Engineering of betaine biosynthesis and transport for abiotic stress tolerance in plants

Drought and salinity are the major factors that decrease crop yield. Organisms thriving in osmotic stress environments need adaptive mechanisms for adjusting their intracellular environment to external osmotic stress conditions. One such mechanism, to prevent water loss from the cells is to accumulate large amounts of low molecular weight organic compatible solutes such as proline, betaine and polyols to balance internal osmolarity of the cells. Accumulation of compatible solutes can be achieved by enhanced synthesis and/or reduced catabolism. Certain plants synthesize betaine in chloroplasts via a two-step oxidation of choline and betaine accumulation is associated with enhanced stress tolerance. Many important crop plants have low levels of betaine or none at all. Hence, betaine biosynthetic pathway is a target for metabolic engineering to enhance stress tolerance in crops. Introduction of betaine synthesis pathway into betaine non-accumulating plants has often improved stress tolerance. However, betaine levels of the engineered plants were generally low. To further enhance the betaine accumulation levels, we need to diagnose factors limitng betaine accumulation in engineered plants. Here we discuss recent progress on metabolic engineering of choline precursors for abiotic stress tolerance in plants.     

Glycinebetaine protects plants against abiotic stress: mechanisms and biotechnological applications

Various compatible solutes enable plants to tolerate abiotic stress, and glycinebetaine (GB) is one of the most-studied among such solutes. Early research on GB focused on the maintenance of cellular osmotic potential in plant cells. Subsequent genetically engineered synthesis of GB-biosynthetic enzymes and studies of transgenic plants demonstrated that accumulation of GB increases tolerance of plants to various abiotic stresses at all stages of their life cycle. Such GB-accumulating plants exhibit various advantageous traits, such as enlarged fruits and flowers and/or increased seed number under non-stress conditions. However, levels of GB in transgenic GB-accumulating plants are relatively low being, generally, in the millimolar range. Nonetheless, these low levels of GB confer considerable tolerance to various stresses, without necessarily contributing significantly to cellular osmotic potential. Moreover, low levels of GB, applied exogenously or generated by transgenes for GB biosynthesis, can induce the expression of certain stress-responsive genes, including those for enzymes that scavenge reactive oxygen species. Thus, transgenic approaches that increase tolerance to abiotic stress have enhanced our understanding of mechanisms that protect plants against such stress.     

Genetic engineering of glycinebetaine synthesis in plants: current status and implications for enhancement of stress tolerance

Metabolic acclimation via the accumulation of compatible solutes is regarded as a basic strategy for the protection and survival of plants in extreme environments. Certain plants accumulate significant amounts of glycinebetaine (betaine), a compatible quaternary amine, in response to high salinity, cold and drought. It is likely that betaine is involved in the protection of macrocomponents of plant cells, such as protein complexes and membranes, under stress conditions. Genetic engineering of the biosynthesis of betaine from choline has been the focus of considerable attention as a potential strategy for increasing stress tolerance in stress-sensitive plants that are incapable of synthesizing this compatible/protective solute. Three distinct pathways for the synthesis of betaine have been identified in spinach, Escherichia coli and Arthrobacter globiformis, and various genes and cDNAs for the proteins involved are available. Moreover, each of the pathways has been exploited to a greater or lesser extent in efforts to convert betaine-deficient plants to betaine accumulators. In this review, the potential of several recent examples of transgenic approaches to the enhancement of stress tolerance in plants is summarized and discussed.     

