Endogenous gibberellin levels influence in-vitro shoot regeneration in Arabidopsis thaliana (L.) Heynh.

The regeneration of shoot buds from callus cells in vitro is an important technique in modern plant genetic manipulation. Whilst it is clear that genetic factors play a major role in determining the ability of callus cells to become organized into regenerating shoot buds, the precise nature of these factors remains unknown. Here we show that callus derived from mutants of Arabidopsis thaliana which have reduced levels of endogenous bioactive gibberellins (GAs), or reduced responsivity to GAs, regenerates shoot buds more readily than does callus derived from wild-type controls. In addition, exogenous GA reduces, and exogenous paclobutrazol (a GA-biosynthesis inhibitor) increases, the frequency of shoot bud regeneration from wild-type callus. These results show that GA levels play a role in regulating shoot bud regeneration from callus, and suggest that variation in endogenous GA levels or responsivity may account for a major component of the genetic variation in shoot bud regeneration frequency described in other species.     

Gene activation in suspension-cultured cells of Arabidopsis thaliana during blue-light-dependent plantlet regeneration

In cell-suspension cultures of Arabidopsis thaliana (L.) Heynh., transfer to auxin-free medium initiates regeneration leading to the formation of numerous rootlets around day 5. This process is promoted by continuous irradiation of the cell cultures with blue light (400–500 nm) while red light (600–700 nm) is ineffective in this respect. During the course of this process, two mRNA species, encoding, respectively, chalcone synthase and a plasmalemma channel protein, transiently accumulate. A second temporary increase in the steady-state level of these mRNAs is correlated with the onset of chloroplast development after 13–17 d of blue-light exposure of the cell cultures. During this cellular differentiation process a number of mRNAs start to accumulate which specify prominent plastid proteins: the small and the large subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (SSU and LSU), respectively the light-harvesting chlorophyll-a/b protein II (LHCPII). These findings are in accordance with those obtained with carrot suspension cultures where a clear sequence of development, i.e. the formation of somatic embryos followed by bluelight-dependent chloroplast differentiation, has also been observed.Abbreviations AthH2 intrinsic membrane protein of Arabidopsis thaliana (gene) - CHS chalcone-synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - EFR energy fluence rate - LHCPII cab light harvesting chlorophyll-a/b protein of photosystem II (gene) - LSU rbcL large subunit of Rubisco - SSU rbcS small subunit of Rubisco - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase Dedicated to Prof. Wolfhart Rüdiger on the occasion of this 60th birthdayThe research was supported by the Deutsche Forschungsgemeinschaft. We thank Mrs. I. Liebscher for her competent assistance. For the generous gift of cloned gene sequences we thank Prof. Dr. G. Link (Pflanzliche Zellphysiologie, Bochum, Germany), Dr. A. Batschauer (Biologisches Institut II/Botanik, Freiburg, Germany) and Dr. B. Weißhaar (MPI für Züchtungsforschung, Köln, Germany).     

A set of Arabidopsis thaliana miRNAs involve shoot regeneration in vitro

Plant miRNAs, the critical regulator of gene expression, involve many development processes in vivo. However, the roles of miRNAs in plant cell proliferation and redifferntiation in vitro remain unknown. To determine better the molecular mechanism of these processes, we have recently reported that a set of miRNAs with different expression patterns between cells of totipotent and non-totipotent Arabidopsis calli. Some of these were specifically up- or downregulated during callus formation or shoot regeneration, and other development. Among them, miR160, and one of its target genes, ARF10, regulated Arabidopsis in vitro shoot regeneration via WUS, CLV3 and CUC1/2. The miR160-overexpressing, 35S transgenic lines, exhibited reduced shoot regeneration efficiency. The mARF10, a miR160-resistant form of ARF10, showed a high level of shoot regeneration ability. In the transgenic, expression of the above shoot meristem-specific genes was elevated, which is consistent with the improved shoot regeneration. In contrast, the ARF10 deficient knockout mutant produced fewer regenerated shoot. However, overexpressors of ARF10 were only marginally more efficient than the wild type with the respect to shoot regeneration. Our observation strongly supports that proper shoot regeneration from in vitro cultured cells requires the miR160-directed negative influence of ARF10. The enhanced expression of ARF10 is likely to have contributed to the improved regeneration ability.     

