Nutrient availability regulates Deschampsia antarctica photosynthetic and stress tolerance performance in Antarctica.

Deschampsia antarctica is one of the only two native vascular plants in Antarctica, mostly located in the ice-free areas of the Peninsula's coast and adjacent islands. This region is characterized by a short growing season, frequent extreme climatic events, and soils with reduced nutrient availability. However, it is unknown whether its photosynthetic and stress tolerance mechanisms are affected by the availability of nutrients to deal with this particular environment. We studied the photosynthetic, primary metabolic, and stress tolerance performance of D. antarctica plants growing on three close sites (<500 m) with contrasting soil nutrient conditions. Plants from all sites showed similar photosynthetic rates, but mesophyll conductance and photobiochemistry were more limiting (~25%) in plants growing on low-nutrient availability soils. Additionally, these plants showed higher stress levels and larger investments in photoprotection and carbon pools, most probably driven by the need to stabilize proteins and membranes, and remodel cell walls. In contrast, when nutrients were readily available, plants shifted their carbon investment towards amino acids related to osmoprotection, growth, antioxidants, and polyamines, leading to vigorous plants without appreciable levels of stress. Taken together, these findings demonstrate that D. antarctica displays differential physiological performances to cope with adverse conditions depending on resource availability, allowing it to maximize stress tolerance without jeopardizing photosynthetic capacity.

The apoplastic antioxidant system and altered cell wall dynamics influence mesophyll conductance and the rate of photosynthesis.

Mesophyll conductance (gm ), the diffusion of CO2 from substomatal cavities to the carboxylation sites in the chloroplasts, is a highly complex trait driving photosynthesis (net CO2 assimilation, AN ). However, little is known concerning the mechanisms by which it is dynamically regulated. The apoplast is considered as a 'key information bridge' between the environment and cells. Interestingly, most of the environmental constraints affecting gm also cause apoplastic responses, cell wall (CW) alterations and metabolic rearrangements. Since CW thickness is a key determinant of gm , we hypothesize that other changes in this cellular compartiment should also influence gm . We study the relationship between the antioxidant apoplastic system and CW metabolism and the gm responses in tobacco plants (Nicotiana sylvestris L.) under two abiotic stresses (drought and salinity), combining in vivo gas-exchange measurements with analyses of antioxidant activities, CW composition and primary metabolism. Stress treatments imposed substantial reductions in AN (58-54%) and gm (59%), accompanied by a strong antioxidant enzymatic response at the apoplastic and symplastic levels. Interestingly, apoplastic but not symplastic peroxidases were positively related to gm . Leaf anatomy remained mostly stable; however, the stress treatments significantly affected the CW composition, specifically pectins, which showed significant relationships with AN and gm . The treatments additionally promoted a differential primary metabolic response, and specific CW-related metabolites including galactose, glucosamine and hydroxycinnamate showed exclusive relationships with gm independent of the stress. These results suggest that gm responses can be attributed to specific changes in the apoplastic antioxidant system and CW metabolism, opening up more possibilities for improving photosynthesis using breeding/biotechnological strategies.

Cytochrome respiration pathway and sulphur metabolism sustain stress tolerance to low temperature in the Antarctic species Colobanthus quitensis.

Understanding the strategies employed by plant species that live in extreme environments offers the possibility to discover stress tolerance mechanisms. We studied the physiological, antioxidant and metabolic responses to three temperature conditions (4, 15, and 23°C) of Colobanthus quitensis (CQ), one of the only two native vascular species in Antarctica. We also employed Dianthus chinensis (DC), to assess the effects of the treatments in a non-Antarctic species from the same family. Using fused LASSO modelling, we associated physiological and biochemical antioxidant responses with primary metabolism. This approach allowed us to highlight the metabolic pathways driving the response specific to CQ. Low temperature imposed dramatic reductions in photosynthesis (up to 88%) but not in respiration (sustaining rates of 3.0-4.2 µmol CO2  m-2  s-1 ) in CQ, and no change in the physiological stress parameters was found. Its notable antioxidant capacity and mitochondrial cytochrome respiratory activity (20 and two times higher than DC, respectively), which ensure ATP production even at low temperature, was significantly associated with sulphur-containing metabolites and polyamines. Our findings potentially open new biotechnological opportunities regarding the role of antioxidant compounds and respiratory mechanisms associated with sulphur metabolism in stress tolerance strategies to low temperature.

