MORE PANICLES 3, a natural allele of OsTB1/FC1, impacts rice yield in paddy fields at elevated CO2 levels.

Improving crop yield potential through an enhanced response to rising atmospheric CO2 levels is an effective strategy for sustainable crop production in the face of climate change. Large-sized panicles (containing many spikelets per panicle) have been a recent ideal plant architecture (IPA) for high-yield rice breeding. However, few breeding programs have proposed an IPA under the projected climate change. Here, we demonstrate through the cloning of the rice (Oryza sativa) quantitative trait locus for MORE PANICLES 3 (MP3) that the improvement in panicle number increases grain yield at elevated atmospheric CO2 levels. MP3 is a natural allele of OsTB1/FC1, previously reported as a negative regulator of tiller bud outgrowth. The temperate japonica allele advanced the developmental process in axillary buds, moderately promoted tillering, and increased the panicle number without negative effects on the panicle size or culm thickness in a high-yielding indica cultivar with large-sized panicles. The MP3 allele, containing three exonic polymorphisms, was observed in most accessions in the temperate japonica subgroups but was rarely observed in the indica subgroup. No selective sweep at MP3 in either the temperate japonica or indica subgroups suggested that MP3 has not been involved and utilized in artificial selection during domestication or breeding. A free-air CO2 enrichment experiment revealed a clear increase of grain yield associated with the temperate japonica allele at elevated atmospheric CO2 levels. Our findings show that the moderately increased panicle number combined with large-sized panicles using MP3 could be a novel IPA and contribute to an increase in rice production under climate change with rising atmospheric CO2 levels.

CURLED LATER1 encoding the largest subunit of the Elongator complex has a unique role in leaf development and meristem function in rice.

The Elongator complex, which is conserved in eukaryotes, has multiple roles in diverse organisms. In Arabidopsis thaliana, Elongator is shown to be involved in development, hormone action and environmental responses. However, except for Arabidopsis, our knowledge of its function is poor in plants. In this study, we initially carried out a genetic analysis to characterize a rice mutant with narrow and curled leaves, termed curled later1 (cur1). The cur1 mutant displayed a heteroblastic change, whereby the mutant leaf phenotype appeared specifically at a later adult phase of vegetative development. The shoot apical meristem (SAM) was small and the leaf initiation rate was low, suggesting that the activity of the SAM seemed to be partially reduced in cur1. We then revealed that CUR1 encodes a yeast ELP1-like protein, the largest subunit of Elongator. Furthermore, disruption of OsELP3 encoding the catalytic subunit of Elongator resulted in phenotypes similar to those of cur1, including the timing of the appearance of mutant phenotypes. Thus, Elongator activity seems to be specifically required for leaf development at the late vegetative phase. Transcriptome analysis showed that genes involved in protein quality control were highly upregulated in the cur1 shoot apex at the later vegetative phase, suggesting the restoration of impaired proteins probably produced by partial defects in translational control due to the loss of function of Elongator. The differences in the mutant phenotype and gene expression profile between CUR1 and its Arabidopsis ortholog suggest that Elongator has evolved to play a unique role in rice development.

Development of a visible marker trait based on leaf sheath-specific anthocyanin pigmentation applicable to various genotypes in rice.

To overcome a limitation to the breeding of autogamous crops, recurrent selection using transgenic male sterility (RSUTMS) has been proposed. In this system, negatively or positively selectable marker traits are required along with dominant transgenic male sterility. Anthocyanin pigmentation is an excellent marker trait. Two regulatory genes for MYB and bHLH and a structural gene for DFR are required for anthocyanin pigmentation in rice. Therefore, to apply anthocyanin pigmentation as a marker trait in various rice genotypes, coordinated expression of the three genes is required. In this study, we developed a leaf sheath-specific promoter and introduced three genes-DFR and C1/Myb, driven by the 35S promoter, and OsB2/bHLH, driven by the leaf sheath-specific promoter-into the rice genome. Leaf sheath-specific pigmentation was confirmed in all seven genotypes tested, which included japonica and indica cultivars. Analysis of genome sequence data from 25 cultivars showed that the strategy of conferring leaf sheath-specific anthocyanin pigmentation by introduction of these three genes would be effective for a wide range of genotypes and will be applicable to RSUTMS.

A high-performance system of multiple gas-exchange chambers with a laser spectrometer to estimate leaf photosynthesis, stomatal conductance, and mesophyll conductance.

