Diversity of Legumes in the Cashew Agroforestry System in East Timor (Southeast Asia).

Cashew is an important export-oriented crop in several tropical countries, often under monocropping systems. Intercropping with legume species is promoted as a sustainable practice, enhancing agricultural productivity and providing nutritional food sources to rural communities. This study aimed to characterize the diversity of Leguminosae (or Fabaceae) in the cashew agroforestry systems of East Timor (Southeast Asia). Fourteen cashew orchards were sampled across the country, and information about leguminous species uses was collected from local populations. About 50 species are commonly part of the country's cashew agroforestry system, many of them simultaneously used as food, fodder, and in traditional medicine. Six bean species-Cajanus cajan (L.) Huth, Phaseolus lunatus L., Phaseolus vulgaris L., Vigna angularis (Willd.) Ohwi and H.Ohashi, Vigna radiata (L.) R.Wilczek and Vigna unguiculata (L.) Walp.-are largely used as food. The mineral contents of these beans revealed relevant differences between species and, in some cases, between types (seed colour) within species. Periods of hunger and low food variety are frequent in East Timor, reflecting a very poor nutritional state of the population. Knowing and using legumes for local nutrition, as well as for healthcare and well-being, adds great value to these species as components of East Timor cashew agroforestry systems.

A proteomic analysis shows the stimulation of light reactions and inhibition of the Calvin cycle in the skin chloroplasts of ripe red grape berries.

The role of photosynthesis in fruits still challenges scientists. This is especially true in the case of mature grape berries of red varieties lined by an anthocyanin-enriched exocarp (skin) almost impermeable to gases. Although chlorophylls are degraded and replaced by carotenoids in several fruits, available evidence suggests that they may persist in red grapes at maturity. In the present study, chloroplasts were isolated from the skin of red grape berries (cv. Vinhão) to measure chlorophyll levels and the organelle proteome. The results showed that chloroplasts (and chlorophylls) are maintained in ripe berries masked by anthocyanin accumulation and that the proteome of chloroplasts from green and mature berries is distinct. Several proteins of the light reactions significantly accumulated in chloroplasts at the mature stage including those of light-harvesting complexes of photosystems I (PSI) and II (PSII), redox chain, and ATP synthase, while chloroplasts at the green stage accumulated more proteins involved in the Calvin cycle and the biosynthesis of amino acids, including precursors of secondary metabolism. Taken together, results suggest that although chloroplasts are more involved in biosynthetic reactions in green berries, at the mature stage, they may provide ATP for cell maintenance and metabolism or even O2 to feed the respiratory demand of inner tissues.

Nitrogen Acquisition and Transport in the Ectomycorrhizal Symbiosis-Insights from the Interaction between an Oak Tree and Pisolithus tinctorius.

In temperate forests, the roots of various tree species are colonized by ectomycorrhizal fungi, which have a key role in the nitrogen nutrition of their hosts. However, not much is known about the molecular mechanisms related to nitrogen metabolism in ectomycorrhizal plants. This study aimed to evaluate the nitrogen metabolic response of oak plants when inoculated with the ectomycorrhizal fungus Pisolithus tinctorius. The expression of candidate genes encoding proteins involved in nitrogen uptake and assimilation was investigated in ectomycorrhizal roots. We found that three oak ammonium transporters were over-expressed in root tissues after inoculation, while the expression of amino acid transporters was not modified, suggesting that inorganic nitrogen is the main form of nitrogen transferred by the symbiotic fungus into the roots of the host plant. Analysis by heterologous complementation of a yeast mutant defective in ammonium uptake and GFP subcellular protein localization clearly confirmed that two of these genes encode functional ammonium transporters. Structural similarities between the proteins encoded by these ectomycorrhizal upregulated ammonium transporters, and a well-characterized ammonium transporter from E. coli, suggest a similar transport mechanism, involving deprotonation of NH4+, followed by diffusion of uncharged NH3 into the cytosol. This view is supported by the lack of induction of NH4+ detoxifying mechanisms, such as the GS/GOGAT pathway, in the oak mycorrhizal roots.

The leaf lipid composition of ectomycorrhizal oak plants shows a drought-tolerance signature.

