Transcriptome analysis reveals the tolerant mechanisms to cobalt and copper in barley.

Cobalt (Co) and copper (Cu) co-exist commonly in the contaminated soils and at excessive levels, they are toxic to plants. However, their joint effect and possible interaction have not been fully addressed. In this work, a hydroponic experiment was performed to investigate the combined effects of Co and Cu on two barley genotypes at transcriptional level by RNA-seq analysis. The results identified 358 genes inclusively expressed in both genotypes under single and combined treatments of Co and Cu, with most of them being related to metal transport, stress response and transcription factor. The combined treatment induced more differently expressed genes (DEGs) than the single treatment, with Yan66, a metal tolerant genotype having more DEGs than Ea52, a sensitive genotype. The pathways associated with anthocyanin biosynthesis, MAPK signaling, glutathione biosynthesis, phenylalanine metabolism, photosynthesis, arginin biosynthesis, fatty acid elongation, and plant hormone signal transduction biosynthesis were induced and inhibited in Yan66 and Ea52, respectively. Furthermore, flavonoid biosynthesis was much more largely enhanced and accordingly more free flavonoid components (naringin, narirutin and neohesperidin) were accumulated in Yan66 than in Ea52. It may be suggested that high tolerance to both Co and Cu in Yan66 is attributed to its high gene regulatory ability.

Physiological and molecular mechanisms of cobalt and copper interaction in causing phyto-toxicity to two barley genotypes differing in Co tolerance.

The combined effects of cobalt (Co) and copper (Cu) in their toxicity to plants is poorly studied although these two metals co-exist commonly in soil. In this study, a hydroponic experiment was carried out to investigate the effect of longer exposure of two barley genotypes differing in Co tolerance to the combined Co and Cu stress. The results confirmed the previous findings that Co accumulation in plant tissues was reduced by Cu presence, while Cu accumulation was stimulated by Co presence. Moreover, both single and combined treatments of Co and Cu reduced the mineral (Mn, Zn and K) uptake. Co and Cu applied alone or in combination at rate of 50 µM resulted in the significant reduction of plant growth and increase of oxidative stress (ROS and MDA), and meanwhile the capacity of scavenging active oxygen species (AOS) was enhanced, reflected by increased phytochelatin (PC) and glutathione (GSH and GSSG) content, as well as expression of the related genes (HvPCS1 and HvGR1). Yan66, a Co tolerant genotype was less affected in oxidative stress, and had higher AOS scavenging capacity in comparison with Ea52, a Co sensitive one. Among three HvSOD isoforms, only HvFeSOD expression was up-regulated in the combined treatment relative to control as well as the treatment of Co or Cu alone, while HvCuZnSOD and HvMnSOD were down-regulated and unaffected, respectively. In addition, the expressions of metal transporter genes (HvHMA2, HvHMA3 and HvHMA5) varied with genotype and metal treatments, with the extent being greater in Yan66 on the whole. The results suggest that upon longer exposure to the combined stress of Co and Cu, the greater phyto-toxicity than each element alone is associated with more Cu accumulation stimulated by Co and that, the higher regulation of transporter genes observed in Yan66 could in part explain for its higher metal tolerance ability.

Copper alleviates cobalt toxicity in barley by antagonistic interaction of the two metals.

Cobalt (Co) commonly co-exists with copper (Cu) in natural soils, but the information about their combined effects on plants is poorly available. In this study, we hydroponically investigated the combined effects of Co and Cu on two barley genotypes differing in Co toxicity tolerance to reveal the interaction pattern of these two metals. The results showed that single treatment of Co or Cu at the dose of 100 µM led to a significant decrease of growth and photosynthetic rate, and a significant increase of lipid peroxidation, ROS radicals as well as anti-oxidative enzyme (SOD, CAT and GR) activities and glutathione content, with the extent of effect being less in Yan66 than Ea52. The combined treatment of Co and Cu alleviated the toxicity of both metals in comparison with each metal treatment alone, as reflected by improved growth and photosynthesis, and much slight oxidative stress. The alleviation of metal toxicity upon combined treatment is mainly attributed to a drastic reduction of Co uptake and its translocation from roots to shoots. It may be suggested that interaction of Co and Cu on their uptake and movement in plants is antagonistic.

Phosphate alleviates arsenate toxicity by altering expression of phosphate transporters in the tolerant barley genotypes.

