Enrique López-Juez

Biogenesis and homeostasis of chloroplasts and other plastids

Chloroplasts are the organelles that define plants, and they are responsible for photosynthesis as well as numerous other functions. They are the ancestral members of a family of organelles known as plastids. Plastids are remarkably dynamic, existing in strikingly different forms that interconvert in response to developmental or environmental cues. The genetic system of this organelle and its coordination with the nucleocytosolic system, the import and routing of nucleus-encoded proteins, as well as organellar division all contribute to the biogenesis and homeostasis of plastids. They are controlled by the ubiquitin–proteasome system, which is part of a network of regulatory mechanisms that integrate plastid development into broader programmes of cellular and organismal development.

Emission of methane from plants

It has been proposed that plants are capable of producing methane by a novel and unidentified biochemical pathway. Emission of methane with an apparently biological origin was recorded from both whole plants and detached leaves. This was the first report of methanogenesis in an aerobic setting, and was estimated to account for 10–45 per cent of the global methane source. Here, we show that plants do not contain a known biochemical pathway to synthesize methane. However, under high UV stress conditions, there may be spontaneous breakdown of plant material, which releases methane. In addition, plants take up and transpire water containing dissolved methane, leading to the observation that methane is released. Together with a new analysis of global methane levels from satellite retrievals, we conclude that plants are not a major source of the global methane production.

Integrative Transcript and Metabolite Analysis of Nutritionally Enhanced DE-ETIOLATED1 Downregulated Tomato Fruit

The downregulation of DE-ETIOLATED1 (DET1) results in tomato fruits with the simultaneous elevation of multiple nutritional antioxidants. Characterization through the analysis and integration of large-scale metabolomic and transcriptomic data sets reveals a coordinated activation of core metabolic processes underlying DET1 loss of function in developing tomato fruit. Fruit-specific downregulation of the DE-ETIOLATED1 (DET1) gene product results in tomato fruits (Solanum lycopersicum) containing enhanced nutritional antioxidants, with no detrimental effects on yield. In an attempt to further our understanding of how modulation of this gene leads to improved quality traits, detailed targeted and multilevel omic characterization has been performed. Metabolite profiling revealed quantitative increases in carotenoid, tocopherol, phenylpropanoids, flavonoids, and anthocyanidins. Qualitative differences could also be identified within the phenolics, including unique formation in fruit pericarp tissues. These changes resulted in increased total antioxidant content both in the polar and nonpolar fractions. Increased transcription of key biosynthetic genes is a likely mechanism producing elevated phenolic-based metabolites. By contrast, high levels of isoprenoids do not appear to result from transcriptional regulation but are more likely related to plastid-based parameters, such as increased plastid volume per cell. Parallel metabolomic and transcriptomic analyses reveal the widespread effects of DET1 downregulation on diverse sectors of metabolism and sites of synthesis. Correlation analysis of transcripts and metabolites independently indicated strong coresponses within and between related pathways/processes. Interestingly, despite the fact that secondary metabolites were the most severely affected in ripe tomato fruit, our integrative analyses suggest that the coordinated activation of core metabolic processes in cell types amenable to plastid biogenesis is the main effect of DET1 loss of function.

Distinct Light-Initiated Gene Expression and Cell Cycle Programs in the Shoot Apex and Cotyledons of Arabidopsis

In darkness, shoot apex growth is repressed, but it becomes rapidly activated by light. We show that phytochromes and cryptochromes play largely redundant roles in this derepression in Arabidopsis thaliana. We examined the light activation of transcriptional changes in a finely resolved time course, comparing the shoot apex (meristem and leaf primordia) and the cotyledon and found >5700 differentially expressed genes. Early events specific to the shoot apices included the repression of genes for Really Interesting New Gene finger proteins and basic domain/leucine zipper and basic helix-loop-helix transcription factors. The downregulation of auxin and ethylene and the upregulation of cytokinin and gibberellin hormonal responses were also characteristic of shoot apices. In the apex, genes involved in ribosome biogenesis and protein translation were rapidly and synchronously induced, simultaneously with cell proliferation genes, preceding visible organ growth. Subsequently, the activation of signaling genes and transcriptional signatures of cell wall expansion, turgor generation, and plastid biogenesis were apparent. Furthermore, light regulates the forms and protein levels of two transcription factors with opposing functions in cell proliferation, E2FB and E2FC, through the Constitutively Photomorphogenic1 (COP1), COP9-Signalosome5, and Deetiolated1 light signaling molecules. These data provide the basis for reconstruction of the regulatory networks for light-regulated meristem, leaf, and cotyledon development.

