Cloning and characterization of a plastidal and a mitochondrial isoform of tobacco protoporphyrinogen IX oxidase

Protoporphyrinogen IX oxidase is the last enzyme in the common pathway of heme and chlorophyll synthesis and provides precursor for the mitochondrial and plastidic heme synthesis and the predominant chlorophyll synthesis in plastids. We cloned two different, full-length tobacco cDNA sequences by complementation of the protoporphyrin-IX-accumulating Escherichia coli hemG mutant from heme auxotrophy. The two sequences show similarity to the recently published Arabidopsis PPOX, Bacillus subtilis hemY, and to mammalian sequences encoding protoporphyrinogen IX oxidase. One cDNA sequence encodes a 548-amino acid residues protein with a putative transit sequence of 50 amino acid residues, and the second cDNA encodes a protein of 504 amino acid residues. Both deduced protein sequences share 27.2% identical amino acid residues. The first in vitro translated protoporphyrinogen IX oxidase could be translocated to plastids, and the approximately 53-kDa mature protein was detected in stroma and membrane fraction. The second enzyme was targeted to mitochondria without any detectable reduction in size. Localization of both enzymes in subcellular fractions was immunologically confirmed. Steady-state RNA analysis indicates an almost synchronous expression of both genes during tobacco plant development, greening of young seedlings, and diurnal and circadian growth. The mature plastidal and the mitochondrial isoenzyme were overexpressed in E. coli. Bacterial extracts containing the recombinant mitochondrial enzyme exhibit high protoporphyrinogen IX oxidase activity relative to control strains, whereas the plastidal enzyme could only be expressed as an inactive peptide. The data presented confirm a compartmentalized pathway of tetrapyrrole synthesis with protoporphyrinogen IX oxidase in plastids and mitochondria.

Developmental changes in DNA methylation of the two tobacco pollen nuclei during maturation

Changes in DNA methylation during tobacco pollen development have been studied by confocal fluorescence microscopy using a monoclonal anti-5-methylcytosine (anti-m5C) antibody and a polyclonal anti-histone H1 (anti-histone) antibody as an internal standard. The specificity of the anti-m5C antibody was demonstrated by a titration series against both single-stranded DNA and double-stranded DNA substrates in either the methylated or unmethylated forms. The antibody was found to show similar kinetics against both double- and single-stranded DNA, and the fluorescence was proportional to the amount of DNA used. No signal was observed with unmethylated substrates. The extent of methylation of the two pollen nuclei remained approximately constant after the mitotic division that gave rise to the vegetative and generative nuclei. However, during the subsequent development of the pollen, the staining of the generative nucleus decreased until it reached a normalized value of \documentclass[12pt]{minimal} \usepackage{wasysym} \usepackage{amsmath} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}\frac{1}{5}\end{equation*}\end{document} of that of the vegetative nucleus. The use of a confocal microscope makes these data independent of possible focusing artefacts. The anti-histone antibody was used as a control to show that, while the antibody staining directed against 5-methylcytosine changed dramatically during pollen maturation, the histone signal did not. We observed the existence of structural dimorphism amongst tobacco pollen grains, the majority having three pollen apertures and the rest with four. However, the methylation changes observed occurred to the same extent in both subclasses.

Transgenic tobacco plants with reduced capability to detoxify reactive oxygen intermediates are hyperresponsive to pathogen infection

