Tobacco Homolog Research Articles

Evaluation of reference genes for miRNA and mRNA normalization in tobacco infected with PVYNTN, PVYN-Wi and PVYZ-NTN strains

This is the first report on identification of the most suitable reference genes for RT-qPCR quantification of miRNA and mRNA in tobacco response to the prevalent recombinant potato virus Y (PVY) strains PVYNTN, PVYN-Wi and the newly identified PVYZ-NTN. Of 10 tested genes, the expression levels of neIF5C, nU2af and nPP2A were the most stable in samples taken from non-inoculated, mock-inoculated, and infected plants at three days post-inoculation (dpi) and 14 dpi. While the homologues of eIF5 were most stably expressed in tobacco in this study and in potato in our previous study (Yin et al., 2021) following inoculation with the same three PVY strains, the homologues of other two genes PP2A and U2af were stably expressed only in tobacco but unstable in potato. The tobacco homologue of PP2A, which was the most stably expressed one in tobacco interaction with PVYNTN, PVYN-Wi and PVYZ-NTN strains in this study, was the least stable one in tobacco interaction with the non-recombinant PVYO strain in a previous study (Baek et al., 2017). This study provides evidence on the influence of host species on expression of housekeeping genes and points out virus strain as a new factor influencing expression stability of reference gene. Caution should be taken when choosing reference genes in gene expression study in Solanaceae hosts response to different PVY strains.

Hypersensitivity-Related Genes HSR201 and HSR203J Are Regulated by Calmodulin-Binding Protein 60-Type Transcription Factors and Required for Pathogen Signal-Induced Salicylic Acid Synthesis.

Salicylic acid (SA) plays a key role in plant resistance to pathogens. In Arabidopsis, the isochorismate synthase pathway mainly contributes to pathogen-induced SA synthesis, and the expression of SA synthesis genes is activated by two calmodulin (CaM)-binding protein 60 (CBP60)-type transcription factors, CBP60g and SARD1. In tobacco, the mechanisms underlying SA synthesis remain largely unknown. SA production is induced by wounding in tobacco plants in which the expression of two stress-related mitogen-activated protein kinases is suppressed. Using this phenomenon, we identified genes whose expression is associated with SA synthesis. One of the genes, NtCBP60g, showed 23% amino acid sequence identity with CBP60g. Transient overexpression of NtCBP60g as well as NtSARD1, a tobacco homolog of SARD1, induced SA accumulation in Nicotiana benthamiana leaves. NtCBP60g and NtSARD1 bound CaM, and CaM enhanced SA accumulation induced by NtCBP60g and NtSARD1. Conversely, mutations in NtCBP60g and NtSARD1 that abolished CaM binding reduced their ability to induce SA. Expression profiling and promoter analysis identified two hypersensitivity-related genes, HSR201 and HSR203J as the targets of NtCBP60g and NtSARD1. Virus-induced gene silencing of both NtCBP60g and NtSARD1 homologs compromised SA accumulation and the expression of HSR201 and HSR203J homologs, which were induced by a pathogen-derived elicitor in N. benthamiana leaves. Moreover, elicitor-induced SA accumulation was compromised by silencing of the HSR201 homolog and the HSR203J homolog. These results suggested that HSR201 and HSR203J are regulated by NtCBP60g and NtSARD1 and are required for elicitor-induced SA synthesis.

The Minus-End-Directed Kinesin OsDLK Shuttles to the Nucleus and Modulates the Expression of Cold-Box Factor 4.

The transition to terrestrial plants was accompanied by a progressive loss of microtubule minus-end-directed dynein motors. Instead, the minus-end-directed class-XIV kinesins expanded considerably, likely related to novel functions. One of these motors, OsDLK (Dual Localisation Kinesin from rice), decorates cortical microtubules but moves into the nucleus in response to cold stress. This analysis of loss-of-function mutants in rice indicates that OsDLK participates in cell elongation during development. Since OsDLK harbours both a nuclear localisation signal and a putative leucin zipper, we asked whether the cold-induced import of OsDLK into the nucleus might correlate with specific DNA binding. Conducting a DPI-ELISA screen with recombinant OsDLKT (lacking the motor domain), we identified the Opaque2 motif as the most promising candidate. This motif is present in the promoter of NtAvr9/Cf9, the tobacco homologue of Cold-Box Factor 4, a transcription factor involved in cold adaptation. A comparative study revealed that the cold-induced accumulation of NtAvr9/Cfp9 was specifically quelled in transgenic BY−2 cells overexpressing OsDLK-GFP. These findings are discussed as a working model, where, in response to cold stress, OsDLK partitions from cortical microtubules at the plasma membrane into the nucleus and specifically modulates the expression of genes involved in cold adaptation.

