Guevara, D. R., Bi, Y.-M. and Rothstein, S. J. 2014. Identification of regulatory genes to improve nitrogen use efficiency. Can. J. Plant Sci. 94: 1009-1012. Crop production on soils containing sub-optimal levels of nitrogen (N) severely compromises yield potential. The development of crop varieties displaying high N use efficiency (NUE) is necessary in order to optimize N fertilizer use, and reduce the environmental damage caused by the current excessive application of N in agricultural areas. Genome-wide microarray analysis of rice plants grown under N-limiting environments was performed to identify NUE candidate genes. An early nodulin gene, OsENOD93-1, was strongly up-regulated during plant growth under low N. A constitutive Ubiquitin promoter was used to drive the expression of the OsENOD93-1 gene in transgenic plants to determine the importance of OsENOD93-1 for rice NUE. Transgenic rice plants over-expressing the OsENOD93-1 gene achieved ~23% and 16% more yield and biomass, respectively, compared with wild-type plants when grown under N-limitation conditions. OsENOD93-1-OX transgenic plants accumulated a higher amount of total amino acids in the roots and xylem sap under N stress, suggesting that OsENOD93-1 plays a role in the transportation of amino acids. Taken together, we demonstrate that an effective way to identify NUE gene candidates involves both transcriptional profiling coupled with a transgenic validation approach to improve complex traits such as NUE in important crops.

Wan, S., Greenham, T., Goto, K., Mottiar, Y., Johnson, A. M., Staebler, J. M., Zaidi, M. A., Shu, Q. and Altosaar, I. 2014. A novel nitrous oxide mitigation strategy: expressing nitrous oxide reductase from Pseudomonas stutzeri in transgenic plants. Can. J. Plant Sci. 94: 1013-1025. As a stable greenhouse gas, nitrous oxide (N₂O) plays a significant role in stratospheric ozone destruction. The primary anthropogenic N₂O source is the use of nitrogen in agriculture. Currently, the annual N₂O emissions from this soil-plant-microbial system is more than 2.6 Tg (1 Tg=1 million metric tonnes) of N₂O-N globally. So it is important to explore some innovative and effective biology-based strategies for N₂O mitigation. If shown to be effective in field trails as well as laboratory-scale experiments, such GMO plants could help guide international policies on adaptation to climate change. The bacterial enzyme nitrous oxide reductase (N₂OR) is the only known enzyme capable of catalyzing the final step of the denitrification pathway, conversion of N₂O to N₂. To “scrub” the N₂O emissions, bacterial N₂OR was heterologously expressed in plants. Structurally, the enzyme N₂OR is encoded by nosZ, but its biosynthesis and assembly in prokaryotes require the products of several nos genes, including a putative ABC-type transporter encoded by nosDFY, and the copper chaperone NosL for biogenesis of the metal centre. We have generated transgenic tobacco plants expressing the nosZ gene, as well as tobacco plants in which the other nos genes were co-expressed under the control of a root-specific promoter (rolD) and a constitutive promoter (d35S). The nosZ gene from Pseudomonas stutzeri heterologously expressed in tobacco produced active recombinant N₂OR. The positive results in the preliminary proof-of-principle experiments indicated that plants heterologously expressing N₂OR could mitigate emissions at the source before N₂O reaches the stratosphere or troposphere.

Walley, F. L., Gillespie, A. W., Adetona, A. B., Germida, J. J. and Farrell, R. E. 2014. Manipulation of rhizosphere organisms to enhance glomalin production and C-sequestration: Pitfalls and promises. Can. J. Plant Sci. 94: 1025-1032. Arbuscular mycorrhizal fungi (AMF) reportedly produce glomalin, a glycoprotein that has the potential to increase soil carbon (C) and nitrogen (N) storage. We hypothesized that interactions between rhizosphere microorganisms, such as plant growth-promoting rhizobacteria (PGPR), and AMF, would influence glomalin production. Our objectives were to determine the effects of AMF/PGPR interactions on plant growth and glomalin production in the rhizosphere of pea (Pisum sativum L.) with the goal of enhancing C and N storage in the rhizosphere. One component of the study focussed on the molecular characterization of glomalin and glomalin-related soil protein (GRSP) using complementary synchrotron-based N and C X-ray absorption near-edge structure (XANES) spectroscopy, pyrolysis field ionization mass spectrometry (Py-FIMS), and proteomics techniques to characterize specific organic C and N fractions associated with glomalin production. Our research ultimately led us to conclude that the proteinaceous material extracted, and characterized in the literature, as GRSP is not exclusively of AMF origin. Our research supports the established concept that GRSP is important to soil quality, and C and N storage, irrespective of origin. However, efforts to manipulate this important soil C pool will remain compromised until we more clearly elucidate the chemical nature and origin of this resource.

