A rare Waxy allele coordinately improves rice eating and cooking quality and grain transparency

In rice (Oryza sativa), amylose content (AC) is the major factor that determines eating and cooking quality (ECQ). The diversity in AC is largely attributed to natural allelic variation at the Waxy (Wx) locus. Here we identified a rare Wx allele, Wx^mw, which combines a favorable AC, improved ECQ and grain transparency. Based on a phylogenetic analysis of Wx genomic sequences from 370 rice accessions, we speculated that Wx^mw may have derived from recombination between two important natural Wx alleles, Wxⁱⁿ and Wx^b. We validated the effects of Wx^mw on rice grain quality using both transgenic lines and near‐isogenic lines (NILs). When introgressed into the japonica Nipponbare (NIP) background, Wx^mw resulted in a moderate AC that was intermediate between that of NILs carrying the Wx^b allele and NILs with the Wx^mp allele. Notably, mature grains of NILs fixed for Wx^mw had an improved transparent endosperm relative to soft rice. Further, we introduced Wx^mw into a high‐yielding japonica cultivar via molecular marker‐assisted selection: the introgressed lines exhibited clear improvements in ECQ and endosperm transparency. Our results suggest that Wx^mw is a promising allele to improve grain quality, especially ECQ and grain transparency of high‐yielding japonica cultivars, in rice breeding programs.     

The origin of Wxla provides new insights into the improvement of grain quality in rice

Appearance and taste are important factors in rice (Oryza sativa) grain quality. Here, we investigated the taste scores and related eating‐quality traits of 533 diverse cultivars to assess the relationships between—and genetic basis of—rice taste and eating‐quality. A genome‐wide association study highlighted the Wx gene as the major factor underlying variation in taste and eating quality. Notably, a novel waxy (Wx) allele, Wx^la, which combined two mutations from Wx^b and Wxⁱⁿ, exhibited a unique phenotype. Reduced GBSSI activity conferred Wx^la rice with both a transparent appearance and good eating quality. Haplotype analysis revealed that Wx^la was derived from intragenic recombination. In fact, the recombination rate at the Wx locus was estimated to be 3.34 kb/cM, which was about 75‐fold higher than the genome‐wide mean, indicating that intragenic recombination is a major force driving diversity at the Wx locus. Based on our results, we propose a new network for Wx evolution, noting that new Wx alleles could easily be generated by crossing genotypes with different Wx alleles. This study thus provides insights into the evolution of the Wx locus and facilitates molecular breeding for quality in rice.     

Cellular determinants of the mutational specificity of 1-nitroso-6-nitropyrene and 1-nitroso-8-nitropyrene in the lacI gene of Escherichia coli.

We have characterized 202 lacI⁻ mutations, and 158 dominant lacI^−d mutations following treatment of Escherichia coli strains NR6112 and EE125 with 1-nitroso-6-nitropyrene (1,6-NONP), an activated metabolite of the carcinogen 1,6-dinitropyrene. In all, 91% of the induced point mutations occurred at G:C residues. The −(G:C) frameshifts were the dominant mutational class in the lacI⁻ collections of both NR6112 and EE125, and in the lacI^−d collection of NR6112. Frameshift mutations occurred preferentially in runs of guanine residues, and their frequency increased with the length of the reiterated sequence. In strain EE125, which contained the plasmid pKM101, there was a marked stimulation in the frequency of base substitution mutations that was particularly apparent in the lacI^−d collection. This study completes a comprehensive analysis of 1194 lacI⁻ and 348 lacI^−d mutations induced by either 1,6-NONP or its positional isomer 1-nitroso-8-nitropyrene (1,8-NONP) in strains of E. coli that differ with regard to their ability to carry out nucleotide excision repair and/or their ability to express the translesion synthesis DNA polymerase RI (MucAB) encoded by plasmid pKM101. Among the mutations are 763 frameshift mutations, 367 base substitutions and 47 deletions; these mutations have been characterized at more than 300 distinct sites in the lacI gene. Our studies provide detailed insight into the DNA sequence alterations and mutational mechanisms associated with dinitropyrene mutagenesis. We review the mutational spectra, and discuss cellular lesion repair or tolerance mechanisms that modulate the observed mutational specificity.     

