Gregory J. Taylor

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.

Modulation of Citrate Metabolism Alters Aluminum Tolerance in Yeast and Transgenic Canola Overexpressing a Mitochondrial Citrate Synthase

Aluminum (Al) toxicity is a major constraint for crop production in acid soils, although crop cultivars vary in their tolerance to Al. We have investigated the potential role of citrate in mediating Al tolerance in Al-sensitive yeast (Saccharomyces cerevisiae; MMYO11) and canola (Brassica napus cv Westar). Yeast disruption mutants defective in genes encoding tricarboxylic acid cycle enzymes, both upstream (citrate synthase [CS]) and downstream (aconitase [ACO] and isocitrate dehydrogenase [IDH]) of citrate, showed altered levels of Al tolerance. A triple mutant of CS (Δcit123) showed lower levels of citrate accumulation and reduced Al tolerance, whereas Δaco1- and Δidh12-deficient mutants showed higher accumulation of citrate and increased levels of Al tolerance. Overexpression of a mitochondrial CS (CIT1) in MMYO11 resulted in a 2- to 3-fold increase in citrate levels, and the transformants showed enhanced Al tolerance. A gene for Arabidopsis mitochondrial CS was overexpressed in canola using an Agrobacterium tumefaciens-mediated system. Increased levels of CS gene expression and enhanced CS activity were observed in transgenic lines compared with the wild type. Root growth experiments revealed that transgenic lines have enhanced levels of Al tolerance. The transgenic lines showed enhanced levels of cellular shoot citrate and a 2-fold increase in citrate exudation when exposed to 150 μm Al. Our work with yeast and transgenic canola clearly suggest that modulation of different enzymes involved in citrate synthesis and turnover (malate dehydrogenase, CS, ACO, and IDH) could be considered as potential targets of gene manipulation to understand the role of citrate metabolism in mediating Al tolerance.

Aluminum Resistance in Triticum aestivum Associated with Enhanced Exudation of Malate

Summary Two aluminum (Al)-resistant (Atlas 66, Maringa) and two Al-sensitive (Roblin, Katepwa) cultivars of Triticum aestivum (wheat) were grown under aseptic conditions in the presence and absence of Al to evaluate the potential role of organic anion exudates in conferring resistance to Al. Five organic anions, α-ketoglutarate, citrate, malate, succinate and fumarate, were commonly detected in the root exudates, but only malate and succinate were consistently exuded in all cultivars under all treatments. Under control conditions, malate was exuded in higher quantities from roots of Al-resistant cultivars (Atlas 66 and Maringa), compared with the Al-sensitive cultivars. Exposure to 100 μM Al increased exudation of malate from roots of Al-resistant cultivars by 100–120 %, while in the Al-sensitive cultivars, exudation of malate was reduced. A decrease in exudation of succinate was observed in Atlas 66 and Maringa with 100 μM Al, while no effect was observed in Roblin and Katepwa. Differences between cultivars in the effect of Al on malate accumulation were detected as early as 24 h after exposure. Addition of exogenous malate (250 μM to 500 μM) to nutrient media containing 100 μM Al restored root elongation in Al-sensitive cultivars, Roblin and Katepwa, to control levels. To determine whether exudation of malate from roots reflected de novo synthesis arising from activity of the TCA cycle, plants were labeled with 14 C-acetate. With the exception of acetate itself, malate was the only organic anion in which 14 C was detected. In Al-resistant cultivars, treatment with Al increased exudation of 14 C into malate by 48 to 54 % when expressed as a percent of total label in root exudates. In Al-sensitive cultivars, incorporation of 14 C into malate declined by 22 to 29 % with exposure to Al. The unique pattern of 14 C labeling and enhanced exudation of malate in the Al-resistant cultivars, Atlas 66 and Maringa, provides strong although indirect evidence for a role of malate in Al-resistance.

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.

