Cloning and overexpression of the ascorbate peroxidase gene from the yam (Dioscorea alata) enhances chilling and flood tolerance in transgenic Arabidopsis

Journal of Plant Research - Minghuai 1 (MH1) is a yam (Dioscorea alata) cultivar with high tolerance to flooding but sensitivity to chilling. MH1 responded differently to chilling and flooding...     

A point mutation in LTT1 enhances cold tolerance at the booting stage in rice

The cold tolerance of rice at the booting stage is a main factor determining sustainability and regional adaptability. However, relatively few cold tolerance genes have been identified that can be effectively used in breeding programmes. Here, we show that a point mutation in the low-temperature tolerance 1 (LTT1) gene improves cold tolerance by maintaining tapetum degradation and pollen development, by activation of systems that metabolize reactive oxygen species (ROS). Cold-induced ROS accumulation is therefore prevented in the anthers of the ltt1 mutants allowing correct development. In contrast, exposure to cold stress dramatically increases ROS accumulation in the wild type anthers, together with the expression of genes encoding proteins associated with programmed cell death and with the accelerated degradation of the tapetum that ultimately leads to pollen abortion. These results demonstrate that appropriate ROS management is critical for the cold tolerance of rice at the booting stage. Hence, the ltt1 mutation can significantly improve the seed setting ability of cold-sensitive rice varieties under low-temperature stress conditions, with little yield penalty under optimal temperature conditions. This study highlights the importance of a valuable genetic resource that may be applied in rice breeding programmes to enhance cold tolerance.     

A quantitative trait locus for cold tolerance at the booting stage on rice chromosome 8

A quantitative trait locus (QTL) for cold tolerance at the booting stage of a cold-tolerant rice breeding line, Hokkai-PL9, was analyzed. A total of 487 simple sequence repeat (SSR) markers distributed throughout the genome were used to survey for polymorphism between Hokkai-PL9 and a cold-sensitive breeding line, Hokkai287, and 54 markers were polymorphic. Single marker analysis revealed that markers on chromosome 8 are associated with cold tolerance. By interval mapping using an F₂ population between Hokkai-PL9 and Hokkai287, a QTL for cold tolerance was detected on the short arm of chromosome 8. The QTL explains 26.6% of the phenotypic variance, and its additive effect is 11.4%. Substitution mapping suggested that the QTL is located in a 193-kb interval between SSR markers RM5647 and PLA61. We tentatively designated the QTL as qCTB8 (quantitative trait locus for cold tolerance at the booting stage on chromosome 8).     

Mapping quantitative trait loci for heat tolerance at the booting stage using chromosomal segment substitution lines in rice

High temperature stress is a major obstacle in rice productivity. Considerable progress has been made on studying heat tolerance (HT) at different stages. However, the genetic basis of HT at the booting stage is poorly understood. In this study, we analyzed the morphological features of a heat-sensitive japonica cultivar Sasanishiki under natural high temperature stress at the booting stage. The anthers became smaller and the number, and fertility, of pollen grains were decreased significantly. As a result, there was a dramatic reduction in spikelet fertility. In contrast, the indica cultivar Habataki showed high HT and normal spikelet fertility under high temperature stress. Additonally, a set of chromosome segment substitution lines, derived from Sasanishiki and Habataki, were evaluated for HT related quantitative trait loci (QTLs) across two environments in the natural field. A total of 12 QTLs associated with HT were detected, of which, 5 were identified in two environments, and 7 in one environment. Furthermore, one of the major-effect QTLs (qHTB3-3) detected on the long arm of chromosome 3, was confirmed using overlapping substituted lines. qHTB3-3 was finally mapped between the two markers RM3525 and 3-M95, approximately 2.8 Mb apart. These findings and further gene cloning of qHTB3-3 will help us better understand the molecular control of HT in rice, and may contribute to the development of high HT rice varieties.     

OsSDIR1 overexpression greatly improves drought tolerance in transgenic rice

Recent genomic and genetic analyses based on Arabidopsis suggest that ubiquitination plays crucial roles in the plant response to abiotic stress and the phytohormone abscisic acid (ABA). However, few such studies have been reported in rice as a monocotyledonous model plant. Taking advantage of strategies in biochemistry, molecular cell biology and genetics, the RING-finger containing E3 ligase OsSDIR1 (Oryza sativa SALT-AND DROUGHT-INDUCED RING FINGER 1) was found to be a candidate drought tolerance gene for engineering of crop plants. The expression of OsSDIR1 was detected in all tissues of rice and up-regulated by drought and NaCl, but not by ABA. In vitro ubiquitination assays demonstrated that OsSDIR1 is a functional E3 ubiquitin ligase and that the RING finger region is required for its activity. OsSDIR1 could complement the drought sensitive phenotype of the sdir1 mutant and overexpressing transgenic Arabidopsis were more sensitive to ABA, indicating that the OsSDIR1 gene is a functional ortholog of SDIR1. Upon drought treatment, the OsSDIR1-transgenic rice showed strong drought tolerance compared to control plants. Analysis of the stomata aperture revealed that there were more closed stomatal pores in transgenic plants than those of control plants. This result was also confirmed by the water loss assay and leaf related water content (RWC) measurements during drought treatment. Thus, we demonstrated that monocot- and dicot- SDIR1s are conserved yet have diverse functions.     

