Novel Insights from Live-imaging in Shoot Meristem Development

Microscopic imaging of fluorescent reporters for key meristem regulators in live tissues is emerging as a powerful technique,enabling researchers to observe dynamic spatial and temporal distribution of hormonal and developmental regulators in living cells.Aided by time-lapse microphotography,new types of imaging acquisition and analysis software,and computational modeling,we are gaining significant insights into shoot apical meristem(SAM) behavior and function.This review is focused on summarizing recent advances in the understanding of SAM organization,development,and behavior derived from live-imaging techniques.This includes the revelation of mechanical forces in microtubule-controlled anisotropic growth,the role of the CLV-WUS network in the specification of peripheral zone and central zone cells,the multiple feedback loops involving cytokinin in controlling WUS expression,auxin dynamics in determining the position of new primordia,and,finally,sequence of regulatory events leading to de novo assembly of shoots from callus in culture.Future studies toward formulating "digital SAM" that incorporates multi-dimensional data ranging from images of SAM morphogenesis to a genome-scale expression map of SAM will greatly enhance our ability to understand,predict,and manipulate SAM,containing the stem cells that give rise to all above ground parts of a plant.     

Shoot inversion-induced ethylene production: a general phenomenon?

Shoot inversion induction of ethylene production was found in inverted shoots of corn, peas, soybean, sunflower, tomato, andPharbitis nil. The increases in ethylene production were found to range from two- to threefold in soybean to eightfold in corn and sunflower. The occurrence of peaks of ethylene production ranged from 16 h following shoot inversion in corn to 72 h inPharbitis. That the enhanced ethylene production was due to activation of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase is supported by the finding of increased ACC content in inverted shoots of all species tested. Shoot inversion inhibition of elongation was found in inverted shoots of pea, soybean, sunflower, tomato, andPharbitis nil. This inhibition is thought to be mediated via increased ethylene production in the inverted shoots. That shoot inversion induction of ethylene is not a persistent effect is supported by the finding that ethylene synthesis could be terminated by reorientation of shoots to the upright position and could be reinitiated by the subsequent inversion of the shoots. The effects of shoot inversion on the enhancement of ethylene production and on the inhibition of elongation of the inverted shoot appear to be general phenomena.     

Is There a Role for the Apex in Shoot Geotropism?

Experiments with horizontal etiolated sunflower (Helianthus annuus L.) seedlings supported centrally such that both apical and basal ends are free to react to geostimulus, revealed that the apical end commences curvature 1 to 2 hours earlier than the basal end. The later curvature in the basal region is a consequence of the absence of growth in the initial period rather than merely slower growth. A comparison of zonal growth rates in a vertical and a horizontal seedling confirmed that geostimulus induces a renewal of growth in a region where growth had ceased. Removing the apical half of the hypocotyl showed that the curvature resulting from this growth initiation in the basal region is dependent on attachment to the apical region. Evidence that this dependence is unlikely to be due to energy deficiency is adduced. The prior response of the apical end to geostimulus and the apically dependent later initiation of new growth in the basal region are compatible with the delay inherent in message transport from apex to base and are considered as evidence for apical involvement in the totality of the seedling's georesponse.     

Shoot δ15N correlates with genotype and salt stress in barley

Given a uniform N source, the ¹⁵N of barley shoots provided a genotypic range within treatments and a separation between control and salt-stress treatments as great as did ¹³C^*. Plant ¹⁵N has been represented in the literature as a bioassay of external source ¹⁵N and used to infer soil N sources, thus precluding consideration of the plant as a major cause in determining its own 8¹⁵N. We believe this to be the first report of plant ¹⁵N as a genetic trait. No mechanistic model is needed for use of ¹⁵N as a trait in controlled studies; however, a qualitative model is suggested for further testing.Symbol ¹⁵N (or ¹³C) the difference between: (1) the ratio of heavy to light isotopes of the element in a sample and (2) that of its reference standard     

