Betaine Aldehyde Dehydrogenase Activity Research Articles

Effects of magnetic field exposure patterns on polyhydroxyalkanoates synthesis by Haloferax mediterranei at extreme hypersaline context: Carbon distribution and salt tolerance

The synthesis of polyhydroxyalkanoates (PHAs) by extremophiles presents a promising alternative to mitigate pollution originating from the use of petroleum-based plastics. This study focuses on the impact of different magnetic field (MF) exposure patterns on PHA production and carbon metabolism, aiming to enhance PHA productivity by Haloferax mediterranei within the extreme hypersaline environment and subsequently reducing production costs. Results indicated that under 300 g/L salinity, the highest PHA productivity (1.45 ± 0.06 g/(L d)) and PHA content (65.91 % PHA/cell dry weight) were achieved with 50 mT MF exposure throughout the fermentation period. Continuous exposure to 50 mT MF proved vital for maximizing cell biomass and PHA productivity. Continuous exposure to 50 mT MF enabled Haloferax mediterranei to channel acetyl-CoA towards the PHA synthesis pathway while maintaining growth and proliferation. Correlation analysis further proved the principal role of carbon flux on PHA accumulation. Due to the demand for balancing osmotic pressure, cellular substances were sacrificed to ensure PHA synthesis as anti-salinity substance. Meanwhile, the observed promotion of MF on PHA production, betaine aldehyde dehydrogenase activity, and K+ uptake contributed to sustaining cellular activity at 300 g/L salinity. This study provides a non-gene editing approach to enhance PHA productivity.

The GRAS transcription factor PtrPAT1 of Poncirus trifoliata functions in cold tolerance and modulates glycine betaine content by regulating the BADH-like gene

Abstract GRAS, termed after GAI (gibberellic acid insensitive), RGA (repressor of GA1), and SCR (scarecrow), is a plant-specific transcription factor crucial for plant development and stress response. However, understanding of the functions played by the GRAS members and their target genes in citrus is limited. In this study, we identified a cold stress-responsive GRAS gene from Poncirus trifoliate, designated as PtrPAT1, by yeast one-hybrid library screening using the promoter of PtrBADH-l, a betaine aldehyde dehydrogenase (BADH)-like gene. PtrPAT1, belonging to the PAT1 subfamily, was localized in the nucleus and plasma membrane, exhibited transactivation activity and showed a remarkable upregulation under cold stress. Overexpression of PtrPAT1 elevated BADH activity, increased glycine betaine (GB) accumulation and conferred enhanced cold tolerance in transgenic tobacco plants compared with wild type, while downregulation in trifoliate orange by virus-induced gene silencing (VIGS) resulted in opposite trends. Furthermore, the activities of two antioxidant enzymes, including peroxidase (POD) and superoxide dismutase (SOD), were significantly increased in the overexpression plants, but remarkably decreased in the VIGS line, consistent with accumulation patterns of the reactive oxygen species (ROS). PtrPAT1 was demonstrated to interact with and activate the PtrBADH-l promoter through the putative PAT1-binding motif with the core sequence of TTTCATGT, indicating that PtrBADH-l is a target gene of PtrPAT1. Taken together, these results demonstrate that PtrPAT1 positively affects cold tolerance through the regulation of GB biosynthesis by modulating PtrBADH-l expression.

Exogenous betaine enhances salt tolerance of Glycyrrhiza uralensis through multiple pathways

