The challenge of remobilisation in plant nitrogen economy. A survey of physio-agronomic and molecular approaches

In this article, we discuss the ways in which our understanding of the controls of nitrogen remobilisation in model species and crop plants have been increased through classical physiological studies and the use of transgenic plants or mutants with modified capacities for nitrogen or carbon assimilation and recycling. An improved understanding of the transition between nitrogen assimilation and nitrogen recycling will be vital, if improvements in crop nitrogen use efficiency are to reduce the need for excessive input of fertilisers and improve or stabilise yield. In this review, we present an overall view of past work and more recent studies on this topic, using different plants systems and models depicting the biochemical and molecular events occurring during the transition between sink leaves and source leaves. These models may provide a way to identify the nature of the metabolic or developmental signals triggering in a coordinate manner nitrogen and carbon recycling during leaf senescence. Another way of developing crop varieties with improved nitrogen use efficiency, and identifying key elements controlling the process of nitrogen remobilisation, is the use of quantitative genetics. We present and discuss recent findings on the genetic variability and basis of nitrogen use efficiency in crops in general and in maize in particular. A genetic approach using maize recombinant inbred lines was undertaken allowing the detection of Quantitative Trait Loci (QTLs) for morphological traits, grain yield and its components under high nitrogen or low nitrogen input. Co‐mapping was observed between genes encoding enzymes involved in nitrogen assimilation (nitrate reductase, glutamine synthetase) and these Quantitative Trait Loci. All coincidences were consistent with the expected physiological function of the corresponding enzyme activities. This work strongly suggests that in maize, nitrogen use efficiency can be improved both by marker‐assisted selection and genetic engineering.     

Manipulating the pathway of ammonia assimilation through genetic engineering and breeding: consequences to plant physiology and plant development

In this article we discuss the ways in which our understanding of the nature of the molecular controls of nitrogen assimilation have been increased by the use of leguminous and non-leguminous plants with modified capacities for ammonium assimilation. These modifications have been achieved through genetic engineering and breeding. An improved understanding of nitrogen assimilation will be vital if improvements in crop nitrogen use efficiency are to be made to reduce the need for excessive input of fertilisers. In this review we present an overall view of past work and more recent studies on this topic. In our work, using tobacco and Lotus as model plants, glutamine synthetase and glutamate synthase activites have been altered by stimulating or inhibiting in an organ- or tissue-specific manner the expression of the corresponding genes. The physiological impact of these genetic manipulations has been studied on plants grown under different nitrogen regimes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Role and function of cytokinin oxidase in plants

Cytokinin oxidase (CK oxidase) is widely distributed in plants and is the only enzyme that has been shown unequivocally to catalyze the catabolism of specific cytokinins (CKs) to inactive products that lack the N⁶-unsaturated side chain. Thus, the enzyme is thought to play a major role in controlling the level or species of CKs in plant tissues. However, despite its discovery more than 25 years ago, little attention has been given to the elucidation of its role and function in plant growth and development. This review seeks to bring in to context the current state of knowledge regarding the biochemical and molecular properties, regulation in undifferentiated and differentiated tissues, and recent results from studies using transgenic plants in an attempt to provide a more comprehensive understanding of the physiological significance of the enzyme in plants. Notwithstanding species, tissue and other specific differences, in general, CK oxidase appears to contribute to CK homeostasis in plants. However, complete clarity as to its function awaits purification of the protein to homogeneity and the ultimate development of requisite molecular probes.     

植物内生菌促进宿主氮吸收与代谢研究进展

内生菌与植物共生能够提高宿主的氮吸收与氮代谢水平,这可能是由于内生菌在植物体内引发的多种效应的综合结果.植物内生菌能够通过促进植物根系发育和固氮作用为宿主植物提供更多的无机氮素;能够通过分泌多种胞外酶系如漆酶、蛋白水解酶等使宿主植物更好地利用有机氮素;能够提高宿主氮代谢关键酶如硝酸还原酶(NR)、谷氨酰胺合成酶(GS)等酶的活性;能够提高宿主植物激素水平和维生素含量从而促进宿主氮代谢;能够通过影响宿主植物氮代谢促进宿主植物分蘖、提高宿主植物叶绿素含量和光合速率等等.综述了国内外关于植物内生菌促进宿主氮代谢的相关报道,归纳了植物内生菌影响宿主氮素吸收与代谢的可能机制,并展望了关于植物内生菌促进宿主氮代谢机制方面的研究方向.     

