Abscisic Acid-Dependent and -Independent Expression of the Carrot Late-Embryogenesis-Abundant-Class Gene Dc3 in Transgenic Tobacco Seedlings1

We studied the expression of three promoter 5′ deletion constructs (−218, −599, and −1312) of the LEA (late embryogenesis abundant)-class gene Dc3 fused to β-glucuronidase (GUS), where each construct value refers to the number of base pairs upstream of the transcription start site at which the deletion occurred. The Dc3 gene is noted for its induction by abscisic acid (ABA), but its response to other plant hormones and various environmental stresses has not been reported previously for vegetative cells. Fourteen-day-old transgenic tobacco (Nicotiana tabacum L.) seedlings were exposed to dehydration, hypoxia, salinity, exogenous ethylene, or exogenous methyl jasmonate (MeJa). GUS activity was quantified fluorimetrically and expression was observed by histochemical staining of the seedlings. An increase in GUS activity was observed in plants with constructs −599 and −1312 in response to dehydration and salinity within 6 h of stress, and at 12 h in response to hypoxia. No increase in endogenous ABA was found in any of the three lines, even after 72 h of hypoxia. An ABA-independent increase in GUS activity was observed when endogenous ABA biosynthesis was blocked by fluridone and plants were exposed to 5 μL L−1 ethylene in air or 100 μm MeJa. Virtually no expression was observed in construct −218 in response to dehydration, salinity, or MeJa, but there was a moderate response to ethylene and hypoxia. This suggests that the region between −218 and −599 is necessary for ABA (dehydration and salinity)- and MeJa-dependent expression, whereas ethylene-mediated expression does not require this region of the promoter.

Spermine Is a Salicylate-Independent Endogenous Inducer for Both Tobacco Acidic Pathogenesis-Related Proteins and Resistance against Tobacco Mosaic Virus Infection1

Intercellular spaces are often the first sites invaded by pathogens. In the spaces of tobacco mosaic virus (TMV)-infected and necrotic lesion-forming tobacco (Nicotiana tabacum L.) leaves, we found that an inducer for acidic pathogenesis-related (PR) proteins was accumulated. The induction activity was recovered in gel-filtrated fractions of low molecular mass with a basic nature, into which authentic spermine (Spm) was eluted. We quantified polyamines in the intercellular spaces of the necrotic lesion-forming leaves and found 20-fold higher levels of free Spm than in healthy leaves. Among several polyamines tested, exogenously supplied Spm induced acidic PR-1 gene expression. Immunoblot analysis showed that Spm treatment increased not only acidic PR-1 but also acidic PR-2, PR-3, and PR-5 protein accumulation. Treatment of healthy tobacco leaves with salicylic acid (SA) caused no significant increase in the level of endogenous Spm, and Spm did not increase the level of endogenous SA, suggesting that induction of acidic PR proteins by Spm is independent of SA. The size of TMV-induced local lesions was reduced by Spm treatment. These results indicate that Spm accumulates outside of cells after lesion formation and induces both acidic PR proteins and resistance against TMV via a SA-independent signaling pathway.

Comparison of Binding Properties and Early Biological Effects of Elicitins in Tobacco Cells1

Elicitins are a family of small proteins secreted by Phytophthora species that have a high degree of homology and elicit defense reactions in tobacco (Nicotiana tabacum). They display acidic or basic characteristics, the acidic elicitins being less efficient in inducing plant necrosis. In this study we compared the binding properties of four elicitins (two basic and two acidic) and early-induced signal transduction events (Ca2+ influx, extracellular medium alkalinization, and active oxygen species production). The affinity for tobacco plasma membrane-binding sites and the number of binding sites were similar for all four elicitins. Furthermore, elicitins compete with one another for binding sites, suggesting that they interact with the same receptor. The four elicitins induced Ca2+ influx, extracellular medium alkalinization, and the production of active oxygen species in tobacco cell suspensions, but the intensity and kinetics of these effects were different from one elicitin to another. As a general observation the concentrations that induce similar levels of biological activities were lower for basic elicitins (with the exception of cinnamomin-induced Ca2+ uptake). The qualitative similarity of early events induced by elicitins indicates a common transduction scheme, whereas fine signal transduction tuning is different in each elicitin.

