Development and field performance of nitrogen use efficient rice lines for Africa

Nitrogen (N) fertilizers are a major input cost in rice production, and its excess application leads to major environmental pollution. Development of rice varieties with improved nitrogen use efficiency (NUE) is essential for sustainable agriculture. Here, we report the results of field evaluations of marker‐free transgenic NERICA4 (New Rice for Africa 4) rice lines overexpressing barley alanine amino transferase (HvAlaAT) under the control of a rice stress‐inducible promoter (pOsAnt1). Field evaluations over three growing seasons and two rice growing ecologies (lowland and upland) revealed that grain yield of pOsAnt1:HvAlaAT transgenic events was significantly higher than sibling nulls and wild‐type controls under different N application rates. Our field results clearly demonstrated that this genetic modification can significantly increase the dry biomass and grain yield compared to controls under limited N supply. Increased yield in transgenic events was correlated with increased tiller and panicle number in the field, and evidence of early establishment of a vigorous root system in hydroponic growth. Our results suggest that expression of the HvAlaAT gene can improve NUE in rice without causing undesirable growth phenotypes. The NUE technology described in this article has the potential to significantly reduce the need for N fertilizer and simultaneously improve food security, augment farm economics and mitigate greenhouse gas emissions from the rice ecosystem.     

Arbuscular mycorrhiza improve growth,nitrogen uptake,and nitrogen use efficiency in wheat grown under elevated CO<Subscript>2</Subscript>

Effects of the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis on plant growth, carbon (C) and nitrogen (N) accumulation, and partitioning was investigated in Triticum aestivum L. plants grown under elevated CO₂ in a pot experiment. Wheat plants inoculated or not inoculated with the AM fungus were grown in two glasshouse cells with different CO₂ concentrations (400 and 700 ppm) for 10 weeks. A ¹⁵N isotope labeling technique was used to trace plant N uptake. Results showed that elevated CO₂ increased AM fungal colonization. Under CO₂ elevation, AM plants had higher C concentration and higher plant biomass than the non-AM plants. CO₂ elevation did not affect C and N partitioning in plant organs, while AM symbiosis increased C and N allocation into the roots. In addition, plant C and N accumulation, ¹⁵N recovery rate, and N use efficiency (NUE) were significantly higher in AM plants than in non-AM controls under CO₂ enrichment. It is concluded that AM symbiosis favors C and N partitioning in roots, increases C accumulation and N uptake, and leads to greater NUE in wheat plants grown at elevated CO₂.     

Nitrogen use efficiencies of spring barley grown under varying nitrogen conditions in the field and growth chamber

Background and Aims

Nitrogen-use efficiency (NUE) of cereals needs to be improved by nitrogen (N) management, traditional plant breeding methods and/or biotechnology, while maintaining or, optimally, increasing crop yields. The aims of this study were to compare spring-barley genotypes grown on different nitrogen levels in field and growth-chamber conditions to determine the effects on N uptake (NUpE) and N utilization efficiency (NUtE) and ultimately, NUE.

Methods

Morphological characteristics, seed yield and metabolite levels of 12 spring barley (Hordeum vulgare) genotypes were compared when grown at high and low nitrogen levels in field conditions during the 2007 and 2008 Canadian growing seasons, and in potted and hydroponic growth-chamber conditions. Genotypic NUpE, NUtE and NUE were calculated and compared between field and growth-chamber environments.

Key Results

Growth chamber and field tests generally showed consistent NUE characteristics. In the field, Vivar, Excel and Ponoka, showed high NUE phenotypes across years and N levels. Vivar also had high NUE in growth-chamber trials, showing NUE across complex to simplistic growth environments. With the high NUE genotypes grown at low N in the field, NUtE predominates over NUpE. N metabolism-associated amino acid levels were different between roots (elevated glutamine) and shoots (elevated glutamate and alanine) of hydroponically grown genotypes. In field trials, metabolite levels were different between Kasota grown at high N (elevated glutamine) and Kasota at low N plus Vivar at either N condition.

Conclusions

Determining which trait(s) or gene(s) to target to improve barley NUE is important and can be facilitated using simplified growth approaches to help determine the NUE phenotype of various genotypes. The genotypes studied showed similar growth and NUE characteristics across field and growth-chamber tests demonstrating that simplified, low-variable growth environments can help pinpoint genetic targets for improving spring barley NUE.     