The Roles of Auxin in Regulating “Shoot Branching” of Cremastra appendiculata

Cremastra appendiculata (D. Don) Makino is a mainly vegetative propagation terrestrial orchid that is a typical representative of the warm-temperate vegetation in China. In this experiment, we investigated the growth and development process of C. appendiculata leaf buds and examined their biochemical components (proteins, auxin, and cytokinin) to gain insight into the “shoot branching” of C. appendiculata pseudobulb string. The results showed that the metabolic activity of C. appendiculata pseudobulbs became lower with the increase of pseudobulb age. However, biennial and triennial pseudobulbs have higher auxin levels than annual pseudobulbs in the intact plant (P < 0.05). After decapitation, the auxin rapidly reduces in biennials. The reduction of auxin level promotes cytokinin biosynthesis, which makes the biennial dormant buds start to germinate 18 days after decapitation. These data and phenomena suggested that auxin plays important roles in regulating shoot branching of C. appendiculata, although further studies are needed to consolidate this viewpoint. Our data indirectly support the classical apical dominance theory whereby biennial pseudobulbs are strongly dependent on reduced auxin to initiate leaf bud outgrowth.

     

Shoot Apex Demand Determines Assimilate and Nutrients Partitioning and Nutrient-uptake Rate in Tobacco Plants

Our previous experiment revealed that apex-removed plants have larger root systems but a lower K＋-uptake rates than intact tobacco plants. Since the apex is not only a center of growth and metabolism, but also an important place of auxin synthesis and export, the aims of this study were to distinguish whether the apex demand or auxin synthesized in the apex regulates assimilate and nutrients partitioning within plant, and to explain the reason for the lower K＋-uptake rate of the apex-removed plant. In comparison with the control plant, covering the shoot apex with a black transparent plastic bag reduced net increases in dry matter and nutrients; however, the distribution of the dry matter and nutrients between shoot and roots and nutrient-uptake rates were not changed. Removal of the shoot apex shifted the dry mass and nutrients distributions to roots, and reduced the rate of nutrient uptake. Application of 1-naphthylacetic acid （NAA） could partly replace the role of the removed apex, stimulated assimilate and nutrient deposition into the treated tissue, and enhanced the reduced plasma membrane ATPase activity of roots to the control level. However, treatment of the apex-removed plants with NAA could not rescue the reduced nutrient uptake rate and the shifted assimilates and nutrients partitioning caused by excision of the apex. Higher nutrient uptake rate of the intact plants could not be explained by root growth parameters, such as total root surface area and number of root tips. The results from the present study indicate that strong apex demand determined assimilates and nutrients partitioning and nutrient-uptake rate in tobacco （Nicotiana tabacum） plants.     

Shoot regeneration from petioles and leaves of Vitis X labruscana ‘Catawba’

Summary Shoot regeneration and normal plants were obtained from leaf and petiole explants derived from in vitro grown shoots of Vitis X labruscana Catawba. Regeneration was induced in the presence of both 6-benzylaminopurine and indole-3-butyric acid; combinations of 2,4-dichlorophenoxyacetic acid or 2-naphthoxyacetic acid with 6-benzylaminopurine did not permit regeneration from leaf explants. Up to 15% of leaf and 70% of petiole explants regenerated shoots on media with 5.0–10.0 M BA and 0.1–0.5 M IBA. Incubation in the dark was required to obtain regeneration. About 50% of shoots developed normally following transfer to light. An average of one shoot regenerated from leaf explants and 3.3 shoots regenerated per petiole explant. Regeneration from petioles and leaves was always from the basipetal end. The interaction of 6-benzylaminopurine with indole-3-butyric acid was also examined.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NN69 Nitsch and Nitsch (1969) medium - NOA 2-naphthoxyacetic acid     

Interaction between a Nanovirus-like Component and the Tobacco Curly Shoot Virus／Satellite Complex

