Ectopic expression of nicotianamine synthase genes results in improved iron accumulation and increased nickel tolerance in transgenic tobacco

Heavy metals are essential for basic cellular processes but toxic in higher concentrations. This requires the precise control of their intracellular concentrations, a process known as homeostasis. The metal-chelating, non-proteinogenous amino acid nicotianamine (NA) is a key component of plant metal assimilation and homeostasis. Its precise function is still unknown. Therefore, this article aims to contribute new information on the in vivo function of NA and to evaluate its potential use for plant nutrition and crop fortification. For this purpose, a nicotianamine synthase gene of Arabidopsis thaliana was ectopically expressed in transgenic tobacco plants. The presence of extra copies of the nicotianamine synthase gene co-segregated with up to 10-fold elevated levels of NA in comparison with wild type. The increased NA level led to: (a) a significantly increased iron level in leaves of adult plants; (b) the accumulation of zinc and manganese, but not copper; (c) an improvement of the iron use efficiency in adult plants grown under iron limitation; and (d) an enhanced tolerance against up to 1 m m nickel. Taken together, the data predict that NA may be a useful tool for improved plant nutrition on adverse soils and possibly for enhanced nutritional value of leaf and seed crops.     

The effects of nutrient supply,predominantly addition of iron,and rhizobial inoculation on the tolerance of Lupinus pilosus genotypes to a calcareous soil

Two glasshouse experiments were conducted to examine the effects of nutrient supply and rhizobial inoculation on the performance of Lupinus pilosus genotypes differing in tolerance to calcareous soils. In experiment 1, plants were grown for 84 days in a calcareous soil (50% CaCO₃; soil water content 90% of field capacity) at four nutrient treatments (no-added nutrients, added nutrients without Fe, added nutrients with soil applied FeEDDHA, added nutrients with foliar applied FeSO₄). In experiment 2, plants were grown for 28 days with supply of NH₄NO₃ without inoculation or inoculated with Bradyrhizobium sp. (Lupinus). Chlorosis in the youngest leaves was a good indicator of the relative tolerance of the genotypes to the calcareous soil in both experiments, except the treatment with FeEDDHA at 5 mg kg^–1 soil which was toxic to all genotypes. Chlorosis scores correlated with chlorophyll meter readings and chlorophyll concentrations. The foliar application of FeSO₄ did not fully alleviate chlorotic symptoms despite concentrations of active or total Fe in the youngest leaves being increased. Adding nutrients and chemical nitrogen did not change the severity of chlorosis or improve the growth of the plant. The nutrient supply did not alter the ranking of tolerance of genotypes to the calcareous soil. The results suggest that nutrient deficiency or poor nodulation was not a major cause of poor plant growth on calcareous soils and that bicarbonate may exert a direct effect on chlorophyll synthesis. The mechanism for tolerance is likely to be related to an ability to exclude bicarbonate or prevent its transport to the leaves.     

A loss-of-function mutation in AtYSL1 reveals its role in iron and nicotianamine seed loading

The Arabidopsis Yellow Stripe 1-Like (YSL) proteins have been identified by homology with the maize (Zea mays) Yellow Stripe 1 (YS1) transporter which is responsible for iron-phytosiderophore (PS) uptake by roots in response to iron shortage. Although dicotyledonous plants do not synthesize PS, they do synthesize the PS precursor nicotianamine, a strong metal chelator essential for maintenance of iron homeostasis and copper translocation. Furthermore, ZmYS1 and the rice (Oryza sativa) protein OsYSL2 have metal-nicotianamine transport activities in heterologous expression systems. In this work, we have characterized the function of AtYSL1 in planta. Two insertional loss-of-function ysl1 mutants of Arabidopsis were found to exhibit increased nicotianamine accumulation in shoots. More importantly, seeds of both ysl1 knockouts contained less iron and nicotianamine than wild-type seeds, even when produced by plants grown in the presence of an excess of iron. This phenotype could be reverted by expressing the wild-type AtYSL1 gene in ysl1 plants. ysl1 seeds germinated slowly, but this defect was rescued by an iron supply. AtYSL1 was expressed in the xylem parenchyma of leaves, where it was upregulated in response to iron excess, as well as in pollen and in young silique parts. This pattern is consistent with long-distance circulation of iron and nicotianamine and their delivery to the seed. Taken together, our work provides strong physiological evidence that iron and nicotianamine levels in seeds rely in part on AtYSL1 function.     

