Computer simulation of osmotic expansion and shrinkage in okra hypocotyl segments.

Okra hypocotyl segments were incubated in solutions of 0.3 or 0.4 M sorbitol at various temperatures and their shrinkage was measured. The result yielded an apparent activation energy for shrinkage of 4.8 kcal/mol, which is close to that of the viscosity of water. This coincidence suggests that the viscosity of water, i.e., the reciprocal function of water conductivity, is a limiting factor for osmotic shrinkage. Abrasion of okra hypocotyl segments with Carborundum substantially increased the rate of their osmotic shrinkage, indicating that the cuticle is the major barrier to water uptake by segments. The apparent activation energy for osmotic shrinkage was 4.5 kcal/mol in abraded segments. By introducing water conductivity into an algorithm, osmotic shrinkage and expansion of hypocotyl segments was successfully predicted by computation with this algorithm. Hence the extent of the contribution of water conductivity in osmotic shrinkage and expansion can be evaluated. Based on this simulation, water conductivity was identified as one of the major factors in governing the elongation growth rate of cells along with the osmotic pressure of the cell sap and the mechanical properties of the cell wall.

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.

Irregular meiosis in a somatic hybrid between S. bulbocastanum and S. tuberosum detected by species-specific PCR markers and cytological analysis.

A system of randomly amplified polymorphic DNA (RAPD) markers was developed to facilitate the transfer of S. bulbocastanum (blb) genes into the S. tuberosum (tbr) genome by hybridization and backcrossing. DNA from tbr, blb and the hexaploid hybrid was used as a template for polymerase chain reaction (PCR) amplification. Polymorphic RAPD products, originating from 10-mer primers, specific for blb were cloned and sequenced at their ends to allow the synthesis of 18-mer primers. The 18-mer primers allowed a more reproducible assay than the corresponding RAPDs. Of eight 18-mer primer pairs, four amplified the expected products specific for blb. However, the stringency of the primer annealing conditions needed to be carefully optimized to avoid amplification of the homeologous tbr product, suggesting that the original RAPD polymorphisms were due to single base-pair changes rather than deletions or insertions. Two primers used for amplification of backcross 2 progeny segregated in a 1∶1 (presence:absence) ratio; the other two were unexpectedly absent. The most likely explanation for the loss of these markers is irregular meiosis in the original hexaploid hybrid and subsequent elimination of chromosomes. Cytological analysis of the meiosis in the hybrid demonstrated widespread irregular pairing and the presence of lagging univalents. In addition, the first backcross individual used as the parent for the second backcross had 54 chromosomes instead of the predicted 60. In conclusion, our results demonstrate that PCR technology can be used for the efficient isolation of taxon-specific markers in Solanum. Furthermore, by the use of these markers we detected the loss of chromosomes that was subsequently shown by cytological analysis to be caused by irregular meiosis of the somatic hybrid.

DNA Replication-Dependent Histone H2A mRNA Expression in Pea Root Tips.

Histone H2A mRNA is selectively expressed in scattered subpopulations of cells in the pea (Pisum sativum) root apical meristem. To study whether this specific expression was associated with the cell cycle, a double-labeling technique was used to identify cells replicating DNA during S phase and those expressing H2A mRNA. Cells in S phase were detected by [3H]thymidine incorporation and autoradiography, whereas cells containing H2A mRNA were identified by in situ hybridization using digoxigenin-labeled probes. Approximately 92% of the [3H]thymidine-labeled S-phase cells expressed H2A mRNA and 85% of cells that expressed H2A mRNA were in S phase. In root tissue located basal to the promeristem, synchronous co-located expression was observed in scattered packets of proliferating cells. Furthermore, neither H2A mRNA nor S-phase cells could be detected within the quiescent center or mature root cap. When DNA synthesis was inhibited with hydroxyurea, a commensurate and specific decrease in steady-state levels of H2A mRNA was found. We conclude that cell-specific expression of pea histone H2A mRNA is replication dependent and that H2A mRNA is transiently accumulated during a period of the cell cycle that mostly overlaps the S phase. We propose that the overlap between H2A expression and S phase could occur if H2A mRNA accumulation began in late G1 and abated in late S.

Cell-specific expression of plant histone H2A genes.

