High Levels Of Glycine Betaine Research Articles

Recovery from Salinity and Drought Stress in the Perennial Sarcocornia fruticosa vs. the Annual Salicornia europaea and S. veneta.

Current agricultural problems, such as the decline of freshwater and fertile land, foster saline agriculture development. Salicornia and Sarcocornia species, with a long history of human consumption, are ideal models for developing halophyte crops. A greenhouse experiment was set up to compare the response of the perennial Sarcocornia fruticosa and the two annual Salicornia europaea and S. veneta to 30 days of salt stress (watering with 700 mM NaCl) and water deficit (complete withholding of irrigation) separate treatments, followed by 15 days of recovery. The three species showed high tolerance to salt stress, based on the accumulation of ions (Na+, Cl−, Ca2+) in the shoots and the synthesis of organic osmolytes. These defence mechanisms were partly constitutive, as active ion transport to the shoots and high levels of glycine betaine were also observed in non-stressed plants. The three halophytes were sensitive to water stress, albeit S. fruticosa to a lesser extent. In fact, S. fruticosa showed a lower reduction in shoot fresh weight than S. europaea or S. veneta, no degradation of photosynthetic pigments, a significant increase in glycine betaine contents, and full recovery after the water stress treatment. The observed differences could be due to a better adaptation of S. fruticosa to a drier natural habitat, as compared to the two Salicornia species. However, a more gradual stress-induced senescence in the perennial S. fruticosa may contribute to greater drought tolerance in this species.

Changes in Bull Semen Metabolome in Relation to Cryopreservation and Fertility.

Simple SummaryAlthough semen cryopreservation has facilitated the diffusion of artificial insemination and in vitro fertilization in cattle, it still represents a major factor affecting sperm fertility. It is known that cryopreservation induces the loss of fertility-associated proteins, while the effect on metabolites has not been evaluated, although several compounds affect sperm physiology and fertility. The aim of the present work was to study the metabolome in bovine sperm and seminal plasma after cryopreservation and to correlate the metabolic profile of high- and low-fertility bulls in order to identify fertility markers. The analysis, carried out by liquid chromatography–mass spectrometry methods, revealed differences in metabolite contents between fresh and cryopreserved semen, both at cellular and plasmatic levels. Interestingly, metabolites showing variation have important functions related to fertility. In addition, the study highlighted the differences in lipid profile between high- and low-fertility bulls. The identification of new potential fertility markers is of high economic impact. In addition, it opens the way for the development of corrective strategies to improve the fertility of low-fertility bulls.Semen cryopreservation determines several sperm damages, including the loss of fertility-associated proteins. The purpose of the study was to compare the metabolite contents in bovine sperm and seminal plasma before and after cryopreservation, and between high- and low-fertility bulls in vitro. Forty-eight ejaculates, collected from eight bulls (six per bull), were analyzed by liquid chromatography–mass spectrometry. Cryopreservation resulted in an over-expression of lysophosphatidylcholine (0:0/18:2(9Z,12Z)) in seminal plasma. In addition, higher levels of glycine betaine and pyro-l-glutaminyl-l-glutamine were observed in cryopreserved compared to fresh spermatozoa. The fresh seminal plasma of high-fertility bulls showed an over-expression of l-acetylcarnitine, glycerol tripropanoate, 2,3-diacetoxypropyl stearate and glycerophosphocholine, and an under-expression of lysophosphatidylcholine and butyrylcarnitine, compared to low-fertility bulls. Higher levels of glycerophosphocholine and lysophosphatidylcholine (16:0/0:0) were recorded in fresh spermatozoa from high-fertility bulls. In high-fertility bulls, a greater content of glycerophosphocholine and lower levels of butyrylcarnitine, glycine betaine and l-carnitine were found in cryopreserved seminal plasma, and lower levels of glycine betaine were detected in cryopreserved spermatozoa. In conclusion, cryopreservation affects bovine semen metabolome at both plasmatic and cellular compartments, and metabolic profile differs between high- and low-fertility bulls.

Spatial and Temporal Profile of Glycine Betaine Accumulation in Plants Under Abiotic Stresses.