Iron-chelates evaluation in a calcareous soil

A greenhouse pot experiment withLolium multiflorum, cv. Tetila, grown in a calcareous soil was carried out to determine the efficiency of iron-chelate (Fe-EDTA,-DTPA,-EDDHA and Rexene) as soil amendments. In the soil solution Fe was displaced more readily from EDTA chelate and less so from EDDHA chelate. A significant increase of Mn, Cu and Zn in the soil solution was observed, with Fe-DTPA; Cu and Zn with Fe-EDTA; Cu with Rexene and, with Fe-EDDHA after 14 days. Plant took up more Fe from Rexene treatment than from the other treatments, Fe-EDDHA was the least efficient. In general, Mn, Cu and Zn concentrations in the leaf diminish in all the treatments compared with the control.     

Overexpression of barley nicotianamine synthase 1 confers tolerance in the sweet potato to iron deficiency in calcareous soil

Background and aims

Iron (Fe) is an essential micronutrient for all higher organisms. Fe is sparingly available in calcareous soils and Fe deficiency is a major agricultural problem worldwide. Nicotianamine (NA) is a metal chelator involved in metal translocation in plants. Sweet potato is an attractive crop that can grow in poor soil and thus is useful for planting in uncultivated soil. In addition, the sweet potato has recently been suggested as a source of bioethanol. Our aim is to increase NA concentration in sweet potato to ameliorate Fe deficiency.

Method

Sweet potato plants expressing the barley NA synthase 1 (HvNAS1) gene under the control of CaMV 35S promoter were produced by Agrobacterium-mediated transformation.

Results

The transgenic sweet potato exhibited tolerance to low Fe availability when grown in calcareous soil. The level of tolerance to low Fe availability was positively correlated with the HvNAS1 expression level. The NA concentration of the transgenic sweet potato leaves was up to 7.9-fold greater than that of the non-transgenic (NT) plant leaves. Furthermore, the Fe and zinc concentrations were 3- and 2.9-fold greater, respectively, in transgenic sweet potato than in NT plant leaves.

Conclusions

Our results suggest that increasing the NA concentration of sweet potato by overexpression of HvNAS1 could significantly improve agricultural productivity and energy source.

     

The effect of soil moisture on the tolerance of Lupinus pilosus genotypes to a calcareous soil

Commercial narrow-leafed lupins (Lupinus angustifolius L.) grown on calcareous soils commonly display chlorotic symptoms resembling Fe deficiency. The severity of chlorosis increases with concurrent increases in soil moisture content. Our research has indicated that the rough-seeded lupin species, Lupinus pilosus Murr., has a range of adaptation to calcareous soils, from tolerant to intolerant. A pot experiment was conducted comparing a tolerant, a moderately tolerant and a moderately intolerant genotype of L. pilosus. Plants were grown for 35 days in a calcareous soil (50% CaCO₃) at three moisture contents (80%, 100% and 120% of field capacity); the growth was compared with that on a fertile black cracking clay control soil at 70% of field capacity. Visual chlorosis score, chlorophyll meter readings, number of leaves and shoot dry weights were recorded at 14, 21, 28 and 35 days after sowing. Concentrations of chlorophyll, active Fe and nutrients in the youngest fully expanded leaves were also measured. Results showed that increased soil moisture increased the severity of chlorotic symptoms (increased chlorosis score) in all genotypes. The tolerant genotype showed significantly less symptoms than other genotypes at all moisture contents. All genotypes were able to recover from chlorosis symptoms at 80% moisture in the calcareous soil. Chlorosis score negatively correlated with chlorophyll meter readings, chlorophyll concentration and foliar active and total Fe, and Mn concentrations. Visual chlorosis score appeared to be a cost effective, accurate and efficient method enabling classification of the tolerance of genotypes. The chlorotic symptoms were likely to be due to HCO₃ ^- induced nutrient deficiencies or a direct effect of HCO₃ ^- on chlorophyll synthesis. This study indicates that the most probable mechanism of tolerance is related to an ability to prevent uptake of HCO₃ ^- or efficiently sequester it once inside the root which prevents increases in internal pH and transport to the shoots.     

