Genetic and physiological requirements for high-level sesquiterpene-production in tomato glandular trichomes.

Type-VI glandular trichomes of wild tomato Solanum habrochaites PI127826 produce high levels of the sesquiterpene 7-epizingiberene and its derivatives, making the plant repellent and toxic to several pest insects and pathogens. How wild tomato trichomes achieve such high terpene production is still largely unknown. Here we show that a cross (F1) with a cultivated tomato produced only minute levels of 7-epizingiberene. In the F2-progeny, selected for the presence of the 7-epizingiberene biosynthesis genes, only three percent produced comparable amounts the wild parent, indicating this trait is recessive and multigenic. Moreover, trichome density alone did not explain the total levels of terpene levels found on the leaves. We selected F2 plants with the "high-production active-trichome phenotype" of PI127826, having trichomes producing about 150 times higher levels of terpenes than F2 individuals that displayed a "low-production lazy-trichome phenotype". Terpene quantities in trichomes of these F2 plants correlated with the volume of the storage cavity and shape of the gland. We found that trichome morphology is not a predetermined characteristic, but cavity volume rather depended on gland-cell metabolic activity. Inhibitor assays showed that the plastidial-precursor pathway (MEP) is fundamental for high-level production of both cytosolic as well as plastid-derived terpenes in tomato trichomes. Additionally, gene expression profiles of isolated secretory cells showed that key enzymes in the MEP pathway were higher expressed in active trichomes. We conclude that the MEP pathway is the primary precursor-supply route in wild tomato type-VI trichomes and that the high-production phenotype of the wild tomato trichome is indeed a multigenic trait.

Multi-Omics Approaches Unravel Specific Features of Embryo and Endosperm in Rice Seed Germination.

Seed germination and subsequent seedling growth affect the final yield and quality of the crop. Seed germination is defined as a series of processes that begins with water uptake by a quiescent dry seed and ends with the elongation of embryonic axis. Rice is an important cereal crop species, and during seed germination, two tissues function in a different manner; the embryo grows into a seedling as the next generation and the endosperm is responsible for nutritional supply. Toward understanding the integrated roles of each tissue at the transcriptional, translational, and metabolic production levels during germination, an exhaustive "multi-omics" analysis was performed by combining transcriptomics, label-free shotgun proteomics, and metabolomics on rice germinating embryo and endosperm, independently. Time-course analyses of the transcriptome and metabolome in germinating seeds revealed a major turning point in the early phase of germination in both embryo and endosperm, suggesting that dramatic changes begin immediately after water imbibition in the rice germination program at least at the mRNA and metabolite levels. In endosperm, protein profiles mostly showed abundant decreases corresponding to 90% of the differentially accumulated proteins. An ontological classification revealed the shift from the maturation to the germination process where over-represented classes belonged to embryonic development and cellular amino acid biosynthetic processes. In the embryo, 19% of the detected proteins are differentially accumulated during germination. Stress response, carbohydrate, fatty acid metabolism, and transport are the main functional classes representing embryo proteome change. Moreover, proteins specific to the germinated state were detected by both transcriptomic and proteomic approaches and a major change in the network operating during rice germination was uncovered. In particular, concomitant changes of hormonal metabolism-related proteins (GID1L2 and CNX1) implicated in GAs and ABA metabolism, signaling proteins, and protein turnover events emphasized the importance of such biological networks in rice seeds. Using metabolomics, we highlighted the importance of an energetic supply in rice seeds during germination. In both embryo and endosperm, starch degradation, glycolysis, and subsequent pathways related to these cascades, such as the aspartate-family pathway, are activated during germination. A relevant number of accumulated proteins and metabolites, especially in embryos, testifies the pivotal role of energetic supply in the preparation of plant growth. This article summarizes the key genetic pathways in embryo and endosperm during rice seed germination at the transcriptional, translational, and metabolite levels and thereby, emphasizes the value of combined multi-omics approaches to uncover the specific feature of tissues during germination.

PlotXpress, a webtool for normalization and visualization of reporter expression data.