Ectomycorrhizas have been reported to increase plant tolerance to drought. However, the mechanisms involved are not yet fully understood. Membranes are the first targets of degradation during drought, and growing evidences support a role for membrane lipids in plant tolerance and adaptation to drought. We have previously shown that improved tolerance of ectomycorrhizal oak plants to drought could be related to leaf membrane lipid metabolism, namely through an increased ability to sustain fatty acid content and composition, indicative of a higher membrane stability under stress. Here, we analysed in deeper detail the modulation of leaf lipid metabolism in oak plants mycorrhized with Pisolithus tinctorius and subjected to drought stress. Results show that mycorrhizal plants show patterns associated with water deficit tolerance, like a higher content of chloroplast lipids, whose levels are maintained upon drought stress. Likewise, mycorrhizal plants show increased levels of unsaturated fatty acids in the chloroplast phosphatidylglycerol lipid fraction. As a common response to drought, the digalactosyldiacyloglycerol/monogalactosyldiacyloglycerol ratio increased in the non-mycorrhizal plants, but not in the mycorrhizal plants, associated to smaller alterations in the expression of galactolipid metabolism genes, indicative of a higher drought tolerance. Under drought, inoculated plants showed increased expression of genes involved in neutral lipids biosynthesis, which could be related to an increased ability to tolerate drought stress. Overall, results from this study provide evidences of the involvement of lipid metabolism in the response of ectomycorrhizal plants to water deficit and point to an increased ability to maintain a stable chloroplast membrane functional integrity under stress.

Ectomycorrhizal inoculation with Pisolithus tinctorius reduces stress induced by drought in cork oak.

We investigated whether the performance of cork oak under drought could be improved by colonization with the ectomycorrhizal fungus Pisolithus tinctorius. Results show that inoculation alone had a positive effect on plant height, shoot biomass, shoot basal diameter, and root growth. Under drought, root growth of mycorrhizal plants was significantly increased showing that inoculation was effective in increasing tolerance to drought. In accordance, mycorrhizal plants subjected to drought showed less symptoms of stress when compared to non-mycorrhizal plants, such as lower concentration of soluble sugars and starch, increased ability to maintain fatty acid content and composition, and increased unsaturation level of membrane lipids. After testing some of the mechanisms suggested to contribute to the enhanced tolerance of mycorrhizal plants to drought, we could not find any by which Pisolithus tinctorius could benefit cork oak, at least under the drought conditions imposed in our experiment. Inoculation did not increase photosynthesis under drought, suggesting no effect in sustaining stomatal opening at low soil water content. Similarly, plant water status was not affected by inoculation suggesting that P. tinctorius does not contribute to an increased plant water uptake during drought. Inoculation did increase nitrogen concentration in plants but it was independent of the water status. Furthermore, no significant mycorrhizal effect on drought-induced ROS production or osmotic adjustment was detected, suggesting that these factors are not important for the improved drought tolerance triggered by P. tinctorius.

Oak protein profile alterations upon root colonization by an ectomycorrhizal fungus.

An increased knowledge on the real impacts of ectomycorrhizal symbiosis in forest species is needed to optimize forest sustainable productivity and thus to improve forest services and their capacity to act as carbon sinks. In this study, we investigated the response of an oak species to ectomycorrhizae formation using a proteomics approach complemented by biochemical analysis of carbohydrate levels. Comparative proteome analysis between mycorrhizal and nonmycorrhizal cork oak plants revealed no differences at the foliar level. However, the protein profile of 34 unique oak proteins was altered in the roots. Consistent with the results of the biochemical analysis, the proteome analysis of the mycorrhizal roots suggests a decreasing utilization of sucrose for the metabolic activity of mycorrhizal roots which is consistent with an increased allocation of carbohydrates from the plant to the fungus in order to sustain the symbiosis. In addition, a promotion of protein unfolding mechanisms, attenuation of defense reactions, increased nutrient mobilization from the plant-fungus interface (N and P), as well as cytoskeleton rearrangements and induction of plant cell wall loosening for fungal root accommodation in colonized roots are also suggested by the results. The suggested improvement in root capacity to take up nutrients accompanied by an increase of root biomass without apparent changes in aboveground biomass strongly re-enforces the potential of mycorrhizal inoculation to improve cork oak forest resistance capacity to cope with coming climate change.

Specific adjustments in grapevine leaf proteome discriminating resistant and susceptible grapevine genotypes to Plasmopara viticola.