Nicotianamine synthase 2 localizes to the vesicles of iron‐deficient rice roots,and its mutation in the YXXφ or LL motif causes the disruption of vesicle formation or movement in rice

Graminaceous plants release mugineic acid family phytosiderophores (MAs) to acquire iron from the soil. Here, we show that deoxymugineic acid (DMA) secretion from rice roots fluctuates throughout the day, and that vesicles accumulate in roots before MAs secretion. We developed transgenic rice plants that express rice nicotianamine (NA) synthase (NAS) 2 (OsNAS2) fused to synthetic green fluorescent protein (sGFP) under the control of its own promoter. In root cells, OsNAS2–sGFP fluorescence was observed in a dot‐like pattern, moving dynamically within the cell. This suggests that these vesicles are involved in NA and DMA biosynthesis. A tyrosine motif and a di‐leucine motif, which have been reported to be involved in cellular transport, are conserved in all identified NAS proteins in plants. OsNAS2 mutated in the tyrosine motif showed NAS activity and was localized to the vesicles; however, these vesicles stuck together and did not move. On the other hand, OsNAS2 mutated in the di‐leucine motif lost NAS activity and did not localize to these vesicles. The amounts of NA and DMA produced and the amount of DMA secreted by OsNAS2–sGFP plants were significantly higher than in non‐transformants and domain‐mutated lines, suggesting that OsNAS2–sGFP, but not the mutated forms, was functional in vivo. Overall, the localization of NAS to vesicles and the transport of these vesicles are crucial steps in NA synthesis, leading to DMA synthesis and secretion in rice.     

两种土壤类型草本植物根系丛枝菌根真菌多样性

将分属12个科的17种宿主植物分别种植于石灰土和紫色土上,120 d后收获取样,通过CTAB法提取共生菌根内AM真菌的DNA,用特异引物U1/U2扩增编码核糖体28S大亚基的rDNA部分序列,并利用聚丙烯酰胺凝胶电泳(PAGE)和银染显影技术显色, 比较了石灰土和紫色土上草本植物根系内丛枝菌根真菌群落的物种多样性.结果表明：生长于石灰土上的17种宿主植物根系内共发现AM真菌条带29种,平均每种宿主植物内8.29种;生长于紫色土上的17种宿主植物根系内发现条带24种,平均每种宿主植物内9.47种.根系内的AM真菌条带包括特异条带和共有条带.经聚类分析发现,AM真菌对宿主植物的侵染有一定的科属专一性,且受土壤环境等外在条件的影响.同时探讨了AM真菌应用于石灰岩地区生态恢复的可能性.     

Co-occurrence of nicotianamine and avenic acids in Avena sativa and Oryza sativa

An amino acid derivative isolated from seedlings of Avena sativa and Oryza sativa, along with avenic acid A and its derivatives which possess a chelating ability with iron ions, has been shown to be nicotianamine. The co-occurrence of nicotianamine and avenic acids in the same plant, as well as their structural similarity, reveals their close biosynthetic relationship.     

