Biosynthesis of Piceatannol from Resveratrol in Grapevine Can Be Mediated by Cresolase-Dependent Ortho-Hydroxylation Activity of Polyphenol Oxidase.

Piceatannol is a naturally occurring hydroxylated analogue of the stilbene phytoalexin resveratrol that can be found in grape fruit and derived products. Piceatannol has aroused great interest as it has been shown to surpass some human health-beneficial properties of resveratrol including antioxidant activity, several pharmacological activities and also bioavailability. The plant biosynthetic pathway of piceatannol is still poorly understood, which is a bottleneck for the development of both plant defence and bioproduction strategies. Cell cultures of Vitis vinifera cv. Gamay, when elicited with dimethyl-ß-cyclodextrin (MBCD) and methyl jasmonate (MeJA), lead to large increases in the accumulation of resveratrol, and after 120 h of elicitation, piceatannol is also detected due to the regiospecific hydroxylation of resveratrol. Therefore, an ortho-hydroxylase must participate in the biosynthesis of piceatannol. Herein, three possible types of resveratrol hydroxylation enzymatic reactions have been tested, specifically, a reaction catalyzed by an NADPH-dependent cytochrome, P450 hydroxylase, a 2-oxoglutarate-dependent dioxygenase and ortho-hydroxylation, similar to polyphenol oxidase (PPO) cresolase activity. Compared with P450 hydoxylase and the dioxygenase activities, PPO displayed the highest specific activity detected either in the crude extract, the particulate or the soluble fraction obtained from cell cultures elicited with MBCD and MeJA for 120 h. The overall yield of PPO activity present in the crude extract (107.42 EU) was distributed mostly in the soluble fraction (66.15 EU) rather than in the particulate fraction (3.71 EU). Thus, partial purification of the soluble fraction by precipitation with ammonium sulphate, dialysis and ion exchange chromatography was carried out. The soluble fraction precipitated with 80% ammonium sulphate and the chromatographic fractions also showed high levels of PPO activity, and the presence of the PPO protein was confirmed by Western blot and LC-MS/MS. In addition, a kinetic characterization of the cresolase activity of partially purified PPO was carried out for the resveratrol substrate, including Vmax and Km parameters. The Km value was 118.35 ± 49.84 µM, and the Vmax value was 2.18 ± 0.46 µmol min-1 mg-1.

Prenylated Flavonoids of the Moraceae Family: A Comprehensive Review of Their Biological Activities.

Prenylated flavonoids (PFs) are natural flavonoids with a prenylated side chain attached to the flavonoid skeleton. They have great potential for biological activities such as anti-diabetic, anti-cancer, antimicrobial, antioxidant, anti-inflammatory, enzyme inhibition, and anti-Alzheimer's effects. Medicinal chemists have recently paid increasing attention to PFs, which have become vital for developing new therapeutic agents. PFs have quickly developed through isolation and semi- or full synthesis, proving their high value in medicinal chemistry research. This review comprehensively summarizes the research progress of PFs, including natural PFs from the Moraceae family and their pharmacological activities. This information provides a basis for the selective design and optimization of multifunctional PF derivatives to treat multifactorial diseases.

Insights into enhancing Centella asiatica organ cell biofactories via hairy root protein profiling.