Low-temperature tolerance of the Antarctic species Deschampsia antarctica: A complex metabolic response associated with nutrient remobilization.

The species Deschampsia antarctica (DA) is one of the only two native vascular species that live in Antarctica. We performed ecophysiological, biochemical, and metabolomic studies to investigate the responses of DA to low temperature. In parallel, we assessed the responses in a non-Antarctic reference species (Triticum aestivum [TA]) from the same family (Poaceae). At low temperature (4°C), both species showed lower photosynthetic rates (reductions were 70% and 80% for DA and TA, respectively) and symptoms of oxidative stress but opposite responses of antioxidant enzymes (peroxidases and catalase). We employed fused least absolute shrinkage and selection operator statistical modelling to associate the species-dependent physiological and antioxidant responses to primary metabolism. Model results for DA indicated associations with osmoprotection, cell wall remodelling, membrane stabilization, and antioxidant secondary metabolism (synthesis of flavonols and phenylpropanoids), coordinated with nutrient mobilization from source to sink tissues (confirmed by elemental analysis), which were not observed in TA. The metabolic behaviour of DA, with significant changes in particular metabolites, was compared with a newly compiled multispecies dataset showing a general accumulation of metabolites in response to low temperatures. Altogether, the responses displayed by DA suggest a compromise between catabolism and maintenance of leaf functionality.

A field portable method for the semi-quantitative estimation of dehydration tolerance of photosynthetic tissues across distantly related land plants.

Desiccation tolerant (DT) plants withstand complete cellular dehydration, reaching relative water contents (RWC) below 30% in their photosynthetic tissues. Desiccation sensitive (DS) plants exhibit different degrees of dehydration tolerance (DHT), never surviving water loss >70%. To date, no procedure for the quantitative evaluation of DHT extent exists that is able to discriminate DS species with differing degrees of DHT from truly DT plants. We developed a simple, feasible and portable protocol to differentiate between DT and different degrees of DHT in the photosynthetic tissues of seed plants and between fast desiccation (< 24 h) tolerant (FDT) and sensitive (FDS) bryophytes. The protocol is based on (1) controlled desiccation inside Falcon tubes equilibrated at three different relative humidities that, consequently, induce three different speeds and extents of dehydration and (2) an evaluation of the average percentage of maximal photochemical efficiency of PSII (Fv /fm) recovery after rehydration. Applying the method to 10 bryophytes and 28 tracheophytes from various locations, we found that (1) imbibition of absorbent material with concentrated salt-solutions inside the tubes provides stable relative humidity and avoids direct contact with samples; (2) for 50 ml capacity tubes, the optimal plant amount is 50-200 mg fresh weight; (3) the method is useful in remote locations due to minimal instrumental requirements; and (4) a threshold of 30% recovery of the initial Fv /fm upon reaching RWC ≤ 30% correctly categorises DT species, with three exceptions: two poikilochlorophyllous species and one gymnosperm. The protocol provides a semi-quantitative expression of DHT that facilitates comparisons of species with different morpho-physiological traits and/or ecological attributes.

Salinity tolerance is related to cyanide-resistant alternative respiration in Medicago truncatula under sudden severe stress.

Salt respiration is defined as the increase of respiration under early salt stress. However, the response of respiration varies depending on the degree of salt tolerance and salt stress. It has been hypothesized that the activity of the alternative pathway may increase preventing over-reduction of the ubiquinone pool in response to salinity, which in turn can increase respiration. Three genotypes of Medicago truncatula are reputed as differently responsive to salinity: TN1.11, A17 and TN6.18. We used the oxygen-isotope fractionation technique to study the in vivo respiratory activities of the cytochrome oxidase pathway (COP) and the alternative oxidase pathway (AOP) in leaves and roots of these genotypes treated with severe salt stress (300 mM) during 1 and 3 days. In parallel, AOX capacity, gas exchange measurements, relative water content and metabolomics were determined in control and treated plants. Our study shows for first time that salt respiration is induced by the triggered AOP in response to salinity. Moreover, this phenomenon coincides with increased levels of metabolites such as amino and organic acids, and is shown to be related with higher photosynthetic rate and water content in TN6.18.

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.

