Pathogen resistance in Sphagneticola trilobata (Singapore daisy): molecular associations and differentially expressed genes in response to disease from a widespread fungus.

Understanding the molecular associations underlying pathogen resistance in invasive plant species is likely to provide useful insights into the effective control of alien plants, thereby facilitating the conservation of native biodiversity. In the current study, we investigated pathogen resistance in an invasive clonal plant, Sphagneticola trilobata, at the molecular level. Sphagneticola trilobata (i.e., Singapore daisy) is a noxious weed that affects both terrestrial and aquatic ecosystems, and is less affected by pathogens in the wild than co-occurring native species. We used Illumina sequencing to investigate the transcriptome of S. trilobata following infection by a globally distributed generalist pathogen (Rhizoctonia solani). RNA was extracted from leaves of inoculated and un-inoculated control plants, and a draft transcriptome of S. trilobata was generated to examine the molecular response of this species following infection. We obtained a total of 49,961,014 (94.3%) clean reads for control (un-inoculated plants) and 54,182,844 (94.5%) for the infected treatment (inoculated with R. solani). Our analyses facilitated the discovery of 117,768 de novo assembled contigs and 78,916 unigenes. Of these, we identified 3506 differentially expressed genes and 60 hormones associated with pathogen resistance. Numerous genes, including candidate genes, were associated with plant-pathogen interactions and stress response in S. trilobata. Many recognitions, signaling, and defense genes were differentially regulated between treatments, which were confirmed by qRT-PCR. Overall, our findings improve our understanding of the genes and molecular associations involved in plant defense of a rapidly spreading invasive clonal weed, and serve as a valuable resource for further work on mechanism of disease resistance and managing invasive plants.

Physiological and Proteomic Signatures Reveal Mechanisms of Superior Drought Resilience in Pearl Millet Compared to Wheat.

Presently, pearl millet and wheat are belonging to highly important cereal crops. Pearl millet, however, is an under-utilized crop, despite its superior resilience to drought and heat stress in contrast to wheat. To investigate this in more detail, we performed comparative physiological screening and large scale proteomics of drought stress responses in drought-tolerant and susceptible genotypes of pearl millet and wheat. These chosen genotypes are widely used in breeding and farming practices. The physiological responses demonstrated large differences in the regulation of root morphology and photosynthetic machinery, revealing a stay-green phenotype in pearl millet. Subsequent tissue-specific proteome analysis of leaves, roots and seeds led to the identification of 12,558 proteins in pearl millet and wheat under well-watered and stress conditions. To allow for this comparative proteome analysis and to provide a platform for future functional proteomics studies we performed a systematic phylogenetic analysis of all orthologues in pearl millet, wheat, foxtail millet, sorghum, barley, brachypodium, rice, maize, Arabidopsis, and soybean. In summary, we define (i) a stay-green proteome signature in the drought-tolerant pearl millet phenotype and (ii) differential senescence proteome signatures in contrasting wheat phenotypes not capable of coping with similar drought stress. These different responses have a significant effect on yield and grain filling processes reflected by the harvest index. Proteome signatures related to root morphology and seed yield demonstrated the unexpected intra- and interspecies-specific biochemical plasticity for stress adaptation for both pearl millet and wheat genotypes. These quantitative reference data provide tissue- and phenotype-specific marker proteins of stress defense mechanisms which are not predictable from the genome sequence itself and have potential value for marker-assisted breeding beyond genome assisted breeding.

Gene Expression Profiling Reveals Enhanced Defense Responses in an Invasive Weed Compared to Its Native Congener During Pathogenesis.

Invasive plants are a huge burden on the environment, and modify local ecosystems by affecting the indigenous biodiversity. Invasive plants are generally less affected by pathogens, although the underlying molecular mechanisms responsible for their enhanced resistance are unknown. We investigated expression profiles of three defense hormones (salicylic acid, jasmonic acid, and ethylene) and their associated genes in the invasive weed, Alternanthera philoxeroides, and its native congener, A. sessilis, after inoculation with Rhizoctonia solani. Pathogenicity tests showed significantly slower disease progression in A. philoxeroides compared to A. sessilis. Expression analyses revealed jasmonic acid (JA) and ethylene (ET) expressions were differentially regulated between A. philoxeroides and A. sessilis, with the former having prominent antagonistic cross-talk between salicylic acid (SA) and JA, and the latter showing weak or no cross-talk during disease development. We also found that JA levels decreased and SA levels increased during disease development in A. philoxeroides. Variations in hormonal gene expression between the invasive and native species (including interspecific differences in the strength of antagonistic cross-talk) were identified during R. solani pathogenesis. Thus, plant hormones and their cross-talk signaling may improve the resistance of invasive A. philoxeroides to pathogens, which has implications for other invasive species during the invasion process.

Proteomics of Heat-Stress and Ethylene-Mediated Thermotolerance Mechanisms in Tomato Pollen Grains.