Several halophytes and a few crop plants, including Poaceae, synthesize and accumulate glycine betaine (GB) in response to environmental constraints. GB plays an important role in osmoregulation, in fact, it is one of the main nitrogen-containing compatible osmolytes found in Poaceae. It can interplay with molecules and structures, preserving the activity of macromolecules, maintaining the integrity of membranes against stresses and scavenging ROS. Exogenous GB applications have been proven to induce the expression of genes involved in oxidative stress responses, with a restriction of ROS accumulation and lipid peroxidation in cultured tobacco cells under drought and salinity, and even stabilizing photosynthetic structures under stress. In the plant kingdom, GB is synthesized from choline by a two-step oxidation reaction. The first oxidation is catalyzed by choline monooxygenase (CMO) and the second oxidation is catalyzed by NAD+-dependent betaine aldehyde dehydrogenase. Moreover, in plants, the cytosolic enzyme, named N-methyltransferase, catalyzes the conversion of phosphoethanolamine to phosphocholine. However, changes in CMO expression genes under abiotic stresses have been observed. GB accumulation is ontogenetically controlled since it happens in young tissues during prolonged stress, while its degradation is generally not significant in plants. This ability of plants to accumulate high levels of GB in young tissues under abiotic stress, is independent of nitrogen (N) availability and supports the view that plant N allocation is dictated primarily to supply and protect the growing tissues, even under N limitation. Indeed, the contribution of GB to osmotic adjustment and ionic and oxidative stress defense in young tissues, is much higher than that in older ones. In this review, the biosynthesis and accumulation of GB in plants, under several abiotic stresses, were analyzed focusing on all possible roles this metabolite can play, particularly in young tissues.

Transgenic poplar expressing codA exhibits enhanced growth and abiotic stress tolerance

Glycine betaine (GB), a compatible solute, effectively stabilizes the structure and function of macromolecules and enhances abiotic stress tolerance in plants. We generated transgenic poplar plants (Populus alba×Populus glandulosa) expressing a bacterial choline oxidase (codA) gene under the control of the oxidative stress-inducible SWPA2 promoter (referred to as SC plants). Among the 13 SC plants generated, three lines (SC4, SC14 and SC21) were established based on codA transcript levels, tolerance to methyl viologen-mediated oxidative stress and Southern blot analysis. Growth was better in SC plants than in non-transgenic (NT) plants, which was related to elevated transcript levels of auxin-response genes. SC plants accumulated higher levels of GB under oxidative stress compared to the NT plants. In addition, SC plants exhibited increased tolerance to drought and salt stress, which was associated with increased efficiency of photosystem II activity. Finally, SC plants maintained lower levels of ion leakage and reactive oxygen species under cold stress compared to the NT plants. These observations suggest that SC plants might be useful for reforestation on global marginal lands, including desertification and reclaimed areas.

Salt effects on proline and glycine betaine levels and photosynthetic performance in Melilotus siculus, Tecticornia pergranulata and Thinopyrum ponticum measured in simulated saline conditions.

