L-ascorbate Peroxidase Research Articles

Natural variation in rice ascorbate peroxidase gene APX9 is associated with a yield-enhancing QTL cluster.

We previously identified a cluster of yield-related quantitative trait loci (QTLs) including plant height in CR4379, a near-isogenic line from a cross between Oryza sativa spp. japonica cultivar ‘Hwaseong’ and the wild relative Oryza rufipogon. Map-based cloning and transgenic approaches revealed that APX9, which encodes an l-ascorbate peroxidase 4, is associated with this cluster. A 3 bp InDel was observed leading to the addition of a valine in Hwaseong compared with O. rufipogon. APX9-overexpressing transgenic plants in the Hwaseong background were taller than Hwaseong. Consistent with these results, APX9 T-DNA insertion mutants in the japonica cultivar Dongjin were shorter. These results confirm that APX9 is the causal gene for the QTL cluster. Sequence analysis of APX9 from 303 rice accessions revealed that the 3 bp InDel clearly differentiates japonica (APX9HS) and O. rufipogon (APX9OR) alleles. indica accessions shared both alleles, suggesting that APX9HS was introgressed into indica followed by crossing. The finding that O. rufipogon accessions with different origins carry APX9OR suggests that the 3 bp insertion was specifically selected in japonica during its domestication. Our findings demonstrate that APX9 acts as a major regulator of plant development by controlling a valuable suite of agronomically important traits in rice.

Proteomic Changes in Antioxidant System in Strawberry During Ripening.

To investigate the strawberry antioxidant defense system during fruit ripening, a targeted quantitative proteomic approach using multiple reaction monitoring (MRM) was developed to investigate targeted proteins in the antioxidant enzyme system in strawberry fruit. We investigated 46 proteins and isoforms with 73 identified peptides which may be involved in this antioxidant enzyme system. Among the proteins that changed during ripening, aldo/keto reductase (AKR), superoxide dismutase (SOD) and glutathione transferase (GT) increased significantly, while dehydroascorbate reductase, 2-Cys peroxiredoxin, catalase (CAT), 1-Cys peroxiredoxin and L-ascorbate peroxidase (APX) decreased significantly. These results suggest that fruit ripening of strawberry activates the enzymes of an SOD/glutathione metabolism system. The methodologies used in this study will be useful for systematically characterizing the role of antioxidant enzymes in fruit ripening of other plants.

Differentially expressed genes related to oxidoreductase activity and glutathione metabolism underlying the adaptation of Phragmites australis from the salt marsh in the Yellow River Delta, China.

The common reed (Phragmites australis) is a dominant species in the coastal wetlands of the Chinese Yellow River Delta, where it tolerates a wide range of salinity. Recent environmental changes have led to the increase of soil salinity in this region, which has degraded much of the local vegetation. Clones of common reeds from the tidal marsh may have adapted to local high salinity habitat through selection on genes and metabolic pathways conferring salt tolerance. This study aims to reveal molecular mechanisms underlying salt tolerance in the tidal reed by comparing them to the salt-sensitive freshwater reed under salt stress. We employed comparative transcriptomics to reveal the differentially expressed genes (DEGs) between these two types of common reeds under different salinity conditions. The results showed that only three co-expressed genes were up-regulated and one co-expressed gene was down-regulated between the two reed types. On the other hand, 1,371 DEGs were exclusively up-regulated and 285 DEGs were exclusively down-regulated in the tidal reed compared to the control, while 115 DEGs were exclusively up-regulated and 118 DEGs were exclusively down-regulated in the freshwater reed compared to the control. From the pattern of enrichment of transcripts involved in salinity response, the tidal reed was more active and efficient in scavenging reactive oxygen species (ROS) than the freshwater reed, with the tidal reed showing significantly higher gene expression in oxidoreductase activity. Furthermore, when the reeds were exposed to salt stress, transcripts encoding glutathione metabolism were up-regulated in the tidal reed but not in the freshwater reed. DEGs related to encoding glutathione reductase (GR), glucose-6-phosphate 1-dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PD), glutathione S-transferase (GST) and L-ascorbate peroxidase (LAP) were revealed as especially highly differentially regulated and therefore represented candidate genes that could be cloned into plants to improve salt tolerance. Overall, more genes were up-regulated in the tidal reed than in the freshwater reed from the Yellow River Delta when under salt stress. The tidal reed efficiently resisted salt stress by up-regulating genes encoding for oxidoreductase activity and glutathione metabolism. We suggest that this type of common reed could be extremely useful in the ecological restoration of degraded, high salinity coastal wetlands in priority.

