The root iron transporter 1 governs cadmium uptake in Vicia sativa roots.

Cadmium is a non-essential element for plants and that inhibits plant growth and development. The Zhangye Mawan (ZM) variety of Vicia sativa is more sensitive to Cd toxicity than that Lanjian 3# (L3) variety, but the underlying mechanism is not fully understood. Here, we demonstrated that ZM showed higher Cd accumulation than L3 based on root Cd content and Cd fluorescence intensity in root protoplasts. VsRIT1, a member of the ZIP (ZRT/IRT-like protein) family, showed expression levels in ZM roots 8-fold higher than those in L3 roots under Cd exposure. VsRIT1 expression increased Cd transport and accumulation in Arabidopsis and yeast. These suggests that VsRIT1 participates in Cd uptake by V. sativa roots. Furthermore, ZM root tips have a higher capacity for transient Cd influx than L3 roots when exposed to Cd alone or Cd and iron (Fe) together, owing to the higher VsRIT1 expression in ZM. Our findings also imply that Cd may compete with Fe or/and zinc (Zn) for uptake via VsRIT1 in V. sativa or yeast.

Effects of different rootstocks on cadmium accumulation characteristics of the post-grafting generations of Galinsoga parviflora.

A pot experiment was conducted to study the effects of different rootstocks on the cadmium (Cd) accumulation characteristics of the post-grafting generations of Cd-hyperaccumulator Galinsoga parviflora plants. Five treatments, ungrafted and G. parviflora seedlings grafted on the rootstocks of Kalimeris indica, Senecio scandens, Conyza canadensis, and Artemisia sieversiana, were utilized. The four rootstock grafts decreased the shoot biomass of the G. parviflora post-grafting generation compared with ungrafted. The K. indica and S. scandens grafts increased the Cd concentration in shoots of the G. parviflora post-grafting generation by 15.06% and 14.40%, respectively, compared with ungrafted, while the C. canadensis and A. sieversiana grafts had no significant effects. K. indica grafts increased the amount of Cd extracted by shoots of the G. parviflora post-grafting generation by 10.59% compared with ungrafted, while the other treatments resulted in decreases. Compared with ungrafted, the different rootstocks had no significant effects on the photosynthetic pigment content of the G. parviflora post-grafting generation, and only C. canadensis grafts increased the superoxide dismutase activity level, while only K. indica grafts increased the peroxidase activity level. Therefore, the K. indica rootstock could increase the phytoremediation capability of G. parviflora post-grafted plants grown in Cd-contaminated soil.

Cutting after grafting affects the growth and cadmium accumulation of Nasturtium officinale.

The growth and cadmium (Cd) accumulation of emergent plant Nasturtium officinale R. Br. cuttings taken from plants grafted onto rootstocks of four terrestrial Cruciferae species were studied in a pot experiment. Scions from N. officinale seedlings were grafted onto rootstocks of Brassica chinensis L., Raphanus sativus L., Brassica napus L., and Rorippa dubia (Pers.) H. Hara. Cuttings were taken after 1 month and grown in Cd-contaminated soil (10 mg Cd kg-1) for 60 days. Compared with non-grafted N. officinale, grafting onto R. sativus and B. napus rootstocks increased the root, shoot, and whole plant biomasses of N. officinale cuttings. Brassica napus rootstock was more effective than R. sativus rootstock for increasing the biomass of N. officinale cuttings. The four rootstocks decreased or had no significant effect on photosynthetic pigment contents in N. officinale cuttings compared with non-grafted N. officinale. Only grafting onto B. napus rootstock enhanced antioxidant enzyme activities. Compared with non-grafted N. officinale, R. sativus and B. napus rootstocks decreased the Cd contents in roots and shoots of N. officinale cuttings, whereas the other rootstocks had no significant effect on the shoot Cd content. The four rootstocks had no increase effects on Cd extraction by N. officinale cuttings. Therefore, cutting after grafting did not enhance the phytoremediation ability of N. officinale for growth in Cd-contaminated soil. However, R. sativus and B. napus rootstocks decreased the Cd content in N. officinale cuttings, which offers a potential approach for N. officinale safety production as a wild vegetable in Cd-contaminated soils.

Effects of mutual grafting on the cadmium accumulation characteristics of two ecotypes of Solanum photeinocarpum.

