Physiological and proteomic alterations in rice (Oryza sativa L.) seedlings under hexavalent chromium stress

Main Conclusion

Rice plants employ two strategies to cope with Cr toxicity: immobilizing Cr ions into cell walls to reduce its translocation and activating antioxidant defense to mitigate Cr-induced oxidative stress. The investigation aimed at understanding the physiological and proteomic responses of rice seedlings to hexavalent chromium (Cr⁶⁺) stress was conducted using two rice genotypes, which differ in Cr tolerance and accumulation. Cr toxicity (200 µM) heavily increased the accumulation of H₂O₂ and \({\text{O}}_{2}^{{ \cdot-}}\) , enhanced lipid peroxidation, decreased cell viability and consequently inhibited rice plant growth. Proteomic analyses suggest that the response of rice proteome to Cr stress is genotype- and Cr dosage-dependent and tissue specific. Sixty-four proteins, which show more than fourfold difference under either two Cr levels, have been successfully identified. They are involved in a range of cellular processes, including cell wall synthesis, energy production, primary metabolism, electron transport and detoxification. Two proteins related to cell wall structure, NAD-dependent epimerase/dehydratase and reversibly glycosylated polypeptide were greatly up-regulated by Cr stress. Their enhancements coupled with callose accumulation by Cr suggest that cell wall is an important barrier for rice plants to resist Cr stress. Some enzymes involved in antioxidant defense, such as ferredoxin-NADP reductase, NADP-isocitrate dehydrogenase, glyoxalase I (Gly I) and glutamine synthetase 1 (GS1) have also been identified in response to Cr stress. However, they were only detected in Cr-tolerant genotype, indicating the genotypic difference in the capacity of activating the defense system to fight against Cr-induced oxidative stress. Overall, two strategies in coping with Cr stress in rice plants can be hypothesized: (i) immobilizing Cr ions into cell walls to reduce its translocation and (ii) activating antioxidant defense to mitigate Cr-induced oxidative stress.     

Silicon-mediated amelioration of zinc toxicity in rice (Oryza sativa L.) seedlings

Background and aims

Silicon (Si) was suggested to enhance plant resistance to toxic elements, and its beneficial role was mainly based on external and internal plant mechanisms. This work aimed at investigating the internal effect of Si on zinc (Zn) detoxification to rice (Oryza sativa L., cv. Tian You 116) seedlings.

Methods

In a hydroponic experiment, we examined the uptake, xylem loading and localization of Zn in rice seedlings under the condition of 200?μM Zn contamination with the additional silicate supply at three levels ( 0, 0.5 and 1.8?mM).

Results

The silicate addition significantly increased the seedling biomass, and decreased Zn concentration in both root and shoot of seedlings and in xylem sap flow. Zinpyr-1 fluorescence test and Energy-dispersive X-ray spectroscopy analysis showed the concentration of biologically active Zn²⁺ decreased, and Zn and Si co-localized in the cell wall of metabolically less active tissues, especially in sclerenchyma of root. The fractionation analysis further supported silicate supply increased about 10% the cell wall bound fraction of Zn.

Conclusions

This study suggests the Si-assisted Zn tolerance of rice is mainly due to the reduction of uptake and translocation of excess Zn, and a stronger binding of Zn in the cell wall of less bioactive tissues might also contribute to some degree.     

Ammonium nutrition increases water absorption in rice seedlings (Oryza sativa L.) under water stress