Chloroplast protection in plum pox virus-infected peach plants by L-2-oxo-4-thiazolidine-carboxylic acid treatments: effect in the proteome.

Sharka, a disease caused by plum pox virus (PPV), has a significant economic impact on fruit tree production. In this work, we analysed the effect of (2,1,3)-benzothiadiazole (BTH) and L-2-oxo-4-thiazolidine-carboxylic acid (OTC) on plant growth and virus content. OTC reduced sharka symptom, stimulated plant growth and alleviated PPV-induced oxidative stress, indicated by a lack of changes in some oxidative stress parameters. PPV infection reduced chloroplast electron transport efficiency. However, in the presence of BTH or OTC, no changes in the chlorophyll fluorescence parameters were observed. PPV produced an alteration in chloroplast ultrastructure, giving rise to a decrease in starch contents that was less dramatic in OTC-treated plants. Furthermore, PPV reduced the abundance of proteins associated with photosynthesis, carbohydrate and amino acid metabolism and photorespiration. These changes did not take place in OTC-treated plants, and increases in the expression of proteins related with the aforementioned processes, including ADP-glucose pyrophosphorylase, were produced, which correlated with the lower decrease in starch contents observed in PPV-infected plants treated with OTC. The results suggested that OTC treatment provides protection to the photosynthetic machinery and/or the chloroplast metabolism in PPV-infected peaches. Thus, OTC could have practical implications in agriculture in improving the vigour of different plant species as well as in immunizing plants against pathogens.

Graft union formation in grapevine induces transcriptional changes related to cell wall modification, wounding, hormone signalling, and secondary metabolism.

Grafting is particularly important to the cultivation of perennial crops such as grapevine (Vitis vinifera) because rootstocks can provide resistance to soil-borne pests and diseases as well as improve tolerance to some abiotic stresses. Successful grafting is a complex biochemical and structural process beginning with the adhesion of the two grafted partners, followed by callus formation and the establishment of a functional vascular system. At the molecular level, the sequence of events underlying graft union formation remains largely uncharacterized. The present study investigates the transcriptome of grapevine rootstock and graft interface tissues sampled 3 d and 28 d after grafting of over-wintering stems in the spring. Many genes were differentially expressed over time, from 3 d to 28 d after grafting, which could be related to the activation of stem growth and metabolic activity in the spring. This hypothesis is supported by the up-regulation of many genes associated with cell wall synthesis, and phloem and xylem development. Generally, there was an up-regulation of gene expression in the graft interface tissue compared with the rootstock, particularly genes involved in cell wall synthesis, secondary metabolism, and signalling. Although there was overlap between the genes differentially expressed over time (from 3 d to 28 d after grafting) with the gene differentially expressed between the rootstock and the graft interface, numerous graft interface-specific genes were identified.

Ecophysiological Differentiation among Two Resurrection Ferns and Their Allopolyploid Derivative.

Theoretically, the coexistence of diploids and related polyploids is constrained by reproductive and competitive mechanisms. Although niche differentiation can explain the commonly observed co-occurrence of cytotypes, the underlying ecophysiological differentiation among cytotypes has hardly been studied. We compared the leaf functional traits of the allotetraploid resurrection fern Oeosporangium tinaei (HHPP) and its diploid parents, O. hispanicum (HH) and O. pteridioides (PP), coexisting in the same location. Our experimental results showed that all three species can recover physiological status after severe leaf dehydration, which confirms their 'resurrection' ability. However, compared with PP, HH had much higher investment per unit area of light-capturing surface, lower carbon assimilation rate per unit mass for the same midday water potential, higher non-enzymatic antioxidant capacity, higher carbon content, and lower contents of nitrogen, phosphorus, and other macronutrients. These traits allow HH to live in microhabitats with less availability of water and nutrients (rock crevices) and to have a greater capacity for resurrection. The higher assimilation capacity and lower antioxidant capacity of PP explain its more humid and nutrient-rich microhabitats (shallow soils). HHPP traits were mostly intermediate between those of HH and PP, and they allow the allotetraploid to occupy the free niche space left by the diploids.

The Lack of Alternative Oxidase 1a Restricts in vivo Respiratory Activity and Stress-Related Metabolism for Leaf Osmoprotection and Redox Balancing Under Sudden Acute Water and Salt Stress in Arabidopsis thaliana.

