Salinity induced oxidative stress and antioxidant system in salt-tolerant and salt-sensitive cultivars of rice (Oryza sativa L.)

Indices of oxidative stress viz., superoxide radical and H₂O₂ content increased in leaves of all the cultivars with the rise in salinity level, the increase was more pronounced and significant in salt-sensitive varieties and non-significant in resistant cultivars. Except for glutathione reductase (GR), basal activities of all other antioxidative enzymes viz. superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX) and glutathione reductase (GR) were significantly higher in leaves of all the resistant cultivars as compared to the sensitive ones. A differential response of salinity was observed on various enzymatic and non-enzymatic components of antioxidant system in leaves of salt-tolerant and salt-sensitive cultivars of rice (Oryza sativa L.). Activities of superoxide dismutase and glutathione reductase enhanced in all the tolerant cultivar while declined in the sensitive cultivars with increasing salinity from 0 to 100 mM. Salt-stress induced the activities of catalase and peroxidase in all the cultivars but the magnitude of increase was more pronounced in the sensitive cultivars than in the tolerant cultivars. Contrarily, APX activity increased in the salt-sensitive cultivars but showed no significant change in the salt-tolerant cultivars. The amount of ascorbic acid content, reduced glutathione (GSH), reduced/oxidized glutathione (GSSG) ratio was higher in leaves of the tolerant cultivars than that of the sensitive cultivars under saline conditions. It is inferred that leaves of salt-tolerant cultivars tend to attain greater capacity to perform reactions of antioxidative pathway under saline conditions to combat salinity-induced oxidative stress.     

Cold-induced cytosolic free calcium ion concentration changes in wheat

Relatively little is known about changes in the cytosolic free calcium ion concentration ([Ca²⁺]_c) in monocotyledonous plants. Therefore, we produced transgenic winter wheat lines stably expressing the calcium-sensitive photoprotein aequorin constitutively in the cytosol. [Ca²⁺]_c was detected in vivo by luminometry, and [Ca²⁺]_c elevations were imaged at video rate. Experiments with the transgenic seedlings focused on potential changes in [Ca²⁺]_c during cold exposure. Temperature-induced changes in [Ca²⁺]_c were found to be more dependent on the change in temperature (dT dt⁻¹) than on the absolute value of temperature. [Ca²⁺]_c increased only at cooling rates higher than 8°C min⁻¹, indicating that an overall cellular [Ca²⁺]_c increase is of minor relevance as a signal for cold acclimation in wheat under ecological conditions. The results are discussed with regard to the so-called ‘calcium signature hypothesis’.     

Aluminium induces rapid changes in cytosolic pH and free calcium and potassium concentrations in root protoplasts of wheat (Triticum aestivum)

Addition of aluminium chloride (50 μM Al) caused different effects on the transmembrane electrical potential (PD) of root cells in Al-tolerant wheat (Triticum aestivum) cv. Kadett and Al-sensitive cv. WW 20299. As changes in PD of plant cells may depend on transient fluxes of protons, potassium and/or calcium through cell membranes, the effect of Al was investigated on the cytosolic concentrations of these ions in protoplasts isolated from root tips of the same cultivars. The tetra[acetoxymethyl] esters of the fluorescent dyes bis-carboxyethyl-carboxyfluorescein, BCECF, K⁺-binding benzofuran isophthalate, PBFI, and the stilbene chromophore Fura 2-AM were used to determine pH, K⁺ and Ca²⁺, respectively. Changes in fluorescence ratios, directly reflecting changes in [H⁺], [K⁺] and [Ca²⁺] in the cytosol, were determined by photometry fluorescence microscopy. Additions and removals of Al to and from both cultivars caused hyperpolarizations and depolarizations, respectively, but only in the sensitive cv. WW 20299 did the resting PD decrease gradually. Addition of Al to the protoplasts caused rapid changes in cytosolic pH, free [K⁺] and [Ca²⁺]. In both cultivars Al caused a transient oscillating increase in cytosolic [Ca²⁺] for 1 or 2 min and a rapid pH-dependent change in cytosolic [K⁺]. At pH 5 the presence of K⁺ in the medium diminished the Al-induced decrease in cytosolic [K⁺]. Aluminium (50 μM) induced a transient increase in cytosolic [H⁺] (pH decreased) in both cultivars, but the cytosolic pH returned to its initial value only in the Al-tolerant cv. Kadett. In the Alsensitive cv. WW 20299, repeated additions of Al caused a gradual decline in pH. Moreover, in the presence of 1 mM KCl, pH recovered completely in both cultivars. Since only the effect on pH differed in the two cultivars, the more toxic effect of Al on the cv. WW 20299 should be related to the change in pH.     