Siciliano, S. D. 2014. Identification of regulatory genes to reduce N₂O production. Can. J. Plant Sci. 94: 1033-1036. The production of nitrous oxide occurs predominantly by microbial activity. This microbial activity can be broadly sub-divided into denitrification, the sequential reduction of nitrate to nitrous oxide or dinitrogen gas, or into nitrification, the sequential oxidation of ammonia to nitrite. The consumption of nitrous oxide occurs by microbial activity as well, but only by a single pathway, i.e., the activity of nitrous oxide reductase (nos). The purpose of this investigation was to determine the dominant producer of nitrous oxide in our agricultural ecosystems, and then explore how these producers interacted with other biological and edaphic factors to regulate overall nitrous oxide production. Finally, we also investigated what controlled nitrous oxide consumption in these agricultural ecosystems. Much to our surprise, the dominant production of nitrous oxide in these upland agricultural soils occurred by nitrification, likely the nitrification-denitrification pathway. In addition, a root exudate, formate, was a large driver of nitrous oxide release via its interaction with the fungal biomass under micro-aerophilic conditions. Despite these unusual sources of production, what became apparent was that the net flux of nitrous oxide in an agricultural soil was linked to denitrifier consumption of nitrous oxide. In conclusion, this project found that there was a wide variety of non-bacterial denitrifier producers of nitrous oxide in an agricultural soil and that they interact not only between themselves but with the plant community. However, the net production of nitrous oxide in agricultural fields was still tightly linked to bacterial denitrification, but through the consumption of nitrous oxide by bacterial denitrifiers.

Flynn, B., Scott, N. and Dong, Z. 2014. Nitrogen fixation, hydrogen production and N₂O emissions. Can. J. Plant Sci. 94: 1037-1041. H₂ is a by-product of the nitrogenase reaction. Exposure to H₂ is linked to increased N₂O production, increased CO₂ fixation and plant growth promotion in soil. The effects of H₂ exposure on soil were observed using controlled H₂ gas treatments and field trials with legumes. In field trials, increased N₂O production was observed in soil adjacent to legume nodules and inoculation of H₂-oxidizing isolates led to increased N₂O emissions in corn fields. Many H₂-oxidizing isolates tested positive for key denitrification genes, indicating a connection between H₂ uptake and N₂O emissions. H₂ treatment significantly increased copy number of the nitrite reductase (nirK) gene suggesting increased denitrification as the source of N₂O. There was also a significant increase in copy number and expression of the RubisCO (cbbL) gene in soil. H₂-oxidizing bacterial isolates (JM63 and JM162a) were found to promote plant growth, increasing tiller number and yield in spring wheat and barley. Combined results of T-RFLP and 16S rDNA clone libraries analysis revealed bacterial community structure changes in response to H₂ treatment, primarily with increases to the Gammaproteobacteria and Betaproteobacteria groups. The results of these studies help provide a better understanding of the soil bacterial community's responses to H₂ exposure and may lead to the development of a commercially viable plant growth promoting inoculant.