We measured proline and glycine betaine levels and photosynthetic performance (net-photosynthetic rate (Pn), stomatal conductance (gs), maximum quantum yield of PSII (Fv/Fm) and non-photochemical quenching (NPQ)) in relation to Na+ and Cl- accumulation in Melilotus siculus (Turra) B.D.Jacks. (Fabaceae), Tecticornia pergranulata (J.M.Black) K.A.Sheph. & Paul G.Wilson (Amaranthaceae: Salicornioideae) and Thinopyrum ponticum (Podp.) Z.-W.Liu & R.-C.Wang (Poaceae) grown under saline conditions in the greenhouse. These plants were selected in this study because of their known salt-tolerance capacity and value as forage plants. Moreover, the pasture legume M. siculus is considered to have particular potential for saline land remediation because of its salinity and waterlogging tolerance. Maximum Na+ and Cl- accumulation occurred in Te. pergranulata shoots. Minimum was in Th. ponticum shoots. Maximum Na+ accumulation occurred in the roots of Te. pergranulata, whereas that of Cl- occurred in the roots of Th. ponticum. Accumulation of both Na+ and Cl- was the least in M. siculus roots. Te. pergranulata metabolized high levels of glycine betaine (110µmolg-1 DW). M. siculus metabolized high levels of proline (6µmolg-1 DW). Th. ponticum accumulated intermediate levels of these organic osmolytes. No significant change occurred in Fv/Fm values. Pn value increased and NPQ value decreased in Te. pergranulata with increasing salinity and the reverse occurred in both M. siculus and Th. ponticum. A negative significant correlation occurred between Pn and glycine betaine in M. siculus and Th. ponticum. A positive significant correlation occurred between NPQ and glycine betaine in M. siculus. No correlation occurred between proline and Pn, proline and NPQ in the tested three plants. Te. pergranulata could maintain cell-osmotic balance by synthesising high levels of organic osmolytes especially glycine betaine and concurrently showing the most efficient photosynthetic performance. Compared with the levels of osmolytes in Te. pergranulata, the levels of osmolytes that occur in M. siculus and Th. ponticum were insufficient to maintain cell-osmotic balance and also that M. siculus and Th. ponticum showed a lower level of photosynthetic performance. We conclude that glycine betaine is potentially the vital organic osmolyte for Te. pergranulata and Th. ponticum enabling salinity stress tolerance. However, in M. siculus, proline appears to be the potential organic osmolyte in salinity stress tolerance. In terms of the potential of these species for stabilising saline soils in central-western New South Wales, Te. pergranulata would be the candidate of choice; however, for greater pasture value Th. ponticum would be the next.

Glycine betaine biosynthesis in saltbushes (Atriplex spp.) under salinity stress

AbstractSoil salinity and drought severely affect all aspects of plant physiology, leading to significant losses of crop productivity and native biodiversity. A key to sustainable land use in such areas is to cultivate well-adapted native plants that are also commercially important and have the appropriate gene pool. Glycine betaine (GB) is an osmoprotectant that imparts salt and drought tolerance to some plants. It is also shown separately to provide significant health benefits to animals and humans. We investigated whether Australian saltbushes, which are extremely salt and drought tolerant and also impart health benefits to grazing animals, may have the genetic basis for GB biosynthesis, explaining the two different observations. Complementary DNAs encoding the two key enzymes of the plant GB biosynthesis pathway, choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), were identified and analysed from Atriplex nummularia and Atriplex semibaccata. The sequences showed the putative CMO proteins exhibited all functionally important features including the Reiske-type cluster (2Fe-2S) and mononuclear non-heme Fe cluster, and the putative BADHs exhibited conservation of active site residues. The expression of both genes was found to be significantly up-regulated in leaf tissues under salt stress. The leaf tissues also showed accumulation of very high levels of GB, at 29.69 mmol/kg fresh weight for A. nummularia and 42.68 mmol/kg fresh weight for A. semibaccata, which is several times higher than in cereal crops. The results demonstrate a strong potential of cultivation of saltbushes for re-vegetation and as a perennial fodder in salinity and drought-affected areas.

Increased Chilling Tolerance Following Transfer of a betA Gene Enhancing Glycinebetaine Synthesis in Cotton (Gossypium hirsutum L.)

A betA gene encoding choline dehydrogenase from Escherichia coli was transformed into cotton (Gossypium hirsutum L.) via Agrobacterium-mediated transformation. Transgenic cotton plants exhibited improved tolerance to chilling due to accumulation of glycinebetaine (GB). The results of our experiment showed that GB contents of leaves of transgenic lines 1, 3, 4, and 5, both before and after chilling stress, were significantly higher than those of wild-type (WT) plants. At 15°C, transgenic lines 1, 3, 4, and 5 exhibited higher germination capacity as determined by the germination speed and final germination percentage and, displayed less inhibition in seedling shoot growth rate than WT plants. Under chilling stress, transgenic lines 4 and 5 maintained higher relative water content, upper carbon dioxide (CO2) fixation capacity and PSII electron transfer rate, better osmotic adjustment (OA), a lower percentage of ion leakage, and less lipid membrane peroxidation when compared with WT plants. Chilling resistance of the transgenic lines was demonstrated to be positively correlated with GB content under chilling stress. The high levels of GB in transgenic cotton plants might not only protect the integrity of cell membrane from chilling damage, but also be involved in OA which alleviated chilling induced water stress. Moreover, under chilling-stressed conditions, transgenic cotton plants enhanced stomatal conductance, PSII electron transport rate, and further leaf photosynthesis through accumulating high levels of GB.

