Genetic and physiological analysis of tolerance to acute iron toxicity in rice

Background

Fe toxicity occurs in lowland rice production due to excess ferrous iron (Fe²⁺) formation in reduced soils. To contribute to the breeding for tolerance to Fe toxicity in rice, we determined quantitative trait loci (QTL) by screening two different bi-parental mapping populations under iron pulse stresses (1,000 mg L⁻¹ = 17.9 mM Fe²⁺ for 5 days) in hydroponic solution, followed by experiments with selected lines to determine whether QTLs were associated with iron exclusion (i.e. root based mechanisms), or iron inclusion (i.e. shoot-based mechanisms).

Results

In an IR29/Pokkali F₈ recombinant inbred population, 7 QTLs were detected for leaf bronzing score on chromosome 1, 2, 4, 7 and 12, respectively, individually explaining 9.2-18.7% of the phenotypic variation. Two tolerant recombinant inbred lines carrying putative QTLs were selected for further experiments. Based on Fe uptake into the shoot, the dominant tolerance mechanism of the tolerant line FL510 was determined to be exclusion with its root architecture being conducive to air transport and thus the ability to oxidize Fe²⁺ in rhizosphere. In line FL483, the iron tolerance was related mainly to shoot-based mechanisms (tolerant inclusion mechanism). In a Nipponbare/Kasalath/Nipponbare backcross inbred population, 3 QTLs were mapped on chromosomes 1, 3 and 8, respectively. These QTLs explained 11.6-18.6% of the total phenotypic variation. The effect of QTLs on chromosome 1 and 3 were confirmed by using chromosome segment substitution lines (SL), carrying Kasalath introgressions in the genetic background on Nipponbare. The Fe uptake in shoots of substitution lines suggests that the effect of the QTL on chromosome 1 was associated with shoot tolerance while the QTL on chromosome 3 was associated with iron exclusion.

Conclusion

Tolerance of certain genotypes were classified into shoot- and root- based mechanisms. Comparing our findings with previously reported QTLs for iron toxicity tolerance, we identified co-localization for some QTLs in both pluse and chronic stresses, especially on chromosome 1.     

Iron Uptake and Loading into Rice Grains

Iron (Fe) is an important micronutrient for living organisms. Fe deficiency severely impairs plant growth and is a widespread human dietary problem, with particularly high numbers of affected children and females. Rice (Oryza sativa) is a source of energy for more than half of the world’s population. Thus, understanding the mechanisms of Fe uptake and translocation in rice is of utmost importance in the development of rice varieties that are tolerant to low Fe availability and with high seed levels of Fe. In recent years, the mechanisms underlying Fe transport and homeostasis have been revealed, providing opportunities to increase the Fe content of rice grain. As excess Fe is toxic to cells, plants have developed sophisticated mechanisms to control Fe flow, making it difficult to alter Fe transport. Thus, choosing appropriate chelators and Fe transporters driven by appropriate promoters seems to be the key in developing rice that is tolerant to low Fe availability and which accumulates high grain levels of Fe. Many recent studies have been aimed at increasing the Fe content of rice. Here, we summarize these efforts and review recent progress in understanding the mechanisms of Fe transport.     

Increase in Iron and Zinc Concentrations in Rice Grains Via the Introduction of Barley Genes Involved in Phytosiderophore Synthesis

Increasing the iron (Fe) and zinc (Zn) concentrations of staple foods, such as rice, could solve Fe and Zn deficiencies, which are two of the most serious nutritional problems affecting humans. Mugineic acid family phytosiderophores (MAs) play a very important role in the uptake of Fe from the soil and Fe transport within the plant in graminaceous plants. To explore the possibility of MAs increasing the Fe concentration in grains, we cultivated three transgenic rice lines possessing barley genome fragments containing genes for MAs synthesis (i.e., HvNAS1, HvNAS1, and HvNAAT-A and HvNAAT-B or IDS3) in a paddy field with Andosol soils. Polished rice seeds with IDS3 inserts had up to 1.40 and 1.35 times higher Fe and Zn concentrations, respectively, compared to non-transgenic rice seeds. Enhanced MAs production due to the introduced barley genes is suggested to be effective for increasing Fe and Zn concentrations in rice grains.     

