Growth control by cell wall pectins

Plant cell growth is controlled by the balance between turgor pressure and the extensibility of the cell wall. Several distinct classes of wall polysaccharides and their interactions contribute to the architecture and the emergent features of the wall. As a result, remarkable tensile strength is achieved without relinquishing extensibility. The control of growth and development does not only require a precisely regulated biosynthesis of cell wall components, but also constant remodeling and modification after deposition of the polymers. This is especially evident given the fact that wall deposition and cell expansion are largely uncoupled. Pectins form a functionally and structurally diverse class of galacturonic acid-rich polysaccharides which can undergo abundant modification with a concomitant change in physicochemical properties. This review focuses on homogalacturonan demethylesterification catalyzed by the ubiquitous enzyme pectin methylesterase (PME) as a growth control module. Special attention is drawn to the recently discovered role of this process in primordial development in the shoot apical meristem.     

Stimulation of elongation growth and cell wall loosening in rice coleoptiles under microgravity conditions in space

We analyzed the growth rate and the cell wall properties of coleoptiles of rice seedlings grown at 23.6 degrees C for 68.5, 91.5 and 136 h during the Space Shuttle STS-95 mission. In space, elongation growth of coleoptiles was stimulated and the cell wall extensibility increased. Also, the levels of the cell wall polysaccharides per unit length of coleoptiles and the relative content of the high molecular mass matrix polysaccharides decreased in space. These differences in the cell wall polysaccharides could be involved in increasing the cell wall extensibility, leading to growth stimulation of rice coleoptiles in space.     

Temperature modulates the cell wall mechanical properties of rice coleoptiles by altering the molecular mass of hemicellulosic polysaccharides

The present study was conducted to investigate the mechanism inducing the difference in the cell wall extensibility of rice ( Oryza sativa L. cv. Koshihikari) coleoptiles grown under various temperature (10–50°C) conditions. The growth rate and the cell wall extensibility of rice coleoptiles exhibited the maximum value at 30–40°C, and became smaller as the growth temperature rose or dropped from this temperature range. The amounts of cell wall polysaccharides per unit length of coleoptile increased in coleoptiles grown at 40°C, but not at other temperature conditions. On the other hand, the molecular size of hemicellulosic polysaccharides was small at temperatures where the cell wall extensibility was high (30–40°C). The autolytic activities of cell walls obtained from coleoptiles grown at 30 and 40°C were substantially higher than those grown at 10, 20 and 50°C. Furthermore, the activities of (1→3),(1→4)- β -glucanases extracted from coleoptile cell walls showed a similar tendency. When oat (1→3),(1→4)- β -glucans with high molecular mass were incubated with the cell wall enzyme preparations from coleoptiles grown at various temperature conditions, the extensive molecular mass downshifts were brought about only by the cell wall enzymes obtained from coleoptiles grown at 30–40°C. There were close correlations between the cell wall extensibility and the molecular mass of hemicellulosic polysaccharides or the activity of β -glucanases. These results suggest that the environmental temperature regulates the cell wall extensibility of rice coleoptiles by modifying mainly the molecular mass of hemicellulosic polysaccharides. Modulation of the activity of β -glucanases under various temperature conditions may be involved in the alteration of the molecular size of hemicellulosic polysaccharides.     

Changes in expansin activity and cell wall susceptibility to expansin action during cessation of internodal elongation in floating rice

We investigated the involvement of expansin action in determining the growth rate of internodes of floating rice (Oryza sativa L.). Floating rice stem segments in which rapid internodal elongation had been induced by submergence for 2 days were exposed to air or kept in submergence for 2 more days. Both treatments reduced the elongation rate of the internodes, and the degree of reduction was much greater in air-exposed stem segments than in continually submerged segments. These rates of internodal elongation were correlated with acid-induced cell wall extensibility and cell wall susceptibility to expansins in the cell elongation zone of the internodes, but not with extractable expansin activity. These results suggest that the reduced growth rate of internodes must be due, at least in part, to the decrease in acid-induced cell wall extensibility, which can be modulated through changes in the cell wall susceptibility to expansins rather than through expansin activity. Analysis of the cell wall composition of the internodes showed that the cellulosic and noncellulosic polysaccharide contents increased in response to exposure to air, but they remained almost constant under continued submergence although the cell wall susceptibility to expansins gradually declined even under continued submergence. The content of xylose in noncellulosic neutral sugars in the cell walls of internodes was closely and negatively correlated with changes in the susceptibility of the walls to expansins. These results suggest that the deposition of xylose-rich polysaccharides into the cell walls may be related to a decrease in acid-induced cell wall extensibility in floating rice internodes through the modulation of cell wall susceptibility to expansins.     

