Showing papers in "Planta in 2006"

Contribution of the arbuscular mycorrhizal symbiosis to heavy metal phytoremediation.

Abstract: High concentrations of heavy metals (HM) in the soil have detrimental effects on ecosystems and are a risk to human health as they can enter the food chain via agricultural products or contaminated drinking water. Phytoremediation, a sustainable and inexpensive technology based on the removal of pollutants from the environment by plants, is becoming an increasingly important objective in plant research. However, as phytoremediation is a slow process, improvement of efficiency and thus increased stabilization or removal of HMs from soils is an important goal. Arbuscular mycorrhizal (AM) fungi provide an attractive system to advance plant-based environmental clean-up. During symbiotic interaction the hyphal network functionally extends the root system of their hosts. Thus, plants in symbiosis with AM fungi have the potential to take up HM from an enlarged soil volume. In this review, we summarize current knowledge about the contribution of the AM symbiosis to phytoremediation of heavy metals.

Brassinosteroid confers tolerance in Arabidopsis thaliana and Brassica napus to a range of abiotic stresses

Abstract: In addition to an essential role in plant development, brassinosteroids (BRs) appear to have the ability to protect plants against various environmental stresses. However, studies confirming the ability of BRs to modulate plant responses to different environmental stresses are lacking. Earlier we had demonstrated that treatment with 24-epibrassinolide (EBR), a BR, increases the basic thermotolerance of Brassica napus and tomato seedlings [Plant Mol Biol 40:333-342, 1999]. Here we demonstrate that EBR treatment enhances seedling tolerance to drought and cold stresses in both Arabidopsis thaliana and B. napus, and helps to overcome a salt-stress-induced inhibition of seed germination. The ability of EBR to confer tolerance in plants to a variety of stresses was confirmed through analysis of expression of a subset of drought and cold stress marker genes. Transcriptional changes in these genes were more apparent in EBR-treated A. thaliana, in particular during earlier time points of stress. To see if BR is essential for the heat stress (HS) response, we made use of BR-deficient mutants. Both det2-1 and dwf4 mutants still expressed heat shock proteins (hsps) to high levels during HS, indicating that although BR augments thermotolerance in plants, it is not necessary for hsp expression during HS.

Nitric oxide enhances salt tolerance in maize seedlings through increasing activities of proton-pump and Na+/H+ antiport in the tonoplast

Abstract: Nitric oxide (NO), an endogenous signaling molecule in animals and plants, mediates responses to abiotic and biotic stresses. Our previous work demonstrated that 100 μM sodium nitroprusside (SNP, an NO donor) treatment of maize seedlings increased K+ accumulation in roots, leaves and sheathes, while decreasing Na+ accumulation (Zhang et al. in J Plant Physiol Mol Biol 30:455–459, 2004b). Here we investigate how NO regulates Na+, K+ ion homeostasis in maize. Pre-treatment with 100 μM SNP for 2 days improved later growth of maize plants under 100 mM NaCl stress, as indicated by increased dry matter accumulation, increased chlorophyll content, and decreased membrane leakage from leaf cells. An NO scavenger, methylene blue (MB-1), blocked the effect of SNP. These results indicated that SNP-derived NO enhanced maize tolerance to salt stress. Further analysis showed that NaCl induced a transient increase in the NO level in maize leaves. Both NO and NaCl treatment stimulated vacuolar H+-ATPase and H+-PPase activities, resulting in increased H+-translocation and Na+/H+ exchange. NaCl-induced H+-ATPase and H+-PPase activities were diminished by MB-1. 1-Butanol, an inhibitor of phosphatidic acid (PA) production by phospholipase D (PLD), reduced NaCl- and NO-induced H+-ATPase activation. In contrast, applied PA stimulated H+-ATPase activity. These results suggest that NO acts as a signal molecule in the NaCl response by increasing the activities of vacuolar H+-ATPase and H+-PPase, which provide the driving force for Na+/H+ exchange. PLD and PA play an important role in this process.

Raman imaging to investigate ultrastructure and composition of plant cell walls: distribution of lignin and cellulose in black spruce wood (Picea mariana).

Abstract: A detailed understanding of the structural organization of the cell wall of vascular plants is important from both the perspectives of plant biology and chemistry and of commercial utilization. A state-of-the-art 633-nm laser-based confocal Raman microscope was used to determine the distribution of cell wall components in the cross section of black spruce wood in situ. Chemical information from morphologically distinct cell wall regions was obtained and Raman images of lignin and cellulose spatial distribution were generated. While cell corner (CC) lignin concentration was the highest on average, lignin concentration in compound middle lamella (CmL) was not significantly different from that in secondary wall (S2 and S2–S3). Images generated using the 1,650 cm−1 band showed that coniferaldehyde and coniferyl alcohol distribution followed that of lignin and no particular cell wall layer/region was therefore enriched in the ethylenic residue. In contrast, cellulose distribution showed the opposite pattern—low concentration in CC and CmL and high in S2 regions. Nevertheless, cellulose concentration varied significantly in some areas, and concentrations of both lignin and cellulose were high in other areas. Though intensity maps of lignin and cellulose distributions are currently interpreted solely in terms of concentration differences, the effect of orientation needs to be carefully considered to reveal the organization of the wood cell wall.

