Reversible changes in galactolipid saturation level and head group composition are associated with tolerance to postharvest chilling in tomato fruit.

BACKGROUND: Chilling injury (CI) is a physiological disorder that results in a limitation for cold storage (CS) of many fruits and vegetables. The low temperature-induced changes in the properties and composition of cell membranes are involved in the response to chilling temperature and in the mechanism of CI and tolerance. RESULTS: We compared the changes in the lipid composition by gas chromatography-mass spectrometry before, immediately after CS, as well as during a 3-day subsequent period, of tomato fruits with different chilling-sensitivity: Micro-Tom (tolerant) and Minitomato (susceptible). The changes in linolenic acid content, double bond index and digalactosyldiacylglycerol/monogalactosyldiacylglycerol ratio (DGDG/MGDG) showed membrane fluidity adjustment, depending on the temperature. By a database search, we identified 18 membrane-bound fatty acid desaturase (FAD) genes and five DGDG synthases (DGD) genes that phylogenetically clustered into four and two subfamilies, respectively. The FAD and DGD genes were differentially expressed in response to CS, as determined by quantitative reverse transcriptase-polymerase chain reaction analysis. CONCLUSION: The data strongly suggest that reversion of CS-induced changes during the recovery period is important for the proper function of the membrane and tolerance to postharvest CI in tomato fruit. © 2021 Society of Chemical Industry.

Mitochondrial Small Heat Shock Proteins Are Essential for Normal Growth of Arabidopsis thaliana.

Mitochondria play important roles in the plant stress responses and the detoxification of the reactive oxygen species generated in the electron transport chain. Expression of genes encoding stress-related proteins such as the mitochondrial small heat shock proteins (M-sHSP) is upregulated in response to different abiotic stresses. In Arabidopsis thaliana, three M-sHSPs paralogous genes were identified, although their function under physiological conditions remains elusive. The aim of this work is to uncover the in vivo function of all three M-sHSPs at the whole plant level. To accomplish this goal, we analyzed the phenotype, proteomic, and metabolic profiles of Arabidopsis knock-down lines of M-sHSPs (single, double, and triple knock-down lines) during normal plant growth. The triple knock-down plants showed the most prominent altered phenotype at vegetative and reproductive stages without any externally applied stress. They displayed chlorotic leaves, growth arrest, and low seed production. Concomitantly, they exhibited increased levels of sugars, proline, and citric, malic, and ascorbic acid, among other metabolites. In contrast, single and double knock-down plants displayed a few changes in their phenotype. A redundant function among the three M-sHSPs is indicated by the impairment in vegetative and reproductive growth associated with the simultaneous loss of all three M-sHSPs genes. The triple knock-down lines showed alteration of proteins mainly involved in photosynthesis and antioxidant defense compared to the control plants. On the other hand, heat stress triggered a distinct cytosolic response pattern and the upregulation of other sHSP members, in the knock-down plants. Overall, depletion of all three M-sHSPs in Arabidopsis severely impacted fundamental metabolic processes, leading to alterations in the correct plant growth and development. These findings expand our knowledge about the contribution of organelle-specific M-sHSPs to healthy plant growth under non-stress conditions.

Integral use of Argentinean Solanum betaceum red fruits as functional food ingredient to prevent metabolic syndrome: effect of in vitro simulated gastroduodenal digestion.

The "red chilto" (Solanum betaceum Cav) is a native fruit from the Yungas forest of Argentina. Red chilto is a neglected and underutilized native species (NUS). The objective of this work was to evaluate the potentiality of pulp, seed and skin from "red chilto" as a functional food ingredient to add value to a native resource of Argentine Yungas to promote sustainable integral use of it. The powders have low carbohydrate and sodium content and are a source of vitamin C, phenolic acids (rosmarinic acid and caffeoylquinic acid), anthocyanins, condensed tannins, carotenoids, potassium, and fiber. The phenolics of chilto powders showed, before and after simulated gastroduodenal digestion, antioxidant activity (ABTS•+; H2O2; O2 •; HO•) and were able to inhibit enzymes related to metabolic syndrome, such as α-glucosidase, α-amylase and lipase. Chilto powder showed hypoglycemic effect by increasing glucose adsorption, decreasing glucose diffusion rate and by promoting glucose transport across the cell membrane. These results suggest the potential of Argentinean "red chilto" fruits as functional food ingredients or dietary supplements to prevent metabolic syndrome principally by its antioxidant, hypoglycemic and hypolipemic effects.

