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.     

Can less yield more? Is reducing nutrient input into the environment compatible with maintaining crop production?

Plant scientists have long recognized the need to develop crops that absorb and use nutrients more efficiently. Two approaches have been used to increase nutrient use efficiency (NUE) in crop plants. The first involves both traditional breeding and marker-assisted selection in an attempt to identify the genes involved. The second uses novel gene constructs designed to improve specific aspects of NUE. Here, we discuss some recent developments in the genetic manipulation of NUE in crop plants and argue that an improved understanding of the transition between nitrogen assimilation and nitrogen recycling will be important in applying this technology to increasing crop yields. Moreover, we emphasize the need to combine genetic and transgenic approaches to make significant improvements in NUE.     

Leaf nitrogen remobilisation for plant development and grain filling

A major challenge of modern agriculture is to reduce the excessive input of fertilisers and, at the same time, to improve grain quality without affecting yield. One way to achieve this goal is to improve plant nitrogen economy through manipulating nitrogen recycling, and especially nitrogen remobilisation, from senescing plant organs. In this review, the contribution of nitrogen remobilisation efficiency (NRE) to global nitrogen use efficiency (NUE), and tools dedicated to the determination of NRE are described. An overall examination of the physiological, metabolic and genetic aspects of nitrogen remobilisation is presented.     

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.     

Prospects for improving nitrogen use efficiency: Insights given by 15N-labelling experiments

In higher plants, recent advances in plant molecular genetics, combined with modern physiological and biochemical studies, have expanded our understanding of the regulatory mechanisms controlling the primary steps of inorganic nitrogen assimilation and the subsequent biochemical pathways involved in nitrogen supply and recycling for higher plant metabolism, growth and development. In this presentation, we describe improvements in our understanding of the molecular controls of nitrogen assimilation through the use of transgenic plants and the study of genetic variability in model and crop species. To illustrate this research programme, the physiological impact of modified gene expression, using either transgenic plants or different genotypes, was studied using ¹⁵N-labelling experiments in order to monitor the influx of nitrate or ammonia and its subsequent incorporation into amino acids.     

Physiology of maize I: A comprehensive and integrated view of nitrogen metabolism in a C4 plant

Maize ( Zea mays L., line F2) plants were grown in the field under high or low fertilization input to monitor the metabolic, biochemical and molecular events occurring in young vegetative leaves and in the different leaf stages along the main axis in plants harvested 15 days after silking. This study shows that in maize which possess large sinks represented by the seeds, nitrogen (N) management is different compared with tobacco in which sink strength is much lower and mostly limited to young developing leaves. Although in young leaves nitrate assimilation predominates in both species, ammonium assimilation exhibits some species-specific differences with respect to inorganic and organic N metabolite accumulation during leaf ageing. These differences are likely to be related to the high sink strength of the ear in maize, which continuously imports carbon and N assimilates during grain filling. Consequently, a number of cytosolic glutamine synthetase isoenzymes are expressed during leaf ageing to maintain a constant flux of reduced N necessary for the synthesis of organic N molecules used either for leaf protein synthesis or directly translocated to the grain. This situation contrasts with that found in tobacco for which leaf ammonium assimilation in the plastids is shifted to the cytosol during the transition from sink leaves to source leaves. These species-specific differences for N assimilation and recycling are discussed in relation to the evolution of leaf photosynthetic activity and leaf senescence, which both seem to be largely dependent on the different sink strength in each species.     

Can genetic manipulation of plant nitrogen assimilation enzymes result in increased crop yield and greater N-use efficiency? An assessment

The literature on the relations between plant nitrogen (N) assimilation enzymes and plant/crop N assimilation, growth and yield is reviewed to assess if genetic manipulation of the activities of N assimilation enzymes can result in increased yield and/or increased N use efficiency. The available data indicate that (I) levels of N assimilation enzymes do not limit primary N assimilation and hence yield; (II) root or shoot nitrate assimilation can have advantages under specific environmental conditions; (III) for cereals, cytosolic glutamine synthetase (GS1) is a key enzyme in the mobilisation of N from senescing leaves and its activity in senescing leaves is positively related to yield; and (TV) for rice (Oryza sativd), NADH-glutamate synthase (NADH-GOGAT) is important in the utilisation of N in grain filling and its activity in developing grains is positively related to yield. In our opinion, selection of plants, from either a genetically manipulated population or genetic resources, with expression of nitrate reductase/nitrite reductase primarily in the root or shoot should increase plant/crop growth and hence yield under specific environmental conditions. In addition for cereals the selection of plants with high GS1 in senescing leaves and in some cases high NADH-GOGAT in developing grains could help maximise the retrieval of plant N in seeds.     