Reactive oxygen intermediates (ROI) play a critical role in the defense of plants against invading pathogens. Produced during the “oxidative burst,” they are thought to activate programmed cell death (PCD) and induce antimicrobial defenses such as pathogenesis-related proteins. It was shown recently that during the interaction of plants with pathogens, the expression of ROI-detoxifying enzymes such as ascorbate peroxidase (APX) and catalase (CAT) is suppressed. It was suggested that this suppression, occurring upon pathogen recognition and coinciding with an enhanced rate of ROI production, plays a key role in elevating cellular ROI levels, thereby potentiating the induction of PCD and other defenses. To examine the relationship between the suppression of antioxidative mechanisms and the induction of PCD and other defenses during pathogen attack, we studied the interaction between transgenic antisense tobacco plants with reduced APX or CAT and a bacterial pathogen that triggers the hypersensitive response. Transgenic plants with reduced capability to detoxify ROI (i.e., antisense APX or CAT) were found to be hyperresponsive to pathogen attack. They activated PCD in response to low amounts of pathogens that did not trigger the activation of PCD in control plants. Our findings support the hypothesis that suppression of ROI-scavenging enzymes during the hypersensitive response plays an important role in enhancing pathogen-induced PCD.

The incompatible interaction between Phytophthora parasitica var. nicotianae race 0 and tobacco is suppressed in transgenic plants expressing antisense lipoxygenase sequences

Nicotiana tabacum 46-8 cultivar displays an incompatible interaction with race 0 of Phytophthora parasitica var. nicotianae (Ppn), a fungal pathogen of most tobacco cultivars. At the plant level, incompatibility is characterized by the induction of lipoxygenase (LOX, EC = 1.13.11.12) activity and localized hypersensitive cell death before defense gene activation. To evaluate the involvement of LOX in the onset of plant defense, tobacco 46-8 plants were genetically engineered using full-length or partial-length antisense (AS) tobacco LOX cDNA constructs. AS expression strongly reduced elicitor- and pathogen-induced LOX activity. Eight independent AS-LOX lines were selected and assayed for their response to Ppn. After root or stem inoculation with race 0, all AS-LOX lines but one displayed a compatible phenotype whereas control transformed plants, not containing the AS-LOX cassette, showed the typical incompatible reaction. The presence of the fungus in transgenic lines was demonstrated by PCR amplification of a Ppn-specific genomic sequence. A linear relationship was found between the extent of LOX suppression and the size of the lesion caused by the fungus. The AS-LOX plants also showed enhanced susceptibility toward the compatible fungus Rhizoctonia solani. The results demonstrate the strong involvement of LOX in the establishment of incompatibility in plant–microorganism interactions, consistent with its role in the defense of host plants.

Transgenically Enhanced Sorbitol Synthesis Facilitates Phloem Boron Transport and Increases Tolerance of Tobacco to Boron Deficiency1

The mobility of elements within plants contributes to a plant species' tolerance of nutrient deficiencies in the soil. The genetic manipulation of within-plant nutrient movement may therefore provide a means to enhance plant growth under conditions of variable soil nutrient availability. In these experiments tobacco (Nicotiana tabacum) was engineered to synthesize sorbitol, and the resultant effect on phloem mobility of boron (B) was determined. In contrast to wild-type tobacco, transgenic tobacco plants containing sorbitol exhibit a marked increase in within-plant B mobility and a resultant increase in plant growth and yield when grown with limited or interrupted soil B supply. Growth of transgenic tobacco could be maintained by reutilization of B present in mature tissues or from B supplied as a foliar application to mature leaves. In contrast, B present in mature leaves of control tobacco lines could not be used to provide the B requirements for new plant growth. 10B-labeling experiments verified that B is phloem mobile in transgenic tobacco but is immobile in control lines. These results demonstrate that the transgenic enhancement of within-plant nutrient mobility is a viable approach to improve plant tolerance of nutrient stress.