Water stress is a primary limitation on plant growth. In previous studies, it has been found that ammonium enhances the tolerance of rice plants to water stress, but how water is related to nitrogen form and water stress remains unknown. To study the effects of nitrogen form (NH ₄ ⁺ , NO ₃ ^? , and a mixture of NH ₄ ⁺ and NO ₃ ^? ) on the growth and water absorption of rice (Oryza sativa L.) seedlings, a hydroponic experiment with water stress, simulated by the addition of polyethylene glycol (PEG, 10% w/v, MW 6000), was conducted in a greenhouse. The results showed that, compared with non-water stress, under water stress, the fresh weight of rice seedlings increased by 14% with NH ₄ ⁺ nutrition, whereas it had decreased by about 20% with either NO ₃ ^? or mixed nitrogen nutrition. No significant difference was found in the transpiration rate of excised shoots or in xylem exudation of excised roots in NH ₄ ⁺ supply between the two water situations, whereas xylem flow decreased by 57% and 24% under water stress in NO ₃ ^? and mixed nutrition, and root hydraulic conductivity decreased by 29% and 54% in plants in NH ₄ ⁺ and NO ₃ ^? nutrition conditions, respectively. Although water absorption ability decreased in both NH ₄ ⁺ and NO ₃ ^? nutrition, aquaporin activity was higher in NH ₄ ⁺ than in NO ₃ ^? nutrition, regardless of water stress. We conclude that NH ₄ ⁺ nutrition can improve water handling in rice seedlings and subsequently enhance their resistance to drought.     

Metabolic responses of etiolated rice (Oryza sativa L.) seedlings to streptomycin

This paper reports experiments performed to investigate the influence of various concentrations of streptomycin sulphate on a few parameters of importance in the metabolism of rice (Oryza sativa L.) seedlings. It was shown that respiration rate was accelerated by streptomycin. The specific activities of catalase and peroxidase decreased whereas IAA oxidase increased with increasing streptomycin concentrations. Increased activities of all these enzymes were apparent on a dry weight basis suggesting increased succulence caused by streptomycin treatment. There was a considerable rise in the water soluble protein content following streptomycin application. It may be suggested that growth inhibition by streptomycin results from reduction in the auxin level owing to enhanced auxin destruction.     

High temperature stress and spikelet fertility in rice (Oryza sativa L.)

In future climates, greater heat tolerance at anthesis will be required in rice. The effect of high temperature at anthesis on spikelet fertility was studied on IR64 (lowland indica) and Azucena (upland japonica) at 29.6 degrees C (control), 33.7 degrees C, and 36.2 degrees C tissue temperatures. The objectives of the study were to: (i) determine the effect of temperature on flowering pattern; (ii) examine the effect of time of day of spikelet anthesis relative to a high temperature episode on spikelet fertility; and (iii) study the interactions between duration of exposure and temperature on spikelet fertility. Plants were grown at 30/24 degrees C day/night temperature in a greenhouse and transferred to growth cabinets for the temperature treatments. Individual spikelets were marked with paint to relate fertility to the time of exposure to different temperatures and durations. In both genotypes the pattern of flowering was similar, and peak anthesis occurred between 10.30 h and 11.30 h at 29.2 degrees C, and about 45 min earlier at 36.2 degrees C. In IR64, high temperature increased the number of spikelets reaching anthesis, whereas in Azucena numbers were reduced. In both genotypesor=33.7 degrees C at anthesis caused sterility. In IR64, there was no interaction between temperature and duration of exposure, and spikelet fertility was reduced by about 7% per degrees C>29.6 degrees C. In Azucena there was a significant interaction and spikelet fertility was reduced by 2.4% degrees Cd-1 above a threshold of 33 degrees C. Marking individual spikelets is an effective method to phenotype genotypes and lines for heat tolerance that removes any apparent tolerance due to temporal escape.     

Seasonal changes in canopy photosynthesis and respiration, and partitioning of photosynthate, in rice (Oryza sativa L.) grown under free-air CO2 enrichment

