David Lalanne

A Regulatory Network-Based Approach Dissects Late Maturation Processes Related to the Acquisition of Desiccation Tolerance and Longevity of Medicago truncatula Seeds

A network analysis approach to gene regulation during seed maturation of Medicago truncatula uncovers distinct temporal regulatory programs related to desiccation tolerance, longevity, and pod abscission and the key regulators governing these programs. In seeds, desiccation tolerance (DT) and the ability to survive the dry state for prolonged periods of time (longevity) are two essential traits for seed quality that are consecutively acquired during maturation. Using transcriptomic and metabolomic profiling together with a conditional-dependent network of global transcription interactions, we dissected the maturation events from the end of seed filling to final maturation drying during the last 3 weeks of seed development in Medicago truncatula. The network revealed distinct coexpression modules related to the acquisition of DT, longevity, and pod abscission. The acquisition of DT and dormancy module was associated with abiotic stress response genes, including late embryogenesis abundant (LEA) genes. The longevity module was enriched in genes involved in RNA processing and translation. Concomitantly, LEA polypeptides accumulated, displaying an 18-d delayed accumulation compared with transcripts. During maturation, gulose and stachyose levels increased and correlated with longevity. A seed-specific network identified known and putative transcriptional regulators of DT, including ABSCISIC ACID-INSENSITIVE3 (MtABI3), MtABI4, MtABI5, and APETALA2/ ETHYLENE RESPONSE ELEMENT BINDING PROTEIN (AtAP2/EREBP) transcription factor as major hubs. These transcriptional activators were highly connected to LEA genes. Longevity genes were highly connected to two MtAP2/EREBP and two basic leucine zipper transcription factors. A heat shock factor was found at the transition of DT and longevity modules, connecting to both gene sets. Gain- and loss-of-function approaches of MtABI3 confirmed 80% of its predicted targets, thereby experimentally validating the network. This study captures the coordinated regulation of seed maturation and identifies distinct regulatory networks underlying the preparation for the dry and quiescent states.

A survey of flowering genes reveals the role of gibberellins in floral control in rose

Exhaustive studies on flowering control in annual plants have provided a framework for exploring this process in other plant species, especially in perennials for which little molecular data are currently available. Rose is a woody perennial plant with a particular flowering strategy—recurrent blooming, which is controlled by a recessive locus (RB). Gibberellins (GA) inhibit flowering only in non-recurrent roses. Moreover, the GA content varies during the flowering process and between recurrent and non-recurrent rose. Only a few rose genes potentially involved in flowering have been described, i.e. homologues of ABC model genes and floral genes from EST screening. In this study, we gained new information on the molecular basis of rose flowering: date of flowering and recurrent blooming. Based on a candidate gene strategy, we isolated genes that have similarities with genes known to be involved in floral control in Arabidopsis (GA pathway, floral repressors and integrators). Candidate genes were mapped on a segregating population, gene expression was studied in different organs and transcript abundance was monitored in growing shoot apices. Twenty-five genes were studied. RoFT, RoAP1 and RoLFY are proposed to be good floral markers. RoSPY and RB co-localized in our segregating population. GA metabolism genes were found to be regulated during floral transition. Furthermore, GA signalling genes were differentially regulated between a non-recurrent rose and its recurrent mutant. We propose that flowering gene networks are conserved between Arabidopsis and rose. The GA pathway appears to be a key regulator of flowering in rose. We postulate that GA metabolism is involved in floral initiation and GA signalling might be responsible for the recurrent flowering character.

Inference of Longevity-Related Genes from a Robust Coexpression Network of Seed Maturation Identifies Regulators Linking Seed Storability to Biotic Defense-Related Pathways

The identification of a gene regulatory network related to seed longevity in both Medicago truncatula and Arabidopsis reveals a role for biotic defense-related genes in acquisition of longevity. Seed longevity, the maintenance of viability during storage, is a crucial factor for preservation of genetic resources and ensuring proper seedling establishment and high crop yield. We used a systems biology approach to identify key genes regulating the acquisition of longevity during seed maturation of Medicago truncatula. Using 104 transcriptomes from seed developmental time courses obtained in five growth environments, we generated a robust, stable coexpression network (MatNet), thereby capturing the conserved backbone of maturation. Using a trait-based gene significance measure, a coexpression module related to the acquisition of longevity was inferred from MatNet. Comparative analysis of the maturation processes in M. truncatula and Arabidopsis thaliana seeds and mining Arabidopsis interaction databases revealed conserved connectivity for 87% of longevity module nodes between both species. Arabidopsis mutant screening for longevity and maturation phenotypes demonstrated high predictive power of the longevity cross-species network. Overrepresentation analysis of the network nodes indicated biological functions related to defense, light, and auxin. Characterization of defense-related wrky3 and nf-x1-like1 (nfxl1) transcription factor mutants demonstrated that these genes regulate some of the network nodes and exhibit impaired acquisition of longevity during maturation. These data suggest that seed longevity evolved by co-opting existing genetic pathways regulating the activation of defense against pathogens.

