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Peanuts grown in saline alkali soil are also subjected to drought stress caused by water scarcity. Therefore, we used HY25 (peanut variety) as an experimental material to investigate the effects of drought on the height of peanut main stems, length of the first lateral branch, leaf area per plant, SPAD value, net photosynthetic rate, and accumulation and distribution of photosynthetic products in saline alkali soil. The results showed that the combined stress of short-term drought and salt significantly reduced the main stem height, first lateral branch length, single plant leaf area, SPAD value, net photosynthetic rate (Pn), intercellular carbon dioxide concentration (Ci), and dry matter accumulation of peanuts, including a decrease in single plant pod yield, 100-pod weight, 100-kernel weight, and peanut yield. And the impact of drought stress on peanut yield varies at different growth stages. For example, under drought stress alone, the sensitive period is the 40th day after planting (40D) > 60th day after planting (60D) > 30th day after planting (30D). Short-term drought has the greatest impact on peanut yield at 40D, while in contrast, resuming watering after drought at 30D results in a slight but not significant increase in peanut yield in comparison with the control. Under the combined stress of drought and salt, the sensitive period of peanuts was 40D > 30D > 60D, and the single pod weight of peanuts was significantly reduced by 15.26% to 57.60% from the flowering stage to the pod stage under drought treatment compared to salt treatment, indicating a significant interaction between drought and salt stress, reducing the single leaf area and net photosynthetic rate of peanut leaves, ultimately leading to a decrease in peanut yield. Therefore, when planting peanuts in saline alkali soil, drought should be avoided, especially early drought, in order to prevent the combined effects of drought and salt stress from harming peanut yield.
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Peanut (Arachis hypogaea L.) is an important oil and cash crop. An efficient utilization of saline-alkali soil resources, the development of peanut planting in saline-alkali soil, and obtaining high and stable yield have become urgent needs to ensure peanut production. Arbuscular mycorrhizal fungi (AMF) have been reported to develop the potential productivity of host plants and improve their salt resistance and tolerance. However, there is still limited research on promoting the growth and morphology of peanut roots. Therefore, in this study, seeds of salt-tolerant peanut variety "HY 25" were coated with commercial AMF inoculant before being planted in saline-alkali and normal soils to investigate the effects of AMF on peanut root growth and rhizosphere soil. The results showed that root morphological characteristics were significantly increased by the use of AMF at the podding stage in saline-alkali soil and from the flowering and pegging stage to the maturity stage in normal soil. Of note, the total root volume of peanut inoculated with AMF significantly increased by 31.57% during the podding stage in saline-alkali soil. Meanwhile, AMF significantly increased the phosphatase and invertase activities in the peanut rhizosphere of saline-alkali soil from the flowering stage to maturity stage and soil CAT activity at the maturity stage (41.16~48.82%). In normal soil, soil phosphatase and urease activities were enhanced by AMF at the flowering stage and the podding stage, respectively. AMF also increased the contents of soil organic matter, available phosphorus, and hydrolysable nitrogen, but decreased soil EC in saline-alkali soil. In addition to the significant increase in soil available phosphorus content, AMF had no significant effect on the physical and chemical properties of the soil and other soil nutrients in normal soil. AMF significantly increased pod biomass and yield in saline-alkali soil and normal soil, and improved their agronomic characteristics. In conclusion, peanut seeds coated with AMF improved the root morphological characteristics of peanuts and improved the physical and chemical properties in peanut rhizosphere, especially in saline-alkali soil. The process of rhizosphere soil nutrient transformation was also enhanced. Finally, AMF improved plant agronomic traits to increase the pod yield (16.5~21.9%). This study provides the theoretical basis and technical support for the application of AMF in peanut production in saline-alkali soil.
