Ascorbate Peroxidase Isoforms Research Articles

GA3 and BAP phytohormone seed priming enhances germination and PEG induced drought stress tolerance in soybean by triggering the expression of osmolytes, antioxidant enzymes and related genes at the early seedling growth stages

Drought stress strongly affects seed germination and seedling development during the early stages of plant growth in soybean (Glycine max L.). Seed priming with phytohormone (PH) is known to regulate various physiological and biochemical processes that govern plant growth and yield under optimal and stress conditions. In the present study, the effects of seed priming with Gibberellic acid (GA3) (0.28 mM), 6-Benzyl aminopurine (BAP) (20 mM) and Methyl Jasmonate (MJ) (2.5 mM) was studied during germination and at early seedling growth stages in JS-335 (drought sensitive) and MAUS-71 (moderately drought tolerant) soybean varieties under PFG induced drought stress conditions. The seed priming effects were studied in terms of germination percentage and rate (Timson Index), accumulation of osmolytes (proline and glycine betaine), antioxidant enzymes activity (superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT)), and the relative expression of genes viz., Cu/Zn-superoxide dismutase copper chaperone (SOD-CCS) , iron-superoxide dismutase (SODB2) , betaine aldehyde dehydrogenase (BADH2) and dehydration-responsive element binding protein (DREB2A). The germination percentage of non-primed soybean seeds decreased under drought stress but significantly increased upon GA3 and BAP priming. Interestingly, seed priming with MJ completely inhibited the germination in both the varieties. GA3 and BAP seed priming increased proline and glycine betaine accumulation under PEG induced drought stress in both the soybean varieties. The results showed that the activities of SOD, APX and CAT were significantly enhanced in the leaves of seedlings raised from GA3 and BAP-primed seeds, as compared to the non-primed seeds under the drought stress conditions. Isozyme profiling of SOD and APX revealed that the GA3 and BAP seed priming treatments enhanced the drought-inducible expression of SOD isozymes rather than inducing novel isoforms. In case of APX isozymes, GA3 seed priming appears to preserve APX isozyme diversity in both varieties, and BAP seed priming elevated the expression of APX isoforms in both soybean varieties under the drought conditions. In response to the drought stress, relative expression of the genes associated with superoxide dismutase and glycine betaine viz., SOD CCS, SODB2, BADH2 and DREB2A was markedly upregulated in the leaves of seedlings raised from the GA3 and BAP primed seeds in both the soybean varieties. The global correlation analysis designated that the osmolyte Proline, antioxidant enzyme APX and CAT, and SOD CCS, SODB2, BADH2 and DREB2A genes were potent regulator for drought stress tolerance mechanism in soybean. This study illuminates the intricate interplay of GA3 and BAP hormonal seed priming during the germination and early seedling stages in soybean plants under the drought stress. This finding emphasizes the critical consideration of genotype specific reactions to the hormonal seed priming under PEG induced drought stress conditions.

The Antioxidant Defense System of Tomato (Solanum lycopersicumL.) Varieties under Drought Stress and upon Post-Drought Rewatering.

Water shortage induces physiological, biochemical, and molecular alterations in plant leaves that play an essential role in plant adaptive response. The effects of drought and post-drought rewatering on the activity of antioxidant enzymes and levels of H2O2, phenolic compounds, ascorbic acid, and proline were studied in six local tomato (Solanum lycopersicumL.) varieties. The contents of H2O2 and ascorbic acid increased in all drought-exposed tomato plants and then decreased upon rewatering. The level of phenolic compounds also decreased in response to water shortage and then recovered upon rehydration, although the extent of this response was different in different varieties. The activities of ascorbate peroxidase (APX) and guaiacol peroxidase (POX) and the content of proline significantly increased in the drought-stressed plants and then decreased when the plants were rewatered. The activities of 8 constitutive APX isoforms and 2 constitutive POX isoforms varied upon exposure to drought and were observed after rewatering in all studied varieties. The information on the response of tomato plants to drought and subsequent rewatering is of great importance for screening and selection of drought-tolerant varieties, as well as for development of strategies for increasing plant productivity under adverse environmental conditions.

Physiological function and regulation of ascorbate peroxidase isoforms.

