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Key message: NtCycB2 negatively regulates the initiation of tobacco long stalk glandular trichomes and influences the expression of diterpenoid biosynthesis- and environmental stress resistance-related genes. Many asterid plants possess multicellular trichomes on their surface, both glandular and non-glandular. The CycB2 gene plays a key role in multicellular trichome initiation, but has distinct effects on different types of trichomes; its mechanisms remain unknown. In tomato (Solanum lycopersicum), SlCycB2 negatively regulates non-glandular trichome formation, but its effects on glandular trichomes are ambiguous. In this study, we cloned the SlCycB2 homolog of Nicotiana tabacum, NtCycB2, and analyzed its effect on three types of trichomes, long stalk glandular trichomes (LGT), short stalk glandular trichomes (SGT), and non-glandular trichomes (NGT). Knocking out NtCycB2 (NtCycB2-KO) promoted LGT formation, while overexpression of NtCycB2 (NtCycB2-OE) decreased LGT density. SGT and NGT were not significantly influenced in either NtCycB2-KO or NtCycB2-OE plants, indicating that NtCycB2 regulated only LGT formation in tobacco. In addition, compared with NtCycB2-OE and control plants, NtCycB2-KO plants produced more trichome exudates, including diterpenoids and sugar esters, and exhibited stronger aphid resistance. To further elucidate the function of NtCycB2, RNA-Seq analysis of the NtCycB2-KO, NtCycB2-OE, and control plants was conducted. 2,552 and 1,933 differentially expressed genes (DEGs) were found in NtCycB2-KO and NtCycB2-OE plants, respectively. Gene Ontology analysis of the common DEGs revealed that ion transport, carbohydrate and amino acid metabolism, photosynthesis, and transcription regulation processes were significantly enriched. Among these DEGs, diterpenoid biosynthesis genes were upregulated in NtCycB2-KO plants and downregulated in NtCycB2-OE plants. Two MYB transcription factors and several stress resistance-related genes were also identified, suggesting they may participate in regulating LGT formation and aphid resistance. [ABSTRACT FROM AUTHOR]

Key message: NtARF6 overexpression represses nicotine biosynthesis in tobacco. Transcriptome analysis suggests that NtARF6 acts as a regulatory hub that connect different phytohormone signaling pathways to antagonize the jasmonic acid-induced nicotine biosynthesis. Plant specialized metabolic pathways are regulated by a plethora of molecular regulators that form complex networks. In Nicotiana tabacum, nicotine biosynthesis is regulated by transcriptional activators, such as NtMYC2 and the NIC2-locus ERFs. However, the underlying molecular mechanism of the regulatory feedback is largely unknown. Previous research has shown that NbARF1, a nicotine synthesis repressor, reduces nicotine accumulation in N. benthamiana. In this study, we demonstrated that overexpression of NtARF6, an ortholog of NbARF1, was able to reduce pyridine alkaloid accumulation in tobacco. We found that NtARF6 could not directly repress the transcriptional activities of the key nicotine pathway structural gene promoters. Transcriptomic analysis suggested that this NtARF6-induced deactivation of alkaloid biosynthesis might be achieved by the antagonistic effect between jasmonic acid (JA) and other plant hormone signaling pathways, such as ethylene (ETH), salicylic acid (SA), abscisic acid (ABA). The repression of JA biosynthesis is accompanied by the induction of ETH, ABA, and SA signaling and pathogenic infection defensive responses, resulting in counteracting JA-induced metabolic reprogramming and decreasing the expression of nicotine biosynthetic genes in vivo. This study provides transcriptomic evidence for the regulatory mechanism of the NtARF6-mediated repression of alkaloid biosynthesis and indicates that this ARF transcription factor might act as a regulatory hub to connect different hormone signaling pathways in tobacco. [ABSTRACT FROM AUTHOR]

Key message: Comprehensive transcriptome analysis suggested that the primary metabolism is modulated to augment the supply of substrates towards secondary metabolism operating in the glandular trichomes of Nicotiana tabacum. The comparative gene expression and co-expression network analysis revealed that certain members of transcription factor genes belonging to the MYB, HD-ZIP, ERF, TCP, SRS, WRKY and DOF families may be involved in the regulation of metabolism and/other aspects in the glandular trichomes of N. tabacum The glandular trichomes of Nicotiana tabacum are highly productive in terms of secondary metabolites and therefore have been projected to be used as a prognostic platform for metabolic engineering of valuable natural products. For obvious reasons, detailed studies pertaining to the metabolic and gene regulatory networks operating in the glandular trichomes of N. tabacum are of pivotal significance to be undertaken. We have carried out next-generation sequencing of glandular trichomes of N. tabcaum and investigated differential gene expression among different tissues, including trichome-free leaves. We identified a total of 37,269 and 37,371 genes, expressing in trichome free leaf and glandular trichomes, respectively, at a cutoff of FPKM ≥ 1. The analysis revealed that different pathways involved with the primary metabolism are modulated in glandular trichomes of N. tabacum, providing a plausible explanation for the enhanced biosynthesis of secondary metabolism in the glandular trichomes. Further, comparative gene expression analysis revealed several genes, which display preferential expression in the glandular trichomes and thereby seem to be potential candidate genes for future studies in connection to the discovery of novel trichome specific promoters. The present study also led to the comprehensive identification of 1750 transcription factor genes expressing at a cutoff of FPKM ≥ 1 in the glandular trichomes of N. tabacum. The clustering and co-expression analysis suggested that transcription factor genes belonging to HD-ZIP, ERF, WRKY, MYB, TCP, SRS and DOF families may be the major players in the regulation of gene expression in the glandular trichomes of N. tabacum. To the best of our knowledge, the present work is the first effort towards detailed identification of genes, especially regulatory genes expressing in the glandular trichomes of N. tabacum. The data resource and the empirical findings from present work in all probability must, therefore, provide a reference and background context for future work aiming at deciphering molecular mechanism of regulation of secondary metabolism and gene expression in the glandular trichomes of N. tabacum. [ABSTRACT FROM AUTHOR]

Key message: The potent anti-HIV microbicide griffithsin was expressed to high levels in tobacco chloroplasts, enabling efficient purification from both fresh and dried biomass, thus providing storable material for inexpensive production and scale-up on demand.Abstract: The global HIV epidemic continues to grow, with 1.8 million new infections occurring per year. In the absence of a cure and an AIDS vaccine, there is a pressing need to prevent new infections in order to curb the disease. Topical microbicides that block viral entry into human cells can potentially prevent HIV infection. The antiviral lectin griffithsin has been identified as a highly potent inhibitor of HIV entry into human cells. Here we have explored the possibility to use transplastomic plants as an inexpensive production platform for griffithsin. We show that griffithsin accumulates in stably transformed tobacco chloroplasts to up to 5% of the total soluble protein of the plant. Griffithsin can be easily purified from leaf material and shows similarly high virus neutralization activity as griffithsin protein recombinantly expressed in bacteria. We also show that dried tobacco provides a storable source material for griffithsin purification, thus enabling quick scale-up of production on demand. [ABSTRACT FROM AUTHOR]

