Overexpression of rice phosphate transporter gene OsPT6 enhances phosphate uptake and accumulation in transgenic rice plants

Background and aims

Whereas the expression patterns and kinetic properties of the rice (Oryza sativa) phosphate transporter gene OsPht1; 6 (OsPT6) are well documented, little is known about the biological functions of this gene. The aim of this study was to investigate the roles of OsPT6 in inorganic phosphate (Pi) acquisition and mobilization, and examine its potential to enhance agricultural production.

Methods

Here, we generated OsPT6 overexpression transgenic plants using Wuyujing 7, a widely cultivated variety of japonica rice, and then treated transgenic lines and wild type with different Pi supply in hydroponic and soil experiments to explore the functions of OsPT6 in rice.

Results

The OsPT6-overexpressing rice lines grew better and accumulated more biomass than wild-type plants, and exhibited significant increases in P accumulation in various tissues, including reproductive tissues under both hydroponic and soil culture conditions. Phosphate-uptake experiment using radiolabeled Pi (³³P) showed that the rate of Pi uptake was 75 % and 73 % greater in transgenic plants grown under Pi-sufficient and -deficient conditions, respectively, than the wild-type controls, and that the shoot/root ratio of ³³P was 104 % and 42 % greater, respectively. In addition, the grain yield per transgenic plant was much higher than that of the wild-type plants under field conditions.

Conclusions

Taken together, our results demonstrate that OsPT6 plays a vital role in Pi acquisition and mobilization in rice and suggest that this gene may be used for genetic engineering rice plants that require less Pi fertilizer.     

Ammonium accumulation in relation to growth of rice cell suspension under phosphate deprivation

Suspension-cultured rice cells growth was markedly inhibited and ammonium content increased when rice cells were deprived of phosphate. When rice cells were cultured at increasing concentrations of ammonium chloride, ammonium content increased, however, no significant inhibition of cell growth was observed. Addition of D-arginine, an inhibitor of putrescine biosynthesis, resulted in a complete recovery of growth in rice cells under phosphate deprivation, but did not decrease the content of ammonium. Our results indicate that the growth inhibition induced by phosphate deprivation is not associated with ammonium accumulation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Differential toxicity and accumulation of inorganic and methylated arsenic in rice

Background and aims

Efficient accumulation of arsenic (As) in rice (Oryza sativa L.) poses a potential health risk to rice consumers. The aim of this study was to investigate the mechanisms of uptake, transport and distribution of inorganic arsenic (As_i) and dimethylarsinic acid (DMA) in rice plants.

Methods

Rice was exposed to As_i (As(V)) and DMA in hydroponics. High-performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS) and synchrotron X-ray fluorescence (SXRF) microprobe were used to determine As concentration and the in situ As distribution.

Results

DMA induced abnormal florets before flowering and caused a sharp decline in the seed setting rate after flowering compared to As_i. Rice grains accumulated 2-fold higher DMA than As_i. The distribution of As_i concentration (root?>?leaf?>?husk?>?caryopsis) in As(V) treatments was different from that of the DMA concentration (caryopsis?>?husk?>?root?≥?leaf) in DMA treatments. SXRF showed that As_i mainly accumulated in the vascular trace of caryopsis with limited distribution to the endosperm, whereas DMA was observed in both tissues.

Conclusions

DMA tended to accumulate in caryopsis and induced higher toxicity to the reproductive tissues resulting in markedly reduced grain yield, whereas As_i mainly remained in the vegetative tissues and had no significant effect on yield. DMA is more toxic than As_i to the reproductive tissues when both of them are at similar concentrations in nutrient solution.     

No contribution of the pentose phosphate pathway in dormancy-breaking of cocklebur seeds

The role of the oxidative pentose phosphate (PP) pathway in the dormancy-breaking of cocklebur (Xanthium pennsylvanicum Wallr.) seeds was investigated. D-[1-¹⁴C]-glucose or D-[6-¹⁴C]-glucose was fed to dormant and non-dormant lower seeds or to their axial or cotyledonary segments which were imbibed for different durations, and C₆/C₁ ratios of respired ¹⁴CO₂ as an index of the PP pathway activity were calculated. Contrary to expectation, there was no significant difference in the C₆/C₁ ratios between the dormant and non-dormant seeds or segments during a water imbition period of 24 h, although the PP pathway actually operated already in an early stage of water imbibition. Also concerning the activities of G6PDH and 6PGDH, the key enzymes of this pathway, no difference between the dormant and non-dormant seeds was found. It was thus concluded that, unlike other seeds, there is no contribution of the PP pathway to the regulation of dormancy of the cocklebur seed.     