An increase in atmospheric CO(2) concentration ( [CO(2)]) is generally expected to enhance photosynthesis and biomass. Rice plants (Oryza sativa L.) were grown in ambient CO(2) (AMB) or free-air CO(2)-enrichment (FACE), in which the target [CO(2)] was 200 micromol mol(-1) above AMB. (13)CO(2) was fed to the plants at different stages so we could examine the partitioning of photosynthates. Furthermore, canopy photosynthesis and respiration were measured at those stages. The ratio of (13)C content in the whole plant to the amount of fixed (13)C under FACE was similar to that under AMB at the vegetative stage. However, the ratio under FACE was greater than the ratio under AMB at the grain-filling stage. At the vegetative stage, plants grown under FACE had a larger biomass than those grown under AMB owing to enhancement of canopy photosynthesis by the increased [CO(2)]. On the other hand, at the grain-filling stage, CO(2) enrichment promoted the partitioning of photosynthate to ears, and plants grown under FACE had a greater weight of ears. However, enhancement of ear weight by CO(2) enrichment was not as great as that of biomass at the vegetative stage. Plants grown under FACE did not necessarily show higher canopy photosynthetic rates at the grain-filling stage. Therefore, we concluded that the ear weight did not increase as much as biomass at the vegetative stage owing to a loss of the advantage in CO(2) gain during the grain-filling period.     

Zinc ameliorates copper-induced oxidative stress in developing rice (Oryza sativa L.) seedlings

Rice (Oryza sativa L.) seedlings were treated with different concentrations of copper (Cu) either in presence or absence of zinc (Zn), and different events were investigated to evaluate the ameliorative effect of Zn on Cu stress. In presence of high Cu concentration, growth of both root and shoots were considerably reduced. Decline in elongation and fresh mass was observed in root and shoot. Zn alone did not show any considerable difference as compared to control, but when supplemented along with high concentration Cu, it prompted the growth of both root and shoot. After 7 days, root growth was 9.36 and 9.59 cm, respectively, at 200 and 500 μM of Cu alone as compared to 10.59 and 12.26 cm at similar Cu concentrations, respectively, in presence of Zn. Cu accumulation was considerably high after 7 days of treatment. In absence of Zn, significant accumulation of Cu was observed. Zn supplementation ameliorated the toxic impact of Cu and minimized its accumulation. Cu treatment for 1 and 7 days resulted in a dose-dependent increase in hydrogen peroxide (H₂O₂). When Cu was added in presence of Zn, the H₂O₂ production in root and shoot was reduced significantly. The increase in H₂O₂ production under Cu stress was accompanied by augmentation of lipid peroxidation. In absence of Zn, Cu alone enhanced the malondialdehyde (MDA) production in both root and shoot after 1 and 7 days of treatment. The MDA content drastically reduced in root and shoot as when Zn was added during Cu treatment. The activities of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (GPX) were elevated under Cu stress both in root and shoot. Addition of Zn further stimulated the activities of these enzymes. Both ascorbate (AsA) and glutathione (GSH) contents were high under Cu stress either in presence or absence of Zn. The results suggests that Zn supplementation improves plant survival capacity under high Cu stress by modulating oxidative stress through stimulation of antioxidant mechanisms and restricts the accumulation of toxic concentrations of Cu.     

Chloroplast downsizing under nitrate nutrition restrained mesophyll conductance and photosynthesis in rice (Oryza sativa L.) under drought conditions

The phenomenon whereby ammonium enhances the tolerance of rice seedlings (Oryza sativa L., cv. 'Shanyou 63' hybrid indica China) to water stress has been reported in previous studies. To study the intrinsic mechanism of biomass synthesis related to photosynthesis, hydroponic experiments supplying different nitrogen (N) forms were conducted; water stress was simulated by the addition of polyethylene glycol. Water stress decreased leaf water potential (Ψ(leaf)) under nitrate nutrition, while it had no negative effect under ammonium nutrition. The decreased Ψ(leaf) under nitrate nutrition resulted in chloroplast downsizing and subsequently decreased mesophyll conductance to CO(2) (g(m)). The decreased g(m) and stomatal conductance (g(s)) under nitrate nutrition with water stress restrained the CO(2) supply to the chloroplast and Rubisco. The relatively higher distribution of leaf N to Rubisco under ammonium nutrition might also be of benefit for photosynthesis under water stress. In conclusion, chloroplast downsizing induced a decline in g(m), a relatively higher decrease in g(s) under nitrate nutrition with water stress, restrained the CO(2) supply to Rubisco and finally decreased the photosynthetic rate.     

