Leaf drought adaptive response in winter oilseed rape is altered at the onset of senescence: a study combining NMR relaxometry, multi-omics and microscopy.

Climate change is bringing more frequent and intense droughts, reducing overall water availability and adversely affecting crops. There is a need to improve our understanding of the tissular and cellular adaptation mechanisms that are critical for plant water conservation strategies. Here, we have used NMR relaxometry in combination with microscopy and multi-omic analysis to study the effects of progressive soil drought on winter oilseed rape (WOSR, Brassica napus L., cv. Aviso) leaves. This study reveals the structural and metabolic adjustments these leaves operate to maintain cell homeostasis. Our results are original in showing that the adaptive responses are altered in leaves at the onset of senescence, associated with changes in metabolic plasticity and mesophyll structures. Thus, long-term responses in young leaves involving osmotic adjustment were combined with the maintenance of tissue hydration and cell growth, contributing to high survival and recovery capacity. For the first time, short-term responses observed in early senescent-old leaves were associated with early drought-induced dehydration of the spongy layer. However, this dehydration was not followed by osmotic adjustment and did not allow maintenance of leaf tissue turgor. These findings open further studies on the genetic variability of drought responses related to identified short- and long-term structural and metabolic plasticity traits in Brassica species.

Identification of transcriptional modules linked to the drought response of Brassica napus during seed development and their mitigation by early biotic stress.

In order to capture the drought impacts on seed quality acquisition in Brassica napus and its potential interaction with early biotic stress, seeds of the 'Express' genotype of oilseed rape were characterized from late embryogenesis to full maturity from plants submitted to reduced watering (WS) with or without pre-occurring inoculation by the telluric pathogen Plasmodiophora brassicae (Pb + WS or Pb, respectively), and compared to control conditions (C). Drought as a single constraint led to significantly lower accumulation of lipids, higher protein content and reduced longevity of the WS-treated seeds. In contrast, when water shortage was preceded by clubroot infection, these phenotypic differences were completely abolished despite the upregulation of the drought sensor RD20. A weighted gene co-expression network of seed development in oilseed rape was generated using 72 transcriptomes from developing seeds from the four treatments and identified 33 modules. Module 29 was highly enriched in heat shock proteins and chaperones that showed a stronger upregulation in Pb + WS compared to the WS condition, pointing to a possible priming effect by the early P. brassicae infection on seed quality acquisition. Module 13 was enriched with genes encoding 12S and 2S seed storage proteins, with the latter being strongly upregulated under WS conditions. Cis-element promotor enrichment identified PEI1/TZF6, FUS3 and bZIP68 as putative regulators significantly upregulated upon WS compared to Pb + WS. Our results provide a temporal co-expression atlas of seed development in oilseed rape and will serve as a resource to characterize the plant response towards combinations of biotic and abiotic stresses.

New insights into chlorophyll-WSCP (water-soluble chlorophyll proteins) interactions : The case study of BnD22 (Brassica napus drought-induced 22 kDa).

The water-soluble chlorophyll-proteins (WSCP) of class II from Brassicaceae are non-photosynthetic proteins that bind chlorophylls (Chls) and chlorophyll derivatives. Their physiological roles, biochemical functions and mode of action are still unclear. It is assumed that the WSCPs have a protection function against Chl photodamage during stressful conditions. WSCPs are subdivided into class IIA and class IIB according to their apparent Chla/b binding ratio. Although their Chla/Chlb binding selectivity has been partly characterized, their Chl affinities are not yet precisely defined. For instance, WSCPs IIA do not show any Chl binding preference while WSCPs IIB have greater affinity to Chlb. In this study, we present a novel method for assessment of Chl binding to WSCPs based on the differences of Chl photobleaching rates in a large range of Chl/protein ratios. The protein we have chosen to study WSCP is BnD22, a WSCP IIA induced in the leaves of Brassica napus under water deficit. BnD22 formed oligomeric complexes upon binding to Chla and/or Chlb allowing a protective effect against photodamage. The binding constants indicate that BnD22 binds with high affinity the Chls and with a strong selectivity to Chla. Moreover, dependending of Chl/protein ratio upon reconstitution, two distinct binding events were detected resulting from difference of Chl stoichiometry inside oligomeric complexes.

RNA sequencing data for responses to drought stress and/or clubroot infection in developing seeds of Brassica napus.