Engineering of cysteine and methionine biosynthesis in potato

Summary. Methionine and cysteine, two amino acids containing reduced sulfur, are not only an important substrate of protein biosynthesis but are also precursors of various other metabolites such as glutathione, phytochelatines, S-adenosylmethionine, ethylene, polyamines, biotin, and are involved as methyl group donor in numerous cellular processes. While methionine is an essential amino acid due to an inability of monogastric animals and human beings to synthesise this metabolite, animals are still able to convert methionine consumed with their diet into cysteine. Thus, a balanced diet containing both amino acids is necessary to provide a nutritionally favourable food or feed source. Because the concentrations of methionine and cysteine are often low in edible plant sources, e.g. potato, considerable efforts in plant breeding and research have been and are still performed to understand the physiological, biochemical, and molecular mechanisms that contribute to their synthesis, transport, and accumulation in plants. During the last decade molecular tools have enabled the isolation of most of the genes involved in cysteine and methionine biosynthesis, and the efficient plant transformation technology has allowed the creation of transgenic plants that are altered in the activity of individual genes. The physiological analysis of these transgenic plants has contributed considerably to our current understanding of how amino acids are synthesised. We focused our analysis on potato (Solanum tuberosum cv. Désirée) as this plant provides a clear separation of source and sink tissues and, for applied purposes, already constitutes a crop plant. From the data presented here and in previous work we conclude that threonine synthase and not cystathionine gamma-synthase as expected from studies of Arabidopsis constitutes the main regulatory control point of methionine synthesis in potato. This article aims to cover the current knowledge in the area of molecular genetics of sulfur-containing amino acid biosynthesis and will provide new data for methionine biosynthesis in solanaceous plants such as potato. Received December 19, 2001 Accepted January 7, 2002     

Assembly of a cytosolic pine glutamine synthetase holoenzyme in leaves of transgenic poplar leads to enhanced vegetative growth in young plants

Over‐expression of glutamine synthetase (GS, EC 6.3.1.2), a key enzyme in nitrogen assimilation, may be a reasonable approach to enhance plant nitrogen use efficiency. In this work phenotypic and biochemical characterizations of young transgenic poplars showing ectopic expression of a pine cytosolic GS transgene in photosynthetic tissue (Gallardo et al., Planta 210, 19–26, 1999) are presented. Analysis of 22 independent transgenic lines in a 6 month greenhouse study indicated that expression of the pine GS transgene affects early vegetative growth and leaf morphology. In comparison with non‐transgenic controls, transgenic trees exhibited significantly greater numbers of nodes and leaves (12%), and higher average leaf length and width resulting in an increase in leaf area (25%). Leaf shape was not altered. Transgenic poplars also exhibited increased GS activity (66%), chlorophyll content (33%) and protein content (21%). Plant height was correlated with GS content in young leaves, suggesting that GS can be considered a marker for vegetative growth. Molecular and kinetic characterization of GS isoforms in leaves indicated that poplar GS isoforms are similar to their counterparts in herbaceous plants. A new GS isoenzyme that displayed molecular and kinetic characteristics corresponding to the octomeric pine cytosolic GS1 was identified in the photosynthetic tissues of transgenic poplar leaves. These results indicate that enhanced growth and alterations in biochemistry during early growth are the consequence of transgene expression and assembly of pine GS1 subunits into a new functional holoenzyme in the cytosol of photosynthetic cells.     

