Rice plants take up iron as an Fe3+-phytosiderophore and as Fe2+

Only graminaceous monocots possess the Strategy II iron (Fe)-uptake system in which Fe is absorbed by roots as an Fe3+-phytosiderophore. In spite of being a Strategy II plant, however, rice (Oryza sativa) contains the previously identified Fe2+ transporter OsIRT1. In this study, we isolated the OsIRT2 gene from rice, which is highly homologous to OsIRT1. Real-time PCR analysis revealed that OsIRT1 and OsIRT2 are expressed predominantly in roots, and these transporters are induced by low-Fe conditions. When expressed in yeast (Saccharomyces cerevisiae) cells, OsIRT2 cDNA reversed the growth defects of a yeast Fe-uptake mutant. This was similar to the effect of OsIRT1 cDNA. OsIRT1- and OsIRT2-green fluorescent protein fusion proteins localized to the plasma membrane when transiently expressed in onion (Allium cepa L.) epidermal cells. OsIRT1 promoter-GUS analysis revealed that OsIRT1 is expressed in the epidermis and exodermis of the elongating zone and in the inner layer of the cortex of the mature zone of Fe-deficient roots. OsIRT1 expression was also detected in the ccompanion cells. Analysis using the positron-emitting tracer imaging system showed that rice plants are able to take up both an Fe3+-phytosiderophore and Fe2+. This result indicates that, in addition to absorbing an Fe3+-phytosiderophore, rice possesses a novel Fe-uptake system that directly absorbs the Fe2+, a strategy that is advantageous for growth in submerged conditions.     

Cloning an iron-regulated metal transporter from rice

Rice cDNA and genomic libraries were screened in order to clone an Fe(II) transporter gene. A cDNA clone highly homologous to the Arabidopsis Fe(II) transporter gene IRT1 was isolated from Fe-deficient rice roots. The cDNA clone was named OsIRT1. A genomic clone corresponding to the cDNA was also obtained, sequenced and analysed. When expressed in yeast cells, OsIRT1 cDNA reversed the growth defects of the yeast iron-uptake mutant. Northern blot analysis revealed that OsIRT1 mRNA was predominantly expressed in roots and was induced by Fe- and Cu-deficiency. This suggests that OsIRT1 is a functional metal transporter for iron, and is actively engaged in Fe uptake from soils, especially under limiting conditions.     

The Soil Type Affects Both the Differential Accumulation of Iron between Wild-type and Ferritin Over-expressor Tobacco Plants and the Sensitivity of their Rhizosphere Bacterioflora to Iron Stress

Transgenic tobacco P6 over-expressing ferritin is known to activate iron transport systems and to have increased iron content. Iron phytoextraction by this transgene is then expected to be higher than that of the wild-type (WT). In the present study, the possibility to modify iron availability for bacteria via the cultivation of the transgene P6 was explored by comparing the sensitivity to iron stress of bacteria isolated from the rhizosphere of the two plant genotypes (WT and P6). This sensitivity was evaluated by measuring the bacterial density when plated on a solid media depleted (supplemented with 8-hydroxiquinoline) or not (supplemented with Fe-8-hydroxyquinoline) in iron. The experimental conditions favorable to the differential iron accumulation between the wild-type and transgenic tobacco were identified. The two plant genotypes were grown in three soils (Hervau, Thory and Oudun) chosen for their differences in iron content, and the plants were yielded at three stages (vegetative, floral bud and flowering). The highest differential accumulation of iron in favor of the over-expressing transgene was found in the plants at the floral bud stage when cultivated in the Oudun and Thory soils. Since at that stage, the plant growth was significantly higher in the Oudun soil, the phytoextraction of iron was the highest in this soil. At the floral bud stage, bacteria isolated from the rhizosphere of the transgene cultivated in the Oudun and Thory soils appeared to be less susceptible to iron stress than those from the wild-type. Bacterial density recovered on agar medium depleted in iron was significantly the highest in the rhizosphere of the transgene cultivated in the Oudun soil. Altogether, these data indicate that the over-expressing ferritin transgenic plants, that accumulate and extract more iron from the rhizosphere than the wild-type plants, select in their rhizosphere bacteria less susceptible to iron stress compared to those selected by the wild-type plants.     

Expression profiling of Oryza sativa metal homeostasis genes in different rice cultivars using a cDNA macroarray.