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Long-term excessive application of chemical fertilizers can cause many problems, such as soil degradation and environmental pollution. Therefore, we reduced conventional nitrogen fertilization and added organic fertilizers in some cases to investigate the response of photosynthetic characteristics, root nodules and yield on reduced nitrogen fertilization. Compared to conventional nitrogen fertilization, the 25% and 35% nitrogen reduction treatments reduced the leaf area index, net photosynthetic rate, 100-fruit weight, 100-kernel weight and the yield of peanut, but had no significant effect on the kernel rate. With constant N fertilizer, adding organic fertilization alone increased leaf area index, chlorophyll, net photosynthetic rate and yield of peanut. In compounded treatments of nitrogen and organic fertilizer, the highest yields were achieved in the 25% N reduction with the 3000 kg/hm-2 organic fertilizer treatment (T3) and the 4500 kg/hm-2 organic fertilizer treatment (T4); furthermore, the net photosynthetic rate, leaf area index, yield and fertilizer contribution were significantly higher in these two treatments than in the conventional fertilizer treatments. Nitrogen fertilizer had significant effects on the quantity and fresh weight of root nodules. Concretely, nitrogen reduction increased the quantity and fresh weight of root nodules of peanut in the early stage of fertility but decreased them in the harvest stage. Nitrogen reduction with an additional organic fertilizer in the late stage of fertility increased the quantity and fresh weight of root nodules of peanut. Considering the property of root nodules was significantly positively correlated with net photosynthetic rate and yield, the arguments above may be the mechanism of the highest yields found in T3 and T4. This work can provide empirical and instructional support for a balanced fertilization strategy in peanut agriculture and high-yielding and efficient cultivation of peanut.
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BACKGROUND: Arbuscular Mycorrhizal Fungi (AMF) are beneficial microorganisms in soil-plant interactions; however, the underlying mechanisms regarding their roles in legumes environmental stress remain elusive. Present trials were undertaken to study the effect of AMF on the ameliorating of salt, drought, and cold stress in peanut (Arachis hypogaea L.) plants. A new product of AMF combined with Rhizophagus irregularis SA, Rhizophagus clarus BEG142, Glomus lamellosum ON393, and Funneliformis mosseae BEG95 (1: 1: 1: 1, w/w/w/w) was inoculated with peanut and the physiological and metabolomic responses of the AMF-inoculated and non-inoculated peanut plants to salt, drought, and cold stress were comprehensively characterized, respectively. RESULTS: AMF-inoculated plants exhibited higher plant growth, leaf relative water content (RWC), net photosynthetic rate, maximal photochemical efficiency of photosystem II (PSII) (Fv/Fm), activities of antioxidant enzymes, and K+: Na+ ratio while lower leaf relative electrolyte conductivity (REC), concentration of malondialdehyde (MDA), and the accumulation of reactive oxygen species (ROS) under stressful conditions. Moreover, the structures of chloroplast thylakoids and mitochondria in AMF-inoculated plants were less damaged by these stresses. Non-targeted metabolomics indicated that AMF altered numerous pathways associated with organic acids and amino acid metabolisms in peanut roots under both normal-growth and stressful conditions, which were further improved by the osmolytes accumulation data. CONCLUSION: This study provides a promising AMF product and demonstrates that this AMF combination could enhance peanut salt, drought, and cold stress tolerance through improving plant growth, protecting photosystem, enhancing antioxidant system, and regulating osmotic adjustment.
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Micorrizas , Micorrizas/fisiologia , Arachis/metabolismo , Antioxidantes/metabolismo , Resposta ao Choque Frio , Secas , Cloreto de SódioRESUMO
Peanut (Arachis hypogaea. L) is an important oil seed crop. Both arbuscular mycorrhizal fungi (AMF) symbiosis and calcium (Ca2+) application can ameliorate the impact of saline soil on peanut production, and the rhizosphere bacterial communities are also closely correlated with peanut salt tolerance; however, whether AMF and Ca2+ can withstand high-salinity through or partially through modulating rhizosphere bacterial communities is unclear. Here, we used the rhizosphere bacterial DNA from saline alkali soil treated with AMF and Ca2+ alone or together to perform high-throughput sequencing of 16S rRNA genes. Taxonomic analysis revealed that AMF and Ca2+ treatment increased the abundance of Proteobacteria and Firmicutes at the phylum level. The nitrogen-fixing bacterium Sphingomonas was the dominant genus in these soils at the genus level, and the soil invertase and urease activities were also increased after AMF and Ca2+ treatment, implying that AMF and Ca2+ effectively improved the living environment of plants under salt stress. Moreover, AMF combined with Ca2+ was better than AMF or Ca2+ alone at altering the bacterial structure and improving peanut growth in saline alkali soil. Together, AMF and Ca2+ applications are conducive to peanut salt adaption by regulating the bacterial community in saline alkali soil.