Identification of candidate MLO powdery mildew susceptibility genes in cultivated Solanaceae and functional characterization of tobacco NtMLO1.

Specific homologs of the plant Mildew Locus O (MLO) gene family act as susceptibility factors towards the powdery mildew (PM) fungal disease, causing significant economic losses in agricultural settings. Thus, in order to obtain PM resistant phenotypes, a general breeding strategy has been proposed, based on the selective inactivation of MLO susceptibility genes across cultivated species. In this study, PCR-based methodologies were used in order to isolate MLO genes from cultivated solanaceous crops that are hosts for PM fungi, namely eggplant, potato and tobacco, which were named SmMLO1, StMLO1 and NtMLO1, respectively. Based on phylogenetic analysis and sequence alignment, these genes were predicted to be orthologs of tomato SlMLO1 and pepper CaMLO2, previously shown to be required for PM pathogenesis. Full-length sequence of the tobacco homolog NtMLO1 was used for a heterologous transgenic complementation assay, resulting in its characterization as a PM susceptibility gene. The same assay showed that a single nucleotide change in a mutated NtMLO1 allele leads to complete gene loss-of-function. Results here presented, also including a complete overview of the tobacco and potato MLO gene families, are valuable to study MLO gene evolution in Solanaceae and for molecular breeding approaches aimed at introducing PM resistance using strategies of reverse genetics.Electronic supplementary materialThe online version of this article (doi:10.1007/s11248-015-9878-4) contains supplementary material, which is available to authorized users.

Erratum to: A tobacco homolog of DCN1 is involved in pollen development and embryogenesis

Erratum to: A tobacco homolog of DCN1 is involved in pollen development and embryogenesis

A tobacco homolog of DCN1 is involved in pollen development and embryogenesis.

We show that DCN1 binds ubiquitin and RUB/NEDD8, associates with cullin, and is functionally conserved. DCN1 activity is required for pollen development transitions and embryogenesis, and for pollen tube growth. Plant proteomes show remarkable plasticity in reaction to environmental challenges and during developmental transitions. Some of this adaptability comes from ubiquitin-mediated protein degradation regulated by cullin-RING E3 ubiquitin ligases (CRLs). CRLs are activated through modification of the cullin subunit with the ubiquitin-like protein RUB/NEDD8 by an E3 ligase called defective in cullin neddylation 1 (DCN1). Here we show that tobacco DCN1 binds ubiquitin and RUB/NEDD8 and associates with cullin. When knocked down by RNAi, tobacco pollen formation was affected and zygotic embryogenesis was blocked around the globular stage. Additionally, we found that RNAi of DCN1 inhibited the stress-triggered reprogramming of cultured microspores from their intrinsic gametophytic mode of development to an embryogenic state. This stress-induced developmental switch is a known feature in many important crops and leads ultimately to the formation of haploid embryos and plants. Compensating the RNAi effect by re-transformation with a promoter-silencing construct restored pollen development and zygotic embryogenesis, as well as the ability for stress-induced formation of embryogenic microspores. Overexpression of DCN1 accelerated pollen tube growth and increased the potential for microspore reprogramming. These results demonstrate that the biochemical function of DCN1 is conserved in plants and that its activity is involved in transitions during pollen development and embryogenesis, and for pollen tube growth.

