An Abscisic Acid-Independent Oxylipin Pathway Controls Stomatal Closure and Immune Defense in Arabidopsis

Plant stomata function in innate immunity against bacterial invasion and abscisic acid (ABA) has been suggested to regulate this process. Using genetic, biochemical, and pharmacological approaches, we demonstrate that (i) the Arabidopsis thaliana nine-specific-lipoxygenase encoding gene, LOX1, which is expressed in guard cells, is required to trigger stomatal closure in response to both bacteria and the pathogen-associated molecular pattern flagellin peptide flg22; (ii) LOX1 participates in stomatal defense; (iii) polyunsaturated fatty acids, the LOX substrates, trigger stomatal closure; (iv) the LOX products, fatty acid hydroperoxides, or reactive electrophile oxylipins induce stomatal closure; and (v) the flg22-mediated stomatal closure is conveyed by both LOX1 and the mitogen-activated protein kinases MPK3 and MPK6 and involves salicylic acid whereas the ABA-induced process depends on the protein kinases OST1, MPK9, or MPK12. Finally, we show that the oxylipin and the ABA pathways converge at the level of the anion channel SLAC1 to regulate stomatal closure. Collectively, our results demonstrate that early biotic signaling in guard cells is an ABA-independent process revealing a novel function of LOX1-dependent stomatal pathway in plant immunity.     

The response of the grape berry moth (Lobesia botrana) to a dietary phytopathogenic fungus (Botrytis cinerea): the significance of fungus sterols

A Tortricidae (Lobesia botrana) has a mutualistic relationship with the fungus (Botrytis cinerea). In this study, we investigated the growth, survival, fecundity and amount of sterols and steroids in larvae of this vineyard pest reared on artificial diets containing mycelium (3%) or purified sterols (0.01%) of the phytopathogenic fungus. Two principal questions related to the physiological and biochemical basis of this mutualistic relationship were addressed: (1) how the fungus influences growth, survival, fecundity, sterol and steroid contents of the insect and (2) are fungal sterols involved in the biochemical basis of mutualism? The presence of fungus in the diet led to a decrease of total duration of larval development (mean gain 5.1-9.4 days compared to the total duration in control of 42.9 days), an increase in survival (mean gain 50-76.3%) and fecundity (gain of 94-102%). These positive effects of the fungus on the biology and physiology of the insect were directly correlated to the presence of fungal sterols in the diet. Fungal sterols are one of the biochemical basis of the mutualistic relationship between L. botrana and B. cinerea.     

A Medicago truncatula Tobacco Retrotransposon Insertion Mutant Collection with Defects in Nodule Development and Symbiotic Nitrogen Fixation

A Tnt1-insertion mutant population of Medicago truncatula ecotype R108 was screened for defects in nodulation and symbiotic nitrogen fixation. Primary screening of 9,300 mutant lines yielded 317 lines with putative defects in nodule development and/or nitrogen fixation. Of these, 230 lines were rescreened, and 156 lines were confirmed with defective symbiotic nitrogen fixation. Mutants were sorted into six distinct phenotypic categories: 72 nonnodulating mutants (Nod-), 51 mutants with totally ineffective nodules (Nod+ Fix-), 17 mutants with partially ineffective nodules (Nod+ Fix+/-), 27 mutants defective in nodule emergence, elongation, and nitrogen fixation (Nod+/- Fix-), one mutant with delayed and reduced nodulation but effective in nitrogen fixation (dNod+/- Fix+), and 11 supernodulating mutants (Nod++Fix+/-). A total of 2,801 flanking sequence tags were generated from the 156 symbiotic mutant lines. Analysis of flanking sequence tags revealed 14 insertion alleles of the following known symbiotic genes: NODULE INCEPTION (NIN), DOESN'T MAKE INFECTIONS3 (DMI3/CCaMK), ERF REQUIRED FOR NODULATION, and SUPERNUMERARY NODULES (SUNN). In parallel, a polymerase chain reaction-based strategy was used to identify Tnt1 insertions in known symbiotic genes, which revealed 25 additional insertion alleles in the following genes: DMI1, DMI2, DMI3, NIN, NODULATION SIGNALING PATHWAY1 (NSP1), NSP2, SUNN, and SICKLE. Thirty-nine Nod- lines were also screened for arbuscular mycorrhizal symbiosis phenotypes, and 30 mutants exhibited defects in arbuscular mycorrhizal symbiosis. Morphological and developmental features of several new symbiotic mutants are reported. The collection of mutants described here is a source of novel alleles of known symbiotic genes and a resource for cloning novel symbiotic genes via Tnt1 tagging.     

