Lina Gallego-Giraldo

Genome-wide analysis of phenylpropanoid defence pathways

Phenylpropanoids can function as preformed and inducible antimicrobial compounds, as well as signal molecules, in plant-microbe interactions. Since we last reviewed the field 8 years ago, there has been a huge increase in our understanding of the genes of phenylpropanoid biosynthesis and their regulation, brought about largely by advances in genome technology, from whole-genome sequencing to massively parallel gene expression profiling. Here, we present an overview of the biosynthesis and roles of phenylpropanoids in plant defence, together with an analysis of confirmed and predicted phenylpropanoid pathway genes in the sequenced genomes of 11 plant species. Examples are provided of phylogenetic and expression clustering analyses, and the large body of underlying genomic data is provided through a website accessible from the article.

Salicylic acid mediates the reduced growth of lignin down-regulated plants

Down-regulation of the enzyme hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) in thale cress (Arabidopsis thaliana) and alfalfa (Medicago sativa) leads to strongly reduced lignin levels, reduced recalcitrance of cell walls to sugar release, but severe stunting of the plants. Levels of the stress hormone salicylic acid (SA) are inversely proportional to lignin levels and growth in a series of transgenic alfalfa plants in which lignin biosynthesis has been perturbed at different biosynthetic steps. Reduction of SA levels by genetically blocking its formation or causing its removal restores growth in HCT–down-regulated Arabidopsis, although the plants maintain reduced lignin levels. SA-mediated growth inhibition may occur via interference with gibberellic acid signaling or responsiveness. Our data place SA as a central component in growth signaling pathways that either sense flux into the monolignol pathway or respond to secondary cell-wall integrity, and indicate that it is possible to engineer plants with highly reduced cell-wall recalcitrance without negatively impacting growth.

Selective lignin downregulation leads to constitutive defense response expression in alfalfa (Medicago sativa L.)

• Downregulation of hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) in alfalfa (Medicago sativa) reduces lignin levels and improves forage quality and saccharification efficiency for bioethanol production. However, the plants have reduced stature. It was previously reported that HCT-down-regulated Arabidopsis have impaired auxin transport, but this has recently been disproved. • To address the basis for the phenotypes of lignin-modified alfalfa, we measured auxin transport, profiled a range of metabolites including flavonoids and hormones, and performed in depth transcriptome analyses. • Auxin transport is unaffected in HCT antisense alfalfa despite increased flavonoid biosynthesis. The plants show increased cytokinin and reduced auxin levels, and gibberellin levels and sensitivity are both reduced. Levels of salicylic, jasmonic and abscisic acids are elevated, associated with massive upregulation of pathogenesis and abiotic stress-related genes and enhanced tolerance to fungal infection and drought. • We suggest that HCT downregulated alfalfa plants exhibit constitutive activation of defense responses, triggered by release of bioactive cell wall fragments and production of hydrogen peroxide as a result of impaired secondary cell wall integrity.

An NAC transcription factor orchestrates multiple features of cell wall development in Medicago truncatula

To identify genes controlling secondary cell wall biosynthesis in the model legume Medicago truncatula, we screened a Tnt1 retrotransposon insertion mutant population for plants with altered patterns of lignin autofluorescence. From more than 9000 R1 plants screened, four independent lines were identified with a total lack of lignin in the interfascicular region. The mutants also showed loss of lignin in phloem fibers, reduced lignin in vascular elements, failure in anther dehiscence and absence of phenolic autofluorescence in stomatal guard cell walls. Microarray and PCR analyses confirmed that the mutations were caused by the insertion of Tnt1 in a gene annotated as encoding a NAM (no apical meristem)-like protein (here designated Medicago truncatula NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1, MtNST1). MtNST1 is the only family member in Medicago, but has three homologs (AtNST1-AtNST3) in Arabidopsis thaliana, which function in different combinations to control cell wall composition in stems and anthers. Loss of MtNST1 function resulted in reduced lignin content, associated with reduced expression of most lignin biosynthetic genes, and a smaller reduction in cell wall polysaccharide content, associated with reduced expression of putative cellulose and hemicellulose biosynthetic genes. Acid pre-treatment and cellulase digestion released significantly more sugars from cell walls of nst1 mutants compared with the wild type. We discuss the implications of these findings for the development of alfalfa (Medicago sativa) as a dedicated bioenergy crop.

