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Lignin, the second most abundant biopolymer, is a promising renewable energy source and chemical feedstock. A key element of lignin biosynthesis is unknown: how do lignin precursors (monolignols) get from inside the cell out to the cell wall where they are polymerized? Modeling indicates that monolignols can passively diffuse through lipid bilayers, but this has not been tested experimentally. We demonstrate significant monolignol diffusion occurs when laccases, which consume monolignols, are present on one side of the membrane. We hypothesize that lignin polymerization could deplete monomers in the wall, creating a concentration gradient driving monolignol diffusion. We developed a two-photon microscopy approach to visualize lignifying Arabidopsis thaliana root cells. Laccase mutants with reduced ability to form lignin polymer in the wall accumulated monolignols inside cells. In contrast, active transport inhibitors did not decrease lignin in the wall and scant intracellular phenolics were observed. Synthetic liposomes were engineered to encapsulate laccases, and monolignols crossed these pure lipid bilayers to form polymer within. A sink-driven diffusion mechanism explains why it has been difficult to identify genes encoding monolignol transporters and why the export of varied phenylpropanoids occurs without specificity. It also highlights an important role for cell wall oxidative enzymes in monolignol export.

Two isolates of Turnip mosaic virus (UK 1 and JPN 1), representative of two different viral strains, induced differential alterations on secondary cell wall (SCW) development in Arabidopsis thaliana, suggesting cell-type specific effects of these viral infections. These potential effects were analyzed in inflorescence stems and flowers of infected plants, together with other possible cellular effects of the infections. Results obtained from macroscopic and histochemical analyses showed that infection with either virus significantly narrowed stem area, but defects in SCW were only found in JPN 1 infections. In flowers, reduced endothecium lignification was also found for JPN 1, while UK 1 infections induced severe floral cell and organ development alterations. A transcriptomic analysis focused on genes controlling and regulating SCW formation also showed notable differences between both viral isolates. UK 1 infections induced a general transcriptional decrease of most regulatory genes, whereas a more complex pattern of alterations was found in JPN 1 infections. The role of the previously identified viral determinant of most developmental alterations, the P3 protein, was also studied through the use of viral chimeras. No SCW alterations or creeping habit growth were found in infections by the chimeras, indicating that if the P3 viral protein is involved in the determination of these symptoms, it is not the only determinant. Finally, considerations as to the possibility of a taxonomical reappraisal of these TuMV viral strains are provided.

The final step of lignin biosynthesis is the polymerization of monolignols in apoplastic cell wall domains. In this process, monolignols secreted by lignifying cells, or occasionally neighboring non-lignifying and/or other lignifying cells, are activated by cell-wall-localized oxidation systems, such as laccase/O2 and/or peroxidase/H2O2, for combinatorial radical coupling to make the final lignin polymers. Plants can precisely control when, where, and which types of lignin polymers are assembled at tissue and cellular levels, but do not control the polymers’ exact chemical structures per se. Recent studies have begun to identify specific laccase and peroxidase proteins responsible for lignin polymerization in specific cell types and during different developmental stages. The coordination of polymerization machinery localization and monolignol supply is likely critical for the spatio-temporal patterning of lignin polymerization. Further advancement in this research area will continue to increase our capacity to manipulate lignin content/structure in biomass to meet our own biotechnological purposes.

