SEIPIN Proteins Mediate Lipid Droplet Biogenesis to Promote Pollen Transmission and Reduce Seed Dormancy.

Lipid droplets (LDs) are ubiquitous organelles in plant cells, but their physiological roles are largely unknown. To gain insight into the function of LDs in plants, we have characterized the Arabidopsis homologs of SEIPIN proteins, which are crucial factors for LD biogenesis in yeast and animals. SEIPIN1 is expressed almost exclusively in embryos, while SEIPIN2 and SEIPIN3 have broader expression profiles with maximal levels in embryos and pollen, where LDs accumulate most abundantly. Genetic analysis demonstrates that all three SEIPINs contribute to proper LD biogenesis in embryos, whereas in pollen, only SEIPIN2 and SEIPIN3 play a significant role. The double seipin2 seipin3 and triple seipin mutants accumulate extremely enlarged LDs in seeds and pollen, which hinders their subsequent mobilization during germination. Interestingly, electron microscopy analysis reveals the presence of nuclear LDs attached to type I nucleoplasmic reticulum in triple seipin mutant embryos, supporting that SEIPINs are essential for maintaining the correct polarity of LD budding at the nuclear envelope, restricting it to the outer membrane. In pollen, the perturbations in LD biogenesis and turnover are coupled to reduced germination in vitro and with lower fertilization efficiency in vivo. In seeds, germination per se is not affected in seipin2 seipin3 and triple seipin mutants, but there is a striking increase in seed dormancy levels. Our findings reveal the relevance of SEIPIN-dependent LD biogenesis in pollen transmission and in adjusting the timing of seed germination, two key adaptive traits of great importance in agriculture.

Jasmonate-dependent modifications of the pectin matrix during potato development function as a defense mechanism targeted by Dickeya dadantii virulence factors.

The plant cell wall constitutes an essential protection barrier against pathogen attack. In addition, cell-wall disruption leads to accumulation of jasmonates (JAs), which are key signaling molecules for activation of plant inducible defense responses. However, whether JAs in return modulate the cell-wall composition to reinforce this defensive barrier remains unknown. The enzyme 13-allene oxide synthase (13-AOS) catalyzes the first committed step towards biosynthesis of JAs. In potato (Solanum tuberosum), there are two putative St13-AOS genes, which we show here to be differentially induced upon wounding. We also determine that both genes complement an Arabidopsis aos null mutant, indicating that they encode functional 13-AOS enzymes. Indeed, transgenic potato plants lacking both St13-AOS genes (CoAOS1/2 lines) exhibited a significant reduction of JAs, a concomitant decrease in wound-responsive gene activation, and an increased severity of soft rot disease symptoms caused by Dickeya dadantii. Intriguingly, a hypovirulent D. dadantii pel strain lacking the five major pectate lyases, which causes limited tissue maceration on wild-type plants, regained infectivity in CoAOS1/2 plants. In line with this, we found differences in pectin methyl esterase activity and cell-wall pectin composition between wild-type and CoAOS1/2 plants. Importantly, wild-type plants had pectins with a lower degree of methyl esterification, which are the substrates of the pectate lyases mutated in the pel strain. These results suggest that, during development of potato plants, JAs mediate modification of the pectin matrix to form a defensive barrier that is counteracted by pectinolytic virulence factors from D. dadantii.

Increasing omega-3 desaturase expression in tomato results in altered aroma profile and enhanced resistance to cold stress.

