Inhibition of ribonuclease and protease activities in arsenic exposed rice seedlings: role of proline as enzyme protectant

When seedlings of two rice (Oryza sativa L.) cvs. Malviya-36 and Pant-12 were raised under 25 and 50 microM As2O3 in the medium an increase in the level of RNA, proteins and proline accompanied with a decline in the level of free amino acid pool was observed under arsenic supplementation compared to controls. In situ As3+ treatment caused a marked inhibition in activities of ribonuclease (RNase, EC 3.1.27.1), protease and leucine aminopeptidase (LAP, EC 3.4.11.1) whereas the activity level of carboxypeptidase (EC 3.4.16.5) was enhanced. In vitro supply of As2O3 in the enzyme assay medium beyond 400 microM resulted in gradual inhibition of RNase and beyond 5 microM inhibition of LAP activities. Addition of 1M proline in the assay medium significantly restored the loss in RNase activity due to in vitro arsenic treatment or due to osmotic stress created by incorporation of polyethylene glycol (PEG). Isoform pattern of RNase extracted from As3+ -exposed seedlings showed a significant alteration compared to its pattern in unexposed seedlings. Results suggest that arsenic exposure impairs hydrolysis of RNA and proteins in rice seedlings due to inhibition of RNase and proteases activities and that proline accumulating under As3+ toxicity appears to serve as enzyme protectant.     

Modulation of nitrate reductase activity in rice seedlings under aluminium toxicity and water stress: role of osmolytes as enzyme protectant

Nitrate reductase (NR) activity in the presence of Mg2+ (NR act) representing the non-phosphorylated NR state and the activity in the presence of EDTA (NR max) representing maximum NR activity was measured in roots and shoots of 15 d grown aluminium and water stressed rice seedlings to examine changes in NR activation state due to these stresses. Seedlings subjected to a moderate water stress level of -0.5 MPa for 24 h or grown in presence of 80 microM Al3+showed decreased level of NR max but resulted in higher NR act and NR activation state. However, seedlings grown in presence of a higher level of 160 microM Al3+ showed a decline in NR act as well as NR max. With a higher water stress Level of -2.0MPa a marked decline in the levels of both NR act and NR max was observed, whereas NR activation state remained almost unaltered with severe water stress. NR activity appeared to be sensitive to H2O2, PEG-6000, NaCl and various metal salts. Incorporation of these components in the enzyme assay medium led to decreased affinity of enzyme towards its substrate with increase in Km and decrease in Vmax values. Addition of each of the osmolytes i.e. 1 mol/L proline, 1 mol/L glycine betaine or 1 mol/L sucrose in the enzyme assay medium caused a considerable protection to the enzyme against the damaging effects of stressful components. An enhanced level of proline and glycine betaine was observed in Al-stressed seedlings and sucrose in Al as well as water stressed seedlings. Results suggest that Al toxicity and water stress decrease total amount of functional NR in rice seedlings and the osmolytes proline, glycine betaine and sucrose appear to have a direct protective action on enzyme NR under stressful conditions     

Nickel toxicity inhibits ribonuclease and protease activities in rice seedlings: protective effects of proline

Seedlings of two rice cvs Malviya-36 and Pant-12, when grown under increasing levels of nickel (Ni²⁺: 200 and 400 μM) in the nutrient medium, showed increased levels of RNA, soluble proteins and free amino acids, especially proline over a total growth period of 5–20 days. Ribonuclease (RNase) and protease activities decreased in both roots and shoots due to Ni treatment in situ. Under in vitro conditions, a gradual inhibition of RNase activity was observed with increasing concentrations of Ni²⁺ (0–2500 μM) in the assay medium. Artificial desiccation simulated by 40% PEG or the presence of 2.5 mM Ni²⁺ in the reaction medium resulted in about 52–53% loss of RNase activity. Such a loss could be partially restored by 1 M proline in the assay medium. The activity staining of RNase revealed seven and four isoforms of RNase in roots and shoots, respectively. The intensity of most of the bands decreased with increasing levels of Ni²⁺ treatment in situ. The results suggest that Ni toxicity in rice seedlings suppresses the hydrolysis of RNA and proteins by inhibiting the activity of RNase and protease, respectively. Proline appears to act as a protectant of the enzyme RNase against metal- and PEG-induced damages.     

