Integrated multi-omics and genetic analyses reveal molecular determinants underlying Arabidopsis snap33 mutant phenotype.

The secretory pathway is essential for plant immunity, delivering diverse antimicrobial molecules into the extracellular space. Arabidopsis thaliana soluble N-ethylmaleimide-sensitive-factor attachment protein receptor SNAP33 is a key actor of this process. The snap33 mutant displays dwarfism and necrotic lesions, however the molecular determinants of its macroscopic phenotypes remain elusive. Here, we isolated several new snap33 mutants that exhibited constitutive cell death and H2O2 accumulation, further defining snap33 as an autoimmune mutant. We then carried out quantitative transcriptomic and proteomic analyses showing that numerous defense transcripts and proteins were up-regulated in the snap33 mutant, among which genes/proteins involved in defense hormone, pattern-triggered immunity, and nucleotide-binding domain leucine-rich-repeat receptor signaling. qRT-PCR analyses and hormone dosages supported these results. Furthermore, genetic analyses elucidated the diverse contributions of the main defense hormones and some nucleotide-binding domain leucine-rich-repeat receptor signaling actors in the establishment of the snap33 phenotype, emphasizing the preponderant role of salicylic acid over other defense phytohormones. Moreover, the accumulation of pattern-triggered immunity and nucleotide-binding domain leucine-rich-repeat receptor signaling proteins in the snap33 mutant was confirmed by immunoblotting analyses and further shown to be salicylic acid-dependent. Collectively, this study unveiled molecular determinants underlying the Arabidopsis snap33 mutant phenotype and brought new insights into autoimmunity signaling.

RNAseq based variant dataset in a black poplar association panel.

OBJECTIVE: Black poplar (Populus nigra L.) is a species native to Eurasia with a wide distribution area. It is an ecologically important species from riparian ecosystems, that is used as a parent of interspecific (P. deltoides x P. nigra) cultivated poplar hybrids. Variant detection from transcriptomics sequences of 241 P. nigra individuals, sampled in natural populations from 11 river catchments (in four European countries) is described here. These data provide new valuable resources for population structure analysis, population genomics and genome-wide association studies. DATA DESCRIPTION: We generated transcriptomics data from a mixture of young differentiating xylem and cambium tissues of 480 Populus nigra trees sampled in a common garden experiment located at Orléans (France), corresponding to 241 genotypes (2 clonal replicates per genotype, at maximum) by using RNAseq technology. We launched on the resulting sequences an in-silico pipeline that allowed us to obtain 878,957 biallelic polymorphisms without missing data. More than 99% of these positions are annotated and 98.8% are located on the 19 chromosomes of the P. trichocarpa reference genome. The raw RNAseq sequences are available at the NCBI Sequence Read Archive SPR188754 and the variant dataset at the Recherche Data Gouv repository under https://doi.org/10.15454/8DQXK5 .

Evolutionary analyses and expression patterns of TCP genes in Ranunculales.

TCP transcription factors play a role in a large number of developmental processes and are at the crossroads of numerous hormonal biosynthetic and signaling pathways. The complete repertoire of TCP genes has already been characterized in several plant species, but not in any species of early diverging eudicots. We focused on the order Ranunculales because of its phylogenetic position as sister group to all other eudicots and its important morphological diversity. Results show that all the TCP genes expressed in the floral transcriptome of Nigella damascena (Ranunculaceae) are the orthologs of the TCP genes previously identified from the fully sequenced genome of Aquilegia coerulea. Phylogenetic analyses combined with the identification of conserved amino acid motifs suggest that six paralogous genes of class I TCP transcription factors were present in the common ancestor of angiosperms. We highlight independent duplications in core eudicots and Ranunculales within the class I and class II subfamilies, resulting in different numbers of paralogs within the main subclasses of TCP genes. This has most probably major consequences on the functional diversification of these genes in different plant clades. The expression patterns of TCP genes in Nigella damascena were consistent with the general suggestion that CIN and class I TCP genes may have redundant roles or take part in same pathways, while CYC/TB1 genes have more specific actions. Our findings open the way for future studies at the tissue level, and for investigating redundancy and subfunctionalisation in TCP genes and their role in the evolution of morphological novelties.