Constitutive arginine-dependent nitric oxide synthase activity in different organs of pea seedlings during plant development

Abstract: Nitric oxide (NO) is an important signalling molecule in different animal and plant physiological processes. Little is known about its biological function in plants and on the enzymatic source or site of NO production during plant development. The endogenous NO production from L-arginine (NO synthase activity) was analyzed in leaves, stems and roots during plant development, using pea seedlings as a model. NOS activity was analyzed using a novel chemiluminescence-based assay which is more sensitive and specific than previous methods used in plant tissues. In parallel, NO accumulation was analyzed by confocal laser scanning microscopy using as fluorescent probes either DAF-2 DA or DAF-FM DA. A strong increase in NOS activity was detected in stems after 11 days growth, coinciding with the maximum stem elongation. The arginine-dependent NOS activity was constitutive and sensitive to aminoguanidine, a well-known irreversible inhibitor of animal NOS, and this NOS activity was differentially modulated depending on the plant organ and seedling developmental stage. In all tissues studied, NO was localized mainly in the vascular tissue (xylem) and epidermal cells and in root hairs. These loci of NO generation and accumulation suggest novel functions for NO in these cell types.

Role of auxin in regulating Arabidopsis flower development

Abstract: To elucidate the role of auxin in flower morphogenesis, its distribution patterns were studied during flower development in Arabidopsis thaliana (L.) Heynh. Expression of DR5::GUS was regarded to reflect sites of free auxin, while immunolocalization with auxin polyclonal antibodies visualized conjugated auxin distribution. The youngest flower bud was loaded with conjugated auxin. During development, the apparent concentration of free auxin increased in gradual patterns starting at the floral-organ tip. Anthers are major sites of high concentrations of free auxin that retard the development of neighboring floral organs in both the acropetal and basipetal directions. The IAA-producing anthers synchronize flower development by retarding petal development and nectary gland activity almost up to anthesis. Tapetum cells of young anthers contain free IAA which accumulates in pollen grains, suggesting that auxin promotes pollen-tube growth towards the ovules. High amounts of free auxin in the stigma induce a wide xylem fan immediately beneath it. After fertilization, the developing embryos and seeds show elevated concentrations of auxin, which establish their axial polarity. This developmental pattern of auxin production during floral-bud development suggests that young organs which produce high concentrations of free IAA inhibit or retard organ-primordium initiation and development at the shoot tip.

Combined effects of water stress and high temperature on photosynthesis, nitrogen metabolism and lipid peroxidation of a perennial grass Leymus chinensis

Abstract: Drought and high-temperature stresses have been extensively studied; however, little is known about their combined impact on plants. In the present study, we determined the photosynthetic gas exchange, chlorophyll fluorescence, nitrogen level, and lipid peroxidation of the leaves of a perennial grass (Leymus chinensis (Trin.) Tzvel.) subjected to three constant temperatures (23, 29 and 32°C), and five soil-moisture levels (75–80%, 60–65%, 50–55%, 35–40% and 25–30% of field capacity, respectively). High temperature significantly decreased plant biomass, leaf green area, leaf water potential, photosynthetic rate (A), maximal efficiency of PSII photochemistry (F v/F m), actual PSII efficiency (ΦPSII), the activities of nitrate reductase (NR; EC 1.6.6.1) and glutamine synthetase (GS; EC 6.3.1.2), but markedly increased the ratio of leaf area to leaf weight (SLA), endopeptidase (EP; EC 3.4.24.11) activity, and malondialdehyde (MDA) content, especially under severe water stress conditions. The A and F v/F m were significantly and positively correlated with leaf-soluble protein content, and the activities of NR and GS. However, both photosynthesis parameters were significantly and negatively correlated with EP activity and MDA content (P < 0.05). It is suggested that high temperature, combined with severe soil drought, might reduce the function of PSII, weaken nitrogen anabolism, strengthen protein catabolism, and provoke lipid peroxidation. The results also indicate that severe water stress might exacerbate the adverse effects of high temperature, and their combination might reduce the plant productivity and distribution range of L. chinensis in the future.

Flavonoid diversity and biosynthesis in seed of Arabidopsis thaliana.

Abstract: Functional characterization of genes involved in the flavonoid metabolism and its regulation requires in-depth analysis of flavonoid structure and composition of seed from the model plant Arabidopsis thaliana. Here, we report an analysis of the diverse and specific flavonoids that accumulate during seed development and maturation in wild types and mutants. Wild type seed contained more than 26 different flavonoids belonging to flavonols (mono and diglycosylated quercetin, kaempferol and isorhamnetin derivatives) and flavan-3-ols (epicatechin monomers and soluble procyanidin polymers with degrees of polymerization up to 9). Most of them are described for the first time in Arabidopsis. Interestingly, a novel group of four biflavonols that are dimers of quercetin-rhamnoside was also detected. Quercetin-3-O-rhamnoside (the major flavonoid), biflavonols, epicatechin and procyanidins accumulated in the seed coat in contrast to diglycosylated flavonols that were essentially observed in the embryo. Epicatechin, procyanidins and an additional quercetin-rhamnoside-hexoside derivative were synthesized in large quantities during seed development, whereas quercetin-3-O-rhamnoside displayed two peaks of accumulation. Finally, 11 mutants affected in known structural or regulatory functions of the pathway and their three corresponding wild types were also studied. Flavonoid profiles of the mutants were consistent with previous predictions based on genetic and molecular data. In addition, they also revealed the presence of new products in seed and underlined the plasticity of this metabolic pathway in the mutants.