NODULE ROOT and COCHLEATA Maintain Nodule Development and Are Legume Orthologs of Arabidopsis BLADE-ON-PETIOLE Genes

During their symbiotic interaction with rhizobia, legume plants develop symbiosis-specific organs on their roots, called nodules, that house nitrogen-fixing bacteria. The molecular mechanisms governing the identity and maintenance of these organs are unknown. Using Medicago truncatula nodule root (noot) mutants and pea (Pisum sativum) cochleata (coch) mutants, which are characterized by the abnormal development of roots from the nodule, we identified the NOOT and COCH genes as being necessary for the robust maintenance of nodule identity throughout the nodule developmental program. NOOT and COCH are Arabidopsis thaliana BLADE-ON-PETIOLE orthologs, and we have shown that their functions in leaf and flower development are conserved in M. truncatula and pea. The identification of these two genes defines a clade in the BTB/POZ-ankyrin domain proteins that shares conserved functions in eudicot organ development and suggests that NOOT and COCH were recruited to repress root identity in the legume symbiotic organ.     

T-DNA tagging in the model legume Medicago truncatula allows efficient gene discovery

The annual legume Medicago truncatula has been proposed as a model plant to study various aspects of legume biology including rhizobial and mycorrhizal symbiosis because it is well suited for the genetic analysis of these processes . To facilitate the characterization of M. truncatula genes participating in various developmental processes we have initiated an insertion mutagenesis program in this plant using three different T-DNAs as tags. To investigate which type of vector is the most suitable for mutagenesis we compared the behavior of these T-DNAs. One T-DNA vector was a derivative of pBin19 and plant selection was based on kanamycin resistance. The two other vectors carried T-DNA conferring Basta resistance in the transgenic plants. For each T-DNA type, we determined the copy number in the transgenic lines, the structure of the T-DNA loci and the sequences of the integration sites. The T-DNA derived from pBin19 generated complex T-DNA insertion patterns. The two others generally gave single copy T-DNA inserts that could result in gene fusions for the pGKB5 T-DNA. Analysis of the T-DNA borders revealed that several M. truncatula genes were tagged in these transgenic lines and in vivo gus fusions were also obtained. These results demonstrate that T-DNA tagging can efficiently be used in M. truncatula for gene discovery.     

The energetic costs of case construction in the caddisfly Limnephilus rhombicus: direct impacts on larvae and delayed impacts on adults

Caddisflies, whose aquatic larvae build a portable case with silk, are a suitable model organism to test the impacts of resource allocation trade-off during development and examine the evolution of life-history strategies. In the caddisfly Limnephilus rhombicus, adult feeding is minimal. Therefore, the whole resources are acquired during the larval phase and must be allocated to case construction, growth and reproduction. In this study, the larval energetic reserves of L. rhombicus were manipulated by forcing larvae to rebuild their cases in the final larval stage. This allowed us to measure the physiological cost of construction. First, we recorded oxygen consumption during case reconstruction. Second, we measured the sugar, protein and lipid contents of larvae forced to rebuild their case and of larvae required only to re-enter on their case. Larvae had their sugar, protein and lipid content measured after the rebuilding event and 72 h later. The same analyses were carried out with adults immediately after emergence. We found that larvae forced to rebuild a case consumed 1.5 times more oxygen than control larvae. This energy expenditure generated a cost that was estimated to be a loss of larval protein of approximately 35%. Insects were unable to compensate for this loss of proteins during the end of the larval stage, and their metamorphosis to adults was also impacted. Therefore, we suggest that loss of larval protein is linked to silk production and may alter fitness.     