In plants salt and water stress result in an induction of respiration and accumulation of stress-related metabolites (SRMs) with osmoregulation and osmoprotection functions that benefit photosynthesis. The synthesis of SRMs may depend on an active respiratory metabolism, which can be restricted under stress by the inhibition of the cytochrome oxidase pathway (COP), thus causing an increase in the reduction level of the ubiquinone pool. However, the activity of the alternative oxidase pathway (AOP) is thought to prevent this from occurring while at the same time, dissipates excess of reducing power from the chloroplast and thereby improves photosynthetic performance. The present research is based on the hypothesis that the accumulation of SRMs under osmotic stress will be affected by changes in folial AOP activity. To test this, the oxygen isotope-fractionation technique was used to study the in vivo respiratory activities of COP and AOP in leaves of wild-type Arabidopsis thaliana plants and of aox1a mutants under sudden acute stress conditions induced by mannitol and salt treatments. Levels of leaf primary metabolites and transcripts of respiratory-related proteins were also determined in parallel to photosynthetic analyses. The lack of in vivo AOP response in the aox1a mutants coincided with a lower leaf relative water content and a decreased accumulation of crucial osmoregulators. Additionally, levels of oxidative stress-related metabolites and transcripts encoding alternative respiratory components were increased. Coordinated changes in metabolite levels, respiratory activities and photosynthetic performance highlight the contribution of the AOP in providing flexibility to carbon metabolism for the accumulation of SRMs.

Oxidative stress induced in tobacco leaves by chloroplast over-expression of maize plastidial transglutaminase.

As part of a project aiming to characterize the role of maize plastidial transglutaminase (chlTGZ) in the plant chloroplast, this paper presents results on stress induced by continuous chlTGZ over-expression in transplastomic tobacco leaves. Thylakoid remodelling induced by chlTGZ over-expression in young leaves of tobacco chloroplasts has already been reported (Ioannidis et al. in Biochem Biophys Acta 1787:1215-1222, 2009). In the present work, we determined the induced alterations in the photosynthetic apparatus, in the chloroplast ultrastructure, and, particularly, the activation of oxidative and antioxidative metabolism pathways, regarding ageing and functionality of the tobacco transformed plants. The results revealed that photochemistry impairment and oxidative stress increased with transplastomic leaf age. The decrease in pigment levels in the transformed leaves was accompanied by an increase in H(2)O(2) and lipid peroxidation. The rise in H(2)O(2) correlated with a decrease in catalase activity, whereas there was an increase in peroxidase activity. In addition, chlTGZ over-expression lead to a drop in reduced glutathione, while Fe-superoxide dismutase activity was higher in transformed than in wild-type leaves. Together with the induced oxidative stress, the over-expressed chlTGZ protein accumulated progressively in chloroplast inclusion bodies. These traits were accompanied by thylakoid scattering, membrane degradation and reduction of thylakoid interconnections. Consequently, the electron transport between photosystems decrease in the old leaves. In spite of these alterations, transplastomic plants can be maintained and reproduced in vitro. These results are discussed in line with chlTGZ involvement in chloroplast functionality.

The Apoplastic and Symplastic Antioxidant System in Onion: Response to Long-Term Salt Stress.

The response of apoplastic antioxidant systems in root and leaf tissues from two onion genotypes ('Texas 502', salt-sensitive and 'Granex 429', salt-resistant) in response to salinity was studied. Electrolyte leakage data indicated the membrane integrity impairing by the effect of salts, especially in 'Texas 502'. We detected superoxide dismutase (SOD) and peroxidase (POX) activity in the root and leaf apoplastic fractions from onion plants. Salinity increased SOD activity in the root symplast of 'Texas 502' and in 'Granex 429' leaves. In contrast, salinity reduced SOD activity in the leaf and root apoplastic fractions from 'Texas 502'. In 'Granex 429', salt-stress increased leaf apoplastic POX activity and symplastic catalase (CAT) activity of both organs, but a decline in root apoplastic POX from 'Texas 502' took place. Salt-stress increased monodehydroascorbate reductase (MDHAR) in root and leaf symplast and in root glutathione reductase GR, mainly in 'Granex 429', but only in this genotype, leaf dehydroascorbate reductase (DHAR) activity increased. In contrast, a decline in leaf GR was produced only in 'Texas 502'. Salinity increased leaf ASC levels, and no accumulation of dehydroascorbate (DHA) was observed in roots in both cases. These responses increased the redox state of ascorbate, especially in roots. In contrast, salinity declined reduced glutathione (GSH), but oxidised glutathione (GSSG) was accumulated in leaves, decreasing the redox state of glutathione. Salinity slightly increased root GSH concentration in the salt-tolerant genotype and was unchanged in the salt-sensitive genotype, but no accumulation of GSSG was produced, favoring the rise and/or maintenance of the redox state of the glutathione. These results suggest that the lower sensitivity to salt in 'Granex 429' could be related to a better performance of the antioxidant machinery under salinity conditions.