Expression of a Chloroplast-Targeted Cyanobacterial Flavodoxin in Tomato Plants Increases Harvest Index by Altering Plant Size and Productivity.

Tomato is the most important horticultural crop worldwide. Domestication has led to the selection of highly fruited genotypes, and the harvest index (HI), defined as the ratio of fruit yield over total plant biomass, is usually employed as a biomarker of agronomic value. Improvement of HI might then result from increased fruit production and/or lower vegetative growth. Reduction in vegetative biomass has been accomplished in various plant species by expression of flavodoxin, an electron shuttle flavoprotein that interacts with redox-based pathways of chloroplasts including photosynthesis. However, the effect of this genetic intervention on the development of reproductive organs has not been investigated. We show herein that expression of a plastid-targeted cyanobacterial flavodoxin in tomato resulted in significant reduction of plant size affecting stems, leaves, and fruit. Decreased size correlated with smaller cells and was accompanied by higher pigment contents and photosynthetic activities per leaf cross-section. Flavodoxin accumulated in green fruit but declined with ripening. Significant increases in HI were observed in flavodoxin-expressing lines due to the production of higher fruit number per plant in smaller plants. Therefore, overall yields can be enhanced by increasing plant density in the field. Metabolic profiling of ripe red fruit showed that levels of sugars, organic acids, and amino acids were similar or higher in transgenic plants, indicating that there was no trade-off between increased HI and fruit metabolite contents in flavodoxin-expressing plants. Taken together, our results show that flavodoxin has the potential to improve major agronomic traits when introduced in tomato.

Fruit metabolic and transcriptional programs differentiate among Andean tomato (Solanum lycopersicum L.) accessions.

MAIN CONCLUSION: Andean tomatoes differed from the wild ancestor in the metabolic composition and the expression of genes related with mitochondrial functions, and environmental stresses, making them potentially suitable for breeding programmes. Traditional landraces or "criollo" tomatoes (Solanum lycopersicum L.) from Andean areas of Argentina, selected for their fruit quality, were analysed in this study. We explored the metabolome and transcriptome of the ripe fruit in nine landrace accessions representing the seven genetic groups and compared them to the mature fruit of the wild progenitor Solanum pimpinellifolium. The content of branched- (isoleucine and valine) and aromatic (phenylalanine and tryptophan) amino acids, citrate and sugars were significantly different in the fruit of several "criollo" tomatoes compared to S. pimpinellifolium. The transcriptomic profile of the ripe fruit showed several genes significantly and highly regulated in all varieties compared to S. pimpinellifolium, like genes encoding histones and mitochondrial proteins. Additionally, network analysis including transcripts and metabolites identified major hubs with the largest number of connections such as constitutive photomorphogenic protein 1 (a RING finger-type ubiquitin E3 ligase), five Zn finger transcription factors, ascorbate peroxidase, acetolactate synthase, and sucrose non-fermenting 1 kinase. Co-expression analysis of these genes revealed a potential function in acquiring tomato fruit quality during domestication.

Contrasting metabolic profiles of tasty Andean varieties of tomato fruit in comparison with commercial ones.

BACKGROUND: The fruits of most commercial tomato cultivars (Solanum lycopersicum L.) are deficient in flavour. In contrast, traditional 'criollo' tomato varieties are appreciated for fruit of excellent organoleptic quality. Small farmers from the Andean valleys in Argentina have maintained their own tomato varieties, which were selected mainly for flavour. This work aims to correlate the chemical composition of the fruit with the sensory attributes of eight heirloom tomato varieties. The long-term goal is to identify potential candidate genes capable of altering the chemicals involved in flavour. RESULTS: A sensory analysis was conducted and the metabolomics of fruit were determined. The data revealed that defined tomato aroma and sourness correlated with citrate and several volatile organic compounds (VOC), such as α-terpineol, p-menth-1-en-9-al, linalool and 3,6-dimethyl-2,3,3a,4,5,7a-hexahydrobenzofuran (DMHEX), a novel volatile recently identified in tomato. Two sensory attributes - sweetness and a not-acidic taste - correlated with the characteristic tomato taste, and also with fructose, glucose, and two VOCs, benzaldehyde, and 2-methyl-2-octen-4-one. CONCLUSIONS: These data provide new evidence of the complex chemical combination that induced the flavour and aroma of the good-tasting 'criollo' tomato fruit. That is, the compounds that correlated with defined tomato aroma and acidic taste did not correlate with sweetness, or with characteristic tomato taste. © 2018 Society of Chemical Industry.