A comparison of nitrate transport in four different rice(Oryza sativa L.) cultivars

As rice can use both nitrate (NO3-) and ammonium (NH4+), we have tested the hypothesis that the shift in the pattern of cultivars grown in Jiangsu Province reflects the ability of the plants to exploit NO3- as a nitrogen (N) source. Four rice cultivars were grown in solution culture for comparison of their growth on NO3- and NH4+ nitrogen sources. All four types of rice,Xian You 63 (XY63), Yang Dao 6 (YD), Nong Keng 57 (NK) and Si You 917 (SY917), grew well and produced similar amounts of shoot biomass with 1 mmol/L NH4+ as the only N source.However, the roots of NK were significantly smaller in comparison with the other cultivars. When supplied with 1 mmol/L NO3-, YD produced the greatest biomass; while NK achieved the lowest growth among the four cultivars. Electrophysiological measurements on root rhizodermal cells showed that the NO3--elicited changes in membrane potential (ΔEm) of these four rice cultivars were significantly different when exposed to low external NO3- (＜1 mmol/L); while they were very similar at high external NO3- (10 mmol/L). The root cell membrane potentials of YD and XY63 were more responsive to low external NO3- than those of NK and SY917. The ΔEm values for YD and XY63 rhizodermal cells were almost the same at both 0.1 mmol/L and 1 mmol/L NO3-;while for the NK and SY917 the values became larger as the external NO3- increased. For YD cultivar, ΔEm was measured over a range of NO3- concentrations and a Michaelis-Menten fit to the data gave a Km value of 0.17 mmol/L. Net NO3- uptake depletion kinetics were also compared and for some cultivars (YD and XY63) a single-phase uptake system with first order kinetics best fitted the data; while other cultivars (ND and SY917) showed a better fit to two uptake systems. These uptake systems had two affinity ranges: one had a similar Km in all the cultivars (0.2 mmol/L); the other much higher affinity system (0.03 mmol/L) was only present in NK and SY917. The expression pattern of twelve different NO3- transporter genes was tested using specific primers, but only OsNRT1.1 and OsNRT2. 1 expression could be detected showing significant differences between the four rice cultivars. The results from both the physiological and molecular experiments do provide some support for the hypothesis that the more popular rice cultivars grown in Jiangsu Province may be better at using NO3- as an N source.     

A comparison of nitrate transport in four different rice (Oryza sativa L.) cultivars

As rice can use both nitrate (NO ⁻₃ ) and ammonium (NH ⁺₄ ), we have tested the hypothesis that the shift in the pattern of cultivars grown in Jiangsu Province reflects the ability of the plants to exploit NO ⁻₃ as a nitrogen (N) source. Four rice cultivars were grown in solution culture for comparison of their growth on NO ⁻₃ and NH ⁺₄ nitrogen sources. All four types of rice, Xian You 63 (XY63), Yang Dao 6 (YD), Nong Keng 57 (NK) and Si You 917 (SY917), grew well and produced similar amounts of shoot biomass with 1 mmol/L NH ⁺₄ as the only N source. However, the roots of NK were significantly smaller in comparison with the other cultivars. When supplied with 1 mmol/L NO ⁻₃ , YD produced the greatest biomass; while NK achieved the lowest growth among the four cultivars. Electrophysiological measurements on root rhizodermal cells showed that the NO ⁻₃ -elicited changes in membrane potential (†E _m) of these four rice cultivars were significantly different when exposed to low external NO ⁻₃ (<1 mmol/L); while they were very similar at high external NO ⁻₃ (10 mmol/L). The root cell membrane potentials of YD and XY63 were more responsive to low external NO ⁻₃ than those of NK and SY917. The †E _m values for YD and XY63 rhizodermal cells were almost the same at both 0.1 mmol/L and 1 mmol/L NO ⁻₃ ; while for the NK and SY917 the values became larger as the external NO ⁻₃ increased. For YD cultivar, †E _m was measured over a range of NO ⁻₃ concentrations and a Michaelis-Menten fit to the data gave aK _m value of 0.17 mmol/L. Net N ⁻₃ uptake depletion kinetics were also compared and for some cultivars (YD and XY63) a single-phase uptake system with first order kinetics best fitted the data; while other cultivars (ND and SY917) showed a better fit to two uptake systems. These uptake systems had two affinity ranges: one had a similarK _m in all the cultivars (0.2 mmol/L); the other much higher affinity system (0.03 mmol/L) was only present in NK and SY917. The expression pattern of twelve different N ⁻₃ transporter genes was tested using specific primers, but onlyOsNRT1. 1 andOsNRT2.1 expression could be detected showing significant differences between the four rice cultivars. The results from both the physiological and molecular experiments do provide some support for the hypothesis that the more popular rice cultivars grown in Jiangsu Province may be better at using NO ⁻₃ as an N source.