免耕与秸秆高留茬还田对抛秧稻田土壤酶活性的影响

利用大田试验,研究了免耕+秸秆还田、免耕、常耕+秸秆还田、常耕4种耕作方式对稻田土壤剖面不同层次土壤酶活性的影响.结果表明:4种耕作方式的酶活性在土壤剖面上表现为上层高于下层,其中免耕+秸秆处理上、下土层间的差异大于其他处理.上层土壤的脲酶、酸性磷酸酶、蛋白酶和纤维素酶活性为免耕处理大干常耕处理,有秸秆还田处理大干无秸秆还田处理,以免耕+秸秆处理最高,常耕处理最低;下层土壤4种酶活性以常耕+秸秆处理最高,免耕+秸秆处理次之,免耕和常耕处理较低.水稻不同生育时期,脲酶和纤维素酶活性在分蘖期较低,增加至孕穗期达到峰值,至成熟时又降至低值;酸性磷酸酶活性以分蘖期较高,拔节期较低;蛋白酶活性在分蘖期和抽穗期分别出现峰值.     

NaCl induced changes in ionically bound peroxidase activity in roots of rice seedlings

The changes in ionically bound peroxidase activity in roots of NaCl-stressed rice seedlings and their correlation with root growth were investigated. Increasing concentrations of NaCl from 50 to 150 mM progressively decreases root growth. The reduction of root growth by NaCl is closely correlated with the increase in ionically bound peroxidase activity. Since proline and ammonium accumulations are associated with root growth inhibition caused by NaCl, we determined the effects of proline or NH4Cl on root growth and ionically bound peroxidase activity in roots. External application of proline or NH4Cl markedly inhibited root growth and increased ionically bound peroxidase activity in roots of rice seedlings in the absence of NaCl. An increase in ionically bound peroxidase activity in roots preceded inhibition of root growth caused by NaCl, NH4Cl or proline. Mannitol inhibited root growth, but decreased rather than increased ionically bound peroxidase activity at the concentration iso-osmotic with NaCl. The inhibition of root growth and the increase in ionically bound peroxidase activity in roots by NaClis reversible and is associated with ionic rather than osmotic component. This revised version was published online in June 2006 with corrections to the Cover Date.     

Role of abscisic acid in cadmium tolerance of rice (Oryza sativa L.) seedlings

Changes in abscisic acid (ABA) contents in Cd-treated rice (Oryza sativa L.) seedlings of two cultivars were investigated. On treatment with CdCl2, the ABA content rapidly increased in the leaves and roots of Cd-tolerant cultivar (cv. Tainung 67, TNG67) but not in the Cd-sensitive cultivar (cv. Taichung Native 1, TN1). The reduction of transpiration rate of TN1 caused by Cd was less than that of TNG67. Exogenous application of ABA reduced transpiration rate, decreased Cd content, and enhanced Cd tolerance of TN1 seedlings. Exogenous application of the ABA biosynthesis inhibitor, fluridone, reduced ABA accumulation, increased transpiration rate and Cd content, and decreased Cd tolerance of TNG67 seedlings. Fluridone effect on Cd toxicity of TNG67 seedlings was reversed by the application of ABA. The roles of endogenous ABA in Cd tolerance of rice seedlings are discussed and suggested.     

Enhancement of NADP-malic enzyme in transgenic rice induced the accumulation of reactive oxygen species

It had been demonstrated that the photosynthetic photodamage, such as photoinhibition and photooxidation, was enhanced in transgenic rice plants overexpressing NADP-malic enzyme (ME). However, its physiological base has not been investigated. In order to elucidate the physiological elements contributed to the enhancement of photodamage in NADP-ME transgenic rice plants, some physiological indices related to reactive oxygen species (ROS) accumulation were studied using the T₁ progeny of transgenic rice plants. The results showed that more ROS such as O ₂ ^−. and H₂O₂ were accumulated in transgenic rice plants, which enhanced photooxidation, while the accumulation of malondialdehyde in transgenic rice plants was not evident as compared with the wild-type plants. The measurement of NADPH/NADP ratios in leaves showed that transgenic rice plants had a higher ratio than untransformed rice plants. Based on these data, we speculated that overexpression of NADP-ME led to the deficiency of NADP and overreduction of photosystem I, which induced the accumulation of ROS in the transgenic rice plants, and just ROS were accounted for plant sensitivity to photooxidation. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 3, pp. 364–370. The text was submitted by the author in English.     

Cd induced changes in proline level and peroxidase activity in roots of rice seedlings

The effects of Cd on changes in proline level and peroxidase activity in roots of rice seedlings were investigated. CdCl₂ was effective in inhibiting root growth and in accumulating proline in roots. The inhibition of root growth by Cd is reversible. The reduction of root growth induced by Cd is closely associated with accumulation of proline in roots. External application of proline markedly inhibited root growth of rice seedlings in the absence of Cd. Ionically bound, but not soluble, peroxidase activity in roots was increased by CdCl₂. Proline treatment also resulted in an increase in ionically bound peroxidase activity in roots. The relationship between growth inhibition of roots induced by Cd and changes of proline level and peroxidase activity is discussed.Abbreviations POX peroxidase     