Recent advancements in plant biotechnology have highlighted the potential of hairy roots as a biotechnological platform, primarily due to their rapid growth and ability to produce specialized metabolites. This study aimed to delve deeper into hairy root development in C. asiatica and explore the optimization of genetic transformation for enhanced bioactive compound production. Previously established hairy root lines of C. asiatica were categorized based on their centelloside production capacity into HIGH, MID, or LOW groups. These lines were then subjected to a meticulous label-free proteomic analysis to identify and quantify proteins. Subsequent multivariate and protein network analyses were conducted to discern proteome differences and commonalities. Additionally, the quantification of rol gene copy numbers was undertaken using qPCR, followed by gene expression measurements. From the proteomic analysis, 213 proteins were identified. Distinct proteome differences, especially between the LOW line and other lines, were observed. Key proteins related to essential processes like photosynthesis and specialized metabolism were identified. Notably, potential biomarkers, such as the Tr-type G domain-containing protein and alcohol dehydrogenase, were found in the HIGH group. The presence of ornithine cyclodeaminase in the hairy roots emerged as a significant biomarker linked with centelloside production capacity lines, indicating successful Rhizobium-mediated genetic transformation. However, qPCR results showed an inconsistency with rol gene expression levels, with the HIGH line displaying notably higher expression, particularly of the rolD gene. The study unveiled the importance of ornithine cyclodeaminase as a traceable biomarker for centelloside production capacity. The strong correlation between this biomarker and the rolD gene emphasizes its potential role in optimizing genetic transformation processes in C. asiatica.

High variability of perezone content in rhizomes of Acourtia cordata wild plants, environmental factors related, and proteomic analysis.

With the aim of exploring the source of the high variability observed in the production of perezone, in Acourtia cordata wild plants, we analyze the influence of soil parameters and phenotypic characteristics on its perezone content. Perezone is a sesquiterpene quinone responsible for several pharmacological effects and the A. cordata plants are the natural source of this metabolite. The chemistry of perezone has been widely studied, however, no studies exist related to its production under natural conditions, nor to its biosynthesis and the environmental factors that affect the yield of this compound in wild plants. We also used a proteomic approach to detect differentially expressed proteins in wild plant rhizomes and compare the profiles of high vs. low perezone-producing plants. Our results show that in perezone-producing rhizomes, the presence of high concentrations of this compound could result from a positive response to the effects of some edaphic factors, such as total phosphorus (Pt), total nitrogen (Nt), ammonium (NH4), and organic matter (O. M.), but could also be due to a negative response to the soil pH value. Additionally, we identified 616 differentially expressed proteins between high and low perezone producers. According to the functional annotation of this comparison, the upregulated proteins were grouped in valine biosynthesis, breakdown of leucine and isoleucine, and secondary metabolism such as terpenoid biosynthesis. Downregulated proteins were grouped in basal metabolism processes, such as pyruvate and purine metabolism and glycolysis/gluconeogenesis. Our results suggest that soil parameters can impact the content of perezone in wild plants. Furthermore, we used proteomic resources to obtain data on the pathways expressed when A. cordata plants produce high and low concentrations of perezone. These data may be useful to further explore the possible relationship between perezone production and abiotic or biotic factors and the molecular mechanisms related to high and low perezone production.

Factors Affecting the Bioproduction of Resveratrol by Grapevine Cell Cultures under Elicitation.

Here we present a study of the characterization and optimization of the production of trans-Resveratrol (t-R) in grape (Vitis vinifera cv. Gamay) cell cultures elicited with methyl jasmonate (MeJA) and dimethyl-ß-cyclodextrin (DIMEB). The aim of this study was to determine the influence of a number of factors of the grapevine cell culture on t-R production level in 250 mL shaken flasks that would enable the better control of this bioproduction system when it is upscaled to a 2 L stirred bioreactor. The factors included the optimal growth phase for elicitation, the concentration of elicitors and of biomass, the order of addition of elicitors, and the illumination regime and ageing of cells. We found out that the optimal biomass density for the production of t-R was 19% (w/v) with an optimal ratio of 0.5 g DIMEB/g biomass. The most productive concentrations of the elicitors tested were 50 mM DIMEB and 100 µM MeJA, reaching maximum values of 4.18 mg·mL-1 and 16.3 mg·g biomass-1 of t-R concentration and specific production, respectively. We found that the order of elicitor addition matters since, as compared with the simultaneous addition of both elicitors, the addition of MeJA 48 h before DIMEB results in ca. 40% less t-R production, whilst there is no significant difference when MeJA is added 48 h after DIMEB. Upon upscaling, the better conditions tested for t-R production were aeration at 1.7 vol/vol/min without agitation, 24 °C, and 30 g·L-1 sucrose, achieving production rates similar to those obtained in shaken flasks.