The biological role of DNA1, a nanovirus-like component shown to be associated with the begomovirus/satellite complex, has not yet been identified. Here, we demonstrated that DNA1 of Tobacco curly shoot virus isolate Y35 (TbCSV-Y35) attenuated leaf-curling symptoms induced by TbCSV-Y35 or TbCSV-Y35 plus Y35 DNAβ in the early stage of symptom development and induced leaf cluster at a later stage of symptom development in Nicotiana benthamiana plants. The leaf disc assay demonstrated that TbCSV-Y35 DNA1 replicated autonomously. Southern blot analysis revealed that TbCSV-Y35 DNA1 reduced viral DNA accumulation. Viral DNA accumulation was not reduced when plants were co-inoculated with TbCSV-Y35 DNAβ, but the TbCSV-Y35 DNAβ level was dramatically reduced in the presence of TbCSV-Y35 DNA1. To determine whether the interaction between TbCSV/satellite complex and DNA1 had isolate specificity, DNA1 of TbCSV isolate Y132 was cloned and sequenced. It was found to have 75% nucleotide sequence identity with TbCSV-Y35 DNA1. Infectivity tests showed that TbCSV-Y132 DNA1 had no effect on the symptoms induced by TbCSV-Y35 or TbCSV-Y35 and Y35 DNAβ in N. benthamiana plants, although Y 132 DNA1 could replicate in these plants.     

Optimized Shoot Regeneration System for the Commercial Korean Radish ‘Jin Ju Dae Pyong’

We have investigated the usefulness of hypocotyl (cultured on N1B2 medium) and cotyledon explants (on CR medium: Murashige and Skoog (MS) medium, 3% sucrose, 20 μm benzylaminopurine, pH 5.8) for the regeneration of shoots of the Korean radish ‘Jin Ju Dae Pyong’. The importance of ethylene (indirectly), polyamines and gelling agent were studied in both media. Although the addition of ethylene-inhibitors and silver nitrate to the culture media were beneficial towards shoot regeneration and agar-based treatments (0.8% w/v) being superior in shoot production compared to agarose treatments (0.4% w/v), both explants responded differently in culture. Hypocotyls cultured in the presence of silver nitrate or aminoethoxyvinylglycine (AVG) regenerated significantly (p < 0.05) more shoots compared to N1B2 medium alone; supplementation of 20 μM AVG to N1B2 medium gave optimal shoot production (40% of explants regenerating shoots). The addition of 10 μM AVG to CR medium produced maximum shoot regeneration from cotyledon explants (60% producing shoots). Plants derived from 3-month-old cultures produced greater seed weights, larger leaves and greater genetic variability (50–80% of cells having 20–40 chromosomes) compared to seed-derived (85–90% diploid) and plants from 1-month-old cultures (78–88% diploid). Our results show, that if prolonged culture of explants is avoided, a large number of phenotypically-normal plants can be produced, which in turn, could be utilized in the genetic improvement of radish.     

Shoot and root proliferation in ‘Williams’ banana: are the topolins better cytokinins?

