The Accumulation of Glycine Betaine Is Dependent on Choline Monooxygenase (OsCMO), Not on Phosphoethanolamine N-Methyltransferase (OsPEAMT1), in Rice (Oryza sativa L. ssp. japonica)

Glycine betaine (GB) is an important osmoprotectant, which improves plant tolerance to various abiotic stresses. In higher plants, GB is synthesized through two-step oxidations of choline, catalyzed by choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), respectively. Choline, the precursor of GB, is synthesized by phosphoethanolamine N-methyltransferase (PEAMT). Rice is known as a typical non-GB-accumulated species. However, the underlying mechanism related to GB accumulation remains elusive. Here, we determined whether the endogenous accumulation of choline is sufficient to GB biosynthesis in rice and whether the rice CMO protein has the function of oxidizing choline to generate betaine aldehyde. The results showed that overexpression of the rice PEAMT1 gene (OsPEAMT1) resulted in increased levels of choline, while GB content remained unchanged in the transgenic rice plants overexpressing OsPEAMT1. However, the intracellular GB level and the tolerance to salt stress of the transgenic lines overexpressing OsCMO were significantly enhanced. Immunoblotting analysis demonstrated that abundant functional OsCMO proteins with correct size were detected in OsCMO-overexpressing transgenic rice plants, but rarely accumulated in the wild type. Collectively, these results implicated that the endogenous accumulation level of choline is not the major factor leading to non-GB accumulation in rice. Instead, the defective expression of OsCMO resulted in non-GB accumulation.     

Osmotic Stress Induces Expression of Choline Monooxygenase in Sugar Beet and Amaranth

Choline monooxygenase (CMO) catalyzes the committing step in the synthesis of glycine betaine, an osmoprotectant accumulated by many plants in response to salinity and drought. To investigate how these stresses affect CMO expression, a spinach (Spinacia oleracea L., Chenopodiaceae) probe was used to isolate CMO cDNAs from sugar beet (Beta vulgaris L., Chenopodiaceae), a salt- and drought-tolerant crop. The deduced beet CMO amino acid sequence comprised a transit peptide and a 381-residue mature peptide that was 84% identical (97% similar) to that of spinach and that showed the same consensus motif for coordinating a Rieske-type [2Fe-2S] cluster. A mononuclear Fe-binding motif was also present. When water was withheld, leaf relative water content declined to 59% and the levels of CMO mRNA, protein, and enzyme activity rose 3- to 5-fold; rewatering reversed these changes. After gradual salinization (NaCl:CaCl₂ = 5.7:1, mol/mol), CMO mRNA, protein, and enzyme levels in leaves increased 3- to 7-fold at 400 mm salt, and returned to uninduced levels when salt was removed. Beet roots also expressed CMO, most strongly when salinized. Salt-inducible CMO mRNA, protein, and enzyme activity were readily detected in leaves of Amaranthus caudatus L. (Amaranthaceae). These data show that CMO most probably has a mononuclear Fe center, is inducibly expressed in roots as well as in leaves of Chenopodiaceae, and is not unique to this family.     

Effects of salinity on growth,membrane permeability and root hydraulic conductivity in three saltbush species

The species of the genus Atriplex have been introduced in West Asia and North Africa to determine their adaptability for use as fodder species. These halophytes are well adapted to extreme environmental conditions and may possess interesting properties for soil rehabilitation. The effect of NaCl stress on growth, water relation and mineral nutrition were investigated in three xero-halophyte species of Atriplex used for rehabilitation of arid steppe in Algeria. Atriplex halimus, Atriplex canescens and Atriplex nummularia, were cultivated in hydroponic conditions and treated with increasing doses of NaCl (0–300 mM). All species showed positive plant growth for low and moderate levels of salinity. A. halimus had higher dry weight production than A. nummularia and A. canescens in high salinity concentration. Increasing concentration of salinity induced decrease in chlorophyll content (Chl a and b) and root hydraulic conductivity (L₀) in all species, especially in A. canescens. All three species showed marked increase in electrolyte leakage across the salinity gradient. In addition all species were able to accumulate a large quantity of sodium (Na), chloride (Cl) and proline and to maintain higher relative water content, which was probably associated with a greater capacity for osmotic adjustment, whereas potassium (K) and calcium (Ca) decreased with increase salinity. The data suggest that salt tolerance strategies in all Atriplex species could involve a delicate balance among ion accumulation, osmotic adjustment, production of osmotica and maintenance of relative water content and growth.     