Stimulation of elongation growth and xyloglucan breakdown in Arabidopsis hypocotyls under microgravity conditions in space

Seedlings of Arabidopsis thaliana (L.) Heynh. (ecotype Columbia and an ethylene-resistant mutant etr1-1) were cultivated for 68.5, 91.5 and 136 h on board during the Space Shuttle STS-95 mission, and changes in the elongation growth and the cell wall properties of hypocotyls were analyzed. Elongation growth of dark-grown hypocotyls of both Columbia and etr1-1 was stimulated under microgravity conditions in space. There were no clear differences in the degree of growth stimulation between Columbia and etr1-1, indicating that the ethylene level was not abnormally high in the cultural environment of this space experiment. Microgravity also increased the mechanical extensibility of cell walls in both cultivars, and such an increase was attributed to the increase in the apparent irreversible extensibility. The levels of cell wall polysaccharides per unit length of hypocotyls decreased in space. Microgravity also reduced the weight-average molecular mass of xyloglucans in the hemicellulose-II fraction. Also, the activity of xyloglucan-degrading enzymes extracted from hypocotyl cell walls increased under microgravity conditions. These results suggest that microgravity reduces the molecular mass of xyloglucans by increasing xyloglucan-degrading activity. Modifications of xyloglucan metabolism as well as the thickness of cell wall polysaccharides seem to be involved in an increase in the cell wall extensibility, leading to growth stimulation of Arabidopsis hypocotyls in space.     

Cell Wall Modifying Proteins Mediate Plant Acclimatization to Biotic and Abiotic Stresses

Cell wall modification is an important aspect of plant acclimation to environmental stresses. Structural changes of the existing cell wall mediated by various cell wall modifying proteins help a plant adjust to environmental changes by regulating growth and policing the entry of biotic agents. For example, accelerated shoot growth during submergence and shading allows some plants to escape these unfavorable conditions. This is mediated by the regulation of wall modifying proteins that alter cell wall structure and allow it to yield to turgor, thus fueling cellular expansion. Regulation of cell wall protein activity results in growth modulation during drought, where maintenance of root growth through changes in wall extensibility is an important adaptation to water deficit. Freeze-tolerant plants adjust their cell wall properties to prevent freezing-induced dehydration and also use the cell wall as a barrier against ice crystal propagation. Cell wall architecture is an important determinant of plant resistance to biotic stresses. A rigid cell wall can fend off pathogen attack by forming an impenetrable, physical barrier. When breached, products released during wall modification can trigger plant defense signaling. This review documents and discusses studies demonstrating the importance of timely cell wall modification during plant stress responses by focusing on a well-researched subset of wall modifying proteins.     

Growth regulation mechanisms in higher plants under microgravity conditions - changes in cell wall metabolism.

During Space Shuttle STS-95 mission, we cultivated seedlings of rice (Oryza sativa L. cv. Koshihikari and cv. Tan-ginbozu) and Arabidopsis (Arabidopsis thaliana L. cv. Columbia and cv. etr1-1) for 68.5, 91.5, and 136 hr on board, and then analyzed changes in the nature of their cell walls, growth, and morphogenesis under microgravity conditions. In space, elongation growth of both rice coleoptiles and Arabidopsis hypocotyls was stimulated. Also, the increase in the cell wall extensibility, especially that in the irreversible extensibility, was observed for such materials. The analyses of the amounts, the structure, and the physicochemical properties of the cell wall constituents indicated that the decreases in levels and molecular masses of cell wall polysaccharides were induced under microgravity conditions, which appeared to contribute to the increase in the wall extensibility. The activity of certain wall enzymes responsible for the metabolic turnover of the wall polysaccharides was increased in space. By the space flight, we also confirmed the occurrence of automorphogenesis of both seedlings under microgravity conditions; rice coleoptiles showed an adaxial bending, whereas Arabidopsis hypocotyls elongated in random directions. Furthermore, it was shown that spontaneous curvatures of rice coleoptiles in space were brought about uneven modifications of cell wall properties between the convex and the concave sides.     

