EFFECT OF HORMONES AND VITAMIN B12 ON GAMETOPHORE DEVELOPMENT IN THE MOSS PYLAISIELLA SELWYNII

Bud formation in the moss Pylaisiella selwynii is greatly enhanced by cytokinins at concentrations as low as 10⁻¹²m , yet these buds usually fail to develop into normal gametophores. Various ratios at different concentrations of the cytokinin N-6-γ,γ-dimethylallylaminopurine to indoleacetic acid failed to enhance bud initiation over that obtained with cytokinin alone or to permit normal gametophore development. Deletion of the cobaltous ions from the culture medium prevented the appearance of the few gametophores usually formed in the complete medium, but different amounts of cobaltous ion did not significantly enhance initiation of gametophore development. Bud initiation was enhanced 3- to 20-fold by vitamin B₁₂ at 10⁻⁵m or by B₁₂ coenzyme at 10⁻⁴m , and the time of appearance of these buds was advanced by 6–12 days compared to control plants. At these concentrations of the B₁₂ compounds the buds formed normal gametophores, but at 10⁻⁴m vitamin B₁₂ they grew into callus-like masses similar to those obtained with cytokinins. Although the effects of B₁₂ on bud initiation and development mimicked those of cytokinins, except in permitting normal development, no additive or synergistic effects were observed when they were tested together. It is suggested that B₁₂ may play a regulatory role in the control of gametophore initiation and development in mosses.     

Expression of the isopentenyl transferase gene is regulated by auxin in transgenic tobacco tissues

The isopentenyl transferase gene (ipt) fromAgrobacterium tumefaciens was isolated and introduced, via a disarmed binary vector, into tobacco using theAgrobacterium tumefaciens-mediated gene transfer system. The expression of theipt gene was monitored by RNA hybridization, western blotting and cytokinin analysis. The addition of auxin to the media rapidly reduced the level of cytokinins in the transgenic tissues and this was associated with a reduction in IPT mRNA and protein levels. It is concluded that the hormone auxin can regulate expression of a gene involved in biosynthesis of the second hormone cytokinin. Although exogenous benzyladenine did not directly affectipt gene expression, it did antagonize the effect of auxin on levels of cytokinins and IPT mRNA and protein.     

Heteroblasty in the moss,Aphanoregma patens (Physcomitrella patens), results from progressive modulation of a single fundamental leaf developmental programme

Abstract

Leaves at the apex of a mature Aphanoregma patens (Hedw.) Lindb. (Physcomitrella patens (Hedw.) Bruch Schimp. in B.S.G.) gametophore differ markedly in size and form from those at its base. To determine how these differences are produced during development, we first examined qualitative and quantitative differences between successive leaves along the stem and among leaves at different developmental stages. Differences between successive leaves were slight and cumulative. Local changes in cell number and size combined to produce a regularly shaped and approximately bilaterally symmetrical leaf suggesting that cell division and cell expansion are regionally regulated and coordinated at the organ level. The midrib and marginal teeth are discrete characters, which were prefigured by changes in cell shape in leaves that lacked these characters. In leaf primordia, cell proliferation was responsible for most of the changes in leaf form and size early in development and may have continued as cell expansion took over as the primary contributor to leaf growth and morphogenesis. Thus, leaf heteroblasty in Physcomitrella probably results from modulation of a single developmental programme by external and/or internal forces, which alter progressively in intensity as a gametophore grows. We applied exogenous cytokinin and auxin separately to growing cultures to explore their effects on leaf growth. Cytokinin and auxin stimulated leaf cell division and leaf cell elongation, respectively. Also, young upper leaves of gametophores exposed to exogenous auxin closely resembled basal leaves of untreated plants. Therefore, endogenous cytokinins and auxins may be among the modulating internal forces involved in leaf morphogenesis and the establishment of leaf heteroblasty.     

