Somaclonal variation in rice – Submergence tolerance and other agronomic characters

The ability to withstand complete submergence was studied in R₂ seedlings raised by self-pollination from 158 R₁ plants regenerated from callus of mature rice seeds (cultivars FR13A and Calrose). Compared to parental controls, significant improvement in submergence tolerance as assessed by percentage survival, number of new leaves and chlorophyll content of the third leaf, was found in 5 of the 115 cv. FR13A somaclones and 3 of the 43 cv. Calrose somaclones tested using an aquarium system in a controlled-temperature glasshouse. With some exceptions these improvements were also observed in the R₃ generation when tested under field conditions in Thailand. Variation in other agronomically important characters was observed in the R₂ plants grown in a temperature glasshouse under 2 contrasting environments.     

High-throughput analysis of fatty acid composition of plasma glycerophospholipids

Plasma FA composition, a marker of FA status and dietary intake, is associated with health outcomes on a short- and long-term basis. Detailed investigation of the relationships between plasma FA composition and health requires the analysis of large numbers of samples, but manual sample preparation is very cumbersome and time consuming. We developed a high-throughput method for the analysis of FAs in plasma glycerophospholipids (GPs) with increased sensitivity. Sample preparation requires two simple steps: protein precipitation and subsequent base catalyzed methyl ester synthesis. Analysis of GP FAs is performed by gas chromatography. Coefficients of variation for FAs contributing more than 1% to total FAs are below 4%. Compared with the established reference method, results of the new method show good agreement and very good correlations (r > 0.9). The new method reduces the manual workload to about 10% of the reference method. Only 100 µl plasma volume is needed, which allows for the analysis of samples from infants. The method is well suitable for application in large clinical trials and epidemiological studies.     

Somaclonal variation does not preclude the use of rice transformants for genetic screening

Rice (Oryza sativa) is one of the world's most important crops. Rice researchers make extensive use of insertional mutants for the study of gene function. Approximately half a million flanking sequence tags from rice insertional mutant libraries are publicly available. However, the relationship between genotype and phenotype is very weak. Transgenic plant assays have been used frequently for complementation, overexpression or antisense analysis, but sequence changes caused by callus growth, Agrobacterium incubation medium, virulence genes, transformation and selection conditions are unknown. We used high‐throughput sequencing of DNA from rice lines derived from Tainung 67 to analyze non‐transformed and transgenic rice plants for mutations caused by these parameters. For comparison, we also analyzed sequence changes for two additional rice varieties and four T‐DNA tagged transformants from the Taiwan Rice Insertional Mutant resource. We identified single‐nucleotide polymorphisms, small indels, large deletions, chromosome doubling and chromosome translocations in these lines. Using standard rice regeneration/transformation procedures, the mutation rates of regenerants and transformants were relatively low, with no significant differences among eight tested treatments in the Tainung 67 background and in the cultivars Taikeng 9 and IR64. Thus, we could not conclusively detect sequence changes resulting from Agrobacterium‐mediated transformation in addition to those caused by tissue culture‐induced somaclonal variation. However, the mutation frequencies within the two publically available tagged mutant populations, including TRIM transformants or Tos17 lines, were about 10‐fold higher than the frequency of standard transformants, probably because mass production of embryogenic calli and longer callus growth periods were required to generate these large libraries.     

Somaclonal variation rate evolution in plant tissue culture: Contribution to understanding through a statistical approach

Summary In order to better understand somaclonal variant rate evolution in plant tissue culture, a statistical approach has been adopted. According to this approach, the variant percentage could be calculated by: %V=[1−(1−p) ⁿ ]×100, where %V is the percentage of variant, p the probability of variation and n the number of multiplication cycles. A numerical estimation was performed to characterize the variance of this function. It has been demonstrated that a wide scale of variance is associated with ‘%V’, due to the occurrence of variations after a variable number of multiplication cycles in the different lines of culture. Two main conclusions can be drawn from this model: (1) a variant rate increase can be expected as an exponential function of the number of multiplication cycles; (2) after a given number of multiplication cycles, variable off-types percentages can be expected. Due to the complexity of biological systems, this statistical approach could obviously not be applied directly for the calculation and forecasting of variant rates in tissue culture. However, this approach results in a better understanding of two apparently confusing experimental features often reported in tissue culture: the increase of the variant rate as a function of the length of the culture period on the one hand, and, on the other hand, the observations of different variant rates among lines cultured for the same lengths of time under strietly identical culture conditions. This approach also underlined that the comparison of somaclonal variant percentage between batches of plants from different in vitro treatments could be, in some cases, insufficient for ascertaining a difference of variability generated by tissue culture.     

