Melatonin mitigates cadmium and aluminium toxicity through modulation of antioxidant potential in Brassica napus L

• Melatonin has emerged as an essential molecule in plants, due to its role in defence against metal toxicity. Aluminium (Al) and cadmium (Cd) toxicity inhibit rapeseed seedling growth.
• In this study, we applied different doses of melatonin (50 and 100 µm ) to alleviate Al (25 µm ) and Cd (25 µm ) stress in rapeseed seedlings. Results show that Al and Cd caused toxicity in rapeseed seedling, as evidenced by a decrease in height, biomass and antioxidant enzyme activity.
• Melatonin increased the expression of melatonin biosynthesis‐related Brassica napus genes for caffeic acid O‐methyl transferase (BnCOMT) under Al and Cd stress. The genes BnCOMT‐1, BnCOMT‐5 and BnCOMT‐8 showed up‐regulated expression, while BnCOMT‐4 and BnCOMT‐6 were down‐regulated during incubation in water. Melatonin application increased the germination rate, shoot length, root length, fresh and dry weight of seedlings. Melatonin supplementation under Al and Cd stress increased superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, proline, chlorophyll and anthocyanin content, as well as photosynthesis rate. Both Cd and Al treatments significantly increased hydrogen peroxide and malondialdehyde levels in rapeseed seedlings, which were strictly counterbalanced by melatonin. Analysis of Cd and Al in different subcellular compartments showed that melatonin enhanced cell wall and soluble fractions, but reduced the vacuolar and organelle fractions in Al‐ and Cd‐treated seedlings.
• These results suggest that melatonin‐induced improvements in antioxidant potential, biomass, photosynthesis rate and successive Cd and Al sequestration play a pivotal role in plant tolerance to Al and Cd stress. This mechanism may have potential implications in safe food production.

     

Potential of efficient and resistant plant growth‐promoting rhizobacteria in lead uptake and plant defence stimulation in Lathyrus sativus under lead stress

• The ability of plant growth‐promoting rhizobacteria (PGPR) to enhance Lathyrus sativus tolerance to lead (Pb) stress was investigated.
• Ten consortia formed by mixing four efficient and Pb‐resistant PGPR strains were assessed for their beneficial effect in improving Pb (0.5 mM) uptake and in inducing the host defence system of L. sativus under hydroponic conditions based on various physiological and biochemical parameters.
• Lead stress significantly decreased shoot (SDW) and root (RDW) dry weight, but PGPR inoculation improved both dry weights, with highest increases in SDW and RDW of plants inoculated with I5 (R. leguminosarum (M5) + P. fluorescens (K23) + Luteibacter sp. + Variovorax sp.) and I9 (R. leguminosarum (M5) + Variovorax sp. + Luteibacter sp. + S. meliloti) by 151% and 94%, respectively. Additionally, inoculation significantly enhanced both chlorophyll and soluble sugar content, mainly in I5 inoculated leaves by 238% and 71%, respectively, despite the fact that Pb decreased these parameters. We also found that PGPR inoculation helps to reduce oxidative damage and enhances antioxidant enzyme activity, phenolic compound biosynthesis, carotenoids and proline content. PGPR inoculation increased Pb uptake in L. sativus, with highest increase in shoots of plants inoculated with I5 and I7, and in roots and nodules of plants inoculated with I1. Moreover, PGPR inoculation enhanced mineral homeostasis for Ca, Cu and Zn under Pb stress, mainly in plants inoculated with I1, I5, I7 and I9.
• Results of our study suggest the potential of efficient and Pb‐resistant PGPR in alleviating harmful effects of metal stress via activation of various defence mechanisms and enhancing Pb uptake that promotes tolerance of L. sativus to Pb stress.

