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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.

The effects of reductions in cell wall lignin content, manifested by RNA interference suppression of coumaroyl 3'-hydroxylase, on plant growth, water transport, gas exchange, and photosynthesis were evaluated in hybrid poplar trees (Populus alba x grandidentata). The growth characteristics of the reduced lignin trees were significantly impaired, resulting in smaller stems and reduced root biomass when compared to wild-type trees, as well as altered leaf morphology and architecture. The severe inhibition of cell wall lignification produced trees with a collapsed xylem phenotype, resulting in compromised vascular integrity, and displayed reduced hydraulic conductivity and a greater susceptibility to wall failure and cavitation. In the reduced lignin trees, photosynthetic carbon assimilation and stomatal conductance were also greatly reduced, however, shoot xylem pressure potential and carbon isotope discrimination were higher and water-use efficiency was lower, inconsistent with water stress. Reductions in assimilation rate could not be ascribed to increased stomatal limitation. Starch and soluble sugars analysis of leaves revealed that photosynthate was accumulating to high levels, suggesting that the trees with substantially reduced cell wall lignin were not carbon limited and that reductions in sink strength were, instead, limiting photosynthesis.

All vascular plants are protected from the environment by a cuticle, a lipophilic layer synthesized by epidermal cells and composed of a cutin polymer matrix and waxes. The mechanism by which epidermal cells accumulate and assemble cuticle components in rapidly expanding organs is largely unknown. We have begun to address this question by analyzing the lipid compositional variance, the surface micromorphology, and the transcriptome of epidermal cells in elongating Arabidopsis (Arabidopsis thaliana) stems. The rate of cell elongation is maximal near the apical meristem and decreases steeply toward the middle of the stem, where it is 10 times slower. During and after this elongation, the cuticular wax load and composition remain remarkably constant (32 microg/cm2), indicating that the biosynthetic flux into waxes is closely matched to surface area expansion. By contrast, the load of polyester monomers per unit surface area decreases more than 2-fold from the upper (8 microg/cm2) to the lower (3 microg/cm2) portion of the stem, although the compositional variance is minor. To aid identification of proteins involved in the biosynthesis of waxes and cutin, we have isolated epidermal peels from Arabidopsis stems and determined transcript profiles in both rapidly expanding and nonexpanding cells. This transcriptome analysis was validated by the correct classification of known epidermis-specific genes. The 15% transcripts preferentially expressed in the epidermis were enriched in genes encoding proteins predicted to be membrane associated and involved in lipid metabolism. An analysis of the lipid-related subset is presented.

Endocytosis was measured along the axis of differentiation of epidermal and cortical root cells of Lobelia erinus from meristematic to fully expanded vacuolate cells. Both lanthanum and lead were tested as markers of endocytosis; lanthanum proved to be more effective. Lanthanum treatment produced electron dense deposits in the apoplast of the root, as well as coated pits, coated vesicles, smooth vesicles and multivesicular bodies within the cells. X-ray microanalysis was used to confirm the lanthanide nature of the deposits. In both secretory (meristematic and elongating cells actively depositing new cell wall material) and non secretory (mature vacuolate) cells the amount of endocytosis occurring was measured by counting the' number of lanthanum labelled vesicles/um² /cell. The amount of endocytosis correlated very well with the cell wall secretory activity. The highest amount of endocytosis was found in the elongating cells, with meristematic having an intermediate value. Mature, vacuolate cells had the least endocytosis. The relationship between endocytosis and secretory activity suggests that endocytosis may be acting to remove excess membrane material added during exocytosis of secretory vesicles. Cytochemical tests for polysaccharides were performed on both conventional transmission electron microscopy preparations and ultrarapidly frozen, freeze substituted preparations. The ultrastructural preservation was superior using the cryotechnique. The organelles involved in secretion of cell wall components were compared with the organelles associated with endocytosis in chapter 1. The Golgi showed distinct dictyosome polarity, small peripheral vesicles and an elaborate trans Golgi network. Vesicles in the cytoplasm displayed diverse staining properties. One population of larger, densely staining vesicles located on the trans Golgi and in the cortical cytoplasm was interpreted to be secretory vesicles. Microtubules were disrupted with colchicine to test the importance of these cytoskeletal elements in endocytosis. Immunofluorescence was used to determine the concentration of colchicine which disrupted cortical microtubules in these cells. After colchicine treatment, there were fewer endocytotic vesicles and less lanthanum label in the multivesicular body, suggesting microtubules are involved in endocytosis during elongation. Cell wall pore size was tested using electron dense apoplast markers. Partial enzyme digestions were used to find which components of the cell wall determine porosity. Pectin was found to be the most important component, while cellulose and protein did not seem to effect porosity in these primary roots. The porosity of the wall was interpreted in terms of the intact wall ultrastructure. Ultrarapid freezing, freeze substitution were used to reexamine endocytosis in elongating cells. Using lanthanum as a marker for endocytosis, the results of the earlier, conventional TEM study were supported and extended. In addition to coated and smooth vesicles, multivesicular bodies were labelled. The instantaneous preservation of the endomembrane system allowed the localization of label in the partially coated reticulum and in vesicles associated with the Golgi as well. The partially coated reticulum and multivesicular body are proposed to represent plant endosomes. When interpreted in conjunction with the secretion study, it can be suggested that the heterogenous populations of vesicles occur in the cytoplasm of the elongating Lobelia erinus root cells.

12 Pág. Centro de Biotecnología y Genómica de Plantas., 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 work at the CBGP was funded by several INIA grants. During the course of the work SL-G was funded by a predoctoral FPI-INIA fellowship/contract. We thank the Spanish Ministry of Science for the Severo Ochoa Excellence Accreditations to the CBGP (SEV-2016-0672).

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.