The contribution of the phosphate transporters (PHTs) in uptake of arsenate (As5+) and phosphate (P) is a widely recognized mechanism. Here we investigated how P regulates the uptake of As5+ and the subsequent effects on growth and relative expression of PHTs. The study was conducted on 3 barley genotypes differing in As tolerance (ZDB160, As-tolerant; ZDB115, moderately tolerant; ZDB475, As-sensitive) using a hydroponic experiment. There were 3 As5+ (0, 10 and 100µM) and 3P (0, 50 and 500µM) levels. The results showed that the negative effect of As stress on plant growth, photosynthesis and cell ultra-structure is As dose and barley genotype dependent, confirming the distinctly genotypic difference in As tolerance. As uptake and accumulation in plant tissues are closely associated with inhibited extent of growth and photosynthesis, with the tolerant genotype ZDB160 having lower As content than other two genotypes. The toxic effect caused by As stress could be alleviated by P addition, mainly due to reduced As uptake. Moreover, the tolerant genotype showed relatively lower expression PHTs than sensitive ones upon exposure to both As stress and P addition, suggesting regulation of PHTs expression is a major mechanism for relative uptake of As and P, in subsequence affecting As tolerance. Moreover, among 6 PHTs examined in this study, the expressions of PHT1.3, PHT1.4 and PHT1.6 showed the marked difference among the three barley genotypes in responses to As stress and P addition, indicating further research on the contribution of phosphate transporters to As and P uptake should be focused on these PHTs.

Alleviating effects of calcium on cobalt toxicity in two barley genotypes differing in cobalt tolerance.

Cobalt (Co) contamination in soils is becoming a severe issue in environment safety and crop production. Calcium (Ca), as a macro-nutrient element, shows the antagonism with many divalent heavy metals and the capacity of alleviating oxidative stress in plants. In this study, the protective role of Ca in alleviating Co stress was hydroponically investigated using two barley genotypes differing in Co toxicity tolerance. Barley seedlings exposed to 100µM Co showed the significant reduction in growth and photosynthetic rate, and the dramatic increase in the contents of reactive oxygen species (ROS), malondialdehyde (MDA), reduced glutathione (GSH) and oxidized glutathione (GSSG), and the activities of anti-oxidative enzymes, with Ea52 (Co-sensitive) being much more affected than Yan66 (Co-tolerant). Addition of Ca in growth medium alleviated Co toxicity by reducing Co uptake and enhancing the antioxidant capacity. The effect of Ca in alleviating Co toxicity was much greater in Yan66 than in Ea52. The results indicate that the alleviation of Co toxicity in barley plants by Ca is attributed to the reduced Co uptake and enhanced antioxidant capacity.

High accumulation of phenolics and amino acids confers tolerance to the combined stress of cobalt and copper in barley (Hordeum vulagare).

Cobalt (Co) and copper (Cu) co-exist in the metal contaminated soils and cause the serious toxicity to crops, while their interactive effect on plant growth and development is still poorly understood. In this work, a hydroponic experiment was carried out to reveal the interactive effect of Co and Cu on photosynthesis and metabolite profiles of two barley genotypes differing in metal tolerance. The results showed that both single and combined treatments of Co and Cu caused a significant reduction in chlorophyll content and photosynthetic rate of the two barley (Hordeum vulgare) genotypes, with the effect being greater for the combined treatment and the sensitive genotype (Ea52) being more affected than the tolerant genotype (Yan66). Compared to Cu or Co treatment alone, the combined treatment significantly increased the levels of phenolic components, including cinnamic derivatives (caffeic, chlorogenic, ferullic, p-coumaric); benzoic derivatives (p-hydroxybenzoic, vanillic, syringic, sallicilic, protocatechuic acid) as well as free amino acids, with Yan66 having more accumulation than Ea52. Meanwhile, under the combined treatment, the phenylalanine ammonialyase-related gene (HvPAL) was highly regulated along with the genes involved in the synthesis of malate (HvMDH) and citrate (HvCSY), with Ya66 showing the higher expression of these genes than Ea52. It can be concluded that higher Cu and Co stress tolerance in Yan66 is attributed to more accumulation of the metabolites including phenolics and amino acids.

Transcriptomic comparison of two barley genotypes differing in arsenic tolerance exposed to arsenate and phosphate treatments.