Effect of exogenous fatty acids on growth,membrane fluidity,and phospholipid fatty acid composition in yeast

The growth response of a double-mutant fatty acid auxotroph of yeast Saccharomyces cerevisiae to exogenous saturated fatty acids of a homologous series from 12:0 to 16:0, each supplied with oleate, linoleate, linolenate, or cis-Δ¹¹- eicosenoate, cannot be explained in terms of the efficiency of incorporation of the fatty acids into phospholipids or alteration of membrane fluidity. There is, however, a negative correlation between growth and levels of 12:0 plus 13:0 in phospholipids, as well as a positive correlation between growth and levels of 14:0, 1 5:0, and 1 6:0. We, therefore, conclude that the predominant factor in these phospholipid fatty acyl chain modifications is maintenance of an optimal concentration of C14:0 through C16:0 in phospholipids of this organism.     

Effect of habitat and motor activity of molluscs on fatty acid composition of triglycerides and phospholipids

A comparative analysis of fatty acids (FA) in neutral lipids and phospholipids of digestive gland and pedal muscle has been performed in molluscs from various ecological groups differing by belonging to sea or fresh water, trophic types or the associated motor activity. In freshwater pulmonary gastropods Lymnaea stagnalis and Lymnaea ovalis and marine prosobranchial molluscs Buccinum undatum and Littorina littorea the total content of ω3-acids in phospholipids of the studied tissues differed more than twice, predominantly due to the combined effect of temperature and salinity of the habitat. The lower viscosity of cell membranes in marine species (ω3/ω6 < 1) is determined to the greatest degree by the presence of eicosapentaenoic acid that accounts for 22–25% of the FA sum in marine species. Comparison of the molluscs by their trophic belonging has revealed the presence of linoleic acid in triglycerides in digestive glands of phytophages (8–12%), but the practically complete absence of this acid in the predator B. undulatum (< 0.8%). By mobility, L. littorea inhabiting the high-low tide littoral was inferior to freshwater pulmonary gastropods and to the marine predator, as it stops moving twice a day during the low tide. In phospholipids of pedal muscle of this mollusc the amount of long-chain polyunsaturated C: 22 FA was 3–6 times lower than that in other studied species, which might possibly indicate the role of these acids in functioning of the pedal muscle contractile tissue. On the whole, use of the FA characteristics as the parameters determining belonging to certain ecological group requires a certain caution due to a complex action of biotic and abiotic factors on the animal metabolism. The exception is the ω3/ω6 ratio in total phospholipids of fresh water and marine gastropods.     

AKR2A interacts with KCS1 to improve VLCFAs contents and chilling tolerance of Arabidopsis thaliana

Arabidopsis thaliana AKR2A plays an important role in plant responses to cold stress. However, its exact function in plant resistance to cold stress remains unclear. In the present study, we found that the contents of very long‐chain fatty acids (VLCFAs) in akr2a mutants were decreased, and the expression level of KCS1 was also reduced. Overexpression of KCS1 in the akr2a mutants could enhance VLCFAs contents and chilling tolerance. Yeast‐2‐hybrid and bimolecular fluorescence complementation (BIFC) results showed that the transmembrane motif of KCS1 interacts with the PEST motif of AKR2A both in vitro and in vivo. Overexpression of KCS1 in akr2a mutants rescued akr2a mutant phenotypes, including chilling sensitivity and a decrease of VLCFAs contents. Moreover, the transgenic plants co‐overexpressing AKR2A and KCS1 exhibited a greater chilling tolerance than the plants overexpressing AKR2A or KCS1 alone, as well as the wild‐type. AKR2A knockdown and kcs1 knockout mutants showed the worst performance under chilling conditions. These results indicate that AKR2A is involved in chilling tolerance via an interaction with KCS1 to affect VLCFA biosynthesis in Arabidopsis.     

New insights into the genetic basis of natural chilling and cold shock tolerance in rice by genome‐wide association analysis

In order to understand cold adaptability and explore additional genetic resources for the cold tolerance improvement of rice, we investigated the genetic variation of 529 rice accessions under natural chilling and cold shock stress conditions at the seedling stage using genome‐wide association studies; a total of 132 loci were identified. Among them, 12 loci were common for both chilling and cold shock tolerance, suggesting that rice has a distinct and overlapping genetic response and adaptation to the two stresses. Haplotype analysis of a known gene OsMYB2, which is involved in cold tolerance, revealed indica–japonica differentiation and latitude tendency for the haplotypes of this gene. By checking the subpopulation and geographical distribution of accessions with tolerance or sensitivity under these two stress conditions, we found that the chilling tolerance group, which mainly consisted of japonica accessions, has a wider latitudinal distribution than the chilling sensitivity group. We conclude that the genetic basis of natural chilling stress tolerance in rice is distinct from that of cold shock stress frequently used for low‐temperature treatment in the laboratory and the cold adaptability of rice is associated with the subpopulation and latitudinal distribution.     

