Mitsuhiro Obara

Changes in nitrogen assimilation, metabolism, and growth in transgenic rice plants expressing a fungal NADP(H)-dependent glutamate dehydrogenase (gdhA)

In plants, glutamine synthetase (GS) is the enzyme that is mainly responsible for the assimilation of ammonium. Conversely, in microorganisms such as bacteria and Ascomycota, NADP(H)-dependent glutamate dehydrogenase (GDH) and GS both have important roles in ammonium assimilation. Here, we report the changes in nitrogen assimilation, metabolism, growth, and grain yield of rice plants caused by an ectopic expression of NADP(H)-GDH (gdhA) from the fungus Aspergillus niger in the cytoplasm. An investigation of the kinetic properties of purified recombinant protein showed that the fungal gdhA had 5.4–10.2 times higher Vmax value and 15.9–43.1 times higher Km value for NH4+, compared with corresponding values for rice cytosolic GS as reported in the literature. These results suggested that the introduction of fungal GDH into rice could modify its ammonium assimilation pathway. We therefore expressed gdhA in the cytoplasm of rice plants. NADP(H)-GDH activities in the gdhA-transgenic lines were markedly higher than those in a control line. Tracer experiments by feeding with 15NH4+ showed that the introduced gdhA, together with the endogenous GS, directly assimilated NH4+ absorbed from the roots. Furthermore, in comparison with the control line, the transgenic lines showed an increase in dry weight and nitrogen content when sufficient nitrogen was present, but did not do so under low-nitrogen conditions. Under field condition, the transgenic line examined showed a significant increase in grain yield in comparison with the control line. These results suggest that the introduction of fungal gdhA into rice plants could lead to better growth and higher grain yield by enhancing the assimilation of ammonium.

Reverse genetics approach to characterize a function of NADH-glutamate synthase1 in rice plants

Rice plants grown in anaerobic paddy soil prefer to use ammonium ion as an inorganic nitrogen source for their growth. The ammonium ions are assimilated by the coupled reaction of glutamine synthetase (GS) and glutamate synthase (GOGAT). In rice, there is a small gene family for GOGAT: there are two NADH-dependent types and one ferredoxin (Fd)-dependent type. Fd-GOGAT is important in the re-assimilation of photorespiratorily generated ammonium ions in chloroplasts. Although cell-type and age-dependent expression of two NADH-GOGAT genes has been well characterized, metabolic function of individual gene product is not fully understood. Reverse genetics approach is a direct way to characterize functions of isoenzymes. We have isolated a knockout rice mutant lacking NADH-dependent glutamate synthase1 (NADH-GOGAT1) and our studies show that this isoenzyme is important for primary ammonium assimilation in roots at the seedling stage. NADH-GOGAT1 is also important in the development of active tiller number, when the mutant was grown in paddy field until the harvest. Expression of NADH-GOGAT2 and Fd-GOGAT in the mutant was identical with that in wild-type, suggesting that these GOGATs are not able to compensate for NADH-GOGAT1 function.

Mapping quantitative trait loci controlling root length in rice seedlings grown with low or sufficient supply using backcross recombinant lines derived from a cross between Oryza sativa L. and Oryza glaberrima Steud.

To understand genetic adaptation of rice to environmental nitrogen status, this study mapped and verified the quantitative trait locus/loci for root elongation in response to exogenous concentration. Significant inhibition of root elongation by increasing concentration in hydroponic culture was observed in seedlings of Oryza sativa cv Taichung 65 (japonica variety) but not in O. glaberrima line IRGC 104038. A total of 161 backcross recombinant lines between these species were employed to map the quantitative trait locus/loci controlling root length of seedlings grown hydroponically with a low (5 µM) or sufficient (500 µM) supply. Five quantitative trait locus/loci were detected: two on chromosome 1 and three singles on chromosomes 2, 3, and 6. The quantitative trait locus/loci qRL1.1 was significant under sufficient supply. Progeny analysis using heterogeneous inbred-type family nearly isogenic lines (HIF-NILs) for the candidate region of qRL1.1 revealed that seminal roots of IRGC 104038-fixed genotypes were significantly longer (12.4 or 11.6%) than those of Taichung 65-fixed genotypes when grown with 500 µM , but not without or with 5 µM . qRL1.1 was confirmed and delimited within a 4-Mbp-long region on the long-arm region of chromosome 1. Comparative mapping with genes for uptake and nitrogen metabolism showed the apparent location of the aspartate aminotransferase gene (OsAAT2) within the candidate region of qRL1.1. Together, the results suggest that qRL1.1 is an adaptive quantitative trait locus/loci for rice root elongation in response to sufficient supply of external and OsAAT2 may be a candidate gene responsible for qRL1.1.

