Reducing phosphorus accumulation in rice grains with an impaired transporter in the node.

Phosphorus is an important nutrient for crop productivity. More than 60% of the total phosphorus in cereal crops is finally allocated into the grains and is therefore removed at harvest. This removal accounts for 85% of the phosphorus fertilizers applied to the field each year. However, because humans and non-ruminants such as poultry, swine and fish cannot digest phytate, the major form of phosphorus in the grains, the excreted phosphorus causes eutrophication of waterways. A reduction in phosphorus accumulation in the grain would contribute to sustainable and environmentally friendly agriculture. Here we describe a rice transporter, SULTR-like phosphorus distribution transporter (SPDT), that controls the allocation of phosphorus to the grain. SPDT is expressed in the xylem region of both enlarged- and diffuse-vascular bundles of the nodes, and encodes a plasma-membrane-localized transporter for phosphorus. Knockout of this gene in rice (Oryza sativa) altered the distribution of phosphorus, with decreased phosphorus in the grains but increased levels in the leaves. Total phosphorus and phytate in the brown de-husked rice were 20-30% lower in the knockout lines, whereas yield, seed germination and seedling vigour were not affected. These results indicate that SPDT functions in the rice node as a switch to allocate phosphorus preferentially to the grains. This finding provides a potential strategy to reduce the removal of phosphorus from the field and lower the risk of eutrophication of waterways.

A decrease in phytic acid content substantially affects the distribution of mineral elements within rice seeds.

Phytic acid (myo-inositol hexakisphosphate; InsP6) is the storage compound of phosphorus and many mineral elements in seeds. To determine the role of InsP6 in the accumulation and distribution of mineral elements in seeds, we performed fine mappings of mineral elements through synchrotron-based X-ray microfluorescence analysis using developing seeds from two independent low phytic acid (lpa) mutants of rice (Oryza sativa L.). The reduced InsP6 in lpa seeds did not affect the translocation of mineral elements from vegetative organs into seeds, because the total amounts of phosphorus and the other mineral elements in lpa seeds were identical to those in the wild type (WT). However, the reduced InsP6 caused large changes in mineral localization within lpa seeds. Phosphorus and potassium in the aleurone layer of lpa greatly decreased and diffused into the endosperm. Zinc and copper, which were broadly distributed from the aleurone layer to the inner endosperm in the WT, were localized in the narrower space around the aleurone layer in lpa mutants. We also confirmed that similar distribution changes occurred in transgenic rice with the lpa phenotype. Using these results, we discussed the role of InsP6 in the dynamic accumulation and distribution patterns of mineral elements during seed development.

Ectopic expression of myo-inositol 3-phosphate synthase induces a wide range of metabolic changes and confers salt tolerance in rice.

Salt stress is an important factor that limits crop production worldwide. The salt tolerance of plants is a complex biological process mediated by changes in gene expression and metabolite composition. The enzyme myo-inositol 3-phosphate synthase (MIPS; EC 5.5.1.4) catalyzes the first step of myo-inositol biosynthesis, and overexpression of the MIPS gene enhances salt stress tolerance in several plant species. In this study, we performed metabolite profiling of both MIPS-overexpressing and wild-type rice. The enhanced salt stress tolerance of MIPS-overexpressing plants was clear based on growth and the metabolites under salt stress. We found that constitutive overexpression of the rice MIPS gene resulted in a wide range of metabolic changes. This study demonstrates for the first time that overexpression of the MIPS gene increases various metabolites responsible for protecting plants from abiotic stress. Activation of both basal metabolism, such as glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle, and inositol metabolism is induced in MIPS-overexpressing plants. We discuss the relationship between the metabolic changes and the improved salt tolerance observed in transgenic rice.

Overexpression of a Gene Involved in Phytic Acid Biosynthesis Substantially Increases Phytic Acid and Total Phosphorus in Rice Seeds.

The manipulation of seed phosphorus is important for seedling growth and environmental P sustainability in agriculture. The mechanism of regulating P content in seed, however, is poorly understood. To study regulation of total P, we focused on phytic acid (inositol hexakisphosphate; InsP6) biosynthesis-related genes, as InsP6 is a major storage form of P in seeds. The rice (Oryza sativa L.) low phytic acid mutant lpa1-1 has been identified as a homolog of archael 2-phosphoglycerate kinase. The homolog might act as an inositol monophosphate kinase, which catalyzes a key step in InsP6 biosynthesis. Overexpression of the homolog in transgenic rice resulted in a significant increase in total P content in seed, due to increases in InsP6 and inorganic phosphates. On the other hand, overexpression of genes that catalyze the first and last steps of InsP6 biosynthesis could not increase total P levels. From the experiments using developing seeds, it is suggested that the activation of InsP6 biosynthesis in both very early and very late periods of seed development increases the influx of P from vegetative organs into seeds. This is the first report from a study attempting to elevate the P levels of seed through a transgenic approach.