Rice is an important food crop, but it is a poor source of essential micronutrients such as iron and zinc. In order to improve the metal ion content of rice grains through breeding or biotechnology, more information is needed on the molecular players that help mobilize metals from leaves to developing seeds. To profile several genes simultaneously, a cDNA macroarray was developed using 36 metal-related genes from rice, including ZIPs, NRAMPs, and YSLs (coding for known or potential metal transporters), as well as NAS, FER, FRO, NAAT, FDH, GSTU, and PDR (involved in metal homeostasis). Because flag leaves are the major source of phloem-delivered photoassimilates and remobilized metals for developing seeds, we analyzed the expression of these metal-related genes in flag and non-flag leaves of four different rice cultivars (Cocodrie, Taipei 309, IR58, and IR68144) during the period of mid-grain fill. Genes (24 of 36) exhibited low to non-detectable signals in the macroarray, while 12 genes (OsIRT1, OsZIP1, OsZIP5, OsZIP8, OsYSL5, OsYSL6, OsYSL7, OsYSL8, OsYSL18, OsNRAMP2, OsNRAMP4 and OsNRAMP7) were found to be highly expressed in both flag and non-flag leaves of all four cultivars. Additional expression analysis using semi-quantitative or quantitative PCR provided results that were generally consistent with the macroarray, but semi-quantitative PCR confirmed that OsFDH, OsFER1, OsNAAT, OsNAS1, OsPDR9, OsYSL12, OsYSL13, OsZIP7, and OsZIP10 were also expressed in leaves. This specialized macroarray has provided a short list of potential candidate genes, expressed in leaves, which might contribute to the process of metal transport to distant sinks, such as seeds.     

A cytosolic NADP-malic enzyme gene from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) confers salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

NADP-malic enzyme (NADP-ME, EC 1.1.1.40) functions in many different pathways in plant and may be involved in plant defense such as wound and UV-B radiation. Here, expression of the gene encoding cytosolic NADP-ME (cytoNADP-ME, GenBank Accession No. AY444338) in rice (Oryza sativa L.) seedlings was induced by salt stress (NaCl). NADP-ME activities in leaves and roots of rice also increased in response to NaCl. Transgenic Arabidopsis plants over-expressing rice cytoNADP-ME had a greater salt tolerance at the seedling stage than wild-type plants in MS medium-supplemented with different levels of NaCl. Cytosolic NADPH/NADP⁺ concentration ratio of transgenic plants was higher than those of wild-type plants. These results suggest that rice cytoNADP-ME confers salt tolerance in transgenic Arabidopsis seedlings.     

Mapping QTLs and candidate genes for iron and zinc concentrations in unpolished rice of Madhukar×Swarna RILs

Identifying QTLs/genes for iron and zinc in rice grains can help in biofortification programs. 168 F(7) RILs derived from Madhukar×Swarna were used to map QTLs for iron and zinc concentrations in unpolished rice grains. Iron ranged from 0.2 to 224ppm and zinc ranged from 0.4 to 104ppm. Genome wide mapping using 101 SSRs and 9 gene specific markers showed 5 QTLs on chromosomes 1, 3, 5, 7 and 12 significantly linked to iron, zinc or both. In all, 14 QTLs were identified for these two traits. QTLs for iron were co-located with QTLs for zinc on chromosomes 7 and 12. In all, ten candidate genes known for iron and zinc homeostasis underlie 12 of the 14 QTLs. Another 6 candidate genes were close to QTLs on chromosomes 3, 5 and 7. Thus the high priority candidate genes for high Fe and Zn in seeds are OsYSL1 and OsMTP1 for iron, OsARD2, OsIRT1, OsNAS1, OsNAS2 for zinc and OsNAS3, OsNRAMP1, Heavy metal ion transport and APRT for both iron and zinc together based on our genetic mapping studies as these genes strictly underlie QTLs. Several elite lines with high Fe, high Zn and both were identified.     

OsNRAMP5, a major player for constitutive iron and manganese uptake in rice

Manganese (Mn) and iron (Fe) are essential mineral micronutrients for plants and their deficiency and or toxicity represents a serious agricultural problem. In rice the information about genes involved in Mn uptake from soil is scarce. Recently, we showed that OsNRAMP5 is a plasma membrane protein involved in Mn and Fe transport. The concentration of Mn in roots, shoots and xylem sap of OsNRAMP5 RNAi (OsNRAMP5i) plants was significantly reduced compared with WT plants. The expression of OsNRAMP5 is not controlled by Fe deficiency in root and was also observed in pistil, ovary, lemma and palea. These data show that rice would utilize OsNRAMP5 for constitutive Fe and Mn uptake, while OsNRAMP5 would also play a role in Fe and Mn transport during flowering and seed development.     