International audience; Lignin is a key secondary cell wall chemical constituent, and is both a barrier to biomass utilization and a potential source of bioproducts. The Arabidopsis transcription factors MYB58 and MYB63 have been shown to upregulate gene expression of the general phenylpropanoid and monolignol biosynthetic pathways. The overexpression of these genes also results in dwarfism. The vascular integrity, soluble phenolic profiles, cell wall lignin, and transcriptomes associated with these MYB-overexpressing lines were characterized. Plants with high expression of MYB58 and MYB63 had increased ectopic lignin and the xylem vessels were regular and open, suggesting that the stunted growth is not associated with loss of vascular conductivity. MYB58 and MYB63 overexpression lines had characteristic soluble phenolic profiles with large amounts of monolignol glucosides and sinapoyl esters, but decreased flavonoids. Because loss of function lac4 lac17 mutants also accumulate monolignol glucosides, we hypothesized that LACCASE overexpression might decrease monolignol glucoside levels in the MYB-overexpressing plant lines. When laccases related to lignification (LAC4 or LAC17) were co-overexpressed with MYB63 or MYB58, the dwarf phenotype was rescued. Moreover, the overexpression of either LAC4 or LAC17 led to wild-type monolignol glucoside levels, as well as wild-type lignin levels in the rescued plants. Transcriptomes of the rescued double MYB63- OX/LAC17-OX overexpression lines showed elevated, but attenuated, expression of the MYB63 gene itself and the direct transcriptional targets of MYB63. Contrasting the dwarfism from overabundant monolignol production with dwarfism from lignin mutants provides insight into some of the proposed mechanisms of lignin modification-induced dwarfism.

Lignin, a critical phenolic polymer in secondary cell walls of plant cells, enables strength in fibers and water transportation in xylem vessel elements. Secreted enzymes, namely laccases (LACs) and peroxidases (PRXs), facilitate lignin polymerization by oxidizing lignin monomers (monolignols). Previous work in Arabidopsis (Arabidopsis thaliana) demonstrated that AtLAC4 and AtPRX64 localized to discrete lignified cell wall domains in fibers, although the spatial distributions of other enzymes in these large gene families are unknown. Here, we show that characteristic sets of putative lignin-associated LACs and PRXs localize to precise regions during stem development, with LACs and PRXs co-occurring in cell wall domains. AtLAC4, AtLAC17, and AtPRX72 localized to the thick secondary cell wall of xylem vessel elements and fibers, whereas AtLAC4, AtPRX64, and AtPRX71 localized to fiber cell corners. Interestingly, AtLAC4 had a transient cell corner localization early in fiber development that disappeared in the mature stem. In contrast with these secondary cell wall localizations, AtLAC10, AtPRX42, AtPRX52, and AtPRX71 were found in nonlignified tissues. Despite ubiquitous PRX occurrence in cell walls, PRX oxidative activity was restricted to lignifying regions during development, which suggested regulated production of apoplastic hydrogen peroxide. Relative amounts of apoplastic reactive oxygen species differed between lignified cell types, which could modulate PRX activity. Together, these results indicate that precise localization of oxidative enzymes and differential distribution of oxidative substrates, such as hydrogen peroxide, provide mechanisms to control spatiotemporal deposition of lignin during development.

The secondary cell wall (SCW) of xylem vessel cells provides rigidity and strength that enables efficient water conduction throughout the plant. To gain insight into SCW deposition, we mutagenized Arabidopsis thaliana VASCULAR-RELATED NAC-DOMAIN7-inducible plant lines, in which ectopic protoxylem vessel cell differentiation is synchronously induced. The baculites mutant was isolated based on the absence of helical SCW patterns in ectopically-induced protoxylem vessel cells, and mature baculites plants exhibited an irregular xylem (irx) mutant phenotype in mature plants. A single nucleic acid substitution in the CELLULOSE SYNTHASE SUBUNIT 7 (CESA7) gene in baculites was identified: while the mutation was predicted to produce a C-terminal truncated protein, immunoblot analysis revealed that cesa7bac mutation results in loss of production of CESA7 proteins, indicating that baculites is a novel cesa7 loss-of-function mutant. In cesa7bac, despite a lack of patterned cellulose deposition, the helically-patterned deposition of other SCW components, such as the hemicellulose xylan and the phenolic polymer lignin, was not affected. Similar phenotypes were found in another point mutation mutant cesa7mur10-2, and an established knock-out mutant, cesa7irx3-4. Taken together, we propose that the spatio-temporal deposition of different SCW components, such as xylan and lignin, is not dependent on cellulose patterning.