Dry matter and nitrogen accumulation and partitioning in rice (Oryza sativa L.) exposed to experimental warming with elevated CO2

Effects of elevated CO₂ concentration ([CO₂]) and air temperature (T_air) on accumulation and intra-plant partitioning of dry matter (DM) and nitrogen in paddy rice were investigated by performing a pot experiment in six natural sunlit temperature gradient chambers (TGCs) with or without CO₂ fumigation. Rice (Oryza sativa L.) plants were grown in TGCs for a whole season under two levels of [CO₂] (ambient, 380 ppm; elevated, 622 ppm) and two daily T_air regimes (ambient, 25.2°C; elevated, 27.3°C) in split-plot design with triplication. The effects of elevated [CO₂] and T_air on DM were most dramatic for grain and shoot with a significant (P?<?0.05) interaction between [CO₂] and T_air. Overall, total grain DM increased with elevated [CO₂] by 69.6% in ambient T_air but decreased with elevated T_air by 33.8% in ambient [CO₂] due to warming-induced floral sterility. Meanwhile, shoot DM significantly increased with elevated T_air by 20.8% in ambient [CO₂] and by 46.6% in elevated [CO₂]. Although no [CO₂]?×?T_air interaction was detected, the greatest total DM was achieved by co-elevation of [CO₂] and T_air (by 42.8% relative to the ambient conditions) via enhanced shoot and root DM accumulation, but not grain. This was attributed largely both to increase in tiller number and to accumulation of photosynthate in the shoot and root due to inhibition of photosynthate allocation to grain caused by warming-induced floral sterility. Distribution of N (both soil N and fertilizer ¹⁵N) among rice parts in responding to climatic variables entirely followed the pattern of DM. Our findings demonstrate that the projected warming is likely to induce a significant reduction in grain yield of rice by inhibiting DM (i.e., photosynthates) allocation to grain, though this may partially be mitigated by elevated [CO₂].     

Intraspecific variation in the response of rice (Oryza sativa L.) to increased CO2 and temperature: growth and yield response of 17 cultivars

Seventeen rice (Oryza sativa L.) cultivars of contrasting ecosystemsand origins were exposed to two CO₂ concentrations (373 [ambient]and 664 µl l^–1 CO₂ [elevated]) at two differentday/night temperatures (29/21 C and 37/29 C) in glasshousesat the International Rice Research Institute phytotron duringthe dry seasons of 1994 and 1995. Growth at elevated CO₂ (asdetermined by total plant biomass at maturity) increased byan average of 70% and 22%, respectively, for all cultivars forgrowth temperatures of 29/21 C and 37/29 C relative to theambient CO₂ treatment. At the 29/21 C optimal growth temperature,grain yield increased on average c. 50% with enriched CO₂. Incontrast, at the higher growth temperature (37/29 C), grainyield was almost zero, presumably due, in part, to temperature-inducedinfertility (i.e. the percentage of filled spikelets was <1%).Among cultivars, lAC 165, a tropical japonica from Brazil, showedthe largest relative increase in both biomass and grain yield.While the range of responses to increased CO₂ and/or temperaturewere quite large (e.g. 10–250%) and may not be applicableto field conditions, data indicate that lines are availablewhich could maximize productivity as CO₂ concentration increases.Additional work, however, would be needed to identify cultivarswhich would maintain maximum yields in a high CO₂, high temperatureenvironment. Key words: Plant growth analysis, experimental design, computational methods, relative growth rate, net assimilation rate     

Nitrate reductase of rice (Oryza sativa L.) under foot-rot disease

Summary Studies reported here reveal a low nitrate reductase activity in the shoots of MTU 9 rice plants while in roots high enzyme activity has been recorded.Under pathogenesis, a low nitrate reductase activity in the roots and a high enzyme activity in the shoots have been recorded in susceptible rice plants. In the resistant rice plants (GEB 24) no such marked difference in the enzyme activity has been observed.The effect of fusaric and gibberellic acid on this enzyme activity has been studied. In the case of fusaric acid, the nitrate reductase activity in the roots is inversely proportional to the concentration whereas in gibberellic acid, it is directly proportional to the concentration in the shoots.     