     

A comparison of nitrate transport in four different rice(Oryza sativa L.) cultivars

As rice can use both nitrate (NO3-) and ammonium (NH4+), we have tested the hypothesis that the shift in the pattern of cultivars grown in Jiangsu Province reflects the ability of the plants to exploit NO3- as a nitrogen (N) source. Four rice cultivars were grown in solution culture for comparison of their growth on NO3- and NH4+ nitrogen sources. All four types of rice, Xian You 63 (XY63), Yang Dao 6 (YD), Nong Keng 57 (NK) and Si You 917 (SY917), grew well and produced similar amounts of shoot biomass with 1 mmol/L NH4+ as the only N source. However, the roots of NK were significantly smaller in comparison with the other cultivars. When supplied with 1 mmol/L NO3- YD produced the greatest biomass; while NK achieved the lowest growth among the four cultivars. Electrophysiological measurements on root rhizodermal cells showed that the NO3- elicited changes in membrane potential (△Em) of these four rice cultivars were significantly different when exposed to low external NO3- (<1 mmol/L); while they were very similar at high external NO3- (10 mmol/L). The root cell membrane potentials of YD and XY63 were more responsive to low external NO3- than those of NK and SY917. The△Em values for YD and XY63 rhizodermal cells were almost the same at both 0.1 mmol/L and 1 mmol/L NO3-; while for the NK and SY917 the values became larger as the external NO3- increased. For YD cultivar,△Em was measured over a range of NO3- concentrations and a Michaelis-Menten fit to the data gave a Km value of 0.17 mmol/L. Net NO3- uptake depletion kinetics were also compared and for some cultivars (YD and XY63) a single-phase uptake system with first order kinetics best fitted the data; while other cultivars (ND and SY917) showed a better fit to two uptake systems. These uptake systems had two affinity ranges: one had a similar Km in all the cultivars (0.2 mmol/L); the other much higher affinity system (0.03 mmol/L) was only present in NK and SY917. The expression pattern of twelve different NO3- transporter genes was tested using specific primers, but only OsNRT1.1 and OsNRT2.1 expression could be detected showing significant differences between the four rice cultivars. The results from both the physiological and molecular experiments do provide some support for the hypothesis that the more popular rice cultivars grown in Jiangsu Province may be better at using NO3- as an N source.     

Erythropoietin increases cytosolic free calcium concentration and thrombin induced changes in cytosolic free calcium in platelets from spontaneously hypertensive rats

Using fura-2 cytosolic free calcium concentrations were measured in intact washed platelets from 9 spontaneously hypertensive rats (SHR) and from 9 age-matched normotensive Wistar-Kyoto rats (WKY). In resting platelets cytosolic free calcium concentration was significantly higher in SHR than in WKY (171.8 +/- 64.4 nM vs 93.1 +/- 59.0 nM, p less than 0.05). After preincubation with erythropoietin cytosolic free calcium concentration was significantly higher in SHR than in WKY (197.5 +/- 83.2 vs 93.0 +/- 60.1, p less than 0.01). Using platelets from SHR erythropoietin increased mean resting cytosolic free calcium concentration by 14.9% (p less than 0.05) and mean thrombin induced changes of cytosolic free calcium by 58.3% (p less than 0.01). In contrast, erythropoietin caused no significant increase in the resting calcium concentration or in thrombin induced changes of cytosolic free calcium in platelets from WKY. It is concluded that erythropoietin is involved in the pathogenesis of hypertension by elevating cytosolic free calcium concentration.     