International audience; BackgroundDedicated lignocellulosic feedstock from grass crops for biofuel production is extensively increasing. However, the access to fermentable cell wall sugars by carbohydrate degrading enzymes is impeded by lignins. These complex polymers are made from reactive oxidized monolignols in the cell wall. Little is known about the laccase-mediated oxidation of monolignols in grasses, and inactivation of the monolignol polymerization mechanism might be a strategy to increase the yield of fermentable sugars.ResultsLACCASE5 and LACCASE8 are inactivated in a Brachypodium double mutant. Relative to the wild type, the lignin content of extract-free mature culms is decreased by 20-30% and the saccharification yield is increased by 140%. Release of ferulic acid by mild alkaline hydrolysis is also 2.5-fold higher. Interfascicular fibers are mainly affected while integrity of vascular bundles is not impaired. Interestingly, there is no drastic impact of the double mutation on plant growth.ConclusionThis work shows that two Brachypodium laccases with clearly identified orthologs in crops are involved in lignification of this model plant. Lignification in interfascicular fibers and metaxylem cells is partly uncoupled in Brachypodium. Orthologs of these laccases are promising targets for improving grass feedstock for cellulosic biofuel production.

Based on mutant phenotypes the MONOPTEROS (MP)/Auxin Response Factor 5 (ARF5) gene acts in several developmental processes including leaf vein development. Since overlapping functions among ARF genes are common, we assessed the related ARF 3-8 and 19 genes for potential overlap in expression during vein development using in-situ hybridization. Like MP/ARF5, ARF3 was expressed in preprocambial and procambial cells. ARF7 was also expressed in procambial cells, close to and during vein differentiation. ARF19 was expressed in differentiating vessel elements. To assess if genes with vein expression have overlapping functions, double mutants were generated. While arf3, 5 and 7 mutants formed leaves normally, double mutant combinations of mp/arf5 with arf3 or arf7 resulted in a breakdown of leaf formation. Instead, novel structures not present in any of the single mutants formed. The results implicate ARF3 and ARF7 in rosette leaf formation and suggest that their functions overlap and act in parallel with MP/ARF5 in this process. The observed vascular expression patterns suggest unique functions (ARF7 and 19) and potentially overlapping functions (ARF3 and 5) in vein development. Since arf3 arf5 double mutants do not form leaves, assessment of their potential combined action in vein development will require the use of conditional mutants.

Background Dedicated lignocellulosic feedstock from grass crops for biofuel production is extensively increasing. However, the access to fermentable cell wall sugars by carbohydrate degrading enzymes is impeded by lignins. These complex polymers are made from reactive oxidized monolignols in the cell wall. Little is known about the laccase-mediated oxidation of monolignols in grasses, and inactivation of the monolignol polymerization mechanism might be a strategy to increase the yield of fermentable sugars. Results LACCASE5 and LACCASE8 are inactivated in a Brachypodium double mutant. Relative to the wild type, the lignin content of extract-free mature culms is decreased by 20–30% and the saccharification yield is increased by 140%. Release of ferulic acid by mild alkaline hydrolysis is also 2.5-fold higher. Interfascicular fibers are mainly affected while integrity of vascular bundles is not impaired. Interestingly, there is no drastic impact of the double mutation on plant growth. Conclusion This work shows that two Brachypodium laccases with clearly identified orthologs in crops are involved in lignification of this model plant. Lignification in interfascicular fibers and metaxylem cells is partly uncoupled in Brachypodium. Orthologs of these laccases are promising targets for improving grass feedstock for cellulosic biofuel production. Electronic supplementary material The online version of this article (10.1186/s13068-019-1525-5) contains supplementary material, which is available to authorized users.