Proteomic and metabolic profile analysis of low-temperature storage responses in Ipomoea batata Lam. tuberous roots

BackgroundSweetpotato (Ipomoea batatas L.) is one of the seven major food crops grown worldwide. Cold stress often can cause protein expression pattern and substance contents variations for tuberous roots of sweetpotato during low-temperature storage. Recently, we developed proteometabolic profiles of the fresh sweetpotatoes (cv. Xinxiang) in an attempt to discern the cold stress-responsive mechanism of tuberous root crops during post-harvest storage.ResultsFor roots stored under 4 °C condition, the CI index, REC and MDA content in roots were significantly higher than them at control temperature (13 °C). The activities of SOD, CAT, APX, O2.- producing rate, proline and especially soluble sugar contents were also significantly increased. Most of the differentially expressed proteins (DEPs) were implicated in pathways related to metabolic pathway, especially phenylpropanoids and followed by starch and sucrose metabolism. L-ascorbate peroxidase 3 and catalase were down-regulated during low temperature storage. α-amylase, sucrose synthase and fructokinase were significantly up-regulated in starch and sucrose metabolism, while β-glucosidase, glucose-1-phosphate adenylyl-transferase and starch synthase were opposite. Furthermore, metabolome profiling revealed that glucosinolate biosynthesis, tropane, piperidine and pyridine alkaloid biosynthesis as well as protein digestion and absorption played a leading role in metabolic pathways of roots. Leucine, tryptophan, tyrosine, isoleucine and valine were all significantly up-regulated in glucosinolate biosynthesis.ConclusionsOur proteomic and metabolic profile analysis of sweetpotatoes stored at low temperature reveal that the antioxidant enzymes activities, proline and especially soluble sugar content were significantly increased. Most of the DEPs were implicated in phenylpropanoids and followed by starch and sucrose metabolism. The discrepancy between proteomic (L-ascorbate peroxidase 3 and catalase) and biochemical (CAT/APX activity) data may be explained by higher H2O2 levels and increased ascorbate redox states, which enhanced the CAT/APX activity indirectly. Glucosinolate biosynthesis played a leading role in metabolic pathways. Leucine, tryptophan, tyrosine, isoleucine and valine were all significantly up-regulated in glucosinolate biosynthesis.

Proteomic responses to progressive dehydration stress in leaves of chickpea seedlings

BackgroundChickpea is an important food legume crop with high protein levels that is widely grown in rainfed areas prone to drought stress. Using an integrated approach, we describe the relative changes in some physiological parameters and the proteome of a drought-tolerant (MCC537, T) and drought-sensitive (MCC806, S) chickpea genotype.ResultsUnder progressive dehydration stress, the T genotype relied on a higher relative leaf water content after 3 and 5 d (69.7 and 49.3%) than the S genotype (59.7 and 40.3%) to maintain photosynthetic activities and improve endurance under stress. This may have been facilitated by greater proline accumulation in the T genotype than the S genotype (14.3 and 11.1 μmol g− 1 FW at 5 d, respectively). Moreover, the T genotype had less electrolyte leakage and lower malondialdehyde contents than the S genotype under dehydration stress, indicating greater membrane stability and thus greater dehydration tolerance. The proteomic analysis further confirmed that, in response to dehydration, the T genotype activated more proteins related to photosynthesis, stress response, protein synthesis and degradation, and gene transcription and signaling than the S genotype. Of the time-point dependent proteins, the largest difference in protein abundance occurred at 5 d, with 29 spots increasing in the T genotype and 30 spots decreasing in the S genotype. Some of the identified proteins—including RuBisCo, ATP synthase, carbonic anhydrase, psbP domain-containing protein, L-ascorbate peroxidase, 6-phosphogluconate dehydrogenase, elongation factor Tu, zinc metalloprotease FTSH 2, ribonucleoproteins and auxin-binding protein—may play a functional role in drought tolerance in chickpea.ConclusionsThis study highlights the significance of genotype- and time-specific proteins associated with dehydration stress and identifies potential resources for molecular drought tolerance improvement in chickpea.

