Evaluation of four different strategies to characterize plasma membrane proteins from banana roots

Plasma membrane proteins constitute a very important class of proteins. They are involved in the transmission of external signals to the interior of the cell and selective transport of water, nutrients and ions across the plasma membrane. However, the study of plasma membrane proteins is challenging because of their poor solubility in aqueous media and low relative abundance. In this work, we evaluated four different strategies for the characterization of plasma membrane proteins from banana roots: (i) the aqueous-polymer two-phase system technique (ATPS) coupled to gelelectrophoresis (gel-based), and (ii) ATPS coupled to LC-MS/MS (gel free), (iii) a microsomal fraction and (iv) a full proteome, both coupled to LC-MS/ MS. Our results show that the gel-based strategy is useful for protein visualization but has major limitations in terms of time reproducibility and efficiency. From the gel-free strategies, the microsomal-based strategy allowed the highest number of plasma membrane proteins to be identified, followed by the full proteome strategy and by the ATPS based strategy. The high yield of plasma membrane proteins provided by the microsomal fraction can be explained by the enrichment of membrane proteins in this fraction and the high throughput of the gel-free approach combined with the usage of a fast high-resolution mass spectrometer for the identification of proteins

Characterizing fruit ripening in plantain and Cavendish bananas: A proteomics approach

The fruit physiology of banana cultivars other than Cavendish is poorly understood. To study the ripening process, samples were taken daily from plantain and Cavendish bananas and the ripening stages were determined. We present data from the green to the fully mature stage. By analyzing the protein abundances during ripening we provide some new insights into the ripening process and how plantains fruits are different. Multivariate analysis of the proteins was performed correlated to the starch dynamics. A drop in sucrose synthase and a rise of acid invertase during ripening indicated a change in the balance of the sucrose fate. During ripening, sugars may no longer be available for respiration since they are stored in the vacuoles, making citrate the preferred respiratory substrate. We found significant cultivar specific differences in granule-bound starch synthase, alpha- and beta amylases and cell wall invertase when comparing the protein content at the same ripening stage. This corroborates the difference in starch content/structure between both banana types. Differences in small heat shock proteins and in the cell wall-modifying enzyme xyloglucan endotransglucosylase/hydrolase support respectively the presumed higher carotenoid content and the firmer fruit structure of plantains

Somatic embryogenesis in coffee: the evolution of biotechnology and the integration of omics technologies offer great opportunities

One of the most important crops cultivated around the world is coffee. There are two main cultivated species, Coffea arabica and C. canephora. Both species are difficult to improve through conventional breeding, taking at least 20 years to produce a new cultivar. Biotechnological tools such as genetic transformation, micropropagation and somatic embryogenesis (SE) have been extensively studied in order to provide practical results for coffee improvement. While genetic transformation got many attention in the past and is booming with the CRISPR technology, micropropagation and SE are still the major bottle neck and urgently need more attention. The methodologies to induce SE and the further development of the embryos are genotype-dependent, what leads to an almost empirical development of specific protocols for each cultivar or clone. This is a serious limitation and excludes a general comprehensive understanding of the process as a whole. The aim of this review is to provide an overview of which achievements and molecular insights have been gained in (coffee) somatic embryogenesis and encourage researchers to invest further in the in vitro technology and combine it with the latest omics techniques (genomics, transcriptomics, proteomics, metabolomics, and phenomics). We conclude that the evolution of biotechnology and the integration of omics technologies offer great opportunities to (i) optimize the production process of SE and the subsequent conversion into rooted plantlets and (ii) to screen for possible somaclonal variation. However, currently the usage of the latest biotechnology did not pass the stage beyond proof of potential and needs to further improve

Elucidation of the compatible interaction between banana and Meloidogyne incognita via high-throughput proteome profiling

With a diverse host range, Meloidogyne incognita (root-knot nematode) is listed as one of the most economically important obligate parasites of agriculture. This nematode species establishes permanent feeding sites in plant root systems soon after infestation. A compatible host-nematode interaction triggers a cascade of morphological and physiological process disruptions of the host, leading to pathogenesis. Such disruption is reflected by altered gene expression in affected cells, detectable using molecular approaches. We employed a high-throughput proteomics approach to elucidate the events involved in a compatible banana- M. incognita interaction. This study serves as the first crucial step in developing natural banana resistance for the purpose of biological-based nematode management programme. We successfully profiled 114 Grand naine root proteins involved in the interaction with M. incognita at the 30th- and 60th- day after inoculation (dai). The abundance of proteins involved in fundamental biological processes, cellular component organisation and stress responses were significantly altered in inoculated root samples. In addition, the abundance of proteins in pathways associated with defence and giant cell maintenance in plants such as phenylpropanoid biosynthesis, glycolysis and citrate cycle were also implicated by the infestation

Genotype-specific growth and proteomic responses of maize toward salt stress

Salt stress in plants triggers complex physiological responses that are genotype specific. Many of these responses are either not yet described or not fully understood or both. In this work, we phenotyped three maize genotypes of the CIMMYT gene bank alongside the reference B73 genotype (NCRPIS – United States) under both control and salt-stressed conditions. We have ranked their growth potential and we observed significant differences in Na+ and Cl- ion accumulation. Genotype CML421 showed the slowest growth, while CML451 had the lowest accumulation of ions in its leaves. The phenotyping defined the right timing for the proteomics analysis, allowing us to compare the contrasting genotypes. In general 1,747 proteins were identified, of which 209 were significantly more abundant in response to salt stress. The five most significantly enriched annotations that positively correlated with stress were oxidation reduction, catabolic process, response to chemical stimulus, translational elongation and response to water. We observed a higher abundance of proteins involved in reactions to oxidative stress, dehydration, respiration, and translation. The five most significantly enriched annotations negatively correlated with stress were nucleosome organization, chromatin assembly, protein-DNA complex assembly, DNA packaging and nucleosome assembly. The genotypic analysis revealed 52 proteins that were correlated to the slow-growing genotype CML421. Their annotations point toward cellular dehydration and oxidative stress. Three root proteins correlated to the CML451 genotype were annotated to protein synthesis and ion compartmentalization. In conclusion, our results highlight the importance of the anti-oxidative system for acclimatization to salt stress and identify potential genotypic marker proteins involved in salt-stress responses

How do roots respond to osmotic stress? A transcriptomic approach to address this question in a non-model crop

Drought is a complex phenomenon that is relevant for many crops. Performing high-throughput transcriptomics in non-model crops is challenging. The non-model crop where our workflow has been tested on is banana (Musa spp.), which ranks among the top ten staple foods (total production over 145 million tons in 2013 (FAOstat)[1]). Bananas need vast amounts of water and even mild-drought conditions are responsible for considerable yield losses[2]. To characterize drought in the roots of different banana genotypes, we designed a lab model based on osmotic stress (5% PEG treatment for 3 days) and performed mRNA-seq analysis[3]. Using Illumina technology, 18 cDNA libraries were sequenced producing around 568 million high quality reads, of which 70-84% were mapped to the diploid reference genome[4]. We show that the applied stress leads to a drop in energy levels inducing a metabolic shift towards (i) higher oxidative respiration, (ii) alternative respiration and (iii) fermentation. We also analyzed the expression patterns of paralogous genes belonging to the same gene families and detected possible cases of sub-functionalization