Identification of Natural Diterpenes that Inhibit Bacterial Wilt Disease in Tobacco, Tomato and Arabidopsis

The soil-borne bacterial pathogen Ralstonia solanacearum invades a broad range of plants through their roots, resulting in wilting of the plant, but no effective protection against this disease has been developed. Two bacterial wilt disease-inhibiting compounds were biochemically isolated from tobacco and identified as sclareol and cis-abienol, labdane-type diterpenes. When exogenously applied to their roots, sclareol and cis-abienol inhibited wilt disease in tobacco, tomato and Arabidopsis plants without exhibiting any antibacterial activity. Microarray analysis identified many sclareol-responsive genes in Arabidopsis roots, including genes encoding or with a role in ATP-binding cassette (ABC) transporters, and biosynthesis and signaling of defense-related molecules and mitogen-activated protein kinase (MAPK) cascade components. Inhibition of wilt disease by sclareol was attenuated in Arabidopsis mutants defective in the ABC transporter AtPDR12, the MAPK MPK3, and ethylene and abscisic acid signaling pathways, and also in transgenic tobacco plants with reduced expression of NtPDR1, a tobacco homolog of AtPDR12. These results suggest that multiple host factors are involved in the inhibition of bacterial wilt disease by sclareol-related compounds.

The TobaccoBLADE-ON-PETIOLE2Gene Mediates Differentiation of the Corolla Abscission Zone by Controlling Longitudinal Cell Expansion

The BLADE-ON-PETIOLE (BOP) genes of Arabidopsis (Arabidopsis thaliana) have been shown to play an essential role in floral abscission by specializing the abscission zone (AZ) anatomy. However, the molecular and cellular mechanisms that underlie differentiation of the AZ are largely unknown. In this study, we identified a tobacco (Nicotiana tabacum) homolog of BOP (designated NtBOP2) and characterized its cellular function. In tobacco plants, the NtBOP2 gene is predominantly expressed at the base of the corolla in an ethylene-independent manner. Both antisense suppression of NtBOP genes and overexpression of NtBOP2 in tobacco plants caused a failure in corolla shedding. Histological analysis revealed that the differentiation of the corolla AZ was blocked in the transgenic flowers. This blockage was due to uncontrolled cell elongation at the region corresponding to wild-type AZ. The role of NtBOP2 in regulating cell elongation was further demonstrated in Bright Yellow 2 single cells: perturbation of NtBOP2 function by a dominant negative strategy led to the formation of abnormally elongated cells. Subcellular localization analysis showed that NtBOP2-green fluorescent protein fusion proteins were targeted to both the nucleus and cytoplasm. Yeast two-hybrid, firefly luciferase complementation imaging, and in vitro pull-down assays demonstrated that NtBOP2 proteins interacted with TGA transcription factors. Taken together, these results indicated that NtBOP2 mediated the differentiation of AZ architecture by controlling longitudinal cell growth. Furthermore, NtBOP2 may achieve this outcome through interaction with the TGA transcription factors and via an ethylene-independent signaling pathway.

Deubiquitinating enzymes AtUBP12 and AtUBP13 and their tobacco homologue NtUBP12 are negative regulators of plant immunity

• Signalling by ubiquitination is implicated in diverse aspects of the plant lifecycle, and enzymes of ubiquitin metabolism are overrepresented in the Arabidopsis genome compared with other model eukaryotes. Despite the importance of ubiquitination in the regulation of signalling, little is known about deubiquitinating enzymes, which reverse the process of ubiquitination. • Transgenic RNA interference-based cosuppression and the isolation of Atubp12/13 double mutants collectively provides the first report that AtUBP12 and AtUBP13 are functionally redundant and are required for immunity against virulent Pseudomonas syringae pv tomato in Arabidopsis. The Solanaceous AtUBP12 orthologue NtUBP12 was identified. Viral-induced gene silencing and transient gain-of-function assays were employed to establish that the NtUBP12 protein functions as a negative regulator of the Cf-9-triggered hypersensitive response. • Here, we demonstrate that NtUBP12 and AtUBP12 are bona fide deubiquitinating enzymes capable of cleaving lysine-48-linked ubiquitin chains. AtUBP12 and NtUBP12 are functionally interchangeable and their deubiquitinating activity is required to suppress plant cell death. • Overall, our data implicate AtUBP12- and NtUBP12-dependent deubiquitination in the stabilization of common substrates across Solanaceae and Brassicaceae which regulate disease resistance.