RNAi-directed down-regulation of RSV results in increased resistance in rice (Oryza sativa L.)

Rice stripe disease (RSD), caused by rice stripe virus (RSV), is a serious disease in temperate rice-growing areas. We have created an RNAi construct containing coat protein gene (CP) and disease specific protein gene (SP) sequences from RSV. The RNAi construct was transformed into two susceptible japonica varieties, Suyunuo and Guanglingxiangjing, to develop resistance against RSD. The homozygous progeny of rice plants in the T(5) and T(7) generations containing RNAi constructs, after self-fertilization were strongly resistant to viral infection. RT-PCR indicated that viral replication of SP and CP in the transgenic plants was significantly inhibited. There were no obvious morphological or developmental differences between the transgenic and wild-type plants from seedling stage to maturity. The excellent agronomic traits of these two varieties, such as high yield and good quality were maintained. Suppression of virus genes using RNAi is therefore a practical and effective strategy for controlling viral infection in crops.     

Effects of abscisic acid on ozone tolerance of rice (Oryza sativa L.) seedlings

Ozone is one of the major gaseous pollutants detrimental to crop growthand metabolism. The objective of this research was to study how ABA amelioratesthe effects of ozone on rice seedlings. Seedlings of two rice cultivars withdifferent sensitivities to ozone (Tainung 67, tolerant; and Taichung Native 1,sensitive) were treated with 400 ppb of ozone or ABA and 400ppb of ozone to determine their effect on growth, stomatalmovement, chlorophyll characteristics, and the activity of antioxidant enzymes.Activities of the enzymes SOD, APOD, GR and POD were significantly higher inthesensitive cultivar, TN 1, than in the tolerant cultivar, TNG 67. Seedlings ofthe sensitive cultivar pretreated with ABA (10 M) weresignificantly more tolerant of ozone than control seedlings. Pretreatment withABA effectively reduced stomatal conductance and the degree of injury. Abscisicacid also increased ascorbate peroxidase and glutathione reductase activity.Ozone increased peroxidase activity in sensitive seedlings, but ABA decreasedperoxidase activity. The sensitive cultivar had a higher density of stomata onits leaves than the tolerant cultivar. The results suggest that ABA inducedtolerance to ozone may be more associated with its effects on stomatal movementthan on the modulation of antioxidant enzyme activity.     

Regulation of sugar metabolism in rice (Oryza sativa L.) seedlings under arsenate toxicity and its improvement by phosphate

The effect of arsenate with or without phosphate on the growth and sugar metabolism in rice seedlings cv. MTU 1010 was studied. Arsenate was found to be more toxic for root growth than shoot growth and water content of the seedlings gradually decreased with increasing concentrations. Arsenate exposure at 20 μM and 100 μM resulted in an increase in reducing sugar content and decrease in non-reducing sugar content. There was a small increase in starch content, the activity of starch phosphorylase was increased but α-amylase activity was found to be decreased. Arsenate toxicity also affected the activities of different carbohydrate metabolizing enzymes. The activities of sucrose degrading enzymes viz., acid invertase and sucrose synthase were increased whereas, the activity of sucrose synthesizing enzyme, viz. sucrose phosphate synthase declined. The combined application of arsenate with phosphate exhibited significant alterations of all the parameters tested under the purview of arsenate treatment alone which was congenial to better growth and efficient sugar metabolism in rice seedlings. Thus, the use of phosphorus enriched fertilizers may serve to ensure the production of healthy rice plants in arsenic contaminated soils.     

