Tomoko Hatanaka

Functional Incorporation of Sorghum Small Subunit Increases the Catalytic Turnover Rate of Rubisco in Transgenic Rice

Rubisco limits photosynthetic CO2 fixation because of its low catalytic turnover rate (kcat) and competing oxygenase reaction. Previous attempts to improve the catalytic efficiency of Rubisco by genetic engineering have gained little progress. Here we demonstrate that the introduction of the small subunit (RbcS) of high kcat Rubisco from the C4 plant sorghum (Sorghum bicolor) significantly enhances kcat of Rubisco in transgenic rice (Oryza sativa). Three independent transgenic lines expressed sorghum RbcS at a high level, accounting for 30%, 44%, and 79% of the total RbcS. Rubisco was likely present as a chimera of sorghum and rice RbcS, and showed 1.32- to 1.50-fold higher kcat than in nontransgenic rice. Rubisco from transgenic lines showed a higher Km for CO2 and slightly lower specificity for CO2 than nontransgenic controls. These results suggest that Rubisco in rice transformed with sorghum RbcS partially acquires the catalytic properties of sorghum Rubisco. Rubisco content in transgenic lines was significantly increased over wild-type levels but Rubisco activation was slightly decreased. The expression of sorghum RbcS did not affect CO2 assimilation rates under a range of CO2 partial pressures. The Jmax/Vcmax ratio was significantly lower in transgenic line compared to the nontransgenic plants. These observations suggest that the capacity of electron transport is not sufficient to support the increased Rubisco capacity in transgenic rice. Although the photosynthetic rate was not enhanced, the strategy presented here opens the way to engineering Rubisco for improvement of photosynthesis and productivity in the future.

Vernonia DGATs increase accumulation of epoxy fatty acids in oil

Vernolic acid (cis-12-epoxy-octadeca-cis-9-enoic acid) is valuable as a renewable chemical feedstock. This fatty acid can accumulate to high levels in the seed oil of some plant species such as Vernonia galamensis and Stokesia laevis which are unsuitable for large-scale production. A cost-effective alternative for production of epoxy fatty acids is to genetically engineer its biosynthesis in commercial oilseeds. An epoxygenase cDNA (SlEPX) responsible for vernolic acid synthesis and two acyl-CoA : diacylglycerol acyltransferase cDNAs (VgDGAT1 and VgDGAT2) catalysing triacylglycerol (TAG) formation were cloned from developing seeds of S. laevis and V. galamensis. Co-expression of SlEPX and VgDGAT1 or VgDGAT2 greatly increases accumulation of vernolic acid both in petunia leaves and soybean somatic embryos. Seed-specific expression of VgDGAT1 and VgDGAT2 in SlEPX mature soybean seeds results in vernolic acid levels of approximately 15% and 26%. Both DGAT1 and DGAT2 increase epoxy fatty acid accumulation with DGAT2 having much greater impact.

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.

Overexpression of Rubisco Activase Decreases the Photosynthetic CO2 Assimilation Rate by Reducing Rubisco Content in Rice Leaves

The effects of overexpression of Rubisco activase on photosynthesis were studied in transgenic rice expressing barley or maize Rubisco activase. Immunoblot and SDS-PAGE analyses showed that transgenic lines from both gene constructs expressed the foreign Rubisco activase at high levels. The activation state of Rubisco in transgenic lines was slightly higher than that in non-transgenic plants (NT). In addition, light activation of Rubisco was significantly more rapid in transgenic lines compared with NT. These findings indicate that the overexpression of Rubisco activase can enhance Rubisco activation. However, despite enhanced activation of Rubisco in these transgenic plants, the CO(2) assimilation rate at ambient CO(2) conditions was decreased. This decrease in CO(2) assimilation rate was observed in both young developing and mature leaves independent of nitrogen nutrition. The contents of nitrogen and Chl did not differ significantly between transformants and NT; however, Rubisco content was substantially decreased in transgenic lines. There was no evidence for reduced transcription of RbcS or RbcL in these transgenic lines; in fact, transcript levels were marginally increased compared with NT. These results indicate that the overexpression of Rubisco activase leads to a decrease in Rubisco content, possibly due to post-transcriptional mechanisms.

Vernonia DGATs can complement the disrupted oil and protein metabolism in epoxygenase-expressing soybean seeds.

Plant oils can be useful chemical feedstocks such as a source of epoxy fatty acids. High seed-specific expression of a Stokesia laevis epoxygenase (SlEPX) in soybeans only results in 3-7% epoxide levels. SlEPX-transgenic soybean seeds also exhibited other phenotypic alterations, such as altered seed fatty acid profiles, reduced oil accumulation, and variable protein levels. SlEPX-transgenic seeds showed a 2-5% reduction in total oil content and protein levels of 30.9-51.4%. To address these pleiotrophic effects of SlEPX expression on other traits, transgenic soybeans were developed to co-express SlEPX and DGAT (diacylglycerol acyltransferase) genes (VgDGAT1 & 2) isolated from Vernonia galamensis, a high accumulator of epoxy fatty acids. These side effects of SlEPX expression were largely overcome in the DGAT co-expressing soybeans. Total oil and protein contents were restored to the levels in non-transgenic soybeans, indicating that both VgDGAT1 and VgDGAT2 could complement the disrupted phenotypes caused by over-expression of an epoxygenase in soybean seeds.