Ascorbate peroxidase (APX) reduces H2O2 to H2O by utilizing ascorbate as a specific electron donor and constitutes the ascorbate-glutathione cycle in organelles of plants including chloroplasts, cytosol, mitochondria, and peroxisomes. It has been almost 40 years since APX was discovered as an important plant-specific H2O2-scavenging enzyme, during which time many research groups have conducted molecular physiological analyses. It is now clear that APX isoforms function not only just as antioxidant enzymes but also as important factors in intracellular redox regulation through the metabolism of reactive oxygen species. The function of APX isoforms is regulated at multiple steps, from the transcriptional level to post-translational modifications of enzymes, thereby allowing them to respond flexibly to ever-changing environmental factors and physiological phenomena such as cell growth and signal transduction. In this review, we summarize the physiological functions and regulation mechanisms of expression of each APX isoform.

An oligonucleotide/oligosaccharide-binding-fold protein enhances the alternative splicing event producing thylakoid membrane-bound ascorbate peroxidase in Nicotiana tabacum.

The stromal and thylakoid membrane-bound ascorbate peroxidase isoforms are produced by the alternative splicing event of the 3′-terminal region of the APXII gene in spinach (Spinacia oleracea) and tobacco (Nicotiana tabacum), but not in Arabidopsis (Arabidopsis thaliana). However, all alternative splicing variants were detected in APXII gene-transformed Arabidopsis, indicating the occurrence of its regulatory mechanisms in Arabidopsis. The efficiency of this alternative splicing event in producing thylakoid membrane-bound ascorbate peroxidase mRNA is regulated by a splicing regulatory cis element, but trans splicing regulatory factor(s) for alternative splicing remain unclear. To identify this factor, we conducted a forward genetic screen using Arabidopsis in combination with a luciferase reporter system to evaluate the alternative splicing efficiency of thylakoid membrane-bound ascorbate peroxidase mRNA production. We isolated 9 mutant lines that showed low efficiency of the AS in producing thylakoid membrane-bound ascorbate peroxidase mRNA compared with that in the control plants. From one mutant [APXII alternative splicing inhibition (apsi1)], the causal gene responsible for the phenotype, AT5G38890 (oligonucleotide/oligosaccharide-binding-fold protein, APSI1), was identified. The levels of thylakoid membrane-bound ascorbate peroxidase mRNA from the transformed APXII gene decreased and increased in APSI1 knockout and APSI1-overexpressing plants, respectively. APSI1 was localized to the nucleus and specifically bound to the splicing regulatory cis element sequence. Tobacco plants that disrupted the closest homologs of APSI1 showed low levels of endogenous thylakoid membrane-bound ascorbate peroxidase mRNA. These results indicate that APSI1 is an enhancing component of the alternative splicing event of APXII.

Exogenous Ca2+ enhances antioxidant defense in rice to simulated acid rain by regulating ascorbate peroxidase and glutathione reductase.

Exogenous calcium enhances rice tolerance to acid rain stress by regulating isozymes composition and transcriptional expression of ascorbate peroxidase and glutathione reductase. Calcium (Ca) participates in signal transduction in plants under abiotic stress, and addition of Ca2+ is beneficial to alleviate damage of plants caused by acid rain. To clarify the effect of exogenous Ca2+ on tolerance of plants to acid rain stress, we investigated regulation of Ca2+ (5mM) on activities, isozymes composition and transcriptional expression of ascorbate peroxidase (APX) and glutathione reductase (GR), redox state, and H2O2 concentration and growth in rice leaves and roots under simulated acid rain (SAR) stress. SAR (pH 3.5/2.5) decreased the total activities of APX and GR in rice by decreasing the concentration of APX isoforms (APXII in leaves and APXIII in roots) as well as activation degree of GR isozymes and transcription level of GR1, indicating that SAR (pH 3.5/2.5) destroyed the redox state in rice cells and induced H2O2 excessive accumulation, and inhibited growth of rice. Exogenous Ca2+ alleviated SAR-induced inhibition on activities of APX and GR by regulating the concentration, activation, and transcription of their isozymes, and then maintained the redox level of cells and protected cells from oxidative damage, being beneficial to the growth of rice. Therefore, the promotion of exogenous Ca2+ on activities of APX and GR can be important to enhance rice tolerance to acid rain by maintaining redox state and avoiding oxidative damage.