Characterization of plant growth-promoting bacteria associated with rice cropped in iron-stressed soils

Plant growth-promoting rhizobacteria (PGPR) are able to promote plant growth using a wide variety of mechanisms as well as provide bioprotection against biotic and abiotic stresses. The objectives of this study were to isolate and characterize putative PGPR associated with rice cultivars with a distinct tolerance to iron toxicity grown in two areas: one area with a well-established history of iron toxicity and another without iron toxicity. Bacterial strains were selectively isolated based on their growth in selective media and were identified by partial sequencing of their 16S rRNA genes. Bacterial isolates were evaluated for their ability to produce indolic compounds, siderophores, and ACC deaminase and to solubilize tricalcium phosphates. In vitro biological nitrogen fixation was evaluated for the bacterial isolates used in the inoculation experiments. A total of 329 bacterial strains were isolated. The composition of the bacterial genera and the occurrence of different plant growth-promoting (PGP) traits were significantly affected by the iron conditions and by the cultivar. Strains belonging to the Burkholderia and Enterobacter genera were the most abundant of all the Gram-negative isolates, and those belonging to the Paenibacillus and Bacillus genera were the most abundant of the Gram-positive isolates. A large number of putative PGPR belonging to different bacterial genera presented several PGP traits. Strains belonging to the Burkholderia, Chryseobacterium, and Ochrobactrum genera contributed to plant growth as well as to enhanced nutrient uptake of the rice plants in in vivo experiments. Growth and nutrient uptake of plants inoculated with isolate FeS53 (Paenibacillus sp.) in the presence of an iron excess were similar to those of plants submitted to the control iron condition, indicating that this bacterium can mitigate the effects caused by iron stress.     

Nicotianamine synthase gene expression differs in barley and rice under Fe-deficient conditions

Nicotianamine (NA) is an intermediate in the biosynthetic pathway of the mugineic acid family phytosiderophores (MAs), which are crucial components of the iron acquisition apparatus of graminaceous plants. In non-graminaceous plants, NA is thought to be an essential chelator for metal cation homeostasis. Thus NA plays a key role in Fe metabolism and homeostasis in all higher plants. Nicotianamine synthase (NAS, EC 2.5.1.43) catalyzes the trimerization of S-adenosylmethionine to form one molecule of NA. Barley, a plant that is resistant to Fe deficiency, secretes large amounts of MAs, whereas rice, a plant that is susceptible to Fe deficiency, secretes only small amounts. In this study we isolated a genomic fragment containing HvNAS1 from barley and three rice cDNA clones, osnas1, osnas2 and osnas3, from Fe-deficient rice roots. We also isolated a genomic fragment containing both OsNAS1 and OsNAS2. In contrast to barley, in which Fe deficiency induces the expression of NAS genes only in roots, Fe deficiency in rice induced NAS gene expression in both roots and chlorotic leaves. The amounts of endogenous NA in both the roots and leaves were higher than in barley. We introduced barley genomic DNA fragments containing HvNAS1 with either 9 or 2 kb of the 5'-flanking region into rice, using Agrobacterium-mediated transformation. Fe deficiency induced HvNAS1 expression in both roots and leaves of the transgenic rice, as occurs with rice NAS genes. Barley and rice NAS genes are compared in a discussion of alteration of the NAS genes during adaptation to Fe deficiency.     

A rice <Emphasis Type="Italic">YABBY</Emphasis> gene, <Emphasis Type="Italic">OsYABBY4</Emphasis>, preferentially expresses in developing vascular tissue

Developmental gene families have diversified during land plant evolution. The primary role of YABBY gene family is promoting abaxial fate in model eudicot, Arabidopsis thaliana. However recent results suggest that roles of YABBY genes are not conserved in the angiosperms. In this paper, a rice YABBY gene was isolated, and its expression patterns were analyzed in detail. Sequence characterization and phylogenetic analyses showed the gene is OsYABBY4, which is group-classified into FIL/YAB3 subfamily. Beta-glucuronidase reporter assay and in situ analysis consistently revealed that OsYABBY4 was expressed in the meristems and developing vascular tissue of rice, predominantly in the phloem tissue, suggesting that the function of the rice gene is different from those of its counterparts in eudicots. OsYABBY4 may have been recruited to regulate the development of vasculature in rice. However, transgenic Arabidopsis plants ectopically expressing OsYABBY4 behaved very like those over-expressing FIL or YAB3 with abaxialized lateral organs, suggesting the OsYABBY4 protein domain is conserved with its Arabidopsis counterparts in sequences. Our results also indicate that the functional diversification of OsYABBY4 may be associated with the divergent spatial-temporal expression patterns, and YABBY family members may have preserved different expression regulatory systems and functions during the evolution of different kinds of species.     