Direct measurements of ecophysiological processes such as leaf photosynthesis are often hampered due to the excessive time required for gas-exchange measurements and the limited availability of multiple gas analyzers. Although recent advancements in commercially available instruments have improved the ability to take measurements more conveniently, the amount of time required for each plant sample to acclimate to chamber conditions has not been sufficiently reduced. Here we describe a system of multiple gas-exchange chambers coupled with a laser spectrometer that employs tunable diode laser absorption spectroscopy (TDLAS) to measure leaf photosynthesis, stomatal conductance, and mesophyll conductance. Using four gas-exchange chambers minimizes the time loss associated with acclimation for each leaf sample. System operation is semiautomatic, and leaf temperature, humidity, and CO2 concentration can be regulated and monitored remotely by a computer system. The preliminary results with rice leaf samples demonstrated that the system is capable of high-throughput measurements, which is necessary to obtain better representativeness of the ecophysiological characteristics of plant samples.

A rice gene that confers broad-spectrum resistance to ß-triketone herbicides.

The genetic variation of rice cultivars provides a resource for further varietal improvement through breeding. Some rice varieties are sensitive to benzobicyclon (BBC), a ß-triketone herbicide that inhibits 4-hydroxyphenylpyruvate dioxygenase (HPPD). Here we identify a rice gene, HIS1 (HPPD INHIBITOR SENSITIVE 1), that confers resistance to BBC and other ß-triketone herbicides. We show that HIS1 encodes an Fe(II)/2-oxoglutarate-dependent oxygenase that detoxifies ß-triketone herbicides by catalyzing their hydroxylation. Genealogy analysis revealed that BBC-sensitive rice variants inherited a dysfunctional his1 allele from an indica rice variety. Forced expression of HIS1 in Arabidopsis conferred resistance not only to BBC but also to four additional ß-triketone herbicides. HIS1 may prove useful for breeding herbicide-resistant crops.

Development of transgenic male-sterile rice by using anther-specific promoters identified by comprehensive screening of the gene expression profile database 'RiceXPro'.

Because genomic selection is designed for the population breeding of allogamous species, a successive outcrossing system is required for efficient use of genomic selection in autogamous crops, such as Oryza sativa L. (rice). Transgenic and dominant male-sterility is a suitable tool for efficient outcrossing of autogamous crops. Though there have been some reports of dominant male-sterile rice developed using transgenic technology, the flowering habit was substandard. Here, to isolate promoters that, when linked to a lethal gene, induce dominant male-sterility while retaining a good flowering habit, we identified 38 candidate genes with anther-specific expression by using the 'RiceXPro' database. We then evaluated the abilities of the near-upstream regions of these genes to induce male-sterility when linked to the lethal gene barnase and introduced into the rice cultivar 'Nipponbare'. Seven of the 38 promoters induced clear dominant male-sterility; promoters expressed in the later stage of anther development induced male-sterility while retaining better flowering habits when compared to ones expressed in the early stage. These seven promoters could potentially be used to facilitate development of an efficient outcross-based breeding system in rice.

Development and characterization of transgenic dominant male sterile rice toward an outcross-based breeding system.

Genomic selection is attracting attention in the field of crop breeding. To apply genomic selection effectively for autogamous (self-pollinating) crops, an efficient outcross system is desired. Since dominant male sterility is a powerful tool for easy and successive outcross of autogamous crops, we developed transgenic dominant male sterile rice (Oryza sativa L.) using the barnase gene that is expressed by the tapetum-specific promoter BoA9. Barnase-induced male sterile rice No. 10 (BMS10) was selected for its stable male sterility and normal growth characteristics. The BMS10 flowering habits, including heading date, flowering date, and daily flowering time of BMS10 tended to be delayed compared to wild type. When BMS10 and wild type were placed side-by-side and crossed under an open-pollinating condition, the seed-setting rate was <1.5%. When the clipping method was used to avoid the influence of late flowering habits, the seed-setting rate of BMS10 increased to a maximum of 86.4%. Although flowering synchronicity should be improved to increase the seed-setting rate, our results showed that this system can produce stable transgenic male sterility with normal female fertility in rice. The transgenic male sterile rice would promote a genomic selection-based breeding system in rice.

Characterization and expression analyses of two plastidic enolase genes in rice.

To verify the presence of enolase related to the chloroplastic glycolysis in rice, database search was carried out and identified seven putative enolase genes in the rice genome. Among them, OsEno1 and OsEno3 encode long proteins with N-terminal extensions. GFP protein fusions of these N-terminal extensions were both targeted to plastids of onion epidermal cell. Promoter::GUS analysis showed that OsEno3 was highly expressed in young developing leaves, but its expression was drastically decreased during leaf development and greening. On the other hand, the expression of OsEno1 was low and detected in limited portions such as leaf sheath at the tiller base. Recombinant OsEno1 protein showed enolase activity with a pH optimum at pH 8.0, whereas OsEno3 did not exhibit detectable activity. Although it remains obscure if OsEno3 encodes a functional enolase in vivo, our results demonstrate that the entire glycolytic pathway does not operate in rice chloroplasts.

