Characterization of the gene for Δ1-pyrroline-5-carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa L.

A cDNA for ¹-pyrroline-5-carboxylate (P5C) synthetase (cOsP5CS), an enzyme involved in the biosynthesis of proline, was isolated and characterized from a cDNA library prepared from 14-day-old seedlings of Oryza sativa cv. Akibare. The deduced amino acid sequence of the P5CS protein (OsP5CS) from O. sativa exhibited 74.2% and 75.5% homology to that of the P5CS from Arabidopsis thaliana and Vigna aconitifolia, respectively. Northern blot analysis revealed that the gene for P5CS (OsP5CS) was induced by high salt, dehydration, treatment of ABA and cold treatment, while it was not induced by heat treatment. Simultaneously, accumulation of proline was observed as a result of high salt treatment in O. sativa. Moreover, the levels of expression of OsP5CS mRNA and content of proline under salt stress condition were compared between a salt-tolerant cultivar, Dee-gee-woo-gen (DGWG) and a salt-sensitive breeding line, IR28. It was observed that the expression of the P5CS gene and the accumulation of proline in DGWG steadily increased, whereas those in IR28 increased slightly.     

Stress-induced Δ1-pyrroline-5-carboxylate synthetase (P5CS) gene confers tolerance to salt stress in transgenic sugarcane

High salinity interferes in sugarcane growth and development, affecting not only crop yield but also reducing sucrose concentration in culms. Sugarcane plants submitted to salt stress can accumulate compatible solutes, such as proline, which may counteract the effects of salt accumulation in the vacuole and scavenge reactive oxygen species. The objective of this study was to evaluate the response to salt stress of sugarcane plants transformed with the Vigna aconitifolia P5CS gene, which encodes ?1-pyrroline-5-carboxylate synthetase, under the control of a stress-induced promoter AIPC (ABA-inducible promoter complex). For this, 4-month-old clonally multiplied sugarcane plants from two transformation events were irrigated every 2 days with 1/10 Hoagland’s solution supplemented with 100, 150 and 200 NaCl, progressively, during 28 days. Transgenic lines showed increased transgene expression in 3.75-fold when compared with the control plants after 9 days of irrigation with saline water, which can explain the higher proline concentration found in these plants. At the end of the experiment (day 28), the transgenic lines accumulated up to 25 % higher amounts of proline when compared with non-transformed control plants. Stress response in transgenic plants was also accompanied by a reduction of malondialdehyde (MDA) derived from cellular lipid peroxidation in leaves, lower Na⁺ accumulation in leaves and maintenance of photochemical efficiency of PSII. Thus, proline contributed to the protection of the photosynthetic apparatus and the prevention of oxidative damage in transgenic sugarcane under salt stress.     

The relationship between proline content, the expression level of P5CS (Δ1-pyrroline-5-carboxylate synthetase), and drought tolerance in Tibetan hulless barley (Hordeum vulgare var. nudum)

Many plants accumulate proline (Pro) when suffered from drought; thus, the relationship between Pro accumulation and plant drought tolerance becomes an increasing concern. Pro is synthesized from either glutamine or ornithine, and the former pathway dominates under osmotic stress conditions. In this study, the dynamic accumulation of free Pro under drought stress in 10 genotypes of Tibetan hulless barley (Hordeum vulgare var. nudum) with water lose rate (WLR) of 0.3304 to 0.5839 g/(h g dry wt) was investigated. However, no correlation between Pro accumulation and drought tolerance was found. Furthermore, the barley stripe mosaic virus establisheding virus-induced gene silencing was employed to suppress the expression of the encoding gene Δ¹-Pyrroline-5-Carboxylate Synthetase (P5CS), which catalyzes the ratelimiting step of Glu pathway in Pro biosynthesis. By the quantitative real-time polymerase chain reaction, the decrease of the P5CS expression was found, and a consequent Pro degradation was also detected in P5CS-silenced plants. However, neither increased WLR of detached leaves nor decreased survival rate under drought stress was found compared with control plants. These results suggested that the repressed expression of P5CS and decreased content of free Pro may not interfere with the drought tolerance of Tibetan hulless barley.     

Isolation and sequence analysis of the cDNA encoding Δ1-pyrroline-5-carboxylate reductase from Zalerion arboricola

A cDNA encoding Δ¹-pyrroline-5-carboxylate reductase (P5CR) was isolated from the pneumocandin(Pmo)-producing fungus, Zalerion arboricola (Za), by complementation of a P5CR-deficient mutant (pro3) of Saccharomyces cerevisiae (Sc). The cloned cDNA was placed under control of the Sc galactokinase (GAL1) promoter and restored P5CR activity to the pro3 mutant. Sequence analysis revealed that the Za P5CR-encoding cDNA encodes an approx. 35 kDa protein with substantial amino acid (aa) identity to P5CR from another filamentous fungus, Neurospora crassa (Nc). Za P5CR exhibits a moderate degree of aa identity to P5CR from plants, bacteria, human and Sc. This is the first gene to be isolated from Za.     

