Phenotypic and transcriptomic responses of diverse rice accessions to transient heat stress during early grain development.

Heat stress (HS) occurring during the grain-filling period has a detrimental effect on grain yield and quality in rice (Oryza sativa). The development of heat-resilient cultivars could partly solve this issue if tolerant alleles can be identified and incorporated into the germplasm. In this study, we posit that some of the phenotypic variations for heat resilience during grain development could be due to variations in gene expression among accessions. To test this, we characterized the HS response of 10 diverse rice accessions from three major sub-populations using physiological and transcriptome analyses. At a single-grain level, grain width and grain thickness emerged as the most heat-sensitive traits. During a transient HS, IND-3 was categorized as highly sensitive, while five accessions exhibited moderate heat sensitivity, and four accessions were tolerant. Only a core set of 29.4% of the differentially expressed genes was common to the three rice sub-populations. Heat-tolerant accession TEJ-5 uniquely triggered an unfolded protein response (UPR) under HS, as evident from the induction of OsbZIP50 and downstream UPR genes. OsbZIP58, a gene that positively regulates grain filling, was more highly induced by HS in IND-2 despite its moderate heat sensitivity. Collectively, our analysis suggests that both unique gene expression responses and variation in the level of responses for a given pathway distinguish diverse accessions. Only some of these responses are associated with single-grain phenotypes in a manner consistent with the known roles of these genes and pathways.

Natural variation in LONELY GUY-Like 1 regulates rice grain weight under warmer night conditions.

Global nighttime temperatures are rising at twice the rate of daytime temperatures and pose a challenge for rice (Oryza sativa) production. High nighttime temperature (HNT) stress affects rice yield by reducing grain weight, size, and fertility. Although the genes associated with these yield parameters have been identified and characterized under normal temperatures, the genetic basis of grain weight regulation under HNT stress remains less explored. We examined the natural variation for rice single grain weight (SGW) under HNT stress imposed during grain development. A genome-wide association analysis identified several loci associated with grain weight under HNT stress. A locus, SGW1, specific to HNT conditions resolved to LONELY GUY-Like 1 (LOGL1), which encodes a putative cytokinin-activation enzyme. We demonstrated that LOGL1 contributes to allelic variation at SGW1. Accessions with lower LOGL1 transcript abundance had higher grain weight under HNT. This was supported by the higher grain weight of logl1-mutants relative to the wild type under HNT. Compared to logl1-mutants, LOGL1 over-expressers showed increased sensitivity to HNT. We showed that LOGL1 regulates the thiamin biosynthesis pathway, which is under circadian regulation, which in turn is likely perturbed by HNT stress. These findings provide a genetic source to enhance rice adaptation to warming night temperatures and improve our mechanistic understanding of HNT stress tolerance pathways.

Ultrasensitive Surface Plasmon Resonance Sensor with a Feature of Dynamically Tunable Sensitivity and High Figure of Merit for Cancer Detection.

Cancer is one of the leading causes of death worldwide, and it is well known that an early detection of cancer in a human body will provide an opportunity to cure the cancer. Early detection of cancer depends on the sensitivity of the measuring device and method, where the lowest detectable concentration of the cancerous cell in a test sample becomes a matter of high importance. Recently, Surface Plasmon Resonance (SPR) has proven to be a promising method to detect cancerous cells. The SPR method is based on the detection of changes in refractive indices of samples under testing and the sensitivity of such a SPR based sensor is related to the smallest detectable change in the refractive index of the sample. There exist many techniques where different combinations of metals, metal alloys and different configurations have been shown to lead to high sensitivities of the SPR sensors. Based on the difference in the refractive index between a normal healthy cell and a cancerous cell, recently, SPR method has been shown to be applicable to detect different types of cancers. In this work, we propose a new sensor surface configuration that comprises of gold-silver-graphene-black phosphorus to detect different cancerous cells based on the SPR method. Additionally, recently we proposed that the application of electric field across gold-graphene layers that form the SPR sensor surface can provide enhanced sensitivity than that is possible without the application of electrical bias. We utilized the same concept and numerically studied the impact of electrical bias across the gold-graphene layers combined with silver and black Phosphorus layers which forms the SPR sensor surface. Our numerical results have shown that electrical bias across the sensor surface in this new heterostructure can provide enhanced sensitivity compared to the original unbiased sensor surface. Not only that, our results have shown that as the electrical bias increases, the sensitivity increases up to a certain value and stabilizes at a still improved sensitivity value. Such dependence of sensitivity on the applied bias provides a dynamic tunability of the sensitivity and figure-of-merit (FOM) of the sensor to detect different types of cancer. In this work, we used the proposed heterostructure to detect six different types of cancers: Basal, Hela, Jurkat, PC12, MDA-MB-231, and MCF-7. Comparing our results to work published recently, we were able to achieve an enhanced sensitivity ranging from 97.2 to 1851.4 (deg/RIU) and FOM values ranging from 62.13 to 89.81 far above the values presented recently by other researchers.