Key message: The topoisomerase II expression varies as a function of cell proliferation. Maximal topoisomerase II expression was tightly coupled to S phase and G2/M phase via both transcriptional and post-transcriptional regulation. Investigation in meiosis using pollen mother cells also revealed that it is not the major component of meiotic chromosomes, it seems to diffuse out once meiotic chromosomal condensation is completed. Abstract: Synchronized tobacco BY-2 cell cultures were used to study the role of topoisomerase II in various stages of the cell cycle. Topoisomerase II transcript accumulation was observed during the S- and G2/M- phase of cell cycle. This biphasic expression pattern indicates the active requirement of topoisomerase II during these stages of the cell cycle. Through immuno-localization of topoisomerase II was observed diffusely throughout the nucleoplasm in interphase nuclei, whereas, the nucleolus region exhibited a more prominent immuno-positive staining that correlated with rRNA transcription, as shown by propidium iodide staining and BrUTP incorporation. The immuno-staining analysis also showed that topoisomerase II is the major component of mitotic chromosomes and remain attached to the chromosomes during cell division. The inhibition of topoisomerase II activity using specific inhibitors revealed quite dramatic effect on condensation of chromatin and chromosome individualization from prophase to metaphase transition. Partially condensed chromosomes were not arranged on metaphase plate and chromosomal perturbations were observed when advance to anaphase, suggesting the importance of topoisomerase II activity for proper chromosome condensation and segregation during mitosis. Contrary, topoisomerase II is not the major component of meiotic chromosomes, even though mitosis and meiosis share many processes, including the DNA replication, chromosome condensation and precisely regulated partitioning of chromosomes into daughter cells. Even if topoisomerase II is required for individualization and condensation of meiotic chromosomes, it seems to diffuse out once meiotic chromosomal condensation is completed. [ABSTRACT FROM AUTHOR]

Key message: A new selectable marker gene for stable transformation of the plastid genome was developed that is similarly efficient as the aadA, and produces no background of spontaneous resistance mutants. Abstract: More than 25 years after its development for Chlamydomonas and tobacco, the transformation of the chloroplast genome still represents a challenging technology that is available only in a handful of species. The vast majority of chloroplast transformation experiments conducted thus far have relied on a single selectable marker gene, the spectinomycin resistance gene aadA. Although a few alternative markers have been reported, the aadA has remained unrivalled in efficiency and is, therefore, nearly exclusively used. The development of new marker genes for plastid transformation is of crucial importance to all efforts towards extending the species range of the technology as well as to those applications in basic research, biotechnology and synthetic biology that involve the multistep engineering of plastid genomes. Here, we have tested a bifunctional resistance gene for its suitability as a selectable marker for chloroplast transformation. The bacterial enzyme aminoglycoside acetyltransferase(6′)-Ie/aminoglycoside phosphotransferase(2″)-Ia possesses an N-terminal acetyltransferase domain and a C-terminal phosphotransferase domain that can act synergistically and detoxify aminoglycoside antibiotics highly efficiently. We report that, in combination with selection for resistance to the aminoglycoside tobramycin, the aac(6′ )-Ie/ aph(2″ )-Ia gene represents an efficient marker for plastid transformation in that it produces similar numbers of transplastomic lines as the spectinomycin resistance gene aadA. Importantly, no spontaneous antibiotic resistance mutants appear under tobramycin selection. [ABSTRACT FROM AUTHOR]

Genome editing is one of the most powerful tools for revealing gene function and improving crop plants. Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been used as a powerful and efficient tool for genome editing in various organisms. Here, we report genome editing in tobacco ( Nicotiana tabacum) mediated by the CRISPR/Cas9 system. Two genes, NtPDS and NtPDR6, were used for targeted mutagenesis. First, we examined the transient genome editing activity of this system in tobacco protoplasts, insertion and deletion (indel) mutations were observed with frequencies of 16.2-20.3 % after transfecting guide RNA (gRNA) and the nuclease Cas9 in tobacco protoplasts. The two genes were also mutated using multiplexing gRNA at a time. Additionally, targeted deletions and inversions of a 1.8-kb fragment between two target sites in the NtPDS locus were demonstrated, while indel mutations were also detected at both the sites. Second, we obtained transgenic tobacco plants with NtPDS and NtPDR6 mutations induced by Cas9/gRNA. The mutation percentage was 81.8 % for NtPDS gRNA4 and 87.5 % for NtPDR6 gRNA2. Obvious phenotypes were observed, etiolated leaves for the psd mutant and more branches for the pdr6 mutant, indicating that highly efficient biallelic mutations occurred in both transgenic lines. No significant off-target mutations were obtained. Our results show that the CRISPR/Cas9 system is a useful tool for targeted mutagenesis of the tobacco genome. [ABSTRACT FROM AUTHOR]

Plastoglobules (PG) are lipid droplets in chloroplasts and other plastid types having important functions in lipid metabolism. Plastoglobulins (PGL) also known as fibrillins (FBN) are evolutionary conserved proteins present at the PG surface but also to various extents at the thylakoid membrane. PGLs are thought to have structural functions in PG formation and maintenance. The targeting of an Arabidopsis PGL (PGL34) to PG required the full protein sequence with the exception of a short C-terminal stretch. This indicated that PGL targeting relies on correct folding rather than a discrete sequence. PGLs lack strongly hydrophic regions and may therefore extrinsically associate with PG and thylakoid membranes via interaction with hydrophilic headgroups of surface lipids. Here, we report on the expression of the Arabidopsis plastoglobulin of 35kD (PGL35 or FBN1a) expressed as a mature protein fused to HIVp24 (human immunodeficiency virus capsid particle p24) or HCV (hepatitis C virus core protein) in transplastomic tobacco. A PGL35-HIVp24 fusion targeted in part to plastoglobules but a larger proportion was recovered in the thylakoid fraction. The findings indicate that transplastomic PGL35-HIVp24 folded correctly after its synthesis inside the chloroplast and then dually targeted to plastoglobules as well as thylakoid membranes. [ABSTRACT FROM AUTHOR]