BackgroundGlycyrrhiza uralensis Fisch., a valuable medicinal plant, shows contrasting salt tolerance between seedlings and perennial individuals, and salt tolerance at seedling stage is very weak. Understanding this difference is crucial for optimizing cultivation practices and maximizing the plant’s economic potential. Salt stress resistance at the seedling stage is the key to the cultivation of the plant using salinized land. This study investigated the physiological mechanism of the application of glycine betaine (0, 10, 20, 40, 80 mM) to seedling stages of G. uralensis under salt stress (160 mM NaCl).ResultsG. uralensis seedlings’ growth was severely inhibited under NaCl stress conditions, but the addition of GB effectively mitigated its effects, with 20 mM GB had showing most significant alleviating effect. The application of 20 mM GB under NaCl stress conditions significantly increased total root length (80.38%), total root surface area (93.28%), and total root volume (175.61%), and significantly increased the GB content in its roots, stems, and leaves by 36.88%, 107.05%, and 21.63%, respectively. The activity of betaine aldehyde dehydrogenase 2 (BADH2) was increased by 74.10%, 249.38%, and 150.60%, respectively. The 20 mM GB-addition treatment significantly increased content of osmoregulatory substances (the contents of soluble protein, soluble sugar and proline increased by 7.05%, 70.52% and 661.06% in roots, and also increased by 30.74%, 47.11% and 26.88% in leaves, respectively.). Furthermore, it markedly enhanced the activity of antioxidant enzymes and the content of antioxidants (SOD, CAT, POD, APX and activities and ASA contents were elevated by 59.55%, 413.07%, 225.91%, 300.00% and 73.33% in the root, and increased by 877.51%, 359.89%, 199.15%, 144.35%, and 108.11% in leaves, respectively.), and obviously promoted salt secretion capacity of the leaves, which especially promoted the secretion of Na+ (1.37 times).ConclusionsIn summary, the exogenous addition of GB significantly enhances the salt tolerance of G. uralensis seedlings, promoting osmoregulatory substances, antioxidant enzyme activities, excess salt discharge especially the significant promotion of the secretion of Na+Future studies should aim to elucidate the molecular mechanisms that operate when GB regulates saline stress tolerance.

The power and versatility of genome editing tools in crop improvement

The power and versatility of genome editing tools in crop improvement

Lanthanum (La) improves growth, yield formation and 2-acetyl-1-pyrroline biosynthesis in aromatic rice (Oryza sativa L.)

BackgroundLanthanum (La) is a rare earth element that can influence plant growth and development. However, the effect of La on growth, yield formation and 2-acetyl-1-pyrroline (2-AP, a key compound responsible for the aroma of rice) biosynthesis in aromatic rice (Oryza sativa L. subsp. japonica Kato) has not been reported. Therefore, the present study investigated the effects of La on growth, photosynthesis, yield formation and 2-AP biosynthesis in aromatic rice through three experiments.ResultsTwo pot experiments and a two-year field trial were conducted with different rates of La application (20–120 LaCl3 mg kg−1 and 12 kg ha−1 LaCl3), and treatments without La application were used as controls. The results showed that the application of LaCl3 at 80 and 100 mg kg−1 and at 12 kg ha−1 greatly increased the 2-AP content (by 6.45–43.03%) in aromatic rice seedlings and mature grains compared with the control. The La treatments also increased the chlorophyll content, net photosynthetic rate and total aboveground biomass of rice seedlings. Higher antioxidant enzyme (superoxide, peroxidase, and catalase) activity was detected in the La treatments than in the control. The La treatments also increased the grain yield, grain number per panicle and seed-setting rate of aromatic rice relative to the control. Moreover, the grain proline and γ-aminobutyric acid contents and the activity of betaine aldehyde dehydrogenase significantly decreased under the La treatment. The application of La to soil enhanced the activity of proline dehydrogenase by 20.62–56.95%.ConclusionsLa improved the growth, yield formation and 2-AP content of aromatic rice and enhanced 2-AP biosynthesis by increasing the conversion of proline to 2-AP and decreasing the conversion of GABald to GABA.

Exogenous proline induces regulation in 2-acetyl-1-pyrroline (2-AP) biosynthesis and quality characters in fragrant rice (Oryza sativa L.)