The challenge of improving nitrogen use efficiency in crop plants: towards a more central role for genetic variability and quantitative genetics within integrated approaches

In this review, recent developments and future prospects of obtaining a better understanding of the regulation of nitrogen use efficiency in the main crop species cultivated in the world are presented. In these crops, an increased knowledge of the regulatory mechanisms controlling plant nitrogen economy is vital for improving nitrogen use efficiency and for reducing excessive input of fertilizers, while maintaining an acceptable yield. Using plants grown under agronomic conditions at low and high nitrogen fertilization regimes, it is now possible to develop whole-plant physiological studies combined with gene, protein, and metabolite profiling to build up a comprehensive picture depicting the different steps of nitrogen uptake, assimilation, and recycling to the final deposition in the seed. A critical overview is provided on how understanding of the physiological and molecular controls of N assimilation under varying environmental conditions in crops has been improved through the use of combined approaches, mainly based on whole-plant physiology, quantitative genetics, and forward and reverse genetics approaches. Current knowledge and prospects for future agronomic development and application for breeding crops adapted to lower fertilizer input are explored, taking into account the world economic and environmental constraints in the next century.     

Changes in nitrogen assimilation, metabolism, and growth in transgenic rice plants expressing a fungal NADP(H)-dependent glutamate dehydrogenase (gdhA)

In plants, glutamine synthetase (GS) is the enzyme that is mainly responsible for the assimilation of ammonium. Conversely, in microorganisms such as bacteria and Ascomycota, NADP(H)-dependent glutamate dehydrogenase (GDH) and GS both have important roles in ammonium assimilation. Here, we report the changes in nitrogen assimilation, metabolism, growth, and grain yield of rice plants caused by an ectopic expression of NADP(H)-GDH (gdhA) from the fungus Aspergillus niger in the cytoplasm. An investigation of the kinetic properties of purified recombinant protein showed that the fungal gdhA had 5.4–10.2 times higher V _max value and 15.9–43.1 times higher K _m value for NH₄ ⁺, compared with corresponding values for rice cytosolic GS as reported in the literature. These results suggested that the introduction of fungal GDH into rice could modify its ammonium assimilation pathway. We therefore expressed gdhA in the cytoplasm of rice plants. NADP(H)-GDH activities in the gdhA-transgenic lines were markedly higher than those in a control line. Tracer experiments by feeding with ¹⁵NH₄ ⁺ showed that the introduced gdhA, together with the endogenous GS, directly assimilated NH₄ ⁺ absorbed from the roots. Furthermore, in comparison with the control line, the transgenic lines showed an increase in dry weight and nitrogen content when sufficient nitrogen was present, but did not do so under low-nitrogen conditions. Under field condition, the transgenic line examined showed a significant increase in grain yield in comparison with the control line. These results suggest that the introduction of fungal gdhA into rice plants could lead to better growth and higher grain yield by enhancing the assimilation of ammonium.     

Transgenic tobacco plants expressing grass <Emphasis Type="Italic">AstEXPA1</Emphasis> gene show improved performance to several stresses

Expansins are cell wall-loosening proteins and now widely accepted to associate with the plant resistance against various abiotic stresses. In this study, we cloned an expansin gene of AstEXPA1 from Agrostis stolonifera, a heat-resistant creeping bentgrass cultivar, and transformed it into tobacco plants. Physiological index test showed that the transgenic lines were resistant to various abiotic stresses of drought, heat, cold, and salt in comparison to non-transgenic plants. Comprehensive analysis of four physiological response indices showed that the transgenic plants performed much better resistance to drought, following to heat, cold and salt stress, respectively. Meanwhile soluble sugar content displayed more weight to plant resistance by over-expressing AstEXPA1 gene, followed as proline content, REL, and MDA content. The results here would expand our understanding of the expansin roles and drive better insights into plant molecular breeding against stress.     

Transgenic plants with cyanobacterial genes

Over the years, cyanobacteria have been regarded as ideal model systems for studying fundamental biochemical processes like oxygenic photosynthesis and carbon and nitrogen assimilation. Additionally, they have been used as human foods, sources for vitamins, proteins, fine chemicals, and bioactive compounds. Aiming to increase plant productivity as well as nutritional values, cyanobacterial genes involved in carbon metabolism, fatty acid biosynthesis, and pigment biosynthesis have been intensively exploited as alternatives to homologous gene sources. In this short review, transgenic plants with cyanobacterial genes generated over the last two decades are examined, and the future prospects for transgenic crops using cyanobacterial genes obtained from functional genomics studies of numerous cyanobacterial genomes information are discussed.     

