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

Cryopreservation of Byrsonima intermedia embryos followed by room temperature thawing

Byrsonima intermedia is a shrub from the Brazilian Cerrado with medicinal properties. The storage of biological material at ultra-low temperatures (-196°C) is termed cryopreservation and represents a promising technique for preserving plant diversity. Thawing is a crucial step that follows cryopreservation. The aim of this work was to cryopreserve B. intermedia zygotic embryos and subsequently thaw them at room temperature in a solution rich in sucrose. The embryos were decontaminated and desiccated in a laminar airflow hood for 0-4 hours prior to plunging into liquid nitrogen. The embryo moisture content (% MC) during dehydration was assessed. Cryopreserved embryos were thawed in a solution rich in sucrose at room temperature, inoculated in a germination medium and maintained in a growth chamber. After 30 days, the embryo germination was evaluated. No significant differences were observed between the different embryo dehydration times, where they were dehydrated for at least one hour. Embryos with a MC between 34.3 and 20.3% were germinated after cryopreservation. In the absence of dehydration, all embryos died following cryopreservation. We conclude that B. intermedia zygotic embryos can be successfully cryopreserved and thawed at room temperature after at least one hour of dehydration in a laminar airflow bench

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

Arbuscular mycorrhizal fungi for the biocontrol of plant-parasitic nematodes: a review of the mechanisms involved

Arbuscular mycorrhizal fungi (AMF) are obligate root symbionts that can protect their host plant against biotic stress factors such as plant-parasitic nematode (PPN) infection. PPN consist of a wide range of species with different life styles that can cause major damage in many important crops worldwide. Various mechanisms have been proposed to play a role in the biocontrol effect of AMF against PPN. This review presents an overview of the different mechanisms that have been proposed, and discusses into more detail the plausibility of their involvement in the biocontrol against PPN specifically. The proposed mechanisms include enhanced plant tolerance, direct competition for nutrients and space, induced systemic resistance (ISR) and altered rhizosphere interactions. Recent studies have emphasized the importance of ISR in biocontrol and are increasingly placing rhizosphere effects on the foreground as well, both of which will be the focal point of this review. Though AMF are not yet widely used in conventional agriculture, recent data help to develop a better insight into the modes of action, which will eventually lead toward future field applications of AMF against PPN. The scientific community has entered an exciting era that provides the tools to actually unravel the underlying molecular mechanisms, making this a timely opportunity for a review of our current knowledge and the challenges ahead

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

In vitro culture of Annona emarginata: a rootstock for commercial annonaceae species

Annona emarginata is a native fruit tree in the Brazilian Cerrado which, unlike the commercial species, does not present a high fruit quality. On the other hand, it stands out based on its rootstock value. However, there are some problems that hinder the largescale production of Annona emarginata seedlings for use as a rootstock. In order to overcome these difficulties, micropropagation has become a viable alternative for the rapid and efficient propagation of Annona spp, but Annona emarginata micropropagation has not yet been reported. Therefore, the aim of this study was to initiate Annona emarginata in vitro growth. For axillary shoot proliferation, in vitro nodal segments of 0.5 cm were transferred to MS or WPM supplemented with BA at different concentrations. Nodal segments were also cultured on WPM medium with ranging concentrations of different plant growth regulators, aiming for either shoot elongation or rooting. The results showed that the use of 1 µM BA in WPM medium is recommended for in vitro multiplication of Annona emarginata. This is based on the low adventitious shoot formation, combined with a higher number of buds and leaves. The use of GA3 at any concentration tested induced the formation of malformed plants. Root formation could not be stimulated, regardless the duration of auxin treatment

Attempts to eradicate two Pelargonium viruses (PFBV and PLPV) by meristem culture and shoot-tip cryotherapy

Attempts to eradicate the Pelargonium flower break virus (PFBV) and Pelargonium line pattern virus (PLPV) by meristem culture and apex “droplet-vitrification” cryopreservation was carried out using 5 different cultivars. A simple meristem culture did not permit to eliminate PFBV and only 15% of Pelargonium x hortorum ‘Stellar Artic’ plants regenerated from meristems was PLPV-ELISA-negative. Plants regenerated from cryopreserved apices were tested by DAS-ELISA after a 3-month growing period. Viruses were not detected in 25 and 50% of the tested plants for PFBV and PLPV respectively. Immunolocalisations were carried out for virus localisation in apices from greenhouse plants (control) and vitroplants regenerated after meristem culture or cryopreservation. Immunolocalisations realised on control explants excised from DAS-ELISA positive plants showed that PFBV and PLPV were present in the apices, even in the meristematic dome. However, viral particles were more numerous in the cells of the basal zone than in the more meristematic ones. Immunolocalisations realised on apices from the DAS-ELISA negative cryoregenerated plants showed the viruses were still present. Our results firstly demonstrated that PFBV and PLPV are even present inside meristematic cells and secondly that cryopreservation could decrease their amount in Pelargonium plants but without eliminating them totally. More knowledge on virus behaviour during cryopreservation processes could optimize the management of genetic resources using this conservation method