Elongation of wood fibers combines features of diffuse and tip growth.

Xylem fibers are highly elongated cells that are key constituents of wood, play major physiological roles in plants, comprise an important terrestrial carbon reservoir, and thus have enormous ecological and economic importance. As they develop, from fusiform initials, their bodies remain the same length while their tips elongate and intrude into intercellular spaces. To elucidate mechanisms of tip elongation, we studied the cell wall along the length of isolated, elongating aspen xylem fibers and used computer simulations to predict the forces driving the intercellular space formation required for their growth. We found pectin matrix epitopes (JIM5, LM7) concentrated at the tips where cellulose microfibrils have transverse orientation, and xyloglucan epitopes (CCRC-M89, CCRC-M58) in fiber bodies where microfibrils are disordered. These features are accompanied by changes in cell wall thickness, indicating that while the cell wall elongates strictly at the tips, it is deposited all over fibers. Computer modeling revealed that the intercellular space formation needed for intrusive growth may only require targeted release of cell adhesion, which allows turgor pressure in neighboring fiber cells to 'round' the cells creating spaces. These characteristics show that xylem fibers' elongation involves a distinct mechanism that combines features of both diffuse and tip growth.

A sustained CYCLINB1;1 and STM expression in the neoplastic tissues induced by Rhodococcus fascians on Arabidopsis underlies the persistence of the leafy gall structure.

RHODOCOCCUS FASCIANS: is a gram-positive phytopathogen that infects a wide range of plant species. The actinomycete induces the formation of neoplastic growths, termed leafy galls, that consist of a gall body covered by small shoots of which the outgrowth is arrested due to an extreme form of apical dominance. In our previous work, we demonstrated that in the developing gall, auxin drives the transdifferentiation of parenchyma cells into vascular elements. In this work, with the use of transgenic Arabidopsis thaliana plants carrying molecular reporters for cell division (pCYCB1;1:GUS) and meristematic activity (pSTM:GUS), we analyzed the fate of cells within the leafy gall. Our results indicate that the size of the gall body is determined by ongoing mitotic cell divisions as illustrated by strong CYCB1;1 expression combined with the de novo formation of new meristematic areas triggered by STM expression. The shoot meristems that develop in the peripheral parts of the gall are originating from high ectopic STM expression. Altogether the presented data provide further insight into the cellular events that accompany the development of leafy galls in response to R. fascians infection.

Arabidopsis XTH4 and XTH9 Contribute to Wood Cell Expansion and Secondary Wall Formation.

Xyloglucan is the major hemicellulose of dicotyledon primary cell walls, affecting the load-bearing framework with the participation of xyloglucan endo-transglycosylase/hydrolases (XTHs). We used loss- and gain-of function approaches to study functions of XTH4 and XTH9 abundantly expressed in cambial regions during secondary growth of Arabidopsis (Arabidopsis thaliana). In secondarily thickened hypocotyls, these enzymes had positive effects on vessel element expansion and fiber intrusive growth. They also stimulated secondary wall thickening but reduced secondary xylem production. Cell wall analyses of inflorescence stems revealed changes in lignin, cellulose, and matrix sugar composition indicating an overall increase in secondary versus primary walls in mutants, indicative of higher xylem production compared with the wild type (since secondary walls were thinner). Intriguingly, the number of secondary cell wall layers compared with the wild type was increased in xth9 and reduced in xth4, whereas the double mutant xth4x9 displayed an intermediate number of layers. These changes correlated with specific Raman signals from the walls, indicating changes in lignin and cellulose. Secondary walls were affected also in the interfascicular fibers, where neither XTH4 nor XTH9 was expressed, indicating that these effects were indirect. Transcripts involved in secondary wall biosynthesis and cell wall integrity sensing, including THESEUS1 and WALL ASSOCIATED KINASE2, were highly induced in the mutants, indicating that deficiency in XTH4 and XTH9 triggers cell wall integrity signaling, which, we propose, stimulates xylem cell production and modulates secondary wall thickening. Prominent effects of XTH4 and XTH9 on secondary xylem support the hypothesis that altered xyloglucan affects wood properties both directly and via cell wall integrity sensing.

Ontogenetic Changes in Auxin Biosynthesis and Distribution Determine the Organogenic Activity of the Shoot Apical Meristem in pin1 Mutants.