The effects of five topolins (meta-Topolin?=?mT; meta-Topolin riboside?=?mTR; meta-Methoxy topolin?=?MemT; meta-Methoxy topolin riboside?=?MemTR and meta-Methoxy topolin 9-tetrahydropyran-2-yl?=?MemTTHP) on shoot and root regeneration of micropropagated ??Williams?? bananas were compared to benzyladenine (BA). Sterile shoot-tip explants were cultured on modified Murashige and Skoog (MS) media containing 10, 20 or 30???M of the above cytokinins (CKs) for 42 days. Using shoot regenerated from optimum CK concentration, rooting experiments involving the use of indole-3-acetic acid, indole-3-butyric acid, naphthalene acetic acid, smoke?Cwater (SW) and karrikinolide (KAR₁) were conducted. Apart from 10???M BA and 30???M MemTTHP treatments, the number of shoots produced in all the CK treatments were significantly higher than the control. In addition, 30???M mT resulted in the highest number of shoots (7.3?±?1.0). Unlike other CK treatments requiring higher concentrations, optimum mean shoot number per explant rate was attained at the lowest concentration in MemT and MemTTHP (10???M) treatments. The use of 10???M MemTTHP had the least root inhibitory effect during the shoot proliferation phase. In terms of abnormality index, mTR-regenerated plantlets were of the best quality across all the CKs tested. In mT- and BA-derived shoots, SW and KAR₁ significantly increased the number and length of roots compared to the control. Overall, when compared to BA, the use of topolin demonstrated higher mean shoot number per explant (MemT and MemTTHP) at lowest CK concentrations and the ease of rooting during the shoot proliferation phase (MemTTHP). During the rooting phase, topolin treatments produced more off-shoots than BA-treated ones. The current finding is a further demonstration of the increasing importance of topolins in micropropagation.     

Is Predetermined Cellular Competence Required for Alternative Embryo or Shoot Induction on Sunflower Zygotic Embryos?

Abstract: Somatic embryos and adventitious shoots can be induced simultaneously on immature zygotic embryos of sunflower, their relative distribution on the explant being constant but their relative number depending on the sucrose concentration of the culture medium. We show here that the cells at the origin of both types of structures are competent for either development and express this potential as a function of their position relative to the medium. Two zones, inductive for somatic embryos or adventitious shoots, have been established under the influence of the culture medium. The precision of their localisation is demonstrated by the existence of tissue chimeras which also indicate the presence of diffusible signal(s) responsible for the determination of cellular identity. We have further determined that commitment to the organogenic pathway occurred very rapidly, i.e. within 12 h after the onset of the culture. A synthetic model is presented in which we suggest a possible sequence of key events involved in the morphogenic processes.     

Shoot regeneration and cryopreservation of shoot tips of apple (Malus) by encapsulation–dehydration

Here, we report an efficient and widely applicable method for cryopreservation of Malus shoot tips by encapsulation–dehydration using adventitious shoots. Shoots were induced from leaf segments cultured on a shoot induction medium containing 2–3 mg L^?1 thidiazuron, depending on genotype, and 0.5 mg L^?1 indole-3-butyric acid. Shoot tips (3 mm in length) containing six leaf primordia excised from 11-wk-old adventitious shoots were encapsulated and precultured with 0.5 M sucrose for 5 d, followed by air-drying for 6 h prior to direct immersion in liquid nitrogen. With our protocol, we obtained a mean organogenesis rate of 100%, a mean of 4.5 adventitious shoots per explant (leaf segment), and a mean shoot recovery of 57.0% from cryopreserved shoot tips in four Malus species. Inter-simple sequence repeat (ISSR) analysis did not reveal any polymorphic bands in regenerants recovered from either leaf segments or cryopreserved shoot tips of ‘Gala’. To the best of our knowledge, this is the first report on cryopreservation of Malus shoot tips using adventitious shoots derived from leaf segments and is the most widely applicable protocol so far reported for cryopreservation of Malus. Establishment of this protocol provides an alternative means for cryopreservation of Malus.     

On Donor–Acceptor Relations and Growth Correlation in Shoot Development of the Pedunculate Oak Germling

We studied the first cycle of shoot growth of the Quercus robur L. germlings in which the donor–acceptor relations were changed by removing a part of cotyledons, growing in darkness, or removing growing leaf blades. In all cases, the greatest changes in growth and growth correlations were observed in the shoot upper metameres carrying leaf blades and the least, in lower metameres preformed in the acorn embryo. The removal of growing leaves changed the rhythm of shoot growth.     

Does Auxin Play a Role in the Release of Apical Dominance by Shoot Inversion in Ipomoea nil?

