was then used to establish whether a combination of lectins is indeed advantageous for the study of plant N-glycoproteomes, and to assess whether any of these structural characteristics result in predictable preferential binding to specific lectins. From these studies it became evident that large dynamic range of N-glycoprotein abundance was a significant limiting factor in the structural determination of the tomato, and that there was a bias toward the detection of highly abundant N-glycopeptides. We therefore evaluated the use of an in planta recombinant expression strategy, combined with affinity purification MS (AP-MS), as a means to characterize the N-glycan structures of glycoproteins whose abundance is too low to be readily determined via the primary shotgun pipeline, using a tomato xyloglucan-specific endoglucanase inhibitor protein (XEGIP) as a test case.