Increase of endogenous zeatin riboside by introduction of the ipt gene in wild type and the lateral suppressor mutant of tomato

We studied axillary meristem formation of the lateral suppressor (ls) mutant of tomato after elevating the endogenous cytokinin levels through introduction of the isopentenyltransferase (ipt) gene from Agrobacterium tumefaciens. Growth and development of several transformants were examined during in vitro culture. Transformants exhibited phenotypes varying in severity and were divided into four classes. A number of the ipt transformants had a normal phenotype, as non-transformed plants. Others showed a mild to severe ‘cytokinin-like’ phenotype. Transformants with a mild phenotype exhibited reduced internode length and reduced root development. Transformants with a severe phenotype showed even shorter internodes, loss of apical dominance, reduction of leaf size, production of callus at the basis of the shoots and absence of root development or development of green non-branching roots. The severity of the phenotype correlated well with the level of ipt gene expression, as measured by northern analysis. Transformants with a severe phenotype also exhibited increased levels of zeatin riboside, but zeatin levels were not elevated. The increase in endogenous zeatin riboside levels in the ls mutant did not restore axillary meristem formation, but sometimes bulbous structures were formed in the initially ‘empty’ leaf axils. Several adventitious meristems and shoots developed from below the surface of these structures. It is concluded that a reduced level of cytokinins in the ls mutant shoots is not responsible for the absence of axillary meristem formation.     

Gibberellins and the procera mutant of tomato

The procera mutant of tomato (Lycopersicon esculentum L.) has a phenotype which is remarkably similar to that of normal tomatoes treated with exogenous gibberellin (GA), indicating that it might be a GA over-producer. However, analysis of endogenous GAs by gas chromatography-mass spectrometry showed that Procera actually has lower levels of GA₂₀ and GA₁ than normal. The reason for these anomalously low GA levels is not clear, as there was no difference between procera and normal plants in their ability to metabolize [³H]GA₂₀. The procera mutant responded to exogenous gibberellic acid with increased extension growth, but the proportional response for a given dose of GA was the same in procera and normal plants. It therefore appears that the procera mutation does not directly affect either the GA status of the plant, or its ability to respond to GA.Abbreviations GA gibberellin - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - MeTMSi methyl trimethylsilyl - SIM selected ion monitoring     

The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene

The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with ³H and auxin was found to promote conversion of zeatin-type cytokinins to ³H-labelled adenine derivatives. When the very rapid metabolism of exogenous [³H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.Abbreviations Ade adenine - Ados adenosine - BA 6-benzylaminopurine - C control - Con A concanavallin A - CP cellulose phosphate - IPT isopentenyl transferase - NAA 1-naphthylacetic acid - NP normal phase - NPPU N-(3-nitrophenyl)-N-phenylurea - RIA radioimmunoassay - RP reversed phase We wish to thank Dr. J. Zwar for supplying phenylurea derivitives.     

Cloning an ipt gene from Agrobacterium tumefaciens: characterisation of cytokinins in derivative transgenic plant tissue

The isopentenyl transferase gene was isolated from Agrobacterium tumefaciens AcH5 using polymerase chain reaction and transformed into Petunia and Kalanchoë using both A. tumefaciens and A. rhizogenes transformation systems. Morphological evidence and elevated endogenous cytokinin levels indicated that the PCR product was an active gene. Accurate quantification of the cytokinins was obtained by radioimmunoassay, following purification and separation of the free bases and ribosides by HPLC. Of the six cytokinins quantified, zeatin riboside and its stabilised dihydro-derivative, dihydrozeatin riboside, showed the greatest increases in the transformed Petunia tissue (up to 600-fold). The importance of measuring changes in individual cytokinins is discussed.     

Immunocytochemical localization of cytokinins in Craigella tomato and a sideshootless mutant

