Appearance and disappearance of proteins in the shoot apical meristem of Sinapis alba in transition to flowering

Vegetative plants of Sinapis alba L. grown under short days were induced to flower by exposure to one long day or continuous long days. Irrespective of the number of long days, the first flower primordia were initiated by the shoot apical meristem 60 h after the start of the inductive treatment. An indirect histoimmunofluorescence technique was used to search in the apical meristem for three antigenic proteins which had been previously detected by immunodiffusion tests in the whole apical bud (Pierard et al. (1977) Physiol. Plant. 41, 254–258). One protein called protein A, present in the vegetative meristem, increased in concentration during the first 48 h following the start of the inductive treatment. It stayed constant up to 96 h and disappeared completely at a later time. Two other proteins called B and C, absent in the vegetative meristem, appeared in the meristem of induced plants between 30 and 36 h after the start of the inductive treatment and progressively accumulated at later times up to 240 h. These proteins appeared 8 h before the irreversible commitment of the meristem to produce flower primordia (point of no return) was reached and 24 h before start of flower production. These observations support an interpretation of floral evocation as consisting, at least partially, of an early and qualitative change in gene expression.Abbreviations AVB anti-vegetative-bud antiserum - ARB antireproductive-bud antiserum - IgG immunoglobulins G - TRITC tetramethylrhodamine isothiocyanate - GAR IgG goat antirabbit IgG - S₀ IgG non-immune rabbit IgG     

Changes in the Pattern of Proteins Synthesized in the Shoot Apical Meristem of Sinapis alba during Floral Transition

Because Sinapis alba can be induced to flower by a single longday (LD), we have sought for changes in gene expression in theshoot meristem during the floral transition induced by a 22h LD. To analyse the pattern of proteins synthesized in themeristem, we have used in vivo labelling of proteins, an improvedextraction procedure specifically designed for green plant tissues,two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)and a strict gel comparison procedure giving a confidence levelof 3%. Following the LD, complex qualitative and quantitative changesaffected about 8% of the proteins. Twelve different patternsof changes were observed. Some quantitative changes and onequalitative change were already found 16 h after the divergenceof light regimes, other changes appeared only later. Exceptfor one protein, the qualitative changes appeared only afterthe production of flower primordia. (Received March 21, 1992; Accepted September 23, 1992)     

ONTOGENY OF THE INFLORESCENCE OF SAURURUS CERNUUS (SAURURACEAE)

The spicate inflorescence of Saururus cernuus L. (Saururaceae) results from the activity of an inflorescence apical meristem which produces 200–300 primordia in acropetal succession. The inflorescence apex arises by conversion of the terminal vegetative apex. During transition the apical meristem increases greatly in height and width and changes its cellular configuration from one of tunica-corpus to one of mantle (with two tunica layers) and core. Primordia are initiated by periclinal divisions in the subsurface layer. These are “common” primordia, each of which subsequently divides to produce a floral apex above and a bract primordium below. The bract later elongates so that the flower appears borne on the bract. All common primordia are formed by the time the inflorescence is about 4.4 mm long; the apical meristem ceases activity at this stage. As cessation approaches, cell divisions become rare in the apical meristem, and height and width of the meristem above the primordia diminish, as primordia continue to be initiated on the flanks. Cell differentiation proceeds acropetally into the apical meristem and reaches the summital tunica layers last of all. Solitary bracts are initiated just before apical cessation, but no imperfect or ebracteate flowers are produced in Saururus. The final event of meristem activity is hair formation by individual cells of the tunica at the summit, a feature not previously reported for apical meristems.     

Growth changes in the shoot apex of Sinapis alba during transition to flowering

The aim of the work was to report morphological changes whichoccur in the shoot apex during the morphogenetic switch to floweringin the model long day (LD) plant, Sinapis alba. During the floraltransition induced by 1 LD the growth rate of all componentsof the shoot apex is modified profoundly. The earliest changes,detected at 24 h after start of LD, include a decrease in plastochronduration and an increase of growth of leaf primordia. One daylater, the meristem dome starts to increase in volume, apicalinternodes have an increased height and there is a precociousoutgrowth of axillary meristems. All these changes precede initiationof flower primordia, which starts at about 60 h after the startof LD. Later changes include meristem doming, a decrease inthe plastochron ratio and a shift to a more complex phyllotaxis.All the changes, except the decreased plastochron ratio, arecharacteristics of an apex with an increased tempo of growth.The stimulation of longitudinal growth (height of apical intemodes)is more marked and occurs earlier than the reduction of radialgrowth (plastochron ratio). Key words: Axillary meristem, internode growth, leaf growth, plastochron ratio, plastochron duration     