Evaluating metabolic and genomic data for predicting grain traits under high night temperature stress in rice.

The asymmetric increase in average nighttime temperatures relative to increase in average daytime temperatures due to climate change is decreasing grain yield and quality in rice. Therefore, a better genome-level understanding of the impact of higher night temperature stress on the weight of individual grains is essential for future development of more resilient rice. We investigated the utility of metabolites obtained from grains to classify high night temperature (HNT) conditions of genotypes, and metabolites and single-nucleotide polymorphisms (SNPs) to predict grain length, width, and perimeter phenotypes using a rice diversity panel. We found that the metabolic profiles of rice genotypes alone could be used to classify control and HNT conditions with high accuracy using random forest or extreme gradient boosting. Best linear unbiased prediction and BayesC showed greater metabolic prediction performance than machine learning models for grain-size phenotypes. Metabolic prediction was most effective for grain width, resulting in the highest prediction performance. Genomic prediction performed better than metabolic prediction. Integrating metabolites and genomics simultaneously in a prediction model slightly improved prediction performance. We did not observe a difference in prediction between the control and HNT conditions. Several metabolites were identified as auxiliary phenotypes that could be used to enhance the multi-trait genomic prediction of grain-size phenotypes. Our results showed that, in addition to SNPs, metabolites collected from grains offer rich information to perform predictive analyses, including classification modeling of HNT responses and regression modeling of grain-size-related phenotypes in rice.

A non-destructive approach for measuring rice panicle-level photosynthetic responses using 3D-image reconstruction.

BACKGROUND: Our understanding of the physiological responses of rice inflorescence (panicle) to environmental stresses is limited by the challenge of accurately determining panicle photosynthetic parameters and their impact on grain yield. This is primarily due to the lack of a suitable gas exchange methodology for panicles and non-destructive methods to accurately determine panicle surface area. RESULTS: To address these challenges, we have developed a custom panicle gas exchange cylinder compatible with the LiCor 6800 Infra-red Gas Analyzer. Accurate surface area measurements were determined using 3D panicle imaging to normalize the panicle-level photosynthetic measurements. We observed differential responses in both panicle and flag leaf for two temperate Japonica rice genotypes (accessions TEJ-1 and TEJ-2) exposed to heat stress during early grain filling. There was a notable divergence in the relative photosynthetic contribution of flag leaf and panicles for the heat-tolerant genotype (TEJ-2) compared to the sensitive genotype (TEJ-1). CONCLUSION: The novelty of this method is the non-destructive and accurate determination of panicle area and photosynthetic parameters, enabling researchers to monitor temporal changes in panicle physiology during the reproductive development. The method is useful for panicle-level measurements under diverse environmental stresses and is sensitive enough to evaluate genotypic variation for panicle physiology and architecture in cereals with compact inflorescences.

Rice Chalky Grain 5 regulates natural variation for grain quality under heat stress.