Efficient transformation of Korean rice cultivars (Oryza sativa L) mediated byAgrobacterium tumefaciens

The purpose of this study was to improve transformation efficiency for three Korean rice cultivars, Ilpum, Dasan, and Namyang. Using two different media with or without light, efficiencies of callus induction, regeneration, and transformation of the Korean cultivars were compared to Japanese cultivar, Nipponbare, as a control. Immature cv. Nipponbare seeds produced 35.5% and 16.1% regeneration efficiency on CIM and N6D media, respectively. Among the Korean cultivars, only cv. Ilpum induced on CIM in the dark was actively regenerated with efficiency of 8.2%. With LBA4404 (pTOK233), no difference for the efficiency of transformation was found between mature and immature seeds of cv. Ilpum. This result reveals that mature seeds can be substituted for this study with no difference. The anther-derived calli of cv. Namyang inoculated with either LBA4404 (pTOK233) or EHA101 (pSMABuba) showed regeneration efficiencies of 14.5% and 20.9%, respectively, even though efficiency of transformation did not differ with these two vectors. We suggest that the anther-derived calli are better-materials for transformation experiment due to their genotype-independent regeneration. In the assay of GUS, all of the calli that survived on the second selection medium were strongly stained. PCR-Southern blot analyses confirmed that T-DNA was stably transformed into all tissues selected. Cvs. Nipponbare and Namyang transformed by LBA4404 (pTOK233) showed positive color in the NPTII ELISA.     

Gene cloning and expression of cytosolic glutathione reductase in rice (Oryza sativa L.)

We have isolated a cDNA (RGRC2) encoding glutathione reductase (GR) from rice (Oryza sativa L.). The comparison of deduced amino acid sequences from RGRC2 and other plant GR cDNAs indicated that RGRC2 encodes a putative cytosolic isoform. The recombinant RGRC2 protein had enzymatic properties comparable to those of GR from rice embryo. Subcellular fractionation showed that the RGRC2 protein is localized primarily in cytosol. mRNA and protein of RGRC2 were observed mainly in roots and calli but little in leaf tissues. Southern blot analysis showed that the RGRC2 gene exists as a single copy gene. Here, we have also isolated a genomic clone completely corresponding to RGRC2. The RGRC2 gene is split into 16 exons spread about 7.4 kb of chromosomal DNA, with coding sequence beginning in the 2nd exon and ending in the 16th exon. From the presence of two ABA-responsive elements in the 5'-flanking region of RGRC2, we examined the expression in rice seedlings treated with ABA and the ABA-related environmental stresses, chilling, drought and salinity. The expression of RGRC2 was strongly induced by all these treatments. We suggest that the expression of the rice cytosolic GR gene is regulated via ABA-mediated signal transduction pathway under environmental stresses.     

Oxygen dynamics in submerged rice (Oryza sativa)

Complete submergence of plants prevents direct O(2) and CO(2) exchange with air. Underwater photosynthesis can result in marked diurnal changes in O(2) supply to submerged plants. Dynamics in pO(2) had not been measured directly for submerged rice (Oryza sativa), but in an earlier study, radial O(2) loss from roots showed an initial peak following shoot illumination. O(2) dynamics in shoots and roots of submerged rice were monitored during light and dark periods, using O(2) microelectrodes. Tissue sugar concentrations were also measured. On illumination of shoots of submerged rice, pO(2) increased rapidly and then declined slightly to a new quasi-steady state. An initial peak was evident first in the shoots and then in the roots, and was still observed when 20 mol m(-3) glucose was added to the medium to ensure substrate supply in roots. At the new quasi-steady state following illumination, sheath pO(2) was one order of magnitude higher than in darkness, enhancing also pO(2) in roots. The initial peak in pO(2) following illumination of submerged rice was likely to result from high initial rates of net photosynthesis, fuelled by CO(2) accumulated during the dark period. Nevertheless, since sugars decline with time in submerged rice, substrate limitation of respiration could also contribute to morning peaks in pO(2) after longer periods of submergence.     