Post-embedding immunocytochemical techniques using peroxidase-antiperoxidase or immunoglobulin G-gold as markers were used for the localization of cytokinins (CKs) in two isogenic lines, Craigella (C) and Craigella lateral suppressor (Cls), of tomato Lycopersicon esculentum Mill. Terminal buds, nodes, hypocotyl segments and root tips were submitted to a periodate-borohydride procedure, to obtain the coupling of isopentenyladeosine and zeatin riboside to cellular proteins, followed by a fixative step with a paraformaldehyde and glutaraldehyde mixture. Enzyme-linked immunosorbent assay tests performed on ovalbumin-coated microtitration plates have shown that this method was effective for CK riboside and base coupling to proteins. Paraffin-wax- or Spurr's-resin-embedded sections were cleared of wax or resin before incubation with anti-zeatin riboside or anti-isopentenyladenosine antibodies. The procedure was thoroughly investigated and many controls were done in order to eliminate artefacts. The immunostaining patterns observed along the plants showed a basipetally decreasing gradient of CKs along the stem and in the roots. Immunolabelling was higher in the actively growing regions of the stem bud and root apices. Terminal buds of Cls appeared to be less immunoreactive than C, whereas no differences were detected in root-tip immunolabelling. The staining patterns are consistent with the idea that root and bud apices have a different CK metabolism. The absence of axillary bud formation in Cls is correlated with low CK levels in the organogensis sites.Abbreviations C Craigella, isogenic line - CK cytokinin - Cls Craigella lateral suppressor - EDC 1-(3-dimethylaminopropyl)3-ethylcarbodiimide hydrochloride - ELISA enzyme-linked immunosorbent assay - 2iP isopentenyladenine - 2iPA isopentenyladenosine - PAP peroxidase-anti-peroxidase - PFAG paraformaldehyde/glutaraldehyde mixture - Z zeatin - ZR zeatin riboside     

Accumulation of an ABA analogue in the wilty tomato mutant,flacca

A new abscisic acid (ABA) analogue has been isolated from tomato plants. High levels of the compound are found in flacca mutants compared with normal isogenic controls. The analogue also accumulates in response to water stress. Three alternative structures, consistent with the mass spectrum, have been proposed. The possibility that the compound may be a biosynthetic precursor of ABA is considered.We are grateful for the financial support of the ARC.     

Somatic instability of carotenoid biosynthesis in the tomato ghost mutant and its effect on plastid development

The tomato (Lycopersicon esculentum (L.) Mill.) ghost plant is a mutant of the San Marzano cultivar affected in carotenoid biosynthesis. ghost plants exhibit a variable pattern of pigment biosynthesis during development. Cotyledons are green but true leaves are white. Green sectors, which appear to be clonal in origin, are frequently observed in the white tissue. Because of the lack of photosynthesis ghost plants have a very low viability in soil. We have developed a strategy for propagating ghost plants that employs organ culture to generate variegated green-white plants which, supported by the photosynthetic green areas, develop in soil to almost wild-type size. These plants were used to analyze the pigment content of the different tissues observed during development and plastid ultrastructure. Cotyledons and green leaves contain both colored carotenoids and chlorophyll but only the colorless carotenoid phytoene accumulates in white leaves. the plastids in the white tissue of ghost leaves lack internal membrane structures but normal chloroplasts can be observed in the green areas. The chromoplasts of white fruits are also impaired in their ability to form thylakoid membranes.     

Fatty acid content in wild type and WZSL mutant of Euglena gracilis

The effects of growth conditions on fatty acid profilewere examined in the photosynthetic wild type and inthe spontaneous non-photosynthetic WZSL mutant of theunicellular flagellate Euglena gracilis. Inthe light, the amount of polyunsaturated fatty acids(PUFAs) is higher in the wild type than in the mutant,independent of the carbon source. Among importantPUFAs, linolenic acid (18:3 3) is present inhigh amount only in wild type cells grown in the lightwith any of the tested carbon sources. The content ofother PUFAs, such as arachidonic acid (20:46), EPA (20:5 3) and DHA (22:63), is not correlated with the presence oflight or chloroplasts.The main effect of the dark in both strains is tolower the content of PUFAs and mono-unsaturated fattyacids and to increase the content of saturated fattyacids with all the carbon sources.     

The SELF-PRUNING gene family in tomato

The SELF PRUNING (SP) gene controls the regularity of the vegetative-reproductive switch along the compound shoot of tomato and thus conditions the 'determinate' (sp/sp) and 'indeterminate' (SP_) growth habits of the plant. SP is a developmental regulator which is homologous to CENTRORADIALIS (CEN) from Antirrhinum and TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT) from Arabidopsis. Here we report that SP is a member of a gene family in tomato composed of at least six genes, none of which is represented in the tomato EST collection. Sequence analysis of the SP gene family revealed that its members share homology along their entire coding regions both among themselves and with the six members of the Arabidopsis family. Furthermore, members of the gene family in the two species display a common genomic organization (intron-exon pattern). In tomato, phylogenetically close homologues diverged considerably with respect to their organ expression patterns while SP2I and its closest homologue from Arabidopsis (MFT) exhibited constitutive expression. This research focusing on a plant of sympodial growth habit sets the stage for a functional analysis of this weakly expressed gene family which plays a key role in determining plant architecture.     

