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.     

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.     

Cell wall changes involved in the automorphic curvature of rice coleoptiles under microgravity conditions in space

Seedlings of rice (Oryza sativa L. cv. Koshihikari and cv. Tan-ginbozu) were cultivated on board the Space Shuttle STS-95 mission and changes in the morphology and the cell wall properties of coleoptiles were analyzed. In space, rice coleoptiles showed a spontaneous (automorphic) curvature toward the caryopsis in the elongating region. The angle of automorphic curvature was larger in Koshihikari than in a gibberellin-deficient dwarf cultivar, Tan-ginbozu, and the angle gradually decreased during the growth of coleoptiles in both cultivars. The more quickly expanding convex side of the bending region of the rice coleoptiles showed a greater extensibility of the cell wall than the opposite side. There was a significant correlation between the angle of curvature and the difference in the cell wall extensibility between the convex and the concave sides. Both the levels of the cell wall polysaccharides per unit length of coleoptile and the ratio of high-molecular-mass polysaccharides in the hemicellulose fraction were lower in the convex side than the concave one. Also, the activity of (13),(14)--glucanases in the cell wall was higher in the convex side than the concave one. These results suggest that the uneven modifications of cell wall metabolism bring about the difference in the levels and the molecular size of the cell wall polysaccharides, thereby causing the difference in capacity of the cell wall to expand between the dorsal and the ventral sides, leading to the automorphic curvature of rice coleoptiles in space. The data also suggest the involvement of gibberellins in inducing the automorphic curvature under microgravity conditions.     

Effects of temperature on the cell wall and osmotic properties in dark-grown rice and azuki bean seedlings

The mechanism inducing the difference in growth rate under various temperature (10–50 °C) conditions was analyzed using rice and azuki bean seedlings. The growth rate of rice coleoptiles and azuki bean epicotyls increased as temperature increased up to 40 and 30 °C, respectively, and the elongation was retarded at a higher temperature. The cell wall extensibility of rice coleoptiles and azuki bean epicotyls also showed the highest value at 40 and 30 °C, respectively, and became smaller as the temperature rose or dropped from the optimum. The opposite tendency was observed in the minimum stress-relaxation time of the cell wall. On the other hand, the cellular osmotic concentration of rice coleoptiles and azuki bean epicotyls was lower at the temperature optimum for growth at 40 and 30 °C, respectively. When rice and azuki bean seedlings grown at 10, 20, 40, or 50 °C were transferred to the initial temperature (30 °C), the growth rate of coleoptiles and epicotyls was mostly elevated, concomitant with an increase in the cell wall extensibility. The growth rate was correlated with the cell wall mechanical parameters in both materials. These results suggest that the environmental temperature modulates the growth rate of plant shoots by affecting mainly the mechanical properties of the cell wall. Electronic Publication     

Cellular basis for the automorphic curvature of rice coleoptiles on a three-dimensional clinostat: possible involvement of reorientation of cortical microtubules

Coleoptiles of rice (Oryza sativa L.) show a spontaneous (automorphic) curvature toward the caryopsis under microgravity conditions. The possible involvement of the reorientation of cortical microtubules in automorphic curvature was studied in rice coleoptiles grown on a three-dimensional clinostat. When rice seedlings that had been grown in the normal gravitational field were transferred to the clinostat in the dark, cortical microtubules of epidermal cells in the dorsal side of the coleoptiles oriented more transversely than the ventral side within 0.5 h. The rotation on the clinostat also increased the cell wall extensibility in the dorsal side and decreased the extensibility in the ventral side, and induced automorphic curvature. The reorientation of cortical microtubules preceded the changes in the cell wall extensibility and the curvature. The irradiation of rice seedlings with white light from above inhibited microtubule reorientation and changes in the cell wall extensibility, as well as curvature of coleoptiles. Also, colchicine, applied to the bending region of coleoptiles, partially inhibited the automorphic curvature. These results suggest that reorientation of cortical microtubules is involved in causing automorphic curvature in rice coleoptiles on the clinostat.     

