Chemical genetics uncovers novel inhibitors of lignification, including p-iodobenzoic acid targeting CINNAMATE-4-HYDROXYLASE

Plant secondary-thickened cell walls are characterized by the presence of lignin, a recalcitrant and hydrophobic polymer that provides mechanical strength and ensures long-distance water transport. Exactly the recalcitrance and hydrophobicity of lignin put a burden on the industrial processing efficiency of lignocellulosic biomass. Both forward and reverse genetic strategies have been used intensively to unravel the molecular mechanism of lignin deposition. As an alternative strategy, we introduce here a forward chemical genetic approach to find candidate inhibitors of lignification. A high-throughput assay to assess lignification in Arabidopsis (Arabidopsis thaliana) seedlings was developed and used to screen a 10-k library of structurally diverse, synthetic molecules. Of the 73 compounds that reduced lignin deposition, 39 that had a major impact were retained and classified into five clusters based on the shift they induced in the phenolic profile of Arabidopsis seedlings. One representative compound of each cluster was selected for further lignin-specific assays, leading to the identification of an aromatic compound that is processed in the plant into two fragments, both having inhibitory activity against lignification. One fragment, p-iodobenzoic acid, was further characterized as a new inhibitor of CINNAMATE 4-HYDROXYLASE, a key enzyme of the phenylpropanoid pathway synthesizing the building blocks of the lignin polymer. As such, we provide proof of concept of this chemical biology approach to screen for inhibitors of lignification and present a broad array of putative inhibitors of lignin deposition for further characterization

Maize tricin-oligolignol metabolites and their implications for monocot lignification

Lignin is an abundant aromatic plant cell wall polymer consisting of phenylpropanoid units in which the aromatic rings display various degrees of methoxylation. Tricin [5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4H-chromen-4-one], a flavone, was recently established as a true monomer in grass lignins. To elucidate the incorporation pathways of tricin into grass lignin, the metabolites of maize (Zea mays) were extracted from lignifying tissues and profiled using the recently developed 'candidate substrate product pair' algorithm applied to ultra-high-performance liquid chromatography and Fourier transform-ion cyclotron resonance-mass spectrometry. Twelve tricin-containing products (each with up to eight isomers), including those derived from the various monolignol acetate and p-coumarate conjugates, were observed and authenticated by comparisons with a set of synthetic tricin-oligolignol dimeric and trimeric compounds. The identification of such compounds helps establish that tricin is an important monomer in the lignification of monocots, acting as a nucleation site for starting lignin chains. The array of tricin-containing products provides further evidence for the combinatorial coupling model of general lignification and supports evolving paradigms for the unique nature of lignification in monocots

Role of gravitational stress in land plant evolution - The gravitational factor in lignification Semiannual report

Gravitational effects on lignification in plant

Studies on lignification in wheat (Triticum aestivum var. Thatcher) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Botany at Massey University

PART I Transections of the stem of Triticum were examined after staining with dyes specific for functional groups within the lignin polymer. Anatomical observations suggest that the basis for the rapid increase in the lignin content of this plant 35 to 40 days after germination, is the differentiation of subepidermal sclerenchyma fibres in the stem at this time. The lignin formed in the fibre walls appears to have a higher methoxyl content than the lignin of the xylem vessels. A comparison of the development of lignification with stem elongation and flowering was made and the interrelationship of these processes discussed. PART II The role of p-hydroxyphenyllactic acid in lignification in wheat was investigated. ¹⁴C-labelled tyrosine, p-hydroxyphenyllactic acid (HPLA), and ³H-labelled HPLA were administered separately to the cut ends of shoots of Triticum and the incorporation of label into ethanol-soluble and ethanol-insoluble ferulic (and in some cases only, p-hydroxycinnamic) acid was measured. On the basis of the pattern of incorporation of label from the ¹⁴C-tyrosine, experiments were carried out to determine the route by which HPLA is converted to lignin precursors. A failure to detect label from ³H-HPLA in the cinnamic acids suggests that HPLA is not dehydrated directly to p-hydroxycinnamic acid and is not of regulatory significance in lignification in either 10 or 40 day-old wheat plants. PART III Information from several levels of organization within the plant is drawn together and discussed. Suggestions for further work investigating the controlling factors in lignification are included