Determination of threshold value of iron in soils and plants for the response of rice and lentil to iron application in calcareous soil

Summary In a pot experiment with 26 calcareous soils, the critical limit of Fe in soils and plants was evaluated. DTPA-extractable Fe was found significanty correlated with Bray's per cent yield in rice. The Fe²⁺ (iron) in rice and lentil was also found significantly correlated with DTPA-extractable Fe as well as Bray's per cent yield showing thereby the superiority of Fe²⁺ (iron) in leaves over DTPA-extractable soil Fe to differentiate Fe responsive soils from non-responsive ones. The total Fe content in plant tissues does not seem correlated with the occurrence of Fe deficiency. The threshold values of DTPA-extractable soil Fe and Fe²⁺ (iron) in rice and lentil leaves were 6.95, 44 and 74.5 ppm, respectively below which appreciable responses to Fe application were observed. The optimum Fe level for these soils was found to be 10 ppm in which the dry matter yield response in all the 19 rice soils and 16 lentil soils ranged from 14.28 to 56.16 (Av. 25.75%) and 13.31 to 53.97 (Av. 22.47%), respectively.     

不同演替阶段岩溶石灰土可培养细菌的群落特征

岩溶土壤是岩溶生态系统的重要组成部分,其母质碳酸盐岩在气候、地形、时间及生物等因素的综合作用下可依次演替出黑色石灰土、棕色石灰土、黄色石灰土和红色石灰土.[目的]了解可培养细菌群落对岩溶石灰土演替过程的响应,可为岩溶石漠化治理、生态恢复和重建等提供理论依据.[方法]以黑色石灰土、棕色石灰土、黄色石灰土和红色石灰土为研究...     

Adaptive strategies of Parietaria diffusa (M.&K.) to calcareous habitat with limited iron availability

The study of native plants growing in hostile environments is useful to understand how these species respond to stress conditions. Parietaria diffusa (M.&K.) is able to survive in highly calcareous soils and extreme environments, such as house walls, without displaying any chlorotic symptoms. Here, we have investigated the existence of Strategy I complementary/alternative mechanism(s) involved in Fe solubilization and uptake and responsible for Parietaria's extraordinary efficiency. After assessing the specific traits involved in a calcicole-behaviour in the field, we have grown plants in conditions of Fe deficiency, either direct (-Fe) or induced by the presence of bicarbonate (+FeBic). Then, the growth performance, physiological and biochemical responses of the plants were investigated. The study shows that in Parietaria+FeBic, the classical responses of Strategy I plants are activated to a lower extent than in -Fe. In addition, there is a greater production of phenolics and organic acids that are both exuded and accumulated in the roots, which in turn show structures similar to 'proteoid-like roots'. We suggest that in the presence of this constraint, Parietaria undergoes some metabolic rearrangements that involve PEP-consuming reactions and an enhancement of the shikimate pathway.     

有机物料对喀斯特地区石灰土有效N、Fe、Zn含量的影响

为研究不同有机物料对喀斯特石灰土元素有效性的影响,采用40 d的室内培养实验,比较了单独添加不同比例（1%、3%、5%）的生物质炭、鸡粪肥、羊粪肥对喀斯特石灰土有效N、Fe、Zn含量的影响。结果表明：添加生物质炭提高了喀斯特石灰土的pH值,而添加鸡粪肥和羊粪肥降低了喀斯特石灰土的pH值;添加3种有机物料均增加了喀斯特石灰土的有机质含量,大小关系为：生物质炭 > 鸡粪肥 > 羊粪肥,但添加鸡粪肥和羊粪肥土壤有机质的化学活性和微生物活性更高。受pH、有机质活性、碳氮比等因素的影响,添加鸡粪肥和羊粪肥能增加土壤有效N含量,但两种有机肥对土壤有效N的提高效果相差不大,而添加生物质炭反而降低了土壤有效N的含量;3种有机物料均能提高土壤的有效Fe和有效Zn含量,其中鸡粪肥效果最佳,其次为羊粪肥和生物质炭。当3种有机物料的添加比例为5%时,生物质炭处理土壤的有效N、Fe、Zn含量分别是对照处理的0.92、1.13、1.21倍;鸡粪肥处理土壤的有效N、Fe、Zn含量分别是对照处理的1.22、1.63和3.39倍;羊粪肥处理土壤的有效N、Fe、Zn含量分别是对照处理的1.27、1.34和2.59倍。所以,相对于生物质炭,有机粪肥对喀斯特地区的石灰土有更好的改良效果。     