A Tobacco Homolog of DCN1 is Involved in Cellular Reprogramming and in Developmental Transitions

AbstractPlant proteomes show remarkable plasticity in reaction to environmental challenges and during developmental transitions. Some of this adaptability comes from ubiquitin-mediated protein destruction regulated by cullin-RING E3 ubiquitin ligases (CRLs). CRLs are activated through modification of the cullin subunit with the ubiquitin-like protein RUB/NEDD8 by an E3 ligase called defective in cullin neddylation 1 (DCN1). Here we show that tobacco DCN1 binds ubiquitin and RUB/NEDD8, and associates with cullin. When knocked down by RNAi, tobacco pollen formation stopped and zygotic embryogenesis was blocked around the globular stage. Additionally, we found that RNAi of DCN1 inhibited the stress-triggered reprogramming of cultured microspores from their intrinsic gametophytic mode of development to an embryogenic state. This stress-induced developmental switch is a known feature in many important crops and leads ultimately to the formation of haploid embryos and plants. Compensating the RNAi effect by re-transformation with a promoter-silencing construct restored pollen development and zygotic embryogenesis, as well as the ability for stress-induced formation of embryogenic microspores. Overexpression of DCN1, however, accelerated pollen tube growth and increased the potential for microspore reprogramming. These results demonstrate that the biochemical function of DCN1 is conserved in plants and that its activity is specifically required for transitions during pollen development and embryogenesis, and for pollen tube tip growth.

A spontaneous dominant-negative mutation within a 35S::AtMYB90 transgene inhibits flower pigment production in tobacco.

BackgroundIn part due to the ease of visual detection of phenotypic changes, anthocyanin pigment production has long been the target of genetic and molecular research in plants. Specific members of the large family of plant myb transcription factors have been found to play critical roles in regulating expression of anthocyanin biosynthetic genes and these genes continue to serve as important tools in dissecting the molecular mechanisms of plant gene regulation.FindingsA spontaneous mutation within the coding region of an Arabidopsis 35S::AtMYB90 transgene converted the activator of plant-wide anthocyanin production to a dominant-negative allele (PG-1) that inhibits normal pigment production within tobacco petals. Sequence analysis identified a single base change that created a premature nonsense codon, truncating the encoded myb protein. The resulting mutant protein lacks 78 amino acids from the wild type C-terminus and was confirmed as the source of the white-flower phenotype. A putative tobacco homolog of AtMYB90 (NtAN2) was isolated and found to be expressed in flower petals but not leaves of all tobacco plants tested. Using transgenic tobacco constitutively expressing the NtAN2 gene confirmed the NtAN2 protein as the likely target of PG-1-based inhibition of tobacco pigment production.ConclusionsMessenger RNA and anthocyanin analysis of PG-1Sh transgenic lines (and PG-1Sh x purple 35S::NtAN2 seedlings) support a model in which the mutant myb transgene product acts as a competitive inhibitor of the native tobacco NtAN2 protein. This finding is important to researchers in the field of plant transcription factor analysis, representing a potential outcome for experiments analyzing in vivo protein function in test transgenic systems that over-express or mutate plant transcription factors.

The tobacco MAP215/Dis1-family protein TMBP200 is required for the functional organization of microtubule arrays during male germline establishment

The haploid microspore division during pollen development in flowering plants is an intrinsically asymmetric division which establishes the male germline for sexual reproduction. Arabidopsis gem1 mutants lack the male germline as a result of disturbed microspore polarity, division asymmetry, and cytokinesis and represent loss-of-function mutants in MOR1/GEM1, a plant orthologue of the conserved MAP215/Dis1 microtubule associated protein (MAP) family. This provides genetic evidence for the role of MAP215/Dis1 in the organization of gametophytic microtubule arrays, but it has remained unknown how microtubule arrays are affected in gem1 mutant microspores. Here, novel male gametophytic microtubule-reporter Nicotiana tabacum plants were constructed, expressing a green fluorescent protein-α-TUBULIN fusion protein (GFP-TUA6) under the control of a microspore-specific promoter. These plants allow effective visualization of all major male gametophytic microtubule arrays and provide useful tools to study the regulation of microtubule arrays by MAPs and other effectors. Depletion of TMBP200, a tobacco homologue of MOR1/GEM1 in gametophytic microtubule-reporter plants using microspore-targeted RNA interference, induced defects in microspore polarity, division asymmetry and cytokinesis that were associated with striking defects in phragmoplast position, orientation, and structure. Our observations further reveal a requirement for TMBP200 in gametophytic spindle organization and a novel role in spindle position and orientation in polarized microspores. These results provide direct evidence for the function of MAP215/Dis1 family protein TMBP200 in the organization of microtubule arrays critical for male germline formation in plants.

