The production and scavenging of chemically reactive species, such as ROS/RNS, are central to a broad range of biotic and abiotic stress and physiological responses in plants. Among the techniques developed for the identification of oxidative stress-induced modifications on proteins, the so-called ‘redox proteome’, proteomics appears to be the best-suited approach. Oxidative or nitrosative stress leaves different footprints in the cell in the form of different oxidatively modified components and, using the redox proteome, it will be possible to decipher the potential roles played by ROS/RNS-induced modifications in stressed cells. The purpose of this review is to present an overview of the latest research endeavours in the field of plant redox proteomics to identify the role of post-translational modifications of proteins in developmental cell stress. All the strategies set up to analyse the different oxidized/nitrosated amino acids, as well as the different reactivities of ROS and RNS for different amino acids are revised and discussed. A growing body of evidence indicates that ROS/RNS-induced protein modifications may be of physiological significance, and that in some cellular stresses they may act causatively and not arise as a secondary consequence of cell damage. Thus, although previously the oxidative modification of proteins was thought to represent a detrimental process in which the modified proteins were irreversibly inactivated, it is now clear that, in plants, oxidatively/nitrosatively modified proteins can be specific and reversible, playing a key role in normal cell physiology. In this sense, redox proteomics will have a central role in the definition of redox molecular mechanisms associated with cellular stresses.

This study was designed to investigate whether thermotolerant roots exhibit respiratory acclimation to elevated temperatures. Root respiratory acclimation traits in response to increasing temperatures were compared between two Agrostis species contrasting in heat tolerance: thermal A. scabra and heat-sensitive A. stolonifera. Roots of both species were exposed to 17, 27, or 37 °C. Root RGR declined with increasing temperatures from 17 °C to 37 °C in both species; however, root growth of A. scabra maintained a significantly higher RGR than A. stolonifera at 27 °C or 37°C. A. scabra exhibited a significantly higher respiration acclimation potential to elevated temperatures, both in the short term (60 min) and in the long term (7–28 d) as compared with A. stolonifera, when temperatures increased from 17 °C to 27 °C or from 27 °C to 37 °C. Thermal A. scabra also maintained a significantly lower maintenance cost than A. stolonifera as temperatures increased to 27 °C or 37 °C. The results suggested that root thermotolerance of thermal A. scabra was associated with both short-term and long-term respiratory acclimation to changes in temperatures. The superior ability of adjusting the rate of root respiration to compensate for increases in carbon demand during short- or long-term temperature increases in the heat-tolerant A. scabra may result in the reduction in carbon expenditure or costs for maintenance, leading to extended root survivability in high temperature soils.

Arabidopsis flowers in long day (LD) in response to signals transported from the photoinduced leaf to the shoot apex. These LD signals may include protein of the gene FLOWERING LOCUS T (FT) while in short day (SD) with its slower flowering, signalling may involve sucrose and gibberellin. Here, it is shown that after 5 weeks growth in SD, a single LD up-regulated leaf blade expression of FT and CONSTANS (CO) within 4–8 h, and flowers were visible within 2–3 weeks. Plants kept in SDs were still vegetative 7 weeks later. This LD response was blocked in ft-1 and a co mutant. Exposure to different LD light intensities and spectral qualities showed that two LD photoresponses are important for up-regulation of FT and for flowering. Phytochrome is effective at a low intensity from far-red (FR)-rich incandescent lamps. Independently, photosynthesis is active in an LD at a high intensity from red (R)-rich fluorescent lamps. The photosynthetic role of a single high light LD is demonstrated here by the blocking of the flowering and FT increase on removal of atmospheric CO₂ or by decreasing the LD light intensity by 10-fold. These conditions also reduced leaf blade sucrose content and photosynthetic gene expression. An SD light integral matching that in a single LD was not effective for flowering, although there was reasonable FT-independent flowering after 12 SD at high light. While a single photosynthetic LD strongly amplified FT expression, the ability to respond to the LD required an additional but unidentified photoresponse. The implications of these findings for studies with mutants and for flowering in natural conditions are discussed.

Signals produced in leaves are transported to the shoot apex where they cause flowering. Protein of the gene FLOWERING LOCUS T (FT) is probably a long day (LD) signal in Arabidopsis. In the companion paper, rapid LD increases in FT expression associated with flowering driven photosynthetically in red light were documented. In a far red (FR)-rich LD, along with FT there was a potential role for gibberellin (GA). Here, with the GA biosynthesis dwarf mutant ga1-3, GA₄-treated plants flowered after 26 d in short days (SD) but untreated plants were still vegetative after 6 months. Not only was FT expression low in SD but applied GA bypassed some of the block to flowering in ft-1. On transfer to LD, ga1-3 only flowered when treated simultaneously with GA, and FT expression increased rapidly (<19.5 h) and dramatically (15-fold). In contrast, in the wild type in LD there was little requirement for GA for FT increase and flowering so its endogenous GA content was near to saturating. Despite this permissive role for endogenous GA in Columbia, RNA interference (RNAi) silencing of the GA biosynthesis gene, GA 20-OXIDASE2, revealed an additional, direct role for GA in LD. Flowering took twice as long after silencing the LD-regulated gene, GA 20-OXIDASE2. Such independent LD input by FT and GA reflects their non-sympatric expression (FT in the leaf blade and GA 20-OXIDASE2 in the petiole). Overall, FT acts as the main LD floral signal in Columbia and GA acts on flowering both via and independently of FT.