Antioxidative defense response of Ulva prolifera under high or low-temperature stimulus

Because marine organisms have evolved to adapt to a wide variety of environmental factors on varying temporal and spatial scales, the antioxidative metabolic pathway are different. The aim of this study was to investigate the different antioxidative response of Ulva Prolifera (U. prolifera) that have been exposed to high temperature (UpHT) or low-temperature (UpLT) stresses. The results of our study suggested that mainly UpHT and not UpLT increased the expression of superoxide dismutase (SOD), catalase (CAT), and L-ascorbate peroxidase (APX) more than those in the medium-temperature (UpMT) controls. In addition, biological index analyses indicated that both SOD and CAT activities increased after culturing for 48h. The chlorophyll levels increased to the highest point after culturing for 4 d in the UpHT group but remained unchanged in the UpLT group. In addition, peroxide (H2O2) and malondialdehyde (MDA) levels increased under UpHT but remained unchanged under UpLT stimuli. We suggest that our findings have significantly improved our understanding of the different molecular mechanisms involved in the tolerance of algae to UpHT or UpLT stimulus, which might explain the seasonal blooms of U. prolifera.

Evaluation of 1-methylcyclopropene (1-MCP) and low temperature conditioning (LTC) to control brown of Huangguan pears

Harvested ‘Huangguan’ pears are popular with consumers, but a series of physiological diseases are likely to occur in Huangguan pears during storage, especially brown. The objective of the study was to evaluate the effectiveness of 1-methylcyclopropene (1-MCP) and low temperature conditioning (LTC) on the control of brown in Huangguan pears. Physiological indexes and enzyme activity including browning index, total phenol content, malondialdehyde (MDA) content, electrolyte leakage, hydrogen peroxide (H2O2) content, superoxide radical (O2−) production rate, flavonoid content, soluble solid content (SSC), l-ascorbate peroxidase (APX), phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO) were measured. Results showed that 1-MCP or LTC treatment suppressed browning index, MDA content, electrolyte leakage, H2O2 content, O2− production rate, PPO activity and improved total phenol content, flavonoid content, SSC, APX and PAL activity in comparison with the control. Furthermore, the combination of 1-MCP and LTC contributed the maximum efficacy compared to the individual treatment. In conclusion, our results indicated the combined treatment may be an efficient way to control brown in Huangguan pears.

Comparative Proteomic Analysis of Two Manilkara Species Leaves Under NaCl Stress.

Background: Salinity is a major environmental limiting factor, which affect agricultural production. The two Manilkara seedlings (M. roxburghiana and M. zapota) with high economic importance, could not adapt well to higher soil salinity and little is known about their proteomic mechanisms.Objectives: The mechanisms responsible for the effects of salinity on the two Manilkara species leaves were examined by means of proteomic analysis.Material and Methods: The seedlings were cultivated in a greenhouse and treated with NaCl. Leaves of control and the salt-stressed seedlings were sampled for phenol protein extraction. Proteins were separated by two-dimensional gel electrophoresis coupled with mass spectroscopy to study the change of proteins under different NaCl concentration.Results: For M. roxburghiana leaves, 21 protein spots exhibited significant abundance variations between the control and the 6‰, 8‰ NaCl treatments, of these 13 proteins were identified. They included L-ascorbate peroxidase, chloroplast carbonic anhydrase, phosphoglycerate kinase, 5 heat-shock proteins(HSPs) which were all down- regulated; For M. zapota leaves, 35 protein spots exhibited significant abundance variations, then 24 proteins were identified, including 7 down-regulated HSPs as well as glyceraldehyde-3-phosphate dehydrogenase, Cell division protein, putative mitochondrial NAD-dependent malate dehydrogenase, ATP synthase, Rubisco large subunit-binding protein, Cytochrome c peroxidase.Conclusions: Based on the common identified proteins between the two M. species, our results indicated that the identificated proteins in the two Manilkara species were involved in carbohydrate metabolism, photosynthesis, defense and stress. HSPs exhibited variation strictly related to NaCl stress. The down-regulated HSPs meant the function to repair cells that have suffered damage weaken during stress process. Furthermore, except for HSP70 in M. zapota leaves, the HSPs in the two species were all small heat shock proteins (sHSPs) with molecular weights ranging from 15 to 42 kDa.

Illumina-based transcriptomic analysis on recalcitrant seeds of Panax notoginseng for the dormancy release during the after-ripening process.

