DataSheet_1_Soil applied silicon and manganese combined with foliar application of 5-aminolevulinic acid mediate photosynthetic recovery in Cd-stressed Salvia miltiorrhiza by regulating Cd-transporter genes.doc

Salvia miltiorrhiza is an important medicinal plant that experiences significant growth and biomass losses when cultivated on cadmium (Cd) contaminated soils. High Cd accumulation in plant tissues also increases the risk of metal entry into the food chain. In this study, we proposed that Cd accumulation in S. miltiorrhiza can be restricted through plant growth regulators and nutrient management. Therefore, S. miltiorrhiza seedlings were transplanted into mixed nutrient soil for two weeks, then treated with 30 mg kg-1 CdCl2, 200 mg kg-1 Na2SiO3·9H2O, and 100 mg kg-1 MnSO4, and simultaneously sprayed with 10 mg L-1 ALA on the leaves one week later. This study showed that elevated Cd accumulation significantly reduced plant growth and biomass. This growth inhibition damaged photosynthetic machinery and impaired carbon assimilation. In contrast, 5-aminolevulinic acid (ALA) significantly promoted the biomass of S. miltiorrhiza, and the dry weight of plants treated with ALA combined with manganese (Mn)/silicon (Si) increased by 42% and 55% as compared with Cd+Mn and Cd+Si treatments. Exogenously applied ALA and Si/Mn significantly activated antioxidant enzymes and promoted the growth recovery of S. miltiorrhiza. Further, exogenous ALA also reduced the Cd concentration in S. miltiorrhiza, especially when combined with Si. Compared with the Cd+Si treatment, the Cd+Si+ALA treatment reduced the Cd concentration in roots and leaves by 59% and 60%, respectively. Gene expression analysis suggested that ALA and Si significantly up-regulated genes associated with Cd transport. Other genes related to heavy metal tolerance mechanisms are also regulated to cope with heavy metal stress. These results indicated that the combined action of ALA and Si/Mn could reduce Cd-toxicity by increasing chlorophyll content and changing oxidative stress and can also affect Cd accumulation by regulating gene expression.</p

Additional file 1: Table S1. of Comparative transcriptome profiling of two Brassica napus cultivars under chromium toxicity and its alleviation by reduced glutathione

DGE generated sequence details (%) under different treatments of chromium (Cr) and reduced glutathione (GSH) in two cultivars of Brassica napus. Table S2 TPA generated sequence read assembly and number of contigs and unigenes in two cultivars of Brassica napus (ZS 758 and Zheda 622). Table S3 Oligonucleotide sequences used in RT-PCR analysis. Table S4 Top 30 comparative transcription factors (TFs) (selected on the basis of RPKM value) under the control between ZS 758 and Zheda 622. While ZS 758 taken as a standard. Table S5 Top 30 comparative transcription factors (TFs) (selected on the basis of RPKM value) under the control in ZS 758 and Zheda 622. While Zheda 622 taken as a standard. Table S6 Top 30 comparative transcription factors (TFs) (selected on the basis of RPKM value) under the Cr 400 μM between ZS758 and Zheda 622. While ZS 758 taken as a standard. Table S7 Top 30 comparative transcription factors (TFs) (selected on the basis of RPKM value) under the Cr 400 μM between ZS 758 and Zheda 622. While Zheda 622 taken as a standard. Table S8 Top 30 comparative transcription factors (TFs) (selected on the basis of RPKM value) under the Cr 400 μM + GSH 1 mM between ZS 758 and Zheda 622. While ZS 758 taken as a standard. Table S9 Top 30 comparative transcription factors (TFs) (selected on the basis of RPKM value) under the Cr 400 μM + GSH 1 mM between ZS 758 and Zheda 622. While Zheda 622 taken as a standard. (DOCX 59 kb

Additional file 2: Figure S1. of Comparative transcriptome profiling of two Brassica napus cultivars under chromium toxicity and its alleviation by reduced glutathione

Pie-chart shows the E-value, similarity and species distribution of Brassica napus L. to other plant species. Figure S2 Scattered plots shows the pair wise comparison of differentially expressed genes, ZS 758 considered as a control and Zheda 622 as a treatment under the different concentrations. In Figure, (A) represents the control, (B) as Cr 400 μM, and (C) represents the Cr 400 μM + GSH 1 mM. Figure S3 Pie-chart (A) shows the number of up-regulated and down-regulated DEGs in ZS 758/Zheda 622 and (B) Zheda 622/ZS 758, respectively. Figure S4 HemI hierarchal cluster shows stress responsive relatively differentially expressed genes (DEGs). Diagram (A) shows the DEGs among treatments i.e. Ck, Cr 400 μM, and Cr 400 μM + 1 mM GSH and (B) between cultivars such as ZS 758 and Zheda 622. Figure S5 Shows the comparative gene ontology functional classification (WEGO) by transcriptome profile analysis in ZS 758 vs Zheda 622. Former cultivar ZS 758 was taken as a control while later cultivar Zheda 622 as a treatment. Figure S6 Diagram showing the transcription factors (TFs) between cultivars and among the treatments. Numbers of each circle show the number of TFs that are uniquely (inside of non overlapping part) or commonly (inside of overlapping part) regulated. (A) diagram shows the TFs between cultivars and (B) shows the TFs among the treatments. (DOCX 1005 kb

Differentially regulated <i>Brassica napus</i> proteins due to Cd²⁺ and/or ALA treatments.