Proteomic analysis of residual proteins in blades and petioles of fallen leaves of Brassica napus

Brassica napus L. is an important crop plant, characterised by high nitrogen (N) levels in fallen leaves, leading to a significant restitution of this element to the soil, with important consequences at the economic and environmental levels. It is now well established that the N in fallen leaves is due to weak N remobilisation that is especially related to incomplete degradation of foliar proteins during leaf senescence. Identification of residual proteins in a fallen leaf (i.e. incompletely degraded in the last step of the N remobilisation process) constitutes important information for improving nutrient use efficiency. Proteome analysis of the vascular system (petioles) and blades from fallen leaves of Brassica napus was performed, and the 30 most abundant residual proteins in each tissue were identified. Among them, several proteins involved in N recycling remain in the leaf after abscission. Moreover, this study reveals that some residual proteins are associated with energy metabolism, protection against oxidative stress, and more surprisingly, photosynthesis. Finally, comparison of blade and petiole proteomes show that, despite their different physiological roles in the non‐senescing leaf, both organs redirect their metabolism in order to ensure catabolic reactions. Taken together, the results suggest that a better degradation of these leaf proteins during the senescence process could enable improvements in the N use efficiency of Brassica napus.     

植物谷氨酰胺合成酶研究进展及其应用前景

氮素是制约作物产量的主要营养元素之一,谷氨酰胺合成酶(Glutamine synthase,GS;EC 6.3.1.2)是氮素代谢途径中的关键酶。目前,拟南芥、水稻、小麦和玉米等植物中的GS成员均已被分离鉴定。研究表明,超表达GS能够提高植物对氮素的利用效率,从而在植株的生长发育特别是产量形成过程中发挥重要作用,但是其功能在不同植物上并不完全一致,可能与GS基因受到转录和翻译后等水平的调控有关。以下综述了植物GS基因分类、QTL定位、对氮素代谢响应、组织表达特异性、生物学功能及其分子调控机制等方面的研究进展,并展望了植物GS基因的应用前景,以期为利用GS基因来提高植物氮素利用效率提供具有参考价值的信息。     

Towards a better understanding of the genetic and physiological basis for nitrogen use efficiency in maize

To enhance our understanding of the genetic basis of nitrogen use efficiency in maize (Zea mays), we have developed a quantitative genetic approach by associating metabolic functions and agronomic traits to DNA markers. In this study, leaves of vegetative recombinant inbred lines of maize, already assessed for their agronomic performance, were analyzed for physiological traits such as nitrate content, nitrate reductase (NR), and glutamine synthetase (GS) activities. A significant genotypic variation was found for these traits and a positive correlation was observed between nitrate content, GS activity and yield, and its components. NR activity, on the other hand, was negatively correlated. These results suggest that increased productivity in maize genotypes was due to their ability to accumulate nitrate in their leaves during vegetative growth and to efficiently remobilize this stored nitrogen during grain filling. Quantitative trait loci (QTL) for various agronomic and physiological traits were searched for and located on the genetic map of maize. Coincidences of QTL for yield and its components with genes encoding cytosolic GS and the corresponding enzyme activity were detected. In particular, it appears that the GS locus on chromosome 5 is a good candidate gene that can, at least partially, explain variations in yield or kernel weight. Because at this locus coincidences of QTLs for grain yield, GS, NR activity, and nitrate content were also observed, we hypothesize that leaf nitrate accumulation and the reactions catalyzed by NR and GS are coregulated and represent key elements controlling nitrogen use efficiency in maize.     

