Zinc deficiency-inducible OsZIP8 encodes a plasma membrane-localized zinc transporter in rice

Zinc is an essential micronutrient for several physiological and biochemical processes. To investigate its transport in rice, we characterized OsZIP8, a rice ZIP (Zrt, Irt-like Protein) gene that is strongly up-regulated in shoots and roots under Zn deficiency. OsZIP8 could complement the growth defect of Zn-uptake yeast mutant. The OsZIP8-GFP fusion proteins were localized to the plasma membrane, suggesting that OsZIP8 is a plasma membrane zinc transporter in rice. Activation and overexpression of this gene disturbed the zinc distribution in rice plants, resulting in lower levels in shoots and mature seeds, but an increase in the roots. Field-grown transgenic plants were shorter than the WT. Under treatment with excess zinc, transgenics contained less zinc in their shoots but accumulated more in the roots. Altogether, these results demonstrate that OsZIP8 is a zinc transporter that functions in Zn uptake and distribution. Furthermore, zinc homeostasis is important to the proper growth and development of rice.     

OsZIP5 is a plasma membrane zinc transporter in rice

Zinc is essential for normal plant growth and development. To understand its transport in rice, we characterized OsZIP5, which is inducible under Zn deficiency. OsZIP5 complemented the growth defect of a yeast Zn-uptake mutant, indicating that OsZIP5 is a Zn transporter. The OsZIP5-GFP fusion protein was localized to the plasma membrane. Transgenic plants overexpressing the gene grew less well. Overexpression of the gene decreased the Zn concentration in shoots, but caused it to rise in the roots. Knockout plants showed no visible phenotypic changes under either normal or deficient conditions. However, they were tolerant to excess Zn and contained less Zn. In contrast, overexpressing transgenics were sensitive to excess Zn. These results indicate that OsZIP5 plays a role in Zn distribution within rice.     

A high activity zinc transporter OsZIP9 mediates zinc uptake in rice

Zinc (Zn) is an essential micronutrient for most organisms including humans, and Zn deficiency is widespread in human populations, particularly in underdeveloped regions. Cereals such as rice (Oryza sativa) are the major dietary source of Zn for most people. However, the molecular mechanism underlying Zn uptake in rice is still not fully understood. Here, we report that a member of the ZIP (ZRT, IRT‐like protein) family, OsZIP9, contributes to Zn uptake in rice. It was expressed in the epidermal and exodermal cells of lateral roots, localized in the plasma membrane and induced during Zn deficiency. Yeast‐expressed OsZIP9 showed much higher Zn influx transport activity than other rice ZIP proteins in a wide range of Zn concentrations. OsZIP9 knockout rice plants showed a significant reduction in growth at low Zn concentrations, but could be rescued by a high Zn supply. Compared with the wild type, accumulation of Zn in root, shoot and grain was much lower in knockout lines, particularly with a low supply of Zn under both hydroponic and paddy soil conditions. OsZIP9 also showed Co uptake activity. Natural variation of OsZIP9 expression level is highly associated with Zn content in milled grain among rice varieties in the germplasm collection. Taken together, these results show that OsZIP9 is an important influx transporter responsible for the take up of Zn and Co from external media into root cells.     

Genomic analysis and expression pattern of <Emphasis Type="Italic">OsZIP1, OsZIP3</Emphasis>, and <Emphasis Type="Italic">OsZIP4</Emphasis> in two rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) genotypes with different zinc efficiency

The ZRT-and IRT-like proteins (ZIP) comprise a large family of transition metal transporters in plants that have diverse functions to transport zinc, iron, copper, etc. Here, we provided a complete overview of this gene family in rice (Oryza sativa L.). Based on the hidden Markov model and BLAST analysis, a total of 17 ZIP-coding genes were identified and further studied by semi-quantitative RT-PCR analysis. Sequence analysis revealed 17 putative genes distributed randomly on eight chromosomes. Although most of the predicted proteins had typical characteristics of the ZIP protein family, the extent of their sequence similarity varied considerably. The expression patterns of OsZIP1, OsZIP3, and OsZIP4, which encode Zn²⁺ transporters in rice, were studied in the Zn-efficient and Zn-inefficient rice genotypes (IR8192 and Erjiufeng) by semi-quantitative RT-PCR analysis of roots, shoots, and panicle from the plants grown under Zn deficiency and normal conditions. OsZIP1 was expressed only in the roots and very weakly if at all in the panicles, while the other two genes were expressed in all parts of plants under study. The Zn-deficient conditions up-regulated the expression of OsZIP1, OsZIP3, and OsZIP4 in the roots and that of OsZIP4 in the shoots of both genotypes, indicating that all these genes may participate in rice zinc nutrition. Furthermore, the expression of OsZIP3 and OsZIP4 was found to be much stronger in the roots of IR8192 than those of Erjiufeng, which suggests that these genes may contribute to high Zn efficiency in rice. The expression patterns and the roles of other OsZIPs are also discussed on the basis of the phylogenetic tree of ZIP proteins and RT-PCR analysis of the two rice genotypes with different zinc efficiency.     