Photochemical efficiency correlated with candidate gene expression promote coffee drought tolerance

The aim of this study was to identify the correlation between photochemical efficiency and candidate genes expression to elucidate the drought tolerance mechanisms in coffee progenies (Icatu Vermelho IAC 3851-2 × Catimor UFV 1602-215) previously identified as tolerant in field conditions. Four progenies (2, 5, 12 and 15) were evaluated under water-deficit conditions (water deficit imposed 8 months after transplanting seedlings to the pots) and under irrigated system. Evaluations of physiological parameters and expression of candidate genes for drought tolerance were performed. Progeny 5 showed capacity to maintain water potential, which contributed to lower qP variation between irrigated and deficit conditions. However, the increases of qN and NPQ in response to stress indicate that this progeny is photochemically responsive to small variations of Ψam protecting the photosystem and maintaining qP. Data obtained for progeny 12 indicated a lower water status maintenance capacity, but with increased qN and NPQ providing maintenance of the ɸPSII and ETR parameters. A PCA analysis revealed that the genes coding regulatory proteins, ABA-synthesis, cellular protectors, isoforms of ascorbate peroxidase clearly displayed a major response to drought stress and discriminated the progenies 5 and 12 which showed a better photochemical response. The genes CaMYB1, CaERF017, CaEDR2, CaNCED, CaAPX1, CaAPX5, CaGolS3, CaDHN1 and CaPYL8a were up-regulated in the arabica coffee progenies with greater photochemical efficiency under deficit and therefore contributing to efficiency of the photosynthesis in drought tolerant progenies.

Characterization and expression analysis of ascorbate peroxidase from the moss Dicranum scoparium during abiotic stresses

Ascorbate peroxidase (APX) is an important antioxidant enzyme responsible for the conversion of H2O2 to H2O and O2. In this study, APX was studied in the widespread boreal cushion moss Dicranum scoparium. Native PAGE of crude extracts of moss thalli revealed the presence of APX isoforms in D. scoparium with a range of molecular masses. An APX complementary DNA (cDNA) gene of 771-bp length was cloned and designated as DsAPX. The cloned coding domain sequence (CDS) encoded a 256 amino acid polypeptide, and the predicted protein product was calculated to have a molecular mass of 28.4 kDa with an isoelectric point of 5.7. Several highly conserved sites important for enzyme activity were predicted using bioinformatic tools. The DsAPX protein has similarities of 91%, 78%, 67% and 66% with APX homologs from Grimmia pilifera, Physcomitrella patens, Zea mays and Nicotiana tomentosiformis, respectively. The high homology with cytosolic APX from G. pilifera strongly suggests that the cloned DsAPX gene encodes a cytosolic APX. We studied the role of APX in the tolerance of this moss to abiotic stresses. Changes in both APX activity and DsAPX gene expression, estimated using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), showed that the gene was up-regulated in response to desiccation/rehydration, and expression was maintained during a heat stress of +50°C. By contrast, DsAPX was down-regulated by a freezing (–20°C) treatment. Results obtained extend our knowledge of the diversity of APX isoforms that occur in bryophytes and suggest that APX may be involved in the tolerance of D. scoparium to abiotic stresses.

Salicylic acid modulates isoenzyme pattern of enzymatic antioxidants in wheat

The isoenzyme profile of leaf and root tissues of wheat plants grown with different concentrations of salicylic acid (SA) was studied by native polyacrylamide gel electrophoresis (PAGE). Wheat plants were co-cultivated with different concentration of SA (0, 50, 500 and 1000 µM) in Hogland solution. Primary leaves and roots from one week old plants were used for the extraction of enzymes. The crude enzyme extracts were subjected to native PAGE and were stained for the enzymatic antioxidants catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX) and superoxide dismutase (SOD). Three isoforms of CAT were observed in both leaf and root tissues of which the intensity of major isoform was reduced in leaf tissues of plants grown with 1000 µM SA where as induction in band intensity of two minor isoforms were noticed in root tissues of plants grown with 500 and 1000 µM SA. The native-PAGE for peroxidase activity revealed five isoforms in root and two in leaf tissues. One of these isoforms in both the tissues showed a gradual reduction in activity with each concentration of SA. Band intensity of APX isoforms were found to be decreased in both leaf and root tissues of plants grown with 500 and 1000 µM SA. A stimulation in band intensity of two of the APX isoforms was noticed in plants grown with 50 µM SA. The isoenzyme profile of SOD revealed two iso-isoforms in leaf tissues and three isoforms in root tissues of both control and SA grown plants. Inhibitory test confirmed these isoenzymes as Cu/Zn-SOD. The study suggested that the effect of SA on the isoenzyme profile of wheat plants is tissue specific and also varies for different enzymes. The low concentration of SA stimulates the synthesis some isoforms where as the higher concentration has negative effect on the synthesis of some isoforms of enzymatic antioxidants.