According to the auxin-inhibition hypothesis of apical dominance,apically produced auxin moves down the stem and inhibits axillarybud outgrowth, either directly or indirectly. This hypothesishas been examined further by monitoring changes in basipetalauxin transport and endogenous auxin concentration in Ipomoeanil caused by shoot inversion, a stimulus that releases apicaldominance. The results indicate that inversion reduces auxintransport in the main stem. In upright shoots of intact plants,a 16-h pretreatment with [³H]IAA 4 cm below the apex resultsin downward movement of label and accumulation in nodes, especiallythe cotyledonary node. Label does not accumulate in the lateralbuds. GC-MS determinations of endogenous free auxin level inthe fourth node, where a lateral bud grows out following inversionof the upper part of the shoot, show no changes at 3 and 8 hafter inversion, the range of times for inversion-induced budrelease, or at 24 h, when bud outgrowth is continuing. However,inversion did cause a just-detectable decrease (approx. 10%)in the IAA level of the shoot's elongation region. Althoughauxin transport in segments of the main stem is partially inhibitedby inversion over a period shorter than the latent time of budrelease, thus providing a means for the expected depletion ofauxin in the fourth node, no depletion could be detected there.These results suggest that either a decrease in IAA level inthe main stem is not causal of bud release or that the decreasedIAA pool responsible for bud release is compartmented and cannotbe measured in whole-tissue extracts.Copyright 1993, 1999 AcademicPress Apical dominance, auxin content, auxin transport, axillary bud release, GC-MS, Ipomoea nil, Pharbitis nil, shoot inversion     

<Emphasis Type="Italic">Rhodococcus fascians</Emphasis>: Shoot Proliferation without Elevated Cytokinins?

In order to determine whether the disease symptoms caused by virulent strains of Rhodococcus fascians are due to increased cytokinin activity in infected tissues, germinating peas (Pisum sativum cv Novella) were inoculated with either a virulent strain or a nonvirulent strain of Rhodococcus fascians. The nonvirulent strain lacked both the ipt gene and the putative cytokinin oxidase/dehydrogenase homologue, fas5. Control peas were not inoculated. Twelve cytokinins were isolated from pea shoots 3, 6 and 9 days post-inoculation. Within 6 days of inoculation the levels of cytokinin free bases, ribosides, O-glucosides and nucleotides were decreased in shoots inoculated with the virulent strain, and were increased in shoots inoculated with the nonvirulent strain relative to the uninoculated control. The results are discussed with respect to the classic Skoog and Miller (1965) model of organogenesis and to the possible involvement of the plant cytokinin oxidase/dehydrogenase during infection by virulent strains of R. fascians.     

How is Regeneration of Plants after Mowing Affected by Shoot Size in Two Species-Rich Meadows with Different Water Supply?

Mowing a meadow is an example of an equalizing process that reduces differences among species by removing aboveground biomass approximately 5 cm above ground. This regular disturbance that affects all plants prevents competitive exclusion of small species and thus allows coexistence of numerous species differing in shoot size. In this paper we search for the mechanism behind this by comparing the shoot biomass of 41 common species in dry and wet species-rich meadows in mown and recently abandoned plots in June (before mowing) and in October. We asked the following questions: i) Do the plants differ in proportion of biomass lost by mowing? ii) Are the mown plants able to compensate for biomass lost by mowing? iii) Is the compensatory ability of mown plants related to their size? iv) Is the compensatory ability of plants related to severity of disturbance (removed biomass)? v) Does water availability in meadows affect these features? Our results revealed that the earlier explanation of equalization of meadow plants after mowing due to the proportionally larger biomass loss in larger plants than small plants does not represent the entire mechanism. Even when larger plants in the wet meadow lost more biomass, the proportion of lost biomass was not dependent on plant size, and compensation ability (growth of mown in comparison with unmown plants) was not related to the lost biomass in this meadow type. On the contrary, the observed pattern could be explained by different compensation abilities of small versus tall plants. In addition, according to our expectations, the compensation for lost biomass in the wet meadow was higher than in the dry one.     

