Puneet Paul

Perspectives on deciphering mechanisms underlying plant heat stress response and thermotolerance

Global warming is a major threat for agriculture and food safety and in many cases the negative effects are already apparent. The current challenge of basic and applied plant science is to decipher the molecular mechanisms of heat stress response (HSR) and thermotolerance in detail and use this information to identify genotypes that will withstand unfavorable environmental conditions. Nowadays X-omics approaches complement the findings of previous targeted studies and highlight the complexity of HSR mechanisms giving information for so far unrecognized genes, proteins and metabolites as potential key players of thermotolerance. Even more, roles of epigenetic mechanisms and the involvement of small RNAs in thermotolerance are currently emerging and thus open new directions of yet unexplored areas of plant HSR. In parallel it is emerging that although the whole plant is vulnerable to heat, specific organs are particularly sensitive to elevated temperatures. This has redirected research from the vegetative to generative tissues. The sexual reproduction phase is considered as the most sensitive to heat and specifically pollen exhibits the highest sensitivity and frequently an elevation of the temperature just a few degrees above the optimum during pollen development can have detrimental effects for crop production. Compared to our knowledge on HSR of vegetative tissues, the information on pollen is still scarce. Nowadays, several techniques for high-throughput X-omics approaches provide major tools to explore the principles of pollen HSR and thermotolerance mechanisms in specific genotypes. The collection of such information will provide an excellent support for improvement of breeding programs to facilitate the development of tolerant cultivars. The review aims at describing the current knowledge of thermotolerance mechanisms and the technical advances which will foster new insights into this process.

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.

Chaperone network composition in Solanum lycopersicum explored by transcriptome profiling and microarray meta‐analysis

Heat shock proteins (Hsps) are molecular chaperones primarily involved in maintenance of protein homeostasis. Their function has been best characterized in heat stress (HS) response during which Hsps are transcriptionally controlled by HS transcription factors (Hsfs). The role of Hsfs and Hsps in HS response in tomato was initially examined by transcriptome analysis using the massive analysis of cDNA ends (MACE) method. Approximately 9.6% of all genes expressed in leaves are enhanced in response to HS, including a subset of Hsfs and Hsps. The underlying Hsp-Hsf networks with potential functions in stress responses or developmental processes were further explored by meta-analysis of existing microarray datasets. We identified clusters with differential transcript profiles with respect to abiotic stresses, plant organs and developmental stages. The composition of two clusters points towards two major chaperone networks. One cluster consisted of constitutively expressed plastidial chaperones and other genes involved in chloroplast protein homeostasis. The second cluster represents genes strongly induced by heat, drought and salinity stress, including HsfA2 and many stress-inducible chaperones, but also potential targets of HsfA2 not related to protein homeostasis. This observation attributes a central regulatory role to HsfA2 in controlling different aspects of abiotic stress response and tolerance in tomato.

HsfA2 controls the activity of developmentally and stress-regulated heat stress protection mechanisms in tomato male reproductive tissues

A heat stress transcription factor is involved in tomato pollen thermotolerance, contributing to stress responses as well as the abundance of chaperones in the priming program activated during microsporogenesis. 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 complexity of vesicle transport factors in plants examined by orthology search.

Vesicle transport is a central process to ensure protein and lipid distribution in eukaryotic cells. The current knowledge on the molecular components and mechanisms of this process is majorly based on studies in Saccharomyces cerevisiae and Arabidopsis thaliana, which revealed 240 different proteinaceous factors either experimentally proven or predicted to be involved in vesicle transport. In here, we performed an orthologue search using two different algorithms to identify the components of the secretory pathway in yeast and 14 plant genomes by using the ‘core-set’ of 240 factors as bait. We identified 4021 orthologues and (co-)orthologues in the discussed plant species accounting for components of COP-II, COP-I, Clathrin Coated Vesicles, Retromers and ESCRTs, Rab GTPases, Tethering factors and SNAREs. In plants, we observed a significantly higher number of (co-)orthologues than yeast, while only 8 tethering factors from yeast seem to be absent in the analyzed plant genomes. To link the identified (co-)orthologues to vesicle transport, the domain architecture of the proteins from yeast, genetic model plant A. thaliana and agriculturally relevant crop Solanum lycopersicum has been inspected. For the orthologous groups containing (co-)orthologues from yeast, A. thaliana and S. lycopersicum, we observed the same domain architecture for 79% (416/527) of the (co-)orthologues, which documents a very high conservation of this process. Further, publically available tissue-specific expression profiles for a subset of (co-)orthologues found in A. thaliana and S. lycopersicum suggest that some (co-)orthologues are involved in tissue-specific functions. Inspection of localization of the (co-)orthologues based on available proteome data or localization predictions lead to the assignment of plastid- as well as mitochondrial localized (co-)orthologues of vesicle transport factors and the relevance of this is discussed.

