Arabidopsis transcriptome dataset of the response of imbibed wild-type and glucosinolate-deficient seeds to nitrogen-containing compounds.

The presented RNAseq data were obtained from Arabidopsis seeds dry and 6h imbibed to describe, in wild-type and glucosinolate (GSL)-deficient genotypes, the response at the RNA level to nitrogen compounds, i.e., potassium nitrate (KNO3, 10mM), potassium thiocyanate (KSCN, 8µM). The cyp79B2 cyp79B3 (cyp79B2/B3) double mutant deficient in Indole GSL, the myb28 myb29 (myb28/29) double mutant deficient in aliphatic GSL, the quadruple mutant cyp79B2 cyp79B3 myb28 myb29 (qko) deficient in total GSL in the seed and the WT reference genotype in Col-0 background were used for the transcriptomic analysis. Total ARN was extracted using NucleoSpin® RNA Plant and Fungi kit. Library construction and sequencing were performed with DNBseq™ technology at Beijing Genomics Institute. FastQC was used to check reads quality and mapping analysis were made using a quasi-mapping alignment from Salmon. Gene expression changes in mutant seeds compared to WT were calculated using DESeq2 algorithms. This comparison with the qko, cyp79B2/B3 and myb28/29 mutants made it possible to identify 30220, 36885 and 23807 differentially expressed genes (DEGs), respectively. Mapping rate result was merge into a single report using MultiQC; graphic results were illustrated through Veen diagrams and volcano plots. Fastq raw data and count files from 45 samples are available in the repository Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI) and can be consulted with the data identification number GSE221567 at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE221567.

Glucose-6-Phosphate Dehydrogenases: The Hidden Players of Plant Physiology.

Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes a metabolic hub between glycolysis and the pentose phosphate pathway (PPP), which is the oxidation of glucose-6-phosphate (G6P) to 6-phosphogluconolactone concomitantly with the production of nicotinamide adenine dinucleotide phosphate (NADPH), a reducing power. It is considered to be the rate-limiting step that governs carbon flow through the oxidative pentose phosphate pathway (OPPP). The OPPP is the main supplier of reductant (NADPH) for several "reducing" biosynthetic reactions. Although it is involved in multiple physiological processes, current knowledge on its exact role and regulation is still piecemeal. The present review provides a concise and comprehensive picture of the diversity of plant G6PDHs and their role in seed germination, nitrogen assimilation, plant branching, and plant response to abiotic stress. This work will help define future research directions to improve our knowledge of G6PDHs in plant physiology and to integrate this hidden player in plant performance.

Dual-transcriptomic datasets evaluating the effect of the necrotrophic fungus Alternaria brassicicola on Arabidopsis germinating seeds.

Many fungal pathogens are carried and transmitted by seeds. These pathogens affect germination and seed quality. Their transmission from the germinating seed to seedling causes many diseases in crops. Seed defense mechanisms during germination are poorly documented. RNA-seq experiments were used to describe the molecular mechanisms involved in seed interaction with a necrotrophic fungus. Here the Arabidopsis thaliana/Alternaria brassicicola pathosystem was used to perform dual-transcriptomic approach. Arabidopsis thaliana seeds and necrotrophic fungus transcripts were identified at critical germination and seedling establishment stages. Total RNA was extracted from healthy and infected germinating seeds and seedlings at 3, 6 and 10 days after sowing. Transcript libraries were made and sequenced, then fungal and plant short reads were mapped and quantified respectively against Arabidopsis thaliana and Alternaria brassicicola reference transcriptomes. This dual-transcriptomic approach revealed that 3409, 7506 and 8589 Arabidopsis thaliana genes showed a differential expression at respectevely 3, 6 and 10 days after sowing between healthy and infected seeds, including 1192 genes differentially expressed at the three studied stages. Moreover, in this experiement, we also identified the dynamic of the transcript changes occurring at the same stages in the necrotrophic fungus concomitantly during germination and seedling establishment.

Seed Transmission of Pathogens: Non-Canonical Immune Response in Arabidopsis Germinating Seeds Compared to Early Seedlings against the Necrotrophic Fungus Alternaria brassicicola.