Telomere homeostasis is regulated at multiple levels, including the local chromatin structure of telomeres and subtelomeres. Recent reports demonstrated that a decrease in repressive chromatin marks, such as levels of cytosine methylation in subtelomeric regions, results in telomere elongation in mouse cells. Here we show that a considerable fraction of cytosines is methylated not only in subtelomeric, but also in telomeric DNA of tobacco BY-2 cells. Drug-induced hypomethylation (demonstrated at subtelomeric, telomeric, and global DNA levels) results in activation of telomerase. However, in contrast to mouse cells, the decrease in 5-methylcytosine levels and upregulation of telomerase do not result in any changes of telomere lengths. These results demonstrate the involvement of epigenetic mechanisms in the multilevel process of regulation of telomerase activity in plant cells and, at the same time, they indicate that changes in telomerase activity can be overridden by other factors governing telomere length stability. [ABSTRACT FROM AUTHOR]

Chloroplast transformation remains a demanding technique and is still restricted to relatively few plant species. The limited availability of selectable marker genes and the lack of selection markers that would be universally applicable to all plant species represent some of the most serious technical problems involved in extending the species range of plastid transformation. Here we report the development of the chloramphenicol acetyltransferase gene cat as a new selectable marker for plastid transformation. We show that, by selecting for chloramphenicol resistance, tobacco chloroplast transformants are readily obtained. Transplastomic lines quickly reach the homoplasmic state (typically in one additional regeneration round), accumulate the chloramphenicol acetyltransferase enzyme to high levels and transmit their plastid transgenes maternally into the next generation. No spontaneous antibiotic resistance mutants appear upon chloramphenicol selection. Several lines of evidence support the assumption that plant mitochondria are also sensitive to chloramphenicol suggesting that the chloramphenicol acetyltransferase may be a good candidate selectable marker for plant mitochondrial transformation. [ABSTRACT FROM AUTHOR]

Identification of a genetically stable Nicotiana tabacum (tobacco) plant with a uniform population of transformed plastid genomes (ptDNA) takes two cycles of plant regeneration from chimeric leaves and analysis of multiple shoots by Southern probing in each cycle. Visual detection of transgenic sectors facilitates identification of transformed shoots in the greenhouse, complementing repeated cycles of blind purification in culture. In addition, it provides a tool to monitor the maintenance of transplastomic state. Our current visual marker system requires two genes: the aurea bar ( bar) gene that confers a golden leaf phenotype and a spectinomycin resistance ( aadA) gene that is necessary for the introduction of the bar gene in the plastid genome. We developed a novel aadA gene that fulfills both functions: it is a conventional selectable aadA gene in culture, and allows detection of transplastomic sectors in the greenhouse by leaf color. Common causes of pigment deficiency in leaves are mutations in photosynthetic genes, which affect chlorophyll accumulation. We use a different approach to achieve pigment deficiency: post-transcriptional interference with the expression of the clpP1 plastid gene by aurea aadA transgene. This interference produces plants with reduced growth and a distinct color, but maintains a wild-type gene set and the capacity for photosynthesis. Importantly, when the aurea gene is removed, green pigmentation and normal growth rate are restored. Because the aurea plants are viable, the new aadA genes are useful to query rare events in large populations and for in planta manipulation of the plastid genome. [ABSTRACT FROM AUTHOR]

The biological conversion of plant biomass into fermentable sugars is key to the efficient production of biofuels and other renewable chemicals from plants. As up to more than 90% of the dry weight of higher plants is fixed in the cell wall, this will require the low-cost production of large amounts of cell wall-degrading enzymes. Transgenic plants can potentially provide an unbeatably cheap production platform for industrial enzymes. Transgene expression from the plastid genome is particularly attractive, due to high-level foreign protein accumulation in chloroplasts, absence of epigenetic gene silencing and improved transgene containment. Here, we have explored the potential of transplastomic plants to produce large amounts of thermostable cell wall-degrading enzymes from the bacterium Thermobifida fusca. We show that a set of four enzymes that are required for efficient degradation of cellulose (and the hemicellulose xyloglucan) could be expressed successfully in transplastomic tobacco plants. However, overexpression of the enzymes (to between approximately 5 and 40% of the plant's total soluble protein) resulted in pigment-deficient mutant phenotypes. We demonstrate that the chloroplast-produced cellulolytic enzymes are highly active. Although further optimization is needed, our data indicate that transgenic plastids offer great potential for the production of enzyme cocktails for the bioconversion of cellulosic biomass. [ABSTRACT FROM AUTHOR]

In higher plants, DNA transfer from the plastid (chloroplast) genome to the nucleus is a frequent, ongoing process. However, there has been uncertainty over whether this transfer occurs by a direct DNA mechanism or whether RNA intermediates are involved. Previous experiments utilising transplastomic Nicotiana tabacum ( tp7 and tp17) enabled the detection of plastid-to-nucleus transfer in real time. To determine whether RNA intermediates are involved in this transfer, transplastomic lines ( tpneoACG) were generated containing, in their plastid genomes, a nuclear promoter-driven kanamycin resistance gene ( neo) with a start codon that required plastid RNA editing but otherwise identical to tp7 and tp17. Therefore it was expected that kanamycin resistance would only be acquired following RNA-mediated transfer of neo to the nucleus. Screening of tpneoACG progeny revealed several kanamycin-resistant plants, each of which contained the neo gene located in the nucleus. Surprisingly, neo was unedited in all these plants, indicating that neoACG was active in the absence of an edited start codon and suggesting that RNA intermediates were not involved in the transfers. However, analysis of tpneoACG revealed that only a low proportion of transcripts potentially able to mediate neo transfer were edited, thus precluding unequivocal conclusions regarding the role of RNA in plastid-to-nucleus transfer. The low proportion of edited transcripts was found to be due to predominant antisense neo transcripts, rather than to low editing efficiency of the sense transcripts. This study highlights a number of important considerations in the design of experiments utilising plastid RNA editing. The results also suggest that RNA editing sites reduce but do not eliminate functional plastid-to-nucleus gene transfer. This is relevant both in an evolutionary context and in placing RNA editing-dependent genes in the plastid genome as a means of transgene containment. [ABSTRACT FROM AUTHOR]

class V chitinase from Nicotiana tabacum (NtChiV) with amino acid sequence similar to that of Serratia marcescens chitinase B (SmChiB) was expressed in E. coli and purified to homogeneity. When N-acetylglucosamine oligosaccharides [(NAG)] were hydrolyzed by the purified NtChiV, the second glycosidic linkage from the non-reducing end was predominantly hydrolyzed in a manner similar to that of SmChiB. NtChiV was shown to hydrolyze partially N-acetylated chitosan non-processively, whereas SmChiB hydrolyzes the same substrate processively. The crystal structure of NtChiV was determined by the single-wavelength anomalous dispersion method at 1.2 Å resolution. The protein adopts a classical (β/α)-barrel fold (residues 1-233 and 303-348) with an insertion of a small (α + β) domain (residues 234-302). This is the first crystal structure of a plant class V chitinase. The crystal structure of the inactive mutant NtChiV E115Q complexed with (NAG) was also solved and exhibited a linear conformation of the bound oligosaccharide occupying −2, +1, +2, and +3 subsites. The complex structure corresponds to an initial state of (NAG) binding, which is proposed to be converted into a bent conformation through sliding of the +1, +2, and +3 sugar units to −1, +1, and +2 subsites. Although NtChiV is similar to SmChiB, the chitin-binding domain is present in the C-terminus of the latter, but not in the former. Aromatic amino acid residues found in the substrate binding cleft of SmChiB, including Trp97, are substituted with aliphatic residues in NtChiV. These structural differences appear to be responsible for NtChiV being a non-processive enzyme. [ABSTRACT FROM AUTHOR]