Isopentenyladenine from mutants of the moss, Physcomitrella patens

The culture media from gametophore over-producing mutants of the moss Physcomitrella patens have been examined for their cytokinin content. Two cytokinins have been detected, one of which has been identified as N⁶-(Δ²-isopentenyl) adenine (2iP).     

Photoaffinity labelling with the cytokinin agonist azido-CPPU of a 34 kDa peptide of the intracellular pathogenesis-related protein family in the moss Physcomitrella patens

As in higher plants, the development of the moss Physcomitrella patens is regulated by environmental signals and phytohormones. At the protonema level transition from chloronema to caulonema cells is under auxin control. The formation on second sub-apical caulonema cells of buds that will give rise to the leafy gametophore requires cytokinins. Using [³H]azidoCPPU (1-(2-azido-6-chloropyrid-4-yl)-3-(4-[³H])phenylurea), a photoactivatable cytokinin agonist, we have specifically photolabelled a soluble 34 kDa protein of P. patens. Urea derivatives were very efficient competitors of photolabelling while purine-type cytokinins were poor competitors. The protein UBP34 was purified by affinity chromatography and the sequences of six internal peptides obtained. A cDNA encoding UBP34 was cloned by screening a P. patens protonema cDNA library with a probe amplified by PCR using degenerate primers designed from the peptide sequences. The UBP34 amino acid sequence shows an average sequence identity of 42% with both intracellular PR proteins and the BetV1-related family of plant allergens. Recombinant UBP34 expressed in Escherichia coli was confirmed to bind azidoCPPU.     

Characterisation of a temperature-sensitive gametophore over-producing mutant of the moss, Physcomitrella patens

Summary A mutant of the moss, Physcomitrella patens, was isolated which was temperature-sensitive for the production of gametophores. At 17° C this mutant, designated ove 409, produced normal leafy shoots. At 24° C ove 409 produced many abnormal buds characteristic of bud-over-producing (ove) mutants. ove 409 produced an intermediate phenotype at 21° C. The cytokinin levels in the culture medium of this mutant, the wild-type and a cytokinin overproducing mutant, oveA78, were measured by combined gas chromatography mass spectrometry at the permissive and nonpermissive temperatures. Production of cytokinin was found to be affected by temperature in all strains; the change in phenotype of ove 409 correlated with the production of N⁶-(²-isopentenyl) adenine. Complementation analysis was performed using this mutant by protoplast fusion. ove 409 was found to be in the same complementation group as a previously isolated ove mutant, oveA78.     

Regulation of cytokinin oxidase activity in tobacco callus expressing the T-DNA ipt gene

There are indications that the cytokinin content in transgenic tissues expressing the cytokinin biosynthetic ipt gene is under metabolic control, which prevents the accumulation of cytokinins to lethal levels. The objective of this study was to investigate the relationships between the content of endogenous cytokinins and the activity of cytokinin oxidase (which is believed to be a copper-containing amine oxidase, EC 1.4.3.6.) in ipt transgenic tobacco callus. In addition, the effect of exogenously applied N-benzyladenine (BA) on this relationship was examined. Endogenous cytokinin concentrations were measured in callus of Nicotiana tabacum L. cv. Petit Havana SRI transformed with the ipt of Agrobacterium tumefaciens under the control of a light-inducible promoter and in non-transformed tissue using LC-tandem mass spectrometry. The activity of cytokinin oxidase was estimated by measuring the conversion of [2,8-³H]N⁶-(Δ²-isopentenyl)adenine to [³H]adenine by enzyme preparations in vitro. The 14-day-old ipt-transformed callus contained a 25-fold higher amount of cytokinins as compared to the non-transformed tissue. Mainly zeatin- and dihydrozeatin-types of cytokinins (free bases, ribosides, nucleotides and O-glucosides) accumulated in the ipt transgenic tissue. The cytokinin pool of both ipt-transformed and non-transformed tissues consisted predominantly of cytokinins that are either resistant to cytokinin oxidase attack (nucleotides and O-glucosides of cytokinins and cytokinins bearing N⁶-saturated side chain) or have a low affinity for the enzyme (zeatin and its riboside). The former represented 71.6 and 74.8% and the latter 27.7 and 24.4% of the pool of endogenous cytokinins in ipt-transformed and non-transformed tissues, respectively. Enzyme preparations from ipt-transformed tissue exhibited 1.5-fold higher cytokinin oxidase activity compared with that observed in control tissues. Application of exogenous BA affected the total levels of cytokinins of the two tissue lines in different ways. The cytokinin content increased by 1.7- and 1.5-fold in ipt-transformed tissues 6 and 12 h after BA application, respectively, while it declined in the non-transformed control by 1.6- to 2.0-fold between 3 and 12 h after BA application. The increase in cytokinin content in the ipt callus is due to an increase of zeatin- and dihydrozeatin-type cytokinins (nucleotides, ribosides and free bases) leading to an enhanced accumulation of O-glucosides after 12 h. Following BA treatment, the cytokinin oxidase activity increased up to 1.8-fold in ipt-transformed and 1.6-fold in non-transformed tissues. The levels of isopentenyl-type cytokinins were near the detection limit; however, the enhancement of cytokinin oxidase activity after BA treatment in both tissue lines was correlated with the content of preferred substrate of the enzyme, N⁶-(Δ²-isopentenyl)adenosine.     