Laccases and peroxidases localize to different wall domains in Arabidopsis stems. These enzymes are tightly anchored in the secondary cell wall, providing a mechanism for spatial control of lignification., Lignin is an important phenolic biopolymer that provides strength and rigidity to the secondary cell walls of tracheary elements, sclereids, and fibers in vascular plants. Lignin precursors, called monolignols, are synthesized in the cell and exported to the cell wall where they are polymerized into lignin by oxidative enzymes such as laccases and peroxidases. In Arabidopsis thaliana, a peroxidase (PRX64) and laccase (LAC4) are shown to localize differently within cell wall domains in interfascicular fibers: PRX64 localizes to the middle lamella whereas LAC4 localizes throughout the secondary cell wall layers. Similarly, laccases localized to, and are responsible for, the helical depositions of lignin in protoxylem tracheary elements. In addition, we tested the mobility of laccases in the cell wall using fluorescence recovery after photobleaching. mCHERRY-tagged LAC4 was immobile in secondary cell wall domains, but mobile in the primary cell wall when ectopically expressed. A small secreted red fluorescent protein (sec-mCHERRY) was engineered as a control and was found to be mobile in both the primary and secondary cell walls. Unlike sec-mCHERRY, the tight anchoring of LAC4 to secondary cell wall domains indicated that it cannot be remobilized once secreted, and this anchoring underlies the spatial control of lignification.

Conifers possess an array of physical and chemical defences against stem-boring insects. Stone cells provide a physical defence associated with resistance against bark beetles and weevils. In Sitka spruce (Picea sitchensis), abundance of stone cells in the cortex of apical shoots is positively correlated with resistance to white pine weevil (Pissodes strobi). We identified histological, biochemical and molecular differences in the stone cell phenotype of weevil resistant (R) or susceptible (S) Sitka spruce genotypes. R trees displayed significantly higher quantities of cortical stone cells near the apical shoot node, the primary site for weevil feeding. Lignin, cellulose, xylan and mannan were the most abundant components of stone cell secondary walls, respectively. Lignin composition of stone cells isolated from R trees contained a higher percentage of G-lignin compared with S trees. Transcript profiling revealed higher transcript abundance in the R genotype of coumarate 3-hydroxylase, a key monolignol biosynthetic gene. Developing stone cells in current year apical shoots incorporated fluorescent-tagged monolignol into the secondary cell wall, while mature stone cells of previous year apical shoots did not. Stone cell development is an ephemeral process, and fortification of shoot tips in R trees is an effective strategy against insect feeding.

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.

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.

The primary inflorescence stem of Arabidopsis thaliana is rich in lignified cell walls, in both vascular bundles and interfascicular fibres. Previous gene expression studies demonstrated a correlation between expression of phenylpropanoid biosynthetic genes and a subset of genes encoding ATP-binding cassette (ABC) transporters, especially in the ABCB/multi-drug resistance/P-glycoprotein (ABCB/MDR/PGP) and ABCG/pleiotropic drug resistance (ABCG/PDR) subfamilies. The objective of this study was to characterize these ABC transporters in terms of their gene expression and their function in development of lignified cells. Based on in silico analyses, four ABC transporters were selected for detailed investigation: ABCB11/MDR8, ABCB14/MDR12, ABCB15/MDR13, and ABCG33/PDR5. Promoter::glucuronidase reporter assays for each gene indicated that promoters of ABCB11, ABCB14, ABCB15, and ABCG33 transporters are active in the vascular tissues of primary stem, and in some cases in interfascicular tissues as well. Homozygous T-DNA insertion mutant lines showed no apparent irregular xylem phenotype or alterations in interfascicular fibre lignification or morphology in comparison with wild type. However, in abcb14-1 mutants, stem vascular morphology was slightly disorganized, with decreased phloem area in the vascular bundle and decreased xylem vessel lumen diameter. In addition, abcb14-1 mutants showed both decreased polar auxin transport through whole stems and altered auxin distribution in the procambium. It is proposed that both ABCB14 and ABCB15 promote auxin transport since inflorescence stems in both mutants showed a reduction in polar auxin transport, which was not observed for any of the ABCG subfamily mutants tested. In the case of ABCB14, the reduction in auxin transport is correlated with a mild disruption of vascular development in the inflorescence stem.