Grapevine downy mildew is an important disease affecting crop production leading to severe yield losses. This study aims to identify the grapevine cultivar-specific adjustments of leaf proteome that allow the discrimination between resistance and susceptibility towards P. viticola (constitutive (0h) and in after inoculation (6, 12 and 24h). Leaf proteome analysis was performed using 2D difference gel electrophoresis followed by protein identification via mass spectrometry. In addition, we analysed ROS production, antioxidant capacity, lipid peroxidation and gene expression. Proteins related to photosynthesis and metabolism allowed the discrimination of resistant and susceptible grapevine cultivars prior to P. viticola inoculation. Following inoculation increase of hydrogen peroxide levels, cellular redox regulation, establishment of ROS signalling and plant cell death seem to be key points differentiating the resistant genotype. Lipid associated signalling events, particularly related to jasmonates appear also to play a major role in the establishment of resistance. The findings from this study contribute to a better understanding of genotype-specific differences that account for a successful establishment of a defence response to the downy mildew pathogen. BIOLOGICAL SIGNIFICANCE: Here, we present for the first time grapevine cultivar-specific adjustments of leaf proteome that allow the discrimination between resistance and susceptibility towards P. viticola (constitutive (0h) and in after inoculation (6, 12 and 24h). We have highlighted that, following inoculation, the major factors differentiating the resistant from the susceptible grapevine cultivars are the establishment of effective ROS signalling together with lipid-associated signalling events, particularly related to jasmonates. It is believed that plants infected with biotrophic pathogens suppress JA-mediated responses, however recent evidences shown that jasmonic acid signalling pathway in grapevine resistance against Plasmopara viticola. Our results corroborate those evidences and highlight the importance of lipid- signalling for an effective resistance response against the downy mildew pathogen.

Metabolite extraction for high-throughput FTICR-MS-based metabolomics of grapevine leaves.

In metabolomics there is an ever-growing need for faster and more comprehensive analysis methods to cope with the increase of biological studies. Direct infusion Fourier-transform ion cyclotron-resonance mass spectrometry (DI-FTICR-MS) is used in non-targeted metabolomics to obtain high-resolution snapshots of the metabolic state of a system. In any metabolic profiling study, the establishment of an effective metabolite extraction protocol is paramount. We developed an improved metabolite extraction method, compatible with DI-FTICR-MS-based metabolomics, using grapevine leaves. This extraction protocol allowed the extraction of polar and non-polar compounds, covering all major classes found in plants and increasing metabolome coverage.

Tracking cashew economically important diseases in the West African region using metagenomics.

During the last decades, agricultural land-uses in West Africa were marked by dramatic shifts in the coverage of individual crops. Nowadays, cashew (Anacardium occidentale L.) is one of the most export-oriented horticulture crops, notably in Guinea-Bissau. Relying heavily on agriculture to increase their income, developing countries have been following a strong trend of moving on from traditional farming systems toward commercial production. Emerging infectious diseases, driven either by adaptation to local conditions or inadvertent importation of plant pathogens, are able to cause tremendous cashew production losses, with economic and social impact of which, in developing countries is often underestimated. Presently, plant genomics with metagenomics as an emergent tool, presents an enormous potential to better characterize diseases by providing extensive knowledge on plant pathogens at a large scale. In this perspective, we address metagenomics as a promising genomic tool to identify cashew fungal associated diseases as well as to discriminate the causal pathogens, aiming at obtaining tools to help design effective strategies for disease control and thus promote the sustainable production of cashew in West African Region.

Oak root response to ectomycorrhizal symbiosis establishment: RNA-Seq derived transcript identification and expression profiling.

Ectomycorrhizal symbiosis is essential for the life and health of trees in temperate and boreal forests where it plays a major role in nutrient cycling and in functioning of the forest ecosystem. Trees with ectomycorrhizal root tips are more tolerant to environmental stresses, such as drought, and biotic stresses such as root pathogens. Detailed information on these molecular processes is essential for the understanding of symbiotic tissue development in order to optimize the benefits of this natural phenomenon. Next generation sequencing tools allow the analysis of non model ectomycorrhizal plant-fungal interactions that can contribute to find the "symbiosis toolkits" and better define the role of each partner in the mutualistic interaction. By using 454 pyrosequencing we compared ectomycorrhizal cork oak roots with non-symbiotic roots. From the two cDNA libraries sequenced, over 2 million reads were obtained that generated 19,552 cork oak root unique transcripts. A total of 2238 transcripts were found to be differentially expressed when ECM roots were compared with non-symbiotic roots. Identification of up- and down-regulated gens in ectomycorrhizal roots lead to a number of insights into the molecular mechanisms governing this important symbiosis. In cork oak roots, ectomycorrhizal colonization resulted in extensive cell wall remodelling, activation of the secretory pathway, alterations in flavonoid biosynthesis, and expression of genes involved in the recognition of fungal effectors. In addition, we identified genes with putative roles in symbiotic processes such as nutrient exchange with the fungal partner, lateral root formation or root hair decay. These findings provide a global overview of the transcriptome of an ectomycorrhizal host root, and constitute a foundation for future studies on the molecular events controlling this important symbiosis.