In plants, transgenes containing Potato spindle tuber viroid (PSTVd) cDNA sequences were efficient targets of PSTVd infection-mediated RNA-directed DNA methylation. Here, we demonstrate that in PSTVd-infected tobacco plants, a 134 bp PSTVd fragment (PSTVd-134) did not become densely methylated when it was inserted into a chimeric Satellite tobacco mosaic virus (STMV) construct. Only about 4–5% of all cytosines (Cs) of the PSTVd-134 were methylated when flanked by satellite sequences. In the same plants, C methylation was approximately 92% when the PSTVd-134 was in a PSTVd full length sequence context and roughly 33% when flanked at its 3′ end by a 19 bp PSTVd and at its 5′ end by a short viroid-unrelated sequence. In addition, PSTVd small interfering RNAs (siRNAs) produced from the replicating viroid failed to target PSTVd-134-containing chimeric STMV RNA for degradation. Satellite RNAs appear to have adopted secondary structures that protect them against RNA interference (RNAi)—mediated degradation. Protection can be extended to short non-satellite sequences residing in satellite RNAs, rendering them poor targets for nuclear and cytoplasmic RNAi induced in trans. [ABSTRACT FROM AUTHOR]

Putrescine N-methyltransferase (PMT) catalyzes the first committed step of nicotine biosynthesis, converting putrescine into N-methylputrescine. A variety of chemical, environmental, and developmental cues have been implicated in its regulation. Here we have examined the differential expression of β-glucuronidase (GUS) transgenes under the control of the transcriptional regulatory sequences of four distinct members of the NtPMT gene family from tobacco ( Nicotiana tabacum L.). BY-2 cell cultures expressing various NtPMT promoter- GUS constructs were examined for their response to treatment with various combinations of methyl jasmonate (MeJA), auxin (AUX), and ethylene (ETH). All four NtPMT gene promoters examined were inducible by MeJA, although the extent of the induction varied dramatically, with the NtPMT1a promoter being the most responsive. High AUX levels in the cell growth media repressed NtPMT::GUS transgene expression and inhibited their MeJA-induced transcription. Treatment of BY-2 cells with ETH alone did not result in a significant alteration in NtPMT::GUS expression. However, similar to AUX, ETH treatment led to the suppression of MeJA-induced transcription. Detailed deletion analysis of the NtPMT1a gene promoter showed that as little as 111 bp upstream of the transcriptional start site were sufficient to confer MeJA-responsiveness. Deletion of a conserved G-box element (GCACGTTG) at −103 to −96 bp completely abolished MeJA-responsiveness. Further mutagenesis studies revealed that in addition to a functional G-box, MeJA-responsiveness of the NtPMT1a promoter also required a TA-rich region and a GCC-motif (TGCGCCC) located at −80 to −69 bp and −62 to −56 bp relative to the start site, respectively. A synthetic G-box tetramer ( 4 X syn G-box) fused to a −83 bp fragment from the NtPMT1apromoter (containing the TA-rich region, GCC-box, and TATA-box) displayed a 30-fold induction by MeJA treatment, whereas when the 4 X syn G-box was fused to a minimal (−46 bp) promoter fragment derived from the CaMV 35S gene, no induction by MeJA treatment was detected. Our results indicate that multiple intersecting signal transduction pathways and different transcriptional regulatory factors are involved in mediating JA-responsiveness of NtPMT expression in tobacco. [ABSTRACT FROM AUTHOR]

This report describes a novel receptor-like kinase gene of tobacco ( Nicotiana tabacum L.) that, in cell culture, is rapidly regulated by very low concentrations of cytokinin. The steady-state transcript level of the CYTOKONIN-REGULATED KINASE 1 gene ( CRK1) was strongly reduced 30 min after cytokinin treatment. At higher concentrations abscisic acid and auxin induced a similar response. None of the other plant hormones tested elicited this response. Further analyses of the cytokinin-dependent regulation showed that the reduction of transcript was transient, and the duration of the recovery phase was dependent on the hormone concentration. CRK1 is not a primary response gene as the simultaneous addition of cycloheximide inhibits its regulation by cytokinin. Inhibitor studies revealed that a protein phosphatase is likely involved in signalling processes upstream of CRK1. CRK1 is expressed at low levels in the leaves, stem and roots of tobacco. It is predicted that the CRK1 protein is located in the plasma membrane. It has in its N-terminal putative receptor sequence a signal peptide, a serine- and a proline-rich region, a six repeat motif similar to the CRINKLY4 protein of Zea mays and several regions homologous to purine-binding motifs. A single transmembrane domain is followed by a highly conserved intracellular Ser/Thr kinase domain. Therefore, CRK1 is a novel type of class I plant receptor kinase. We hypothesize that CRK1 is involved in an early step of hormone signalling and that transcript down-regulation reflects a desensitization step in reaction to the signalling molecule. [ABSTRACT FROM AUTHOR]

Endochitinases contribute to the defence response of plants against chitin-containing pathogens. The vacuolar class I chitinases consist of an N-terminal cysteine-rich domain (CRD) linked by a glycine-threonine-rich spacer with 4-hydroxylated prolyl residues to the catalytic domain. We examined the functional role of the CRD and spacer region in class I chitinases by comparing wild-type chitinase A (CHN A) of Nicotiana tabacum with informative recombinant forms. The chitinases were expressed in transgenic N. sylvestris plants, purified to near homogeneity, and their structures confirmed by mass spectrometry and partial sequencing. The enzymes were tested for their substrate preference towards chitin, lipo-chitooligosaccharide Nod factors of Rhizobium, and bacterial peptidoglycans (lysozyme activity) as well as for their capacity to inhibit hyphal growth of Trichoderma viride. Deletion of the CRD and spacer alone or in combination resulted in a modest <50% reduction of hydrolytic activity relative to CHN A using colloidal chitin or M. lysodeikticus walls as substrates; whereas, antifungal activity was reduced by up to 80%. Relative to CHN A, a variant with two spacers in tandem, which binds chitin, showed very low hydrolytic activity towards chitin and Nod factors, but comparable lysozyme activity and enhanced antifungal activity. Neither hydrolytic activity, substrate specificity nor antifungal activity were strictly correlated with the CRD-mediated capacity to bind chitin. This suggests that the presence of the chitin-binding domain does not have a major influence on the functions of CHN A examined. Moreover, the results with the tandem-spacer variant raise the possibility that substantial chitinolytic activity is not essential for inhibition of T. viride growth by CHN A. [ABSTRACT FROM AUTHOR]

