Growth stimulation in inflorescences of an Arabidopsis tubulin mutant under microgravity conditions in space.

Cortical microtubules are involved in plant resistance to hypergravity, but their roles in resistance to 1 g gravity are still uncertain. To clarify this point, we cultivated an Arabidopsis α-tubulin 6 mutant (tua6) in the Cell Biology Experiment Facility on the Kibo Module of the International Space Station, and analyzed growth and cell wall mechanical properties of inflorescences. Growth of inflorescence stems was stimulated under microgravity conditions, as compared with ground and on-orbit 1 g conditions. The stems were 10-45% longer and their growth rate 15-55% higher under microgravity conditions than those under both 1 g conditions. The degree of growth stimulation tended to be higher in the tua6 mutant than the wild-type Columbia. Under microgravity conditions, the cell wall extensibility in elongating regions of inflorescences was significantly higher than the controls, suggesting that growth stimulation was caused by cell wall modifications. No clear differences were detected in any growth or cell wall property between ground and on-orbit 1 g controls. These results support the hypothesis that cortical microtubules generally play an important role in plant resistance to the gravitational force.

Xyloglucan endotransglucosylase and cell wall extensibility.

Transgenic tomato hypocotyls with altered levels of an XTH gene were used to study how XET activity could affect the hypocotyl growth and cell wall extensibility. Transgenic hypocotyls showed significant over-expression (line 13) or co-suppression (line 33) of the SlXTH1 in comparison with the wild type, with these results being correlated with the results on specific soluble XET activity, suggesting that SlXTH1 translates mainly for a soluble XET isoenzyme. A relationship between XET activity and cell wall extensibility was found, and the highest total extensibility was located in the apical hypocotyl segment of the over-expressing SlXTH1 line, where the XET-specific activity and hypocotyl growth were also highest compared with the wild line. Also, in the co-suppression SlXTH1 line, total extensibility values were lower than in the wild type line. The study of linkages between cell wall polysaccharides by FTIR showed that hypocotyls over-expressing SlXTH1 and having a higher XET-specific activity, were grouped away from the wild line, indicating that the linkages between pectins and between cellulose and xyloglucans might differ. These results suggested that the action of the increased XET activity in the transgenic line could be responsible for the cell wall structural changes, and therefore, alter the cell wall extensibility. On the other hand, results on xyloglucan oligosaccharides composition of the xyloglucan by MALDI TOF-MS showed no differences between lines, indicating that the xyloglucan structure was not affected by the XET action. These results provide evidences that XTHs from group I are involved mainly in the restructuring of the cell wall during growth and development, but they are not the limiting factor for plant growth.

Hypergravity-induced changes in gene expression in Arabidopsis hypocotyls.

Under hypergravity conditions, the cell wall of stem organs becomes mechanically rigid and elongation growth is suppressed, which can be recognized as the mechanism for plants to resist gravitational force. The changes in gene expression by hypergravity treatment were analyzed in Arabidopsis hypocotyls by the differential display method, for identifying genes involved in hypergravity-induced growth suppression. Sixty-two cDNA clones were expressed differentially between the control and 300 g conditions: the expression levels of 39 clones increased, whereas those of 23 clones decreased under hypergravity conditions. Sequence analysis and database searching revealed that 12 clones, 9 up-regulated and 3 down-regulated, have homology to known proteins. The expression of these genes was further analyzed using RT-PCR. Finally, six genes were confirmed to be up-regulated by hypergravity. One of such genes encoded 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGR), which catalyzes a reaction producing mevalonic acid, a key precursor of terpenoids such as membrane sterols and several types of hormones. The expression of HMGR gene increased within several hours after hypergravity treatment. Also, compactin, an inhibitor of HMGR, prevented hypergravity-induced growth suppression, suggesting that HMGR is involved in suppression of Arabidopsis hypocotyl growth by hypergravity. In addition, hypergravity increased the expression levels of genes encoding CCR1 and ERD15, which were shown to take part in the signaling pathway of environmental stimuli such as temperature and water, and those of the alpha-tubulin gene. These genes may be involved in a series of cellular events leading to growth suppression of stem organs under hypergravity conditions.