Proline accumulation and the expression of Δ1-pyrroline-5-carboxylate synthetase in two safflower cultivars

Proline (Pro) accumulation under water stress was measured in safflower (Carthamus tinctorius L.) drought tolerant cv. A1 and sensitive cv. Nira. Activities of pyrroline-5-carboxylate reductase (P5C reductase) and pyrroline-5-carboxylate synthetase (P5C synthetase), two enzymes involved in the Pro biosynthetic pathway were also estimated. Water stress resulted in a reduction in the leaf dry mass and chlorophyll content along with a gradual accumulation of Pro. RT-PCR results show higher expression of Δ¹-pyrroline-5-carboxylate synthetase (p5cs) gene in correlation with up-regulated Pro accumulation in cv. A1. P5C reductase was found to be the Pro synthesis rate limiting whereas P5C synthetase did not show any specific response to the drought stress in both cultivars.     

Δ-9-Tetrahydrocannabinol increases prodynorphin and proenkephalin gene expression in the spinal cord of the rat

Hypoalgesia induced by cannabinoid drugs has been found to implicate the opioid system. The effect of five days treatment with Δ-9-tetrahydrocannabinol (THC) was examined on prodynorphin (PDYN) and proenkephalin (PENK) gene expression in the spinal cord of male rats. PDYN and PENK gene expression was estimated measuring by northern blot analysis mRNA levels in the whole spinal cord, containing perikarya of these neurons. The subchronic treatment with THC (5 mg/kg/day; 5 days; i.p.) produced an increase in PDYN (39%) and PENK (34%) gene expression when compared with the vehicle treated group. These results suggest that the effects of THC in the spinal cord involve an increase in opioid activity, and therefore sustain the hypothesis of an interaction between the cannabinoid and opioid systems in this region.     

Enhanced synthesis of poly gamma glutamic acid by increasing the intracellular reactive oxygen species in the Bacillus licheniformis Δ1-pyrroline-5-carboxylate dehydrogenase gene ycgN-deficient strain

Poly gamma glutamic acid (γ-PGA) is an anionic polyamide with numerous applications. Previous studies revealed that L-proline metabolism is implicated in a wide range of cellular processes by increasing intercellular reactive oxygen species (ROS) generation. However, the relationship between L-proline metabolism and γ-PGA synthesis has not yet been analyzed. In this study, our results confirmed that deletion of Δ1-pyrroline-5-carboxylate dehydrogenase gene ycgN in Bacillus licheniformis WX-02 increased γ-PGA yield to 13.91 g L⁻¹, 85.22% higher than that of the wild type (7.51 g L⁻¹). However, deletion of proline dehydrogenase gene ycgM had no effect on γ-PGA synthesis. Furthermore, a 2.92-fold higher P5C content (19.24 μmol gDCW⁻¹) was detected in the ycgN deficient strain WXΔycgN, while the P5C levels of WXΔycgM and the double mutant strain WXΔycgMN showed no difference, compared to WX-02. Moreover, the ROS level of WXΔycgN was increased by 1.18-fold, and addition of n-acetylcysteine (antioxidant) decreased its ROS level, which further reduced γ-PGA synthesis capability of WXΔycgN. Collectively, our results demonstrated that proline catabolism played an important role in maintaining ROS homeostasis, and deletion of ycgN-enhanced P5C accumulation, which induced a transient ROS signal to promote γ-PGA synthesis in B. licheniformis.

     

Genetic structure and gene flow in French populations of two Ostrinia taxa: host races or sibling species?

Most models of ecological speciation concern phytophagous insects in which speciation is thought to be driven by host shifts and subsequent adaptations of populations. Despite the ever-increasing number of studies, the current evolutionary status of most models remains incompletely resolved, as estimates of gene flow between taxa remain extremely rare. We studied the population genetics of two taxa of the Ostrinia genus--one feeding mainly on maize and the other on mugwort and hop--occurring in sympatry throughout France. The actual level of divergence of these taxa was unknown because the genetic structure of populations had been investigated over a limited geographical area and the magnitude of gene flow between populations had not been estimated. We used 11 microsatellite markers to investigate the genetic structure of populations throughout France and the extent of gene flow between the two Ostrinia taxa at several sites at which they are sympatric. We observed clear genetic differentiation between most populations collected on the typical respective hosts of each taxon. However, populations displaying intermediate allelic frequencies were found on hop plants in southern France. Individual assignments revealed that this result could be accounted for by the presence of both taxa on the same host. Gene flow, estimated by determining the proportion of hybrids detected, was low: probably<1% per generation, regardless of site. This indicates that the two Ostrinia taxa have reached a high level of genetic divergence and should be considered sibling species rather than host races.     