In molecular cell biology, reporter assays are frequently used to investigate gene expression levels. Reporter assays employ a gene that encodes a light-emitting protein, of which the luminescence is quantified as a proxy of gene expression. Commercial parties provide reporter assay kits that include protocols and specialized detection machinery. However, downstream analysis of the output data and their presentation are not standardized. We have developed plotXpress to fill this gap, providing a free, open-source platform for the semi-automated analysis and standardized visualisation of experimental gene reporter data. Users can upload raw luminescence data acquired from a reporter gene assay with an internal control. In plotXpress, the data is corrected for sample variation with the internal control and the average for each condition is calculated. When a reference condition is selected the fold change is calculated for all other conditions, based on the selected reference. The results are shown as dot plots with a statistical summary, which can be adjusted to create publication-grade plots without requiring coding skills. Altogether, plotXpress is an open-source, low-threshold, web-based tool, that promotes a standardized and reproducible analysis while providing an appealing visualization of reporter data. The webtool can be accessed at: https://huygens.science.uva.nl/PlotXpress/.

Natural variation in wild tomato trichomes; selecting metabolites that contribute to insect resistance using a random forest approach.

BACKGROUND: Plant-produced specialised metabolites are a powerful part of a plant's first line of defence against herbivorous insects, bacteria and fungi. Wild ancestors of present-day cultivated tomato produce a plethora of acylsugars in their type-I/IV trichomes and volatiles in their type-VI trichomes that have a potential role in plant resistance against insects. However, metabolic profiles are often complex mixtures making identification of the functionally interesting metabolites challenging. Here, we aimed to identify specialised metabolites from a wide range of wild tomato genotypes that could explain resistance to vector insects whitefly (Bemisia tabaci) and Western flower thrips (Frankliniella occidentalis). We evaluated plant resistance, determined trichome density and obtained metabolite profiles of the glandular trichomes by LC-MS (acylsugars) and GC-MS (volatiles). Using a customised Random Forest learning algorithm, we determined the contribution of specific specialised metabolites to the resistance phenotypes observed. RESULTS: The selected wild tomato accessions showed different levels of resistance to both whiteflies and thrips. Accessions resistant to one insect can be susceptible to another. Glandular trichome density is not necessarily a good predictor for plant resistance although the density of type-I/IV trichomes, related to the production of acylsugars, appears to correlate with whitefly resistance. For type VI-trichomes, however, it seems resistance is determined by the specific content of the glands. There is a strong qualitative and quantitative variation in the metabolite profiles between different accessions, even when they are from the same species. Out of 76 acylsugars found, the random forest algorithm linked two acylsugars (S3:15 and S3:21) to whitefly resistance, but none to thrips resistance. Out of 86 volatiles detected, the sesquiterpene α-humulene was linked to whitefly susceptible accessions instead. The algorithm did not link any specific metabolite to resistance against thrips, but monoterpenes α-phellandrene, α-terpinene and ß-phellandrene/D-limonene were significantly associated with susceptible tomato accessions. CONCLUSIONS: Whiteflies and thrips are distinctly targeted by certain specialised metabolites found in wild tomatoes. The machine learning approach presented helped to identify features with efficacy toward the insect species studied. These acylsugar metabolites can be targets for breeding efforts towards the selection of insect-resistant cultivars.

ZNF91 deletion in human embryonic stem cells leads to ectopic activation of SVA retrotransposons and up-regulation of KRAB zinc finger gene clusters.

Transposable element (TE) invasions have shaped vertebrate genomes over the course of evolution. They have contributed an extra layer of species-specific gene regulation by providing novel transcription factor binding sites. In humans, SINE-VNTR-Alu (SVA) elements are one of three still active TE families; approximately 2800 SVA insertions exist in the human genome, half of which are human-specific. TEs are often silenced by KRAB zinc finger (KZNF) proteins recruiting corepressor proteins that establish a repressive chromatin state. A number of KZNFs have been reported to bind SVAs, but their individual contribution to repressing SVAs and their roles in suppressing SVA-mediated gene-regulatory effects remains elusive. We analyzed the genome-wide binding profile for ZNF91 in human cells and found that ZNF91 interacts with the VNTR region of SVAs. Through CRISPR-Cas9-mediated deletion of ZNF91 in human embryonic stem cells, we established that loss of ZNF91 results in increased transcriptional activity of SVAs. In contrast, SVA activation was not observed upon genetic deletion of the ZNF611 gene encoding another strong SVA interactor. Epigenetic profiling confirmed the loss of SVA repression in the absence of ZNF91 and revealed that mainly evolutionary young SVAs gain gene activation-associated epigenetic modifications. Genes close to activated SVAs showed a mild up-regulation, indicating SVAs adopt properties of cis-regulatory elements in the absence of repression. Notably, genome-wide derepression of SVAs elicited the communal up-regulation of KZNFs that reside in KZNF clusters. This phenomenon may provide new insights into the potential mechanisms used by the host genome to sense and counteract TE invasions.