Heat stress occurring during rice (Oryza sativa) grain development reduces grain quality, which often manifests as increased grain chalkiness. Although the impact of heat stress on grain yield is well-studied, the genetic basis of rice grain quality under heat stress is less explored as quantifying grain quality is less tractable than grain yield. To address this, we used an image-based colorimetric assay (Red, R; and Green, G) for genome-wide association analysis to identify genetic loci underlying the phenotypic variation in rice grains exposed to heat stress. We found the R to G pixel ratio (RG) derived from mature grain images to be effective in distinguishing chalky grains from translucent grains derived from control (28/24°C) and heat stressed (36/32°C) plants. Our analysis yielded a novel gene, rice Chalky Grain 5 (OsCG5) that regulates natural variation for grain chalkiness under heat stress. OsCG5 encodes a grain-specific, expressed protein of unknown function. Accessions with lower transcript abundance of OsCG5 exhibit higher chalkiness, which correlates with higher RG values under stress. These findings are supported by increased chalkiness of OsCG5 knock-out (KO) mutants relative to wildtype (WT) under heat stress. Grains from plants overexpressing OsCG5 are less chalky than KOs but comparable to WT under heat stress. Compared to WT and OE, KO mutants exhibit greater heat sensitivity for grain size and weight relative to controls. Collectively, these results show that the natural variation at OsCG5 may contribute towards rice grain quality under heat stress.

HyperSeed: An End-to-End Method to Process Hyperspectral Images of Seeds.

High-throughput, nondestructive, and precise measurement of seeds is critical for the evaluation of seed quality and the improvement of agricultural productions. To this end, we have developed a novel end-to-end platform named HyperSeed to provide hyperspectral information for seeds. As a test case, the hyperspectral images of rice seeds are obtained from a high-performance line-scan image spectrograph covering the spectral range from 600 to 1700 nm. The acquired images are processed via a graphical user interface (GUI)-based open-source software for background removal and seed segmentation. The output is generated in the form of a hyperspectral cube and curve for each seed. In our experiment, we presented the visual results of seed segmentation on different seed species. Moreover, we conducted a classification of seeds raised in heat stress and control environments using both traditional machine learning models and neural network models. The results show that the proposed 3D convolutional neural network (3D CNN) model has the highest accuracy, which is 97.5% in seed-based classification and 94.21% in pixel-based classification, compared to 80.0% in seed-based classification and 85.67% in seed-based classification from the support vector machine (SVM) model. Moreover, our pipeline enables systematic analysis of spectral curves and identification of wavelengths of biological interest.

Allelic variation in rice Fertilization Independent Endosperm 1 contributes to grain width under high night temperature stress.

A higher minimum (night-time) temperature is considered a greater limiting factor for reduced rice yield than a similar increase in maximum (daytime) temperature. While the physiological impact of high night temperature (HNT) has been studied, the genetic and molecular basis of HNT stress response remains unexplored. We examined the phenotypic variation for mature grain size (length and width) in a diverse set of rice accessions under HNT stress. Genome-wide association analysis identified several HNT-specific loci regulating grain size as well as loci that are common for optimal and HNT stress conditions. A novel locus contributing to grain width under HNT conditions colocalized with Fie1, a component of the FIS-PRC2 complex. Our results suggest that the allelic difference controlling grain width under HNT is a result of differential transcript-level response of Fie1 in grains developing under HNT stress. We present evidence to support the role of Fie1 in grain size regulation by testing overexpression (OE) and knockout mutants under heat stress. The OE mutants were either unaltered or had a positive impact on mature grain size under HNT, while the knockouts exhibited significant grain size reduction under these conditions.

Structural and Functional Heat Stress Responses of Chloroplasts of Arabidopsis thaliana.

Temperature elevations constitute a major threat to plant performance. In recent years, much was learned about the general molecular mode of heat stress reaction of plants. The current research focuses on the integration of the knowledge into more global networks, including the reactions of cellular compartments. For instance, chloroplast function is central for plant growth and survival, and the performance of chloroplasts is tightly linked to the general status of the cell and vice versa. We examined the changes in photosynthesis, chloroplast morphology and proteomic composition posed in Arabidopsisthaliana chloroplasts after a single or repetitive heat stress treatment over a period of two weeks. We observed that the acclimation is potent in the case of repetitive application of heat stress, while a single stress results in lasting alterations. Moreover, the physiological capacity and its adjustment are dependent on the efficiency of the protein translocation process as judged from the analysis of mutants of the two receptor units of the chloroplast translocon, TOC64, and TOC33. In response to repetitive heat stress, plants without TOC33 accumulate Hsp70 proteins and plants without TOC64 have a higher content of proteins involved in thylakoid structure determination when compared to wild-type plants.