The transmission of seed-borne pathogens by the germinating seed is responsible for major crop diseases. The immune responses of the seed facing biotic invaders are poorly documented so far. The Arabidopsis thaliana/Alternaria brassicicola patho-system was used to describe at the transcription level the responses of germinating seeds and young seedling stages to infection by the necrotrophic fungus. RNA-seq analyses of healthy versus inoculated seeds at 3 days after sowing (DAS), stage of radicle emergence, and at 6 and 10 DAS, two stages of seedling establishment, identified thousands of differentially expressed genes by Alternaria infection. Response to hypoxia, ethylene and indole pathways were found to be induced by Alternaria in the germinating seeds. However, surprisingly, the defense responses, namely the salicylic acid (SA) pathway, the response to reactive oxygen species (ROS), the endoplasmic reticulum-associated protein degradation (ERAD) and programmed cell death, were found to be strongly induced only during the latter post-germination stages. We propose that this non-canonical immune response in early germinating seeds compared to early seedling establishment was potentially due to the seed-to-seedling transition phase. Phenotypic analyses of about 14 mutants altered in the main defense pathways illustrated these specific defense responses. The unexpected germination deficiency and insensitivity to Alternaria in the glucosinolate deficient mutants allow hypothesis of a trade-off between seed germination, necrosis induction and Alternaria transmission to the seedling. The imbalance of the SA and jasmonic acid (JA) pathways to the detriment of the JA also illustrated a non-canonical immune response at the first stages of the seedling.

The peptide SCOOP12 acts on reactive oxygen species homeostasis to modulate cell division and elongation in Arabidopsis primary root.

Small secreted peptides have been described as key contributors to complex signalling networks that control plant development and stress responses. The Brassicaceae-specific PROSCOOP family encodes precursors of Serine riCh endOgenOus Peptides (SCOOPs). In Arabidopsis SCOOP12 has been shown to promote the defence response against pathogens and to be involved in root development. Here, we explore its role as a moderator of Arabidopsis primary root development. We show that the PROSCOOP12 null mutation leads to longer primary roots through the development of longer differentiated cells while PROSCOOP12 overexpression induces dramatic plant growth impairments. In comparison, the exogenous application of synthetic SCOOP12 peptide shortens roots through meristem size and cell length reductions. Moreover, superoxide anion (O2·-) and hydrogen peroxide (H2O2) production in root tips vary according to SCOOP12 abundance. By using reactive oxygen species scavengers that suppress the proscoop12 phenotype, we showed that root growth regulation by SCOOP12 is associated with reactive oxygen species metabolism. Furthermore, our results suggest that peroxidases act as potential SCOOP12 downstream targets to regulate H2O2 production, which in turn triggers cell wall modifications in root. Finally, a massive transcriptional reprogramming, including the induction of genes from numerous other pathways, including ethylene, salicylic acid, and glucosinolates biosynthesis, was observed, emphasizing its dual role in defence and development.

Outgrowth of the axillary bud in rose is controlled by sugar metabolism and signalling.

Shoot branching is a pivotal process during plant growth and development, and is antagonistically orchestrated by auxin and sugars. In contrast to extensive investigations on hormonal regulatory networks, our current knowledge on the role of sugar signalling pathways in bud outgrowth is scarce. Based on a comprehensive stepwise strategy, we investigated the role of glycolysis/the tricarboxylic acid (TCA) cycle and the oxidative pentose phosphate pathway (OPPP) in the control of bud outgrowth. We demonstrated that these pathways are necessary for bud outgrowth promotion upon plant decapitation and in response to sugar availability. They are also targets of the antagonistic crosstalk between auxin and sugar availability. The two pathways act synergistically to down-regulate the expression of BRC1, a conserved inhibitor of shoot branching. Using Rosa calluses stably transformed with GFP-fused promoter sequences of RhBRC1 (pRhBRC1), glycolysis/TCA cycle and the OPPP were found to repress the transcriptional activity of pRhBRC1 cooperatively. Glycolysis/TCA cycle- and OPPP-dependent regulations involve the -1973/-1611 bp and -1206/-709 bp regions of pRhBRC1, respectively. Our findings indicate that glycolysis/TCA cycle and the OPPP are integrative parts of shoot branching control and can link endogenous factors to the developmental programme of bud outgrowth, likely through two distinct mechanisms.

Convergence and Divergence of Sugar and Cytokinin Signaling in Plant Development.