Building a local community of practice in scientific programming for life scientists.

In this paper, we describe why and how to build a local community of practice in scientific programming for life scientists who use computers and programming in their research. A community of practice is a small group of scientists who meet regularly to help each other and promote good practices in scientific programming. While most life scientists are well trained in the laboratory to conduct experiments, good practices with (big) data sets and their analysis are often missing. We propose a model on how to build such a community of practice at a local academic institution, present two real-life examples, and introduce challenges and implemented solutions. We believe that the current data deluge that life scientists face can benefit from the implementation of these small communities. Good practices spread among experimental scientists will foster open, transparent, and sound scientific results beneficial to society.

An Integrated "Multi-Omics" Comparison of Embryo and Endosperm Tissue-Specific Features and Their Impact on Rice Seed Quality.

Although rice is a key crop species, few studies have addressed both rice seed physiological and nutritional quality, especially at the tissue level. In this study, an exhaustive "multi-omics" dataset on the mature rice seed was obtained by combining transcriptomics, label-free shotgun proteomics and metabolomics from embryo and endosperm, independently. These high-throughput analyses provide a new insight on the tissue-specificity related to rice seed quality. Foremost, we pinpointed that extensive post-transcriptional regulations occur at the end of rice seed development such that the embryo proteome becomes much more diversified than the endosperm proteome. Secondly, we observed that survival in the dry state in each seed compartment depends on contrasted metabolic and enzymatic apparatus in the embryo and the endosperm, respectively. Thirdly, it was remarkable to identify two different sets of starch biosynthesis enzymes as well as seed storage proteins (glutelins) in both embryo and endosperm consistently with the supernumerary embryo hypothesis origin of the endosperm. The presence of a putative new glutelin with a possible embryonic favored abundance is described here for the first time. Finally, we quantified the rate of mRNA translation into proteins. Consistently, the embryonic panel of protein translation initiation factors is much more diverse than that of the endosperm. This work emphasizes the value of tissue-specificity-centered "multi-omics" study in the seed to highlight new features even from well-characterized pathways. It paves the way for future studies of critical genetic determinants of rice seed physiological and nutritional quality.

A Combination of Histological, Physiological, and Proteomic Approaches Shed Light on Seed Desiccation Tolerance of the Basal Angiosperm Amborella trichopoda.

Desiccation tolerance allows plant seeds to remain viable in a dry state for years and even centuries. To reveal potential evolutionary processes of this trait, we have conducted a shotgun proteomic analysis of isolated embryo and endosperm from mature seeds of Amborella trichopoda, an understory shrub endemic to New Caledonia that is considered to be the basal extant angiosperm. The present analysis led to the characterization of 415 and 69 proteins from the isolated embryo and endosperm tissues, respectively. The role of these proteins is discussed in terms of protein evolution and physiological properties of the rudimentary, underdeveloped, Amborella embryos, notably considering that the acquisition of desiccation tolerance corresponds to the final developmental stage of mature seeds possessing large embryos.

Small RNAs from Bemisia tabaci Are Transferred to Solanum lycopersicum Phloem during Feeding.