Strong expression of the rice catalase gene CatB promoter in protoplasts and roots of both a monocot and dicots.

The rice (Oryza sativa L.) catalase (EC 1.11.1.6) gene CatB is expressed in roots and cultured cells. We examined the promoter activity of its 5'-flanking region in a monocot and in two dicots. Transient expression assays in rice Oc and tobacco BY-2 suspension cell protoplasts showed that CatB's 5'-flanking DNA fragments (nucleotides -1066 to +298) had about 20 and 3-4 times as much promoter activity, respectively, as the CaMV 35S promoter. Serial deletion analyses of the CatB promoter region revealed that the shortest fragment (-56 to +298) still had about 10 times as much promoter activity as the CaMV 35S promoter in rice protoplasts. In tobacco protoplasts, the activity of the fragment (-56 to +298) was about half of the CaMV 35S promoter. Transgenic rice and Arabidopsis plants carrying GUS genes driven by the 5'-truncated CatB promoters were generated and their GUS activity was examined. The region ranging from -329 to +298 showed preferential expression in the roots of rice and Arabidopsis, and in the shoot apical meristems of Arabidopsis. In situ hybridization revealed that CatB was highly expressed in branch root primordia and root apices of rice. Fusion of the GUS gene to the region (-329 to +298) conferred strong expression in these same areas, indicating that the presence of this region was sufficient to express CatB specifically in the roots. There may be new regulatory element(s) in this region, because it contained no previously known cis-regulatory elements specific for gene expression in roots.     

Long-term expression of the uidA gene in Gladiolus plants under control of either the ubiquitin,rolD, mannopine synthase,or cauliflower mosaic virus promoters following three seasons of dormancy

UidA silencing did not occur following three seasons of dormancy for 23 independently transformed lines of Gladiolus plants carrying the bar- uidA fusion gene under control of either the cauliflower mosaic virus 35S (CaMV 35S), ubiquitin ( UBQ3), mannopine synthase ( mas2), or rolD promoters. The highest levels of GUS (beta-glucuronidase) expression were observed in callus, shoots, and roots of plants carrying the bar- uidA fusion gene under control of the CaMV 35S promoter and in shoots and roots of greenhouse-grown plants that contained the rolD promoter. There was no major difference in GUS expression when plants carrying the fusion gene driven by either the CaMV 35S, mas2, or UBQ3 promoters were grown in vitro as compared to growth in the greenhouse, although plants containing the rolD promoter expressed at 4- to 11-fold higher levels in shoots and roots, respectively, when grown in the greenhouse. The levels of GUS expression in greenhouse-grown plants were higher in roots than shoots for all four promoters. Of the 21 plants analyzed, 20 contained one to three copies of the bar- uidA fusion gene. Of the 23 plants analyzed, 11 had rearrangements of the transgene, but without apparent effects on levels of GUS expression.     

Comparing constitutive promoters using CAT activity in transgenic tobacco plants

The effectiveness of different promoters for use in transgenic tobacco was compared using a reporter gene expressing chloramphenicol acetyl transferase (CAT). Plasmids with CAT gene controlled by cauliflower mosaic virus 35S (CaMV 35S), rice actin1 (Ract1) and tobacco polyubiquitin (Tubi.u4) promoters were delivered into tobacco plants by Agrobacterium-mediated transformation. The Ract1 promoter, previously shown to be a strong promoter in rice and other monocots, failed to promote strong expression in tobacco. CAT expression was greatest from the vector carrying Tubi.u4 with a 5'UTR and leader intron without a ubiquitin monomer. In transgenic plants harboring the Tubi.u4 promoter, CAT expression was approximately twice that of the CaMV 35S promoter. Our results suggest that foreign genes under the control of a ubiquitin promoter devoid of monomer will be useful for high-level gene expression in tobacco.     

Silicon suppresses zinc uptake through down-regulating zinc transporter gene in rice

One of the beneficial effects of silicon (Si) is to improve nutrient imbalance including deficiency and excess of nutrients, however the molecular mechanisms underlying this effect are still poorly understood. In this study, we investigated the interaction between Si and zinc (Zn) in rice by using a mutant (lsi1) defective in Si uptake and its wild-type (WT, cv. Oochikara) at different Zn levels. High Zn inhibited the root elongation of both WT and lsi1 mutant, but Si did not alleviate this inhibition in both lines. By contrast, Si supply decreased Zn concentration in both the roots and shoots of the WT, but not in the lsi1 mutant. A short-term (24 h) labeling experiment with stable isotope ⁶⁷Zn showed that Si decreased ⁶⁷Zn uptake, but did not affect the root-to-shoot translocation and distribution ratio to different organs of ⁶⁷Zn in the WT. Furthermore, Si accumulated in the shoots, rather than Si in the external solution, is required for suppressing Zn uptake, but this was not caused by Si-decreased transpiration. A kinetic study showed that Si did not affect K_m value of root Zn uptake, but decreased V_max value in the WT. Analysis of genes related with Zn transport showed that among ZIP family genes, the expression of only OsZIP1 implicated in Zn uptake, was down-regulated by Si in the WT, but not in the lsi1 mutant. These results indicate that Si accumulated in the shoots suppresses the Zn uptake through down-regulating the transporter gene involved in Zn uptake in rice.     