The effects of mutual grafting on the cadmium (Cd) accumulation characteristics of two ecotypes (farmland and mining) of the potential Cd-hyperaccumulator Solanum photeinocarpum were studied through a pot experiment for one month. Four treatments were used in the experiment: ungrafted farmland ecotype (F-CK), ungrafted mining ecotype (M-CK), the farmland ecotype as the scion grafted onto rootstocks of the mining ecotype (F-Scion), and the mining ecotype as the scion grafted onto rootstocks of the farmland ecotype (M-Scion). Mutual grafting increased the rootstock biomass of both S. photeinocarpum ecotypes. However, mutual grafting decreased the scion biomass of F-Scion compared with F-CK and M-CK, and the scion biomass of M-Scion was higher than that of M-CK and lower than that of F-CK. The Cd content in the rootstock of M-Scion increased compared with F-CK, and the Cd content in the rootstock of F-Scion increased compared with M-CK, but mutual grafting decreased the Cd content in scions of both S. photeinocarpum ecotypes. Mutual grafting increased Cd extraction by rootstocks of both S. photeinocarpum ecotypes, but decreased extraction by scions. Therefore, mutual grafting can increase Cd accumulation in S. photeinocarpum rootstocks but not increase Cd accumulation in S. photeinocarpum scions in a short period.

iTRAQ-Based Comparative Proteomic Analysis of the Roots of TWO Winter Turnip Rapes (Brassica rapa L.) with Different Freezing-Tolerance.

The freezing tolerance of roots is crucial for winter turnip rape (Brassica rapa L.) survival in the winter in Northwest China. Cold acclimation (CA) can alleviate the root damage caused by freezing stress. To acknowledge the molecular mechanisms of freezing tolerance in winter turnip rape, two Brassica rapa genotypes, freezing stressed after the induction of cold acclimation, were used to compare the proteomic profiles of roots by isobaric tags for relative and absolute quantification (iTRAQ). Under freezing stress (-4 °C) for 8 h, 139 and 96 differentially abundant proteins (DAPs) were identified in the roots of "Longyou7" (freezing-tolerant) and "Tianyou4" (freezing-sensitive), respectively. Among these DAPs, 91 and 48 proteins were up- and down-accumulated in "Longyou7", respectively, and 46 and 50 proteins were up- and down-accumulated in "Tianyou4", respectively. Under freezing stress, 174 DAPs of two varieties were identified, including 9 proteins related to ribosome, 19 DAPs related to the biosynthesis of secondary metabolites (e.g., phenylpropanoid and the lignin pathway), and 22 down-accumulated DAPs enriched in oxidative phosphorylation, the pentose phosphate pathway, fructose and mannose metabolism, alpha-linolenic acid metabolism, carbon fixation in photosynthetic organisms, ascorbate and aldarate metabolism. The expressional pattern of the genes encoding the 15 significant DAPs were consistent with the iTRAQ data. This work indicates that protein biosynthesis, lignin synthesis, the reduction of energy consumption and a higher linolenic acid content contribute to the freezing tolerance of winter turnip rape. Functional analyses of these DAPs would be helpful in dissecting the molecular mechanisms of the stress responses in B. rapa.

iTRAQ-Based Quantitative Proteome Revealed Metabolic Changes in Winter Turnip Rape (Brassica rapa L.) under Cold Stress.

Winter turnip rape (Brassica rapa L.) is a large-scale winter-only oil crop cultivated in Northwest China. However, its cold-resistant molecular mechanism remains inadequate. Studying the cold adaptation mechanisms of winter turnip rape based on the proteomic technique of isobaric tags for relative and absolute quantification (iTRAQ) offers a solution to this problem. Under cold stress (-4 °C for eight hours), 51 and 94 differently accumulated proteins (DAPs) in Longyou 7 (cold-tolerant) and Tianyou 4 (cold-sensitive) were identified, respectively. These DAPs were classified into 38 gene ontology (GO) term categories, such as metabolic process, cellular process, catalytic activity, and binding. The 142 DAPs identified between the two cold-stressed cultivars were classified into 40 GO terms, including cellular process, metabolic process, cell, catalytic activity, and binding. Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that the DAPs participated in 10 pathways. The abundance of most protein functions in ribosomes, carbon metabolism, photosynthesis, and energy metabolism including the citrate cycle, pentose phosphate pathway, and glyoxylate and dicarboxylate metabolism decreased, and the proteins that participate in photosynthesis⁻antenna and isoflavonoid biosynthesis increased in cold-stressed Longyou 7 compared with those in cold-stressed Tianyou 4. The expression pattern of genes encoding the 10 significant DAPs was consistent with the iTRAQ data. This study provides new information on the proteomic differences between the leaves of Longyou 7 and Tianyou 4 plants and explains the possible molecular mechanisms of cold-stress adaptation in B. rapa.

Identification of cold stress responsive microRNAs in two winter turnip rape (Brassica rapa L.) by high throughput sequencing.