Growth restoration in azuki bean and maize seedlings by removal of hypergravity stimuli.

Hypergravity stimuli, gravitational acceleration of more than 1 x g, decrease the growth rate of azuki bean epicotyls and maize coleoptiles and mesocotyls by decreasing the cell wall extensibility via an increase in the molecular mass of matrix polysaccharides. An increase in the pH in the apoplastic fluid is hypothesized to be involved in the processes of the increase in the molecular mass of matrix polysaccharides due to hypergravity. However, whether such physiological changes by hypergravity are induced by normal physiological responses or caused by physiological damages have not been elucidated. In the present study, we examined the effects of the removal of hypergravity stimuli on growth and the cell wall properties of azuki bean and maize seedlings to clarify whether the effects of hypergravity stimuli on growth and the cell wall properties are reversible or irreversible. When the seedlings grown under hypergravity conditions at 300 x g for several hours were transferred to 1 x g conditions, the growth rate of azuki bean epicotyls and maize coleoptiles and mesocotyls greatly increased within a few hours. The recovery of growth rate of these organs was accompanied by an immediate increase in the cell wall extensibility, a decrease in the molecular mass of matrix polysaccharides, and an increase in matrix polysaccharide-degrading activities. The apoplastic pH also decreased promptly upon the removal of hypergravity stimuli. These results suggest that plants regulate the growth rate of shoots reversibly in response to hypergravity stimuli by changing the cell wall properties, by which they adapt themselves to different gravity conditions. This study also revealed that changes in growth and the cell wall properties under hypergravity conditions could be recognized as normal physiological responses of plants. In addition, the results suggest that the effects of microgravity on plant growth and cell wall properties should be reversible and could disappear promptly when plants are transferred from microgravity to 1 x g. Therefore, plant materials should be fixed or frozen on orbit for detecting microgravity-induced changes in physiological parameters after recovering the materials to earth in space experiments.

Gravitational force regulates elongation growth of Arabidopsis hypocotyls by modifying xyloglucan metabolism.

Growth of dark-grown Arabidopsis hypocotyls was suppressed under hypergravity conditions (300 g), or was stimulated under microgravity conditions in space (Space Shuttle STS-95). The mechanical extensibility of cell walls decreased and increased under hypergravity and microgravity conditions, respectively. The amounts of cell wall polysaccharides (pectin, hemicellulose-I, hemicellulose-II and cellulose) per unit length of hypocotyls increased under hypergravity conditions, and decreased under microgravity conditions. The amount and the molecular mass of xyloglucans also increased under the hypergravity conditions, while those decreased under microgravity conditions. The activity of xyloglucan-degrading enzymes extracted from hypocotyl cell walls decreased and increased under hypergravity and microgravity conditions, respectively. These results indicate that the amount and the molecular mass of xyloglucans are affected by the magnitude of gravity and that such changes are caused by changes in xyloglucan-degrading activity. Modifications of xyloglucan metabolism as well as the thickness of cell walls by gravity stimulus may be the primary event determining the cell wall extensibility, thereby regulating the growth rate of Arabidopsis hypocotyls.

Automorphogenesis and gravitropism of plant seedlings grown under microgravity conditions.

Plant seedlings exhibit automorphogenesis on clinostats. The occurrence of automorphogenesis was confirmed under microgravity in Space Shuttle STS-95 flight. Rice coleoptiles showed an inclination toward the caryopsis in the basal region and a spontaneous curvature in the same adaxial direction in the elongating region both on a three-dimensional (3-D) clinostat and in space. Both rice roots and Arabidopsis hypocotyls also showed a similar morphology in space and on the 3-D clinostat. In rice coleoptiles, the mechanisms inducing such an automorphic curvature were studied. The faster-expanding convex side of rice coleoptiles showed a higher extensibility of the cell wall than the opposite side. Also, in the convex side, the cell wall thickness was smaller, the turnover of the matrix polysaccharides was more active, and the microtubules oriented more transversely than the concave side, and these differences appear to be causes of the curvature. When rice coleoptiles grown on the 3-D clinostat were placed horizontally, the gravitropic curvature was delayed as compared with control coleoptiles. In clinostatted coleoptiles, the corresponding suppression of the amyloplast development was also observed. Similar results were obtained in Arabidopsis hypocotyls. Thus, the induction of automorphogenesis and a concomitant decrease in graviresponsiveness occurred in plant shoots grown under microgravity conditions.

