Parallel analysis of global garlic gene expression and alliin content following leaf wounding.

BACKGROUND: Allium sativum (garlic) is an economically important food source and medicinal plant rich in sulfides and other protective substances such as alliin, the precursor of allicin biosynthesis. Cysteine, serine and sulfur is the precursor of alliin biosynthesis. However, little is known about the alliin content under abiotic stress or the mechanism by which it is synthesized. RESULTS: The findings revealed that the content of alliin was lowest in the garlic roots, and highest in the buds. Furthermore, alliin levels decreased in mature leaves following wounding. Transcriptome data generated over time after wounding further revealed significant up-regulation of genes integral to the biosynthetic pathways of cysteine and serine in mature garlic leaves. CONCLUSIONS: The findings suggest that differential expression of cysteine, serine and sulfide-related genes underlies the accumulation of alliin and its precursors in garlic, providing a basis for further analyses of alliin biosynthesis.

A forty year journey: The generation and roles of NO in plants.

In this year there is the 40th anniversary of the first publication of plant nitric oxide (NO) emission by Lowell Klepper. In the decades since then numerous milestone discoveries have revealed that NO is a multifunctional molecule in plant cells regulating both plant development and stress responses. Apropos of the anniversary, these authors aim to review and discuss the developments of past concepts in plant NO research related to NO metabolism, NO signaling, NO's action in plant growth and in stress responses and NO's interactions with other reactive compounds. Despite the long-lasting research efforts and the accumulating experimental evidences numerous questions are still needed to be answered, thus future challenges and research directions have also been drawn up.

The promoter of a basic PR1-like gene, AtPRB1, from Arabidopsis establishes an organ-specific expression pattern and responsiveness to ethylene and methyl jasmonate.

Antimicrobial proteins are a key feature underlying the deployment of both pre-formed and inducible defence responses. Probably the most well characterised class are the pathogenesis-related (PR) proteins, which are found in both basic and acidic isoforms. Here we describe the isolation and characterisation of a gene, designated AtPRB1, encoding a basic PR1-like protein from Arabidopsis. This protein showed high amino acid sequence identity with basic and acidic PR1 proteins from other plant species, for example PRB1 from Nicotiana tabacum and PR1 from Brassica napus, at 64% and 78% identity respectively. A genomic DNA fragment containing 2345 bp upstream from the putative transcriptional start site was fused to the gene encoding the luciferase (LUC) gene from Photinus pyralis in order to test for promoter activity. The resulting construct was transformed into Arabidopsis accession Col-0 and analysis of LUC activity, using an ultra-low-light imaging camera system, revealed that the AtPRB1 promoter established an exquisite organ-specific expression pattern. LUC activity was observed in flowers, stems and roots but not in leaf tissue. Superimposed upon this organ-specific expression pattern was responsiveness, in root tissue, to ethylene and methyl jasmonate (MeJA), important cues during the establishment of plant disease resistance. In contrast, AtPRB1::LUC gene expression was repressed in response to salicylic acid treatment. Analysis of a limited series of AtPRB1 5'-promoter deletion mutants, identified a number of promoter regions important for both the establishment of organ-specific expression and responsiveness to ethylene and MeJA. While AtPRB1 gene expression was not induced in response to an avirulent isolate of Peronospora parasitica in leaf tissue, this gene may contribute to horizontal resistance in other tissues and/or to MeJA- and ethylene-dependent defence responses engaged against necrotrophic pathogens in root tissue. It is anticipated that transgenic plants containing AtPRB1-based promoter::reporter constructs will provide useful tools for the future dissection of the cognate signalling networks regulating the expression of this gene.

Oxidative burst and cognate redox signalling reported by luciferase imaging: identification of a signal network that functions independently of ethylene, SA and Me-JA but is dependent on MAPKK activity.

