Subapical Stem Research Articles

Shade signals alter the expression of circadian clock genes in newly-formed bioenergy sorghum internodes.

Stem internodes of bioenergy sorghum inbred R.07020 are longer at high plant density (shade) than at low plant density (control). Initially, the youngest newly‐formed subapical stem internodes of shade‐treated and control plants are comparable in length. However, full‐length internodes of shade‐treated plants are three times longer than the internodes of the control plants. To identify the early molecular events associated with internode elongation in response to shade, we analyzed the transcriptome of the newly‐formed internodes of shade‐treated and control plants sampled between 4 and 6 hr after the start of the light period (14 hr light/10 hr dark). Sorghum genes homologous to the Arabidopsis shade marker genes ATHB2 and PIL1 were not differentially expressed. The results indicate that shade signals promote internode elongation indirectly because sorghum internodes are not illuminated and grow while enclosed with leaf sheaths. Sorghum genes homologous to the Arabidopsis morning‐phased circadian clock genes LHY, RVE, and LNK were downregulated and evening‐phased genes such as TOC1, PRR5, and GI were upregulated in young internodes in response to shade. We hypothesize that a change in the function or patterns of expression of the circadian clock genes is the earliest molecular event associated with internode elongation in response to shade in bioenergy sorghum. Increased expression of CycD1, which promotes cell division, and decreased expression of cell wall‐loosening and MBF1‐like genes, which promote cell expansion, suggest that shade signals promote internode elongation in bioenergy sorghum in part through increasing cell number by delaying transition from cell division to cell expansion.

The actinobacteria-specific gene wblA controls major developmental transitions in Streptomyces coelicolor A3(2)

The Streptomyces coelicolor A3(2) sporulation gene whiB is the paradigm of a family of genes (wbl, whiB-like) that are confined to actinobacteria. The chromosome of S. coelicolor contains 11 wbl genes, among which five are conserved in many actinobacteria: whiB itself; whiD, a sporulation gene; wblC, which is required for multi-drug resistance; and wblA and wblE, whose roles had previously been little studied. We succeeded in disrupting wblA and the six non-conserved genes, but could not disrupt wblE. Although mutations in the six non-conserved wbl genes (including some multiple wbl mutants) produced no readily detectable phenotype, mutations in wblA had novel and complex effects. The aerial mycelium of wblA mutants was coloured red, because of the ectopic presence of pigmented antibiotics (actinorhodin and undecylprodigiosin) normally confined to lower parts of wild-type colonies, and consisted almost entirely of non-sporulating, thin, straight filaments, often bundled together in a fibrillar matrix. Rare spore chains were also formed, which exhibited wild-type properties but were genetically still wblA mutants. A wblA mutant achieved higher biomass than the wild-type. Microarray analysis indicated major transcriptional changes in a wblA mutant: using a relatively stringent cut-off, 183 genes were overexpressed, including genes for assimilative primary metabolism and actinorhodin biosynthesis, and 103 were underexpressed, including genes associated with stages of aerial hyphal growth. We suggest that WblA is important in both the slow-down of biomass accumulation and the change from aerial hyphal initial cells to the subapical stem and apical compartments that precede sporulation; and that the mutant aerial mycelium consists of recapitulated defective aerial hyphal initial cells.

“Lateral Control”: Phytohormone Relations in the Conifer Treetop and the Short- and Long-Term Effects of Bud Excision in Abies nordmanniana

In a conifer tree, such as Nordmann fir, Abies nordmanniana Spach, the leader bud and its immediate surroundings play a decisive role in crown architecture. As subapical branch buds are segregated from the leader meristem, resource allocation between ortho- and plagiotropic growth is determined. The relationship between treetop buds in young trees was studied in the natural state and after surgical removal in early July of either the leader bud (decapitation) or the subapical whorl branch buds (destipitation). The two bud types showed consistent cytokinin profile differences but similar seasonal dynamics in cytokinins and auxin (IAA). After bud excision, ZRP increased dramatically in the subapical stem within 1 h, followed by ZR within 1 week. Supernormal levels of ZR were maintained through autumn and persisted in spring in the destipitated trees, but had returned to normal in the decapitated trees. The treetop buds remaining after bud excision experienced an immediate decrease in most cytokinins, followed, however, by a large surplus later in the season. The following spring this high level persisted in the leader bud of destipitated trees, but not in whorl buds of decapitated trees. Conspicuous growth pattern changes followed from destipitation, but few from decapitation. Growth reactions suggest that resource allocation to main branch buds inhibits leader growth in normal trees, a kind of “lateral control.” Auxin and ABA content in buds and stems was largely unaffected by treatments. Data suggest that subapical leader tissues beneath the apical bud group are a primary source of cytokinin regulation.