Regulation of ethylene levels in canola (Brassica campestris by 1 -aminocyclopropane -1 -carboxylate deaminase-containing Methylobacterium fujisawaense

Abstract: We report the presence of ACC deaminase in Methylobacterium fujisawaense and its lowering of ethylene levels and promotion of root elongation in canola seedlings under gnotobiotic conditions. To test a part of the previous model proposed for ACC deaminase producing bacteria with Methylobacterium, ACC levels and various enzyme activities were monitored in canola. Lower amounts of ACC were present in the tissues of seeds treated with M. fujisawaense strains than in control seeds treated with MgSO(4). Though the increased activities of ACC synthase in the tissue extracts of the treated seedlings might be due to bacterial indole-3-acetic acid, the amount of ACC was reduced due to bacterial ACC deaminase activity. The activities of ACC oxidase, the enzyme catalyzing conversion of ACC to ethylene remained lower in M. fujisawaense treated seedlings. This consequently lowered the ethylene in plants and prevented ethylene inhibition of root elongation. Our results collectively suggest that Methylobacterium commonly found in soils, as well as on the surfaces of leaves, seeds, and in the rhizosphere of a wide variety of plants could be better exploited to promote plant growth.

Fungal and plant gene expression during the colonization of cacao seedlings by endophytic isolates of four Trichoderma species.

Abstract: Endophytic isolates of Trichoderma species are being considered as biocontrol agents for diseases of Theobroma cacao (cacao) Gene expression was studied during the interaction between cacao seedlings and four endophytic Trichoderma isolates, T ovalisporum-DIS 70a, T hamatum-DIS 219b, T harzianum-DIS 219f, and Trichoderma sp-DIS 172ai Isolates DIS 70a, DIS 219b, and DIS 219f were mycoparasitic on the pathogen Moniliophthora roreri, and DIS 172ai produced metabolites that inhibited growth of M roreri in culture ESTs (116) responsive to endophytic colonization of cacao were identified using differential display and their expression analyzed using macroarrays Nineteen cacao ESTs and 17 Trichoderma ESTs were chosen for real-time quantitative PCR analysis Seven cacao ESTs were induced during colonization by the Trichoderma isolates These included putative genes for ornithine decarboxylase (P1), GST-like proteins (P4), zinc finger protein (P13), wound-induced protein (P26), EF-calcium-binding protein (P29), carbohydrate oxidase (P59), and an unknown protein (U4) Two plant ESTs, extensin-like protein (P12) and major intrinsic protein (P31), were repressed due to colonization The plant gene expression profile was dependent on the Trichoderma isolate colonizing the cacao seedling The fungal ESTs induced in colonized cacao seedlings also varied with the Trichoderma isolate used The most highly induced fungal ESTs were putative glucosyl hydrolase family 2 (F3), glucosyl hydrolase family 7 (F7), serine protease (F11), and alcohol oxidase (F19) The pattern of altered gene expression suggests a complex system of genetic cross talk occurs between the cacao tree and Trichoderma isolates during the establishment of the endophytic association

Effect of sugar-induced senescence on gene expression and implications for the regulation of senescence in Arabidopsis.

Abstract: There has been some debate whether leaf senescence is induced by sugar starvation or by sugar accumulation. External supply of sugars has been shown to induce symptoms of senescence such as leaf yellowing. However, it was so far not clear if sugars have a signalling function during developmental senescence. Glucose and fructose accumulate strongly during senescence in Arabidopsis thaliana (L.) Heynh. leaves. Using Affymetrix GeneChip analysis we determined the effect of sugar-induced senescence on gene expression. Growth on glucose in combination with low nitrogen supply induced leaf yellowing and changes in gene expression that are characteristic of developmental senescence. Most importantly, the senescence-specific gene SAG12, which was previously thought to be sugar-repressible, was induced over 900-fold by glucose. Induction of SAG12, which is expressed during late senescence, demonstrates that processes characteristic for late stages are sugar-inducible. Two MYB transcription factor genes, PAP1 and PAP2, were identified as senescence-associated genes that are induced by glucose. Moreover, growth on glucose induced genes for nitrogen remobilisation that are typically enhanced during developmental senescence, including the glutamine synthetase gene GLN1;4 and the nitrate transporter gene AtNRT2.5. In contrast to wild-type plants, the hexokinase-1 mutant gin2-1 did not accumulate hexoses and senescence was delayed. Induction of senescence by externally supplied glucose was partially abolished in gin2-1, indicating that delayed senescence was a consequence of decreased sugar sensitivity. Taken together, our results show that Arabidopsis leaf senescence is induced rather than repressed by sugars.

Glyphosate, paraquat and ACCase multiple herbicide resistance evolved in a Lolium rigidum biotype

Abstract: Glyphosate is the world’s most widely used herbicide. A potential substitute for glyphosate in some use patterns is the herbicide paraquat. Following many years of successful use, neither glyphosate nor paraquat could control a biotype of the widespread annual ryegrass (Lolium rigidum), and here the world’s first case of multiple resistance to glyphosate and paraquat is confirmed. Dose–response experiments established that the glyphosate rate causing 50% mortality (LD50) for the resistant (R) biotype is 14 times greater than for the susceptible (S) biotype. Similarly, the paraquat LD50 for the R biotype is 32 times greater than for the S biotype. Thus, based on the LD50 R/S ratio, this R biotype of L. rigidum is 14-fold resistant to glyphosate and 32-fold resistant to paraquat. This R biotype also has evolved resistance to the acetyl-coenzyme A carboxylase (ACCase) inhibiting herbicides. The mechanism of paraquat resistance in this biotype was determined as restricted paraquat translocation. Resistance to ACCase-inhibiting herbicides was determined as due to an insensitive ACCase. Two mechanisms endowing glyphosate resistance were established: firstly, a point mutation in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, resulting in an amino acid substitution of proline to alanine at position 106; secondly, reduced glyphosate translocation was found in this R biotype, indicating a co-occurrence of two distinct glyphosate resistance mechanisms within the R population. In total, this R biotype displays at least four co-existing resistance mechanisms, endowing multiple resistance to glyphosate, paraquat and ACCase herbicides. This alarming case in the history of herbicide resistance evolution represents a serious challenge for the sustainable use of the precious agrochemical resources such as glyphosate and paraquat.