The induction of sun and shade leaves of the European beech (Fagus sylvatica L.): anatomical studies

Summary Primordia from buds of sun and shade twigs of European beech (Fagus sylvatica L.) were collected six times a year for anatomical investigations. Differentiation into sun-leaf and shade-leaf primordia was first observed in early August. Sun-leaf primordia had five, and shade-leaf primordia four layers of mesophyll meristem cells. With potted graft unions of beeches possible structural changes of leaf primordia were investigated. Trees adapted to shade develop sun-leaf primordia when put into full daylight, provided the transfer happened before July. Trees adapted to full daylight developed leaf primordia which remained structurally sun-leaf primordia when the plant was kept under shade conditions. Shadeleaf branches of young beech trees cut in February in order to expose the shade buds to full daylight developed either shade leaves or intermediate shade/sun leaves. These experiments show that the subtending leaf may provide the developing axillary bud with photoassimilates, but its character, whether sun or shade leaf, has no influence on the character of the developing leaf primordia.     

The Shoot Apex as a Marker of the Responsivity to Photoperiodic Treatment Inducing Flowering of Chenopodium rubrum L.

Two maxima in flowering response to one inductive dark period of 13 h were found in the short day plant Chenopodium rubrum within three weeks of cultivation under continuous illumination either in vitro or in vivo. These maxima correlated with the number of leaf primordia and their relation to the size of the apical meristem. The first maximum in flowering responsivity corresponded with the stage when primordia of the second leaf pair had not yet overtopped the apical meristem, the second one when the primordia of the fourth leaf overgrew the meristem. Maximum responsivity to flowering reached by a mother plant was reflected in explants derived from it. The above morphological markers of responsiveness to floral induction were not linked to plant age and/or to general growth habit. The explants flowered only when part of the stem was present.     

In situ localization of storage protein mRNAs in developing meristems of Brassica napus embryos

Probes derived from cDNA clones of napin and cruciferin, the major storage proteins of Brassica napus, and in situ hybridization techniques were used to examine changes in the spatial and temporal distribution of storage protein messages during the course of embryogeny, with a special emphasis on the developing apical meristems. Napin mRNAs begin to accumulate in the cortex of the axis during late heart stage, in the outer faces of the cotyledons during torpedo stage and in the inner faces of the cotyledons during cotyledon stage. Cruciferin mRNAs accumulate in a similar pattern but approximately 5 days later. Cells in the apical regions where root and shoot meristems develop do not accumulate storage protein messages during early stages of embryogeny. In the upper axis, the boundary between these apical cells and immediately adjacent cells that accumulate napin and cruciferin mRNAs is particularly distinct. Our analysis indicates that this boundary is not related to differences in tissue or cell type, but appears instead to be coincident with the site of a particular set of early cell divisions. A major change in the mRNA accumulation patterns occurs halfway through embryogeny, as the embryos enter maturation stage and start drying down. Final maturation of the shoot apical meristem is associated with the development of leaf primordia and the accumulation of napin mRNAs in the meristem, associated leaf primordia and vascular tissue. Cruciferin mRNAs accumulate only in certain zones of the shoot apical meristem and on the flanks of leaf primordia. Neither type of mRNA accumulates in the root apical meristem at any stage.     

