Characterization and Subcellular Compartmentation of Recombinant 4-Hydroxyphenylpyruvate Dioxygenase from Arabidopsis in Transgenic Tobacco1

4-Hydroxyphenylpyruvate dioxygenase (4HPPD) catalyzes the formation of homogentisate (2,5-dihydroxyphenylacetate) from p-hydroxyphenylpyruvate and molecular oxygen. In plants this enzyme activity is involved in two distinct metabolic processes, the biosynthesis of prenylquinones and the catabolism of tyrosine. We report here the molecular and biochemical characterization of an Arabidopsis 4HPPD and the compartmentation of the recombinant protein in chlorophyllous tissues. We isolated a 1508-bp cDNA with one large open reading frame of 1338 bp. Southern analysis strongly suggested that this Arabidopsis 4HPPD is encoded by a single-copy gene. We investigated the biochemical characteristics of this 4HPPD by overproducing the recombinant protein in Escherichia coli JM105. The subcellular localization of the recombinant 4HPPD in chlorophyllous tissues was examined by overexpressing its complete coding sequence in transgenic tobacco (Nicotiana tabacum), using Agrobacterium tumefaciens transformation. We performed western analyses for the immunodetection of protein extracts from purified chloroplasts and total leaf extracts and for the immunocytochemistry on tissue sections. These analyses clearly revealed that 4HPPD was confined to the cytosol compartment, not targeted to the chloroplast. Western analyses confirmed the presence of a cytosolic form of 4HPPD in cultured green Arabidopsis cells.

Decrease in Phosphoribulokinase Activity by Antisense RNA in Transgenic Tobacco. Relationship between Photosynthesis, Growth, and Allocation at Different Nitrogen Levels1

To study the direct effects of photosynthesis on allocation of biomass by altering photosynthesis without altering leaf N or nitrate content, phosphoribulokinase (PRK) activity was decreased in transgenic tobacco (Nicotiana tabacum L.) with an inverted tobacco PRK cDNA and plants were grown at different N levels (0.4 and 5 mm NH4NO3). The activation state of PRK increased as the amount of enzyme was decreased genetically at both levels of N. At high N a 94% decrease in PRK activity had only a small effect (20%) on photosynthesis and growth. At low N a 94% decrease in PRK activity had a greater effect on leaf photosynthesis (decreased by up to 50%) and whole-plant photosynthesis (decreased by up to 35%) than at high N. These plants were up to 35% smaller than plants with higher PRK activities because they had less structural dry matter and less starch, which was decreased by 3- to 4-fold, but still accumulated to 24% to 31% of dry weight; young leaves contained more starch than older leaves in older plants. Leaves had a higher ion and water content, and specific leaf area was higher, but allocation between shoot and root was unaltered. In conclusion, low N in addition to a 94% decrease in PRK by antisense reduces the activity of PRK sufficient to diminish photosynthesis, which limits biomass production under conditions normally considered sink limited.

Use of Ubiquitin Fusions to Augment Protein Expression in Transgenic Plants1

A major goal of plant biotechnology is the production of genetically engineered crops that express natural or foreign proteins at high levels. To enhance protein accumulation in transgenic plants, we developed a set of vectors that express proteins and peptides as C-terminal translational fusions with ubiquitin (UBQ). Studies of several proteins in tobacco (Nicotiana tabacum) showed that: (a) proteins can be readily expressed in plants as UBQ fusions; (b) by the action of endogenous UBQ-specific proteases (Ubps), these fusions are rapidly and precisely processed in vivo to release the fused protein moieties in free forms; (c) the synthesis of a protein as a UBQ fusion can significantly augment its accumulation; (d) proper processing and localization of a protein targeted to either the apoplast or the chloroplast is not affected by the N-terminal UBQ sequence; and (e) single amino acid substitutions surrounding the cleavage site can inhibit in vivo processing of the fusion by Ubps. Noncleavable UBQ fusions of β-glucuronidase became extensively modified, with additional UBQs in planta. Because multiubiquitinated proteins are the preferred substrates of the 26S proteasome, noncleavable fusions may be useful for decreasing protein half-life. Based on their ability to augment protein accumulation and the sequence specificity of Ubps, UBQ fusions offer a versatile way to express plant proteins.

