Prunus persica apoplastic proteome analysis reveals candidate proteins involved in the resistance and defense against Taphrina deformans.

Taphrina deformans is the fungus responsible for the peach leaf curl disease. To gain insight into the molecular mechanisms involved in plant resistance and response to the fungus, apoplastic differentially abundant proteins (DAPs) in a resistant (DR) and/or in a susceptible genotype (FL) were identified after 12 and 96 h post inoculation (hpi) and compared to those at 0 hpi. The Prunus persica apoplastic proteome was assessed by LC-MS/MS analysis. Altogether 332 proteins were identified, and their molecular and biological functions were classified. In both genotypes, major changes occurred at 96 hpi when the fungus had achieved the filamentous form. However, at 96 hpi, DR exhibited a greater number of increased proteins than FL. DAPs were enriched in biotic stress response, with most of the proteins belonging to the pathogenesis related (PR)-type. PRs exhibited the greatest fold changes of induction in DR. While PRs acting on pathogen cell wall (PR2, PR3 and PR4) were increased in both susceptible and resistant genotypes, others were exclusively induced in DR, such as some isoforms of PR5, defensin and PR17. Proteins exclusively induced in DR upon T.deformans inoculation such as four berberine bridge enzymes, two snakins and a GDS-lipase were identified. Moreover, upon inoculation cuticle was thickened to a greater extent in DR than in FL. This work reveals the active role of the apoplast against T. deformans and not only contributes to the elucidation of responses involved in resistance to leaf curl disease but also improves the knowledge on peach defenses against pathogens.

Stone Fruit as Biofactories of Phytochemicals With Potential Roles in Human Nutrition and Health.

Phytochemicals or secondary metabolites present in fruit are key components contributing to sensory attributes like aroma, taste, and color. In addition, these compounds improve human nutrition and health. Stone fruits are an important source of an array of secondary metabolites that may reduce the risk of different diseases. The first part of this review is dedicated to the description of the main secondary organic compounds found in plants which include (a) phenolic compounds, (b) terpenoids/isoprenoids, and (c) nitrogen or sulfur containing compounds, and their principal biosynthetic pathways and their regulation in stone fruit. Then, the type and levels of bioactive compounds in different stone fruits of the Rosaceae family such as peach (Prunus persica), plum (P. domestica, P. salicina and P. cerasifera), sweet cherries (P. avium), almond kernels (P. dulcis, syn. P. amygdalus), and apricot (P. armeniaca) are presented. The last part of this review encompasses pre- and postharvest treatments affecting the phytochemical composition in stone fruit. Appropriate management of these factors during pre- and postharvest handling, along with further characterization of phytochemicals and the regulation of their synthesis in different cultivars, could help to increase the levels of these compounds, leading to the future improvement of stone fruit not only to enhance organoleptic characteristics but also to benefit human health.

Identification of genes involved in the drought adaptation and recovery in Portulaca oleracea by differential display.

Portulaca oleracea is one of the richest plant sources of ω-3 and ω-6 fatty acids and other compounds potentially valuable for nutrition. It is broadly established in arid, semiarid and well-watered fields, thus making it a promising candidate for research on abiotic stress resistance mechanisms. It is capable of withstanding severe drought and then of recovering upon rehydration. Here, the adaptation to drought and the posterior recovery was evaluated at transcriptomic level by differential display validated by qRT-PCR. Of the 2279 transcript-derived fragments amplified, 202 presented differential expression. Ninety of them were successfully isolated and sequenced. Selected genes were tested against different abiotic stresses in P. oleracea and the behavior of their orthologous genes in Arabidopsis thaliana was also explored to seek for conserved response mechanisms. In drought adapted and in recovered plants changes in expression of many protein metabolism-, lipid metabolism- and stress-related genes were observed. Many genes with unknown function were detected, which also respond to other abiotic stresses. Some of them are also involved in the seed desiccation/imbibition process and thus would be of great interest for further research. The potential use of candidate genes to engineer drought tolerance improvement and recovery is discussed.

Deciphering the mechanisms involved in Portulaca oleracea (C4) response to drought: metabolic changes including crassulacean acid-like metabolism induction and reversal upon re-watering.