Expression profiling of the Arabidopsis ferric chelate reductase (FRO) gene family reveals differential regulation by iron and copper

Abstract: The Arabidopsis FRO2 gene encodes the iron deficiency-inducible ferric chelate reductase responsible for reduction of iron at the root surface; subsequent transport of iron across the plasma membrane is carried out by a ferrous iron transporter (IRT1). Genome annotation has identified seven additional FRO family members in the Arabidopsis genome. We used real-time RT-PCR to examine the expression of each FRO gene in different tissues and in response to iron and copper limitation. FRO2 and FRO5 are primarily expressed in roots while FRO8 is primarily expressed in shoots. FRO6 and FRO7 show high expression in all the green parts of the plant. FRO3 is expressed at high levels in roots and shoots, and expression of FRO3 is elevated in roots and shoots of iron-deficient plants. Interestingly, when plants are Cu-limited, the expression of FRO6 in shoot tissues is reduced. Expression of FRO3 is induced in roots and shoots by Cu-limitation. While it is known that FRO2 is expressed at high levels in the outer layers of iron-deficient roots, histochemical staining of FRO3-GUS plants revealed that FRO3 is predominantly expressed in the vascular cylinder of roots. Together our results suggest that FRO family members function in metal ion homeostasis in a variety of locations in the plant.

Infection with virulent and avirulent P. syringae strains differentially affects photosynthesis and sink metabolism in Arabidopsis leaves

Abstract: Infection of plants with pathogens leads not only to the induction of defence reactions but also to changes in carbohydrate metabolism. In this study, the effects of infection by a virulent and an avirulent strain of P. syringae on spatio-temporal changes in photosynthesis were compared using chlorophyll fluorescence imaging. The maximum PSII quantum yield, effective PSII quantum yield and nonphotochemical quenching were decreased in Arabidopsis leaves infected with either strain. At the same time, the quantum yield of nonregulated energy dissipation was increased. These changes could be detected by chlorophyll fluorescence imaging before symptoms were visible by eye. The effects were restricted to the vicinity of the infection site and did not spread to uninfected areas of the leaf. Qualitatively similar changes in photosynthetic parameters were observed in both interactions. Major differences between the responses to both strains were evident in the onset and time course of changes. A decrease in photosynthesis was detectable already at 3 h only after challenge with the avirulent strain while after 48 h the rate of photosynthesis was lower with the virulent strain. In contrast to photosynthesis, the regulation of marker genes for source/sink relations and the activities of invertase isoenzymes showed qualitative differences between both interactions. Inoculation of the virulent but not the avirulent strain resulted in downregulation of photosynthetic genes and upregulation of vacuolar invertases. The activity of vacuolar invertases transiently increased upon infection with the virulent strain but decreased with the avirulent strain while extracellular invertase activity was downregulated in both interactions.

Interactions between ethylene, gibberellin and abscisic acid regulate emergence and growth rate of adventitious roots in deepwater rice

Abstract: Growth of adventitious roots is induced in deepwater rice (Oryza sativa L.) when plants become submerged. Ethylene which accumulates in flooded plant parts is responsible for root growth induction. Gibberellin (GA) is ineffective on its own but acts in a synergistic manner together with ethylene to promote the number of penetrating roots and the growth rate of emerged roots. Studies with the GA biosynthesis inhibitor paclobutrazol revealed that root emergence was dependent on GA activity. Abscisic acid (ABA) acted as a competitive inhibitor of GA activity. Root growth rate on the other hand was dependent on GA concentration and ABA acted as a potent inhibitor possibly of GA but also of ethylene signaling. The results indicated that root emergence and elongation are distinct phases of adventitious root growth that are regulated through different networking between ethylene, GA and ABA signaling pathways. Adventitious root emergence must be coordinated with programmed death of epidermal cells which cover root primordia. Epidermal cell death is also controlled by ethylene, GA and ABA albeit with cell-type specific cross-talk. Different interactions between the same hormones may be a means to ensure proper timing of cell death and root emergence and to adjust the growth rate of emerged adventitious roots.

Sodium nitroprusside, cyanide, nitrite, and nitrate break Arabidopsis seed dormancy in a nitric oxide-dependent manner.

Abstract: The seeds of many plant species are dormant at maturity and dormancy loss is a prerequisite for germination. Numerous environmental and chemical treatments are known to lessen or remove seed dormancy, but the biochemical changes that occur during this change of state are poorly understood. Several lines of research have implicated nitric oxide (NO) as a participant in this process. Here, we show that dormant seeds of Arabidopsis thaliana (L.) Heynh. will germinate following treatment with the NO donor sodium nitroprusside (SNP), cyanide (CN), nitrite or nitrate. In all cases, the NO scavenger c-PTIO effectively promotes the maintenance of seed dormancy. c-PTIO does not, however, inhibit germination of fully after-ripened seeds, and c-PTIO does not interact directly with nitrite, nitrate or CN. We also show that volatile CN effectively breaks dormancy of Arabidopsis seeds, and that CN is the volatile compound in SNP that promotes dormancy loss. Our data support the hypothesis that NO is a signaling molecule that plays an important role in the loss of seed dormancy.