Enhanced tolerance of transgenic potato plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against oxidative stress and high temperature

Oxidative stress is a major damaging factor for plants exposed to environmental stresses. In order to develop transgenic potato plants with enhanced tolerance to environmental stress, the genes of both Cu/Zn superoxide dismutase and ascorbate peroxidase were expressed in chloroplasts under the control of an oxidative stress-inducible SWPA2 promoter (referred to as SSA plants). SSA plants showed enhanced tolerance to 250 μM methyl viologen, and visible damage in SSA plants was one-fourth that of non-transgenic (NT) plants that were almost destroyed. In addition, when SSA plants were treated with a high temperature of 42°C for 20 h, the photosynthetic activity of SSA plants decreased by only 6%, whereas that of NT plants decreased by 29%. These results suggest that the manipulation of the antioxidative mechanism of the chloroplasts may be applied in the development of industrial transgenic crop plants with increased tolerance to multiple environmental stresses.Communicated by I. S. Chung     

QTLs of cold tolerance-related traits at the booting stage for NIL-RILs in rice revealed by SSR

QTLs for cold tolerance-related traits at the booting stage using balanced population for 1525 recombinant inbred lines of near-isogenic lines (viz.NIL-RILs for BC5F3 and BC₅F₄ and BC₅F₅) over 3 years and two locations by backcrossing the strongly cold-tolerant landrace (Kunmingxiaobaigu) and a cold-sensitive cultivar (Towada) was analyzed. In this study, 676 microsatellite markers were employed to identify QTLs conferring cold tolerance at booting stage. Single marker analysis revealed that 12 markers associated with cold tolerance on chromosome 1, 4 and 5. Using a LOD significance threshold of 3.0,compositive interval mapping based on a mixed linear model revealed eight QTLs for 10 cold tolerance-related traits on chromosomes 1, 4, and 5. They were tentatively designatedqCTB-1-1, qCTB-4-1, qCTB-4-2, qCTB-4-3, qCTB-4-4, qCTB-4-5, qCTB-4-6, andqCTB-5-1. The marker intervals of them were narrowed to 0.3-6.8 cM. Genetic distances between the peaks of the QTL and nearest markers varied from 0 to 0.04 cM. We were noticed in some traits associated cold tolerance, such asqCTB-1-1 for 5 traits (plant height, panicle exsertion, spike length, blighted grains per spike and spikelet fertility),qCTB-4-1 for 8 traits (plant height, node length under spike, leaf length, leaf width, spike length, full grains per spike, total grains per spike and spikelet fertility),qCTB-4-2 for 3 traits (spike length, full grains per spike and spikelet fertility),qCTB-5-1 for 5 traits (plant height, panicle exsertion, blighted grains per spike, full grains per spike and spikelet fertility). The variance explained by a single QTL ranged from 0.80 to 16.80%. Three QTLs (qCTB-1-1, qCTB-4-1, qCTB-4-2) were detected in two or more trials. Our study sets a foundation for cloning cold-tolerance genes and provides opportunities to understand the mechanism of cold tolerance at the booting stage.     

三种酰胺类新农药对水稻孕穗期稻纵卷叶螟的防效试验

单季杂交晚稻孕穗期稻纵卷叶螟Cnaphalocrocis medinalis Guenée发生不整齐,为害重,在主治药剂氟虫腈即将禁用之际,急需防治高龄幼虫的长效药剂,酰胺类农药是满足这一条件的新一代农药。3种酰胺类农药试验结果,在单季稻孕穗期防治稻纵卷叶螟,20%氟虫双酰胺(WDG)150g/hm2处理速效性与持效性表现最好,药后3d防效达90.3%,药后15d防效高达96.2%;20%氯虫苯甲酰胺(SC)150mL/hm2处理速效性略低于氟虫双酰胺,持效接近,药后3d、15d防效分别达75.2%、91.2%;40%氯虫·噻虫嗪(WDG)120mL/hm2处理速效介于氟虫双酰胺和氯虫苯甲酰胺之间,持效略低于前二者,药后3d、15d防效分别为83.2%、88.3%。对照药剂5%氟虫腈(SC)750mL/hm2和90%杀虫单3000g/hm2处理药后3d、15d的防效均低于上述3种酰胺类新药剂。保叶效果以氯虫苯甲酰胺、氟虫双酰胺处理最高,药后15d保叶率分别为83.6%和85.0%,二者无显著差异,其次为氯虫·噻虫嗪处理为59.6%,3种酰胺类新药剂保叶效果均显著高于对照药剂氟虫腈和杀虫单。结果表明,3种酰胺类农药孕穗期防治稻纵卷叶螟的药效、持效性和保叶效果均高于当前主治药剂氟虫腈、杀虫单,可在生产上推广应用。     