Panax notoginseng (Burk) F.H. Chen is an economically and medicinally important plant of the family Araliacease, with seed dormancy being a key factor limiting the extended cultivation of P. notoginseng. The seeds belong to the morphophysiological dormancy (MPD) group, and it has also been described as the recalcitrant seed. To date, the molecular mechanism of dormancy release in the recalcitrant seed of P. notoginseng is unknown. In the present study, the transcript profiles of seeds from different after-ripening stages (0, 20, 40 and 60 days) were investigated using Illumina Hiseq 2500 technology. 91 979946 clean reads were generated, and 81 575 unigenes were annotated in at least one database. In addition, the differentially expressed genes (DEGs) were identified by the pairwise comparisons. We screened out 2483 DEGs by the three key groups of 20 days vs 0 d, 40 d vs 0 d and 60 d vs 0 d. The DEGs were analyzed by gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway annotation. Meanwhile, we obtained 78 DEGs related to seeds dormancy release at different after-ripening stages of P. notoginseng, of which 15 DEGs were associated with abscisic acid and gibberellin. 26 DEGs that encode late embryogenesis abundant protein and antioxidant enzyme were correlated with desiccation tolerance in seeds. In summary, the results obtained here showed that PECTINESTERASE-2-LIKE, GA-INSENSITIVE, ENT-KAURENE SYNTHASE, PROTEIN PHOSPHATASE 2C, GIBBERELLIN 2-BETA-DIOXYGENASE, SUPEROXIDE DISMUTASE, L-ASCORBATE PEROXIDASE, CATALASE, LATE EMBRYOGENESIS ABUNDANT PROTEIN DC3 and DEHYDRIN 9 were potentially involved in dormancy release and desiccation sensitivity of P. notoginseng seeds. The data might provide a basis for researches on MPD.

ITRAQ-based analysis of leaf proteome identifies important proteins in secondary metabolite biosynthesis and defence pathways crucial to cross-protection against TMV

Cross-protection is a phenomenon in which infection with a mild virus strain protects host plants against subsequent infection with a closely related severe virus strain. This study showed that a mild strain mutant virus, Tobacco mosaic virus (TMV)-43A could cross protect Nicotiana benthamiana plants against wild-type TMV. Furthermore, we investigated the host responses at the proteome level to identify important host proteins involved in cross-protection. We used the isobaric tags for relative and absolute quantification (iTRAQ) technique to analyze the proteome profiles of TMV, TMV-43A and cross-protected plants at different time-points. Our results showed that TMV-43A can cross-protect N. benthamiana plants from TMV. In cross-protected plants, photosynthetic activities were augmented, as supported by the increased accumulation of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) and geranylgeranyl diphosphate synthase (GGPS) enzymes, which are crucial for chlorophyll biosynthesis. The increased abundance of ROS scavenging enzymes like thioredoxins and L-ascorbate peroxidase would prevent oxidative damage in cross-protected plants. Interestingly, the abundance of defence-related proteins (14–3-3 and NbSGT1) decreased, along with a reduction in virus accumulation during cross-protection. In conclusion, we have identified several important host proteins that are crucial in cross-protection to counter TMV infection in N. benthamiana plants. Biological significanceTMV is the most studied model for host-virus interaction in plants. It can infect wide varieties of plant species, causing significant economic losses. Cross protection is one of the methods to combat virus infection. A few cross-protection mechanisms have been proposed, including replicase/coat protein-mediated resistance, RNA silencing, and exclusion/spatial separation between virus strains. However, knowledge on host responses at the proteome level during cross protection is limited. To address this knowledge gap, we have leveraged on a global proteomics analysis approach to study cross protection. We discovered that TMV-43A (protector) protects N. benthamiana plants from TMV (challenger) infection through multiple host pathways: secondary metabolite biosynthesis, photosynthesis, defence, carbon metabolism, protein translation and processing and amino acid biosynthesis. In the secondary metabolite biosynthesis pathway, enzymes 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) and geranylgeranyl diphosphate synthase (GGPS) play crucial roles in chlorophyll biosynthesis during cross protection. In addition, accumulation of ROS scavenging enzymes was also found in cross-protected plants, providing rescues from excessive oxidative damage. Reduced abundance of plant defence proteins is correlated to reduced virus accumulation in host plants. These findings have increased our knowledge in host responses during cross-protection.

