<Emphasis Type="Italic">EcFLO</Emphasis>, a<Emphasis Type="Italic"> FLORICAULA</Emphasis>-like gene from<Emphasis Type="Italic"> Eschscholzia californica</Emphasis> is expressed during organogenesis at the vegetative shoot apex

FLORICAULA/ LEAFY-like genes were initially characterized as flower meristem identity genes. In a range of angiosperms, expression occurs also in vegetative shoot apices and developing leaves, and in some species with dissected leaves expression is perpetuated during organogenesis at the leaf marginal blastozone. The evolution of these expression patterns and associated functions is not well understood. We have isolated and characterized a FLORICAULA-like gene from California Poppy, Eschscholzia californica Cham. (Papaveraceae), a species belonging to the basal eudicot clade Ranunculales. EcFLO encodes a putative 416-amino-acid protein with highest similarity to homologous genes from Trochodendron and Platanus. We show that EcFLO mRNA is expressed during the vegetative phase of the shoot apical meristem and in developing dissected leaves in a characteristic manner. This pattern is compared to that of other eudicots and discussed in terms of evolution of FLORICAULA expression and function.     

The pattern of histone H4 expression in the tomato shoot apex changes during development

Two histone H4 cDNA clones were isolated from a tomato (Lycopersicon esculentum Mill.) shoot-tip cDNA library using a heterologous probe from barley (Hordeum vulgare L.). Both cDNAs, which are 81% identical in the coding region, are polyadenylated and belong to a small gene family in the tomato genome. Histone H4 message is abundant in young tissues and rare in older tissues. In the shoot apical meristem, the distribution of H4-expressing cells changes during development. In a juvenile vegetative apex, H4 message is detectable in the central region and the peripheral parts of the meristem. In a mature vegetative apical meristem, H4-expressing cells are localized in the peripheral zone extending into the provascular strands and the rib meristem whereas the central zone is almost devoid of H4 mRNA. After floral transition, H4 mRNA is found throughout the floral meristem, indicating a second change in the pattern of H4 expression. The observed changes in H4 expression are indicative of changes in the distribution of mitotic activity in the shoot apical meristem during plant development. In addition, H4-expressing cells were found to occur frequently in clusters, which may indicate a partial synchronization of cell divisions in the shoot apex.     

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.     

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.     

Changes in RNA levels in the shoot apex of Silene during the transition to flowering

Changes in RNA concentration in the shoot apical meristem during induction and the transition to flowering were measured histochemically in Silene coeli-rosa (L.) Godron, a long-day plant. In the apices of plants induced by 7 long days the RNA concentration increased to about 25 per cent higher than in non-induced plants. Three long days did not induce flowering but resulted in a transient rise in RNA concentration. When plants were given long days interrupted by varying numbers of short days successful induction was accompanied by a sustained increase in RNA concentration but those treatments which were not inductive gave only transient increases in RNA. Gibberellic acid had no effect on induction or apical growth rates but increased the RNA concentration by 50 per cent or more in both induced and non-induced plants. Plants induced to flower at 13° C had the same RNA concentration and growth rate at the apex as in non-induced plants at 20° C. Since changes in RNA concentration in the apex could occur without changes in growth rate and without flowering, and induction could occur without a change in RNA concentration or growth rate, it is suggested that the increase in RNA and growth rate which normally occur at the transition to flowering might not be essential for the formation of a flower but may be more closely related to the rapid growth associated with the formation of the inflorescence.Abbreviations LD long day - SD short-day     

Occurrence of two cell subpopulations with different cell-cycle durations in the central and peripheral zones of the vegetative shoot apex of Sinapis alba L.

The cell-cycle duration and the growth fraction were estimated in the vegetative shoot apical meristem of Sinapis alba L. The length of the cell cycle was about 86 h, i.e. 2.5 times shorter than the cell-doubling time (M. Bodson, 1975, Ann. Bot. 39, 547–554) and the growth fraction was between 32 to 41%. These data demonstrated that the cell population of this meristem was heterogeneous, including one subpopulation of rapidly cycling cells and one subpopulation of non-cycling cells, i.e. cells with a very long cell cycle compared with that of the rapidly cycling cells. Non-cycling cells had no particular localization within the meristem. Both the central and peripheral zones of the meristem were mosaics of rapidly cycling and non-cycling cells.Abbreviations G₁ pre-DNA-synthesis phase - G₂ post-DNA-synthesis phase - GF growth fraction - M mitosis phase - PLM pulse-labelled-mitoses method - S DNA-synthesis phase - T cell-cycle duration - TdR thymidine     

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.     