植物耐盐基因工程研究进展

盐害是影响植物生长和作物产量的主要因素之一。用于提高植物耐盐性的基因工程方法很多,最常见的就是在植物中过量表达抗盐相关的功能基因,包括植物信号传导蛋白基因、植物离子通道蛋白基因和合成小分子渗透剂的酶基因等。归纳了近年来植物耐盐基因工程的研究进展,并展望了植物耐盐基因工程的研究前景。     

Compatible solute biosynthesis in cyanobacteria

Compatible solutes are a functional group of small, highly soluble organic molecules that demonstrate compatibility in high amounts with cellular metabolism. The accumulation of compatible solutes is often observed during the acclimation of organisms to adverse environmental conditions, particularly to salt and drought stress. Among cyanobacteria, sucrose, trehalose, glucosylglycerol and glycine betaine are used as major compatible solutes. Interestingly, a close correlation has been discovered between the final salt tolerance limit and the primary compatible solute in these organisms. In addition to the dominant compatible solutes, many strains accumulate mixtures of these compounds, including minor compounds such as glucosylglycerate or proline as secondary or tertiary solutes. In particular, the accumulation of sucrose and trehalose results in an increase in tolerance to general stresses such as desiccation and high temperatures. During recent years, the biochemical and molecular basis of compatible solute accumulation has been characterized using cyanobacterial model strains that comprise different salt tolerance groups. Based on these data, the distribution of genes involved in compatible solute synthesis among sequenced cyanobacterial genomes is reviewed, and thereby, the major compatible solutes and potential salt tolerance of these strains can be predicted. Knowledge regarding cyanobacterial salt tolerance is not only useful to characterize strain-specific adaptations to ecological niches, but it can also be used to generate cells with increased tolerance to adverse environmental conditions for biotechnological purposes.     

Glycinebetaine in saline conditions: an assessment of the current state of knowledge

Salt stress is one of the environmental threats that have devastating impacts on plant distribution, growth and production. Different plants are believed to have salt tolerance mechanisms that occur at the cellular level. One facet of the cellular mechanisms of adaptation to salinity stress is to accumulate either inorganic and/or organic solutes. Glycinebetaine (GB), as well as other organic solutes, has been referred to as compatible solutes, for the reason that they are innocent with essential biochemical reactions even at high concentrations. GB has been assumed to be involved in osmotic adjustment and/or osmoprotection of cellular functional macromolecules and, hence, can improve tolerance to saline conditions. However, the exact mechanism and direct evidences for such correlative data are still lacking despite many attempts to improve growth under saline conditions by exogenous application as well as genetic engineering of metabolic pathways involved in metabolism of GB. Despite the enormous amount of information accumulated in this regard, the exact function of GB in the adaptation to saline environments is not fully clear to this point, and even GB functions have been argued. Because of that, inconsistencies exist in the published data regarding GB accumulation and functions under salt stress. In this review, we provide an update on evidence supporting each of these arguments to reassess how GB affects plant growth and physiological traits under salt imposition, and whether its effects correlate with salt tolerance.     

植物耐冷性基因工程

温度决定物种的分布,同时还影响作物的产量和品质。植物耐冷的机制涉及到许多方面,包括膜脂组成的变化、可混溶溶质的积累、抗氧化酶活性的提高、低温相关基因的诱导表达等。由于植物的耐冷性状由多基因控制,采用传统的育种方法往往难以取得理想的结果,而植物基因工程技术的发展及应用则提供了另外可能的途径,可以通过转移耐冷性状形成的关键基因从而对植物进行改良。本文从膜脂组成、可混溶溶质、抗冻蛋白、抗氧化酶和诱导植物低温相关基因的转录因子等方面对植物耐冷性的基因工程研究进行了综述,以期为植物育种者和从事冷胁迫机制研究的工作者提供参考。     

Protective mechanisms of heat tolerance in crop plants

High temperature (HT) has become a global concern because it severely affects the growth and production of crops. Heat stress causes an abrupt increase in the expression of stress-associated proteins which provide tolerance by stimulating the defense response in plants. Heat-shock proteins (Hsps) and antioxidant enzymes are important in encountering heat stress in plants. The heat-shock response is characterized by repression of normal cellular protein synthesis and induction of Hsp synthesis. Under HT stress, upregulation of various enzymatic and nonenzymatic antioxidants, maintenance of cell membrane stability, production of various compatible solutes and hormonal changes occurs. Reactive oxygen species involving several pathways such as water–water cycle, Halliwell–Asada, glutathione peroxidase, Haber–Weiss and Fenton reactions helps in protecting plants against toxic radicals which otherwise could cause damage to lipophilic protein. Genetic approaches to elucidate and map genes or quantitative trait loci conferring thermotolerance will facilitate marker-assisted breeding for heat tolerance and also pave the way for characterizing genetic factors which could be useful for engineering plants with improved heat tolerance. This review discusses the protective mechanism of heat stress responses encompassing different pathways that provide tolerance during HT stress.     