A natural variant of NAL1, selected in high-yield rice breeding programs, pleiotropically increases photosynthesis rate.

Improvement of leaf photosynthesis is an important strategy for greater crop productivity. Here we show that the quantitative trait locus GPS (GREEN FOR PHOTOSYNTHESIS) in rice (Oryza sativa L.) controls photosynthesis rate by regulating carboxylation efficiency. Map-based cloning revealed that GPS is identical to NAL1 (NARROW LEAF1), a gene previously reported to control lateral leaf growth. The high-photosynthesis allele of GPS was found to be a partial loss-of-function allele of NAL1. This allele increased mesophyll cell number between vascular bundles, which led to thickened leaves, and it pleiotropically enhanced photosynthesis rate without the detrimental side effects observed in previously identified nal1 mutants, such as dwarf plant stature. Furthermore, pedigree analysis suggested that rice breeders have repeatedly selected the high-photosynthesis allele in high-yield breeding programs. The identification and utilization of NAL1 (GPS) can enhance future high-yield breeding and provides a new strategy for increasing rice productivity.

Selecting genetic transformants of indica and indica-derived rice cultivars using bispyribac sodium and a mutated ALS gene.

Bispyribac sodium (BS), a pyrimidinyl carboxy herbicide, is a well-known inhibitor of acetolactate synthase (ALS) activity. ALS is an enzyme in the biosynthetic pathway for branched-chain amino acids. A mutant form of rice ALS (OsmALS [W548L/S627I]) that confers resistance to BS can be used as an in vitro selection marker gene for plant transformation. Since indica and indica-derived cultivars are thought to have lower BS sensitivity than japonica rice, the application of BS as a selectable reagent for genetic transformation in indica and indica-derived cultivars is more challenging than for japonica cultivars. In this study, callus and seedlings of eight different rice cultivars (five indica-derived cultivars, two indica cultivars and one japonica cultivar) were tested for BS sensitivity. Our study indicates for the first time that callus shows a higher sensitivity to BS than seedlings in indica and indica-derived cultivars. We used BS with OsmALS [W548L/S627I] to select transformed calli, and transgenic rice plants from indica and indica-derived cultivars were successfully obtained.

Phosphoenolpyruvate carboxylase intrinsically located in the chloroplast of rice plays a crucial role in ammonium assimilation.

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of primary metabolism in bacteria, algae, and vascular plants, and is believed to be cytosolic. Here we show that rice (Oryza sativa L.) has a plant-type PEPC, Osppc4, that is targeted to the chloroplast. Osppc4 was expressed in all organs tested and showed high expression in the leaves. Its expression in the leaves was confined to mesophyll cells, and Osppc4 accounted for approximately one-third of total PEPC protein in the leaf blade. Recombinant Osppc4 was active in the PEPC reaction, showing V(max) comparable to cytosolic isozymes. Knockdown of Osppc4 expression by the RNAi technique resulted in stunting at the vegetative stage, which was much more marked when rice plants were grown with ammonium than with nitrate as the nitrogen source. Comparison of leaf metabolomes of ammonium-grown plants suggested that the knockdown suppressed ammonium assimilation and subsequent amino acid synthesis by reducing levels of organic acids, which are carbon skeleton donors for these processes. We also identified the chloroplastic PEPC gene in other Oryza species, all of which are adapted to waterlogged soil where the major nitrogen source is ammonium. This suggests that, in addition to glycolysis, the genus Oryza has a unique route to provide organic acids for ammonium assimilation that involves a chloroplastic PEPC, and that this route is crucial for growth with ammonium. This work provides evidence for diversity of primary ammonium assimilation in the leaves of vascular plants.

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.

Characterization and functional analysis of phosphoenolpyruvate carboxylase kinase genes in rice.