Improved salt tolerance and seed cotton yield in cotton (Gossypium hirsutum L.) by transformation with betA gene for glycinebetaine synthesis

Homozygous transgenic cotton (Gossypium hirsutum L.) plants that accumulated glycinebetaine (GB) in larger quantities were more tolerant to salt than wild-type (WT) plants. Four transgenic lines, namely 1, 3, 4, and 5, accumulated significantly higher levels of GB than WT plants did both before and after salt stress. At 175 and 275 mM NaCl, seeds of all the transgenic lines germinated earlier and recorded a higher final germination percentage, and the seedlings grew better, than those of the WT. Under salt stress, all the lines showed some characteristic features of salt tolerance, such as higher leaf relative water content (RWC), higher photosynthesis, better osmotic adjustment (OA), lower percentage of ion leakage, and lower peroxidation of the lipid membrane. Levels of endogenous GB in the transgenic plants were positively correlated with RWC and OA. The results indicate that GB in transgenic cotton plants not only maintains the integrity of cell membranes but also alleviates osmotic stress caused by high salinity. Lastly, the seed cotton yield of transgenic lines 4 and 5 was significantly higher than that of WT plants in saline soil. This research indicates that betA gene has the potential to improve crop’s salt tolerance in areas where salinity is limiting factors for agricultural productivity.

Improved salt tolerance of transgenic wheat by introducing betA gene for glycine betaine synthesis

A betA gene encoding choline dehydrogenase from Escherichia coli (E. coli) was transformed into wheat (Triticum aestivum L.) via Agrobacterium-mediated transformation. PCR amplification and Southern blotting confirmed the existence of transgene in transformed plants and their progeny. Levels of expression of the betA gene varied among the different transgenic lines based on RT-PCR analysis. Under salt stress conditions, transgenic lines L2 and L3 had higher levels of glycine betaine and chlorophyll, lower Na+/K+ ratios and solute potential, and less cell membrane damage. These lines also retained moderately higher photosynthesis rates and more vigorous growth than the wild-type line at 200 mM NaCl. In a field trial in a high salt field, transgenic lines L2 and L3 had higher germination rates, more tillers and higher grain yields in comparison to the wild-type plants. This suggested that the transgenic plants were more tolerant to salt stress and have potential for breeding salt-tolerant wheat.

The codA transgene for glycinebetaine synthesis increases the size of flowers and fruits in tomato

The tolerance of various species of plant to abiotic stress has been enhanced by genetic engineering with certain genes. However, the use of such transgenes is often associated with negative effects on growth and productivity under non-stress conditions. The codA gene from Arthrobacter globiformis is of particular interest with respect to the engineering of desirable productive traits in crop plants. The expression of this gene in tomato plants resulted in significantly enlarged flowers and fruits under non-stress conditions. The enlargement of flowers and fruits was associated with high levels of glycinebetaine that accumulated in reproductive organs, such as flower buds and fruits. The enlargement of flowers was related to an increase in the size and number of cells, and reflected the pleiotropic effect of the codA transgene on the expression of genes involved in the regulation of cell division.

Increase of glycinebetaine synthesis improves drought tolerance in cotton

The tolerance to drought stress of the homozygous transgenic cotton (Gossypium hirsutum L.) plants with enhanced glycinebetaine (GB) accumulation was investigated at three development stages. Among the five transgenic lines investigated, lines 1, 3, 4, and 5 accumulated significantly higher levels of GB than the wild-type (WT) plants either before or after drought stress, and the transgenic plants were more tolerant to drought stress than the wild-type counterparts from young seedlings to flowering plants. Under drought stress conditions, transgenic lines 1, 3, 4, and 5 had higher relative water content, increased photosynthesis, better osmotic adjustment (OA), a lower percentage of ion leakage, and less lipid membrane peroxidation than WT plants. The GB levels in transgenic plants were positively correlated with drought tolerance under water stress. The results suggested that GB may not only protect the integrity of the cell membrane from drought stress damage, but also be involved in OA in transgenic cotton plants. Most importantly, the seedcotton yield of transgenic line 4 was significantly greater than that of WT plants after drought stress, which is of great value in cotton production.

