Showing papers in "Journal of Experimental Botany in 2005"

Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress

Abstract: Plant stress studies are more and more based on gene expression. The analysis of gene expression requires sensitive, precise, and reproducible measurements for specific mRNA sequences. Real-time RT-PCR is at present the most sensitive method for the detection of low abundance mRNA. To avoid bias, real-time RT-PCR is referred to one or several internal control genes, which should not fluctuate during treatments. Here, the non-regulation of seven housekeeping genes (b-tubulin, cyclophilin, actin, elongation factor 1-a (ef1a), 18S rRNA, adenine phosphoribosyl transferase (aprt), and cytoplasmic ribosomal protein L2) during biotic (late blight) and abiotic stresses (cold and salt stress) was tested on potato plants using geNorm software. Results from the three experimental conditions indicated that ef1a was the most stable among the seven tested. The expression of the other housekeeping genes tested varied upon stress. In parallel, a study of the variability of expression of hsp20.2, shown to be implicated in late blight stress, was realized. The relative quantification of the hsp20.2 gene varied according to the internal control and the number of internal controls used, thus highlighting the importance of the choice of internal controls in such experiments.

Microbial co-operation in the rhizosphere

Abstract: Soil microbial populations are immersed in a framework of interactions known to affect plant fitness and soil quality. They are involved in fundamental activities that ensure the stability and productivity of both agricultural systems and natural ecosystems. Strategic and applied research has demonstrated that certain co-operative microbial activities can be exploited, as a low-input biotechnology, to help sustainable, environmentally-friendly, agro-technological practices. Much research is addressed at improving understanding of the diversity, dynamics, and significance of rhizosphere microbial populations and their cooperative activities. An analysis of the co-operative microbial activities known to affect plant development is the general aim of this review. In particular, this article summarizes and discusses significant aspects of this general topic, including (i) the analysis of the key activities carried out by the diverse trophic and functional groups of micro-organisms involved in cooperative rhizosphere interactions; (ii) a critical discussion of the direct microbe–microbe interactions which results in processes benefiting sustainable agroecosystem development; and (iii) beneficial microbial interactions involving arbuscular mycorrhiza, the omnipresent fungus–plant beneficial symbiosis. The trends of this thematic area will be outlined, from molecular biology and ecophysiological issues to the biotechnological developments for integrated management, to indicate where research is needed in the future.

Chemical derivatization and mass spectral libraries in metabolic profiling by GC/MS and LC/MS/MS

Abstract: An overview is presented of gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), the two major hyphenated techniques employed in metabolic profiling that complement direct 'fingerprinting' methods such as atmospheric pressure ionization (API) quadrupole time-of-flight MS, API Fourier transform MS, and NMR. In GC/MS, the analytes are normally derivatized prior to analysis in order to reduce their polarity and facilitate chromatographic separation. The electron ionization mass spectra obtained are reproducible and suitable for library matching, mass spectral collections being readily available. In LC/MS, derivatization and library matching are at an early stage of development and mini-reviews are provided. Chemical derivatization can dramatically increase the sensitivity and specificity of LC/MS methods for less polar compounds and provides additional structural information. The potential of derivatization for metabolic profiling in LC/MS is demonstrated by the enhanced analysis of plant extracts, including the potential to measure volatile acids such as formic acid, difficult to achieve by GC/MS. The important role of mass spectral library creation and usage in these techniques is discussed and illustrated by examples.

Uptake system of silicon in different plant species.

Abstract: The accumulation of silicon (Si) in the shoots varies considerably among plant species, but the mechanism responsible for this variation is poorly understood. The uptake system of Si was investigated in terms of the radial transport from the external solution to the root cortical cells and the release of Si from the cortical cells to the xylem in rice, cucumber, and tomato, which differ greatly in shoot Si concentration. Symplasmic solutions of the root tips were extracted by centrifugation. The concentrations of Si in the root-cell symplast in all species were higher than that in the external solution, although the concentration in rice was 3- and 5-fold higher than that in cucumber and tomato, respectively. A kinetic study showed that the radial transport of Si was mediated by a transporter with a Km value of 0.15 mM in all species, but with different Vmax values in the order of rice>cucumber>tomato. In the presence of the metabolic inhibitor 2,4-dinitrophenol, and at low temperature, the Si concentration in the root-cell symplast decreased to a level similar to that of the apoplasmic solution. These results suggest that both transporter-mediated transport and passive diffusion of Si are involved in the radial transport of Si and that the transporter-mediated transport is an energy-dependent process. The Si concentration of xylem sap in rice was 20- and 100-fold higher than that in cucumber and tomato, respectively. In contrast to rice, the Si concentration in the xylem sap was lower than that in the external solution in cucumber and tomato. A kinetic study showed that xylem loading of Si was also mediated by a kind of transporter in rice, but by passive diffusion in cucumber and tomato. These results indicate that a higher density of transporter for radial transport and the presence of a transporter for xylem loading are responsible for the high Si accumulation in rice.