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.     

Growth promotion and an increase in cell wall extensibility by silicon in rice and some other Poaceae seedlings

The effect of silicon on organ growth and its mechanisms of action were studied in rice ( Oryza sativa L. cv. Koshihikari), oat ( Avena sativa L. cv. Victory), and wheat ( Triticum aestivum L. cv. Daichino-Minori) seedlings grown in the dark. Applying silicon in the form of silicic acid to these seedlings via culture solution resulted in growth promotion of third (rice) or second (oat and wheat) leaves. The optimal concentration of silicon was 5-10 mM. No growth promotion was observed in early organs, such as coleoptiles or first leaves. In silicon-treated rice third leaves, the epidermal cell length increased, especially in the basal regions, without any effect on the number of cells, showing that silicon promoted cell elongation but not cell division. Silicon also increased the cell wall extensibility significantly in the basal regions of rice third leaves. These results indicate that silicon stimulates growth of rice and some other Poaceae leaves by increasing cell wall extensibility.     

Phytochrome action in Oryza sativa L.

Summary The mechanical properties of the cell wall were measured in coleoptiles of totally etiolated rice seedlings. Coleoptiles were either decapitated or briefly exposed to red (R) and/or far-red (FR) light. The elastic and plastic extensibilities of the cell wall changed with age (length) of the coleoptiles. Decapitation and exposure to R induced changes in these properties, and the time-courses were similar. Following decapitation or R irradiation, the plastic extensibility of the cell wall decreased more conspicuously than elastic extensibility. Exogenous application of auxin immediately following decapitation alleviated the effect of removal of the tip. FR irradiation reduced both kinds of extensibilities, but its effect was much less than that of R, and it reversed the R-induced effect to the level of tissue treated with FR only. In repeated R-FR treatments, the decrease of elastic extensibility by R and its reversal by FR could be repeated, but the effect of a second irradiation with R after FR on plastic extensibility was not as apparent as that of the first. Reduction of cell-wall extensibility of etiolated rice coleoptiles caused by R light appeared, at least partly, to be due to a reduced auxin supply in the elongating region from the tip, similar to that caused by decapitation.     

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.     

Growth promotion and an increase in cell wall extensibility by silicon in rice and some other Poaceae seedlings

The effect of silicon on organ growth and its mechanisms of action were studied in rice (Oryza sativa L. cv. Koshihikari), oat (Avena sativa L. cv. Victory), and wheat (Triticum aestivum L. cv. Daichino-Minori) seedlings grown in the dark. Applying silicon in the form of silicic acid to these seedlings via culture solution resulted in growth promotion of third (rice) or second (oat and wheat) leaves. The optimal concentration of silicon was 5–10 mM. No growth promotion was observed in early organs, such as coleoptiles or first leaves. In silicon-treated rice third leaves, the epidermal cell length increased, especially in the basal regions, without any effect on the number of cells, showing that silicon promoted cell elongation but not cell division. Silicon also increased the cell wall extensibility significantly in the basal regions of rice third leaves. These results indicate that silicon stimulates growth of rice and some other Poaceae leaves by increasing cell wall extensibility. Received: July 31, 2001 / Accepted: September 18, 2001     

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.     

Correlation between cell wall extensibility and the content of diferulic and ferulic acids in cell walls of Oryza sativa coleoptiles grown under water and in air

Rice ( Oryza sativa L. cv. Sasanishiki) coleoptiles grown under water achieved greater length than those grown either in air or under water with constant air bubbling. The extensibility of cell walls in coleoptiles grown under water was larger than that in the other treatments. Per unit length of the coleoptile, the content of ferulic and diferulic acids ester-linked to hemicelluloses was higher in air and bubbling type coleoptiles than in water type ones. The extensibility of the coleoptile cell walls correlated with the content of diferulic acids per unit length and per hemicellulose, suggesting that the enhancement of the formation of diferulic acid bridges in hemicelluloses in air or under water with air bubbling makes the cell walls mechanically rigid; thereby inhibiting cell elongation in rice coleoptiles. In addition, the ratio of diferulic acid to ferulic acid was almost constant irrespective of coleoptile age, zone and growth conditions, suggesting that the feruloylation of hemicelluloses is rate-limiting in the formation of diferulic acid bridges in the cell walls of rice coleoptiles.     