High content of cytosolic glutamine synthetase does not accompany a high activity of the enzyme in rice (Oryza sativa) leaves of indica cultivars

Reproductive stages of 5 japonica, 9 indica, and 2 javanica cultivars of rice ( Oryza sativa L.) were provided to compare the contents of protein for cytosolic glutamine synthetase (GS1; EC 6.3.1.2) in the lowest position of the attached leaf blade (position 6 from the primary leaf) and those for NADH-glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in non-green portion of the expanding 10th leaf blade. Some of the indica cultivars, including Kasalath, contained GS1 protein twice as high as other japonica and javanica cultivars based on total leaf nitrogen. Most of the indica cultivars, on the other hand, contained less NADH-GOGAT protein than japonica and javanica cultivars. Immunostaining proved that GS1 protein was located in vascular tissues of the leaf blades of Kasalath, which was identical to our previous results with a japonica cultivar [Sakurai et al. (1996) Planta 200: 306–311]. Although relative contents of GS1 protein in the leaf blade of Kasalath increased as a function of leaf age, GS1 activity remained relatively constant. In addition, Kasalath showed lower activity than other japonica and javanica cultivars, especially during leaf expansion. GS1 activity, based on GS1 protein amount, changed during the life span of the leaf blade and we thus assume that GS1 activity was modulated post-translationally in rice leaves.     

Uptake of sodium in protoplasts of salt-sensitive and salt-tolerant cultivars of rice, Oryza sativa L. determined by the fluorescent dye SBFI

In this study, the uptake of Na+ into the cytosol of rice (Oryza sativa L. cvs Pokkali and BRRI Dhan29) protoplasts was measured using the acetoxy methyl ester of the fluorescent sodium-binding benzofuran isopthalate, SBFI-AM, and fluorescence microscopy. By means of inhibitor analyses the mechanisms for uptake and sequestration of Na+ in the salt-sensitive indica rice cv. BRRI Dhan29 and in the salt-tolerant indica rice cv. Pokkali were detected. Less Na+ was taken up into the cytosol of Pokkali than into BRRI Dhan29. The results indicate that K+-selective channels do not contribute to the Na+ uptake in Pokkali, whereas they are the major pathways for Na+ uptake in BRRI Dhan29 along with non-selective cation channels. However, non-selective cation channels seem to be the main pathways for Na+ uptake in Pokkali. Protoplasts from Pokkali leaves took up Na+ only transiently in the presence of extracellular Na+ at 5-100 mM. Therefore, it is likely that the protoplasts have a mechanism for fast extrusion of Na+ out of the cytoplasm. Experiments with protoplasts pretreated with NH4NO3 and NH4VO3 suggest that the salt-tolerant Pokkali extrudes Na+ mainly into the vacuole. After cultivation of both cultivars in the presence of 10 or 50 mM NaCl for 72 h, the isolated protoplasts from Pokkali took up less Na+ than the control protoplasts. The results suggest that the salt-tolerance in Pokkali depends on reduced uptake through K+-selective channels and a fast extrusion of Na+ into the vacuoles.     

Aluminium effects on growth, nutrient net uptake and transport in 3 rice (Oryza sativa) cultivars with different sensitivity to aluminium

Varietal differences in net nutrient uptake rate and transport efficiency in the presence of aluminium have seldom been investigated in rice. Therefore, effects of Al on growth, uptake and transport of macronutrients (K, P, Ca, and Mg) and micronutrients (Fe, Zn, Cu, and Mn) were evaluated in 3 rice cultivars (BG35, DA14 and IR45) with different Al sensitivity. The plants were grown in nutrient solution at pH 4.1. An initial growth was completed in the time interval 1 to 5 days immediately before the addition of Al. The final growth period with Al (0, 140, 280 or 560 μ M ) was completed on day 26. With Al, a comparatively high P accumulation occurred in shoots and roots of the Al tolerant cultivar BG35. In contrast, the Al sensitive cultivar IR45 maintained a relatively high Ca accumulation during the Al treatment. A reduced total net uptake rate of P and Ca by IR45 in the time period 5 to 26 days was due to both a reduced root fresh weight and a reduced net uptake rate per g fresh weight of root. Moreover, net Ca transport to the shoots higher than net uptake rate in DA14 and IR45 at > 140 μ M Al during the test period suggests restricted Ca uptake by the roots in combination with a continuous net loss of Ca from the roots to the shoots as time proceeds. In the case of Mg and Mn, there was a general reduction of net uptake rates, irrespective of Al sensitivity of cultivars. With Al treatment, comparatively high accumulation of Fe, Zn and Cu occurred in the roots of IR45, concomitant with a high net Zn and Cu uptake rate. It is concluded that differences in Al sensitivity among rice cultivars BG35, DA14 and IR45 are not primarily linked to the depressed internal Mg or Mn status of the plants but rather to changes in the uptake and distribution of Ca and P.     