Light quantity controls leaf-cell and chloroplast development in Arabidopsis thaliana wild type and blue-light-perception mutants

Abstract. Plants acclimate to changes in light quantity by altering leaf-cell development and the accumulation of chloroplast components, such that light absorption is favoured under limiting illumination, and light utilisation under non-limiting conditions. Previous evidence suggests an involvement of a high-light photosynthetic redox signal in the down-regulation of the accumulation of the light-harvesting complexes of photosystem II (Lhcb) and the expression of the Lhcb genes, and of a blue-light signal in the control of leaf development and in the increase in photosynthetic capacity, as affected by the accumulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). We examined the internal anatomy of leaves, the ultrastructure of chloroplasts and accumulation of light-harvesting complexes and Rubisco in wild-type Arabidopsis thaliana (L.) Heynh. and in mutants in each of the three known blue-light photoreceptors, cryptochrome 1, cryptochrome 2 and phototropin, as well as a mutant in both cryptochromes. Our results indicate an extensive capacity of the Arabidopsis mesophyll cells to adapt to high light fluence rate with an increase in palisade elongation. Under high light, chloroplasts showed increased starch accumulation and reductions in the amount of granal thylakoids per chloroplast, in the proportion of chlorophyll b relative to chlorophyll a, and in the accumulation of the major Lhcb polypeptides. The responses were similar for all four mutants, with respect to their wild types. The results are consistent with either a complete redundancy in function between cryptochromes and phototropin, or their absence of involvement in the light-quantity responses tested. We observed minimal effects of light quantity on Rubisco accumulation over the range of fluence rates used, and conclude that elongation of palisade mesophyll cells and accumulation of Rubisco are controlled separately. This indicates that light acclimation must be the result of a number of individual elementary responses. Quantitative differences in the acclimatory responses were observed between the Landsberg erecta and Columbia wild-type ecotypes used.

Vitellogenin: a review of analytical methods to detect (anti) estrogenic activity in fish.

Vitellogenin, the fish egg yolk precursor protein, is the most common biochemical endpoint in general use for the detection of (anti) estrogen active substances in fish and other oviparous species. This review aims to cover the major methods (both protein and nucleic acid) for vitellogenin determination. Comparisons are drawn between vitellogenin and other endpoint effect levels. This review highlights strengths and weaknesses of the various techniques, concentrating on practical considerations and problems in evaluating the data they generate.

Phytochrome, gibberellins, and hypocotyl growth : a study using the cucumber (Cucumis sativus L.) long hypocotyl mutant

The possible involvement of gibberellins (GAs) in the regulation of hypocotyl elongation by phytochrome was examined. Under white light the tall long hypocotyl (lh) cucumber (Cucumis sativus L.) mutant, deficient in a type B-like phytochrome, shows an increased “responsiveness” (defined as response capability) to applied GA4 (the main endogenous active GA) compared to the wild type. Supplementing far-red irradiation results in a similar increase in responsiveness in the wild type. Experiments involving application of the precursor GA9 and of an inhibitor of GA4 inactivation suggest that both the GA4 activation and inactivation steps are phytochrome independent. Endogenous GA levels of whole seedlings were analyzed by combined gas chromatography-mass spectrometry using deuterated internal standards. The levels of GA4 (and those of GA34, the inactivated GA4) were lower in the lh mutant under low-irradiance fluorescent light compared with the wild type, similar to wild type under higher irradiance light during the initial hypocotyl extension phase, and higher during the phase of sustained growth, in which extension involved an increase in the number of cells in the upper region. In all cases, growth of the lh mutant was more rapid than that of the wild type. It is proposed that GA4 and phytochrome control cell elongation primarily through separate mechanisms that interact at a step close to the terminal response.