     

Calcium acetate‐induced reduction of cadmium accumulation in Oryza sativa: Expression of auto‐inhibited calcium‐ATPase and cadmium transporters

• Calcium (Ca) signalling has an essential role in regulating plant responses to various abiotic stresses.
• This study applied Ca in various forms (Ca acetate and CaCl₂) and concentrations to reduce cadmium (Cd) concentration in rice and propose a possible mechanism through which Ca acts to control the Cd concentration in rice.
• The results showed that supplementation of Cd‐contaminated soil with Ca acetate reduced the Cd concentration in rice after exposure for 7 days in both hydroponic and soil conditions. The possible involvement of the auto‐inhibited Ca²⁺‐ATPase gene (ACA) might act to control the primary signal of the Cd stress response. The messages from ACA3 and ACA13 tended to up‐regulate the low‐affinity cation transporter (OsLCT1) and down‐regulate Cd uptake and the Cd translocation transporter, including the genes, natural resistance‐associated macrophage protein 5 (Nramp5) and Zn/Cd‐transporting ATPase 2 (HMA2), which resulted in a reduction in the Cd concentration in rice. After cultivation for 120 days, the application of Ca acetate into Cd‐contaminated soil inhibited Cd uptake of rice.
• Increasing the Ca acetate concentration in the soil lowered the Cd concentration in rice shoots and grains. Moreover, Ca acetate maintained rice productivity and quality whereas both aspects decreased under Cd stress.

     

Ultrastructural and metabolic disorders induced by short‐term cadmium exposure in Avicennia schaueriana plants and its excretion through leaf salt glands

• Environmental cadmium (Cd) sources have increased in mangrove sediments in recent decades, inducing cellular damage to many plants. Avicennia schaueriana is abundant in mangrove sites and has been subject to Cd contamination. The possible effects of Cd toxicity and the structural and physiological disturbances to this plant were studied. Can this plant express early cellular tolerance mechanisms to such metal contamination?
• Seedlings of A. schaueriana were collected from sites of their natural occurrence, placed in plastic pots containing nutrient solution for 60 days, and subsequently exposed to increasing Cd concentrations for 5 days under experimental conditions. The anatomical, ultrastructural and physiological changes induced by Cd were analysed.
• Cd accumulated mainly in the root system and in pneumatophores, stems and leaves, induced differential accumulation of mineral nutrients, but did not induce necrosis or changes in leaf anatomy. However, there was a decrease in starch grains and an increase in deposited electron‐dense material in the cortex and vascular bundles. Cd induced both increases in calcium (Ca) content in shoots and Ca oxalate crystal precipitation in leaf mesophyll and was detected in crystals and in the secretion of salt glands.
• Our observations and experimental results provide evidence of Cd tolerance in A. schaueriana. As a new feature, despite the clear cellular physiological disorders, this plant is able to eliminate Cd through leaf salt glands and immobilise it in Ca crystals, representing fast mechanisms for Cd exclusion and complexation in leaves in heavy metal coastal polluted marine ecosystems.

     

Characterisation of plant growth‐promoting rhizobacteria from rhizosphere soil of heat‐stressed and unstressed wheat and their use as bio‐inoculant

• High temperature induces several proteins in plants that enhance tolerance to high temperature shock. The fate of proteins synthesised in microbial cells or secreted into culture media by interacting microbes has not been fully elucidated. The present investigation aimed to characterise plant growth‐promoting rhizobacteria (PGPR) isolated from the rhizosphere of wheat genotypes (differing in tolerance to high temperature stress) and evaluate their performance as bioinoculant for use in wheat.
• Four bacterial strains, viz. Pseudomonas brassicacearum, Bacillus thuringiensis, Bacillus cereus strain W6 and Bacillus subtilis, were isolated from the rhizosphere of heat‐stressed and unstressed wheat genotypes. The wheat genotypes were exposed to high temperature stress at 45 °C for 10 days (3 h daily) at pre‐anthesis phase. Isolates were identified on the basis of morphology and biochemical characteristics, 16S rRNA gene sequencing and whole cell protein profiles. Results were further complemented by size exclusion chromatography (SEC) with fast protein liquid chromatography (FPLC) and SDS PAGE of 80% ammonium sulphate precipitates of the cell‐free supernatants.
• Isolates were positive for catalase, oxidases and antimicrobial activity . P. brassicacearum from the rhizosphere of the heat‐tolerant genotype was more efficient in phosphate solubilisation, bacteriocin production, antifungal and antibacterial activity against Helminthosporium sativum, Fusarium moniliforme and Klebsiella pneumonia, respectively. The inoculated seedlings had significantly higher root and shoot fresh weight, enhanced activity of antioxidant enzymes, proline and protein content. Total profiling of the culture with SDS‐PAGE indicated expression of new protein bands in 95 kDa in P. brassicacearum.
• Temperature‐induced changes in PGPR isolates are similar to those in the host plant. P. brassicacearum may be a good candidate for use in biofertiliser production for plants exposed to high temperature stress.