Comparative proteomic analysis of salt-responsive proteins in canola roots by 2-DE and MALDI-TOF MS

Salinity stress is a major abiotic stress that affects plant growth and limits crop production. Roots are the primary site of salinity perception, and salt sensitivity in roots limits the productivity of the entire plant. To better understand salt stress responses in canola, we performed a comparative proteomic analysis of roots from the salt-tolerant genotype Safi-7 and the salt-sensitive genotype Zafar. Plants were exposed to 0, 150, and 300 mM NaCl. Our physiological and morphological observations confirmed that Safi-7 was more salt-tolerant than Zafar. The root proteins were separated by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry was applied to identify proteins regulated in response to salt stress. We identified 36 and 25 protein spots whose abundance was significantly affected by salt stress in roots of plants from the tolerant and susceptible genotype, respectively. Functional classification analysis revealed that the differentially expressed proteins from the tolerant genotype could be assigned to 14 functional categories, while those from the susceptible genotype could be classified into 9 functional categories. The most significant differences concerned proteins involved in glycolysis (Glyceraldehyde-3-phosphate dehydrogenase, Fructose-bisphosphate aldolase, Phosphoglycerate kinase 3), stress (heat shock proteins), Redox regulation (Glutathione S-transferase DHAR1, L-ascorbate peroxidase), energy metabolism (ATP synthase subunit B), and transport (V-type proton ATPase subunit B1) which were increased only in the tolerant line under salt stress. Our results provide the basis for further elucidating the molecular mechanisms of salt-tolerance and will be helpful for breeding salt-tolerant canola cultivars.

Proteomic Identification of Differentially Altered Proteins During Regeneration from Nodular Cluster Cultures in Vriesea reitzii (Bromeliaceae)

Tissue culture techniques have been employed for bromeliad mass propagation by means of the morphogenetic route of nodular cluster cultures (NC), which exhibit high regenerative potential. This study aimed to identify proteins differentially altered during the regeneration of shoots from NC in the bromeliad Vriesea reitzii. Proteomic analysis was performed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Image analysis from NC, microshoots and shoots resulted in the detection of 345, 482, and 209 protein spots, respectively. Proteins related to the stress response (catalase, probable l-ascorbate peroxidase 3 and heat shock protein 81-1) and amino acid metabolism (glutamine synthetase, 4-hydroxy-tetrahydrodipicolinate reductase 1, serine hydroxymethyltransferase 1 and 14-3-3-like protein GF14-F) and other proteins (elongation factor Ts, probable signal peptidase complex subunit 2, calcium-dependent protein kinase 23, and ubiquitin-like protein-NEDD8-like protein RUB3) exhibited increased abundance in the early stages of development. On the other hand, proteins related to carbohydrate metabolism (ribulose bisphosphate carboxylase/oxygenase activase, ribulose bisphosphate carboxylase, and phosphoglycerate kinase 1) and ATP synthesis (ATP synthase subunit alpha and ATP synthase subunit beta) exhibited increased abundance in later stages. Changes in proteins related to cellular metabolic processes, the assembly of the photosynthetic mechanism, the response to medium stress and the dynamics of the cell cytoskeleton contribute to a high regenerative potential, which characterizes NC. To our knowledge, this is the first proteomic analysis performed during the regeneration process from NC in bromeliads. This study may provide important insights into proteins and cellular events involved in the micropropagation of this group of plants.

Title: Enhanced salt tolerance and photosynthetic performance: Implication of ɤ-amino butyric acid application in salt-exposed lettuce (Lactuca sativa L.) plants