Heat stress is a major cause for yield loss in many crops, including vegetable crops. Even short waves of high temperature, becoming more frequent during recent years, can be detrimental. Pollen development is most heat-sensitive, being the main cause for reduced productivity under heat-stress across a wide range of crops. The molecular mechanisms involved in pollen heat-stress response and thermotolerance are however, not fully understood. Recently, we have demonstrated that ethylene, a gaseous plant hormone, plays a role in tomato (Solanum lycopersicum) pollen thermotolerance. These results were substantiated in the current work showing that increasing ethylene levels by using an ethylene-releasing substance, ethephon, prior to heat-stress exposure, increased pollen quality. A proteomic approach was undertaken, to unravel the mechanisms underlying pollen heat-stress response and ethylene-mediated pollen thermotolerance in developing pollen grains. Proteins were extracted and analyzed by means of a gel LC-MS fractionation protocol, and a total of 1,355 proteins were identified. A dataset of 721 proteins, detected in three biological replicates of at least one of the applied treatments, was used for all analyses. Quantitative analysis was performed based on peptide count. The analysis revealed that heat-stress affected the developmental program of pollen, including protein homeostasis (components of the translational and degradation machinery), carbohydrate, and energy metabolism. Ethephon-pre-treatment shifted the heat-stressed pollen proteome closer to the proteome under non-stressful conditions, namely, by showing higher abundance of proteins involved in protein synthesis, degradation, tricarboxylic acid cycle, and RNA regulation. Furthermore, up-regulation of protective mechanisms against oxidative stress was observed following ethephon-treatment (including higher abundance of glutathione-disulfide reductase, glutaredoxin, and protein disulfide isomerase). Taken together, the findings identified systemic and fundamental components of pollen thermotolerance, and serve as a valuable quantitative protein database for further research.

Ethylene production and signaling in tomato (Solanum lycopersicum) pollen grains is responsive to heat stress conditions.

KEY MESSAGE: Tomato pollen grains have the capacity for ethylene production, possessing specific components of the ethylene-biosynthesis and -signaling pathways, being affected/responsive to high-temperature conditions. Exposure of plants to heat stress (HS) conditions reduces crop yield and quality, mainly due to sensitivity of pollen grains. Recently, it was demonstrated that ethylene, a gaseous plant hormone, plays a significant role in tomato pollen heat-tolerance. It is not clear, however, whether, or to what extent, pollen grains are dependent on the capacity of the surrounding anther tissues for ethylene synthesis and signaling, or can synthesize this hormone and possess an active signaling pathway. The aim of this work was (1) to investigate if isolated, maturing and mature, tomato pollen grains have the capacity for ethylene production, (2) to find out whether pollen grains possess an active ethylene-biosynthesis and -signaling pathway and characterize the respective tomato pollen components at the transcript level, (3) to look into the effect of short-term HS conditions. Results from accumulation studies showed that pollen, anthers, and flowers produced ethylene and HS affected differentially ethylene production by (rehydrated) mature pollen, compared to anthers and flowers, causing elevated ethylene levels. Furthermore, several ethylene synthesis genes were expressed, with SlACS3 and SlACS11 standing out as highly HS-induced genes of the pollen ethylene biosynthesis pathway. Specific components of the ethylene-signaling pathway as well as several ethylene-responsive factors were expressed in pollen, with SlETR3 (ethylene receptor; named also NR, for never ripe) and SlCTR2 (constitutive triple response2) being HS responsive. This work shows that tomato pollen grains have the capacity for ethylene production, possessing active ethylene-biosynthesis and -signaling pathways, highlighting specific pollen components that serve as a valuable resource for future research on the role of ethylene in pollen thermotolerance.

Survey of Genes Involved in Biosynthesis, Transport, and Signaling of Phytohormones with Focus on Solanum lycopersicum.

Phytohormones control the development and growth of plants, as well as their response to biotic and abiotic stress. The seven most well-studied phytohormone classes defined today are as follows: auxins, ethylene, cytokinin, abscisic acid, jasmonic acid, gibberellins, and brassinosteroids. The basic principle of hormone regulation is conserved in all plants, but recent results suggest adaptations of synthesis, transport, or signaling pathways to the architecture and growth environment of different plant species. Thus, we aimed to define the extent to which information from the model plant Arabidopsis thaliana is transferable to other plants such as Solanum lycopersicum. We extracted the co-orthologues of genes coding for major pathway enzymes in A. thaliana from the translated genomes of 12 species from the clade Viridiplantae. Based on predicted domain architecture and localization of the identified proteins from all 13 species, we inspected the conservation of phytohormone pathways. The comparison was complemented by expression analysis of (co-) orthologous genes in S. lycopersicum. Altogether, this information allowed the assignment of putative functional equivalents between A. thaliana and S. lycopersicum but also pointed to some variations between the pathways in eudicots, monocots, mosses, and green algae. These results provide first insights into the conservation of the various phytohormone pathways between the model system A. thaliana and crop plants such as tomato. We conclude that orthologue prediction in combination with analysis of functional domain architecture and intracellular localization and expression studies are sufficient tools to transfer information from model plants to other plant species. Our results support the notion that hormone synthesis, transport, and response for most part of the pathways are conserved, and species-specific variations can be found.

Heat-Treatment-Responsive Proteins in Different Developmental Stages of Tomato Pollen Detected by Targeted Mass Accuracy Precursor Alignment (tMAPA).

Recently, we have developed a quantitative shotgun proteomics strategy called mass accuracy precursor alignment (MAPA). The MAPA algorithm uses high mass accuracy to bin mass-to-charge (m/z) ratios of precursor ions from LC-MS analyses, determines their intensities, and extracts a quantitative sample versus m/z ratio data alignment matrix from a multitude of samples. Here, we introduce a novel feature of this algorithm that allows the extraction and alignment of proteotypic peptide precursor ions or any other target peptide from complex shotgun proteomics data for accurate quantification of unique proteins. This strategy circumvents the problem of confusing the quantification of proteins due to indistinguishable protein isoforms by a typical shotgun proteomics approach. We applied this strategy to a comparison of control and heat-treated tomato pollen grains at two developmental stages, post-meiotic and mature. Pollen is a temperature-sensitive tissue involved in the reproductive cycle of plants and plays a major role in fruit setting and yield. By LC-MS-based shotgun proteomics, we identified more than 2000 proteins in total for all different tissues. By applying the targeted MAPA data-processing strategy, 51 unique proteins were identified as heat-treatment-responsive protein candidates. The potential function of the identified candidates in a specific developmental stage is discussed.