Rice caryopsis structure in relation to distribution of micronutrients (iron, zinc, β-carotene) of rice cultivars including transgenic indica rice

The transgenic indica rice lines of IR68144 and BR29, developed using endosperm-specific promoters were analyzed for their iron, zinc and β-carotene content in the endosperm. Biochemical analysis clearly revealed the presence of higher accumulation of iron, zinc and β-carotene in transgenic rice grains in comparison with control. Prussian blue staining reaction evidenced the presence of iron in the endosperm cells of transgenic rice grains in comparison with control where iron is restricted only to aleurone and embryo. The rice grain structure of IR64, IR72, IR68144, Swarna, BRRI Dhan 29 (BR29), BR28, Taipai 309 (T309) and New Plant Type-3 (NPT3) indicated that the number of aleurone layers, size of the embryo and size of the caryopsis determines the quantity of important micronutrients (iron, zinc) in the grains. Biochemical analysis revealed that iron and zinc content drastically varies in polished and unpolished rice and among the varieties examined. During the polishing process almost entire aleurone and most part of the embryo is removed which are the main storehouse for major micronutrients. It is estimated that more than 70% of micronutrients are lost during polishing process.     

Genetic effect on uptake of selected nutrients in some rice (O. sativa L.) varieties in phosphorus deficient soils

The influence of genes on the uptake by rice plants of certain macro- and micronutrients (calcium, magnesium, iron, manganese and zinc) was studied, by diallel (7 × 7) analysis, in P deficient upland soil. Both additive and dominant gene effects, with a preponderance of the former, were found to be responsible for the uptake of all the elements studied. Local varieties were found to be not only good yielders but also much more efficient in element uptake. Heterotic effects were observed in various crosses with respect to the uptake of all the aforementioned nutrients. Statistical analysis indicated that while the uptake of iron and zinc were negatively correlated, the uptake of manganese and calcium, manganese and zinc, calcium and magnesium and calcium and zinc were positively correlated.     

Phytosiderophore efflux transporters are crucial for iron acquisition in graminaceous plants

Eukaryotic organisms have developed diverse mechanisms for the acquisition of iron, which is required for their survival. Graminaceous plants use a chelation strategy. They secrete phytosiderophore compounds, which solubilize iron in the soil, and then take up the resulting iron-phytosiderophore complexes. Bacteria and mammals also secrete siderophores to acquire iron. Although phytosiderophore secretion is crucial for plant growth, its molecular mechanism remains unknown. Here, we show that the efflux of deoxymugineic acid, the primary phytosiderophore from rice and barley, involves the TOM1 and HvTOM1 genes, respectively. Xenopus laevis oocytes expressing TOM1 or HvTOM1 released (14)C-labeled deoxymugineic acid but not (14)C-labeled nicotianamine, a structural analog and biosynthetic precursor of deoxymugineic acid, indicating that the TOM1 and HvTOM1 proteins are the phytosiderophore efflux transporters. Under conditions of iron deficiency, rice and barley roots express high levels of TOM1 and HvTOM1, respectively, and the overexpression of these genes increased tolerance to iron deficiency. In rice roots, the efficiency of deoxymugineic acid secretion was enhanced by overexpression of TOM1 and decreased by its repression, providing further evidence that TOM1 encodes the efflux transporter of deoxymugineic acid. We have also identified two genes encoding efflux transporters of nicotianamine, ENA1 and ENA2. Our identification of phytosiderophore efflux transporters has revealed the final piece in the molecular machinery of iron acquisition in graminaceous plants.     

植物缺铁应答bHLH转录因子研究进展

铁是植物生命活动必需的微量元素之一,土壤中有效铁含量较低,易导致植物缺铁。bHLH转录因子家族多个成员参与植物缺铁响应,发挥重要的调控作用。为深入了解植物对缺铁的反应机制,文中对植物缺铁胁迫应答的bHLH转录因子的结构、分类和功能及其调控机制、介导的缺铁胁迫信号通路进行综述,为应用bHLH转录因子培育缺铁耐受作物或富铁作物提供理论依据和设计策略。