Tissue Print Hybridization Research Articles

IDENTIFICATION AND CHARACTERIZATION OF POTATO SPINDLE TUBER VIROID INFECTING SOLANUM JASMINOIDES AND S. RANTONNETII IN ITALY

Potato spindle tuber viroid (PSTVd), a quarantine pathogen included in the EPPO A2 list was identified in plants of Solanum jasminoides and S. rantonnetii in Italy. Molecular characterization of four S. jasminoides and one S. rantonnetii PSTVd isolates showed limited sequence variability. A tissue-printing hybridization method for detecting PSTVd in ornamental Solanaceae was successfully tested and its potential for large scale surveys is discussed. This appears to be the first report of PSTVd in Italy and the first report of PSTVd naturally infecting S. rantonnetii.

First Report of Cucurbit aphid-borne yellows virus in Spain.

In late spring 2003, field-grown melon plants (Cucumis melo L.) showing bright yellowing of older leaves were observed near Valladolises in Campo de Cartagena, Murcia, Spain. Symptoms resembled those caused by viruses of the genus Crinivirus (family Closteroviridae), but absence or very low populations of whiteflies were observed. However, diseased foci showed clear indications of heavy aphid infestations. Later, during the fall of 2003, squash plants (Cucurbita pepo L.) grown in open fields in the same area showed similar symptoms. Tissue print hybridizations to detect Cucurbit yellow stunting disorder virus (CYSDV) and Beet pseudo yellows virus (BPYV) in symptomatic samples were negative. CYSDV and BPYV are two yellowing-inducing criniviruses previously described in Spain. In contrast, standard double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) with antiserum against Cucurbit aphid-borne yellows virus (CABYV; genus Polerovirus, family Luteoviridae) that was kindly provided by H. Lecoq (INRA-Montfavet Cedex, France) were consistently positive. Definitive confirmation of CABYV associated with symptomatic samples was obtained by performing reverse-transcription polymerase chain reaction (RT-PCR) analyses for the CABYV coat protein gene. Total RNA extracts (TRI reagent; Sigma Chemical, St. Louis, MO) were obtained from symptomatic and asymptomatic leaf samples and RT-PCR reactions were carried out using the primers 5'-GAATACGGTCGCGGCTAGAAATC-3' (CE9) and 5'-CTATTTCGGGTTCTGGACCTGGC-3' (CE10) based on the CABYV sequence published by Guilley et al. (2). A single DNA product of approximately 600 bp was obtained only from symptomatic samples. Amplified DNA fragments from two independent samples (samples 36-2 and 37-5) were cloned in E. coli and sequenced (GenBank Accession Nos. AY529653 and AY529654). Sequence comparisons showed a 95% nucleotide sequence identity between the two sequences. A 97% and 94% nucleotide sequence identity was found among 36-2 and 37-5, respectively and the CABYV sequence published by Guilley et al. (2). CABYV seems to be widespread throughout the Mediterranean Basin (1,3) but to our knowledge, it has not previously been described in Spain. Additionally, our data suggest that significant genetic variability might be present in the Spanish CABYV populations.

Detection of Citrus Leaf Blotch Virus Using Digoxigenin-Labeled cDNA Probes and RT–PCR

Citrus leaf blotch virus (CLBV) was detected by dot-blot hybridization (DBH), and tissue print hybridization (TPH) and by one-step RT–PCR in citrus plants growing both in the greenhouse and in the field. DBH with digoxigeninlabeled cDNA probes allowed CLBV detection in dsRNA-rich and total RNA preparations equivalent to 5 and 0.1 mg of infected tissue, respectively. DBH gave intense signals with RNA extracts from young bark, tender shoots and young leaves, whereas the best hybridization signals with TPH were obtained using tender shoots and young leaf petioles. One-step RT–PCR was 10-fold more sensitive than DBH and amplification was obtained with all infected tissues. CLBV was readily detected in young leaves of infected Eureka lemon, Marsh grapefruit, Nules clementine, Navelina orange and Nagami kumquat in the greenhouse, using either hybridization or RT–PCR, but not in leaves of Pineapple sweet orange. Detection in field trees was less consistent and was only achieved by RT–PCR and DBH. CLBV was detected by DBH and RT–PCR in different citrus varieties from several geographic areas showing bud union crease on trifoliate rootstocks, but not in neighbor trees with the same symptoms or in other varieties showing bud union crease on those rootstocks. Failure to detect CLBV in trees with bud union crease could be due to low virus titer or uneven distribution within the plant. Alternatively, a different agent could be involved in causing bud union crease. Abbreviations: CLBV – Citrus leaf blotch virus; DBH – dot-blot hybridization; DIG-probes – digoxigenin-labeled cDNA probes; TPH – tissue print hybridization.