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.     

Analysis of gametophytic development in the moss,Physcomitrella patens,using auxin and cytokinin resistant mutants

Mutants altered in their response to auxins and cytokinins have been isolated in the moss Physcomitrella patens either by screening clones from mutagenized spores for growth on high concentrations of cytokinin or auxin, in which case mutants showing altered sensitivities can be recognized 3–4 weeks later, or by non-selective isolation of morphologically abnormal mutants, some of which are found to have altered sensitivities. Most of the mutants obtained selectively are also morphologically abnormal. The mutants are heterogeneous in their responses to auxin and cytokinin, and the behaviour of some is consistent with their being unable to make auxin, while that of others may be due to their being unable to synthesize cytokinin. Physiological analysis of the mutants has shown that both endogenous auxin and cytokinin are likely to play important and interdependent roles in several steps of gametophytic development. Although their morphological abnormalities lead to sterility, genetic analysis of some of the mutants has been possible by polyethyleneglycol induced protoplast fusion.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - NAA 1-naphthalene acetic acid - 2,4D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - IAP 6-( ²isopentenyl) aminopurine - NAR NAA resistant mutants - BAR BAP resistant mutants     

DEVELOPMENT AND GAMETOPHORE INITIATION IN THE MOSS PYLAISIELLA SELWYNII AS INFLUENCED BY AGROBACTERIUM TUMEFACIENS

Spores of the heterotrichous moss Pylaisiella selwynii Kindb. were sown in a defined inorganic liquid culture medium and incubated at 27 C with a 16-hr photoperiod. They germinated at 7–10 days, and formed a few caulonemal buds at 27–30 days which developed into gametophores by 40 days. Bud formation and gametophore development followed a pattern common to many mosses. Addition of a virulent strain of Agrobacterium tumefaciens (B6) to the moss cultures increased bud formation and hastened the time of their appearance by 5–6 days. With 10⁹ or more bacteria per ml of moss culture medium the percentage of plants with gametophores at day 35 after the spores were sown was 96 % or greater, as opposed to 0–24 % in the controls. The mean number of gametophores per responding plant was also increased from one per plant in controls to 4–6 per plant in inoculated cultures. Addition of the bacterium at day 17–18 of culture was as effective as early additions of the bacterium, suggesting that the moss must become ready to bud before the bacterium can influence its development. The promotion of gametophore formation was directly related to the number of bacteria added and depended upon the presence of viable bacteria. The supernatant from bacterial cultures did not promote gametophore formation. The changes induced by A. tumefaciens were similar to those reported for cytokinins.     