The effects of exogenous proline and proline analogues on in vitro shoot organogenesis in Arabidopsis

In vitro shoot organogenesis from Arabidopsis hypocotyl explants was stimulated by 1 mMproline, and to a lesser extent by 5 mM proline, butinhibited by inclusion of 10 mM proline in thehormonally-supplemented regeneration medium. Theability of low concentrations of the prolineanalogues, azetidine-2-carboxylate and thioproline toovercome the stimulatory effect of 1 mM proline, anda slight increase in the stimulative effects of 1 mMproline by D-proline, are consistent with an importantrole for the interconversions of proline and itsprecursors in regulating cell division anddifferentiation.     

Direct shoot organogenesis and plant regeneration in safflower

Summary  Adventitious shoot buds were induced directly on the adaxial surface of the cotyledons of eight safflower cultivars after 14 d of culture initiation on Murashige and Skoog's (MS) medium supplemented with various levels of 6-benzylaminopurine (BA). Maximum shoot organogenesis of 54.4% with 10.2 shoots per responding cotyledon was obtained with 8.87 μM BA in the cv. S-144. Regenerated shoots were classified into three groups on the basis of their morphological features and were found to be correlated with the levels of BA. The highest number of normal shoots was obtained from 2.2 μM BA treatment. The regenerated shoots of group I (normal shoots) were rooted on half-strength MS medium supplemented with 5.3 μM α-naphthaleneacetic acid, 3% sucrose and 0.8% bacto-agar. Rooted plantlets were successfully transferred to soil and appeared morphologically normal. Histological studies revealed that shoot buds originated adventitiously from subepidermal cells.     

Ethylene advances the transition from vegetative growth to flowering in Arabidopsis thaliana

The transition from vegetative growth to flowering is the most drastic change in plant development. In order to examine the involvement of ethylene in growth transition, we compared the development of ethylene-related mutants, eto1, etr1, ein2-1 and ein3-1, with the wild type (WT) in Arabidopsis thaliana. The ethylene sensitivity of two WT and the mutants is decreased in the following order: eto1 = WT < ein3-1 < ein2-1 = etr1-1. Bolting time was also delayed in nearly the same order: eto1 < WT < ein3-1 < ein2-1 < etr1. Leaf numbers increased according to the delay of bolting time, indicating that the delay of bolting time was caused by the delay of transition from vegetative to reproductive growth. Other growth parameters, including leaf area and number of flowers opening at the same time, increased in the same order, indicating that these changes were caused by a single factor, the amount of ethylene signal which was transferred though an ethylene signal transduction pathway. These results suggest that ethylene is involved in the transition from vegetative to reproductive growth in Arabidopsis thaliana.     

Enhanced shoot regeneration from Brassica campestris by silver nitrate

Summary The morphogenetic response of Brassica campestris genotype R500 to inhibitors of ethylene biosynthesis and action was investigated. A medium containing 1.0 mg.l^–1 NAA, 2.0 mg.l^–1 BAP, and 30 or 60 M AgNO₃ significantly enhanced both the percentage shoot regeneration and the number of shoots per cotyledon expiant. Although callus proliferation occurred on hypocotyl segments, no shoots were formed in response to AgNO₃ with expiants older than five days. Cotyledons older than six days formed shoots only with AgNO₃. Cobalt chloride at 20 and 30 M increased cotyledon shoot regeneration but was inferior to AgNO₃. Hypocotyl segments were unresponsive. Salicylic acid at 25 and 50 M prevented both shoot regeneration and callusing without any obvious toxic effects. Removal of expiants from AgNO₃ after 12 days did not alter the percentage of shoot regeneration but increased the number of shoots per expiant. This response was dependent on the level of BAP. Percentage shoot regeneration and number of shoots per cotyledon explant were not affected by removal of CoCl₂. These results indicate that the poor regenerative capacity of this genotype may be related to ethylene biosynthesis or metabolism.Abbreviations NAA Naphthalene Acetic Acid - BAP 6-Benzylamino Purine - MS Murashige and Skoog Medium     

