Guy Kiddle

Leaf Vitamin C Contents Modulate Plant Defense Transcripts and Regulate Genes That Control Development through Hormone Signaling

Vitamin C deficiency in the Arabidopsis mutant vtc1 causes slow growth and late flowering. This is not attributable to changes in photosynthesis or increased oxidative stress. We have used the vtc1 mutant to provide a molecular signature for vitamin C deficiency in plants. Using statistical analysis, we show that 171 genes are expressed differentially in vtc1 compared with the wild type. Many defense genes are activated, particularly those that encode pathogenesis-related proteins. Furthermore, transcript changes indicate that growth and development are constrained in vtc1 by the modulation of abscisic acid signaling. Abscisic acid contents are significantly higher in vtc1 than in the wild type. Key features of the molecular signature of ascorbate deficiency can be reversed by incubating vtc1 leaf discs in ascorbate. This finding provides evidence that many of the observed effects on transcript abundance in vtc1 result from ascorbate deficiency. Hence, through modifying gene expression, vitamin C contents not only act to regulate defense and survival but also act via phytohormones to modulate plant growth under optimal conditions.

Control of Ascorbate Synthesis by Respiration and Its Implications for Stress Responses

We show for the first time that respiration can control ascorbate (AA) synthesis in plants. Evidence for this control is provided by (a) the localization of l-galactono-1,4-lactone dehydrogenase (GalLDH), the terminal enzyme in AA biosynthesis, with mitochondrial complex I, and its regulation by

Ascorbic Acid Deficiency Activates Cell Death and Disease Resistance Responses in Arabidopsis

Programmed cell death, developmental senescence, and responses to pathogens are linked through complex genetic controls that are influenced by redox regulation. Here we show that the Arabidopsis (Arabidopsis thaliana) low vitamin C mutants, vtc1 and vtc2, which have between 10% and 25% of wild-type ascorbic acid, exhibit microlesions, express pathogenesis-related (PR) proteins, and have enhanced basal resistance against infections caused by Pseudomonas syringae. The mutants have a delayed senescence phenotype with smaller leaf cells than the wild type at maturity. The vtc leaves have more glutathione than the wild type, with higher ratios of reduced glutathione to glutathione disulfide. Expression of green fluorescence protein (GFP) fused to the nonexpressor of PR protein 1 (GFP-NPR1) was used to detect the presence of NPR1 in the nuclei of transformed plants. Fluorescence was observed in the nuclei of 6- to 8-week-old GFP-NPR1 vtc1 plants, but not in the nuclei of transformed GFP-NPR1 wild-type plants at any developmental stage. The absence of senescence-associated gene 12 (SAG12) mRNA at the time when constitutive cell death and basal resistance were detected confirms that elaboration of innate immune responses in vtc plants does not result from activation of early senescence. Moreover, H2O2-sensitive genes are not induced at the time of systemic acquired resistance execution. These results demonstrate that ascorbic acid abundance modifies the threshold for activation of plant innate defense responses via redox mechanisms that are independent of the natural senescence program.

Plant homologs of the Plasmodium falciparum chloroquine-resistance transporter, PfCRT, are required for glutathione homeostasis and stress responses

In Arabidopsis thaliana, biosynthesis of the essential thiol antioxidant, glutathione (GSH), is plastid-regulated, but many GSH functions, including heavy metal detoxification and plant defense activation, depend on cytosolic GSH. This finding suggests that plastid and cytosol thiol pools are closely integrated and we show that in Arabidopsis this integration requires a family of three plastid thiol transporters homologous to the Plasmodium falciparum chloroquine-resistance transporter, PfCRT. Arabidopsis mutants lacking these transporters are heavy metal-sensitive, GSH-deficient, and hypersensitive to Phytophthora infection, confirming a direct requirement for correct GSH homeostasis in defense responses. Compartment-specific measurements of the glutathione redox potential using redox-sensitive GFP showed that knockout of the entire transporter family resulted in a more oxidized glutathione redox potential in the cytosol, but not in the plastids, indicating the GSH-deficient phenotype is restricted to the cytosolic compartment. Expression of the transporters in Xenopus oocytes confirmed that each can mediate GSH uptake. We conclude that these transporters play a significant role in regulating GSH levels and the redox potential of the cytosol.

