Elucidation of salt-tolerance metabolic pathways in contrasting rice genotypes and their segregating progenies

Key message

Differentially expressed antioxidant enzymes, amino acids and proteins in contrasting rice genotypes, and co-location of their genes in the QTLs mapped using bi-parental population, indicated their role in salt tolerance.

Abstract

Soil salinity is a major environmental constraint limiting rice productivity. Salt-tolerant ‘CSR27’, salt-sensitive ‘MI48’and their extreme tolerant and sensitive recombinant inbred line (RIL) progenies were used for the elucidation of salt stress tolerance metabolic pathways. Salt stress-mediated biochemical and molecular changes were analyzed in the two parents along with bulked-tolerant (BT) and bulked-sensitive (BS) extreme RILs. The tolerant parent and BT RILs suffered much lower reduction in the chlorophyll as compared to their sensitive counterparts. Activities of antioxidant enzymes superoxide dismutase (SOD) and peroxidase (POD) and non-enzymatic antioxidant ascorbic acid were much higher in salt-stressed CSR27 and BT RILs than MI48 and BS RILs. Further, the tolerant lines showed significant enhancement in the levels of amino acids methionine and proline in response to salt stress in comparison to the sensitive lines. Similarly, the tolerant genotypes showed minimal reduction in cysteine content whereas sensitive genotypes showed a sharp reduction. Real time PCR analysis confirmed the induction of methionine biosynthetic pathway (MBP) enzymes cystathionine-β synthase (CbS), S-adenosyl methionine synthase (SAMS), S-adenosyl methionine decarboxylase (SAMDC) and serine hydroxymethyl transferase (SHMT) genes in tolerant lines, suggesting potential role of the MBP in conferring salt tolerance in rice variety CSR27. Proteome profiling also confirmed higher expression of SOD, POD and plastidic CbS and other proteins in the tolerant lines, whose genes were co-located in the QTL intervals for salt tolerance mapped in the RIL population. The study signifies integrated biochemical-molecular approach for identifying salt tolerance genes for genetic improvement for stress tolerant rice varieties.

     

Sequencing and Expression of the Gene that Encodes a 20-kDa Polypeptide of the PSI Complex from Cucumber Cotyledon

A cDNA clone encoding the polypeptide from cucumber PS I thatmigrates with an apparent molecular weight of 20 kDa on SDS-polyacrylamidegels has been isolated. The 907-bp sequence of this clone hasbeen determined and contains one large open reading frame thatencodes a 22,720-Da precursor polypeptide (207 amino acid residues).The molecular weight of the mature polypeptide was predictedto be 17,037-Da (153 amino acid residues). The deduced aminoacid sequence of this protein indicates that it is routed towardsthe stromal side of the thylakoid membrane and has no membrane-spanningregions. The sequence also confirmed the identity of the proteinas the product of the psa D gene. Chemical cross-linking offerredoxin to the PS I complex identified the 20-kDa subunitas the ferredoxin-binding protein. Northern hybridization experimentsrevealed that the mRNA of approximately 1,100 nucleotides forthe 20-kDa polypeptide was present in etiolated cucumber cotyledons,and its level increased about 5-fold during greening. The 20-kDapolypeptide was not detected by immunoblotting in etiolatedcotyledons, and it accumulated only after illumination. Labelingexperiments in vivo showed the absence of incorporation of [³⁵S]Metinto the polypeptide in etiolated cotyledons. These resultssuggest that the expression of the psa D gene is controlledat the translational level. (Received April 5, 1990; Accepted June 28, 1990)     

Frozen-thawed human blood monocytes respond reproducibly to activation stimuli: Implications for screening of BRMs