盐胁迫下三色苋甜菜碱及有关酶含量的变化

三色苋(Amaranthus tricolor)不同器官中的甜菜碱(GB)含量显著不同.除子叶外,根、茎和叶的GB含量和茎、叶中的胆碱单加氧酶(CMO)含量都因300 mmol/L的NaCl处理而增加.甜菜碱醛脱氢酶(BADH)的表达无论盐处理与否在所有器官中都能检测到,其含量变化不大.当种子发芽时,具备合成GB的能力,CMO含量增加;在此之前未能检测到CMO,也不能合成GB.研究结果表明三色苋响应盐胁迫而合成GB的关键酶是CMO.     

Overexpression of the betaine aldehyde dehydrogenase gene from Atriplex hortensis enhances salt tolerance in the transgenic trifoliate orange (Poncirus trifoliata L. Raf.)

Trifoliate orange (Poncirus trifoliata L. Raf.), a rootstock widely used for citrus species, is salt-sensitive. Worldwide, salinity is a major abiotic stress affecting citrus growth and yield. Glycinebetaine (GB) is an important osmoprotectant involved in responses to salt stress. However, current evidence regarding the effect of salt stress on GB accumulation in trifoliate orange is limited, and the GB synthesis gene has not yet been shown to confer enhanced salt stress tolerance to this species in a transgenic context. In the current study, we first examined the change in GB level of trifoliate orange seedlings exposed to salt stress, and found that salt increased endogenous GB level in a concentration-dependent manner. A betaine aldehyde dehydrogenase gene (AhBADH) cloned from Atriplex hortensis was introduced into the trifoliate orange by means of Agrobacterium-mediated transformation. RT-PCR analysis on three selected transgenic lines showed that the AhBADH gene was overexpressed in each of them. GB levels in these lines were also higher than those in untransformed wild-type (WT) plants. In the transgenic lines, exposure to 200 mM NaCl resulted in significantly less serious leaf burning and defoliation, lower MDA accumulation, and higher chlorophyll contents than those in the WT plants. Moreover, when exposed to salt, shoots of transgenic plants contained lower levels of Na⁺ and Cl⁻, higher levels of K⁺, and a higher K/Na ratio, while the same was true for the roots in most cases. Taken together, the data suggest that overexpression of the AhBADH gene in transgenic trifoliate orange enhanced salt stress tolerance. This may be correlated with the low levels of lipid peroxidation, protection of the photosynthetic machinery, and increase in K⁺ uptake.     

The effects of salt stress on growth,water relations and ion accumulation in two halophyte Atriplex species

Atriplex halimus is found in the Mediterranean Basin along the coastal areas of Sardinia, but few data are available on its adaptability to salinity. The effects of drought and salinity under controlled conditions on two clones of A. halimus, designated MOR2 and SOR4, originating from southern and northern Sardinia, respectively, were compared with those of seedlings of A. nummularia, an Australian species widely used in the restoration of arid areas. The effects of increasing NaCl salinity above seawater concentrations and of increasing the KCl concentration gradient were tested. Plants were harvested and analysed after 10 and 20 days of NaCl and KCl treatments. All plants remained alive until the end of treatment, although growth was strongly reduced, mainly for the A. halimus MOR2 clone, under increasing concentrations of KCl. The leaves and roots of both species responded positively to increasing NaCl concentrations up to 600 mM NaCl for A. halimus, whereas the optimal growth of A. nummularia was recorded at 300 mM NaCl. SOR4 was more sensitive to KCl toxicity. The Na⁺ concentration in the plants increased with increased salinity and was higher in A. halimus than in A. nummularia, suggesting that A. halimus is an ion accumulator and may be used for phytoremediation. The sodium accumulation in the roots of the A. halimus MOR2 clone was far greater than in its leaves. This suggests that MOR2 is an Na⁺ excluder, either by minimising the entry of salt into the plant or by an excretion mechanism via the vesiculated hairs that play a significant role in the removal of salt from the remainder of the leaf, thereby preventing its accumulation to toxic levels in the leaves, whereas SOR4 acted as an Na⁺ includer. Higher levels of proline were detected in the MOR2 clone under NaCl treatments, suggesting a more developed adaptative mechanism for the selection of this characteristic in the southern part of the island, which is more exposed to abiotic stresses, particularly water stress that is either generated by salinity or by other causes.     

