Michael Lawton

Programmed cell death, mitochondria and the plant hypersensitive response

The plant response to attempted infection by microbial pathogens is often accompanied by rapid cell death in and around the initial infection site, a reaction known as the hypersensitive response. This response is associated with restricted pathogen growth and represents a form of programmed cell death (PCD). Recent pharmacological and molecular studies have provided functional evidence for the conservation of some of the basic regulatory mechanisms underlying the response to pathogens and the activation of PCD in animal and plant systems. In animals, the mitochondrion integrates diverse cellular stress signals and initiates the death execution pathway, and studies indicate a similar involvement for mitochondria in regulating PCD in plants. But many of the cell-death regulators that have been characterized in humans, worms and flies are absent from the Arabidopsis genome, indicating that plants probably use other regulators to control this process.

Hydrogen Peroxide Stimulates Salicylic Acid Biosynthesis in Tobacco

Hydrogen peroxide induced the accumulation of free benzoic acid (BA) and salicylic acid (SA) in tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaves. Six hours after infiltration with 300 mM H2O2, the levels of BA and SA in leaves increased 5-fold over the levels detected in control leaves. The accumulation of BA and SA was preceded by the rapid activation of benzoic acid 2-hydroxylase (BA2H) in the H2O2-infiltrated tissues. This enzyme catalyzes the formation of SA from BA. Enzyme activation could be reproduced in vitro by addition of H2O2 or cumene hydroperoxide to the assay mixture. H2O2 was most effective in vitro when applied at 6 mM. In vitro activation of BA2H by peroxides was inhibited by the catalase inhibitor 3-amino-1,2,4-triazole. We suggest that H2O2 activates SA biosynthesis via two mechanisms. First, H2O2 stimulates BA2H activity directly or via the formation of its substrate, molecular oxygen, in a catalase-mediated reaction. Second, higher BA levels induce the accumulation of BA2H protein in the cells and provide more substrate for this enzyme.

Pathway of Salicylic Acid Biosynthesis in Healthy and Virus-Inoculated Tobacco

Salicylic acid (SA) is a likely endogenous regulator of localized and systemic disease resistance in plants. During the hypersensitive response of Nicotiana tabacum L. cv Xanthi-nc to tobacco mosaic virus (TMV), SA levels rise dramatically. We studied SA biosynthesis in healthy and TMV-inoculated tobacco by monitoring the levels of SA and its likely precursors in extracts of leaves and cell suspensions. In TMV-inoculated leaves, stimulation of SA accumulation is accompanied by a corresponding increase in the levels of benzoic acid. 14C-Tracer studies with cell suspensions and mock-or TMV-inoculated leaves indicate that the label moves from trans-cinnamic acid to SA via benzoic acid. In healthy and TMV-inoculated tobacco leaves, benzoic acid induced SA accumulation. o-Coumaric acid, which was previously reported as a possible precursor of SA in other species, did not increase SA levels in tobacco. In healthy tobacco tissue, the specific activity of newly formed SA was equal to that of the supplied [14C]benzoic acid, whereas in TMV-inoculated leaves some isotope dilution was observed, presumably because of the increase in the pool of endogenous benzoic acid. We observed accumulation of pathogen-esis-related-1 proteins and increased resistance to TMV in benzoic acid- but not in o-coumaric acid-treated tobacco leaves. This is consistent with benzoic acid being the immediate precursor of SA. We conclude that in healthy and virus-inoculated tobacco, SA is formed from cinnamic acid via benzoic acid.