CHANGES IN EPILOBIUM PHYLLOTAXY INDUCED BY N-1-NAPHTHYLPHTHALAMIC ACID AND α-4-CHLOROPHENOXYISOBUTYRIC ACID

Preliminary studies establishing relationships between leaf plastochron index and Epilobium hirsutum L. shoot growth provide a method for rigorous selection of plants utilized in experiments designed to test the working hypothesis that endogenous auxin gradient interactions are factors of phyllotactic control in this species. Application of N-1-naphthylphthalamic acid (NPA), an auxin transport inhibitor, to one of the youngest bijugate primordia on the shoot meristem results in increased growth of the treated primordium. Fasciation between the treated primordium and one of the next primordia to be initiated alters relative vertical spacing of primordia. Angular shifts between subsequent primordia result in spiral transformation of Epilobium bijugate phyllotaxy. Application of α-4-chlorophenoxyisobutyric acid (CPIB), an auxin antagonist, to one of the youngest bijugate primordia on the shoot meristem results in decreased growth of the treated primordium that alters both radial and vertical spacing of primordia. This is followed by angular shifts between subsequent primordia resulting in spiral transformation of the bijugate phyllotaxy. Changes in the growth parameters of NPA- and CPIB-treated shoots are similar. Relative plastochron rates of radial and vertical shoot growth of induced spiral shoots are about half those of lanolin paste control shoots, as are the plastochrons and relative plastochron rates of leaf elongation. Treated shoot meristems have eccentricities of 0.5 as compared to bijugate control meristem eccentricities of 0.7. No significant difference is apparent between basal transverse areas of treated and control shoot meristems. The relative chronological rates of growth of treated shoots are not significantly different from those rates of control shoots. Spiral transformation results from changes in relative positions of leaf primordia insertion on the shoot meristem, not from changes in growth of treated shoots. These changes are accompanied by an increased rate of leaf initiation on a more circular shoot meristem. Existing theoretical models of phyllotaxy are discussed in relation to these chemically induced changes of Epilobium leaf arrangement.     

Changes in the Polypeptide Composition during the Ontogenetic Development of the Shoot Apex of Crocus sativus

The shoot apex development during the life cycle of Crocus sativus L. was characterized by light microscopy and two-dimensional gel electrophoresis. Using silver staining of polyacrylamide gels, numerous quantitative and qualitative changes in the populations of polypeptides were observed during transition from vegetative to prefloral and from prefloral to floral stages. Using 80 g protein, we were able to detect 352 polypeptidic spots. In comparison with the vegetative apex, 89 new polypeptides were identified in the prefloral meristem and 29 polypeptides were missing. In the reproductive meristem, 94 new spots were identified and 44 spots were missing. Thus, substantial quantitative and qualitative changes in the populations of polypeptides occurred during the prefloral stage, a point of no return in plant development, i.e., and before floral primordia initiation.     

A new way of achieving plant regeneration of Solanum lycopersicoides Dun. from root primordia culture

Cell aggregates with root primordia were formed in root primordia culture (RPC) of Solanum lycopersicoides grown in modified liquid MS medium containing 15 mg/l NAA. After transfer to liquid medium containing 1 mg/l 2,4-D, the aggregates dissociated into single root primordia (RP) which had an organizing root meristem at the apical pole. Oval structures called pseudoembryos were formed from single RP. After passage to liquid MS medium without phyto-hormones and organic compounds (with the exception of sucrose), an apical root meristem developed and the shoot apical meristem was initiated. The pseudoembryos developed into elongated pseudoseedlings which formed plants after transfer to a 1/2MSV medium. The development of pseudoembryos occurred without the callus phase. Moreover, the induction of the shoot meristem occurred without exogenous cytokinins. Received: 30 August 1999 / Revision received: 20 December 1999 / Accepted: 3 January 2000     

Shoot Apical Meristems of Picea sitchensis Seedlings Accelerate in Growth Following Bud-set

First-year seedlings of Picea sitchensis were induced to setbuds by transferring them from long days (17 h) to short days(10 h at 20 °C, 12 h night at 12 °C). After 20 shortdays, the seedlings stopped growing in height, the shoot apicaldomes began enlarging and the transverse growth rates of theshoot apical dome tissues increased. For about 25 days thereafterprimordia were produced faster within the developing buds thanat the apices of seedlings which had been kept in long daysto prevent bud-set. Accelerated primordia production after bud-setenables the largest possible buds to be formed during the frost-freeperiod before winter. Picea sitchensis, apical meristem, bud-set     

Bi-directional inflorescence development inArabidopsis thaliana: Acropetal initiation of flowers and basipetal initiation of paraclades

In this study we investigated Arabidopsis thaliana (L.) Heynh. inflorescence development by characterizing morphological changes at the shoot apex during the transition to flowering. Sixteen-hour photoperiods were used to synchronously induce flowering in vegetative plants grown for 30 d in non-inductive 8-h photoperiods. During the first inductive cycle, the shoot apical meristem ceased producing leaf primordia and began to produce flower primordia. The differentiation of paraclades (axillary flowering shoots), however, did not occur until after the initiation of multiple flower primordia from the shoot apical meristem. Paraclades were produced by the basipetal activation of buds from the axils of leaf primordia which had been initiated prior to photoperiodic induction. Concurrent with the activation of paraclades was the partial suppression of paraclade-associated leaf primordia, which became bract leaves. The suppression of bract-leaf primordia and the abrupt initiation of flower primordia during the first inductive photoperiod is indicative of a single phase change during the transition to flowering in photoperiodically induced Arabidopsis. Morphogenetic changes characteristic of the transition to flowering in plants grown continuously in 16-h photoperiods were qualitatively equivalent to the changes observed in plants which were photoperiodically induced after 30 d. These results suggest that Arabidopsis has only two phases of development, a vegetative phase and a reproductive phase; and that the production of flower primordia, the differentiation of paraclades from the axils of pre-existing leaf primordia and the elongation of internodes all occur during the reproductive phase.     