Haplotyping of Rice Genotypes Using Simple Sequence Repeat Markers Associated with Salt Tolerance

Salt stress is a major problem in most of the rice growing areas in the world. A major QTL Saltol associated with salt tolerance at the seedling stage has been mapped on chromosome 1 in rice. This study aimed to characterize the haplotype diversity at Saltol and additional QTLs associated with salt tolerance. Salt tolerance at the seedling stage was assessed in 54 rice genotypes in the scale of 1 to 9 score at EC = 10 d Sm-1 under controlled environmental conditions. Seven new breeding lines including three KMR3/O. rufipogon introgression lines showed similar salt tolerant ability as FL478 and can be good sources of new genes/alleles for salt tolerance. Simple sequence repeat(SSR) marker RM289 showed only two alleles and RM8094 showed seven alleles. Polymorphic information content value varied from 0.55 for RM289 to 0.99 for RM8094 and RM493. Based on 14 SSR markers, the 54 lines were clearly separated into two major clusters. Fourteen haplotypes were identified based on Saltol linked markers with FL478 as the reference. Alleles of RM8094 and RM3412 can discriminate between the salt tolerant and susceptible genotypes clearly and hence can be useful in marker-assisted selection at the seedling stage. Other markers RM10720 on chromosome 1 and RM149 and RM264 on chromosome 8 can also distinguish tolerant and susceptible lines but with lesser stringency.     

磷对水稻高Fe2+胁迫的缓解作用

采用溶液培养法研究了高Fe2+胁迫下不同磷水平对粳稻Azucena（耐铁毒基因型）和籼稻IR64（铁毒敏感基因型）的生长和生理特性的影响。结果表明,与正常供Fe2+相比,高Fe2+胁迫抑制了水稻地上部和根系的生长,降低了干物质积累量和叶片叶绿素含量。外源供磷水平的提高,水稻地上部和根系生长受铁毒抑制程度有所减弱,叶片丙二醛含量和质膜透性下降,POD活性和叶绿素含量增加,而SOD活性则有所下降。表明在一定程度上,磷营养对提高水稻的耐Fe2+毒害具有重要作用,而磷对缓解Azucena的Fe2+毒害的效果较为明显。     

Using stress tolerance indicator (STI) to select high grain yield iron-deficiency tolerant wheat genotypes in calcareous soils

Despite large variation among crop genotypes in response to Fe fertilization, there is no reliable indicator for identifying Fe-deficiency tolerant wheat genotypes with high grain yield. The aim of this investigation was to compare the grain yield response of 20 spring and 30 winter bread wheat genotypes to Fe fertilization under field conditions and to select high grain yield Fe-deficiency tolerant genotypes using a stress tolerance indicator (STI). Two individual trials, each one consisting two field plot experiments, were conducted during 2006–2007 and 2007–2008 growing seasons. Spring wheat genotypes (Trial l) and winter wheat genotypes (Trial 2) were planted at two different locations. Two Fe rates (0 and 20 kg Fe ha⁻¹ as Fe-EDTA) were applied. Spring and winter wheat genotypes differed significantly (P < 0.01) in the grain yield both with and without added Fe treatments. Application of Fe fertilizer increased grain yield of spring wheat genotypes by an average of 211 and 551 kg ha⁻¹ in Karaj and Isfahan locations, respectively. By Fe application, the mean grain yield of winter wheat genotypes increased 532 and 798 kg ha⁻¹ in Karaj and Isfahan sites, respectively. Iron efficiency (Fe-EF) significantly differed among wheat genotypes and ranged from 65% to 113% for spring wheat and from 69% to 125% for winter wheat genotypes. No significant correlation was found between Fe-EF and grain yield of spring wheat genotypes under Fe deficient conditions. For winter wheat genotypes grown in Mashhad, Fe-efficiency was not significantly correlated with the grain yield produced without added Fe treatment. The STI was significantly (P < 0.01) varied among spring and winter wheat genotypes. The interaction between location and genotype had no significant effect on the STI. According to these results, the STI should be considered as an effective criterion for screening programs, if a high potential grain yield together with more stable response to Fe fertilization in different environments is desired.     