Corrigendum: Genotypic Variation of Nitrogen Use Efficiency and Amino Acid Metabolism in Barley.

[This corrects the article DOI: 10.3389/fpls.2021.807798.].

A guanosine tetraphosphate (ppGpp) mediated brake on photosynthesis is required for acclimation to nitrogen limitation in Arabidopsis.

Guanosine pentaphosphate and tetraphosphate (together referred to as ppGpp) are hyperphosphorylated nucleotides found in bacteria and the chloroplasts of plants and algae. In plants and algae artificial ppGpp accumulation can inhibit chloroplast gene expression, and influence photosynthesis, nutrient remobilization, growth, and immunity. However, it is so far unknown whether ppGpp is required for abiotic stress acclimation in plants. Here, we demonstrate that ppGpp biosynthesis is necessary for acclimation to nitrogen starvation in Arabidopsis. We show that ppGpp is required for remodeling the photosynthetic electron transport chain to downregulate photosynthetic activity and for protection against oxidative stress. Furthermore, we demonstrate that ppGpp is required for coupling chloroplastic and nuclear gene expression during nitrogen starvation. Altogether, our work indicates that ppGpp is a pivotal regulator of chloroplast activity for stress acclimation in plants.

A derived ZW chromosome system in Amborella trichopoda, representing the sister lineage to all other extant flowering plants.

The genetic basis and evolution of sex determination in dioecious plants is emerging as an active area of research with exciting advances in genome sequencing and analysis technologies. As the sole species within the sister lineage to all other extant flowering plants, Amborella trichopoda is an important model for understanding the evolution and development of flowers. Plants typically produce only male or female flowers, but sex determination mechanisms are unknown for the species. Sequence data derived from plants of natural origin and an F1 mapping population were used to identify sex-linked genes and the nonrecombining region. Amborella trichopoda has a ZW sex determination system. Analysis of genes in a 4 Mb nonrecombining sex-determination region reveals recent divergence of Z and W gametologs, and few Z- and W-specific genes. The sex chromosomes of A. trichopoda evolved less than 16.5 Myr ago, long after the divergence of the extant angiosperms.

The Genomic Impact of Mycoheterotrophy in Orchids.

Mycoheterotrophic plants have lost the ability to photosynthesize and obtain essential mineral and organic nutrients from associated soil fungi. Despite involving radical changes in life history traits and ecological requirements, the transition from autotrophy to mycoheterotrophy has occurred independently in many major lineages of land plants, most frequently in Orchidaceae. Yet the molecular mechanisms underlying this shift are still poorly understood. A comparison of the transcriptomes of Epipogium aphyllum and Neottia nidus-avis, two completely mycoheterotrophic orchids, to other autotrophic and mycoheterotrophic orchids showed the unexpected retention of several genes associated with photosynthetic activities. In addition to these selected retentions, the analysis of their expression profiles showed that many orthologs had inverted underground/aboveground expression ratios compared to autotrophic species. Fatty acid and amino acid biosynthesis as well as primary cell wall metabolism were among the pathways most impacted by this expression reprogramming. Our study suggests that the shift in nutritional mode from autotrophy to mycoheterotrophy remodeled the architecture of the plant metabolism but was associated primarily with function losses rather than metabolic innovations.

Transcriptome Analysis Reveals Putative Target Genes of APETALA3-3 During Early Floral Development in Nigella damascena L.