The tobacco gene g10 is preferentially and maximally expressed in mature pollen, shows homology to pectate lyases, and is the putative homologue of the tomato gene lat56. Analysis of regulatory elements within the g10 promoter was carried out to verify the importance of putative regulatory sequence motifs. Analysis of transgenic plants showed that 1190 bp of g10 5′ sequence directed preferential expression of GUS in pollen, with bimodal peaks of expression just before and during pollen mitosis I, and in mature anthers. This was confirmed by northern analysis of native g10 transcripts in isolated spores. Transient expression analysis defined the minimal g10 promoter region capable of directing expression in pollen as −86 to +217. Three upstream regions within −427 bp modulate the expression from g10. Gain-of-function analyses showed that the region from −106 to −53 could enhance pollen-specific expression of a minimal CaMV 35S promoter. These analyses further showed that sequences upstream of −86 modulate expression in pollen, but are not essential for preferential pollen expression. The function of a conserved GTGA motif shared between the tobacco g10 and tomato lat56 promoters was demonstrated in g10. Thus, further functional evidence is provided for the conservation of mechanisms for the regulation of late pollen genes across species. [ABSTRACT FROM AUTHOR]

The promoter of the maize glyceraldehyde-3-phosphate dehydrogenase 4 gene ( GapC4) confers strong, specific and ubiquitous anaerobic reporter gene expression in tobacco. To identify factors required for heterologous anaerobic gene expression, 19 progressive 5′ and 3′ promoter deletions were linked to a chimeric GapC4 TATA box-β-glucuronidase (GUS) reporter gene construct and transformed into tobacco. In all transgenic lines aerobic expression values were in the range obtained for negative controls while histochemical GUS assays reveal some weak expression in roots only. Anaerobic induction of about 100-fold to more than 1000-fold above unspecific background is mediated by a region of about 190 bp of the GapC4 promoter. Anaerobic reporter gene induction strongly decreases upon deletion of a 20 bp fragment from −286 to −266 relative to the transcription start point. This fragment harbours putative cis-acting sequences. Electrophoretic mobility shift assays with a 50 bp fragment harbouring these cis sequences reveal a high-mobility complex that is formed with nuclear extracts from aerobic and anaerobic leaf tissue while an additional low-mobility complex is anaerobiosis-specific. The formation of the high-mobility complex requires the sequence GTGGGCCCG. The 50 bp fragment alone confers weak and orientation-dependent anaerobic induction to a GapC4 TATA box-β-glucuronidase (GUS) reporter gene. [ABSTRACT FROM AUTHOR]

Cultured cells of tobacco BY2 secrete more than 100 proteins into culture medium. Six major proteins were purified, and partial protein sequences were determined. Five of them were found to be similar to an ascorbic acid oxidase, three peroxidase isozymes and a β-1,3-exoglucanase, respectively. A cDNA clone encoding the remaining polypeptide, whose amino acid sequence showed no similarity with earlier reported proteins, was isolated. It encoded a putative 27 kDa protein of 242 amino acids with resemblance to WCI-5, a wheat protein induced by benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) which activates genes involved in systemic acquired resistance. Transcripts of this clone accumulated upon tobacco mosaic virus infection, mechanical wounding and drought treatment, an induction profile that satisfies the definition of pathogenesis-related (PR) proteins by van Loon et al. (Plant Mol. Biol. Rep. 12 (1994) 245). No similar PR proteins have so far been reported, and therefore our newly designated NtPRp27 points to the existence of a novel PR protein family in tobacco plants. [ABSTRACT FROM AUTHOR]

The structure and nuclear genomic organization of the gene family encoding putrescine N-methyltransferase (PMT), the key enzyme in diverting polyamine metabolism towards the biosynthesis of nicotine and related alkaloids, was examined in Nicotiana tabacum. Five genes encoding PMT are present in the N. tabacum genome and all are expressed. The complete coding region and immediate 5′- and 3′- flanking regions were characterized for four members of the gene family and the Exon 1 region of the fifth member of the family was determined. Comparison of the nucleotide and deduced amino acid sequences of the N. tabacum PMT genes with those of presumed progenitor species, N. sylvestris, N. tomentosiformis and N. otophora, revealed that three members of the N. tabacum PMT gene family were most similar to the three genes present in N. sylvestris, whereas the two remaining PMT genes were similar to PMT genes present in N. tomentosiformis and N. otophora genomes, respectively. These data are consistent with an evolutionary origin of N. tabacum resulting from a cross involving N. sylvestris and an introgressed hybrid between N. tomentosiformis and N. otophora. The five PMT genes present in N. tabacum are expressed in the roots of wild-type plants, but not in other organs. The steady-state level of all five PMT transcripts is transiently increased in roots following topping (removal of the floral meristem), although the maximum level of induction for the individual transcripts varies considerably. In contrast to wild-type plants, no increase in PMT transcript levels was observed in a low-alkaloid (nic1nic2) mutant of Burley 21. These data support a role for nic1 and nic2 in the global regulation of alkaloid formation in tobacco and provide for the first time molecular confirmation of the presumed origin of cultivated tobacco. [ABSTRACT FROM AUTHOR]

Transgenic tobacco plants were produced that contained single-copy pART54 T-DNA, with a 35S-uidA gene linked to loxP-flanked kanamycin resistance (nptII) and cytosine deaminase (codA) genes. Retransformation of these plants with pCre1 (containing 35S transcribed cre recombinase and hygromycin (hpt) resistance genes) resulted in excision of the loxP-flanked genes from the genome. Phenotypes of progeny from selfed-retransformed plants confirmed nptII and codA excision and integration of the cre-linked hpt gene. To avoid integration of the hpt gene, and thereby generate plants totally free of marker genes, we attempted to transiently express the cre recombinase. Agrobacterium tumefaciens (pCre1) was cocultivated with leaf discs of two pART54-transformed lines and shoots were regenerated in the absence of hygromycin selection. Nineteen of 773 (0.25%) shoots showed tolerance to 5-fluorocytosine (5-fc) which is converted to the toxic 5-fluorouracil by cytosine deaminase. 5-fc tolerance in six shoots was found to be due to excision of the loxP-flanked region of the pART54 T-DNA. In four of these shoots excision could be attributed to cre expression from integrated pCre1 T-DNA, whereas in two shoots excision appeared to be a consequence of transient cre expression from pCre1 T-DNA molecules which had been transferred to the plant cells but not integrated into the genome. The absence of selectable marker genes was confirmed by the phenotype of the T1 progeny. Therefore, through transient cre expression, marker-free transgenic plants were produced without sexual crossing. This approach could be applicable to the elimination of marker genes from transgenic crops which must be vegetatively propagated to maintain their elite genotype. [ABSTRACT FROM AUTHOR]