Gamma-Amino Butyric Acid (GABA) is a substantial component of the free amino acid pool with low concentration in plant tissues. Enhanced GABA content occurs during plant growth and developmental processes like seed germination. GABA level, basically, alters in response to many endogenous and exogenous stimuli. In the current study, GABA effects were studied on germination, photosynthetic performance and oxidative damages in salt-exposed lettuce plants. Three NaCl (0, 40 and 80 mM) and two GABA (0 and 25 μM) concentrations were applied on lettuce during two different developmental (seed germination and seedlings growth) stages. Negative effects of salinity on germination and plant growth were removed by GABA application. GABA significantly reduced mean germination time (MGT) in salt-exposed lettuce seeds. Although, salinity caused a significant decline in maximum quantum yield of photosystem II (Fv/Fm) during distinct steps of plant growth, GABA application improved Fv/Fm particularly on high salinity level. GABA decreased specific energy fluxes per reaction center (RC) for energy absorption and dissipation, while enhanced-electron transport flux in photosynthetic apparatus of lettuce plants was observed in GABA-supplemented plants. Moreover, decline in non-photochemical quenching (NPQ) and quenching coefficients (qP, qL, qN) by salt stress were recovered by GABA application. Elevated electrolyte leakage considerably decreased by GABA exposure on salt-treated plants. Although, proline level increased by NaCl treatments in a concentration dependent manner, combined application of salt with GABA caused a significant reduction in proline content. Catalase; EC 1.11.1.6 (CAT), l-ascorbate peroxidase; EC 1.11.1.11 (APX), and superoxide dismutase; EC 1.15.1.1 (SOD) activities were increased by GABA exposure in salt-supplemented plants that resulted in regulated hydrogen peroxide level. In conclusion, a multifaceted role for GABA is suggested for minimizing detrimental effects of salinity on lettuce through improvement of photosynthetic functionality and regulation of oxidative stress.

Stress and damage mechanisms in Dendrobium nobile Lindl. protocorm-like bodies during pre- and post-liquid nitrogen exposure in cryopreservation revealed by iTRAQ proteomic analysis

Liquid nitrogen (LN) exposure, a crucial step in cryopreservation, is mainly responsible for the reduced viability of protocorm-like bodies (PLBs) in Dendrobium nobile Lindl. To characterize the stress and damage mechanisms during pre- and post-LN exposure, the isobaric tags for relative and absolute quantification (iTRAQ)-labeling method was used to evaluate proteome profiles of PLBs in D. nobile ‘Hamana Lake Dream’ undergoing the cooling and rewarming process. In total, 1747 proteins were detected; a cluster of orthologous group (COG) analysis of the identified proteins revealed that posttranslational modification, protein turnover, and chaperone-related proteins were the predominant protein classes. Of these, 108 proteins were differentially changed and grouped into different functional categories, which included protein turnover, stress and defense, carbohydrate and energy metabolism, signaling transduction, metabolism, membrane, and transport. Furthermore, a quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that some proteins, such as annexin, l-ascorbate peroxidase 1, copper/zinc superoxide dismutase, glutathione S-transferase dehydroascorbate reductase 2-like, and an adenosine triphosphate (ATP) synthase, were regulated at their transcriptional levels. Based on functional analysis of these proteins, it was found that protein synthesis, processing, and degradation might be the main strategies used to reestablish cell balance after the cooling and rewarming process. Moreover, the production of reactive oxygen species (ROS) and the decline in energy production, signaling transduction, and membrane transport during pre- and post-LN exposure might be responsible for the viability loss of the PLBs. This work provides potential protein candidates for exploring the stress and cryo-injury mechanism in cryopreservation.

A Novel L-ascorbate Peroxidase 6 Gene, ScAPX6, Plays an Important Role in the Regulation of Response to Biotic and Abiotic Stresses in Sugarcane.

The L-ascorbate peroxidase 6 gene (APX6) is one of the most important genes for scavenging H2O2 and plays a vital role in plant resistance to environmental stresses. In this study, a novel ScAPX6 gene (GenBank Accession No. KT907352) was obtained from a sugarcane variety (ROC22). Bioinformatics analysis showed that ScAPX6 has a cDNA length of 1,086 bp and encoded 333 amino acid residues. Subcellular localization confirmed that ScAPX6 was located in the chloroplast. Enhanced growth of Escherichia coli BL21 cells that expressed ScAPX6 showed high tolerance under copper (Cu) stress. Real-time quantitative PCR analysis revealed that ScAPX6 was constitutively expressed wherein with the highest expression levels in sugarcane pith and leaf and the lowest in the root. ScAPX6 was down-regulated by salicylic acid (SA), hydrogen peroxide (H2O2), polyethylene glycol (PEG) and sodium chloride (NaCl) stimuli. Interestingly, it was significantly up-regulated under the stresses of abscisic acid (ABA) and methyl jasmonate (MeJA) wherein with the highest inducible expression levels at 6 h at 6.0- and 70.0-times higher, respectively than that of control. Overexpression of ScAPX6 in Nicotiana benthamiana leaves enhanced the resistance to the infection of tobacco pathogens Pseudomonas solanacearum and Fusarium solani var. coeruleum. These results implied that ScAPX6 might positively respond to ABA, MeJA, and Cu, but might negatively respond to the stresses of SA, H2O2, PEG, and NaCl. Keeping in view the current investigation, ScAPX6 could be associated with the hypersensitive response (HR) or immunity of sugarcane, which will provide a baseline for the function identification of sugarcane ScAPX6.