Distinct Cyclin D Genes Show Mitotic Accumulation or Constant Levels of Transcripts in Tobacco Bright Yellow-2 Cells1

The commitment of eukaryotic cells to division normally occurs during the G1 phase of the cell cycle. In mammals D-type cyclins regulate the progression of cells through G1 and therefore are important for both proliferative and developmental controls. Plant CycDs (D-type cyclin homologs) have been identified, but their precise function during the plant cell cycle is unknown. We have isolated three tobacco (Nicotiana tabacum) CycD cyclin cDNAs: two belong to the CycD3 class (Nicta;CycD3;1 and Nicta;CycD3;2) and the third to the CycD2 class (Nicta;CycD2;1). To uncouple their cell-cycle regulation from developmental control, we have used the highly synchronizable tobacco cultivar Bright Yellow-2 in a cell-suspension culture to characterize changes in CycD transcript levels during the cell cycle. In cells re-entering the cell cycle from stationary phase, CycD3;2 was induced in G1 but subsequently remained at a constant level in synchronous cells. This expression pattern is consistent with a role for CycD3;2, similar to mammalian D-type cyclins. In contrast, CycD2;1 and CycD3;1 transcripts accumulated during mitosis in synchronous cells, a pattern of expression not normally associated with D-type cyclins. This could suggest a novel role for plant D-type cyclins during mitosis.

Overexpression of an Arabidopsis cDNA Encoding a Sterol-C241-Methyltransferase in Tobacco Modifies the Ratio of 24-Methyl Cholesterol to Sitosterol and Is Associated with Growth Reduction

Higher plants synthesize 24-methyl sterols and 24-ethyl sterols in defined proportions. As a first step in investigating the physiological function of this balance, an Arabidopsis cDNA encoding an S-adenosyl-l-methionine 24-methylene lophenol-C241-methyltransferase, the typical plant enzyme responsible for the production of 24-ethyl sterols, was expressed in tobacco (Nicotiana tabacum L.) under the control of a constitutive promoter. Transgenic plants displayed a novel 24-alkyl-Δ5-sterol profile: the ratio of 24-methyl cholesterol to sitosterol, which is close to 1 in the wild type, decreased dramatically to values ranging from 0.01 to 0.31. In succeeding generations of transgenic tobacco, a high S-adenosyl-l-methionine 24-methylene lophenol-C241-methyltransferase enzyme activity and, consequently, a low ratio of 24-methyl cholesterol to sitosterol, was associated with reduced growth compared with the wild type. However, this new morphological phenotype appeared only below the threshold ratio of 24-methyl cholesterol to sitosterol of approximately 0.1. Because the size of cells was unchanged in small, transgenic plants, we hypothesize that a radical decrease of 24-methyl cholesterol and/or a concomitant increase of sitosterol would be responsible for a change in cell division through as-yet unknown mechanisms.

Characterization of Transgenic Tobacco with an Increased α-Linolenic Acid Level1

Microsomal ω-3 fatty acid desaturase catalyzes the conversion of 18:2 (linoleic acid) to 18:3 (α-linolenic acid) in phospholipids, which are the main constituents of extrachloroplast membranes. Transgenic tobacco (Nicotiana tabacum) plants with increased 18:3 contents (designated SIIn plants) were produced through the introduction of a construct with the tobacco microsomal ω-3 fatty acid desaturase gene under the control of the highly efficient promoter containing the E12Ω sequence. 18:3 contents in the SIIn plants were increased by about 40% in roots and by about 10% in leaves compared with the control plants. With regard to growth at 15°C and 25°C and the ability to tolerate chilling at 1°C and 5°C, there were no discernible differences between the SIIn and the control plants. Freezing tolerance in leaves and roots, which was assessed by electrolyte leakage, was almost the same between the SIIn and the control plants. The fluidity of plasma membrane from the SIIn plants was almost the same as that of the control plants. These results indicate that an increase in the 18:3 level in phospholipids is not directly involved in compensation for the diminishment in growth or membrane properties observed under low temperatures.