Molecular factors involved in the formation of secondary vascular tissues and lignification in higher plants

The formation of secondary vascular tissues involves complex processes and many steps, a number of which have been examined in detail in this study. A novel CuZn-SOD, with a high pI and thus denoted hipI-SOD, was identified and characterized in Pinus sylvestris. Results from immunolocalisation analyses indicated that it is localised in lignified structures, suggesting that SOD might participate in the formation of secondary cell walls and lignification. To further investigate its role in these processes, a Zinnia mesophyll cell system was set up. This enabled us to follow the differentiation from mesophyll cell to tracheary element. Various inhibitors against SOD and H2O2-production were applied. The results suggested that HipI-SOD might have a novel and important function in secondary cell wall formation and lignification processes. The expression pattern and localization of the protein during formation of tracheary elements support this assumption. The other part of this study involved analysis of transcription factors and their regulation, especially in secondary vascular tissues. The genes encoding three MYB-transcription factors and one novel Zinc-finger transcription factor were found in an EST-library from the cambial region of hybrid aspen (Populus tremula L. x tremuloides Michx.). The genes were cloned and characterized and their regulation by hormones, sucrose and gravity was investigated. The genes were found to be under hormone and sucrose control, and their expression altered during tension wood formation. Transgenic plants were constructed, carrying one of two antisense constructs of MYB-genes, PttMYB46 or PttMYB76, which were strongly expressed in lignified tissues. Analysis of plants with either of these constructs displayed a complex phenotype, including reduced growth, increased concentration of some phenolic acids and changes in lignin composition. Some of the phenotypic traits were indicative of strong investment in defensive characters

Progeny from the crosses of two antisense potato plants exhibit ectopic xylem differentiation

Progeny from the crosses of two transgenic potato lines csr2-1 and csr4-8, containing two different antisense constructs, csr2 and csr4 had been previously characterized to exhibit altered tuber production. Histochemical staining and microscopic examinations of the tubers were made to investigate cellular phenotype in the tubers. We observed ectopic proliferation of xylem, which is most pronounced in the csr2 tubers. Light microscopy of csr2 tubers revealed that the proliferation of xylem was associated with lignification of their cell walls. This paper provides evidence of cellular phenotype as a consequence of the presence of the antisense construct

Role of gravitational stress in land plant evolution - The gravitational factor in lignification Semiannual status report, period ending 31 Oct. 1968

Gravitational and mechanical stress effects on lignification in land plant

Ecology and structure of Drosophyllum lusitanicum (l.) Links populations in the south-west of the Iberian Peninsula

Ecología y estructura de poblaciones de Drosophyllum lusitanicum (L.) Link en el suroeste de la Península Ibérica. Drosophyllum lusitanicum es una planta autógama y nautocórica cuyo tamaño y estructura de población han sido estudiados en dos áreas del suroeste de la Península Ibérica. Su carácter cenológico se documenta mediante una tabla fitosociológica sintética basada en inventarios ya publicados y datos nuevos. En la Sierra de Monchique (Portugal) se cartografían 21 poblaciones cuyo tamaño medio es de 165 individuos. Sin embargo, en el Campo de Gibraltar (España), se encontró la población de mayor tamaño, con varios miles de individuos en la Sierra del Aljibe. Aquí se estudió el tamaño de las plantas, el diámetro de la roseta basal y el grado de lignificación de 700 individuos, lo que se utilizó como indicador indirecto de la estructura de edades de 19 poblaciones.Drosophyllum lusitanicum tiene un cierto carácter pionero. Se presenta mayoritariamente en los brezales aclarados de Ericion umbellatae (Stauracantho-Drosophylletum, Querco lusitanicae-Stauracanthetum, Genisto tridentis-Stauracanthetum, etc.). Es capaz de colonizar medios alterados, como cunetas de carreteras y cortafuegos, y es un apófito en las comunidades basales de Drosophyllo-[Stauracanthenion], siendo además esta especie favorecida por los frecuentes incendios de la zona. En los alcornocales abiertos de Myrto-Quercetum suberis y en los brezales densos de Erica australis predominan los individuos grandes, siendo éstas poblaciones maduras. Finalmente, se aportan algunos datos poco conocidos de su reproducción y ecología que indican la necesidad de un mayor esfuerzo para la conservación del área portuguesa estudiada