Subtilisin-like proteases in plant-pathogen recognition and immune priming: a perspective.

Subtilisin-like proteases (subtilases) are serine proteases that fulfill highly specific functions in plant development and signaling cascades. Over the last decades, it has been shown that several subtilases are specifically induced following pathogen infection and very recently an Arabidopsis subtilase (SBT3.3) was hypothesized to function as a receptor located in the plasma membrane activating downstream immune signaling processes. Despite their prevalence and potential relevance in the regulation of plant defense mechanisms and crop improvement, our current understanding of subtilase function is still very limited. In this perspective article, we overview the current status and highlight the involvement of subtilases in pathogen recognition and immune priming.

Reference gene selection and validation for the early responses to downy mildew infection in susceptible and resistant Vitis vinifera cultivars.

The pivotal role of cultivated grapevine (Vitis vinifera L.) in many countries economy is compromised by its high susceptibility to Plasmopara viticola, the causal agent of downy mildew disease. Recent research has identified a set of genes related to resistance which may be used to track downy mildew infection. Quantification of the expression of these resistance genes requires normalizing qPCR data using reference genes with stable expression in the system studied. In this study, a set of eleven genes (VATP16, 60 S, UQCC, SMD3, EF1α, UBQ, SAND, GAPDH, ACT, PsaB, PTB2) was evaluated to identify reference genes during the first hours of interaction (6, 12, 18 and 24 hpi) between two V. vinifera genotypes and P. viticola. Two analyses were used for the selection of reference genes: direct comparison of susceptible, Trincadeira, and resistant, Regent, V. vinifera cultivars at 0 h, 6, 12, 18 and 24 hours post inoculation with P. viticola (genotype effect); and comparison of each genotype with mock inoculated samples during inoculation time-course (biotic stress effect). Three statistical methods were used, GeNorm, NormFinder, and BestKeeper, allowing to identify UBQ, EF1α and GAPDH as the most stable genes for the genotype effect. For the biotic stress effect, EF1α, SAND and SMD3 were the most constant for the susceptible cultivar Trincadeira and EF1α, GAPDH, UBQ for the resistant cultivar Regent. In addition, the expression of three defense-related transcripts, encoding for subtilisin-like protein, CYP and PR10, was analysed, for both datasets, during inoculation time-course. Taken together, our results provide guidelines for reference gene(s) selection towards a more accurate and widespread use of qPCR to study the first hours of interaction between different grapevine cultivars and P. viticola.

A possible approach for gel-based proteomic studies in recalcitrant woody plants.

Woody plants are particularly difficult to investigate due to high phenolic, resin, and tannin contents and laborious sample preparation. In particular, protein isolation from woody plants for two-dimensional gel electrophoresis (2-DE) is challenging as secondary metabolites negatively interfere with protein extraction and separation. In this study, three protein extraction protocols, using TCA, phenol and ethanol as precipitation or extraction agents, were tested in order to select the more efficient for woody recalcitrant plant gel-based proteomics. Grapevine leaves, pine needles and cork oak ectomycorrhizal roots were used to represent woody plant species and tissues. The phenol protocol produced higher quality 2-DE gels, with increased number of resolved spots, better spot focusing and representation of all molecular mass and isoelectric point ranges tested. In order to test the compatibility of the phenol extracted proteomes with protein identification several spots were excised from the phenol gels and analyzed by mass spectrometry (MALDI-TOF/TOF). Regardless the incomplete genome/protein databases for the plant species under analysis, 49 proteins were identified by Peptide Mass Fingerprint (PMF). Proteomic data have been deposited to the ProteomeXchange with identifier PXD000224. Our results demonstrate the complexity of protein extraction from woody plant tissues and the suitability of the phenol protocol for obtaining high quality protein extracts for efficient 2-DE separation and downstream applications such as protein identification by mass spectrometry.

Labellum transcriptome reveals alkene biosynthetic genes involved in orchid sexual deception and pollination-induced senescence.

One of the most remarkable pollination strategy in orchids biology is pollination by sexual deception, in which the modified petal labellum lures pollinators by mimicking the chemical (e.g. sex pheromones), visual (e.g. colour and shape/size) and tactile (e.g. labellum trichomes) cues of the receptive female insect species. The present study aimed to characterize the transcriptional changes occurring after pollination in the labellum of a sexually deceptive orchid (Ophrys fusca Link) in order to identify genes involved on signals responsible for pollinator attraction, the major goal of floral tissues. Novel information on alterations in the orchid petal labellum gene expression occurring after pollination demonstrates a reduction in the expression of alkene biosynthetic genes using O. fusca Link as the species under study. Petal labellum transcriptional analysis revealed downregulation of transcripts involved in both pigment machinery and scent compounds, acting as visual and olfactory cues, respectively, important in sexual mimicry. Regulation of petal labellum senescence was revealed by transcripts related to macromolecules breakdown, protein synthesis and remobilization of nutrients.