Manipulation of in Vivo Sorbitol Production Alters Boron Uptake and Transport in Tobacco1

Recent evidence that some species can retranslocate boron as complexes with sugar alcohols in the phloem suggests a possible mechanism for enhancing boron efficiency. We investigated the relationship between sugar alcohol (sorbitol) content, boron uptake and distribution, and translocation of foliar-applied, isotopically enriched 10B in three lines of tobacco (Nicotiana tabacum) plants differing in sorbitol production. In tobacco line S11, transformed with sorbitol-6-phosphate dehydrogenase, the production of sorbitol was accompanied by an increase in the concentration of boron in plant tissues and an increased uptake of boron compared with either tobacco line A4, transformed with antisense orientation of sorbitol-6-phosphate dehydrogenase, or wild-type tobacco (line SR1, zero-sorbitol producer). Foliar application of 10B to mature leaves was translocated to the meristematic tissues only in line S11. These results demonstrate that the concentration of the boron-complexing sugar alcohol in the plant tissue has a significant effect on boron uptake and distribution in plants, whereas the translocation of the foliar-applied 10B from the mature leaves to the meristematic tissues verifies that boron is mobile in sorbitol-producing plants (S11) as we reported previously. This suggests that selection or transgenic generation of cultivars with an increased sugar alcohol content can result in increased boron uptake, with no apparent negative effects on short-term growth.

Dynamics of Cytokinins in Apical Shoot Meristems of a Day-Neutral Tobacco during Floral Transition and Flower Formation1

This study considered cytokinin distribution in tobacco (Nicotiana tabacum L.) shoot apices in distinct phases of development using immunocytochemistry and quantitative tandem mass spectrometry. In contrast to vegetative apices and flower buds, we detected no free cytokinin bases (zeatin, dihydrozeatin, or isopentenyladenine) in prefloral transition apices. We also observed a 3-fold decrease in the content of cytokinin ribosides (zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine) during this transition phase. The group concluded that organ formation (e.g. leaves and flowers) is characterized by enhanced cytokinin content, in contrast to the very low endogenous cytokinin levels found in prefloral transition apices, which showed no organogenesis. The immunocytochemical analyses revealed a differing intracellular localization of the cytokinin bases. Dihydrozeatin and isopentenyladenine were mainly cytoplasmic and perinuclear, whereas zeatin showed a clear-cut nuclear labeling. To our knowledge, this is the first time that this phenomenon has been reported. Cytokinins do not seem to act as positive effectors in the prefloral transition phase in tobacco shoot apices. Furthermore, the differences in distribution at the cellular level may be indicative of a specific physiological role of zeatin in nuclear processes.

Plastome Engineering of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Tobacco to Form a Sunflower Large Subunit and Tobacco Small Subunit Hybrid1

Targeted gene replacement in plastids was used to explore whether the rbcL gene that codes for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, the key enzyme of photosynthetic CO2 fixation, might be replaced with altered forms of the gene. Tobacco (Nicotiana tabacum) plants were transformed with plastid DNA that contained the rbcL gene from either sunflower (Helianthus annuus) or the cyanobacterium Synechococcus PCC6301, along with a selectable marker. Three stable lines of transformants were regenerated that had altered rbcL genes. Those containing the rbcL gene for cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase produced mRNA but no large subunit protein or enzyme activity. Those tobacco plants expressing the sunflower large subunit synthesized a catalytically active hybrid form of the enzyme composed of sunflower large subunits and tobacco small subunits. A third line expressed a chimeric sunflower/tobacco large subunit arising from homologous recombination within the rbcL gene that had properties similar to the hybrid enzyme. This study demonstrated the feasibility of using a binary system in which different forms of the rbcL gene are constructed in a bacterial host and then introduced into a vector for homologous recombination in transformed chloroplasts to produce an active, chimeric enzyme in vivo.

