Two functionally distinct members of the MATE (multi‐drug and toxic compound extrusion) family of transporters potentially underlie two major aluminum tolerance QTLs in maize

Crop yields are significantly reduced by aluminum (Al) toxicity on acidic soils, which comprise up to 50% of the world’s arable land. Al‐activated release of ligands (such as organic acids) from the roots is a major Al tolerance mechanism in plants. In maize, Al‐activated root citrate exudation plays an important role in tolerance. However, maize Al tolerance is a complex trait involving multiple genes and physiological mechanisms. Recently, transporters from the MATE family have been shown to mediate Al‐activated citrate exudation in a number of plant species. Here we describe the cloning and characterization of two MATE family members in maize, ZmMATE1 and ZmMATE2, which co‐localize to major Al tolerance QTL. Both genes encode plasma membrane proteins that mediate significant anion efflux when expressed in Xenopus oocytes. ZmMATE1 expression is mostly concentrated in root tissues, is up‐regulated by Al and is significantly higher in Al‐tolerant maize genotypes. In contrast, ZmMATE2 expression is not specifically localized to any particular tissue and does not respond to Al. [¹⁴C]‐citrate efflux experiments in oocytes demonstrate that ZmMATE1 is a citrate transporter. In addition, ZmMATE1 expression confers a significant increase in Al tolerance in transgenic Arabidopsis. Our data suggests that ZmMATE1 is a functional homolog of the Al tolerance genes recently characterized in sorghum, barley and Arabidopsis, and is likely to underlie the largest maize Al tolerance QTL found on chromosome 6. However, ZmMATE2 most likely does not encode a citrate transporter, and could be involved in a novel Al tolerance mechanism.     

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.

     

A multidrug and toxic compound extrusion (MATE) transporter modulates auxin levels in root to regulate root development and promotes aluminium tolerance

MATE (multidrug and toxic compound extrusion) transporters play multiple roles in plants including detoxification, secondary metabolite transport, aluminium (Al) tolerance, and disease resistance. Here we identify and characterize the role of the Arabidopsis MATE transporter DETOXIFICATION30. AtDTX30 regulates auxin homeostasis in Arabidopsis roots to modulate root development and Al-tolerance. DTX30 is primarily expressed in roots and localizes to the plasma membrane of root epidermal cells including root hairs. dtx30 mutants exhibit reduced elongation of the primary root, root hairs, and lateral roots. The mutant seedlings accumulate more auxin in their root tips indicating role of DTX30 in maintaining auxin homeostasis in the root. Al induces DTX30 expression and promotes its localization to the distal transition zone. dtx30 seedlings accumulate more Al in their roots but are hyposensitive to Al-mediated rhizotoxicity perhaps due to saturation in root growth inhibition. Increase in expression of ethylene and auxin biosynthesis genes in presence of Al is absent in dtx30. The mutants exude less citrate under Al conditions, which might be due to misregulation of AtSTOP1 and the citrate transporter AtMATE. In conclusion, DTX30 modulates auxin levels in root to regulate root development and in the presence of Al indirectly modulates citrate exudation to promote Al tolerance.     

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.

     

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.

     

mlo‐based powdery mildew resistance in hexaploid bread wheat generated by a non‐transgenic TILLING approach

Wheat is one of the most widely grown cereal crops in the world and is an important food grain source for humans. However, wheat yields can be reduced by many abiotic and biotic stress factors, including powdery mildew disease caused by Blumeria graminis f.sp. tritici (Bgt). Generating resistant varieties is thus a major effort in plant breeding. Here, we took advantage of the non‐transgenic Targeting Induced Lesions IN Genomes (TILLING) technology to select partial loss‐of‐function alleles of TaMlo, the orthologue of the barley Mlo (Mildew resistance locus o) gene. Natural and induced loss‐of‐function alleles (mlo) of barley Mlo are known to confer durable broad‐spectrum powdery mildew resistance, typically at the expense of pleiotropic phenotypes such as premature leaf senescence. We identified 16 missense mutations in the three wheat TaMlo homoeologues, TaMlo‐A1, TaMlo‐B1 and TaMlo‐D1 that each lead to single amino acid exchanges. Using transient gene expression assays in barley single cells, we functionally analysed the different missense mutants and identified the most promising candidates affecting powdery mildew susceptibility. By stacking of selected mutant alleles we generated four independent lines with non‐conservative mutations in each of the three TaMlo homoeologues. Homozygous triple mutant lines and surprisingly also some of the homozygous double mutant lines showed enhanced, yet incomplete, Bgt resistance without the occurrence of discernible pleiotropic phenotypes. These lines thus represent an important step towards the production of commercial non‐transgenic, powdery mildew‐resistant bread wheat varieties.     