Transformation and regeneration of loblolly pine: shoot apex inoculation with Agrobacterium

Loblolly pine (Pinus taeda L.) is the mostimportant tree species in US commerce and has much to gain through geneticengineering. This species can be transformed using particle bombardment andAgrobacterium; however, the regeneration of plants fromtransgenic tissues has been difficult and the recovery of transgenic plants hasbeen rare. A shoot-based and genotype-independent transformation methodemploying Agrobacterium tumefaciens was used to facilitaterecovery of plants and permit the transformation of elite germplasm. Shootsfrom4–6 week old seedlings and adventitious shoots from culture wereinoculated with A. tumefaciens EHA101 (pGUS3), or EHA105(pSSLa.3), subjected to selection and regenerated. Shoots that survivedexhibited expression of the uidA gene (GUS) in a patterncharacteristic of the either the CaMV35S promoter (pGUS3), or the larch RbcSpromoter (pSSLa.3) transferred. Recovered plants were screened using PCRamplification. Southern DNA analyses and amplification of the T-DNA borderjunction confirmed genomic integration of both transferreduidA and nptII genes. In this proofofconcept study, the overall recovery of P. taeda shoots wasfair (10–20%), while recovery of intact rooted plants was poor (>1%)due to difficulty in rooting. Recovery of intact rooted plants from inoculatedshoots of P. eldarica and P. radiatawas more efficient (10–30%). The addition of a shoot multiplication stepand effective rooting protocols will improve the efficiency of this genotypeindependent transformation method in P. taeda, and inotherPinus spp.     

Regeneration of Gossypium hirsutum and G. barbadense from shoot apex tissues for transformation

A method of regenerating cotton plants from the shoot apical meristem of seedlings was developed for use with particle gun and Agrobacterium-mediated transformation. This method was developed to circumvent the problems of genotype restriction and chromosomal damage frequently encountered in cotton regeneration in tissue culture through somatic embryogenesis. In this procedure, the cells of the shoot meristem are targeted for transformation. Normal and fertile plants of Gossypium barbadense Pima S-6, and 19 cultivars of G. hirsutum were regenerated using this method. Shoot regeneration from these tissues was direct and relatively rapid. A MS based, hormone-free medium could be used with all the varieties tested.This project was funded by grants from Cotton Incorporated, Nisshinbo Industries, and a grant from the Texas Agricultural Experiment Station to RHS. Texas Agricultural Experiment Station Technical Article TA-25667.     

Early effects of floral induction on cell division in the shoot apex of Silene

The changes in cell number, the relative proportions of interphase nuclei with different amounts of DNA, mitotic index and labelling index have been investigated in the shoot apex of Silene coeli-rosa L. (a long-day plant) during the first long day of photoinduction, and compared with the corresponding changes in plants in short days. 3 h after the start of induction the proportion of nuclei in the G2 phase of the cell cycle had increased, the mitotic index tended to be higher, and the labelling index was lower than in plants in short days. 8–9 h later the values for plants in the long day had become similar to those for plants in short days. No evidence was obtained for a synchronisation of cells in one phase of the cell cycle as a result of photoinduction. The results obtained were consistent with a temporary shortening of the cell cycle in the induced apices over the first long day which resulted in a greater increase in cell number by the end of the first day of photoinduction than in plants in short days.Abbreviations LD long day - SD short day     

Ageing of theSilene coeli-rosa L. shoot apex under non-inductive conditions: Changes in morphology,mitotic index,and polypeptide composition

Summary Ageing was studied in the shoot apex of the long day plant,Silene coeli-rosa by maintaining it in non-inductive short day conditions for 170 days. The dimensions, the zonation, and the polypeptidic pattern of the shoot apex, and the rate of leaf initiation were altered in 170-day-old plants compared with young plants grown under the same conditions (28 days). In aged plants, the number of cells increased in all shoot apical zones and, notably, the mitotic index increased in the axial zone; however, the rate of leaf initiation slowed down. These changes showed some similarities to those during the intermediate phase in quantitative photoperiodic species. The two-dimensional mini-gel electrophoretic study of protein extracts from shoot apices of young and ageing plants maintained in non-inductive conditions revealed 489 common polypeptidic spots, 13 unique to the young state and 24 new ones specific to aged plants. The spots characteristic for each state represented only 3.6% of the total identified polypeptides, but apical development under non-inductive conditions was characterized by qualitative changes in the polypeptide complement.Abbreviations C nuclear DNA complement - 1D gel first dimension gel - LD long day - NEPHGE non equilibrium pH gradient electrophoresis - SD short day - SDS sodium dodecyl sulfate - TCA trichloracetic acid     

Arrangement of cortical microtubules at the surface of the shoot apex inVinca major L.: Observations by immunofluorescence microscopy