Thylakoidal APX modulates hydrogen peroxide content and stomatal closure in rice (Oryza sativa L.)

In rice (Oryza sativa L.) there are two chloroplast ascorbate peroxidases isoforms, OsAPX7 (stromal) and OsAPX8 (thylakoidal). Previous works demonstrated that double silencing of OsAPX7 and OsAPX8 led to differences in photosynthetic parameters in stress conditions. Nevertheless, both APX encoding genes present contrasting expression patterns in response to abiotic stress and the individual role of them in chloroplast redox metabolism and in stress response remains largely unknown.Here, we showed that plants silenced to OsAPX8 (RNAiOsAPX8) have a lower growth rate and a higher H2O2 content, when compared to non-transformed (NT) plants. Furthermore, RNAiOsAPX8 plants show a decrease in stomatal opening with consequent increase in leaf surface temperature. However, these plants were not more susceptible to drought stress. RNAseq analysis showed that the silencing of OsAPX8 has impact on plant gene expression, mainly affecting genes related to photosynthesis and signal transduction. Our results indicate that the reduction of OsAPX8 expression led to a stress-like phenotype but it does not alter drought tolerance.

Ascorbic Acid-A Potential Oxidant Scavenger and Its Role in Plant Development and Abiotic Stress Tolerance.

Over-production of reactive oxygen species (ROS) in plants under stress conditions is a common phenomenon. Plants tend to counter this problem through their ability to synthesize ROS neutralizing substances including non-enzymatic and enzymatic antioxidants. In this context, ascorbic acid (AsA) is one of the universal non-enzymatic antioxidants having substantial potential of not only scavenging ROS, but also modulating a number of fundamental functions in plants both under stress and non-stress conditions. In the present review, the role of AsA, its biosynthesis, and cross-talk with different hormones have been discussed comprehensively. Furthermore, the possible involvement of AsA-hormone crosstalk in the regulation of several key physiological and biochemical processes like seed germination, photosynthesis, floral induction, fruit expansion, ROS regulation and senescence has also been described. A simplified and schematic AsA biosynthetic pathway has been drawn, which reflects key intermediates involved therein. This could pave the way for future research to elucidate the modulation of plant AsA biosynthesis and subsequent responses to environmental stresses. Apart from discussing the role of different ascorbate peroxidase isoforms, the comparative role of two key enzymes, ascorbate peroxidase (APX) and ascorbate oxidase (AO) involved in AsA metabolism in plant cell apoplast is also discussed particularly focusing on oxidative stress perception and amplification. Limited progress has been made so far in terms of developing transgenics which could over-produce AsA. The prospects of generation of transgenics overexpressing AsA related genes and exogenous application of AsA have been discussed at length in the review.

Abiotic Stress Tolerance in Plants: Myriad Roles of Ascorbate Peroxidase.

One of the most significant manifestations of environmental stress in plants is the increased production of Reactive Oxygen Species (ROS). These ROS, if allowed to accumulate unchecked, can lead to cellular toxicity. A battery of antioxidant molecules is present in plants for keeping ROS levels under check and to maintain the cellular homeostasis under stress. Ascorbate peroxidase (APX) is a key antioxidant enzyme of such scavenging systems. It catalyses the conversion of H2O2 into H2O, employing ascorbate as an electron donor. The expression of APX is differentially regulated in response to environmental stresses and during normal plant growth and development as well. Different isoforms of APX show differential response to environmental stresses, depending upon their sub-cellular localization, and the presence of specific regulatory elements in the upstream regions of the respective genes. The present review delineates role of APX isoforms with respect to different types of abiotic stresses and its importance as a key antioxidant enzyme in maintaining cellular homeostasis.

Diversity and Evolution of Ascorbate Peroxidase Functions in Chloroplasts: More Than Just a Classical Antioxidant Enzyme?