Glycine Betaine Biosynthesis Is Induced by Salt Stress but Repressed by Auxinic Herbicides in <Emphasis Type="Italic">Kochia scoparia</Emphasis>.

Kochia scoparia biotypes that are susceptible or resistant to the auxinic herbicide dicamba were used to characterize expression levels of choline monooxygenase (CMO) and glycine betaine accumulation in response to salt stress and herbicide treatment. A 1180-bp cDNA was isolated using differential display and 3 RACE with a deduced amino acid sequence that was more than 90% similar to the carboxy terminal 290 residues of CMOs from four related plant species. Salt stress led to a substantial increase in CMO mRNA and enzyme levels in K. scoparia biotypes, and the accumulation of up to 80 mol g^–1 fresh weight glycine betaine. In contrast, dicamba treatment was followed by the rapid attenuation of CMO message and protein levels, with a recovery of expression in the resistant but not the susceptible biotype. CMO mRNA and enzyme levels similarly declined, and recovered in the resistant biotype, after dicamba treatment of plants that were previously salt stressed for 4 days. The opposing effects of these two stresses may represent a regulatory scheme in which competition for the substrate choline leads to a repression of glycine betaine biosynthesis to make sufficient choline available for auxin-mediated growth processes.     

Isolation of a choline monooxygenase cDNA clone from Amaranthus tricolor and its expressions under stress conditions

INTRODUCTIONAmaranth is a C4 dicotyledonous mesophytecrop plant. A. tricofor is a major variety for veg-etable and ornamental crops, and is widely culti-vated in the wor1d. Osmoprotectant glycine betaine(GB) was detected in Amaranthaceae, A. HyPochon-driacus L[2] and A. Caudatus L[3, 4]. GB iswidespread and an effective osmoprotectant in manyplants[3]. We studied the photosynthetic adaptationmechanism of A. trico1or under salt stress due to ac-cumulation of GB[5].GB is synthesized …     

Accumulation of glycinebetaine in rice plants that overexpress choline monooxygenase from spinach and evaluation of their tolerance to abiotic stress

BACKGROUND AND AIMS: Glycinebetaine (GB), a quaternary ammonium compound, is a very effective compatible solute. In higher plants, GB is synthesized from choline (Cho) via betaine aldehyde (BA). The first and second steps in the biosynthesis of GB are catalysed by choline monooxygenase (CMO) and by betaine aldehyde dehydrogenase (BADH), respectively. Rice (Oryza sativa), which has two genes for BADH, does not accumulate GB because it lacks a functional gene for CMO. Rice plants accumulate GB in the presence of exogenously applied BA, which leads to the development of a significant tolerance to salt, cold and heat stress. The goal in this study was to evaluate and to discuss the effects of endogenously accumulated GB in rice. METHODS: Transgenic rice plants that overexpressed a gene for CMO from spinach (Spinacia oleracea) were produced by Agrobacterium-mediated transformation. After Southern and western blotting analysis, GB in rice leaves was quantified by (1)H-NMR spectroscopy and the tolerance of GB-accumulating plants to abiotic stress was investigated. KEY RESULTS: Transgenic plants that had a single copy of the transgene and expressed spinach CMO accumulated GB at the level of 0.29-0.43 micromol g(-1) d. wt and had enhanced tolerance to salt stress and temperature stress in the seedling stage. CONCLUSIONS: In the CMO-expressing rice plants, the localization of spinach CMO and of endogenous BADHs might be different and/or the catalytic activity of spinach CMO in rice plants might be lower than it is in spinach. These possibilities might explain the low levels of GB in the transgenic rice plants. It was concluded that CMO-expressing rice plants were not effective for accumulation of GB and improvement of productivity.     