Arrangements of cortical microtubules (MTs) and of cellulose microfibrils at the surface of the vegetative shoot apex ofVinca major L. were examined by immunofluorescence microscopy and polarizing microscopy, respectively. Cortical MTs adjacent to the outermost walls of the apex were arranged more or less randomly in individual cells: especially in cells in the central region of the apex the arrangement was almost completely random. However, in the peripheral region MTs tended to show parallel alignment in individual cells, and an overall pattern that was roughly concentric around the apical dome was discerned. Observations of birefringence of cell walls indicated that cellulose microfibrils in the peripheral region of the apex were also arranged in a pattern which was roughly concentric around the apical dome. These patterns of arrangements of MTs and microfibrils are understood to be perpendicular to the radial cell files observed in the peripheral region of the apex, and can be related to the radial expansion of the surface of the apex.     

Cytochemistry and immunolocalisation of polysaccharides and proteoglycans in the endosperm of green Arabica coffee beans

Summary. The major noncellulosic polysaccharides and proteoglycans in the coffee bean (Coffea arabica) cell wall are (galacto)mannans and arabinogalactan proteins. Immunological and chemical probes demonstrated that the mannans and arabinogalactan proteins were located continuously across the width of the cell wall, but that the concentration of different structural epitopes within these polysaccharide types showed considerable spatial variation. For the mannans this was implied by the striated pattern demonstrated by fluctuation of the affinity between the mannan monoclonal antibody BGM C6 and (galacto)mannan. The arabinogalactan proteins labelled by the Yariv reagent and the arabinogalactan protein-specific antibody LM2 appeared to be located in all regions of the wall except the middle lamella, but showed some differences in intensity of labelling. However, the LM6 antibody, specific for (15)--arabinan epitopes, was located only as a compact region adjacent to the cell lumen in the body of the endosperm; though, it did label throughout the wall of epidermal cells. This implied that either some of the more highly arabinosylated arabinogalactan proteins contained contiguous 5-arabinosyl residues or that a rhamnogalacturonan which contained 5-arabinosyl residues as side chains existed in the cell wall. In either case the polymers were very restricted in their distribution. A second category of pectin, a homogalacturonan detected by JIM7, was located only in the middle lamella region. The architecture of the wall, as revealed by resin etching, appeared to reflect the chemical heterogeneity, with three distinct physical zones identifiable in a cross section across a single wall.Correspondence and reprints: Nestlé Research Center, Nestec Ltd., Vers-chez-les-Blanc, P.O. Box 44, 1000 Lausanne 26, Switzerland     

Mutations in the <Emphasis Type="Italic">TORNADO2</Emphasis> gene affect cellular decisions in the peripheral zone of the shoot apical meristem of <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">thaliana</Emphasis>

Summary An EMS (ethyl methanesulfonate) mutagenesis effector screen performed with the STM:GUS marker line in Arabidopsis thaliana identified a loss-of-function allele of the TORNADO2 gene. The histological and genetic analyses described here implicate TRN2 in SAM function, where the peripheral zone in trn2 mutants is enlarged relative to the central stem cell zone. The trn2 mutant allele partially rescues the phenotype of shoot meristemless mutants but behaves additively to wuschel and clavata3 alleles during the vegetative phase and in the outer floral whorls. The development of carpels in trn2 wus-1 double mutant flowers indicates that pluripotent cells persist in floral meristems in the absence of TRN2 function and can be recruited for carpel anlagen. The data implicate a membrane-bound plant tetraspanin protein in cellular decisions in the peripheral zone of the SAM.     

Plant meristems: <Emphasis Type="Italic">CLAVATA3/ESR</Emphasis>-related signaling in the shoot apical meristem and the root apical meristem

The plant meristems, shoot apical meristem (SAM) and root apical meristem (RAM), are unique structures made up of a self-renewing population of undifferentiated pluripotent stem cells. The SAM produces all aerial parts of postembryonic organs, and the RAM promotes the continuous growth of roots. Even though the structures of the SAM and RAM differ, the signaling components required for stem cell maintenance seem to be relatively conserved. Both meristems utilize cell-to-cell communication to maintain proper meristematic activities and meristem organization and to coordinate new organ formation. In SAM, an essential regulatory mechanism for meristem organization is a regulatory loop between WUSCHEL (WUS) and CLAVATA (CLV), which functions in a non-cell-autonomous manner. This intercellular signaling network coordinates the development of the organization center, organ boundaries and distant organs. The CLAVATA3/ESR (CLE)-related genes produce signal peptides, which act non-cell-autonomously in the meristem regulation in SAM. In RAM, it has been suggested that a similar mechanism can regulate meristem maintenance, but these functions are largely unknown. Here, we overview the WUS–CLV signaling network for stem cell maintenance in SAM and a related mechanism in RAM maintenance. We also discuss conservation of the regulatory system for stem cells in various plant species. S. Sawa is the recipient of the BSJ Award for Young Scientist, 2007.     