Effect of chilling and subsequent storage at 20°C on electrolyte leakage and phospholipid fatty acid composition of tomato pericarp

Mature green tomato fruit ( Lycopersicon esculentum cv. Caruso) were stored at 1°C or 20°C and analyzed on day 0, 18 and 22 for electrolyte leakage, ripening-associated changes in pigmentation and phospholipid fatty acid composition. Chilled fruit were also analyzed 4 days after they were returned to 20°C. Fruit did not ripen significantly during chilling and subsequent storage at 20°C, and showed visible chilling injury symptoms only at 20°C. Electrolyte leakage increased in control and chilled fruit, indicating enhanced membrane permeability during both ripening and chilling. Returning the fruit to ambient temperature gave an apparent decrease in electrolyte leakage. Phospholipid and linolenic acid content and double bond index decreased during ripening at 20°C. The small changes in phospholipid fatty acid composition during chilling cannot account for the enhanced membrane permeability. The significant decrease in percentage of linolenic acid and in double bond index in the total lipids, but not in the phospholipids, upon returning the fruit to 20°C suggests loss of galactolipid polyunsaturated fatty acids     

Comparison of phospholipid and fatty acid composition of wild and cultured striped bass eggs

The dominant fatty acids in all neutral lipid fractions of non-water hardened eggs from two wild and one cultured stock of striped bass Morone saxatilis were the monoenes, 18 : 1n9/n7>16 : 1n7>17 : 1. The dominant fatty acids in the phospholipid fraction of all eggs, regardless of origin, were 22 : 6n3>18 : 1n9/n7>20 : 5n3>16 : 1n7>16 : 0>18 : 0. Arachadonic acid (AA, 20 : 4n6) was significantly lower (2·0%) in cultured fish eggs compared to either wild stock (5·8–6·1%). Fatty acids from the liver and eggs of wild Shubenacadie fish were similar to one another with respect to both neutral and phospholipid fractions. However, the AA and eicosapentaenoic acid (EPA, 20 : 5n3) content of the phospholipid fraction varied according to the hypothesized migration behaviour of Shubenacadie fish. The total lipid content of wild fish eggs was significantly greater than that of cultured fish. The total phospholipid content of Shubenacadie eggs was significantly higher than either Roanoke or cultured fish eggs. Phosphotidylinositol (PI) was the dominant phospholipid found ins all egg samples from all origins as opposed to phosphotidylcholine, which is usually the dominant phospholipid. These data indicate that PI and AA may have important and as yet unidentified roles in fertilization and embryonic development in these fish.     

Introduction of the cDNA for shape Arabidopsis glycerol-3-phosphate acyltransferase (GPAT) confers unsaturation of fatty acids and chilling tolerance of photosynthesis on rice

The chilling sensitivity of several plant species is closely correlated with the levels of unsaturation of fatty acids in the phosphatidylglycerol (PG) of chloroplast membranes. Plants with a high proportion of unsaturated fatty acids, such as Arabidopsis thaliana, are resistant to chilling, whereas species like squash with only a low proportion are rather sensitive to chilling. The glycerol-3-phosphate O-acyltransferase (GPAT) enzyme of chloroplasts plays an important role in determining the levels of PG fatty acid desaturation.A cDNA for oleate-selective GPAT of Arabidopsis under the control of a maize Ubiquitin promoter was introduced into rice (Oryza sativa L.) using the Agrobacterium-mediated gene transfer method. The levels of unsaturated fatty acids in the phosphatidylglycerol of transformed rice leaves were found to be 28% higher than that of untransformed controls. The net photosynthetic rate of leaves of transformed rice plants was 20% higher than that of the wild type at 17°C. Thus, introduction of cDNA for the Arabidopsis GPAT causes greater unsaturation of fatty acids and confers chilling tolerance of photosynthesis on rice.     