Overcoming barriers to understanding the cellular basis of aluminium resistance

There appears to be an emerging consensus that resistance to aluminium (Al) is mediated at the cellular level. Virtually all current hypotheses which seek to explain the basis of Al resistance have a cellular focus, including those which postulate that external mechanisms limit the rate of Al entry across the membrane and/or protect sensitive extracellular sites, as well as those which postulate that internal mechanisms detoxify Al in the cytoplasm. If Al resistance is a cellular phenomenon, it should be expressed in single cells. Attempts to demonstrate resistance in cell culture systems, however, have not been uniformly satisfying. Considerable uncertainty has arisen from use of experimental conditions which favour formation of insoluble or non-toxic Al species. This problem has plagued research which has attempted to select for Al resistance in cell culture systems, as well as research which has attempted to express existing patterns of differential resistance in cell culture systems. Despite technical problems such as this, work at the cellular level has provided some important contributions. Most importantly, we now know resistance to Al can be expressed at the cellular level. We have discovered also that plant cells accumulate Al much more rapidly in cell culture systems than in intact roots and that isolated cells are more sensitive to Al than complex tissues. While this type of research is still hampered by a number of technical barriers, it would appear that more rapid progress could be achieved if greater emphasis was placed on true “experimental” work. Furthermore, we need to begin evaluating experimental data in the context of an integrated Al stress response if we are to achieve a full understanding of the cellular basis of Al resistance.

Characterization of 1,3-β-D-Glucan (Callose) Synthesis in Roots of Triticum aestivum in Response to Aluminum Toxicity

Summary To develop a sensitive marker for aluminum (Al) toxicity, synthesis of 1,3-β-D-glucan (callose) in 5-mm root tips of Triticum aestivum was examined using spectrofluorometry. In a time course study where an Al-sensitive cultivar, Neepawa, was exposed to 75 µM Al, a rapid phase of callose synthesis was observed in the first 6–12 h, followed by a slower linear phase with no saturation up to 48 h. Treatment with Al increased accumulation of callose by 86% within 30 min and by 3821% after 48 h. In experiments comparing genotypes, more callose accumulated in roots of Al-sensitive cultivars than in Al-resistant cultivars/lines after 2-h and 24-h exposure. Accumulation of callose increased at concentrations as low as 5 µM Al and continued to increase with saturation occurring between 250 and 500 M Al. Callose synthesis was negatively correlated with root growth. An Al-resistant experimental line, PT 741, and an Al-sensitive cultivar, Neepawa, accumulated callose to a similar extent when faced with concentrations of Al that produced equal reductions in root growth. Because accumulation of callose reflects cumulative exposure to Al, a second technique based upon labeling of callose with [ 14 C]glucose was developed to measure current synthesis. Treatment with Al increased incorporation of 14 C into laminarinase (a 1,3(1,3;1,4)-β-D-glucan 3(4)-glucanohydrolase, EC 3.2.1.6) digestion products from cell-wall material isolated from both the Al-sensitive Neepawa and Al-resistant PT 741. Greater incorporation of 14 C was observed in Neepawa over a broad range of Al concentrations (0-250 µM). Results of both spectrofluorometric and 14 C labeling studies support the use of callose synthesis as a rapid and sensitive marker for Al-induced injury.

Cadmium uptake and translocation in seedlings of near isogenic lines of durum wheat that differ in grain cadmium accumulation

BackgroundCadmium (Cd) concentrations in durum wheat (Triticum turgidum L. var durum) grain grown in North American prairie soils often exceed proposed international trade standards. To understand the physiological processes responsible for elevated Cd accumulation in shoots and grain, Cd uptake and translocation were studied in seedlings of a pair of near-isogenic durum wheat lines, high and low for Cd accumulation in grain.ResultsIn short-term studies (<3 h) using 109Cd-labelled nutrient solutions, there were no differences between lines in time- or concentration-dependent 109Cd accumulation by roots. In contrast, rates of 109Cd translocation from roots to shoots following longer exposure (48–60 h) were 1.8-fold higher in the high Cd-accumulating line, despite equal whole-plant 109Cd accumulation in the lines. Over the same period, the 109Cd concentration in root-pressure xylem exudates was 1.7 to 1.9-fold higher in the high Cd-accumulating line. There were no differences between the lines in 65Zn accumulation or partitioning that could account for the difference between lines in 109Cd translocation.ConclusionThese results suggest that restricted root-to-shoot Cd translocation may limit Cd accumulation in durum wheat grain by directly controlling Cd translocation from roots during grain filling, or by controlling the size of shoot Cd pools that can be remobilised to the grain.