Effect of Phytohormone Biosynthesis Genes (ipt and iaaM + iaaH) on the Sexual Reproduction of Transgenic Tobacco Plants

Transformation of Nicotiana silvestris Spegar et Comes plants by the ipt or iaaM + iaaH genes changed the hormonal status of plant reproductive organs. The total content of cytokinins and ABA increased, whereas IAA content in the pistils and anthers of the ipt-plants did not change. Reduced fertility of the ipt plants correlated with an elevated cytokinin and ABA contents of their reproductive organs. Pollen tubes of these plants showed defective growth in pistils in situ, and ovaries manifested a low metabolic activity. The transgenic (iaaM + iaaH)-plants were characterized by an elevated IAA content and reduced ABA content, whereas the total content of cytokinins did not change. The fertility of these plants did not differ from that in the wild type.     

The unique bryophyte-specific repeat-containing protein SHORT-LEAF regulates gametophore development in moss

Convergent evolution of shoot development across plant lineages has prompted numerous comparative genetic studies. Though functional conservation of gene networks governing flowering plant shoot development has been explored in bryophyte gametophore development, the role of bryophyte-specific genes remains unknown. Previously, we have reported Tnt1 insertional mutants of moss defective in gametophore development. Here, we report a mutant (short-leaf; shlf) having two-fold shorter leaves, reduced apical dominance, and low plasmodesmata frequency. UHPLC-MS/MS-based auxin quantification and analysis of soybean (Glycine max) auxin-responsive promoter (GH3:GUS) lines exhibited a striking differential auxin distribution pattern in the mutant gametophore. Whole-genome sequencing and functional characterization of candidate genes revealed that a novel bryophyte-specific gene (SHORT-LEAF; SHLF) is responsible for the shlf phenotype. SHLF represents a unique family of near-perfect tandem direct repeat (TDR)-containing proteins conserved only among mosses and liverworts, as evident from our phylogenetic analysis. Cross-complementation with a Marchantia homolog partially recovered the shlf phenotype, indicating possible functional specialization. The distinctive structure (longest known TDRs), absence of any known conserved domain, localization in the endoplasmic reticulum, and proteolytic cleavage pattern of SHLF imply its function in bryophyte-specific cellular mechanisms. This makes SHLF a potential candidate to study gametophore development and evolutionary adaptations of early land plants.     

A CELLULOSE SYNTHASE (CESA) gene essential for gametophore morphogenesis in the moss Physcomitrella patens

In seed plants, different groups of orthologous genes encode the CELLULOSE SYNTHASE (CESA) proteins that are responsible for cellulose biosynthesis in primary and secondary cell walls. The seven CESA sequences of the moss Physcomitrella patens (Hedw.) B. S. G. form a monophyletic sister group to seed plant CESAs, consistent with independent CESA diversification and specialization in moss and seed plant lines. The role of PpCESA5 in the development of P. patens was investigated by targeted mutagenesis. The cesa5 knockout lines were tested for cellulose deficiency using carbohydrate-binding module affinity cytochemistry and the morphology of the leafy gametophores was analyzed by 3D reconstruction of confocal images. No defects were identified in the development of the filamentous protonema or in production of bud initials that normally give rise to the leafy gametophores. However, the gametophore buds were cellulose deficient and defects in subsequent cell expansion, cytokinesis, and leaf initiation resulted in the formation of irregular cell clumps instead of leafy shoots. Analysis of the cesa5 knockout phenotype indicates that a biophysical model of organogenesis can be extended to the moss gametophore shoot apical meristem.     