石灰性土壤拉恩式溶磷细菌的筛选鉴定及溶磷特性

从山西石灰性土壤作物根际分离筛选出多株溶磷细菌,经过多次分离纯化得到一株溶磷能力较强的菌株W25,通过菌落形态、生理生化特性和16S rRNA序列分析,确定溶磷菌W25为拉恩式菌属.对W25溶解磷特性进一步研究表明:其对磷酸三钙、磷酸铝和磷酸铁最高溶磷能力分别为385.5、110.4、216.6 mg·L-1;在磷酸铝和磷酸铁培养液中,W25溶磷量与培养液pH的相关系数分别为0.56和0.81,呈极显著负相关;在不同碳氮源条件下,W25以葡萄糖为碳源和NH4NO3为氮源时对磷酸三钙的溶磷量最高,对碳源的利用顺序依次为葡萄糖＞乳糖＞蔗糖＞甘露醇＞淀粉,对氮源的利用顺序依次为NH4NO3 ＞NH4Cl＞(NH4)2SO4＞NaNO3＞KNO3.不同氮源对W25产生有机酸的种类影响较大,以铵态氮为氮源产生甲酸和乙酸,以硝态氮为氮源产生草酸和琥珀酸,以硝酸铵为氮源还产生柠檬酸.     

Mobilisation of iron and manganese by plant-borne and synthetic metal chelators

Phytosiderophores released by roots of iron-deficient grasses mobilise Fe, Zn, Mn and Cu in calcareous soils. Mobilisation of Fe, Zn and Cu can be explained as the chelation of these metal cations by phytosiderophores. Mobilisation of Mn could not be so explained because phytosiderophores have a much smaller affinity for Mn than for Fe, Cu and Zn. Model experiments have been made with freshly precipitated Fe(OH)₃ and different soils to study the mobilisation of iron and manganese by plant-borne chelating phytosiderophores, the synthetic metal chelators DTPA and the microbial metal chelator sulphonated ferrioxamine B (FOB). Compared with the synthetic chelator DTPA, the plant-borne chelating phytosiderophores mobilised Fe very efficiently, but no change was observed in the Mn mobilisation by phytosiderophores.Different phytosiderophores, as well as the microbial metal chelator FOB, were used to compare the mobilisation of iron and manganese in a calcareous soil.     

Influence of nicotianamine and iron supply on formation and elongation of adventitious roots in hypocotyl cuttings of the tomato mutant 'chloronerva' (Lycopersicon esculentum)

The influence of nicotianamine (NA) on formation and elongation of adventitious roots in hypocotyls of de-rooted NA-less mutant seedlings of Lycopersicon esculentum Mill, was examined in relation to the iron supply [ferric N-N'-ethylenediaminedi-(2-hydroxyphenylacetate) (FEDDHA), ferric ethylenediaminetetracetate (FeEDTA), ferric N-(2-hydroxyethyl)-ethylenediaminetriacetate (FeHEDTA, Fe-citrate and FeCl₃] in the nutrient solution. The initiation of root primordia in hypocotyl cuttings was independent of NA and occurred with about the same frequency in both, mutant and wild-type. In the mutant the development of primordia to adventitious roots was blocked at all iron sources used, except FeEDTA. Addition of NA (5x 10⁻⁶ to 2 × 10⁻⁵ M ) to the rooting medium resulted in a fast growth of adventitious roots in mutant cuttings with all iron sources tested. Rooting of wild-type cuttings was independent from NA application and iron sources. We suppose that NA is involved in the intracellular transport of iron. Its function is possibly linked with chelation of ferrous iron in the cell.     