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Arachis/crescimento & desenvolvimento , Bactérias/metabolismo , Cálcio/metabolismo , Fungos/fisiologia , Álcalis/análise , Arachis/microbiologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , DNA Bacteriano/genética , Fungos/classificação , Fungos/genética , Fungos/isolamento & purificação , Filogenia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , RNA Ribossômico 16S , Rizosfera , Salinidade , Solo/química , Microbiologia do Solo , SimbioseRESUMO
A pot experiment with Huayu 25 as experimental material was conducted, with treatments of drought and salt stresses. The effects of drought and salt stresses at the flowering stage on the plant morphology, pod yield, and soil bacterial community structure in the rhizosphere were examined. The results showed that Proteobacteria, Actinobacteria, Saccharibacteria, Chloroflexi, Cyanobacteria, and Acidobacteria were the dominant phyla in the rhizosphere soil of peanut. Compared with that under normal conditions, the relative abundance of Proteobacteria and Actinobacteria dramatically decreased, while that of Cyanobacteria evidently increased in drought-treated and salt-treated soil. Moreover, the variation of Cyanobacteria abundance caused by combined drought and salt stresses was stronger than that caused by single drought or salt stress. Functional meta-genomic profiling indicated that a series of sequences related to signaling transduction, defense mechanism and post-translational modification, protein turnover, chaperones were enriched in rhizosphere soil under stressed conditions, which might have implications for plant survival and stress tolerance. Drought and salt stress affectedpeanut growth and reduced pod yield. Results from this study would present reference on the future improvement of stress tolerance of peanuts via modifying soil microbial community.
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Arachis , Rizosfera , Secas , RNA Ribossômico 16S , Solo , Microbiologia do SoloRESUMO
Green leaf volatiles (GLVs) are released by plants when they encounter biotic stress, but their functions in the response to abiotic stress have not been determined. We have previously shown that exogenous application of (Z)-3-hexeny-1-yl acetate (Z-3-HAC), a kind of GLV, could alleviate salt stress in peanut (Arachis hypogaea L.) seedlings; however, notably little is known concerning the transcription regulation mechanisms of Z-3-HAC. In this study, we comprehensively characterized the transcriptomes and physiological indices of peanut seedlings exposed to Z-3-HAC and/or salt stress. Analysis of transcriptome data showed that 1420 genes were upregulated in the seedlings primed with Z-3-HAC under salt stress compared with the non-primed treatment. Interestingly, these genes were significantly enriched in the photosynthetic and ascorbate metabolism-related categories, as well as several plant hormone metabolism pathways. The physiological data revealed that Z-3-HAC significantly increased the net photosynthetic rate, SPAD value, plant height and shoot biomass compared with the non-primed peanut seedlings under salt stress. A significantly higher ratio of K+ :Na+ , reduced-to-oxidized glutathione (GSH:GSSG), and ascorbate-to-dehydroascorbate (AsA:DHA) were also observed for the plants primed with Z-3-HAC compared with the salt stress control. Meanwhile, Z-3-HAC significantly increased the activity of enzymes in the AsA-GSH cycle. Taken together, these results highlight the importance of Z-3-HAC in protecting peanut seedlings against salt stress by affecting photosynthesis, cellular redox homeostasis, K+ :Na+ homeostasis, and phytohormones.
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Arachis , Fotossíntese , Acetatos , Glutationa , Homeostase , Oxirredução , Estresse Salino , Plântula , Estresse FisiológicoRESUMO
BACKGROUND: Exposure of seeds to high salinity can cause reduced germination and poor seedling establishment. Improving the salt tolerance of peanut (Arachis hypogaea L.) seeds during germination is an important breeding goal of the peanut industry. Bacterial communities in the spermosphere soils may be of special importance to seed germination under salt stress, whereas extant results in oilseed crop peanut are scarce. METHODS: Here, bacterial communities colonizing peanut seeds with salt stress were characterized using 16S rRNA gene sequencing. RESULTS: Peanut spermosphere was composed of four dominant genera: Bacillus, Massilia, Pseudarthrobacter, and Sphingomonas. Comparisons of bacterial community structure revealed that the beneficial bacteria (Bacillus), which can produce specific phosphatases to sequentially mineralize organic phosphorus into inorganic phosphorus, occurred in relatively higher abundance in salt-treated spermosphere soils. Further soil enzyme activity assays showed that phosphatase activity increased in salt-treated spermosphere soils, which may be associated with the shift of Bacillus. CONCLUSION: This study will form the foundation for future improvement of salt tolerance of peanuts at the seed germination stage via modification of the soil microbes.