Histone H2A is a component of eukaryotic chromatin whose expression has not been studied in plants. We isolated and characterized a tomato and a pea cDNA encoding histone H2A. We found that in tomato H2A is encoded by a small gene family and that both the pea and the tomato mRNAs are polyadenylated. Tomato H2A has 82% amino acid residue identity to pea H2A, 83% to wheat, and 65% to human and yeast H2A. Plant H2As differ from fungal and animal H2As in their amino-terminal and carboxy-terminal regions. Carboxy-terminal plant H2A regions contain the motif SPKK, a peptide implicated in binding of A/T-rich DNA regions. By using RNA gel blot analysis, we determined that the steady-state mRNA level of these genes was abundant in apices and early developing fruit and very low in mature tissues. In situ RNA hybridization showed strong spatial regulation because the mRNA was abundant in some cells and not detectable in others. In tomato shoot tips, H2A-expressing cells were distributed irregularly in or near meristems. In tomato or pea root tips, expressing cells were concentrated near the apex, and their distribution was consistent with that expected of cycling cells. Other H2A transcripts were found in nondividing cortical cells that are known to undergo endoduplication during the late maturation phase of primary development.

Inhibition of Acid-Enhanced Elongation of Zea mays Root Segments by Galactose.

The effect of sugars and metabolic inhibitors on the elongation of Zea mays root segments was analyzed by a rhizometer which records the elongation of each of 32 root segments at the same time. Galactose suppressed the acid-enhanced rapid elongation after a lag period of 1.5 hours, but it did not inhibit the slow elongation at pH 7. Mannose was less inhibitory than galactose. Arabinose, xylose, glucose, sucrose, mannitol, and sorbitol caused no inhibition. When galactose was removed after a 1-hour treatment, the elongation was partially recovered. Cycloheximide and 2-deoxyglucose suppressed acid-enhanced elongation when these were applied at the same time as acid treatments, whereas cordycepin (3'-deoxyadenosine) inhibited elongation only if it was applied prior to acid treatment. Over the 9-hour period of elongation studied, the inhibition by galactose was comparable to that of cycloheximide. Since galactose has been reported to suppress the sugar metabolism necessary for the cell wall synthesis, the later phase of acid-enhanced elongation of root segments may at least partially depend on the synthesis or metabolism of cell wall components. The inhibition of root growth by galactose may be partially ascribed to a direct effect on the elongation process in roots, an effect that is enhanced by the acidification of the cell walls.

Factors Affecting Crown Gall Tumorigenesis in Tuber Slices of Jerusalem Artichoke (Helianthus tuberosus, L.).

Agrobacterium tumefaciens can induce tumors on thin slices which are excised from Jerusalem artichoke (Helianthus tuberosus) tubers and grown in culture on medium containing minerals and a carbon source. A comparative study was made of the kinetics of cell division in slices under three conditions: (a) slices which were untreated and showed only spontaneous (wound-induced) cell divisions; (b) slices treated with indoleacetic acid at several concentrations; and (c) slices treated with virulent or avirulent bacteria. The earliest spontaneous cell divisions were completed (as detected by the appearance of new daughter cell pairs) by about 3 hours. These cells divide only once. In indoleacetic acid-treated tissue, more cells divide, with the first cell pairs being detected slightly earlier than in slices not subjected to the hormone. The number of cells which divide is roughly proportional to auxin concentration. Tissue treated with virulent bacteria showed only the pattern of spontaneous cell division until about 72 hours, after which another burst of cell division commenced and continued indefinitely. The bacteria-induced growths produced the unusual amino acids which are characteristic of crown gall tumors. The percentage of slices with tumors was sharply reduced if certain avirulent A. tumefaciens strains were applied prior to virulent strains.

α-and ß-Glycosidases in maize roots.

Four glycosidases were analyzed in 10 mm apical segments prepared from growing roots (15 mm) of Zea mays L. The pH optima were found to be 5.8 for ß-glucosidase, 4.4 for ß-galactosidase, 6.4 for α-glucosidase and 6.0 for α-galactosidase. The ß-glucosidase showed 4-fold higher activity than the ß-galactosidase. The distribution of the ß-glucosidase activity was signifcantly different from that of the ß-galactosidase, α-glucosidase and α-galactosidase.