In the shoot apical meristem (SAM) of Arabidopsis, PIN1-dependent polar auxin transport (PAT) regulates two crucial developmental processes: organogenesis and vascular system formation. However, the knockout mutation in the PIN1 gene does not fully inhibit these two processes. Therefore, we investigated a potential source of auxin for organogenesis and vascularization during inflorescence stem development. We analyzed auxin distribution in wild-type (WT) and pin1 mutant plants using a refined protocol of auxin immunolocalization; auxin activity, with the response reporter pDR5:GFP; and expression of auxin biosynthesis genes YUC1 and YUC4. Our results revealed that regardless of the functionality of PIN1-mediated PAT, auxin is present in the SAM and vascular strands. In WT plants, auxin always accumulates in all cells of the SAM, whereas in pin1 mutants, its localization within the SAM changes ontogenetically and is related to changes in the structure of the vascular system, organogenic activity of SAM, and expression levels of YUC1 and YUC4 genes. Our findings indicate that the presence of auxin in the meristem of pin1 mutants is an outcome of at least two PIN1-independent mechanisms: acropetal auxin transport from differentiated tissues with the use of vascular strands and auxin biosynthesis within the SAM.

Auxin-mediated regulation of vascular patterning in Arabidopsis thaliana leaves.

The vascular system develops in response to auxin flow as continuous strands of conducting tissues arranged in regular spatial patterns. However, a mechanism governing their regular and repetitive formation remains to be fully elucidated. A model system for studying the vascular pattern formation is the process of leaf vascularization in Arabidopsis. In this paper, we present current knowledge of important factors and their interactions in this process. Additionally, we propose the sequence of events leading to the emergence of continuous vascular strands and point to significant problems that need to be resolved in the future to gain a better understanding of the regulation of the vascular pattern development.

Neovascularization during leafy gall formation on Arabidopsis thaliana upon Rhodococcus fascians infection.

MAIN CONCLUSION: Extensive de novo vascularization of leafy galls emerging upon Rhodococcus fascians infection is achieved by fascicular/interfascicular cambium activity and transdifferentiation of parenchyma cells correlated with increased auxin signaling. A leafy gall consisting of fully developed yet growth-inhibited shoots, induced by the actinomycete Rhodococcus fascians, differs in structure compared to the callus-like galls induced by other bacteria. To get insight into the vascular development accompanying the emergence of the leafy gall, the anatomy of infected axillary regions of the inflorescence stem of wild-type Arabidopsis thaliana accession Col-0 plants and the auxin response in pDR5:GUS-tagged plants were followed in time. Based on our observations, three phases can be discerned during vascularization of the symptomatic tissue. First, existing fascicular cambium becomes activated and interfascicular cambium is formed giving rise to secondary vascular elements in a basipetal direction below the infection site in the main stem and in an acropetal direction in the entire side branch. Then, parenchyma cells in the region between both stems transdifferentiate acropetally towards the surface of the developing symptomatic tissue leading to the formation of xylem and vascularize the hyperplasia as they expand. Finally, parenchyma cells in the developing gall also transdifferentiate to vascular elements without any specific direction resulting in excessive vasculature disorderly distributed in the leafy gall. Prior to any apparent anatomical changes, a strong auxin response is mounted, implying that auxin is the signal that controls the vascular differentiation induced by the infection. To conclude, we propose the "sidetracking gall hypothesis" as we discuss the mechanisms driving the formation of superfluous vasculature of the emerging leafy gall.

Expression of fungal acetyl xylan esterase in Arabidopsis thaliana improves saccharification of stem lignocellulose.

Cell wall hemicelluloses and pectins are O-acetylated at specific positions, but the significance of these substitutions is poorly understood. Using a transgenic approach, we investigated how reducing the extent of O-acetylation in xylan affects cell wall chemistry, plant performance and the recalcitrance of lignocellulose to saccharification. The Aspergillus niger acetyl xylan esterase AnAXE1 was expressed in Arabidopsis under the control of either the constitutively expressed 35S CAMV promoter or a woody-tissue-specific GT43B aspen promoter, and the protein was targeted to the apoplast by its native signal peptide, resulting in elevated acetyl esterase activity in soluble and wall-bound protein extracts and reduced xylan acetylation. No significant alterations in cell wall composition were observed in the transgenic lines, but their xylans were more easily digested by a ß-1,4-endoxylanase, and more readily extracted by hot water, acids or alkali. Enzymatic saccharification of lignocellulose after hot water and alkali pretreatments produced up to 20% more reducing sugars in several lines. Fermentation by Trametes versicolor of tissue hydrolysates from the line with a 30% reduction in acetyl content yielded ~70% more ethanol compared with wild type. Plants expressing 35S:AnAXE1 and pGT43B:AnAXE1 developed normally and showed increased resistance to the biotrophic pathogen Hyaloperonospora arabidopsidis, probably due to constitutive activation of defence pathways. However, unintended changes in xyloglucan and pectin acetylation were only observed in 35S:AnAXE1-expressing plants. This study demonstrates that postsynthetic xylan deacetylation in woody tissues is a promising strategy for optimizing lignocellulosic biomass for biofuel production.