Isolation and characterization of a Δ1-pyrroline-5-carboxylate synthetase (NtP5CS) from Nitraria tangutorum Bobr. and functional comparison with its Arabidopsis homologue

Several functional and regulatory proteins play important roles in controlling plant stress tolerance. Proline (Pro) is one of the most accumulated osmolytes correlated with tolerance to stresses. Δ¹-Pyrroline-5-carboxylate synthetase (P5CS) is a rate-limiting enzyme in Pro biosynthesis. In the present study, we isolated the cDNA for a P5CS gene (NtP5CS) from the halophyte Nitraria tangutorum. Phylogenetic analysis and subcellular localization analysis of NtP5CS-GFP protein in onion cells showed that NtP5CS was a new P5CS gene and was involved in Pro synthesis in N. tangutorum. Expression of the NtP5CS gene was induced by salt stress, dehydration, and high and low temperatures. Escherichia coli overexpressing AtP5CS or NtP5CS exhibited better growth in all treatments, including high salinity, high alkalinity, dehydration, osmotic, heat and cold stresses. Additionally, NtP5CS recombinant E. coli cells grew better than did AtP5CS recombinant cells in response to abiotic stresses. Our data demonstrate that the P5CS from a halophytic species functions more efficiently than its homologue from a glycophytic species in improving the stress tolerance of E. coli.     

Effect of ploidy levels on the activities of Δ1-pyrroline-5-carboxylate synthetase,superoxide dismutase and peroxidase in Cenchrus species grown under water stress

The effects of ploidy levels on the activities of Δ¹-pyrroline-5-carboxylate synthetase (P5CS; EC not assigned), superoxide dismutase (SOD; EC 1.15.1.1) and guaiacol peroxidase (POX; EC 1.11.1.7), as well as malondialdehyde (MDA) and proline contents were studied in two months old plants of Cenchrus species. The Cenchrus species represent three ploidy levels: diploid, tetraploid, hexaploid and two life spans: annual and perennial. Plants were subjected to water stress for 2, 4, 6 and 8 d by withholding water under glasshouse conditions. Although the levels of proline increased with the magnitude of water stress, the P5CS activity did not show a corresponding increase in all species. Peroxidase and superoxide dismutase activities showed an increase or steady state in the early phase of drought and then declined with the further increase in the magnitude of water stress, indicating differing behaviors of species towards drought tolerance. Under drought, diploid Cenchrus species had a higher POX activity, MDA accumulation and lower proline content than tetraploid species. Lower POX and higher P5CS activities and proline contents, however, were observed in hexaploid and tetraploid species. Taken together, our findings suggest that diploid species have a less efficient antioxidant system to scavenge reactive oxygen species than tetra and hexaploid Cenchrus. This may result in a corresponding variability in growth and persistence under natural grasslands. The study also paves the way for investigations on the molecular events associated with drought in Cenchrus species differing in ploidy and life span.     

Characterization of the rat carbonic anhydrase II gene structure: sequence analysis of the 5′ flanking region and 3′ UTR

The rat carbonic anhydrase II gene was characterized and found to be approximately 15.5 kb in length and to contain 7 exons and 6 introns. All intron/exon junction and branch point sequences conform to consensus sequences, and the overall rat CA II genomic structure appears to be conserved upon comparison with mouse, human, and chicken CA II genes. The putative cis-acting elements within the analyzed 1014 bp 5′ flanking region include: TATA box, 4 Sp1 binding sites, 2 AP2 sites and putative tissue-specific β-globin-like repeat elements. A CpG island of approximately 800 bp was identified that begins about 600 bp upstream of exon 1 and extends about 200 bp into intron 1. In the 3′ UTR, two polyadenylation signals (AATAAA) are present, the second of which is believed to be utilized. Northern blot analysis reveals that the 1.7 kb rat CA II mRNA is abundantly expressed in adult male brain and kidney, while negligible amounts are detected in heart and liver.     

Ecophysiology of selected tree species in different plant communities at the periphery of the Atlantic Forest of SE—Brazil III. Three legume trees in a semi-deciduous dry forest

Three legume tree species (Fabaceae) occurring abundantly in a semi-deciduous tropical dry forest of the Atlantic forest complex in southeastern Brazil were subjected to a comparative ecophysiological study at the end of the dry season/beginning of the wet season. The trees chosen were morphologically very similar: Caesalpinia echinata Lam. and Caesalpinia ferrea Mart. ex. Tul., both 10–20 m of height, of the sub-family Caesalpinioideae, and the somewhat smaller, 2–4 m tall, Machaerium obovatum Kuhlm. & Hoehne of the sub-family Faboideae. Despite their similarities with respect to their geographic distribution restricted to Brazilian dry forests, their comparable abundance in the study site and their phylogenetic proximity, the three species display distinctly different ecophysiological behaviour. Compared to the other two species, C. ferrea had the highest photosynthetic capacity (maximum apparent photosynthetic electron transport rate, ETR_max) and higher saturation light-intensity, was less subject to photoinhibition as indicated by potential quantum yield of photosystem II (F _v/F _m) and had the lowest bulk N content of which soluble non-protein N compounds were only 1.5%. It showed stronger sun plant characteristics. C. echinata had lower photosynthetic capacity, was under chronic photoinhibition and had high bulk N content of which 6.1% were soluble N compounds with high concentrations of proline. In addition to proline, high concentrations of sugars may serve as osmoprotectants. M. obovatum also showed lower photosynthetic capacity and was under chronic photoinhibition. Here, arginine may have a function as osmoprotectant. The ecophysiological differences between the three species are not related to local abundance. However, the observations presented highlight a contrasting behaviour of the otherwise very similar compatriot species.