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.

A Conserved Acidic Motif in the N-Terminal Domain of Nitrate Reductase Is Necessary for the Inactivation of the Enzyme in the Dark by Phosphorylation and 14-3-3 Binding1

It has previously been shown that the N-terminal domain of tobacco (Nicotiana tabacum) nitrate reductase (NR) is involved in the inactivation of the enzyme by phosphorylation, which occurs in the dark (L. Nussaume, M. Vincentz, C. Meyer, J.P. Boutin, and M. Caboche [1995] Plant Cell 7: 611–621). The activity of a mutant NR protein lacking this N-terminal domain was no longer regulated by light-dark transitions. In this study smaller deletions were performed in the N-terminal domain of tobacco NR that removed protein motifs conserved among higher plant NRs. The resulting truncated NR-coding sequences were then fused to the cauliflower mosaic virus 35S RNA promoter and introduced in NR-deficient mutants of the closely related species Nicotiana plumbaginifolia. We found that the deletion of a conserved stretch of acidic residues led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivation by phosphorylation in the dark. Therefore, the removal of this acidic stretch seems to have the same effects on NR activation state as the deletion of the N-terminal domain. A hypothetical explanation for these observations is that a specific factor that impedes inactivation remains bound to the truncated enzyme. A synthetic peptide derived from this acidic protein motif was also found to be a good substrate for casein kinase II.

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.

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.

A Nitrilase-Like Protein Interacts with GCC Box DNA-Binding Proteins Involved in Ethylene and Defense Responses1

Ethylene-responsive element-binding proteins (EREBPs) of tobacco (Nicotiana tabacum L.) bind to the GCC box of many pathogenesis-related (PR) gene promoters, including osmotin (PR-5). The two GCC boxes on the osmotin promoter are known to be required, but not sufficient, for maximal ethylene responsiveness. EREBPs participate in the signal transduction pathway leading from exogenous ethylene application and pathogen infection to PR gene induction. In this study EREBP3 was used as bait in a yeast two-hybrid interaction trap with a tobacco cDNA library as prey to isolate signal transduction pathway intermediates that interact with EREBPs. One of the strongest interactors was found to encode a nitrilase-like protein (NLP). Nitrilase is an enzyme involved in auxin biosynthesis. NLP interacted with other EREBP family members, namely tobacco EREBP2 and tomato (Lycopersicon esculentum L.) Pti4/5/6. The EREBP2-EREBP3 interaction with NLP required part of the DNA-binding domain. The specificity of interaction was further confirmed by protein-binding studies in solution. We propose that the EREBP-NLP interaction serves to regulate PR gene expression by sequestration of EREBPs in the cytoplasm.

Acid-Growth Response and α-Expansins in Suspension Cultures of Bright Yellow 2 Tobacco1

The possibility that Bright Yellow 2 (BY2) tobacco (Nicotiana tabacum L.) suspension-cultured cells possess an expansin-mediated acid-growth mechanism was examined by multiple approaches. BY2 cells grew three times faster upon treatment with fusicoccin, which induces an acidification of the cell wall. Exogenous expansins likewise stimulated BY2 cell growth 3-fold. Protein extracted from BY2 cell walls possessed the expansin-like ability to induce extension of isolated walls. In western-blot analysis of BY2 wall protein, one band of 29 kD was recognized by anti-expansin antibody. Six different classes of α-expansin mRNA were identified in a BY2 cDNA library. Northern-blot analysis indicated moderate to low abundance of multiple α-expansin mRNAs in BY2 cells. From these results we conclude that BY2 suspension-cultured cells have the necessary components for expansin-mediated cell wall enlargement.