Purification and characterization of an anaerobically induced alanine aminotransferase from barley roots

Alanine aminotransferase (AlaAT, EC 2.6.1.2) is an enzyme that is induced under anaerobic conditions in cereal roots. In barley (Hordeum vulgare L.) roots, there are a number of isoforms of AlaAT. We have identified the anaerobically induced isoform and have purified it to homogeneity. The isolation procedure involved a two-step ammonium sulfate precipitation, gel filtration, ion-exchange chromatography, and chromatofocusing. The enzyme was purified approximately 350-fold to a specific activity of 2231 units/milligram protein. The apparent molecular masses of the native and sodium dodecyl sulfate-denatured AlaAT proteins are 97 and 50 kilodaltons, respectively, indicating that the native enzyme is probably a homodimer. AlaAT has a number of interesting characteristics when compared with other plant aminotransferases. AlaAT does not require the presence of pyridoxyl-5-phosphate to retain its activity, and it appears to be very specific in the reactions that it will catalyze.     

Cisgenic overexpression of cytosolic glutamine synthetase improves nitrogen utilization efficiency in barley and prevents grain protein decline under elevated CO2

Cytosolic glutamine synthetase (GS1) plays a central role in nitrogen (N) metabolism. The importance of GS1 in N remobilization during reproductive growth has been reported in cereal species but attempts to improve N utilization efficiency (NUE) by overexpressing GS1 have yielded inconsistent results. Here, we demonstrate that transformation of barley (Hordeum vulgare L.) plants using a cisgenic strategy to express an extra copy of native HvGS1‐1 lead to increased HvGS1.1 expression and GS1 enzyme activity. GS1 overexpressing lines exhibited higher grain yields and NUE than wild‐type plants when grown under three different N supplies and two levels of atmospheric CO₂. In contrast with the wild‐type, the grain protein concentration in the GS1 overexpressing lines did not decline when plants were exposed to elevated (800–900 μL/L) atmospheric CO₂. We conclude that an increase in GS1 activity obtained through cisgenic overexpression of HvGS1‐1 can improve grain yield and NUE in barley. The extra capacity for N assimilation obtained by GS1 overexpression may also provide a means to prevent declining grain protein levels under elevated atmospheric CO₂.     

Biochemical characterization and kinetic properties of alanine aminotransferase homologues partially purified from wheat (Triticum aestivum L.)

Four homologues of alanine aminotransferase have been isolated from shoots of wheat seedlings and purified by saline precipitation, gel filtration, preparative electrophoresis and anion exchange chromatography on Protein-Pak Q 8HR column attached to HPLC. Alanine aminotransferase 1 (AlaAT1) and 2 (AlaAT2) were purified 303- and 452-fold, respectively, whereas l-glutamate: glyoxylate aminotransferase 1 (GGAT1) and 2 (GGAT2) were purified 485- and 440-fold, respectively. Consistent inhibition of AlaAT (EC 2.6.1.2) and GGAT (EC 2.6.1.4) activities by p-hydroxymercuribenzoate points on participation of cysteine residues in the enzyme activity. The molecular weight of AlaAT1 and AlaAT2 was estimated to be 65 kDa and both of them are monomers in native state. Nonsignificant differences between K_m using alanine as substrate and catalytic efficiency (k_cat/K_m) for l-alanine in reaction with 2-oxoglutarate indicate comparable kinetic constants for AlaAT1 and AlaAT2. Similar kinetic constants for l-alanine in reaction with 2-oxoglutarate and for l-glutamate in reaction with pyruvate for all four homologues suggest equally efficient reaction in both forward and reverse directions. GGAT1 and GGAT2 were able to catalyze transamination between l-glutamate and glyoxylate, l-alanine and glyoxylate and reverse reactions between glycine and 2-oxoglutarate or pyruvate. Both GGATs also consisted of a single subunit with molecular weight of about 50 kDa. The estimated K_m for GGAT1 (3.22 M) and GGAT2 (1.27 M) using l-glutamate as substrate was lower in transamination with glyoxylate than with pyruvate (9.52 and 9.09 mM, respectively). Moreover, distinctively higher values of catalytic efficiency for l-glutamate in reaction with glyoxylate than for l-glutamate in reaction with pyruvate confirm involvement of these homologues into photorespiratory metabolism.     

Biochemical and genetic analyses of N metabolism in maize testcross seedlings: 1. Leaves

Key message

Aside from the identification of 32 QTL for N metabolism in the seedling leaves of a maize testcross population, alanine aminotransferase was found to be a central enzyme in N assimilation.

Abstract

Excessive application of nitrogen (N) fertilizer to grow commercial crops like maize is a cause of concern because of the runoff of excess N into streams and rivers. Breeding maize with improved N use efficiency (NUE) would reduce environmental pollution as well as input costs for the farmers. An understanding of the genetics underlying N metabolism is key to breeding for NUE. From a set of 176 testcrosses derived from the maize IBMsyn10 population grown in hydroponics, we analyzed the youngest fully expanded leaf at four-leaf stage for enzymes and metabolites related to N metabolism. Three enzymes, along with one metabolite explained 24% of the variation in shoot dry mass. Alanine aminotransferase (AlaAT) stood out as the key enzyme in maintaining the cellular level of glutamate as it alone explained 58% of the variation in this amino acid. Linkage mapping revealed 32 quantitative trait loci (QTL), all trans to the genomic positions of the structural genes for various enzymes of N assimilation. The QTL models for different traits accounted for 7–31% of the genetic variance, whereas epistasis was generally not significant. Five coding regions underlying 1-LOD QTL confidence intervals were identified for further validation studies. Our results provide evidence for the key role of AlaAT in N assimilation likely through homeostatic control of glutamate levels in the leaf cells. The two QTL identified for this enzyme would help to select desirable recombinants for improved N assimilation.