Recognition of avirulent microbial pathogens activates an oxidative burst leading to the accumulation of reactive oxygen intermediates (ROIs), which are thought to integrate a diverse set of defence mechanisms resulting in the establishment of plant disease resistance. A novel transgenic Arabidopsis line containing a gst1:luc transgene was developed and employed to report the temporal and spatial dynamics of ROI accumulation and cognate redox signalling in response to attempted infection by avirulent strains of Pseudomonas syringae pv. tomato (Pst). Strong engagement of the oxidative burst was dependent on the presence of functional Pst hrpS and hrpA gene products. Experiments employing pharmacological agents suggested that at least two distinct sources, including an NADPH oxidase and a peroxidase-type enzyme, contributed to the generation of redox cues. The analysis of gst1 and pal1 gene expression in nahG, coi1 and etr1 plants suggested that engagement of the oxidative burst and cognate redox signalling functioned independently of salicylic acid, methyl jasmonate and ethylene. In contrast, studies using a panel of protein kinase and phosphatase inhibitors and in-gel kinase assays in these mutant backgrounds suggested that a 48 kDa mitogen-activated protein kinase (MAPK) activity was required for the activation of gst1 and pal1 in response to redox cues. Thus the engagement of a bifurcating redox signalling pathway possessing a MAPK module may contribute both to the establishment of plant disease resistance, and to the development of cellular protectant mechanisms.

Rapid stimulation of a soybean protein-serine kinase that phosphorylates a novel bZIP DNA-binding protein, G/HBF-1, during the induction of early transcription-dependent defenses.

The G-box (CACGTG) and H-box (CCTACC) cis elements function in the activation of phenylpropanoid biosynthetic genes involved in the elaboration of lignin precursors, phytoalexins and the secondary signal salicylic acid as early responses to pathogen attack. We have isolated a soybean cDNA encoding a novel bZIP protein, G/HBF-1, which binds to both the G-box and adjacent H-box in the proximal region of the chalcone synthase chs15 promoter. While G/HBF-1 transcript and protein levels do not increase during the induction of phenylpropanoid biosynthetic genes, G/HBF-1 is phosphorylated rapidly in elicited soybean cells, almost exclusively on serine residues. Using recombinant G/HBF-1 as a substrate, we identified a cytosolic protein-serine kinase that is rapidly and transiently stimulated in cells elicited with either glutathione or an avirulent strain of the soybean pathogen Pseudomonas syringae pv. glycinea. Phosphorylation of G/HBF-1 in vitro enhances binding to the chs15 promoter and we conclude that stimulation of G/HBF-1 kinase activity and G/HBF-1 phosphorylation are terminal events in a signal pathway for activation of early transcription-dependent plant defense responses.

Combination of H-box [CCTACC(N)7CT] and G-box (CACGTG) cis elements is necessary for feed-forward stimulation of a chalcone synthase promoter by the phenylpropanoid-pathway intermediate p-coumaric acid.

The phenylpropanoid pathway intermediate p-coumaric acid (4-CA) stimulates expression of the bean (Phaseolus vulgaris L.) chalcone synthase (malonyl-CoA:4-coumaroyl-CoA, EC 2.3.1.74) chs15 gene promoter in electroporated protoplasts of alfalfa (Medicago sativa L.). We have analyzed the effects of 5' deletions, mutations, and competition with promoter sequences in trans on the expression of a chs15 promoter-chloramphenicol acetyltransferase gene fusion in elicited alfalfa protoplasts. Two distinct sequence elements, the H-box (consensus CCTACC(N)7CT) and the G-box (CACGTG), are required for stimulation of the chs15 promoter by 4-CA. Furthermore, a 38-base-pair chs15 promoter sequence containing both cis elements conferred responsiveness to 4-CA on the cauliflower mosaic virus 35S minimal promoter. The H-box and G-box in combination establish the complex developmental pattern of chs15 expression and are also involved in stress induction. Hence, potential internal pathway regulation through feed-forward stimulation by 4-CA operates by modulation of the signal pathways for developmental and environmental regulation.

Phenylpropanoid pathway intermediates regulate transient expression of a chalcone synthase gene promoter.