Mutagenesis and carcinogenesis in nucleotide excision repair-deficient XPA knock out mice

Mice with a defect in the xeroderma pigmentosum group A (XPA) gene have a complete deficiency in nucleotide excision repair (NER). As such, these mice mimic the human XP phenotype in that they have a >1000-fold higher risk of developing UV-induced skin cancer. Besides being UV-sensitive, XPA^−/− mice also develop internal tumors when they are exposed to chemical carcinogens. To investigate the effect of a total NER deficiency on the induction of gene mutations and tumor development, we crossed XPA^−/− mice with transgenic lacZ/pUR288 mutation-indicator mice. The mice were treated with various agents and chemicals like UV-B, benzo[a]pyrene and 2-aceto-amino-fluorene. Gene mutation induction in several tumor target- and non-target tissues was determined in both the bacterial lacZ reporter gene and in the endogenous Hprt gene. Furthermore, alterations in the p53- and ras genes were determined in UV-induced skin tumors of XPA^−/− mice. In this work, we review these results and discuss the applicability and reliability of enhanced gene mutant frequencies as early indicators of tumorigenesis.     

优质稻米‘青香软粳’低直链淀粉含量形成分子机制的初步研究

Waxy（Wx）基因在调控直链淀粉含量形成过程中起着重要的作用,是稻米蒸煮食味品质的一个关键决定因素。在本项研究中,我们以直链淀粉含量为8.7%和10.2%的优质稻品种‘青香软粳’和‘南粳46’为主要研究对象,初步分析并探讨了这两个优质稻品种稻米低直链淀粉形成的分子调控机理。结果显示,虽然都为Wx-II型粳稻品种,‘南粳46’的Wx基因在启动子1773位置存在AA/CT的变异,‘南粳46’和‘青香软粳’的Wx基因在启动子＋693位置具有G/A位点多态性。转录水平分析表明,在‘南粳46’和‘青香软粳’稻米发育过程中,Wx基因的表达模式有较大的差异,可能与其启动子序列的多态性相关,AA/CT变异处于Wx基因的转录调控区。Wx基因启动子后＋693位点的多态性与已报道Wxmq多态性位点之一相同,引起Arg158/His158的变异;生物信息学软件分析表明,该位点处于底物进入Wx蛋白催化中心的袋口上,Arg158/His158位点的变异可能影响到底物进入Wx蛋白催化活性中心的速率,从而影响稻米直链淀粉的合成过程和含量。文章丰富了Wx基因多态性的研究,为进一步验证其核苷酸变异与稻米直链淀粉含量的相关性奠定了基础。     

An Escherichia coli plasmid-based, mutational system in which supF mutants are selectable: insertion elements dominate the spontaneous spectra.

A new system is described to determine the mutational spectra of mutagens and carcinogens in Escherichia coli; data on a limited number (142) of spontaneous mutants is presented. The mutational assay employs a method to select (rather than screen) for mutations in a supF target gene carried on a plasmid. The E. coli host cells (ES87) are lacI⁻ (am26), and carry the lacZΔM15 marker for -complementation in β-galactosidase. When these cells also carry a plasmid, such as pUB3, which contains a wild-type copy of supF and lacZ-, the lactose operon is repressed (off). Furthermore, supF suppression of lacl^um26 results in a lactose repressor that has an uninducible, lacl^S genotype, which makes the cells unable to grow on lactose minimal plates. In contrast, spontaneous or mutagen-induced supF⁻ mutations in pUB3 prevent suppresion of lacl^am26 and result in constitutive expression of the lactose operon, which permits growth on lactose minimal plates. The spontaneous mutation frequency in the supF gene is 0.7 and 1.0 × 10⁻⁶ without and with SOS induction, respectively. Spontaneous mutations are dominated by large insertions (67% in SOS-uninduced and 56% in SOS-induced cells), and their frequency of appearance is largely unaffected by SOS induction. These are identified by DNA sequencing to be Insertion Element: IS1 dominates, but IS4, IS5, gamma-delta and IS10 are also obtained. Large deletions also contribute significantly (19% and 15% for - SOS and +SOS, respectively), where a specific deletion between a 10 base pair direct repeat dominates; the frequency of appearance of these mutations also appears to be unaffected by SOS induction. In contrast, SOS induction increases base pairing mutations (13% and 27% for -SOS and +SOS, respectively), The ES87/pUB3 system has many advantages for determining mutational spectra, including the fact that mutant isolation is fast and simple, and the determination of mutational changes is rapid because of the small size of supF.     