Cloning and functional analysis of two type 1 diacylglycerol acyltransferases from Vernonia galamensis.

Vernonia galamensis accumulates vernolic acid (cis-12-epoxyoctadeca-cis-9-enoic acid) as the major fatty acid in its seed oil. Such epoxy fatty acids are useful in a number of industrial applications. Successful genetic engineering of commercial oilseed crops to produce high levels of vernolic acid depends on a better understanding of the source plant enzymes for vernolic acid accumulation. Developing V. galamensis seed microsome assays demonstrate that diacylglycerol acyltransferase (DGAT), an enzyme for the final step of triacylglycerol synthesis, has a strong substrate preference for vernolic acid bearing substrates including acyl-CoA and diacylglycerol. There are two classes of DGATs known as DGAT1 and DGAT2. Here we report on the isolation, characterization, and functional analysis of two DGAT1 cDNAs from V. galamensis (VgDGAT1a and VgDGAT1b). VgDGAT1a and VgDGAT1b are expressed in all plant tissues examined with highest expression in developing seeds. Enzymatic assays using isolated microsomes from transformed yeast show that VgDGAT1a and VgDGAT1b have the same DGAT activity levels and substrate specificities. Oleoyl-CoA and sn-1,2-dioleoylglycerol are preferred substrates over vernoloyl-CoA and sn-1,2-divernoloylglycerol. This data indicates that the two VgDGAT1s are functional, but not likely to be responsible for the selective accumulation of vernolic acid in V. galamensis seed oil.

Unusual Small Subunit That Is Not Expressed in Photosynthetic Cells Alters the Catalytic Properties of Rubisco in Rice

Rice utilizes different type of Rubisco small subunit in nonphotosynthetic cells. Rubisco small subunits (RbcSs) are encoded by a nuclear multigene family in plants. Five RbcS genes, OsRbcS1, OsRbcS2, OsRbcS3, OsRbcS4, and OsRbcS5, have been identified in rice (Oryza sativa). Among them, the amino acid sequence of OsRbcS1 differs notably from those of other rice RbcSs. Phylogenetic analysis showed that OsRbcS1 is genetically distant from other rice RbcS genes and more closely related to RbcS from a fern and two woody plants. Reverse transcription-PCR and promoter β-glucuronidase analyses revealed that OsRbcS1 was not expressed in leaf blade, a major photosynthetic organ in rice, but was expressed in leaf sheath, culm, anther, and root central cylinder. In leaf blade of transgenic rice overexpressing OsRbcS1 and leaf sheath of nontransgenic rice, OsRbcS1 was incorporated into the Rubisco holoenzyme. Incorporation of OsRbcS1 into Rubisco increased the catalytic turnover rate and Km for CO2 of the enzyme and slightly decreased the specificity for CO2, indicating that the catalytic properties were shifted to those of a high-activity type Rubisco. The CO2 assimilation rate at low CO2 partial pressure was decreased in overexpression lines but was not changed under ambient and high CO2 partial pressure compared with nontransgenic rice. Although the Rubisco content was increased, Rubisco activation state was decreased in overexpression lines. These results indicate that the catalytic properties of Rubisco can be altered by ectopic expression of OsRbcS1, with substantial effects on photosynthetic performance in rice. We believe this is the first demonstration of organ-specific expression of individual members of the RbcS gene family resulting in marked effects on Rubisco catalytic activity.

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.

Screening of High kcat Rubisco among Poaceae for Improvement of Photosynthetic CO2 Assimilation in Rice

Abstract The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a major limitation of photosynthetic CO2 assimilation in C3 plants. In order to find useful Rubisco for improvement of photosynthesis in rice under elevated CO2, we analyzed the catalytic turnover rate (kcat) of Rubisco in Poaceae including C3 alpine plants, C3 cold-resistant plants and C4 plants. Rubisco in these plants showed 1.1- to 2.8-fold higher kcat than that in rice. However, the most of high kcat Rubisco also showed a higher km for CO2 (Kc) than that of rice, indicating that increase in kcat led to decrease in the affinity for CO2. Rubisco in Festuca ovina, Phleum pratense and Sorghum bicolor showed relatively high kcat to Kc. Although the kcat of Rubisco in F. ovina and P. pratense was not so high (1.5-1.6 fold relative to rice), the Kc was comparable to that in rice and the amino acid sequence of RbcL shared higher identity to that in rice than that in S. bicolor. By contrast, Rubisco of S. bicolor showed considerably high kcat (2.5-fold relative to rice), which is considered to be the most important factor for improvement of photosynthesis. In our estimation, the expression of high kcat Rubisco of F. ovina and S. bicolor in rice could significantly enhance CO2 assimilation at Ci of 50 Pa, the level assumed to be reached by the middle of this century.