Developmental phases and STM expression during Arabidopsis shoot organogenesis

Shoot organogenesis in Arabidopsis thaliana wasstudied with regard to the timing of key developmental phases and expression ofthe SHOOTMERISTEMLESS (STM) gene.Shoot regeneration in the highly organogenic ecotype C24 was affected byexplanttype and age. The percentage of C24 cotyledon explants producing shootsdecreased from 90% to 26% when donor seedlings were more than 6 dold, but 96% of root explants produced shoots regardless of the age of thedonorplant. Using explant transfer experiments, it was shown that C24 cotyledonexplants required about 2 days to become competent and another 8-10 days tobecome determined for shoot organogenesis. A C24 line containing the promoterofthe SHOOTMERISTEMLESS (STM) genelinked to the -glucuronidase(GUS) gene was used as a tool for determining the timingofde novo shoot apical meristem (SAM) development incotyledon and root explants. Cotyledon and root explants from anSTM:GUS transgenic C24 line were placed on shoot inductionmedium and GUS expression was examined after 6-16 days ofculture. GUS expression could be found in localizedregionsof callus cells on root and cotyledon explants after 12 days indicating thatthese groups of cells were expressing the STM gene, hadreached the key time point of determination, and were producing an organizedSAM. This was consistent with the timing of determination as indicated byexplant transfer experiments. Root explants from anSTM:GUStransgenic Landsberg erecta line and a two-step tissue culture method revealedasimilar pattern of localized GUS expression duringde novo shoot organogenesis. This is the first studydocumenting the timing and pattern of expression of theSTMgene during de novo shoot organogenesis.     

Meta-topolin stimulates de novo shoot organogenesis and plant regeneration in cassava

A novel protocol for de novo shoot organogenesis from cassava has been developed utilizing meta-topolin to stimulate shoot regeneration from leaf, petiole and stem internode explants. While use of meta-topolin alone was capable of inducing shoot regeneration, a two-stage system combining meta-topolin with 2,4-D in a first stage medium, followed by subculture onto elevated levels of meta-topolin, was superior for inducing multiple shoot regeneration events in more than 35% of explants in cultivar TME 7. Caulogenesis was achieved in eleven additional cultivars. Meta-topolin was also found to be beneficial for stimulating shoot regeneration from somatic embryos and cotyledon explants. The shoot organogenesis techniques described enhance the capacity of existing embryogenic systems and present previously unavailable morphogenic pathways for developing genetic transformation and gene editing technologies in cassava.     

Thidiazuron-induced de novo shoot organogenesis on seedlings,etiolated hypocotyls and stem segments of Huang-qin

Development of an efficient in vitro propagation system for Huang-qin (Scutellaria baicalensis), a traditional Chinese medicinal plant used in the treatment of a wide range of human ailments, is described. Thidiazuron [TDZ: N-phenyl-N′- (1,2,3-thidiazol-5-ylurea)] effectively induced regeneration on cultured intact seedlings, etiolated hypocotyl explants and sterile stem segments of Huang-qin. Histological examinations of excised hypocotyl or nodal explants revealed that adventitious shoots formed through an intermediate callus. Comparison of TDZ-induced regeneration in the three tissue types indicated that isolation of explants was not essential for optimal regenerative efficiency. Significantly more regenerants formed along hypocotyls of intact seedlings (20 shoots/explant) than were observed on excised hypocotyls (9.7 shoots/explant) indicating that endogenous metabolites produced in adjacent tissues provided resources for the shoot initiation. More than 95% of de novo regenerants formed roots and then intact plantlets under either sterile culture or greenhouse conditions. Regeneration protocols developed in this study may provide the basis for improvement of this crop through the identification of medicinally active constituents and eventual development optimized pharmaceutical products. This revised version was published online in June 2006 with corrections to the Cover Date.     

1-Methylcyclopropene and ethylene as regulators of in vitro organogenesis in kiwi explants

In this paper, we study the influence of ACC, AVG and 1-MCP on in vitro organogenesis of kiwi (Actinidia deliciosa) explants and on ethylene metabolism. Results indicated that increasing ethylene production and accumulation in the head space of the culture vessel by adding ACC to the culture medium inhibited organogenesis, except for rooting, which increased and stimulated ACC oxidase activity threefold, whereas AVG increased the length and number of shoots and leaves and inhibited about 80% ACC synthase activity compared with the reference explants. 1-MCP exerts a stimulatory effect analogous to AVG, increasing ACC synthase activity and organogenesis of kiwi explants. This effect is not reverted by the addition of ACC to the culture medium. Therefore, ethylene production and its accumulation in the headspace of the culture vessels seems to be responsible for the inhibition of shoot development as well as increasing rooting in in vitro cultured kiwi explants.     

