Rebecca A. Smith

Xylem tissue specification, patterning, and differentiation mechanisms

Vascular plants (Tracheophytes) have adapted to a variety of environments ranging from arid deserts to tropical rainforests, and now comprise >250,000 species. While they differ widely in appearance and growth habit, all of them share a similar specialized tissue system (vascular tissue) for transporting water and nutrients throughout the organism. Plant vascular systems connect all plant organs from the shoot to the root, and are comprised of two main tissue types, xylem and phloem. In this review we examine the current state of knowledge concerning the process of vascular tissue formation, and highlight important mechanisms underlying key steps in vascular cell type specification, xylem and phloem tissue patterning, and, finally, the differentiation and maturation of specific xylem cell types.

Laccases Direct Lignification in the Discrete Secondary Cell Wall Domains of Protoxylem

The discrete localization of laccases to secondary cell walls directs lignification in protoxylem. Plants precisely control lignin deposition in spiral or annular secondary cell wall domains during protoxylem tracheary element (TE) development. Because protoxylem TEs function to transport water within rapidly elongating tissues, it is important that lignin deposition is restricted to the secondary cell walls in order to preserve the plasticity of adjacent primary wall domains. The Arabidopsis (Arabidopsis thaliana) inducible VASCULAR NAC DOMAIN7 (VND7) protoxylem TE differentiation system permits the use of mutant backgrounds, fluorescent protein tagging, and high-resolution live-cell imaging of xylem cells during secondary cell wall development. Enzymes synthesizing monolignols, as well as putative monolignol transporters, showed a uniform distribution during protoxylem TE differentiation. By contrast, the oxidative enzymes LACCASE4 (LAC4) and LAC17 were spatially localized to secondary cell walls throughout protoxylem TE differentiation. These data support the hypothesis that precise delivery of oxidative enzymes determines the pattern of cell wall lignification. This view was supported by lac4lac17 mutant analysis demonstrating that laccases are necessary for protoxylem TE lignification. Overexpression studies showed that laccases are sufficient to catalyze ectopic lignin polymerization in primary cell walls when exogenous monolignols are supplied. Our data support a model of protoxylem TE lignification in which monolignols are highly mobile once exported to the cell wall, and in which precise targeting of laccases to secondary cell wall domains directs lignin deposition.

Neighboring Parenchyma Cells Contribute to Arabidopsis Xylem Lignification, while Lignification of Interfascicular Fibers Is Cell Autonomous

Lignification provides strength to cells with secondary cell walls, such as fibers and water-conducting tracheary elements. In this study, the timing of lignification was examined using autoradiography, and targeted knockdown of lignin biosynthesis indicated that fibers undergo cell autonomous lignifications, while neighboring nonlignified cells can contribute to xylem cell lignification. Lignin is a critical structural component of plants, providing vascular integrity and mechanical strength. Lignin precursors (monolignols) must be exported to the extracellular matrix where random oxidative coupling produces a complex lignin polymer. The objectives of this study were twofold: to determine the timing of lignification with respect to programmed cell death and to test if nonlignifying xylary parenchyma cells can contribute to the lignification of tracheary elements and fibers. This study demonstrates that lignin deposition is not exclusively a postmortem event, but also occurs prior to programmed cell death. Radiolabeled monolignols were not detected in the cytoplasm or vacuoles of tracheary elements or neighbors. To experimentally define which cells in lignifying tissues contribute to lignification in intact plants, a microRNA against CINNAMOYL CoA-REDUCTASE1 driven by the promoter from CELLULOSE SYNTHASE7 (ProCESA7:miRNA CCR1) was used to silence monolignol biosynthesis specifically in cells developing lignified secondary cell walls. When monolignol biosynthesis in ProCESA7:miRNA CCR1 lines was silenced in the lignifying cells themselves, but not in the neighboring cells, lignin was still deposited in the xylem secondary cell walls. Surprisingly, a dramatic reduction in cell wall lignification of extraxylary fiber cells demonstrates that extraxylary fibers undergo cell autonomous lignification.