Piperonylic Acid, a Selective, Mechanism-Based Inactivator of the trans-Cinnamate 4-Hydroxylase: A New Tool to Control the Flux of Metabolites in the Phenylpropanoid Pathway1

Piperonylic acid (PA) is a natural molecule bearing a methylenedioxy function that closely mimics the structure of trans-cinnamic acid. The CYP73A subfamily of plant P450s catalyzes trans-cinnamic acid 4-hydroxylation, the second step of the general phenylpropanoid pathway. We show that when incubated in vitro with yeast-expressed CYP73A1, PA behaves as a potent mechanism-based and quasi-irreversible inactivator of trans-cinnamate 4-hydroxylase. Inactivation requires NADPH, is time dependent and saturable (KI = 17 μm, kinact = 0.064 min−1), and results from the formation of a stable metabolite-P450 complex absorbing at 427 nm. The formation of this complex is reversible with substrate or other strong ligands of the enzyme. In plant microsomes PA seems to selectively inactivate the CYP73A P450 subpopulation. It does not form detectable complexes with other recombinant plant P450 enzymes. In vivo PA induces a sharp decrease in 4-coumaric acid concomitant to cinnamic acid accumulation in an elicited tobacco (Nicotiana tabacum) cell suspension. It also strongly decreases the formation of scopoletin in tobacco leaves infected with tobacco mosaic virus.

Expression of the Yeast FRE Genes in Transgenic Tobacco

Two yeast genes, FRE1 and FRE2 (encoding Fe(III) reductases) were placed under the control of the cauliflower mosaic virus 35S promoter and introduced into tobacco (Nicotiana tabacum L.) via Agrobacterium tumefaciens-mediated transformation. Homozygous lines containing FRE1, FRE2, or FRE1 plus FRE2 were generated. Northern-blot analyses revealed mRNA of two different sizes in FRE1 lines, whereas all FRE2 lines had mRNA only of the expected length. Fe(III) reduction, chlorophyll contents, and Fe levels were determined in transgenic and control plants under Fe-sufficient and Fe-deficient conditions. In a normal growth environment, the highest root Fe(III) reduction, 4-fold higher than in controls, occurred in the double transformant (FRE1 + FRE2). Elevated Fe(III) reduction was also observed in all FRE2 and some FRE1 lines. The increased Fe(III) reduction occurred along the entire length of the roots and on shoot sections. FRE2 and double transformants were more tolerant to Fe deficiency in hydroponic culture, as shown by higher chlorophyll and Fe concentrations in younger leaves, whereas FRE1 transformants did not differ from the controls. Overall, the beneficial effects of FRE2 were consistent, suggesting that FRE2 may be used to improve Fe efficiency in crop plants.

The H+-Sucrose Cotransporter NtSUT1 Is Essential for Sugar Export from Tobacco Leaves1

In many species translocation of sucrose from the mesophyll to the phloem is carrier mediated. A sucrose/H+-symporter cDNA, NtSUT1, was isolated from tobacco (Nicotiana tabacum) and shown to be highly expressed in mature leaves and at low levels in other tissues, including floral organs. To study the in vivo function of NtSUT1, tobacco plants were transformed with a SUT1 antisense construct under control of the cauliflower mosaic virus 35S promoter. Upon maturation, leaves of transformants expressing reduced amounts of SUT1 mRNA curled downward, and strongly affected plants developed chloroses and necroses that led to death. The leaves exhibited impaired ability to export recently fixed 14CO2 and were unable to export transient starch during extended periods of darkness. As a consequence, soluble carbohydrates accumulated and photosynthesis was reduced. Autoradiographs of leaves show a heterogenous pattern of CO2 fixation even after a 24-h chase. The 14C pattern does not change with time, suggesting that movement of photosynthate between mesophyll cells may also be impaired. The affected lines show a reduction in the development of the root system and delayed or impaired flowering. Taken together, the effects observed in a seed plant (tobacco) demonstrate the importance of SUT1 for sucrose loading into the phloem via an apoplastic route and possibly for intermesophyll transport as well.