磷、铝胁迫对玉米幼苗生长和养分吸收的影响

选取5个基因型玉米品种,采用营养液培养,研究低磷和铝毒条件下不同基因型玉米苗期生长状况及对磷、钾、钙、镁、铁、锌的吸收。结果表明,耐低磷基因型玉米品种适应低磷的能力较强,具有较长的根系和较大的根干重,株高受低磷的影响明显小于敏感基因型玉米品种。低磷胁迫增大了植株的根冠比,改变了植株对营养元素的吸收及其地上部和根系的分配。铝胁迫下,铝敏感基因型玉米品种根伸长受到铝的抑制作用大于耐铝基因型玉米品种,各种营养元素的吸收累积明显受抑制,耐铝基因型玉米品种地上部和根系相对干重下降较少,而敏感基因型玉米品种相对干重显著下降。     

Morpho-Physiological Response of Oryza glaberrima to Gradual Soil Drying

Soil drought occurrence during dry season has been the main constraint, besides prolonged flooding during rainy season, in increasing cropping intensity and rice productivity in tropical riparian wetland. Use of drought tolerant rice genotype might be a suitable option for overcoming such problem. This study focused on the effects of gradual soil drying during early vegetative growth stage on morphological and physiological traits of five Oryza glaberrima genotypes, namely RAM12, RAM14, RAM59, RAM97 and RAM101, and two Oryza sativa subsp japonica genotypes, i.e. Koshihikari and Minamihatamochi. The plants were subjected to 6 d of gradual soil drying condition from 15 days after transplanting (DAT) to 20 DAT, and were allowed to recover until 22 DAT. Gradual soil drying reduced plant growth as indicated by dry mass accumulation. Drought reduced stomatal conductance and increased leaf rolling score of all the genotypes. All the genotypes showed comparable response on stomatal conductance, but O. glaberrima genotypes performed higher in leaf rolling recovery. Meanwhile, O. sativa genotypes decreased total leaf area and specific leaf area, but increased specific leaf weight in order to avoid further damages due to drought stress. Drought tolerance mechanisms in RAM101, RAM12, RAM59 and RAM14 were associated with leaf morpho-physiological responses, root traits and dry biomass accumulation.     

QTL Analysis for Grain Iron and Zinc Concentrations in Two O. nivara Derived Backcross Populations

Identification of quantitative trait loci (QTLs) for grain mineral elements can assist in faster and more precise development of micronutrient dense rice varieties through marker-assisted breeding. In the present study, QTLs were mapped for Fe and Zn concentrations in two BC₂F₃ mapping populations derived from the crosses of O. sativa cv Swarna with two different accessions of O. nivara. In all, 10 and 8 QTLs were identified for grain Fe and Zn concentrations in population 1, and 7 and 5 QTLs were identified in population 2, respectively. Eighty percent of the QTLs detected in both populations were derived from O. nivara. Five QTLs for Fe and three QTLs for Zn explained more than 15% phenotypic variance either in interval or composite interval mapping. The locations of O. nivara derived QTLs such as qFe2.1, qFe3.1, qFe8.2 and qZn12.1 were consistently identified in both the populations. Epistatic interaction was observed only between RM106 and RM6 on chromosome 2 and between RM22 and RM7 on chromosome 3 for Fe concentration in population 1. Sixteen candidate genes for metal homeostasis were found to co-locate with 10 QTLs for Fe and Zn concentrations in both the populations. Most of the Fe and Zn QTLs were found to co-locate with QTLs for grain yield and grain quality traits. Some of the major effect QTLs identified can be used to improve rice grain Fe and Zn concentrations.     