Normal and Abnormal Development in the Arabidopsis Vegetative Shoot Apex

Vegetative development in the Arabidopsis shoot apex follows both sequential and repetitive steps. Early in development, the young vegetative meristem is flat and has a rectangular shape with bilateral symmetry. The first pair of leaf primordia is radially symmetrical and is initiated on opposite sides of the meristem. As development proceeds, the meristem changes first to a bilaterally symmetrical trapezoid and then to a radially symmetrical dome. Vegetative development from the domed meristem continues as leaves are initiated in a repetitive manner. Abnormal development of the vegetative shoot apex is described for a number of mutants. The mutants we describe fall into at least three classes: (1) lesions in the shoot apex that do not show an apparent alteration in the shoot apical meristem, (2) lesions in the apical meristem that also (directly or indirectly) alter leaf primordia, and (3) lesions in the apical meristem that alter meristem size and leaf number but not leaf morphology. These mutations provide tools both to genetically analyze vegetative development of the shoot apex and to learn how vegetative development influences floral development.     

CHANGE IN EPILOBIUM PHYLLOTAXY DURING REPRODUCTIVE TRANSITION

The ontogeny of Epilobium hirsutum grown under natural summer photoperiod in a glasshouse was divided into vegetative, early transitional, transitional, and floral stages. Bijugate phyllotaxy, common to both the vegetative and early transitional stages, is transformed into spiral phyllotaxy during the transitional stage by an initial change in the divergence angle of a single primordium inserted at a unique level on the shoot. Leaf primordia subsequently are inserted in a spiral arrangement in the indeterminate floral shoot apex. The early transitional shoot apical meristem is about 1.5 times the volume of the vegetative meristem but expands at about two-thirds the relative plastochron rate of volume increment of the vegetative meristem. There are progressive decreases in the plastochron and relative plastochron rates of radial and vertical shoot growth through ontogeny. Relative chronological rates of shoot growth, however, are not altered during ontogeny. Spiral transformation results from changes in the relative points of insertion of leaf primordia on the shoot meristem. These changes are accompanied by an increased rate of primordia initiation on a more circular shoot meristem. The change in phyllotaxy during ontogeny is similar to that which was artificially induced by chemical modification of auxin concentration gradients in the shoot apex, with the additional feature that there is an initial increase in the volume of the shoot meristem prior to the natural spiral transformation. Size of the shoot apical meristem, however, appears to have little influence on Epilobium phyllotaxy; but the geometric shape of the meristem is well correlated with bijugate to spiral transformations. This suggests that geometric parameters of the shoot meristem should be considered in theoretical models of phyllotaxy.     

Gibberellin-induced reorganization of spatial relationships of emerging leaf primordia at the shoot apical meristem in Hedera helix L.

The transition from spiral to distichous leaf arrangement during gibberellic-acid (GA₃)-induced rejuvenation in Hedera was studied in detail by scanning electron microscopy of the shoot apical meristem. The transition, which involves the initiation of about 14 new leaf primordia, is accomplished by progressive increments in the divergence angle between the leaf primordia from an initial average value of 138.9 ° until it approaches 180 °. This process is preceded, as well as accompanied, by an increased radial displacement of young leaf primordia away from the apical meristem. Although the width of the leaf primordia also increases, this is unlikely to be a causal factor since it occurs only late in the transition. The size of the primordium-free area of the apical meristem is also unlikely to be involved. Quantitative analysis shows that the divergence angle of consecutive leaf primordia commonly fluctuates between relatively large and small values. Thus the transitional stages form a spirodistichous arrangement in which the divergence angle within each pair of leaves is large relative to that between leaf pairs. The stimulation of the radial displacement of the leaf primordia and the associated phyllotactic transition may involve GA₃-induced modification in the spatial organization of cortical microtubules in the apical meristem and related changes in directional cell expansion.Abbreviations DA divergence angle - GA₃ gibberellic acid We thank Mr. Gilbert Ahlstrand for his advice regarding scanning electron microscopy. This paper is contribution of the University of Minnesota Agricultural Experimental Station No. 18,726.     