Biological costs and benefits to plant–microbe interactions in the rhizosphere

Abstract: This review looks briefly at plants and their rhizosphere microbes, the chemical communications that exist, and the biological processes they sustain. Primarily it is the loss of carbon compounds from roots that drives the development of enhanced microbial populations in the rhizosphere when compared with the bulk soil, or that sustains specific mycorrhizal or legume associations. The benefits to the plant from this carbon loss are discussed. Overall the general rhizosphere effect could help the plant by maintaining the recycling of nutrients, through the production of hormones, helping to provide resistance to microbial diseases and to aid tolerance to toxic compounds. When plants lack essential mineral elements such as P or N, symbiotic relationships can be beneficial and promote plant growth. However, this benefit may be lost in well-fertilized (agricultural) soils where nutrients are readily available to plants and symbionts reduce growth. Since these rhizosphere associations are commonplace and offer key benefits to plants, these interactions would appear to be essential to their overall success.

Expression profiling of genes involved in starch synthesis in sink and source organs of rice

Abstract: A comprehensive analysis of the transcript levels of genes which encode starch-synthesis enzymes is fundamental for the assessment of the function of each enzyme and the regulatory mechanism for starch biosynthesis in source and sink organs. Using quantitative real-time RT-PCR, an examination was made of the expression profiles of 27 rice genes encoding six classes of enzymes, i.e. ADPglucose pyrophosphorylase (AGPase), starch synthase, starch branching enzyme, starch debranching enzyme, starch phosphorylase, and disproportionating enzyme in developing seeds and leaves. The modes of gene expression were tissueand developmental stage-specific. Four patterns of expression in the seed were identified: group 1 genes, which are expressed very early in grain formation and are presumed to be involved in the construction of fundamental cell machineries, de novo synthesis of glucan primers, and initiation of starch granules; group 2 genes, which are highly expressed throughout endosperm development; group 3 genes, which have transcripts that are low at the onset but which rise steeply at the start of starch synthesis in the endosperm and are thought to play essential roles in endosperm starch synthesis; and group 4 genes, which are expressed scantly, mainly at the onset of grain development, and might be involved in synthesis of starch in the pericarp. The methodology also revealed that the defect in the cytosolic AGPase small subunit2b (AGPS2b) transcription from the AGPS2 gene in endosperm sharply enhanced the expressions of endosperm and leaf plastidial AGPS1, the endosperm cytosolic AGPase large subunit2 (AGPL2), and the leaf plastidial AGPL1.

Effects of free proline accumulation in petunias under drought stress

Abstract: Petunias (Petunia hybrida cv. ‘Mitchell’) accumulate free proline (Pro) under drought-stress conditions. It is therefore believed that Pro acts as an osmoprotectant in plants subjected to drought conditions. Petunia plants were transformed by D 1 -pyrroline-5-carboxylate synthetase genes (AtP5CS from Arabidopsis thaliana L. or OsP5CS from Oryza sativa L.). The transgenic plants accumulated Pro and their drought tolerance was tested. The Pro content amounted to 0.57–1.01% of the total amino acids in the transgenic plants, or 1.5–2.6 times that in wild-type plants grown under normal conditions. The transgenic plant lines tolerated 14 d of drought stress, which confirms that both P5CS transgenes had full functionality. Exogenous L-Pro treatment caused the plants to accumulate Pro; plants treated with 5 mM L-Pro accumulated up to 18 times more free Pro than untreated plants. Exogenous L-Pro restricted the growth of wild-type petunias more than that of Arabidopsis plants. The capacity for free Pro accumulation might depend on the plant species. The growth of petunia plants was influenced not only by the Pro concentration in the plants, but by the ratio of the Pro content to the total amino acids, because the growth of the transgenic petunia plants appeared normal.

Metabolic profiling of Medicago truncatula cell cultures reveals the effects of biotic and abiotic elicitors on metabolism

Abstract: GC-MS-based metabolite profiling was used to analyse the response of Medicago truncatula cell cultures to elicitation with methyl jasmonate (MeJa), yeast elicitor (YE), or ultraviolet light (UV). Marked changes in the levels of primary metabolites, including several amino acids, organic acids, and carbohydrates, were observed following elicitation with MeJa. A similar, but attenuated response was observed following YE elicitation, whereas little response was observed following UV elicitation. MeJa induced the accumulation of the triterpene beta-amyrin, a precursor to the triterpene saponins, and LC-MS analysis confirmed the accumulation of triterpene saponins in MeJa-elicited samples. In addition, YE induced a slight, but significant accumulation of shikimic acid, an early precursor to the phenylpropanoid pathway, which was also demonstrated to be YE-inducible by LC-MS analyses. Correlation analyses of metabolite relationships revealed perturbation of the glycine, serine, and threonine biosynthetic pathway, and suggested the induction of threonine aldolase activity, an enzyme as yet uncharacterized from plants. Members of the branched chain amino acid pathway accumulated in a concerted fashion, with the strongest correlation being that between leucine and isoleucine (r2=0.941). While UV exposure itself had little effect on primary metabolites, the experimental procedure, as revealed by control treatments, induced changes in several metabolites which were similar to those following MeJa elicitation. Sucrose levels were lower in MJ- and YE-elicited samples compared with control samples, suggesting that a portion of the effects observed on the primary metabolic pool are a consequence of fundamental metabolic repartitioning of carbon resources rather than elicitor-specific induction. In addition, beta-alanine levels were elevated in all elicited samples, which, when viewed in the context of other elicitation responses, suggests the altered metabolism of coenzyme A and its esters, which are essential in secondary metabolism.