Down-Regulation of Phosphoenolpyruvate Carboxylase Kinase in Grapevine Cell Cultures and Leaves Is Linked to Enhanced Resveratrol Biosynthesis.

In grapevine, trans-Resveratrol (tR) is produced as a defence mechanism against stress or infection. tR is also considered to be important for human health, which increases its interest to the scientific community. Transcriptomic analysis in grapevine cell cultures treated with the defence response elicitor methyl-ß-cyclodextrin (CD) revealed that both copies of PHOSPHOENOLPYRUVATE CARBOXYLASE KINASE (PPCK) were down-regulated significantly. A role for PPCK in the defence response pathway has not been proposed previously. We therefore analysed the control of PPCK transcript levels in grapevine cell cultures and leaves elicited with CD. Moreover, phosphoenolpyruvate carboxylase (PPC), stilbene synthase (STS), and the transcription factors MYB14 and WRKY24, which are involved in the activation of STS transcription, were also analysed by RT-qPCR. The results revealed that under CD elicitation conditions PPCK down-regulation, increased stilbene production and loss of PPC activity occurs in both tissues. Moreover, STS transcripts were co-induced with MYB14 and WRKY24 in cell cultures and leaves. These genes have not previously been reported to respond to CD in grape leaves. Our findings thus support the hypothesis that PPCK is involved in diverting metabolism towards stilbene biosynthesis, both for in vitro cell culture and whole leaves. We thus provide new evidence for PEP being redirected between primary and secondary metabolism to support tR production and the stress response.

Role of a cryptic tRNA gene operon in survival under translational stress.

As compared to eukaryotes, bacteria have a reduced tRNA gene set encoding between 30 and 220 tRNAs. Although in most bacterial phyla tRNA genes are dispersed in the genome, many species from distinct phyla also show genes forming arrays. Here, we show that two types of arrays with distinct evolutionary origins exist. This work focuses on long tRNA gene arrays (L-arrays) that encompass up to 43 genes, which disseminate by horizontal gene transfer and contribute supernumerary tRNA genes to the host. Although in the few cases previously studied these arrays were reported to be poorly transcribed, here we show that the L-array of the model cyanobacterium Anabaena sp. PCC 7120, encoding 23 functional tRNAs, is largely induced upon impairment of the translation machinery. The cellular response to this challenge involves a global reprogramming of the transcriptome in two phases. tRNAs encoded in the array are induced in the second phase of the response, directly contributing to cell survival. Results presented here show that in some bacteria the tRNA gene set may be partitioned between a housekeeping subset, which constantly sustains translation, and an inducible subset that is generally silent but can provide functionality under particular conditions.

Rational Design of Resveratrol O-methyltransferase for the Production of Pinostilbene.

Pinostilbene is a monomethyl ether analog of the well-known nutraceutical resveratrol. Both compounds have health-promoting properties, but the latter undergoes rapid metabolization and has low bioavailability. O-methylation improves the stability and bioavailability of resveratrol. In plants, these reactions are performed by O-methyltransferases (OMTs). Few efficient OMTs that monomethylate resveratrol to yield pinostilbene have been described so far. Here, we report the engineering of a resveratrol OMT from Vitis vinifera (VvROMT), which has the highest catalytic efficiency in di-methylating resveratrol to yield pterostilbene. In the absence of a crystal structure, we constructed a three-dimensional protein model of VvROMT and identified four critical binding site residues by applying different in silico approaches. We performed point mutations in these positions generating W20A, F24A, F311A, and F318A variants, which greatly reduced resveratrol's enzymatic conversion. Then, we rationally designed eight variants through comparison of the binding site residues with other stilbene OMTs. We successfully modified the native substrate selectivity of VvROMT. Variant L117F/F311W showed the highest conversion to pinostilbene, and variant L117F presented an overall increase in enzymatic activity. Our results suggest that VvROMT has potential for the tailor-made production of stilbenes.