Promotion of shoot development and tuberisation in potato by expression of a chimaeric cytokinin synthesis gene at normal and elevated CO2 levels

The bacterial cytokinin biosynthesis gene ipt under control of a chalcone synthase promoter (PCHS) was introduced into potato (Solanum tuberosum L.). Two transgenic lines were selected for detailed study, because in these, root development was reduced only moderately, thus, enabling the plants to be grown in pots. Expression of the PCHS-ipt gene elevated the level of zeatin cytokinins markedly in the apical bud, subapical stems and leaves. The transgenic (IPT) plants exhibited a lower and denser leaf canopy relative to wild-type (WT) plants owing to reduction in main stem length, increase in node number per stem and promotion of lateral shoot development. Main stem diameter was increased markedly due to promotion of cell division associated with activation of cyclin-dependent kinase in the subapical stem. Expression of the PCHS-ipt gene induced aerial stolons, promoted growth of underground stolons and increased tuber number but reduced tuber weight and nitrogen content. The gene expression also increased pinnae and pinnule number per leaf, increased thickness of pinnae and promoted transpiration, photosynthesis and stomatal conductance – effects monitored by gas exchange and 18O and 13C analysis. The elevation of [CO2] to 900 μmol mol–1 promoted growth of both WT and IPT plants, ameliorated the negative effect of high cytokinin on tuber weight and interacted additively with ipt gene expression to promote stem growth.

Cytokinin Profiles in the Conifer Tree Abies nordmanniana: Whole-Plant Relations in Year-Round Perspective

Conifer trees are routinely manipulated hormonally to increase flowering, branching, or adjust crown shape for production purposes. This survey of internal cytokinin levels provides a background for such treatments in Abies nordmanniana, a tree of great economic interest. Reference points in the crown and root system were sampled destructively in 4- and 6-year-old trees and analyzed for a range of cytokinins by LC-MS/MS. No seasonal patterns were detected in the root samples, and a major portion of cytokinin was in conjugated forms. Dramatic and consistent seasonal changes occurred in the crown, at levels 17–65 times higher than in the root. Predominant among crown cytokinins was ZR, except in the needles where IPR was also prominent. Within the crown, cytokinin profiles in different organs differed consistently. The leader bud showed a pronounced mid-June minimum, and a maximum later in summer. Subapical buds showed the same June minimum but peaked in mid autumn at a much lower level. Maxima in these buds were preceded by peaks in the subapical stem. Parallel patterns were observed in homologous tissues on branches.This pattern is consistent with two surges beginning in the uppermost stem tissues leading to subsequent accumulation or stimulated production within the buds. Strong differential hormonal profiles between adjacent buds with different fates agree with recent evidence of localized cytokinin production. The data suggest a reduced role of root-derived cytokinins in crown development. Practical cytokinin treatments for crown-shape regulation require close attention to dosage as well as precise timing and positioning.

Selective Deactivation of Gibberellins below the Shoot Apex is Critical to Flowering but Not to Stem Elongation of Lolium

Gibberellins (GAs) cause dramatic increases in plant height and a genetic block in the synthesis of GA(1) explains the dwarfing of Mendel's pea. For flowering, it is GA(5) which is important in the long-day (LD) responsive grass, Lolium. As we show here, GA(1) and GA(4) are restricted in their effectiveness for flowering because they are deactivated by C-2 hydroxylation below the shoot apex. In contrast, GA(5) is effective because of its structural protection at C-2. Excised vegetative shoot tips rapidly degrade [14C]GA(1), [14C]GA(4), and [14C]GA(20) (>80% in 6 h), but not [14C]GA(5). Coincidentally, genes encoding two 2beta-oxidases and a putative 16-17-epoxidase were most expressed just below the shoot apex (<3 mm). Further down the immature stem (>4 mm), expression of these GA deactivation genes is reduced, so allowing GA(1) and GA(4) to promote sub-apical stem elongation. Subsequently, GA degradation declines in florally induced shoot tips and these GAs can become active for floral development. Structural changes which stabilize GA(4) confirm the link between florigenicity and restricted GA 2beta-hydroxylation (e.g. 2alpha-hydroxylation and C-2 di-methylation). Additionally, a 2-oxidase inhibitor (Trinexapac Ethyl) enhanced the activity of applied GA(4), as did limiting C-16,17 epoxidation in 16,17-dihydro GAs or after C-13 hydroxylation. Overall, deactivation of GA(1) and GA(4) just below the shoot apex effectively restricts their florigenicity in Lolium and, conversely, with GA(5), C-2 and C-13 protection against deactivation allows its high florigenicity. Speculatively, such differences in GA access to the shoot apex of grasses may be important for separating floral induction from inflorescence emergence and thus could influence their survival under conditions of herbivore predation.