硝基苯酚胁迫下外源褪黑素对水稻幼苗生长及生理特性的影响

采用液体培养实验方法,研究硝基苯酚胁迫对水稻(Oryza sativa L.)幼苗生长、抗氧化特性、光系统Ⅱ(PSⅡ)光合特性的影响,以及添加外源褪黑素对缓解硝基苯酚胁迫的作用。结果显示,随着硝基苯酚胁迫浓度的升高,水稻幼苗株高、根长、地下部干重、地上部干重、全株干重和叶片PSⅡ实际光化学效率[Y(Ⅱ)]、光化学淬灭系数(q P)、PSⅡ电子传递速率(ETR)、叶绿素含量均有所下降,而叶片非光化学淬灭系数(qN、NPQ)上升;同时,根系活性氧[过氧化氢(H_2O_2)和超氧阴离子(O·-2)]积累量、抗氧化酶[超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)]活性,以及渗透调节物质(可溶性蛋白和可溶性糖)含量呈先升高后降低的趋势。在非硝基苯酚胁迫下,与对照组相比,添加外源褪黑素显著提高了幼苗地下部干重、根系可溶性糖含量和SOD活性、叶片PSⅡ光化学效率和叶绿素含量。与单独添加硝基苯酚处理相比,硝基苯酚+褪黑素复合处理显著缓解了硝基苯酚胁迫对幼苗生长、叶片PSⅡ光化学效率和叶绿素合成的抑制作用;降低了根系活性氧水平、抗氧化酶活性和渗透调节物质含量。研究结果表明添加外源褪黑素能够显著缓解硝基苯酚胁迫对水稻幼苗生长、根系活性氧水平、抗氧化酶活性、叶片PSⅡ光化学效率及叶绿素合成的不良影响,提高水稻幼苗对硝基苯酚胁迫的适应性。     

Root morphology and cadmium uptake kinetics of the cadmium-sensitive rice mutant

To understand the physiological mechanism that confers Cd sensitivity, root morphology and Cd uptake kinetics of the Cd-sensitive mutant and wild type rice were investigated. The root length, root surface area, and root number of mutant rice decreased more significantly with increasing Cd concentration in growth media compared with the wild type rice. The uptake kinetics for ¹⁰⁹Cd²⁺ in roots of both the mutant and wild type rice were characterized by a rapid linear phase during the first 6 h and a slower linear phase during the subsequent period. Concentration-dependent Cd²⁺ influx in both species could be characterized by the Michaelis-Menten equation, with similar apparent K_m values for mutant and wild type rice (2.54 and 2.37 μM, respectively). However, the V_max for Cd²⁺ influx in mutant root cells was nearly 2-fold higher than that for wild type rice, indicating that enhanced absorption into the root is one of the mechanisms involved in Cd sensitivity in mutant rice.     

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.     

The effect of NaCl on proline accumulation in rice leaves

The regulation of proline accumulation in detached leaves of rice(Oryza sativa cv. Taichung Native 1) was investigated.Increasing concentrations of NaCl from 50 to 200 mM progressivelyincreased proline content in detached rice leaves. NaCl induced prolineaccumulation was mainly due to the effect of both Na⁺ andCl^– ions. Proline accumulation caused by NaCl was related toprotein proteolysis, an increase in ornithine--aminotransferaseactivity,a decrease in proline dehydrogenase activity, a decrease in prolineutilisation,and an increase in the content of the precursors of proline biosynthesis,ornithine and arginine. Results also show that proline accumulation caused byNaCl was associated with ammonium ion accumulation.     

Regulation of ammonium-induced proline accumulation in detached rice leaves

Accumulation of proline in response to NH₄Cl was studied indetached leaves of rice (Oryza sativa cv. Taichung Native1). Increasing concentrations of NH₄Cl from 50 to 200mMprogressively increased proline content and this was correlated with theincrease in ammonium content. Proline accumulation induced by NH₄Clwas related to proteolysis, an increase in ornithine--aminotransferaseactivity, a decrease in proline dehydrogenase activity, and a decrease inproline utilisation and could not be explained by NH₄Cl-inducedmodification in ¹-pyrroline-5-carboxylate reductase activity.The content of glutamic acid was decreased by NH₄Cl, whereas theincrease in arginine and ornithine contents was found to be associated with theincrease in proline content in NH₄Cl-treated detached rice leaves.     

光质对水稻幼苗生长及生理特性的影响

以荧光灯为对照,采用发光二极管（light emitting diode,LED）精确调制不同光谱能量分布,研究了光质对‘武运粳7号’和‘抗优63’两种水稻幼苗生长及生理特性的影响.结果表明：光质对两个品种水稻幼苗生长有显著影响且存在差异.蓝光显著抑制幼苗株高,提高‘武运粳7号’叶片的可溶性蛋白含量及两个品种水稻五叶期幼苗的壮苗指数;红光显著提高三叶期幼苗的茎基直径、壮苗指数以及五叶期叶片的可溶性糖和淀粉含量;红蓝组合光显著提高三叶期幼苗的根数、茎基直径、壮苗指数、根系活力和可溶性糖含量,以及五叶期幼苗的鲜、干质量、壮苗指数、叶片可溶性糖和蔗糖含量;黄光可在幼苗生长初期明显增加株高,提高叶片色素含量.总体上,红蓝组合光有利于培育水稻壮苗.     