Assessment of Variation in Morpho-Physiological Traits and Genetic Diversity in Relation to Submergence Tolerance of Five Indigenous Lowland Rice Landraces

The present study evaluated submergence responses in 88 lowland indigenous rice(Oryza sativa L.) landraces from Koraput, India, to identify submergence-tolerant rice genotypes. In pot experiments, variations in survival rate, shoot elongation, relative growth index, dry matter, chlorophyll, soluble sugar and starch contents were evaluated in two consecutive years under well-drained and completely submerged conditions. Principal component analysis showed that the first three axes contributed 96.820% of the total variation among the landraces, indicating wide variation between genotypes. Major traits such as survival rate, relative growth index, soluble sugar and starch contents appeared to be important determinants of phenotypic diversity among the landraces. Phenotypic coefficient of variance was higher than genotypic coefficient of variance for all the traits and all showed high heritability(90.38%–99.54%). Five rice landraces(Samudrabali, Basnamundi, Gadaba, Surudaka and Dokarakuji) were the most tolerant to submergence. When submerged for up to 14 d, Samudrabali, Basnamundi and Godoba were notable for having greater survival rates than a standard submergence tolerant variety FR13 A, and also notable for elongating more vigorously and accumulating more biomass. These three landraces may therefore be especially useful in lowland rice growing areas that are affected by both moderate stagnant water and flash flooding. Molecular genotyping revealed that the submergence tolerance of Samudrabali, Basnamundi and Godoba is linked to the presence of one or more different Sub1 loci and it may well prove useful for breeding improved submergence tolerant rice varieties, thereby assising to improve yield stability in the rainfed lowland agro-ecosystem.     

磷对水稻耐铝性及根尖细胞壁组分的影响

 为阐明外源磷供应引起水稻体内磷代谢和根尖细胞壁组分变化进而阐述磷、铝间的相互作用,以水稻菲优多系1号(耐铝毒基因型)和红良优166(铝毒敏感基因型)为材料,水培条件下先用0.5、10和30 mg/L磷预处理9 d,然后用50 μmol/L Al处理48 h,研究铝毒胁迫下磷对水稻根尖的防护效应及磷作用下根尖细胞壁组分变化与水稻耐铝性的关系。结果表明,50 μmol/L Al处理抑制水稻总根长,尤其是在0.5 mg/L磷预处理后用铝交替处理时该作用更为明显。铝毒胁迫下,0.5 mg/L磷预处理时两基因型水稻叶片的丙二醛(MDA)、抗坏血酸(ASA)、游离脯氨酸(Pro)含量显著高于其他处理,10 mg/L和30 mg/L磷预处理显著降低两基因型水稻叶片的MDA、ASA和Pro含量,表明充足的磷供应减轻了铝对水稻的伤害。耐铝毒水稻根尖的果胶和半纤维素2含量在30 mg/L磷与铝交替处理时显著低于0.5 mg/L 和10 mg/L 磷与铝交替处理,根系的酸性磷酸酶(ACP)活性在0.5 mg/L磷与铝交替处理时显著高于10 mg/L与30 mg/L磷与铝交替处理。而铝毒敏感水稻的根尖细胞壁多糖组分含量、ACP活性在不同浓度的磷、铝交替处理间无显著差异。表明铝耐性水稻在缺磷条件下通过提高ACP活性以提供更多的Pi与铝结合钝化铝,在磷充足条件下通过降低细胞壁多糖含量以减少铝结合位点,进而提高铝毒耐性。     

Soil-based screening for iron toxicity tolerance in rice using pots

The objective of this study was to assess the reliability of pot-based screening method for iron (Fe) toxicity tolerance in rice using soils from hot spots. Five lowland rice varieties with known reaction to Fe toxicity were grown in pots in a screen house for three seasons. Fe-toxic soils from two hot spot fields – Edozighi, Nigeria and Niaouli, Benin were used and soil from Africa Rice Center (AfricaRice) experimental farm, Cotonou, Benin was included as control. Leaf bronzing score (LBS) was determined at different stages, and grain yield was determined at maturity. Heritability was estimated using data across the three seasons. High heritability was recorded for LBS and grain yield. Grain yield reduction in stress treatment relative to control varied from 15 to 56% depending on the variety and soil. Bao Thai, Suakoko 8, and WITA 4 had better performance under Fe toxicity in terms of LBS, yield and relative yield reduction, whereas Bouake 189 and IR64 had poorer performance. Grain yield and LBS were significantly correlated but negatively at 60 days after sowing (DAS). Overall, the results found in this experiment were consistent with previous field studies. Therefore, pot screening using soils from hot spots can be used by rice breeding programs to reliably assess Fe toxicity tolerance ex situ.     

