Naotsugu Uchida

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.

Effect of plant growth regulators on somatic embryogenesis in leaf cultures of Coffea canephora

The effects of plant growth regulators on somatic embryogenesis were studied in leaf cultures of Coffea canephora. The maximum number of somatic embryos were obtained on media that contained only cytokinin as a plant growth regulator. All of the auxins tested (NAA, IBA, IAA and 2, 4-D) inhibited the formation of embryos. The optimal concentration of each cytokinin (2-iP, BA and kinetin) for somatic embryogenesis was 5 μM. Under optimal conditions, each explant formed more than 100 embryoids with little callus and few adventitious roots. Embryoids were formed only at the cut edges of the leaf discs. Cytokinins were absorbed only at the cut edges of leaf discs that were in contact with the medium, and were not transported to other parts of the explant.

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.

Enhancement of Rice Leaf Photosynthesis by Crossing between Cultivated Rice, Oryza sativa and Wild Rice Species, Oryza rufipogon

Abstract To study whether wild rice species have genes that may increase potential photosynthetic capacities of rice cultivars, we generated BC2 populations by reciprocally backcrossing Oryza rufipogon (W630) with O. sativa cv. Nipponbare and IR36; N-BC2 populations and IR-BC2 populations, respectively. We measured the oxygen evolution rates (OER) of single leaves under saturating light and CO2 as the maximum photosynthetic rates and the contents of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase. Several lines in each BC2 population had significantly higher OERs than parental cultivars, and 14~25% of plants inBC2 populations had higher OERs than the highest values in parental cultivars. The highest OERs in BC2 populations were about 60% higher than average OERs in parental cultivars. The BC2 populations contained 30~40% more Rubisco than parental cultivars. The Rubisco activase contents in N-BC2 populations were 15~30% lower than that in Nipponbare. Cytoplasms derived from O. rufipogon and O. sativa had different effects on the contents of Rubisco and Rubisco activase particularly in N-BC2 populations. In several lines of each BC2 population the OERs had positive correlations with the contents of Rubisco and/or Rubisco activase. These results suggest that O. rufipogon can be used as a source of germplasm to enhance the photosynthetic capacity of O. sativa.

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.

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.

SDS-dependent proteases induced by ABA and its relation to Rubisco and Rubisco activase contents in rice leaves

Protease activities and its relation to the contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase were investigated in detached leaves of rice (Oryza sativa L.) floated on the solutions containing abscisic acid (ABA) or benzyladenine (BA). Rubisco and Rubisco activase contents were decreased during the time course and the decreases were enhanced by ABA and suppressed by BA. The decrease in Rubisco activase was faster than that in Rubisco. SDS-dependent protease activities at 50-70 kDa (rice SDS-dependent protease: RSP) analyzed by the gelatin containing PAGE were significantly enhanced by ABA. RSPs were also increased in attached leaves during senescence. RSPs had the pH optimum of 5.5, suggesting that RSPs are vacuolar protease. Both decrease in Rubisco and Rubisco activase contents and increase in RSPs activities were suppressed by cycloheximide. These findings indicate that the activities of RSPs are well correlated with the decrease in these protein contents. Immunoblotting analysis showed that Rubisco in the leaf extracts was completely degraded by 5h at pH 5.5 with SDS where it was optimal condition for RSPs. However, the degradation of Rubisco did not proceed at pH 7.5 without SDS where it is near physiological condition for stromal proteins. Rubisco activase was degraded at similar rate under both conditions. These results suggest that RSPs can functions in a senescence related degradation system of chloroplast protein in rice leaves. Rubisco activase would be more susceptible to proteolysis than Rubisco under physiological condition and this could affect the contents of these proteins in leaves.

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.

Photosynthetic Characteristics of Antisense Transgenic Rice Expressing Reduced Levels of Rubisco Activase

Abstract The activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which catalyses CO2 fixation in photosynthesis, requires the assistance of the regulatory protein Rubisco activase. Rubisco activase promotes carbamylation of Rubisco by releasing inhibitory sugar phosphates bound to the catalytic site of Rubisco in the light. To clarify the effects of Rubisco activase contents on the photosynthesis of rice, we investigated the steady-state photosynthesis and light-induction of photosynthesis in transgenic rice plants, in which leaf Rubisco activase levels were reduced. The reduction in Rubisco activase did not affect steady-state photosynthesis under high light intensity until the Rubisco activase was about 15% of that in control plants. However, light-induction of photosynthesis, namely, increase in photosynthetic rate following a transition from a low to high light intensity, was considerably low in transgenic rice plants with 20−25% Rubisco activase, which was sufficient to support the steady-state photosynthesis. In addition, the Rubisco activase content was highly correlated with the initial rate of Rubisco activation after the increase in light intensity. These results suggest that Rubisco activase in rice leaves largely limits the light-induction of photosynthesis, but not steady-state photosynthesis.

Mechanism of high photosynthetic capacity in BC2F4 lines derived from a cross between Oryza sativa and wild relatives O. rufipogon.

Abstract We found that several BC2F4 lines had high leaf photosynthetic rates under light-saturated and ambient CO2 conditions. These lines are progenies of BC2F1 plants with high photosynthetic capacities which were generated by backcrossing between Oryza rufipogon (W630) and O. sativa cv. Nipponbare, as a recurrent parent. Some photosynthetic characteristics of the BC2F4 lines were investigated to identify the factors increasing photosynthetic rates. Photosynthetic rates of these lines under light-saturated conditions at 50 to 700 ppm CO2 concentrations were higher than those in Nipponbare. The estimated-maximum photosynthetic rates under light-saturated and CO2-saturated conditions in BC2F4 lines were also higher than that in Nipponbare. The photosynthetic rate under light-saturated and ambient CO2 conditions was positively correlated with the carboxylation efficiency as an indicator of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity in vivo rather than stomatal conductance. Initial and total Rubisco activities in vitro tended to be higher in the BC2F4 lines than in Nipponbare. The content of active Rubisco calculated from the activation state of Rubisco was also higher in the BC2F4 lines than in Nipponbare. These results suggest that high photosynthetic capacities of BC2F1 plants can be maintained high in their progenies and high photosynthetic rates under light-saturated and ambient CO2 conditions in the BC2F4 lines are achieved mainly by the high activity of Rubisco due to the high active Rubisco content.