Portulaca oleracea is a C(4) plant; however, under drought it can change its carbon fixation metabolism into a crassulacean acid metabolism (CAM)-like one. While the C(3) -CAM shift is well known, the C(4) -CAM transition has only been described in Portulaca. Here, a CAM-like metabolism was induced in P. oleracea by drought and then reversed by re-watering. Physiological and biochemical approaches were undertaken to evaluate the drought and recovery responses. In CAM-like plants, chlorophyll fluorescence parameters were transitory affected and non-radiative energy dissipation mechanisms were induced. Induction of flavonoids, betalains and antioxidant machinery may be involved in photosynthetic machinery protection. Metabolic analysis highlights a clear metabolic shift, when a CAM-like metabolism is induced and then reversed. Increases in nitrogenous compounds like free amino acids and urea, and of pinitol could contribute to withstand drought. Reciprocal variations in arginase and urease in drought-stressed and in re-watered plants suggest urea synthesis is strictly regulated. Recovery of C(4) metabolism was accounted by CO(2) assimilation pattern and malate levels. Increases in glycerol and in polyamines would be of importance of re-watered plants. Collectively, in P. oleracea multiple strategies, from induction of several metabolites to the transitory development of a CAM-like metabolism, participate to enhance its adaptation to drought.

Role of photosynthesis and analysis of key enzymes involved in primary metabolism throughout the lifespan of the tobacco flower.

Although the physiological and economical relevance of flowers is recognized, their primary metabolism during development has not been characterized, especially combining protein, transcript, and activity levels of the different enzymes involved. In this work, the functional characterization of the photosynthetic apparatus, pigment profiles, and the main primary metabolic pathways were analysed in tobacco sepals and petals at different developmental stages. The results indicate that the corolla photosynthetic apparatus is functional and capable of fixing CO(2); with its photosynthetic activity mainly involved in pigment biosynthesis. The particular pattern of expression, across the tobacco flower lifespan, of several proteins involved in respiration and primary metabolism, indicate that petal carbon metabolism is highest at the anthesis stage; while some enzymes are activated at the later stages, along with senescence. The first signs of corolla senescence in attached flowers are observed after anthesis; however, molecular data suggest that senescence is already onset at this stage. Feeding experiments to detached flowers at anthesis indicate that sugars, but not photosynthetic activity of the corolla, are capable of delaying the senescence process. On the other hand, photosynthetic activity and CO(2) fixation is active in sepals, where high expression levels of particular enzymes were detected. Sepals remained green and did not show signs of senescence in all the flower developmental stages analysed. Overall, the data presented contribute to an understanding of the metabolic processes operating during tobacco flower development, and identify key enzymes involved in the different stages.

Does Bienertia cycloptera with the single-cell system of C(4) photosynthesis exhibit a seasonal pattern of delta (13)C values in nature similar to co-existing C (4) Chenopodiaceae having the dual-cell (Kranz) system?

Family Chenopodiaceae is an intriguing lineage, having the largest number of C(4) species among dicots, including a number of anatomical variants of Kranz anatomy and three single-cell C(4) functioning species. In some previous studies, during the culture of Bienertia cycloptera Bunge ex Boiss., carbon isotope values (delta(13)C values) of leaves deviated from C(4) to C(3)-C(4) intermediate type, raising questions as to its mode of photosynthesis during growth in natural environments. This species usually co-occurs with several Kranz type C(4) annuals. The development of B. cycloptera morphologically and delta(13)C values derived from plant samples (cotyledons, leaves, bracts, shoots) were analyzed over a complete growing season in a salt flat in north central Iran, along with eight Kranz type C(4) species and one C(3) species. For a number of species, plants were greenhouse-grown from seeds collected from the site, in order to examine leaf anatomy and C(4) biochemical subtype. Among the nine C(4) species, the cotyledons of B. cycloptera, and of the Suaeda spp. have the same respective forms of C(4) anatomy occurring in leaves, while cotyledons of members of tribe Caroxyloneae lack Kranz anatomy, which is reflected in the delta(13)C values found in plants grown in the natural habitat. The nine C(4) species had average seasonal delta(13)C values of -13.9 per thousand (with a range between species from -11.3 to -15.9 per thousand). The measurements of delta(13)C values over a complete growing season show that B. cycloptera performs C(4) photosynthesis during its life cycle in nature, similar to Kranz type species, with a seasonal average delta(13)C value of -15.2 per thousand.

Leaf development in the single-cell C4 system in Bienertia sinuspersici: expression of genes and peptide levels for C4 metabolism in relation to chlorenchyma structure under different light conditions.