Fungal transcript pattern during the preinfection stage (12 h) of ectomycorrhiza formed between Pisolithus tinctorius and Castanea sativa roots, identified using cDNA microarrays.

Transcriptional changes in Pisolithus tinctorius leading to ectomycorrhizal formation in P. tinctorius- Castanea sativa were investigated using a 12-h fungal interaction in vitro system. Using a 3107-cDNA clone microarray, 34 unique expressed sequence tags (ESTs) were found to be differentially expressed. These ESTs represent 14 known genes, 5 upregulated and 9 downregulated, and 20 orphan sequences. Some transcripts of upregulated genes (with unknown function) were previously identified in other mycorrhizal Pisolithus spp. associations. ESTs for S-adenosyl-L-homocysteine hydrolase and several orphan sequences were identified in our system. The identified transcript of downregulated genes involved hydrophobins, 5S, 18S, and 28S ribosomal RNA genes, large subunits of ribosomal RNA (mitochondrial gene), and two types of heat shock proteins. This study demonstrates the high complexity of molecular events involved in the preinfection steps and suggests the utilization of different fungal gene repertories before ectomycorrhizal formation. These data constitute a first contribution for the molecular understanding of early signaling events between P. tinctorius and C. sativa roots during ectomycorrhizal formation.

Organogenic nodule development in hop (Humulus lupulus L.): transcript and metabolic responses.

BACKGROUND: Hop (Humulus lupulus L.) is an economically important plant forming organogenic nodules which can be used for genetic transformation and micropropagation. We are interested in the mechanisms underlying reprogramming of cells through stress and hormone treatments. RESULTS: An integrated molecular and metabolomic approach was used to investigate global gene expression and metabolic responses during development of hop's organogenic nodules. Transcript profiling using a 3,324-cDNA clone array revealed differential regulation of 133 unigenes, classified into 11 functional categories. Several pathways seem to be determinant in organogenic nodule formation, namely defense and stress response, sugar and lipid metabolism, synthesis of secondary metabolites and hormone signaling. Metabolic profiling using 1H NMR spectroscopy associated to two-dimensional techniques showed the importance of metabolites related to oxidative stress response, lipid and sugar metabolism and secondary metabolism in organogenic nodule formation. CONCLUSION: The expression profile of genes pivotal for energy metabolism, together with metabolites profile, suggested that these morphogenic structures gain energy through a heterotrophic, transport-dependent and sugar-degrading anaerobic metabolism. Polyamines and auxins are likely to be involved in the regulation of expression of many genes related to organogenic nodule formation. These results represent substantial progress toward a better understanding of this complex developmental program and reveal novel information regarding morphogenesis in plants.

Transcriptional and metabolic profiling of grape (Vitis vinifera L.) leaves unravel possible innate resistance against pathogenic fungi.

Grapevine species (Vitis sp.) are prone to several diseases, fungi being the major pathogens compromising its cultivation and economic profit around the world. Knowledge of the complexity of mechanisms responsible for resistance to fungus infection of cultivars, such as Regent, is necessary for strategies to be defined which will improve resistance in highly susceptible crop species. Transcript and metabolic profiles of the Vitis vinifera cultivars Regent and Trincadeira (resistant and susceptible to fungi, respectively) were analysed by cDNA microarray, quantitative real-time PCR, and nuclear magnetic resonance spectroscopy. The integration of datasets obtained through transcriptome and metabolome analysis revealed differences in transcripts and metabolites between both cultivars. These differences are probably associated with the innate resistance of Regent towards the mildews. Several transcripts related to stress and defence, namely a subtilisin-like protease, phenylalanine ammonia lyase, S-adenosylmethionine synthase, WD-repeat protein like, and J2P, were up-regulated in Regent suggesting an intrinsic resistance capability of this cultivar. A metabolic profile revealed an accumulation of compounds such as inositol and caffeic acid, which are known to confer resistance to fungi. The differences in transcripts and metabolites detected are discussed in terms of the metabolic pathways and their possible role in plant defence against pathogen attack, as well as their potential interest to discriminate among resistant and susceptible grapevine cultivars.