Overexpression of AtERF019 delays plant growth and senescence, and improves drought tolerance in Arabidopsis.

The transcription factor superfamily, APETALA2/ethylene response factor, is involved in plant growth and development, as well as in environmental stress responses. Here, an uncharacterized gene of this family, AtERF019, was studied in Arabidopsis thaliana under abiotic stress situations. Arabidopsis plants overexpressing AtERF019 showed a delay in flowering time of 7 days and a delay in senescence of 2 weeks when comparison with wild type plants. These plants also showed increased tolerance to water deficiency that could be explained by a lower transpiration rate, owing to their smaller stomata aperture and lower cuticle and cell wall permeability. Furthermore, using a bottom-up proteomic approach, proteins produced in response to stress, namely branched-chain-amino-acid aminotransferase 3 (BCAT3) and the zinc finger transcription factor oxidative stress 2, were only identified in plants overexpressing AtERF019. Additionally, a BCAT3 mutant was more sensitive to water-deficit stress than wild type plants. Predicted gene targets of AtERF019 were oxidative stress 2 and genes related to cell wall metabolism. These data suggest that AtERF019 could play a primary role in plant growth and development that causes an increased tolerance to water deprivation, so strengthening their chances of reproductive success.

Estimation of Stomatal Aperture in Arabidopsis thaliana Using Silicone Rubber Imprints.

Estimation of stomatal aperture using low viscosity silicone-base impression material has the advantage of working with the whole leaf. The developmental stage and the environment strongly affect the stomatal aperture. Therefore, it is mandatory to have accurate estimations of the stomatal aperture of intact leaves under different situations. With this technique, it is possible to get the real picture at any moment. The outputs of the data include studies on cell area and morphology, epidermis cell and stomata lineages, among others. This protocol is useful for the accurate estimation of stomatal aperture in many samples of intact leaves in Arabidopsis thaliana.

Small heat shock proteins and the postharvest chilling tolerance of tomato fruit.

Plants have the largest number of small heat shock proteins (sHsps) (15-42 kDa) among eukaryotes, but little is known about their function in vivo. They accumulate in response to different stresses, and specific sHsps are also expressed during developmental processes such as seed development, germination, and ripening. The presence of organelle-specific sHsps appears to be unique to plants. The sHsps expression is regulated by heat stress transcription factors (Hsfs). In this work, it was explored the role of sHsps in the chilling injury of tomato fruit. The level of transcripts and proteins of cytoplasmic and organellar sHsps was monitored in fruit during ripening and after cold storage (4 weeks at 4°C). Expression of HsfA1, HsfA2, HsfA3, and HsfB1 was also examined. Two cultivars of tomato (Solanum lycopersicum) contrasting in chilling tolerance were assayed: Micro-Tom (chilling-tolerant) and Minitomato (chilling-sensitive). Results showed that sHsps were induced during ripening in fruit from both cultivars. However, sHsps were induced in Micro-Tom fruit but not in Minitomato fruit after storage at a low temperature. In particular, sHsp 17.4-CII and sHsp23.8-M transcripts strongly accumulated in Micro-Tom fruit and HsfA3 transcript diminished after cold storage. These data suggest that sHsps may be involved in the protection mechanisms against chilling stress and substantiate the hypothesis that sHsps may participate in the mechanism of tomato genotype chilling tolerance.

Data on polyphenols and biological activity analyses of an Andean tomato collection and their relationships with tomato traits and geographical origin.

Data provide information about a tomato collection composed of accessions from the Andean Valley, commercial accessions and wild species. Antioxidant metabolites were measured in mature fruits of this collection, and their biological activities were assessed by both in vitro and in vivo methods. In this work, the parameters used to identify and quantify polyphenols compounds in tomato fruit by liquid chromatography coupled to diode array detector and quadrupole time of flight mass spectrometer are described. Moreover, data supporting a procedure to characterize the properties of tomato fruits to revert death by thermal stress in Caenorhabditis elegans are explained in detail. Lastly, principal component analysis and hierarchical cluster analysis of metabolites composition, antioxidant activities (in vivo and in vitro), tomato traits and geographical origin of the tomatoes collection are shown. The data presented here are related to the research article entitled "Hydrophilic antioxidants from Andean Tomato Landraces assessed by their bioactivities in vitro and in vivo" [1].

Hydrophilic antioxidants from Andean tomato landraces assessed by their bioactivities in vitro and in vivo.