Quantitative trait loci for flowering time and inflorescence architecture in rose

The pattern of development of the inflorescence is an important characteristic in ornamental plants, where the economic value is in the flower. The genetic determinism of inflorescence architecture is poorly understood, especially in woody perennial plants with long life cycles. Our objective was to study the genetic determinism of this characteristic in rose. The genetic architectures of 10 traits associated with the developmental timing and architecture of the inflorescence, and with flower production were investigated in a F1 diploid garden rose population, based on intensive measurements of phenological and morphological traits in a field. There were substantial genetic variations in inflorescence development traits, with broad-sense heritabilities ranging from 0.82 to 0.93. Genotypic correlations were significant for most (87%) pairs of traits, suggesting either pleiotropy or tight linkage among loci. However, non-significant and low correlations between some pairs of traits revealed two independent developmental pathways controlling inflorescence architecture: (1) the production of inflorescence nodes increased the number of branches and the production of flowers; (2) internode elongation connected with frequent branching increased the number of branches and the production of flowers. QTL mapping identified six common QTL regions (cQTL) for inflorescence developmental traits. A QTL for flowering time and many inflorescence traits were mapped to the same cQTL. Several candidate genes that are known to control inflorescence developmental traits and gibberellin signaling in Arabidopsis thaliana were mapped in rose. Rose orthologues of FLOWERING LOCUS T (RoFT), TERMINAL FLOWER 1 (RoKSN), SPINDLY (RoSPINDLY), DELLA (RoDELLA), and SLEEPY (RoSLEEPY) co-localized with cQTL for relevant traits. This is the first report on the genetic basis of complex inflorescence developmental traits in rose.

An emerging picture of the seed desiccome: confirmed regulators and newcomers identified using transcriptome comparison

Desiccation tolerance (DT) is the capacity to withstand total loss of cellular water. It is acquired during seed filling and lost just after germination. However, in many species, a germinated seed can regain DT under adverse conditions such as osmotic stress. The genes, proteins and metabolites that are required to establish this DT is referred to as the desiccome. It includes both a range of protective mechanisms and underlying regulatory pathways that remain poorly understood. As a first step toward the identification of the seed desiccome of Medicago truncatula, using updated microarrays we characterized the overlapping transcriptomes associated with acquisition of DT in developing seeds and the re-establishment of DT in germinated seeds using a polyethylene glycol treatment (−1.7 MPa). The resulting list contained 740 and 2829 transcripts whose levels, respectively, increased and decreased with DT. Fourty-eight transcription factors (TF) were identified including MtABI3, MtABI5 and many genes regulating flowering transition and cell identity. A promoter enrichment analysis revealed a strong over-representation of ABRE elements together with light-responsive cis-acting elements. In Mtabi5 Tnt1 insertion mutants, DT could no longer be re-established by an osmotic stress. Transcriptome analysis on Mtabi5 radicles during osmotic stress revealed that 13 and 15% of the up-regulated and down-regulated genes, respectively, are mis-regulated in the mutants and might be putative downstream targets of MtABI5 implicated in the re-establishment of DT. Likewise, transcriptome comparisons of the desiccation sensitive Mtabi3 mutants and hairy roots ectopically expressing MtABI3 revealed that 35 and 23% of the up-regulated and down-regulated genes are acting downstream of MtABI3. Our data suggest that ABI3 and ABI5 have complementary roles in DT. Whether DT evolved by co-opting existing pathways regulating flowering and cellular phase transition and cell identity is discussed.

ABI5 Is a Regulator of Seed Maturation and Longevity in Legumes

ABI5 is a prominent regulator of late seed maturation in legumes, coupling seed vigor with raffinose family oligosaccharide and late embryogenesis abundant protein accumulation and degreening. The preservation of our genetic resources and production of high-quality seeds depends on their ability to remain viable and vigorous during storage. In a quantitative trait locus analysis on seed longevity in Medicago truncatula, we identified the bZIP transcription factor ABSCISIC ACID INSENSITIVE5 (ABI5). Characterization of Mt-abi5 insertion mutant seeds revealed that both the acquisition of longevity and dormancy were severely impaired. Using transcriptomes of developing Mt-abi5 seeds, we created a gene coexpression network and revealed ABI5 as a regulator of gene modules with functions related to raffinose family oligosaccharide (RFO) metabolism, late embryogenesis abundant (LEA) proteins, and photosynthesis-associated nuclear genes (PhANGs). Lower RFO contents in Mt-abi5 seeds were linked to the regulation of SEED IMBIBITION PROTEIN1. Proteomic analysis confirmed that a set of LEA polypeptides was reduced in mature Mt-abi5 seeds, whereas the absence of repression of PhANG in mature Mt-abi5 seeds was accompanied by chlorophyll and carotenoid retention. This resulted in a stress response in Mt-abi5 seeds, evident from an increase in α-tocopherol and upregulation of genes related to programmed cell death and protein folding. Characterization of abi5 mutants in a second legume species, pea (Pisum sativum), confirmed a role for ABI5 in the regulation of longevity, seed degreening, and RFO accumulation, identifying ABI5 as a prominent regulator of late seed maturation in legumes.