A new type of endo-xyloglucan transferase devoted to xyloglucan hydrolysis in the cell wall of azuki bean epicotyls.

A new type of xyloglucan-degrading enzyme was isolated from the cell wall of azuki bean (Vigna angularis Ohwi et Ohashi cv. Takara) epicotyls and its characteristics were determined. The enzyme was purified to apparent homogeneity by Concanavalin A (Con A)-Sepharose, cation exchange, and gel filtration columns from a cell wall protein fraction extracted with 1 M sodium chloride. The purified enzyme gave a single protein band of 33 kDa on SDS-PAGE. The enzyme specifically cleaved xyloglucans and showed maximum activity at pH 5.0 when assayed by the iodine-staining method. An increase in reducing power in xyloglucan solution was clearly detected after treatment with the purified enzyme. Xyloglucans with molecular masses of 500 and 25 kDa were gradually hydrolyzed to 5 kDa for 96 h without production of any oligo- or monosaccharide with the purified enzyme. The purified enzyme did not show an endo-type transglycosylation reaction, even in the presence of xyloglucan oligosaccharides. Partial amino acid sequences of the enzyme shared an identity with endo-xyloglucan transferase (EXGT) family, especially with xyloglucan endotransglycosylase (XET) from nasturtium. These results suggest that the enzyme is a new member of EXGT devoted solely to xyloglucan hydrolysis.

[Usefulness of the Centrifuge Accommodation Module for analyzing gravity responses in plant seedlings].

Onboard centrifuges are indispensable tools for clarifying the effects of microgravity on various physiological processes in plant seedlings. Centrifuges are basically attached to the incubators designed for the International Space Station (ISS). However, because of the limitation in size, that loaded to the Cell Biology Experiment Facility (CBEF) is usable only to some small seedlings such as Arabidopsis. The Centrifuge Accommodation Module (CAM) has great advantages in the size and the amounts of plant materials feasible to load, the quality of acceleration produced, and the easiness of operation on it. The CAM is an apparatus that characterizes the ISS most and its construction on schedule is highly expected.

Changes in the apoplastic pH are involved in regulation of xyloglucan breakdown of azuki bean epicotyls under hypergravity conditions.

Hypergravity inhibited elongation growth of azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls by decreasing the mechanical extensibility of cell walls via the increase in the molecular mass of xyloglucans [Soga et al. (1999) Plant Cell Physiol. 40: 581]. Here, we report that the pH value of the apoplastic fluid in epicotyls increased from 5.8 to 6.6 by hypergravity (300 x g) treatment. When the xyloglucan-degrading enzymes extracted from cell walls of the 1 x g control epicotyls were assayed in buffer at pH 6.6 and 5.8, the activity at pH 6.6 was almost half of that at pH 5.8. In addition, when enzymically active cell wall preparations obtained from 1 x g control epicotyls were autolyzed in buffer at pH 5.8 and 6.6 and then xyloglucans were extracted from the autolyzed cell walls, the molecular mass of xyloglucans incubated at pH 5.8 decreased during the autolysis, while that at pH 6.6 did not change. Thus, the xyloglucans were not depolymerized by autolysis at the pH value (6.6) observed in the hypergravity-treated epicotyls. These findings suggest that in azuki bean epicotyls, hypergravity decreases the activities of xyloglucan-degrading enzymes by increasing the pH in the apoplastic fluid, which may be involved in the processes of the increase in the molecular mass of xyloglucans, leading to the decrease in the cell wall extensibility.

Involvement of ethylene and gibberellin signalings in chromosaponin I-induced cell division and cell elongation in the roots of Arabidopsis seedlings.