GTP Is Required for the Microtubule Catastrophe-Inducing Activity of MAP200, a Tobacco Homolog of XMAP215

Widely conserved among eukaryotes, the microtubule-associated protein 215 (MAP215) family enhances microtubule dynamic instability. The family member studied most extensively, Xenopus laevis XMAP215, has been reported to enhance both assembly and disassembly parameters, although the mechanism whereby one protein can exert these apparently contradictory effects has not been clarified. Here, we analyze the activity of a plant MAP215 homolog, tobacco (Nicotiana tabacum) MAP200 on microtubule behavior in vitro. We show that, like XMAP215, MAP200 promotes both assembly and disassembly parameters, including microtubule growth rate and catastrophe frequency. When MAP200 is added to tubulin and taxol, strikingly long-coiled structures form. When GDP partially replaces GTP, the increase of catastrophe frequency by MAP200 is strongly diminished, even though this replacement stimulates catastrophe in the absence of MAP200. This implies that MAP200 induces catastrophes by a specific, GTP-requiring pathway. We hypothesize that, in the presence of MAP200, a catastrophe-prone microtubule lattice forms occasionally when elongated but nonadjacent protofilaments make lateral contacts.

The MYB305 Transcription Factor Regulates Expression of Nectarin Genes in the Ornamental Tobacco Floral Nectary

We have isolated and characterized the cDNA encoding the ornamental tobacco (Nicotiana langsdorffii X N. sanderae) homolog of the antirrhinum (Antirrhinum majus) MYB305. This transcription factor was robustly expressed at Stage 12 of nectary development but was only weakly expressed in the earlier Stage 6 nectaries. The ornamental tobacco MYB305 contains a conserved R2R3 MYB DNA binding domain with 76 amino acids in the activation domain. A green fluorescent protein-MYB305 fusion localized to nucleus of tobacco protoplasts and yeast one-hybrid assays demonstrated that it functions as a transcription activator. A conserved 23-amino acid C-terminal domain is required to activate gene expression. The coding region of the myb305 cDNA was expressed in Escherichia coli as a glutathione S-transferase fusion protein and was purified to homogeneity. This protein shows binding to two consensus MYB binding sites on the ornamental tobacco Nectarin I (nec1) promoter as well as to the single site located on the Nectarin V (nec5) promoter. Deletions of either of the binding sites from the nec1 promoter significantly reduced expression in nectary tissues. Temporally, MYB305 expression precedes that of nec1 and nec5, as would be expected if the MYB305 factor regulates expression of the nec1 and nec5 genes. Ectopic expression of MYB305 in foliage was able to induce expression of both nec1 and nec5, as well as two flavonoid biosynthetic genes in the foliage. Finally, RNA interference knockdown of MYB305 resulted in reduced expression of both nectarins and flavonoid biosynthetic genes. We conclude that expression of MYB305 regulates expression of the major nectarin genes in the floral nectary.

Tobacco Arp3 is localized to actin-nucleating sites in vivo

The polarity of actin is a central determinant of intracellular transport in plant cells. To visualize actin polarity in living plant cells, the tobacco homologue of the actin-related protein 3 (ARP3) was cloned and a fusion with the red fluorescent protein (RFP) was generated. Upon transient expression of these fusions in the tobacco cell line BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2), punctate structures were observed near the nuclear envelope and in the cortical plasma. These dots could be shown to decorate actin filaments by expressing RFP–ARP3 in a marker line, where actin was tagged by GFP (green fluorescent protein)–FABD (fimbrin actin-binding domain 2). When actin filaments were disrupted by latrunculin B or by prolonged cold treatment, and subsequently allowed to recover, the actin filaments reformed from the RFP–ARP3 structures, that therefore represented actin nucleation sites. The intracellular distribution of these sites was followed during the formation of pluricellular files, and it was observed that the density of RFP–ARP3 increased in the apex of the polarized, terminal cells of a file, whereas it was equally distributed in the central cells of a file. These findings are interpreted in terms of position-dependent differences of actin organization.