Diferulic and ferulic acid in the cell wall of Avena coleoptiles—Their relationships to mechanical properties of the cell wall

Alkaline hydrolysis liberated ferulic and diferulic acid from polysaccharides of the Avena coleoptile ( Avena sativa L. cv. Victory I) cell walls. The amount of the two phenolic acids bound to cell walls increased substantially at day 4–5 after sowing, when the growth rate of the coleoptile started to decrease. The level of these acids was almost constant from the tip to base in 3-day-old coleoptiles, but increased toward the basal zone in 4- and 5-day-old ones. The ratio of diferulic acid to ferulic acid was almost constant irrespective of coleoptile age and zone. An increase in the amount of ferulic and diferulic acids bound to cell wall polysaccharides correlated with a decrease in extensibility and with an increase in minimum stress-relaxation time and relaxation rate of the cell wall. The level of lignin in the cellulose fraction increased as coleoptiles aged, but this increase did not correlate with changes in mechanical properties of the cell walls. These results suggest that ferulic acid, ester-linked to cell wall polysaccharides, is oxidized to give diferulic acid, which makes the cell wall mechanically rigid by cross-linking matrix polysaccharides and results in limited cell extension growth. In addition, it is probable that the step of feruloylation of cell wall polysaccharides is rate-limiting in the formation of in-termolecular bridges by diferulic acid in Avena coleoptile cell walls.     

Trans-Golgi Network—An Intersection of Trafficking Cell Wall Components

The cell wall, a crucial cell compartment, is composed of a network of polysaccharides and proteins, providing structural support and protection from external stimuli. While the cell wall structure and biosynthesis have been extensively studied, very little is known about the transport of polysaccharides and other components into the developing cell wall. This review focuses on endomembrane trafficking pathways involved in cell wall deposition. Cellulose synthase complexes are assembled in the Golgi, and are transported in vesicles to the plasma membrane. Non-cellulosic polysaccharides are synthesized in the Golgi apparatus, whereas cellulose is produced by enzyme complexes at the plasma membrane. Polysaccharides and enzymes that are involved in cell wall modification and assembly are transported by distinct vesicle types to their destinations; however, the precise mechanisms involved in selection, sorting and delivery remain to be identified. The endomembrane system orchestrates the delivery of Golgi-derived and possibly endocytic vesicles carrying cell wall and cell membrane components to the newly-formed cell plate. However, the nature of these vesicles, their membrane compositions, and the timing of their delivery are largely unknown. Emerging technologies such as chemical genomics and proteomics are promising avenues to gain insight into the trafficking of cell wall components.     

Redox reactions in apoplast of growing cells

Redox reactions affecting the cell wall extensibility proceed in the apoplast of growing cells. The reactions involve dozens of oxidoreductases localized in cell walls (Class I and III heme peroxidases, FAD- and Cu-dependent amine oxidases, oxalate oxidase, ascorbate oxidase, superoxide dismutase, etc.) together with NADPH oxidase and quinone reductase of the plasma membrane. The cell wall extensibility decreases due to peroxidase-catalyzed phenolic cross-links of polymers. Cell growth is proven to be directly dependent on production of reactive oxygen species (ROS) in the apoplast. A special value is attached to hydroxyl radical OH?, which is able to locally cleave polysaccharides and, thus, increase wall extensibility. Generation of OH? results from one-electron reduction of H₂O₂ and, consequently, is related to the complex of enzymatic and spontaneous reactions of H₂O₂ turnover in the apoplast. The extensibility also depends on an ascorbate concentration in the apoplast and on a ratio of its oxidized to reduced forms. This dependence is expressed not only in the well-known down-regulation of phenols oxidation but also through pro-oxidant and signal activities. There is only indirect evidence of a role of apoplast-originated redox signaling in the cell growth regulation. In addition to ascorbate, the signaling may supposedly involve ROS, glutathione recycling reactions, numerous redox-sensitive peptides, and proteins localized in the cell wall and the plasma membrane.     

Collapsed xylem phenotype of Arabidopsis identifies mutants deficient in cellulose deposition in the secondary cell wall.

Recessive mutations at three loci cause the collapse of mature xylem cells in inflorescence stems of Arabidopsis. These irregular xylem (irx) mutations were identified by screening plants from a mutagenized population by microscopic examination of stem sections. The xylem cell defect was associated with an up to eightfold reduction in the total amount of cellulose in mature inflorescence stems. The amounts of cell wall-associated phenolics and polysaccharides were unaffected by the mutations. Examination of the cell walls by using electron microscopy demonstrated that the decreases in cellulose content of irx lines resulted in an alteration of the spatial organization of cell wall material. This suggests that a normal pattern of cellulose deposition may be required for assembly of lignin or polysaccharides. The reduced cellulose content of the stems also resulted in a decrease in stiffness of the stem material. This is consistent with the irregular xylem phenotype and suggests that the walls of irx plants are not resistant to compressive forces. Because lignin was implicated previously as a major factor in resistance to compressive forces, these results suggest either that cellulose has a direct role in providing resistance to compressive forces or that it is required for the development of normal lignin structure. The irx plants had a slight reduction in growth rate and stature but were otherwise normal in appearance. The mutations should be useful in facilitating the identification of factors that control the synthesis and deposition of cellulose and other cell wall components.     