Escherichia coli DnaK (Hsp70) cooperates with DnaJ and GrpE in its essential role as a molecular chaperone. Function of mitochondrial Hsp70 (mHsp70) in protein folding and organellar import in eukaryotes is critically dependent on GrpE. We cloned two genes from tobacco (Nicotiana tabacum) BY2 cells based on peptide sequences from a purified protein. The predicted amino acid sequences of both clones resembled that of GrpE from E. coli and its homologues from eukaryotes, and a cDNA clone from Arabidopsis thaliana. One gene (Type 1) encoded a deduced protein that was identical to the purified protein while the other (Type 2) encoded a deduced protein that has 80% sequence identity to Type 1. Both tobacco and Arabidopsis thaliana GrpE homologues bound to DnaK and ATP inhibited this binding. The tobacco GrpE homologue contained a typical N-terminal mitochondrial target presequence of 64 residues and the presequence directed the green fluorescent protein to tobacco mitochondria. The tobacco GrpE homologue also associated with mHsp70 when reintroduced into BY2 protoplasts, and this association was disrupted by ATP. A three-dimensional structure for the tobacco GrpE homologue was modeled based on the X-ray structure of E. coli GrpE complexed with DnaK. The modeled structure has the same overall structure as E. coli GrpE. We propose that the tobacco GrpE homologue interacts with mHsp70 in a manner analogous to E. coli GrpE with DnaK and designate it as tobacco mitochondrial GrpE (NtmGrpE). [ABSTRACT FROM AUTHOR]

Lysine synthesis in prokaryotes, some phycomycetes and higher plants starts with the condensation of L-aspartate-β-semialdehyde (L-ASA) and pyruvate into dihydrodipicolinic acid. The enzyme that catalyses this step, dihydrodipicolinate synthase (DHDPS), is inhibited by the end-product lysine and is therefore thought to have a regulatory control on lysine synthesis. We have cloned and sequenced an Arabidopsis thaliana DNA fragment containing 900 bases upstream of the dhdps coding sequence. A transcriptional fusion of this fragment with the β-glucuronidase reporter gene (uidA, Gus) was used to study the transcription properties of this promoter fragment (DS). No lysine-induced repression on transcription could be detected. Expression of DS-Gus activity in transformed Arabidopsis thaliana and Nicotiana tabacum was found to be cell type-specific. In the vegetative parts of the plant, GUS activity was located in meristems and young vasculature of roots, in vasculature of stem and leaves and in the meristems of young shoots. In flowers, high expression was found in the carpels, style, stigma, developing embryos, tapetum of young anthers and pollen. We demonstrated that the Arabidopsis DS promoter can direct its cell type-specific expression in a heterologous plant, Nicotiana tabacum. The importance of transcriptional regulation of the dhdps gene, and in more general genes involved in amino acid biosynthesis, is discussed. [ABSTRACT FROM AUTHOR]

A cDNA of tobacco BY-2 cells corresponding to an mRNA species which was rapidly induced by methyl jasmonate (MeJA) in the presence of cycloheximide (CHX) was found to encode ornithine decarboxylase (ODC). Another cDNA from a MeJA-inducible mRNA encoded S-adenosylmethionine synthase (SAMS). Although these enzymes could be involved in the biosynthesis of polyamines, the level of putrescine, a reaction product of ODC, increased slowly and while the levels of spermidine and spermine did not change following treatment of cells with MeJA. However, N-methylputrescine, which is a precursor of pyrrolidine ring of nicotine, started to increase shortly after MeJA-treatment of cells and the production of nicotine occured thereafter. The levels of mRNA for arginine decarboxylase (ADC), an alternative enzyme for putrescine synthesis, and that for S-adenosylmethionine decarboxylase (SAMDC), required for polyamine synthesis, were not affected by MeJA. In addition to mRNAs for ODC and SAMS, mRNA for putrescine N-methyltransferase (PMT) was also induced by MeJA. Unlike the MeJA-induction of ODC mRNA, MeJA-induction of SAMS and PMT mRNAs were blocked by CHX. The level of ODC mRNA declined after 1 to 4 h following MeJA treatment, while the levels of mRNAs for SAMS and PMT continued to increase. Auxin significantly reduced the MeJA-inducible accumulation of mRNAs for ODC, SAMS and PMT. These results indicate that MeJA sequentially induces expression of a series of genes involved in nicotine biosynthesis by multiple regulatory mechanisms.p> [ABSTRACT FROM AUTHOR]

A Nicotiana tabacum cDNA sequence encoding histidinol phosphate aminotransferase (HPA) was isolated by functional complementation of an Escherichia coli histidine auxotroph (UTH780). The enzymatic assay has confirmed that the isolated cDNA encodes a functional HPA protein. Amino acid sequence alignment of the HPA protein from N. tabacum, Saccharomyces cerevisiae and E. coli revealed that, despite the low degree of identity, some residues were found to be highly conserved. The predicted protein contains a transit peptide sequence at the amino-terminal end, suggesting a chloroplastic localization of the HPA enzyme. Western blot analysis demonstrated that the deduced HPA protein and the mature HPA protein have an apparent molecular mass of about 45 kDa and 40 kDa respectively. Gene copy number estimation by Southern analysis indicates the presence of at least two genes per haploid genome coding for this protein in Nicotiana sp. From northern analysis results, the gene seems to be highly expressed in green tissues and the detected transcript showed a single band of expected molecular size. [ABSTRACT FROM AUTHOR]

A new binary vector encoding for Candida albicans dihydrofolate reductase (DFR1) has been constructed and used as a dominant selectable marker for plant transformation. Transgenic tobacco plants with an increased resistance to methotrexate (Mtx) were obtained by co-transformation of tobacco leaf discs with Agrobacterium tumefaciens strains carrying two new binary vectors: pTI20 and pTI18. Co-transformants of Nicotiana tabacum were directly selected for and rooted on medium containing both kanamycin (kan) and Mtx. Leaf discs of transgenic plants were assayed for capacity of regeneration at different Mtx concentrations. Analysis of transcripts was performed on total RNA extracted from two Mtx-resistant plants. The transgenic plants increased resistance to Mtx can be explained by the exceptionally low capacity of Mtx to bind C. albicans dihydrofolate reductase, accountable by the presence of two amino acid residues strategically important in Mtx binding. [ABSTRACT FROM AUTHOR]