Involvement of EIN3 homologues in basic PR gene expression and flower development in tobacco plants

The TEIL (Tobacco EIN3-Like) gene is a tobacco homologue of arabidopsis Ethylene Insensitive 3 (EIN3), and the gene product binds an 8 bp sequence in the tobacco PR1a promoter in a sequence specific manner. It was found here that accumulation of TEIL transcript was induced by wounding and preceded basic PR gene expression. To study the downstream signalling pathway of TEIL, TEIL was overexpressed under the control of the constitutive 35S promoter in tobacco plants. In 35S::TEIL lines, basic PR genes, which are wound-, jasmonate-, and ethylene-inducible, were expressed constitutively. Next, the conserved 781 bp sequence among tobacco EIN3-like (EIL) protein genes was introduced as an inverted-repeat (IR) into tobacco to suppress expression of these genes. In two independent IRTEIL lines, the TEIL transcript was not found and transcripts of other tobacco EILs, NtEIL3, and NtEIL5, were reduced. In IRTEIL plants, wound-, jasmonate-, and ACC-induced accumulation of basic PR gene transcripts was significantly inhibited. These results indicate that TEIL functions upstream of tobacco basic PR genes in wound signalling via not only ethylene but also jasmonate. In 35S::TEIL plants, the pistil length of the flower was longer with a slight protrusion of the stigma compared with the control. In IRTEIL plants, the length of the stamens was shorter than the control with significant protrusion of the stigma in the flower. These observations indicate the involvement of tobacco EILs in flower development.

MAPK Cascade in Stomatal Specification

Stomata are special structures in plant leaves that allow gas and water vapor exchange between plants and the environment. Stomata are separated by pavement cells. Wang et al . used genetic approaches to show that in Arabidopsis thaliana stomatal specification involves a mitogen-activated protein kinase (MAPK) cascade. Because double knockout of MPK3 and MPK6 is embryonic-lethal and single loss-of-function mutants do not exhibit any phenotype, Wang et al . created single-knockout mpk6 −/− plants in which MPK3 was reduced by RNA interference. These plants arrested as seedlings and showed clustered stomata instead of the normal arrangement in which stomata are always separated by one pavement cell. A similar phenotype was observed in mpk3 , mpk6 double-mutant plants that were rescued with a steroid-inducible MPK6 gene. MKK4 and MKK5 are the upstream kinases to MPK3 and MPK6 in stress response pathways, and RNA interference of these two MAPKKs simultaneously caused the seedling leaves to have all stomata with no pavement cells. Gain-of-function experiments were performed using a steroid-inducible, constitutively activated mutant of the tobacco homolog of MKK4 and MKK5 (GVG-Nt-MEK DD ). These plants had no stomata, but when crossed into either mpk6 −/− or mpk3 −/− mutant background, then normal stomata and stomatal arrangement were restored. The dose of MAPK signaling appears important for proper specification of stomatal cell fate. Plants deficient in the MAPKKK Yoda (YDA) also have a clustered stomata phenotype, and GVG-Nt-MEK DD suppressed this clustered phenotype in a dose-dependent manner, with higher concentrations of steroid suppressing the clustering more effectively than lower concentrations. In-gel kinase assays showed that the activity of MPK3 and MPK6 was increased in plants in which YDA was constitutively activated. Examination of cotyledons in the various mutant plants at various times during development showed that the stomatal phenotypes were associated with aberrations in asymmetric cell divisions. Fewer asymmetric cell divisions occurred in plants in which the pathway was hyperactivated (GVG-Nt-MEK DD ) and ectopic cell divisions occurred, all producing small stomatal cells in plants in which the pathway was inactivated (rescued mpk3 , mpk6 double mutants). H. Wang, N. Ngwenyama, Y. Liu, J. C. Walker, S. Zhang, Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis . Plant Cell 19 , 63-73 (2007). [Abstract] [Full Text]