Oilseed rape (Brassica napus L.) is the third largest oil crop worldwide. Like other crops, oilseed rape faces unfavorable environmental conditions resulting from multiple and combined actions of abiotic and biotic constraints that occur throughout the growing season. In particular drought severely reduces seed yield but also impacts seed quality in oilseed rape. In addition, clubroot disease, caused by the pathogen Plasmodiophora brassicae, limits the yield of the oilseed rape crops grown in infected areas. Clubroot induces swellings or galls on the roots that decrease the flow of water and nutrients within the plant. Furthermore, combinations of different stresses lead to complex plant responses that can not be predicted by the simple addition of individual stress responses. Indeed, an abiotic constraint can either reduce or stimulate the plant response to a pathogen or pest. Transcriptome datasets from different conditions are key resources to improve our knowledge of environmental stress-resistance mechanisms in plant organs. Here, we describe a RNA-seq dataset consisting of 72 samples of immature B. napus seeds from plants grown either under drought, infected with P. brassicae, or a combination of both stresses. A total of 67.6 Gb of transcriptome paired-end reads were filtered, mapped onto the B. napus reference genome Darmor-bzh and used for identification of differentially expressed genes and gene ontology enrichment. The raw reads are available under accession PRJNA738318 at NCBI Sequence Read Archive (SRA) repository. The dataset is a resource for the scientific community exploring seed plasticity.

Leaf status and environmental signals jointly regulate proline metabolism in winter oilseed rape.

Proline metabolism is an essential component of plant adaptation to multiple environmental stress conditions that is also known to participate in specific developmental phases, particularly in reproductive organs. Recent evidence suggested a possible role for proline catabolism in Brassica napus for nitrogen remobilization processes from source leaves at the vegetative stage. Here, we investigate transcript levels of Δ1-PYRROLINE-5-CARBOXYLATE SYNTHASE (P5CS) and PROLINE DEHYDROGENASE (ProDH) genes at the vegetative stage with respect to net proline biosynthesis and degradation fluxes in leaves having a different sink/source balance. We showed that the underexpression of three P5CS1 genes in source leaves was accompanied by a reduced commitment of de novo assimilated 15N towards proline biosynthesis and an overall depletion of free proline content. We found that the expression of ProDH genes was strongly induced by carbon starvation conditions (dark-induced senescence) compared with early senescing leaves. Our results suggested a role for proline catabolism in B. napus, but acting only at a late stage of senescence. In addition, we also identified some P5CS and ProDH genes that were differentially expressed during multiple processes (leaf status, dark to light transition, and stress response).

Molecular evolution and transcriptional regulation of the oilseed rape proline dehydrogenase genes suggest distinct roles of proline catabolism during development.

MAIN CONCLUSION: Six BnaProDH1 and two BnaProDH2 genes were identified in Brassica napus genome. The BnaProDH1 genes are mainly expressed in pollen and roots' organs while BnaProDH2 gene expression is associated with leaf vascular tissues at senescence. Proline dehydrogenase (ProDH) catalyzes the first step in the catabolism of proline. The ProDH gene family in oilseed rape (Brassica napus) was characterized and compared to other Brassicaceae ProDH sequences to establish the phylogenetic relationships between genes. Six BnaProDH1 genes and two BnaProDH2 genes were identified in the B. napus genome. Expression of the three paralogous pairs of BnaProDH1 genes and the two homoeologous BnaProDH2 genes was measured by real-time quantitative RT-PCR in plants at vegetative and reproductive stages. The BnaProDH2 genes are specifically expressed in vasculature in an age-dependent manner, while BnaProDH1 genes are strongly expressed in pollen grains and roots. Compared to the abundant expression of BnaProDH1, the overall expression of BnaProDH2 is low except in roots and senescent leaves. The BnaProDH1 paralogs showed different levels of expression with BnaA&C.ProDH1.a the most strongly expressed and BnaA&C.ProDH1.c the least. The promoters of two BnaProDH1 and two BnaProDH2 genes were fused with uidA reporter gene (GUS) to characterize organ and tissue expression profiles in transformed B. napus plants. The transformants with promoters from different genes showed contrasting patterns of GUS activity, which corresponded to the spatial expression of their respective transcripts. ProDHs probably have non-redundant functions in different organs and at different phenological stages. In terms of molecular evolution, all BnaProDH sequences appear to have undergone strong purifying selection and some copies are becoming subfunctionalized. This detailed description of oilseed rape ProDH genes provides new elements to investigate the function of proline metabolism in plant development.

Sixteen cytosolic glutamine synthetase genes identified in the Brassica napus L. genome are differentially regulated depending on nitrogen regimes and leaf senescence.

