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BackgroundLead (Pb) and mercury (Hg) are persistent hazardous metals in industrially polluted soils which can be toxic in low quantities. Metal toxicity can cause changes at cellular and molecular level which should be studied for better understanding of tolerance mechanism in plants. Soybean (Glycine max L.) is an important oilseed crop of the world including India. Indian soils growing soybean are often contaminated by Pb and Hg. The aim of this study was to explore how soybean root nodule responds to Pb and Hg through proteomic and ecophysiological alterations in order to enhance tolerance to metal stress.ResultsSoybean plants were exposed to Pb (30 ppm PbCl2) and Hg (0.5 ppm HgCl2) to study histological, histochemical, biochemical and molecular response of N2-fixing symbiotic nodules. Both Pb and Hg treatment increased the level of oxidative stress in leaves and nodules. Chlorosis in leaves and morphological/anatomical changes in nodules were observed. Activities of ascorbate peroxidase, glutathione reductase and catalase were also modulated. Significant changes were observed in abundance of 76 proteins by Pb and Hg. Pb and Hg influenced abundance of 33 proteins (17 up and 16 down) and 43 proteins (33 up and 10 down), respectively. MS/MS ion search identified 55 proteins which were functionally associated with numerous cellular functions. Six crucial proteins namely catalase (CAT), allene oxide synthase (AOS), glutathione S-transferase (GST), calcineurin B like (CBL), calmodulin like (CML) and rapid alkalinisation factor (RAF) were selected for transcript abundance estimation. The qRT-PCR based real time expression exhibited a positive correlation with proteomics expression except for GST and RAF.ConclusionSoybean root nodule responds to metal stress by increased abundance of defence, development and repair related proteins. An efficient proteomic modulation might lead to metal-induced stress tolerance in N2-fixing nodules. Although concentrations of Pb and Hg used in the study cannot be considered equimolar, yet Hg seems to induce more changes in nodule proteomic profile, and higher damage to both bacteroides and root anatomy.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1499-7) contains supplementary material, which is available to authorized users.

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Isolation of high-quality RNA from weed plants such as is a difficult task due to the hindrance caused by numerous secondary metabolites. Such metabolites not only affect the quality and yield of RNA, but also limit the quality of downstream applications. Therefore, the present study was undertaken to design a protocol for yielding RNA with better quality and quantity from leaf which could be suitable for functional genomics. To achieve the objective, four different important RNA extraction protocols, viz. acid guanidinium thiocyanate-phenol-chloroform, phenol-LiCl precipitation, TRIzol, and PVP-ethanol were tested. The PVP-ethanol method proved to be best among the tested protocols. This method was further modified for obtaining improved quality and yield of RNA. The modified method successfully enhanced the yield of RNA from 280 to 334 µg g fresh weight. The absorbance ratio (/) was in the purity range of 1.9 that indicated the good quality of RNA. To prove the feasibility of the extracted RNA in PCR-based cDNA synthesis, actin transcripts were targeted and successfully amplified using suitable primers. The improved protocol thus not only improved the yield and quality of RNA, but also gave better results in reverse transcriptase PCR.

The phytotoxic actions of two herbicides, glyphosate and amitrole, were compared using corn seedlings. Six days after treatment with amitrole at 1.68 kg/ha, plant heights, leaf lengths, and shoot fresh weights were reduced 20, 20, and 25% respectively, whereas with glyphosate applied at 1.12 kg/ha the inhibition was 62, 60, and 84% respectively. The lowest concentration of glyphosate (0.28 kg/ha) inhibited plant growth more than the inhibition caused by the highest concentration of amitrole (3.36 kg/ha). A purple colour appeared on the whole shoot of plants treated with glyphosate, and with amitrole, only the new growth of shoots was white. Both treatments reduced chlorophyll levels and the amitrole treatment reduced the carotenoid levels more than the chlorophyll levels. The root growth of plants treated with 1.12 kg/ha glyphosate was inhibited more than 80%, whereas with amitrole at 1.68 kg/ha, the inhibition was 40%. Within 6 h glyphosate severely reduced the rate of root respiration and 12 h after treatment respiration was reduced more than 70%. This rapid effect on root respiration was not observed with amitrole, and even 48 h after treatment, the decrease in respiration was less than 30%. We conclude that the primary site of action of glyphosate in corn seedlings is in the roots whereas the effect of amitrole is in the shoot.

Alcoholic extract of Hippophae rhamnoides, RH-3, reported to render >80% survival against lethal whole body Co-60-gamma irradiation (10 Gy) in mice, was investigated for its immunostimulatory effects. In comparison with un-irradiated control, whole body irradiation did not reduce peritoneal macrophage counts at 24 h post-irradiation. RH-3 treatment (30 mg kg(-1) body weight) alone or 30 min before whole-body irradiation enhanced viable counts of macrophages significantly (P< or =0.05) compared with both un-irradiated control and irradiated groups. Whole-body irradiation reduced the number of viable splenocytes significantly (P<0.05) compared with un-irradiated control at 24 h post-irradiation. RH-3 treatment alone or before whole-body irradiation appreciably countered radiation-induced decrease in splenocyte count. 3H-thymidine uptake method revealed that whole-body irradiation reduced splenocyte proliferation significantly (159 +/- 45 counts min(-1)/10(6) cells; P< or =0.05) in comparison with control (607 +/- 142 counts min(-1)) at 24 h after irradiation but RH3 treatment before irradiation reduced the steep decrease and maintained it as 444+/-153 counts min(-1). After whole-body irradiation, the ratio of spleen weight/mouse weight decreased to 1.5 +/- 04 compared with 2.9 +/- 0.32 in un-irradiated control at 24 h post-irradiation. Similarly, total protein content in splenocytes also decreased to 48 +/- 6 microg/10(6) cells in comparison with 368 +/- 16 microg/10(6) cells of un-irradiated control. RH-3 treatment before irradiation countered radiation-induced decrease in both spleen weight/mouse weight ratio (4.0 +/- 0.35) and total protein content (360 +/- 13 mug/10(6) splenocytes). In the supernatant of peritoneal macrophage cultures exposed to 2 Gy Co-60-gamma radiation ex-vivo, the total nitrite content was enhanced significantly (P<0.05) to 5.72 +/- 0.09 microM in comparison with un-irradiated control (1.64 +/- 0.09 microM). RH-3 treatment (30 microg mL(-1)) before irradiation reduced total nitrite significantly (0.93 +/- 0.3; P< or =0.05) in comparison with irradiated control group. At 24 h after whole body irradiation, the CD4+/CD8+ ratio reduced to 1.5 in comparison with un-irradiated control (1.9) but RH-3 treatment before irradiation restored the ratio to 2.1. These findings explicitly reveal the immunostimulatory activity of RH-3, which may play an important role in the manifestation of its radioprotective efficacy.

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