Mitochondrial functions under hypoxic conditions: The steady states of cytochrome c reduction and of energy metabolism

1. The effect of low oxygen concentration on the oxidation-reduction states of cytochrome c and of pyridine nucleotide, on Ca²⁺ uptake, on the energy-linked reduction of pyridine nucleotide by succinate, and on the rate of oxygen consumption have been examined under various metabolic conditions, using pigeon heart mitochondria.

2. The oxygen concentration required to provide half-maximal reduction of cytochrome c (p50_c) ranges from 0.27 to 0.03 μM (0.2-0.02 Torr) depending upon the metabolic activity. There is a linear increase of the p50_c value with increasing respiratory rate.

3. The fraction of the normoxic respiration that is observed at p50_c is 70–90% under State 4 conditions, but is 30% under State 3 conditions.

4. The oxygen requirement for half-maximal reduction of pyridine nucleotide (p50_PN) varies less than p50_c, being 0.08 μM in State 3 and 0.06 μM in the uncoupled state.

5. The ability of the mitochondria to exhibit an energy-linked reduction of pyridine nucleotide by succinate disappears at an oxygen concentration of 0.09 μM (0.06 Torr). Below this oxygen concentration, endogenous Ca²⁺ begins to be released from the mitochondria. Thus, the critical oxygen concentration for bioenergetic function of mitochondria corresponds approximately to 50% reduction of pyridine nucleotide (p50_PN).     

Mutations at the W locus affect survival of neural crest-derived melanocytes in the mouse

The development of melanoblasts in normally pigmented and dominant spotting (W) embryos was followed by in situ hybridisation to TRP-2/DT mRNA, which labels migratory melanoblasts from 10 days post coitum. Numerous melanoblasts migrate to the inner ear around 11 days. In contrast, few migratory melanoblasts are associated with the eye or skin at this stage and melanoblast distribution within the trunk and tail is patchy. The distribution of melanoblasts in 10.5–11-day-old W^v/W^v, W^sh/W^sh and W⁴¹/W⁴¹ mutants was similar to that in controls but melanoblast density was lower and by 12 days was severely reduced. These results suggest that mutations of the c-kit receptor tyrosine kinase encoded at the W locus do not alter early migration or differentiation of melanoblasts but severely affect melanoblast survival.     

