Postharvest changes in LIN5-down-regulated plants suggest a role for sugar deficiency in cuticle metabolism during ripening.

The cell wall invertase gene (LIN5) was reported to be a key enzyme influencing sugar uptake of tomato (Solanum lycopersicum) fruit. It was additionally revealed to be a key regulator of total soluble solids content in fruit as well as for reproductive development, being mainly involved in flower development, early fruit and seed development but also in ripening. Here, we demonstrate that silencing of the LIN5 gene promotes changes affecting fruit cuticle development which has a direct effect on postharvest properties. Transformants were characterized by reduced transpirational water loss in mature fruits accompanied by several other changes in the cuticle. Quantitative chemical composition, coupled with microscopy of isolated cuticle fruits revealed that the cuticle of the transformants were characterized by an increase of the thickness as well as significant increase in the content of cuticle components (cutin, phenolic compounds, and waxes). Furthermore, detailed analysis of the waxes revealed that the transformants displayed changes in waxes composition, showing higher levels of n-alkanes and triterpenoids which can shift the proportion of crystalline and amorphous waxes and change the water flux through the cuticle. Expression of the genes involved in cuticle biosynthesis indicated that LIN5 influences the biosynthesis of components of the cuticle, indicating that this process is coupled to sugar uploading via a mechanism which links carbon supply with the capacity for fruit expansion.

Durum Wheat Roots Adapt to Salinity Remodeling the Cellular Content of Nitrogen Metabolites and Sucrose.

Plants are currently experiencing increasing salinity problems due to irrigation with brackish water. Moreover, in fields, roots can grow in soils which show spatial variation in water content and salt concentration, also because of the type of irrigation. Salinity impairs crop growth and productivity by inhibiting many physiological and metabolic processes, in particular nitrate uptake, translocation, and assimilation. Salinity determines an increase of sap osmolality from about 305 mOsmol kg-1 in control roots to about 530 mOsmol kg-1 in roots under salinity. Root cells adapt to salinity by sequestering sodium in the vacuole, as a cheap osmoticum, and showing a rearrangement of few nitrogen-containing metabolites and sucrose in the cytosol, both for osmotic adjustment and oxidative stress protection, thus providing plant viability even at low nitrate levels. Mainly glycine betaine and sucrose at low nitrate concentration, and glycine betaine, asparagine and proline at high nitrate levels can be assumed responsible for the osmotic adjustment of the cytosol, the assimilation of the excess of ammonium and the scavenging of ROS under salinity. High nitrate plants with half of the root system under salinity accumulate proline and glutamine in both control and salt stressed split roots, revealing that osmotic adjustment is not a regional effect in plants. The expression level and enzymatic activities of asparagine synthetase and Δ1-pyrroline-5-carboxylate synthetase, as well as other enzymatic activities of nitrogen and carbon metabolism, are analyzed.

Reproductive failure in Arabidopsis thaliana under transient carbohydrate limitation: flowers and very young siliques are jettisoned and the meristem is maintained to allow successful resumption of reproductive growth.

The impact of transient carbon depletion on reproductive growth in Arabidopsis was investigated by transferring long-photoperiod-grown plants to continuous darkness and returning them to a light-dark cycle. After 2 days of darkness, carbon reserves were depleted in reproductive sinks, and RNA in situ hybridization of marker transcripts showed that carbon starvation responses had been initiated in the meristem, anthers and ovules. Dark treatments of 2 or more days resulted in a bare-segment phenotype on the floral stem, with 23-27 aborted siliques. These resulted from impaired growth of immature siliques and abortion of mature and immature flowers. Depolarization of PIN1 protein and increased DII-VENUS expression pointed to rapid collapse of auxin gradients in the meristem and inhibition of primordia initiation. After transfer back to a light-dark cycle, flowers appeared and formed viable siliques and seeds. A similar phenotype was seen after transfer to sub-compensation point irradiance or CO2 . It also appeared in a milder form after a moderate decrease in irradiance and developed spontaneously in short photoperiods. We conclude that Arabidopsis inhibits primordia initiation and aborts flowers and very young siliques in C-limited conditions. This curtails demand, safeguarding meristem function and allowing renewal of reproductive growth when carbon becomes available again.

