Tetsushi Azuma

The role of ethylene in somatic embryogenesis from leaf discs of Coffea canephora

The role of ethylene in the formation of somatic embryos from leaf discs of Coffea canephora was studied on a medium that contained a cytokinin, iso-pentenyladenine, as the sole plant growth regulator. During incubation under these conditions, explants always produced a small amount of ethylene. Removal of this ethylene by an absorbent reduced the number of somatic embryos induced by the cytokinin. Application of inhibitors of the production of ethylene (Co2+ ions) and of the action of ethylene (Ag+ ions) inhibited the formation of embryos. Exogenous ethylene gas (12 μl/l) partially overcame the effect of Co2+ ions. These results indicate that ethylene plays an important role in regulating somatic embryogenesis in leaf cultures of Coffea canephora.

Involvement of the Decrease in Levels of Abscisic Acid in the Internodal Elongation of Submerged Floating Rice

Summary Submergence induces rapid internodal elongation in floating rice ( Oryza sativa L.). The involvement of abscisic acid (ABA) in such internodal elongation has been examined. Application of ABA to stem sections reduced the extent of internodal elongation induced by submergence, ethylene, or gibberellic acid (GA 3 ). Submergence and treatment with ethylene decreased the levels of ABA in plants and stem sections. This result indicates that a decrease in levels of ABA is involved in the internodal elongation induced by ethylene, the level of which increases in internodes during submergence. However, when plants or stem sections were treated with ethylene at low relative humidity, the levels of ABA decreased but internodes barely increased in length. Moist surroundings, such as submerged conditions, may be necessary if the decrease in levels of ABA is to lead to internodal elongation in floating rice.

Activation of Arabidopsis MAPK kinase kinase (AtMEKK1) and induction of AtMEKK1–AtMEK1 pathway by wounding

We have constructed a series of deletion mutants of Arabidopsis MAPK kinase kinase (AtMEKK1) and obtained a constitutively active mutant, AtMEKK1Δ166, which lacks in self-inhibitory sequence of N-terminal 166 amino acids but still has substrate specificity. AtMEKK1Δ166 predominantly phosphorylates AtMEK1, an Arabidopsis MAPKK, but not its double mutant (AtMEK1T218A/S224E), suggesting that Thr-218 and Ser-224 are the phosphorylation sites. In wounded seedlings, AtMEKK1 was activated and phosphorylated its downstream AtMEK1. Furthermore, analysis using anti-AtMEKK1 and anti-AtMEK1 antibodies revealed that the interaction between the two proteins was signal dependent. These results suggest the presence of AtMEKK1–AtMEK1 pathway induced by wounding.

Posttranscriptional regulation by the upstream open reading frame of the phosphoethanolamine N-methyltransferase gene.

Phosphoethanolamine N-methyltransferase (PEAMT) is involved in choline biosynthesis in plants. The 5′ untranslated region (UTR) of several PEAMT genes was found to contain an upstream open reading frame (uORF). We generated transgenic Arabidopsis calli that expressed a chimeric gene constructed by fusing the 5′ UTR of the Arabidopsis PEAMT gene (AtNMT1) upstream of the β-glucuronidase gene. The AtNMT1 uORF was found to be involved in declining levels of the chimeric gene mRNA and repression of downstream β-glucuronidase gene translation in the calli when the cells were treated with choline. Further, we discuss the role of the uORF.

Relationship between the Deposition of Phenolic Acids in the Cell Walls and the Cessation of Rapid Growth in Internodes of Floating Rice

Abstract We examined the involvement of p-coumaric, ferulic and 5-5-coupled diferulic acids ester-linked to cell walls in determining the elongation rate of internodes of floating rice (Oryza sativa L.). When floating rice stem segments were exposed to air after 2 days of submergence, the elongation rate of internodes was reduced and the degree of reduction was greater in the light than in the dark, while the internodes of stem segments submerged further for a comparable period continued rapid elongation. The amounts of ferulic and 5-5-coupled diferulic acids in the cell walls in the elongation zone of internodes significantly increased during the first day after exposure to air either in light or darkness. The increase of these phenolics in the cell walls after exposure to air was also observed on the second day in light, but not in darkness. On the other hand, the amount of p-coumaric acid increased only slightly on the first day after exposure to air, but rapidly on the second day in light. This pattern of change in the amounts of p-coumaric acid resembled that in the cell-wall mass (dry weight). The application of sucrose to the segments in darkness increased the amounts of phenolics in the cell walls of internodes to almost the same amount as those in light. These results indicate that the accumulation of ferulic and 5-5-coupled diferulic acids in cell walls may be related to the cessation of internodal elongation in floating rice and that the synthesis of phenolics in the cell wall is caused partially by the provision of sugar in light.