Identification of proteins related to microspore embryogenesis responsiveness in anther cultures of winter triticale (\xd7Triticosecale Wittm.)

For a better understanding of the physiological background of microspore embryogenesis (ME), the protein profile was analyzed in four winter triticale DH lines, which show extremely different embryogenic potential. The analysis were conducted with anthers at the phase of development optimal for ME induction and then after low temperature (LT, 3 weeks at 4 °C) ME-inducing tillers treatment. The sub-proteome of anthers was mapped by two-dimensional gel electrophoresis (2-DE). The protein species significantly more abundant (at least 2-fold) in responsive DH lines after LT treatment were chosen for identification by MALDI-TOF/TOF analysis. In total, 31 protein species were successfully identified as involved in the determination of microspore competence, stress response and in the regulation of ME induction. Microspore competence required sufficient energy supply and efficient system of cell protection that determine survival under prolonged LT stress treatment. LT stress was associated with increased accumulation of proteins typical for cell defence against oxidative stress (e.g., l-ascorbate peroxidase), chaperons (e.g., HSP70) and other enzymes/factors ensuring protein biosynthesis, stability and active cell divisions. Also here, effective cell defence required undisturbed energy supply. Among proteins that accumulated differentially in accordance with microspore embryogenic potential again the most important role seems to be played by the enzymes ensuring energy production and determining ability of plant stress adaptation. Two protein species (enolase, 12S storage protein), proposed earlier as candidates for markers of embryogenesis in other in vitro plant culture systems confirmed their utility for triticale anther cultures.

Analysis of chlorophyll a fluorescence and proteomic differences of rice leaves in response to photooxidation

This study investigated the effects of increased sunlight on photooxidation of rice leaf mutant 812HS and its wild-type 812S under field conditions. Light is important for plant growth and development. However, when the absorbed energy exceeds the capacity of utilization of photosynthesis, it leads to the accumulation of singlet oxygen molecules and other reactive oxygen species, which causes oxidative damage. Chlorophyll a fluorescence was applied to examine photosystem II photochemistry. The results demonstrated that intensive light had a negative influence on plant photosynthetic processes. However, the electron transport chain was inhibited and energy dissipation was increased, which can minimize photooxidative damage to the optical system. Meanwhile, proteomic analysis showed that the differential expression of proteins in response to photooxidation participated in multiple pathways, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit, RuBisCO large chain precursor, RuBisCO activase, flavodoxin-like quinone reductase 1, l-ascorbate peroxidase S, oxygen-evolving complex protein 1, and glycolate oxidase. The results indicated that photooxidation induced a response in the rice via the stress-related pathway. The aforementioned proteins, identified by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), may be very useful in comprehending how plants respond to photooxidation and can be used as characteristics of stress-induced signals. The results of chlorophyll fluorescence parameter analysis demonstrated the negative influence of intense light on plant photosynthetic processes. This was evidenced by the dissipation of excessive energy and the suppression of the electron transport chain to minimize photooxidative damage to the proteins. Future studies should compare the proteomic difference with parallel gene expression and metabolite profiles.

Differential proteomic analysis of embryogenic lines in oil palm (Elaeis guineensis Jacq)