Reactive oxygen species (ROS) have dual functions in plant cells as cytotoxic molecules and emergency signals. The balance between the production and scavenging of these molecules in chloroplasts, major sites for the production of ROS, is one of the key determinants for plant acclimation to stress conditions. The water-water cycle is a crucial regulator of ROS levels in chloroplasts. In this cycle, the stromal and thylakoid membrane-attached isoforms of ascorbate peroxidase (sAPX and tAPX, respectively) are involved in the metabolism of H2O2 Current genome and phylogenetic analyses suggest that the first monofunctional APX was generated as sAPX in unicellular green algae, and that tAPX occurred in multicellular charophytes during plant evolution. Chloroplastic APXs, especially tAPX, have been considered to be the source of a bottleneck in the water-water cycle, at least in higher plants, because of their high susceptibility to H2O2 A number of studies have succeeded in improving plant stress resistance by reinforcing the fragile characteristics of the enzymes. However, researchers have unexpectedly failed to find a 'stress-sensitive phenotype' among loss-of-function mutants, at least in laboratory conditions. Interestingly, the susceptibility of enzymes to H2O2 may have been acquired during plant evolution, thereby allowing for the flexible use of H2O2 as a signaling molecule in plants, and this is supported by growing lines of evidence for the physiological significance of chloroplastic H2O2 as a retrograde signal in plant stress responses. By overviewing historical, biochemical, physiological and genetic studies, we herein discuss the diverse functions of chloroplastic APXs as antioxidant enzymes and signaling modulators.

Nitric oxide induces specific isoforms of antioxidant enzymes in soybean leaves subjected to enhanced ultraviolet-B radiation

Antioxidant enzymes play a key role in plant tolerance to different types of stress, including ultraviolet-B (UV-B) radiation. Here we report that nitric oxide (NO) enhances antioxidant enzymes gene expression and increases the activity of specific isoforms protecting against UV-B radiation. Pre-treatments with sodium nitroprussiate (SNP), a NO-donor, prevented lipid peroxidation, ion leakage and H2O2 and superoxide anion accumulation in leaves of UV-B-treated soybean plants. Transcripts levels of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) were significantly induced by SNP. These data correlated with the enhancement of particular antioxidant enzyme isoforms, such as one CAT isoform and two APX isoforms. Moreover, SNP induced the expression of three new isoforms of SOD, identified as Mn-SOD subclass. Further results showed that total activities of SOD, CAT and APX significantly increased by 2.2-, 1.8- and 2.1-fold in SNP-treated plants compared to controls, respectively. The protective effect of SNP against UV-B radiation was negated by addition of the specific NO scavenger cPTIO, indicating that NO released by SNP mediates the enhancement of antioxidant enzymes activities. In conclusion, NO is involved in the signaling pathway that up-regulates specific isoforms of antioxidant enzymes protecting against UV-B-induced oxidative stress.

Investigating the expression pattern of the OsAPx1 gene promoter in rice

Background Ascorbate peroxidase (APx) is a key enzyme of the antioxidant metabolism, catalyzing the decomposition of hydrogen peroxide (H2O2) in water, using ascorbate as an electron donor. The H2O2 is a reactive oxygen species (ROS) produced constantly by aerobic metabolism. Under biotic and abiotic stress the level of H2O2 increases and, in large quantities, can cause cellular damage. In rice, there are eight APx genes that encode products target to different subcellular compartments: cytosol, peroxisoma, mitochondria and chloroplast. OsAPx1 gene encodes a cytosolic isoform of APx. The study of promoters is an important tool that allows to analyze the overall expression pattern of genes in plants.

Dose-Dependent Reactive Species Accumulation and Preferential Double-Strand Breaks Repair are Featured in the γ-ray Response in Medicago truncatula Cells