Lignin engineering in forest trees

Wood is a renewable resource that is mainly composed of lignin and cell wall polysaccharides. The polysaccharide fraction is valuable as it can be converted into pulp and paper, or into fermentable sugars. On the other hand, the lignin fraction is increasingly being considered a valuable source of aromatic building blocks for the chemical industry. The presence of lignin in wood is one of the major recalcitrance factors in woody biomass processing, necessitating the need for harsh chemical treatments to degrade and extract it prior to the valorization of the cell wall polysaccharides, cellulose and hemicellulose. Over the past years, large research efforts have been devoted to engineering lignin amount and composition to reduce biomass recalcitrance toward chemical processing. We review the efforts made in forest trees, and compare results from greenhouse and field trials. Furthermore, we address the value and potential of CRISPR-based gene editing in lignin engineering and its integration in tree breeding programs

Geochemical support for a climbing habit within the Paleozoic seed fern genus Medullosa

A long-standing problem in paleobotany is the accurate identification of the growth habits and statures of fossil plants. Tissue-specific analysis of stable carbon isotope ratios in plant fossils can provide an independent perspective on this issue. Lignin, a fundamental biopolymer providing structural support in plant tissues and the second most abundant organic material in plants, is ^(13)C depleted by several parts per thousand, averaging 4.1‰, relative to other plant constructional materials (e.g. cellulose). With this isotopic difference, the biochemical structural composition of ancient plants (and inferred stature) can be interrogated using microscale in situ isotope analysis between different tissues in fossils. We applied this technique to a well-preserved specimen of the Late Paleozoic seed plant Medullosa, an extinct genus with a variety of growth habits that includes several enigmatic yet abundant small-stemmed species widely found in calcium carbonate concretions (“coal balls”) in the Pennsylvanian coal beds of Iowa, USA. It remains unclear which of the medullosans were freestanding, and recent analysis of the medullosan vascular system has shown that this system provided little structural support to the whole plant. The leading hypothesis for small-stemmed medullosan specimens predicts that cortical tissues could have provided additional structural support, but only if they were lignified. The expected isotopic difference between lignified tissue and unlignified tissue is smaller than that expected from pure extracts, for the simple reason that even woody tissues maximally contain 40% lignin (by mass). This reduces the expected maximum difference between weakly and heavily lignified tissues by 60%, down to ~0.5‰–2‰. Analysis of the medullosan stem reveals a consistent difference in isotope ratios of 0.7‰–1.0‰ between lignified xylem and cortical tissues. This implies low abundances of lignin (between 0% and 11%) within the cortex. This inferred structural biochemistry supports hypotheses that the peripheral portions of these medullosan stems were not biomechanically reinforced to permit the plants to grow as freestanding, arborescent trees. A number of climbing or scandent medullosans have been identified in the fossil record, and this mode of growth has been suggested to be common within the group on the basis of observations from comparative biomechanics, hydraulics, and development. Finally, this mode of growth is common in several clades of stem group seed plants, including Lyginopteris and Callistophyton, along with Medullosa. This study provides further support for ideas that place a great portion of early seed plant diversity under the canopy, rather than forming it