Fine mapping a QTL qCTB7 for cold tolerance at the booting stage on rice chromosome 7 using a near-isogenic line

Low temperature at the booting stage is a serious abiotic stress in rice, and cold tolerance is a complex trait controlled by many quantitative trait loci (QTL). A QTL for cold tolerance at the booting stage in cold-tolerant near-isogenic rice line ZL1929-4 was analyzed. A total of 647 simple sequence repeat (SSR) markers distributed across 12 chromosomes were used to survey for polymorphisms between ZL1929-4 and the cold-sensitive japonica cultivar Towada, and nine were polymorphic. Single marker analysis revealed that markers on chromosome 7 were associated with cold tolerance. By interval mapping using an F₂ population from ZL1929-4 × Towada, a QTL for cold tolerance was detected on the long arm of chromosome 7. The QTL explained 9 and 21% of the phenotypic variances in the F₂ and F₃ generations, respectively. Recombinant plants were screened for two flanking markers, RM182 and RM1132, in an F₂ population with 2,810 plants. Two-step substitution mapping suggested that the QTL was located in a 92-kb interval between markers RI02905 and RM21862. This interval was present in BAC clone AP003804. We designated the QTL as qCTB7 (quantitative trait locus for cold tolerance at the booting stage on chromosome 7), and identified 12 putative candidate genes.     

A novel functional gene associated with cold tolerance at the seedling stage in rice

Identification and cloning of cold‐tolerant genes that can stably express under different cold environments are crucial for molecular rice breeding for cold tolerance. In the previous study, we identified a cold‐tolerant QTL at the seedling stage, qCTS‐9 which could be detected under different cold environments using a recombinant inbred line (RIL) population derived from a cold‐tolerant variety Lijiangxintuanheigu (LTH) and a cold‐sensitive variety Shanhuangzhan 2 (SHZ‐2). In this study, eight candidate genes within the qCTS‐9 interval were identified through integrated analysis of QTL mapping with genomewide differential expression profiling of LTH. The qRT‐PCR assay showed that only Os09g0410300 exhibited different expression patterns between LTH and SHZ‐2 during cold stress, and significantly positive correlation was found between cold induction of Os09g0410300 and seedling cold tolerance in the RI lines. Five SNPs and one InDel in the promoters of Os09g0410300 were detected between LTH and SHZ‐2, and the InDel marker ID410300 designed based on the insertion–deletion polymorphism in the promoter was significantly associated with seedling cold tolerance in RIL population. Further, Os09g0410300 over‐expression plants exhibited enhanced cold tolerance at the seedling stage compared with the wild‐type plants. Thus, our results suggest that Os09g0410300 is the functional gene underlying qCTS‐9. To our knowledge, it is a novel gene contributed to enhance cold tolerance at the seedling stage in rice. Identification of the functional gene underlying qCTS‐9 and development of the gene‐specific marker will facilitate molecular breeding for cold tolerance at the seedling stage in rice through transgenic approach and marker‐assisted selection (MAS).     

Revision of the relationship between anther morphology and pollen sterility by cold stress at the booting stage in rice

Background and AimsCold stress in rice (Oryza sativa) plants at the reproductive stage prevents normal anther development and causes pollen sterility. Tapetum hypertrophy in anthers has been associated with pollen sterility in response to cold at the booting stage. Here, we re-examined whether the relationships between anther abnormality and pollen sterility caused by cold stress at the booting stage in rice can be explained by a monovalent factor such as tapetum hypertrophy.MethodsAfter exposing plants to a 4-d cold treatment at the booting stage, we collected and processed anthers for transverse sectioning immediately and at the flowering stage. We anatomically evaluated the effect of cold treatment on anther internal morphologies, pollen fertilities and pollen numbers in the 13 cultivars with various cold sensitivities.Key ResultsWe observed four types of morphological anther abnormalities at each stage. Pollen sterility was positively correlated with the frequency of undeveloped locules, but not with tapetum hypertrophy as commonly believed. In cold-sensitive cultivars grown at low temperatures, pollen sterility was more frequent than anther morphological abnormalities, and some lines showed remarkably high pollen sterility without any anther morphological alterations. Most morphological anomalies occurred only in specific areas within large and small locules. Anther length tended to shorten in response to cold treatment and was positively correlated with pollen number. One cultivar showed a considerably reduced pollen number, but fertile pollen grains under cold stress. We propose three possible relationships to explain anther structure and pollen sterility and reduction due to cold stress.ConclusionsThe pollen sterility caused by cold stress at the booting stage was correlated with the frequency of entire locule-related abnormalities, which might represent a phenotypic consequence, but not a direct cause of pollen abortion. Multivalent factors might underlie the complicated relationships between anther abnormality and pollen sterility in rice.     