龙眼叶片膜脂脂肪酸组分与龙眼耐寒性的关系

龙眼叶片膜脂不饱和脂肪酸含量和脂肪酸不饱和度与龙眼不同品种的耐寒性呈正相关;在年周期中,不饱和脂肪酸含量的变化与龙眼耐寒性的变化呈现平行关系;龙眼叶片膜脂脂肪酸组分含量反映着龙眼品种间耐寒性遗传上的差异,可作为耐寒性的鉴定指标。     

Changes in the composition of membrane lipids in relation to differentiation in Aegle marmelos callus cultures

Changes in fatty acid, phospholipid and galactolipid contents during cellular and organ differentiation in Aegle marmelos have been described. Decrease in phosphatidylinositol content and presence of 3-trans-hexadecenoic acid in phosphatidylglycerol were related to greening and shoot buds differentiation. The galactolipids level, the monogalactosyl diglyceride/digalactosyl diglyceride ratio and the linolenic acid level (mainly in monogalactosyl diglyceride) increased with the degree of differentiation, indicating the possible biogenesis of functional chloroplasts.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - BA benzylaminopurine - DW dry weight - FW fresh weight - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PG phosphatidylglycerol - PS phosphatidyl serine - MGDG monogalactosyl diglyceride - DGDG digalactosyl diglyceride - 16:0 palmatic acid - 18:0 stearic acid - 18:1 oleic acid - 18:2 linoleic acid - 18:3 linolenic acid - trans-16:1 3-trans-hexadecenoic acid     

Engineering membrane asymmetry to increase medium-chain fatty acid tolerance in Saccharomyces cerevisiae

Saccharomyces cerevisiae is an attractive chassis for the production of medium-chain fatty acids, but the toxic effect of these compounds often prevents further improvements in titer, yield, and productivity. To address this issue, Lem3 and Sfk1 were identified from adaptive laboratory evolution mutant strains as membrane asymmetry regulators. Co-overexpression of Lem3 and Sfk1 [Lem3(M)-Sfk1(H) strain] through promoter engineering remodeled the membrane phospholipid distribution, leading to an increased accumulation of phosphatidylethanolamine in the inner leaflet of the plasma membrane. As a result, membrane potential and integrity were increased by 131.5% and 29.2%, respectively; meanwhile, the final OD₆₀₀ in the presence of hexanoic acid, octanoic acid, and decanoic acid was improved by 79.6%, 73.4%, and 57.7%, respectively. In summary, this study shows that membrane asymmetry engineering offers an efficient strategy to enhance medium-chain fatty acids tolerance in S. cerevisiae, thus generating a robust industrial strain for producing high-value biofuels.     

Modification of phospholipids fatty acid composition in reuber H35 hepatoma cells: effect on HMG-CoA reductase activity

There is controversy about the effect of saturated and polyunsaturated fats on 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, the main regulatory enzyme of cholesterogenic pathway. Results from dietary studies are difficult to interpret because diets normally contain a mixture of fatty acids. Therefore, we have used Reuber H35 hepatoma cells whose phospholipids were enriched in different individual fatty acids and have studied their effects on the cellular reductase activity. Lauric, myristic, eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids were supplemented to the culture medium coupled to bovine serum albumin. The four fatty acids were incorporated into phospholipids from cells grown in media containing whole serum or lipoprotein-poor serum (LPPS). Reductase activity of cells cultivated in a medium with LPPS was three to four times higher than those cultivated in medium with whole serum. Saturated fatty acids increased reductase activity of cells grown in medium with whole serum, whereas n-3 polyunsaturated fatty acids (PUFA) decreased it. However, both saturated and polyunsaturated fatty acids increased reductase activity when serum lipoproteins were removed. In conclusion, this is one of the first reports demonstrating that saturated and n-3 PUFA only show differential effects on HMG-CoA reductase activity in the presence of lipoproteins.     

Role of abscisic acid in chilling tolerance of rice (Oryza sativa L.) seedlings. I. Endogenous abscisic acid levels

Changes of ABA levels in chilled rice (Oryza sativa L.) seedlings of two varieties were determined. On exposure to chilling, ABA concentration rapidly increased in the chilling-tolerant cultivar (cv. Tainung 67, TNG.67) but not in the chilling-sensitive cultivar (cv. Taichung Native 1, TN.1). Both detached shoots and roots of TNG.67 seedlings showed a significant ABA increase after exposure to chilling. TN.1 seedlings could not accumulate ABA under low temperature but well-watered status. Exogenous application of the ABA biosynthetic inhibitor, fluridone, reduced ABA accumulation, as well as survival ratio of chilled TNG.67 seedlings. Electrolyte leakage and leaf conductance were also increased by the inhibitor and the effects could be reversed by exogenously applied ABA. ABA concentrations in xylem sap of TNG.67 seedlings increased within 4 h after chilling, and this was temporally coincident with the reduction of leaf conductance. The roles of endogenous ABA in the tolerance of rice seedlings to chilling on a whole plant basis are discussed and suggested.     