In plants, there are issues related to the effects of gamma (γ)-rays that are still poorly explored, particularly as concerns the biological response to irradiation delivered at a low dose rate. In the present work, the effects of exposure to increasing γ-ray total doses (6, 12, 25, and 50 Gy) delivered at 0.29 Gy min−1, were evaluated in terms of DNA damage sensing and repair in Medicago truncatula proliferating cell suspension cultures. The profiles of reactive oxygen species (ROS) and nitric oxide (NO) production, monitored by fluorescent staining, were in agreement with the transcriptional response of genes encoding the respiratory burst oxidase-like protein C (MtRBOHC), nitrite reductase (MtNR), the cytosolic isoform of ascorbate peroxidase (MtAPX), and a type 2 metallothionein (MtMT2). The genotoxic effects were assessed using the alkaline and neutral version of single cell gel electrophoresis (SCGE), detecting the occurrence of SSBs and DSBs. Independent on the irradiation dose, M. truncatula cells always revealed the preferential repair of DSBs, while the SSB repair was less effective. The DNA repair response was investigated focusing on genes representative of different DNA repair pathways. The overall picture derived from gene profiling analysis highlights that M. truncatula cells can produce an active response to IR-mediated genotoxic stress, as indicated by the up-regulation of the DSB sensor MtMRE11 gene, the MtTop2 gene, and the MtOGG1gene, an essential component of the base excision repair pathway. In contrast, the MtTop1, MtTdp1 and MtTFIIS genes, believed to be part of the nucleotide excision repair pathway, were significantly down-regulated.

Gamma irradiation with different dose rates induces different DNA damage responses in Petunia x hybrida cells

In plants, there is evidence that different dose rate exposures to gamma (γ) rays can cause different biological effects. The dynamics of DNA damage accumulation and molecular mechanisms that regulate recovery from radiation injury as a function of dose rate are poorly explored. To highlight dose-rate dependent differences in DNA damage, single cell gel electrophoresis was carried out on regenerating Petunia x hybrida leaf discs exposed to LDR (total dose 50Gy, delivered at 0.33Gymin−1) and HDR (total doses 50 and 100Gy, delivered at 5.15Gymin−1) γ-ray in the 0–24h time period after treatments. Significant fluctuations of double strand breaks and different repair capacities were observed between treatments in the 0–4h time period following irradiation. Dose-rate-dependent changes in the expression of the PhMT2 and PhAPX genes encoding a type 2 metallothionein and the cytosolic isoform of ascorbate peroxidase, respectively, were detected by Quantitative RealTime-Polymerase Chain Reaction. The PhMT2 and PhAPX genes were significantly up-regulated (3.0- and 0.7-fold) in response to HDR. The results are discussed in light of the potential practical applications of LDR-based treatments in mutation breeding.

Ultrastructural and antioxidative changes in lupine embryo axes in response to salt stress

Embryo axes of lupine (<em>Lupinus luteus</em> L. ‘Mister’) were subjected to 0.1 M NaCl salt stress for 24 and 48 h. The ultrastructure modification and adjustment of antioxidant enzymes activities and izoenzymes profiles were observed. In cells of lupine embryo axes grown for 48 hours in medium with 0.1 M NaCl mitochondria took the forked shape and bulges of the outer mitochondrial membranes appeared. Moreover, the inflating and swelling of rough endoplasmic reticulum (RER) lumen and fragmentation of RER were noticed. The level of H<sub>2</sub>O<sub>2</sub> was higher in salt treated embryo axes after 24 hours and increase of thiobarbituric acid reactive substances was observed after both 24 and 48 h of salt treatment. Native gel electrophoresis showed increased intensities of bands for catalase isozymes in response to salt stress, whereas activity of catalase was higher only in embryo axes grown for 48 h in control conditions. Appearance of two new isoforms of ascorbate peroxidase was observed after 48 h only under control condition, however increased activities were stated for both control and salt-stress condition after 48 h. No changes in isozymes pattern for superoxide dismutase were observed, but significant decrease in superoxide dismutase activity was noticed in relation to time and salt stress. Possible role of these enzymes in salt stress tolerance is discussed. The 0.1 M salt stress is regarded as a middle stress for lupine embryo axes and the efficiency of stress prevention mechanisms is proposed.

Ascorbate as seen through plant evolution: the rise of a successful molecule?

Ascorbate is a widespread and efficient antioxidant that has multiple functions in plants, traditionally associated with the reactions of photosynthesis. This review aims to look at ascorbate from an evolutionary perspective. Cyanobacteria, algae, and bryophytes contain lower concentrations of ascorbate than higher plants, where the molecule accumulates in high concentrations in both photosynthetic and non-photosynthetic organs and tissues. This increase in ascorbate concentration is paralleled by an increase in the number of isoforms of ascorbate peroxidase and the ascorbate regenerating enzymes mono- and dehydroascorbate reductase. One way of understanding the rise in ascorbate concentrations is to consider ascorbate as a molecule among others that has been subject to selection pressures during evolution, due to its cost or benefit for the cell and the organism. Ascorbate has a low cost in terms of synthesis and toxicity, and its benefits include protection of the glutathione pool and proper functioning of a range of enzymes. The hypothesis presented here is that these features would have favoured increasing roles for the molecule in the development and growth of multicellular organisms. This review then focuses on this diversity of roles for ascorbate in both photosynthetic and non-photosynthetic tissues of higher plants, including fruits and seeds, as well as further functions the molecule may possess by looking at other species. The review also highlights one of the trade-offs of domestication, which has often reduced or diluted ascorbate content in the quest for increased fruit growth and yield, with unknown consequences for the corresponding functional diversity, particularly in terms of stress resistance and adaptive responses to the environment.