Changes in the pattern of cell arrangement at the surface of the shoot apical meristem in Hedera helix L. following gibberellin treatment

The changes in the pattern of cell arrangement and surface topography at the shoot apical meristem of Hedera helix L., which occur during gibberellic acid (GA₃)-induced transition from spiral to distichous phyllotaxis, were examined by scanning electron microscopy of rapidly frozen tissue. The technique preserves the original shape of the cells in their turgid state. It reveals distinct sets of radially oriented cell files, about four to eight cells wide, which extend from the central region of the meristem toward leaf primordia on the meristem flanks. In apices with spiral phyllotaxis, a new emerging primordium (0) appears as an acropetal bulge between the radial files adjacent to the third (3) and the second (2) older primordia. The bulging is associated with radial or oblique cell divisions while those located at the meristem flanks and in the radial files are oriented tangentially. As the displacement of existing primordia away from the central region increases following the GA₃ treatment, radial and oblique divisions as well as acropetal bulging invade the radial files adjacent to the primordium 2; consequently the angular divergence of the emerging primordium from the youngest existing primordium (1) increases. In apices with distichous phyllotaxis, the earliest bulging appears on both sides of the radial files facing primordium 2, with a slight depression at the files. The radial files therefore correspond to regions of the meristem where acropetal bulging is generally delayed, although this effect apparently diminishes with increasing distance of existing primordia from the meristem center.Abbreviations GA₃ gibberellic acid We thank Mr. Gilbert Ahlstrand, University of Minnesota, for his advice and assistance with the scanning electron microscopy. Contribution of the University of Minnesota Agricultural Experimental Station No. 19032.     

Specification of chimeric flowering shoots in wild-type Arabidopsis

Within wild-type Arabidopsis populations, a subset of the plants were found to have a single chimeric shoot on their primary shoot axes. The chimeric shoots were located below the lowest primary-axis flower; and they exhibited features of both flowers and paraclades (lateral flowering shoots). Morphological analyses of chimeric shoots indicated that they developed from single primordia. In each chimeric shoot, the side furthest from the apical meristem was specified as 'flower'—while the side closest to the meristem was specified as 'paraclade'—suggesting that a stimulus from outside the apical meristem can directly induce primordia to develop as flowers. It is concluded that the development of the teratological chimeric shoots resulted from the overlap of the vegetative and floral specification processes within single primordia.     

Behavior of organelles and their nucleoids in the shoot apical meristem during leaf development in Arabidopsis thaliana L.

The behavior of organelle nucleoids and cell nuclei was studied in the shoot apical meristem and developing first foliage leaves of Arabidopsis thaliana. Samples were embedded in Technovit 7100 resin, cut into thin sections and stained with 4-6-diamidino-2-phenylindole to observe DNA. Fluorimetry was performed using a video-intensified microscope photon-counting system. The DNA content of individual mitochondria was more than 1 Mbp in the shoot apical meristem and the young leaf primordium, and decreased to approximately 170 kbp in the mature foliage leaf. In contrast, the DNA content of individual plastids was low in the shoot apical meristem and increased until day 7 after sowing. Application of 5-bromo-2-deoxyuridine, an analogue of thymidine, was usesd to investigate DNA synthesis in situ. The activities of DNA synthesis in the mitochondria and plastids changed according to the stage of development. Mitochondrial DNA was actively synthesized in the shoot apical meristem and young leaf primordia. This strongly suggests that the amount of mitochondrial DNA per mitochondrion, which has been synthesized in the shoot apical meristem and young leaf primordium, is gradually reduced due to continual divisions of the mitochondria during low levels of mitochondrial DNA synthesis. Synthesis of DNA in the plastid became active in the leaf primordia following DNA synthesis in the mitochondria, and the small plastids were filled with large plastid nucleotids. This enlargement of the plastid nucleoids occurred before the synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase and the development of thylakoids.Abbreviations BrdU 5-bromo-2-deoxyuridine - DAPI 4-6-diamidino-2-phenylindole - DiOC₆a 3,3-dihexyloxacarbocyanine - mtDNA mitochondrial DNA - mt-nucleoid mitochondrial nucleoid - ptDNA plastid DNA - pt-nucleoid plastid nucleoid - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This work was supported by grant No. 2553 to M.F. and Nos. 04454019, 03304005 and 06262204 to T.K. from the Ministry of Education, Science and Culture of Japan, and by a grant for a pioneering research project in biotechnology from the Ministry of Agriculture, Forestry and Fisheries of Japan.