Suppression and promotion of growth by ethylene in rice seedlings depends on ambient humidity

We examined the effect of ethylene on the growth of rice seedlings (Oryza sativa L.) at various degrees of humidity. Ethylene significantly suppressed the growth of shoots when applied to seedlings grown under 30% relative humidity (RH), but promoted the growth of shoots when applied to seedlings grown under 100% RH. The application of gibberellic acid (GA₃) promoted the elongation of shoots in seedlings grown under 30% and 100% RH. Ethylene inhibited the shoot elongation induced by GA₃ at 30% RH, but enhanced the elongation induced by GA₃ at 100% RH. These results indicate that ethylene can either promote or suppress the growth of rice shoots depending on ambient humidity, and that these actions of ethylene may be mediated through modulating the responsiveness of shoots to gibberellin.     

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.     

A loss‐of‐function allele of OsHMA3 associated with high cadmium accumulation in shoots and grain of Japonica rice cultivars

Excessive cadmium (Cd) accumulation in rice poses a risk to food safety. OsHMA3 plays an important role in restricting Cd translocation from roots to shoots. A non‐functional allele of OsHMA3 has been reported in some Indica rice cultivars with high Cd accumulation, but it is not known if OsHMA3 allelic variation is associated with Cd accumulation in Japonica cultivars. In this study, we identified a Japonica cultivar with consistently high Cd accumulation in shoots and grain in both field and greenhouse experiments. The cultivar possesses an OsHMA3 allele with a predicted amino acid mutation at the 380^th position from Ser to Arg. The haplotype had no Cd transport activity when the gene was expressed in yeast, and the allele did not complement a known nonfunctional allele of OsHMA3 in F₁ test. The allele is present only in temperate Japonica cultivars among diversity panels of 1483 rice cultivars. Different cultivars possessing this allele showed greatly increased root‐to‐shoot Cd translocation and a shift in root Cd speciation from Cd―S to Cd―O bonding determined by synchrotron X‐ray absorption spectroscopy. Our study has identified a new loss‐of‐function allele of OsHMA3 in Japonica rice cultivars leading to high Cd accumulation in shoots and grain.     

Parameter determination involved in phytotoxicity and transport of cadmium in rice seedlings

Hydroponic experiments were conducted with rice seedlings to determine parameters involved in phytotoxicity and transport of Cadmium (Cd). Results showed that a linear decrease in relative growth rate and water use efficiency was observed in rice seedlings with increasing Cd concentrations. More severe inhibitory effects on both parameters were found at the 4-d treatment. The effective concentrations (EC) were estimated using the Levenberg–Marquardt Algorithm. The EC values obtained from the relative growth rate were always smaller than these from water use efficiency, indicating that the former was more sensitive to change of Cd than the latter. Phyto-transport of Cd was apparent, but the distribution of Cd in different parts of rice seedlings was different and roots were the dominant sink for Cd accumulation (p < 0.05). Although the translocation of Cd into shoots was detectable, more Cd was detected in shoots at the 4-d treatments than that at the 2-d treatments (p < 0.05). The partial correlation analysis suggested that the total accumulation rate of Cd was highly related to the doses of Cd supplied (p < 0.05). In conclusion, Cd is problematic at relatively low concentrations for rice seedlings, and inhibitory effects are highly dependent on the selected response endpoints and the duration of exposure periods.     

Physiology of rice tungro virus disease: Proline accumulation due to infection

Tungro virus infection stimulates proline accumulation in leaves of rice ( Oryza sativa L.), especially in a sensitive cultivar, Taichung Native 1. Disease-induced proline accumulation increases with the severity of the disease. Proline also accumulates in senescing, detached healthy rice leaves. The magnitude of proline accumulation in these leaves was further accentuated by ABA and retarded by kinetin. In the absence of drought stress, virus infection induces severe symptoms (stunting) in a drought tolerant cultivar (Lalnakanda 41) when compared to cultivars with intermediate (MW 10) and high sensitivity (Cauvery) to drought. Thus tungro virus mimics water stress in inducing proline accumulation in rice leaves. In both cases a common factor, ABA, may mediate proline accumulation. In drought stress, proline accumulation is associated with tolerance, while in virus stress, proline accumulation is connected with sensitivity. It is, therefore, clear that proline cannot always act to relieve physiological stress.