Initiation of leaves at the flanks of the shoot apical meristem occurs at sites of auxin accumulation and pronounced expression of auxin-inducible PIN-FORMED1 (PIN) genes, suggesting a feedback loop to progressively focus auxin in concrete spots. Because PIN expression is regulated by auxin response factor activity, including MONOPTEROS (MP), it appeared possible that MP affects leaf formation as a positive regulator of PIN genes and auxin transport. Here, we analyze a novel, completely leafless phenotype arising from simultaneous interference with both auxin signaling and auxin transport. We show that mp pin1 double mutants, as well as mp mutants treated with auxin-efflux inhibitors, display synergistic abnormalities not seen in wild type regardless of how strongly auxin transport was reduced. The synergism of abnormalities indicates that the role of MP in shoot meristem organization is not limited to auxin transport regulation. In the mp mutant background, auxin transport inhibition completely abolishes leaf formation. Instead of forming leaves, the abnormal shoot meristems dramatically increase in size, harboring correspondingly enlarged expression domains of CLAVATA3 and SHOOTMERISTEMLESS, molecular markers for the central stem cell zone and the complete meristem, respectively. The observed synergism under conditions of auxin efflux inhibition was further supported by an unrestricted PIN1 expression in mp meristems, as compared to a partial restriction in wild-type meristems. Auxin transport-inhibited mp meristems also lacked detectable auxin maxima. We conclude that MP promotes the focusing of auxin and leaf initiation in part through pathways not affected by auxin efflux inhibitors.

Vascular tissues are of particular importance to terrestrial plants as they allow long-distance transport within the plant and also provide support for upright growth. Nowhere are these traits more obvious than in tree species. Here we have evaluated the role of auxin transport in the differentiation of primary and secondary vascular tissues in a tree species, trembling aspen ( Populus tremuloides Michx). We found that a partial inhibition of auxin transport resulted in increased width and numbers of veins in leaves. A similar vascular overgrowth was observed during early secondary vascular differentiation of stems. This stem overgrowth consisted almost entirely of early differentiation of metaxylem and fibers. We hypothesize that the early differentiation of metaxylem and fibers results from inhibitor-induced accumulation of auxin in stems and that the differentiation of these tissues requires higher levels of auxin exposure than protoxylem. The controlled conditions used in this study also provide a framework for reverse genetics approaches to identify genes involved in vascular differentiation based on elevated expression in tissues developing vascular overgrowth.

Genetic evidence links the Arabidopsis MONOPTEROS (MP) and PIN-FORMED1 (PIN1) genes to the patterning of leaf veins. To elucidate their potential functions and interactions in this process, we have assessed the dynamics of MP and PIN1 expression during vascular patterning in Arabidopsis leaf primordia. Both genes undergo a dynamic process of gradual refinement of expression into files one to two cells wide before overt vascular differentiation. The subcellular distribution of PIN1 is also gradually refined from a non-polar distribution in isodiametric cells to strongly polarized in elongated procambial cells and provides an indication of overall directions of auxin flow. We found evidence that MP expression can be activated by auxin exposure and that PIN1 as well as DR5::GUS expression is defective in mp mutant leaves. Taken together the results suggest a feedback regulatory loop that involves auxin, MP and PIN1 and provide novel experimental support for the canalization-of-auxin-flow hypothesis.

Many plant genes are known to be involved in the development of cambium and wood, but how the expression and functional interaction of these genes determine the unique biology of wood remains largely unknown. We used the soc1ful loss of function mutant - the woodiest genotype known in the otherwise herbaceous model plant Arabidopsis - to investigate the expression and interactions of genes involved in secondary growth (wood formation). Detailed anatomical observations of the stem in combination with mRNA sequencing were used to assess transcriptome remodeling during xylogenesis in wild-type and woody soc1ful plants. To interpret the transcriptome changes, we constructed functional gene association networks of differentially expressed genes using the STRING database. This analysis revealed functionally enriched gene association hubs that are differentially expressed in herbaceous and woody tissues. In particular, we observed the differential expression of genes related to mechanical stress and jasmonate biosynthesis/signaling during wood formation in soc1ful plants that may be an effect of greater tension within woody tissues. Our results suggest that habit shifts from herbaceous to woody life forms observed in many angiosperm lineages could have evolved convergently by genetic changes that modulate the gene expression and interaction network, and thereby redeploy the conserved wood developmental program. ispartof: The Plant Journal vol:86 issue:5 pages:376-390 ispartof: location:England status: published