Plants adjust their growth and development through a sophisticated regulatory system integrating endogenous and exogenous cues. Many of them rely on intricate crosstalk between nutrients and hormones, an effective way of coupling nutritional and developmental information and ensuring plant survival. Sugars in their different forms such as sucrose, glucose, fructose and trehalose-6-P and the hormone family of cytokinins (CKs) are major regulators of the shoot and root functioning throughout the plant life cycle. While their individual roles have been extensively investigated, their combined effects have unexpectedly received little attention, resulting in many gaps in current knowledge. The present review provides an overview of the relationship between sugars and CKs signaling in the main developmental transition during the plant lifecycle, including seed development, germination, seedling establishment, root and shoot branching, leaf senescence, and flowering. These new insights highlight the diversity and the complexity of the crosstalk between sugars and CKs and raise several questions that will open onto further investigations of these regulation networks orchestrating plant growth and development.

ScreenSeed as a novel high throughput seed germination phenotyping method.

A high throughput phenotyping tool for seed germination, the ScreenSeed technology, was developed with the aim of screening genotype responsiveness and chemical drugs. This technology was presently used with Arabidopsis thaliana seeds to allow characterizing seed samples germination behavior by incubating seeds in 96-well microplates under defined conditions and detecting radicle protrusion through the seed coat by automated image analysis. This study shows that this technology provides a fast procedure allowing to handle thousands of seeds without compromising repeatability or accuracy of the germination measurements. Potential biases of the experimental protocol were assessed through statistical analyses of germination kinetics. Comparison of the ScreenSeed procedure with commonly used germination tests based upon visual scoring displayed very similar germination kinetics.

Posttranscriptional Regulation of RhBRC1 (Rosa hybrida BRANCHED1) in Response to Sugars is Mediated via its Own 3' Untranslated Region, with a Potential Role of RhPUF4 (Pumilio RNA-Binding Protein Family).

The shoot branching pattern is a determining phenotypic trait throughout plant development. During shoot branching, BRANCHED1 (BRC1) plays a master regulator role in bud outgrowth, and its transcript levels are regulated by various exogenous and endogenous factors. RhBRC1 (the homologous gene of BRC1 in Rosa hybrida) is a main branching regulator whose posttranscriptional regulation in response to sugar was investigated through its 3'UTR. Transformed Rosa calluses containing a construction composed of the CaMV35S promoter, the green fluorescent protein (GFP) reporter gene, and the 3'UTR of RhBRC1 (P35S:GFP::3'UTRRhBRC1) were obtained and treated with various combinations of sugars and with sugar metabolism effectors. The results showed a major role of the 3'UTR of RhBRC1 in response to sugars, involving glycolysis/the tricarboxylic acid cycle (TCA) and the oxidative pentose phosphate pathway (OPPP). In Rosa vegetative buds, sequence analysis of the RhBRC1 3'UTR identified six binding motifs specific to the Pumilio/FBF RNA-binding protein family (PUF) and probably involved in posttranscriptional regulation. RhPUF4 was highly expressed in the buds of decapitated plants and in response to sugar availability in in-vitro-cultured buds. RhPUF4 was found to be close to AtPUM2, which encodes an Arabidopsis PUF protein. In addition, sugar-dependent upregulation of RhPUF4 was also found in Rosa calluses. RhPUF4 expression was especially dependent on the OPPP, supporting its role in OPPP-dependent posttranscriptional regulation of RhBRC1. These findings indicate that the 3'UTR sequence could be an important target in the molecular regulatory network of RhBRC1 and pave the way for investigating new aspects of RhBRC1 regulation.

The SCOOP12 peptide regulates defense response and root elongation in Arabidopsis thaliana.

Small secreted peptides are important players in plant development and stress response. Using a targeted in silico approach, we identified a family of 14 Arabidopsis genes encoding precursors of serine-rich endogenous peptides (PROSCOOP). Transcriptomic analyses revealed that one member of this family, PROSCOOP12, is involved in processes linked to biotic and oxidative stress as well as root growth. Plants defective in this gene were less susceptible to Erwinia amylovora infection and showed an enhanced root growth phenotype. In PROSCOOP12 we identified a conserved motif potentially coding for a small secreted peptide. Exogenous application of synthetic SCOOP12 peptide induces various defense responses in Arabidopsis. Our findings show that SCOOP12 has numerous properties of phytocytokines, activates the phospholipid signaling pathway, regulates reactive oxygen species response, and is perceived in a BAK1 co-receptor-dependent manner.

Genome Sequence of the Necrotrophic Plant Pathogen Alternaria brassicicola Abra43.

Alternaria brassicicola causes dark spot (or black spot) disease, which is one of the most common and destructive fungal diseases of Brassicaceae spp. worldwide. Here, we report the draft genome sequence of strain Abra43. The assembly comprises 29 scaffolds, with an N50 value of 2.1 Mb. The assembled genome was 31,036,461 bp in length, with a G+C content of 50.85%.