Cell wall components regulate photosynthesis and leaf water relations of Vitis vinifera cv. Grenache acclimated to contrasting environmental conditions.

Environmental conditions determine plants performance as they shape - among other key factors - leaf features and physiology. However, little is known regarding to the changes occurring in leaf cell wall composition during the acclimation to an environmental stress and, specially, if these changes have an impact on other leaf physiology aspects. In order to induce changes in photosynthesis, leaf water relations and cell wall main components (i.e., cellulose, hemicelluloses and pectins) and see how they co-vary, Vitis vinifera cv. Grenache was tested under four different conditions: (i) non-stress conditions (i.e., control, with high summer temperature and irradiance), (ii) growth chamber conditions, (iii) growth chamber under water stress and (iv) cold growth chamber. Plants developed in growth chambers decreased net CO2 assimilation (AN) and mesophyll conductance (gm) compared to control. Although cold did not change the bulk modulus of elasticity (ε), it decreased in growth chamber conditions and water stress. Control treatment showed the highest values for photosynthetic parameters and ε as well as for leaf structural traits such as leaf mass area (LMA) and leaf density (LD). Whereas cellulose content correlated with photosynthetic parameters, particularly AN and gm, pectins and the amount of alcohol insoluble residue (AIR) - an approximation of the isolated cell wall fraction - correlated with leaf water parameters, specifically, ε. Although preliminary, our results suggest that cell wall modifications due to environmental acclimations can play a significant role in leaf physiology by affecting distinctly photosynthesis and water relations in a manner that might depend on environmental conditions.

Photosynthesis Optimized across Land Plant Phylogeny.

Until recently, few data were available on photosynthesis and its underlying mechanistically limiting factors in plants, other than crops and model species. Currently, a new large pool of data from extant representatives of basal terrestrial plant groups is emerging, allowing exploration of how photosynthetic capacity (Amax) increases from minimum values in bryophytes to maximum in tracheophytes, which is associated to an optimization of the balance between its limiting factors. From predominant mesophyll conductance limitation (lm) in bryophytes and lycophytes (fern allies) to stomatal conductance (ls) and lm colimitation in pteridophytes (ferns) and gymnosperms, a balanced colimitation by the three limitations is finally reached in angiosperms. We discuss the implications of this new knowledge for future biotechnological attempts to improve crop photosynthesis.

Alteration in the chloroplastic metabolism leads to ROS accumulation in pea plants in response to plum pox virus.

In this work, a recombinant plum pox virus (PPV, Sharka) encoding green fluorescent protein is used to study its effect on antioxidant enzymes and protein expression at the subcellular level in pea plants (cv. Alaska). PPV had produced chlorotic spots as well as necrotic spots in the oldest leaves at 13-15 d post-inoculation. At 15 d post-inoculation, PPV was present in the chlorotic and necrotic areas, as shown by the fluorescence signal produced by the presence of the green fluorescent protein. In the same areas, an accumulation of reactive oxygen species was noticed. Studies with laser confocal and electron microscopy demonstrated that PPV accumulated in the cytosol of infected cells. In addition, PPV infection produced an alteration in the chloroplast ultrastructure, giving rise to dilated thylakoids, an increase in the number of plastoglobuli, and a decreased amount of starch content. At 3 d post-inoculation, although no changes in the oxidative stress parameters were observed, an increase in the chloroplastic hydrogen peroxide levels was observed that correlated with a decrease in the enzymatic mechanisms involved in its elimination (ascorbate peroxidase and peroxidase) in this cell compartment. These results indicate that an alteration in the chloroplastic metabolism is produced in the early response to PPV. This oxidative stress is more pronounced during the development of the disease (15 d post-inoculation) judging from the increase in oxidative stress parameters as well as the imbalance in the antioxidative systems, mainly at the chloroplastic level. Finally, proteomic analyses showed that most of the changes produced by PPV infection with regard to protein expression at the subcellular level were related mainly to photosynthesis and carbohydrate metabolism. It seems that PPV infection has some effect on PSII, directly or indirectly, by decreasing the amount of Rubisco, oxygen-evolving enhancer, and PSII stability factor proteins. The results indicate that Sharka symptoms observed in pea leaves could be due to an imbalance in antioxidant systems as well as to an increased generation of reactive oxygen species in chloroplasts, induced probably by a disturbance of the electron transport chain, suggesting that chloroplasts can be a source of oxidative stress during viral disease development.