Flavonoid diversity and biosynthesis in seed of Arabidopsis thaliana

Functional characterization of genes involved in the flavonoid metabolism and its regulation requires in-depth analysis of flavonoid structure and composition of seed from the model plant Arabidopsis thaliana. Here, we report an analysis of the diverse and specific flavonoids that accumulate during seed development and maturation in wild types and mutants. Wild type seed contained more than 26 different flavonoids belonging to flavonols (mono and diglycosylated quercetin, kaempferol and isorhamnetin derivatives) and flavan-3-ols (epicatechin monomers and soluble procyanidin polymers with degrees of polymerization up to 9). Most of them are described for the first time in Arabidopsis. Interestingly, a novel group of four biflavonols that are dimers of quercetin-rhamnoside was also detected. Quercetin-3-O-rhamnoside (the major flavonoid), biflavonols, epicatechin and procyanidins accumulated in the seed coat in contrast to diglycosylated flavonols that were essentially observed in the embryo. Epicatechin, procyanidins and an additional quercetin-rhamnoside-hexoside derivative were synthesized in large quantities during seed development, whereas quercetin-3-O-rhamnoside displayed two peaks of accumulation. Finally, 11 mutants affected in known structural or regulatory functions of the pathway and their three corresponding wild types were also studied. Flavonoid profiles of the mutants were consistent with previous predictions based on genetic and molecular data. In addition, they also revealed the presence of new products in seed and underlined the plasticity of this metabolic pathway in the mutants.     

ARR12 promotes de novo shoot regeneration in Arabidopsis thaliana via activation of WUSCHEL expression

Auxin and cytokinin direct cell proliferation and differentiation during the in vitro culture of plant cells, but the molecular basis of these processes, especially de novo shoot regeneration, has not been fully elucidated. Here, we describe the regulatory control of shoot regeneration in Arabidopsis thaliana (L.) Heynh, based on the interaction of ARABIDOPSIS RESPONSE REGULATOR12 (ARR12) and WUSCHEL (WUS). The major site of ARR12 expression coincided with the location where the shoot apical meristem (SAM) initiated. The arr12 mutants showed severely impaired shoot regeneration and reduced responsiveness to cytokinin; consistent with this, the overexpression of ARR12 enhanced shoot regeneration. Certain shoot meristem specification genes, notably WUSCHEL (WUS) and CLAVATA3, were significantly downregulated in the arr12 explants. Chromatin immunoprecipitation (ChIP) and transient activation assays demonstrated that ARR12 binds to the promoter of WUS. These observations indicate that during shoot regeneration, in vitro, ARR12 functions as a molecular link between cytokinin signaling and the expression of shoot meristem specification genes.     

Structure of Flower in Arabidopsis thaliana: Spatial Pattern Formation

Morphological analysis of flowers was carried out in Arabidopsis thaliana wild type plants and agamous and apetala2 mutants. No direct substitution of organs takes place in the mutants, since the number and position of organs in them do not correspond to the structure of wild type flower. In order to explain these data, a notion of spatial pattern formation in the meristem was introduced, which preceded the processes of appearance of organ primordia and formation of organs. Zones of acropetal and basipetal spatial pattern formation in the flower of wild type plants were postulated. It was shown that the acropetal spatial pattern formation alone took place in agamous mutants and basipetal spatial pattern formation alone, in apetala2 mutants. Different variants of flower structure are interpreted as a result of changes in the volume of meristem (space) and order of spatial pattern formation (time).     