Phosphoenolpyruvate carboxylase (PEPC), a key enzyme of primary metabolism of higher plants, is regulated by reversible phosphorylation, which is catalyzed by PEPC kinase (PPCK). Rice has three functional PPCK genes, OsPPCK1, OsPPCK2 and OsPPCK3, all of which have an intron close to the 3' end of the coding region. A novel control mechanism was found for expression of OsPPCK2, namely alternative transcription initiation, and two different transcripts were detected. The four different transcripts of the OsPPCK genes showed different expression patterns. While OsPPCK1 and OsPPCK3 were highly expressed in roots and at low levels in other organs, the two OsPPCK2 transcripts were expressed in all organs. OsPPCK3 was expressed mostly at night, while the long OsPPCK2 transcripts were present in the leaves only in the daytime. Nitrate supplementation of leaves selectively induced expression of both OsPPCK2 transcripts, while phosphate starvation only induced the shorter one. Such diverse expression patterns of OsPPCK genes suggest the importance and variety of strict activity regulation of PEPC in rice. From the correlation between gene expression and the phosphorylation level of PEPC, which was monitored as that of the maize PEPC expressed in transgenic rice plants, it was concluded that the short OsPPCK2 transcripts were expressed in rice leaf mesophyll cells upon nitrogen supplementation and phosphate starvation, whereas OsPPCK3 participated in the nocturnal phosphorylation of PEPC in these cells. Expression of PPCK proteins in rice leaves was detected by immunoblotting using a specific antiserum, and the expression of two different OsPPCK2 proteins derived from alternative transcription initiation was confirmed.

Differentiation of dicarboxylate transporters in mesophyll and bundle sheath chloroplasts of maize.

In NADP-malic enzyme-type C(4) plants such as maize (Zea mays L.), efficient transport of oxaloacetate and malate across the inner envelope membranes of chloroplasts is indispensable. We isolated four maize cDNAs, ZmpOMT1 and ZmpDCT1 to 3, encoding orthologs of plastidic 2-oxoglutarate/malate and general dicarboxylate transporters, respectively. Their transcript levels were upregulated by light in a cell-specific manner; ZmpOMT1 and ZmpDCT1 were expressed in the mesophyll cell (MC) and ZmpDCT2 and 3 were expressed in the bundle sheath cell (BSC). The recombinant ZmpOMT1 protein expressed in yeast could transport malate and 2-oxoglutarate but not glutamate. By contrast, the recombinant ZmpDCT1 and 2 proteins transported 2-oxoglutarate and glutamate at similar affinities in exchange for malate. The recombinant proteins could also transport oxaloacetate at the same binding sites as those for the dicarboxylates. In particular, ZmpOMT1 transported oxaloacetate at a higher efficiency than malate or 2-oxoglutarate. We also compared the activities of oxaloacetate transport between MC and BSC chloroplasts from maize leaves. The K(m) value for oxaloacetate in MC chloroplasts was one order of magnitude lower than that in BSC chloroplasts, and was close to that determined with the recombinant ZmpOMT1 protein. Southern analysis revealed that maize has a single OMT gene. These findings suggest that ZmpOMT1 participates in the import of oxaloacetate into MC chloroplasts in exchange for stromal malate. In BSC chloroplasts, ZmpDCT2 and/or ZmpDCT3 were expected to import malate that is transported from MC.

Identifying and characterizing plastidic 2-oxoglutarate/malate and dicarboxylate transporters in Arabidopsis thaliana.

We characterized three Arabidopsis genes, AtpOMT1, AtpDCT1 and AtpDCT2, localized on chromosome 5 and homologous to spinach chloroplastic 2-oxoglutarate/malate transporter (OMT) gene. The yeast-expressed recombinant AtpOMT1 protein transported malate and 2-oxoglutarate but not glutamate. By contrast, the recombinant AtpDCT1 protein transported 2-oxoglutarate and glutamate at similar affinities in exchange for malate. These findings suggested that AtpOMT1 is OMT and AtpDCT1 is a general dicarboxylate transporter (DCT). The recombinant proteins could also transport oxaloacetate at the same binding sites for dicarboxylates. In particular, the AtpOMT1 had a K(m) value for oxaloacetate one order of magnitude lower than those for malate and 2-oxoglutarate. Although the transcripts for the three genes were accumulated in all tissues examined, the expression of the genes in leaf tissues was light inducible. The expression of the three genes was also induced by nitrate supplement but the induction was most prominent and transient in AtpOMT1 similar to nitrate reductase gene. These findings lead to a proposition that AtpOMT1 functions as an oxaloacetate transporter in the malate-oxaloacetate shuttle across chloroplast membranes. We identified T-DNA insertional mutants of AtpOMT1 and AtpDCT1. Although the AtpOMT1 mutants could grow normally in normal air, the AtpDCT1 mutants were non-viable under the same conditions. The AtpDCT1 mutants were able to grow under the high CO2 condition to suppress photorespiration. These findings suggested that at least AtpDCT1 is a necessary component for photorespiratory nitrogen recycling.