Even though petals are homoplastic structures, their identity consistently involves genes of the APETALA3 (AP3) lineage. However, the extent to which the networks downstream of AP3 are conserved in species with petals of different evolutionary origins is unknown. In Ranunculaceae, the specificity of the AP3-III lineage offers a great opportunity to identify the petal gene regulatory network in a comparative framework. Using a transcriptomic approach, we investigated putative target genes of the AP3-III ortholog NdAP3-3 in Nigella damascena at early developmental stages when petal identity is determined, and we compared our data with that from selected eudicot species. We generated a de novo reference transcriptome to carry out a differential gene expression analysis between the wild-type and mutant NdAP3-3 genotypes differing by the presence vs. absence of petals at early stages of floral development. Among the 1,620 genes that were significantly differentially expressed between the two genotypes, functional annotation suggested a large involvement of nuclear activities, including regulation of transcription, and enrichment in processes linked to cell proliferation. Comparing with Arabidopsis data, we found that highly conserved genes between the two species are enriched in homologs of direct targets of the AtAP3 protein. Integrating AP3-3 binding site data from another Ranunculaceae species, Aquilegia coerulea, allowed us to identify a set of 18 putative target genes that were conserved between the three species. Our results suggest that, despite the independent evolutionary origin of petals in core eudicots and Ranunculaceae, a small conserved set of genes determines petal identity and early development in these taxa.

RNAi suppression of DNA methylation affects the drought stress response and genome integrity in transgenic poplar.

Trees are long-lived organisms that continuously adapt to their environments, a process in which epigenetic mechanisms are likely to play a key role. Via downregulation of the chromatin remodeler DECREASED IN DNA METHYLATION 1 (DDM1) in poplar (Populus tremula × Populus alba) RNAi lines, we examined how DNA methylation coordinates genomic and physiological responses to moderate water deficit. We compared the growth and drought response of two RNAi-ddm1 lines to wild-type (WT) trees under well-watered and water deficit/rewatering conditions, and analyzed their methylomes, transcriptomes, mobilomes and phytohormone contents in the shoot apical meristem. The RNAi-ddm1 lines were more tolerant to drought-induced cavitation but did not differ in height or stem diameter growth. About 5000 differentially methylated regions were consistently detected in both RNAi-ddm1 lines, colocalizing with 910 genes and 89 active transposable elements. Under water deficit conditions, 136 differentially expressed genes were found, including many involved in phytohormone pathways; changes in phytohormone concentrations were also detected. Finally, the combination of hypomethylation and drought led to the mobility of two transposable elements. Our findings suggest major roles for DNA methylation in regulation of genes involved in hormone-related stress responses, and the maintenance of genome integrity through repression of transposable elements.

Genotypic Variation of Nitrogen Use Efficiency and Amino Acid Metabolism in Barley.

Owing to the large genetic diversity of barley and its resilience under harsh environments, this crop is of great value for agroecological transition and the need for reduction of nitrogen (N) fertilizers inputs. In the present work, we investigated the diversity of a North African barley genotype collection in terms of growth under limiting N (LN) or ample N (HN) supply and in terms of physiological traits including amino acid content in young seedlings. We identified a Moroccan variety, Laanaceur, accumulating five times more lysine in its leaves than the others under both N nutritional regimes. Physiological characterization of the barley collection showed the genetic diversity of barley adaptation strategies to LN and highlighted a genotype x environment interaction. In all genotypes, N limitation resulted in global biomass reduction, an increase in C concentration, and a higher resource allocation to the roots, indicating that this organ undergoes important adaptive metabolic activity. The most important diversity concerned leaf nitrogen use efficiency (LNUE), root nitrogen use efficiency (RNUE), root nitrogen uptake efficiency (RNUpE), and leaf nitrogen uptake efficiency (LNUpE). Using LNUE as a target trait reflecting barley capacity to deal with N limitation, this trait was positively correlated with plant nitrogen uptake efficiency (PNUpE) and RNUpE. Based on the LNUE trait, we determined three classes showing high, moderate, or low tolerance to N limitation. The transcriptomic approach showed that signaling, ionic transport, immunity, and stress response were the major functions affected by N supply. A candidate gene encoding the HvNRT2.10 transporter was commonly up-regulated under LN in the three barley genotypes investigated. Genes encoding key enzymes required for lysine biosynthesis in plants, dihydrodipicolinate synthase (DHPS) and the catabolic enzyme, the bifunctional Lys-ketoglutarate reductase/saccharopine dehydrogenase are up-regulated in Laanaceur and likely account for a hyperaccumulation of lysine in this genotype. Our work provides key physiological markers of North African barley response to low N availability in the early developmental stages.