Water extraction under terminal drought explains the genotypic differences in yield, not the anti-oxidant changes in leaves of pearl millet (Pennisetum glaucum).

Pearl millet (Pennisetum glaucum (L.) R.Br.) is a resilient crop suiting the harshest conditions of the semi-arid tropics, in which we assessed possible relationships between crop tolerance, anti-oxidative enzyme activity and plant/soil water status. Biochemical acclimation and cell homeostasis traits have been proposed as critical for the drought tolerance of crops, but their limited practical application in breeding so far suggests that the role of biochemical acclimation for drought tolerance is still unclear. Previous research may have been of limited value because it has not approached biochemical acclimation from the angle of plant water relations. Four pearl millet genotypes, contrasting for terminal drought tolerance, were evaluated (sensitive H77/833-2, tolerant PRLT2/89-33 and two near isogenic lines carrying a terminal drought tolerance quantitative trait locus) under water-stress (WS) and well-watered (WW) conditions in a lysimetric system that simulates field-like conditions. We assessed the genotypic variation and relationship between photosynthetic pigments (chlorophylls a and b and carotenoids), antioxidative isoenzymatic spectrum (superoxide dismutase, ascorbate peroxidase and catalase), physiological traits (soil moisture available, normalised transpiration, stay-green score and water extraction) and biomass and yield. Biochemical traits investigated were tightly related among each other under WS conditions but not under WW conditions. Two major ascorbate peroxidase isoforms (APX6&7), whose variation in both water regimes reflected the presence/absence of the drought tolerance quantitative trait locus, were identified, but these did not relate to yield. Both, yield and biochemical traits under terminal drought stress were closely related to the traits linked to plant/soil water status (soil moisture available, normalised transpiration, stay-green score and water extraction), whereas yield and the biochemical indicators were not correlated, except for one. It is concluded that there is no direct effect of biochemical traits on yield parameters since both are consequences of soil-plant water status and their putative relation appear to be secondary - through plant/soil water status.

Cloning, expression and functional validation of drought inducible ascorbate peroxidase (Ec-apx1) from Eleusine coracana

Eleusine coracana (finger millet) is a stress-hardy but under-utilized cereal crop that possesses an efficient antioxidant defense system. The plant is capable of enduring long durations of water deficit stress. Experiments were conducted to clone a potent stress responsive isoform of ascorbate peroxidase and validate its role under drought stress. Reverse transcriptase PCR was used to obtain the partial cDNA of apx1 gene, from a meticulously screened drought tolerant genotype of E. coracana (PR202). Using RACE strategy, the full length apx1 cDNA was cloned and sequenced. The cDNA length of the E. coracana apx1 (Ec-apx1) gene is 1,047bp with a 750bp ORF, encoding a 250 amino acid protein having a molecular weight of 28.5kDa. The identity of the amino acid sequence, deduced from the cDNA, with the APX family homologs was about 74-97%. The full-length apx1 ORF was sub-cloned in a prokaryotic expression vector pET23b. The recombinant fusion protein, Ec-apx1, had high expression level in BL21 strain of E. coli and exhibited APX enzyme activity. The structure-function relationship of the protein was deduced by modelling a three-dimensional structure of Ec-apx1, on the basis of comparative homology using SWISS-MODEL. Real time PCR analysis of Ec-apx1 expression at mRNA level showed that the transcript increased under drought stress, with maximum levels attained 5-days after imposition of stress. Our results suggest that Ec-apx1 has a distinct pattern of expression and plays a pivotal role in drought stress tolerance. Therefore, the cloned isoform of ascorbate peroxidase can be used for developing stress tolerant genotypes of important crops, through transgenic approach.