Cold stratification and exogenous nitrates entail similar functional proteome adjustments during Arabidopsis seed dormancy release.

Despite having very similar initial pools of stored mRNAs and proteins in the dry state, mature Arabidopsis seeds can either proceed toward radicle protrusion or stay in a dormant state upon imbibition. Dormancy breaking, a prerequisite to germination completion, can be induced by different treatments though the underlying mechanisms remain elusive. Thus, we investigated the consequence of such treatments on the seed proteome. Two unrelated dormancy-releasing treatments were applied to dormant seeds, namely, cold stratification and exogenous nitrates, in combination with differential proteomic tools to highlight the specificities of the imbibed dormant state. The results reveal that both treatments lead to highly similar proteome adjustments. In the imbibed dormant state, enzymes involved in reserve mobilization are less accumulated and it appears that several energetically costly processes associated to seed germination and preparation for subsequent seedling establishment are repressed. Our data suggest that dormancy maintenance is associated to an abscisic-acid-dependent recapitulation of the late maturation program resulting in a higher potential to cope with environmental stresses. The comparison of the present results with previously published -omic data sets reinforces and extends the assumption that post-transcriptional, translational, and post-translational regulations are determinant for seed germination.

Proteomics and posttranslational proteomics of seed dormancy and germination.

The seed is the dispersal unit of plants and must survive the vagaries of the environment. It is the object of intense genetic and genomic studies because processes related to seed quality affect crop yield and the seed itself provides food for humans and animals. Presently, the general aim of postgenomics analyses is to understand the complex biochemical and molecular processes underlying seed quality, longevity, dormancy, and vigor. Due to advances in functional genomics, the recent past years have seen a tremendous progress in our understanding of several aspects of seed development and germination. Here, we describe the proteomics protocols (from protein extraction to mass spectrometry) that can be used to investigate several aspects of seed physiology, including germination and its hormonal regulation, dormancy release, and seed longevity. These techniques can be applied to the study of both model plants (such as Arabidopsis) and crops.

Protein damage and repair controlling seed vigor and longevity.

The formation of abnormal isoaspartyl residues derived from aspartyl or asparaginyl residues is a major source of spontaneous protein misfolding in cells. The repair enzyme protein L: -isoaspartyl methyltransferase (PIMT) counteracts such damage by catalyzing the conversion of abnormal isoaspartyl residues to their normal aspartyl forms. Thus, this enzyme contributes to the survival of many organisms, including plants. Analysis of the accumulation of isoaspartyl-containing proteins and its modulation by the PIMT repair pathway, using germination tests, immunodetection, enzymatic assays, and HPLC analysis, gives new insights in understanding controlling mechanisms of seed longevity and vigor.

New proteomic developments to analyze protein isomerization and their biological significance in plants.

Spontaneous isoaspartyl formation from aspartyl dehydration or asparaginyl deamidation is a major source of modifications in protein structures. In cells, these conformational changes could be reverted by the protein L-isoaspartyl methyltransferase (PIMT) repair enzyme that converts the isoaspartyl residues into aspartyl. The physiological importance of this metabolism has been recently illustrated in plants. Recent developments allowing peptide isomer identification and quantification at the proteome scale are portrayed. The relevance of these new proteomic approaches based on 2-D electrophoresis or electron capture dissociation analysis methods was initially documented in mammals. Extended use to Arabidopsis model systems is promising for the discovery of controlling mechanisms induced by these particular post-translational modifications and their biological role in plants.

Arabidopsis seed secrets unravelled after a decade of genetic and omics-driven research.

Seeds play a fundamental role in colonization of the environment by spermatophytes, and seeds harvested from crops are the main food source for human beings. Knowledge of seed biology is therefore important for both fundamental and applied issues. This review on seed biology illustrates the important progress made in the field of Arabidopsis seed research over the last decade. Access to 'omics' tools, including the inventory of genes deduced from sequencing of the Arabidopsis genome, has speeded up the analysis of biological functions operating in seeds. This review covers the following processes: seed and seed coat development, seed reserve accumulation, seed dormancy and seed germination. We present new insights in these various fields and describe ongoing biotechnology approaches to improve seed characteristics in crops.

[Seed aging and survival mechanisms]. / Vieillissement des semences et mécanismes de survie.