Aspen Tension Wood Fibers Contain ß-(1---> 4)-Galactans and Acidic Arabinogalactans Retained by Cellulose Microfibrils in Gelatinous Walls.

Contractile cell walls are found in various plant organs and tissues such as tendrils, contractile roots, and tension wood. The tension-generating mechanism is not known but is thought to involve special cell wall architecture. We previously postulated that tension could result from the entrapment of certain matrix polymers within cellulose microfibrils. As reported here, this hypothesis was corroborated by sequential extraction and analysis of cell wall polymers that are retained by cellulose microfibrils in tension wood and normal wood of hybrid aspen (Populus tremula × Populus tremuloides). ß-(1→4)-Galactan and type II arabinogalactan were the main large matrix polymers retained by cellulose microfibrils that were specifically found in tension wood. Xyloglucan was detected mostly in oligomeric form in the alkali-labile fraction and was enriched in tension wood. ß-(1→4)-Galactan and rhamnogalacturonan I backbone epitopes were localized in the gelatinous cell wall layer. Type II arabinogalactans retained by cellulose microfibrils had a higher content of (methyl)glucuronic acid and galactose in tension wood than in normal wood. Thus, ß-(1→4)-galactan and a specialized form of type II arabinogalactan are trapped by cellulose microfibrils specifically in tension wood and, thus, are the main candidate polymers for the generation of tensional stresses by the entrapment mechanism. We also found high ß-galactosidase activity accompanying tension wood differentiation and propose a testable hypothesis that such activity might regulate galactan entrapment and, thus, mechanical properties of cell walls in tension wood.

Glycoside Hydrolase Activities in Cell Walls of Sclerenchyma Cells in the Inflorescence Stems of Arabidopsis thaliana Visualized in Situ.

Techniques for in situ localization of gene products provide indispensable information for understanding biological function. In the case of enzymes, biological function is directly related to activity, and therefore, knowledge of activity patterns is central to understanding the molecular controls of plant development. We have previously developed a novel type of fluorogenic substrate for revealing glycoside hydrolase activity in planta, based on resorufin ß-glycosides Here, we explore a wider range of such substrates to visualize glycoside hydrolase activities in Arabidopsis inflorescence stems in real time, especially highlighting distinct distribution patterns of these activities in the secondary cell walls of sclerenchyma cells. The results demonstrate that ß-1,4-glucosidase, ß-1,4-glucanase and ß-1,4-galactosidase activities accompany secondary wall deposition. In contrast, xyloglucanase activity follows a different pattern, with the highest signal observed in mature cells, concentrated in the middle lamella. These data further the understanding of the process of cell wall deposition and function in sclerenchymatic tissues of plants.

Putative dual pathway of auxin transport in organogenesis of Arabidopsis.

In Arabidopsis, damage to the superficial acropetal polar auxin transport (PAT) inhibits generative but not vegetative organ initiation. In order to verify whether in a vegetative phase auxin can be transported to the meristem in a different way, the research on wild-type and plants with defective PAT was performed. Distance from the differentiated vascular elements to the shoot apical meristem (SAM) was measured for Arabidopsis cultured in different experimental systems. The influence of this distance on the ability to induce organogenesis as well as transport of the fluorescent dye to the SAM, and the LEAFY gene expression were analyzed. The youngest protoxylem elements were used as a marker of the vascular tissues. The distance of protoxylem to the SAM and organogenesis were interrelated. Organ initiation occurred only when protoxylem was localized near to the SAM. Experimental elongation of internodes in a vegetative rosette caused an increase in the distance between protoxylem and the SAM organogenic zone. Thus, the inhibition of organ initiation took place already during the vegetative phase. The results suggest the presence of at least two pathways of acropetal transport of auxin inducing organogenesis: one superficial way through PAT, and the second, putative one, internal through the vascular system. Possibly, organogenesis is completely blocked only when both these pathways are dysfunctional.

A real-time fluorogenic assay for the visualization of glycoside hydrolase activity in planta.