Oil palm tissue culture is one way to produce superior oil palm planting materials. However, the low rate of embryogenesis is a major hindrance for the adoption of this technology in oil palm tissue culture laboratories. In this study, we use proteomic technologies to compare differential protein profiles in leaves from palms of high and low proliferation rates in tissue culture in order to understand the underlying biological mechanism for the low level of embryogenesis. Two protein extraction methods, namely trichloroacetic acid/acetone precipitation and polyethylene glycol fractionation were used to produce total proteins and fractionated protein extracts respectively, with the aim of improving the resolution of protein species using two-dimensional gel electrophoresis. A total of 40 distinct differential abundant protein spots were selected from leaf samples collected from palms with proven high and low proliferation rates. The variant proteins were subsequently identified using mass spectrometric analysis. Twelve prominent protein spots were then characterised using real-time polymerase chain reaction to compare the mRNA expression and protein abundant profiles. Three proteins, namely triosephosphate isomerase, l-ascorbate peroxidase, and superoxide dismutase were identified to be potential biomarker candidates at both the protein abundant and mRNA expression levels. Biological significanceIn this study, proteomic analysis was used to identify abundant proteins from total protein extracts. PEG fractionation was used to reveal lower abundant proteins from both high and low proliferation embryogenic lines of oil palm samples in tissue culture. A total of 40 protein spots were found to be significant in abundance and the mRNA levels of 12 of these were assessed using real time PCR. Three proteins namely, triosephosphate isomerase, l-ascorbate peroxidase and superoxide dismutase were found to be concordant in their mRNA expression and protein abundance. Triosephosphate isomerase is a key enzyme in glycolysis. Both l-ascorbate peroxidase and superoxide dismutase play a role in anti-oxidative scavenging defense systems. These proteins have potential for use as biomarkers to screen for high and low embryogenic oil palm samples.

Proteomic analysis of chromium stress and sulfur deficiency responses in leaves of two canola (Brassica napus L.) cultivars differing in Cr(VI) tolerance

Sulfur (S) is an essential macronutrient for plant growth and development, and it plays an essential role in response to environmental stresses. Plants suffer with combined stress of S deficiency and hexavalent chromium [Cr(VI)] in the rhizosphere. Little is known about the impact of S deficiency on leaf metabolism of canola (Brassica napus L.) under Cr(VI) stress. Therefore, this study is the first to examine the effects of Cr(VI) stress and S deficiency in canola at a molecular level. A comparative proteomic approach was used to investigate the differences in protein abundance between Cr-tolerant NK Petrol and Cr-sensitive Sary cultivars. The germinated seeds were grown hydroponically in S-sufficient (+S) nutrient solution for 7 days and then subjected to S-deficiency (−S) for 7 days. S-deficient and +S seedlings were then exposed to 100μM Cr(VI) for 3 days. Protein patterns analyzed by two-dimensional electrophoresis (2-DE) revealed that 58 protein spots were differentially regulated by Cr(VI) stress (+S/+Cr), S-deficiency (−S/−Cr) and combined stress (−S/+Cr). Of these, 39 protein spots were identified by MALDI-TOF/TOF mass spectrometry. Differentially regulated proteins predominantly had functions not only in photosynthesis, but also in energy metabolism, stress defense, protein folding and stabilization, signal transduction, redox regulation and sulfur metabolism. Six stress defense related proteins including 2-Cys peroxiredoxin BAS1, glutathione S-transferase, ferritin-1, l-ascorbate peroxidase, thiazole biosynthetic enzyme and myrosinase-binding protein-like At3g16470 exhibited a greater increase in NK Petrol. The stress-related proteins play an important role in the detoxification of Cr(VI) and maintaining cellular homeostasis under variable S nutrition.

SRO1 regulates heavy metal mercury stress response in Arabidopsis thaliana

Heavy metals in the environment are harmful limiting factors for the normal growth and development of plants. Here, we isolated and identified an Arabidopsis thaliana T-DNA insertion mutant, named sro1-1, which showed a hyper-sensitive response to HgCl2. The SRO1 protein contains a WWE domain that mediates protein-protein interactions. Under HgCl2 treatment, when compared with the wild-type plants, the growth of sro1-1 was repressed dramatically and the number of true leaves was reduced and etiolated. The electrolyte leakage rates showed that cell membrane integrity in sro1-1 was damaged more severely than in the wild type. DAB (3,5-diaminobenzidine) staining and confocal microscopy showed that Hg2+ stress induced more hydrogen peroxide accumulation in sro1-1 than in the wild type. The qRT-PCR results indicated that the expression of some abiotic stress-induced genes, such as L-ascorbate peroxidase (APX1), was reduced under oxidative or Hg2+ stress. Transgenic plants containing a GFP::SRO1 fusion protein showed that SRO1 was localized in the nucleus of the cells. SRO1 was shown to be expressed in various tissues, and was most highly expressed in the vigorous tissues. Our results suggest that SRO1 may play an important role in the stress response of A. thaliana to heavy metals.