A Conserved Mechanism of GABA Binding and Antagonism Is Revealed by Structure-Function Analysis of the Periplasmic Binding Protein Atu2422 in Agrobacterium tumefaciens

Bacterial periplasmic binding proteins (PBPs) and eukaryotic PBP-like domains (also called as Venus flytrap modules) of G-protein-coupled receptors are involved in extracellular GABA perception. We investigated the structural and functional basis of ligand specificity of the PBP Atu2422, which is implicated in virulence and transport of GABA in the plant pathogen Agrobacterium tumefaciens. Five high-resolution x-ray structures of Atu2422 liganded to GABA, Pro, Ala, and Val and of point mutant Atu2422-F77A liganded to Leu were determined. Structural analysis of the ligand-binding site revealed two essential residues, Phe⁷⁷ and Tyr²⁷⁵, the implication of which in GABA signaling and virulence was confirmed using A. tumefaciens cells expressing corresponding Atu2422 mutants. Phe⁷⁷ restricts ligand specificity to α-amino acids with a short lateral chain, which act as antagonists of GABA signaling in A. tumefaciens. Tyr²⁷⁵ specifically interacts with the GABA γ-amino group. Conservation of these two key residues in proteins phylogenetically related to Atu2422 brought to light a subfamily of PBPs in which all members could bind GABA and short α-amino acids. This work led to the identification of a fingerprint sequence and structural features for defining PBPs that bind GABA and its competitors and revealed their occurrence among host-interacting proteobacteria.     

Importance of sterols acquired through host feeding in synovigenic parasitoid oogenesis

Eupelmus vuilleti (Hymenoptera; Eupelmidae) is a host feeding ectoparasitoid of fourth-instar larvae or pupae of Callosobruchus maculatus (Coleoptera; Bruchidae) infecting Vigna unguiculata seed and pods (Fabacae). Parasitoid females are synovigenic, i.e. they are born with immature eggs and need to feed from the host in order to sustain egg production. In this study, the role of sterols obtained through host feeding in parasitoid oogenesis are examined. Quantitative and qualitative analyses of the sterol contents in each partner of the tritrophic interaction show that a parasitoid female's larval sterol contents is sufficient to produce only 30% of the total number of eggs laid throughout a female's life cycle. In a second step, by manipulating the composition of the sterols hemolymph in the host, it is shown that cholesterol obtained through adult nutrition plays a crucial role in the eggs viability but does not affect the egg production quantitatively. This result has important implications for understanding both the nutrient allocation strategy in this species and the impact of cholesterol in parasitoid reproduction.     

Extending the growth rate hypothesis to species development: Can stoichiometric traits help to explain the composition of macroinvertebrate communities?

Ecological stoichiometry seeks to understand the ecological consequences of elemental imbalances between consumers and their resources. Therein, the well-accepted growth rate hypothesis (GRH) states that organisms exhibiting rapid growth have higher phosphorus (P) demand – and thus lower C:P and N:P ratios – than slow growing ones, due to a higher allocation to P-rich rRNA. However, GRH has rarely been extended to other biological traits than growth, especially at the community level. In this study, we investigated whether macroinvertebrate stoichiometric traits (e.g. C:P and N:P ratios) can be linked to their development traits, and whether these stoichiometric traits are related to macroinvertebrate community assemblage under different nutrient conditions. We allocated more than 400 European taxa to different groups, defined using available information about three development-related traits: ‘life span', ‘voltinism' and ‘number of reproductive cycles per individual'. We sampled 18 invertebrate taxa in six streams exhibiting different levels of nutrient concentration and measured their stoichiometric traits. Further, we quantified invertebrate taxon abundances in these streams during an annual survey. Based on these data, we tested whether community composition regarding the developmental groups differs, depending on nutrient concentration. We found significant differences in the proportions of the developmental groups along a gradient of water N:P, in relation to their stoichiometric traits. Taxa with low C:P and N:P ratios were generally associated with faster development groups, and these taxa tended to occur at higher proportions in streams exhibiting low dissolved N:P ratios. In contrast, communities from P-poor, high dissolved N:P streams, were dominated by slowly developing taxa with high N:P ratios. Our results highlight that extending the GRH to species development rate might give some insights about the mechanisms by which nutrient concentrations in ecosystems influence consumers' community composition.