<p><b>(A)</b> Functional distribution of the 16 up-regulated proteins that were differentially produced in response to Cd^<b>2+</b> and/or ALA treatments.<b>(B)</b> Functional distribution of the 18 down-regulated proteins that were differentially produced in response to Cd^<b>2+</b> and/or ALA treatments.</p

Effects of different treatments of 5-aminolevulinic acid (ALA) (mg/L) and cadmium (Cd²⁺) (μM) on (A) osmotic potential (MPa), (B) relative water contents (%), (C) soluble sugar contents (mg g^-1 FW), (D) free amino acid contents (mg g^-1 FW) and (E) proline contents (mg g^-1 FW) in the leaves of <i>Brassica napus</i> cv. ZS758.

<p>Values are the means ± SD of three replications. Variants possessing the same letter are not statistically significant at P < 0.05.</p

Down-regulated proteins in the leaves of <i>Brassica napus</i> cv. ZS 758 under 500 μM Cd²⁺ alone as compared to control (with A lettering), under the combine treatment of ALA and 500 μM Cd²⁺ as compared to Cd²⁺ alone (with E lettering) and under the combine treatment of ALA and 500 μM Cd²⁺ (with C lettering) as compared to control conditions.

<p>Down-regulated proteins in the leaves of <i>Brassica napus</i> cv. ZS 758 under 500 μM Cd²⁺ alone as compared to control (with A lettering), under the combine treatment of ALA and 500 μM Cd²⁺ as compared to Cd²⁺ alone (with E lettering) and under the combine treatment of ALA and 500 μM Cd²⁺ (with C lettering) as compared to control conditions.</p

Representative 2-DE maps comparing <i>Brassica napus</i> cv. ZS 758 leaf proteins isolated from normal (a, d) and under 500 μM Cd²⁺ alone (b, e) and under combine application of ALA and 500 μM Cd²⁺ (c, f) along with protein marker.

<p>Differentially accumulated protein spots are indicated by green sashes. Twenty-nine down-regulated spots (C01-C29) and twenty-four up-regulated spots (D01-D24) under control conditions (a, d); twenty-seven down-regulated spots (A01-A27) and fifty-three up-regulated spots (B01-B53) under Cd^<b>2+</b> alone (b, e); fifty-three down-regulated spots (E01-E53) and twenty-two up-regulated spots (F01-F22) under the combine treatment of ALA and Cd^<b>2+</b> (c, f) are indicated on the map.</p

Effects of different treatments of 5-aminolevulinic acid (ALA) (mg/l) and cadmium (Cd²⁺) (μM) on the expression of ascorbate peroxidase gene (APX), catalase gene (CAT), superoxide dismutase gene (SOD), peroxidase gene (POD) and glutathione reductase gene (GR) in the leaves of <i>Brassica napus</i> cv. ZS758.

<p>The RT-qPCR analysis was performed to examine mRNA levels of five antioxidant enzyme genes in plants treated with CK, 100 μM Cd^<b>2+</b> alone, 500 μM Cd^<b>2+</b> alone, 25 mg/l ALA alone, 100 μM Cd^<b>2+</b> + 25 mg/l ALA and 500 μM Cd^<b>2+</b> + 25 mg/l ALA. Values are the means ± SD of three replications. Means followed by the same letter did not significantly differ at P<0.05 according to Duncan’s multiple range test.</p

Up-regulated proteins in the leaves of <i>Brassica napus</i> cv. ZS 758 under 500 μM Cd²⁺ alone as compared to control (with B lettering), under the combine treatment of ALA and 500 μM Cd²⁺ as compared to Cd²⁺ alone (with F lettering) and under the combine treatment of ALA and 500 μM Cd²⁺ (with D lettering) as compared to control conditions.

<p>Up-regulated proteins in the leaves of <i>Brassica napus</i> cv. ZS 758 under 500 μM Cd²⁺ alone as compared to control (with B lettering), under the combine treatment of ALA and 500 μM Cd²⁺ as compared to Cd²⁺ alone (with F lettering) and under the combine treatment of ALA and 500 μM Cd²⁺ (with D lettering) as compared to control conditions.</p

DataSheet_1_Bio-fabrication of Zinc Oxide nanoparticles to rescue Mung Bean against Cercospora leaf spot disease.docx

Plant disease management using nanotechnology is evolving continuously across the world. The purpose of this study was to determine the effect of different concentrations of green synthesized zinc oxide nanoparticles (ZnO NPs) using Trachyspermum ammi seed extract on Cercospora leaf spot disease in mung bean plants under in-vitro and in-planta conditions. Additionally, the effects on mung bean agronomic and physiological parameters were also assessed. The green synthesized ZnO NPs were characterized using UV-visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and Scanning electron microscopy (SEM). Green synthesized NPs were tested for their ability to inhibit fungal growth at five different concentrations under in-vitro experiment. After 7 days of inoculation, ZnO NPs (1200 ppm) inhibited mycelial growth substantially (89.86% ± 0.70). The in-planta experiment showed statistically significant result of disease control (30% ± 11.54) in response to 1200 ppm ZnO NPs. The same treatment showed statistically significant improvements in shoot length, root length, number of leaves, number of pods, shoot fresh weight (28.62%), shoot dry weight (85.18%), root fresh weight (38.88%), and root dry weight (38.88%) compared to the control. Our findings show that green synthesized ZnO NPs can control Cercospora canescens in mung bean, pointing to their use in plant disease control and growth enhancement.</p