模拟降雨格局变化对亚热带地区两树种液流特征的影响

在全球变化的影响下, 中国亚热带地区近几十年降水格局发生了急剧变化。这种变化对亚热带常绿阔叶林植物的生长和森林水分平衡的影响尚不清楚。为此, 该研究从植物整树蒸腾角度出发, 通过在天然次生林中进行人工隔除降雨模拟降水格局变化, 研究降水变化对植物水分利用的影响。试验于2012年9月至2014年12月在广东鹤山森林生态系统国家野外科学观测研究站内的常绿阔叶林内进行, 通过林下搭建遮雨棚, 截留干季(10月至次年3月)的降雨, 并在湿季(次年4至9月)等量返还到样地中, 在保证总降水量不变的前提下模拟干季更干、湿季更湿(DD)的降雨格局变化。在此期间对样地内的木荷(Schima superba)与火力楠(Michelia macclurei)树干液流特征进行连续监测。运用独立样本t检验对对照组(AC)两个树种间的平均最大液流通量密度(¯J_S)差异性进行分析, 并将DD处理下两个树种的¯J_S与AC进行对比, 来检验隔除降雨对森林蒸腾的效应。结果表明: 当光合有效辐射(PAR)大于1 100 μmol·m ^-2·s ^-1时, 对照样地火力楠和木荷的¯J_S分别为(49.5 ± 1.7)和(43.6 ± 2.0) mL∙m ^-2∙s ^-1, 且前者表现出对光合有效辐射(PAR)更强的敏感性。截留降雨处理开始后(2012-10), 两个树种DD与AC处理的¯J_S比值(DD:AC)均先减小后增加, 其中木荷的比值从处理前的0.74下降到了第1次截留降雨处理期(2012-10至2013-03)的0.68, 增加到了第2次截留降雨处理期(2013-10至2014-03)的0.93以及第3次截留降雨处理期(2014-10至2014-11)的1.04; 火力楠则从处理前的1.00下降到了第1次截留降雨处理期的0.94, 在第2次截留降雨处理期增长到1.06, 变化幅度小于木荷。此外, 在第3次截留降雨处理期, 木荷在相同的水汽压亏缺及PAR下能够保持更高的¯J_S。这些结果表明, 短期干旱事件会促使森林蒸腾急剧下降, 然而在长期干旱下, 植物会通过提高¯J_S来弥补干旱带来的损失, 而木荷由于具有较大的¯J_S可塑性, 从而使其在干旱条件下维持更高的水分传输速率。     

Investigations on radiosensitive and radioresistant populations of Drosophila melanogaster: XI. The resistance factor rar-3: effects in inmature oocytes

The effects of the radioresistance factor rar-3 on the X-ray induction of various types of genetic damage in immature oocytes (about stage7) of Drosophila melanogaster were studied.

The dose-reduction factors previously postulated for rar-3 with respect to dominant lethals (1.58), sex-linked recessive lethals (1.87), non-disjunction of major chromosomes (1.58), and homologous interchanges (1.58)_were confirmed experimentally. It is concluded that all effects attributed arbitrarily to rar-3 are contributed by the single genetic factor rar-3.

No difference were found in quality of sex-linked recessive lethals (Y suppression, distribution over the X) induced in either rar-3 or rar-3⁺. Recombination frequencies were normal in unirradiated rar-3.     

Effect of O6-alkylguanine-DNA alkyltransferase on the frequency and spectrum of mutations induced by N-methyl-N''-nitro-N-nitrosoguanidine in the HPRT gene of diploid human fibroblasts

N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) reacts with 12 nucleophilic sites in DNA to induce a variety of lesions, but O⁶-methylguanine (O⁶-MeG) and O⁴-methylthymine are the most effective premutagenic lesions produced, mispairing with thymine and guanine, respectively. O⁶-MeG is repaired by O⁶-alkylguanine-DNA alkyltransferase (AGT), which removes the methyl group from the O⁶ position and transfers it to itself, rendering the transferase inactive. When diploid human fibroblasts were exposed to 25 μM, O⁶-benzylguanine (O⁶-BzG) in the medium for 3 h, their level of AGT activity was dramatically reduced, to a level of at most 1.6% of the control. Populations of cells pretreated with this level of O⁶-BzG for 2 h or not pretreated, were exposed to MNNG at a concentration of 2, 4 or 6 μM in the presence or absence of O⁶-BzG and assayed for survival of colony-forming ability and the frequency of 6-thioguanine-resistant cells (mutations induced in the HPRT gene). O⁶-BzG (25 μM) was also present in the appropriate half of the cells during the 24 h immediately follwing exposure to MNNG. This 27-h exposure to O⁶-BzG alone had no cytotoxic or mutagenic effect on the cells but significantly increased the cytotoxicity and mutagenecity of MNNG, increasing the mutant frequency to that found previously in human cells constitutively devoid of AGT activity. At doses of 2 μM and 4 μM MNNG, the mutant frequency observed with the AGT-depleted cells was 120 × 10⁻⁶ and 240 × 10⁻⁶, respectively; in the cells with abundant AGT activity, these values were 10 × 10⁻⁶ and 20 × 10⁻⁶, respectively. DNA-sequence analysis of the coding region of the HPRT gene in 36 independent mutants obtained from MNNG-treated AGT-depleted populations and 36 from the control populations showed that even though AGT repair lowered the frequency of mutants by more than 90%, it did not affect the kinds of mutations induced by MNNG nor the strand distribution of the premutagenic guanine lesions. In mutants from the AGT-depleted cells, there were 26 base substitutions and 13 putative splice site mutations; in the control, there were 25 base substitutions and 11 splice site mutations. All but two substitutions involved G · C with 92% being G · C → A · T. In both sets, of the premutagenic lesions were located in the nontranscribed strand. Many ‘hot spots’ were seen, and there was evidence that AGT repaired more lesions from the 5′ half of the gene than from the 3′ half.     