Divergent phenotypic response of rice accessions to transient heat stress during early seed development.

Increasing global surface temperatures is posing a major food security challenge. Part of the solution to address this problem is to improve crop heat resilience, especially during grain development, along with agronomic decisions such as shift in planting time and increasing crop diversification. Rice is a major food crop consumed by more than 3 billion people. For rice, thermal sensitivity of reproductive development and grain filling is well-documented, while knowledge concerning the impact of heat stress (HS) on early seed development is limited. Here, we aim to study the phenotypic variation in a set of diverse rice accessions for elucidating the HS response during early seed development. To explore the variation in HS sensitivity, we investigated aus (1), indica (2), temperate japonica (2), and tropical japonica (4) accessions for their HS (39/35°C) response during early seed development that accounts for transition of endosperm from syncytial to cellularization, which broadly corresponds to 24 and 96 hr after fertilization (HAF), respectively, in rice. The two indica and one of the tropical japonica accessions exhibited severe heat sensitivity with increased seed abortion; three tropical japonicas and an aus accession showed moderate heat tolerance, while temperate japonicas exhibited strong heat tolerance. The accessions exhibiting extreme heat sensitivity maintain seed size at the expense of number of fully developed mature seeds, while the accessions showing relative resilience to the transient HS maintained number of fully developed seeds but compromised on seed size, especially seed length. Further, histochemical analysis revealed that all the tested accessions have delayed endosperm cellularization upon exposure to the transient HS by 96 HAF; however, the rate of cellularization was different among the accessions. These findings were further corroborated by upregulation of cellularization-associated marker genes in the developing seeds from the heat-stressed samples.

MADS78 and MADS79 Are Essential Regulators of Early Seed Development in Rice.

MADS box transcription factors (TFs) are subdivided into type I and II based on phylogenetic analysis. The type II TFs regulate floral organ identity and flowering time, but type I TFs are relatively less characterized. Here, we report the functional characterization of two type I MADS box TFs in rice (Oryza sativa), MADS78 and MADS79 Transcript abundance of both these genes in developing seed peaked at 48 h after fertilization and was suppressed by 96 h after fertilization, corresponding to syncytial and cellularized stages of endosperm development, respectively. Seeds overexpressing MADS78 and MADS 79 exhibited delayed endosperm cellularization, while CRISPR-Cas9-mediated single knockout mutants showed precocious endosperm cellularization. MADS78 and MADS 79 were indispensable for seed development, as a double knockout mutant failed to make viable seeds. Both MADS78 and 79 interacted with MADS89, another type I MADS box, which enhances nuclear localization. The expression analysis of Fie1, a rice FERTILIZATION-INDEPENDENT SEED-POLYCOMB REPRESSOR COMPLEX2 component, in MADS78 and 79 mutants and vice versa established an antithetical relation, suggesting that Fie1 could be involved in negative regulation of MADS78 and MADS 79 Misregulation of MADS78 and MADS 79 perturbed auxin homeostasis and carbon metabolism, as evident by misregulation of genes involved in auxin transport and signaling as well as starch biosynthesis genes causing structural abnormalities in starch granules at maturity. Collectively, we show that MADS78 and MADS 79 are essential regulators of early seed developmental transition and impact both seed size and quality in rice.

SeedExtractor: An Open-Source GUI for Seed Image Analysis.