Dimethyl Labeling-Based Quantitative Proteomics of Recalcitrant Cocoa Pod Tissue.

Dimethyl labeling is a type of stable-isotope labeling suitable for creating isotopic variants of peptides and thus be utilized for quantitative proteomics experiments. Labeling is achieved through a reductive amination/alkylation reaction using the low-cost reagents formaldehyde and cyanoborohydride, resulting in dimethylation of free amine groups of Lys and N-termini. Availability of isotopomeric forms of these reagents allows for the generation of up to six different isotopic variants. Here we describe the application of dimethylation to create two isotopic variants, light and heavy, differing in 4 Da, to label the total tryptic digest peptides of cocoa pod extracted from healthy pods from cultivars susceptible and resistant to the fungal disease called "frosty pod" caused by Moniliophthora roreri.

A Deep Proteomics Perspective Into Grape Berry Quality Traits During Ripening.

Discovery-based proteomics studies have an important role in the understanding of the biochemical processes that occur during grape berry ripening. The ripening process is relevant in determining grape berry quality. For a proteome analysis of grape berry ripening, Kambiranda et al. (2018) applied a label-free mass spectrometry-based quantitative approach. The authors reported the identification of proteins associated with the production flavor, aroma and ethylene production. Despite the valuable contribution of discovery-based proteomics studies, the picture is still incomplete. Future efforts in gaining proteome coverage would benefit the identification of proteins associated with grape berry quality traits.

Rosa hybrida orcinol O-methyl transferase-mediated production of pterostilbene in metabolically engineered grapevine cell cultures.

Stilbenes are naturally scarce high-added-value plant compounds with chemopreventive, pharmacological and cosmetic properties. Bioproduction strategies include engineering the metabolisms of bacterial, fungal and plant cell systems. Strikingly, one of the most effective strategies consists in the elicitation of wild grapevine cell cultures, which leads to vast stilbene resveratrol accumulation in the extracellular medium. The combination of both cell culture elicitation and metabolic engineering strategies to produce resveratrol analogs proved more efficient for the hydroxylated derivative piceatannol than for the dimethylated derivative pterostilbene, for which human hydroxylase HsCYP1B1- and grapevine O-methyltransferase VvROMT-transformed cell cultures were respectively used. Rose orcinol O-methyltransferase (OOMT) displays enzymatic properties, which makes it an appealing candidate to substitute VvROMT in the combined strategy to enhance the pterostilbene production level by engineered grapevine cells upon elicitation. Here we cloned a Rosa hybrida OOMT gene, and created a genetic construction suitable for Agrobacterium-mediated plant transformation. OOMT's ability to catalyze the conversion of resveratrol into pterostilbene was first assessed in vitro using protein extracts of agroinfiltrated N. benthamiana leaves and transformed grapevine callus. The grapevine cell cultures transformed with RhOOMT produced about 16 mg/L culture of pterostilbene and reached an extracellular distribution of up to 34% of total production at the best, which is by far the highest production reported to date in a plant system. A bonus large resveratrol production of ca. 1500-3000 mg/L was simultaneously obtained. Our results demonstrate a viable successful metabolic engineering strategy to produce pterostilbene, a resveratrol analog with enhanced pharmacological properties.

Targeted Quantification of Isoforms of a Thylakoid-Bound Protein: MRM Method Development.

Targeted mass spectrometric methods such as selected/multiple reaction monitoring (SRM/MRM) have found intense application in protein detection and quantification which competes with classical immunoaffinity techniques. It provides a universal procedure to develop a fast, highly specific, sensitive, accurate, and cheap methodology for targeted detection and quantification of proteins based on the direct analysis of their surrogate peptides typically generated by tryptic digestion. This methodology can be advantageously applied in the field of plant proteomics and particularly for non-model species since immunoreagents are scarcely available. Here, we describe the issues to take into consideration in order to develop a MRM method to detect and quantify isoforms of the thylakoid-bound protein polyphenol oxidase from the non-model and database underrepresented species Eriobotrya japonica Lindl.