Developments at the graft interface in homo- and hetero-grafts: Gene expression and histological changes during the first month after grafting.

Gene expression changes induced during graft union formation (the first month after grafting) in grapevine have been studied using whole genome microarrays. The genes differentially expressed between the rootstock and graft interface tissues of homo-grafts (Cabernet Sauvignon (CS) grafted onto CS) were compared at 3 and 28 days after grafting (dag). Graft union formation was associated with the upregulation of genes involved in secondary metabolism, cell wall, wound responses and hormone signaling. These gene expression differences were associated with the accumulation of lignin, cellulose and callose in the callus cells. Superimposed upon this, hetero-grafting between two different grapevine genotypes resulted in the further upregulation of stress and/or defense responses at the graft interface. Here we discuss the limitations of the techniques used to study the developments at the graft interface to date and future research directions to understand graft union formation in plants.

Cu/Zn superoxide dismutase and ascorbate peroxidase enhance in vitro shoot multiplication in transgenic plum.

In this study we examined the role of antioxidant metabolism in in vitro shoot multiplication. We generated transgenic plum plantlets overexpressing the cytsod and cytapx genes in cytosol under the control of the constitutive promoter CaMV35S. Three transgenic lines with up-regulated sod at transcriptional levels that showed silenced cytapx expression displayed an elevated in vitro multiplication rate. By contrast, a transgenic line harboring several copies of cytapx and with elevated APX enzymatic activity did not show any improvement in plant vigor, measured as the number of axillary shoots and shoot length. All of the lines with elevated micropropagation ability exhibited intensive H2O2 accumulation, monitored by 3,3'-diaminobenzidine (DAB) staining as well as by colorimetric analysis, providing direct in vitro evidence of the role of H2O2 and antioxidant genes in in vitro shoot multiplication.

Plant growth stimulation in Prunus species plantlets by BTH or OTC treatments under in vitro conditions.

The effects of benzothiadiazole (BTH) and L-2-oxothiazolidine-4-carboxylic acid (OTC) on the growth and viral content of micropropagated, Plum pox virus (PPV)-infected peach [(Prunus persica (L.) Batsch] 'GF305' plantlets were analyzed. Low BTH and OTC concentrations resulted in a significant increase in the growth of GF305 peach and plum plants, with greater effects in PPV-infected than in healthy GF305 peach plantlets. Neither BTH nor OTC reduced the virus content. In fact, the highest growth and viral contents coincided, especially with the 10 µM BTH treatment. Differing effects on the antioxidative metabolism of PPV-infected GF305 peach plantlets were observed, depending on the compound and the concentration used: BTH decreased GSH, whereas OTC increased it. In PPV-infected plants, the 50 µM OTC treatment produced a decrease in ascorbate peroxidase, catalase, and glutathione peroxidase, but an increase in superoxide dismutase. However, BTH produced a rise in peroxidase activity. Both 10 µM BTH and 50 µM OTC produced H2O2 accumulation that was correlated with the histochemical detection of H2O2 by 3,3'-diaminobenzidine staining. PPV infection induced NPR1 expression and a synergistic effect occurred in the presence of 50 µM OTC, since this compound produced an up-regulation of NPR1 in both healthy and PPV-infected GF305 peach plantlets. The results showed that GSH, as previously suggested, and/or H2O2 could be involved in the regulation of NPR1 expression. Globally, the results show that both OTC and BTH improved the vigor of Prunus species, including peach and plum, under in vitro conditions, producing positive effects on growth, antioxidative metabolism and NPR1 expression. All of these improvements could be critical for more successful ex vitro acclimatization as well as for improved responses to different stresses.