Accurate measurement of seed size parameters is essential for both breeding efforts aimed at enhancing yields and basic research focused on discovering genetic components that regulate seed size. To address this need, we have developed an open-source graphical user interface (GUI) software, SeedExtractor that determines seed size and shape (including area, perimeter, length, width, circularity, and centroid), and seed color with capability to process a large number of images in a time-efficient manner. In this context, our application takes ∼2 s for analyzing an image, i.e., significantly less compared to the other tools. As this software is open-source, it can be modified by users to serve more specific needs. The adaptability of SeedExtractor was demonstrated by analyzing scanned seeds from multiple crops. We further validated the utility of this application by analyzing mature-rice seeds from 231 accessions in Rice Diversity Panel 1. The derived seed-size traits, such as seed length, width, were used for genome-wide association analysis. We identified known loci for regulating seed length (GS3) and width (qSW5/GW5) in rice, which demonstrates the accuracy of this application to extract seed phenotypes and accelerate trait discovery. In summary, we present a publicly available application that can be used to determine key yield-related traits in crops.

Metabolic Dynamics of Developing Rice Seeds Under High Night-Time Temperature Stress.

High temperature stress during rice reproductive development results in yield losses. Reduced grain yield and grain quality has been associated with high temperature stress, and specifically with high night-time temperatures (HNT). Characterizing the impact of HNT on the phenotypic and metabolic status of developing rice seeds can provide insights into the mechanisms involved in yield and quality decline. Here, we examined the impact of warmer nights on the morphology and metabolome during early seed development in six diverse rice accessions. Seed size was sensitive to HNT in four of the six genotypes, while seed fertility and seed weight were unaffected. We observed genotypic differences for negative impact of HNT on grain quality. This was evident from the chalky grain appearance due to impaired packaging of starch granules. Metabolite profiles during early seed development (3 and 4 days after fertilization; DAF) were distinct from the early grain filling stages (7 and 10 DAF) under optimal conditions. We observed that accumulation of sugars (sucrose, fructose, and glucose) peaked at 7 DAF suggesting a major flux of carbon into glycolysis, tricarboxylic acid cycle, and starch biosynthesis during grain filling. Next, we determined hyper (HNT > control) and hypo (HNT < control) abundant metabolites and found 19 of the 57 metabolites to differ significantly between HNT and control treatments. The most prominent changes were exhibited by differential abundance of sugar and sugar alcohols under HNT, which could be linked to a protective mechanism against the HNT damage. Overall, our results indicate that combining metabolic profiles of developing grains with yield and quality parameters under high night temperature stress could provide insight for exploration of natural variation for HNT tolerance in the rice germplasm.

PI-Plat: a high-resolution image-based 3D reconstruction method to estimate growth dynamics of rice inflorescence traits.

BACKGROUND: Recent advances in image-based plant phenotyping have improved our capability to study vegetative stage growth dynamics. However, more complex agronomic traits such as inflorescence architecture (IA), which predominantly contributes to grain crop yield are more challenging to quantify and hence are relatively less explored. Previous efforts to estimate inflorescence-related traits using image-based phenotyping have been limited to destructive end-point measurements. Development of non-destructive inflorescence phenotyping platforms could accelerate the discovery of the phenotypic variation with respect to inflorescence dynamics and mapping of the underlying genes regulating critical yield components. RESULTS: The major objective of this study is to evaluate post-fertilization development and growth dynamics of inflorescence at high spatial and temporal resolution in rice. For this, we developed the Panicle Imaging Platform (PI-Plat) to comprehend multi-dimensional features of IA in a non-destructive manner. We used 11 rice genotypes to capture multi-view images of primary panicle on weekly basis after the fertilization. These images were used to reconstruct a 3D point cloud of the panicle, which enabled us to extract digital traits such as voxel count and color intensity. We found that the voxel count of developing panicles is positively correlated with seed number and weight at maturity. The voxel count from developing panicles projected overall volumes that increased during the grain filling phase, wherein quantification of color intensity estimated the rate of panicle maturation. Our 3D based phenotyping solution showed superior performance compared to conventional 2D based approaches. CONCLUSIONS: For harnessing the potential of the existing genetic resources, we need a comprehensive understanding of the genotype-to-phenotype relationship. Relatively low-cost sequencing platforms have facilitated high-throughput genotyping, while phenotyping, especially for complex traits, has posed major challenges for crop improvement. PI-Plat offers a low cost and high-resolution platform to phenotype inflorescence-related traits using 3D reconstruction-based approach. Further, the non-destructive nature of the platform facilitates analyses of the same panicle at multiple developmental time points, which can be utilized to explore the genetic variation for dynamic inflorescence traits in cereals.