磷水平对不同磷效率水稻生长及磷、锌养分吸收的影响

 选择4个耐低磷水稻基因型508、99011、580、99112和2个磷敏感基因型99056、99012，用土培盆栽进行全生育期试验，研究不同磷水平对它们生长及吸收利用磷和锌的影响。结果表明，不同耐低磷水稻基因型不仅吸收利用磷的能力不同，对锌的吸收利用也存在差异。耐低磷基因型580、99011和508都具有较大的生物量，且受磷水平影响较小；而耐低磷基因型99112和磷敏感基因型99056、99012生物量都较小且受磷水平影响较大。耐低磷基因型580和99011吸收的磷较多；而508的利用效率更占优势。99056吸收的磷较少，尤其在低磷处理时吸收磷的能力较差；99012对磷的利用效率最低。30 mg/kg的磷处理显著提高植株体锌含量。从吸收锌的总量来看，580占绝对优势，其次是99011和508；99012吸收的锌最少，其次是99056。此外，在取样的3个时期，相同磷处理的P/Zn比均以99012最大，99056最小。     

Effects of Different Nitrogen Fertilizer Levels and Native Soil Properties on Rice Grain Fe, Zn and Protein Contents

Deposition of protein and metal ions (Fe, Zn) in rice grains is a complex polygenic trait showing considerable environmental effect. To analyze the effect of nitrogen application levels and native soil properties on rice grain protein, iron (Fe) and zinc (Zn) contents, 32 rice genotypes were grown at three different locations each under 80 and 120 kg/hm2 nitrogen fertilizer applications. In treatments with nitrogen fertilizer application, the brown rice grain protein content (GPC) increased significantly (1.1% to 7.0%) under higher nitrogen fertilizer application (120 kg/hm2) whereas grain Fe/Zn contents showed non-significant effect of nitrogen application level, thus suggesting that the rate of uptake and translocation of macro-elements does not influence the uptake and translocation of micro-elements. The pH, organic matter content and inherent Fe/Zn levels of native soil showed significant effects on grain Fe and Zn contents of all the rice genotypes. Grain Zn content of almost all the tested rice genotypes was found to increase at Location III having loamy soil texture, neutral pH value (pH 6.83) and higher organic matter content than the other two locations (Locations I and II), indicating significant influence of native soil properties on brown rice grain Zn content while grain Fe content showed significant genotype × environment interaction effect. Genotypic difference was found to be the most significant factor to affect grain Fe/Zn contents in all the tested rice genotypes, indicating that although native soil properties influence phyto-availability of micronutrients and consequently influencing absorption, translocation and grain deposition of Fe/Zn ions, yet genetic makeup of a plant determines its response to varied soil conditions and other external factors. Two indica rice genotypes R-RF-31 (27.62 μg/g grain Zn content and 7.80% GPC) and R1033-968-2-1 (30.05 μg/g grain Zn content and 8.47% GPC) were identified as high grain Zn and moderate GPC rice genotypes. These results indicate that soil property and organic matter content increase the availability of Fe and Zn in rhizosphere, which in turn enhances the uptake, translocation and redistribution of Fe/Zn into rice grains.     