A new approach to evaluate relative resistance and tolerance of tomato cultivars to begomoviruses causing the tomato yellow leaf curl disease in Spain

A simple procedure to evaluate relative resistance and tolerance of tomato cultivars to the begomoviruses causing tomato yellow leaf curl (TYLC) disease in Spain was developed. To estimate the resistance and tolerance levels of a cultivar, several formulae were developed based on the ratio of infected plants, virus titre (estimated by tissue–print hybridization) and symptom intensity. The formulae were applied to five commercial tomato cultivars (Amoretto, Birloque, Royesta, Tovigreen and Ulises) naturally infected by TYLC viruses. The analyses showed that Ulises, Birloque and Tovigreen exhibited a moderate resistance, and Ulises was also highly tolerant. There was a positive correlation between symptom intensity and virus titre in infected plants, suggesting that the hybridization technique could also be used as an early estimator of tolerance. Finally, molecular hybridization and nucleotide sequence analyses of the begomovirus intergenic region showed that the local TYLC virus population consisted of a single species, Tomato yellow leaf curl virus (TYLCV, formerly TYLCV‐Israel), with low genetic variation (nucleotide identity between isolates higher than 97%).

Detection and typing of tomato yellow leaf curl viruses

The denomination Tomato yellow leaf curl virus (TYLCV) comprises several viruses that cause severe damage to tomato crops in warm and temperate regions worldwide. TYLCV viruses are widespread in the Mediterranean Basin, in which two species have been reported: Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV, previously TYLCV‐Is). The availability of methods convenient for the diagnosis of these viruses is essential. We have investigated several alternatives for reliable detection and differentiation of TYLCSV and TYLCV. Triple‐antibody sandwich enzyme‐linked immunosorbent assay (TAS‐ELISA) proved to be very useful for large‐scale diagnosis in field situations, but lacked discriminating capacity and sensitivity in the stages of infection in which low virus titre is present. The DNA‐based methods are suited to laboratory operations and plant disease clinics, where accuracy of detection and discrimination of viruses is required. Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) was the most reliable method to discriminate between TYLCSV and TYLCV, but is not suited to high sample turnover. For large‐scale testing, tissue print hybridization assay provides a reliable and sensitive alternative to PCR.

Genomic organization and expression properties of the VfENOD5 gene from broad bean ( Vicia faba L.)

A full-length cDNA encoding the broad bean ( Vicia faba L.) early nodulin VfENOD5 was isolated from a nodule cDNA library. In addition to the ENOD5 homologues from other legumes the derived VfENOD5 amino acid sequence also displayed homologies to the phytocyanin-related nodulins GmENOD55-2, MtENOD16, and MtENOD20. A close inspection of the ENOD5 proteins from broad bean, pea and vetch indicated that all these nodulins possess a putative C-terminal GPI-anchor signal sequence. This novel finding supports the hypothesis that ENOD5 is an arabinogalactan protein. Tissue print hybridizations revealed that the broad bean ENOD5 gene was not only expressed in the central tissues of root nodules. In contrast to other legumes hybridizing transcripts were also be detected in a narrow zone within the peripheral nodule tissues. Sequence analysis of a genomic clone indicated the presence of a single intron interrupting the VfENOD5 coding region at a position precisely corresponding to the MtENOD16 and MtENOD20 introns.

Tissue print hybridisation and reverse transcriptase PCR in the detection of infectious bursal disease viruses in bursal tissues

The genome segments of infectious bursal disease viruses (IBDV) in the bursa of Fabricius from experimently infected chickens or field samples were detected by tissue print hybridization (TPH) with subsequent reverse transcriptase (RT)- polymerase chain reaction (PCR). Bursae were imprinted onto nylon membrane and then hybridized with a cloned digoxigenin (DIG)-labeled cDNA probe. Tissue prints on nylon membrane were readily distinguished from control prints by color development and differences in signal intensity. In order to verify the TPH test, RT - PCR was used to amplify a 643-base pair fragment on the VP 2 gene of IBDV in the bursa of Fabricius. With all isolates, a c DNA fragment of 643 bp long was generated as expected and further confirmed the specificity ofTPH . Our results suggest that a large number of field samples or selected tissues can be rapidly examined by TPH technique when combined with a cloned DIG -labeled c DNA probe.

