Haruto Sasaki

Overproduction of C4 photosynthetic enzymes in transgenic rice plants: an approach to introduce the C4-like photosynthetic pathway into rice

Four enzymes, namely, the maize C(4)-specific phosphoenolpyruvate carboxylase (PEPC), the maize C(4)-specific pyruvate, orthophosphate dikinase (PPDK), the sorghum NADP-malate dehydrogenase (MDH), and the rice C(3)-specific NADP-malic enzyme (ME), were overproduced in the mesophyll cells of rice plants independently or in combination. Overproduction individually of PPDK, MDH or ME did not affect the rate of photosynthetic CO(2) assimilation, while in the case of PEPC it was slightly reduced. The reduction in CO(2) assimilation in PEPC overproduction lines remained unaffected by overproduction of PPDK, ME or a combination of both, however it was significantly restored by the combined overproduction of PPDK, ME, and MDH to reach levels comparable to or slightly higher than that of non-transgenic rice. The extent of the restoration of CO(2) assimilation, however, was more marked at higher CO(2) concentrations, an indication that overproduction of the four enzymes in combination did not act to concentrate CO(2) inside the chloroplast. Transgenic rice plants overproducing the four enzymes showed slight stunting. Comparison of transformants overproducing different combinations of enzymes indicated that overproduction of PEPC together with ME was responsible for stunting, and that overproduction of MDH had some mitigating effects. Possible mechanisms underlying these phenotypic effects, as well as possibilities and limitations of introducing the C(4)-like photosynthetic pathway into C(3) plants, are discussed.

Cultivar differences in leaf photosynthesis of rice bred in Japan

The grain yield of rice (Oryza sativa L.), as well as of other cereal crops, is limited to a large extent, by the supply of photosynthates produced during grain filling period. In this study, flag leaf photosynthesis (LPS) after heading was compared among 32 cultivars bred during the past century in Japan, to determine if the improvement of LPS has occurred with the breeding advance of high yielding cultivars. Measurement of LPS was made for 5 consecutive years in the paddy field, on the flag leaf of the main stem, at heading (LPS-0), and 2 weeks (LPS-2) and 4 weeks (LPS-4) after heading. LPS decreased with advance of leaf senescence from LPS-0 to LPS-2, and then to LPS-4. However, if nitrogen was top-dressed at the heading time, high LPS-2 was maintained, particularly in the newer cultivars. A significant positive correlation between LPS and the released year of cultivar was found at LPS-2, especially in the nitrogen top-dressed plot, but not at LPS-0 or LPS-4. Cultivar difference in LPS of the senescing leaves were not stable through the different years, whereas LPS-0 was stable over years, suggesting that the LPS in the senescent leaf is susceptible to the environmental variation due to the effects on leaf senescence. Cultivar difference in LPS at any stage was closely associated with mesophyll conductance to CO2, and stomatal conductance was also associated with cultivar difference in such a high LPS as LPS-0 and nitrogen top-dressed LPS-2. Significant correlation between LPS and specific leaf weight was not observed at any stage of the flag leaf.

Factors Responsible for Decreasing Sturdiness of the Lower Part in Lodging of Rice (Oryza sativa L.)

Abstract Here, we propose new improvement targets capable of decreasing loss of the sturdiness of the lower part in the rice plant (Oryza sativa L.), thereby improving lodging resistance. In nine rice cultivars with various plant lengths, we analyzed the factors responsible for sturdiness of the lower part and, thus, for resistance to lodging. The ratio of lodging resistance to sturdiness of the lower part (RLS) was calculated. The difference in pushing resistance between the lower part and the whole plant varied among cultivars. Among the morphological traits, plant length and the weight of the upper part of plant were not correlated with RLS, but the difference between plant length and length from the ground to the ear (DPE), as well as the weight of the lower stem, were positively correlated with RLS. DPE and the weight of the lower stem were not significantly correlated with ear weight. These results suggest that improvements in DPE and in the weight of the lower stem could be primary targets for improving RLS, thus increasing lodging resistance, without affecting yield.

Simultaneous analysis of amino acids and carboxylic acids by capillary electrophoresis-mass spectrometry using an acidic electrolyte and uncoated fused-silica capillary.