Tailoring tobacco hairy root metabolism for the production of stilbenes.

Tobacco hairy root (HR) cultures, which have been widely used for the heterologous production of target compounds, have an innate capacity to bioconvert exogenous t-resveratrol (t-R) into t-piceatannol (t-Pn) and t-pterostilbene (t-Pt). We established genetically engineered HR carrying the gene encoding stilbene synthase (STS) from Vitis vinifera and/or the transcription factor (TF) AtMYB12 from Arabidopsis thaliana, in order to generate a holistic response in the phenylpropanoid pathway and coordinate the up-regulation of multiple metabolic steps. Additionally, an artificial microRNA for chalcone synthase (amiRNA CHS) was utilized to arrest the normal flux through the endogenous chalcone synthase (CHS) enzyme, which would otherwise compete for precursors with the STS enzyme imported for the flux deviation. The transgenic HR were able to biosynthesize the target stilbenes, achieving a production of 40 µg L-1 of t-R, which was partially metabolized into t-Pn and t-Pt (up to 2.2 µg L-1 and 86.4 µg L-1, respectively), as well as its glucoside piceid (up to 339.7 µg L-1). Major metabolic perturbations were caused by the TF AtMYB12, affecting both primary and secondary metabolism, which confirms the complexity of biotechnological systems based on seed plant in vitro cultures for the heterologous production of high-value molecules.

A Tau Class Glutathione-S-Transferase is Involved in Trans-Resveratrol Transport Out of Grapevine Cells.

Vitis vinifera cell cultures respond to pathogens and elicitors by synthesizing and extracellularly accumulating stilbenoid phytoalexins. Large amounts of trans-resveratrol (t-R) are produced when a cell culture is elicited with methylated cyclodextrins (MBCD), either alone or combined with methyl jasmonate (MeJA). t-R transport to the extracellular medium, which represents the apoplastic space, would place this antifungal defense right in the battlefield to efficiently fight against pathogen attack. Yet despite their physiological relevance, these transport pathways are mostly unknown. A broad hypothesis-free DIGE-based proteomic experiment of a temporal series of elicited grapevine cell cultures was performed to explore the expression profiles of t-R biosynthetic proteins and other co-expressing proteins potentially involved in such a cell response. A correlation between two tau class glutathione-S-transferases (GSTs) with several stilbene synthase and phenylalanine ammonia-lyase isoforms, and with the t-R metabolite itself, was found and further assessed by a qRT-PCR gene expression analysis. The best candidate, GSTU-2, was cloned from the cDNA of the MBCD + MeJA-elicited grapevine cells and used for Agrobacterium-mediated grapevine cell transformation. The non-elicited lines that overexpressed GSTU-2 displayed an extracellular t-R accumulating phenotype, but stabilization of t-R required the addition to culture medium of adsorbent compounds, e.g., PVP or ß-cyclodextrin. The wild-type cell cultures accumulated no t-R, not even in the presence of adsorbents. The transient expression of the GSTU-2-GFP fusion proteins in grapevine cells showed localisation in the plasma membrane, and the immunoprecipitation of HA-tagged GSTU-2 revealed its interaction with HIR, a plasma membrane-bound protein. These findings are consistent with a functional role in transport. This is the first report providing several pieces of experimental evidence for the involvement of a specific tau class GST in t-R transport to the extracellular medium.

Biotechnological production of recombinant tissue plasminogen activator protein (reteplase) from transplastomic tobacco cell cultures.

Transplastomic plants are a system of choice for the mass production of biopharmaceuticals due to the polyploidy of the plastid genome and the low risk of pollen-mediated outcrossing because of maternal inheritance. However, as field-grown plants, they can suffer contamination by agrochemicals and fertilizers, as well as fluctuations in yield due to climatic changes and infections. Tissue-type plasminogen activator (tPA), a protein used to treat heart attacks, converts plasminogen into plasmine, which digests fibrin and induces the dissolution of fibrin clots. Recently, we obtained transplastomic tobacco plants carrying the K2S gene encoding truncated human tPA (reteplase) with improved biological activity, and confirmed the presence of the target protein in the transgenic plant leaves. Considering the advantages of plant cell cultures for biopharmaceutical production, we established a cell line derived from the K2S tobacco plants. The active form of reteplase was quantified in cultures grown in light or darkness, with production 3-fold higher in light.