黑龙江省水稻空壳率与孕穗期低温的关系

对黑龙江省6个主要水稻品种(龙稻3号、垦稻12号、空育131、龙稻7号、龙粳16号和松粳6号)进行孕穗期低温(处理温度分别为15℃、17℃、19℃,低温持续时间分别为2、4、6和8d)处理,采用线性内插和统计回归方法,分析了水稻单穗空壳率与孕穗期低温的关系.结果表明:研究区敏感性水稻品种和耐冷性较强水稻品种的障碍型冷害临界温度分别为17℃和16℃;孕穗期水稻对低温最敏感的时期为抽穗前14～18d.15℃低温处理8d时,松粳6号、垦稻12号的空壳率明显增加,空育131空壳率小幅升高,表明松粳6号和垦稻12号对低温反应较敏感、耐冷性较弱,而空育131对低温反应迟钝,耐冷性较强;垦稻12号、龙粳16号、空育131的冷积温与空壳率存在显著的相关关系(P0.01),随着冷积温的增加,水稻空壳率明显升高,但品种间的增幅不同.     

Enhanced tolerance to salt stress in transgenic rice that overexpresses chloroplast glutamine synthetase

The potential role of photorespiration in the protection against salt stress was examined with transgenic rice plants. Oryza sativa L. cv. Kinuhikari was transformed with a chloroplastic glutamine synthetase (GS2) gene from rice. Each transgenic rice plant line showed a different accumulation level of GS2. A transgenic plant line, G39-2, which accumulated about 1.5-fold more GS2 than the control plant, had an increased photorespiration capacity. In another line, G241-12, GS2 was almost lost and photorespiration activity could not be detected. Fluorescence quenching analysis revealed that photorespiration could prevent the over-reduction of electron transport systems. When exposed to 150 mM NaCl for 2 weeks, the control rice plants completely lost photosystem II activity, but G39-2 plants retained more than 90% activity after the 2-week treatment, whereas G241-12 plants lost these activities within one week. In the presence of isonicotinic acid hydrazide, an inhibitor of photorespiration, G39-2 showed the same salt tolerance as the control plants. The intracellular contents of NH₄ ⁺ and Na⁺ in the stressed plants correlated well with the levels of GS2. Thus, the enhancement of photorespiration conferred resistance to salt in rice plants. Preliminary results suggest chilling tolerance in the transformant.     

Calcium is involved in the abscisic acid-induced ascorbate peroxidase, superoxide dismutase and chilling resistance in Stylosanthes guianensis

The objective of this work was to test whether Ca²⁺, a second messenger in stress response, is involved in ABA-induced antioxidant enzyme activities in Stylosanthes guianensis. Plants were sprayed with abscisic acid (ABA), calcium channel blocker, LaCl₃, calcium chelator, ethylene glycol-bis(β-amino ethyl ether)-N,N,N′, N′-tetraacetid acid (EGTA), and ABA in combination with LaCl₃ or EGTA. Their effects on superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities and chilling resistance were compared. The results showed that ABA decreased electrolyte leakage and lipid peroxidation but increased maximum photochemical efficiency measured as variable to maximum fluorescence ratio (F_v/F_m) under chilling stress. Treatment with LaCl₃ or EGTA alone and in combination with ABA increased electrolyte leakage and lipid peroxidation, decreased Fv/Fm, suggesting that the block in Ca²⁺ signalling decreased chilling resistance of S. guianensis and the ABA-enhanced chilling resistance. ABA-induced SOD and APX activities were suppressed by LaCl₃ or EGTA. The results suggested that Ca²⁺ is involved in the ABA-enhanced chilling resistance and the ABA-induced SOD and APX activities in S. guianensis.     

Enhanced axial symmetry at the Fe(3+)-heme center of peroxidase by ascorbate: a basis for the ascorbate-dependent peroxidase action.

In the absence of its substrate hydrogen peroxide, peroxidase exhibits perturbations in its Fe(3+)-heme center, when incubated with ascorbic acid. The electron paramagnetic pattern sprang towards a higher g-value side, denoting a sharpening of the rhombic axial symmetry around the heme-center. The interpretation is that the ascorbate dependent peroxidase action starts with the formation of an Fe(3+)-ascorbate charge transfer complex intermediate.