Potential nutraceutical properties of hydrophilic antioxidants in fruits of tomato landraces collected in Andean valleys were characterised. Antioxidant metabolites were measured by HPLC-DAD-MS/MS in mature fruits and their biological activities were assessed by in vitro and in vivo methods. In vitro antioxidant capacities were established by TEAC and FRAP methods. For in vivo biological activities we used a procedure based on Caenorhabditis elegans subjected to thermal stress. In addition, Saccharomyces cerevisiae was also used as a rapid screening system to evaluate tomato antioxidant capacity. All tomato accessions displayed significant differences regarding metabolic composition, biological activity and antioxidant capacity. Metabolite composition was associated with geographical origin and fruit size. Antioxidant activities showed significant association with phenolic compounds, such as caffeoylquinic acids, ferulic acid-O-hexosides and rutin. Combination of in vitro and in vivo methods applied here allowed evaluation of the variability in nutraceutical properties of tomato landraces, which could be applied to other fruits or food products.

Reduced levels of NADH-dependent glutamate dehydrogenase decrease the glutamate content of ripe tomato fruit but have no effect on green fruit or leaves.

Glutamate (Glu) is a taste enhancer that contributes to the characteristic flavour of foods. In fruit of tomato (Solanum lycopersicum L.), the Glu content increases dramatically during the ripening process, becoming the most abundant free amino acid when the fruit become red. There is also a concomitant increase in NADH-dependent glutamate dehydrogenase (GDH) activity during the ripening transition. This enzyme is located in the mitochondria and catalyses the reversible amination of 2-oxoglutarate to Glu. To investigate the potential effect of GDH on Glu metabolism, the abundance of GDH was altered by artificial microRNA technology. Efficient silencing of all the endogenous SlGDH genes was achieved, leading to a dramatic decrease in total GDH activity. This decrease in GDH activity did not lead to any clear morphological or metabolic phenotype in leaves or green fruit. However, red fruit on the transgenic plants showed markedly reduced levels of Glu and a large increase in aspartate, glucose and fructose content in comparison to wild-type fruit. These results suggest that GDH is involved in the synthesis of Glu in tomato fruit during the ripening processes. This contrasts with the biological role ascribed to GDH in many other tissues and species. Overall, these findings suggest that GDH has a major effect on the control of metabolic composition during tomato fruit ripening, but not at other stages of development.

Overexpression of AtWRKY30 enhances abiotic stress tolerance during early growth stages in Arabidopsis thaliana.

AtWRKY30 belongs to a higher plant transcription factor superfamily, which responds to pathogen attack. In previous studies, the AtWRKY30 gene was found to be highly and rapidly induced in Arabidopsis thaliana leaves after oxidative stress treatment. In this study, electrophoretic mobility shift assays showed that AtWRKY30 binds with high specificity and affinity to the WRKY consensus sequence (W-box), and also to its own promoter. Analysis of the AtWRKY30 expression pattern by qPCR and using transgenic Arabidopsis lines carrying AtWRKY30 promoter-ß-glucuronidase fusions showed transcriptional activity in leaves subjected to biotic or abiotic stress. Transgenic Arabidopsis plants constitutively overexpressing AtWRKY30 (35S::W30 lines) were more tolerant than wild-type plants to oxidative and salinity stresses during seed germination. The results presented here show that AtWRKY30 is responsive to several stress conditions either from abiotic or biotic origin, suggesting that AtWRKY30 could have a role in the activation of defence responses at early stages of Arabidopsis growth by binding to W-boxes found in promoters of many stress/developmentally regulated genes.

Off-the-Vine Ripening of Tomato Fruit Causes Alteration in the Primary Metabolite Composition.

The influence of postharvest fruit ripening in the composition of metabolites, transcripts and enzymes in tomato (Solanum lycopersicum L.) is poorly understood. The goal of this work was to study the changes in the metabolite composition of the tomato fruit ripened off-the-vine using the cultivar Micro-Tom as model system. Proton nuclear magnetic resonance (1H NMR) was used for analysis of the metabolic profile of tomato fruits ripened on- and off-the-vine. Significant differences under both ripening conditions were observed principally in the contents of fructose, glucose, aspartate and glutamate. Transcript levels and enzyme activities of -amino butyrate transaminase (EC 2.6.1.19) and glutamate decarboxylase (EC 4.1.1.15) showed differences in fruits ripened under these two conditions. These data indicate that the contents of metabolites involved in primary metabolism, and conferring the palatable properties of fruits, are altered when fruits are ripened off-the-vine.