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Arachis/microbiologia , Bactérias/crescimento & desenvolvimento , Microbiota , Estresse Salino , Bactérias/genética , Biodiversidade , Filogenia , RNA Ribossômico 16S/genética , Solo/química , Microbiologia do SoloRESUMO
Soil salinity is regarded as severe environmental stress that can change the composition of rhizosphere soil bacterial community and import a plethora of harms to crop plants. However, relatively little is known about the relationship between salt stress and root microbial communities in groundnuts. The goal of this study was to assess the effect of salt stress on groundnut growth performance and rhizosphere microbial community structure. Statistical analysis exhibited that salt stress indeed affected groundnut growth and pod yield. Further taxonomic analysis showed that the bacterial community predominantly consisted of phyla Proteobacteria, Actinobacteria, Saccharibacteria, Chloroflexi, Acidobacteria, and Cyanobacteria. Among these bacteria, numbers of Cyanobacteria and Acidobacteria mainly increased, while that of Actinobacteria and Chloroflexi decreased after salt treatment via taxonomic and qPCR analysis. Moreover, Sphingomonas and Microcoleus as the predominant genera in salt-treated rhizosphere soils might enhance salt tolerance as plant growth-promoting rhizobacteria. Metagenomic profiling showed that series of sequences related to signaling transduction, posttranslational modification, and chaperones were enriched in the salt-treated soils, which may have implications for plant survival and salt tolerance. These data will help us better understand the symbiotic relationship between the dominant microbial community and groundnuts and form the foundation for further improvement of salt tolerance of groundnuts via modification of soil microbial community.
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Arachis/microbiologia , Microbiota/genética , Rizosfera , Estresse Salino , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Arachis/crescimento & desenvolvimento , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Cianobactérias/genética , Cianobactérias/isolamento & purificação , DNA Bacteriano , Metagenômica , Filogenia , Raízes de Plantas/microbiologia , RNA Ribossômico 16S , Salinidade , Solo/química , Microbiologia do Solo , Sphingomonas/genética , Sphingomonas/isolamento & purificaçãoRESUMO
Green leaf volatiles play vital roles in plant biotic stress; however, their functions in plant responses to abiotic stress have not been determined. The aim of this study was to investigate the possible role of (Z)-3-hexeny-1-yl acetate (Z-3-HAC), a kind of green leaf volatile, in alleviating the salinity stress of peanut (Arachis hypogaea L.) seedlings and the underlying physiological mechanisms governing this effect. One salt-sensitive and one salt-tolerant peanut genotype were primed with 200 µM Z-3-HAC at the 4-week-old stage before they were exposed to salinity stress. Physiological measurements showed that the primed seedlings possessed higher relative water content, net photosynthetic rate, maximal photochemical efficiency of photosystem II, activities of the antioxidant enzymes, and osmolyte accumulation under salinity conditions. Furthermore, the reactive oxygen species, electrolyte leakage, and malondialdehyde content in the third fully expanded leaves were significantly lower than in nonprimed plants. Additionally, we found that application of Z-3-HAC increased the total length, surface area, and volume of the peanut roots under salinity stress. These results indicated that the green leaf volatile Z-3-HAC protects peanut seedlings against damage from salinity stress through priming for modifications of photosynthetic apparatus, antioxidant systems, osmoregulation, and root morphology.