Overaccumulation of glycine betaine enhances tolerance to drought and heat stress in wheat leaves in the protection of photosynthesis

We investigated the different responses of wheat (Triticum aestivum L.) plants to drought- (DS) and heat stress (HS), and analyzed the physiological mechanisms of glycine betaine (GB) involved in the improvement of wheat tolerance to the combination of these stresses. The transgenic wheat T6 line was generated by introducing a gene encoding betaine aldehyde dehydrogenase (BADH) into the wild-type (WT) Shi4185 line. The gene was cloned from the Garden Orache plant (Atriplex hortensis L.). Wheat seedlings were subjected to drought stress (30%, PEG-6000), heat stress (40°C), and their combination. Photosynthetic gas exchange, water status and lipid peroxidation of wheat leaves were examined under different stresses. When subjected to a combination of drought and heat, the inhibition of photosynthesis was significantly increased compared to that under DS or HS alone. The increased inhibition of photosynthesis by the combined stresses was not simply the additive stress effect of separate heat- and drought treatments; different responses in plant physiology to DS and HS were also found. HS decreased the chlorophyll (Chl) content, net photosynthetic rate (P _N), carboxylation efficiency (CE) and apparent quantum yield (AQY) more than DS but DS decreased the transpiration rate (E), stomata conductance (g _s) and intercellular CO₂ concentration (C _i) more than HS. GB over-accumulation led to increased photosynthesis not only under individual DS or HS but also under their combination. The enhancement of antioxidant activity and the improvement of water status may be the mechanisms underlying the improvement of photosynthesis by GB in wheat plants.     

山菠菜胆碱单氧化物酶基因(CMO)的克隆与分析

甜菜碱是一类广泛存在于生物体内的渗透保护剂。高等植物中,甜菜碱的生物合成经由胆碱→甜菜碱醛→甜菜碱两步反应完成,其中第一步反应,也是甜菜碱生物合成的限速反应,由胆碱单氧化物酶(CMO)催化。本研究以耐盐植物山菠菜(Atriplex hortensis)为材料构建了盐胁迫下的cDNA文库,用菠菜CMO cDNA为探针从中筛选获得一个长1.77kb的cDNA克隆,测序结果表明该克隆包含一个完整的开放读码框,编码一个由438个氨基酸构成的多肽,与菠菜和甜菜CMO的氨基酸序列同源性分别为81%和72%。同菠菜和甜菜中的CMO序列相比,山菠菜CMO基因(AhCMO)也具有保守的RieskeType［2Fe2S］簇结合区和保守的多铁原子核结合域。对盐处理条件下山菠菜CMO基因转录水平的研究表明CMO基因在盐胁迫情况下表达量增加约3倍。将CMO与35S启动子连接后转化烟草(Nictiana tabacumvar.Xanthi),获得了具有一定耐盐性状的转基因植株,在1.2%NaCl的盐浓度下生长良好。     

Increased glycine betaine synthesis and salinity tolerance in AhCMO transgenic cotton lines

Glycine betaine is an osmoprotectant that plays an important role and accumulates rapidly in many plants during salinity or drought stress. Choline monooxygenase (CMO) is a major catalyst in the synthesis of glycine betaine. In our previous study, a CMO gene (AhCMO) cloned from Atriplex hortensis was introduced into cotton (Gossypium hirsutum L.) via Agrobacterium mediation to enhance resistance to salinity stress. However, there is little or no knowledge of the salinity tolerance of the transgenic plants, particularly under saline-field conditions. In the present study, two transgenic AhCMO cotton lines of the T₃ generation were used to study the AhCMO gene expression, and to determine their salinity tolerance in both greenhouse and field under salinity stress. Molecular analysis confirmed that the transgenic plants expressed the AhCMO gene. Greenhouse study showed that on average, seedlings of the transgenic lines accumulated 26 and 131% more glycine betaine than those of non-transgenic plants (SM3) under normal and salt-stress (150 mmol l⁻¹ NaCl) conditions, respectively. The osmotic potential, electrolyte leakage and malondialdehyde (MDA) accumulation were significantly lower in leaves of the transgenic lines than in those of SM3 after salt stress. The net photosynthesis rate and Fv/Fm in transgenic cotton leaves were less affected by salinity than in non-transgenic cotton leaves. Therefore, transgenic cotton over-expressing AhCMO was more tolerant to salt stress due to elevated accumulation of glycine betaine, which provided greater protection of the cell membrane and photosynthetic capacity than in non-transgenic cotton. The seed cotton yield of the transgenic plants was lower under normal conditions, but was significantly higher than that of non-transgenic plants under salt-stressed field conditions. The results indicate that over-expression of AhCMO in cotton enhanced salt stress tolerance, which is of great value in cotton production in the saline fields.     

