Evaluation of salt tolerance at the seedling stage in rice genotypes by growth performance, ion accumulation, proline and chlorophyll content

Aims

The objectives of this study were to evaluate salt tolerance level of rice genotypes using the well-established screening criteria; the salt injury score, survival percentage and ratio between Na⁺ and K⁺, as well as the contents of proline and chlorophyll, and to identify the relationship between salt tolerance and physiological characters.

Methods

One hundred and six rice genotypes were grown in hydroponic solutions subjected to salt stress and evaluated for salt tolerance ability and the physiological parameters. Multivariate cluster analysis was performed based on salinity tolerance scores (ST scores; score 1 being the most tolerant, score 9 the most sensitive), survival percentage and Na⁺/K⁺ ratio.

Results

ST scores based on salt injury symptoms were negatively correlated with survival percentage and chlorophyll concentration in the stressed seedlings but positively correlated with Na⁺/K⁺ ratio and proline content. Rice genotypes were classified into five salt tolerance groups: tolerant (T), moderately tolerant (MT), moderately sensitive (MS), sensitive (S) and highly sensitive (HS). The means of ST scores were significantly different among the five tolerance groups indicating that the ST score was the most reliable index for identifying salt tolerance. The means of Na⁺/K⁺ ratio and proline content in stressed seedlings were distinctively different between the extreme T and HS groups, but the means among the intermediate groups (MT, MS and S) were not significantly different. Chlorophyll content, on the other hand, was not related to the levels of salt tolerance.

Conclusions

In addition to the commonly used Na⁺/K⁺ ratio, proline content is suggested to be another useful criterion to differentiate salt-tolerant from salt-sensitive rice. This study also identified several Thai improved and local cultivars with the level of salt tolerance and physiological characters comparable to Pokkali, the standard salt-tolerant donor and may be utilized as alternative sources of salt tolerance alleles.     

Chronic mild salinity affects source leaves physiology and productivity parameters of rice plants (Oryza sativa L., cv. Taipei 309)

Aim

In rice, the top two leaves are the major carbohydrate source during grain filling. Physiological performance of these leaves under salinity may allow estimate stress-induced yield loss.

Methods

Greenhouse grown rice plants (cv. Taipei 309) were subjected to 10 and 20 mM NaCl stress levels from germination till maturity. Plant development was measured at the flowering stage and yield parameters were quantified after complete ripening of panicles.

Results

Gas exchange in the main source leaves were not significantly affected by any of the stress levels. However, growth parameters as well as total metabolizable carbohydrates content, chlorophyll content (CCI), maximal efficiency of PSII photochemistry in dark-adapted state (F _v/F _m) and lipid peroxidation were significantly affected. Rice yield, measured as total panicle production, declined to 78 and 21 % of controls in 10 and 20 mM NaCl stress, respectively. Stress-induced yield loss was positively related with the decline in CCI, F _v/F _m and K⁺/Na⁺ ratio as well as with the increase in lipid peroxidation and total soluble carbohydrate contents.

Conclusions

Though the stress levels used in this work are below what is considered the minimal critical threshold of toxicity for rice, they induce significant negative effects on plant development and yield, when present along the whole plant life cycle.     

Variation in responses to boron in rice

Background and aims

Boron (B) deficiency depresses grain set and grain yield of wheat and maize while having little effect on their vegetative growth. This paper describes effects of B deficiency in rice and how these vary with planting season and variety.

Methods

Three rice varieties (KDML105, CNT1, SPR1) were grown in sand culture without (B0) and with 10 μM (B10) B added to the nutrient solution, in the cool season of 2007/08 and 2008/09 and the hot season of 2011 in Chiang Mai, Thailand (18°47′N, 98°59′E). Boron responses were measured in growth and yield parameters, pollen viability and B concentration of the flag leaf and anthers at anthesis.

Results

Grain weight was strongly depressed by B deficiency ranging from 28 % in SPR1 to 79 % in CNT1, and the yield was much lower in the cool season than in the hot season plantings. The variation in grain weight was closely associated with grain set and number of spikelets but not with shoot dry weight or tillering. Grain set was closely related to pollen viability, and both were increased with increasing anther B concentration at >20 mg B kg^?1. In addition to its adverse effect on grain set, B deficiency also depressed grain filling and weight of individual grains in rice.