Engineering of enhanced glycine betaine synthesis improves drought tolerance in maize

Glycine betaine plays an important role in some plants, including maize, in conditions of abiotic stress, but different maize varieties vary in their capacity to accumulate glycine betaine. An elite maize inbred line DH4866 was transformed with the betA gene from Escherichia coli encoding choline dehydrogenase (EC 1.1.99.1), a key enzyme in the biosynthesis of glycine betaine from choline. The transgenic maize plants accumulated higher levels of glycine betaine and were more tolerant to drought stress than wild-type plants (non-transgenic) at germination and the young seedling stage. Most importantly, the grain yield of transgenic plants was significantly higher than that of wild-type plants after drought treatment. The enhanced glycine betaine accumulation in transgenic maize provides greater protection of the integrity of the cell membrane and greater activity of enzymes compared with wild-type plants in conditions of drought stress.

The role of glycine betaine in the protection of plants from stress: clues from transgenic plants.

The acclimation of a plant to a constantly changing environment involves the accumulation of certain organic compounds of low molecular mass, known collectively as compatible solutes, in the cytoplasm. The evidence from numerous investigations of the physiology, genetics, biophysics and biochemistry of plants strongly suggests that glycine betaine (GB), an amphoteric quaternary amine, plays an important role as a compatible solute in plants under various types of environmental stress, such as high levels of salts and low temperature. Plant species vary in their capacity to synthesize GB and some plants, such as spinach and barley, accumulate relatively high levels of GB in their chloroplasts while others, such as Arabidopsis and tobacco, do not synthesize this compound. Genetic engineering has allowed the introduction into GB-deficient species of biosynthetic pathways to GB from both micro-organisms and higher plants; this approach has facilitated investigations of the importance of GB in stress protection. In this review, we summarize recent progress in the genetic manipulation of the synthesis of GB, with special emphasis on the relationship between the protective effects of GB in vivo and those documented in vitro.

Study on the pathway of glycinebetaine biosynthesis in a monocotyledonous grass, Aneurolepidium chinense, grown in semiarid area

In some higher plants, glycinebetaine is known to be synthesized and accumulated in cells in response to salt stress or drought stress. Glycinebetaine is a compatible solute and acts as an excellent osmoprotectant. High levels of glycinebetaine are present in leaves of diverse families of dicotyledons and of some monocotyledons. The pathway of glycinebetaine biosynthesis in higher plants is as follows: Choline ? Betaine aldehyde ? Glycinebetaine. The first step is catalyzed by choline monooxygenase (CMO), being recently purified and characterized. The final step is catalyzed by betaine aldehyde dehydrogenase (BADH), which has been well characterized. Existence of BADH isozymes was suggested in spinach, mangrove, sorghum and barleys, and we reported that all those monocotyledonous plants have BADHs with a C-terminal tripeptide SKL that is known to be a signal for targeting preproteins to microbodies. We cloned and characterized a new type of sheep grass (Aneurolepidium chinense) BADH gene more homologous with dicotyledonous BADHs. Differential expression pattern was observed between the two types of sheep grass BADH genes under various stresses. Although the SKL type of BADH gene was inducible in sheep grass leaves, the new type of BADH gene was expressed constitutively under both heat and salt stresses. We also examine the localization of the new type of BADH protein. Furthermore we examine, whether or not sheep grass has chloroplastic CMO to protect photosynthetic machineries under heat or salt stresses.