Physiological and molecular analysis of aluminum tolerance in selected Kenyan maize lines

Aims

Aluminum (Al) toxicity is an important limitation to maize production in many tropical and sub-tropical acid soil areas. The aim of this study was to survey the variation in Al tolerance in a panel of maize lines adapted for Kenya and look for novel sources of Al tolerance.

Methods

112 Kenyan maize accessions were phenotyped for Al tolerance in solution culture. Several Al tolerance-related parameters including relative net root growth (RNRG), root apex Al accumulation, Al-activated root organic acid exudation, and expression of the maize Al tolerance gene, ZmMATE1, were used to classify Kenyan maize accessions.

Results

Based on RNRG, 42 %, 28 %, and 30 % of the lines were classified as highly tolerant, moderately tolerant and sensitive, respectively. Tolerant accessions accumulated less Al in their root apices compared to sensitive lines. The Kenyan maize line, CON 5, and the Brazilian standard for tolerance, Cateto, exhibited the greatest Al tolerance based on RNRG, but CON 5 had only about 50 % of ZmMATE1 gene expression relative to Cateto. CON 5 also had low root apex Al content and high citrate exudation, suggesting that it may employ a citrate transporter other than ZmMATE1.

Conclusions

We identified a very Al tolerant Kenyan maize line whose Al tolerance may be based in part on a novel tolerance gene. The maize lines identified in this study are useful germplasm for the development of varieties suitable for agriculture on acid soils in Kenya.

     

The speciation and distribution characteristics of Cu in Phragmites australis (Cav.) Trin ex. Steudel

• Heavy metal allocation and the mechanism(s) of metal sequestration in different clonal organs, micro‐domains and subcellular structures has not been systematically studied for rhizomatous perennial plants. It is thus pertinent to investigate knowledge of the speciation and distribution characteristics of Cu in Phragmites australis to elucidating the mobility of metals in wetland plants after their uptake via root systems so as to facilitate development of strategies to enhance Cu tolerance.
• This study investigated the distributions of Cu in P. australis root, stem and leaf using ICP‐MS, synchrotron‐based X‐ray micro‐fluorescence and X‐ray absorption spectroscopy, then evaluated the effects of Cu on cellular structure and ultrastructure via transmission electron microscopy.
• The results indicate a clear preferential localisation of Cu in the roots as compared with the shoots (stems and leaves). The intensity of Cu in the vascular bundles was higher than that in the surrounding epidermis and the endodermis and parenchyma outside the medullary cavity. The dominant chemical form of Cu in P. australis was similar to Cu citrate.
• The results suggest that although Cu can be easily transported into the vascular tissues in roots and stems via Cu citrate, most of the metal absorbed by plants is retained in the roots because if its high binding to the cell wall, thus preventing metal translocation to aerial parts of the plants. Therefore, P. australis showed a high capacity to accumulate Cu in roots, being therefore a suitable species for phytostabilisation interventions.

     

TaCYP81D5, one member in a wheat cytochrome P450 gene cluster,confers salinity tolerance via reactive oxygen species scavenging

As one of the largest gene families in plants, the cytochrome P450 monooxygenase genes (CYPs) are involved in diverse biological processes including biotic and abiotic stress response. Moreover, P450 genes are prone to expanding due to gene tandem duplication during evolution, resulting in generations of novel alleles with the neo‐function or enhanced function. Here, the bread wheat (Triticum aestivum) gene TaCYP81D5 was found to lie within a cluster of five tandemly arranged CYP81D genes, although only a single such gene (BdCYP81D1) was present in the equivalent genomic region of the wheat relative Brachypodium distachyon. The imposition of salinity stress could up‐regulate TaCYP81D5, but the effect was abolished in plants treated with an inhibitor of reactive oxygen species synthesis. In SR3, a wheat cultivar with an elevated ROS content, the higher expression and the rapider response to salinity of TaCYP81D5 were related to the chromatin modification. Constitutively expressing TaCYP81D5 enhanced the salinity tolerance both at seedling and reproductive stages of wheat via accelerating ROS scavenging. Moreover, an important component of ROS signal transduction, Zat12, was proven crucial in this process. Though knockout of solely TaCYP81D5 showed no effect on salinity tolerance, knockdown of BdCYP81D1 or all TaCYP81D members in the cluster caused the sensitivity to salt stress. Our results provide the direct evidence that TaCYP81D5 confers salinity tolerance in bread wheat and this gene is prospective for crop improvement.     