Purification and Characterization of Plant Dynamin from Tobacco BY-2 Cells

We purified an 84 kDa polypeptide from the MAP (microtubule-associated protein) fraction of tobacco BY-2 cultured cells. LC/MS/MS (liquid chromatography-tandem mass spectrometry) analysis revealed that this polypeptide is a tobacco homolog of AtDRP3 (Arabidopsis thaliana dynamin-related protein 3). Electron microscopy revealed that NtDRP3 (Nicotiana tabacum dynamin-related protein 3) assembles to form a filamentous structure. When GDP was added to the NtDRP3 fraction, the filaments disappeared and many particles appeared. Biochemical analysis revealed that NtDRP3 could bind to and bundle both microtubules and actin filaments in vitro.

A Mitogen-activated Protein Kinase NtMPK4 Activated by SIPKK is Required for Jasmonic Acid Signaling and Involved in Ozone Tolerance via Stomatal Movement in Tobacco

The mitogen-activated protein kinase (MAPK) cascade is involved in responses to biotic and abiotic stress in plants. In this study, we isolated a new MAPK, NtMPK4, which is a tobacco homolog of Arabidopsis MPK4 (AtMPK4). NtMPK4 was activated by wounding along with two other wound-responsive tobacco MAPKs, WIPK and SIPK. We found that NtMPK4 was activated by salicylic acid-induced protein kinase kinase (SIPKK), which has been isolated as an SIPK-interacting MAPK kinase. In NtMPK4 activity-suppressed tobacco, wound-induced expression of jasmonic acid (JA)-responsive genes was inhibited. NtMPK4-silenced plants showed enhanced sensitivity to ozone. Inversely, transgenic tobacco plants, in which SIPKK or the constitutively active type SIPKK(EE) was overexpressed, exhibited greater responsiveness to wounding with enhanced resistance to ozone. We further found that NtMPK4 was expressed preferentially in epidermis, and the enhanced sensitivity to ozone in NtMPK4-silenced plants was caused by an abnormal regulation of stomatal closure in an ABA-independent manner. These results suggest that NtMPK4 is involved in JA signaling and in stomatal movement.

A Tobacco Calcium/Calmodulin-binding Protein Kinase Functions as a Negative Regulator of Flowering

A tobacco calcium/calmodulin-binding protein kinase (NtCBK1) was isolated and identified. The predicted NtCBK1 protein has 599 amino acids, an N-terminal kinase domain, and shares high homology with other calmodulin (CaM)-related kinases. Whereas NtCBK1 phosphorylates itself and substrates such as histone IIIS and syntide-2 in the absence of CaM, its kinase activity can be stimulated by tobacco CaMs. However, unlike another tobacco protein kinase designated NtCBK2, NtCBK1 was not differentially regulated by the different CaM isoforms tested. The CaM-binding domain of NtCBK1 was located between amino acids 436 and 455, and this domain was shown to be necessary for CaM modulation of kinase activity. RNA in situ hybridization showed that NtCBK1 was highly regulated in the transition to flowering. Whereas NtCBK1 mRNA was accumulated in the shoot apical meristem during vegetative growth, its expression was dramatically decreased in the shoot apical meristem after floral determination, and in young flower primordia. The expression of NtCBK1 was up-regulated to high levels in floral organ primordia. Fluctuations in NtCBK1 expression were verified by analysis of tobacco plants expressing green fluorescent protein under the control of the NtCBK1 promoter, suggesting a role of NtCBK1 in the transition to flowering. This conclusion was confirmed by overexpressing NtCBK1 in transgenic tobacco plants, where maintenance of high levels of NtCBK1 in the shoot apical meristem delayed the switch to flowering and extended the vegetative phase of growth. Further work indicated that overexpression of NtCBK1 in transgenic tobacco did not affect the expression of NFL, a tobacco homologue of the LFY gene that controls meristem initiation and floral structure in tobacco. In addition, the promotion of tobacco flowering time by DNA demethylation cannot be blocked by the overexpression of NtCBK1.