Ascorbate oxidase-dependent changes in the redox state of the apoplast modulate gene transcript accumulation leading to modified hormone signaling and orchestration of defense processes in tobacco

The role of the redox state of the apoplast in hormone responses, signaling cascades, and gene expression was studied in transgenic tobacco (Nicotiana tabacum) plants with modified cell wall-localized ascorbate oxidase (AO). High AO activity specifically decreased the ascorbic acid (AA) content of the apoplast and altered plant growth responses triggered by hormones. Auxin stimulated shoot growth only when the apoplastic AA pool was reduced in wild-type or AO antisense lines. Oxidation of apoplastic AA in AO sense lines was associated with loss of the auxin response, higher mitogen-activated protein kinase activities, and susceptibility to a virulent strain of the pathogen Pseudomonas syringae. The total leaf glutathione pool, the ratio of reduced glutathione to glutathione disulfide, and glutathione reductase activities were similar in the leaves of all lines. However, AO sense leaves exhibited significantly lower dehydroascorbate reductase and ascorbate peroxidase activities than wild-type and antisense leaves. The abundance of mRNAs encoding antioxidant enzymes was similar in all lines. However, the day/night rhythms in the abundance of transcripts encoding the three catalase isoforms were changed in response to the AA content of the apoplast. Other transcripts influenced by AO included photorespiratory genes and a plasma membrane Ca2+ channel-associated gene. We conclude that the redox state of the apoplast modulates plant growth and defense responses by regulating signal transduction cascades and gene expression patterns. Hence, AO activity, which modulates the redox state of the apoplastic AA pool, strongly influences the responses of plant cells to external and internal stimuli.

Effects of Leaf Ascorbate Content on Defense and Photosynthesis Gene Expression in Arabidopsis thaliana

Ascorbate deficiency in the Arabidopsis thaliana vtc1 mutant had no effect on photosynthesis, but modified defense pathways. The ascorbate content of vtc1 leaves was increased 14-fold after 10 mM ascorbate was supplied, without a concomitant change in redox state. High ascorbate modified the abundance of 495 transcripts. Transcripts encoding dehydroascorbate reductase, pathogenesis-related protein 1, and a peroxiredoxin were decreased, whereas those encoding salicylate induction-deficient protein 1, Cu,Zn superoxide dismutase, iron superoxide dismutase, metallothionein, and glutathione transferases were increased. Catalase transcripts were unaffected, but ascorbate peroxidase isoforms APX1 and tAPX were slightly decreased and sAPX transcripts increased. A number of nuclear encoded transcripts for photosynthetic electron transport components were repressed as a result of ascorbate accumulation, whereas those that were chloroplast-encoded were increased. High ascorbate caused decreases in mRNAs encoding chloroplast enzymes such as fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase that are activated by reduced thioredoxin. In contrast, others, such as glucose 6-phosphate dehydrogenase, whose activity is inactivated by reduced thioredoxin, were repressed. Together, these results show that ascorbate is involved in metabolic cross-talk between redox-regulated pathways. The abundance of this antioxidant provides information on redox buffering capacity that coordinates redox processes associated with the regulation of photosynthesis and plant defense.

The Transcription Factor ABI4 Is Required for the Ascorbic Acid–Dependent Regulation of Growth and Regulation of Jasmonate-Dependent Defense Signaling Pathways in Arabidopsis

This work demonstrates that low ascorbate triggers abscisic acid-, salicylic acid-, and jasmonate-dependent signaling pathways in leaves that together regulate plant growth and defense responses. It provides insights into how cellular redox state regulates the expression of transcription factors and controls plant growth. Cellular redox homeostasis is a hub for signal integration. Interactions between redox metabolism and the ABSCISIC ACID-INSENSITIVE-4 (ABI4) transcription factor were characterized in the Arabidopsis thaliana vitamin c defective1 (vtc1) and vtc2 mutants, which are defective in ascorbic acid synthesis and show a slow growth phenotype together with enhanced abscisic acid (ABA) levels relative to the wild type (Columbia-0). The 75% decrease in the leaf ascorbate pool in the vtc2 mutants was not sufficient to adversely affect GA metabolism. The transcriptome signatures of the abi4, vtc1, and vtc2 mutants showed significant overlap, with a large number of transcription factors or signaling components similarly repressed or induced. Moreover, lincomycin-dependent changes in LIGHT HARVESTING CHLOROPHYLL A/B BINDING PROTEIN 1.1 expression were comparable in these mutants, suggesting overlapping participation in chloroplast to nucleus signaling. The slow growth phenotype of vtc2 was absent in the abi4 vtc2 double mutant, as was the sugar-insensitive phenotype of the abi4 mutant. Octadecanoid derivative-responsive AP2/ERF-domain transcription factor 47 (ORA47) and AP3 (an ABI5 binding factor) transcripts were enhanced in vtc2 but repressed in abi4 vtc2, suggesting that ABI4 and ascorbate modulate growth and defense gene expression through jasmonate signaling. We conclude that low ascorbate triggers ABA- and jasmonate-dependent signaling pathways that together regulate growth through ABI4. Moreover, cellular redox homeostasis exerts a strong influence on sugar-dependent growth regulation.