Loss of function of cinnamyl alcohol dehydrogenase 1 leads to unconventional lignin and a temperature-sensitive growth defect in Medicago truncatula

There is considerable debate over the capacity of the cell wall polymer lignin to incorporate unnatural monomer units. We have identified Tnt1 retrotransposon insertion mutants of barrel medic (Medicago truncatula) that show reduced lignin autofluorescence under UV microscopy and red coloration in interfascicular fibers. The phenotype is caused by insertion of retrotransposons into a gene annotated as encoding cinnamyl alcohol dehydrogenase, here designated M. truncatula CAD1. NMR analysis indicated that the lignin is derived almost exclusively from coniferaldehyde and sinapaldehyde and is therefore strikingly different from classical lignins, which are derived mainly from coniferyl and sinapyl alcohols. Despite such a major alteration in lignin structure, the plants appear normal under standard conditions in the greenhouse or growth chamber. However, the plants are dwarfed when grown at 30 °C. Glycome profiling revealed an increased extractability of some xylan and pectin epitopes from the cell walls of the cad1-1 mutant but decreased extractability of others, suggesting that aldehyde-dominant lignin significantly alters cell wall structure.

Inhibition of Auxin Transport from the Ovary or from the Apical Shoot Induces Parthenocarpic Fruit-Set in Tomato Mediated by Gibberellins

Fruit-set in tomato (Solanum lycopersicum) depends on gibberellins and auxins (GAs). Here, we show, using the cv MicroTom, that application of N-1-naphthylphthalamic acid (NPA; an inhibitor of auxin transport) to unpollinated ovaries induced parthenocarpic fruit-set, associated with an increase of indole-3-acetic acid (IAA) content, and that this effect was negated by paclobutrazol (an inhibitor of GA biosynthesis). NPA-induced ovaries contained higher content of GA1 (an active GA) and transcripts of GA biosynthetic genes (SlCPS, SlGA20ox1, and -2). Interestingly, application of NPA to pollinated ovaries prevented their growth, potentially due to supraoptimal IAA accumulation. Plant decapitation and inhibition of auxin transport by NPA from the apical shoot also induced parthenocarpic fruit growth of unpollinated ovaries. Application of IAA to the severed stump negated the plant decapitation effect, indicating that the apical shoot prevents unpollinated ovary growth through IAA transport. Parthenocarpic fruit growth induced by plant decapitation was associated with high levels of GA1 and was counteracted by paclobutrazol treatment. Plant decapitation also produced changes in transcript levels of genes encoding enzymes of GA biosynthesis (SlCPS and SlGA20ox1) in the ovary, quite similar to those found in NPA-induced fruits. All these results suggest that auxin can have opposing effects on fruit-set, either inducing (when accumulated in the ovary) or repressing (when transported from the apical shoot) that process, and that GAs act as mediators in both cases. The effect of NPA application and decapitation on fruit-set induction was also observed in MicroTom lines bearing introgressed DWARF and SELF-PRUNING wild-type alleles.

Integrative Analysis of Transgenic Alfalfa (Medicago sativa L.) Suggests New Metabolic Control Mechanisms for Monolignol Biosynthesis