A novel compartment, the ‘subapical stem’ of the aerial hyphae, is the location of a sigN‐dependent, developmentally distinct transcription in Streptomyces coelicolor

Streptomyces coelicolor has nine SigB-like RNA polymerase sigma factors, several of them implicated in morphological differentiation and/or responses to different stresses. One of the nine, SigN, is the focus of this article. A constructed sigN null mutant was delayed in development and exhibited a bald phenotype when grown on minimal medium containing glucose as carbon source. One of two distinct sigN promoters, sigNP1, was active only during growth on solid medium, when its activation coincided with aerial hyphae formation. Transcription from sigNP1 was readily detected in several whi mutants (interrupted in morphogenesis of aerial mycelium into spores), but was absent from all bld mutants tested, suggesting that sigNP1 activity was restricted to the aerial hyphae. It also depended on sigN, thus sigN was autoregulated. Mutational and transcription studies revealed no functional significance to the location of sigN next to sigF, encoding another SigB-like sigma factor. We identified another potential SigN target, nepA, encoding a putative small secreted protein. Transcription of nepA originated from a single, aerial hyphae-specific and sigN-dependent promoter. While in vitro run-off transcription using purified SigN on the Bacillus subtilis ctc promoter confirmed that SigN is an RNA polymerase sigma factor, SigN failed to initiate transcription from sigNP1 and from the nepA promoter in vitro. Additional in vivo data indicated that further nepA upstream sequences, which are likely to bind a potential activator, are required for successful transcription. Using a nepA-egfp transcriptional fusion we located nepA transcription to a novel compartment, the 'subapical stem' of the aerial hyphae. We suggest that this newly recognized compartment defines an interface between the aerial and vegetative parts of the Streptomyces colony and might also be involved in communication between these two compartments.

A florigenic effect of sucrose in Fuchsia hybrida is blocked by gibberellin-induced assimilate competition.

The use of gas chromatography-mass spectrometry-selected ion monitoring along with a (13)C internal standard has allowed sensitive measurements of the sucrose (Suc) content of individual shoot apices of Fuchsia hybrida. With intact plants, as the photosynthetic irradiance increased, so did shoot apex Suc content, reaching saturation at about 500 micromol m(-2) s(-1). These same plants flowered at the higher irradiances, remaining vegetative in 10-h short days at an irradiance of 230 micromol m(-2) s(-1). The strong correlation (r = 0.93) in these studies between flowering and shoot apex Suc content indicates a role for Suc as a stimulus to flowering in this species. However, Suc is not the long-day (LD) "florigen" of F. hybrida because 2 to 4 LD given as a 14-h low-irradiance photoperiod extension (10-15 micromol m(-2) s(-1)) induced flowering but without increase in shoot apex Suc content. Flowering induced by either pathway, the LD- or the Suc-mediated one, was inhibited by applying gibberellin (GA) to the shoot tip. Such inhibition of flowering by GA, at least for the LD pathway, was associated with a reduced apex Suc content, enhanced elongation of subapical stem tissue, and a reduced import into the shoot apex of leaf-sourced assimilate. Thus, our findings show how GA inhibits flowering of F. hybrida and confirm the importance of nutrient diversion in regulating flowering.

Recognition of 5'-terminal TAR structure in human immunodeficiency virus-1 mRNA by eukaryotic translation initiation factor 2.