     

Effects of grassland management on plant nitrogen use efficiency (NUE): Evidence from a long-term experiment

Grassland management intensification can greatly influence nitrogen (N) dynamics between aboveground and belowground compartments mainly because of the large amount of available N forms, which are repeatedly added to soils. A better understanding of how chronic fertilisation might affect N use efficiency (NUE) in plants can contribute to reducing N losses from soils and improve the sustainability of managed grasslands. Here we address how NUE might be affected by (1) the addition of key nutrients (e.g. N, P, K, Mg) in different combinations, (2) grazing by rabbits, and (3) liming (i.e. CaCO₃ applications) in a 22-year-old permanent grassland experiment established in Berkshire, UK, in 1991. We first calculate seven different NUE indexes, which are known to respond either to changes in soil N availability (i.e. endogenous N inputs from soil N mineralization processes) or to exogenous N inputs (i.e. synthetic N fertiliser). We found that plant NUE calculated as plant biomass produced per unit of N acquired significantly decreased under the chronic addition of multiple nutrients (NPKMg) and was even lower under N-only applications. Most NUE indexes significantly decreased under grazing but greatly increased under liming applications. We found evidence that NUE indexes, which accounted for endogenous N sources decreased at increased rates of soil N mineralization. Finally, we found no significant relationships between any of the NUE indexes and estimates of soil N losses (Mg N ha⁻¹) or N retention in soils (i.e. units of soil N retained per unit of N added) calculated from changes in net soil N budget over 22 years. Our study carried out on relatively acidic sandy soils suggests how liming applications in combination with low levels of multi-nutrient additions (NPKMg) can significantly improve plant biomass production per unit of N added thus contributing to enhance the sustainability of managed grassland ecosystems.     

Tradeoffs between nitrogen- and water-use efficiency in dominant species of the semiarid steppe of Inner Mongolia

In water-limited environments, photosynthetic carbon gain and loss of water by transpiration are in a permanent tradeoff as both are contrarily regulated by stomata conductance. In semiarid steppe grasslands water limitation may covary with nitrogen limitation. Steppe grassland species are capable of optimizing their use of limiting resources, water and nitrogen, but regulation is still poorly understood. In a two-year experiment with addition of water (irrigation simulating a wet year) and nitrogen (0, 25, and 50 kg urea-N?ha^?1) we assessed intrinsic water use efficiency (WUE_i), nitrogen use efficiency (NUE), and related plant functional traits (PFTs) of four dominant C₃ species (Leymus chinensis, Agropyron cristatum, Stipa grandis, and Artemisia frigida). Water and N fertilizer supplementation significantly increased plant primary production, and N effect was more pronounced under irrigated conditions. Parallel with the responses of plant production, a strong tradeoff between WUE_i and NUE was detected: water supply increased NUE but decreased WUE_i, whereas N addition slightly increased WUE_i at the expense of NUE. This tradeoff occurred at the leaf level, and involved the responses of leaf N concentration and specific leaf area. WUE_i of species changed among treatments in a predictable manner by the parameter of leaf N content per area. Dominant plant species commonly achieved a higher utilization efficiency of the more limiting resource via altering PFTs, which was an important mechanism of adaptation to variable resource limitation in semiarid grasslands.     

Functional conservation between mammalian MGRN1 and plant LOG2 ubiquitin ligases

Plant LOSS OF GDU 2 (LOG2) and Mammalian Mahogunin Ring Finger 1 (MGRN1) proteins are RING-type E3 ligases sharing similarity N-terminal to the RING domain. Deletion of this region disrupts the interaction of LOG2 with the plant membrane protein GLUTAMINE DUMPER1 (GDU1). Phylogenetic analysis identified two clades of LOG2/MGRN1-like proteins in vertebrates and plants. The ability of MGRN1 to functionally replace LOG2 was tested. MGRN1 ubiquitylates GDU1 in vitro and can partially substitute for LOG2 in the plant, partially restoring amino acid resistance to a GDU1-myc over-expression, log2-2 background. Altogether, these results suggest a conserved function for the N-terminal domain in evolution.     