染色体片段导入系在作物遗传育种中的应用

准确而有效的定位农作物数量性状基因座(Quantitative Trait Loci,QTLs)是植物分子育种的核心,传统的QTL定位群体遗传背景复杂,受群体大小和统计方法等多方面的限制,难以达到QTL精细定位。随着分子标记技术、计算机统计软件及分子辅助选择的飞速发展,一种新的QTL定位群体脱颖而出,这就是染色体片段导入系(Chromosome Segment Introgression Lines,CSILs)。它不但能有效消除"遗传背景噪音"对QTL定位的干扰,还能够在群体中挖掘出大量的有利隐蔽基因,对农作物遗传育种的进一步发展有巨大贡献。对染色体片段导入系的优越性,应用范围以及应用前景作以综述。     

DCMU inhibits in vivo nitrate reduction in illuminated barley (C(3)) leaves but not in maize (C(4)): a new mechanism for the role of light?

The leaves of C(4) plants possess a superior metabolic efficiency not only in terms of photosynthetic carbon assimilation, but also in terms of inorganic nitrogen assimilation, when compared to C(3)plants. In vivo nitrate assimilation efficiency of leaves is dependent on light, but the obligatory presence of light has been debated and its role remains confounded. This problem has not been addressed from the standpoint of the C(3) vs. C(4) nature of the species investigated, which may actually hold the key to resolve the controversy. Here, we present the first report providing evidence for differential photo-regulation of leaf nitrate reduction in barley ( Hordeum vulgare L.) vs. maize ( Zea mays L.) plants, which may help explain the superior nitrogen-use efficiency (and hence superior productivity) of maize plants. The novel finding that carbohydrate-depleted maize leaves were able to reduce nitrate when photosynthesis was inhibited by 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea (DCMU) in the presence of light, raises a very important question about the possibilities of a new photo-regulatory mechanism for supporting nitrate reduction in maize leaves operating independently of photosynthetic carbon dioxide fixation. On the other hand, leaves of barley could not carry out any in vivo nitrate assimilation, whatsoever, under these conditions. We find another fundamental difference between the two species in terms of differential regulation of nitrate reductase (NR; EC 1.6.6.1). In barley leaves, NR activity and activation state remained unaffected due to DCMU, but in sharp contrast, both were appreciably upregulated in maize. Collectively, the results indicate that enzyme capacity is not limiting for nitrate reduction in leaves, as the NR activity was higher in barley than in maize. The maize leaves may have had a selective advantage due to C(4) morphology/metabolism in terms of maintaining a better reductant/carbon skeleton supply for nitrate reduction.     

Water Use Efficiency of Field-grown Maize during Moisture Stress

Theoretical analysis of the CO₂ assimilation and water loss by single leaves suggests that the water use efficiency of C₄ species decreases as stomatal resistance increases. To confirm this hypothesis for a complete maize crop, results from computer simulations and a field experiment were compiled for varying stomatal resistances. A soil-plant-atmosphere model allowed simulations of the many simultaneous interactions between a crop canopy and its environment. The simulations for varying stomatal resistances clearly indicated that as stomatal resistance increased, water use efficiency of the maize crop decreased. The field experiment data also confirmed that water use efficiency was significantly decreased under water stress conditions when stomatal resistance increased. We concluded that management practices for maize, which induce moisture stress conditions resulting in increased stomatal resistance, reduce both crop photosynthetic productivity and water use efficiency.     

Genetic Variants in REC8, RNF212, and PRDM9 Influence Male Recombination in Cattle

We use >250,000 cross-over events identified in >10,000 bovine sperm cells to perform an extensive characterization of meiotic recombination in male cattle. We map Quantitative Trait Loci (QTL) influencing genome-wide recombination rate, genome-wide hotspot usage, and locus-specific recombination rate. We fine-map three QTL and present strong evidence that genetic variants in REC8 and RNF212 influence genome-wide recombination rate, while genetic variants in PRDM9 influence genome-wide hotspot usage.     