BACKGROUND: Low temperature is a major abiotic stress affecting the production of rapeseed in China by impeding plant growth and development. A comprehensive knowledge of small-RNA expression pattern in Brassica rapa under cold stress could improve our knowledge of microRNA-mediated stress responses. RESULTS: A total of 353 cold-responsive miRNAs, 84 putative novel and 269 conserved miRNAs, were identified from the leaves and roots of two winter turnip rape varieties 'Longyou 7' (cold-tolerant) and 'Tianyou 4' (cold-sensitive), which were stressed under - 4 °C for 8 h. Eight conserved (miR166h-3p-1, miR398b-3p, miR398b-3p-1, miR408d, miR156a-5p, miR396h, miR845a-1, miR166u) and two novel miRNAs (Bra-novel-miR3153-5p and Bra-novel-miR3172-5p) were differentially expressed in leaves of 'Longyou 7' under cold stress. Bra-novel-miR3936-5p was up-regulated in roots of 'Longyou 7' under cold stress. Four and five conserved miRNAs were differentially expressed in leaves and roots of 'Tianyou 4' after cold stress. Besides, we found two conserved miRNAs (miR319e and miR166m-2) were down-regulated in non-stressed roots of 'Longyou 7' compared with 'Tianyou 4'. After cold stress, we found two and eight miRNAs were differentially expressed in leaves and roots of 'Longyou 7' compared with 'Tianyou 4'. The differentially expressed miRNAs between two cultivars under cold stress include novel miRNAs and the members of the miR166 and miR319 families. A total of 211 target genes for 15 known miRNAs and two novel miRNAs were predicted by bioinformatic analysis, mainly involved in metabolic processes and stress responses. Five differentially expressed miRNAs and predicted target genes were confirmed by quantitative reverse transcription PCR, and the expressional changes of target genes were negatively correlated to differentially expressed miRNAs. Our data indicated that some candidate miRNAs (e.g., miR166e, miR319, and Bra-novel-miR3936-5p) may play important roles in plant response to cold stress. CONCLUSIONS: Our work indicates that miRNA and putative target genes mediated metabolic processes and stress responses are significant to cold tolerance in B. rapa.

Overexpression of a Functional Vicia sativa PCS1 Homolog Increases Cadmium Tolerance and Phytochelatins Synthesis in Arabidopsis.

Phytochelatins (PCs) catalyzed by phytochelatin synthases (PCS) are important for the detoxification of metals in plants and other living organisms. In this study, we isolated a PCS gene (VsPCS1) from Vicia sativa and investigated its role in regulating cadmium (Cd) tolerance. Expression of VsPCS1 was induced in roots of V. sativa under Cd stress. Analysis of subcellular localization showed that VsPCS1 was localized in the cytoplasm of mesophyll protoplasts of V. sativa. Overexpression of VsPCS1 (35S::VsPCS1, in wild-type background) in Arabidopsis thaliana could complement the defects of Cd tolerance of AtPCS1-deficent mutant (atpcs1). Compared with atpcs1 mutants, 35S::VsPCS1/atpcs1 (in AtPCS1-deficent mutant background) transgenic plants significantly lowered Cd-fluorescence intensity in mesophyll cytoplasm, accompanied with enhanced Cd-fluorescence intensity in the vacuoles, demonstrating that the increased Cd tolerance may be attributed to the increased PC-based sequestration of Cd into the vacuole. Furthermore, overexpressing VsPCS1 could enhance the Cd tolerance in 35S::VsPCS1, but have no effect on Cd accumulation and distribution, showing the same level of Cd-fluorescence intensity between 35S::VsPCS1 and wild-type (WT) plants. Further analysis indicated this increased tolerance in 35S::VsPCS1 was possibly due to the increased PCs-chelated Cd in cytosol. Taken together, a functional PCS1 homolog from V. sativa was identified, which hold a strong catalyzed property for the synthesis of high-order PCs that retained Cd in the cytosol rather the vacuole. These findings enrich the original model of Cd detoxification mediated by PCS in higher plants.

Cd-induced oxidative stress and lignification in the roots of two Vicia sativa L. varieties with different Cd tolerances.

We examined the effects of Cd on growth, lipid peroxidation, reactive oxygen species (ROS) accumulation, antioxidant enzymatic activity, and lignin content in the roots of two varieties of Vicia sativa. Treatment with Cd decreased plant growth and increased ROS and lipid peroxidation levels to a greater extent in the Cd-sensitive variety ZM than in the Cd-tolerant variety L3. Most hydrogen peroxide (H2O2) and superoxide anion (O2(•-)) were accumulated in the cell walls and extracellular spaces in response to Cd treatments. Chemical assays and experiments using inhibitors showed that larger increases in H2O2 and O2(•-) production in ZM than in L3 were probably attributed to elevated Cd-induced nicotinamide adenine dinucleotide-peroxidase (NADH-POD) activity. Cd treatment increased the accumulation of lignin and the guaiacol peroxidase (GPOD) activities in the apoplast more significantly in ZM root than in L3. Howerver, root laccase activity was higher in L3 than in ZM. Thus Cd toxicity induced significant lignification in the roots of V. sativa, and increases in H2O2 accumulation and apoplastic GPOD activity were likely responsible for this effect.