A role for SENSITIVE TO FREEZING2 in protecting chloroplasts against freeze-induced damage in Arabidopsis.

SUMMARY The sensitive to freezing2 (SFR2) gene has an important role in freezing tolerance in Arabidopsis thaliana. We show that homologous genes are present, and expressed, in a wide range of terrestrial plants, including species not able to tolerate freezing. Expression constructs derived from the cDNAs of a number of different plant species, including examples not tolerant to freezing, are able to complement the freezing sensitivity of the Arabidopsis sfr2 mutant. In Arabidopsis the SFR2 protein is localized to the chloroplast outer envelope membrane, as revealed by the analysis of transgenic plants expressing SFR2 fusions to GFP, by confocal microscopy, and by the immunological analysis of isolated chloroplasts treated with thermolysin protease. Moreover, the chloroplasts of the sfr2 mutant show clear evidence of rapid damage after a freezing episode, suggesting a role for SFR2 in the protection of the chloroplast.

Response of light-grown wild-type and long hypocotyl mutant cucumber plants to end-of-day far-red light.

Abstract— A long‐hypocotyl mutant (lh) of cucumber (Cucumis sativus L.) has been studied which has previously been shown to lack phytochrome control of growth in de‐etiolated seedlings and thought to be modified with respect to the light‐stable type of phytochrome. We have analyzed the response of lh mutant and isogenic wild‐type (WT) plants to daily treatment with end‐of‐day far‐red light (EODFR). Only the WT responded to this treatment resulting in a large increase in internode length; an increase in petiole length; changes in leaf development (increased area, decreased thickness and reduction in indentation); redistribution of dry matter from leaf blades to stem; increased apical dominance and promotion of tendril formation. There were only small or no significant effects on chlorophyll and total carotenoid content, chlorophyll alb ratio, soluble protein levels and net photosyn‐thetic rates. The lh mutant failed to respond to EODFR treatment, and had the appearance of a shade‐avoiding plant growing in extreme shade. The lh mutant appears to completely lack the phytochrome responses attributable to the type of phytochrome that is active in shade detection. A discussion of the possible roles of the stable and labile types of phytochrome in light grown plants follows.

Response of light-grown wild-type and aurea-mutant tomato plants to end-of-day far-red light.

Abstract The effect of end-of-day (EOD) far-red (FR) light treatment on tomato (Lycopersicon esculentum Mill.) plants of the wild type (WT) and an aurea (au) mutant (which is deficient in the spectrophotometrically detectable light-labile phytochrome pool in etiolated seedlings) was studied. Both the WT and au mutant exhibit a quantitatively similar EOD-FR response (i.e. stimulation of elongation growth) indicating that phytochrome is functional in light-grown seedlings of the au mutant. However, no dramatic effects of EOD-FR light on leaf elongation, chlorophyll, carotenoid and soluble protein levels were observed in either the WT or au mutant. The au mutant contained less chlorophyll and more soluble protein per unit fresh weight than the WT. Although practically no anthocyanin could be detected in leaves of the au mutant, the WT showed a tenfold reduction in anthocyanin in response to EOD-FR light. The lower productivity of the au mutant compared with the WT results from its reduced leaf surface area, since its maximum rate of photosynthesis or photosynthetic efficiency appears to be slightly higher than that of the WT. Phytochrome was extracted and partially purified from light-grown plants of the WT and au mutant and was quantified by spectrophotometry. On a fresh weight basis the phytochrome content of au-mutant plants was 66% of that of WT plants. It is proposed that the light-labile phytochrome pool regulates the synthesis of the photosynthetic apparatus in light-grown plants and that the light-stable phytochrome pool functions in the EOD-FR elongation response, the latter pool being present and functional in the au mutant.