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Objective: Three soil types in different salt contents were taken as the experiment objectives. We evaluated the effect of various saline alkali soil types on diversity of bacterial community structure in spermosphere soil during water absorption and germination of peanut seeds. Methods: The V3-V4 region of 16S ribosomal RNA genes was amplified using PCR, and the PCR products were then analyzed using Illumina high-throughput sequencing technology. Results: (1) The diversity of soil bacterial community in saline alkali soil was higher than that in non-saline alkali soil. Especially, the highest diversity was in spermosphere soil from Qingtuo. (2) The microflora structures in different soils were distinct at the class level. Soil bacteria in four samples were classified into six classes, including Proteobacteria, Actinobacteria, Actinobacteria, Bacteroidetes, Acidobacteria and Firmicutes. Proteobacteria and Actinobacteria groups were dominant in colonies. The analysis of whole samples colony structure showed that the difference of type and abundance at phylum and genus level during different adsorption time was most significant (P<0.05). (3) The analysis of beta diversity and phylogenetic distances of constructed phylogenetic trees revealed that the sequenced clones fell into two major groups within the domain bacteria. Conclusion: The diversity of bacteria community compositions in the high salt content soil was higher. There were obvious differences in microbial community structure of different soil types at class level, primarily in the Proteobacteria and Actinobacteria. The type and abundance of microbial colonies at both phylum and genus levels were affected by the seed germination time. However, there was no influence on the genetic distance between the samples from the same soil type.
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Bactérias/isolamento & purificação , Biodiversidade , Microbiologia do Solo , Solo/química , Álcalis/análise , Álcalis/metabolismo , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , China , FilogeniaRESUMO
Taking 'Huayu 22' peanut as test material, effect of soil water content and nitrogen fertilization on the leaf physiological activities and root morphological characteristics of peanut plants were analyzed. Two levels of soil water condition were: (1) well-watered condition and (2) moderate water stress, and three levels of nitrogen were: (1) none nitrogen (N0), (2) moderate nitrogen (N1, 90 kg · hm(-2)) and (3) high nitrogen (N2, 180 kg · hm(-2)). The results showed that N1 significantly increased the peanut yield under two water conditions, but showed no significant effect on harvest index compared with N0. Under water stress condition, N1 had no significant effects on total root biomass and total root length, but the total root surface area was remarkably increased. The nitrogen fertilization significantly increased the root length and root surface area in 20-40 cm soil layer, and N2 significantly increased the root biomass and root surface area in the soil layer below 40 cm. The application of nitrogen remarkably increased CAT and POD activities in leaf, while MDA content was decreased with the increase of nitrogen level. Under well-watered condition, the root biomass, root length and root surface area in the soil layer below 40 cm and total root surface area were significantly reduced by nitrogen application, however, only N1 could increase leaf protective enzyme activities. Correlation analysis showed that the root length in 20-40 cm soil layer and SOD, CAT, POD activities in leaf were highly significantly related with peanut yield.
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Arachis/fisiologia , Desidratação , Folhas de Planta/fisiologia , Raízes de Plantas/crescimento & desenvolvimento , Água/fisiologia , Biomassa , Nitrogênio/química , Solo/químicaRESUMO
Taking two peanut varieties Huayu 17 and Tangke 8 as test objects, a soil column culture experiment was conducted in a rainproof tank to study the peanut root morphological development and physiological characteristics at late growth stages under moderate drought and well-watered conditions. Tanke 8 had more developed root system and higher yield and drought coefficient, while Huayu 17 had poorer root adaptability to drought stress. For the two varieties, their root length density and root biomass were mainly distributed in 0-40 cm soil layer, whereas their root traits differed in the same soil layer. The total root length, total root surface area, and total root volume of Huayu 17 at each growth stage were smaller under drought stress than under well-balanced water treatment, while these root characteristics of Tangke 8 under drought stress only decreased at flowering-pegging stage. Drought stress increased the root biomass, surface area, and volume of the two varieties in 20-40 cm soil layer, but decreased these root traits in the soil layers below 40 cm. Under drought stress, the root activity of the two varieties in the soil layers below 40 cm at pod filling stage decreased, and the decrement was larger for Huayu 17. The differences in the root system development and physiological characteristics of the two varieties at late growth stages under drought stress suggested that the root system of the two varieties had different water absorption and utilization under drought stress.