Exogenous application of salicylic acid alleviates cadmium toxicity and reduces hydrogen peroxide accumulation in root apoplasts of Phaseolus aureus and Vicia sativa.

We examined ameliorative effects of salicylic acid (SA) on two cadmium (Cd)-stressed legume crops with different Cd tolerances, viz. Phaseolus aureus (Cd sensitive) and Vicia sativa (Cd tolerant). Cd at 50 µM significantly increased the production of hydrogen peroxide (H(2)O(2)) and superoxide anion (O(2)(·-) ) in root apoplasts of P. aureus and V. sativa. When comparing the two species, we determined that Cd-induced production of H(2)O(2) and O(2)(·-) was more pronounced in P. aureus root apoplasts than in V. sativa root apoplasts. V. sativa had higher activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) than P. aureus in root symplasts and apoplasts. Seed-soaking pretreatment with 100 µM SA decreased Cd-induced production of H(2)O(2) and O(2)(·-) in apoplasts of both species, and increased activities of symplastic and apoplastic SOD, symplastic APX, and apoplastic CAT under Cd stress. Hence, SA-induced Cd tolerances in P. aureus and V. sativa are likely associated with increases in symplastic and apoplastic antioxidant enzyme activities.

Excess copper induces production of hydrogen peroxide in the leaf of Elsholtzia haichowensis through apoplastic and symplastic CuZn-superoxide dismutase.

The effects of excess copper (Cu) on the production of reactive oxygen species (ROS) and antioxidant enzyme activities in leaves of the Cu-accumulator Elsholtzia haichowensis Sun were investigated. The addition of 100 microM of copper significantly increased the accumulation of hydrogen peroxide (H(2)O(2)) and the activities of superoxide dismutase (SOD) and other antioxidant enzymes. The increase in SOD activity was attributable to an increase in apoplastic and symplastic copper-zinc superoxide dismutase (CuZn-SOD) activity. Induction of CuZn-SOD proteins was demonstrated by immunoblot analysis. This study also provides the first cytochemical evidence of an accumulation of superoxide anion in the chloroplasts of mesophyll cells, and H(2)O(2) in the mesophyll cell walls and extracellular space, as a consequence of Cu treatment. Experiments with diphenyleneiodonium as an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and N-N-diethyldithiocarbamate as an inhibitor of SOD showed that the source of H(2)O(2) in the cell wall could be, in part, NADPH oxidase. Apoplastic guaiacol peroxidase (POD) and symplastic ascorbate peroxidase (APX) activities were induced in leaves of E. haichowensis with 100 microM Cu, suggesting that apoplastic POD and symplastic APX may be important in avoiding the buildup of toxic H(2)O(2) concentrations.

Cadmium-induced accumulation of hydrogen peroxide in the leaf apoplast of Phaseolus aureus and Vicia sativa and the roles of different antioxidant enzymes.

The effects of cadmium (Cd) on the accumulation of hydrogen peroxide (H(2)O(2)) and superoxide anion (O(2)(-)) in leaves of Phaseolus aureus and Vicia sativa were investigated. Cadmium at 100 microM significantly increased the production of O(2)(-) and H(2)O(2), as well as the activities of plasma membrane-bound nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and the symplastic and apoplastic activities of superoxide dismutase and ascorbate peroxidase in the leaves of both species. Apoplastic guaiacol peroxidase activity was significantly induced in the leaves of both species, particularly in P. aureus exposed to 100 microM Cd. Experiments with diphenylene iodonium as an inhibitor of NADPH oxidase and NaN(3) as an inhibitor of peroxidase showed that the majority of Cd-induced reactive oxygen species production in the leaves of both species may involve plasma membrane-bound NADPH oxidase and apoplastic peroxidase. Compared to V. sativa, increases in Cd-induced production of O(2)(-) and H(2)O(2) and activities of NADPH oxidase and apoplastic peroxidase were more pronounced in P. aureus. In contrast, V. sativa had higher leaf symplastic superoxide dismutase and ascorbate peroxidase activities than P. aureus. The results indicated that V. sativa was more tolerant to Cd than P. aureus.