Fixation of mutations in the viral genome during an outbreak of foot-and-mouth disease: heterogeneity and rate variations

Rates of fixation of mutations during the evolution of the foot-and-mouth disease virus (FMDV) C₁ in nature have been estimated by hybridization of viral RNA to cloned cDNAs representing defined FMDV genome segments, and comparison of the selected RNAs by T₁ RNase oligonucleotide fingerprinting. Values ranged from <0.04 × 10⁻² to 4.5 × 10⁻² substitutions per nucleotide per year (s/nt/yr), depending on the time period and the genomic segment considered. Rates for viral structural protein genes were up to sixfold higher than for nonstructural protein genes. Values in excess of 10⁻² s/nt/yr have been measured for the RNA region that encodes VP1–VP3. The nucleotide sequences of the major immunogenic region of capsid protein VP1 have been determined for six new FMDV C₁ isolates, and they are compared with the two previously known sequences of FMDV C₁ (C-S8 and C₁-O). Both oligonucleotide fingerprinting of selected RNA fragments and direct nucleotide sequencing demonstrate that genetic heterogeneity exists among three viruses isolated on the same day, introducing a significant indetermination in the evaluation of fixation rates of mutations. During the FMDV C₁ outbreak, amino acid substitutions did occur that are known to affect the immunological properties of the virus. The proportion of mutations between two viral RNAs does not increase significantly with the time elapsed between the two isolations, suggesting a cocirculation of multiple, related, nonidentical FMDVs (‘evolving quasispecies’) as the mode of evolution of this agent.     

Loss of Gn1a/OsCKX2 confers heavy-panicle rice with excellent lodging resistance

Significant achievements have been made in breeding programs for the heavy-panicle-type(HPT)rice(Oryza sativa) in Southwest China. The HPT varieties now exhibit excellent lodging resistance,allowing them to overcome the greater pressures caused by heavy panicles. However, the genetic mechanism of this lodging resistance remains elusive. Here, we isolated a major quantitative trait locus, Panicle Neck Diameter 1(PND1), andidentified the causal gene as GRAIN NUMBER 1 A/CYTOKININ OXIDASE 2(Gn1 A/Os...     

New mutations in cloned Escherichia coli umuDC genes: novel phenotypes of strains carrying a umuC125 plasmid