Overexpression of <Emphasis Type="Italic">Citrus junos</Emphasis> mitochondrial citrate synthase gene in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> confers aluminum tolerance

Aluminum (Al) toxicity is one of the major factors that limit plant growth in acid soils. Al-induced release of organic acids into rhizosphere from the root apex has been identified as a major Al-tolerance mechanism in many plant species. In this study, Al tolerance of Yuzu (Citrus Junos Sieb. ex Tanaka) was tested on the basis of root elongation and the results demonstrated that Yuzu was Al tolerant compared with other plant species. Exposure to Al triggered the exudation of citrate from the Yuzu root. Thus, the mechanism of Al tolerance in Yuzu involved an Al-inducible increase in citrate release. Aluminum also elicited an increase of citrate content and increased the expression level of mitochondrial citrate synthase (CjCS) gene and enzyme activity in Yuzu. The CjCS gene was cloned from Yuzu and overexpressed in Nicotiana benthamiana using Agrobacterium tumefaciens-mediated methods. Increased expression level of the CjCS gene and enhanced enzyme activity were observed in transgenic plants compared with the wild-type plants. Root growth experiments showed that transgenic plants have enhanced levels of Al tolerance. The transgenic Nicotiana plants showed increased levels of citrate in roots compared to wild-type plants. The exudation of citrate from roots of the transgenic plants significantly increased when exposed to Al. The results with transgenic plants suggest that overexpression of mitochondrial CS can be a useful tool to achieve Al tolerance.     

Distinct seasonal dynamics of responses to elevated CO2 in two understorey grass species differing in shade‐tolerance

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Impact of the TaMATE1B gene on above and below-ground growth of durum wheat grown on an acid and Al3+-toxic soil

Background and aims

Subsoil acidity with a high aluminium (Al³⁺) soil content inhibits root growth and proliferation of durum wheat (tetraploid AABB, Triticum turgidum) leading to poor nutrient and water uptake. This study evaluated the impact of Al³⁺-tolerantTaMATE1B allele on root and shoot traits of durum wheat grown in an acidic soil with a high Al³⁺concentration.

Methods

Two durum wheat lines, Jandaroi–TaMATE1B with the TaMATE1B gene introgressed from Al³⁺-tolerant bread wheat and Jandaroi–null (a sister line lacking the Al³⁺-tolerant TaMATE1B allele), were grown in rhizoboxes in a glasshouse. We mapped root growth and proliferation over time and measured shoot traits and grain yield.

Results

Introgression of the Al³⁺-tolerant TaMATE1B allele into durum wheat enabled root growth and proliferation below 0.25 m of the soil profile, where the soil pH was low (4.1, CaCl₂ extract) with high Al³⁺ content (16.5 mg kg⁻¹), and increased total root length and biomass at 42 days after sowing (DAS; Z₃₃) by 38.3 and 22%, respectively, relative to the Jandaroi–null. Differences in root growth between the two lines were apparent from tillering stage (Z₃₃) and by 50% anthesis (Z₆₄), respectively. Jandaroi–TaMATE1B had 69.2% greater root biomass, 76.2% greater root length, 5.89% greater leaf area and 18% greater shoot biomass than Jandaroi–null at 50% anthesis (Z₆₄). Time to anthesis and physiological maturity was delayed 6–7 days in Jandaroi–TaMATE1B, compared to Jandaroi–null. Jandaroi–TaMATE1B tended to have relatively greater, but not significantly different, shoot biomass, grain yield and yield components than Jandaroi–null.

Conclusions

Introgression of the Al³⁺-tolerant TaMATE1B allele into durum wheat enabled root growth and proliferation down an acidic soil profile with a high Al³⁺ concentration. We assume that in the field where plants need to acquire water at depth differences in above-ground parameters would be amplified.

     

Several lanthanides activate malate efflux from roots of aluminium‐tolerant wheat

Many elements of the lanthanide series exist as trivalent cations in solution below pH 6. The present study was carried out to investigate whether lanthanides could stimulate malate efflux from wheat (Triticum aestivum L.) roots, as has been found for trivalent aluminium (Al) cations. Excised root apices treated with 100 µm of each of seven different lanthanide elements (lanthanum, praseodymium, europium, gadolinium, terbium, erbium, and ytterbium) stimulated malate efflux, with five‐ to fifty‐fold more malate being released from an Al‐tolerant wheat line than from a near‐isogenic Al‐sensitive line. As erbium stimulated the greatest malate efflux of the lanthanides tested, this response was characterized further. The characteristics of the erbium‐activated efflux were similar to the Al‐activated efflux described previously suggesting that both of these ions activate the same transport mechanism. The capacity for erbium‐activated malate efflux cosegregated with Al tolerance in wheat seedlings derived from a cross between Al‐sensitive and Al‐tolerant near‐isogenic lines. This is the first study to identify cations, other than Al, which can activate malate release from wheat roots. It also provides additional evidence that malate efflux from root apices is the primary mechanism for Al tolerance in wheat.