Schuetz, M., Douglas, C., Samuels, L. and Ellis, B. 2014. Manipulating lignin deposition. Can. J. Plant Sci. 94: 1043-1049. Since lignin represents one of most durable forms of fixed carbon in plant biomass, we hypothesized that increasing root lignin content for crops whose root systems remained in the soil after harvest would elevate the total amount of carbon retained in the soil in Canadian agroecosystems. The immediate goal of this Greencrop project was, therefore, to gain a better understanding of the molecular mechanisms that control deposition of the lignin polymer in plant cell walls, with a view to eventually manipulating the quantity and location of lignin in crop plant root systems. To this end, we examined two classes of Arabidopsis thaliana proteins - transcription factors, which are believed to play crucial roles in regulating lignin biosynthesis, and ATP binding cassette transporters, which are putative lignin precursor transporters. These studies revealed that a complex network of interacting transcriptional regulators is involved in activating and suppressing the expression of key genes required for secondary cell wall deposition and lignification.

Mabood, F., Zhou, X. and Smith, D. L. 2014. Microbial signaling and plant growth promotion. Can. J. Plant Sci. 94: 1051-1063. The rhizosphere offers a complex microhabitat where root exudates provide a diverse mixture of organic compounds that are used as nutrients or signals by the soil microbial population. On the other hand, these soil microorganisms produce compounds that directly or indirectly assist in plant growth promotion. The widely recognized mechanisms of plant growth promotion are biofertilization, production of phytohormones, suppression of diseases through biocontrol, induction of disease resistance and production of volatile signal compounds. During the past few decades our understanding of the interaction between rhizobacteria and plants has expanded enormously and this has resulted in application of microbial products used as crop inoculants (as biofertilizers), for increased crop biomass and disease suppression. However, this plant-microbe interaction is affected by adverse environmental conditions, and recent work has suggested that inoculants carrying plant-to-bacteria or bacteria-to-plant signals can overcome this and promote plant productivity under stressful environmental conditions. Very recent work has also shown that some plant growth-promoting rhizobacteria secrete novel signaling molecules that also promote plant growth. The use of rhizobacterial signaling in promoting plant growth offers a new window of opportunity, especially when we are looking at plants to provide biofuels and novel bioproducts. Developing technologies that can enhance plant growth and productivity is imperative.

Whalen, J. K., Gul, S., Poirier, V., Yanni, S. F., Simpson, M. J., Clemente, J. S., Feng, X., Grayston, S. J., Barker, J., Gregorich, E. G., Angers, D. A., Rochette, P. and Janzen, H. H. 2014. Transforming plant carbon into soil carbon: Process-level controls on carbon sequestration. Can. J. Plant Sci. 94: 1065-1073. Plants figure prominently in efforts to promote C sequestration in agricultural soils, and to mitigate greenhouse gas (GHG) emissions. The objective of the project was to measure the transformations of plant carbon in soil through controlled laboratory experiments, to further understand (1) root-associated CO₂ and N₂O production during a plant's life cycle, (2) decomposition of plant residues leading to CO₂ production, and (3) stabilization and retention of undecomposed plant residues and microbial by-products in the resistant soil C fraction. Experimental plant materials included transgenic near isolines of Zea mays L. and cell wall mutants of Arabidopsis thaliana, selected for their diverse residue chemistry. Phenology, morphology and above-ground biomass affected soil respiration and N₂O production in root-associated soils. Mineralization of C and N from incubated plant-soil mixtures was complemented with stable isotope tracing (¹³C, ¹⁵N) and ¹³C-phospholipid fatty acid analysis. Advanced chemical techniques such as nuclear magnetic resonance spectroscopy and physical separation (particle size and density separation) were used to track the transformations of plant C into stable soil C compounds. Conceptual models were proposed to explain how the plant residue chemistry×soil physico-chemical interaction affects C sequestration. Incorporating single gene mutations affecting lignin biosynthesis into agricultural and bioenergy crops has the potential to alter short- and long-term C cycling in agroecosystems.