Viscoelastic versus plastic cell wall extensibility in growing seedling organs: a contribution to avoid some misconceptions

In a recent publication (Kutschera, 1996), it was reported thatthe cell walls of growing rye coleoptiles exhibit irreversible(plastic) extensibility in a rheological extension test. Basicallysimilar measurements with cell walls of maize coleoptiles hadpreviously shown that the apparent plastic extensibility determinedin this material is in reality due to the slowly reversible(viscoelastic) extensibility of the walls. A recent reinvestigationof this discrepancy showed that rye coleoptile walls also behaveas a perfectly viscoelastic material if precautions are takento prevent measuring artefacts. Similar results were obtainedwith cell walls from the growing zone of various other seedlingorgans (maize mesocotyl, maize root, cucumber hypocotyl). Itis concluded that plastic extensibility has not yet been convincinglydemonstrated by rheological tests that determine the intrinsicmaterial properties of cell walls. Reported changes in mechanicalmaterial properties of cell walls produced by growth-controllingfactors such as auxin or light may generally be attributed tochanges in viscoelasticity which are not directly related tothe chemo-rheological processes controlling wall extension ofgrowing cells. Key words: Cell wall extensibility, extension growth, plastic cell wall extensibility, viscoelastic cell wall extensibility     

Role of Polysaccharide Synthesis in Elongation Growth and Cell Wall Loosening in Intact Rice Coleoptiles

2,6-Dichlorobenzonitrile (DCB) inhibited only increases in levelsof the cellulosic polysac-charides while monensin and galactoseinhibited increases in levels of both the cellulosic and thematrix polysaccharides in intact rice coleoptiles that weresubmerged in water. Elongation growth of rice coleoptiles wassuppressed by DCB at 10^–6 M, by monensin at 10^–7M, and by galactose at 3 ? 10^–3 M and above. Thus, thesynthesis of both the cellulosic and the matrix polysaccharidesis essential for the elongation of intact rice coleoptiles.These inhibitors increased the minimum stress-relaxation timeand the relaxation rate and they decreased the mechanical extensibilityof the cell wall, indicating that they inhibited cell wall loosening.The concentrations of the inhibitors required for inhibitionof cell wall loosening were higher than those for suppressionof elongation. The data suggest that polysaccharides synthesisplays two roles in elongation. It keeps the cell wall in a "loosened"condition by producing new extensible cell walls, while itsother role is probably related to the fixation or extensionof polymers already present in the cell wall. (Received November 15, 1990; Accepted May 23, 1991)     

Increased Molecular Mass of Hemicellulosic Polysaccharides is Involved in Growth Inhibition of Maize Coleoptiles and Mesocotyls under Hypergravity Conditions

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 β-glucanases extracted from coleoptile and mesocotyl cell walls were decreased by hypergravity. These results suggest that the decreased activities of β-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. Received 22 February 1999/ Accepted in revised form 20 April 1999     

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.     