Potassium nutrition of rice (Oryza sativa L.) varieties under NaCl salinity

A salt-tolerant (Pokkali) and a salt-sensitive (IR28) variety of rice (Oryza sativa L.) were grown in a phytotron to investigate the effect of K (0, 25, 50 and 75 mg K kg^–1 soil) application on their salt tolerance. Potassium application significantly increased potential photosynthetic activity (Rfd value), percentage of filled spikelets, yield and K concentration in straw. At the same time, it also significantly reduced Na and Mg concentrations and consequently improved the K/Na, K/Mg and K/Ca ratios. IR28 responded better to K application than Pokkali. Split application of K failed to exert any beneficial effect over basal application.     

Effects of elevated CO2 on plant growth and nutrient partitioning of rice (Oryza sativa L.) at rapid tillering and physiological maturity

Abstract

This work investigates the relationship between plant growth, grain yield, nutrient acquisition and partitioning in rice (Oryza sativa L.) under elevated CO₂. Plants were grown hydroponically in growth chambers with a 12-h photoperiod at either 370 or 700 µmol CO₂ mol^?1 concentration. Plant dry mass (DM), grain yield and macro- and micronutrient concentrations of vegetative organs and grains were determined. Elevated CO₂ increased biomass at tillering, and this was largely due to an increase in root mass by 160%. Elevated CO₂ had no effect on total nutrient uptake (N, P, K, Mg and Ca). However, nutrient partitioning among organs was significantly altered. N partitioning to leaf blades was significantly decreased, whereas the N partitioning into the leaf sheaths and roots was increased. Nutrient use efficiency of N, P, K, and Mg in all organs was significantly increased at elevated CO₂. At harvest maturity, grain yield was increased by 27% at elevated CO₂ while grain (protein) concentration was decreased by a similar magnitude (28%), suggesting that critical nutrient requirements for rice might need to be reassessed with global climate change.     

Genotype and environment effects on rice (Oryza sativa L.) grain arsenic concentration in Bangladesh

Genetic analysis of 38 rice varieties released by the Bangladesh Rice Research Institute (BRRI) identified 34 as indica, 2 as admixed between indica and aus, and 4 as belonging to the aromatic/Group V subpopulation. Indica varieties developed for the two major rice-growing seasons, the wet monsoon (aman) and the dry winter (boro), were not genetically differentiated. The Additive Main Effect and Multiplicative Interaction (AMMI) model was used to assess the effect of genotype (G), environment (E) and genotype-environment interaction (GEI) on grain arsenic (As) concentration when these rice varieties were grown at ten BRRI research stations located across diverse agro-ecological zones in Bangladesh. G, E and GEI, significantly influenced grain As concentration in both seasons. Overall, E accounted for 69%–80%, G 9%–10% and GEI 10%–21% of the observed variability in grain As. One site, Satkhira had the highest mean grain As concentration and the largest interaction principle component analysis (IPCA) scores in both seasons, indicating maximum interaction with genotypes. Site effects were more pronounced in the boro than in the aman season. The soil level of poorly crystalline Fe-oxide (AOFe), the ratio of AOFe to associated As, soil phosphate extractable As and soil pH were important sub-components of E controlling rice grain As concentration. Irrespective of environment, the mean grain As concentration was significantly higher in the boro (0.290 mg As kg^?1) than in the aman (0.154 mg As kg^?1) season (p?<?0.0001), though the reasons for this are unclear. Based on mean grain As concentration and stability across environments, the variety BR3 is currently the best choice for the boro season, while BR 23 and BRRI dhan 38 are the best choices for the aman season. Popular varieties BR 11 (aman) and BRRI dhan 28 and 29 (boro) had grain As concentrations close to the mean value and were fairly stable across environments, while high-yielding, short-duration aman season varieties (BRRI dhan 32, 33 and 39) developed for intensified cropping had relatively high grain As concentrations. Results suggest that genetic approaches to reducing As in rice grain will require the introduction of novel genetic variation and must be accompanied by appropriate management strategies to reduce As availability and uptake by rice.