Dahal, K., Weraduwage, S. M., Kane, K., Rauf, S. A., Leonardos, E. D., Gadapati, W., Savitch, L., Singh, J., Marillia, E.-F., Taylor, D. C., Micallef, M. C., Knowles, V., Plaxton, W., Barron, J., Sarhan, F., Hüner, N., Grodzinski, B. and Micallef, B. J. 2014. Enhancing biomass production and yield by maintaining enhanced capacity for CO₂ uptake in response to elevated CO₂. Can. J. Plant Sci. 94: 1075-1083. Using four model plants, two members of the Gramineae, rye and wheat, and two Brassicaceae, Brassica napus and Arabidopsis thaliana, two fundamental approaches were exploited to determine how regulating source-sink development would alter photosynthesis, productivity and yield during long-term acclimation to elevated CO_2. In one approach we exploited the cold acclimation response of winter wheat, rye and B. napus. In the other approach we modified the dark respiration in A. thaliana to alter availability of respiratory substrates required for anabolic processes, such as fatty acid metabolism, thus reducing sink limitations on canopy photosynthesis at elevated CO₂. Taken together, the data show the importance of maintaining strong demand from active sinks when the above-ground canopy is being exposed to elevated levels of the primary substrate of photosynthesis, CO₂.

Britto, D. T., Balkos, K. D., Becker, A., Coskun, D., Huynh, W. Q. and Kronzucker, H. J. 2014. Potassium and nitrogen poising: Physiological changes and biomass gains in rice and barley. Can. J. Plant Sci. 94: 1085-1089. Soil nitrogen, potassium, and water are three of the most important factors influencing, often interdependently, the growth of plants. Maximizing plant growth is not simply a matter of maximizing the availability of these and other nutrients; indeed, excess supply can be deleterious to plant performance. Rather, optimal performance may come about by adjusting the supply of each of the disparate factors required for plant growth, not only individually, but in relation to one another. In our work investigating the nutritional maximization of plant growth, we have found that altering the ratios of N and K provided to seedlings of cereal grasses can result in very substantial increases in vegetative biomass accrual, e.g., >220% of low-K controls, in short-term studies with rice, the world's most important cereal grain, and even greater gains in grain yield, in the longer term. Hence, the findings in our laboratory are of direct relevance to the aim of NSERC's Green Crop Network, which was to contribute to the amelioration of climate change by improvement of carbon capture and sequestration in crop plants. In addition, these findings may help to increase the world's food supply, the security of which is sometimes at odds with proposed means to thwart climate change. Our work in this area has also led to a potential breakthrough of a more fundamental sort in plant nutritional biology, which may in itself have important practical implications: evidence that aquaporin-type transport proteins conduct rapid NH₃ fluxes into roots at toxic levels of external ammonia/ammonium.

Wang, J., Cheung, M., Rasooli, L., Amirsadeghi, S. and Vanlerberghe, G. C. 2014. Plant respiration in a high CO₂ world: How will alternative oxidase respond to future atmospheric and climatic conditions? Can. J. Plant Sci. 94: 1091-1101. Plant mitochondria contain an alternative oxidase (AOX) that reduces the energy yield of respiration. While respiration and photosynthesis are known to interact, the role of AOX in the light remains poorly understood. This gap in our understanding of leaf metabolism extends to future conditions of high CO₂ and climate change. While studies indicate that AOX respiration is quite responsive to growth conditions, few studies have examined AOX respiration at high CO₂ and little is known regarding the combined impact of changes in both CO₂ and other climatic factors such as temperature and water availability. Given its non-energy conserving nature, a fundamental response by AOX to these future conditions could impact the net carbon gain that results from the combined processes of photosynthesis and respiration. Here, we show that leaf AOX protein amount in Nicotiana tabacum is dependent upon growth irradiance and CO₂ level, that AOX is subject to biochemical control by intermediates of photorespiration, and that photosynthesis is impacted in transgenic plants lacking AOX. We also review findings that tobacco AOX respiration is responsive to climatic variables (temperature, water availability), thus providing an excellent experimental system to investigate the interplay between AOX, photosynthesis at high CO₂, and climate change.