Rice ascorbate peroxidase gene family encodes functionally diverse isoforms localized in different subcellular compartments

Abstract: Aerobic organisms evolved a complex antioxidant system, which protect the cells against oxidative damage caused by partially reduced oxygen intermediates, also known as reactive oxygen species. In plants, ascorbate peroxidases (EC, 1.11.1.11) catalyze the conversion of H(2)O(2) to H(2)O, using ascorbate as the specific electron donor in this enzymatic reaction. Previously, eight APx genes were identified in the rice (Oryza sativa L.) genome through in silico analysis: two cytosolic isoforms, two putative peroxisomal isoforms, and four putative chloroplastic ones. Using gene-specific probes, we confirmed the presence of the eight APx genes in the rice genome by Southern blot hybridization. Transcript accumulation analysis showed specific expression patterns for each member of the APx family according to developmental stage and in response to salt stress, revealing the complexity of the antioxidant system in plants. Finally, the subcellular localization of rice APx isoforms was determined using GFP-fusion proteins in BY-2 tobacco cells. In agreement with the initial prediction, OSAPX3 was localized in the peroxisomes. On the other hand, the OSAPX6-GFP fusion protein was found in mitochondria of the BY-2 cells, in contrast to the chloroplastic location predicted by sequence analysis. Our findings reveal the functional diversity of the rice APx genes and suggest complementation and coordination of the antioxidant defenses in different cellular compartments during development and abiotic stress.

Overexpression of Arabidopsis phytochelatin synthase in tobacco plants enhances Cd2+ tolerance and accumulation but not translocation to the shoot

Abstract: Phytochelatins (PCs) are metal binding peptides involved in heavy metal detoxification. To assess whether enhanced phytochelatin synthesis would increase heavy metal tolerance and accumulation in plants, we overexpressed the Arabidopsis phytochelatin synthase gene (AtPCS1) in the non-accumulator plant Nicotiana tabacum. Wild-type plants and plants harbouring the Agrobacterium rhizogenes rolB oncogene were transformed with a 35S AtPCS1 construct. Root cultures from rolB plants could be easily established and we demonstrated here that they represent a reliable system to study heavy metal tolerance. Cd2+ tolerance in cultured rolB roots was increased as a result of overexpression of AtPCS1, and further enhanced when reduced glutathione (GSH, the substrate of PCS1) was added to the culture medium. Accordingly, HPLC analysis showed that total PC production in PCS1-overexpressing rolB roots was higher than in rolB roots in the presence of GSH. Overexpression of AtPCS1 in whole seedlings led to a twofold increase in Cd2+ accumulation in the roots and shoots of both rolB and wild-type seedlings. Similarly, a significant increase in Cd2+ accumulation linked to a higher production of PCs in both roots and shoots was observed in adult plants. However, the percentage of Cd2+ translocated to the shoots of seedlings and adult overexpressing plants was unaffected. We conclude that the increase in Cd2+ tolerance and accumulation of PCS1 overexpressing plants is directly related to the availability of GSH, while overexpression of phytochelatin synthase does not enhance long distance root-to-shoot Cd2+ transport.

Gametophytic self-incompatibility: understanding the cellular mechanisms involved in “self” pollen tube inhibition

Abstract: Self-incompatibility (SI) prevents the production of "self" seed and inbreeding by providing a recognition and rejection system for "self," or genetically identical, pollen. Studies of gametophytic SI (GSI) species at a molecular level have identified two completely different S-genes and SI mechanisms. One GSI mechanism, which is found in the Solanaceae, Rosaceae and Scrophulariaceae, has S-RNase as the pistil S-component and an F-box protein as the pollen S-component. However, non-S-locus factors are also required. In an incompatible situation, the S-RNases degrade pollen RNA, thereby preventing pollen tube growth. Here, in the light of recent evidence, we examine alternative models for how compatible pollen escapes this cytotoxic activity. The other GSI mechanism, so far found only in the Papaveraceae, has a small secreted peptide, the S-protein, as its pistil S-component. The pollen S-component remains elusive, but it is thought to be a transmembrane receptor, as interaction of the S-protein with incompatible pollen triggers a signaling network, resulting in rapid actin depolymerization and pollen tube inhibition and programmed cell death (PCD). Here, we present an overview of what is currently known about the mechanisms involved in regulating pollen tube inhibition in these two GSI systems.

Involvement of AtLAC15 in lignin synthesis in seeds and in root elongation of Arabidopsis

Abstract: Laccase, EC 1.10.3.2 or p-diphenol:dioxygen oxidoreductase, has been proposed to be involved in lignin synthesis in plants based on its in vitro enzymatic activity and a close correlation with the lignification process in plants. Despite many years of research, genetic evidence for the role of laccase in lignin synthesis is still missing. By screening mutants available for the annotated laccase gene family in Arabidopsis, we identified two mutants for a single laccase gene, AtLAC15 (At5g48100) with a pale brown or yellow seed coat which resembled the transparent testa (tt) mutant phenotype. A chemical component analysis revealed that the mutant seeds had nearly a 30% decrease in extractable lignin content and a 59% increase in soluble proanthocyanidin or condensed tannin compared with wild-type seeds. In an in vitro enzyme assay, the developing mutant seeds showed a significant reduction in polymerization activity of coniferyl alcohol in the absence of H(2)O(2). Among the dimers formed in the in vitro assay using developing wild-type seeds, 23% of the linkages were beta-O-4 which resembles the major linkages formed in native lignin. The evidence strongly supports that AtLAC15 is involved in lignin synthesis in plants. To our knowledge, this is the first genetic evidence for the role of laccase in lignin synthesis. Changes in seed coat permeability, seed germination and root elongation were also observed in the mutant.

