Auxin signaling is closely associated with Zn-efficiency in rice (Oryza sativa L.)

This study elucidates the involvement of auxin with Zn-efficiency (ZE) in Zn-efficient rice var. Pokkali. Pokkali showed no significant decrease in morpho-physiological features, electrolyte leakage and total soluble proteins due to Zn deficiency as compared with Zn-sufficient seedlings. However, auxin inhibitor under Zn deficiency severely affected these characteristics, suggesting that ZE is associated with auxin signaling in rice. Results further revealed significant decreases in the expression of Zn transporter genes (OsIRT1, OsZIP4 and OsZIP1), OsDMAS1 (deoxymugeneic acid synthase) and phytochelatin in roots due to auxin inhibitor. It implies that auxin signaling may trigger Zn uptake, transport and chelation in rice seedlings to withstand Zn-deficiency. Further, significant reduction of major S-metabolites (cysteine, methionine, glutathione) and antioxidant enzymes (superoxide dismutase and glutathione reductase) along with increased H₂O₂ content, due to auxin inhibitor under Zn deficiency compared with controls. Taken together, these findings reveal that mechanisms associated with ZE in Pokkali are dependent on auxin signaling.     

Biochemical and molecular changes in rice seedlings (Oryza sativa L.) to cope with chromium stress

Chromium (Cr) is very toxic to both humans and plants. This investigation aimed to understand the physiological and molecular responses of rice seedlings to Cr stress. Cr toxicity did not significantly affect morphological features and Cr accumulation in roots and shoots in Pokkali but not in BRRI 51, although there was a reduction in chlorophyll concentration in leaves of both genotypes. These results imply that Pokkali has mechanisms to cope with Cr supplementation. We therefore performed quantitative real‐time PCR on the expression pattern of two chelator genes, OsPCS1 and OsMT1, but there were no significant changes in expression in roots and shoots of Pokkali and BRRI 51 following Cr stress. This suggests that there was no metal sequestration following heavy metal stress in roots of these genotypes. Moreover, no expression of two heavy metal transporter genes, OsHMA3 and OsNRAMP1, was induced after Cr stress in roots and shoots, suggesting that these transporter genes are not induced by Cr stress or might not be involved in Cr uptake in rice. We also performed a targeted study on the effect of Cr on Fe uptake mechanisms. Our studies showed a consistent reduction in Fe uptake, Fe reductase activity and expression of Fe‐related genes (OsFRO1 and OsIRT1) under Cr stress in both roots and leaves of Pokkali. In contrast, these parameters and genes were significantly increased in Cr‐sensitive BRRI 51 under Cr stress. The results confirm that limiting Fe uptake through the down‐regulation of Fe reductase and Fe transporter genes is the main strategy of Cr‐tolerant Pokkali to cope with Cr stress. Finally, increased CAT, POD and GR activity and elevated glutathione and proline synthesis might provide strong antioxidant defence against Cr stress in Pokkali. Taken together, our findings reveal that Cr stress tolerance in rice (Pokkali) is not related to metal sequestration but is associated with reduced Fe transport and increased antioxidant defence.     

Expression patterns of QTL based and other candidate genes in Madhukar × Swarna RILs with contrasting levels of iron and zinc in unpolished rice grains

Background

Identifying QTLs/genes for iron and zinc in rice grains can help in biofortification programs. Genome wide mapping showed 14 QTLs for iron and zinc concentration in unpolished rice grains of F7 RILs derived from Madhukar × Swarna. One line (HL) with high Fe and Zn and one line (LL) with low Fe and Zn in unpolished rice were compared with each other for gene expression using qPCR. 7 day old seedlings were grown in Fe + and Fe − medium for 10 days and RNA extracted from roots and shoots to determine the response of 15 genes in Fe − conditions.

Results

HL showed higher upregulation than LL in shoots but LL showed higher upregulation than HL in roots. YSL2 was upregulated only in HL roots and YSL15 only in HL shoots and both up to 60 fold under Fe − condition. IRT2 and DMAS1 were upregulated 100 fold and NAS2 1000 fold in HL shoot. NAS2, IRT1, IRT2 and DMAS1 were upregulated 40 to 100 fold in LL roots. OsZIP8, OsNAS3, OsYSL1 and OsNRAMP1 which underlie major Fe QTL showed clear allelic differences between HL and LL for markers flanking QTL. The presence of iron increasing QTL allele in HL was clearly correlated with high expression of the underlying gene. OsZIP8 and OsNAS3 which were within major QTL with increasing effect from Madhukar were 8 fold and 4 fold more expressed in HL shoot than in LL shoot. OsNAS1, OsNAS2, OsNAS3, OsYSL2 and OsYSL15 showed 1.5 to 2.5 fold upregulation in flag leaf of HL when compared with flag leaf of Swarna.