The purpose of this study was to identify the optimal freezing conditions for human blood monocytes to allow their recovery and use forin vitro screening of activation stimuli. Human monocytes separated from buffy coats of healthy blood donors were suspended at a density of 1 × 10⁷ cells/ml in freezing medium consisting of 70% medium: 20% fetal bovine serum: 10% DMSO frozen in a stepdown freezer, and stored at –180°C. Monocytes were thawed at different times up to 4 months later. Viability was >90%. Fresh monocytes from different donors and frozen monocytes thawed at different times were incubated with different concentrations of lipopolysaccharide, muramyl tripeptide, muramyl dipeptide, or lipopeptide. Tumoricidal activity and IL-1 production of fresh monocytes varied greatly among the 5 different preparations. In contrast, the frozen monocytes (thawed at different times) produced uniform levels of antitumor activity and IL-1 production. These results show that monocytes recovered from frozen storage maintain their ability to respond to activation stimuli in a uniform and reproducible manner. Thus, the use of frozen-thawed monocytes is recommended for screening of macrophage activating agents.     

Organic bases from Brazilian Piptadenia species

Basic compounds have been detected in five Brazilian Piptadenia species by means of electrophoresis. Bufotenine was detected in P. contorta Benth., and P. moniliformis Benth. The seeds of P. leprostachya Benth. contained theobromine, a new constituent in Leguminosae.     

Expression and functional analysis of putative vacuolar Ca2+-transporters (CAXs and ACAs) in roots of salt tolerant and sensitive rice cultivars

Vacuolar Ca²⁺-transporters could play an important role for salt tolerance in rice (Oryza sativa L.) root. Here, we compared the expression profiles of putative vacuolar cation/H⁺ exchanger (CAX) and calmodulin-regulated autoinhibited Ca²⁺-ATPase (ACA) in rice roots of salt tolerant cv. Pokkali and salt sensitive cv. IR29. In addition to five putative vacuolar CAX genes in the rice genome, a new CAX gene (OsCAX4) has been annotated. In the present study, we isolated the OsCAX4 gene and showed that its encoded protein possesses a unique transmembrane structure and is potentially involved in transporting not only Ca²⁺ but also Mn²⁺ and Cu²⁺. These six OsCAX genes differed in their mRNA expression pattern in roots of tolerant versus sensitive rice cultivars exposed to salt stress. For example, OsCAX4 showed abundant expression in IR29 (sensitive) upon prolonged salt stress. The mRNA expression profile of four putative vacuolar Ca²⁺-ATPases (OsACA4-7) was also examined. Under control conditions, the mRNA levels of OsACA4, OsACA5, and OsACA7 were relatively high and similar among IR29 and Pokkali. Upon salt stress, only OsACA4 showed first a decrease in its expression in Pokkali (tolerant), followed by a significant increase. Based on these results, a role of vacuolar Ca²⁺ transporter for salt tolerance in rice root was discussed.     

Responses of Nipa palm (Nypa fruticans) seedlings,a mangrove species,to salt stress in pot culture

Nipa palm (Nypa fruticans) is the only palm that grows in mangrove vegetation. We investigated the effect of salt stress on the growth and physiology of 6-month-old seedlings of Nipa palm exposed to different degrees of salt stress (as NaCl) in pot culture. The overall growth performance of Nipa palm was unaffected by mild salt stress (8.9−16.6 dS m⁻¹), whereas seedlings grown under severe salt stress (EC = 57.2 dS m⁻¹) had lower chlorophyll content and fluorescence, reduced net photosynthesis and transpiration, which resulted in reduced growth of the plants. Na⁺ contents in leaf, petiole, and root tissues increased considerably under salt stress, depending upon the NaCl levels in the soil solution. Under salt-stress K⁺ content declined, whereas Ca²⁺ content increased somewhat, in parallel to Na⁺. Free proline accumulated in plants growing under high salt stress (EC = 57.2 dS m⁻¹). In contrast, soluble sugars were enriched under intermediate levels of salt stress (EC = 16.6 dS m⁻¹). The results obtained in the present study suggest that, based on ecophysiological data, N. fruticans is a species best adapted to grow in mangrove coastal areas with moderate only salt load, and circumscribing quite well the actual areas of occurrence of this palm in the gradient from seawater habitats to inland sites. © 2014 Elsevier GmbH