L-alanine:2-oxoglutarate aminotransferase isoenzymes from Arabidopsis thaliana leaves

The occurrence of four l-alanine:2-oxoglutarate aminotransferase (AOAT) isoenzymes (AOAT-like proteins): alanine aminotransferase 1 and 2 (AlaAT1 and AlaAT2, EC 2.6.1.2) and l-glutamate:glyoxylate aminotransferase 1 and 2 (GGAT1 and GGAT2, EC 2.6.1.4) was demonstrated in Arabidopsis thaliana leaves. These enzymes differed in their substrate specificity, susceptibility to pyridoxal phosphate inhibitors and behaviour during molecular sieving on Zorbax SE-250 column. A difference was observed in the electrostatic charge values at pH 9.1 between GGAT1 and GGAT2 as well as between AlaAT1 and AlaAT2, despite high levels of amino acid sequence identity (93 % and 85 %, respectively). The unprecedented evidence for the monomeric structure of both AlaAT1 and AlaAT2 is presented. The molecular mass of each enzyme estimated by molecular sieving on Sephadex G-150 and Zorbax SE-250 columns and SDS/PAGE was approximately 60 kDa. The kinetic parameters: K_m (Ala)=1.53 mM, K_m (2-oxoglutarate)=0.18 mM, k_cat=124.6 s⁻¹, k_cat/K_m=8.1 × 10⁴ M⁻¹·s⁻¹ of AlaAT1 were comparable to those determined for other AlaATs isolated from different sources. The two studied GGATs also consisted of a single subunit with molecular mass of 47.3–70 kDa. The estimated K_m values for l-glutamate (1.2 mM) and glyoxylate (0.42 mM) in the transamination catalyzed by putative GGAT1 contributed to indentification of the enzyme. Based on these results we concluded that each of four AOAT genes in Arabidopsis thaliana leaves expresses different AOAT isoenzyme, functioning in a native state as a monomer.     

Structural and functional analysis of new plant promoter pro-SmAMP1 from <Emphasis Type="Italic">Stellaria media</Emphasis>

The nucleotide sequence of a fragment of the promoter region of pro-SmAMP1 gene, having a length of 1257 bp and encoding antifungal peptides, was determined in chickweed (Stellaria media (L.) Vill.). Computer analysis of the nucleotide sequence revealed a number of cis-elements that are typical strong plant promoters. Five 5′-deletion variants were created taking into account the distribution of cis-elements:–1235,–771,–714,–603, and–481 bp of pro-SmAMP1 gene promoter, which were fused to the coding region of the uidA reporter gene in pCambia1381Z plant expression vector. The efficacy of pro-SmAMP1 promoter deletion variants was determined by transient expression in plants of Nicotiana benthamiana and using sequential generations of transgenic Nicotiana tabacum plants. It was found that the levels of GUS reporter protein activity in the extracts from transgenic and agroinfiltrated plants using all deletion variants of pro-SmAMP1 gene promoter were 3–5 times higher than those of 35S CaMV viral promoter. The highest activity of GUS protein was observed in the leaves of transgenic tobacco plants and closely correlated with the mRNA level of encoding gene. The levels of GUS activity did not differ significantly among 11 independent homozygous lines of T₂ generation of N. tabacum plants with different deletion variants of pro-SmAMP1 promoter. The results give reason to assume that all deletion variants of pro-SmAMP1 promoter provide stable and high level of expression of controlled genes. The shortest deletion variant–481 bp of pro-SmAMP1 promoter should be viewed as a potentially strong plant promoter for the genetic engineering of plants.     

Trigalactosyldiacylglycerol 3 protein orthologs are required for basal disease resistance in Nicotiana benthamiana

Phosphatidic acid plays an important role in Nicotiana benthamiana immune responses against phytopathogenic bacteria. We analyzed the contributions of endoplasmic reticulum-derived chloroplast phospholipids, including phosphatidic acid, to the resistance of N. benthamiana against Ralstonia solanacearum. Here, we focused on trigalactosyldiacylglycerol 3 (TGD3) protein as a candidate required for phosphatidic acid signaling. On the basis of Arabidopsis thaliana TGD3 sequences, we identified two putative TGD3 orthologs in the N. benthamiana genome, NbTGD3-1 and NbTGD3-2. To address the role of TGD3s in plant defense responses, we created double NbTGD3-silenced plants using virus-induced gene silencing. The NbTGD3-silenced plants showed a moderately reduced growth phenotype. Bacterial growth and the appearance of bacterial wilt disease were accelerated in NbTGD3-silenced plants, compared with control plants, challenged with R. solanacearum. The NbTGD3-silenced plants showed reduced both expression of allene oxide synthase that encoded jasmonic acid biosynthetic enzyme and NbPR-4, a marker gene for jasmonic acid signaling, after inoculation with R. solanacearum. Thus, NbTGD3-mediated endoplasmic reticulum—chloroplast lipid transport might be required for jasmonic acid signaling-mediated basal disease resistance in N. benthamiana.