Influence of sulphur on arsenic accumulation and metabolism in rice seedlings

The influence of sulphur on the accumulation and metabolism of arsenic in rice was investigated. Rice seedlings were grown in nutrient solutions with low sulphate (1.8 μM SO₄²⁻) or high sulphate (0.7 mM SO₄²⁻) for 12 or 14 d, before being exposed to 10 μM arsenite or arsenate for 2 or 1 d, respectively. In the arsenite exposure treatment, low sulphate-pretreated rice accumulated less arsenite than high sulphate pretreated plants, but the arsenite concentrations in shoots of low sulphate pretreated rice were higher than those of high sulphate pretreated. In the arsenate exposure treatment, the low sulphate pre-treatments also resulted in less arsenite accumulation in rice roots. Sulphur deprivation in nutrient solution decreased the concentrations of non-protein thiols in rice roots exposed to either arsenite or arsenate. The low sulphate-pretreated plants had a higher arsenic transfer factor than the high sulphate-pretreated plants. The results suggest that rice sulphate nutrition plays an important role in regulating arsenic translocation from roots to shoots, possibly through the complexation of arsenite-phytochelatins.     

Expressing ScACR3 in rice enhanced arsenite efflux and reduced arsenic accumulation in rice grains

Arsenic (As) accumulation in rice grain poses a serious health risk to populations with high rice consumption. Extrusion of arsenite [As(III)] by ScAcr3p is the major arsenic detoxification mechanism in Saccharomyces cerevisiae. However, ScAcr3p homolog is absent in higher plants, including rice. In this study, ScACR3 was introduced into rice and expressed under the control of the Cauliflower mosaic virus (CaMV) 35S promoter. In the transgenic lines, As concentrations in shoots and roots were about 30% lower than in the wild type, while the As translocation factors were similar between transgenic lines and the wild type. The roots of transgenic plants exhibited significantly higher As efflux activities than those of the wild type. Within 24 h exposure to 10 μM arsenate [As(V)], roots of ScACR3-expressing plants extruded 80% of absorbed As(V) to the external solution as As(III), while roots of the wild type extruded 50% of absorbed As(V). Additionally, by exposing the As-containing rice plants to an As-lacking solution for 24 h, about 30% of the total As derived from pre-treatment was extruded to the external solution by ScACR3-expressing plants, while about 15% of As was extruded by wild-type plants. Importantly, ScACR3 expression significantly reduced As accumulation in rice straws and grains. When grown in flooded soil irrigated with As(III)-containing water, the As concentration in husk and brown rice of the transgenic lines was reduced by 30 and 20%, respectively, compared with the wild type. This study reports a potential strategy to reduce As accumulation in the food chain by expressing heterologous genes in crops.     

Systematic engineering of phytochelatin synthesis and arsenic transport for enhanced arsenic accumulation in E. coli

Phytochelatin (PC) is a naturally occurring peptide with high affinity towards arsenic (As). In this article, we demonstrated the systematic engineering of PC‐producing E. coli for As accumulation by addressing different bottlenecks in PC synthesis as well as As transport. Phytochelatin synthase from Schizosaccharomyces pombe (SpPCS) was expressed in E. coli resulting in 18 times higher As accumulation. PC production was further increased by co‐expressing a feedback desensitized γ‐glutamylcysteine synthetase (GshI*), resulting in 30‐fold higher PC levels and additional 2‐fold higher As accumulation. The significantly increased PC levels were exploited further by co‐expressing an arsenic transporter GlpF, leading to an additional 1.5‐fold higher As accumulation. These engineering steps were finally combined in an arsenic efflux deletion E. coli strain to achieve an arsenic accumulation level of 16.8 µmol/g DCW, a 80‐fold improvement when compared to a control strain not producing phytochelatins. Biotechnol. Bioeng. 2010. 105: 780–785. © 2009 Wiley Periodicals, Inc.     

The contribution of atmospheric deposition of cadmium and lead to their accumulation in rice grains

Plant and Soil - Over-accumulation of cadmium and lead in rice grain is a global concern as it has adverse health impacts. Atmospheric deposition is an important source of heavy metal accumulation...     

Evidence for a role of phytochelatins in regulating arsenic accumulation in rice grain

Phytochelatins (PCs) play a crucial role in detoxifying cellular arsenic (As) through complexation of arsenite. Here, we investigated whether PCs influence As accumulation in rice grain by using six rice cultivars varying in grain As accumulation. The cultivars with low grain As had significantly higher PCs concentration in the shoots than the cultivars with high grain As, but lower glutathione concentration. Shoot PCs concentration correlated negatively with grain As accumulation. Foliar sprays with 0.5 mM l-buthionine-sulphoxime (BSO) on rice leaves at grain filling stage decreased GSH and PC accumulation in rice shoots by 40-63% and 20-55%, respectively, but did not significantly affect plant growth. Foliar sprays with BSO decreased shoot As concentration, while increased As concentrations in husk and brown rice significantly. These results suggest that PC complexation of arsenite in rice leaves reduces As translocation from leaves to grains, and implicate that manipulation of PC synthesis might mitigate As accumulation in rice grain.     

Silicon has opposite effects on the accumulation of inorganic and methylated arsenic species in rice

Background and aims

Rice (Oryza sativa) is a main source of human exposure to inorganic arsenic and mitigation measures are needed to decrease As accumulation in this staple crop. It has been shown that silicon decreases the accumulation of arsenite but, unexpectedly, increases the accumulation of dimethylarsinic acid (DMA) in rice grain. The aim of this study was to investigate why Si increases DMA accumulation.