Chromosaponin I (CSI), a triterpenoid saponin isolated from pea, stimulates the growth of roots in Arabidopsis thaliana seedlings on wetted filter paper in the light for 14 d. The growth rates of roots in Columbia (Col) and Landsberg erecta (Ler) wild-types were 0.92 and 0.26 mm d(-1), respectively, and they were accelerated to 3.46 (Col) and 2.20 (Ler) mm d(-1) by treating with 300 microM CSI. The length of mature epidermal cells was increased by 1.8-fold (Col) and 2.81-fold (Ler) compared with control and the number of epidermal cells was increased by a factor of 1.65 (Col) and 2.12 (Ler). Treatment with 2-aminoethoxyvinylglycine (AVG), an inhibitor of ethylene biosynthesis, also increased cell length but not cell number. The effects of CSI on root growth were not detected in the ethylene-insensitive mutant ein2-1. CSI did not inhibit ethylene production but stimulated the growth of roots in ctr1-1, the constitutive triple response mutant for ethylene, indicating that CSI inhibits ethylene signaling, especially downstream of CTR1. In the GA-insensitive mutant gai and the mutant spy-3, in which the basal level of GA signaling is activated, CSI did not increase cell number, although both CSI and AVG stimulated cell elongation in these mutants. These results suggest that the inhibition of ethylene signaling is the cause of CSI-induced cell elongation. A possible involvement of both GA and ethylene signalings is discussed for the CSI-induced cell division.

Flower stalk segments of Arabidopsis thaliana ecotype Columbia lack the capacity to grow in response to exogenously applied auxin.

Exogenously applied IAA stimulated cell elongation of segments excised from flower stalks of Arabidopsis thaliana ecotype Landsberg erecta (Ler) by increasing the cell wall extensibility, but it did not affect that of ecotype Columbia (Col). Treatment with a low pH buffer solution (pH 4.0) or fusicoccin (FC), a reagent activating H(+)-ATPases, significantly increased the cell wall extensibility and promoted elongation growth of flower stalk segments of both ecotypes, indicating that the flower stalk segments of Col possess the capacity to grow under acidic pH conditions. IAA promoted the proton excretion in segments of Ler but not of Col. On the other hand, FC increased the proton excretion in segments of Col as much as that of Ler. These results suggest that IAA activates the plasma membrane H(+)-ATPases in the segments of Ler but not those of Col, while FC activates them in both ecotypes. Flower stalks of Col may lack the mechanisms of activation by IAA of the plasma membrane H(+)-ATPases.

Hypergravity-induced increase in the apoplastic pH and its possible involvement in suppression of beta-glucan breakdown in maize seedlings.

Elongation growth of both coleoptiles and mesocotyls of maize (Zea mays L. cv. Cross Bantam T51) seedlings was inhibited under basipetal hypergravity (300 g) conditions. Hypergravity increased the pH of the apoplastic fluid of coleoptiles from 5.0 to 5.5 and mesocotyls from 5.2 to 5.7. When beta-1,3:1,4-D-glucanases (beta-glucanases) extracted from cell walls of the 1-g control coleoptiles and mesocotyls were assayed at pH 5.0 and 5.5 for coleoptiles, and at 5.2 and 5.7 for mesocotyls, respectively, the activity in the increased pH conditions was significantly lower than that in the control pH conditions. During the autolysis of the enzymically active cell wall preparations obtained from 1-g control organs, a molecular mass downshift of hemicellulosic polysaccharides occurred in cell walls. This downshift was suppressed in the increased pH conditions as compared with the control pH conditions. It was reported that hypergravity increased the molecular mass of hemicellulosic polysaccharides by decreasing the beta-glucanase activity, and thereby decreased the mechanical extensibility of cell walls in maize coleoptiles and mesocotyls. These results suggest that, in maize coleoptiles and mesocotyls, hypergravity-induced increase in the pH in the apoplastic fluid is involved in the reduction of the activity of beta-glucanases which, in turn, causes an increase in the molecular mass of hemicellulosic polysaccharides and inhibits elongation growth.