The entanglement of lignin polymers with cellulose and hemicellulose in plant cell walls is a major biological barrier to the economically viable production of biofuels from woody biomass. Recent efforts of reducing this recalcitrance with transgenic techniques have been showing promise for ameliorating or even obviating the need for costly pretreatments that are otherwise required to remove lignin from cellulose and hemicelluloses. At the same time, genetic manipulations of lignin biosynthetic enzymes have sometimes yielded unforeseen consequences on lignin composition, thus raising the question of whether the current understanding of the pathway is indeed correct. To address this question systemically, we developed and applied a novel modeling approach that, instead of analyzing the pathway within a single target context, permits a comprehensive, simultaneous investigation of different datasets in wild type and transgenic plants. Specifically, the proposed approach combines static flux-based analysis with a Monte Carlo simulation in which very many randomly chosen sets of parameter values are evaluated against kinetic models of lignin biosynthesis in different stem internodes of wild type and lignin-modified alfalfa plants. In addition to four new postulates that address the reversibility of some key reactions, the modeling effort led to two novel postulates regarding the control of the lignin biosynthetic pathway. The first posits functionally independent pathways toward the synthesis of different lignin monomers, while the second postulate proposes a novel feedforward regulatory mechanism. Subsequent laboratory experiments have identified the signaling molecule salicylic acid as a potential mediator of the postulated control mechanism. Overall, the results demonstrate that mathematical modeling can be a valuable complement to conventional transgenic approaches and that it can provide biological insights that are otherwise difficult to obtain.

Gibberellin homeostasis in tobacco is regulated by gibberellin metabolism genes with different gibberellin sensitivity

Gibberellins are phytohormones that regulate growth and development of plants. Gibberellin homeostasis is maintained by feedback regulation of gibberellin metabolism genes. To understand this regulation, we manipulated the gibberellin pathway in tobacco and studied its effects on the morphological phenotype, gibberellin levels and the expression of endogenous gibberellin metabolism genes. The overexpression of a gibberellin 3-oxidase (biosynthesis gene) in tobacco (3ox-OE) induced slight variations in phenotype and active GA(1) levels, but we also found an increase in GA(8) levels (GA(1) inactivation product) and a conspicuous induction of gibberellin 2-oxidases (catabolism genes; NtGA2ox3 and -5), suggesting an important role for these particular genes in the control of gibberellin homeostasis. The effect of simultaneous overexpression of two biosynthesis genes, a gibberellin 3-oxidase and a gibberellin 20-oxidase (20ox/3ox-OE), on phenotype and gibberellin content suggests that gibberellin 3-oxidases are non-limiting enzymes in tobacco, even in a 20ox-OE background. Moreover, the expression analysis of gibberellin metabolism genes in transgenic plants (3ox-OE, 20ox-OE and hybrid 3ox/20ox-OE), and in response to application of different GA(1) concentrations, showed genes with different gibberellin sensitivity. Gibberellin biosynthesis genes (NtGA20ox1 and NtGA3ox1) are negatively feedback regulated mainly by high gibberellin levels. In contrast, gibberellin catabolism genes which are subject to positive feedback regulation are sensitive to high (NtGA2ox1) or to low (NtGA2ox3 and -5) gibberellin concentrations. These two last GA2ox genes seem to play a predominant role in gibberellin homeostasis under mild gibberellin variations, but not under large gibberellin changes, where the biosynthesis genes GA20ox and GA3ox may be more important.

Lignin Modification Leads to Increased Nodule Numbers in Alfalfa

Reducing lignin content in stems and roots of alfalfa results in an increased nodule number phenotype. Reduction of lignin levels in the forage legume alfalfa (Medicago sativa) by down-regulation of the monolignol biosynthetic enzyme hydroxycinnamoyl coenzyme A:shikimate hydroxycinnamoyl transferase (HCT) results in strongly increased digestibility and processing ability of lignocellulose. However, these modifications are often also associated with dwarfing and other changes in plant growth. Given the importance of nitrogen fixation for legume growth, we evaluated the impact of constitutively targeted lignin modification on the belowground organs (roots and nodules) of alfalfa plants. HCT down-regulated alfalfa plants exhibit a striking reduction in root growth accompanied by an unexpected increase in nodule numbers when grown in the greenhouse or in the field. This phenotype is associated with increased levels of gibberellins and certain flavonoid compounds in roots. Although HCT down-regulation reduced biomass yields in both the greenhouse and field experiments, the impact on the allocation of nitrogen to shoots or roots was minimal. It is unlikely, therefore, that the altered growth phenotype of reduced-lignin alfalfa is a direct result of changes in nodulation or nitrogen fixation efficiency. Furthermore, HCT down-regulation has no measurable effect on carbon allocation to roots in either greenhouse or 3-year field trials.