Proline is one of the precursors of the biosynthesis of 2-acetyl-1-pyrroline (2-AP) which is the key and characteristic volatile component of fragrant rice aroma. In order to study the effects of exogenous proline on 2-AP biosynthesis and other grain quality attributes in fragrant rice, two indica fragrant rice cultivars, “Meixiangzhan-2” and “Xiangyaxiangzhan”, and one japonica fragrant rice, “Yunjingyou”, were used in present study. At initial heading stage, proline solutions at 0 (CK), 0.10 (Pro1), 0.20 (Pro2) and 0.50 (Pro3) g L-1 were applied as foliar spray solution to fragrant rice plants. Compared with CK, Pro1, Pro2 and Pro3 treatments significantly increased the grain 2-AP content. The significant up-regulation effects due to proline treatments were observed in the contents of proline, △1-pyrrolidine-5-carboxylic acid (P5C) and △1-pyrroline which involved in 2-AP formation. Exogenous proline application also significantly decreased the grain γ-aminobutyric acid (GABA) content. Furthermore, proline treatments enhanced the activity of proline dehydrogenase (ProDH) as well as transcript level of gene PRODH. On the other hand, the transcript level of gene BADH2 and activity of betaine aldehyde dehydrogenase (BADH) decreased under proline treatments. Proline treatments (Pro2 and Pro3) also increased the grain protein content by 3.57–6.51%. Moreover, 32.03–34.25% lower chalky rice rate and 30.80–48.88% lower chalkiness were recorded in proline treatments (Pro2 and Pro3) for both Meixiangzhan and Xiangyaxiangzhan whilst for Yunjingyou, foliar application of proline had no significant effect on chalky rice rate and chalkiness. There was no remarkable difference observed in grain milled quality (brown rice rate, milled rice rate and head rice rate) and amylose content between CK and proline treatments. In conclusion, exogenous proline enhanced the 2-AP biosynthesis and promoted some grain quality characters of fragrant rice.

Sequential expression of key genes in proline, glycine betaine and artemisinin biosynthesis of Artemisia aucheri Boiss using salicylic acid under in vitro osmotic stress

Artemisia aucheri belongs to the Asteraceae family, which is known for its medicinal and aromatic properties. The present study was conducted to elucidate the influence of salicylic acid (SA) pretreatment on proline and glycine betaine (GB) metabolism as well as artemisinin biosynthesis in A. aucheri under in vitro osmotic stress (˗0.6 MPa) induced by polyethylene glycol (PEG). The expression of ∆1-pyrroline-5-carboxylate synthetase (P5CS) and betaine aldehyde dehydrogenase (BADH) genes and related enzymes were enhanced under osmotic stress. SA pretreatment improved accumulation of proline and GB and increased the expression of BADH and P5CS enzyme activity under osmotic stress. However, proline dehydrogenase (PDH) gene expression and PDH activity were not affected by osmotic stress and SA pretreatments. In addition, SA in combination with or without PEG induced expression of the key genes involved in artemisinin biosynthesis and increased artemisinin content. These findings indicated that, higher accumulation of proline and GB under osmotic stress by SA is possibly due to increasing of osmoprotectants biosynthesis. This study suggested that A. aucheri is one of the potential species for artemisinin biosynthesis under environmental stress and exogenous SA.

Effect of salinity on the synthesis and concentration of glycine betaine in osmoregulatory tissues from juvenile shrimps Litopenaeus vannamei

In marine animals, glycine betaine is one of the main osmolytes accumulated under osmotic stress conditions; nevertheless, in penaeids, shrimps little is known about the pathways involved in glycine betaine biosynthesis. In animal cells, glycine betaine is synthesized by the enzyme betaine aldehyde dehydrogenase (BADH). We herein investigated the salinity effect on the synthesis and concentration of glycine betaine on white shrimp Litopenaeus vannamei. Shrimps were subjected to 10, 20, 35, 40, 50, and 60 ppt salinity conditions for seven days. BADH activity increased in hepatopancreas and gills of shrimps subjected to salinities above 35 ppt salinity. In muscle, the BADH activity decreased at 35 ppt salinity. In hepatopancreas from shrimps subjected to 50 and 60 ppt salinities, BADH activity increased 1.1 and 1.7-fold. At 60 ppt salinity, BADH activity increased 1.5-fold respect to 35 ppt in gills. Glycine betaine concentration increased in hepatopancreas, gills, muscle, and hemolymph in shrimps subjected to salinities above 35 ppt. Glycine betaine concentration also increased at 20 ppt salinity, while at 10 ppt, not detected significant differences. The catch of glycine betaine from hemolymph by the cell likely is carried out to avoid protein denaturalization. Ammonia concentration in the aquarium's water only increased at salinities of 20 ppt and 10 ppt (1.1-fold relative to 35 ppt). Our data demonstrated that in L. vannamei, salinity regulates BADH activity and glycine betaine content in a tissue-specific manner.