One of the drawbacks in improving the aroma properties of tomato (Solanum lycopersicum) fruit is the complexity of this organoleptic trait, with a great variety of volatiles contributing to determine specific quality features. It is well established that the oxylipins hexanal and (Z)-hex-3-enal, synthesized through the lipoxygenase pathway, are among the most important aroma compounds and impart in a correct proportion some of the unique fresh notes in tomato. Here, we confirm that all enzymes responsible for the synthesis of these C6 compounds are present and active in tomato fruit. Moreover, due to the low odor threshold of (Z)-hex-3-enal, small changes in the concentration of this compound could modify the properties of the tomato fruit aroma. To address this possibility, we have overexpressed the omega-3 fatty acid desaturases FAD3 and FAD7 that catalyze the conversion of linoleic acid (18:2) to linolenic acid (18:3), the precursor of hexenals and its derived alcohols. Transgenic OE-FAD tomato plants exhibit altered fatty acid composition, with an increase in the 18:3/18:2 ratio in leaves and fruits. These changes provoke a clear variation in the C6 content that results in a significant alteration of the (Z)-hex-3-enal/hexanal ratio that is particularly important in ripe OE-FAD3FAD7 fruits. In addition to this effect on tomato volatile profile, OE-FAD tomato plants are more tolerant to chilling. However, the different behaviors of OE-FAD plants underscore the existence of separate fatty acid fluxes to ensure plant survival under adverse conditions.

Differential distribution of the lipoxygenase pathway enzymes within potato chloroplasts.

The lipoxygenase pathway is responsible for the production of oxylipins, which are important compounds for plant defence responses. Jasmonic acid, the final product of the allene oxide synthase/allene oxide cyclase branch of the pathway, regulates wound-induced gene expression. In contrast, C6 aliphatic aldehydes produced via an alternative branch catalysed by hydroperoxide lyase, are themselves toxic to pests and pathogens. Current evidence on the subcellular localization of the lipoxygenase pathway is conflicting, and the regulation of metabolic channelling between the two branches of the pathway is largely unknown. It is shown here that while a 13-lipoxygenase (LOX H3), allene oxide synthase and allene oxide cyclase proteins accumulate upon wounding in potato, a second 13-lipoxygenase (LOX H1) and hydroperoxide lyase are present at constant levels in both non-wounded and wounded tissues. Wound-induced accumulation of the jasmonic acid biosynthetic enzymes may thus commit the lipoxygenase pathway to jasmonic acid production in damaged plants. It is shown that all enzymes of the lipoxygenase pathway differentially localize within chloroplasts, and are largely found associated to thylakoid membranes. This differential localization is consistently observed using confocal microscopy of GFP-tagged proteins, chloroplast fractionation, and western blotting, and immunodetection by electron microscopy. While LOX H1 and LOX H3 are localized both in stroma and thylakoids, both allene oxide synthase and hydroperoxide lyase protein localize almost exclusively to thylakoids and are strongly bound to membranes. Allene oxide cyclase is weakly associated with the thylakoid membrane and is also detected in the stroma. Moreover, allene oxide synthase and hydroperoxide lyase are differentially distributed in thylakoids, with hydroperoxide lyase localized almost exclusively to the stromal part, thus closely resembling the localization pattern of LOX H1. It is suggested that, in addition to their differential expression pattern, this segregation underlies the regulation of metabolic fluxes through the alternative branches of the lipoxygenase pathway.

Molecular characterization and expression studies during melon fruit development and ripening of L-galactono-1,4-lactone dehydrogenase.

The last step of ascorbic acid (AA) biosynthesis is catalysed by the enzyme L-galactono-1,4-lactone dehydrogenase (GalLDH, EC 1.3.2.3), located on the inner mitochondrial membrane. The enzyme converts L-galactono-1,4-lactone to ascorbic acid (AA). In this work, the cloning and characterization of a GalLDH full-length cDNA from melon (Cucumis melo L.) are described. Melon genomic DNA Southern analysis indicated that CmGalLDH was encoded by a single gene. CmGalLDH mRNA accumulation was detected in all tissues studied, but differentially expressed during fruit development and seed germination. It is hypothesized that induction of CmGalLDH gene expression in ripening melon fruit contributes to parallel increases in the AA content and so playing a role in the oxidative ripening process. Higher CmGalLDH message abundance in light-grown seedlings compared with those raised in the dark suggests that CmGalLDH expression is regulated by light. Finally, various stresses and growth regulators resulted in no significant change in steady state levels of CmGalLDH mRNA in 20-d-old melon seedlings. To the authors' knowledge, this is the first report of GalLDH transcript induction in seed germination and differential gene expression during fruit ripening.