Amino Acid Polymorphisms in Strictly Conserved Domains of a P-Type ATPase HMA5 Are Involved in the Mechanism of Copper Tolerance Variation in Arabidopsis

Copper (Cu) is an essential element in plant nutrition, but it inhibits the growth of roots at low concentrations. Accessions of Arabidopsis (Arabidopsis thaliana) vary in their tolerance to Cu. To understand the molecular mechanism of Cu tolerance in Arabidopsis, we performed quantitative trait locus (QTL) analysis and accession studies. One major QTL on chromosome 1 (QTL1) explained 52% of the phenotypic variation in Cu tolerance in roots in a Landsberg erecta/Cape Verde Islands (Ler/Cvi) recombinant inbred population. This QTL regulates Cu translocation capacity and involves a Cu-transporting P1B-1-type ATPase, HMA5. The Cvi allele carries two amino acid substitutions in comparison with the Ler allele and is less functional than the Ler allele in Cu tolerance when judged by complementation assays using a T-DNA insertion mutant. Complementation assays of the ccc2 mutant of yeast using chimeric HMA5 proteins revealed that N923T of the Cvi allele, which was identified in the tightly conserved domain N(x)6YN(x)4P (where the former asparagine was substituted by threonine), is a cause of dysfunction of the Cvi HMA5 allele. Another dysfunctional HMA5 allele was identified in Chisdra-2, which showed Cu sensitivity and low capacity of Cu translocation from roots to shoots. A unique amino acid substitution of Chisdra-2 was identified in another strictly conserved domain, CPC(x)6P, where the latter proline was replaced with leucine. These results indicate that a portion of the variation in Cu tolerance of Arabidopsis is regulated by the functional integrity of the Cu-translocating ATPase, HMA5, and in particular the amino acid sequence in several strictly conserved motifs.

Induction of microsomal membrane proteins in roots of an aluminum-resistant cultivar of Triticum aestivum L. under conditions of aluminum stress

Three-day-old seedlings of an Al-sensitive (Neepawa) and an Al-resistant (PT741) cultivar of Triticum aestivum were subjected to Al concentrations ranging from 0 to 100 [mu]M for 72 h. At 25 [mu]M Al, growth of roots was inhibited by 57% in the Al-sensitive cultivar, whereas root growth in the Al-resistant cultivar was unaffected. A concentration of 100 [mu]M Al was required to inhibit root growth of the Al-resistant cultivar by 50% and resulted in almost total inhibition of root growth in the sensitive cultivar. Cytoplasmic and microsomal membrane fractions were isolated from root tips (first 5 mm) and the adjacent 2-cm region of roots of both cultivars. When root cytoplasmic proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, no changes in polypeptide patterns were observed in response to Al stress. Analysis of microsomal membrane proteins revealed a band with an apparent molecular mass of 51 kD, which showed significant accumulation in the resistant cultivar following Al exposure. Two-dimensional gel analysis revealed that this band comprises two polypeptides, each of which is induced by exposure to Al. The response of the 51-kD band to a variety of experimental conditions was characterized to determine whether its pattern of accumulation was consistent with a possible role in Al resistance. Accumulation was significantly greater in root tips when compared to the rest of the root. When seedlings were subjected to Al concentrations ranging from 0 to 150 [mu]M, the proteins were evident at 25 [mu]M and were fully accumulated at 100 [mu]M. Time-course studies from 0 to 96 h indicated that full accumulation of the 51-kD band occurred within 24 h of initiation of Al stress. With subsequent removal of stress, the polypeptides gradually disappeared and were no longer visible after 72 h. When protein synthesis was inhibited by cycloheximide, the 51-kD band disappeared even when seedlings were maintained in Al-containing media. Other metals, including Cu, Zn, and Mn, failed to induce this band, and Cd and Ni resulted in its partial accumulation. These results indicate that synthesis of the 51-kD microsomal membrane proteins is specifically induced and maintained during Al stress in the Al-resistant cultivar, PT741.

Exclusion of metals from the symplasm: A possible mechanism of metal tolerance in higher plants

Abstract Hypotheses concerning the tolerance of plants to metal stress have been grouped into two classes, internal mechanisms where metals enter the symplasm but are subsequently rendered harmless, and exclusion mechanisms where tolerance is based on the plants ability to prevent entry of metals into the symplam. While most authors accept that metal tolerance can be achieved through exclusion, the role of exclusion in tolerance has been downplayed in the literature. This may be due to the lack of a clear definition of exclusion as a phenomenon, and to the popularity of new emerging hypotheses such as complexation by chelatins or metallothionein‐like proteins. Despite these problems, data which support the role of exclusion in metal tolerance nave been published. Possible mechanisms might include: 1) immobilization of metals at the cell wall, 2) complexation of metals by chelates exuded from plant roots, 3) formation of a redox barrier at the plasma membrane, and 4) formation of a pH barrier at the plasm...