A chimeric gene construct containing a bean chalcone synthase (CHS) promoter fused to the chloramphenicol acetyltransferase (CAT) reporter gene was strongly expressed when electroporated into alfalfa protoplasts that were then exposed to a fungal elicitor. Low concentrations (5 x 10(-6) to 10(-4) M) of exogenously applied trans-cinnamic acid (CA), the first intermediate of the phenylpropanoid pathway, slightly stimulated elicitor-induced CAT expression, whereas high concentrations (greater than 10(-4) M) severely reduced expression to below the levels observed in the absence of elicitor. In contrast, trans-p-coumaric acid (4-CA, the second intermediate in the pathway) stimulated expression from the CHS promoter up to 4.5-fold at 5 x 10(-4) M. Expression of CAT driven by the promoters of other elicitor-inducible defense response genes was not markedly affected by CA or 4-CA. Stimulation of CHS promoter expression by low concentrations of CA and 4-CA was completely abolished by 5' deletion to position -130, but not -174. When the -180 to -130 region of the CHS15 promoter was coelectroporated into elicited protoplasts on a separate plasmid along with the intact -326 CHS-CAT construct, the decreased CAT expression as a function of CA or 4-CA concentration was consistent with the coelectroporated sequence competing in trans with the intact promoter for the binding of a factor(s) involved in the up regulation of CHS transcription by 4-CA and low concentrations of CA. Our data support the hypothesis that phenylpropanoid compounds may act as natural and specific regulators of plant gene expression and define the location of a cis-acting element in the CHS15 promoter involved in the induction by phenylpropanoid pathway intermediates.

Ti plasmid-specified chemotaxis of Agrobacterium tumefaciens C58C1 toward vir-inducing phenolic compounds and soluble factors from monocotyledonous and dicotyledonous plants.

Twelve phenolic compounds with related structures were analyzed for their ability to act as chemoattractants for Agrobacterium tumefaciens C58C1 and as inducers of the Ti plasmid virulence operons. The results divided the phenolic compounds into three groups: compounds that act as strong vir inducers and are chemoattractants for A. tumefaciens C58C1 harboring the nopaline Ti plasmid pDUB1003 delta 31, but not the isogenic cured strain; compounds that are at best weak vir inducers and are weak chemoattractants for Ti plasmid-harboring and cured A. tumefaciens C58C1; and compounds that are vir noninducers and are also nonattractants. A strong correlation between vir-inducing ability and Ti plasmid requirement for chemotaxis is thus established. In addition, chemical structure rules for vir induction and chemotaxis are outlined. Positive chemotaxis toward root and shoot homogenates from monocotyledonous and dicotyledonous plants was observed. At low extract concentrations, chemotaxis was enhanced by the presence of Ti plasmid. The chemoattractants do not derive from intact cell walls. Lack of attraction is not responsible for the apparent block to monocot transformation by A. tumefaciens.

virA and virG are the Ti-plasmid functions required for chemotaxis of Agrobacterium tumefaciens towards acetosyringone.

Octopine and nopaline Ti-plasmids confer upon Agrobacterium tumefaciens C58C1 the ability to respond chemotactically to the vir-inducing phenolic wound exudate, acetosyringone. A. tumefaciens C58C1 containing Ti-plasmids with Tn5 insertions in virB, C, D or E exhibited marked chemotaxis towards acetosyringone. However, Ti-plasmids with mutations in virA or virG were unable to confer the responsive phenotype. Of the cosmid clones pVK219 (virAB) pVK221 (virBGC) pVK225 (virGCDE) and pVK257 (virABGC) mobilized to cured A. tumefaciens C58C1, only pVK257 bestowed acetosyringone chemotaxis. virA and virG are thus required for chemotaxis of A. tumefaciens towards acetosyringone. This suggests a multifunctional role for virA and virG: at low concentrations of acetosyringone they mediate chemotaxis and at higher concentrations they effect vir-induction.