Aging and death are universal to living systems. In temperate climate latitudes the mature seeds of higher plants are exposed to aging and have developed resistance mechanisms allowing survival and plant propagation. In addition to the physicochemical properties of the seed that confer stress resistance, the protein metabolism contributes importantly to longevity mechanisms. Recently, genetic studies have demonstrated the occurrence of the Protein L-isoaspartyl methyltransferase repair enzyme in controlling age-related protein damages and seed survival. These protective mechanisms by protein repair are widespread in all kingdoms, so that the use of seeds as models to study these controlling processes offers the prospect of understanding longevity mechanisms better.

Protein repair L-isoaspartyl methyltransferase 1 is involved in both seed longevity and germination vigor in Arabidopsis.

The formation of abnormal amino acid residues is a major source of spontaneous age-related protein damage in cells. The protein l-isoaspartyl methyltransferase (PIMT) combats protein misfolding resulting from l-isoaspartyl formation by catalyzing the conversion of abnormal l-isoaspartyl residues to their normal l-aspartyl forms. In this way, the PIMT repair enzyme system contributes to longevity and survival in bacterial and animal kingdoms. Despite the discovery of PIMT activity in plants two decades ago, the role of this enzyme during plant stress adaptation and in seed longevity remains undefined. In this work, we have isolated Arabidopsis thaliana lines exhibiting altered expression of PIMT1, one of the two genes encoding the PIMT enzyme in Arabidopsis. PIMT1 overaccumulation reduced the accumulation of l-isoaspartyl residues in seed proteins and increased both seed longevity and germination vigor. Conversely, reduced PIMT1 accumulation was associated with an increase in the accumulation of l-isoaspartyl residues in the proteome of freshly harvested dry mature seeds, thus leading to heightened sensitivity to aging treatments and loss of seed vigor under stressful germination conditions. These data implicate PIMT1 as a major endogenous factor that limits abnormal l-isoaspartyl accumulation in seed proteins, thereby improving seed traits such as longevity and vigor. The PIMT repair pathway likely works in concert with other anti-aging pathways to actively eliminate deleterious protein products, thus enabling successful seedling establishment and strengthening plant proliferation in natural environments.

Proteomic analysis of seed dormancy in Arabidopsis.

The mechanisms controlling seed dormancy in Arabidopsis (Arabidopsis thaliana) have been characterized by proteomics using the dormant (D) accession Cvi originating from the Cape Verde Islands. Comparative studies carried out with freshly harvested dormant and after-ripened non-dormant (ND) seeds revealed a specific differential accumulation of 32 proteins. The data suggested that proteins associated with metabolic functions potentially involved in germination can accumulate during after-ripening in the dry state leading to dormancy release. Exogenous application of abscisic acid (ABA) to ND seeds strongly impeded their germination, which physiologically mimicked the behavior of D imbibed seeds. This application resulted in an alteration of the accumulation pattern of 71 proteins. There was a strong down-accumulation of a major part (90%) of these proteins, which were involved mainly in energetic and protein metabolisms. This feature suggested that exogenous ABA triggers proteolytic mechanisms in imbibed seeds. An analysis of de novo protein synthesis by two-dimensional gel electrophoresis in the presence of [(35)S]-methionine disclosed that exogenous ABA does not impede protein biosynthesis during imbibition. Furthermore, imbibed D seeds proved competent for de novo protein synthesis, demonstrating that impediment of protein translation was not the cause of the observed block of seed germination. However, the two-dimensional protein profiles were markedly different from those obtained with the ND seeds imbibed in ABA. Altogether, the data showed that the mechanisms blocking germination of the ND seeds by ABA application are different from those preventing germination of the D seeds imbibed in basal medium.

cDNA-AFLP analysis of seed germination in Arabidopsis thaliana identifies transposons and new genomic sequences.

A cDNA-AFLP experiment was designed to identify and clone nucleotide sequences induced during seed germination in Arabidopsis thaliana. Sequences corresponding to known genes involved in processes important for germination, such as mitochondrial biogenesis, protein synthesis and cell cycle progression, were isolated. Other sequences correspond to Arabidopsis BAC clones in regions where genes have not been annotated. Notably, a number of the sequences cloned did not correspond to available sequences in the databases from the Arabidopsis genome, but instead present significant similarity with DNA from other organisms, for example fish species; among them, some may encode transposons. A number of the sequences isolated showed no significant similarity with any sequences in the public databases. Oligonucleotides derived from these new sequences were used to amplify genomic DNA of Arabidopsis. Expression analysis of representative sequences is presented. This work suggests that, during germination, there may be a massive transposon mobilization that may be useful in the annotation of new genome sequences and identification of regulatory mechanisms.