     

Overexpression of AtPAD4 in transgenic Brachypodium distachyon enhances resistance to Puccinia brachypodii

• Brachypodium distachyon (L.) has recently emerged as a model for temperate grasses for investigating the molecular basis of plant–pathogen interactions. Phytoalexin deficient 4 (PAD4) plays a regulatory role in mediating expression of genes involved in plant defence.
• In this research, we generated transgenic B. distachyon plants constitutively overexpressing AtPAD4. Two transgenic B. distachyon lines were verified using PCR and GUS phenotype.
• Constitutive expression of AtPAD4 in B. distachyon enhanced resistance to Puccinia brachypodii. P. brachypodii generated less urediniospores on transgenic than on wild‐type plants. AtPAD4 overexpression enhanced salicylic acid (SA) levels in B. distachyon‐infected tissues. qRT‐PCR showed that expression of pathogenesis‐related 1 (PR1) and other defence‐related genes were up‐regulated in transformed B. distachyon following infection with P. brachypodii.
• Our results indicate that AtPAD4 overexpression in B. distachyon plants led to SA accumulation and induced PR gene expression that reduced the rate of colonisation by P. brachypodii.

     

Intermittent lead‐induced stress on antioxidant enzyme activity and subcellular distribution of Pb in Pogonatherum crinitum seedlings

• Pogonatherum crinitum is a promising lead (Pb) hyperaccumulator due to its high Pb tolerance and accumulation ability. However, the mechanisms that support Pb accumulation and tolerance in P. crinitum are not yet clearly understood.
• An indoor hydroponic experiment was conducted by cultivating P. crinitum seedlings exposed to intermittent Pb stress for 60 days, divided into four stages (T1, T2, T3 and T4), with a 15‐day duration per stage. The following concentrations of Pb were used: 0, 500, 0, 500 mg·l^?1 and 0, 1000, 0, 1000 mg·l^?1). Antioxidant enzyme activity, Pb concentration and subcellular distribution of Pb were measured at each of the above stages.
• The results showed that superoxide dismutase (SOD) activity in shoots, and SOD, peroxidase (POD) and malondialdehyde (MDA) activity in shoots and roots significantly increased from T1 (no Pb stress) to T2 (Pb stress) in both 500 mg·l^?1 and 1000 mg·l^?1 treatments; however, no significant difference was noted between stages T3 (no Pb stress) and T4 (Pb stress). There was no obvious effect of Pb stress on catalase (CAT) activity in shoots and roots among different stages. The Pb concentration in shoots was up to 5090.90 mg·kg^?1 and 7573.57 mg·kg^?1, and the bioconcentration factor (BFC) was 10.18 and 7.57 for the 500 mg·l^?1 and 1000 mg·l^?1 treatments, respectively, which confirmed the Pb hyperaccumulator characteristics of P. crinitum. For plants under Pb stress, most of the Pb was fixed in the cell walls, with a smaller amount in leaves and root vacuoles.
• Both SOD and POD scavenging of reactive oxygen radicals and fixing and compartmentalisation of Pb in the cell wall might play important roles in detoxification of P. crinitum seedlings in response to Pb stress. There was no phased response of P. crinitum to intermittent Pb stress and the physiological response to Pb stress may be contiguous.

     

Chromosome‐level genome assembly of the predator Propylea japonica to understand its tolerance to insecticides and high temperatures