Dynamic changes in the distribution of minerals in relation to phytic acid accumulation during rice seed development.

Phytic acid (inositol hexakisphosphate [InsP(6)]) is the storage compound of phosphorus in seeds. As phytic acid binds strongly to metallic cations, it also acts as a storage compound of metals. To understand the mechanisms underlying metal accumulation and localization in relation to phytic acid storage, we applied synchrotron-based x-ray microfluorescence imaging analysis to characterize the simultaneous subcellular distribution of some mineral elements (phosphorus, calcium, potassium, iron, zinc, and copper) in immature and mature rice (Oryza sativa) seeds. This fine-imaging method can reveal whether these elements colocalize. We also determined their accumulation patterns and the changes in phosphate and InsP(6) contents during seed development. While the InsP(6) content in the outer parts of seeds rapidly increased during seed development, the phosphate contents of both the outer and inner parts of seeds remained low. Phosphorus, calcium, potassium, and iron were most abundant in the aleurone layer, and they colocalized throughout seed development. Zinc was broadly distributed from the aleurone layer to the inner endosperm. Copper localized outside the aleurone layer and did not colocalize with phosphorus. From these results, we suggest that phosphorus translocated from source organs was immediately converted to InsP(6) and accumulated in aleurone layer cells and that calcium, potassium, and iron accumulated as phytic acid salt (phytate) in the aleurone layer, whereas zinc bound loosely to InsP(6) and accumulated not only in phytate but also in another storage form. Copper accumulated in the endosperm and may exhibit a storage form other than phytate.

Molecular cloning and characterization of plasma membrane- and vacuolar-type Na⁺/H⁺ antiporters of an alkaline-salt-tolerant monocot, Puccinellia tenuiflora.

A better understanding of salt tolerance in plants might lead to the genetic engineering of crops that can grow in saline soils. Here we cloned and characterized plasma membrane and vacuolar Na⁺/H⁺ antiporters of a monocotyledonous alkaline-tolerant halophyte, Puccinellia tenuiflora. The predicted amino acid sequence of the transporters were very similar to those of orthologs in monocotyledonous crops. Expression analysis showed that (1) NHA was more strongly induced by NaCl in the roots of P. tenuiflora while in rice it was rather induced in the shoots, suggesting that the role of NHA in salt excretion from the roots partly accounts for the difference in the tolerance of the two species, and that (2) NHXs were specifically induced by NaHCO3 but not by NaCl in the roots of both species, suggesting that vacuolar-type Na⁺/H⁺ antiporters play roles in pH regulation under alkaline salt conditions. Overexpression of the antiporters resulted in increased tolerance of shoots to NaCl and roots to NaHCO3. Overexpression lines exhibited a lower Na⁺ content and a higher K⁺ content in shoots under NaCl treatments, leading to a higher Na⁺/H⁺ ratio.

A novel endosperm transfer cell-containing region-specific gene and its promoter in rice.

The endosperm of cereal grains is an important resource for both food and feed. It contains three major types of tissue: starchy endosperm, the aleurone layer, and transfer cells. To improve grain quality and quantity using molecular methods, control of transgene expression directed by distinct temporal and spatial promoter activity is necessary. To identify aleurone layer-specific and/or transfer cell-specific promoters in rice, microarray analyses were performed, comparing the aleurone layer containing transfer cells and the other reproductive and vegetative tissues. After confirmation by RT-PCR analysis, we identified two putative aleurone layer and/or transfer cell-specific genes, AL1 and AL2. The promoter regions of these genes and ß-glucuronidase (GUS) fusion constructs were stably transformed into rice. The GUS expression patterns indicated that the AL1 promoter was active exclusively in the dorsal aleurone layer adjacent to the main vascular bundle. In rice, transfer cells are differentiated in this region. Therefore, the promoter of the AL1 gene exhibits transfer cell-containing region-specific activity. The AL1 gene encodes a putative anthranilate N-hydroxycinnamoyl/benzoyltransferase. The promoter of this gene will be useful for enhancing uptake of nutrients from the mother cells and protecting filial seeds from pathogen attack.

Molecular characterization of the intercalated cell masses of the amygdala: implications for the relationship with the striatum.