Compatibility of glycinebetaine in rice plants: evaluation using transgenic rice plants with a gene for peroxisomal betaine aldehyde dehydrogenase from barley

ABSTRACTGlycinebetaine is synthesized in plants by the two‐step oxidation of choline, with betaine aldehyde as the intermediate. The reactions are catalyzed by choline mono‐oxygenase and betaine aldehyde dehydrogenase. Rice plants, which do not accumulate glycinebetaine, possess a gene encoding betaine aldehyde dehydrogenase, whose activity is detectable at low levels. To evaluate the compatibility in rice of glycinebetaine on growth and tolerance to salt, cold and heat, we produced transgenic rice plants by introduction of a cDNA for betaine aldehyde dehydrogenase of barley, which is localized in peroxisomes unlike the chloroplast‐specific localization of betaine aldehyde dehydrogenase in spinach and sugar beet. The transgenic rice plants converted high levels of exogenously applied betaine aldehyde (up to 10 mol m–3) to glycinebetaine more efficiently than did wild‐type plants. The elevated level of glycinebetaine in transgenic plants conferred significant tolerance to salt, cold and heat stress. However, very high levels of glycinebetaine, resulting from conversion of applied betaine aldehyde to glycinebetaine or from exogenous application, inhibited increases in length of rice plants but not increases in dry weight. Our results suggested that the benefits of accumulation of glycinebetaine by rice plants might be considerable under high light conditions.

Possible Involvement of Greening in Cell Growth of Suaeda japonica under Salt Stress

Suaeda japonica, a member of the family Chenopodiaceae, is a halophyte that grows on the shores of the Ariake Sea in Japan. Using yellowish and selected green callus tissues of S. japonica, we examined the correlation between cell growth and glycinebetaine content, as well as the activity of betaine aldehyde dehydrogenase (BADH), under salt stress. Although the growth of yellowish callus tissues was markedly inhibited by 0.1 M NaCl, that of green callus tissues was not. However, in 0.3 M NaCl, the fresh weight of green callus tissues was decreased by 15%. Both the endogenous level of glycinebetaine and the activity of BADH in yellowish callus tissues were very low, but green callus tissues contained very high levels of glycinebetaine and high BADH activity. The activity in green callus increased significantly with increases in the concentration of NaCl. The increase in levels of Chl was also observed, but the increase occurred earlier than that in the level of glycinebetaine. Functional development of chloroplasts (an increase in the level of Chl) seems to be involved in the increase in the activity of BADH, and the resultant increase in levels of glycinebetaine seems to facilitate callus growth under salt stress.

Solute distribution in Suaeda maritima

The distribution of sodium, potassium and glycinebetaine in shoot tissues of salt-treated Suaeda maritima was examined by semi-micro techniques after extraction into toluene-water. Much higher K/Na ratios were observed in the apical regions and in axillary buds than in more mature, fully vacuolated tissues. The younger tissues also contained very high levels of glycinebetaine. Electron-probe X-ray microanalysis of bulkfrozen and fractured preparations showed higher K/Na ratios and higher levels of sulphur and phosphorus in the cytoplasm of leaf primordial cells than in vacuoles of either young or old leaves, although the total counts were higher in the vacuolar samples. The results are discussed in relation to current models of subcellular solute compartmentation and salt tolerance in the Chenopodiaceae.

Accumulation of free proline and glycine betaine in Aster tripolium subjected to a saline shock: A kinetic study related to light period

On transferring three‐week‐old plants of Aster tripolium L. growing in a half strength Hoagland's medium to the same medium containing 333 mM NaCl a very quick uptake of salt and, after a lag phase of 3 to 5 h, an increase in free proline level was observed. During the time course of imino acid storage, the accumulation rates were higher in the light than in the dark, thereby suggesting some kind of photocontrol on solute metabolism. At zero time, high levels of glycine betaine were present in young plants grown without salt. However, after the application of saline shock, the betaine level also increased significantly. The highest rate of betaine accumulation was detected during the third day of treatment when the rate of proline storage decreased. Glycine betaine storage could also be linked to light dependent processes; whatever its importance in response to salt shock was, the levels observed were lower than those of plants directly grown on 333 mM NaCl for three weeks. When saline stressed plants were transferred to a medium without NaCl, the proline level quickly decreaed while that of glycine betaine remained stable.