35S驱动Pib基因启动区和编码区的转基因初步分析

将包含Pib基因启动区及下游完整编码区的9.9 kb DNA片段克隆到双元载体pPZP2Ha3(+)中, 构建了35S驱动的正义表达载体pNAR701(20.3 kb); 同时将Pib基因编码区6 986~9 392 bp之间的DNA片段, 克隆到双元载体pPZP2Ha3(-)中, 构建了35S驱动的反义表达载体pNAR703(12.8 kb); 用农杆菌介导法转入中感稻瘟病水稻品种R109中。PCR、Southern blot鉴定以及转基因T0代种子的潮霉素抗性鉴定证明, 目的基因已经整合到R109基因组中, 并能在后代稳定遗传。Northern blot分析表明含有启动区及下游完整编码的Pib基因片段在35S驱动下能够在转基因后代中表达。对T1代苗期转基因植株和分蘖期离体叶片进行抗稻瘟病初步分析, 结果显示pNAR701转基因植株对稻瘟病生理小种ZD1和ZG1的抗性较对照增强, 而转反义片段的pNAR703转基因植株对稻瘟病的抗性较对照减弱。     

Overexpression of phytochrome A enhances the light-induced formation of adventitious shoots on horseradish hairy roots

A full-length cDNA of the gene for phytochrome A from Arabidopsis thaliana was fused with the 35S promoter of cauliflower mosaic virus (CaMV35S-PHYA) and introduced into horseradish (Armoracia rusticana Gaert., Mey. et Scherb.) hairy roots. The phytochrome level in hairy roots transformed with CaMV35SPHYA was about three times greater than that in normal hairy roots and the rate of light-induced formation of adventitious shoots was also higher in the hairy roots transformed with CaMV35SPHYA. These results indicate that the light-induced formation of adventitious shoots on horseradish hairy roots is closely related to the phytochrome level. Received: 11 August 1998 / Revision received: 21 October 1998 / Accepted: 20 November 1998     

Cloning and functional identification of two members of the ZIP (Zrt, Irt-like protein) gene family in rice (Oryza sativa L.)

Two ZIP (Zrt, Irt-like Protein) cDNAs were isolated from rice (Oryza sativa L.) by RT-PCR approach, and named as OsZIP7a and OsZIP8 respectively. The predicted proteins of OsZIP7a and OsZIP8 consist of 384 and 390 amino acid residues respectively, and display high similarity to other plant ZIP proteins. Each protein contains eight transmembrane (TM) domains and a highly conserved ZIP signature motif, with a histidine-rich region in the variable region between TM domains III and IV. By semi-quantitative RT-PCR approach, it was found that the expression of OsZIP7a was significantly induced in rice roots by iron-deficiency, while that of OsZIP8 induced in both rice roots and shoots by zinc-deficiency. When expressed in yeast cells, OsZIP7a and OsZIP8 could complement an iron-uptake-deficient yeast mutant and a zinc-uptake-deficient yeast mutant respectively. It suggested that the OsZIP7a and OsZIP8 might encode an iron and a zinc transporter protein in rice respectively. Xia Yang and Ji Huang are contributed equally to this work.     

Cloning, functional characterization and heterologous expression of TaLsi1, a wheat silicon transporter gene

Silicon (Si) is known to be beneficial to plants, namely in alleviating biotic and abiotic stresses. The magnitude of such positive effects is associated with a plant's natural ability to absorb Si. Many grasses can accumulate as much as 10% on a dry weight basis while most dicots, including Arabidopsis, will accumulate less than 0.1%. In this report, we describe the cloning and functional characterization of TaLsi1, a wheat Si transporter gene. In addition, we developed a heterologous system for the study of Si uptake in plants by introducing TaLsi1 and OsLsi1, its ortholog in rice, into Arabidopsis, a species with a very low innate Si uptake capacity. When expressed constitutively under the control of the CaMV 35S promoter, both TaLsi1 and OsLsi1 were expressed in cells of roots and shoots. Such constitutive expression of TaLsi1 or OsLsi1 resulted in a fourfold to fivefold increase in Si accumulation in transformed plants compared to WT. However, this Si absorption caused deleterious symptoms. When the wheat transporter was expressed under the control of a root-specific promoter (a boron transporter gene (AtNIP5;1) promoter), a similar increase in Si absorption was noted but the plants did not exhibit symptoms and grew normally. These results demonstrate that TaLsi1 is indeed a functional Si transporter as its expression in Arabidopsis leads to increased Si uptake, but that this expression must be confined to root cells for healthy plant development. The availability of this heterologous expression system will facilitate further studies into the mechanisms and benefits of Si uptake.