The intercalated cell masses of the amygdala consist of cell clusters located between the basolateral complex of the amygdala and its surrounding structures including the central nucleus of the amygdala and the external capsule. Although recent studies have revealed that the intercalated cell masses may play an important role in emotional learning and memory, there are only a few reports on its molecular characterization. We examined the expression patterns of transcription factors in the intercalated cell masses in late embryonic stage and postnatal rats, and non-human primates. Dlx5, Foxp2, Pbx3 and Meis2 were expressed in all subdivisions of the intercalated cell masses, while Ebf1, Nkx2.1 and Foxp1 were not. In contrast, Pax6 was only expressed in a small population of the main intercalated islands, but not in the medial or lateral cell clusters. In addition, few Pax6-positive neurons co-expressed Foxp2. Thus the intercalated cell masses do not contain a homogeneous population of neurons, in terms of their molecular constituents. Given that Foxp2, Pbx3 and Meis2 are preferentially expressed in distinct cell populations in the developing striatum, and that the intercalated cell masses of the amygdala appear to be a ventrocaudal expansion of the striatum, the intercalated neurons may share a common origin with some types of neurons located in the dorsal striatum.

Differential effects of a transgene to confer low phytic acid in caryopses located at different positions in rice panicles.

In previous studies, we attempted to reduce phytic acid in rice seeds by silencing the 1d-myo-inositol 3-phosphate synthase gene, RINO1, using an antisense sequence under the control of the rice glutelin GluB-1 promoter. The stable transgenic line showed a weak low phytic acid phenotype. In this study, we show that the position of the caryopsis in the panicle might affect the level of gene silencing through a difference in temporal and spatial expression patterns between RINO1 and GluB-1 promoters, resulting in a large variation in Pi levels and a small increase in Pi in the transgenic seeds.

Generation of stable 'low phytic acid' transgenic rice through antisense repression of the 1D-myo-inositol 3-phosphate synthase gene (RINO1) using the 18-kDa oleosin promoter.

Phytic acid acts as the major storage form of phosphorus in plant seeds and is poorly digested by monogastric animals. The degradation of phytic acid in animal diets is necessary to overcome both environmental and nutritional issues. The enzyme 1D-myo-inositol 3-phosphate [Ins(3)P(1)] synthase (EC 5.5.1.4) catalyses the first step of myo-inositol biosynthesis and directs phytic acid biosynthesis in seeds. The rice Ins(3)P(1) synthase gene (RINO1) is highly expressed in developing seed embryos and in the aleurone layer, where phytic acid is synthesized and stored. In rice seeds, 18-kDa oleosin (Ole18) is expressed in a seed-specific manner, and its transcripts are restricted to the embryo and the aleurone layer. Therefore, to effectively suppress phytic acid biosynthesis, antisense RINO1 cDNA was expressed under the control of the Ole18 promoter, directing the same spatial pattern in seeds as RINO1 in transgenic rice plants. The generated transgenic rice plants showed strong 'low phytic acid' (lpa) phenotypes, in which seed phytic acid was reduced by 68% and free available phosphate was concomitantly increased. No negative effects on seed weight, germination or plant growth were observed. The available phosphate levels of the stable transgenic plants surpassed those of currently available rice lpa mutants.

Expression pattern of inositol phosphate-related enzymes in rice (Oryza sativa L.): implications for the phytic acid biosynthetic pathway.

Phytic acid, myo-inositol-hexakisphosphate (InsP(6)), is a storage form of phosphorus in plants. Despite many physiological investigations of phytic acid accumulation and storage, little is known at the molecular level about its biosynthetic pathway in plants. Recent work has suggested two pathways. One is an inositol lipid-independent pathway that occurs through the sequential phosphorylation of 1D-myo-inositol 3-phosphate (Ins(3)P). The second is a phospholipase C (PLC)-mediated pathway, in which inositol 1,4,5-tris-phosphate (Ins(1,4,5)P(3)) is sequentially phosphorylated to InsP(6). We identified 12 genes from rice (Oryza sativa L.) that code for the enzymes that may be involved in the metabolism of inositol phosphates. These enzymes include 1D-myo-inositol 3-phosphate synthase (MIPS), inositol monophosphatase (IMP), inositol 1,4,5-tris-phosphate kinase/inositol polyphosphate kinase (IPK2), inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1), and inositol 1,3,4-triskisphosphate 5/6-kinase (ITP5/6K). The quantification of absolute amounts of mRNA by real-time RT-PCR revealed the unique expression patterns of these genes. Outstanding up-regulation of the four genes, a MIPS, an IPK1, and two ITP5/6Ks in embryos, suggested that they play a significant role in phytic acid biosynthesis and that the lipid-independent pathway was mainly active in developing seeds. On the other hand, the up-regulation of a MIPS, an IMP, an IPK2, and an ITP5/6K in anthers suggested that a PLC-mediated pathway was active in addition to a lipid-independent pathway in the anthers.