Nitric oxide and light co-regulate glycine betaine homeostasis in sunflower seedling cotyledons by modulating betaine aldehyde dehydrogenase transcript levels and activity

ABSTRACTGlycine betaine (GB), an osmolyte, is produced in chloroplasts by the action of betaine aldehyde dehydrogenase (BADH) on its precursor betaine aldehyde. The present work highlights the significance of nitric oxide (NO) in GB homeostasis as a long-distance salt (120 mM NaCl) stress-elicited response. In light-grown seedling cotyledons, both the activity and transcript levels of BADH are much higher than in dark-grown seedlings irrespective of salt stress. Significantly high accumulation of GB in dark-grown seedling cotyledons indicates its preferential mobilization from cotyledons to other plant parts in light-grown seedlings. NO donor application (diethylenetriamine) maintains high BADH activity in light, although in dark it is brought down marginally. BADH levels are maintained high in light than in dark in respective treatments. Reversal of the effect of NO donor on age-dependent GB content, BADH activity, and transcript levels by NO scavenger (diethyldithiocarbamate) further demonstrates the impact of NO on GB homeostasis in light- and dark-grown seedlings in an age-dependent manner, major modulation being observed in 4-d-old seedlings. The present work, thus, provides new information on co-regulation of GB homeostasis by NO and light. It also puts forward new information of GB–NO crosstalk in maneuvering salt stress sensing as a long-distance response in seedlings.

Overexpression of TaBADH increases salt tolerance in Arabidopsis

Soil salinization is an important threat to wheat growth and production. Previous transcriptome analysis showed that the expression of the betaine aldehyde dehydrogenase (BADH) gene differed significantly between cultivars with strong or weak salinity tolerance. Herein, the BADH gene from the wheat cultivar Dongnongdongmai 1 was cloned and transformed into wild-type Arabidopsis to identify its function in salt tolerance. Root length was calculated at 0, 50, 100, 150, and 200 mmol L−1 NaCl for 7 d. The relative electrolytic leakage (REL), GB content, and BADH activity were measured at 150 mmol L−1 NaCl for 1 and 3 d. It was determined that BADH activity and the GB content of TaBADH-overexpressed transgenic (TaBADHOE) lines were significantly higher than in wild-type lines. Salt stress analysis showed that the root length of TaBADHOE lines 4, 18, and 19 were 0.44, 0.54, and 0.35 cm, respectively, which were significantly longer than the 0.24 cm roots of the wild-type line in the media containing 150 mmol L−1 NaCl for 7 d. In addition, the RELs of transgenic lines 4, 18, and 19 were 0.37, 0.33, and 0.42, respectively, which is significantly lower than the 0.63 of the wild-type line in media containing 150 mmol L−1 NaCl for 3 d. These results demonstrate that TaBADH significantly increased plant salt tolerance, indicating that genetic transformation of TaBADH may be an effective and sustainable breeding method for increasing salt tolerance in wheat cultivars.

Bona fide choline monoxygenases evolved in Amaranthaceae plants from oxygenases of unknown function: Evidence from phylogenetics, homology modeling and docking studies.