Proteomics survey of Solanaceae family: Current status and challenges ahead.

Solanaceae is one of the major economically important families of higher plants and has played a central role in human nutrition since the dawn of human civilization. Therefore, researchers have always been interested in understanding the complex behavior of Solanaceae members to identify key transcripts, proteins or metabolites, which are potentially associated with major traits. Proteomics studies have contributed significantly to understanding the physiology of Solanaceae members. A compilation of all the published reports showed that both gel-based (75%) and gel-free (25%) proteomic technologies have been utilized to establish the proteomes of different tissues, organs, and organelles under normal and adverse environmental conditions. Among the Solanaceae members, most of the research has been focused on tomato (42%) followed by potato (28%) and tobacco (20%), owing to their economic importance. This review comprehensively covers the progress made so far in the field of Solanaceae proteomics including novel methods developed to isolate the proteins from different tissues. Moreover, key proteins presented in this review can serve as a resource to select potential targets for crop improvement. We envisage that information presented in this review would enable us to design the stress tolerant plants with enhanced yields.

Protocol for Enrichment of the Membrane Proteome of Mature Tomato Pollen.

We established and elaborated on a method to enrich the membrane proteome of mature pollen from economically relevant crop using the example of Solanum lycopersicum (tomato). To isolate the pollen protein fraction enriched in membrane proteins, a high salt concentration (750 mM of sodium chloride) was used. The membrane protein-enriched fraction was then subjected to shotgun proteomics for identification of proteins, followed by in silico analysis to annotate and classify the detected proteins.

Importance of organellar proteins, protein translocation and vesicle transport routes for pollen development and function.

KEY MESSAGE: Protein translocation. Cellular homeostasis strongly depends on proper distribution of proteins within cells and insertion of membrane proteins into the destined membranes. The latter is mediated by organellar protein translocation and the complex vesicle transport system. Considering the importance of protein transport machineries in general it is foreseen that these processes are essential for pollen function and development. However, the information available in this context is very scarce because of the current focus on deciphering the fundamental principles of protein transport at the molecular level. Here we review the significance of protein transport machineries for pollen development on the basis of pollen-specific organellar proteins as well as of genetic studies utilizing mutants of known organellar proteins. In many cases these mutants exhibit morphological alterations highlighting the requirement of efficient protein transport and translocation in pollen. Furthermore, expression patterns of genes coding for translocon subunits and vesicle transport factors in Arabidopsis thaliana are summarized. We conclude that with the exception of the translocation systems in plastids-the composition and significance of the individual transport systems are equally important in pollen as in other cell types. Apparently for plastids only a minimal translocon, composed of only few subunits, exists in the envelope membranes during maturation of pollen. However, only one of the various transport systems known from thylakoids seems to be required for the function of the "simple thylakoid system" existing in pollen plastids. In turn, the vesicle transport system is as complex as seen for other cell types as it is essential, e.g., for pollen tube formation.

HsfA2 Controls the Activity of Developmentally and Stress-Regulated Heat Stress Protection Mechanisms in Tomato Male Reproductive Tissues.