A small gene family of broad bean codes for late nodulins containing conserved cysteine clusters

Five transcripts encoding different members of a nodulin family with conserved cysteine clusters (Cys-X 4-Asp-Cys and Cys-X 4-Cys) were identified in broad bean root nodules. They displayed homologies to the early nodulins PsENOD3 and PsENOD14 and the late nodulin PsNOD6 from pea. In addition to the occurence of putative secretory signal peptides, the spatial distribution of the cysteine residues was comparable in both the broad bean and the pea nodulins. Based on tissue print hybridizations, we found that the corresponding broad bean genes VfNOD-CCP1, VfNOD-CCP3 and VfNOD-CCP5 were expressed in the interzone II–III and the nitrogen fixing zone III of mature nodules whereas the gene VfNOD-CCP4 was first induced in the prefixing zone II. A strong expression of the VfNOD-CCP2 gene only could be detected the interzone II–III region. Sequence analysis of a genomic VfNOD-CCP1 clone isolated revealed the presence of one intron seperating a first exon encoding the signal peptide from a second exon encoding the cysteine cluster domain of this nodulin. Apart from the multiple presence of the common nodulin motifs AAAGAT and CTCTT on both DNA strands of the putative VfNOD-CCP1 promoter region a sequence element resembling the organ specific element of the soybean lbc3 gene promoter was identified.

Expression of Bifunctional Caffeoyl-CoA 3-O-Methyltransferase in Stress Compensation and Lignification

Abstract: Caffeate and caffeoyl-CoA O-methyltransferases (COMTs and CCoAOMTs) catalyze the formation of ferulic acid and feruloyl-CoA, respectively, in many plants, and their physiological significance is under investigation. CCoAOMT was proposed to play a pivotal role in cell wall reinforcement during the induced disease resistance response, as exemplified in elici-tor-treated parsley cells, as well as in the formation of guaiacyl-and syringyl-type lignins. This requires selective substrate and tissue specificities. Parsley CCoAOMT expressed in E. coli methylated caffeoyl- or 5-hydroxyferuloyl-CoA to feruloyl- and sinap-oyl-CoA, whereas neither caffeate nor 5-hydroxyferulate was accepted. Tissue print hybridizations of parsley stem and root sections revealed, furthermore, that CCoAOMT mRNA is consti-tutively associated with the vascular tissues, but is also expressed in the surface cell layers upon wounding. In order to study the promoter activity of the parsley CCoAOMT gene, tobacco plantlets were transformed with parsley CCoAOMT promoter-GUS reporter gene constructs; these transformants, at the very young stage, expressed GUS activity in a narrow subapical root zone only extending later to the vascular tissue at the onset of xylem differentiation. GUS activity of the mature transgenic tobacco plants was observed exclusively in the parenchyma lining the differentiated xylem elements and xylem ray cells of root, stem or leaf tissues. Thus, parsley CCoAOMT is a bifunctional enzyme which appears to serve in both stress compensation and lignification. This was supported by the ontogenetic activity profile of tobacco endogeneous CCoAOMT, which correlated closely with the GUS expression under the control of parsley CCoAOMT promoter, while the proportion of CCoAOMT vs. COMT activities varied substantially during growth of the transgenic tobacco plants.

Expression of BifunctionaI Caffeoyl‐CoA 3‐O‐Methyltransferase in Stress Compensation and Lignification