Restoration of shooty morphology of a nontumorous mutant of Nicotiana glauca x N. langsdorffii by cytokinin and the isopentenyltransferase gene

The shooty morphology of a nontumorous amphidiploid mutant of Nicotiana glauca Grah. x N. langsdorffii Weinm. was restored by cytokinins, whether exogenously applied or endogenously produced by transformation of the mutant with a transfer DNA (T-DNA) cytokinin-biosynthesis gene (isopentenyltransferase; ipt). Auxins alone did not confer this effect. Similar transformation was not achieved for the parental species. In the case of transformation with the ipt gene, selection of the transformed tissues was based on its hormone-independent growth in the presence of the antibiotic kanamycin. Transformed tissues exhibited a shooty morphology, indistinguishable from that of wildtype genetic tumors N. glauca x N. langsdorffii. This altered phenotype was caused by the presence and constitutive expression of the ipt gene. The insertion and expression of this gene in transformed tissues was confirmed by using the polymerase chain reaction (PCR) technique as well as conventional molecular hybridization analysis. Expression of the ipt gene led to an elevated level of cytokinin in the transformed mutant tissues. This evidence supports the notion that genetic tumors are caused, at least in part, by elevated levels of cytokinin in interspecific hybrids.     

Genetic analysis of a mutant class of Physcomitrella patens in which the polarity of gravitropism is reversed.

Summary In the moss Physcomitrella patens, single-cell protonemata and multicellular gametophores respond to reorientation relative to the gravity vector by growing negatively gravitropically. A mutant class in which the protonemata, but not the gametophores, respond by growing towards gravity has been identified. In this paper, we describe the isolation of additional mutants of this class. Complementation and segregation ratio analyses were carried out on these mutants, which indicate that a single gene may mutate to switch the polarity of gravitropism.     

Fatty acid composition of mutants of the moss Physcomitrella patens

The fatty acid composition of various mutant strains of the moss Physcomitrella patens has been compared to the wild-type. These included strains defective in their responses to auxins and/or cytokinins, one which releases much more cytokinin into the medium than the wild-type, and two aphototropic strains. The lipids of the aphototropic mutants were also studied after culture in different light regimes. Although some differences in fatty acid composition have been found between strains, these alone are probably not responsible for their physiological differences. Considerable changes occur in many fatty acids in senescent or dark-grown material, including changes in the proportion of C₂₀ polyenoic fatty acids.     

Genetic analysis by somatic hybridization of cytokinin overproducing developmental mutants of the moss, Physcomitrella patens

Summary Cytokinins are important regulators of growth and development in lower and higher eukaryotic plants. Genetic analysis by means of somatic hybridization, achieved through protoplast fusion, revealed that, of 15 independently isolated gametophore and cytokinin over-producing (OVE) mutants in the model system,Physcomitrella patens, 14 carry recessive mutations responsible for this abnormal phenotype. Seven of these strains have been assigned to three complementation groups:OVEA, OVEB andOVEC. A further three strains have been demonstrated not to belong to theOVEA group and another mutant does not fall into groupOVEB. Phenotypic segregation ratios among progeny obtained following self-fertilization of a number of different somatic hybrids showed that severalOVE mutations behave as recessive alleles of single Mendelian genes.     

Gravitropic responses of wild-type and mutant strains of the moss Physcomitrella patens

The gravitropic responses of dark-grown caulonemata and gametophores of wild-type and mutant strains of the moss Physcomitrella patens have been investigated. In the wild-type both caulonemata and gametophores show negative orthogravitropism. No gravitropic response is observed when plants are rotated slowly on a clinostat and the inductive effect of gravity can be replaced by centrifugal force. The gravitropic response of caulonemanta is biphasic, consisting of an initial phase producing a bend of about 20 degrees within 12 h of 90 degrees reorientation and a subsequent slower phase leading to completion of the 90 degrees curvature. No obvious sedimentation of statoliths accompanies this response. Several mutants have been isolated that are either partially or completely impaired in caulonemal gravitropism and one mutant shows a positive gravitropic response. Complementation analysis using somatic hybrids obtained following protoplast fusion indicates that at least three genes can mutate to give an altered gravitropic phenotype. None of these mutants is altered in gametophore gravitropism, suggesting that the gravitropic response of caulonemal filaments may require at least some gene products that are not required for the response of the multicellular gametophores. One class of mutant with impaired caulonemal gravitropism shows a pleiotropic alteration in leaf shape.