Heme oxygenase up-regulation in ultraviolet-B irradiated soybean plants involves reactive oxygen species.

Abstract: Ultraviolet-B (UV-B) radiation has a negative impact on plant cells, and leads to the generation of reactive oxygen species (ROS). Heme oxygenase (HO, EC 1.14.99.3) plays a protective role against oxidative stress in mammals, but little is known about this issue in plants. Here, we report for the first time the response of HO in leaves of soybean (Glycine max L.) plants subjected to UV-B radiation. Under 7.5 and 15 kJ m−2 UV-B doses, HO, catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11) activities were increased and the production of thiobarbituric acid reactive substances (TBARS) regain control values after 4 h of plant recuperation. Treatment with 30 kJ m−2 UV-B provoked a decrease in these antioxidant enzyme activities. Immunoblot analysis showed a 4.3 and 3.7-fold increase in HO-1 protein expression after irradiation with 7.5 and 15 kJ m−2, respectively. HO-1 transcript levels were enhanced (up to 77%) at these doses, as assessed by semi-quantitative RT-PCR. These data demonstrated that increased HO activity was associated with augmented protein expression and transcript levels. Plants pre-treated with the antioxidant ascorbic acid did not show the UV-B-induced up-regulation of HO-1 mRNA, but hydrogen peroxide treatment could mimic this reaction. Our results indicate that HO is up-regulated in a dose-depending manner as a mechanism of cell protection against oxidative damage and that such response occurred as a consequence of HO-1 mRNA enhancement involving ROS.

Identification of expression profiles of sorghum genes in response to greenbug phloem-feeding using cDNA subtraction and microarray analysis

Abstract: The phloem-feeding by greenbug (Schizaphis graminum) elicits unique interactions with their host plants. To investigate the expression profiles of sorghum genes responsive to greenbug feeding, two subtractive cDNA libraries were constructed through different combinatorial subtractions in a strong greenbug resistance sorghum M627 line and a susceptible Tx7000 line with or without greenbug infestation. A total of 3,508 cDNAs were selected from the two cDNA libraries, and subsequent cDNA microarray and northern blot analyses were performed for identification of sorghum genes responsive to greenbugs. In total, 157 sorghum transcripts were identified to be differentially expressed by greenbug feeding. The greenbug responsive genes were isolated and classified into nine categories according to the functional roles in plant metabolic pathways, such as defense, signal transduction, cell wall fortification, oxidative burst/stress, photosynthesis, development, cell maintenance, abiotic stress, and unknown function. Overall, the profiles of sorghum genes, responsive to greenbug phloem-feeding shared common identities with other expression profiles known to be elicited by diverse stresses, including pathogenesis, abiotic stress, and wounding. In addition to well-known defense related regulators such as salicylic acid, jasmonic acid, and abscisic acid, auxin and gibberellic acid were also involved in mediation of the defense responses against greenbug phloem-feeding in sorghum.

How does glutamine synthetase activity determine plant tolerance to ammonium

Abstract: The wide range of plant responses to ammonium nutrition can be used to study the way ammonium interferes with plant metabolism and to assess some characteristics related with ammonium tolerance by plants. In this work we investigated the hypothesis of plant tolerance to ammonium being related with the plants' capacity to maintain high levels of inorganic nitrogen assimilation in the roots. Plants of several species (Spinacia oleracea L., Lycopersicon esculentum L., Lactuca sativa L., Pisum sativum L. and Lupinus albus L.) were grown in the presence of distinct concentrations (0.5, 1.5, 3 and 6 mM) of nitrate and ammonium. The relative contributions of the activity of the key enzymes glutamine synthetase (GS; under light and dark conditions) and glutamate dehydrogenase (GDH) were determined. The main plant organs of nitrogen assimilation (root or shoot) to plant tolerance to ammonium were assessed. The results show that only plants that are able to maintain high levels of GS activity in the dark (either in leaves or in roots) and high root GDH activities accumulate equal amounts of biomass independently of the nitrogen source available to the root medium and thus are ammonium tolerant. Plant species with high GS activities in the dark coincide with those displaying a high capacity for nitrogen metabolism in the roots. Therefore, the main location of nitrogen metabolism (shoots or roots) and the levels of GS activity in the dark are an important strategy for plant ammonium tolerance. The relative contribution of each of these parameters to species tolerance to ammonium is assessed. The efficient sequestration of ammonium in roots, presumably in the vacuoles, is considered as an additional mechanism contributing to plant tolerance to ammonium nutrition.

Genetic and biochemical evidence for involvement of HOTHEAD in the biosynthesis of long-chain alpha-,omega-dicarboxylic fatty acids and formation of extracellular matrix.