High-Resolution Mapping of a Fruit Firmness-Related Quantitative Trait Locus in Tomato Reveals Epistatic Interactions Associated with a Complex Combinatorial Locus

Fruit firmness in tomato (Solanum lycopersicum) is determined by a number of factors including cell wall structure, turgor, and cuticle properties. Firmness is a complex polygenic trait involving the coregulation of many genes and has proved especially challenging to unravel. In this study, a quantitative trait locus (QTL) for fruit firmness was mapped to tomato chromosome 2 using the Zamir Solanum pennellii interspecific introgression lines (ILs) and fine-mapped in a population consisting of 7,500 F2 and F3 lines from IL 2-3 and IL 2-4. This firmness QTL contained five distinct subpeaks, Firs.p.QTL2.1 to Firs.p.QTL2.5, and an effect on a distal region of IL 2-4 that was nonoverlapping with IL 2-3. All these effects were located within an 8.6-Mb region. Using genetic markers, each subpeak within this combinatorial locus was mapped to a physical location within the genome, and an ethylene response factor (ERF) underlying Firs.p.QTL2.2 and a region containing three pectin methylesterase (PME) genes underlying Firs.p.QTL2.5 were nominated as QTL candidate genes. Statistical models used to explain the observed variability between lines indicated that these candidates and the nonoverlapping portion of IL 2-4 were sufficient to account for the majority of the fruit firmness effects. Quantitative reverse transcription-polymerase chain reaction was used to quantify the expression of each candidate gene. ERF showed increased expression associated with soft fruit texture in the mapping population. In contrast, PME expression was tightly linked with firm fruit texture. Analysis of a range of recombinant lines revealed evidence for an epistatic interaction that was associated with this combinatorial locus.

Monolignol ferulate conjugates are naturally incorporated into plant lignins

Plants have convergently evolved to use monolignol ferulate conjugates to produce lignins containing chemically labile backbone esters. Angiosperms represent most of the terrestrial plants and are the primary research focus for the conversion of biomass to liquid fuels and coproducts. Lignin limits our access to fibers and represents a large fraction of the chemical energy stored in plant cell walls. Recently, the incorporation of monolignol ferulates into lignin polymers was accomplished via the engineering of an exotic transferase into commercially relevant poplar. We report that various angiosperm species might have convergently evolved to natively produce lignins that incorporate monolignol ferulate conjugates. We show that this activity may be accomplished by a BAHD feruloyl–coenzyme A monolignol transferase, OsFMT1 (AT5), in rice and its orthologs in other monocots.

Engineering monolignol p-coumarate conjugates into Poplar and Arabidopsis lignins

Novel monolignol p-coumarate conjugates in eudicot lignin result from the introduction of a monocot acyltransferase. Lignin acylation, the decoration of hydroxyls on lignin structural units with acyl groups, is common in many plant species. Monocot lignins are decorated with p-coumarates by the polymerization of monolignol p-coumarate conjugates. The acyltransferase involved in the formation of these conjugates has been identified in a number of model monocot species, but the effect of monolignol p-coumarate conjugates on lignification and plant growth and development has not yet been examined in plants that do not inherently possess p-coumarates on their lignins. The rice (Oryza sativa) p-COUMAROYL-Coenzyme A MONOLIGNOL TRANSFERASE gene was introduced into two eudicots, Arabidopsis (Arabidopsis thaliana) and poplar (Populus alba × grandidentata), and a series of analytical methods was used to show the incorporation of the ensuing monolignol p-coumarate conjugates into the lignin of these plants. In poplar, specifically, the addition of these conjugates did not occur at the expense of the naturally incorporated monolignol p-hydroxybenzoates. Plants expressing the p-COUMAROYL-Coenzyme A MONOLIGNOL TRANSFERASE transgene can therefore produce monolignol p-coumarate conjugates essentially without competing with the formation of other acylated monolignols and without drastically impacting normal monolignol production.

Silencing CHALCONE SYNTHASE in Maize Impedes the Incorporation of Tricin into Lignin and Increases Lignin Content