Enhanced tolerance of rice to low iron availability in alkaline soils using barley nicotianamine aminotransferase genes

One of the widest ranging abiotic stresses in world agriculture arises from low iron (Fe) availability due to high soil pH, with 30% of arable land too alkaline for optimal crop production. Rice is especially susceptible to low iron supply, whereas other graminaceous crops such as barley are not. A barley genomic DNA fragment containing two naat genes, which encode crucial enzymes involved in the biosynthesis of phytosiderophores, was introduced into rice using Agrobacterium-mediated transformation and pBIGRZ1. Phytosiderophores are natural iron chelators that graminaceous plants secrete from their roots to solubilize iron in the soil. The two transgenes were expressed in response to low iron nutritional status in both the shoots and roots of rice transformants. Transgenic rice expressing the two genes showed a higher nicotianamine aminotransferase activity and secreted larger amounts of phytosiderophores than nontransformants under iron-deficient conditions. Consequently, the transgenic rice showed an enhanced tolerance to low iron availability and had 4.1 times greater grain yields than that of the nontransformant rice in an alkaline soil.     

The effects of cropping corn on the extractability of zinc added to a calcareous soil

The effects of cropping corn on the decrease in the extractability of Zn added to a calcareous soil were studied by a pot experiment and chemical extractions. The results show that the concentrations of Zn exchangeable with MgCl₂ (EXC-Zn) and extractable with DTPA (DTPA-Zn) in the soils with added Zn decreased with time. The processes associated with the decrease in extractability in DTPA of Zn added to soil can be described aptly by a diffusion equation which gives the proportion of added Zn in the non-DTPA fraction as a function of the square root of incubation time. This result suggests that the diffusion of Zn cations into microporous solids is a rate-limiting reaction. The relative diffusion rate coefficients (D/r²) were found to be 1.95×10^-10 and 3.34×10^-10 sec^-1 in the soils with added Zn of 20 and 60 mg kg^-1, respectively. Compared with uncropped soil, the concentrations of DTPA-Zn in the soils with added Zn were decreased by cropping. The decrease of DTPA-Zn in the soils in the presence of corn can be attributed to both its acquisition by corn and other processes associated with the growing of corn. The activity of plant roots would appear to enhance the process of decrease in the extractability in DTPA of Zn added to the soil. The source of Zn uptake by corn was affected by the loading or activity of Zn in soil. In the soil with low available Zn, the DTPA non-extractable Zn (non-DTPA-Zn) was mobilized and taken up by corn. In the soils with high available Zn, e.g. the recently added Zn, only EXC-Zn and a part of the DTPA-Zn were taken up by corn.     

Relationships of nicotianamine and other amino acids with nickel, zinc and iron in Thlaspi hyperaccumulators

Experimental evidence suggests that nicotianamine (NA) is involved in the complexation of metal ions in some metal-hyperaccumulating plants. Closely-related nickel (Ni)- and zinc (Zn)-hyperaccumulating species were studied to determine whether a correlation exists between the Ni and Zn concentrations and NA in foliar tissues. A liquid chromatography-mass spectrometry (LC-MS) procedure was developed to quantify the NA and amino acid contents using the derivatizing agent 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. A strong correlation emerged between Ni and NA, but not between Zn and NA. Concentrations of NA and L-histidine (His) also increased in response to higher Ni concentrations in the hydroponic solution supplied to a serpentine population of Thlaspi caerulescens. An inversely proportional correlation was found between the iron (Fe) and Ni concentrations in the leaves. Correlations were also found between Zn and asparagine. The results obtained in this study suggest that NA is involved in hyperaccumulation of Ni but not Zn. The inverse proportionality between the Ni and Fe concentrations in the leaf may suggest that Ni and Fe compete for complexation to NA.