The phloem-feeding whitefly Bemisia tabaci is a serious pest to a broad range of host plants, including many economically important crops such as tomato. These insects serve as a vector for various devastating plant viruses. It is known that whiteflies are capable of manipulating host-defense responses, potentially mediated by effector molecules in the whitefly saliva. We hypothesized that, beside putative effector proteins, small RNAs (sRNA) are delivered by B. tabaci into the phloem, where they may play a role in manipulating host plant defenses. There is already evidence to suggest that sRNAs can mediate the host-pathogen dialogue. It has been shown that Botrytis cinerea, the causal agent of gray mold disease, takes advantage of the plant sRNA machinery to selectively silence host genes involved in defense signaling. Here we identified sRNAs originating from B. tabaci in the phloem of tomato plants on which they are feeding. sRNAs were isolated and sequenced from tomato phloem of whitefly-infested and control plants as well as from the nymphs themselves, control leaflets, and from the infested leaflets. Using stem-loop RT-PCR, three whitefly sRNAs have been verified to be present in whitefly-infested leaflets that were also present in the whitefly-infested phloem sample. Our results show that whitefly sRNAs are indeed present in tomato tissues upon feeding, and they appear to be mobile in the phloem. Their role in the host-insect interaction can now be investigated.

Compartmentation and dynamics of flavone metabolism in dry and germinated rice seeds.

Among secondary metabolites, flavonoids are particularly important for the plant life cycle and could be beneficial for human health. The study of Arabidopsis thaliana transparent testa mutants showed that seed flavonoids are important for environmental adaptation, reactive oxygen species homeostasis, dormancy and longevity. Compared with Arabidopsis and maize (Zea mays L.), far less research has been conducted on rice (Oryza sativa L.) particularly for cultivars with non-pigmented seeds. In this study, we describe the localization, nature and relative abundance of flavonoids in mature and germinated non-pigmented Nipponbare seeds using a combination of confocal microscopy, mass spectrometry and gene expression analysis. The mature seed exclusively accumulates flavones mostly in the embryo and to a lesser extent in the pericarp/testa. Due to the variety of flavone conjugation patterns, 21 different flavones were identified, including sulfated flavones never mentioned before in cereals. Schaftoside (apigenin-6-C-glucoside-8-C-arabinoside) and its two isomers represent nearly 50% of all rice seed flavones and are the only flavonoids accumulated in the pericarp/testa seed compartment. These 21 conjugated flavones showed a very stable profile during rice seed germination sensu stricto, while expression of key flavone synthesis genes strongly increases before the completion of germination. We discuss the potential roles of these rice seed flavones in a seed biology context.

Dynamic proteomics emphasizes the importance of selective mRNA translation and protein turnover during Arabidopsis seed germination.

During seed germination, the transition from a quiescent metabolic state in a dry mature seed to a proliferative metabolic state in a vigorous seedling is crucial for plant propagation as well as for optimizing crop yield. This work provides a detailed description of the dynamics of protein synthesis during the time course of germination, demonstrating that mRNA translation is both sequential and selective during this process. The complete inhibition of the germination process in the presence of the translation inhibitor cycloheximide established that mRNA translation is critical for Arabidopsis seed germination. However, the dynamics of protein turnover and the selectivity of protein synthesis (mRNA translation) during Arabidopsis seed germination have not been addressed yet. Based on our detailed knowledge of the Arabidopsis seed proteome, we have deepened our understanding of seed mRNA translation during germination by combining two-dimensional gel-based proteomics with dynamic radiolabeled proteomics using a radiolabeled amino acid precursor, namely [(35)S]-methionine, in order to highlight de novo protein synthesis, stability, and turnover. Our data confirm that during early imbibition, the Arabidopsis translatome keeps reflecting an embryonic maturation program until a certain developmental checkpoint. Furthermore, by dividing the seed germination time lapse into discrete time windows, we highlight precise and specific patterns of protein synthesis. These data refine and deepen our knowledge of the three classical phases of seed germination based on seed water uptake during imbibition and reveal that selective mRNA translation is a key feature of seed germination. Beyond the quantitative control of translational activity, both the selectivity of mRNA translation and protein turnover appear as specific regulatory systems, critical for timing the molecular events leading to successful germination and seedling establishment.

Nitric oxide implication in the control of seed dormancy and germination.