Molecular analysis of the phytochrome deficiency in an aurea mutant of tomato

Summary The au ^w mutant allele of the aurea locus in tomato has previously been shown to cause deficiency for the phytochrome polypeptide (Parks et al. 1987). We have begun to characterize the molecular basis and consequences of this deficiency. Genomic Southern blot analysis indicates that there are at least two and probably more phytochrome polypeptide structural genes in tomato. RNA blot analysis shows that the au ^w mutant contains normal levels of phytochrome mRNA and in vitro translation of au ^w poly(A)⁺ RNA yields a phytochrome apoprotein that is quantitatively and qualitatively indistinguishable on SDS-polyacrylamide gels from that synthesized from wild-type RNA. These results indicate that the phytochrome deficiency in aurea is not the result of lack of expression of phytochrome genes but is more likely due to instability of the phytochrome polypeptide in planta. Possible reasons for such instability are discussed. Analysis of the molecular phenotype of aurea indicates that the phytochrome-mediated increase in the abundance of the mRNA encoding chlorophyll a/b binding protein (cab) is severely restricted in the mutant as compared with wild-type tomato. Thus, the au w strain exhibits defective photoregulation of gene expression consistent with its very reduced level of the phytochrome photoreceptor.     

The genetic relationship between the tomato mutants,flacca and lateral suppressor,with reference to abscisic acid accumulation

Two tomato mutants, Lycopersicon esculentum flacca and lateral suppressor, are assigned to map position 59 of chromosome 7. The tight linkage between these two gene loci was detected as a result of attempts to establish whether they would exhibit phenotypic interaction. The possibility that both mutants result in abnormalities of abscisic acid (ABA) accumulation is considered. ABA analysis supports the suggestion that plants homozygous for flacca have a substantially lower concentration but indicates that lateral suppressor homozygotes do not differ from normal in ABA content. An attempt is made to reconcile the results with those of Tucker (1976, New. Phytol. 77, 561–568) by suggesting that lateral suppressor plants may accumulate high levels of an ABA metabolite which is indistinguishable from ABA using the Commelina epidermal strip bioassay.Abbreviations ABA abscisic acid - flc flacca - ls lateral suppressor - La Lanceolate     

Analysis of insecticidal activity in transgenic plants carrying the ipt plant growth hormone gene

The expression of a bacterial cytokinin biosynthesis gene (PI-II-ipt) in Nicotiana plumbaginifolia Viviani plants has been correlated with enhanced resistance to Manduca sexta and Myzus persicae. We expressed the PI-II-ipt gene in N. tabacum and Lycopersicon esculentum and observed similar antifeedent effects with the transgenic tobacco but not tomato. A 30 to 50 % reduction in larval weight gain was observed with some of the tomato plants but these results could not be repeated consistently. Leaf surface extracts from transgenic N. plumbaginifolia leaves killed 100 % of M. sexta second instars at concentrations of 0.05 % (w/v) whereas the N. tabacum extracts were at least 20 times less active. Extract suspensions were stable for up to 2 days at ambient temperatures below 42 °C and for at least 3 months at 4 °C when stored in the dark. HPLC analysis of the N. plumbaginifolia extracts yielded an active fraction that reduced hatching of M. sexta eggs by 30 % and killed first, second and third instars within 24, 48 and 72 hours of exposure, respectively. The activity appears to be associated with oxygen-containing aliphatic compounds, possibly diterpenes, as analyzed by TLC, UV absorption and fragmentation with EIMS. Based on the partial characterization of this activity, the production, secretion or accumulation of secondary metabolites in leaves of cytokinin producing PI-II-ipt N. plumbagini-folia plants appears to be responsible for the observed insect resistance.     