Embryo sac development in Arabidopsis thaliana

Summary Aspects of megasporogenesis in Arabidopsis thaliana have been investigated using a variety of histochemical techniques to visualize general cell organization, DNA and callose in whole ovules and sections by bright field, fluorescence, differential interference contrast and scanning electron microscopy. The microtubular cytoskeleton has been studied using immunofluorescence localization of tubulin in sections and whole cells. The observations deviate from reports of preceding studies in that the megasporocyte was found to undergo both meiotic divisions followed by simultaneous cytokinesis (i.e. without an intermediate dyad stage) to give a multiplanar tetrad of megaspores. This represents a variation of monosporic development not previously described. Polarized distribution of organelles prior to meiosis ensures that the functional megaspore receives the largest share. Aberrant wall formation is common between degenerating megaspores. Localized callose deposition in the tetrad separates these cells from the active megaspore. Their pattern of degeneration and displacement is extremely flexible within the embryo sac space. The microtubular cytoskeleton is extensive and largely cytoplasmic, as distinct from cortical, throughout megasporogenesis. In the megasporocyte, megaspores and one-nucleate embryo sac, randomly oriented microtubules throughout the cells may serve to maintain cytoplasmic integrity and position organelles. Numerous microtubules (MTs) associate closely with the nucleus and some radiate from it, perhaps functioning in nuclear positioning. During meiosis MTs are restricted to the spindle configurations and later to the phragmoplasts which form between daughter nuclei. The lack of interphase cortical arrays suggests that the role of internal influences on cell shape is small.     

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.     

Analysis of storage proteins in normal and aborted seeds from embryo-lethal mutants of Arabidopsis thaliana

The major storage proteins isolated from wild-type seeds of Arabidopsis thaliana (L.) Heynh., strain Columbia, were studied by sucrose gradient centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both the hypocotyl and cotyledons of mature embryos contained abundant 12 S (cruciferin) and 2 S (arabin) proteins that appeared similar in size and subunit composition to the cruciferin (12 S) and napin (1.7 S) seed-storage proteins of Brassica napus. The 12 S protein from Arabidopsis was resolved by SDS-PAGE into two groups of subunits with approximate relative molecular weights of 22–23 kDa (kilodalton) and 30–34 kDa. These polypeptides accumulated late in embryo development, disappeared early in germination, and were not detected in other vegetative or reproductive tissues. Accumulation of the 12 S proteins in aborted seeds from nine embryo-lethal mutants with different patterns of abnormal development was studied to determine the extent of cellular differentiation in arrested embryos from each mutant line. Abundant 12 S proteins were found in arrested embryos from two mutants with late lethal phases, but not in seven other mutants with lethal phases ranging from the globular to the cotyledon stages of embryo development. These results indicate that the accumulation of seed-storage proteins in wild-type embryos of Arabidopsis is closely tied to morphogenetic changes that occur during embryo development. Embryo-lethal mutants may therefore be useful in future studies on the developmental regulation of storage-protein synthesis.Abbreviations kDa kilodalton - M_r relative molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

A novel plastid-targeted J-domain protein in Arabidopsis thaliana

Arabidopsis cDNAs encoding ATJ11, the smallest known J-domain protein, have been isolated and characterized. The precursor protein of 161 amino acid residues was synthesized in vitro and imported by isolated pea chloroplasts where it was localized to the stroma and cleaved to a mature protein of 125 amino acid residues. The mature protein consists of an 80 amino acid J-domain, and N- and C-terminal extensions of 24 and 21 amino acid residues, respectively, which show no similarity to regions in other DnaJ-related proteins. ATJ11 produced in Escherichia coli stimulated the weak ATPase activity of E. coli DnaK, but was unable to stimulate refolding of firefly luciferase by DnaK, and inhibited refolding by DnaK, DnaJ and GrpE. ATJ11 is encoded by a single-copy gene on chromosome 4, and is expressed in all plant organs examined. A paralogue of ATJ11, showing 72% identity, is encoded in a 4.5 Mb duplication of chromosome 4 on chromosome 2. These proteins represent a novel class of J-domain proteins.     

Phosphate acquisition heterosis in Arabidopsis thaliana: a morphological and physiological analysis

Although phosphate acquisition efficiency (PAE) is of considerable agricultural importance, little is known about its inheritance. The objective of this study was to determine the inheritance of PAE-related morphological and physiological traits in Arabidopsis thaliana. C24 and Col-O, two Arabidopsis accessions differing in their abilities to acquire phosphate from hydroxyl phosphate, were crossed. The resulting hybrid showed superior acquisition of phosphate from hydroxylapatite when compared with either parent. The data suggest that the superiority of the F1 hybrid is due to the accumulation of favourable dominant genes at numerous loci. The hybrid inherited the long root hair length trait from C24 and the long root length trait of Col-O. In addition, the hybrid inherited enhanced phosphate transporter expression from C24. The analysis of morphological and physiological traits in this hybrid will be useful for evaluating and predicting PAE performance in other plant species.