Chromosomal regions with quantitative trait loci controlling cadmium concentration in brown rice (Oryza sativa)

A novel mapping population consisting of 39 chromosome segment substitution lines (CSSLs) was used to locate the putative quantitative trait loci (QTLs) for cadmium (Cd) concentration in brown rice (Oryza sativa). The mapping population carried a single chromosome segment of 'Kasalath' (indica) in each line overlapping with neighbouring segments in a 'Koshihikari' (japonica) genetic background. The parents and CSSLs were grown in pots filled with Cd-polluted soil until grain filling. The brown rice of three of the 39 CSSLs had significantly lower Cd concentrations than that of Koshihikari, and the brown rice of a further three had significantly higher concentrations. On the basis of graphical genotypes of CSSLs, putative QTLs controlling the Cd concentration in brown rice were detected on chromosomes 3, 6 and 8. Each of the CSSLs was nearly isogenic to Koshihikari, which is the most popular rice cultivar in Japan: they carried > 90% of the Koshihikari genetic background. Therefore, the development of a new Koshihikari with less Cd concentration in brown rice would be feasible in the near future.     

Expression profiling of Oryza sativa metal homeostasis genes in different rice cultivars using a cDNA macroarray.

Rice is an important food crop, but it is a poor source of essential micronutrients such as iron and zinc. In order to improve the metal ion content of rice grains through breeding or biotechnology, more information is needed on the molecular players that help mobilize metals from leaves to developing seeds. To profile several genes simultaneously, a cDNA macroarray was developed using 36 metal-related genes from rice, including ZIPs, NRAMPs, and YSLs (coding for known or potential metal transporters), as well as NAS, FER, FRO, NAAT, FDH, GSTU, and PDR (involved in metal homeostasis). Because flag leaves are the major source of phloem-delivered photoassimilates and remobilized metals for developing seeds, we analyzed the expression of these metal-related genes in flag and non-flag leaves of four different rice cultivars (Cocodrie, Taipei 309, IR58, and IR68144) during the period of mid-grain fill. Genes (24 of 36) exhibited low to non-detectable signals in the macroarray, while 12 genes (OsIRT1, OsZIP1, OsZIP5, OsZIP8, OsYSL5, OsYSL6, OsYSL7, OsYSL8, OsYSL18, OsNRAMP2, OsNRAMP4 and OsNRAMP7) were found to be highly expressed in both flag and non-flag leaves of all four cultivars. Additional expression analysis using semi-quantitative or quantitative PCR provided results that were generally consistent with the macroarray, but semi-quantitative PCR confirmed that OsFDH, OsFER1, OsNAAT, OsNAS1, OsPDR9, OsYSL12, OsYSL13, OsZIP7, and OsZIP10 were also expressed in leaves. This specialized macroarray has provided a short list of potential candidate genes, expressed in leaves, which might contribute to the process of metal transport to distant sinks, such as seeds.     

Genetics of amylose content in rice (Oryza sativa L)

Inheritance of amylose content was studied in crosses involving very low-, intermediate-, and high-amylose parents. The single-grain analysis of parents, F₁, F₂, B₁F₁, and B₂F₁ seed from a single-season harvest, showed that the parental mean difference of 14–17 % in IR37307-8/BPI 121-407 or IR37307-8/IR24632-34 and about 20% in the cross IR37307-8/IR8 were controlled by a single gene with major effect, along with some minor genes and/or modifiers. The appearance of segregants inbetween the two parents was attributed to gene dosage effects in the endosperm. The results indicate that selection for amylose level can effectively be done in early segregating generations. Selection for intermediary segregants would be ineffective because the dosage effects would dissipate in further generations.