A total of 16 BnaGLN1 genes coding for cytosolic glutamine synthetase isoforms (EC 6.3.1.2.) were found in the Brassica napus genome. The total number of BnaGLN1 genes, their phylogenetic relationships, and genetic locations are in agreement with the evolutionary history of Brassica species. Two BnaGLN1.1, two BnaGLN1.2, six BnaGLN1.3, four BnaGLN1.4, and two BnaGLN1.5 genes were found and named according to the standardized nomenclature for the Brassica genus. Gene expression showed conserved responses to nitrogen availability and leaf senescence among the Brassiceae tribe. The BnaGLN1.1 and BnaGLN1.4 families are overexpressed during leaf senescence and in response to nitrogen limitation. The BnaGLN1.2 family is up-regulated under high nitrogen regimes. The members of the BnaGLN1.3 family are not affected by nitrogen availability and are more expressed in stems than in leaves. Expression of the two BnaGLN1.5 genes is almost undetectable in vegetative tissues. Regulations arising from plant interactions with their environment (such as nitrogen resources), final architecture, and therefore sink-source relations in planta, seem to be globally conserved between Arabidopsis and B. napus. Similarities of the coding sequence (CDS) and protein sequences, expression profiles, response to nitrogen availability, and ageing suggest that the roles of the different GLN1 families have been conserved among the Brassiceae tribe. These findings are encouraging the transfer of knowledge from the Arabidopsis model plant to the B. napus crop plant. They are of special interest when considering the role of glutamine synthetase in crop yield and grain quality in maize and wheat.

Homeologous recombination plays a major role in chromosome rearrangements that occur during meiosis of Brassica napus haploids.

Chromosomal rearrangements can be triggered by recombination between distinct but related regions. Brassica napus (AACC; 2n = 38) is a recent allopolyploid species whose progenitor genomes are widely replicated. In this article, we analyze the extent to which chromosomal rearrangements originate from homeologous recombination during meiosis of haploid B. napus (n = 19) by genotyping progenies of haploid x euploid B. napus with molecular markers. Our study focuses on three pairs of homeologous regions selected for their differing levels of divergence (N1/N11, N3/N13, and N9/N18). We show that a high number of chromosomal rearrangements occur during meiosis of B. napus haploid and are transmitted by first division restitution (FDR)-like unreduced gametes to their progeny; half of the progeny of Darmor-bzh haploids display duplications and/or losses in the chromosomal regions being studied. We demonstrate that half of these rearrangements are due to recombination between regions of primary homeology, which represents a 10- to 100-fold increase compared to the frequency of homeologous recombination measured in euploid lines. Some of the other rearrangements certainly result from recombination between paralogous regions because we observed an average of one to two autosyndetic A-A and/or C-C bivalents at metaphase I of the B. napus haploid. These results are discussed in the context of genome evolution of B. napus.

Characterization of a radish introgression carrying the Ogura fertility restorer gene Rfo in rapeseed, using the Arabidopsis genome sequence and radish genetic mapping.

The radish Rfo gene restores male fertility in radish or rapeseed plants carrying Ogura cytoplasmic male-sterility. This system was first discovered in radish and was transferred to rapeseed for the production of F1 hybrid seeds. We aimed to identify the region of the Arabidopsis genome syntenic to the Rfo locus and to characterize the radish introgression in restored rapeseed. We used two methods: amplified consensus genetic markers (ACGMs) in restored rapeseed plants and construction of a precise genetic map around the Rfo gene in a segregating radish population. The use of ACGMs made it possible to detect radish orthologs of Arabidopsis genes in the restored rapeseed genome. We identified radish genes, linked to Rfo in rapeseed and whose orthologs in Arabidopsis are carried by chromosomes 1, 4 and 5. This indicates several breaks in colinearity between radish and Arabidopsis genomes in this region. We determined the positions of markers relative to each other and to the Rfo gene, using the progeny of a rapeseed plant with unstable meiotic transmission of the radish introgression. This enabled us to produce a schematic diagram of the radish introgression in rapeseed. Markers which could be mapped both on radish and restored rapeseed indicate that at least 50 cM of the radish genome is integrated in restored rapeseed. Using markers closely linked to the Rfo gene in rapeseed and radish, we identified a contig spanning six bacterial artificial chromosome (BAC) clones on Arabidopsis chromosome 1, which is likely to carry the orthologous Rfo gene.

Identification of the fertility restoration locus, Rfo, in radish, as a member of the pentatricopeptide-repeat protein family.

Ogura cytoplasmic male sterility (CMS) in radish (Raphanus sativus) is caused by an aberrant mitochondrial gene, Orf138, that prevents the production of functional pollen without affecting female fertility. Rfo, a nuclear gene that restores male fertility, alters the expression of Orf138 at the post-transcriptional level. The Ogura CMS/Rfo two-component system is a useful model for investigating nuclear-cytoplasmic interactions, as well as the physiological basis of fertility restoration. Using a combination of positional cloning and microsynteny analysis of Arabidopsis thaliana and radish, we genetically and physically delimited the Rfo locus to a 15-kb DNA segment. Analysis of this segment shows that Rfo is a member of the pentatricopeptide repeat (PPR) family. In Arabidopsis, this family contains more than 450 members of unknown function, although most of them are predicted to be targeted to mitochondria and chloroplasts and are thought to have roles in organellar gene expression.