Few land plants can synthesize and accumulate the osmoprotectant glycine betaine (GB) even though this metabolic trait has major adaptive importance given the prevalence of drought, hypersaline soils or cold. GB is synthesized from choline in two reactions catalyzed by choline monooxygenases (CMOs) and enzymes of the family 10 of aldehyde dehydrogenases (ALDH10s) that gained betaine aldehyde dehydrogenase activity (BADH). Homolog genes encoding CMO and ALDH10 enzymes are present in all known land plant genomes, but since GB-non-accumulators plants lack the BADH-type ALDH10 isozyme, they would be expected to also lack the CMO activity to avoid accumulation of the toxic betaine aldehyde. To explore CMOs substrate specificity, we performed amino acid sequence alignments, phylogenetic analysis, homology modeling and docking simulations. We found that plant CMOs form a monophyletic subfamily within the Rieske/mononuclear non-heme oxygenases family with two clades: CMO1 and CMO2, the latter diverging from CMO1 after gene duplication. CMO1 enzymes are present in all plants; CMO2s only in the Amaranthaceae high-GB-accumulators plants. CMO2s, and particularly their mononuclear non-heme iron domain where the active site is located, evolved at a faster rate than CMO1s, which suggests positive selection. The homology model and docking simulations of the spinach CMO2 enzyme showed at the active site three aromatic residues forming a box with which the trimethylammonium group of choline could interact through cation-π interactions, and a glutamate, which also may interact with the trimethylammonium group through a charge-charge interaction. The aromatic box and the carboxylate have been shown to be critical for choline binding in other proteins. Interestingly, these residues are conserved in CMO2 proteins but not in CMO1 proteins, where two of these aromatic residues are leucine and the glutamate is asparagine. These findings reinforce our proposal that the CMO1s physiological substrate is not choline but a still unknown metabolite.

Isolation and characterization of a salt stress-responsive betaine aldehyde dehydrogenase in Lycium ruthenicum Murr.

As compatible solute, glycine betaine (GB) plays a significant role in salinity tolerance in GB accumulating plants. Solanaceous crops such as tomato (Solanum lycopersicum) and tobacco (Nicotiana tabacum) are salt sensitive and naturally GB non-accumulators. In Solanaceae, only the Lycium genus has been recorded as halophytes in China, and several Lycium species have been reported as GB accumulators. The last biosynthetic step of GB is catalyzed by aminoaldehyde dehydrogenase (AMADH) with betaine aldehyde dehydrogenase (BADH) activities. Failure of GB synthesis in tomato and tobacco was attributed to lack of BADH activity. Here, by comparing the BADH functional residues of AMADHs between the Lycium genus and solanaceous crops, we predict that all studied AMADH1s have low BADH activities while only LbAMADH2 from L. barbarum has high BADH activity. For two AMADHs in L. ruthenicum, results from substrate enzyme assays confirmed low BADH activity of LrAMADH1 and no BADH activity of LrAMADH2. Despite the very low GB contents in L. ruthenicum seedlings (< 0.5μmol g-1 fresh weight), GB contents in fruits are up to 150 μmol g-1 FW, inferring fruits of L. ruthenicum as good GB sources. In NaCl treated seedlings, accompanied by elevated GB accumulation, expression of LrAMADH1 was up-regulated, indicating response of LrAMADH1 to salt stress in L. ruthenicum. Virus-induced silence of LrAMADH1 leads to less GB accumulation than control, revealing that LrAMADH1 participates in GB synthesis in planta. Collectively, our results show that LrAMADH1 is the bona fide BADH, which responds to salt stress in L. ruthenicum.

Reversible, partial inactivation of plant betaine aldehyde dehydrogenase by betaine aldehyde: mechanism and possible physiological implications.

In plants, the last step in the biosynthesis of the osmoprotectant glycine betaine (GB) is the NAD(+)-dependent oxidation of betaine aldehyde (BAL) catalysed by some aldehyde dehydrogenase (ALDH) 10 enzymes that exhibit betaine aldehyde dehydrogenase (BADH) activity. Given the irreversibility of the reaction, the short-term regulation of these enzymes is of great physiological relevance to avoid adverse decreases in the NAD(+):NADH ratio. In the present study, we report that the Spinacia oleracea BADH (SoBADH) is reversibly and partially inactivated by BAL in the absence of NAD(+)in a time- and concentration-dependent mode. Crystallographic evidence indicates that the non-essential Cys(450)(SoBADH numbering) forms a thiohemiacetal with BAL, totally blocking the productive binding of the aldehyde. It is of interest that, in contrast to Cys(450), the catalytic cysteine (Cys(291)) did not react with BAL in the absence of NAD(+) The trimethylammonium group of BAL binds in the same position in the inactivating or productive modes. Accordingly, BAL does not inactivate the C(450)SSoBADH mutant and the degree of inactivation of the A(441)I and A(441)C mutants corresponds to their very different abilities to bind the trimethylammonium group. Cys(450)and the neighbouring residues that participate in stabilizing the thiohemiacetal are strictly conserved in plant ALDH10 enzymes with proven or predicted BADH activity, suggesting that inactivation by BAL is their common feature. Under osmotic stress conditions, this novel partial and reversible covalent regulatory mechanism may contribute to preventing NAD(+)exhaustion, while still permitting the synthesis of high amounts of GB and avoiding the accumulation of the toxic BAL.