Multiple RNA processing defects and impaired chloroplast function in plants deficient in the organellar protein-only RNase P enzyme.

Transfer RNA (tRNA) precursors undergo endoribonucleolytic processing of their 5' and 3' ends. 5' cleavage of the precursor transcript is performed by ribonuclease P (RNase P). While in most organisms RNase P is a ribonucleoprotein that harbors a catalytically active RNA component, human mitochondria and the chloroplasts (plastids) and mitochondria of seed plants possess protein-only RNase P enzymes (PRORPs). The plant organellar PRORP (PRORP1) has been characterized to some extent in vitro and by transient gene silencing, but the molecular, phenotypic and physiological consequences of its down-regulation in stable transgenic plants have not been assessed. Here we have addressed the function of the dually targeted organellar PRORP enzyme in vivo by generating stably transformed Arabidopsis plants in which expression of the PRORP1 gene was suppressed by RNA interference (RNAi). PRORP1 knock-down lines show defects in photosynthesis, while mitochondrial respiration is not appreciably affected. In both plastids and mitochondria, the effects of PRORP1 knock-down on the processing of individual tRNA species are highly variable. The drastic reduction in the levels of mature plastid tRNA-Phe(GAA) and tRNA-Arg(ACG) suggests that these two tRNA species limit plastid gene expression in the PRORP1 mutants and, hence, are causally responsible for the mutant phenotype.

Consequences of induced brassinosteroid deficiency in Arabidopsis leaves.

BACKGROUND: The identification of brassinosteroid (BR) deficient and BR insensitive mutants provided conclusive evidence that BR is a potent growth-promoting phytohormone. Arabidopsis mutants are characterized by a compact rosette structure, decreased plant height and reduced root system, delayed development, and reduced fertility. Cell expansion, cell division, and multiple developmental processes depend on BR. The molecular and physiological basis of BR action is diverse. The BR signalling pathway controls the activity of transcription factors, and numerous BR responsive genes have been identified. The analysis of dwarf mutants, however, may to some extent reveal phenotypic changes that are an effect of the altered morphology and physiology. This restriction holds particularly true for the analysis of established organs such as rosette leaves. RESULTS: In this study, the mode of BR action was analysed in established leaves by means of two approaches. First, an inhibitor of BR biosynthesis (brassinazole) was applied to 21-day-old wild-type plants. Secondly, BR complementation of BR deficient plants, namely CPD (constitutive photomorphogenic dwarf)-antisense and cbb1 (cabbage1) mutant plants was stopped after 21 days. BR action in established leaves is associated with stimulated cell expansion, an increase in leaf index, starch accumulation, enhanced CO2 release by the tricarboxylic acid cycle, and increased biomass production. Cell number and protein content were barely affected. CONCLUSION: Previous analysis of BR promoted growth focused on genomic effects. However, the link between growth and changes in gene expression patterns barely provided clues to the physiological and metabolic basis of growth. Our study analysed comprehensive metabolic data sets of leaves with altered BR levels. The data suggest that BR promoted growth may depend on the increased provision and use of carbohydrates and energy. BR may stimulate both anabolic and catabolic pathways.

Downregulation of the δ-subunit reduces mitochondrial ATP synthase levels, alters respiration, and restricts growth and gametophyte development in Arabidopsis.

The mitochondrial ATP synthase (F(1)F(o) complex) is an evolutionary conserved multimeric protein complex that synthesizes the main bulk of cytosolic ATP, but the regulatory mechanisms of the subunits are only poorly understood in plants. In yeast, the δ-subunit links the membrane-embedded F(o) part to the matrix-facing central stalk of F(1). We used genetic interference and an inhibitor to investigate the molecular function and physiological impact of the δ-subunit in Arabidopsis thaliana. Delta mutants displayed both male and female gametophyte defects. RNA interference of delta resulted in growth retardation, reduced ATP synthase amounts, and increased alternative oxidase capacity and led to specific long-term increases in Ala and Gly levels. By contrast, inhibition of the complex using oligomycin triggered broad metabolic changes, affecting glycolysis and the tricarboxylic acid cycle, and led to a successive induction of transcripts for alternative respiratory pathways and for redox and biotic stress-related transcription factors. We conclude that (1) the δ-subunit is essential for male gametophyte development in Arabidopsis, (2) a disturbance of the ATP synthase appears to lead to an early transition phase and a long-term metabolic steady state, and (3) the observed long-term adjustments in mitochondrial metabolism are linked to reduced growth and deficiencies in gametophyte development.