Bioconversion of stilbenes in genetically engineered root and cell cultures of tobacco.

It is currently possible to transfer a biosynthetic pathway from a plant to another organism. This system has been exploited to transfer the metabolic richness of certain plant species to other plants or even to more simple metabolic organisms such as yeast or bacteria for the production of high added value plant compounds. Another application is to bioconvert substrates into scarcer or biologically more interesting compounds, such as piceatannol and pterostilbene. These two resveratrol-derived stilbenes, which have very promising pharmacological activities, are found in plants only in small amounts. By transferring the human cytochrome P450 hydroxylase 1B1 (HsCYP1B1) gene to tobacco hairy roots and cell cultures, we developed a system able to bioconvert exogenous t-resveratrol into piceatannol in quantities near to mg L-1. Similarly, after heterologous expression of resveratrol O-methyltransferase from Vitis vinifera (VvROMT) in tobacco hairy roots, the exogenous t-resveratrol was bioconverted into pterostilbene. We also observed that both bioconversions can take place in tobacco wild type hairy roots (pRiA4, without any transgene), showing that unspecific tobacco P450 hydroxylases and methyltransferases can perform the bioconversion of t-resveratrol to give the target compounds, albeit at a lower rate than transgenic roots.

A Focused Multiple Reaction Monitoring (MRM) Quantitative Method for Bioactive Grapevine Stilbenes by Ultra-High-Performance Liquid Chromatography Coupled to Triple-Quadrupole Mass Spectrometry (UHPLC-QqQ).

Grapevine stilbenes are a family of polyphenols which derive from trans-resveratrol having antifungal and antimicrobial properties, thus being considered as phytoalexins. In addition to their diverse bioactive properties in animal models, they highlight a strong potential in human health maintenance and promotion. Due to this relevance, highly-specific qualitative and quantitative methods of analysis are necessary to accurately analyze stilbenes in different matrices derived from grapevine. Here, we developed a rapid, sensitive, and specific analysis method using ultra-high-performance liquid chromatography coupled to triple-quadrupole mass spectrometry (UHPLC-QqQ) in MRM mode to detect and quantify five grapevine stilbenes, trans-resveratrol, trans-piceid, trans-piceatannol, trans-pterostilbene, and trans-ε-viniferin, whose interest in relation to human health is continuously growing. The method was optimized to minimize in-source fragmentation of piceid and to avoid co-elution of cis-piceid and trans-resveratrol, as both are detected with resveratrol transitions. The applicability of the developed method of stilbene analysis was tested successfully in different complex matrices including cellular extracts of Vitis vinifera cell cultures, reaction media of biotransformation assays, and red wine.

Silybum marianum cell cultures stably transformed with Vitis vinifera stilbene synthase accumulate t-resveratrol in the extracellular medium after elicitation with methyl jasmonate or methylated ß-cyclodextrins.

The growing demand for t-resveratrol for industrial uses has generated considerable interest in its production. Heterologous resveratrol production in plant cell suspensions, apart from requiring the introduction of only one or two genes, has the advantage of high biomass yield and a short cultivation time, and thus could be an option for large-scale production. Silybum marianum is the source of the flavonolignan silymarin. Phenylpropanoid synthesis in cultures of this species can be activated by elicitation with methyl jasmonate and methylated ß-cyclodextrins, with products of the pathway (coniferyl alcohol and some isomers of the silymarin complex) being released into the medium. Given that stilbene synthase shares the same key precursors involved in flavonoid and /or monolignol biosynthesis, we explored the potential of metabolically engineered S. marianum cultures for t-resveratrol production. Cell suspensions were stably transformed with Vitis vinifera stilbene synthase 3 and the expression of the transgene led to extracellular t-resveratrol accumulation at the level of milligrams per litre under elicitation. Resveratrol synthesis occurred at the expense of coniferyl alcohol. Production of silymarin was less affected in the transgenic cultures, since the flavonoid pathway is limiting for its synthesis, due to the preferred supply of precursors for the monolignol branch. The fact that the expressed STS gene took excessively produced precursors of non-bioactive compounds (coniferyl alcohol), while keeping the metabolic flow for target secondary compounds (i.e. silymarin) unaltered, opens a way to extend the applications of plant cell cultures for the simultaneous production of both constitutive and foreign valuable metabolites.