Enhanced salinity tolerance in transgenic maize plants expressing a BADH gene from Atriplex micrantha

Salinity is an adverse environmental stress that limits the yield and quality of maize. As one of the most important osmolytes present in higher plants, glycinebetaine helps stabilize metabolism in plant cells and protects the constituents of cells from damage. In this study, a gene from Atriplex micrantha that encodes betaine aldehyde dehydrogenase was introduced by Agrobacterium-mediated transformation into maize inbred lines Zheng58 and Qi319 under the control of the maize ubiquitin promoter. Putative transgenic plants were confirmed by PCR and Southern blotting analysis. The transgenic maize plants expressed higher amounts of betaine aldehyde dehydrogenase activity and also grew better than the WT plants under NaCl stress. Compared with the wild type, the transgenic plants had increased fresh weight, lower malondialdehyde content, lower relative electrical conductivity, higher chlorophyll content, taller plant height, and higher grain yield under salt stress, which indicated that the expression of BADH gene in maize seedlings enhanced the salt tolerance of these plants.

Osmoregulation as a key factor in drought hardening-induced drought tolerance in Jatropha curcas

Jatropha curcas L. is a plant with various commercial uses, and drought is an important limiting factor for its distribution and production. In this study, we investigated the role of drought hardening in an increased drought tolerance in J. curcas, and the involvement of osmoregulation and biochemical pathways in this enhanced tolerance. Results show that a drought hardening treatment with 10 % (m/v) polyethylene glycol 6000 for two days significantly increased a survival rate, decreased the content of malondialdehyde, and alleviated electrolyte leakage in the J. curcas seedlings under the drought stress. Measurements of leaf water potential, osmotic potential, and pressure potential show that this drought hardening treatment can improve the water status of J. curcas seedlings during the early phase of drought stress. In addition, the drought hardening treatment gradually increased the concentrations of compatible solutes proline, glycinebetaine, and soluble sugars during drought hardening and subsequent drought stress. It also clearly raised the activity of betaine aldehyde dehydrogenase, a key enzyme for the glycinebataine biosynhthesis as well as the activity of enzymes Δ1-pyrroline-5-carboxylate synthetase (P5CS), glutamate dehydrogenase, arginase, and ornithine aminotransferase, all key enzymes in the proline biosynthesis. The expression of P5CS gene in the J. curcas seedlings also increased during drought hardening and subsequent drought stress, but the activity of proline dehydrogenase decreased. These results show that the drought hardening treatment can enhance drought tolerance in J. curcas, and osmoregulation is a key factor in this increased drought tolerance.

Hydrogen sulfide donor sodium hydrosulfide-induced accumulation of betaine is involved in the acquisition of heat tolerance in maize seedlings

Hydrogen sulfide (H2S) which is involved in plant growth, development, and the acquisition of stress tolerance including heat tolerance, is considered as the third signal molecule after nitric oxide and reactive oxygen species, while betaine is an important osmolyte with multiple physiological functions, but interaction between H2S and betaine in the acquisition of heat tolerance in plants is not clear. In this study, pretreatment with H2S donor sodium hydrosulfide (NaHS), as comparison to the control seedlings without NaHS treatment, significantly improved the activity of betaine aldehyde dehydrogenase (BADH), a key enzyme in the biosynthesis of betaine, which in turn induced the accumulation of endogenous betaine, eventually enhanced the survival percentage of maize seedlings under heat stress. In contrast, these effects induced by NaHS were eliminated by application of H2S scavenger hypotaurine and inhibitor of BADH disulfiram, respectively, indicating that H2S-improved heat tolerance of maize seedlings may be closely associated with the accumulation of endogenous betaine by activating the activity of BADH. In addition, exogenous betaine treatment enhanced the content of endogenous betaine, followed by improved the survival percentage of maize seedlings compared with the control without betaine treatment. All of the above-mentioned results showed that NaHS pretreatment could induce the accumulation of endogenous betaine by increasing BADH activity, and this accumulation may be involved in the acquisition of heat tolerance of maize seedlings, bridging a gap between H2S and betaine in the acquisition of heat tolerance in plants.