Abstract: Caffeate and caffeoyl‐CoA O‐methyltransferases (COMTs and CCoAOMTs) catalyze the formation of ferulic acid and feruloyl‐CoA, respectively, in many plants, and their physiological significance is under investigation. CCoAOMT was proposed to play a pivotal role in cell wall reinforcement during the induced disease resistance response, as exemplified in elici‐tor‐treated parsley cells, as well as in the formation of guaiacyl‐and syringyl‐type lignins. This requires selective substrate and tissue specificities. Parsley CCoAOMT expressed in E. coli methylated caffeoyl‐ or 5‐hydroxyferuloyl‐CoA to feruloyl‐ and sinap‐oyl‐CoA, whereas neither caffeate nor 5‐hydroxyferulate was accepted. Tissue print hybridizations of parsley stem and root sections revealed, furthermore, that CCoAOMT mRNA is consti‐tutively associated with the vascular tissues, but is also expressed in the surface cell layers upon wounding. In order to study the promoter activity of the parsley CCoAOMT gene, tobacco plantlets were transformed with parsley CCoAOMT promoter‐GUS reporter gene constructs; these transformants, at the very young stage, expressed GUS activity in a narrow subapical root zone only extending later to the vascular tissue at the onset of xylem differentiation. GUS activity of the mature transgenic tobacco plants was observed exclusively in the parenchyma lining the differentiated xylem elements and xylem ray cells of root, stem or leaf tissues. Thus, parsley CCoAOMT is a bifunctional enzyme which appears to serve in both stress compensation and lignification. This was supported by the ontogenetic activity profile of tobacco endogeneous CCoAOMT, which correlated closely with the GUS expression under the control of parsley CCoAOMT promoter, while the proportion of CCoAOMT vs. COMT activities varied substantially during growth of the transgenic tobacco plants.

Differential regulation and distribution of Acridone Synthase in Ruta graveolens in honour of Professor G. H. Neil Towers 75th birthday

Cell suspension cultures of Ruta graveolens L. accumulate polyketide metabolites such as acridone alkaloids and flavonoid pigments. Whereas flavonoid synthesis is induced by light, the production of alkaloids can be enhanced in dark-cultured cells by treatment with fungal elicitors. Acridone synthase (ACS) catalyzes the committed condensing reaction of acridone biosynthesis yielding 1,3-dihydroxy-N-methylacridone from N-methylanthraniloyl- and malonyl-CoAs. The reaction proceeds in a manner analogous to that of chalcone synthase (CHS) which catalyzes the first committed step in flavonoid biosynthesis and cDNA and protein sequences of Ruta ACS possess a high degree of sequence homology to heterologous CHSs. ACS transcript abundance and specific activity were monitored in cultured R. graveolens cells irradiated either continuously with white light or treated with fungal elicitor over a period of 24 h and found to increase transiently upon elicitor treatment and to decrease upon light irradiation. Immunodetection with a rabbit polyclonal ACS antiserum revealed that the amounts of ACS polypeptide decreased slightly in light-irradiated cells but increased in elicitor-treated Ruta cells. Fluorescence microscopy and tissue print hybridizations were employed to aid in localizing the sites of storage and biosynthesis of acridone alkaloids in Ruta plants. Yellow fluorescing alkaloids were detected particularly in root tissue adjacent to the rhizodermis, but also in the endodermis and vascular tissue of the hypocotyl. ACS transcript abundance in situ followed the same spatial pattern, indicating that the synthesis of acridones likely proceeds at all sites of deposition rather than exclusively in the root. Expression in planta and the induction response of ACS suggest that the alkaloids serve as phytoanticipins or phytoalexins in the defense of Ruta particularly to soil-borne pathogens or as feeding deterrents.

Detection and tissue distribution of potato spindle tuberviroid in infected tomato plants by tissue print hybridization.

Potato spindle tuber viroid (PSTVd) was detected in two cultivars of tomato (Lycopersicon esculentum Mill.) by tissue print hybridization of cross-sections of stem and rhachis, using a 35S-labeled PSTVd RNA probe. PSTVd was detectable in the viroid-sensitive and symptom-developping cv "Rutgers" 2 weeks p.i., and in the viroid-tolerant and practically symptomless cv "Goldkugel" 3 weeks p.i. In both tomato cultivars, PSTVd accumulated in the upper parts of the plants newly grown after inoculation. It was predominantly found in association with the ring formed by the vascular tissue. The final accumulation of PSTVd as well as its spatial distribution were similar in the sensitive and in the tolerant tomato cultivar, as estimated from the tissue print autoradiographs. Thus, tissue print hybridization provides a rapid and sensitive means for viroid diagnosis and for the assessment of tissue-specific localization of the viroid RNA.