The tomato lat52 gene encodes an essential cysteine-rich protein preferentially transcribed in the vegetative cell during pollen maturation. Detailed analyses of the identity, organization and role of cis-regulatory elements in controlling the precise developmental and tissue-specific expression of lat52 during pollen development were performed. Analysis of a series of 5′ promoter deletion mutants stably introduced into tobacco demonstrated differential developmental activation of deletion mutants during pollen development. All major cis-regulatory elements required for pollen-specific transcription were located within the upstream region −492 to −52. This region was shown to comprise three independent activator domains A, B and C, each sufficient to activate the minimal CaMV 35S promoter in a pollen-specific manner. 5′ deletion and gain of function approaches were used to show that domain A and the previously defined motif PBII (sub-domain B1) were largely redundant in the presence of downstream sequences in mature pollen. Within domain B two novel pollen-specific sub-domains B2 and B3 were identified. Within domain C, the activity of the PBI motif (sub-domain C1) was shown to be strictly dependent upon a downstream 20 bp pollen-specific activator unit −72 to −52 (sub-domain C2), containing two novel co-dependent regulatory elements AGAAA and TCCACCATA. These results demonstrate that transcriptional activation of lat52 is controlled by a complex of pollen-specific cis-regulatory elements which cooperate to achieve maximum levels of gene expression throughout pollen maturation. Alternative models of the interaction of identified cis-regulatory elements with putative trans-acting factors within the lat52 promoter and their developmental utilization are presented. [ABSTRACT FROM AUTHOR]

Agrobacterium rhizogenes strains of the agropine type harbor on their Ri-plasmid two T-DNAs, a left TL-DNA and a right TR-DNA. The rolB gene of the TL-DNA is the major factor in the pathogenesis of the hairy-root disease and its constitutive expression interferes profoundly with plant morphogenesis. We have tested whether the expression of its sequence related putative homologue from the TR-DNA (rolBTR) may cause also bacterial virulence or affect plant development. Unlike rolB, rolBTR is unable to induce root formation on tobacco leaf discs. Tobacco plants expressing a chimeric 35S::rolBTR gene have reduced stature, off-shoots at the stem base and bent and wrinkled leaves with epinastic growth. 14 N-terminal amino acids which are absent in the rolB protein are indispensable to rolBTR protein activity. The characteristic tyrosine phosphatase super family motif CX5R is absent in the rolBTR protein. For rolB this motif is possibly functionally relevant. We conclude that the rolBTR gene product has morphogenic activity but is not a functional homologue of the rolB protein. [ABSTRACT FROM AUTHOR]

Transcriptionally regulated expression of tobacco anionic peroxidase was investigated with regard to tissue specificity and developmental regulation. Two tobacco species, Nicotiana sylvestris and Nicotiana tabacum cv. Xanthi, were stably transformed with a gene chimera composed of 3 kb of the tobacco anionic peroxidase promoter, the Escherichia coli β-glucuronidase (GUS) coding region and the nopaline synthase terminator. Gene expression was regulated spatially and developmentally in all organs, and generally increased with age and maturity of the plant, tissue or organ. In the aerial portions of the plant, GUS activity was strongly expressed in trichomes and epidermis at nearly all developmental stages. In later stages of development, activity was also detected in ground tissue and parenchyma cells associated with vascular tissues. Activity in roots was limited to cortical cells and vascular-associated parenchyma cells. In reproductive tissue, expression was observed in sepals and petals before anthesis, and in all floral organs after anthesis. Expression was never detected in vascular tissue and was poorly correlated with lignification except in the cells surrounding primary xylem and pericyclic fibers in N. sylvestris. These studies suggest that this peroxidase isoenzyme is only limitedly involved in lignification but may be important in plant defense, growth and development. [ABSTRACT FROM AUTHOR]

Identification of pistil-expressed genes is an important step in understanding pistil development and function in plant reproduction. A tobacco stigma/style cDNA library was differentially screened and several cDNA clones were isolated. One of these tobacco genes, designated tobP1, is characterized here. TobP1 encodes a protein highly homologous to plant polyphenol oxidases. Northern blot analysis of total RNA extracted from different organs and probed with tobP1 cDNA identified a single transcript that is exclusively present in flower organs (petals, stamens, and predominantly in pistils). The tobP1 gene is co-ordinately regulated during development in pistils and stamens, and is not induced in mature leaves even under stress conditions. TobP1 belongs to a multigene family, as reported for PPO in other plant species. [ABSTRACT FROM AUTHOR]

Five cDNA clones encoding the catalytic subunits of Ser/Thr protein phosphatases (PP) of Nicotiana tabacum were described. Among them, three clones (NPP1, NPP2, and NPP3) encoded type 1 PP (PP1), whereas the rest of the clones (NPP4 and NPP5) encoded type 2A PP (PP2A). These cDNA clones exhibited high sequence identity in the PP core region to the corresponding genes from animals and plants. NPP1 mRNA was predominantly expressed in flowers, whereas NPP5 mRNA was mainly detected in leaves and flowers. In contrast, the transcripts of NPP2, NPP3, and NPP4 genes were present in all tissues examined. Throughout flower development, NPP1, NPP2, NPP5 mRNAs were expressed without any significant variation at the steady-state level. Genomic Southern blot showed that tobacco genome contained multiple genes and/or pseudogenes for both type 1 and type 2A phosphatases. [ABSTRACT FROM AUTHOR]

In order to modify the early stages of pollen development in a transgenic context microspore-specific promoters are required. We tested two putatively microspore-specific promoters, the Bp4 promoter from rapeseed and the NTM19 promoter from tobacco. Expression of the gus and barnase reporter genes under the control of these two promoters was studied in transgenic tobacco. Contrary to expectations, the Bp4 promoter became active only after the first pollen mitosis, and not in the microspores. The NTM19 promoter turned out to be highly microspore-specific and directed very high levels of gus expression to the unicellular microspores. The NTM19-barnase transgene caused cell-autonomous death at the mid-unicellular microspore stage, whereas Bp4-barnase induced cell ablation of early to mid-bicellular pollen. Both promoter-barnase transgenes did not affect the sporophyte and were inherited through the female germline. These results show that both the NTM19 and Bp4 promoters are expressed only in the male germline, and that the NTM19 promoter is an excellent tool to direct high levels of transgene expression exclusively to the microspores. This may have important biotechnological applications. [ABSTRACT FROM AUTHOR]