The maize C2-Idf mutant has reduced levels of soluble flavonoids, reduced incorporation of tricin into the lignin polymer, and higher total lignin that is enriched in β-β and β-5 interunit linkages. Lignin is a phenolic heteropolymer that is deposited in secondary-thickened cell walls, where it provides mechanical strength. A recent structural characterization of cell walls from monocot species showed that the flavone tricin is part of the native lignin polymer, where it is hypothesized to initiate lignin chains. In this study, we investigated the consequences of altered tricin levels on lignin structure and cell wall recalcitrance by phenolic profiling, nuclear magnetic resonance, and saccharification assays of the naturally silenced maize (Zea mays) C2-Idf (inhibitor diffuse) mutant, defective in the CHALCONE SYNTHASE Colorless2 (C2) gene. We show that the C2-Idf mutant produces highly reduced levels of apigenin- and tricin-related flavonoids, resulting in a strongly reduced incorporation of tricin into the lignin polymer. Moreover, the lignin was enriched in β-β and β-5 units, lending support to the contention that tricin acts to initiate lignin chains and that, in the absence of tricin, more monolignol dimerization reactions occur. In addition, the C2-Idf mutation resulted in strikingly higher Klason lignin levels in the leaves. As a consequence, the leaves of C2-Idf mutants had significantly reduced saccharification efficiencies compared with those of control plants. These findings are instructive for lignin engineering strategies to improve biomass processing and biochemical production.

Defining the Diverse Cell Populations Contributing to Lignification in Arabidopsis thaliana Stems

Xylem vessel lignification in young Arabidopsis stems requires neighboring xylary parenchyma, but, as stems mature, fibers primarily produce their own lignin monomers. Many land plants evolved tall and sturdy growth habits due to specialized cells with thick lignified cell walls: tracheary elements that function in water transport and fibers that function in structural support. The objective of this study was to define how and when diverse cell populations contribute lignin precursors, monolignols, to secondary cell walls during lignification of the Arabidopsis (Arabidopsis thaliana) inflorescence stem. Previous work demonstrated that, when lignin biosynthesis is suppressed in fiber and tracheary element cells with thickened walls, fibers become lignin-depleted while vascular bundles still lignify, suggesting that nonlignifying neighboring xylem cells are contributing to lignification. In this work, we dissect the contributions of different cell types, specifically xylary parenchyma and fiber cells, to lignification of the stem using cell-type-specific promoters to either knock down an essential monolignol biosynthetic gene or to introduce novel monolignol conjugates. Analysis of either reductions in lignin in knockdown lines, or the addition of novel monolignol conjugates, directly identifies the xylary parenchyma and fiber cell populations that contribute to the stem lignification and the developmental timing at which each contribution is most important.

Highly Decorated Lignins in Leaf Tissues of the Canary Island Date Palm Phoenix canariensis

Phoenix canariensis leaf lignins vary between tissue region and contain an array of pendent groups. The cell walls of leaf base tissues of the Canary Island date palm (Phoenix canariensis) contain lignins with the most complex compositions described to date. The lignin composition varies by tissue region and is derived from traditional monolignols (ML) along with an unprecedented range of ML conjugates: ML-acetate, ML-benzoate, ML-p-hydroxybenzoate, ML-vanillate, ML-p-coumarate, and ML-ferulate. The specific functions of such complex lignin compositions are unknown. However, the distribution of the ML conjugates varies depending on the tissue region, indicating that they may play specific roles in the cell walls of these tissues and/or in the plant’s defense system.

Suppression of CINNAMOYL-CoA REDUCTASE increases the level of monolignol ferulates incorporated into maize lignins

BackgroundThe cell wall polymer lignin provides structural support and rigidity to plant cell walls, and therefore to the plant body. However, the recalcitrance associated with lignin impedes the extraction of polysaccharides from the cell wall to make plant-based biofuels and biomaterials. The cell wall digestibility can be improved by introducing labile ester bonds into the lignin backbone that can be easily broken under mild base treatment at room temperature. The FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) enzyme, which may be naturally found in many plants, uses feruloyl-CoA and monolignols to synthesize the ester-linked monolignol ferulate conjugates. A mutation in the first lignin-specific biosynthetic enzyme, CINNAMOYL-CoA REDUCTASE (CCR), results in an increase in the intracellular pool of feruloyl-CoA.ResultsMaize (Zea mays) has a native putative FMT enzyme, and its ccr mutants produce an increased pool of feruloyl-CoA that can be used for conversion to monolignol ferulate conjugates. The decreased lignin content and monomers did not, however, impact the plant growth or biomass. The increase in monolignol conjugates correlated with an improvement in the digestibility of maize stem rind tissue.ConclusionsTogether, increased monolignol ferulates and improved digestibility in ccr1 mutant plants suggests that they may be superior biofuel crops.