Role of Grafting in Resistance to Water Stress in Tomato Plants: Ammonia Production and Assimilation

In general, drought depresses nutrient uptake by the root and transport to the shoot due to a restricted transpiration rate, which may contribute to growth limitation under water deprivation. Moreover, water stress may also restrict the ability of plants to reduce and assimilate nitrogen through the inhibition of enzymes implicated in nitrogen metabolism. The assimilation of nitrogen has marked effects on plant productivity, biomass, and crop yield, and nitrogen deficiency leads to a decrease in structural components. Plants produce significant quantities of NH₄ ⁺ through the reduction of NO₃ ^? and photorespiration, which must be rapidly assimilated into nontoxic organic nitrogen compounds. The aim of the present work was to determine the response of reciprocal grafts made between one tomato tolerant cultivar (Lycopersicon esculentum), Zarina, and a more sensitive cultivar, Josefina, to nitrogen reduction and ammonium assimilation under water stress conditions. Our results show that when cv. Zarina (tolerant cultivar) was used as rootstock grafted with cv. Josefina (ZarxJos), these plants showed an improved N uptake and NO₃ ^? assimilation, triggering a favorable physiological and growth response to water stress. On the other hand, when Zarina was used as the scion (JosxZar), these grafted plants showed an increase in the photorespiration cycle, which may generate amino acids and proteins and could explain their better growth under stress conditions. In conclusion, grafting improves N uptake or photorespiration, and increases leaf NO₃ ^? photoassimilation in water stress experiments in tomato plants.     

The phosphorylated pathway of serine biosynthesis links plant growth with nitrogen metabolism

Because it is the precursor for various essential cellular components, the amino acid serine is indispensable for every living organism. In plants, serine is synthesized by two major pathways: photorespiration and the phosphorylated pathway of serine biosynthesis (PPSB). However, the importance of these pathways in providing serine for plant development is not fully understood. In this study, we examine the relative contributions of photorespiration and PPSB to providing serine for growth and metabolism in the C3 model plant Arabidopsis thaliana. Our analyses of cell proliferation and elongation reveal that PPSB-derived serine is indispensable for plant growth and its loss cannot be compensated by photorespiratory serine biosynthesis. Using isotope labeling, we show that PPSB-deficiency impairs the synthesis of proteins and purine nucleotides in plants. Furthermore, deficiency in PPSB-mediated serine biosynthesis leads to a strong accumulation of metabolites related to nitrogen metabolism. This result corroborates ¹⁵N-isotope labeling in which we observed an increased enrichment in labeled amino acids in PPSB-deficient plants. Expression studies indicate that elevated ammonium uptake and higher glutamine synthetase/glutamine oxoglutarate aminotransferase (GS/GOGAT) activity causes this phenotype. Metabolic analyses further show that elevated nitrogen assimilation and reduced amino acid turnover into proteins and nucleotides are the most likely driving forces for changes in respiratory metabolism and amino acid catabolism in PPSB-deficient plants. Accordingly, we conclude that even though photorespiration generates high amounts of serine in plants, PPSB-derived serine is more important for plant growth and its deficiency triggers the induction of nitrogen assimilation, most likely as an amino acid starvation response.

The phosphorylated pathway of serine biosynthesis is required to synthesize serine for plant growth; and its deficiency triggers an amino acid starvation response by inducing nitrogen assimilation.     

Enhanced salinity tolerance and improved yield properties in Bangladeshi rice Binnatoa through <Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated transformation of <Emphasis Type="Italic">PgNHX1</Emphasis> from <Emphasis Type="Italic">Pennisetum glaucum</Emphasis>

Rice yield is severely affected by high-salt concentration in the vicinity of the plant. In an effort to engineer rice for improved salt tolerance Agrobacterium-mediated transformation of rice cv. Binnatoa was accomplished with the Pennisetum glaucum vacuolar Na⁺/H⁺ antiporter gene (PgNHX1) under the constitutive CaMV35S promoter. For the molecular analysis of putative transgenic plants, PCR and RT-PCR were performed. Transgenic rice plants expressing PgNHX1 showed better physiological status and completed their life cycle by setting flowers and seeds in salt stress, while wild-type plants exhibited rapid chlorosis and growth inhibition. Moreover, transgenic rice plants produced higher grain yields than wild-type plants under salt stress. Assessment of the salinity tolerance of the transgenic plants at seedling and reproductive stages demonstrated the potential of PgNHX1 for imparting enhanced salt tolerance capabilities and improved yield.     