Methods

Pot and incubation experiments were conducted to investigate how the addition of sparingly soluble silicate gel affected As speciation in the soil solution and the accumulation of different As species in rice tissues.

Results

Silicon addition significantly decreased the concentration of inorganic As (mainly arsenite) but increased the concentration of DMA in both the vegetative and reproductive tissues of rice. Silicon increased the concentration of DMA in the soil solution, whereas autoclaving soil decreased DMA concentration. Less DMA was adsorbed by the soil than arsenate and Si addition significantly inhibited DMA adsorption.

Conclusions

Silicon increased DMA accumulation and decreased arsenite accumulation in rice through different mechanisms. Silicic acid released from the silicate gel increased the availability of DMA for rice uptake by inhibiting DMA adsorption on the soil solid phase or by displacing adsorbed DMA. Although silicic acid also increased the concentration of inorganic As in the soil solution, this effect was much smaller than the inhibitory effect of Si on arsenite uptake by rice roots.     

Interaction of arsenic and phosphate on their uptake and accumulation in Chinese brake fern

Interactive effects of arsenate (As (V)) and phosphate (Pi) were investigated under hydroponic culture. Arsenic concentrations in fronds and roots of Chinese brake fern (Pteris vittata L.) significantly (p < 0.05) increased with increasing As (V), but decreased (p < 0.05) with increasing Pi in nutrient solution. Phosphate uptake was significantly (p < 0.05) inhibited by 1000 micromol L(-1) As (V). Under 100 micromol L(-1) As (V), frond phosphorus (P) increased at 100 and 1000 micromol L(-1) Pi, and root P increased at 250 micromol L(-1) Pi exposures. Arsenic and P concentrations in fronds and roots of Chinese brake fern were negatively correlated (p < 0.05). Arsenate treatments enhanced As and P transport to fronds, while increasing Pi inhibited their transportation, with highest frond P and As (%) obtained under 100 micromol L(-1) treatment. pH values in nutrient solution increased with increasing exposure time, but decreased with increasing Pi levels. Dissolved organic carbon (DOC) contents (dry weight) in nutrient solution decreased with increasing Pi levels, both for treatments with and without As (V). Arsenate at 1000 micromol L(-1) significantly (p < 0.05) increased DOC contents, especially for treatment without Pi. Six organic acids were detected in root exudates of Chinese brake fern, with oxalic and malic acids being most dominant.     

The effects of iron plaque and phosphorus on yield and arsenic accumulation in rice

The effects of arsenate, Fe²⁺, and phosphate on amount and composition of Fe-oxide plaque at the rice-root surface and on the yield and arsenic accumulation in rice (cv. BRRI dhan33) were studied in a replicated pot-culture experiment. Arsenic in the form of Na₂HAsO₄ was applied at concentrations of 0, 15 and 30 mg kg^?1 in combination with P and/or Fe at 0 and 50 mg kg^?1, from KH₂PO₄ and FeSO₄, respectively. Root, grain and straw yields and their As, Fe and P concentrations were determined. The Fe-oxide plaque was extracted from the plant roots using dithionite-citrate-bicarbonate (DCB) and NH₄-oxalate extractions. The addition of Fe²⁺ reduced the toxic effect of As in flooded-rice culture and resulted in reduced grain-As accumulation and increased grain yields. The effect of applied phosphate was the opposite, in that it resulted in higher As concentrations in both grain and straw and lower grain yields. The effects of both Fe and P can be explained based on their impacts on adsorption of As onto soil and rice-plaque Fe-oxides and the subsequent As solubility and availability for uptake by rice. These reactions have important implications to rice-crop management and the natural variability in soils and irrigation-water characteristics that might impact As uptake by rice.     

Mapping quantitative trait loci associated with arsenic accumulation in rice (Oryza sativa)

The quantitative trait loci (QTLs) associated with arsenic (As) accumulation in rice were mapped using a doubled haploid population established by anther culture of F1 plants from a cross between a Japonica cultivar CJ06 and an Indica cultivar TN1 (Oryza sativa). Four QTLs for arsenic (As) concentrations were detected in the map. At the seedling stage, one QTL was mapped on chromosome 2 for As concentrations in shoots with 24.4% phenotypic variance and one QTL for As concentrations in roots was detected on chromosome 3. At maturity, two QTLs for As concentrations in grains were found on chromosomes 6 and 8, with 26.3 and 35.2% phenotypic variance, respectively. No common loci were detected among these three traits. Interestingly, the QTL on chromosome 8 was found to be colocated for As concentrations in grain at maturity and shoot phosphorus (P) concentrations at seedling stage. These results provide an insight into the genetic basis of As uptake and accumulation in rice, and will be useful in identifying genes associated with As accumulation.     

Deoxyxylulose phosphate pathway to terpenoids

Recently, a mevalonate-independent pathway was discovered in bacteria and plants that leads to the formation of isopentenyl diphosphate and dimethylallyl diphosphate, the two basic precursors of isoprenoids. Although many details of the widely distributed pathway are unknown, some intermediates, mechanisms, enzymes and genes of this novel route have been identified. Information on this pathway could provide the basis for the development of new antibiotics, herbicides and antimalarials.