Induction of Benzoic Acid 2-Hydroxylase in Virus-Inoculated Tobacco

Salicylic acid (SA) plays an important role in the induction of plant resistance to pathogens. An accompanying article (N. Yalpani, J. Leon, M.A. Lawton, I. Raskin [1993] Plant Physiol 103: 315-321) shows that SA is synthesized via the decarboxylation of cinnamic acid to benzoic acid (BA), which is, in turn, hydroxylated to SA. Leaf extracts of tobacco (Nicotiana tabacum L. cv Xanthi-nc) catalyze the 2-hydroxylation of BA to SA. The monooxygenase catalyzing this reaction, benzoic acid 2-hydroxylase (BA2H), required NAD(P)H or reduced methyl viologen as an electron donor. BA2H activity was detected in healthy tobacco leaf extracts (1-2 nmol h-1 g-1 fresh weight) and was significantly increased upon inoculation with tobacco mosaic virus (TMV). This increase paralleled the levels of free SA in the leaves. Induction of BA2H activity was restricted to tissue expressing a hypersensitive response at 24[deg]C. TMV induction of BA2H activity and SA accumulation were inhibited when inoculated tobacco plants were incubated at 32[deg]C. However, when inoculated plants were incubated for 4 d at 32[deg]C and then transferred to 24[deg]C, they showed a 15-fold increase in BA2H activity and a 65-fold increase in free SA content compared with healthy plants incubated at 24[deg]C. Treatment of leaf tissue with the protein synthesis inhibitor cycloheximide blocked the induction of BA2H activity by TMV. The effect of TMV inoculation on BA2H could be duplicated by infiltrating leaf discs of healthy plants with BA. This response was observed even when applied levels of BA were much lower than the levels observed in vivo after virus inoculation. Feeding tobacco leaves with phenylalanine, cinnamic acid, or o-coumaric acid (putative precursors of SA) failed to trigger the induction of BA2H activity. BA2H appears to be a pathogen-inducible protein with an important regulatory role in SA accumulation during the development of induced resistance to TMV in tobacco.

Improving sugarcane for biofuel: engineering for an even better feedstock

Sugarcane is a proven biofuel feedstock and accounts for about 40% of the biofuel production worldwide. It has a more favorable energy input/output ratio than that of corn, the other major biofuel feedstock. The rich resource of genetic diversity and the plasticity of autopolyploid genomes offer a wealth of opportunities for the application of genomics and technologies to address fundamental questions in sugarcane towards maximizing biomass production. In a workshop on sugarcane engineering held at Rutgers University, we identified research areas and emerging technologies that could have significant impact on sugarcane improvement. Traditional plant physiological studies and standardized phenotypic characterization of sugarcane are essential for dissecting the developmental processes and patterns of gene expression in this complex polyploid species. Breeder friendly DNA markers associated with target traits will enhance selection efficiency and shorten the long breeding cycles. Integration of cold tolerance from Saccharum spontaneum and Miscanthus has the potential to expand the geographical range of sugarcane production from tropical and subtropical regions to temperate zones. The Flex‐stock and mix‐stock concepts could be solutions for sustaining local biorefineries where no single biofuel feedstock could provide consistent year‐round supplies. The ever increasing capacities of genomics and biotechnologies pave the way for fully exploring these potentials to optimize sugarcane for biofuel production.

The chromatin landscape of the moss Physcomitrella patens and its dynamics during development and drought stress.

The moss Physcomitrella patens is an important model organism for evo-devo studies. Here, we determined the genome-wide chromatin landscape of five important histone three (H3) modifications (H3K4me3, H3K27me3, H3K27Ac, H3K9Ac and H3K9me2) and describe the changes to these histone marks in two contrasted situations, developmental transition and abiotic (drought) stress. Integrative analysis of these histone H3 modifications revealed their preferential association into 15 chromatin states (CS) in genic regions of the P.xa0patens genome. Synergistic relationships that influence expression levels were revealed for the three activating marks H3K4me3, H3K27Ac and H3K9Ac, while an antagonistic relationship was found between CS containing the H3K27me3 and H3K27Ac marks, suggesting that H3K27 is a key indexing residue regarding transcriptional output. Concerning the alteration of histone marks in response to developmental transition (juvenile to adult) and drought stress, the three activating marks H3K4me3, H3K27Ac and H3K9Ac show significant changes in both situations. However, changes to H3K27me3 are central only for genes differentially expressed during development. Interestingly, genes induced during drought stress show significant histone mark toggling during developmental transition. This situation suggests that drought induced adult (gametophore expressed) genes are primed to respond to this stress during the juvenile to adult transition.

