The roles of Arg548 and Gln552 residues in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase were investigated using site-directed mutagenesis. Mutation of Arg548 to alanine or glutamine resulted in the destabilisation of the quaternary structure of the enzyme, suggesting that this residue has a structural role. The mutations R548K, Q552N and Q552A resulted in loss of the ability to catalyse pyruvate carboxylation, biotin-dependent decarboxylation of oxaloacetate and proton exchange between pyruvate and water. These mutants retained the ability to catalyse reactions that occur at the active site of the biotin carboxylase domain, i.e. bicarbonatedependent ATP cleavage and ADP phosphorylation by carbamoyl phosphate. The effects of oxamate on the catalysis in the biotin carboxylase domain by the R548K and Q552N mutants were similar to those on the catalysis of reactions by the wild-type enzyme. However, the presence of oxamate had no effect on the reactions catalysed by the Q552A mutant. We propose that Arg548 and Gln552 facilitate the binding of pyruvate and subsequent proton transfer between pyruvate and biotin in the partial reaction catalysed in the active site of the carboxyl-transferase domain of Rhizobium etli pyruvate carboxylase.Pyruvate carboxylase (PC) (EC 6.4.1.1), a biotin-containing enzyme, catalyses pyruvate carboxylation through a two-step reaction shown in Figure 1 (1,2). In reaction [1] (Fig. 1), the carboxylation of biotin in the biotin carboxylase (BC) domain is thought to proceed by an initial reaction between HCO 3 − and MgATP to form MgADP and a reactive carboxyphosphate intermediate (1,3,4 NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptRecently, structures of several pyruvate carboxylases have been determined (5-7). Hybrid tetramers, composed of mutated subunits of Rhizobium etli (RePC), that either lack biotin or have a carboxyl-transferase (CT) domain mutation, which greatly reduces its ability to catalyse the full forward reaction, nevertheless retain the ability to catalyse the carboxylation of pyruvate, albeit at a reduced rate as compared to wild-type RePC (5), demonstrating that reaction [1] occurs in the BC domain of one subunit whilst reaction [2] occurs in the CT domain of its partner subunit on the same face of the tetrameric enzyme.Previously, the catalytic mechanism and those residues involved in the reactions of both the BC and CT domains have been studied by mutating amino acid residues that are conserved in homologous enzymes (8,9) where the structures of the subunits of these enzymes, such as the biotin carboxylase subunit of acetyl-CoA carboxylase (10) and the 5S subunit of transcarboxylase (11) have been determined. However, with the recent determination of several structures of the PC holoenzyme from various organisms (5-7), a more structure-directed mutational analysis to determine the detailed mechanism of PC has commenced (12).Based on kinetic isotope effect studies, the catalytic reaction in th...

Insertional mutagenesis using transfer DNA or transposable elements, which is an important tool in functional genomics and is well established in several crops, has not been developed in potato (Solanum tuberosum). Here, we report the application of the tobacco (Nicotiana tabacum) Tnt1 retrotransposon as an insertional mutagen in potato. The Tnt1 retrotransposon was introduced into a highly homozygous and self-compatible clone, 523-3, of the diploid wild potato species Solanum chacoense. Transposition of the Tnt1 elements introduced into 523-3 can be efficiently induced by tissue culture. Tnt1 preferentially inserted into genic regions in the potato genome and the insertions were stable during sexual reproduction, making Tnt1 an ideal mutagen in potato. Several distinct phenotypes associated with plant stature and leaf morphology were discovered in mutation screening from a total of 38 families derived from Tnt1-containing lines. We demonstrate that the insertional mutagenesis system based on Tnt1 and the 523-3 clone can be expanded to the genome-wide level to potentially tag every gene in the potato genome.

Drought is a major constraint to oil palm plantation in many regions relying on rainfall. One strategy to overcome this problem is to develop drought-tolerant cultivars through screening and selection. Drought tolerance assessment based on growth and physiological traits provides an alternative approach for rapid screening especially in tree crops. The aims of this study were to determine the contribution of some growth and physiological parameters to drought response variation in oil palm seedling and to use those parameters to evaluate relative drought stress tolerance in tenera oil palm progenies. Eight D × P progenies were grown under well-watered, moderate and severe stressed conditions for a total of 6 months. Data on growth traits, maximum quantum yield (Fv/Fm), SPAD value and proline content were recorded. Principal component analysis (PCA) on studied traits revealed that PC1 and PC2 explained 74.57 and 76.80% of total variance in moderate and severe drought treatments, respectively. Major contributions towards the variation of control and drought-treated plants were identified and appeared to be growth traits in moderate drought stress and growth trait plus proline content in severe drought stress. According to drought tolerance index, PCA analysis and ranking method using integrated PCA values, PSU-106 and PSU-206 were identified to be relatively high tolerant to severe drought stress, while PSU-106, PSU-128, PSU-206 and PSU-208 were tolerant to moderate drought stress. Field validation of D × P progenies selected as drought tolerance is recommended to establish this indirect selection approach in oil palm breeding programme.