Bienertia sinuspersici performs C(4) photosynthesis in individual chlorenchyma cells by the development of two cytoplasmic domains (peripheral and central) with dimorphic chloroplasts, an arrangement that spatially separates the fixation of atmospheric CO(2) into C(4) acids and the donation of CO(2) from C(4) acids to Rubisco in the C(3) cycle. In association with the formation of these cytoplasmic domains during leaf maturation, developmental stages were analyzed for the expression of a number of photosynthetic genes, including Rubisco small and large subunits and key enzymes of the C(4) cycle. Early in development, Rubisco subunits and Gly decarboxylase and Ser hydroxymethyltransferase of the glycolate pathway accumulated more rapidly than enzymes associated with the C(4) cycle. The levels of pyruvate,Pi dikinase and phosphoenolpyruvate carboxylase were especially low until spatial cytoplasmic domains developed and leaves reached maturity, indicating a developmental transition toward C(4) photosynthesis. In most cases, there was a correlation between the accumulation of mRNA transcripts and the respective peptides, indicating at least partial control of the development of photosynthesis at the transcriptional level. During growth under moderate light, when branches containing mature leaves were enclosed in darkness for 1 month, spatial domains were maintained and there was high retention of a number of photosynthetic peptides, including Rubisco subunits and pyruvate,Pi dikinase, despite a reduction in transcript levels. When plants were transferred from moderate to low light conditions for 1 month, there was a striking shift of the central cytoplasmic compartment toward the periphery of chlorenchyma cells; the mature leaves showed strong acclimation with a shade-type photosynthetic response to light while retaining C(4) features indicative of low photorespiration. These results indicate a progressive development of C(4) photosynthesis with differences in the control mechanisms for the expression of photosynthetic genes and peptide synthesis during leaf maturation and in response to light conditions.

Nicotiana tabacum NADP-malic enzyme: cloning, characterization and analysis of biological role.

NADP-malic enzyme (NADP-ME) catalyzes the oxidative decarboxylation of L-malate, producing pyruvate, CO2 and NADPH. The photosynthetic role of this enzyme in C(4) and Crassulacean acid metabolism (CAM) plants has been well established; however, the biological role of several non-photosynthetic isoforms described in C(3), C(4) and CAM plants is still speculative. In this study, the characterization of the NADP-ME isoforms from Nicotiana tabacum was performed. Three different nadp-me transcripts were identified in this C(3) plant, two of which encode for putative cytosolic isoforms (DQ923118 and EH663836), while the third encodes for a plastidic counterpart (DQ923119). Although the three transcripts are expressed in vegetative as well as in reproductive tissues, they display different levels of expression. With regards to enzyme activity, root is the tissue that displays the highest NADP-ME activity. Recombinant NADP-MEs encoded by DQ923118 and DQ923119 were expressed in Escherichia coli and their kinetic parameters and response to different metabolic effectors were analyzed. Studies carried out with crude extracts and with the recombinant proteins indicate that the cytosolic and plastidic isoforms aggregate as tetramers of subunits of 65 and 63 kDa, respectively. Real-time reverse transcription-PCR studies show that the three nadp-me tobacco transcripts respond differently to several biotic and abiotic stress stimuli. Finally, the physiological role of each isoform is discussed in terms of the occurrence, kinetic properties and response to stress. The structure of the NADP-ME family in tobacco is compared with those of other C(3) species.

Species having C4 single-cell-type photosynthesis in the Chenopodiaceae family evolved a photosynthetic phosphoenolpyruvate carboxylase like that of Kranz-type C4 species.

Spatial and temporal regulation of phosphoenolpyruvate carboxylase (PEPC) is critical to the function of C(4) photosynthesis. The photosynthetic isoform of PEPC in the cytosol of mesophyll cells in Kranz-type C(4) photosynthesis has distinctive kinetic and regulatory properties. Some species in the Chenopodiaceae family perform C(4) photosynthesis without Kranz anatomy by spatial separation of initial fixation of atmospheric CO(2) via PEPC from C(4) acid decarboxylation and CO(2) donation to Rubisco within individual chlorenchyma cells. We studied molecular and functional features of PEPC in two single-cell functioning C(4) species (Bienertia sinuspersici, Suaeda aralocaspica) as compared to Kranz type (Haloxylon persicum, Salsola richteri, Suaeda eltonica) and C(3) (Suaeda linifolia) chenopods. It was found that PEPC from both types of C(4) chenopods displays higher specific activity than that of the C(3) species and shows kinetic and regulatory characteristics similar to those of C(4) species in other families in that they are subject to light/dark regulation by phosphorylation and display differential malate sensitivity. Also, the deduced amino acid sequence from leaf cDNA indicates that the single-cell functioning C(4) species possesses a Kranz-type C(4) isoform with a Ser in the amino terminal. A phylogeny of PEPC shows that isoforms in the two single-cell functioning C(4) species are in a clade with the C(3) and Kranz C(4) Suaeda spp. with high sequence homology. Overall, this study indicates that B. sinuspersici and S. aralocaspica have a C(4)-type PEPC similar to that in Kranz C(4) plants, which likely is required for effective function of C(4) photosynthesis.