植物耐盐性机理研究进展

在盐胁迫下环境中某些植物会在发生一些变化。从生理学、生物化学、盐胁迫分子学机制的角度对植物对盐胁迫的反应研究进行了回顾,并提供了一些目前知识水平上能增加植物盐耐性的方法。解释了在盐胁迫下植物的离子吸收、相溶性物质、抗氧化酶、植物激素、光合作用等方面的变化规律,其中也有耐盐植物功能调节的研究,这有助于从多学科研究的角度评估盐胁迫的生态重要性。     

Effects of citric acid as an important component of the responses to saline and alkaline stress in the halophyte <Emphasis Type="Italic">Leymus chinensis</Emphasis> (Trin.)

Some plants accumulate some compatible solutes and exude various organic acids when exposed to environmental stress. These compatible solutes including proline have been suggested to be involved in stress tolerance by maintaining sufficient cell turgor for growth, thereby improving plant growth, protecting enzymes, and membranes. However, less evidence exists regarding the protective roles of organic acids under stress conditions. Here, we investigate the effects of citric acid as a component of the response to stress on plant growth and antioxidant enzyme activities in two genotypes of halophyte Leymus chinensis (Trin.) genotypes, LcWT07 and LcJS0107. Data showed that both saline stress (200 mM NaCl) and alkaline stress (100 mM Na₂CO₃) reduced plant growth on the relative growth rate and CO₂ assimilation rate, but increased the citric acid concentrations in 6-week-old plants over the 72 h experimental period. When 50 mg l⁻¹ citric acid was exogenously applied under stress conditions, it significantly improved the plant growth and internal citric acid concentration, and also induced defense mechanisms by increasing the activities of antioxidant enzymes. To compare with the mitigative effects of exogenous citric acid on stress, exogenous application of proline was also performed under same conditions, and similar effects on the improvement of growth were observed. Based on these results, we suggested that citric acid is an important component of the stress response in L. chinensis, and exogenous application of 50 mg l⁻¹ citric acid might play a positive role on stress tolerance.     

Accumulation of the compatible solutes, glycine-betaine and ectoine, in osmotic stress adaptation and heat shock cross-protection in the biocontrol agent Pantoea agglomerans CPA-2

AIMS: The effect of modifying the water activity (a(w)) of Pantoea agglomerans growth medium with the ionic solute NaCl on water stress resistance, heat-shock survival and intracellular accumulation of the compatible solutes glycine-betaine and ectoine were determined. METHODS AND RESULTS: The bacterium was cultured in an unmodified liquid medium or that modified with NaCl to 0.98 and 0.97 a(w), and viability of cells evaluated on a 0.96 a(w)-modified solid media to check water stress tolerance. Cells grown under ionic stress had better water stress tolerance than control cells. These cells also had cross-protection to heat stress (30 min, 45 degrees C). The modified cells accumulated substantial amounts of the compatible solutes glycine-betaine and ectoine in contrast to the control cells, which contained little or none of these two compounds. CONCLUSIONS: Improvement in osmotic and thermal tolerance of cells of the biocontrol agent P. agglomerans by modifying growth media with the ionic solute NaCl was achieved. The compatible solutes glycine-betaine and ectoine play a critical role in environmental stress tolerance improvement. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach provides a method for improving the physiological quality of inocula and could have implications for formulation and shelf-life of biocontrol agents.     