Small GTP-binding genes play an essential regulatory role in a multitude of cellular processes such as vesicle-mediated intracellular trafficking, signal transduction, cytoskeletal organization, and cell division in plants and animals. Medicago truncatula and Lotus japonicus are important model plants for studying legume-specific biological processes such as nodulation. The publicly available online resources for these plants from websites such as http://www.ncbi.nih.gov, http://www.medicago.org, http://www.tigr.org, and related sites were searched to collect nucleotide sequences that encode GTP-binding protein homologues. A total of 460 small GTPase sequences from several legume species including Medicago and Lotus, Arabidopsis, human, and yeast were phyletically analysed to shed light on the evolution and functional characteristics of legume-specific homologues. One of the main emphases of this study was the elucidation of the possible involvement of some members of small GTPase homologues in the establishment and maintenance of symbiotic associations in root nodules of legumes. A high frequency of vesicle-mediated trafficking in nodules led to the idea of a probable subfunctionalization of some members of this family in legumes. As a result of the analyses, a group of 10 small GTPases that are likely to be mainly expressed in nodules was determined. The sequences determined as a result of this study could be used in more detailed molecular genetic analyses such as creation of RNA inteference silencing mutants for further clarification of the role of GTPases in nodulation. This study will also assist in furthering our understanding of the evolutionary history of small GTPases in legume species.

Ca²⁺, Mg²⁺, and K⁺ activities in red beet (Beta vulgaris L.) vacuoles were evaluated using conventional ion-selective microelectrodes and, in the case of Ca²⁺, by non-invasive ion flux measurements (MIFE) as well. The mean vacuolar Ca²⁺ activity was ~0.2 mM. Modulation of the slow vacuolar (SV) channel voltage dependence by Ca²⁺ in the absence and presence of other cations at their physiological concentrations was studied by patch-clamp in excised tonoplast patches. Lowering pH at the vacuolar side from 7.5 to 5.5 (at zero vacuolar Ca²⁺) did not affect the channel voltage dependence, but abolished sensitivity to luminal Ca²⁺ within a physiological range of concentrations (0.1–1.0 mM). Aggregation of the physiological vacuolar Na⁺ (60 mM) and Mg²⁺ (8 mM) concentrations also results in the SV channel becoming almost insensitive to vacuolar Ca²⁺ variation in a range from nanomoles to 0.1 mM. At physiological cation concentrations at the vacuolar side, cytosolic Ca²⁺ activates the SV channel in a voltage-independent manner with K_d=0.7–1.5 µM. Comparison of the vacuolar Ca²⁺ fluxes measured by both the MIFE technique and from estimating the SV channel activity in attached patches, suggests that, at resting membrane potentials, even at elevated (20 µM) cytosolic Ca²⁺, only 0.5% of SV channels are open. This mediates a Ca²⁺ release of only a few pA per vacuole (~0.1 pA per single SV channel). Overall, our data suggest that the release of Ca²⁺ through SV channels makes little contribution to a global cytosolic Ca²⁺ signal.

Ascorbate is the most abundant small molecule antioxidant in plants and is proposed to function, along with other members of an antioxidant network, in controlling reactive oxygen species. A biochemical and molecular characterization of four ascorbate-deficient (vtc) Arabidopsis thaliana mutants has been carried out to determine if ascorbate deficiency is compensated by changes in the other major antioxidants. Seedlings grown in vitro were used to minimize stress and longer term developmental differences. Comparison was made with the low glutathione cad2 mutant and vtc2-1 treated with D,L-buthionine-[S,R]-sulphoximine to cause combined ascorbate and glutathione deficiency. The pool sizes and oxidation state of ascorbate and glutathione were not altered by deficiency of the other. -Tocopherol and activities of monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, and catalase were little affected. Ascorbate peroxidase activity was higher in vtc1, vtc2-1, and vtc2-2. Ionically bound cell wall peroxidase activity was increased in vtc1, vtc2-1, and vtc4. Supplementation with ascorbate increased cell wall peroxidase activity. 2,6-Dichlorobenzonitrile, an inhibitor of cellulose synthesis, increased cell wall peroxidase activity in the wild type and vtc1. The transcript level of an endochitinase, PR1, and PR2, but not GST6, was increased in vtc1, vtc2-1, and vtc-2-2. Endochitinase transcript levels increased after ascorbate, paraquat, salicylic acid, and UV-C treatment, PR1 after salicylic acid treatment, and PR2 after paraquat and UV-C treatment. Camalexin was higher in vtc1 and the vtc2 alleles. Induction of PR genes, cell wall peroxidase activity, and camalexin in vtc1, vtc2-1, and vtc2-2 suggests that the mutants are affected in pathogen response signalling pathways.