A simple, low-cost capillary electrophoresis-mass spectrometry (CE-MS) method is demonstrated for the simultaneous analysis of amino acids and small carboxylic acids (glycerate, lactate, fumarate, succinate, malate, tartrate, citrate, iso-citrate, cis-aconitate, and shikimate). All CE-MS experiments were performed using a single uncoated fused-silica capillary and with a single separation electrolyte, formic acid. For CE polarity, the CE inlet was set as the anode, and the MS side was set as the cathode. By using high-speed sheath gas flow, the apparent mobilities of all compounds were sped up; thus, the migration times of the carboxylic acids were reduced. In positive ion mode ESI-MS detection, small carboxylic acids were detected faintly as m/z = [M + 18](+) or [M + 23](+), after protonated molecule detection (m/z = [M + 1](+)) of the amino acids. In negative ion mode, all of these small carboxylic acids were detected clearly as deprotonated molecules (m/z = [M - 1](-)), after detection of the amino acids. By changing the polarity of the MS during CE separation, both amino acids and small carboxylic acids were detectable in a single electrophoresis analysis run. With this method, the diurnal metabolic changes of pineapple leaves were observed as reflecting Crassulacean acid metabolism.

Effect of CO2 Enrichment on the Translocation and Partitioning of Carbon at the Early Grain-filling Stage in Rice (Oryza sativa L.)

Abstract Rice plants (Oryza sativa L.) were grown under normal (350 µL L-1 CO2) and CO2-enriched (660 µ L L-1 CO2) conditions, and 13CO2 was supplied to the rice plants after heading to examine the translocation and partitioning of photosynthate at the early grain-filling stage. At 2 days after supplying 13CO2, no difference in the 13C content of the whole plant was observed between the plants grown under normal and CO2-enriched conditions, but translocation of 13C from the leaf blade to other plant organs seemed to be accelerated by CO2 enrichment. Up to 9 days after supplying, 13CO2 fixed into sucrose was mainly used to synthesize starch in the stem rather than translocated to the ear in plants grown under normal conditions. In contrast, the supplied 13C was rapidly translocated to the ear, and 13C stored as starch in the stem was also translocated to the ear in plants grown under CO2-enriched conditions. Therefore, we concluded that CO2 enrichment accelerated the translocation of carbohydrates to the ear.

Effects of Elevated CO2 Concentration on Photosynthetic Carbon Metabolism in Flag-Leaf Blades of Rice before and after Heading

Abstract We monitored the effects of elevated atmospheric C02 concentrations on the photosynthetic carbon metabolism in the flag leaves of rice plant (Oryza sativaL. cv. Akitakomachi) before and after heading. The plants were grown under ambient (350 ppm : control) or elevated (650 ppm) C02 conditions. Flag-leaf blades grown under high C02 accumulated more starch than control leaf blades before heading, but the level of starch declined to almost zero under both C02 concentrations as soon as the development of ears began. Before heading, the transcript level of sucrose-phosphate synthase (SPS) (EC 2.4.1.14), a key enzyme in the sucrose synthesis in flag-leaf blades was significantly higher under elevated C02 conditions than under elevated C02 (P<0.01). The difference in the expression of SPS decreased after heading, coinciding with a change in starch contents in both groups. These results showed that the effects of elevated C02 concentration on rice plants might vary with the growth stage of the leaf blades. We also discussed the influence of the changes in the carbohydrate metabolism of rice plants caused by elevated C02 concentration on yield.

The QTL Analysis of RuBisCO in Flag Leaves and Non-Structural Carbohydrates in Leaf Sheaths of Rice Using Chromosome Segment Substitution Lines and Backcross Progeny F2 Populations

Abstract In rice (Oryza sativa L.), the maintenance of high photosynthetic rate of flag leaves and the carbon remobilization from leaf sheaths after heading is a critical physiological component affecting the yield. To clarify the genetic basis of RuBisCO content of the flag leaf, a major determinant of photosynthetic rate, and non-structural carbohydrate (NSC) concentration in the third leaf sheath at heading, we carried out quantitative trait loci (QTL) analysis with 39 Koshihikari/Kasalath chromosome segment substitution lines (CSSLs) and backcross progeny F2 population derived from target CSSL holding the QTL/Koshihikari in the field. QTLs for RuBisCO content and NSC concentration at heading were detected between R2447-C1286 and R2447-R716 on chromosome 10, respectively, by comparing Koshihikari with four CSSLs for chromosome 10 (SL-229, -230, -231 and -232). The progeny QTL for RuBisCO content and for NSC concentration at heading qRCH-10 and qNSCLSH-10-1, respectively, were detected at similar marker intervals between RM8201 and RM5708. In addition, QTLs for RuBisCO content at 14 d after heading, qRCAH-10-1 and qRCAH-10-2, were detected in regions different from that of qRCH-10. No QTL for NSC concentration at 14 d after heading was detected between RM8201 and R716, the region analyzed in this study. The QTLs qRCH-10 and qRCAH-10-1 for RuBisCO content would have additive effects. These QTLs for RuBisCO content and NSC concentration newly found using CSSLs and their backcross progeny F2 population should be useful for better understanding the genetic basis of source and temporary-sink functions in rice and for genetic improvement of Koshihikari in terms of their functions.