Sub-Cellular Localization and Complex Formation by Aminoacyl-tRNA Synthetases in Cyanobacteria: Evidence for Interaction of Membrane-Anchored ValRS with ATP Synthase.

tRNAs are charged with cognate amino acids by aminoacyl-tRNA synthetases (aaRSs) and subsequently delivered to the ribosome to be used as substrates for gene translation. Whether aminoacyl-tRNAs are channeled to the ribosome by transit within translational complexes that avoid their diffusion in the cytoplasm is a matter of intense investigation in organisms of the three domains of life. In the cyanobacterium Anabaena sp. PCC 7120, the valyl-tRNA synthetase (ValRS) is anchored to thylakoid membranes by means of the CAAD domain. We have investigated whether in this organism ValRS could act as a hub for the nucleation of a translational complex by attracting other aaRSs to the membranes. Out of the 20 aaRSs, only ValRS was found to localize in thylakoid membranes whereas the other enzymes occupied the soluble portion of the cytoplasm. To investigate the basis for this asymmetric distribution of aaRSs, a global search for proteins interacting with the 20 aaRSs was conducted. The interaction between ValRS and the FoF1 ATP synthase complex here reported is of utmost interest and suggests a functional link between elements of the gene translation and energy production machineries.

Transformation of plum plants with a cytosolic ascorbate peroxidase transgene leads to enhanced water stress tolerance.

BACKGROUND AND AIMS: Water deficit is the most serious environmental factor limiting agricultural production. In this work, the tolerance to water stress (WS) of transgenic plum lines harbouring transgenes encoding cytosolic antioxidant enzymes was studied, with the aim of achieving the durable resistance of commercial plum trees. METHODS: The acclimatization process was successful for two transgenic lines: line C3-1, co-expressing superoxide dismutase (two copies) and ascorbate peroxidase (one copy) transgenes simultaneously; and line J8-1, harbouring four copies of the cytosolic ascorbate peroxidase gene (cytapx). Plant water relations, chlorophyll fluorescence and the levels of antioxidant enzymes were analysed in both lines submitted to moderate (7 d) and severe (15 d) WS conditions. Additionally, in line J8-1, showing the best response in terms of stress tolerance, a proteomic analysis and determination of the relative gene expression of two stress-responsive genes were carried out. KEY RESULTS: Line J8-1 exhibited an enhanced stress tolerance that correlated with better photosynthetic performance and a tighter control of water-use efficiency. Furthermore, this WS tolerance also correlated with a higher enzymatic antioxidant capacity than wild-type (WT) and line C3-1 plum plants. On the other hand, line C3-1 displayed an intermediate phenotype between WT plants and line J8-1 in terms of WS tolerance. Under severe WS, the tolerance displayed by J8-1 plants could be due to an enhanced capacity to cope with drought-induced oxidative stress. Moreover, proteomic analysis revealed differences between WT and J8-1 plants, mainly in terms of the abundance of proteins related to carbohydrate metabolism, photosynthesis, antioxidant defences and protein fate. CONCLUSIONS: The transformation of plum plants with cytapx has a profound effect at the physiological, biochemical, proteomic and genetic levels, enhancing WS tolerance. Although further experiments under field conditions will be required, it is proposed that J8-1 plants would be an interesting Prunus rootstock for coping with climate change.