GMO detection using a bioluminescent real time reporter (BART) of loop mediated isothermal amplification (LAMP) suitable for field use

BackgroundThere is an increasing need for quantitative technologies suitable for molecular detection in a variety of settings for applications including food traceability and monitoring of genetically modified (GM) crops and their products through the food processing chain. Conventional molecular diagnostics utilising real-time polymerase chain reaction (RT-PCR) and fluorescence-based determination of amplification require temperature cycling and relatively complex optics. In contrast, isothermal amplification coupled to a bioluminescent output produced in real-time (BART) occurs at a constant temperature and only requires a simple light detection and integration device.ResultsLoop mediated isothermal amplification (LAMP) shows robustness to sample-derived inhibitors. Here we show the applicability of coupled LAMP and BART reactions (LAMP-BART) for determination of genetically modified (GM) maize target DNA at low levels of contamination (0.1-5.0% GM) using certified reference material, and compare this to RT-PCR. Results show that conventional DNA extraction methods developed for PCR may not be optimal for LAMP-BART quantification. Additionally, we demonstrate that LAMP is more tolerant to plant sample-derived inhibitors, and show this can be exploited to develop rapid extraction techniques suitable for simple field-based qualitative tests for GM status determination. We also assess the effect of total DNA assay load on LAMP-BART quantitation.ConclusionsLAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading within a reaction must be controlled to ensure quantification at low target concentrations.

Glutathione – linking cell proliferation to oxidative stress

SIGNIFICANCE The multifaceted functions of reduced glutathione (gamma-glutamyl-cysteinyl-glycine; GSH) continue to fascinate plants and animal scientists, not least because of the dynamic relationships between GSH and reactive oxygen species (ROS) that underpin reduction/oxidation (redox) regulation and signalling. Here we consider the respective roles of ROS and GSH in the regulation of plant growth, with a particular focus on regulation of the plant cell cycle. Glutathione is discussed not only as a crucial low molecular weight redox buffer that shields nuclear processes against oxidative challenge but also a flexible regulator of genetic and epigenetic functions. RECENT ADVANCES The intracellular compartmentalization of GSH during the cell cycle is remarkably consistent in plants and animals. Moreover, measurements of in vivo glutathione redox potentials reveal that the cellular environment is much more reducing than predicted from GSH/GSSG ratios measured in tissue extracts. The redox potential of the cytosol and nuclei of non-dividing plant cells is about -300 mV. This relatively low redox potential maintained even in cells experiencing oxidative stress by a number of mechanisms including vacuolar sequestration of GSSG. We propose that regulated ROS production linked to glutathione-mediated signalling events are the hallmark of viable cells within a changing and challenging environment. CRITICAL ISSUES The concept that the cell cycle in animals is subject to redox controls is well established but little is known about how ROS and GSH regulate this process in plants. However, it is increasingly likely that redox controls exist in plants, although possibly through different pathways. Moreover, redox-regulated proteins that function in cell cycle checkpoints remain to be identified in plants. While GSH-responsive genes have now been identified, the mechanisms that mediate and regulate protein glutathionylation in plants remain poorly defined. FUTURE DIRECTIONS The nuclear GSH pool provides an appropriate redox environment for essential nuclear functions. Future work will focus on how this essential thiol interacts with the nuclear thioredoxin system and nitric oxide to regulate genetic and epigenetic mechanisms. The characterization of redox-regulated cell cycle proteins in plants, and the elucidation of mechanisms that facilitate GSH accumulation in the nucleus are keep steps to unravelling the complexities of nuclear redox controls.

A Temperature-sensitive Mutation in the Arabidopsis thaliana Phosphomannomutase Gene Disrupts Protein Glycosylation and Triggers Cell Death

Eukaryotic phosphomannomutases (PMMs) catalyze the interconversion of mannose 6-phosphate to mannose 1-phosphate and are essential to the biosynthesis of GDP-mannose. As such, plant PMMs are involved in ascorbic acid (AsA) biosynthesis and N-glycosylation. We report on the conditional phenotype of the temperature-sensitive Arabidopsis thaliana pmm-12 mutant. Mutant seedlings were phenotypically similar to wild type seedlings when grown at 16–18 °C but died within several days after transfer to 28 °C. This phenotype was observed throughout both vegetative and reproductive development. Protein extracts derived from pmm-12 plants had lower PMM protein and enzyme activity levels. In vitro biochemical analysis of recombinant proteins showed that the mutant PMM protein was compromised in its catalytic efficiency (Kcat/Km). Despite significantly decreased AsA levels in pmm-12 plants, AsA deficiency could not account for the observed phenotype. Since, at restrictive temperature, total glycoprotein patterns were altered and glycosylation of protein-disulfide isomerase was perturbed, we propose that a deficiency in protein glycosylation is responsible for the observed cell death phenotype.