The ladybird beetle Propylea japonica is an important natural enemy in agro‐ecological systems. Studies on the strong tolerance of P. japonica to high temperatures and insecticides, and its population and phenotype diversity have recently increased. However, abundant genome resources for obtaining insights into stress‐resistance mechanisms and genetic intra‐species diversity for P. japonica are lacking. Here, we constructed the P. japonica genome maps using Pacific Bioscience (PacBio) and Illumina sequencing technologies. The genome size was 850.90 Mb with a contig N50 of 813.13 kb. The Hi‐C sequence data were used to upgrade draft genome assemblies; 4,777 contigs were assembled to 10 chromosomes; and the final draft genome assembly was 803.93 Mb with a contig N50 of 813.98 kb and a scaffold N50 of 100.34 Mb. Approximately 495.38 Mb of repeated sequences was annotated. The 18,018 protein‐coding genes were predicted, of which 95.78% were functionally annotated, and 1,407 genes were species‐specific. The phylogenetic analysis showed that P. japonica diverged from the ancestor of Anoplophora glabripennis and Tribolium castaneum ~ 236.21 million years ago. We detected that some important gene families involved in detoxification of pesticides and tolerance to heat stress were expanded in P. japonica, especially cytochrome P450 and Hsp70 genes. Overall, the high‐quality draft genome sequence of P. japonica will provide invaluable resource for understanding the molecular mechanisms of stress resistance and will facilitate the research on population genetics, evolution and phylogeny of Coccinellidae. This genome will also provide new avenues for conserving the diversity of predator insects.     

Enhancement of cadmium accumulation in sweet sorghum as affected by nitrate

• The Cadmium (Cd)‐polluted soils are is an increasing concern worldwide. Phytoextraction of Cd pollutants by high biomass plants, such as sweet sorghum, is considered an environmentally‐friendly, cost‐effective and sustainable strategy for remediating this problem. Nitrogen (N) is a macronutrient essential for plant growth, development and stress resistance. Nevertheless, how nitrate, as an important form of N, affects Cd uptake, translocation and accumulation in sweet sorghum is still unclear.
• In the present study, a series of nitrate levels (N1, 0.5 mm ; N2, 2 mm ; N3, 4 mm ; N4, 8 mm and N5, 16 mm ) with or without added 5 μm CdCl₂ treatment in sweet sorghum was investigated hydroponically.
• The results indicate that Cd accumulation in the aboveground parts of sweet sorghum was enhanced by optimum nitrate supply, resulting from both increased dry weight and Cd concentration. Although root‐to‐shoot Cd translocation was not enhanced by increased nitrate, some Cd was transferred from cell walls to vacuoles in leaves. Intriguingly, expression levels of Cd uptake and transport genes, SbNramp1, SbNramp5 and SbHMA3, were not closely related to increased Cd as affected by nitrate supply. The expression of SbNRT1.1B in relation to nitrate transport showed an inverted ‘U’ shape with increasing nitrate levels under Cd stress, which was in agreement with trends in Cd concentration changes in aboveground tissues.
• Based on the aforementioned results, nitrate might regulate Cd uptake and accumulation through expression of SbNRT1.1B rather than SbNramp1, SbNramp5 or SbHMA3, the well‐documented genes related to Cd uptake and transport in sweet sorghum.

     

Functional characterisation of two phytochelatin synthases in rice (Oryza sativa cv. Milyang 117) that respond to cadmium stress

• Cadmium (Cd) is one of the most toxic heavy metals and a non‐essential element to all organisms, including plants; however, the genes involved in Cd resistance in plants remain poorly characterised.
• To identify Cd resistance genes in rice, we screened a rice cDNA expression library treated with CdCl₂ using a yeast (Saccharomyces cerevisiae) mutant ycf1 strain (DTY167) and isolated two rice phytochelatin synthases (OsPCS5 and OsPCS15).
• The genes were strongly induced by Cd treatment and conferred increased resistance to Cd when expressed in the ycf1 mutant strain. In addition, the Cd concentration was twofold higher in yeast expressing OsPCS5 and OsPCS15 than in vector‐transformed yeast, and OsPCS5 and OsPCS15 localised in the cytoplasm. Arabidopsis thaliana plants overexpressing OsPCS5/‐15 paradoxically exhibited increased sensitivity to Cd, suggesting that overexpression of OsPCS5/‐15 resulted in toxicity due to excess phytochelatin production in A. thaliana.
• These data indicate that OsPCS5 and OsPCS15 are involved in Cd tolerance, which may be related to the relative abundances of phytochelatins synthesised by these phytochelatin synthases.