Exploring the evolutionary route of the acquisition of betaine aldehyde dehydrogenase activity by plant ALDH10 enzymes: implications for the synthesis of the osmoprotectant glycine betaine

BackgroundPlant ALDH10 enzymes are aminoaldehyde dehydrogenases (AMADHs) that oxidize different ω-amino or trimethylammonium aldehydes, but only some of them have betaine aldehyde dehydrogenase (BADH) activity and produce the osmoprotectant glycine betaine (GB). The latter enzymes possess alanine or cysteine at position 441 (numbering of the spinach enzyme, SoBADH), while those ALDH10s that cannot oxidize betaine aldehyde (BAL) have isoleucine at this position. Only the plants that contain A441- or C441-type ALDH10 isoenzymes accumulate GB in response to osmotic stress. In this work we explored the evolutionary history of the acquisition of BAL specificity by plant ALDH10s.ResultsWe performed extensive phylogenetic analyses and constructed and characterized, kinetically and structurally, four SoBADH variants that simulate the parsimonious intermediates in the evolutionary pathway from I441-type to A441- or C441-type enzymes. All mutants had a correct folding, average thermal stabilities and similar activity with aminopropionaldehyde, but whereas A441S and A441T exhibited significant activity with BAL, A441V and A441F did not. The kinetics of the mutants were consistent with their predicted structural features obtained by modeling, and confirmed the importance of position 441 for BAL specificity. The acquisition of BADH activity could have happened through any of these intermediates without detriment of the original function or protein stability. Phylogenetic studies showed that this event occurred independently several times during angiosperms evolution when an ALDH10 gene duplicate changed the critical Ile residue for Ala or Cys in two consecutive single mutations. ALDH10 isoenzymes frequently group in two clades within a plant family: one includes peroxisomal I441-type, the other peroxisomal and non-peroxisomal I441-, A441- or C441-type. Interestingly, high GB-accumulators plants have non-peroxisomal A441- or C441-type isoenzymes, while low-GB accumulators have the peroxisomal C441-type, suggesting some limitations in the peroxisomal GB synthesis.ConclusionOur findings shed light on the evolution of the synthesis of GB in plants, a metabolic trait of most ecological and physiological relevance for their tolerance to drought, hypersaline soils and cold. Together, our results are consistent with smooth evolutionary pathways for the acquisition of the BADH function from ancestral I441-type AMADHs, thus explaining the relatively high occurrence of this event.

Improved Tolerance to Various Abiotic Stresses in Transgenic Sweet Potato (Ipomoea batatas) Expressing Spinach Betaine Aldehyde Dehydrogenase

Abiotic stresses are critical delimiters for the increased productivity and cultivation expansion of sweet potato (Ipomoea batatas), a root crop with worldwide importance. The increased production of glycine betaine (GB) improves plant tolerance to various abiotic stresses without strong phenotypic changes, providing a feasible approach to improve stable yield production under unfavorable conditions. The gene encoding betaine aldehyde dehydrogenase (BADH) is involved in the biosynthesis of GB in plants, and the accumulation of GB by the heterologous overexpression of BADH improves abiotic stress tolerance in plants. This study is to improve sweet potato, a GB accumulator, resistant to multiple abiotic stresses by promoted GB biosynthesis. A chloroplastic BADH gene from Spinacia oleracea (SoBADH) was introduced into the sweet potato cultivar Sushu-2 via Agrobacterium-mediated transformation. The overexpression of SoBADH in the transgenic sweet potato improved tolerance to various abiotic stresses, including salt, oxidative stress, and low temperature. The increased BADH activity and GB accumulation in the transgenic plant lines under normal and multiple environmental stresses resulted in increased protection against cell damage through the maintenance of cell membrane integrity, stronger photosynthetic activity, reduced reactive oxygen species (ROS) production, and induction or activation of ROS scavenging by the increased activity of free radical-scavenging enzymes. The increased proline accumulation and systemic upregulation of many ROS-scavenging genes in stress-treated transgenic plants also indicated that GB accumulation might stimulate the ROS-scavenging system and proline biosynthesis via an integrative mechanism. This study demonstrates that the enhancement of GB biosynthesis in sweet potato is an effective and feasible approach to improve its tolerance to multiple abiotic stresses without causing phenotypic defects. This strategy for trait improvement in sweet potato not only stabilizes yield production in normal soils in unpredictable climates but also provides a novel germplasm for sweet potato production on marginal lands.