Involvement of Cell Wall-Bound Diferulic Acid in Light-Induced Decrease in Growth Rate and Cell Wall Extensibility of Oryza Coleoptiles

Irradiation of white fluorescent light (5 W m^–2) inhibitedthe growth of Oryza coleoptiles. Light irradiation increasedstress-relaxation parameters of coleoptile cell walls, minimumstressrelaxationtime and relaxation rate, and decreased cellwall extensibility (strain/load). Under light conditions, thecontents of ferulic and diferulic acids ester-linked to thehemicellulosic arabinose residue in cell walls increased andcorrelated with the modification of the cell wall mechanicalproperties. These results suggest that light irradiation enhancesthe formation of diferulic acid bridges in hemicelluloses, makingcell walls mechanically rigid and thus inhibits cell elongationin rice coleoptiles. Also, irrespective of coleoptile age orthe presence of light, the ratio of diferulic acid to ferulicacid was almost constant, suggesting that the rate limitingstep in the formation of diferulic acid bridges in Oryza cellwalls is in the step of feruloylation. (Received September 24, 1991; Accepted December 3, 1991)     

Phenylalanine ammonia-lyase and cell wall peroxidase are cooperatively involved in the extensive formation of ferulate network in cell walls of developing rice shoots

The relationship between the formation of cell wall-bound ferulic acid (FA) and diferulic acid (DFA) and the change in activities of phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) was studied in rice shoots. The length and the fresh mass of shoots increased during the growth period from day 4 to 6, while coleoptiles ceased elongation growth on day 5. The amounts of FA and DFA isomers as well as cell wall polysaccharides continued to increase during the whole period. The activities of PAL and CW-PRX greatly increased in the same manner during the period. There were close correlations between the PAL activity and ferulate content or between the CW-PRX activity and DFA content. The expression levels of investigated genes for PAL and putative CW-PRX showed good accordance with the activities of these enzymes. These results suggest that increases in PAL and CW-PRX activities are cooperatively involved in the formation of ferulate network in cell walls of rice shoots and that investigated genes may be, at least in part, associated with the enzyme activities. The substantial increase in such network probably causes the maturation of cell walls and thus the cessation of elongation growth of coleoptiles.     

Analysis of the Growth Response of Air-Grown Rice Coleoptiles to Submergence

The effect of submergence of air-grown rice seedlings (Oryza sativa L. var. Sasanishiki) on coleoptile growth and ultrastructure, extensibility and chemical composition of the cell walls was investigated. The lag-time between start of submergence and the onset of the enhancement of growth was less than 4 h. The growth response was associated with a drastic thinning of the cell walls and an increase in wall extensibility. At the outer epidermal wall of both air-grown and submerged coleoptiles electron-dense (osmiophilic) particles were detected. During submergence, the net accumulation of cellulose and hemicellulose was reduced, but the increase in pectic substances was unaffected. Submergence caused an 80% inhibition of the net accumulation of wall-bound phenolics (ferulic- and diferulic acid) compared with air-grown controls. The osmotic concentration of the tissue saps was not affected by submergence. Our results support the hypothesis that rapid coleoptile elongation under water is caused by an inhibition of the formation of phenolic cross-links between matrix polysaccharides via diferulate, which results in a mechanical stiffening of the cell walls in the air-grown coleoptile.     

Role of hydroxyproline-rich cell wall protein in growth regulation of rice coleoptiles grown on or under water

The elongation of both intact and excised rice coleoptiles waspromoted when they were submerged in water. Amino acid analysisof the cell wall revealed that air-type coleoptiles (grown onthe surface of water) contained more hydroxyproline than water-typeones (grown under water). The suppression of hydroxylation ofpeptidyl proline under water was confirmed with air-type sectionsby examining the imino acid content, ¹⁴C-proline incorporationinto the cell wall and its modification by ,'-dipyridyl. Also,dipyridyl significantly promoted the growth of floated sectionsto the level of submerged sections. Therefore, the lower hydroxyprolinecontent caused by lower oxygen tension in water is concludedto be one of the factors promoting growth of rice coleoptilesunder water. However, the hydroxyproline content in the cellwall decreased with growth of both air-and water-type coleoptiles;thus hydroxyproline-rich cell wall protein can not be regardedas the final growth cessation factor in rice coleoptiles. (Received December 17, 1979; )