Conclusions

Boron deficiency depressed rice grain yield through adverse effects on reproductive growth, panicle and spikelet formation and grain filling, in addition to grain set as in wheat and maize.     

Novel screening protocol for precise phenotyping of salt-tolerance at reproductive stage in rice

The present study reports an unequivocal and improved protocol for efficient screening of salt tolerance at flowering stage in rice, which can aid phenotyping of population for subsequent identification of QTLs associated with salinity stress, particularly at reproductive stage. To validate the new method, the selection criteria, level and time of imposition of stress; plant growth medium were standardized using three rice genotypes. The setup was established with a piezometer placed in a perforated pot for continuous monitoring of soil EC and pH throughout the period of study. Further, fertilizer enriched soil was partially substituted by gravels for stabilization and maintaining the uniformity of soil EC in pots without hindering its buffering capacity. The protocol including modified medium (Soil:Stone, 4:1) at 8 dS m^?1 salinity level was validated using seven different genotypes possessing differential salt sensitivity. Based on the important selection traits such as high stability index for plant yield, harvest index and number of grains/panicle and also high K⁺ concentration and low Na⁺– K⁺ ratio in flag leaf at grain filling stage were validated and employed in the evaluation of a mapping population in the modified screening medium. The method was found significantly efficient for easy maintenance of desired level of soil salinity and identification of genotypes tolerant to salinity at reproductive stage.     

Steady state proline levels in salt-shocked barley leaves

Excised barley (Hordeum vulgare var Larker) leaves were treated with salt solutions or wilted. After the treatment period, the leaves were allowed to recover in a 50 millimolar sucrose and 1 millimolar glutamate solution, and proline, Na⁺, and K⁺ were measured at intervals. Na⁺ and K⁺ concentrations stayed at a constant high level after the salt treatments, and proline increased to a steady state concentration in response. The relationship between the maximum rate of proline accumulation and the Na⁺ concentration reached in each experiment was linear. The final steady state proline concentration reached was also directly proportional to the Na⁺ concentration. For a given Na⁺ concentration in the leaves, the steady state proline level was greater when 410 millimolar NaCl was added to the leaves than when 205 millimolar NaCl was added. These results are consistent with proline acting as a compatible cytoplasmic solute, balancing an accumulation of salts outside of the cytoplasm.

In contrast to the proline levels in salt-shocked leaves, the concentrations in wilted leaves decreased to near control levels within 24 hours of relief of stress.

     

Influence of reproductive organs on plant senescence in rice and wheat

The chlorophyll and protein contents of the flag, second and third leaves gradually decreased during the reproductive development of rice (Oryza sativa L. cv. Rasi) and wheat (Triticum aestivum L. cv. Sonalika) plants, whereas proline accumulation increased up to the grain maturation stage and slightly decreased thereafter. In rice plant, the rate of decrease in chlorophyll and protein and increase in proline level were higher in the flag leaf than in the second leaf. It was opposite in wheat plant. The export of [³²P]-phosphate from leaves to grains gradually increased reaching a maximal stage at the grain development stage, and then declined. The export of this radioisotope was greater in rice than in wheat. Removal of panicle at the anthesis and grainfilling stages delayed leaf senescence of rice plant, while in wheat the ponicle removal at any stage did not have a marked effect on delaying leaf senescence. The contents of chlorophyll and protein of glumes were higher in wheat than in rice. The variation of such source-sink relationship might be one of the possible reasons for the above effect on leaf senescence.     

Salt accumulation and depletion in the root-zone of the halophyte Atriplex nummularia Lindl.: influence of salinity,leaf area and plant water use

Background and aims

Salt is known to accumulate in the root-zone of Na⁺ excluding glycophytes under saline conditions. We examined the effect of soil salinity on Na⁺ and Cl^? depletion or accumulation in the root-zone of the halophyte (Atriplex nummularia Lindl).