Contribution of a harpin protein from Xanthomonas axonopodis pv. citri to pathogen virulence.

Xanthomonas axonopodis pv. citri (Xac), the bacterium that causes citrus canker, contains a gene in the hrp [for hypersensitive response (HR) and pathogenicity] cluster that encodes a harpin protein called Hpa1. Hpa1 produced HR in the nonhost plants tobacco, pepper and Arabidopsis, whereas, in the host plant citrus, it elicited a weak defence response with no visible phenotype. Co-infiltrations of Xac with or without the recombinant Hpa1 protein in citrus leaves produced a larger number of cankers in the presence of the protein. To characterize the effect of Hpa1 during the disease, an XacΔhpa1 mutant was constructed, and infiltration of this mutant caused a smaller number of cankers. In addition, the lack of Hpa1 hindered bacterial aggregation both in solution and in planta. Analysis of citrus leaves infiltrated with Hpa1 revealed alterations in mesophyll morphology caused by the presence of cavitations and crystal idioblasts, suggesting the binding of the harpin to plant membranes and the elicitation of signalling cascades. Overall, these results suggest that, even though Hpa1 elicits the defence response in nonhost plants and, to a lesser extent, in host plants, its main roles in citrus canker are to alter leaf mesophyll structure and to aggregate bacterial cells, and thus increase virulence and pathogen fitness. We expressed the N-terminal and C-terminal regions and found that, although both regions elicited HR in nonhost plants, only the N-terminal region showed increased virulence and bacterial aggregation, supporting the role of this region of the protein as the main active domain.

Novel glutamate dehydrogenase genes show increased transcript and protein abundances in mature tomato fruits.

NAD(P)H-glutamate dehydrogenase (GDH, EC 1.4.1.3) contributes to the control of glutamate homeostasis in all living organisms. In bacteria and animals, GDH is a homohexamer allosterically regulated, whereas in plants NADH-GDH (EC 1.4.1.2) is also found as heterohexamer of α- and ß-subunits, but its regulation remains undefined. In tomato (Solanum lycopersicum), GDH activity increases during the fruit ripening along with the content of free glutamate, the most abundant amino acid of ripe fruit involved in conferring the genuine tomato flavour. In this work, novel Slgdh-NAD genes were identified in the recently deciphered tomato genome: three encoding the α-subunit (Slgdh-NAD;A1-3) and one additional gene encoding the ß-subunit of GDH (Slgdh-NAD;B1) isolated from a genomic library. These genes are located in different chromosomes. Slgdh-NAD;A1-3 show conserved structures, whereas Slgdh-NAD;B1 includes a novel 5'-untranslated exon. Slgdh-NAD;A1-3 transcripts were detected in all tomato tissues examined, showing the highest levels in mature green fruits, contrasting with Slgdh-NAD;B1 transcripts which were detected mainly in roots or in mature fruits when treated with glutamate, NaCl or salicylic acid. Analyses of GDH activity and protein distribution in different tissues of the Micro-Tom cultivar showed that only the active homohexamer of GDH ß-subunits was detected in roots while heterohexamers of GDH α- and ß-subunits were found in fruits. These results indicate that GDH ß-subunit could modulate the heteromeric isoforms of GDH in response to the environment and physiology of the tomato fruit. This information is relevant to manipulate glutamate contents in tomato fruits genetically.

Ripening tomato fruit after chilling storage alters protein turnover.

BACKGROUND: Tomato fruit is of prime importance owing to its qualities for human nutrition and its economic value. In order to extend its commercial life, it is harvested at mature but unripe stages and stored at low temperatures. The goal of this work was to study the influence of harvest and chilling storage of mature green tomato fruit (cv. Micro-Tom) on the protein pattern, amino acid content and protease activity during fruit ripening. RESULTS: Fruits were sampled during ripening in three different conditions: 1, on the vine; 2, off the vine; 3, off the vine after 4 weeks at 4 °C. During all fruit ripening conditions, protein level decreased while amino acid content increased. Chilling storage of mature green fruit led to a reduction in protein content. Ripening off the vine (conditions 2 and 3) resulted in a threefold increase in red fruit amino acid levels when compared with red fruit on the vine. Protease activities (autoproteolytic, azocaseinolytic and gelatinolytic) were detected in all fruits evaluated and were differently affected by ripening stage, ripening conditions and the presence of specific inhibitors. CONCLUSION: Harvest and chilling storage increased endogenous substrate proteolysis, azocaseinolytic activity and free amino acid levels, which could be related to fruit quality.