Nitric oxide is involved in abscisic acid-induced antioxidant activities in Stylosanthes guianensis

Abstract: Previous studies suggest that abscisic acid (ABA) stimulates the activities of antioxidant enzymes under normal and chilling temperature and enhanced chilling resistance in Stylosanthes guianensis. The objective of this study was to test whether nitric oxide (NO) is involved in the ABA-induced activities of the antioxidant enzymes in Stylosanthes guianensis due to its nature as a second messenger in stress responses. Plants were treated with NO donors, ABA, ABA in combination with NO scavengers or the nitric oxide synthase (NOS) inhibitor and their effects on the activity of antioxidant enzymes and NO production were compared. The results showed that ABA increased the activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX). The effect of ABA on antioxidant enzyme activities was suppressed by the NOS inhibitor, N x -nitro-L-arginine (L-NNA), and the NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline1-oxyl3-oxide (PTIO). NO content increased after 5 h of ABA treatment. The NO-scavenger, PTIO, and the NOS-inhibitor, L-NNA, inhibited the accumulation of NO in ABA-treated Stylosanthes guianensis. NO donor treatment enhanced the activities of SOD, CAT, and APX. The results suggested that NO was involved in the ABA-induced activities of SOD, CAT, and APX in Stylosanthes guianensis. ABA triggered NO production that may lead to the stimulation of antioxidant enzyme activities.

Increased sensitivity to salt stress in an ascorbate-deficient Arabidopsis mutant

Abstract: The Arabidopsis thaliana ascorbate-deficient vtc-1 mutant has only 30% ascorbate contents of the wild type (WT). This ascorbate-deficient mutant was used here to study the physiological roles of ascorbate under salt stress in vivo. Salt stress resulted in a more significant decrease in CO2 assimilatory capacity in the vtc-1 mutant than in the WT. Photosystem II function in the Arabidopsis vtc-1 mutant also showed an increased sensitivity to salt stress. Oxidative stress, indicated by the hydrogen peroxide content, increased more dramatically in the vtc-1 mutant than in the WT under salt stress. To clarify the reason for the increased oxidative stress in the vtc-1 mutant, the contents of small antioxidant compounds and the activities of several antioxidant enzymes in the ascorbate–glutathione cycle were measured. Despite an elevated glutathione pool in the vtc-1 mutant, the ascorbate contents and the reduced form of ascorbate decreased very rapidly under salt stress. These results showed that the activities of MDAR and DHAR were lower in the vtc-1 mutant than in the WT under salt stress. Thus, low intrinsic ascorbate and an impaired ascorbate–glutathione cycle in the vtc-1 mutant under salt stress probably induced a dramatic decrease in the reduced form of ascorbate, which resulted in both enhanced ROS contents and decreased NPQ in the vtc-1 mutant.

OsZIP4, a novel zinc-regulated zinc transporter in rice

Abstract: Zinc (Zn) is an essential element for the normal growth of plants but information is scarce on the mechanisms whereby Zn is transported in rice (Oryza sativa L.) plants. Four distinct genes, OsZIP4, OsZIP5, OsZIP6, and OsZIP7 that exhibit sequence similarity to the rice ferrous ion transporter, OsIRT1, were isolated. Microarray and northern blot analysis revealed that OsZIP4 was highly expressed under conditions of Zn deficiency in roots and shoots. Real-time-PCR revealed that the OsZIP4 transcripts were more abundant than those of OsZIP1 or OsZIP3 in Zn-deficient roots and shoots. OsZIP4 complemented a Zn-uptake-deficient yeast (Saccharomyces cerevisiae) mutant, Deltazrt1,Deltazrt2, indicating that OsZIP4 is a functional transporter of Zn. OsZIP4-synthetic green fluorescent protein (sGFP) fusion protein was transiently expressed in onion epidermal cells localized to the plasma membrane. In situ hybridization analysis revealed that OsZIP4 in Zn-deficient rice was expressed in shoots and roots, especially in phloem cells. Furthermore, OsZIP4 transcripts were detected in the meristem of Zn-deficient roots and shoots. These results suggested that OsZIP4 is a Zn transporter that may be responsible for the translocation of Zn within rice plants.

Estimation of leaf water potential by thermal imagery and spatial analysis

Abstract: Canopy temperature has long been recognized as an indicator of plant water status and as a potential tool for irrigation scheduling. In the present study, the potential of using thermal images for an in-field estimation of the water status of cotton under a range of irrigation regimes was investigated. Thermal images were taken with a radiometric infrared video camera. Specific leaves that appeared in the camera field of view were sampled, their LWP was measured and their temperature was calculated from the images. Regression models were built in order to predict LWP according to the crop canopy temperature and to the empirical formulation of the crop water stress index (CWSI). Statistical analysis revealed that the relationship between CWSI and LWP was more stable and had slightly higher correlation coefficients than that between canopy temperature and LWP. The regression models of LWP against CWSI and against leaf temperatures were used to create LWP maps. The classified LWP maps showed that there was spatial variability in each treatment, some of which may be attributed to the difference between sunlit and shaded leaves. The distribution of LWP in the maps showed that irrigation treatments were better distinguished from each other when the maps were calculated from CWSI than from leaf temperature alone. Furthermore, the inclusion of the spatial pattern in the classification enhanced the differences between the treatments and was better matched to irrigation amounts. Optimal determination of the water status from thermal images should be based on an overall view of the physical status as well as on the analysis of the spatial structure. Future study will involve investigating the robustness of the models and the potential of using water status maps, derived from aerial thermal images, for irrigation scheduling and variable management in commercial fields.