Identification of genes expressed in the shoot apex ofBrassica campestris during floral transition

The vegetative-to-floral transition ofBrassica campestris cv. Osome was induced by vernalization. Poly(A)+RNA was isolated from the transition shoot apex after 6 weeks of vernalization, the floral apex after 12 weeks of vernalization and the expanded leaves just before vernalization, and cDNAs were synthesized. These cDNAs were used for subtraction and differential screening to select cDNA preferentially present in the transition and floral apices. Nucleotide sequences of the resulting 14 cDNA clones were determined, and northern blot analysis was carried out on six cDNAs. Two cDNA clones which did not show significant similarity to known genes were shown to be preferentially expressed in the floral apex.     

Mean rate of DNA replication and replicon size in the shoot apex ofSilene coelirosa L. During the initial 120 minutes of the first day of floral induction

Summary 28-day-old plants ofSilene coeli-rosa were exposed, at 1,700 hours, to long day (LD) conditions comprising light of low fluence rate provided by tungsten bulbs, or maintained in darkness as short day (SD) controls. All plants were exposed at 1,700 hours to tritiated-(methyl-³H)-thymidine for 30, 45, 60, 90, or 120 minutes. Apical domes were isolated and prepared as fiber autoradiographs from which replicon size and rates of DNA replication, per single replication fork were recorded. In SD, replicon size was between 15–20 m and exposure to LD conditions altered neither replicon size nor the pattern of deployment of replicons during S-phase relative to the SD controls. However, the mean rate of replication in LD was 8.7 m h^–1 compared with 5.2 m h^–1 in SD. Thus, exposure to LD resulted in a 1.7-fold increase in the rate of DNA replication relative to the SD controls. This rapid increase in replication rate, detectable within 30 minutes of the start of the LD is discussed in relation to changes known to occur to the cell cycle inSilene during the first day of floral induction.     

Inhibition of growth and synchronised cell division in the shoot apex in relation to flowering in Silene

When plants of Silene coeli-rosa (L.) Godron were induced by seven long days, then exposed to darkness for 48 h before being returned to short days, they went on to initiate flowers with a delay of about 2 d. The synchronisation of cell division which normally occurs before flower initiation was suppressed, showing that it is not essential for flowering. Periods of darkness of up to 240 h inhibited apical growth and leaf initiation but did not prevent eventual flowering in short days. The commitment of the apex to flower was therefore maintained while apical growth was inhibited.Abbreviations SD short day(s) - LD long day(s)     

Phenotypical and structural characterization of the Arabidopsis mutant involved in shoot apical meristem

An Arabidopsis mutant induced by T-DNA insertion was studied with respect to its phenotype, microstructure of shoot apical meristem (SAM) and histochemical localization of the GUS gene in comparison with the wild type. Phenotypical observation found that the mutant exhibited a dwarf phenotype with smaller organs (such as smaller leaves, shorter petioles), and slower development and flowering time compared to the wild type. Optical microscopic analysis of the mutant showed that it had a smaller and more flattened SAM, with reduced cell layers and a shortened distance between two leaf primordia compared with the wild type. In addition, analysis of the histo-chemical localization of the GUS gene revealed that it was specifically expressed in the SAM and the vascular tissue of the mutant, which suggests that the gene trapped by T-DNA may function, in the SAM, and T-DNA insertion could influence the functional activity of the related gene in the mutant, leading to alterations in the SAM and a series of phenotypes in the mutant. __________ Translated from Acta Botanica Boreali-Occidentalia Sinica, 2007, 27(2): 228–232 [译自: 西北植物学报]     

Effects of red and far-red light on cell division in the shoot apex ofSilene coeli-rosa L.

Summary 28-day-old plant ofSilene coeli-rosa were exposed at 1,700 hours to 5 or 10 minutes red light, 5 or 10 minutes far-red light, red followed by far-red, far-red followed by red or maintained in darkness. Measurements of the proportions of cells with the 2 C and 4 C amounts of DNA in the shoot apex of the plants, sampled at 2,000 hours, showed that far-red light promoted an increase in the G2 proportion whereas red light resulted in an increase in the G1 proportion of the cell cycle, relative to the dark controls. Moreover these changes were red, far-red reversible. All light treatments resulted in increases in the mitotic index in the apex compared with the dark controls, suggesting increases in the growth rate. The data implicate phytochrome in a low energy response and suggest that, in the shoot apex, G1 is shortened markedly following exposure to farred light, whilst G2 is shortened the most following exposure to red light. The results are discussed in relation to flower-initiation.