Mutational analysis of chilling tolerance in plants

A mutational approach was taken to identify genes required for low-temperature growth of the chilling-tolerant plant Arabidopsis thaliana. The screen identified mutants that were specifically compromised in their ability to grow at 5°C but were indistinguishable from wild type when grown at 22°C. The populations screened were mutated either by ethyl methanesulphonate or by T-DNA insertion. In both cases symptoms at 5°C included chlorosis, reduced growth, necrosis and death. This diversity of phenotypes demonstrates roles for chilling-tolerance responses in such diverse processes as organdie biogenesis, cell metabolism and cell and organ development. Co-segregation analysis on the first five mutants isolated from the T-DNA lines indicated that in three of them, pfc1, pfc2 and sop1, the chilling phenotype is the result of T-DNA insertion in a gene required for chilling tolerance rather than the creation of a temperature-conditional mutation in an essential housekeeping gene. This identification of T-DNA tagged alleles will facilitate cloning of the PFC1, PFC2 and SOP1 loci and allow for the biochemical and molecular genetic characterization of these chilling-tolerance genes and the proteins that they encode.     

低温胁迫期间水稻光合膜色素与蛋白水平的变化

对４℃和１１℃两种低温胁迫过程中水稻类囊体膜色素与蛋白组成的变化进行了比较研究。结果表明：４℃低温不仅使类囊体膜中的光合色素（叶绿素、类胡萝卜素）含量降低,而且还引起膜蛋白组成的深刻变化,表现在大部分原有膜蛋白组分的含量在低温下明显降低,同时在低温处理的第３天诱导出一条３２．５ＫＤ的新蛋白带。与４℃处理相比,１１℃低温处理只引起了光合色素含量的降低,而对类囊体膜蛋白组成的影响不大,另外发现,两种低     

Somatic cell culture of rice cultivars with different grain types: Somaclonal variation in some grain and quality characters

Variations in some grain and quality characters in progenies of regenerated rice plants were studied. Grain length and weight decreased significantly, yet gel consistency increased. Variations in these quantitative characters of all cultivars studied were consistent, showing the tendencies of the variations. Grain protein contents of the somaclones were higher in one cultivar. Variability of most traits was increased by combining low-dosage radiation and tissue culture.     

Effect of oilseed type on milk fatty acid composition of individual cows,and also bulk tank milk fatty acid composition from commercial farms

Supplementing dairy cow diets with oilseed preparations has been shown to replace milk saturated fatty acids (SFA) with mono- and/or polyunsaturated fatty acids (MUFA, PUFA), which may reduce risk factors associated with cardio-metabolic diseases in humans consuming milk and dairy products. Previous studies demonstrating this are largely detailed, highly controlled experiments involving small numbers of animals, but in order to transfer this feeding strategy to commercial situations further studies are required involving whole herds varying in management practices. In experiment 1, three oilseed supplements (extruded linseed (EL), calcium salts of palm and linseed oil (CPLO) and milled rapeseed (MR)) were included in grass silage-based diets formulated to provide cows with ~350 g oil/day, and compared with a negative control (Control) diet containing no supplemental fat, and a positive control diet containing 350 g/cow per day oil as calcium salt of palm oil distillate (CPO). Diets were fed for 28-day periods in a 5×4 Latin Square design, and milk production, composition and fatty acid (FA) profile were analysed at the end of each period. Compared with Control, all lipid supplemented diets decreased milk fat SFA concentration by an average of 3.5 g/100 g FA, by replacement with both cis- and trans-MUFA/PUFA. Compared with CPO, only CPLO and MR resulted in lower milk SFA concentrations. In experiment 2, 24 commercial dairy farms (average herd size±SEM 191±19.3) from the south west of the United Kingdom were recruited and for a 1 month period asked to supplement their herd diets with either CPO, EL, CPLO or MR at the same inclusion level as the first study. Bulk tank milk was analysed weekly to determine FA concentration by Fourier Transform mid-IR spectroscopy prediction. After 4 weeks, EL, CPLO and MR all decreased herd milk SFA and increased MUFA to a similar extent (average −3.4 and +2.4 g/100 g FA, respectively) when compared with CPO. Differing responses observed between experiments 1 and 2 may be due in part to variations in farm management conditions (including basal diet) in experiment 2. This study demonstrates the importance of applying experimental research into commercial practice where variations in background conditions can augment different effects to those obtained under controlled conditions.