Antioxidant responses of hyper-accumulator and sensitive fern species to arsenic

Abstract: Plant species capable of hyper-accumulating heavy metals are of considerable interest for phytoremediation, and differ in their ability to accumulate metals from the environment. This work aims to examine (i) arsenic accumulation in three fern species [Chinese brake fern (Pteris vittata L.), slender brake fern (Pteris ensiformis Burm. f.), and Boston fern (Nephrolepis exaltata L.)], which were exposed to 0, 150, or 300 muM of arsenic (Na(2)HAsO(4).7H(2)O), and (ii) the role of anti-oxidative metabolism in arsenic tolerance in these fern species. Arsenic accumulation increased with an increase in arsenic concentration in the growth medium, the most being found in P. vittata fronds showing no toxicity symptoms. In addition, accumulation was highest in the fronds, followed by the rhizome, and finally the roots, in all three fern species. Thiobarbituric acid-reacting substances, indicators of stress in plants, were found to be lowest in P. vittata, which corresponds with its observed tolerance to arsenic. All three ferns responded differentially to arsenic exposure in terms of anti-oxidative defence. Higher levels of superoxide dismutase, catalase, and ascorbate peroxidase were observed in P. vittata than in P. ensiformis and N. exaltata, showing their active involvement in the arsenic detoxification mechanism. However, no significant increase was observed in either guaiacol peroxides or glutathione reductase in arsenic-treated P. vittata. Higher activity of anti-oxidative enzymes and lower thiobarbituric acid-reacting substances in arsenic-treated P. vittata correspond with its arsenic hyper-accumulation and no symptoms of toxicity.

Cellular damage induced by cadmium and mercury in Medicago sativa

Abstract: Alfalfa (Medicago sativa) plantlets were exposed to Cd or Hg to study the kinetics of diverse stress indexes. In the so-called beaker-size hydroponic system, plantlets were grown in 30 microM of Cd or Hg for 7 d. Oxidative stress took place and increased over time, a linear response being observed with Cd but not with Hg. To improve the sensitivity of the stress assays used, a micro-assay system, in which seedlings were exposed for 24 h, was developed. Phytotoxicity of metals, quantified as growth inhibition, was observed well before there was any change in the non-protein thiol tissue concentration. When measured with conventional techniques, oxidative stress indexes did not show significant variation. To trace early and small plant responses to Cd and Hg, a microscopic analysis with novel fluorescent dyes, which had not yet been exploited to any significant extent for use in plants, was conducted. These fluorescent probes, which allowed minute cellular responses to 0, 3, 10, and 30 microM of both metals to be visualized in the roots of the alfalfa seedlings, were: (i) 2',7'-dichlorofluorescin diacetate that labels peroxides; (ii) monochlorobimane that stains reduced glutathione/homoglutathione (GSH/hGSH); and (iii) propidium iodide that marks nuclei of dead cells. Oxidative stress and cell death increased after exposure for 6-24 h to Cd and Hg, but labelling of GSH/hGSH decreased acutely. This diminution might be the result of direct interaction of GSH/hGSH with both Cd and Hg, as inferred from an in vitro conjugation assay. Therefore, both Cd and Hg not only compromised severely the cellular redox homeostasis, but also caused cell necrosis. In plants treated with 1 mM L-buthionine sulphoximine, a potent inhibitor of GSH/hGSH synthesis, only the oxidative stress symptoms appeared, indicating that the depletion of the GSH/hGSH pool was not sufficient to promote cell death, and that other phytotoxic mechanisms might be involved.

In higher plants, only root mitochondria, but not leaf mitochondria reduce nitrite to NO, in vitro and in situ.

Abstract: At oxygen concentrations of < or =1%, even completely nitrate reductase (NR)-free root tissues reduced added nitrite to NO, indicating that, in roots, NR was not the only source for nitrite-dependent NO formation. By contrast, NR-free leaf slices were not able to reduce nitrite to NO. Root NO formation was blocked by inhibitors of mitochondrial electron transport (Myxothiazol and SHAM), whereas NO formation by NR-containing leaf slices was insensitive to the inhibitors. Consistent with that, mitochondria purified from roots, but not those from leaves, reduced nitrite to NO at the expense of NADH. The inhibitor studies suggest that, in root mitochondria, both terminal oxidases participate in NO formation, and they also suggest that even in NR-containing roots, a large part of the reduction of nitrite to NO was catalysed by mitochondria, and less by NR. The differential capacity of root and leaf mitochondria to reduce nitrite to NO appears to be common among higher plants, since it has been observed with Arabidopsis, barley, pea, and tobacco. A specific role for nitrite to NO reduction in roots under anoxia is discussed.