An Arabidopsis mutant showing an altered ability to green on illumination after extended periods of darkness has been isolated in a screen for genomes uncoupled (gun) mutants. Following illumination for 24 h, 10-day-old dark-grown mutant seedlings accumulated five times more chlorophyll than wild-type seedlings and this was correlated with differences in plastid morphology observed by transmission electron microscopy. The mutant has been named greening after extended darkness 1 (ged1). Microarray analysis showed much lower amounts of transcripts of genes encoding seed storage proteins, oleosins, and late embryogenesis abundant (LEA) proteins in 7-day-old seedlings of ged1 compared with the wild type. RNA gel-blot analyses confirmed very low levels of transcripts of seed protein genes in ged1 seedlings grown for 2–10 d in the dark, and showed higher amounts of transcripts of photosynthesis-related genes in illuminated 10-day-old dark-grown ged1 seedlings compared with the wild type. Consensus elements similar to abscisic acid (ABA) response elements (ABREs) were detected in the upstream regions of all genes highly affected in ged1. Germination of ged1 seeds was hypersensitive to ABA, although no differences in ABA content were detected in 7-day-old seedlings. This suggests the mutant may have an altered responsiveness to ABA, affecting expression of ABA-responsive genes and plastid development during extended darkness.

The annotation of novel plant genes is frequently based on sequence and structural similarity to known protein motifs. Understanding the biological function of these genes is dependent on identifying conditions under which they are activated, however. The resurrection plant, Xerophyta humilis is a good model system for identifying and characterizing genes which are important for desiccation tolerance. Desiccation induced-1 (dsi-1^VOC), a previously uncharacterized plant gene, is up-regulated during desiccation in leaves, roots, and seeds in X. humilis. The X. humilis desiccation induced-1 gene, Xhdsi-1^VOC, shares structural homology with the vicinal oxygen chelate (VOC) metalloenzyme superfamily. Proteins in this superfamily share little sequence similarity, but are characterized by a common ββββ structural fold. A number of plant orthologues of XhDsi-1^VOC have been identified, including Arabidopsis thaliana At1g07645, which is currently annotated as a glyoxalase I-like gene, and many ESTs derived from seed cDNA libraries. Xhdsi-1^VOC and its orthologues do not, however, contain the glutathione and zinc binding sites conserved in glyoxalase I genes. Furthermore, expression of Xhdsi-1^VOC in yeast failed to rescue a yeast glyoxalase I mutant. Messenger RNA transcripts for At1g07645 accumulate during seed maturation, but are not induced by water loss, salt or mannitol stress in vegetative tissue in Arabidopsis. It is concluded that dsi-1^VOC is a seed-specific gene in desiccation-sensitive plants that is activated by water loss in vegetative tissues in the resurrection plant X. humilis and plays an important role in allowing plant tissues to survive loss of 95% of their relative water content.

The optical properties of leaves from five species, Norway maple (Acer platanoides L.), cotoneaster (Cotoneaster alaunica Golite), hazel (Corylus avellana L.), Siberian dogwood (Cornus alba L.), and Virginia creeper (Parthenocissus quinquefolia (L.) Planch.), differing in pigment composition and at different stages of ontogenesis, were studied. Anthocyanin absorption maxima in vivo, as estimated with spectrophotometry of intact anthocyanic versus acyanic leaves and microspectrophotometry of vacuoles in the leaf cross-sections, were found between 537 nm and 542 nm, showing a red shift of 5–20 nm compared with the corresponding maxima in acidic water–methanol extracts. In non-senescent leaves, strong anthocyanin absorption was found between 500 nm and 600 nm (with a 70–80 nm apparent bandwidth). By and large, absorption by anthocyanin in leaves followed a modified form of the Lambert–Beer law, showing a linear trend up to a content of nearly 50 nmol cm^–2, and permitting thereby a non-invasive determination of anthocyanin content. The apparent specific absorption coefficients of anthocyanins at 550 nm showed no substantial dependence on the species. Anthocyanin contribution to total light absorption at 550 nm was followed in maple leaves in the course of autumn senescence. Photoprotection by vacuolar anthocyanins is discussed with special regard to their distribution within a leaf; radiation screening by anthocyanins predominantly localized in the epidermal cells in A. platanoides and C. avellana leaves was also evaluated.