Modulation Role of Abscisic Acid (ABA) on Growth, Water Relations and Glycinebetaine Metabolism in Two Maize (Zea mays L.) Cultivars under Drought Stress

The role of plant hormone abscisic acid (ABA) in plants under drought stress (DS) is crucial in modulating physiological responses that eventually lead to adaptation to an unfavorable environment; however, the role of this hormone in modulation of glycinebetaine (GB) metabolism in maize particularly at the seedling stage is still poorly understood. Some hydroponic experiments were conducted to investigate the modulation role of ABA on plant growth, water relations and GB metabolism in the leaves of two maize cultivars, Zhengdan 958 (ZD958; drought tolerant), and Jundan 20 (JD20; drought sensitive), subjected to integrated root-zone drought stress (IR-DS) simulated by the addition of polyethylene glycol (PEG, 12% w/v, MW 6000). The IR-DS substantially resulted in increased betaine aldehyde dehydrogenase (BADH) activity and choline content which act as the key enzyme and initial substrate, respectively, in GB biosynthesis. Drought stress also induced accumulation of GB, whereas it caused reduction in leaf relative water content (RWC) and dry matter (DM) in both cultivars. The contents of ABA and GB increased in drought-stressed maize seedlings, but ABA accumulated prior to GB accumulation under the drought treatment. These responses were more predominant in ZD958 than those in JD20. Addition of exogenous ABA and fluridone (Flu) (ABA synthesis inhibitor) applied separately increased and decreased BADH activity, respectively. Abscisic acid application enhanced GB accumulation, leaf RWC and shoot DM production in both cultivars. However, of both maize cultivars, the drought sensitive maize cultivar (JD20) performed relatively better than the other maize cultivar ZD958 under both ABA and Flu application in view of all parameters appraised. It is, therefore, concluded that increase in both BADH activity and choline content possibly resulted in enhancement of GB accumulation under DS. The endogenous ABA was probably involved in the regulation of GB metabolism by regulating BADH activity, and resulting in modulation of water relations and plant growth under drought, especially in the drought sensitive maize cultivar JD20.

Enhanced drought and salinity tolerance in transgenic potato plants with a BADH gene from spinach

Drought and salinity are the most important abiotic stresses that affect the normal growth and development of plants. Glycine betaine is one of the most important osmolytes present in higher plants that enable them to cope with environmental stresses through osmotic adjustment. In this study, a betaine aldehyde dehydrogenase (BADH) gene from spinach under the control of the stress-induced promoter rd29A from Arabidopsis thaliana was introduced into potato cultivar Gannongshu 2 by the Agrobacterium tumefaciens system. Putative transgenic plants were confirmed by Southern blot analysis. Northern hybridization analysis demonstrated that expression of BADH gene was induced by drought and NaCl stress in the transgenic potato plants. The BADH activity in the transgenic potato plants was between 10.8 and 11.7 U. There was a negative relationship (y = −2.2083x + 43.329, r = 0.9495) between BADH activity and the relative electrical conductivity of the transgenic potato plant leaves. Plant height increased by 0.4–0.9 cm and fresh weight per plant increased by 17–29% for the transgenic potato plants under NaCl and polyethylene glycol stresses compared with the control potato plants. These results indicated that the ability of transgenic plants to tolerate drought and salt was increased when their BADH activity was increased.