Grafting raises the salt tolerance of tomato through limiting the transport of sodium and chloride to the shoot

Abstract: With the aim of determining whether grafting could improve salinity tolerance of tomato (Lycopersicon esculentum Mill.), and what characteristics of the rootstock were required to increase the salt tolerance of the shoot, a commercial tomato hybrid (cv. Jaguar) was grafted onto the roots of several tomato genotypes with different potentials to exclude saline ions. The rootstock effect was assessed by growing plants at different NaCl concentrations (0, 25, 50, and 75 mM NaCl) under greenhouse conditions, and by determining the fruit yield and the leaf physiological changes induced by the rootstock after 60 d and 90 d of salt treatment. The grafting process itself did not affect the fruit yield, as non-grafted plants of cv. Jaguar and those grafted onto their own root showed the same yield over time under non-saline conditions. However, grafting raised fruit yield in Jaguar on most rootstocks, although the positive effect induced by the rootstock was lower at 25mMNaCl than at 50 and 75mM NaCl. At these higher levels, the plants grafted onto Radja, Pera and the hybrid Volgogradskij3Pera increased their yields by ~80%, with respect to the Jaguar plants. The tolerance induced by the rootstock in the shoot was related to ionic rather than osmotic stress caused by salinity, as the differential fruit yield responses among graft combinations were mainly related to the different abilities of rootstocks to regulate the transport of saline ions. This was corroborated by the high negative correlation found between fruit yield and the leaf Na1 or Cl2 concentrations in salt-treated plants after 90 d of salt treatment. In conclusion, grafting provides an alternative way to enhance salt tolerance, determined as fruit yield, in the tomato, and evidence is reported that the rootstock is able to reduce ionic stress.

Salt tolerance in wild Hordeum species is associated with restricted entry of Na+ and Cl− into the shoots

Abstract: Eight wild Hordeum species: H. bogdanii, H. intercedens, H. jubatum, H. lechleri, H. marinum, H. murinum, H. patagonicum, and H. secalinum, and cultivated barley (H. vulgare) were grown in nutrient solution containing 0.2 (control), 150, 300, or 450 mol m(-3) NaCl. In saline conditions, the wild Hordeum species (except H. murinum) had better Na+ and Cl- 'exclusion', and maintained higher leaf K+, compared with H. vulgare. For example, at 150 mol m(-3) NaCl, the K+:Na+ in the youngest, fully expanded leaf blades of the wild Hordeum species was, on average, 5.2 compared with 0.8 in H. vulgare. In H. marinum grown in 300 mol m(-3) NaCl, K+ contributed 35% to leaf psi(pi), whereas Na+ and Cl- accounted for only 6% and 10%, respectively. By comparison, in H. vulgare grown at 300 mol m(-3) NaCl, K+ accounted for 19% and Na+ and Cl- made up 21% and 25% of leaf psi(pi), respectively. At 300 mol m(-3) NaCl, glycinebetaine and proline together contributed almost 15% to psi(pi) in the expanding leaf blades of H. marinum, compared with 8% in H. vulgare. Decreased tissue water content under saline conditions made a substantial contribution to declines in leaf psi(pi) in the wild Hordeum species, but not in H. vulgare. A number of the wild Hordeum species were markedly more salt tolerant than H. vulgare. H. marinum and H. intercedens, as examples, had relative growth rates 30% higher than H. vulgare in 450 mol m(-3) NaCl. Hordeum vulgare also suffered up to 6-fold more dead leaf material (as a proportion of shoot dry mass) than the wild Hordeum species. Thus, several salt-tolerant wild Hordeum species were identified, and these showed an exceptional capacity to 'exclude' Na+ and Cl- from their shoots.

Stress responses to polycyclic aromatic hydrocarbons in Arabidopsis include growth inhibition and hypersensitive response-like symptoms.

Abstract: Polycyclic aromatic hydrocarbons (PAHs) are of global environmental concern because they cause many health problems including cancer and inflammation of tissue in humans. Plants are important in removing PAHs from the atmosphere; yet, information on the physiology, cell and molecular biology, and biochemistry of PAH stress responses in plants is lacking. The PAH stress response was studied in Arabidopsis (Arabidopsis thaliana) exposed to the three-ring aromatic compound, phenanthrene. Morphological symptoms of PAH stress were growth reduction of the root and shoot, deformed trichomes, reduced root hairs, chlorosis, late flowering, and the appearance of white spots, which later developed into necrotic lesions. At the tissue and cellular levels, plants experienced oxidative stress. This was indicated by localized H 2 O 2 production and cell death, which were detected using 3, 3'-diaminobenzidine and trypan blue staining, respectively. Gas chromatography-mass spectrometry and fluorescence spectrometry analyses showed that phenanthrene is internalized by the plant. Gene expression of the cell wall-loosening protein expansin was repressed, whereas gene expression of the pathogenesis related protein PR1 was induced in response to PAH exposure. These findings show that (i) Arabidopsis takes up phenanthrene, suggesting possible degradation in plants, (ii) a PAH response in plants and animals may share similar stress mechanisms, since in animal cells detoxification of PAHs also results in oxidative stress, and (iii) plant specific defence mechanisms contribute to PAH stress response in Arabidopsis.

Methyl jasmonate stimulates the de novo biosynthesis of vitamin C in plant cell suspensions.

Abstract: Vitamin C (L-ascorbic acid) is an important primary metabolite of plants that functions as an antioxidant, an enzyme cofactor, and a cell-signalling modulator in a wide array of crucial physiological processes, including biosynthesis of the cell wall, secondary metabolites and phytohormones, stress resistance, photoprotection, cell division, and growth. Plants synthesize ascorbic acid via de novo and salvage pathways, but the regulation of its biosynthesis and the mechanisms behind ascorbate homeostasis are largely unknown. Jasmonic acid and its methyl ester ( jasmonates) mediate plant responses to many biotic and abiotic stresses by triggering a transcriptional reprogramming that allows cells to cope with pathogens and stress. By using 14 C-mannose radiolabelling combined with HPLC and transcript profiling analysis, it is shown that methyl jasmonate treatment increases the de novo synthesis of ascorbic acid in Arabidopsis and tobacco Bright Yellow-2 (BY-2) suspension cells. In BY-2 cells, this stimulation coincides with enhanced transcription of at least two late methyl jasmonate-responsive genes encoding enzymes for vitamin C biosynthesis: the GDP-mannose 3$,5$-epimerase and a putative Lgulono-1,4-lactone dehydrogenase/oxidase. As far as is known, this is the first report of a hormonal regulation of vitamin C biosynthesis in plants. Finally, the role of ascorbic acid in jasmonate-regulated stress responses is reviewed.