Over-expression of a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene improves salt tolerance in an upland rice

To develop a salt-tolerant upland rice cultivar (Oryza sativa L.), OsNHX1, a vacuolar-type Na⁺/H⁺ antiporter gene from rice was transferred into the genome of an upland rice cultivar (IRAT109), using an Agrobacterium-mediated method. Seven independent transgenic calli lines were identified by polymerase chain reaction (PCR) analysis. These 35S::OsNHX1 transgenic plants displayed a little accelerated growth during seedling stage but showed delayed flowering time and a slight growth retardation phenotype during late vegetative stage, suggesting that the OsNHX1 has a novel function in plant development. Northern and western blot analyses showed that the expression levels of OsNHX1 mRNA and protein in the leaves of three independent transgenic plant lines were significantly higher than in the leaves of wild type (WT) plants. T₂ generation plants exhibited increased salt tolerance, showing delayed appearance and development of damage or death caused by salt stress, as well as improved recovery upon removal from this condition. Several physiological traits, such as increased Na⁺ content, and decreased osmotic potential in transgenic plants grown in high saline concentrations, further indicated that the transgenic plants had enhanced salt tolerance. Our results suggest the potential use of these transgenic plants for further agricultural applications in saline soil.     

HCO-3对植物生长发育及代谢的促进作用

重碳酸盐(bicarbonate, HCO^-₃)是碳酸盐岩经岩溶作用风化的产物,它深刻地影响着植物的生长发育和岩溶地区的生态环境。以往研究大都关注HCO^-₃对植物生长代谢的负面影响,如抑制植物的光合作用、降低碳氮代谢关键酶活性、破坏离子平衡等,少有人关注其对植物生长代谢的积极作用。该文依据前人的研究结果,综述了HCO^-₃对植物生长代谢的促进作用。已有的研究工作显示,HCO^-₃不仅在干旱等逆境胁迫下为植物提供短期的碳源和水源,促进气孔打开,恢复光合作用,而且通过调节碳氮代谢关键酶活性促进植物的碳氮代谢,参与调控植物的碳同化和氮还原等复杂的生理过程; 此外,HCO^-₃还通过影响葡萄糖代谢歧化,改变植物糖酵解途径和磷酸戊糖途径的分配,以增强植物的抗逆能力,从而获取生存机会。HCO^-₃的这些积极作用不仅使之成为促进植物生理代谢的关键因子,而且成为连接光合作用和岩溶作用的纽带。阐明HCO^-₃对植物生长发育的积极作用,可为维护喀斯特生态系统的生物多样性和稳定性、优化喀斯特生态系统功能提供理论依据。     

转大肠杆菌过氧化氢酶基因棉花的抗旱生理研究

以导入大肠杆菌过氧化氢酶基因KatE的T3代转基因棉花为供试材料,经卡那霉素检测和PCR鉴定,将筛选出的阳性转基因植株与对照棉花进行整个生育期的持续水分胁迫处理直至收获,比较材料间的生理生化指标的差异,鉴定转基因植株的耐旱能力。结果显示:(1)干旱胁迫持续至初蕾期时,转基因棉花与对照植株间各项抗旱生理指标差异均未达到显著水平。(2)水分胁迫持续至盛蕾和盛花期时,转基因棉花叶片相对含水量、光系统Ⅱ最大光化学效率(Fv/Fm)、CAT活性,以及叶片的净光合速率(Pn)、气孔导度(Gs)和蒸腾速率(Tr)均显著或极显著高于对照植株,叶绿素含量也都明显高于对照植株。干旱胁迫持续至吐絮期时,转基因棉花的株高、果枝数和铃数均显著或极显著高于对照植株,且转基因棉花和对照的籽棉产量分别比正常灌溉处理降低57.5%和60.1%,全生育期的水分胁迫严重影响了棉花籽棉产量,但转基因棉花的籽棉产量仍显著高于对照。研究表明,在新疆石河子当地自然降水(干旱胁迫)条件下,转KatE基因棉花表现出了较好的生理和生长优势,KatE基因有助于提高棉花的抗旱性。