The protein translocation systems in plants – composition and variability on the example of Solanum lycopersicum

BackgroundProtein translocation across membranes is a central process in all cells. In the past decades the molecular composition of the translocation systems in the membranes of the endoplasmic reticulum, peroxisomes, mitochondria and chloroplasts have been established based on the analysis of model organisms. Today, these results have to be transferred to other plant species. We bioinformatically determined the inventory of putative translocation factors in tomato (Solanum lycopersicum) by orthologue search and domain architecture analyses. In addition, we investigated the diversity of such systems by comparing our findings to the model organisms Saccharomyces cerevisiae, Arabidopsis thaliana and 12 other plant species.ResultsThe literature search end up in a total of 130 translocation components in yeast and A. thaliana, which are either experimentally confirmed or homologous to experimentally confirmed factors. From our bioinformatic analysis (PGAP and OrthoMCL), we identified (co-)orthologues in plants, which in combination yielded 148 and 143 orthologues in A. thaliana and S. lycopersicum, respectively. Interestingly, we traced 82% overlap in findings from both approaches though we did not find any orthologues for 27% of the factors by either procedure. In turn, 29% of the factors displayed the presence of more than one (co-)orthologue in tomato. Moreover, our analysis revealed that the genomic composition of the translocation machineries in the bryophyte Physcomitrella patens resemble more to higher plants than to single celled green algae. The monocots (Z. mays and O. sativa) follow more or less a similar conservation pattern for encoding the translocon components. In contrast, a diverse pattern was observed in different eudicots.ConclusionsThe orthologue search shows in most cases a clear conservation of components of the translocation pathways/machineries. Only the Get-dependent integration of tail-anchored proteins seems to be distinct. Further, the complexity of the translocation pathway in terms of existing orthologues seems to vary among plant species. This might be the consequence of palaeoploidisation during evolution in plants; lineage specific whole genome duplications in Arabidopsis thaliana and triplications in Solanum lycopersicum.

The membrane proteome of male gametophyte in Solanum lycopersicum

UNLABELLED 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.

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.

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

Key messageProtein translocation.Abstract 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.

Plant Heat Stress Response and Thermotolerance

Climate change and global warming are considered to be major threats for agricultural production and food safety. Decreased yield of several important crops has already been related to frequently occurring extreme environmental conditions such as heat waves. Since most of the economically and dietary important crops are sensitive to high temperatures, the development of cultivars that can withstand adverse temperatures is a prerequisite for meeting the demands for increased food production. The processes of sensing and responding to heat are complex phenomena in plants which comprise the activation of numerous regulatory and signaling pathways that eventually lead to a fine metabolic adjustment to ensure cell survival. Currently, our knowledge of heat stress response is greatly advanced by the massive production of datasets derived from -omics studies which supplement the current models with new genes, proteins, and metabolites or even introduce whole new pathways. This information is essential for the improvement of plant thermotolerance either through breeding programs or approaches using genetic engineering. This chapter contains an assembly of several aspects regarding heat stress response and thermotolerance. The effects of high temperatures on major aspects of plant growth and development are described, and different methods for thermotolerance screening are presented. In addition, putative heat sensing mechanisms are discussed and the most important metabolic changes are elaborated. Last, a summary of efforts and strategies to improve thermotolerance by breeding or genetic engineering is given.