     

Unravelling the structure and function of the petal appendages in the tribe Schwenckieae (Solanaceae)

• The tribe Schwenckieae (Solanaceae) is characterised by the presence of appendages on the corolla, a diagnostic trait for the group. These appendages constitute a median distal projection of the three‐lobed petal and occur in the genera Melananthus and Schwenckia but are absent in Heteranthia.
• We investigated the micromorphology and anatomical structure of the appendages and lateral petal lobes of Schwenckia americana (two varieties), S. angustifolia, S. curviflora and S. novaveneciana, and Melananthus fasciculatus. We also performed histochemical tests to determine if the appendages are involved in the production of volatiles, acting as a fragrance secretory structure (osmophore).
• The appendages have a uniseriate epidermis, whose cells store phenolics and lipids. The parenchyma is starch‐rich just prior to anthesis in all species studied. The sensory test and anatomical analyses identified scent‐secreting tissues, not only in the appendages, but also in the lateral petal lobes, whose cells are papillose with a sculptured surface. The α‐naphthol p‐phenylenediamine (NADI) reaction detected volatile (essential oils) compounds in S. americana var. americana and S. americana var. angustifolia.
• We demonstrated the secretory tissues and the production of lipids in the corolla appendages of Schwenckia and Melananthus, which indicate their osmogenic function and probable scent emission to attract pollinators.

     

Involvement of LeMRP,an ATP‐binding cassette transporter,in shikonin transport and biosynthesis in Lithospermum erythrorhizon

• Shikonin and its derivatives are important medicinal secondary metabolites accumulating in roots of Lithospermum erythrorhizon. Although some membrane proteins have been identified as transporters of secondary metabolites, the mechanisms underlying shikonin transport and accumulation in L. erythrorhizon cells still remain largely unknown.
• In this study, we isolated a cDNA encoding LeMRP, an ATP‐binding cassette transporter from L. erythrorhizon, and further investigated its functions in the transport and biosynthesis of shikonin using the yeast transformation and transgenic hairy root methods, respectively. Real‐time PCR was applied for expression analyses of LeMRP and shikonin biosynthetic enzyme genes.
• Functional analysis of LeMRP using the heterologous yeast cell expression system showed that LeMRP could be involved in shikonin transport. Transgenic hairy roots of L. erythrorhizon demonstrated that LeMRP overexpressing hairy roots produced more shikonin than the empty vector (EV) control. Real‐time PCR results revealed that the enhanced shikonin biosynthesis in the overexpression lines was mainly caused by highly up‐regulated expression of genes coding key enzymes (LePAL, HMGR, Le4CL and LePGT) involved in shikonin biosynthesis. Conversely, LeMRP RNAi decreased the accumulation of shikonin and effectively down‐regulated expression level of the above genes. Typical inhibitors of ABC proteins, such as azide and buthionine sulphoximine, dramatically inhibited accumulation of shikonin in hairy roots.
• Our findings provide evidence for the important direct or indirect role of LeMRP in transmembrane transport and biosynthesis of shikonin.

     

Zinc deficiency tolerance in maize is associated with the up‐regulation of Zn transporter genes and antioxidant activities

• Zinc (Zn) is an essential micronutrient for the growth and development of plants. However, Zn deficiency is a common abiotic stress causing yield loss in crop plants. This study elucidates the mechanisms of Zn deficiency tolerance in maize through physiological and molecular techniques.
• Maize lines tolerant (PAC) and sensitive (DAC) to Zn deficiency were examined physiologically and by atomic absorption spectrometry (AAS). Proteins, H₂O₂, SOD, POD, membrane permeability and gene expression (using real‐time PCR) of roots and shoots of both maize lines were assessed.
• Zn deficiency had no significant effect on root parameters compared with control plants in PAC and DAC but showed a substantial reduction in shoot parameters in DAC. AAS showed a significant decrease in Zn concentrations in both roots and shoots of DAC but not PAC under Zn deficiency, implying that Zn deficiency tolerance mechanisms exist in PAC. Consistently, total protein and membrane permeability were significantly reduced in DAC but not PAC in both roots and shoots under Zn deficiency in comparison with Zn‐sufficient plants. Real‐time PCR showed that expression of ZmZIP1, ZmZIP4 and ZmIRT1 transporter genes significantly increased in roots of PAC, but not in DAC due to Zn deficiency compared with controls. The H₂O₂ concentration dramatically increased in roots of DAC but not PAC. Moreover, tolerant PAC showed a significant increase in POD and SOD activity due to Zn deficiency, suggesting that POD‐ and SOD‐mediated antioxidant defence might provide tolerance, at least in part, under Zn deficiency in PAC.
• This study provides an essential background for improving Zn biofortification of maize.