Compatible solute accumulation and stress-mitigating effects in barley genotypes contrasting in their salt tolerance

The accumulation of compatible solutes is often regarded as a basic strategy for the protection and survival of plants under abiotic stress conditions, including both salinity and oxidative stress. In this work, a possible causal link between the ability of contrasting barley genotypes to accumulate/synthesize compatible solutes and their salinity stress tolerance was investigated. The impact of H(2)O(2) (one of the components of salt stress) on K(+) flux (a measure of stress 'severity') and the mitigating effects of glycine betaine and proline on NaCl-induced K(+) efflux were found to be significantly higher in salt-sensitive barley genotypes. At the same time, a 2-fold higher accumulation of leaf and root proline and leaf glycine betaine was found in salt-sensitive cultivars. The total amino acid content was also less affected by salinity in salt-tolerant cultivars. In these, potassium was found to be the main contributor to cytoplasmic osmolality, while in salt-sensitive genotypes, glycine betaine and proline contributed substantially to cell osmolality, compensating for reduced cytosolic K(+). Significant negative correlations (r= -0.89 and -0.94) were observed between Na(+)-induced K(+) efflux (an indicator of salt tolerance) and leaf glycine betaine and proline. These results indicate that hyperaccumulation of known major compatible solutes in barley does not appear to play a major role in salt-tolerance, but rather, may be a symptom of salt-susceptibility.     

Effect of pre-treatment with ethanolamine on the response of Helianthus annuus L. to salt stress

The accumulation of compatible solutes is one of the strategies that plants have developed to tolerate salt stress. Proline and betaine are the main metabolites that accumulate in various species of higher plants in response to salt stress. In Helianthus annuus L., pre-treatment of seeds with ethanolamine led to enhanced seedling tolerance to conditions of saline stress during germination, as evidenced by the greater growth of pretreated seedlings (EAS group) versus untreated seedlings (S group), evaluated through such parameters as length, water and chlorophyll content. During the germination period, a considerable increase was observed in proline levels (up to 300%) in seedlings subjected to saline stress, whereas in the EAS group, the proline increment was much smaller (20%). Starting from the fourth day of germination, betaine levels in seedlings pretreated with ethanolamine and then with water (EAW group) and in EAS showed a significant increase versus C (control) and S seedlings, possibly because such a precursor promotes betaine biosynthesis. This could be responsible for the enhanced growth observed in EAS versus S seedlings, as well as for preventing the decrease in chlorophyll content in the EAS group. The accumulation of betaine seems to correlate with the greater tolerance of these seedlings against stress induced by sodium chloride.     

细胞相容性溶质对水分胁迫下玉米根系SOD活性的促进作用

分别用脯氨酸,甜菜碱,蔗糖,甘露醇饲喂玉米根系,PEG－6000模拟水分胁迫,测定外源相容性溶质对根系SOD活性的影响。结果表明,4种溶质对水分胁迫下玉米SOD活性有不同程度的促进作用,其大小顺序为：甘露醇＞蔗糖＞甜菜碱＞脯氨酸。饲喂植株的MDA含量明显降低,降低程度的大小顺序同SOD活性一致。胁迫过程中SOD活性与MDA含量呈极显著负相关,有力地说明了SOD在干旱胁迫下对活性氧的清除和细胞膜结构的保护作用。细胞相容性物质可促进保护酶活性升高,提高植物的干旱适应性。     

Diversity and biosynthesis of compatible solutes in hyper/thermophiles.

The accumulation of compatible solutes, either by uptake from the medium or by de novo synthesis, is a general response of microorganisms to osmotic stress. The diversity of compatible solutes is large but falls into a few major chemical categories, such as carbohydrates or their derivatives and amino acids or their derivatives. This review deals with compatible solutes found in thermophilic or hyperthermophilic bacteria and archaea that have not been commonly identified in microorganisms growing at low and moderate temperatures. The response to NaCl stress of Thermus thermophilus is an example of how a thermophilic bacterium responds to osmotic stress by compatible solute accumulation. Emphasis is made on the pathways leading to the synthesis of mannosylglycerate and glucosylglycerate that have been recently elucidated in several hyper/thermophilic microorganisms. The role of compatible solutes in the thermoprotection of these fascinating microorganisms is also discussed.     