Methods

A pot experiment was conducted in soil to examine Na⁺ and Cl^? concentrations adjacent to roots at four initial NaCl treatments (20, 50, 200 or 400 mM NaCl in the soil solution). Plant water use was manipulated by leaving plants with all leaves intact, removing approximately 50 % of leaves, or removing all leaves. Daily evapotranspiration was replaced by watering undrained pots to weight with deionised water. After 35-38 days, samples were taken of the bulk soil and of soil loosely- and closely-adhering to the roots.

Results

In plants with leaves intact grown with 200 and 400 mM NaCl, average Na⁺ and Cl^? concentrations in the closely adhering soil were about twice the concentrations of the bulk soil. Ion accumulation increased with final leaf area and with cumulative transpiration over the duration of the trial. By contrast, in plants grown with the lowest salinity treatment (20 mM NaCl), Na⁺ and Cl^? concentrations decreased in the closely adhering soil with increasing leaf area and increasing cumulative water use.

Conclusions

Our data show that Na⁺ and Cl^? are depleted from the root-zone of A. nummularia at low salinity but accumulate in the root-zone at moderate to high salinity, and that the ions are drawn towards the plant in the transpiration stream.     

Differential growth and yield responses of salt-tolerant and susceptible rice cultivars to individual (Na<Superscript>+</Superscript> and Cl<Superscript>−</Superscript>) and additive stress effects of NaCl

Negative impacts exerted by sodium (Na⁺) and chloride (Cl^?) ions individually as well their possible additive effects (under NaCl) were evaluated on growth and yield reductions in rice, besides investigating whether salt-tolerant genotypes respond differentially than their sensitive counterparts. Though both Na⁺ and Cl^? ions get accumulated in plant tissues under NaCl stress, most research has historically been aimed to decipher harmful effects induced by Na⁺ ions. Accordingly, physiological and molecular mechanisms involved in Cl^? toxicity are not clearly understood in crop plants. To address these issues, 65-day-old plants of two rice cultivars, Panvel-3 (tolerant) and Sahyadri-3 (sensitive) were subjected to Cl^?, Na⁺ and NaCl (each with 100 mM concentration and electrical conductivity of ≈10 dS m^?1) stress using soil-based systems. Stress conditions were maintained till harvesting of mature (128-day-old) plants. All three treatments induced substantial antagonistic effects on growth, dry mass, yield components (number of grains per panicle, length, width, thickness and weight of grain, along with the percentage of grains filled) and overall crop yield, with greater impacts under NaCl than its constituent ions. Salinity treatments caused an imbalance in reducing sugars, protein, starch and proline contents, with the greatest magnitude under NaCl. A negative correlation between Cl^?/Na⁺ accumulation and crop yield was witnessed, with higher severity on the sensitive cultivar. The overall magnitude of toxicity was observed highest in NaCl followed by Na⁺ and Cl^?, respectively, suggesting additive effects of constituent ions under NaCl. Both cultivars responded similarly; however, the tolerant cultivar, unlike the sensitive one, kept Na⁺:K⁺ ratio <1.0 and accumulated proline in response to salinity treatments used in this study.     

Ecosystem changes in Galápagos highlands by the invasive tree Cinchona pubescens

Background and aims

Salinity is an increasing problem for agricultural production worldwide. Understanding how Na⁺ enters plants is important if reducing Na⁺ influx, a key component of the regulation of Na⁺ accumulation in plants and improving salt tolerance of crop plants, is to be achieved. Our previous work indicated that two distinct low-affinity Na⁺ uptake pathways exist in the halophyte Suaeda maritima. Here, we report the external NaCl concentration at which uptake switches from pathway 1 to pathway 2 and the kinetics of the interaction between external K⁺ concentration and Na⁺ uptake and accumulation in S. maritima in order to determine the roles of K⁺ transporters or channels in low-affinity Na⁺ uptake.

Methods

Na⁺ influx, Na⁺ and K⁺ accumulations in S. maritima exposed to various concentrations of NaCl (0–200 mM) were analyzed in the absence and presence of the inhibitors TEA and Ba⁺ (5 mM TEA or 3 mM Ba²⁺) or KCl (0, 10 or 50 mM).