Arsenic (As) is a ubiquitous metalloid and toxic to plants. Chemical similarity between arsenate and phosphate (P) indicates possible antagonism between them in uptake and transportation. However, there is little study to reveal the interaction of As and P at transcriptional level. In this study RNA-sequencing was conducted on the two barley genotypes differing in As tolerance. A total of 2942 differentially expressed genes (DEGs) were inclusively expressed in both genotypes under As (100 µM) and As (100 µM) + P (50 µM), and these DEGs included hormonal signaling, stress responsive, transport related and transcription factors. P addition in the culture solution inhibited the KEGG pathways related to ABC transporters, ether lipid metabolism, linolenic acid metabolism, endocytosis and RNA transport. ZDB160 had a higher expression of DEGs associated with hormone signaling, secondary metabolites and stress defense under P conditions compared to ZDB475, which might explain its tolerance mechanism to As under P condition. The abscisic acid, jasmonic acid and salicylic acid signaling pathways were also significantly regulated under As + P conditions, which may also account for genotypic differences. Finally we drew up a hypothetical model of high As + P stress tolerance mechanism in ZDB160. It may be concluded that ZDB160 achieves its tolerance to As under P by up-regulating P transporters, resulting in more P uptake and less As translocation. The identified candidate genes related to As + P tolerance may provide insights into understanding As tolerance under limited P conditions.

Elucidating the physiological and biochemical responses of different tobacco (Nicotiana tabacum) genotypes to lead toxicity.

In the present study, the effects of lead (Pb) uptake and toxicity were investigated in a hydroponic culture using 7 tobacco (Nicotiana tabacum L.) genotypes (Bina 1 [B1], Kutsaga Mammoth 10 [KM10], Nanjing 3 [N3], Kutsaga 35 [K35], Kutsaga E1 [KE1], Cocker 176 [C176], and Kutsaga RK6 [KRK6]) that differed in Pb tolerance. Lead was applied as a solution of Pb nitrate at concentrations of 0 µM, 10 µM, 250 µM, and 500 µM. After 4 wk of Pb treatment, tissue biomass and photosynthetic parameters were measured and elemental analysis was performed. The results showed decreases in growth and photosynthetic parameters with increases in Pb concentration compared with the control. The least reduction in the recorded physiological parameters was noted in K35, whereas the greatest reduction was observed in N3, which is an obvious indication of genotypic differences. Activities of peroxidase, catalase, and malondialdehyde increased significantly with increases in Pb concentration, with genotypes K35 and N3 showing the least and the greatest reduction, respectively. The results demonstrate the phototoxic nature of Pb on plants, and it can be concluded that in Pb-prone areas genotypes K35 and B1 can be used for cultivation because they can grow efficiently in the presence of high Pb concentrations while restricting Pb uptake in the aboveground parts, as seen by the higher Pb tolerance index. Environ Toxicol Chem 2017;36:175-181. © 2016 SETAC.

The effects of phosphate on arsenic uptake and toxicity alleviation in tobacco genotypes with differing arsenic tolerances.

Phosphate (PO4 (3-) ) has been reported to suppress arsenate (As(v) ) uptake in plants. However, its effects on controlling the availability of As(v) in tobacco genotypes with different arsenic (As) tolerances has not been fully explored. In the present study, the effects of PO4 (3-) on As(v) uptake were investigated in a hydroponic culture using 2 tobacco (Nicotiana tabacum) genotypes (ZY90 and FSMY) that differed in As(v) tolerance. A total of 9 treatment combinations comprising As(v) treatments of 0 µM, 10 µM, and 100 µM and PO4 (3-) treatments of 0 µM, 50 µM, and 500 µM were used. The results showed that ZY90 had greater reductions in leaf photosynthetic parameters, root and shoot dry weight, length, and nutrient content than did FSMY when exposed to As(v) stress. The addition of 500 µM external PO4 (3-) significantly suppressed As(v) (100 µM) uptake in both FSMY and ZY90, with the effect being more pronounced in FSMY. Greater PO4 (3-) uptake in plants significantly reduced the influx of As(v) , causing an increase in photosynthesis and nutrient uptake. Phosphate supply increased superoxide dismutase activity, catalase activity, and malondialdehyde content. The present study showed that PO4 (3-) is an effective competitive inhibitor of As(v) , and it can be effectively used to control As(v) accumulation in tobacco plants.