Evidence for an evolutionary force that prevents epigenetic silencing between tail-to-tail rice genes with a short spacer.

During the course of evolution, the genome should have toned down various types of genomic noise, such as those that cause the unstable expression or gene silencing observed in transgenic organisms. We found a rice genomic segment where two genes, encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) and ribosomal protein small subunit 20 (rps20), are located in a tail-to-tail orientation and separated by only 300 bp of spacer. It is possible that this kind of structure would give rise to unstable expression due to antisense RNA derived from the neighboring gene. We examined this possibility using Northern blot, reverse transcription-polymerase chain reaction (RT-PCR), and 3' RACE analyses, but obtained no evidence for instability or antisense RNAs of these housekeeping genes. Comparison of the sequences in the corresponding regions among related rice species revealed a lower level of genetic divergence of both the 3'-untranslated region (3'-UTRs) than of the other noncoding regions; in particular both of the boundaries between the 3'-UTRs and the spacer were markedly conserved. The conservation of both the terminal regions is most likely the result of purifying selection, implying a functional role for the strict termination of the transcription of these genes to prevent gene-silencing-related events.

The synergistic effects of sugar and abscisic acid on myo-inositol-1-phosphate synthase expression.

1L-myo-inositol-1-phosphate [Ins(1)P1] synthase (EC 5.5.1.4) catalyses the formation of Ins(1)P1 from glucose-6-phosphate, the first step in the biosynthesis of myo-inositol. Ins(1)P1 is a precursor of phytin (inositol hexakisphosphate), a storage form of phosphate and cations in seeds. Since sucrose and abscisic acid (ABA) are known to affect synthesis of storage compounds in seeds, we investigated the effects of ABA and sucrose on Ins(1)P1 synthase gene (RINO1) expression in cultured cells derived from the scutellum of mature rice seeds. Higher levels of RINO1 transcript accumulation were evident after treatment with either sucrose (10-100 mM) or ABA (10-8 M to 10-4 M). Glucose was also effective in the upregulation, whereas mannitol was not, suggesting that sucrose and glucose acted as metabolizable sugars and not as osmotica. Treatment with ABA and sucrose together resulted in much higher levels of transcript accumulation, suggesting a synergistic induction of the Ins(1)P1 synthase gene.

The effect of cis-Diamminedichloroplatinum II on Na+ and K+ transport in the rabbit cortical collecting duct.

cis-Diamminedichloroplatinum II (CDDP) is an antineoplastic drug against solid malignant tumors. However, its clinical use is limited by nephrotoxicity. CDDP also causes hypokalemia and in vivo microperfusion method have demonstrated that luminal CDDP increases K+ secretion by hyperpolarization of the transepithelial voltage difference through stimulating Na+ transport in the distal segments. However, there is no direct evidence for this mechanism. We therefore examined the effect of luminal CDDP on Na+ and K+ transport in the rabbit cortical collecting duct (CCD) using in vitro isolated tubular microperfusion. Luminal CDDP hyperpolarized the transepithelial voltage difference (V(T)) in a dose-dependent manner at concentrations from 10(-5) M to 10(-3) M and at 10(-3) M CDDP, V(T) was hyperpolarized from -11.6+/-2.3 mV to -16.6+/-3.3 mV (P<0.001). A concentration of 10(-5) M ouabain, 10(-4) M amiloride and 2 mM BaCl2 all completely abolished CDDP-induced hyperpolarization. To confirm the mechanism, Na+ and K+ flux were measured in the presence of 10(-3) M CDDP. CDDP decreased net K+ secretion from -22.2+/-5.7 to -15.2+/-2.9 pmol mm(-1) min(-1) (P<0.01) without any effect on the lumen-to-bath isotope flux of Na+ (52.6+/-10.6 to 52.1+/-10.7 pmol mm(-1) min(-1)). These data suggest that luminal CDDP hyperpolarizes V(T) primarily by inhibiting K+ conductance but did not influence Na+ transport of the luminal membrane. We conclude that the CCD does not play a role in CDDP-induced hypokalemia when CDDP is applied from the luminal side.