Results

Our earlier proposal was confirmed and extended that there are two distinct low-affinity Na⁺ uptake pathways in S. maritima: pathway 1 might be mediated by a HKT-type transporter under low salinity conditions and pathway 2 by an AKT1-type channel or a KUP/HAK/KT type transporter under high salinity conditions. The external NaCl concentration at which two distinct low-affinity Na⁺ uptake switches from pathway 1 to pathway 2, the ‘turning point’, is between 90 and 95 mM. Over a short period (12 h) of Na⁺ and K⁺ treatments, a low concentration of K⁺ (10 mM) facilitated Na⁺ uptake by S. maritima under high salinity (100–200 mM NaCl), whether or not the plants had been subjected to a longer (3 d) period of K⁺ starvation. The kinetics suggests that low concentration of K⁺ (10 mM) might activate AKT1-type channels or KUP/HAK/KT-type transporters under high salinity (100–200 mM NaCl).

Conclusions

The turning-point of external NaCl concentrations for the two low-affinity Na⁺ uptake pathways in Suaeda maritima is between 90 and 95 mM. A low concentration of K⁺ (10 mM) might activate AKT1 or KUP/HAK/KT and facilitate Na⁺ uptake under high salinity (100–200 mM NaCl). The kinetics of K⁺ on Na⁺ uptake and accumulation in S maritima are also consistent with there being two low-affinity Na⁺ uptake pathways.     

YSL16 Is a Phloem-Localized Transporter of the Copper-Nicotianamine Complex That Is Responsible for Copper Distribution in Rice

Cu is an essential element for plant growth, but the molecular mechanisms of its distribution and redistribution within the plants are unknown. Here, we report that Yellow stripe-like16 (YSL16) is involved in Cu distribution and redistribution in rice (Oryza sativa). Rice YSL16 was expressed in the roots, leaves, and unelongated nodes at the vegetative growth stage and highly expressed in the upper nodes at the reproductive stage. YSL16 was expressed at the phloem of nodes and vascular tissues of leaves. Knockout of this gene resulted in a higher Cu concentration in the older leaves but a lower concentration in the younger leaves at the vegetative stage. At the reproductive stage, a higher Cu concentration was found in the flag leaf and husk, but less Cu was present in the brown rice, resulting in a significant reduction in fertility in the knockout line. Isotope labeling experiments with ⁶⁵Cu showed that the mutant lost the ability to transport Cu-nicotianamine from older to younger leaves and from the flag leaf to the panicle. Rice YSL16 transported the Cu-nicotianamine complex in yeast. Taken together, our results indicate that Os-YSL16 is a Cu-nicotianamine transporter that is required for delivering Cu to the developing young tissues and seeds through phloem transport.     

Saline-induced changes of epicuticular waxy layer on the Puccinellia tenuiflora and Oryza sativa leave surfaces

Background

The epicuticular waxy layer of plant leaves enhances the extreme environmental stress tolerance. However, the relationship between waxy layer and saline tolerance was not established well. The epicuticular waxy layer of rice (Oryza sativa L.) was studied under the NaHCO₃ stresses. In addition, strong saline tolerance Puccinellia tenuiflora was chosen for comparative studies.

Results

Scanning electron microscope (SEM) images showed that there were significant changes in waxy morphologies of the rice epicuticular surfaces, while no remarkable changes in those of P. tenuiflora epicuticular surfaces. The NaHCO₃-induced morphological changes of the rice epicuticular surfaces appeared as enlarged silica cells, swollen corns-shapes and leaked salt columns under high stress. Energy dispersive X-ray (EDX) spectroscopic profiles supported that the changes were caused by significant increment and localization of [Na⁺] and [Cl⁻] in the shoot. Atomic absorption spectra showed that [Na⁺]_shoot/[Na⁺]_root for P. tenuiflora maintained stable as the saline stress increased, but that for rice increased significantly.

Conclusion

In rice, NaHCO₃ stress induced localization and accumulation of [Na⁺] and [Cl⁻] appeared as the enlarged silica cells (MSC), the swollen corns (S-C), and the leaked columns (C), while no significant changes in P. tenuiflora.     

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.     