Merging methods in molecular and ecological genetics to study the adaptation of plants to anthropogenic metal-polluted sites: implications for phytoremediation

Metallophyte species that occur naturally on metal-enriched soils represent major biological resources for the improvement of phytoremediation, a benign and cost-effective technology that uses plants to clean up anthropogenic metal-polluted soils. Within the last decade, molecular genetic studies carried out on several model organisms (including Arabidopsis halleri) have considerably enhanced our understanding of metal tolerance and hyperaccumulation in plants, but the identification of the genes of interest for phytoremediation purposes remains a challenge. To meet this challenge, we propose to combine '-omics' with molecular ecology methods. Using A. halleri, we confronted molecular genetic results with: (i) within-species polymorphism and large-scale population differentiation for zinc tolerance; (ii) the demographical context (e.g. migration pattern) of the species for zinc tolerance evolution; (iii) the Quantitative Trait Loci (QTL) analysis of the genetic architecture for zinc tolerance; and (iv) the fine-scale dissection of identified QTL regions, to discuss more precisely the nature of the genes potentially involved in the adaptation to zinc-polluted soils.     

Identification and characterization of QTL controlling Agrobacterium-mediated transient and stable transformation of Brassica oleracea

A commonly encountered difficulty with the genetic engineering of crop plants is that different varieties of a particular species can show great variability in the efficiency with which they can be transformed. This increases the effort required to introduce transgenes into particular genetic backgrounds. The use of Substitution Lines has allowed the finer mapping of three Quantitative Trait Loci (tf1, tf2 and tf3) that explain 26% of the variation in the efficiency of Agrobacterium-mediated transformation in Brassica oleracea. Use of an 'orthogonal set' of genotypes (containing all eight possible combinations of 'positive' and 'negative' alleles at the three QTL), along with time course studies of transgene expression, has allowed the determination of the stages at which these genes have their effects during transformation. With regard to control of the level of transient transgene expression, tf1 (on LGO1) alone has no detectable effect, whilst tf2 (on LGO3) and tf3 (on LGO7) have highly significant effects (P < 0.001). All three loci have highly significant (P < 0.001) effects on the levels of expression of stably integrated transgene. The use of RFLP markers has shown that tf1 and tf2 are in duplicated regions of the B. oleracea genome and appear to be paralogous in origin. Colinearity of these regions with the A. thaliana genome has been identified. The results allow the selection of progeny Brassica oleracea genotypes that are more efficiently transformed than either parent used in the original cross.     

密度与氮肥对关中灌区夏玉米(Zea mays L.)群体光合生理指标的影响

合理的密度是玉米构建良好群体结构、优化群体光合生理功能的基础,适宜的氮肥施用量是玉米进行光合生产的营养物质保障.运用作物群体生理学的方法,采用二次饱和D试验设计,研究了关中灌区夏玉米密度与氮肥耦合和群体光合生理指标的关系及其效应,明确了在关中灌区夏播条件下,在45000～75000株/hm2的密度范围内,密度与玉米籽粒产量、总干物质积累量(DMA)、吐丝期叶面积指数(MLAI)、总光合势(LAD)、平均作物生长率(CGR)为正相关,在45000～60000株/hm2的密度范围内,密度与玉米叶片平均净同化率(NAR)为正相关,而在60000～75000株/hm2的密度范围内,密度与玉米NAR为负相关;在氮素施用量≤600.0 kg/hm2的范围内,氮素施用量与玉米籽粒产量、DMA、CGR为正相关,在氮素施用量≤260.55 kg/hm2的范围内,氮素施用量与玉米MLAI、LAD为正相关,与玉米NAR为负相关,而在氮素施用量260.55～600.0kg/hm2的范围内,氮素施用量与玉米MLAI、LAD为负相关,与玉米NAR为正相关.密度对其影响较大的指标为:籽粒产量、DMA、LAD、CGR和MLAI , 影响较小的指标为:NAR;氮肥对其影响较大的指标为:DMA、CGR、籽粒产量、NAR,影响较小的指标为:LAD和MLAI .对籽粒产量和群体生理指标的综合影响效应,密度显著大于氮肥,玉米生产中,尤其要重视合理密植.通过对回归模型的解析,筛选确定了陕单8806玉米在关中灌区夏播中,实现高产的密度与氮肥耦合优化技术方案:合理密度为61713～66177株/hm2,适宜纯氮施用量为309.88～569.02kg/hm2.