The plastid-specific ribosomal proteins of Arabidopsis thaliana can be divided into non-essential proteins and genuine ribosomal proteins.

Plastid translation occurs on bacterial-type 70S ribosomes consisting of a large (50S) subunit and a small (30S) subunit. The vast majority of plastid ribosomal proteins have orthologs in bacteria. In addition, plastids also possess a small set of unique ribosomal proteins, so-called plastid-specific ribosomal proteins (PSRPs). The functions of these PSRPs are unknown, but, based on structural studies, it has been proposed that they may represent accessory proteins involved in translational regulation. Here we have investigated the functions of five PSRPs using reverse genetics in the model plant Arabidopsis thaliana. By analyzing T-DNA insertion mutants and RNAi lines, we show that three PSRPs display characteristics of genuine ribosomal proteins, in that down-regulation of their expression led to decreased accumulation of the 30S or 50S subunit of the plastid ribosomes, resulting in plastid translational deficiency. In contrast, two other PSRPs can be knocked out without visible or measurable phenotypic consequences. Our data suggest that PSRPs fall into two types: (i) PSRPs that have a structural role in the ribosome and are bona fide ribosomal proteins, and (ii) non-essential PSRPs that are not required for stable ribosome accumulation and translation under standard greenhouse conditions.

Colonization of roots of cultivated Solanum lycopersicum by dark septate and other ascomycetous endophytes.

Tomato (Solanum lycopersicum L.) roots from four different crop sites in Colombia were surface sterilized and 51 fungal isolates were obtained and conserved for further analysis. Based on microscopical observations and growth characteristics, 20 fungal isolates corresponded to genus Fusarium, six presented asexual conidia different from Fusarium, eight were sterile mycelia, seven of which had dark septate hyphae and 17 did not continue to grow on plates after being recovered from conservation. Growth on different media, detailed morphological characterization and ITS region sequencing of the six sporulating and eight sterile isolates revealed that they belonged to different orders of Ascomycota and that the sterile dark septate endophytes did not correspond to the well known Phialocephala group. Interactions of nine isolates with tomato plantlets were assessed in vitro. No effect on shoot development was revealed, but three isolates caused brown spots in roots. Colonization patterns as analyzed by confocal microscopy differed among the isolates and ranged from epidermal to cortical penetration. Altogether 11 new isolates from root endophytic fungi were obtained, seven of which showed features of dark septate endophytes. Four known morphotypes were represented by five isolates, while six isolates belonged to five morphotypes of putative new unknown species.

Tricarboxylic acid cycle activity regulates tomato root growth via effects on secondary cell wall production.

Transgenic tomato (Solanum lycopersicum 'Moneymaker') plants independently expressing fragments of various genes encoding enzymes of the tricarboxylic acid cycle in antisense orientation have previously been characterized as exhibiting altered root growth. In this study, we evaluate the rates of respiration of roots from these lines in addition to determining their total dry weight accumulation. Given that these features were highly correlated, we decided to carry out an evaluation of the cell wall composition in the transformants that revealed a substantial reduction in cellulose. Since the bulk of cellulose is associated with the secondary cell walls in roots, we reasoned that the transformants most likely were deficient in secondary wall cellulose production. Consistent with these findings, cross-sections of the root collar (approximately 15 mm from the junction between root and stem) displayed reduced lignified secondary cell walls for the transformants. In contrast, cell and cell wall patterning displayed no differences in elongating cells close to the root tip. To further characterize the modified cell wall metabolism, we performed feeding experiments in which we incubated excised root tips in [U-(14)C]glucose in the presence or absence of phosphonate inhibitors of the reaction catalyzed by 2-oxoglutarate dehydrogenase. Taken together, the combined results suggest that restriction of root respiration leads to a deficit in secondary cell wall synthesis. These data are discussed in the context of current models of biomass partitioning and plant growth.