Expression of Tobacco Carbonic Anhydrase in the C4 Dicot Flaveria bidentis Leads to Increased Leakiness of the Bundle Sheath and a Defective CO2-Concentrating Mechanism

Flaveria bidentis (L.) Kuntze, a C4 dicot, was genetically transformed with a construct encoding the mature form of tobacco (Nicotiana tabacum L.) carbonic anhydrase (CA) under the control of a strong constitutive promoter. Expression of the tobacco CA was detected in transformant whole-leaf and bundle-sheath cell (bsc) extracts by immunoblot analysis. Whole-leaf extracts from two CA-transformed lines demonstrated 10% to 50% more CA activity on a ribulose-1,5-bisphosphate carboxylase/oxygenase-site basis than the extracts from transformed, nonexpressing control plants, whereas 3 to 5 times more activity was measured in CA transformant bsc extracts. This increased CA activity resulted in plants with moderately reduced rates of CO2 assimilation (A) and an appreciable increase in C isotope discrimination compared with the controls. With increasing O2 concentrations up to 40% (v/v), a greater inhibition of A was found for transformants than for wild-type plants; however, the quantum yield of photosystem II did not differ appreciably between these two groups over the O2 levels tested. The quantum yield of photosystem II-to-A ratio suggested that at higher O2 concentrations, the transformants had increased rates of photorespiration. Thus, the expression of active tobacco CA in the cytosol of F. bidentis bsc and mesophyll cells perturbed the C4 CO2-concentrating mechanism by increasing the permeability of the bsc to inorganic C and, thereby, decreasing the availability of CO2 for photosynthetic assimilation by ribulose-1,5-bisphosphate carboxylase/oxygenase.

Effect of ATP Sulfurylase Overexpression in Bright Yellow 2 Tobacco Cells1 : Regulation of ATP Sulfurylase and SO42− Transport Activities

To determine if the ATP sulfurylase reaction is a regulatory step for the SO42−-assimilation pathway in plants, an Arabidopsis thaliana ATP sulfurylase cDNA, APS2, was fused to the 35S promoter of the cauliflower mosaic virus and introduced by Agrobacterium tumefaciens-mediated transformation into isolated Bright Yellow 2 tobacco (Nicotiana tabacum) cells. The ATP sulfurylase activity in transgenic cells was 8-fold that in control cells, and was correlated with the expression of a specific polypeptide revealed by western analysis using an anti-ATP sulfurylase antibody. The molecular mass of this polypeptide agreed with that for the overexpressed mature protein. ATP sulfurylase overexpression had no effect on [35S]SO42− influx or ATP sulfurylase activity regulation by S availability, except that ATP sulfurylase activity variations in response to S starvation in transgenic cells were 8 times higher than in the wild type. There were also no differences in cell growth or sensitivity to SeO42− (a toxic SO42− analog) between transgenic and wild-type cells. We propose that in Bright Yellow 2 tobacco cells, the ATP sulfurylase derepression by S deficiency may involve a posttranscriptional mechanism, and that the ATP sulfurylase abundance is not limiting for cell metabolism.

Controlled Cytokinin Production in Transgenic Tobacco Using a Copper-Inducible Promoter

The cytokinin group of plant hormones regulates aspects of plant growth and development, including the release of lateral buds from apical dominance and the delay of senescence. In this work the native promoter of a cytokinin synthase gene (ipt) was removed and replaced with a Cu-controllable promoter. Tobacco (Nicotiana tabacum L. cv tabacum) transformed with this Cu-inducible ipt gene (Cu-ipt) was morphologically identical to controls under noninductive conditions in almost all lines produced. However, three lines grew in an altered state, which is indicative of cytokinin overproduction and was confirmed by a full cytokinin analysis of one of these lines. The in vitro treatment of morphologically normal Cu-ipt transformants with Cu2+ resulted in delayed leaf senescence and an increase in cytokinin concentration in the one line analyzed. In vivo, inductive conditions resulted in a significant release of lateral buds from apical dominance. The morphological changes seen during these experiments may reflect the spatial aspect of control exerted by this gene expression system, namely expression from the root tissue only. These results confirmed that endogenous cytokinin concentrations in tobacco transformants can be temporally and spatially controlled by the induction of ipt gene expression through the Cu-controllable gene-expression system.