Transcriptomics at Maize Embryo/Endosperm Interfaces Identifies a Transcriptionally Distinct Endosperm Subdomain Adjacent to the Embryo Scutellum.

Seeds are complex biological systems comprising three genetically distinct tissues nested one inside another (embryo, endosperm, and maternal tissues). However, the complexity of the kernel makes it difficult to understand intercompartment interactions without access to spatially accurate information. Here, we took advantage of the large size of the maize (Zea mays) kernel to characterize genome-wide expression profiles of tissues at different embryo/endosperm interfaces. Our analysis identifies specific transcriptomic signatures in two interface tissues compared with whole seed compartments: the scutellar aleurone layer and the newly named endosperm adjacent to scutellum (EAS). The EAS, which appears around 9 d after pollination and persists for around 11 d, is confined to one to three endosperm cell layers adjacent to the embryonic scutellum. Its transcriptome is enriched in genes encoding transporters. The absence of the embryo in an embryo specific mutant can alter the expression pattern of EAS marker genes. The detection of cell death in some EAS cells together with an accumulation of crushed cell walls suggests that the EAS is a dynamic zone from which cell layers in contact with the embryo are regularly eliminated and to which additional endosperm cells are recruited as the embryo grows.

Silicon supply affects the root transcriptome of Brassica napus L.

MAIN CONCLUSION: Modulation of gene expression in roots of Brassica napus by silicon (Si) supply could allow plants to cope with future stresses. The origin of the beneficial effects of silicon (Si) in plants, especially when they are subject to stress, remains poorly understood. Some authors have shown that Si alleviates plant stress and consider that this is mainly due to a mechanical effect on the cell wall. In addition, the other studies have shown that Si can also affect gene expression and modulate a number of metabolic pathways, especially in plants cultivated under stress conditions. Previously, Haddad et al. (Front Plant Sci 9:5-16, 2018) showed that a pretreatment of Brassica napus plants with Si (1.7 mM) for 1 week alleviated the stress induced by N privation. These results suggest that this improved resistance in Si-treated plants might be due to the establishment of defense mechanisms prior to exposure to the N stress. The aim of the current work was to test this assumption in Brassica napus roots (where Si is mainly stored) using a transcriptomic approach via the RNA sequencing. Our results indicated that the Si supply leads to a modulation of the expression of genes in Brassica napus roots. Functional categorization of the differentially expressed genes demonstrated that numerous genes are involved in different metabolic pathways and especially in cell wall synthesis, phytohormone metabolism, and stress responses. All these results show that Si modifies the root metabolism of B. napus, which could allow a better adaptation to future stresses.

Accuracy of RNAseq based SNP discovery and genotyping in Populusnigra.

BACKGROUD: Populus nigra is a major tree species of ecological and economic importance for which several initiatives have been set up to create genomic resources. In order to access the large number of Single Nucleotide Polymorphisms (SNPs) typically needed to carry out a genome scan, the present study aimed at evaluating RNA sequencing as a tool to discover and type SNPs in genes within natural populations of P. nigra. RESULTS: We have devised a bioinformatics pipeline to call and type SNPs from RNAseq reads and applied it to P. nigra transcriptomic data. The accuracy of the resulting RNAseq-based SNP calling and typing has been evaluated by (i) comparing their position and alleles to those previously reported in candidate genes, (ii) assessing their genotyping accuracy with respect to a previously available SNP chip and (iii) evaluating their inter-annual repeatability. We found that a combination of several callers yields a good compromise between the number of variants type and the accuracy of genotyping. We further used the resulting genotypic data to carry out basic genetic analyses whose results confirm the quality of the RNAseq-based SNP dataset. CONCLUSIONS: We demonstrated the potential and accuracy of RNAseq as an efficient way to genotype SNPs in P. nigra. These results open prospects towards the use of this technology for quantitative and population genomics studies.