Overexpression of soybean ubiquitin-conjugating enzyme gene GmUBC2 confers enhanced drought and salt tolerance through modulating abiotic stress-responsive gene expression in Arabidopsis

Previous studies have shown that ubiquitination plays important roles in plant abiotic stress responses. In the present study, the ubiquitin-conjugating enzyme gene GmUBC2, a homologue of yeast RAD6, was cloned from soybean and functionally characterized. GmUBC2 was expressed in all tissues in soybean and was up-regulated by drought and salt stress. Arabidopsis plants overexpressing GmUBC2 were more tolerant to salinity and drought stresses compared with the control plants. Through expression analyses of putative downstream genes in the transgenic plants, we found that the expression levels of two ion antiporter genes AtNHX1 and AtCLCa, a key gene involved in the biosynthesis of proline, AtP5CS, and the copper chaperone for superoxide dismutase gene AtCCS, were all increased significantly in the transgenic plants. These results suggest that GmUBC2 is involved in the regulation of ion homeostasis, osmolyte synthesis, and oxidative stress responses. Our results also suggest that modulation of the ubiquitination pathway could be an effective means of improving salt and drought tolerance in plants through genetic engineering.     

Response to non-uniform salinity in the root zone of the halophyte Atriplex nummularia: growth, photosynthesis, water relations and tissue ion concentrations

Background and Aims

Soil salinity is often heterogeneous, yet the physiology of halophytes has typically been studied with uniform salinity treatments. An evaluation was made of the growth, net photosynthesis, water use, water relations and tissue ions in the halophytic shrub Atriplex nummularia in response to non-uniform NaCl concentrations in a split-root system.

Methods

Atriplex nummularia was grown in a split-root system for 21 d, with either the same or two different NaCl concentrations (ranging from 10 to 670 mm), in aerated nutrient solution bathing each root half.

Key Results

Non-uniform salinity, with high NaCl in one root half (up to 670 mm) and 10 mm in the other half, had no effect on shoot ethanol-insoluble dry mass, net photosynthesis or shoot pre-dawn water potential. In contrast, a modest effect occurred for leaf osmotic potential (up to 30 % more solutes compared with uniform 10 mm NaCl treatment). With non-uniform NaCl concentrations (10/670 mm), 90 % of water was absorbed from the low salinity side, and the reduction in water use from the high salinity side caused whole-plant water use to decrease by about 30 %; there was no compensatory water uptake from the low salinity side. Leaf Na⁺ and Cl⁻ concentrations were 1·9- to 2·3-fold higher in the uniform 670 mm treatment than in the 10/670 mm treatment, whereas leaf K⁺ concentrations were 1·2- to 2·0-fold higher in the non-uniform treatment.

Conclusions

Atriplex nummularia with one root half in 10 mm NaCl maintained net photosynthesis, shoot growth and shoot water potential even when the other root half was exposed to 670 mm NaCl, a concentration that inhibits growth by 65 % when uniform in the root zone. Given the likelihood of non-uniform salinity in many field situations, this situation would presumably benefit halophyte growth and physiology in saline environments.Key words: Split-root system, salinity heterogeneity, root zone heterogeneity, water potential, water use, stomatal conductance, saltbush, leaf ions, photosynthesis, NaCl     

Phytoextraction of salts by Atriplex Nummularia Lindl. irrigated with reject brine under varying water availability

Abstract

Reverse osmosis is a widely known technology used to produce fresh water from brackish waters. However, the reject brine from desalination plants poses a serious threat to the environment due to soil and groundwater salinization. The aim of this study was to investigate the potential of Atriplex nummularia to extract salts from a soil irrigated with reverse osmosis brine, at varying moisture levels. A field experiment was conducted in a split-plot design, with randomized complete blocks replicated four times. Treatments consisted of irrigation with reject brine in the main plots, with four relative percentages of the soil moisture at field capacity (100, 85, 70, and 50%), and two levels of organic fertilization in the subplots (0 and 1.5?L plant^?1 of goat manure). The mineral composition of leaves and stems indicated that the highest salt extraction by plants occurred when soil moisture was maintained at 100% field capacity. The salt extraction capacity of A. nummularia indicates a high potential for phytoremediation of soils affected by brine disposal from reverse osmosis plants.