The gene encoding the antifungal protein osmotin is induced by several hormonal and environmental signals. In this study, tissue-specific and inducer-mediated expression of the reporter gene β-glucuronidase (uidA) fused to different fragment lengths of the osmotin promoter was evaluated in transgenic tobacco (Nicotiana tabacum). The region of the promoter between −248 to −108 (Fragment A) was found to be essential and sufficient for inducer (abscisic acid (ABA), C2H4 and NaCl)-mediated expression of the reporter gene. Expression of the reporter gene was developmentally regulated and increased with maturity of leaves, stem and flowers. Expression also was tissue-specific being most highly expressed in epidermis and vascular parenchyma of the stem. The regulators ABA, C2H4 and NaCl exhibited tissue-specific induction of this promoter. The promoter was specifically responsive to C2H4 in flowers at virtually all stages of development, but not responsive in these tissues to ABA or NaCl. Conversely, ABA and NaCl were able to induce reporter gene activity using promoter Fragment A in specific tissues of root where C2H4 was unable to induce activity. Further dissection of the promoter Fragment A into fragments containing either the conserved GCC element (PR); PR/AT; or G/AT sequences, and subsequent testing of these fragments fused to GUS in transgenic plants was performed. These experiments revealed that the promoter fragment containing PR element alone, although required, was barely able to allow responsiveness to C2H4. However, significant C2H4-induced activity was obtained with a promoter fragment containing the AT and PR elements together. [ABSTRACT FROM AUTHOR]

Transgenic tobacco plants that overproduce the tobacco anionic peroxidase wilt upon reaching maturity, although having functional stomata and normal vascular anatomy and physiology. These plants were examined further to determine the cause for wilting, and thus better understand how the anionic peroxidase functions in plant growth and development. Shoots from young peroxidase overproducing plants were grafted onto wild-type tobacco root stock to determine if the roots could absorb and transmit sufficient water to maintain leaf turgidity. These grafted plants never wilted when grown in the greenhouse though shoot peroxidase activity remained ten-fold greater than in control plants, thus indicating that wilting is a consequence of peroxidase expression in the roots. Close examination of root systems revealed considerably less root mass in the transformed plant, primarily exhibited through a decrease in branching. At flowering, root growth rate and total root mass in transformed plants were less than 50% of control plants although shoot mass and growth rate were unchanged. This is in contrast to root growth in young seedlings where transformed plants performed equivalently to controls. Root hydraulic conductivity was measured to evaluate the effect of elevated peroxidase expression on water absorption and transport; however, no significant change in hydraulic conductivity was found in transformed plants. The consequence of anionic peroxidase overexpression on indoleacetic acid (IAA) metabolism was also examined. No significant difference in IAA levels was observed; however, root elongation in plants overexpressing peroxidase was insensitive to exogenous IAA. It can be concluded that the overexpression of the tobacco anionic peroxidase in transformed plants results in diminished root mass from fewer root branches, which contributes to the wilting phenomenon seen in these plants. Further, this developmental change in transformed plants may be a consequence of the metabolism of IAA by the anionic peroxidase. [ABSTRACT FROM AUTHOR]

The isolation and the characterization of two tobacco cDNAs, Nt-ERabp1 and Nt-ERabp2, homologous to Zm-ERabp1, encoding the major auxin-binding protein from maize coleoptiles, are described. Their predicted amino acid sequences correspond to proteins of ca. 21 kDa, in which the characteristic regions common to ABP1-related polypeptides are well-conserved. Southern analysis indicates that the genes corresponding to Nt-ERabp1 cDNA and Nt-ERabp2 cDNA derive respectively from Nicotiana tomentosiformis and Nicotiana sylvestris, the diploid progenitors of Nicotiana tabacum. Analysis of mRNA distribution in tobacco plants indicates that these two genes are preferentially expressed in flowers and growing seedlings. Whatever the tissue tested, Nt-ERabp1 mRNA is more abundant than Nt-ERabp2 mRNA. Furthermore, RT-PCR reveals developmental and organ-specific expression of these two genes in flower parts of tobacco plants. In particular, regulation of Nt-ERabp1 mRNA accumulation appears to be correlated with elongation growth of each floral organ. Recombinant Nt-ERabp1, produced in Escherichia coli, is recognized by antibodies raised against Zm-ERabp1. [ABSTRACT FROM AUTHOR]

A genomic clone (Ext 1.4) encoding an extensin was isolated from a Nicotiana tabacum genomic library. The encoded polypeptide showed features characteristic of extensins such as Ser-(Pro)4 repeats and a high content in Tyr and Lys residues. The presence of one Tyr-Leu-Tyr-Lys motif suggests the possibility for one intramolecular isodityrosine cross-link whereas numerous Val-Tyr-Lys motifs may participate in intermolecular cross-links. This extensin appears to be close to an extensin already characterized in N. tabacum but very different from the Ext 1.2 extensin of N. sylvestris. The analysis of genomic DNA gel blots using probes spanning different parts of the gene showed that the Ext 1.4 gene belongs to a complex multigene family having one member originating from N. sylvestris and three members from N. tomentosiformis. The Ext 1.4 specific probe found a 1.4 kb mRNA in stems, roots, ovaries and germinating seeds of healthy plants. The Ext 1.4 gene family is strongly induced in actively dividing cell suspension cultures and after wounding of leaves or stems in conditions where root formation occurs. On the contrary, it is not induced in leaves in response to a hyperensitive reaction to a viral infection or after elicitor treatment. [ABSTRACT FROM AUTHOR]

We have previously reported the isolation of a cDNA encoding a mitotic cyclin, NTCYC1, from a tobacco cell suspension library. Here we describe the expression patterns of NTCYC1 and of Ntsuc1, a suc1 plant homologue, in synchronized tobacco cell suspensions. Furthermore, the expression pattern of this cyclin is compared to that of Ntcdc2-1, a Nicotiana tabacum homologue of cdc2. While no NTCYC1 transcript was detected in cells synchronized in the G1 and S phases, NTCYC1 expression was observed in late G2 and early M phases, disappearing in the G1′ of a new cell cycle. On the other hand, Ntsuc1 and Ntcdc2-1 exhibited a constitutive expression during the cell cycle. A functional analysis performed by microinjecting NTCYC1 mRNA into immature Xenopus oocytes, indicates that NTCYC1 could participate in the control of the G2/M transition in plant cells. Subsequently NTCYC1 expression was used to assess the status of mesophyll cells in expanded leaves of N. tabacum. Depending on leaf position along the shoot axis, a large population of mesophyll cells appeared with a 4C DNA content, suggesting a G2 arrest. It was found that leaves with such a population also contained high levels of NTCYC1 transcripts. With respect to these results concerning a naturally occurring G2-arrested cell population, the regulation of NTCYC1 expression in planta is discussed. [ABSTRACT FROM AUTHOR]