Identification of a molecular dialogue between developing seeds of Medicago truncatula and seedborne xanthomonads

Plant pathogenic bacteria disseminate and survive mainly in association with seeds. This study addresses whether seeds are passive carriers or engage a molecular dialogue with pathogens during their development. We developed two pathosystems using Medicago truncatula with Xanthomonas alfalfae subsp. alfalfae (Xaa), the natural Medicago sp. pathogen and Xanthomonas campestris pv. campestris (Xcc), a Brassicaceae pathogen. Three days after flower inoculation, the transcriptome of Xcc-infected pods showed activation of an innate immune response that was strongly limited in Xcc mutated in the type three secretion system, demonstrating an incompatible interaction of Xcc with the reproductive structures. In contrast, the presence of Xaa did not result in an activation of defence genes. Transcriptome profiling during development of infected seeds exhibited time-dependent and differential responses to Xcc and Xaa. Gene network analysis revealed that the transcriptome of Xcc-infected seeds was mainly affected during seed filling whereas that of Xaa-infected seeds responded during late maturation. The Xcc-infected seed transcriptome exhibited an activation of defence response and a repression of targeted seed maturation pathways. Fifty-one percent of putative ABSCISIC ACID INSENSITIVE3 targets were deregulated by Xcc, including oleosin, cupin, legumin and chlorophyll degradation genes. At maturity, these seeds displayed decreased weight and increased chlorophyll content. In contrast, these traits were not affected by Xaa infection. These findings demonstrate the existence of a complex molecular dialogue between xanthomonads and developing seeds and provides insights into a previously unexplored trade-off between seed development and pathogen defence.

Variability within a pea core collection of LEAM and HSP22, two mitochondrial seed proteins involved in stress tolerance

LEAM, a late embryogenesis abundant protein, and HSP22, a small heat shock protein, were shown to accumulate in the mitochondria during pea (Pisum sativum L.) seed development, where they are expected to contribute to desiccation tolerance. Here, their expression was examined in seeds of 89 pea genotypes by Western blot analysis. All genotypes expressed LEAM and HSP22 in similar amounts. In contrast with HSP22, LEAM displayed different isoforms according to apparent molecular mass. Each of the 89 genotypes harboured a single LEAM isoform. Genomic and RT-PCR analysis revealed four LEAM genes differing by a small variable indel in the coding region. These variations were consistent with the apparent molecular mass of each isoform. Indels, which occurred in repeated domains, did not alter the main properties of LEAM. Structural modelling indicated that the class A α-helix structure, which allows interactions with the mitochondrial inner membrane in the dry state, was preserved in all isoforms, suggesting functionality is maintained. The overall results point out the essential character of LEAM and HSP22 in pea seeds. LEAM variability is discussed in terms of pea breeding history as well as LEA gene evolution mechanisms.

Molecular characterization of the acquisition of longevity during seed maturation in soybean

Seed longevity, defined as the ability to remain alive during storage, is an important agronomic factor. Poor longevity negatively impacts seedling establishment and consequently crop yield. This is particularly problematic for soybean as seeds have a short lifespan. While the economic importance of soybean has fueled a large number of transcriptome studies during embryogenesis and seed filling, the mechanisms regulating seed longevity during late maturation remain poorly understood. Here, a detailed physiological and molecular characterization of late seed maturation was performed in soybean to obtain a comprehensive overview of the regulatory genes that are potentially involved in longevity. Longevity appeared at physiological maturity at the end of seed filling before maturation drying and progressively doubled until the seeds reached the dry state. The increase in longevity was associated with the expression of genes encoding protective chaperones such as heat shock proteins and the repression of nuclear and chloroplast genes involved in a range of chloroplast activities, including photosynthesis. An increase in the raffinose family oligosaccharides (RFO)/sucrose ratio together with changes in RFO metabolism genes was also associated with longevity. A gene co-expression network analysis revealed 27 transcription factors whose expression profiles were highly correlated with longevity. Eight of them were previously identified in the longevity network of Medicago truncatula, including homologues of ERF110, HSF6AB, NFXL1 and members of the DREB2 family. The network also contained several transcription factors associated with auxin and developmental cell fate during flowering, organ growth and differentiation. A transcriptional transition occurred concomitant with seed chlorophyll loss and detachment from the mother plant, suggesting the activation of a post-abscission program. This transition was enriched with AP2/EREBP and WRKY transcription factors and genes associated with growth, germination and post-transcriptional processes, suggesting that this program prepares the seed for the dry quiescent state and germination.