Javed, N., Tahir, M., Geng, J., Li, G. and McVetty, P. B. E. 2014. Identification of Brassica genotypes and molecular markers for increased seed oil content. Can. J. Plant Sci. 94: 1103-1108. Carbon dioxide emissions by the transportation sector are major contributors to global climate change. Lower CO₂ emissions by the transportation sector are linked to the use of renewable fuels including biodiesel. Canola has high seed oil content, adaptation to temperate climates and favorable fatty acid composition, which make it a preferred feedstock for biodiesel production. Doubled haploid (DH) line, random inbred (RI) line and consensus genetics maps for mapping populations derived from Polo × Topas were developed. The DH line-based genetic map was then used for the identification and tagging of quantitative trait loci (QTL) controlling seed oil biosynthesis. This genetic map consisted of 620 loci identified using several different types of molecular markers, and covered a map distance of 2241.1 cM with marker saturation of 3.7 cM. The phenotypic data on the mapping population for seed oil content and component fatty acids were collected from four-environment replicated field trials. One hundred and thirty-one QTL for various fatty acids in canola oil and 14 QTL for oil content were identified. These QTL, combined with marker-assisted selection, may assist breeders in their attempts to develop canola lines with improved oil quality, oil content and oil production per hectare for biodiesel production.

Katavic, V., Shi, L., Yu, Y., Zhao, L., Haughn, G. W. and Kunst, L. 2014. Investigation of the contribution of oil biosynthetic enzymes to seed oil content in Brassica napus and Arabidopsis thaliana. Can. J. Plant Sci. 94: 1109-1112. One of the critical reactions in triacylglycerol (TAG) biosynthesis is activation of fatty acyl chains to fatty acyl CoAs, catalyzed by long-chain acyl CoA synthetases (LACS). In Arabidopsis thaliana there is a family of nine genes that encode LACSs. Studies to determine whether the products of two of these genes, LACS8 and LACS9, function together to contribute acyl-CoAs for storage oil biosynthesis in A. thaliana resulted in discovery that it is not LACS8 but LACS1 that functionally overlaps with LACS9 in TAG biosynthesis (published in Plant Journal). To elucidate regulatory mechanisms of seed oil synthesis, the potential roles of phospholipase D zeta (PLDZ) and rhamnose synthase 2 (RHM2/MUM4) in transcription factor GLABRA2 (GL2)-mediated regulation of seed oil biosynthesis and deposition were investigated. Results demonstrated that PLDZ genes are not involved in GL2-mediated seed oil accumulation and that GL2 regulates seed oil production, at least in part, through its influence on expression of the gene RHM2/MUM4 required for the seed coat mucilage biosynthesis (published in Plant Journal). A novel Arabidopsis mutant with speckled seed coat and reduced seed oil phenotypes resulting from a mutation in a single unknown gene was identified, but attempts to isolate the gene by positional cloning have not been successful to date (unpublished results). Finally, seed oil content in near-isogenic double haploid Brassica napus lines was analyzed, “low oil” and “high oil” lines were identified, and developing seeds for expression profiling of target seed oil biosynthesis/bioassembly genes in selected double haploid lines were collected (unpublished results).

Vessey, J. K., Fei, H., Burton, D. L., Bradley, R. L. and Smith, D. L. 2014. The bilateral influence of plant and rhizosphere characteristics in brassicas varying in seed oil productivity. Can. J. Plant Sci. 94: 1113-1116. It is important that increasing seed oil yield in species of Brassica to improve the crops as biodiesel feedstocks does not result in unforeseen increases in greenhouse gas (GHG) emissions. Studies were conducted to determine if genotypes of Brassica napus and Arabidopsis thaliana varying in seed oil content (SOC) potential had differences in plant and rhizospheric characteristics that could impact GHG emissions. Varying SOC productivity in B. napus resulted in changes in C and N partitioning within the plant, and in some cases had effects on N₂O emission in the field. Although changes were observed in the composition of the rhizosphere of A. thaliana with modified SOC, there was also evidence that rhizospheric bacteria-to-plant signals could be used to improve growth and stress resistance in the plants. Project 4c in the Green Crop Network (GCN) investigated the possible ramifications of varying SOC on various plant growth, rhizospheric and agronomic characteristic of Brassica napus L. and Arabidopsis thaliana (L.) Heynh. The influence of certain bacteria-to-plant signals (i.e., lipo-chitooligosaccharides) was also investigated in these species. The rationale for these investigations was based on the fact that very little is known about how changing seed oil productivity in brassicas might affect other plants processes (e.g., C and N partitioning, root exudations, rhizospheric conditions) that might affect GHG emission from biodiesel feedstock crops designed specifically for maximized SOC.