Conclusion

HL and LL differed in root length, Fe concentration and expression of several genes under Fe deficiency. The major distinguishing genes were NAS2, IRT2, DMAS1, and YSL15 in shoot and NAS2, IRT1, IRT2, YSL2, and ZIP8 in roots. The presence of iron increasing QTL allele in HL at marker locus close to genes also increased upregulation in HL.     

Deoxymugineic acid increases Zn translocation in Zn-deficient rice plants

Deoxymugineic acid (DMA) is a member of the mugineic acid family phytosiderophores (MAs), which are natural metal chelators produced by graminaceous plants. Rice secretes DMA in response to Fe deficiency to take up Fe in the form of Fe(III)–MAs complex. In contrast with barley, the roots of which secrete MAs in response to Zn deficiency, the amount of DMA secreted by rice roots was slightly decreased under conditions of low Zn supply. There was a concomitant increase in endogenous DMA in rice shoots, suggesting that DMA plays a role in the translocation of Zn within Zn-deficient rice plants. The expression of OsNAS1 and OsNAS2 was not increased in Zn-deficient roots but that of OsNAS3 was increased in Zn-deficient roots and shoots. The expression of OsNAAT1 was also increased in Zn-deficient roots and dramatically increased in shoots; correspondingly, HPLC analysis was unable to detect nicotianamine in Zn-deficient shoots. The expression of OsDMAS1 was increased in Zn-deficient shoots. Analyses using the positron-emitting tracer imaging system (PETIS) showed that Zn-deficient rice roots absorbed less ⁶²Zn-DMA than ⁶²Zn²⁺. Importantly, supply of ⁶²Zn-DMA rather than ⁶²Zn²⁺ increased the translocation of ⁶²Zn into the leaves of Zn-deficient plants. This was especially evident in the discrimination center (DC). These results suggest that DMA in Zn-deficient rice plants has an important role in the distribution of Zn within the plant rather than in the absorption of Zn from the soil. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Motofumi Suzuki and Takashi Tsukamoto equally contributed to this work.     

Oxidative Stress Tolerance Mechanism in Rice under Salinity

The research was conducted to investigate comparative oxidative damage including probable protective roles of antioxidant and glyoxalase systems in rice (Oryza sativa L.) seedlings under salinity stress. Seedlings of two rice genotypes: Pokkali (tolerant) and BRRI dhan28 (sensitive) were subjected to 8 dSm⁻¹ salinity stress for seven days in a hydroponic system. We observed significant variation between Pokkali and BRRI dhan28 in phenotypic, biochemical and molecular level under salinity stress. Carotenoid content, ion homeostasis, antioxidant enzymes, ascorbate and glutathione redox system and proline accumulation may help Pokkali to develop defense system during salinity stress. However, the activity antioxidant enzymes particularly superoxide dismutase (SOD), catalase (CAT) and non-chloroplastic peroxidase (POD) were observed significantly higher in Pokkali compared to salt-sensitive BRRI dhan28. Higher glyoxalase (Gly-I) and glyoxalase (Gly-II) activity might have also accompanied Pokkali genotype to reduce potential cytotoxic MG through non-toxic hydroxy acids conversion. However, the efficient antioxidants and glyoxalase system together increased adaptability in Pokkali during salinity stress.     

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.     

A transporter for delivering zinc to the developing tiller bud and panicle in rice

Tiller number is one of the most important agronomic traits that determine rice (Oryza sativa) yield. Active growth of tiller bud (TB) requires high amount of mineral nutrients; however, the mechanism underlying the distribution of mineral nutrients to TB with low transpiration is unknown. Here, we found that the distribution of Zn to TB is mediated by OsZIP4, one of the ZIP (ZRT, IRT-like protein) family members. The expression of OsZIP4 was highly detected in TB and nodes, and was induced by Zn deficiency. Immunostaining analysis revealed that OsZIP4 was mainly expressed in phloem of diffuse vascular bundles in the nodes and the axillary meristem. The mutation of OsZIP4 did not affect the total Zn uptake, but altered Zn distribution; less Zn was delivered to TB and new leaf, but more Zn was retained in the basal stems at the vegetative growth stage. Bioimaging analysis showed that the mutant aberrantly accumulated Zn in enlarged and transit vascular bundles of the basal node, whereas in wild-type high accumulation of Zn was observed in the meristem part. At the reproductive stage, mutation of OsZIP4 resulted in delayed panicle development, which is associated with decreased Zn distribution to the panicles. Collectively, OsZIP4 is involved in transporting Zn to the phloem of diffuse vascular bundles in the nodes for subsequent distribution to TBs and other developing tissues. It also plays a role in transporting Zn to meristem cells in the TBs.     