Enhancement of porosity and aerenchyma formation in nitrogen-deficient rice roots.

Root aerenchyma provides oxygen from plant shoots to roots. In upland crops, aerenchyma formation is induced mainly by oxygen or nutrient deficiency. Unlike upland crops, rice forms root aerenchyma constitutively and also inductively in response to oxygen deficiency. However, the effects of nitrogen deficiency on aerenchyma formation in rice remain unknown although nitrogen deficiency is common in most of the worlds soils. We aimed to clarify the spatiotemporal patterns of aerenchyma formation induced in rice roots by nitrogen deficiency upon establishment of reliable growth conditions. Rice was grown hydroponically to evaluate porosity and aerenchyma formation induced by nitrogen and oxygen deficiency. Reliable growth conditions for nitrogen and oxygen deficiency were successfully established, because seedling root elongation was significantly promoted by nitrogen deficiency and inhibited by oxygen deficiency. Porosity was higher in whole roots grown under nitrogen and oxygen deficiency than in the controls. Root aerenchyma production was induced extensively by nitrogen deficiency but partially by oxygen deficiency. Thus the physiological roles of aerenchyma induced by nitrogen deficiency likely differ from those under oxygen deficiency. It indicates a possibility that inducible aerenchyma formation in nitrogen deficiency might promote adaptation to this deficiency by reducing respiration and remobilizing nitrogen, or both.

Isolation of a novel mutant gene for soil-surface rooting in rice (Oryza sativa L.)

BackgroundRoot system architecture is an important trait affecting the uptake of nutrients and water by crops. Shallower root systems preferentially take up nutrients from the topsoil and help avoid unfavorable environments in deeper soil layers. We have found a soil-surface rooting mutant from an M2 population that was regenerated from seed calli of a japonica rice cultivar, Nipponbare. In this study, we examined the genetic and physiological characteristics of this mutant.ResultsThe primary roots of the mutant showed no gravitropic response from the seedling stage on, whereas the gravitropic response of the shoots was normal. Segregation analyses by using an F2 population derived from a cross between the soil-surface rooting mutant and wild-type Nipponbare indicated that the trait was controlled by a single recessive gene, designated as sor1. Fine mapping by using an F2 population derived from a cross between the mutant and an indica rice cultivar, Kasalath, revealed that sor1 was located within a 136-kb region between the simple sequence repeat markers RM16254 and 2935-6 on the terminal region of the short arm of chromosome 4, where 13 putative open reading frames (ORFs) were found. We sequenced these ORFs and detected a 33-bp deletion in one of them, Os04g0101800. Transgenic plants of the mutant transformed with the genomic fragment carrying the Os04g0101800 sequence from Nipponbare showed normal gravitropic responses and no soil-surface rooting.ConclusionThese results suggest that sor1, a rice mutant causing soil-surface rooting and altered root gravitropic response, is allelic to Os04g0101800, and that a 33-bp deletion in the coding region of this gene causes the mutant phenotypes.

Comparison of contents for cytosolic-glutamine synthetase and NADH-dependent glutamate synthase proteins in leaves of japonica, indica, and javanica rice plants

As reported previously with a Japonica-type rice, Sasanishki, cytosolic glutamine synthetase (GS1; EC 6.3.1.2) was detected in companion cells as well as vascular-parenchyma cells of vascular bundles of senescing blades (Sakurai et al., 1996), while NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in the developing young tissues was located in cell types where the solutes are transported from phloem and xylem (Hayakawa et al., 1994). These results suggest that GS1 is important for the export of leaf nitrogen from senescing leaves, whereas NADH-GOGAT is involved in the synthesis of glutamate from the glutamine that is transported through the vascular system. To evaluate their functions further in the nitrogen remobilization and re-utilization in rice, several cultivars of indica and javanica rice were tested to estimate the contents for GS1 and NADH-GOGAT proteins in senescing and developing leaf blades, respectively. Chinsurah Boro I and Blue Stick, indica cultivars tested in the current study, contained more GS1 protein than Sasanishiki, a japonica, in the senescing leaf blades, when the content was compared on an unit of g fresh weight basis. On the other hand, NADH-GOGAT content in the young leaf blades of Sasanishiki was the highest.