Physiological and biochemical parameters for evaluation and clustering of rice cultivars differing in salt tolerance at seedling stage

Salinity tolerance levels and physiological changes were evaluated for twelve rice cultivars, including four white rice and eight black glutinous rice cultivars, during their seedling stage in response to salinity stress at 100 mM NaCl. All the rice cultivars evaluated showed an apparent decrease in growth characteristics and chlorophyll accumulation under salinity stress. By contrast an increase in proline, hydrogen peroxide, peroxidase (POX) activity and anthocyanins were observed for all cultivars. The K⁺/Na⁺ ratios evaluated for all rice cultivars were noted to be highly correlated with the salinity scores thus indicating that the K⁺/Na⁺ ratio serves as a reliable indicator of salt stress tolerance in rice. Principal component analysis (PCA) based on physiological salt tolerance indexes could clearly distinguish rice cultivars into 4 salt tolerance clusters. Noteworthy, in comparison to the salt-sensitive ones, rice cultivars that possessed higher degrees of salt tolerance displayed more enhanced activity of catalase (CAT), a smaller increase in anthocyanin, hydrogen peroxide and proline content but a smaller drop in the K⁺/Na⁺ ratio and chlorophyll accumulation.     

Peroxidase activities of two rice cultivars differing in salinity tolerance as affected by proline and NaCl

Proline content, ion accumulation, cell wall and soluble peroxidase activities were determined in control and salt-treated calli (150 nM NaCl) and whole plants (30 mM NaCl) of two rice cultivars (salt sensitive cv. IKP and salt tolerant cv. Aiwu). Under salinity, the highest accumulation of Na⁺, Cl^? and proline occurred in calli, roots and younger leaves of cv. IKP, coupled with the highest decrease in K⁺ content; accumulations of Na⁺ and Cl^? were restricted to older leaves in cv. Aiwu. Relative growth rates of calli and roots or shoots from both cultivars were not linked to peroxidase activities. High concentrations (1 M) of exogenously applied glycerol did not inhibitin vitro activities of soluble peroxidase extracted from control and salt-treated calli or plants. Conversely, 35–55% (in cv. IKP) or 60–80% (in cv. Aiwu) of soluble peroxidase activities were found in presence of isosmotic proline concentration. There were no differences between proline and glycerol effects onin vitro cell wall peroxidase activities.     

Phosphorus accumulation in grains of japonica rice as affected by nitrogen fertilizer

Background and aims

This study aims to investigate the effect of nitrogen (N) on grain phosphorus (P) accumulation in japonica rice.

Methods

Six cultivars with contrasting agronomic traits were grown for 3 years (from 2008 to 2010) of field experiments under seven N treatments and 1 year (in 2010) of pot experiments with five N treatments to study the effect of N on grain phosphorus accumulation and to explore its physiological foundation.

Results

Grain total P and phytic acid concentration showed a clearly decreasing trend as N rate increased for both field and pot experiments. Pot experiment revealed that application of N increase plant biomass, but tended to lower plant P uptake, especially for the split topdressing treatments. Both harvest index (HI) and P harvest index (PHI) increased with N rate, but PHI was consistently higher than HI, indicating the larger proportion of P translocation to grain than that of dry matter by N. Further, ratio of PHI/HI differed significantly among genotypes, but was stable across contrasting N treatments.

Conclusions

The combination of decreased plant P uptake and dilution effect of increased grain yield by N is proposed as underlying mechanism of the decreased grain P concentration by high N.     

Physiological and proteomic analysis of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) in flag leaf during flowering stage and milk stage under drought stress

Hybrid rice has the inferiority of early senescence during reproductive stage. Early senescence of leaves induced by drought causes a constraint on crop productivity. Hence, to better understand traits of drought responsive mechanism in hybrid rice flag leaves during sensitive reproductive stage, we performed physiological measurements and conducted 2-D electrophoresis to investigate proteomic profile of rice flag leaves at flowering and milk stage. 43 proteins showed significant changes in silver stained gels were identified by MS/MS at flowering stage and 54 proteins at milk stage. For flowering stage, inactive CO₂ assimilation and ATP synthesis as well abnormal floral development could be postulated from decreased proteins. Additionally, higher levels of defense-related proteins and antioxidases in drought-stressed leaves suggested active ROS scavenging system. For milk stage, both photosynthesis and CO₂ assimilation were impaired. Disrupted oxidative defense and proteolysis system indicated redox imbalance. Upregulation of NADP-MDH facilitated for CO₂ assimilation and antioxidant system. In conclusion, drought stress during reproductive stage impacted a lot on biological processes in rice leaves at protein level, especially energy metabolism and redox balance.     