Pectin Modification in Cell Walls of Ripening Tomatoes Occurs in Distinct Domains

The class of cell wall polysaccharides that undergoes the most extensive modification during tomato (Lycopersicon esculentum) fruit ripening is pectin. De-esterification of the polygalacturonic acid backbone by pectin methylesterase facilitates the depolymerization of pectins by polygalacturonase II (PGII). To investigate the spatial aspects of the de-esterification of cell wall pectins and the subsequent deposition of PGII, we have used antibodies to relatively methylesterified and nonesterified pectic epitopes and to the PGII protein on thin sections of pericarp tissue at different developmental stages. De-esterification of pectins and deposition of PGII protein occur in block-like domains within the cell wall. The boundaries of these domains are distinct and persistent, implying strict, spatial regulation of enzymic activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins strongly associated with cell walls of pericarp tissue at each stage of fruit development show ripening-related changes in this protein population. Western blots of these gels with anti-PGII antiserum demonstrate that PGII expression is ripening-related. The PGII co-extracts with specific pectic fractions extracted with imidazole or with Na2CO3 at 0[deg]C from the walls of red-ripe pericarp tissue, indicating that the strong association between PGII and the cell wall involves binding to particular pectic polysaccharides.     

Acetylesterase-mediated deacetylation of pectin impairs cell elongation, pollen germination, and plant reproduction

Pectin is a major component of the primary cell wall of higher plants. Some galacturonyl residues in the backbone of pectinaceous polysaccharides are often O-acetylated at the C-2 or C-3 position, and the resulting acetylesters change dynamically during the growth and development of plants. The processes involve both enzymatic acetylation and deacetylation. Through genomic sequence analysis, we identified a pectin acetylesterase (PAE1) from black cottonwood (Populus trichocarpa). Recombinant Pt PAE1 exhibited preferential activity in releasing the acetate moiety from sugar beet (Beta vulgaris) and potato (Solanum tuberosum) pectin in vitro. Overexpressing Pt PAE1 in tobacco (Nicotiana tabacum) decreased the level of acetyl esters of pectin but not of xylan. Deacetylation engendered differential changes in the composition and/or structure of cell wall polysaccharides that subsequently impaired the cellular elongation of floral styles and filaments, the germination of pollen grains, and the growth of pollen tubes. Consequently, plants overexpressing PAE1 exhibited severe male sterility. Furthermore, in contrast to the conventional view, PAE1-mediated deacetylation substantially lowered the digestibility of pectin. Our data suggest that pectin acetylesterase functions as an important structural regulator in planta by modulating the precise status of pectin acetylation to affect the remodeling and physiochemical properties of the cell wall's polysaccharides, thereby affecting cell extensibility.     

Changes in cell-wall properties of wheat (Triticum aestivum) roots during aluminum-induced growth inhibition

The effects of aluminum (Al) on root elongation, the mechanical extensibility of the cell wall, and the amount of cell-wall polysaccharides in the roots of Al-resistant (Atlas 66) and Al-sensitive (Scout 66) cultivars of wheat ( Triticum aestivum L.) were examined. Exposure to 10 μ M AlCl₃ for 6 h inhibited root elongation in Scout 66 but not in Atlas 66. It also decreased the mechanical extensibility of the cell wall in the roots of both cultivars, but prominently only in the roots of Scout 66. The amount of hemicellulose in the 10-mm region of root apex of Scout 66 was increased by the exposure to Al, especially in the apical regions. Al did not influence the neutral sugar composition of either pectin or hemicellulose in Scout 66 roots. However, Al increased the weight-average molecular mass of hemicellulosic polysaccharides and the amounts of wall-bound ferulic and diferulic acids in Scout 66 roots. These findings suggest that Al modifies the metabolism of cell-wall components and thus makes the cell wall thick and rigid, thereby inhibiting the growth of wheat roots.     

Role of xyloglucan in gravitropic bending of azuki bean epicotyl

The mechanism of the gravitropic bending was studied in azuki bean epicotyls. The cell wall extensibility of the lower side became higher than that of the upper side in the epicotyl bending upward. The contents of matrix polysaccharides of the cell wall (pectin and xyloglucan in hemicellulose-II) in the lower side became smaller than those in the upper side. The molecular mass of xyloglucans in the lower side decreased. After an epicotyl was fixed to a metal rod to prevent the bending, gravistimulation was applied. Fundamentally the same results were obtained with respect to rheological and chemical characteristics of the cell wall as those of epicotyls showing gravitropic bending. The present results suggested that the initial gravitropic bending was caused by the increase in extensibility of the lower side and the decrease in extensibility of the upper side via the change of the cell wall matrix, especially xyloglucans.     