Enhancement of transpiration by ethylene is responsible for absence of internodal elongation in floating rice at low humidity

Internodal elongation in floating rice (Oryza sativa) is known to be enhanced by treatment with ethylene or gibberellic acid (GA3) at high relative humidity (RH). However, ethylene-induced internodal elongation is inhibited at low RH, while GA3-induced internodal elongation is hardly affected by humidity. We examined the effects of ethylene and GA3 on the rate of transpiration in stem segments incubated at 30% or 100% RH. Ethylene promoted the transpiration of stem segments at 30% RH, but not at 100% RH, while GA3 had little effect on transpiration at either 30% or 100% RH. We propose that the absence of ethylene-induced internodal elongation at low RH is due, at least in part, to ethylene-induced transpiration.

Changes in cell wall polysaccharides in the internodes of submerged floating rice

In excised stem segments of floating rice (Oryza sativa L.), as well as in intact plants, submergence greatly stimulates the elongation of internodes. The differences in the composition of cell wall polysaccharides along the highest internodes of submerged and air-grown stem segments were examined. The newly elongated parts of internodes that had been submerged for two days contained considerably less cellulosic and noncellulosic polysaccharides than air-grown internodes, an indication that the cell walls of the newly elongated parts of submerged internodes are extremely thin. In the young parts of both air-grown and submerged internodes, the relative amounts of noncellulosic polysaccharides were equal to those of α-cellulose, whereas the relative amounts of α-cellulose were higher than those of noncellulosic polysaccharides in the upper, old parts. In the cell-elongation zones of both air-grown and submerged internodes, glucose was predominant among the noncellulosic neutral sugars of cell wall. The relative amount of glucose in noncellulosic neutral sugars decreased toward the upper, old parts of internodes, whereas that of xylose increased.

Interactions between abscisic acid, ethylene and gibberellin in internodal elongation in floating rice: the promotive effect of abscisic acid at low humidity

The enhancement of internodal elongation in floating or deepwater rice (Oryza sativa L. cv. Habiganj Aman II) by treatment with ethylene or gibberellic acid (GA3) at high relative humidity (RH) is inhibited by abscisic acid (ABA). Here, we examined the interactive effects of ethylene, gibberellin (GA) and ABA at low RH on internodal elongation of deepwater rice stem segments. Although ethylene alone hardly promoted internodal elongation of stem sections at 30% RH, it enhanced the internodal elongation induced by GA3. Application of ABA alone to stem segments had no effect on internodal elongation. However, in the presence of ethylene and GA3 at 30% RH, ABA further promoted internodal elongation. This promotive effect of ABA was not found in the internodes of stem segments treated either with ethylene or with GA3 at 30% RH or in the internodes of stem segments treated with ethylene and/or GA3 at 100% RH.

Low levels of oxygen promote internodal elongation in floating rice independently of enhanced ethylene production

Submergence induces rapid elongation of internodes in floating rice(Oryza sativa L. cv. Habiganj Aman II). The initial signalfor such internodal elongation has been considered to be the reduced partialpressure of oxygen in submerged internodal cavities, which promotes theelongation of internodes through the enhancement of ethylene synthesis. Weexamined the relationship between low oxygen pressure and ethylene production inthe rapid elongation of floating rice internodes using ethylene biosynthesisinhibitors, aminooxyacetic acid (AOA) and CoCl2. When floating ricestem segments were incubated in an atmosphere of low O2, internodalelongation accelerated and ethylene production increased. However, in stemsegments treated with AOA or CoCl2, low O2 stillstimulated the elongation of internodes although the ethylene production by theinternodes was less than by those in control stem segments where internodalelongation was not promoted. These results indicate that low O2 iscapable of causing rapid elongation of internodes of floating rice independentlyof enhanced production of ethylene. In addition to low O2,submergence, ethylene and gibberellic acid each enhanced the production ofethylene by internodal tissues, suggesting that enhanced ethylene production isa common phenomenon accompanied by the acceleration of internodal elongation infloating rice.

Changes in Cell Wall-Bound Phenolic Acids in the Internodes of Submerged Floating Rice

Abstract Submergence induces rapid elongation of internodes in floating rice (Oryza sativa L.). We examined the distributions of p-coumaric, ferulic and 5-5-coupled diferulic acids ester-linked to cell walls along the axis of highest internodes of submerged and air-grown floating rice stem segments. The amounts of ferulic and 5-5-diferulic acids per cell wall weight were lowest around the intercalary meristem, and increased as the distance from the meristematic zone increased toward the upper part of the internode in both air-grown and submerged stem segments. The ratio of 5-5-diferulic acid to ferulic acid also increased toward the upper, old parts of internodes in both air-grown and submerged stem segments. These observations suggest that the feruloylation of cell wall polysaccharides and the formation of diferulic acids contribute to the cessation of internodal cell elongation and that the formation of diferulic acids in cell walls is controlled by the coupling reaction in addition to the feruloylation. The amounts of p-coumaric acid per unit length and per cell-wall weight were markedly low in the newly elongated region of submerged internodes, and closely correlated with cell-wall dry mass in both air-grown and submerged internodes, suggesting that the deposition of p-coumaric acid in cell walls is related to the formation of secondary cell walls in floating rice internodes.