Conifers possess an array of physical and chemical defences against stem-boring insects. Stone cells provide a physical defence associated with resistance against bark beetles and weevils. In Sitka spruce (Picea sitchensis), abundance of stone cells in the cortex of apical shoots is positively correlated with resistance to white pine weevil (Pissodes strobi). We identified histological, biochemical and molecular differences in the stone cell phenotype of weevil resistant (R) or susceptible (S) Sitka spruce genotypes. R trees displayed significantly higher quantities of cortical stone cells near the apical shoot node, the primary site for weevil feeding. Lignin, cellulose, xylan and mannan were the most abundant components of stone cell secondary walls, respectively. Lignin composition of stone cells isolated from R trees contained a higher percentage of G-lignin compared with S trees. Transcript profiling revealed higher transcript abundance in the R genotype of coumarate 3-hydroxylase, a key monolignol biosynthetic gene. Developing stone cells in current year apical shoots incorporated fluorescent-tagged monolignol into the secondary cell wall, while mature stone cells of previous year apical shoots did not. Stone cell development is an ephemeral process, and fortification of shoot tips in R trees is an effective strategy against insect feeding.

The TALE homeodomain transcription factor KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) is part of a regulatory network governing the commitment to secondary cell wall biosynthesis of Arabidopsis thaliana, where it contributes to negative regulation of this process. Here, we report that BLH6, a BELL1-LIKE HOMEODOMAIN protein, specifically interacts with KNAT7, and this interaction influences secondary cell wall development. BLH6 is a transcriptional repressor, and BLH6-KNAT7 physical interaction enhances KNAT7 and BLH6 repression activities. The overlapping expression patterns of BLH6 and KNAT7 and phenotypes of blh6, knat7, and blh6 knat7 loss-of-function mutants are consistent with the existence of a BLH6-KNAT7 heterodimer that represses commitment to secondary cell wall biosynthesis in interfascicular fibers. BLH6 and KNAT7 overexpression results in thinner interfascicular fiber secondary cell walls, phenotypes that are dependent on the interacting partner. A major impact of the loss of BLH6 and KNAT7 function is enhanced expression of the homeodomain-leucine zipper transcription factor REVOLUTA/INTERFASCICULAR FIBERLESS1 (REV/IFL1). BLH6 and KNAT7 bind to the REV promoter and repress REV expression, while blh6 and knat7 interfascicular fiber secondary cell wall phenotypes are suppressed in blh6 rev and knat7 rev double mutants, suggesting that BLH6/KNAT7 signaling acts through REV as a direct target.

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.

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.

Genetic evidence links the Arabidopsis MONOPTEROS (MP) and PIN-FORMED1 (PIN1) genes to the patterning of leaf veins. To elucidate their potential functions and interactions in this process, we have assessed the dynamics of MP and PIN1 expression during vascular patterning in Arabidopsis leaf primordia. Both genes undergo a dynamic process of gradual refinement of expression into files one to two cells wide before overt vascular differentiation. The subcellular distribution of PIN1 is also gradually refined from a non-polar distribution in isodiametric cells to strongly polarized in elongated procambial cells and provides an indication of overall directions of auxin flow. We found evidence that MP expression can be activated by auxin exposure and that PIN1 as well as DR5::GUS expression is defective in mp mutant leaves. Taken together the results suggest a feedback regulatory loop that involves auxin, MP and PIN1 and provide novel experimental support for the canalization-of-auxin-flow hypothesis.