Root-derived cytokinins as long-distance signals for NO3−-induced stimulation of leaf growth

Abstract: Leaf growth of many plant species shows rapid changes in response to alterations of the form and the level of N supply. In hydroponically-grown tomato (Lycopersicon esculentum L.), leaf growth was rapidly stimulated by NO(3)(-) application to NH(4)(+) precultured plants, while NH(4)(+) supply or complete N deprivation to NO(3)(-) precultured plants resulted in a rapid inhibition of leaf growth. Just 10 microM NO(3)(-) supply was sufficient to stimulate leaf growth to the same extent as 2 mM. Furthermore, continuous NO(3)(-) supply induced an oscillation of leaf growth rate with a 48 h interval. Since changes in NO(3)(-) levels in the xylem exudate and leaves did not correlate with NO(3)(-)-induced alterations of leaf growth rate, additional signals such as phytohormones may be involved. Levels of a known inhibitor of leaf growth, abscisic acid (ABA), did not consistently correspond to leaf growth rates in wild-type plants. Moreover, leaf growth of the ABA-deficient tomato mutant flacca was inhibited by NH(4)(+) without an increase in ABA concentration and was stimulated by NO(3)(-) despite its excessive ethylene production. These findings suggest that neither ABA nor ethylene are directly involved in the effects of N form on leaf growth. However, under all experimental conditions, stimulation of leaf growth by NO(3)(-) was consistently associated with increased concentration of the physiologically active forms of cytokinins, zeatin and zeatin riboside, in the xylem exudate. This indicates a major role for cytokinins as long-distance signals mediating the shoot response to NO(3)(-) perception in roots.

Chloroplasts as source and target of cellular redox regulation: a discussion on chloroplast redox signals in the context of plant physiology.

Abstract: During the evolution of plants, chloroplasts have lost the exclusive genetic control over redox regulation and antioxidant gene expression. Together with many other genes, all genes encoding antioxidant enzymes and enzymes involved in the biosynthesis of low molecular weight antioxidants were transferred to the nucleus. On the other hand, photosynthesis bears a high risk for photo-oxidative damage. Concomitantly, an intricate network for mutual regulation by anthero- and retrograde signals has emerged to co-ordinate the activities of the different genetic and metabolic compartments. A major focus of recent research in chloroplast regulation addressed the mechanisms of redox sensing and signal transmission, the identification of regulatory targets, and the understanding of adaptation mechanisms. In addition to redox signals communicated through signalling cascades also used in pathogen and wounding responses, specific chloroplast signals control nuclear gene expression. Signalling pathways are triggered by the redox state of the plastoquinone pool, the thioredoxin system, and the acceptor availability at photosystem I, in addition to control by oxolipins, tetrapyrroles, carbohydrates, and abscisic acid. The signalling function is discussed in the context of regulatory circuitries that control the expression of antioxidant enzymes and redox modulators, demonstrating the principal role of chloroplasts as the source and target of redox regulation.

Strategies to maintain redox homeostasis during photosynthesis under changing conditions

Abstract: Plants perform photosynthesis and assimilatory processes in a continuously changing environment. Energy production in the various cell compartments and energy consumption in endergonic processes have to be well adjusted to the varying conditions. In addition, dissipatory pathways are required to avoid any detrimental effects caused by over-reduction. A large number of short-term and long-term mechanisms interact with each other in a flexible way, depending on intensity and the type of impact. Therefore, all levels of regulation are involved, starting from energy absorption and electron flow events through to post-transcriptional control. The simultaneous presence of strong oxidants and strong reductants during oxygenic photosynthesis is the basis for regulation. However, redox-dependent control also interacts with other signal transduction pathways in order to adapt metabolic processes and redox-control to the developmental state. Examples are given here for short-term and long-term control following changes of light intensity and photoperiod, focusing on the dynamic nature of the plant regulatory systems. An integrating network of all these mechanisms exists at all levels of control. Cellular homeostasis will be maintained as long as the mechanisms for acclimation are present in sufficiently high capacities. If an impact is too rapid, and acclimation on the level of gene expression cannot occur, cellular damage and cell death are initiated.

Root cell patterning: a primary target for aluminium toxicity in maize

Abstract: The short-term influence (5-180 min) of 50 microM Al on cell division was investigated in root tips of two Zea mays L. varieties differing in Al-resistance. The incorporation of bromodeoxyuridine into S-phase nuclei was visualized by immunofluorescence staining using confocal laser fluorescence microscopy. In Al-sensitive plants 5 min Al exposure was enough to inhibit cell division in the proximal meristem (250-800 microm from the tip). After 10 or 30 min with Al only, a few S-phase nuclei were found in the cortical initials. By contrast, cell division was stimulated in the distal elongation zone (2.5-3.1 mm). After 180 min the protrusion of an incipient lateral root was observed in this zone. These observations suggest a fast change in cell patterning rather than a general cariotoxic effect after exposure to Al for a short time. No such changes were found in Al-resistant maize. This is the first report showing such fast Al-induced alterations in the number and the position of dividing cells in root tips. The observation that similar changes were induced by a local supply of naphthylphthalamic acid to the distal transition zone suggests that inhibition of auxin transport plays a role in the Al-induced alteration of root cell patterning.