Characterization of the growth and auxin physiology of roots of the tomato mutant,diageotropica

Roots of the tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibit an altered phenotype. These roots are agravitropic and lack lateral roots. Relative to wild-type (VFN8) roots, dgt roots are less sensitive to growth inhibition by exogenously applied IAA and auxin transport inhibitors (phytotropins), and the roots exhibit a reduction in maximal growth inhibition in response to ethylene. However, IAA transport through roots, binding of the phytotropin, tritiated naphthylphthalamic acid ([³H]NPA), to root microsomal membranes, NPA-sensitive IAA uptake by root segments, and uptake of [³H]NPA into root segments are all similar in mutant and wild-type roots. We speculate that the reduced sensitivity of dgt root growth to auxin-transport inhibitors and ethylene is an indirect result of the reduction in sensitivity to auxin in this single gene, recessive mutant. We conclude that dgt roots, like dgt shoots, exhibit abnormalities indicating they have a defect associated with or affecting a primary site of auxin perception or action.Abbreviations BCA bicinchoninic acid - IAA indole 3-acetic acid - dgt diageotropica - IC₅₀ concentration for 50% inhibition of growth - NPA N-1-naphthylphthalamic acid - SCB-1 semicarbazone 1 This research was supported by grants from Sandoz Agro, Inc. (G.K.M), the National Aeronautics and Space Administration (NASA) and the National Science Foundation (T.L.L), and NASA (D.L.R.).     

Identification and quantitation by GC-MS of zeatin and zeatin riboside in xylem sap from rootstock and scion of grafted apple trees

Zeatin and zeatin riboside were identified by full-scan gas chromatography-mass spectrometry (GC-MS) in xylem sap of clonal apple rootstocks (M.27, M.9 and MM.106). These rootstocks exhibit a wide range of control over tree size when grafted to a common scion. The concentrations of zeatin and zeatin riboside were measured by GC-MS selected ion monitoring (SIM) in shoot xylem sap and root pressure exudate obtained from these rootstocks and from trees of Fiesta scion grafted onto the rootstocks. Zeatin was the predominant cytokinin in xylem sap from the dwarfing rootstocks, M.27 and M.9, while zeatin riboside was the predominant cytokinin in xylem sap from the more invigorating rootstock MM.106. Cytokinin concentrations (ng ml^–1) in root pressure exudate and shoot xylem sap, (i.e. from above the graft union in composite trees), increased with increasing vigour of the rootstock, irrespective of whether the plants were non-grafted rootstocks, or were composite plants of Fiesta scion grafted onto the rootstocks. Cytokinin content (ng shoot^–1) of shoot sap differed with rootstock; the more invigorating (MM.106) had greater amounts of cytokinins than the more dwarfing (M.9 and M.27) rootstocks. These results are discussed in relation to possible influences of roots on the growth of shoots via cytokinin supplies in the xylem sap.     

Plant growth regulator and graft control of axillary bud formation and development in the TO-2 mutant tomato

The torosa-2 tomato mutant is characterized by a strong inhibition of release of axillary shoots, that is not under the control of the main apex and IAA. Microscopic examination indicated that about 70% of leaf axils do not have axillary buds. Of the growth regulators tested, gibberellic acid and cytokinins were able to modify the to-2 phenotype: increasing bud number (GA₃ treated) and developing shoots (both substances). Sequential application of growth regulators demonstrated that bud production was only affected by treatments given between sowing time and 32 days after germination. Grafting experiments indicated that endogenous root factors have no essential role in the lateral branching of the genotypes investigated. The control of axillary bud differentiation and the branching pattern in the to-2 appears to be dependent of a complex mechanism involving gibberellins and cytokinins.     

Flowering in the uniflora mutant of tomato (Lycopersicon esculentum Mill.): description of the reproductive structure and manipulation of flowering time

The uniflora (uf) mutant of tomato (Lycopersicon esculentum Mill.) is known to produce solitary, normal, fertile flowers instead of inflorescences. Histological and SEM studies revealed that this unusual reproductive structure resulted from the inability of the plant to produce an inflorescence and not from post-initiation abortion processes affecting young flower buds. Development prior to floral transition was apparently not affected by the mutation since rates of germination and leaf initiation were identical in both uf and the Ailsa Craig (AC) initial cultivar. However, the time of flowering of the mutant was always delayed as compared to AC. In uf, environmental conditions markedly influenced flowering time which occurred early in all individuals in summer, but was strongly delayed during winter, with less than 20% plants reaching flowering before having initiated 40 leaves. Defoliation treatments stimulated floral transition in uf plants since 100% flowering occurred whatever the season and since the time of floral transition was usually advanced in comparison to the non-defoliated control plants. Similarly, compared to intact uf plants, flowering of terminal meristem of cuttings and upper axillary bud of decapitated plants was promoted. The involvement of correlative influences and assimilate availability in the control of flowering in tomato is suggested by these findings.