Modification of chemical properties of cell walls by silicon and its role in regulation of the cell wall extensibility in oat leaves

Effects of silicon on the mechanical and chemical properties of cell walls in the second leaf of oat (Avena sativa L.) seedlings were investigated. The cell wall extensibility in the basal region of the second leaf was considerably higher than that in the middle and subapical regions. Externally applied silicon increased the cell wall extensibility in the basal region, but it did not affect the extensibility in the middle and subapical regions. The amounts of cell wall polysaccharides and phenolic compounds, such as diferulic acid (DFA) and ferulic acid (FA), per unit length were lower in the basal region than in the middle and subapical regions of the leaf, and silicon altered these amounts in the basal region. In this region, silicon decreased the amounts of matrix polymers and cellulose per unit length and of DFA and FA, both per unit length and unit matrix polymer content. Silicon treatment also lowered the activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) in the basal region. In contrast, the amount of silicon in cell walls increased in response to silicon treatment in three regions. These results suggest that in the basal region, silicon reduces the net wall mass and the formation of phenolic acid-mediated cross-linkages between wall polysaccharides. Such modifications of wall architecture may be responsible for the silicon-induced increase in the cell wall extensibility in oat leaves.     

Increased molecular mass of hemicellulosic polysaccharides is involved in growth inhibition of maize coleoptiles and mesocotyls under hypergravity conditions.

Elongation growth of dark grown maize (Zea mays L cv. Cross Bantam T51) coleoptiles and mesocotyls was suppressed by hypergravity at 30 g and above. Acceleration at 300 g significantly decreased the mechanical extensibility of cell walls of both organs. Hypergravity increased the amounts of hemicellulose and cellulose per unit length in mesocotyl walls, but not in coleoptile walls. The weight average molecular masses of hemicellulosic polysaccharides were also increased by hypergravity in both organs. On the other hand, the activities of beta-glucanases extracted from coleoptile and mesocotyl cell walls were decreased by hypergravity. These results suggest that the decreased activities of beta-glucanases by hypergravity cause an increase in the molecular mass of hemicellulosic polysaccharides of both organs. The upshift of molecular mass of hemicellulosic polysaccharides as well as the thickening of cell walls under hypergravity conditions seems to be involved in making the cell wall mechanically rigid, thereby inhibiting elongation growth of maize coleoptiles and mesocotyls.     

Cell wall and enzyme changes during the graviresponse of the leaf-sheath pulvinus of oat (Avena sativa)

The graviresponse of the leaf-sheath pulvinus of oat (Avena sativa) involves an asymmetric growth response accompanied by several asymmetric processes, including degradation of starch and cell wall synthesis. To understand further the cellular and biochemical events associated with the graviresponse, changes in cell walls and their constituents and the activities of related enzymes were investigated in excised pulvini. Asymmetric increases in dry weight with relatively symmetric increases in wall weight accompanied the graviresponse. Starch degradation could not account for increases in wall weight. However, a strong asymmetry in invertase activity indicated that hydrolysis of exogenous sucrose could contribute significantly to the increases in wall and dry weights. Most cell wall components increased proportionately during the graviresponse. However, beta-D-glucan did not increase symmetrically, but rather increased in proportion in lower halves of gravistimulated pulvini. This change resulted from an increase in glucan synthase activity in lower halves. The asymmetry of beta-D-glucan content arose too slowly to account for initiation of the graviresponse. A similar pattern in change in wall extensibility was also observed. Since beta-D-glucan was the only wall component to change, it is hypothesized that this change is the basis for the change in wall extensibility. Since wall extensibility changed too slowly to account for growth initiation, it is postulated that asymmetric changes in osmotic solutes act as the driving factor for growth promotion in the graviresponse, while wall extensibility acts as a limiting factor during growth.     

Cell wall integrity: Targeted post-synthetic modifications to reveal its role in plant growth and defense against pathogens

The plant cell wall, a dynamic network of polysaccharides and glycoproteins of significant compositional and structural complexity, functions in plant growth, development and stress responses. In recent years, the existence of plant cell wall integrity (CWI) maintenance mechanisms has been demonstrated, but little is known about the signaling pathways involved, or their components. Examination of key mutants has shed light on the relationships between cell wall remodeling and plant cell responses, indicating a central role for the regulatory network that monitors and controls cell wall performance and integrity. In this review, we present a short overview of cell wall composition and discuss post-synthetic cell wall modification as a valuable approach for studying CWI perception and signaling pathways.