Up-regulation of the mitochondrial alternative oxidase pathway enhances photosynthetic electron transport under drought conditions

Abstract: The aim of this study was to explore the role of the mitochondrial alternative oxidase (AOX) in the protection of photosynthesis during drought in wheat leaves. The relative water contents of water-replete and drought-exposed wheat plants were 97.2+/-0.3 and 75+/-2, respectively. Drought increased the amount of leaf AOX protein and also enhanced the rate of AOX-dependent O(2) uptake by the respiratory electron transport chain. The amount of the reduced, active form of the AOX protein was specifically increased by drought. The AOX inhibitor salicylhydroxamic acid (1 mM; SHAM) inhibited 70% of AOX activity in vivo in both water-replete and drought-exposed plants. Plants treated with SHAM were then exposed to low (100), high (350), or excess light (800 mumol photons m(-2) s(-1)) for 90 min. SHAM did not modify chlorophyll a fluorescence quenching parameters in water-replete controls after any of these treatments. However, while the maximal quantum yield of photosystem II (PSII) electron transport (F(v)/F(m)) was not affected by SHAM, the immediate quantum yield of PSII electron transport (Phi(PSII)) and photochemical quenching (qP) were gradually reduced by increasing irradiance in SHAM-treated drought-exposed plants, the decrease being most pronounced at the highest irradiance. Non-photochemical quenching (NPQ) reached near maximum levels in plants subjected to drought at high irradiance. However, a combination of drought and low light caused an intermediate increase in NPQ, which attained higher values when AOX was inhibited. Taken together, these results show that up-regulation of the respiratory AOX pathway protects the photosynthetic electron transport chain from the harmful effects of excess light.

Redox regulation of carbon storage and partitioning in response to light and sugars

Abstract: Redox signals generated by the photosynthetic electron transport chain are known to be involved in regulating the Calvin cycle, ATP synthesis, and NADPH export from chloroplasts in response to light. The signal cascade involves transfer of electrons from photosystem I via the ferredoxin-thioredoxin system to target enzymes that are activated by reduction of regulatory disulphide bonds. The purpose of this review is to discuss recent findings showing that this concept can be extended to the regulation of carbon storage and partitioning in plants. Starch is the major carbon store in plants, and ADP-glucose pyrophosphorylase (AGPase) is the key regulatory enzyme of starch synthesis in the plastid. It has been shown that AGPase from potato tubers is subject to post-translational redox modification, and here experimental data will be provided showing that the isozyme from pea leaf chloroplasts is activated by reduced thioredoxin f or m in a similar way. Recent reports will be summarized providing in planta evidence that this mechanism regulates storage starch synthesis in response to light and sugars. Post-translational redox activation of AGPase in response to sugars is part of a signalling mechanism linking the rate of starch synthesis to the availability of carbon in diverse plant tissues. Some of the components of the signalling pathway reporting changes in the cytosolic sugar status to the plastid have been postulated, but detailed work is in progress to confirm the exact mode of action. Recent evidence will be discussed showing that key enzymes of de novo fatty acid synthesis (acetyl-CoA carboxylase) and ammonium assimilation (glutamine synthetase and glutamine:oxoglutarate amino transferase) are regulated by reversible disulphide-bond formation similar to AGPase. Redox regulation is proposed to be the preferred strategy of plastidial enzymes to regulate various metabolic processes such as carbon fixation, starch metabolism, lipid synthesis, and amino acid synthesis in response to physiological and environmental inputs.

Interactive effects of carbon dioxide, temperature, and ultraviolet-B radiation on soybean (Glycine max L.) flower and pollen morphology, pollen production, germination, and tube lengths

Abstract: Plant reproduction is highly vulnerable to global climate change components such as carbon dioxide concentration ([CO(2)]), temperature (T), and ultraviolet-B (UV-B) radiation. The objectives of this study were to determine the effects of season-long exposure to treatments of [CO(2)] at 360 (control) and 720 micromol mol(-1) (+CO(2)), temperature at 30/22 degrees C (control) and 38/30 degrees C (+T) and UV-B radiation 0 (control) and 10 kJ m(-2) d(-1) (+UV-B) on flower and pollen morphology, pollen production, germination, and tube lengths of six soybean genotypes (D 88-5320, D 90-9216, Stalwart III, PI 471938, DG 5630RR, and DP 4933RR) in sunlit, controlled environment chambers. The control treatment had 360 micromol mol(-1) [CO(2)] at 30/22 degrees C and 0 kJ UV-B. Plants grown either at +UV-B or +T, alone or in combination, produced smaller flowers with shorter standard petal and staminal column lengths. Flowers so produced had less pollen with poor pollen germination and shorter tube lengths. Pollen produced by the flowers of these plants appeared shrivelled without apertures and with disturbed exine ornamentation even at +CO(2) conditions. The damaging effects of +T and +UV-B were not ameliorated by +CO(2) conditions. Based on the total stress response index (TSRI), pooled individual component responses over all the treatments, the genotypes were classified as tolerant (DG 5630RR, D 88-5320: TSRI >-790), intermediate (D 90-9216, PI 471938: TSRI -1026), and sensitive (Stalwart III, DP 4933RR: TSRI <-1026). The differences in sensitivity identified among genotypes imply the options for selecting genotypes with tolerance to environmental stresses projected to occur in the future climates.

