| Arsenic is a ubiquitous and non-essential carcinogenic metalloid. Excess arsenic accumulated in soils beyond the safety threshold, which not only lead to soil degradation, but also threaten human health via food chain. Phytoremediation gets great concern worldwide due to the characteristics of green, cost-effective and in-situ remediation. However, phytoremediation effect greatly depends on the choice of suitable plants. Numerous studies demonstrated that fast-growth woody plants such as Populus, Salix are potential candidates, and rice can accumulate arsenic approximately 10-fold of that in other cereals due to highly efficient arsenic transportation. In present study, to evaluate the phytoremediation capabilities of six poplar clones (Zhonglin’2025’,I-45/51,I-214, I-63, Flevo, Danhong), a hydroponics screening for arsenic tolerance, absorption, accumulation and transportation was carried out under three levels of arsenate-treatment (Na3AsO4·12H2O:0μM,50μM,100μM). Then, physiological and comparative proteomics analysis of two contrasting poplar clones (arsenate sensitive and tolerant clones) also performed. In addition, a comparative proteomic analysis of rice shoots has also been performed in this study.1. A hydroponics screening of arsenate sensitive and tolerant poplar clonesExcess arsenate inhibited the growth of six poplar clones, caused toxicity symptoms appeared on the young leaves such as chlorosis, shrink and necrosis. Among six clones, the more severe symptoms were found in clone 45/51 while no symptom in clone 2025. Moreover, clone Danhong showed the lowest reduction of plant growth as well as the lowest inhibition of photosynthesis and transpiration, followed by clone 2025,214 and Harvord, finally clone 45/51 and Flevo.Arsenic accumulation is always in an order of roots> leaves> shoots, and the roots held 95-99% of total arsenic accumulated by whole plants, to a lesser degree in the aerial parts. Upon the exposure to arsenate solutions, the relatively highest accumulation in roots, shoots and leaves, on average, was determined in clone Flevo,2025 and Danhong, which absorbed 1701.99 mg/kg,21.72 mg/kg and 30.07 mg/kg of arsenic, respectively.On average, roots BF, aerial parts BF and Tf of six clones exposed to arsenate solutions is 78.69-131.21,2.14-3.18 and 0.019～0.046, respectively. They are homogeneous among six clones with the relatively lowest roots BF, the highest leaves BF and Tf existed in clone 2025.In conclusion, clone 2025 was considered as a good individual for the remediation of arsenate-polluted soils due to the stronger arsenate tolerance and the higher arsenic accumulated in aerial parts. In contrast, clone 45/51 exhibited the most severe toxicity symptoms as well as the lower photosynthesis, was regarded as an arsenate sensitive clone.2. Physiological and biochemical investigation of arsenate sensitive and tolerant poplar clones exposed to arsenate solutionThe content of photosynthetic pigments in clone 2025 and 45/51 leaves was reduced under arsenate stress in a dose-dependent manner. Moreover, the content of chlorophyll a and chlorophyll b is significant different between clone 2025 and clone 45/51 under 100 μM arsenate stress.The visualization of histochemical staining in situ indicated arsenate treatment triggered the generation of O2- and H2O2 in the roots of clone 45/51, with mainly accumulated in the root tips whose possessing vigorous metabolism. Whereas, there is no obvious accumulation of O2- and H2O2 in the leaves.Upon exposure to arsenate solutions, the activities of CAT, GR and the content of GSH in the clone 2025 and 45/51 roots all increased. Moreover, the activities of POD in the roots and leaves as well as the activities of APX in the leaves decreased. In addition, there is no obvious alternation in SOD activities of roots and leaves.In gereral, the response of two clones to arsenate stress is different at physiological level. In this study, the activities of GR are inconsistent with the content of GSH, which perhaps implied GSH play an important role in arsenic detoxification. This may be the essentially reason that explains the difference of arsenate tolerance between two clones.3. Comparative proteomic profiling of sensitive and tolerant poplar roots exposed to arsenate solutionsOn the 2-D gel images of 2025 and 45/51 roots, about 650 and 670 protein spots could be reproducibly detected in triplicate experiments, respectively. Among them,51 and 74 were differentially displayed (>2 fold changes in abundance and P< 0.05) during the arsenate stress treatment, respectively. After MALDI-TOF-MS analysis and UniProt database searching,34 and 48 differentially expressed proteins were successfully identified. According to MapMan ontology, all the identified proteins could be divided into several different functional groups. They mainly involved in basic metabolism, stress defense, redox regulation, protein metabolism, cell organization, signal conduction, etc.There are 7 overlapped proteins in clone 2025 and 45/51, which indicate that there has shared response of two clones to arsenate stress.Function analysis of some differential expressed proteins such as POD, SOD, HSP70, smHSP, Proteasome subunit alpha type, HDAs etc revealed that clone 2025 have more stronger arsenate perception, antioxidative stress, rubbish clear and repair ablilities than 45/51. In addition, up-regulation of proteins involved in EMP, TCA and ATP synthesis perhaps suggested that 45/51 suffered more severe stress from arsenate.4. Comparative proteomic analysis in combination with physiological and biochemical study of rice shoots exposed to arsenateExcess arsenate inhibited the growth of rice seedlings, caused toxicity symptoms appeared on the root tips such as spare root hairs, yellow-brown spots. The ultra-structure of roots and leaves was destroyed by arsenate through the observation of transmission electronic microscopy.Similarity, about 660 protein spots could be reproducibly detected in triplicate experiments of rice seedling. Among them,38 were differentially displayed (>1.5 fold changes in abundance) during the arsenate stress treatment. After MALDI-TOF-MS analysis and UniProt database searching,38 differentially expressed proteins were all successfully identified,12 of them were up-regulated and 26 down-regulated.The results of function analysis on differential expressed proteins indicated that basic metabolisms, especially photosynthesis metabolism were suppressed by arsenate stress. Moreover, arsenate stress also induced oxidative stress, the degradation of cell wall and the impairment of cell structure.Integration of above-mentioned results provided new insights for comprehensive understanding of arsenate stress-elicited changes in poplar and rice at morphological, physiological and proteomic level and offers a stepping stone toward the cultivation of transgenic poplar with stronger arsenate tolerance, higher accumulation and translocation capabilities as well as transgenic rice with lower arsenate accumulation. |
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