Abstract: In plants, extracellular matrix polymers built from polysaccharides and cuticular lipids have structural and protective functions. The cuticle is found to be ten times thinner in Arabidopsis thaliana (L.) Heynh than in many other plants, and there is evidence that it is unusual in having a high content of alpha-,omega-dicarboxylic fatty acids (FAs) in its polyesters. We designated the new organ fusion mutant hth-12 after it appeared to be allelic to adhesion of calyx edges (ace) and hothead (hth), upon molecular cloning of the gene by transposon tagging. This mutant is deficient in its ability to oxidize long-chain omega-hydroxy FAs to omega-oxo FAs, which results in leaf polyesters in decreased alpha-,omega-dicarboxylic FAs and increased omega-hydroxy FAs. These chemical phenotypes lead to disorder of the cuticle membrane structure in hth-12. ACE/HTH is a single-domain protein showing sequence similarity to long-chain FA omega-alcohol dehydrogenases from Candida species, and we hypothesize that it may catalyze the next step after cytochrome P450 FA omega-hydroxylases in the omega-oxidation pathway. We show that ACE/HTH is specifically expressed in epidermal cells. It appears very likely therefore that the changes in the amount of alpha-,omega-dicarboxylic FAs in hth-12 reflect the different composition of cuticular polyesters. The ACE/HTH gene is also expressed in root epidermal cells which do not form a polyester membrane on the exterior surface, thereby making it possible that the end products of the pathway, alpha-,omega-dicarboxylic FAs, are generally required for the cross-linking that ensures the integrity of the outer epidermal cell wall.

Do nitric oxide donors mimic endogenous NO-related response in plants?

Abstract: Huge advances achieved recently in elucidating the role of NO in plants have been made possible by the application of NO donors. However, the application of NO to plants in various forms and doses should be subjected to detailed verification criteria. Not all metabolic responses induced by NO donors are reliable and reproducible in other experimental designs. The aim of the presented studies was to investigate the half-life of the most frequently applied donors (SNP, SNAP and GSNO), the rate of NO release under the influence of light and reducing agents. At a comparable donor concentration (500 μM) and under light conditions the highest rate of NO generation was found for SNAP, followed by GSNO and SNP. The measured half-life of the donor in the solution was 3 h for SNAP, 7 h for GSNO and 12 h for SNP. A temporary lack of light inhibited NO release from SNP, both in the solution and SNP-treated leaf tissue, which was measured by the electrochemical method. Also a NO, selective fluorescence indicator DAF-2DA in leaves supplied with different donors showed green fluorescence spots in the epidermal cells mainly in the light. SNP as a NO donor was the most photosensitive. The activity of PAL, which plays an important role in plant defence, was also activated by SNP in the light, not in the dark. S-nitrosothiols (SNAP and GSNO) also underwent photodegradation, although to a lesser degree than SNP. Additionally, NO generation capacity from S-nitrosothiols was shown in the presence of reducing agents, i.e. ascorbic acid and GSH, and the absence of light. The authors of this paper would like to polemicize with the commonly cited statement that “donors are compounds that spontaneously break down to release NO” and wish to point out the fact that the process of donor decomposition depends on the numerous external factors. It may be additionally stimulated or inhibited by live plant tissue, thus it is necessary to take into consideration these aspects and monitor the amount of NO released by the donor.

RNAi-mediated silencing of the myo-inositol-1-phosphate synthase gene (GmMIPS1) in transgenic soybean inhibited seed development and reduced phytate content

Abstract: Inositol plays a role in membrane trafficking and signaling in addition to regulating cellular metabolism and controlling growth. In plants, the myo-inositol-1-phosphate is synthesized from glucose 6-phosphate in a reaction catalyzed by the enzyme myo-inositol-1-phosphate synthase (EC 5.5.1.4). Inositol can be converted into phytic acid (phytate), the most abundant form of phosphate in seeds. The path to phytate has been suggested to proceed via the sequential phosphorylation of inositol phosphates, and/or in part via phosphatidylinositol phosphate. Soybean [Glycine max (L.) Merrill] lines were produced using interfering RNA (RNAi) construct in order to silence the myo-inositol-1-phosphate (GmMIPS1) gene. We have observed an absence of seed development in lines in which the presence of GmMIPS1 transcripts was not detected. In addition, a drastic reduction of phytate (InsP6) content was achieved in transgenic lines (up to 94.5%). Our results demonstrated an important correlation between GmMIPS1 gene expression and seed development.

Two cell wall associated peroxidases from Arabidopsis influence root elongation.

Abstract: Two class III peroxidases from Arabidopsis, AtPrx33 and Atprx34, have been studied in this paper. Their encoding genes are mainly expressed in roots; AtPrx33 transcripts were also found in leaves and stems. Light activates the expression of both genes in seedlings. Transformed seedlings producing AtPrx33-GFP or AtPrx34-GFP fusion proteins under the control of the CaMV 35S promoter exhibit fluorescence in the cell walls of roots, showing that the two peroxidases are localized in the apoplast, which is in line with their affinity for the Ca2+-pectate structure. The role they can play in cell wall was investigated using (1) insertion mutants that have suppressed or reduced expression of AtPrx33 or AtPrx34 genes, respectively, (2) a double mutant with no AtPrx33 and a reduced level of Atprx34 transcripts, (3) a mutant overexpressing AtPrx34 under the control of the CaMV 35S promoter. The major phenotypic consequences of these genetic manipulations were observed on the variation of the length of seedling roots. Seedlings lacking AtPrx33 transcripts have shorter roots than the wild-type controls and roots are still shorter in the double mutant. Seedlings overexpressing AtPrx34 exhibit significantly longer roots. These modifications of root length are accompanied by corresponding changes of cell length. The results suggest that AtPrx33 and Atprx34, two highly homologous Arabidopsis peroxidases, are involved in the reactions that promote cell elongation and that this occurs most likely within cell walls.

