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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., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Secondary cell wall (SCW) production during xylem development requires massive up-regulation of hemicellulose (e.g. glucuronoxylan) biosynthesis in the Golgi. Although mutant studies have revealed much of the xylan biosynthetic machinery, the precise arrangement of these proteins and their products in the Golgi apparatus is largely unknown. We used a fluorescently tagged xylan backbone biosynthetic protein (IRREGULAR XYLEM9; IRX9) as a marker of xylan production in the Golgi of developing protoxylem tracheary elements in Arabidopsis ( Arabidopsis thaliana ). Both live-cell confocal and transmission electron microscopy (TEM) revealed SCW deposition is accompanied by a significant proliferation of Golgi stacks. Furthermore, although Golgi stacks were randomly distributed, the organization of the cytoplasm ensured their close proximity to developing SCWs. Quantitative immuno-TEM revealed IRX9 is present in a specific subdomain of the Golgi stack and was most abundant in the ring of the inner margins of medial cisternae where fenestrations are abundant. Conversely, the xylan product accumulated in swollen trans cisternal margins and the Trans-Golgi network (TGN). The irx9 mutant lacked this expansion for both the cisternal margins and the TGN, whereas Golgi stack proliferation was unaffected. Golgi in irx9 also displayed dramatic changes in their structure, with increases in cisternal fenestration and tubulation. Our data support a new model where xylan biosynthesis and packaging into secretory vesicles are localized in distinct structural and functional domains of the Golgi. Rather than polysaccharide biosynthesis occurring in the center of the cisternae, IRX9 and the xylan product are arranged in successive concentric rings in Golgi cisternae., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

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., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

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., (© 2014 American Society of Plant Biologists. All Rights Reserved.)

Lipid secretion from epidermal cells to the plant surface is essential to create the protective plant cuticle. Cuticular waxes are unusual secretory products, consisting of a variety of highly hydrophobic compounds including saturated very-long-chain alkanes, ketones, and alcohols. These compounds are synthesized in the endoplasmic reticulum (ER) but must be trafficked to the plasma membrane for export by ATP-binding cassette transporters. To test the hypothesis that wax components are trafficked via the endomembrane system and packaged in Golgi-derived secretory vesicles, Arabidopsis (Arabidopsis thaliana) stem wax secretion was assayed in a series of vesicle-trafficking mutants, including gnom like1-1 (gnl1-1), transport particle protein subunit120-4, and echidna (ech). Wax secretion was dependent upon GNL1 and ECH. Independent of secretion phenotypes, mutants with altered ER morphology also had decreased wax biosynthesis phenotypes, implying that the biosynthetic capacity of the ER is closely related to its structure. These results provide genetic evidence that wax export requires GNL1- and ECH-dependent endomembrane vesicle trafficking to deliver cargo to plasma membrane-localized ATP-binding cassette transporters.

The highly resistant biopolymer, sporopollenin, gives the outer wall (exine) of spores and pollen grains their unparalleled strength, shielding these structures from terrestrial stresses. Despite a limited understanding of the composition of sporopollenin, it appears that the synthesis of sporopollenin occurs in the tapetum and requires the transport of one or more sporopollenin constituents to the surface of developing microspores. Here, we describe ABCG26, a member of the ATP-binding cassette (ABC) transporter superfamily, which is required for pollen exine formation in Arabidopsis (Arabidopsis thaliana). abcg26 mutants are severely reduced in fertility, with most siliques failing to produce seeds by self-fertilization and mature anthers failing to release pollen. Transmission electron microscopy analyses revealed an absence of an exine wall on abcg26-1 mutant microspores. Phenotypic abnormalities in pollen wall formation were first apparent in early uninucleate microspores as a lack of exine formation and sporopollenin deposition. Additionally, the highest levels of ABCG26 mRNA were in the tapetum, during early pollen wall formation, sporopollenin biosynthesis, and sporopollenin deposition. Accumulations resembling the trilamellar lipidic coils in the abcg11 and abcg12 mutants defective in cuticular wax export were observed in the anther locules of abcg26 mutants. A yellow fluorescent protein-ABCG26 protein was localized to the endoplasmic reticulum and plasma membrane. Our results show that ABCG26 plays a critical role in exine formation and pollen development and are consistent with a model by which ABCG26 transports sporopollenin precursors across the tapetum plasma membrane into the locule for polymerization on developing microspore walls.