The umuDC locus of Escherichia coli is required for most mutagenesis by UV and many chemicals. Mutations in E. coli umuDC genes cloned on pBR322-derived plasmids wer e isolated by two methods. First, spontaneously-arising mutant umuDC plasmids that failed to confe cold-sensitive growth on a lexA51(Def) strain were isolated by selection. Second, mutant umuDC plasmids that affected apparent mutant yield after UV-irradiation in a strain carrying umuD⁺C⁺ in the chromosome were isolated by screening hydroxylamine-mutagenized umuD⁺C⁺ plasmids. pBR322-derived umuD⁺C⁺ plasmids inhibited the induction of the SOS response of lexA⁺ strains as measured by expression of din::Mu dl(lac) Ap) fusionsbut most mutant plasmids did not. Mutant plasmids defective in complementation of chromosomal umuD44, umuC36, or both were found among those selected for failure to confer cold-sensitivity, whereas those identified by the screening procedure yielded mostly mutant plasmids with more complex phenotypes. We studied in greater detail a plasmid pLM109, carrying the umuC125 mutation. This plasmid increased the sensitivity of lexA⁺ strainsto killing by UV-irradiation but was able to complement the deficiencies of umuC mutants in UV mutagenesis. pLM109 failed to confer cold-sensitive growth on lexA(Def) strains but inhibited SOS induction in lexA⁺ strains. The effect of pLM109 on the UV sensitivity of lexA(Def)strains was similar to that of the parental umuD⁺C⁺ plasmid. The mutation responsible for the phenotypes of pLM109 was localized to a 615-bp fragment. DNA sequencing revealed that the umuC125 mutation was a G:C → A:T transition that changed codon 39 of umuC from GCC → GTC thus changing Ala39 to Val39. The implications of the umuC125 mutation for umuDC-dependent effects on UV-mutagenesis and cell survival after UV damage are discussed.     

内蒙古和青藏高原草原植物叶片与根系氮利用效率空间格局及影响因素

氮利用效率是植物的关键功能性状, 同时紧密关联生态系统功能, 但是目前对氮利用效率的区域格局及影响因素仍然不清楚。该研究分析了内蒙古和青藏高原草原82个调查地点、139种植物叶片和根系的氮利用效率及其与环境因素、植物功能群之间的关系, 实验结果显示: 1)草甸草原植物叶片的氮利用效率为53 g·g ^-1, 显著大于高寒草甸(46 g·g ^-1)、荒漠草原(41 g·g ^-1)和典型草原(39 g·g ^-1)。高寒草甸根系氮利用效率为108 g·g ^-1, 显著高于其他生态系统。2)叶片氮利用效率比根系对温度更加敏感, 但随着干旱指数的增加, 两者均表现出显著的降低趋势。3)杂类草叶片和根系氮利用效率低于莎草科和禾本科植物, 豆科植物叶片和根系氮利用效率分别比非豆科植物低48%和60%。4)植物氮利用效率与土壤氮含量之间没有显著关系。总体上, 内蒙古和青藏高原草原植物叶片和根系氮利用效率的空间格局存在差异, 主要影响因素为植物功能群和干旱指数。本研究系统揭示内蒙古和青藏高原草原植物氮利用效率的空间格局及关键驱动因子, 有助于在全球变化背景下了解我国草地生产力维持机制, 同时为草原生态系统管理提供科学依据。     

Labeling of v-Src and BCR-ABL tyrosine kinases with [C]herbimycin A and its use in the elucidation of the kinase inactivation mechanism

The ansamycin antibiotic, herbimycin A, selectively inactivates cytoplasmic tyrosine kinases, most likely by binding irreversibly to the reactive SH group(s) of kinases. To further investigate the mechanism of herbimycin A action, we attempted to label tyrosine kinases with [¹⁴C]herbimycin A. p60^v-src and p2 10^BCR-ABL in immune complexes were labeled with [¹⁴C]herbimycin A, demonstrating that the antibiotic binds directly to tyrosine kinases. Digestion of [¹⁴C]herbimycin A-labeled p60^v-src with Staphylococcus taureus V8 protease revealed that the herbimycin A binding site is within the C-terminal 26-kDa fragment of p60^v-src, which contains the tyrosine kinase domain. Herbimycin A treatment inhibited labeling of p60^v-src by [¹⁴]C]fluorosulfonylbenzoyl adenosine, an affinity labeling reagent of nucleotide binding sites, indicating that herbimycin A-modified p60^v-src cannot interact with ATP. The results suggest that herbimycin A inactivates tyrosine kinases by binding directly to the kinase domain, thereby inhibiting access to ATP.     