Synthetic data sets for the identification of key ingredients for RNA-seq differential analysis.

Numerous statistical pipelines are now available for the differential analysis of gene expression measured with RNA-sequencing technology. Most of them are based on similar statistical frameworks after normalization, differing primarily in the choice of data distribution, mean and variance estimation strategy and data filtering. We propose an evaluation of the impact of these choices when few biological replicates are available through the use of synthetic data sets. This framework is based on real data sets and allows the exploration of various scenarios differing in the proportion of non-differentially expressed genes. Hence, it provides an evaluation of the key ingredients of the differential analysis, free of the biases associated with the simulation of data using parametric models. Our results show the relevance of a proper modeling of the mean by using linear or generalized linear modeling. Once the mean is properly modeled, the impact of the other parameters on the performance of the test is much less important. Finally, we propose to use the simple visualization of the raw P-value histogram as a practical evaluation criterion of the performance of differential analysis methods on real data sets.

Translatome profiling in dormant and nondormant sunflower (Helianthus annuus) seeds highlights post-transcriptional regulation of germination.

Seed dormancy, which blocks germination in apparently favourable conditions, is a key regulatory control point of plant population establishment. As germination requires de novo translation, its regulation by dormancy is likely to be related to the association of individual transcripts to polysomes. Here, the polysome-associated mRNAs, that is, the translatome, were fractionated and characterized with microarrays in dormant and nondormant sunflower (Helianthus annuus) embryos during their imbibition at 10°C, a temperature preventing germination of dormant embryos. Profiling of mRNAs in polysomal complexes revealed that the translatome differs between germinating and nongerminating embryos. Association of transcripts with polysomes reached a maximum after 15 h of imbibition; at this time-point 194 polysome-associated transcripts were specifically found in nondormant embryos and 47 in dormant embryos only. The proteins corresponding to the polysomal mRNAs in nondormant embryos appeared to be very pertinent for germination and were involved mainly in transport, regulation of transcription or cell wall modifications. This work demonstrates that seed germination results from a timely regulated and selective recruitment of mRNAs to polysomes, thus opening novel fields of investigation for the understanding of this developmental process.

Constitutive salicylic acid accumulation in pi4kIIIß1ß2 Arabidopsis plants stunts rosette but not root growth.

Phospholipids have recently been found to be integral elements of hormone signalling pathways. An Arabidopsis thaliana double mutant in two type III phosphatidylinositol-4-kinases (PI4Ks), pi4kIIIß1ß2, displays a stunted rosette growth. The causal link between PI4K activity and growth is unknown. Using microarray analysis, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and multiple phytohormone analysis by LC-MS we investigated the mechanism responsible for the pi4kIIIß1ß2 phenotype. The pi4kIIIß1ß2 mutant accumulated a high concentration of salicylic acid (SA), constitutively expressed SA marker genes including PR-1, and was more resistant to Pseudomonas syringae. pi4kIIIß1ß2 was crossed with SA signalling mutants eds1 and npr1 and SA biosynthesis mutant sid2 and NahG. The dwarf phenotype of pi4kIIIß1ß2 rosettes was suppressed in all four triple mutants. Whereas eds1 pi4kIIIß1ß2, sid2 pi4kIIIß1ß2 and NahG pi4kIIIß1ß2 had similar amounts of SA as the wild-type (WT), npr1pi4kIIIß1ß2 had more SA than pi4kIIIß1ß2 despite being less dwarfed. This indicates that PI4KIIIß1 and PI4KIIIß2 are genetically upstream of EDS1 and need functional SA biosynthesis and perception through NPR1 to express the dwarf phenotype. The slow root growth phenotype of pi4kIIIß1ß2 was not suppressed in any of the triple mutants. The pi4kIIIß1ß2 mutations together cause constitutive activation of SA signalling that is responsible for the dwarf rosette phenotype but not for the short root phenotype.