Metabolic Engineering for Stress Tolerance: Installing Osmoprotectant Synthesis Pathways

Abiotic environmental stresses such as drought, salinity andlow temperature are major limitations for plant growth and cropproductivity. Certain plants, marine algae and bacteria haveevolved a number of adaptations to such abiotic stresses: someof these adaptations are metabolic and others structural. Accumulationof certain organic solutes (known as osmoprotectants) is a commonmetabolic adaptation found in diverse taxa. These solutes protectproteins and membranes against damage by high concentrationsof inorganic ions. Some osmoprotectants also protect the metabolicmachinery against oxidative damage. Many major crops lack theability to synthesize the special osmoprotectants that are naturallyaccumulated by stress-tolerant organisms. Therefore, it washypothesized that installing osmoprotectant synthesis pathwaysis a potential route to breed stress-tolerant crops. Provingthis, recent engineering efforts in model species led to modestbut significant improvements in stress tolerance of transgenicplants. Synthetic pathways to two kinds of osmoprotectants—polyolsand quaternary ammonium compounds—are discussed here.Results from the metabolic engineering experiments emphasizethe need for a greater understanding of primary metabolic pathwaysfrom which osmoprotectant synthesis pathways branch. Futureresearch avenues include the identification and exploitationof diverse osmoprotectants in naturally stress-tolerant organisms,and the use of multiple genes and reiterative engineering toincrease osmoprotectant flux in response to stress. High-throughputgenomic technologies offer a number of tools to refine thisby rapidly identifying genes, pathways, and regulatory controls.Copyright 2000 Annals of Botany Company Review, abiotic stress, osmoprotectant, compatible solute, genetic engineering     

Recent methods of drought stress tolerance in plants

In the climate change scenario the drought has been diagnosed as major stress affecting crop productivity. This review demonstrates some recent findings on the amelioration of drought stress. Nanoparticles, synthetic growth regulators viz. Trinexapac-ethyl, and Biochar addition helps to economize the water budget of plants, enhances the bioavailability of water and nutrients as well as overcomes drought induced osmotic and oxidative stresses. Besides ABA, SA and JA are also involved in inducing tolerance to drought stress through modulation of physiological and biochemical processes in plants. Plant growth promoting rhizobacteria (PGPR) offer new opportunities in agricultural biotechnology. These beneficial microorganisms colonize the rhizosphere/endo-rhizosphere of plants and impart drought tolerance by improving root architechture, enhancing water use efficiency, producing exopolysaccharides, phytohormones viz, ABA, SA and IAA and volatile compounds. Further PGPR also play positive role in combating osmotic and oxidative stresses induced by drought stress through enhancing the accumulation of osmolytes, antioxidants and upregulation or down regulation of stress responsive genes. In transgenic plants stress inducible genes enhanced abiotic stress tolerance by encoding key enzymes regulating biosynthesis of compatible solutes. The role of genes/cDNAs encoding proteins involved in regulating other genes/proteins, signal transduction process and strategies to improve drought stress tolerance have also been discussed.     

Osmoadaptation mechanisms in prokaryotes: distribution of compatible solutes

Microorganisms respond to osmotic stress mostly by accumulating compatible solutes, either by uptake from the medium or by de novo synthesis. These osmotically active molecules preserve the positive turgor pressure required for cell division. The diversity of compatible solutes is large but falls into a few major chemical categories; they are usually small organic molecules such as amino acids or their derivatives, and carbohydrates or their derivatives. Some are widely distributed in nature while others seem to be exclusively present in specific groups of organisms. This review discusses the diversity and distribution of known classes of compatible solutes found in prokaryotes as well as the increasing knowledge of the genes and pathways involved in their synthesis. The alternative roles of some archetypal compatible solutes not subject to osmoregulatory constraints are also discussed.