Male reproductive tissues are more sensitive to heat stress (HS) compared to vegetative tissues, but the basis of this phenomenon is poorly understood. Heat stress transcription factors (Hsfs) regulate the transcriptional changes required for protection from HS In tomato (Solanum lycopersicum), HsfA2 acts as coactivator of HsfA1a and is one of the major Hsfs accumulating in response to elevated temperatures. The contribution of HsfA2 in heat stress response (HSR) and thermotolerance was investigated in different tissues of transgenic tomato plants with suppressed HsfA2 levels (A2AS). Global transcriptome analysis and immunodetection of two major Hsps in vegetative and reproductive tissues showed that HsfA2 regulates subsets of HS-induced genes in a tissue-specific manner. Accumulation of HsfA2 by a moderate HS treatment enhances the capacity of seedlings to cope with a subsequent severe HS, suggesting an important role for HsfA2 in regulating acquired thermotolerance. In pollen, HsfA2 is an important coactivator of HsfA1a during HSR HsfA2 suppression reduces the viability and germination rate of pollen that received the stress during the stages of meiosis and microspore formation but had no effect on more advanced stages. In general, pollen meiocytes and microspores are characterized by increased susceptibility to HS due to their lower capacity to induce a strong HSR This sensitivity is partially mitigated by the developmentally regulated expression of HsfA2 and several HS-responsive genes mediated by HsfA1a under nonstress conditions. Thereby, HsfA2 is an important factor for the priming process that sustains pollen thermotolerance during microsporogenesis.

The membrane proteome of male gametophyte in Solanum lycopersicum.

Pollen cells possess specialized cellular compartments separated by membranes. Consequently, mature pollen contains proteinaceous factors for inter- and intracellular transport of metabolites or ions to facilitate the upcoming energy exhausting processes - germination and fertilization. Despite the current advancement in the understanding of pollen development little is known about the role and molecular nature of the membrane proteome that participates in functioning and development of male gametophyte. We dissected the membrane proteome of mature pollen from economically important crop Solanum lycopersicum (tomato). Isolated membrane fractions from mature pollen of two tomato cultivars (cv. Moneymaker and cv. Red setter) were subjected to shotgun proteomics (GEL-LC-Orbitrap-MS). The global tomato protein assignment was achieved by mapping the peptides on reference genome (cv. Heinz 1706) and de novo assembled transcriptome based on mRNA sequencing from the respective cultivar. We identified 687 proteins, where 176 were assigned as putative membrane proteins. About 58% of the identified membrane proteins participate in transport processes. In depth analysis revealed proteins corresponding to energy related pathways (Glycolysis and Krebs cycle) as prerequisite for mature pollen, thereby revealing a reliable model of energy reservoir of the male gametophyte. BIOLOGICAL SIGNIFICANCE: Mature pollen plays an indispensable role in plant fertility and crop production. To decipher the functionality of pollen global proteomics studies have been undertaken. However, these datasets are deficient in membrane proteins due to their low abundance and solubility. The work presented here provides a comprehensive investigation of membrane proteome of male gametophyte of an agriculturally important crop plant tomato. The analysis of membrane enriched fractions from two tomato cultivars ensured an effective profiling of the pollen membrane proteome. Particularly proteins of the Krebs cycle or the glycolysis process have been detected and thus a model for the energy dynamics and preparedness of the male gametophyte for the upcoming events - germination and fertilization is provided.

Identification and Expression Analysis of Ribosome Biogenesis Factor Co-orthologs in Solanum lycopersicum.

Ribosome biogenesis involves a large inventory of proteinaceous and RNA cofactors. More than 250 ribosome biogenesis factors (RBFs) have been described in yeast. These factors are involved in multiple aspects like rRNA processing, folding, and modification as well as in ribosomal protein (RP) assembly. Considering the importance of RBFs for particular developmental processes, we examined the complexity of RBF and RP (co-)orthologs by bioinformatic assignment in 14 different plant species and expression profiling in the model crop Solanum lycopersicum. Assigning (co-)orthologs to each RBF revealed that at least 25% of all predicted RBFs are encoded by more than one gene. At first we realized that the occurrence of multiple RBF co-orthologs is not globally correlated to the existence of multiple RP co-orthologs. The transcript abundance of genes coding for predicted RBFs and RPs in leaves and anthers of S. lycopersicum was determined by next generation sequencing (NGS). In combination with existing expression profiles, we can conclude that co-orthologs of RBFs by large account for a preferential function in different tissue or at distinct developmental stages. This notion is supported by the differential expression of selected RBFs during male gametophyte development. In addition, co-regulated clusters of RBF and RP coding genes have been observed. The relevance of these results is discussed.