Metal binding by citrus dehydrin with histidine-rich domains

Abstract: Dehydrins are hydrophilic proteins that are responsive to osmotic stress, such as drought, cold, and salinity in plants. Although they have been hypothesized to stabilize macromolecules in stressed cells, their functions are not fully understood. Citrus dehydrin, which accumulates mainly in response to cold stress, enhances cold tolerance in transgenic tobacco by reducing lipid peroxidation. It has been demonstrated that citrus dehydrin scavenges hydroxyl radicals. In this study, the metal binding of citrus dehydrin is reported and the specific domain responsible is identified. The metal binding property of citrus dehydrin was tested using immobilized metal ion affinity chromatography (IMAC). Fe3+, Co2+, Ni2+, Cu2+, and Zn2+ bound to citrus dehydrin, but Mg2+, Ca2+, and Mn2+ did not. Among the bound metals, the highest affinity was detected for Cu(2+)-dehydrin binding, which showed a dissociation constant of 1.6 microM. Citrus dehydrin was able to bind up to 16 Cu2+ ions. IMAC indicated that His residues contributed to Cu(2+)-dehydrin binding. The amino acid sequence of CuCOR15 was divided into five domains, of which domain 1 bound Cu2+ most strongly. One portion of domain 1, HKGEHHSGDHH, was the core sequence for the binding. These results suggest that citrus dehydrin binds metals using a specific sequence containing His. Since citrus dehydrin is a radical-scavenging protein, it may reduce metal toxicity in plant cells under water-stressed conditions.

Physiological characterization of Mg deficiency in Arabidopsis thaliana

Abstract: Although the symptoms of magnesium deficiency are well documented in plants, the primary physiological effects of low Mg availability remain largely unknown. This paper describes the physiological responses of Mg starvation in Arabidopsis thaliana. Growth characteristics, Mg and sugar concentration, and photochemical performance were measured at regular intervals during the induction of Mg deficiency. These data show that Mg deficiency increased the sugar concentration and altered sucrose export from young source leaves before any noticeable effect on photosynthetic activity was seen. The decline in photosynthetic activity might be elicited by increased leaf sugar concentrations. Transcript levels of Cab2 (encoding a chlorophyll a/b protein) were lower in Mg-deficient plants before any obvious decrease in the chlorophyll concentration. These transcriptional data suggest that the reduction of chlorophyll is a response to sugar levels, rather than a lack of Mg atoms for chelating chlorophyll.

Differential responses of maize MIP genes to salt stress and ABA

Abstract: Salt stress is known to reduce root hydraulic conductivity and growth. To examine a concomitant regulation of aquaporins, the expression of the maize MIP gene family in response to NaCl was analysed by DNA array hybridization. Plants responded differentially to 100 versus 200 mM NaCl treatments. Leaf water content was reduced rapidly and persistently after the application of 200 mM NaCl in contrast to 100 mM NaCl. Endogenous ABA strongly accumulated in roots after 2 h; it remained at a highly elevated level for 48 h after the addition of 200 mM NaCl, but rapidly declined in plants treated with 100 mM NaCl, indicating an early recovery from water deficit. Interestingly, 2 h after the addition of 100 mM NaCl, when maize regained the osmotic potential allowing water uptake, three highly expressed, specific isoforms ZmPIP1;1, ZmPIP1;5, and ZmPIP2;4 were transiently induced. They were preferentially transcribed in the outer root tissue suggesting a role in cellular water transport. None of the ZmTIP genes was altered. By contrast, after the addition of 200 mM NaCl these responses were missing. Instead, multiple ZmPIP and ZmTIP genes were repressed by 200 mM NaCl after 24 h. After 48 h, deregulations were overridden in both cases indicating homeostasis. ABA (1 lM) exogenously applied to the roots transiently induced ZmPIP2;4 similar to 100 mM NaCl as well as ZmPIP1;2. Thus, the early induction of ZmPIP2;4 by NaCl may be mediated by ABA. Previously, an increase in root hydraulic conductivity had been observed upon ABA application. By contrast, 100 lM ABA led to a complete, possibly non-specific repression of all detected ZmPIP and ZmTIP genes after 24 h.

A Carotenoid Cleavage Dioxygenase from Vitis vinifera L.: functional characterization and expression during grape berry development in relation to C13-norisoprenoid accumulation

Abstract: A potential Carotenoid Cleavage Dioxygenase (CCD) gene was identified among a Vitis vinifera L. EST collection and a full-length cDNA (VvCCD1) was isolated. Recombinant expression of VvCCD1 confirmed that the gene encoded a functional CCD. Experimental evidence was obtained that VvCCD1 cleaves zeaxanthin symmetrically yielding 3-hydroxy-beta-ionone, a C(13)-norisoprenoidic compound, and a C(14)-dialdehyde. Expression of the gene was studied by real-time PCR at different developmental stages of grape berries from Muscat of Alexandria and Shiraz cultivars. A significant induction of the gene expression approaching veraison was observed in both cultivars. In parallel, the C(13)-norisoprenoid level increased from veraison to maturity in both cultivars.