Phenotypic and molecular characterization of selected tomato recombinant inbred lines derived from the cross Solanum lycopersicum x S. pimpinellifolium.

An important trait defining fresh tomato marketability is fruit shelf life. Exotic germplasm of Solanum pimpinellifolium is able to prolong shelf life. Sixteen recombinant inbred lines with differing values of shelf life and fruit weight were derived by antagonistic-divergent selection from an interspecific cross involving Solanum pimpinellifolium. The objective of this study was to evaluate these recombinant inbred lines for many fruit quality traits such as diameter, height, size, acidity, colour, firmness, shelf life and weight, and to characterize them by amplified fragment length polymorphism markers. For most traits, a wide range of genetic variability was found and a wide range of molecular variation was also detected. Both sets of data allowed the identification of recombinant inbred lines by means of cluster analysis and principal component analysis. Genetic association among some amplified fragment length polymorphism markers and fruit quality traits, suggested by the principal component analysis, could be identified by single point analysis. Potential molecular markers underlying agronomical traits were detected in these recombinant inbred lines.

Free amino acid production during tomato fruit ripening: a focus on L-glutamate.

In tomato, free amino acids increase dramatically during fruit ripening and their abundance changed differentially. More evident is L-glutamate which gives the characteristic "umami" flavor. Glutamate is the principal free amino acid of ripe fruits of cultivated varieties. In this paper, we examined the capacity of tomato fruits to process endogenous as well as exogenous polypeptides during the ripening transition, in order to analyze their contribution to the free amino acid pool. In addition, the activity of some enzymes involved in glutamate metabolism such as gamma-glutamyl transpeptidase (gamma-GTase), glutamate dehydrogenase (GDH), alpha-ketoglutarate-dependent gamma-aminobutyrate transaminase (GABA-T), alanine and aspartate aminotransferases was evaluated. Results showed that peptidases were very active in ripening fruits, and they were able to release free amino acids from endogenous proteins and glutamate from exogenously added glutamate-containing peptides. In addition, red fruit contained enough gamma-GTase activity to sustain glutamate liberation from endogenous substrates such as glutathione. From all the glutamate metabolizing enzymes, GDH and GABA-T showed the higher increase in activities when the ripening process starts. In summary, tomato fruits increase free amino acid content during ripening, most probably due to the raise of different peptidase activities. However, glutamate level of ripe fruit seems to be mostly related to GDH and GABA-T activities that could contribute to increase L-glutamate level during the ripening transition.

A small intergenic region drives exclusive tissue-specific expression of the adjacent genes in Arabidopsis thaliana.

BACKGROUND: Transcription initiation by RNA polymerase II is unidirectional from most genes. In plants, divergent genes, defined as non-overlapping genes organized head-to-head, are highly represented in the Arabidopsis genome. Nevertheless, there is scarce evidence on functional analyses of these intergenic regions. The At5g06290 and At5g06280 loci are head-to-head oriented and encode a chloroplast-located 2-Cys peroxiredoxin B (2CPB) and a protein of unknown function (PUF), respectively. The 2-Cys peroxiredoxins are proteins involved in redox processes, they are part of the plant antioxidant defence and also act as chaperons. In this study, the transcriptional activity of a small intergenic region (351 bp) shared by At5g06290 and At5g06280 in Arabidopsis thaliana was characterized. RESULTS: Activity of the intergenic region in both orientations was analyzed by driving the beta-glucuronidase (GUS) reporter gene during the development and growth of Arabidopsis plants under physiological and stressful conditions. Results have shown that this region drives expression either of 2cpb or puf in photosynthetic or vascular tissues, respectively. GUS expression driven by the promoter in 2cpb orientation was enhanced by heat stress. On the other hand, the promoter in both orientations has shown similar down-regulation of GUS expression under low temperatures and other stress conditions such as mannitol, oxidative stress, or fungal elicitor. CONCLUSION: The results from this study account for the first evidence of an intergenic region that, in opposite orientation, directs GUS expression in different spatially-localized Arabidopsis tissues in a mutually exclusive manner. Additionally, this is the first demonstration of a small intergenic region that drives expression of a gene whose product is involved in the chloroplast antioxidant defence such as 2cpb. Furthermore, these results contribute to show that 2cpb is related to the heat stress defensive system in leaves and roots of Arabidopsis thaliana.