Acetylcholineestarase-Inhibiting Alkaloids from Lycoris radiata Delay Paralysis of Amyloid Beta-Expressing Transgenic C. elegans CL4176

The limited symptom relief and side effects of current Alzheimer’s disease (AD) medications warrant urgent discovery and study of new anti-AD agents. The “cholinergic hypothesis” of AD prompts us to search for plant-derived acetylcholineesterase (AChE) inhibitors such as galanthamine that has been licensed in Europe for AD treatment. We used the unique amyloid β-expressing transgenic C. elegans CL4176, which exhibits paralysis when human Aβ1–42 is induced, to study two natural benzylphenethylamine alkaloids isolated from Lycoris radiata (L’ Her.) Herb, galanthamine and haemanthidine, and their synthetic derivatives 1,2-Di-O-acetyllycorine and 1-O-acetyllycorine for their anti-paralysis effects. Our data indicate that these Lycoris compounds effectively delay the paralysis of CL4176 worms upon temperature up-shift, and prolong the lives of these transgenic worms. Lycoris compounds were shown to significantly inhibit the gene expression of ace-1 and ace-2. Additionally, the Lycoris compounds may modulate inflammatory and stress-related gene expressions to combat the Aβ-toxicity in C. elegans.

Functional characterization of two new members of the caffeoyl CoA O-methyltransferase-like gene family from Vanilla planifolia reveals a new class of plastid-localized O-methyltransferases.

Caffeoyl CoA O-methyltransferases (OMTs) have been characterized from numerous plant species and have been demonstrated to be involved in lignin biosynthesis. Higher plant species are known to have additional caffeoyl CoA OMT-like genes, which have not been well characterized. Here, we identified two new caffeoyl CoA OMT-like genes by screening a cDNA library from specialized hair cells of pods of the orchid Vanilla planifolia. Characterization of the corresponding two enzymes, designated Vp-OMT4 and Vp-OMT5, revealed that in vitro both enzymes preferred as a substrate the flavone tricetin, yet their sequences and phylogenetic relationships to other enzymes are distinct from each other. Quantitative analysis of gene expression indicated a dramatic tissue-specific expression pattern for Vp-OMT4, which was highly expressed in the hair cells of the developing pod, the likely location of vanillin biosynthesis. Although Vp-OMT4 had a lower activity with the proposed vanillin precursor, 3,4-dihydroxybenzaldehyde, than with tricetin, the tissue specificity of expression suggests it may be a candidate for an enzyme involved in vanillin biosynthesis. In contrast, the Vp-OMT5 gene was mainly expressed in leaf tissue and only marginally expressed in pod hair cells. Phylogenetic analysis suggests Vp-OMT5 evolved from a cyanobacterial enzyme and it clustered within a clade in which the sequences from eukaryotic species had predicted chloroplast transit peptides. Transient expression of a GFP-fusion in tobacco demonstrated that Vp-OMT5 was localized in the plastids. This is the first flavonoid OMT demonstrated to be targeted to the plastids.

Specific activation of soybean defense genes by the phosphoprotein phosphatase inhibitor okadaic acid

The protein phosphatase inhibitor, okadaic acid (OKA) increased phenylalanine ammonia lyase (PAL) activity by 30-fold in soybean (Glycine max L.) cell suspension cultures. The increase in PAL activity was preceded by an increase in PAL mRNA. In addition, mRNA for chalcone synthase and a hydroxyproline-rich glycoprotein also increased concomitantly with PAL mRNA. In contrast, mRNA levels for the cytoskeletal proteins tubulin and actin, decreased in response to OKA treatment. The induction of the typical transcriptionally activated plant defense response to pathogens by OKA, suggests that the signal transduction pathway includes a protein phosphorylation cascade in which a protein phosphatase plays a key role.

Recognition and Signaling in Plant-Pathogen Interactions: Implications for Genetic Engineering

Plants are continually exposed to potential pathogens yet for the most part they remain healthy. The ability to resist disease reflects the operation of highly effective defense mechanisms. Plant defenses make use of pre-existing, static mechanisms (1) as well as responses that are invoked during attempted infection (2). To successfully infect a plant, a pathogen must overcome these defenses or ensure that they are not triggered. In this review, I discuss recent developments in our understanding of this process, with a focus on the molecular basis for pathogen recognition and subsequent signaling events. I have focused largely on bacterial and fungal pathogens although many of the concepts discussed also apply to viral pathogens.