Contribution of Nitrogen Absorbed during Ripening Period to Grain Filling in a High-Yielding Rice Variety, Takanari

Absract High-yielding rice varieties require a large accumulation of N in panicles. The objectives of this study were to clarify the change in N allocation during the ripening period (Exp. 1) and to quantify the contribution of N absorbed during the ripening period to panicle N at maturity (Exp. 2) in the high-yielding variety Takanari in comparison with that in Nipponbare as a control. In Exp. 1, 15N-labeled N (15N) was applied at heading to investigate the distribution of newly absorbed N as well as the allocation of plant N. In Exp. 2, split 15N application was performed during the filling period to estimate the above contribution. In Exp. 1, the allocation of plant N and absorbed 15N to the panicles was larger and that to the leaves was smaller in Takanari than in Nipponbare during the ripening period, although Takanari accumulated more N at maturity. The difference in N allocation suggested that the difference in N demand in panicles would be larger than that in N uptake. In Exp. 2, the varietal difference in the grain filling duration was observed: Nipponbare accumulated little N in the panicles after 28 d after heading (DAH), while Takanari accumulated about a quarter of its panicle N during that time. An estimate showed that in Takanari, 13.5% of the panicle N was derived from N absorbed after 28 DAH. These results suggest that the utilization of newly absorbed N until a later period after heading is important for the achievement of high yields.

Identification of QTLs for Improvement of Plant Type in Rice (Oryza sativa L.) Using Koshihikari / Kasalath Chromosome Segment Substitution Lines and Backcross Progeny F2 Population

Abstract Thirty-nine chromosome segment substitution lines (CSSLs) population derived from a Koshihikari / Kasalath cross was used for quantitative trait locus (QTL) analysis of plant type in rice (Oryza sativa L.). Putative rough QTLs (26.2∼60.3cM of Kasalath chromosomal segments) for culm length, plant height, panicle number, chlorophyll content of flag leaf blade at heading and specific leaf weight, were mapped on the several chromosomal segments based on the comparison of CSSLs with Koshihikari in the field experiment for 3 years. In order to verify and narrow QTLs detected in CSSLs, we conducted QTL analyses using F2 populations derived from a cross between Koshihikari and target CSSL holding a putative rough QTL. The qPN-2, QTL for panicle number was mapped on chromosome 2. In traits of flag leaf, the qCHL-4-1 and qCHL-4-2 for chlorophyll content was mapped on chromosome 4, and the qSLW-7 for specific leaf weight on chromosome 7. All QTLs were detected in narrow marker intervals, compared with rough QTLs in CSSLs. The qPN-2, qCHL-4-1 and qCHL-4-2 had only additive effect. On the other hand, the qSLW-7 showed over-dominance. It could be emphasized that QTL analysis in the present study with the combination of CSSLs and backcross progeny F2 population can not only verify the rough QTLs detected in CSSLs but also estimate allelic effects on the QTL.

Changes in Photosynthetic Activity and Export of Carbon by Overexpressing a Maize Sucrose-Phosphate Synthase Gene under Elevated CO2 in Transgenic Rice

Abstract To investigate whether increased sucrose-phosphate synthase (SPS) activity alters photosynthetic activity and/or the export of carbon from leaves under elevated C02 partial pressure ([C02]), we raised two lines of transgenic rice (H54-9 and H69-7), each overexpressing a maize SPS gene, and wild-type rice under ambient [C02] (35 Pa) and elevated [0O2] (100 Pa). Under ambient [C02], no significant difference was observed between the transgenic and wild-type plants in the levels of sucrose or starch in leaves or the photosynthetic activity; but the carbon export rate was higher in H69-7 than in the wild-type. Under elevated [C02], photosynthetic activity increased in all plants, but the accumulation of starch was significantly repressed in H54-9, whose SPS activity was about 12.5 times higher than that of the wild-type. The carbon export rate was higher in both transgenic lines than the wild-type. We considered that increased SPS activity in rice plants would promote the export of carbon from leaves and, as a result, starch accumulation in the leaves would be suppressed and/or photosynthetic activity would be promoted under elevated [CO2].