Accelerated Early Growth of Rice at Elevated CO2 (Is It Related to Developmental Changes in the Shoot Apex?)

The influence of elevated CO2 on the development of the shoot apex and on subsequent vegetative growth and grain yield was investigated using rice (Oryza sativa L. cv Jarrah) grown in flooded soil at either 350 or 700 [mu]L CO2 L-1. At 8 d after planting (DAP), elevated CO2 increased the height and diameter of the apical dome and lengths of leaf primordia and tiller buds but had no effect on their numbers. By 16 DAP, there were five tiller buds in the apex at 700 [mu]L CO2 L-1 compared with only three tiller buds at 350 [mu]L CO2 L-1. These changes in development of the shoot apex at high CO2 were forerunners to faster development of the vegetative shoot at elevated CO2 between 11 and 26 DAP as evidenced by increases in the relative growth rates of the shoot and tillers. Accelerated development at high CO2 was responsible for the 42% increase in tiller number at the maximum tillering stage and the 57% enhancement of grain yield at the final harvest. The link between high CO2 effects on development during the first 15 DAP and final tiller number and grain yield was demonstrated by delaying exposure of plants to high CO2 for 15 d. The delay totally inhibited the tillering response to high CO2, and the increase in grain yield of 20% arose from a greater number of grains per panicle. Consequently, it can be concluded that accelerated development in the shoot apex early in development is crucial for obtaining maximum increases in grain yield at elevated atmospheric CO2 concentrations.     

Competition Strategies of Resources Between Stipa grandis and Cleistogenes squarrosa. Ⅰ. Morphological Response of Shoot and Root on Sulfur Supply

Effects of nutrients (compounds of macro- and micro-elements) supply without sulfur (T₁) and with sulfur (T₂) on the competition between Stipa grandis L. (C₃) and Cleistogenes squarrosa L. (C₄) were examined using a replacement series design in a greenhouse experiment over a period of 80 d. Blank treatment (nothing applied) (T₀) was conducted simultaneously. It seems to be no inter-competition between S. grandis and C. squarrosa in all treatments. There was also no intra-competition for S. grandis in all treatments, and for C. squarrosa under T₀ treatment because of low productivity. However, the intra-competition of aboveground of C. squarrosa was significant when nutrients were supplied regardless of sulfur (S) application. The interaction on dry weight of C. squarrosa per pot between S supply and proportion of C. squarrosa was observed. Under the treatments of T₁ and T₂ the shoot growth of S. grandis increased significantly compared with T₀ treatment, but there was no significant difference between T₁ and T₂ treatments, indicating S had no effect on it. Root morphologies of S. grandis was not sensitive to nutrients added. Nutrients supply (T₁ and T₂ treatments) not only increased significantly the shoot growth of C. squarrosa, but also increased significantly its root growth. Sulfur increased significantly growth of the shoot and root of C. squarrosa. Nutrients supply decreased significantly ratio of root to shoot dry matter (RRS) of C. squarrosa regardless of S application, but the RRS of S. grandis was not affected by nutrients applied. Sulfur also decreased significantly the RRS of C. squarrosa in 100% and 75% proportions of C. squarrosa. The RRS of C. squarrosa was greater significantly in the 100% proportion than that in the 25% proportion of C. squarrosa for all treatments. Therefore, photosynthesis is more allocated to root in infertility soils than in fertility soils and the competition for nutrient resources stimulates root production. The degree of leaf greenness of C. squarrosa in T₁ treatment was less significantly than that in T₀ and T₂ treatments, and it was greater significantly in the 25% proportion than in the 100% proportion of C. squarrosa under T₁ and T₂ treatments, indicating that nutrients supply increase chlorophyll content in plant and may accentuate S deficiency or low plant productivity alleviate S deficiency.