Regulation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase by Carbamylation and 2-Carboxyarabinitol 1-Phosphate in Tobacco: Insights from Studies of Antisense Plants Containing Reduced Amounts of Rubisco Activase1

The regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity by 2-carboxyarabinitol 1-phosphate (CA1P) was investigated using gas-exchange analysis of antisense tobacco (Nicotiana tabacum) plants containing reduced levels of Rubisco activase. When an increase in light flux from darkness to 1200 μmol quanta m−2 s−1 was followed, the slow increase in CO2 assimilation by antisense leaves contained two phases: one represented the activation of the noncarbamylated form of Rubisco, which was described previously, and the other represented the activation of the CA1P-inhibited form of Rubisco. We present evidence supporting this conclusion, including the observation that this second phase, like CA1P, is only present following darkness or very low light flux. In addition, the second phase of CO2 assimilation was correlated with leaf CA1P content. When this novel phase was resolved from the CO2 assimilation trace, most of it was found to have kinetics similar to the activation of the noncarbamylated form of Rubisco. Additionally, kinetics of the novel phase indicated that the activation of the CA1P-inhibited form of Rubisco proceeds faster than the degradation of CA1P by CA1P phosphatase. These results may be significant with respect to current models of the regulation of Rubisco activity by Rubisco activase.

Molecular Characterization of Photomixotrophic Tobacco Cells Resistant to Protoporphyrinogen Oxidase-Inhibiting Herbicides1

Peroxidizing herbicides inhibit protoporphyrinogen oxidase (Protox), the last enzyme of the common branch of the chlorophyll- and heme-synthesis pathways. There are two isoenzymes of Protox, one of which is located in the plastid and the other in the mitochondria. Sequence analysis of the cloned Protox cDNAs showed that the deduced amino acid sequences of plastidial and mitochondrial Protox in wild-type cells and in herbicide-resistant YZI-1S cells are the same. The level of plastidial Protox mRNA was the same in both wild-type and YZI-1S cells, whereas the level of mitochondrial Protox mRNA YZI-1S cells was up to 10 times the level of wild-type cells. Wild-type cells were observed by fluorescence microscopy to emit strong autofluorescence from chlorophyll. Only a weak fluorescence signal was observed from chlorophyll in YZI-1S cells grown in the Protox inhibitor N-(4-chloro-2-fluoro-5-propagyloxy)-phenyl-3,4,5,6-tetrahydrophthalimide. Staining with DiOC6 showed no visible difference in the number or strength of fluorescence between wild-type and YZI-1S mitochondria. Electron micrography of YZI-1S cells showed that, in contrast to wild-type cells, the chloroplasts of YZI-1S cells grown in the presence of N-(4-chloro-2-fluoro-5-propagyloxy)-phenyl-3,4,5,6-tetrahydrophthalimide exhibited no grana stacking. These results suggest that the herbicide resistance of YZI-1S cells is due to the overproduction of mitochondrial Protox.

Plasma Membrane-Associated Actin in Bright Yellow 2 Tobacco Cells1 : Evidence for Interaction with Microtubules

Plasma membrane ghosts form when plant protoplasts attached to a substrate are lysed to leave a small patch of plasma membrane. We have identified several factors, including the use of a mildly acidic actin stabilization buffer and the inclusion of glutaraldehyde in the fixative, that allow immunofluorescent visualization of extensive cortical actin arrays retained on membrane ghosts made from tobacco (Nicotiana tabacum L.) suspension-cultured cells (line Bright Yellow 2). Normal microtubule arrays were also retained using these conditions. Membrane-associated actin is random; it exhibits only limited coalignment with the microtubules, and microtubule depolymerization in whole cells before wall digestion and ghost formation has little effect on actin retention. Actin and microtubules also exhibit different sensitivities to the pH and K+ and Ca2+ concentrations of the lysis buffer. There is, however, strong evidence for interactions between actin and the microtubules at or near the plasma membrane, because both ghosts and protoplasts prepared from taxol-pretreated cells have microtubules arranged in parallel arrays and an increased amount of actin coaligned with the microtubules. These experiments suggest that the organization of the cortical actin arrays may be dependent on the localization and organization of the microtubules.