Low-temperature tolerance and genetic potential in wheat (Triticum aestivum L.): response to photoperiod, vernalization, and plant development.

Abstract: It is frequently observed that winter habit types are more low-temperature (LT) tolerant than spring habit types. This raises the question of whether this is due to pleiotropic effects of the vernalization loci or to the linkage of LT-tolerance genes to these vernalization loci. Reciprocal near-isogenic lines (NILs) for alleles at the Vrn-A1 locus, Vrn-A1 and vrn-A1, determining spring and winter habit respectively, in two diverse genetic backgrounds of wheat (Triticum aestivum L.) were used to separate the effects of vernalization, photoperiod, and development on identical, or near identical, genetic backgrounds. The vrn-A1 allele in the winter lines allowed full expression of genotype dependent LT tolerance potential. The winter allele (vrn-A1) in a very cold tolerant genetic background resulted in 11°C, or a 2.4-fold, greater LT tolerance compared to the spring allele. Similarly, the delay in development caused by short-day (SD) versus long-day (LD) photoperiod in the identical spring habit NIL resulted in an 8.5°C or 2.1-fold, increase in LT tolerance. The duration of time in early developmental stages was shown to underlie full expression of genetic LT-tolerance potential. Therefore, pleiotropic effects of the vernalization loci can explain the association of LT tolerance and winter habit irrespective of either the proposed closely linked Fr-A1 or the more distant Fr-A2 LT-tolerance QTLs. Plant development progressively reduced LT-acclimation ability, particularly after the main shoot meristem had advanced to the double ridge reproductive growth stage. The Vrn-1 genes, or other members of the flowering induction pathway, are discussed as possible candidates for involvement in LT-tolerance repression.

Timecourse microarray analyses reveal global changes in gene expression of susceptible Glycine max (soybean) roots during infection by Heterodera glycines (soybean cyst nematode)

Abstract: Changes in gene expression within roots of Glycine max (soybean), cv. Kent, susceptible to infection by Heterodera glycines (the soybean cyst nematode [SCN]), at 6, 12, and 24 h, and 2, 4, 6, and 8 days post-inoculation were monitored using microarrays containing more than 6,000 cDNA inserts. Replicate, independent biological samples were examined at each time point. Gene expression was analyzed statistically using T-tests, ANOVA, clustering algorithms, and online analytical processing (OLAP). These analyses allow the user to query the data in several ways without importing the data into third-party software. RT-PCR confirmed that WRKY6 transcription factor, trehalose phosphate synthase, EIF4a, Skp1, and CLB1 were differentially induced across most time-points. Other genes induced across most timepoints included lipoxygenase, calmodulin, phospholipase C, metallothionein-like protein, and chalcone reductase. RT-PCR demonstrated enhanced expression during the first 12 h of infection for Kunitz trypsin inhibitor and sucrose synthase. The stress-related gene, SAM-22, phospholipase D and 12-oxophytodienoate reductase were also induced at the early time-points. At 6 and 8 dpi there was an abundance of transcripts expressed that encoded genes involved in transcription and protein synthesis. Some of those genes included ribosomal proteins, and initiation and elongation factors. Several genes involved in carbon metabolism and transport were also more abundant. Those genes included glyceraldehyde 3-phosphate dehydrogenase, fructose-bisphosphate aldolase and sucrose synthase. These results identified specific changes in gene transcript levels triggered by infection of susceptible soybean roots by SCN.

Structural analysis of wheat wax (Triticum aestivum, c.v. ‘Naturastar’ L.): from the molecular level to three dimensional crystals

Abstract: In order to elucidate the self assembly process of plant epicuticular waxes, and the molecular arrange- ment within the crystals, re-crystallisation of wax platelets was studied on biological and non-biological surfaces. Wax platelets were extracted from the leaf blades of wheat (Triticum aestivum L., c.v. 'Naturastar', Poaceae). Waxes were analysed by gas chromatography (GC) and mass spectrometry (MS). Octacosan-1-ol was found to be the most abundant chemical component of the wax mixture (66 m%) and also the determining compound for the shape of the wax platelets. The elec- tron diffraction pattern showed that both the wax mix- ture and pure octacosan-1-ol are crystalline. The re- crystallisation of the natural wax mixture and the pure octacosan-1-ol were studied by scanning tunnelling microscopy (STM), atomic force microscopy (AFM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Crystallisation of wheat waxes and pure octacosano-1-ol on the non polar highly ordered pyrolytic graphite (HOPG) led to the formation of platelet structures similar to those found on the plant surface. In contrast, irregular wax morphologies and flat lying plates were formed on glass, silicon, salt crystals (NaCl) and mica surfaces. Movement of wheat wax through isolated Convallaria majalis cuticles led to typ- ical wax platelets of wheat, arranged in the complex patterns typical for C. majalis. STM of pure octacosan- 1-ol monolayers on HOPG showed that the arrangement of the molecules strictly followed the hexagonal struc- ture of the substrate crystal. Re-crystallisation of wheat waxes on non-polar crystalline HOPG substrate showed that technical surfaces could be used to generate mi- crostructured, biomimetic surfaces. AFM and SEM studies proved that a template effect of the substrate determined the orientation of the re-grown crystals. These effects of the structure and polarity of the sub- strate on the morphology of the epicuticular waxes are relevant for understanding interactions between biolog- ical as well as technical surfaces and waxes.