Allen G. Good

Pollen flow between herbicide-resistant Brassica napus is the cause of multiple-resistant B. napus volunteers1

Abstract A field in which Brassica napus volunteers were not controlled by several applications of glyphosate was investigated in 1998. This field had been planted with glufosinate-resistant and imidazolinone-resistant B. napus in 1997 and was adjacent to a field that had grown glyphosate-resistant B. napus. Mature volunteer B. napus were collected on a 50- by 100-m grid in the field. Progeny from 34 volunteers were sprayed with glyphosate at 440 g ae ha−1, and the survivors were sprayed with either glufosinate or imazethapyr at 400 or 50 g ai ha−1, respectively. Where seed numbers permitted (14 volunteers), seedlings were also sprayed sequentially with glyphosate, glufosinate, and imazethapyr, at 440 g ae ha−1, 400 g ai ha−1, and 50 g ai ha−1, respectively. In total, 15 volunteers had progeny that were between 66 and 82% resistant to glyphosate, consistent with the predicted 3:1 resistant : susceptible ratio. Volunteer B. napus plants with glyphosate-resistant seedlings were most common close to the putative pollen source; however, a plant with glyphosate-resistant progeny was collected 500 m from the adjacent field edge. Seedlings from all nine volunteers collected from the glufosinate-resistant area showed multiple resistance to glyphosate and glufosinate, whereas seedlings from 10 of 20 volunteers collected from the imidazolinone-resistant area showed resistance to imazethapyr and glyphosate. DNA extraction and restriction fragment length polymorphism (RFLP) analysis of seedlings confirmed that mature B. napus volunteers were hybrids resulting from pollen transfer rather than inadvertent seed movement between fields. Two seedlings from the 924 screened were resistant to all three herbicides. Progeny from these self-pollinated individuals were resistant to glyphosate and glufosinate at the predicted 3:1 resistant : susceptible ratio and resistant to imazethapyr at the predicted 15:1 resistant : susceptible ratio. Sequential crossing of three herbicide-resistant varieties is the most likely explanation for the observed multiple herbicide resistance. Integrated management techniques, including suitable crop and herbicide rotations, herbicide mixtures, and nonchemical controls should be used to reduce the incidence and negative effect of B. napus volunteers with multiple herbicide resistance. Nomenclature:Brassica napus L., oil-seed rape; ALS, acetolactate synthase; EPSP, enolpyruvylshikimate-3-phosphate; glufosinate; glyphosate; imazethapyr.

Engineering nitrogen use efficiency with alanine aminotransferase

Nitrogen (N) is the most important factor limiting crop productivity worldwide. The ability of plants to acquire N from applied fertilizers is one of the critical steps limiting the efficient use of nitrogen. To improve N use efficiency, genetically modified plants that overexpress alanine aminotransferase (AlaAT) were engineered by introducing a barley AlaAT cDNA driven by a canola root specific promoter (btg26). Compared with wild-type canola, transgenic plants had in- creased biomass and seed yield both in the laboratory and field under low N conditions, whereas no differences were ob- served under high N. The transgenics also had increased nitrate influx. These changes resulted in a 40% decrease in the amount of applied nitrogen fertilizer required under field conditions to achieve yields equivalent to wild-type plants.

Genetic engineering of improved nitrogen use efficiency in rice by the tissue‐specific expression of alanine aminotransferase

Summary Nitrogen is quantitatively the most essential nutrient for plants and a major factor limiting crop productivity. One of the critical steps limiting the efficient use of nitrogen is the ability of plants to acquire it from applied fertilizer. Therefore, the development of crop plants that absorb and use nitrogen more efficiently has been a long-term goal of agricultural research. In an attempt to develop nitrogen-efficient plants, rice (Oryza sativa L.) was genetically engineered by introducing a barley AlaAT (alanine aminotransferase) cDNA driven by a rice tissue-specific promoter (OsAnt1). This modification increased the biomass and grain yield significantly in comparison with control plants when plants were well supplied with nitrogen. Compared with controls, transgenic rice plants also demonstrated significant changes in key metabolites and total nitrogen content, indicating increased nitrogen uptake efficiency. The development of crop plants that take up and assimilate nitrogen more efficiently would not only improve the use of nitrogen fertilizers, resulting in lower production costs, but would also have significant environmental benefits. These results are discussed in terms of their relevance to the development of strategies to engineer enhanced nitrogen use efficiency in crop plants.

Enhanced low oxygen survival in Arabidopsis through increased metabolic flux in the fermentative pathway.

We manipulated the enzyme activity levels of the alcohol fermentation pathway, pyruvate decarboxylase (PDC), and alcohol dehydrogenase (ADH) in Arabidopsis using sense and antisense overexpression of the corresponding genes (PDC1, PDC2, and ADH1). Transgenic plants were analyzed for levels of fermentation and evaluated for changes in hypoxic survival. Overexpression of either Arabidopsis PDC1 or PDC2 resulted in improved plant survival. In contrast, overexpression of Arabidopsis ADH1 had no effect on flooding survival. These results support the role of PDC as the control step in ethanol fermentation. Although ADH1 null mutants had decreased hypoxic survival, attempts to reduce the level of PDC activity enough to see an effect on plant survival met with limited success. The combination of flooding survival data and metabolite analysis allows identification of critical metabolic flux points. This information can be used to design transgenic strategies to improve hypoxic tolerance in plants.