Germination ability is regulated by a combination of environmental and endogenous signals with both synergistic and antagonistic effects. Nitric oxide (NO) is a potent dormancy-releasing agent in many species, including Arabidopsis, and has been suggested to behave as an endogenous regulator of this physiological blockage. Distinct reports have also highlighted a positive impact of NO on seed germination under sub-optimal conditions. However, its molecular mode of action in the context of seed biology remains poorly documented. This review aims to focus on the implications of this radical in the control of seed dormancy and germination. The consequences of NO chemistry on the investigations on both its signaling and its targets in seeds are discussed. NO-dependent protein post-translational modifications are proposed as a key mechanism underlying NO signaling during early seed germination.

The seed proteome web portal.

The Seed Proteome Web Portal (SPWP; http://www.seed-proteome.com/) gives access to information both on quantitative seed proteomic data and on seed-related protocols. Firstly, the SPWP provides access to the 475 different Arabidopsis seed proteins annotated from two dimensional electrophoresis (2DE) maps. Quantitative data are available for each protein according to their accumulation profile during the germination process. These proteins can be retrieved either in list format or directly on scanned 2DE maps. These proteomic data reveal that 40% of seed proteins maintain a stable abundance over germination, up to radicle protrusion. During sensu stricto germination (24 h upon imbibition) about 50% of the proteins display quantitative variations, exhibiting an increased abundance (35%) or a decreasing abundance (15%). Moreover, during radicle protrusion (24-48 h upon imbibition), 41% proteins display quantitative variations with an increased (23%) or a decreasing abundance (18%). In addition, an analysis of the seed proteome revealed the importance of protein post-translational modifications as demonstrated by the poor correlation (r(2) = 0.29) between the theoretical (predicted from Arabidopsis genome) and the observed protein isoelectric points. Secondly, the SPWP is a relevant technical resource for protocols specifically dedicated to Arabidopsis seed proteome studies. Concerning 2D electrophoresis, the user can find efficient procedures for sample preparation, electrophoresis coupled with gel analysis, and protein identification by mass spectrometry, which we have routinely used during the last 12 years. Particular applications such as the detection of oxidized proteins or de novo synthesized proteins radiolabeled by [(35)S]-methionine are also given in great details. Future developments of this portal will include proteomic data from studies such as dormancy release and protein turnover through de novo protein synthesis analyses during germination.

The ethylene pathway contributes to root hair elongation induced by the beneficial bacteria Phyllobacterium brassicacearum STM196.

In Arabidopsis roots, some epidermal cells differentiate into root hair cells. Auxin regulates root hair positioning, while ethylene controls cell elongation. Phyllobacterium brassicacearum STM196, a beneficial strain of plant growth promoting rhizobacteria (PGPR) isolated from the roots of field-grown oilseed rape, stimulates root hair elongation in Arabidopsis thaliana seedlings. We investigated the role of ethylene in the response of root hair cells to STM196 inoculation. While we could not detect a significant increase in ethylene biosynthesis, we could detect a slight activation of the ethylene signalling pathway. Consistent with this, an exhaustive survey of the root hair elongation response of mutants and transgenic lines affected in the ethylene pathway showed contrasting root hair sensitivities to STM196. We propose that local ethylene emission contributes to STM196-induceed root hair elongation.

Reboot the system thanks to protein post-translational modifications and proteome diversity: How quiescent seeds restart their metabolism to prepare seedling establishment.

Once liberated in their environment, orthodox seeds live in a quiescent dehydrated state not totally exempt of essential molecular events as, for example, the capacity of breaking dormancy during after-ripening. Upon imbibition, if internal regulatory padlocks are released and given adequate external conditions, the quiescent seed is able to "reboot" its system and, thus, germinate. Recent studies unraveled the crucial importance of protein PTMs in seed dormancy, longevity and vigor. As compared to other plant developmental stages, the seed proteome appears quite unique and diverse. Seed proteins encompass several functional classes from primary and secondary metabolism to structural and antimicrobial defense. In the dry state, oxidative damages can occur due to reactive oxygen and nitrogen species produced by non-enzymatic reactions. These reactive species can affect proteins by the oxidation of their amino acids in a post-translational manner. The hormone abscisic acid regulates major aspects of seed life including dormancy and germination. This signaling pathway has been shown to rely on several PTMs such as protein phosphorylation or ubiquitination.