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Arachis/fisiologia , Secas , Raízes de Plantas/crescimento & desenvolvimento , Estresse Fisiológico/fisiologia , Arachis/classificação , Arachis/crescimento & desenvolvimento , China , Raízes de Plantas/fisiologiaRESUMO
A total of two hundred peanut varieties (lines) were exposed to different salt concentrations under pot cultivation, to evaluate salinity tolerance by indices such as emergence, morphology and biomass accumulation from emergence to seedling stage. The results showed that, as the salinity concentration increased, the emergence time was prolonged, plant morphology establishment was inhibited seriously, and biomass accumulation was reduced. The optimal concentration for evaluating salinity tolerance was 0.30%-0.45%. Ten indices were contributed to the mean membership function value by the membership function analysis. According to the correlation coefficient between indices and the mean membership function value, plant fresh mass, shoot fresh mass, root fresh mass, root dry mass, plant height and stem height could be the first selected indices for evaluating salinity tolerance of peanut plant. Plant dry mass, shoot dry mass, taproot length and emergence speed could be the second selected indices to comprehensively evaluate salinity tolerance of peanut plant. The 200 varieties were divided into 4 groups at different salinity concentrations, i. e. high salinity tolerance, salinity tolerance, salinity sensitivity, and high salinity sensitivity. Number of salinity tolerant varieties was decreased with increasing salinity concentration while the salinity sensitive one was increased. Salinity tolerance of some varieties showed the similarity (tolerant or sensitive) under different salinity stresses. Some varieties showed different tolerance under different salinity stresses, i. e. tolerance at low salinity concentration while sensitivity at high salinity concentration.
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Arachis/fisiologia , Salinidade , Plantas Tolerantes a Sal , Arachis/classificação , Biomassa , Plântula , Cloreto de SódioRESUMO
In order to identify the variation of cadmium (Cd) tolerance and accumulation in wheat (Triticum aestivum L.), a study was conducted in hydroponic culture with or without Cd using recombinant inbred lines (RILs) consisting of 103 RILs derived from a cross of Chuan 35050 × Shannong 483 at seedling stage. The parameters of shoot height, secondary roots numbers, tiller numbers, shoot dry weights, root dry weights, and maximum efficiency of photosystem II photochemistry under dark-adopted conditions were measured. Cd-tolerant indexes were then calculated as relative the above traits under Cd stress to those under the control. Cd concentration in shoot or root was determined and Cd accumulation and translocation were calculated. Based on the Cd-tolerant indexes, membership function analysis was used to determine the variation of the above parameters. The results showed a continuous distribution among the RILs and then the RILs were divided into five groups according to their tolerance. Lines 76 and 17 were considered as the most Cd-tolerant lines while lines 103 and 51 were as the most Cd-sensitive lines. Meanwhile, lines 38 and 79 were with minimum Cd translocation ratio while lines 88 and 53 were with maximum Cd translocation ratio, respectively. The relationship between Cd tolerance and accumulation was not significant, indicating Cd tolerance and accumulation may be independent traits in the RILs. Thus, lines with high Cd tolerance and less Cd accumulation could be selected for wheat breeding.
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Cádmio/toxicidade , Plântula/efeitos dos fármacos , Triticum/efeitos dos fármacos , Plântula/metabolismo , Triticum/metabolismoRESUMO
Four wheat (Triticum aestivum L.) lines differing in cadmium (Cd) tolerance previously identified from a recombinant inbred line population were subjected to 50 microM CdCl2 from the three-leaf stage for 24 d, to investigate the responses of wheat seedlings to Cd toxicity. Under Cd stress, most growth parameters and root morphological traits were reduced, except for secondary root numbers and average root diameter. Cd enhanced leaf cell peroxidation due to increased malondialdehyde (MDA) content and reduced activities of superoxide dismutase (SOD) and catalase (CAT) in leaves. In addition, CAT activity decreased in the Cd-sensitive lines while increased in the tolerant lines. Leaf photosystem II (PSII) was damaged, since the maximum efficiency of PSII photochemistry (Fv/Fm) and potential efficiency of PSII photochemistry under dark-adapted (Fv/Fo) decreased, while the initial fluorescence (Fo) increased in all lines under Cd stress. Then, total soluble sugar concentration decreased while free amino acids concentration increased in both shoot and root. We concluded that Cd-tolerant lines accumulated less Cd in plant and contained low Cd concentration in shoot (less translocation of Cd to shoot), maintained higher CAT activity in leaf and higher PS II function than the Cd-sensitive lines under Cd toxicity, thus could be related to their tolerant capacity to Cd in the present study.