Developmental Regulation of Intercellular Protein Trafficking through Plasmodesmata in Tobacco Leaf Epidermis1

Plasmodesmata mediate direct cell-to-cell communication in plants. One of their significant features is that primary plasmodesmata formed at the time of cytokinesis often undergo structural modifications, by the de novo addition of cytoplasmic strands across cell walls, to become complex secondary plasmodesmata during plant development. Whether such modifications allow plasmodesmata to gain special transport functions has been an outstanding issue in plant biology. Here we present data showing that the cucumber mosaic virus 3a movement protein (MP):green fluorescent protein (GFP) fusion was not targeted to primary plasmodesmata in the epidermis of young or mature leaves in transgenic tobacco (Nicotiana tabacum) plants constitutively expressing the 3a:GFP fusion gene. Furthermore, the cucumber mosaic virus 3a MP:GFP fusion protein produced in planta by biolistic bombardment of the 3a:GFP fusion gene did not traffic between cells interconnected by primary plasmodesmata in the epidermis of a young leaf. In contrast, the 3a MP:GFP was targeted to complex secondary plasmodesmata and trafficked from cell to cell when a leaf reached a certain developmental stage. These data provide the first experimental evidence, to our knowledge, that primary and complex secondary plasmodesmata have different protein-trafficking functions and suggest that complex secondary plasmodesmata may be formed to traffic specific macromolecules that are important for certain stages of leaf development.

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.

Evidence That Auxin-Induced Growth of Tobacco Leaf Tissues Does Not Involve Cell Wall Acidification1

Interveinal strips (10 × 1.5 mm) excised from growing tobacco (Nicotiana tabacum L. cv Xanthi) leaves have an auxin-specific, epinastic growth response that is developmentally regulated and is not the result of ethylene induction (C.P. Keller, E. Van Volkenburgh [1997] Plant Physiol 113: 603–610). We report here that auxin (10 μm naphthalene acetic acid) treatment of strips does not result in plasma membrane hyperpolarization or detectable proton efflux. This result is in contrast to the expected responses elicited by 1 μm fusicoccin (FC) treatment, which in other systems mimics auxin growth promotion through stimulation of the plasma membrane H+-ATPase and resultant acid wall loosening; FC produced both hyperpolarization and proton efflux in leaf strips. FC-induced growth was much more inhibited by a strong neutral buffer than was auxin-induced growth. Measurements of the osmotic concentration of strips suggested that osmotic adjustment plays no role in the auxin-induced growth response. Although cell wall loosening of some form appears to be involved, taken together, our results suggest that auxin-induced growth stimulation of tobacco leaf strips results primarily from a mechanism not involving acid growth.

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.

Prenylcysteine α-Carboxyl Methyltransferase in Suspension-Cultured Tobacco Cells1

Isoprenylation is a posttranslational modification that is believed to be necessary, but not sufficient, for the efficient association of numerous eukaryotic cell proteins with membranes. Additional modifications have been shown to be required for proper intracellular targeting and function of certain isoprenylated proteins in mammalian and yeast cells. Although protein isoprenylation has been demonstrated in plants, postisoprenylation processing of plant proteins has not been described. Here we demonstrate that cultured tobacco (Nicotiana tabacum cv Bright Yellow-2) cells contain farnesylcysteine and geranylgeranylcysteine α-carboxyl methyltransferase activities with apparent Michaelis constants of 73 and 21 μm for N-acetyl-S-trans,trans-farnesyl-l-cysteine and N-acetyl-S-all-trans-geranylgeranyl-l-cysteine, respectively. Furthermore, competition analysis indicates that the same enzyme is responsible for both activities. These results suggest that α-carboxyl methylation is a step in the maturation of isoprenylated proteins in plants.

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.

Second Messengers Mediate Increases in Cytosolic Calcium in Tobacco Protoplasts

Addition of membrane-permeable cyclic GMP (cGMP) and cyclic AMP (cAMP) were shown to cause elevation of cytosolic Ca2+ concentration ([Ca2+]cyt) in tobacco (Nicotiana plumbaginofolia) protoplasts. Under the same conditions these cyclic nucleotides were shown to provoke a physiological swelling response in the protoplasts. Nonmembrane-permeable cAMP and cGMP were unable to trigger a detectable [Ca2+]cyt response. Cyclic-nucleotide-mediated elevations in [Ca2+]cyt involved both internal and external Ca2+ stores. Both cAMP- and cGMP-mediated [Ca2+]cyt elevations could be inhibited by the Ca2+-channel blocker verapamil. Addition of inhibitors of phosphodiesterases (isobutylmethylxanthine and zaprinast) and the adenylate cyclase agonist forskolin to the protoplasts (predicted to elevate in vivo cyclic-nucleotide concentrations) caused elevations in [Ca2+]cyt. Addition of the adenylate cyclase inhibitor 2′,5′-dideoxyadenosine before forskolin significantly inhibited the forskolin-induced [Ca2+]cyt elevation. Taken together, these data suggest that a potential communication point for cross-talk between signal transduction pathways using cyclic nucleotides in plants is at the level of Ca2+ signaling.

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.