The asparagine synthetase gene VfAS1 is strongly expressed in the nitrogen-fixing zone of broad bean ( Vicia faba L.) root nodules

A full-length transcript sequence encoding the broad bean ( Vicia faba L.) asparagine synthetase (AS) VfAS1 was isolated from a nodule cDNA library. Sequence homologies indicated that VfAS1 belonged to the glutamine-dependent type of AS enzymes. The corresponding gene was highly expressed in root nodules and at a three-fold lower level in uninfected roots. Additionally, traces of VfAS1 transcripts were detected in epicotyl and stem tissues. Tissue-print hybridizations revealed that the VfAS1 gene was strongly expressed in the nitrogen-fixing zone III of root nodules. VfAS1 transcripts were absent from the meristem, the prefixing zone II as well as from peripheral nodule tissues.

The modular nodulins Nvf-28/32 of broad bean (Vicia faba L.): alternative exon combinations account for different modular structures.

The broad bean late nodulins, Nvf-28/32, are composed of two types of repetitively occurring sequence modules flanked by unique N- and C-terminal modules. Six isoforms of these nodulins were characterized by a specific modular structure resulting from a different individual order of repetitive sequence modules. A detailed analysis of genomic PCR fragments revealed that the repetitive modules and the N-terminal unique module exactly corresponded to exons, whereas the C-terminal module was specified by two exons. Since those exons encoding the repetitive modules missing in specific Nvf-28/32 isoforms were consistently present within genomic sequences, a post-transcriptional generation of VfNOD28/32 transcripts specifying six Nvf-28/32 nodulins was concluded. Using tissue-print hybridizations, these transcripts were localized in the interzone II-III and the nitrogen-fixing zone III of root nodules. From this and from cDNA-cDNA hybridizations demonstrating a comparable timing of expression of VfNOD28/32 and of leghemoglobin transcripts in root nodules, a function of the modular nodulins Nvf-28/32 in late developmental stages of broad bean nodules was inferred.

Genes Encoding 11S-Globulin-Like Proteins are Expressed in the Megagametophyte soon after Fertilization in Eastern White Pine (Pinus strobus L.)

The nucleotide sequence of two cDNA clones synthesized from eastern white pine (Pinus strobus L.) ovule mRNA showed significant sequence homology to the legumins, which correspond to the 11S-globulins of several angiosperms. Northern hybridizations with one of the cDNA clones, named Glb-2 (P. strobus 11S-globulin-like clone 2), revealed that the mRNA was present in the ovules from the early globular stage of embryogenesis through the mature seed stage but not in unfertilized ovules. Glb mRNA was found in the megagametophyte of ovules containing late-stage globular embryos by filter-bound RNA and tissue print hybridization experiments. No Glb mRNA was found in in vitro-cultured globular stage embryos with attached suspensors, sporophytic ovule tissues, whole seedlings, or mature needles.

Isolation and characterization of cDNAs encoding xylogenesis-associated and wounding-induced ribonucleases in Zinnia elegans

The study of plant ribonuclease (RNase) functions is complicated by a complex profile of RNase activities detected in tissues. Thus, isolation of individual RNase genes will be desirable for the further understanding of function of each RNase. Here, we describe the isolation of cDNAs encoding two RNases, ZRNaseI and ZRNaseII, in differentiating tracheary elements (TEs) induced from isolated mesophyll cells of Zinnia elegans. Both the ZRNaseI and ZRNaseII exhibit putative secretion signal sequences at the amino-terminal ends with predicted molecular masses of 24 247 Da and 22 448 Da as mature proteins, respectively. DNA gel blot analysis showed that both RNases in Zinnia appear to be encoded by a small gene family. RNA gel blot analysis showed that the expression of the ZRNaseI gene was associated with the late stage of in vitro TE differentiation, whereas the ZRNaseII gene was mainly induced in response to stress. Neither RNase gene was induced in response to phosphate starvation, or to H2O2 challenge in the cultured mesophyll cells, or to senescence in the leaves. In young leaves, the ZRNaseI gene was not induced in response to wounding. But the ZRNaseII gene was markedly induced by 6 h after wounding. Tissue print hybridization showed that the expression of the ZRNaseI gene was preferentially associated with the differentiation TEs in Zinnia stems, while the ZRNaseII mRNA was not detected in unwounded Zinnia organs. Taken together, the results indicated that the ZRNaseI gene is expressed during the process of xylogenesis both in vitro and in the plant, whereas the ZRNaseII gene is predominantly induced in response to wounding. The identification of these RNase genes provides molecular tools for the dissection of the process of autolysis during xylogenesis, and for the dissection of the role of RNase in wounding response.