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.     

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.     

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.     

Activation of Rice <Emphasis Type="Italic">nicotianamine synthase 2</Emphasis> (<Emphasis Type="Italic">OsNAS2</Emphasis>) enhances iron availability for biofortification

Because micronutrients in human diets ultimately come from plant sources, malnutrition of essential minerals is a significant public health concern. By increasing the expression of nicotianamine synthase (NAS), we fortified the level of bioavailable iron in rice seeds. Activation of iron deficiency-inducible OsNAS2 resulted in a rise in Fe content (3.0-fold) in mature seeds. Its ectopic expression also increased that content. Enhanced expression led to higher tolerance of Fe deficiency and better growth under elevated pH. Mice fed with OsNAS2-D1 seeds recovered more rapidly from anemia, indicating that bioavailable Fe contents were improved by this increase in OsNAS2 expression.     

A node‐localized transporter OsZIP3 is responsible for the preferential distribution of Zn to developing tissues in rice

Developing tissues such as meristem with low transpiration require high Zn levels for their active growth, but the molecular mechanisms underlying the preferential distribution to these tissues are poorly understood. We found that a member of the ZIP (ZRT, IRT‐like protein), OsZIP3, showed high expression in the nodes of rice (Oryza sativa). Immunostaining revealed that OsZIP3 was localized at the xylem intervening parenchyma cells and xylem transfer cells of the enlarged vascular bundle in both basal and upper nodes. Neither OsZIP3 gene expression nor encoded protein was affected by either deficiency or toxic levels of Zn. Knockdown of OsZIP3 resulted in significantly reduced Zn levels in the shoot basal region containing the shoot meristem and elongating zone, but increased Zn levels in the transpiration flow. A short‐term experiment with the ⁶⁷Zn stable isotope showed that more Zn was distributed to the lower leaves, but less to the shoot elongating zone and nodes in the knockdown lines compared with the wild‐type rice at both the vegetative and reproductive growth stages. Taken together, OsZIP3 located in the node is responsible for unloading Zn from the xylem of enlarged vascular bundles, which is the first step for preferential distribution of Zn to the developing tissues in rice.     

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.

     

Bio-available zinc in rice seeds is increased by activation tagging of nicotianamine synthase

We generated rice lines with increased content of nicotianamine (NA), a key ligand for metal transport and homeostasis. This was accomplished by activation tagging of rice nicotianamine synthase 2 (OsNAS2). Enhanced expression of the gene resulted in elevated NA levels, greater Zn accumulations and improved plant tolerance to a Zn deficiency. Expression of Zn-uptake genes and those for the biosynthesis of phytosiderophores (PS) were increased in transgenic plants. This suggests that the higher amount of NA led to greater exudation of PS from the roots, as well as stimulated Zn uptake, translocation and seed-loading. In the endosperm, the OsNAS2 activation-tagged line contained up to 20-fold more NA and 2.7-fold more zinc. Liquid chromatography combined with inductively coupled plasma mass spectrometry revealed that the total content of zinc complexed with NA and 2'-deoxymugineic acid was increased 16-fold. Mice fed with OsNAS2-D1 seeds recovered more rapidly from a zinc deficiency than did control mice receiving WT seeds. These results demonstrate that the level of bio-available zinc in rice grains can be enhanced significantly by activation tagging of OsNAS2.     

The expression of iron homeostasis-related genes during rice germination

To characterize Fe homeostasis during the early stages of seed germination, a microarray analysis was performed. mRNAs extracted from fully mature seeds or seeds harvested 1–3 days after sowing were hybridized to a rice microarray containing approximately 22,000 cDNA oligo probes. Many Fe deficiency-inducible genes were strongly expressed throughout early seed germination. These results suggest that the demand for Fe is extremely high during germination. Under Fe-deficient conditions, rice produces and secretes a metal-cation chelator called deoxymugineic acid (DMA) to acquire Fe from the soil. In addition, DMA and its intermediate nicotianamine (NA) are thought to be involved in long distance Fe transport in rice. Using promoter-β-glucuronidase (GUS) analysis, we investigated the expression patterns during seed germination of the Fe deficiency-inducible genes OsNAS1, OsNAS2, OsNAS3, OsNAAT1, and OsDMAS1, which encode enzymes that participate in the biosynthesis of DMA, and the transporter genes OsYSL2 and OsIRT1, which are involved in Fe transport. All of these genes were expressed in germinating seeds prior to protrusion of the radicle. These results suggest that DMA and NA are produced and involved in Fe transport during germination. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.