Evaluations of shallot genotypes for resistance against fusarium basal rot (Fusarium oxysporum f. sp. cepae) disease

Fusarium basal rot (FBR) caused by Fusarium oxysporum f. sp. cepae (Foc) is one of the most significant production constraint to shallot. Field experiment was conducted in a naturally Foc infested soil at Debre Zeit Agricultural Research Center during 2006 and 2007 cropping seasons to evaluate the level of resistance of sixteen shallot genotypes against FBR disease. Treatments were arranged in randomized complete block design with four replications. The genotypes significantly varied in their susceptibility to FBR and yield. They were grouped into tolerant, moderately and highly susceptible types. Five genotypes (DZ-Sht-168-1A, DZ-Sht-157-1B, Huruta, Negelle and DZ-Sht-169-1b) were identified to be tolerant as they had reduced disease severity levels from 26.8 to 32.5% and increased mean yield by more than 5 t ha⁻¹ compared to highly susceptible genotypes (DZ-Sht-076-4, DZ-Sht-201-1C and DZ-Sht-054-3A). Among the tolerant genotypes, DZ-Sht-169-1b had greatly reduced bulb rot incidence by 48% in ground storage and 30% in wire mesh shelf as compared to highly susceptible genotype DZ-Sht-201-1C. The tolerant genotypes have high yielding characteristic, and farmers could adopt them for cultivation where FBR is a problem.     

Effect of Supplied P Levels on Rice Growth and Uptake of P and Zn in Different P-Efficiency Genotypes

A soil pot culture experiment with four supplied P levels （i.e. P30, P50, P100, P200, representing supplemental P 30, 50, 100, 200 mg/kg, respectively） was conducted to investigate uptake and use ability to P and Zn in the rice genotypes with different P-efficiency, of which rice genotypes 508, 99011, 580, 99112 were Iow-P tolerant and 99056, 99012 were Iow-P sensitive. Low-P tolerant rice 580 and 99011 absorbed more P than the others, and rice genotype 580 had stronger uptake ability especially at Iow-P level such as P50 and P30. 508 could absorb considerable P, and had the lowest P percentage of shoot, indicating it had good performance in P-use efficiency. These three rice genotypes had larger biomass and less response to changed P level than rice genotype 99112, 99056 and 99012. Rice genotype 99112 showed Low-P tolerance mainly by sacrificing biomass to maintain high relative grain yield. The least amount of P absorbed by 99056 showed it had the lowest P uptake efficiency, and the highest P percentage in shoot of 99012 meant it had the lowest P use efficiency. So they two showed Iow-P sensitivity. Zn contents in shoot under P200, P100 and P50 were similar, but P30 increased Zn content in shoot significantly. The Zn contents in shoot of 99112, 99056 and 99012 were higher than those of 508, 99011 and 580, especially at tillering stage and booting stage. As for total Zn content in shoot, Low-P tolerant rice genotype 580 had the largest amount and followed by 99011 and 508, Iow-P tolerant rice genotype 99012 had the smallest amount at the three sampling stage and followed by 99056. Furthermore, P/Zn in shoot of 99012 was the highest, and that of 99056 was the smallest at the same P level.     

Cadmium Accumulation and Its Toxicity in Brittle Culm 1 （bcl）, a Fragile Rice Mutant

Cadmium （Cd） accumulation and toxicity in rice plants were characterized and identified by using brittle culm 1 （bcl）, a fragile rice mutant and its wild type （Shuangkezao, an indica rice） as materials by hydroponics. The low Cd level didn＇t obviously affect the growth parameters in both rice genotypes, but under high Cd levels （1.0 and 5.0 μmol/L）, the growth of both rice plants were substantially inhibited. Moreover, bcl tended to suffer more seriously from Cd toxicity than Shuangkezao. Cd accumulation in both rice plants increased with the increase of Cd levels. There was a significant difference in Cd accumulation between the two rice genotypes with constantly higher Cd concentration in bcl, which also accumulated more Cd at 0, 0.1, and 1.0 μmol/L Cd levels. The same case was found in the two rice plants grown on Cd-contaminated soil. This suggested that cell wall might play an important role in Cd accumulation in rice plants by the physiological mechanisms. The malondialdehyde （MDA） content, superoxide dismutase （SOD） and peroxidase （POD） activities in rice plants were affected differently under Cd treatments, and which implied that POD might play the main role in detoxifying active oxygen free radical. A significant difference in antioxidative system between the two rice genotypes was found with constantly higher MDA content, SOD and POD activities in bcl. In summary, bcl accumulated more Cd and appeared to be more sensitive to Cd stress compared with its wild type.