Fine mapping of a quantitative trait locus for spikelet number per panicle in a new plant type rice and evaluation of a near-isogenic line for grain productivity

qTSN12.1 and qTSN12.2 genes for total spikelet number per panicle were detected, qTSN12.2 on rice chromosome 12. Grain yield of a near-isogenic line carrying fine-mapped qTSN12.2 was increased by 18–36%.

Genetic variation of root angle distribution in rice (Oryza sativa L.) seedlings

We developed a new method of using seedling trays to evaluate root angle distribution in rice (Oryza sativa. L), and found a wide genetic variation among cultivars. The seedling tray method can be used to evaluate in detail the growth angles of rice crown roots at the seedling stage by allocating nine scores (10° to 90°). Unlike basket methods, it can handle large plant populations over a short growth period (only 14 days). By using the method, we characterized the root angle distributions of 97 accessions into two cluster groups: A and B. The numbers of accessions in group A were limited, and these were categorized as shallow rooting types including soil-surface root. Group B included from shallow to deep rooting types; both included Indica and Japonica Group cultivars, lowland and upland cultivars, and landraces and improved types. No relationship between variation in root vertical angle and total root number was found. The variation in root angle distribution was not related to differentiation between the Japonica and Indica Groups, among ecosystems used for rice cultivation, or among degrees of genetic improvement. The new evaluation method and associated information on genetic variation of rice accessions will be useful in root architecture breeding of rice.

Identification of a low tiller gene from a new plant type cultivar in rice (Oryza sativa L.)

We characterized a rice introgression line, YTH34, harboring a chromosome segment from a New Plant Type (NPT) cultivar, IR65600-87-2-2-3, in the genetic background of an Indica Group elite rice cultivar, IR 64, under upland and irrigated lowland conditions in Japan. The number of panicles (as an indicator of tiller number) and number of spikelets per panicle of YTH34 were lower than those of IR 64 under irrigated lowland conditions, but both of those as well as culm length, panicle length, seed fertility, panicle weight, whole plant weight, and harvest index were dramatically reduced under upland conditions. And the low tiller of YTH34 was confirmed to start after the maximum tiller stage. In particular, the decrease of panicle number was remarkable in upland, so we tried to identify the chromosome location of the relevant gene. Through segregation and linkage analyses using F3 family lines derived from a cross between IR 64 and YTH34, and SSR markers, we found that low tiller number was controlled by a single recessive gene, ltn2, and mapped with the distance of 2.1 cM from SSR marker RM21950, in an introgressed segment on chromosome 7. YTH34 harboring ltn2 and the genetic information for DNA markers linked will be useful for genetic modification of plant architectures of Indica Group rice cultivar.

Genetic variation of rice (Oryza sativa L.) germplasm in Myanmar based on genomic compositions of DNA markers

The genetic diversity of 175 rice accessions from Myanmar, including landraces and improved types from upland and lowland ecosystems in five different areas—Western (hilly), Northern (mountainous), North and South-eastern (plateau), and Southern (plain)—was evaluated on the basis of polymorphism data for 65 DNA markers and phenol reactions. On the basis of the DNA polymorphism data, high genetic diversity was confirmed to conserve in the accessions from each ecosystem and area. And the accessions were classified into two cluster groups I and II, which corresponded to Indica Group and Japonica Group, respectively. Cluster group I accessions were distributed mainly in upland ecosystems; group II were distributed in lowland in the Southern area, and the distributions of dominant groups differed among areas. Rice germplasm in Myanmar has maintained high genetic diversity among ecosystems and areas. This information will be used for advanced studies in germplasm and rice breeding in Myanmar.