Differential superoxide dismutase expression in ryegrass cultivars in response to short term aluminium stress

Background and aims

Saline soils limit plant production worldwide through osmotic stress, specific-ion toxicities, and nutritional imbalances.

Methods

The ability of Ca²⁺ and K⁺ to alleviate toxicities of Na⁺ and Mg²⁺ was examined using 89 treatments in short-term (48 h) solution culture studies for cowpea (Vigna unguiculata (L.) Walp.) roots. Root elongation was related to ionic activities at the outer surface of the root plasma membrane.

Results

The addition of K⁺ was found to alleviate the toxic effects of Na⁺, and supplemental Ca²⁺ improved growth further in these partially-alleviated solutions where K⁺ was present. Therefore, Na⁺ appears to interfere with K⁺ metabolism, and Ca²⁺ reduces this interference. Interestingly, the ability of Ca²⁺ to improve K-alleviation of Na⁺ toxicity is non-specific, with Mg²⁺ having a similar effect. In contrast, the addition of Ca²⁺ to Na-toxic solutions in the absence of K⁺ did not improve growth, suggesting that Ca²⁺ does not directly reduce Na⁺ toxicity in these short-term studies (for example, by reducing Na⁺ uptake) when supplied at non-deficient levels. Finally, K⁺ did not alleviate Mg²⁺ toxicity, suggesting that Mg²⁺ is toxic by a different mechanism to Na⁺.

Conclusions

Examination of how the toxic effects of salinity are alleviated provides clues as to the underlying mechanisms by which growth is reduced.     

Alteration of nitrogen metabolism in rice variety 'Nipponbare' induced by alkali stress

Aims

Alkali stress (AS) is an important agricultural contaminant and has complex effects on plant metabolism, specifically root physiology. The aim of this study was to test the role of nitrogen metabolism regulation in alkali tolerance of rice variety 'Nipponbare'.

Methods

In this study, the rice seedlings were subjected to salinity stress (SS) or AS. Growth, the contents of inorganic ions, NH ₄ ⁺ -nitrogen (free amino acids), and NO ₃ ^? -nitrogen in the stressed seedlings were then measured. The expression of some critical genes involved in nitrogen metabolism were also assayed to test their roles in the regulation of nitrogen metabolism during adaptation of rice variety 'Nipponbare' to AS.

Results

AS showed a stronger inhibiting effect on rice variety 'Nipponbare' growth than SS. AS may have more complex effects on nitrogen metabolism than SS.

Conclusions

Effects of AS on the nitrogen metabolism of rice variety 'Nipponbare' mainly comprised two mechanisms. Firstly, in roots, AS caused the reduction of NO ₃ ^? content, which caused two harmful consequences, the large downregulation of OsNR1 expression and the subsequent reduction of NH ₄ ⁺ production in roots. On the other hand, under AS (pH, 9.11), almost all the NH ₄ ⁺ was changed to NH₃, which caused a severe deficiency of NH ₄ ⁺ surrounding the roots. Both events might cause a severe deficiency of NH ₄ ⁺ in roots. Under AS, the increased expression of several OsAMT family members in roots might be an adaptative response to the reduction of NH ₄ ⁺ content in roots or the NH ₄ ⁺ deficiency in rhizosphere. Also, the down-regulation of OsNADH-GOGAT and OsGS1;2 in roots might be due to NH ₄ ⁺ deficiency in roots. Secondly, in shoots, AS caused a larger acuumulatiuon of Na⁺, which possibly affected photorespiration and led to a continuous decrease of NH ₄ ⁺ production in shoots, and inhibited the expression of OsFd-GOGAT and OsGS2 in chloroplasts.