The nodule-specific VfENOD-GRP3 gene encoding a glycine-rich early nodulin is located on chromosome I of Vicia faba L. and is predominantly expressed in the interzone II-III of root nodules

A nodule-specific cDNA was isolated from a Vicia faba L. nodule cDNA library. Since time course experiments revealed an early expression of this transcript in the nodule, this cDNA coded for an early nodulin and was designated VfENOD-GRP3. Based on tissue print hybridizations, we found a predominant expression of VfENOD-GRP3 transcripts in the interzone II-III region of broad bean root nodules. The encoded early nodulin ENOD-GRP3 was characterized by an N-terminal signal peptide and a C-terminal domain displaying a glycine content of 31%. Sequence analysis of a genomic VfENOD-GRP3 clone revealed that the signal peptide and the glycine-rich domain were specified by two separate exons. Primer extension experiments identified two adjacent transcription start sites for VfENOD-GRP3 transcripts. The common nodulin sequences 'AAAGAT' and 'CTCTT' were present five and three times on both DNA strands of the putative VfENOD-GRP3 promoter, respectively. Additionally, three sequence motifs resembling organ-specific elements of the soybean lbc3 gene promoter and a sequence similar to the binding site 1 for the nodule trans-acting factor Nat2 were identified. From Southern blot data and from sequence analysis of genomic PCR fragments, the presence of a VfENOD-GRP3 gene family was inferred. By PCR experiments using sequence-specific primers and DNA of microisolated chromosomes as a template, this family was located on the long arm of chromosome I.

Non-isotopic tissue-printing hybridization: a new technique to study long-distance plant virus movement.

A non-isotopic tissue-print hybridization technique was developed to study long-distance plant virus movement. By using digoxigenin-labeled RNA probes the distribution pattern of the viral RNA was observed in leaf, stem and petiole tissues. In leaf tissue viral RNA was confined preferentially to symptoms and veins, and in stem and petiole sections, the hybridization signal was observed in vascular tissue. Both chemiluminescent and colorigenic detection methods were used. The colorigenic method, though less sensitive, is advantageous in that it gives some anatomical information on the signal distribution. This non-isotopic tissue-print hybridization technique can provide considerable information about the spatial and temporal virus expression with regard to its symptoms.

Expression patterns of three genes in the stem of lucerne (Medicago sativa)

We have identified three stem abundantly expressed genes in lucerne (alfalfa, Medicago sativa). A cDNA library, constructed from lucerne stem polyadenylated RNA, was screened by differential hybridization. From this screening, cDNA clones that correspond to genes which are preferentially, or specifically, expressed in the stem were isolated. MsaS1 encodes an unidentified protein, MsaS2 encodes an S-adenosyl-homocysteine hydrolase and MsaS3 encodes an extensin-like protein. Northern blot analysis of RNA isolated from individual stem internodes indicated that the three corresponding genes show differing developmental patterns of expression. The expression of MsaS1 was confined to the youngest stem tissue and may be regulated by sucrose. In stem tissue the level of RNA for the three genes decreased in response to wounding. Tissue print hybridization analysis was used to localize the expression of the genes to the xylem side of vascular bundles in lucerne stems.

Expression of an auxin- and cytokinin-regulated gene in cambial region in Zinnia.

The expression patterns of a cDNA clone, p48h-10, of an auxin-induced gene were examined in isolated mesophyll cells of Zinnia and in the organs of Zinnia plants. In the isolated mesophyll cells, the mRNA accumulates in 48 hr of culture with 1-naphthaleneacetic acid alone. Because the first cell division occurs before 36 hr of culture, the gene probably is not involved in cell division. Benzyladenine does not induce expression of this gene, but the combination of 1-naphthaleneacetic acid and benzyladenine induces the mRNA accumulation about 24 hr earlier than does 1-naphthaleneacetic acid alone. Tissue print hybridization shows that the mRNA is present predominantly in the cambial region in stems, leaves, and roots and in the vascular bundles in flower buds but does not occur in the apical regions of shoot or root. The characteristics of the gene expression, including auxin- and cytokinin-regulated induction and cambial region localization, encourage us to suggest that the gene is involved in the early process of vascular differentiation.