There currently exists a diverse array of molecular probes for the in situ localization of polysaccharides, nucleic acids, and proteins in plant cells, including reporter enzyme strategies (e.g. protein-glucuronidase fusions). In contrast, however, there is a paucity of methods for the direct analysis of endogenous glycoside hydrolases and transglycosidases responsible for cell wall remodeling. To exemplify the potential of fluorogenic resorufin glycosides to address this issue, a resorufin beta-glycoside of a xylogluco-oligosaccharide (XXXG-beta-Res) was synthesized as a specific substrate for in planta analysis of XEH activity. The resorufin aglycone is particularly distinguished for high sensitivity in muro assays due to a low pK(a) (5.8) and large extinction coefficient (epsilon 62,000 M(-1) cm(-1)), long-wavelength fluorescence (excitation 571 nm/emission 585 nm), and high quantum yield (0.74) of the corresponding anion. In vitro analyses demonstrated that XXXG-beta-Res is hydrolyzed by the archetypal plant XEH, nasturtium (Tropaeolum majus) NXG1, with classical Michaelis-Menten substrate saturation kinetics and a linear dependence on both enzyme concentration and incubation time. Further, XEH activity could be visualized in real time by observing the localized increase in fluorescence in germinating nasturtium seeds and Arabidopsis (Arabidopsis thaliana) inflorescent stems by confocal microscopy. Importantly, this new in situ XEH assay provides an essential complement to the in situ xyloglucan endotransglycosylase assay, thus allowing delineation of the disparate activities encoded by xyloglucan endotransglycosylase/hydrolase genes directly in plant tissues. The observation that XXXG-beta-Res is also hydrolyzed by diverse microbial XEHs indicates that this substrate, and resorufin glycosides in general, may find broad applicability for the analysis of wall restructuring by polysaccharide hydrolases during morphogenesis and plant-microbe interactions.

KORRIGAN1 and its aspen homolog PttCel9A1 decrease cellulose crystallinity in Arabidopsis stems.

KORRIGAN1 (KOR1) is a membrane-bound cellulase implicated in cellulose biosynthesis. PttCel9A1 from hybrid aspen (Populus tremula L. x tremuloides Michx.) has high sequence similarity to KOR1 and we demonstrate here that it complements kor1-1 mutants, indicating that it is a KOR1 ortholog. We investigated the function of PttCel9A1/KOR1 in Arabidopsis secondary growth using transgenic lines expressing 35S::PttCel9A1 and the KOR1 mutant line irx2-2. The presence of elevated levels of PttCel9A1/KOR1 in secondary walls of 35S::PttCel9A1 lines was confirmed by in muro visualization of cellulase activity. Compared with the wild type, 35S::PttCel9A1 lines had higher trifluoroacetic acid (TFA)-hydrolyzable glucan contents, similar Updegraff cellulose contents and lower cellulose crystallinity indices, as determined by (13)C solid-state nuclear magnetic resonance (NMR) spectroscopy. irx2-2 mutants had wild-type TFA-hydrolyzable glucan contents, but reduced Updegraff cellulose contents and higher than wild-type cellulose crystallinity indices. The data support the hypothesis that PttCel9A1/KOR1 activity is present in cell walls, where it facilitates cellulose biosynthesis in a way that increases the amount of non-crystalline cellulose.

Xyloglucan endo-transglycosylase (XET) functions in gelatinous layers of tension wood fibers in poplar--a glimpse into the mechanism of the balancing act of trees.

Tension wood is a specialized tissue of deciduous trees that functions in bending woody stems to optimize their position in space. Tension wood fibers that develop on one side of the stem have an increased potency to shrink compared with fibers on the opposite side, thus creating a bending moment. It is believed that the gelatinous (G) cell wall layer containing almost pure cellulose of tension wood fibers is pivotal to their shrinking. By analyzing saccharide composition and linkage in isolated G-layers of poplar, we found that they contain some matrix components in addition to cellulose, of which xyloglucan is the most abundant. Xyloglucan, xyloglucan endo-transglycosylase (XET) activity and xyloglucan endo-transglycosylase/hydrolase (XTH) gene products were detected in developing G-layers by labeling using CCRC-M1 monoclonal antibody, in situ incorporation of XXXG-SR and the polyclonal antibody to poplar PttXET16-34, respectively, indicating that xyloglucan is incorporated into the G-layer during its development. Moreover, several XTH transcripts were altered and were generally up-regulated in developing tension wood compared with normal wood. In mature G-fibers, XTH gene products were detected in the G-layers while the XET activity was evident in the adjacent S(2) wall layer. We propose that XET activity is essential for G-fiber shrinking by repairing xyloglucan cross-links between G- and S(2)-layers and thus maintaining their contact. Surprisingly, XTH gene products and XET activity persisted in mature G-fibers for several years, suggesting that the enzyme functions after cell death repairing the cross-links as they are being broken during the shrinking process.