Lowland rice is an important cereal crop that plays a key role in the food security and the economy of Thailand. Terminal water stress (TWS) in rainfed lowland areas poses threats to rice productivity due to stress occurrence at terminal crop stages and extreme sensitivity of rice to TWS. A two-year study was conducted to characterize the performance of yield and yield attributes of twelve Thai lowland rice genotypes under TWS, to identify stress-tolerant genotypes using stress response indices and to identify promising stress indices which are correlated with grain yield (GY) under well-watered (WW) and TWS conditions for their use as rapid identifiers in a rice crop breeding program for enhancing drought stress tolerance. Measurements were recorded under WW and TWS conditions. Highly significant variations were observed amongst assessed genotypes for their yield productivity responses. According to stress response indices, genotypes were categorized into stress-tolerant and stress susceptible genotypes. Genotypes Hom Pathum, Sang Yod, Dum Ja and Pathum Thani-1 were found highly stress tolerant and relatively high yielding; genotypes Look Pla and Lep Nok were stress tolerant, whereas genotypes Chor Lung, Hom Nang Kaew and Hom Chan were moderately tolerant genotypes. Hence, stress-tolerant genotypes could be potentially used for cultivation under rainfed and water-limited conditions, where TWS is predicted particularly in southern Thailand to stabilize rice productivity. Stress tolerance indices, including stress tolerance index (STI), geometric mean productivity (GMP), mean productivity index (MPRO) and harmonic mean index (MHAR), indicated strong and positive associations with GY under WW and TWS; thus, these indices could be used to indicate stress tolerance in rice crop breeding program aimed at a rapid screening of lowland rice genotypes for stress tolerance.

Leaf spot disease, particularly Curvularia leaf spot (CLS), can devastatingly damage both quality and quantity of oil palm seedlings in Thailand. Chemical fungicides have been intensively applied to control transmission of this disease. However, this has relatively low efficiency as the causative pathogen gains resistance to fungicides with selection pressure. An alternative or complementary cost-effective and environmentally friendly approach is necessary to find the resistant varieties for disease management. In this present study, a total of 122 lines of Dura female plant, 2 lines of Pisifera male plant, and 4 Tenera commercial varieties (A, B, C and SUP-PSU1) were screened by Curvularia inoculation using detached leaf method. Two weeks after inoculation, the results showed 13 Dura lines as highly resistant to CLS (0% disease incidence), whereas one line (129) and the commercial variety B were highly susceptible (100% disease incidence). Nine Tenera hybrid lines from 13 Dura highly resistant lines were selected to test for high resistance and subjected to Curvularia inoculation in greenhouse conditions, and compared with the susceptible and highly susceptible lines. Three Tenera hybrid lines (138, 187 and 203) showed high resistance to CLS significant difference from susceptible lines. These were the most highly resistant varieties to CLS and should be considered for breeding programs of oil palm stock among the cases tested.

Bioenergy from rice biomass feedstock is considered one of the potential clean energy resources and several small biomass-based powerplants have been established in rice–growing areas of Thailand. Rice production is significantly affected by drought occurrence which results in declined biomass production and quality. The impact of water stress (WS) was evaluated on six rice cultivars for biomass quality, production and bioenergy potential. Rice cultivars were experimented on in the field under well–watered (WW) and WS conditions. Data for biomass contributing parameters were collected at harvest whereas rice biomass samples were analyzed for proximate and lignocellulosic contents. Results indicated that WS negatively influenced crop performance resulting in 11–41% declined biomass yield (BY). Stability assessment indicated that cultivars Hom Pathum and Dum Ja were stress–tolerant as they exhibited smaller reductions by 11% in their BY under WS. Statistics for proximate components indicated a significant negative impact influencing biomass quality as ash contents of Hom Chan, Dum Ja and RD-15 were increased by 4–29%. Lignocellulosic analysis indicated, an increase in lignin contents of Hom Nang Kaew, Hom Pathum, Dum Ja and RD–15 ranging 7–39%. Reduced biomass production resulted in a 10–42% reduction in bioenergy potential (E). Results proved that cultivation of stress-susceptible cultivars or farmer’s choice and occurrence of WS during crop growth will reduce biomass production, biomass feedstock availability to biomass-based powerplants and affect powerplant’s conversion efficiency resulting in declined bioenergy production.

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Vigorous and well-established nursery seedlings are an important component of sustainable oil palm production. We postulated that Si fertilization at the seedling stage could help to achieve improved performance of oil palm seedlings leading to healthy and vigorous nursery establishment. In this study, we evaluated the growth and physiological responses of oil palm Tenera hybrid seedlings under three Si fertilization treatments and a control including (i) 0 g Ca2SiO4 (T0), (ii) 0.5 g Ca2SiO4 (T1), (iii) 3.5 g Ca2SiO4 (T2), and (iv) 7.0 g Ca2SiO4 (T3) per plant per month. Ca2SiO4 was used as the Si fertilizer source and was applied for four consecutive months. Nondestructive data including stem diameter, plant height, leaf length, photosynthetic rate, leaf angle, and leaf thickness and destructive data including leaf, stem, and root fresh weight and dry weight, as well as chlorophyll a, Si, and nitrogen contents, were recorded before treatment (0 DAT), as well as 60 (60 DAT) and 120 days after treatment (120 DAT). Results indicated that Si fertilization enhanced Si accumulation in oil palm seedlings, and maximum accumulation was observed in the aerial parts especially the leaves with the highest accumulation of 0.89 % dry weight at T3. Higher Si accumulation stimulated the growth of seedlings; a total fresh weight of 834.28 g and a total dry weight of 194.34 g were observed at T3. Chlorophyll a content (0.83 gm−2) and net photosynthetic rate (4.98 µM CO2·m−2·s−1) were also observed at T3. Leaf morphology was not significantly influenced under Si fertilization, whereas the nitrogen content of seedlings was significantly increased. Correlation analysis revealed a highly significant and positive association among Si accumulation, chlorophyll a content, photosynthetic rate, total fresh weight, total dry weight, and nitrogen content of seedlings, indicating that Si fertilization enhanced the performance of these attributes. On the basis of the research evidence, it was concluded that Si fertilization should be considered for improved nutrient management for oil palm seedling and nursery production.