Fertilizing Nature: A Tragedy of Excess in the Commons

Why has nitrogen fertilizer use declined in some countries while increasing in others, despite significant environmental harm? Proper crop management strategies offer environmental and economic benefits without sacrificing yields.

Large-scale Identification of Tubulin-binding Proteins Provides Insight on Subcellular Trafficking, Metabolic Channeling, and Signaling in Plant Cells

Microtubules play an essential role in the growth and development of plants and are known to be involved in regulating many cellular processes ranging from translation to signaling. In this article, we describe the proteomic characterization of Arabidopsis tubulin-binding proteins that were purified using tubulin affinity chromatography. Microtubule co-sedimentation assays indicated that most, if not all, of the proteins in the tubulin-binding protein fraction possessed microtubule-binding activity. Two-dimensional gel electrophoresis of the tubulin-binding protein fraction was performed, and 86 protein spots were excised and analyzed for protein identification. A total of 122 proteins were identified with high confidence using LC-MS/MS. These proteins were grouped into six categories based on their predicted functions: microtubule-associated proteins, translation factors, RNA-binding proteins, signaling proteins, metabolic enzymes, and proteins with other functions. Almost one-half of the proteins identified in this fraction were related to proteins that have previously been reported to interact with microtubules. This study represents the first large-scale proteomic identification of eukaryotic cytoskeleton-binding proteins, and provides insight on subcellular trafficking, metabolic channeling, and signaling in plant cells.

Long-Term Anaerobic Metabolism in Root Tissue' Metabolic Products of Pyruvate Metabolism

The onset of anaerobiosis in barley root tissue (Hordeum vulgare L. cv Himalaya) results in the following metabolic responses. There are rapid increases in the levels of pyruvate, lactate, and ethanol. Malate and succinate concentrations increase over the first 12 h, after which they return to the levels found in oxygenated root tissue. Alanine concentration increases over the first 12 h, and this is matched by a corresponding decrease in aspartate. The initial stoichiometric decline in aspartate and increase in alanine suggests that the amino group of aspartate is conserved by transaminating pyruvate to alanine. Aspartate catabolism also probably provides the initial source of carbon for reduction to succinate under anoxic conditions. Under long-term anaerobiosis (>24 h), there is no further accumulation of any of the fermentative end products other than ethanol, which also represents the major metabolic end product during long-term anaerobiosis. Although a number of the enzymes involved in fermentative respiration have been found to be induced under anaerobic conditions, neither aspartate amino-transferase nor malate dehydrogenase is induced in barley root tissue. The observations suggest that the long-term adaptations to hypoxic conditions may be quite different than the more well-characterized short-term adaptations.

NAD(H)-dependent glutamate dehydrogenase is essential for the survival of Arabidopsis thaliana during dark-induced carbon starvation.

Interconversion between glutamate and 2-oxoglutarate, which can be catalysed by glutamate dehydrogenase (GDH), is a key reaction in plant carbon (C) and nitrogen (N) metabolism. However, the physiological role of plant GDH has been a controversial issue for several decades. To elucidate the function of GDH, the expression of GDH in various tissues of Arabidopsis thaliana was studied. Results suggested that the expression of two Arabidopsis GDH genes was differently regulated depending on the organ/tissue types and cellular C availability. Moreover, Arabidopsis mutants defective in GDH genes were identified and characterized. The two isolated mutants, gdh1-2 and gdh2-1, were crossed to make a double knockout mutant, gdh1-2/gdh2-1, which contained negligible levels of NAD(H)-dependent GDH activity. Phenotypic analysis on these mutants revealed an increased susceptibility of gdh1-2/gdh2-1 plants to C-deficient conditions. This conditional phenotype of the double knockout mutant supports the catabolic role of GDH and its role in fuelling the TCA cycle during C starvation. The reduced rate of glutamate catabolism in the gdh2-1 and gdh1-2/gdh2-1 plants was also evident by the growth retardation of these mutants when glutamate was supplied as the alternative N source. Furthermore, amino acid profiles during prolonged dark conditions were significantly different between WT and the gdh mutant plants. For instance, glutamate levels increased in WT plants but decreased in gdh1-2/gdh2-1 plants, and aberrant accumulation of several amino acids was detected in the gdh1-2/gdh2-1 plants. These results suggest that GDH plays a central role in amino acid breakdown under C-deficient conditions.

Future Prospects for Cereals That Fix Nitrogen

What are the next steps in engineering crop plants that fix nitrogen and make their own fertilizer? Nitrogen availability is limiting to plant growth and has long been overcome through applications of synthetic nitrogen-rich fertilizer. This has revolutionized crop yield and food production worldwide, but at substantial economic and environmental cost (fixed N2; a

Molecular cloning and expression of a turgor-responsive gene in Brassica napus

100 billion per year global industry and environmental nitrogen pollution). Indeed, in April, the Edinburgh Declaration on Reactive Nitrogen was the most recent call for global action to address this source of nitrogen pollution. At the same time, the Bill and Melinda Gates Foundation convened a small meeting of international researchers to assess the way forward in reducing dependence on fertilizers through engineering crop plants that “fix” nitrogen themselves to sustain their growth and yield. The discussions of recent advances indicate that there are indeed viable options to achieve this goal.