Mechanisms of spontaneous mutation in DNA repair-proficient Escherichia coli

This paper describes the DNA sequence analysis of 729 independent spontaneous lacI⁻ mutation This total is comprised of 478 novel mutations and 251 previously described events, and therefore should allow a more comprehensive view of spontaneous mutation in Escherichia coli. The spectrum is dominated by a hotspot (71% of all events). Mutations at this site consist of related addition and deletion events involving a number of repetitive sequences. Here we discuss how the frequency and proportion of these events vary in different DNA repair-deficient genetic backgrounds. The distribution of non-hotspot events includes base substitutions (38%), deletions (35%), frameshifts (14%), duplications (4%) and insertion elements (4%). G:C → A:T events dominate among base substitutions, while G:C → C:G events are the least common; the remaining types of base substitution are equally represented. Among deletions, a significant number do not display repeated sequences at their endpoints (26/72). However, almost all multiply recovered events (15/17) possess repeated sequences capable of accounting for the deletion endpoints. Similarily, over of all duplications recovered (5/7) display repeated endpoints. Single-base frameshifts are equally divided between A:T and G:C sites, in each case (−) 1 events occur 3-fold more frequently that (+)1 events. A comparative analysis of each mutational class recovered to lacI⁻ spectra available in a variety of DNA repair/metabolism-deficient strains is presented here in an attempt to assess possible contributions from chemical, physical and enzymic sources of damage.     

Anatomy of the stimulative sequences flanking the ARS consensus sequence of chromosome VI in Saccharomyces cerevisiae

We have analyzed the relationship between autonomously replicating sequence (ARS) structure and function for three ARS (ARS605, ARS607 and ARS609) from chromosome VI of Saccharomyces cerevisiae by systematic XhoI-linker mutation in the ARS consensus sequence (ACS) and flanking sequences. All mutations that encroached upon the ACS destroyed ARS activity. DNA sequences stimulative for ARS function were identified on either side of the ACS of ARS605 and only on the 3'-side of the ACS of ARS607. In ARS609, however, no such stimulative sequences were observed. Base substitutions complementary to the wild-type sequence of those stimulative regions, in ARS605 and ARS607, that did not change the AG of unwinding nor affected ARS activity suggests that these regions have, at least, a function as DNA-unwinding elements (DUE). ARS605, ARS607 and ARS609 DNA are of low AG value and showed hypersensitivity to single-strand-specific nuclease when inserted in negatively supercoiled plasmid. Linker mutations inhibitory for ARS activity (5L11 and 7L14) also caused significant changes in local nucleotide (nt) sensitivity within the ACS and its adjoining regions. Complementary base substitutions, however, did not affect these changes in local nt sensitivity. These results imply that the stimulative regions flanking the ACS are necessary to produce an optimum conformation around the ACS which may be important for full ARS activity.     

Natural alleles of a uridine 5ʹ-diphospho-glucosyltransferase gene responsible for differential endosperm development between upland rice and paddy rice

Traditional upland rice generally exhibits insufficient grains resulting from abnormal endosperm development compared to paddy rice. However, the underlying molecular mechanism of this trait is poorly understood. Here, we cloned the uridine 5ʹ-diphospho (UDP)-glucosyltransferase gene EDR1 (Endosperm Development in Rice) responsible for differential endosperm development between upland rice and paddy rice by performing quantitative trait loci analysis and map-based cloning. EDR1 was highly expressed in developing seeds during grain filling. Natural variations in EDR1 significantly reduced the UDP-glucosyltransferase activity of EDR1^YZN compared to EDR1^YD1, resulting in abnormal endosperm development in the near-isogenic line, accompanied by insufficient grains and changes in grain quality. By analyzing the distribution of the two alleles EDR1^YD1 and EDR1^YZN among diverse paddy rice and upland rice varieties, we discovered that EDR1 was conserved in upland rice, but segregated in paddy rice. Further analyses of grain chalkiness in the alleles of EDR1^YD1 and EDR1^YZN varieties indicated that rice varieties harboring EDR1^YZN and EDR1^YD1 preferentially showed high chalkiness, and low chalkiness, respectively. Taken together, these results suggest that the UDP-glucosyltransferase gene EDR1 is an important determinant controlling differential endosperm development between upland rice and paddy rice.