TAR, a 59 nt 5'-terminal hairpin in human immuno-deficiency virus 1 (HIV-1) mRNA, binds viral Tat and several cellular proteins. We report that eukaryotic translation initiation factor 2 (eIF2) recognizes TAR. TAR and the AUG initiation codon domain, located well downstream from TAR, both contribute to the affinity of HIV-1 mRNA for eIF2. The affinity of TAR for eIF2 was insensitive to lower stem mutations that modify sequence and structure or to sequence changes throughout the remainder that leave the TAR secondary structure intact. Hence, eIF2 recognizes structure rather than sequence in TAR. The affinity for eIF2 was severely reduced by a 3 nt change that converts the single A bulge into a 7 nt internal loop. T1 footprinting showed that eIF2 protects nucleotides in the loop as well as in the strand opposite the A bulge. Thus, eIF2 recognizes the TAR loop and lower part of the sub-apical stem. Though not contiguous, these regions are brought into proximity in TAR by a bend in the helical structure induced by the UCU bulge; binding of eIF2 opens up the bulge context and apical stem. The ability to bind eIF2 suggests a function for TAR in HIV-1 mRNA translation. Indeed, the 3 nt change that reduces the affinity of TAR for eIF2 impairs the ability of reporter mRNA to compete in translation. Interaction of TAR with eIF2 thus allows HIV-1 mRNA to compete more effectively during protein synthesis.

A localised decrease of GA1 in shoot tips of Salix pentandra seedings precedes cessation of shoot elongation under short photoperiod

By application of a recently developed method allowing analysis of gibberellins (GAs) in mg amounts of tissue, the effect of photoperiod on levels of GAs in shoot tips of individual seedlings of the woody species Salix pentandra was studied. In elongating long day‐grown seedlings, maximum levels of GA1 were found 5–20 mm below the apex, approximately twice the levels in other segments. After exposure of plants to 5 or 15 short days, the levels of GA1 were about 50% lower within this specific region of the stem, as compared with seedlings grown under long days. Short day‐induced cessation of shoot elongation also correlated with overall declines in the levels of GA53, GA19, GA20 and GA8, Within each photoperiodic treatment the levels of these GAs were generally relatively similar throughout the upper 35 mm of stems. No differences in internode lengths or in lengths of pith or epidermal cells were found in plants grown under long days compared with those exposed to 5 short days. In both cases, cells in mitosis were observed in the subapical stem tissues of shoot tips. After 15 short days, stem elongation was completed, and dividing cells were generally not found in the subapical part of the stem. However, short day exposure did not prevent elongation of internodes and cells differentiated before the treatment was started. Thus, the localised decrease in level of GA1 in shoot tips under short days precedes the morphological and anatomical changes connected with the short day‐induced cessation of elongation growth. This supports the hypothesised role for GA1 in photoperiodic control of shoot elongation in S. pentandra.

Regulation of ethylene biosynthesis after short‐term heat treatment in etiolated pea stems

Incubation of plant tissues at a constant elevated temperature greatly inhibits both basal and wound ethylene production. However, recovery from heat treatment is relatively rapid and is followed by stimulated ethylene production. The present investigation examines the kinetics of ethylene production after short‐term heal treatment and the regulation of heat‐altered ethylene production. Subapical stem segments of 7‐day‐old etiolated pea L. cv. Alaska) seedlings were analyzed for ethylene production, 1‐aminocyclopropane‐l‐carboxylic acid (ACC) oxidation, and ACC and l‐(malonylamino)cyclopropane‐l‐carboxylic acid (MACC) content after a 2‐min 40°C heat pulse. The short‐term heat pulse transiently inhibited ethylene production and ACC oxidation accompanied by a slight ACC accumulation within a 30‐min time period. Conjugation to MACC did not appear to play an integral role in heat‐regulated ethylene production. It was concluded that the major factor affecting ethylene production after heat treatment is the temporary inactivation of ACC oxidation. The possible roles of ACC synthase, ACC oxidase and lipoxygenase in regulating ethylene production after heat treatment are discussed.

Hormones andCuscuta development: interaction of cytokinin and indole-3-acetic acid in the growth and curvature of subapical stem segments

Cuscuta stem (vines) exhibits two modes of growth—longitudinal elongation forming free-hanging vines, or coiling growth to twine around the host. The elongation zone of free-hanging vine extended up to 160 mm from the stem apex and in vivo growth rate (during 8 h of growth) was maximal in the 20-to-40-mm region. While gibberellic acid (GA3) or fusicoccin (FC) could maintain (GA3) or enhance (FC) the growth rate of apical (10 or 25 mm) segments, indole-3-acetic acid (IAA) (10 μM) induced growth only in subapical (5–160 mm) segments. In vitro growth rate induced by IAA (10 μM) was similar to the in vivo growth rate up to 40 mm. Thereafter, up to 100 mm, IAA induced growth rate exceeded in vivo growth. p ]Subapical segments (∼13 mm) from 5- to 40-mm regions responded to a cytokinin (BA, Z, or iP) or to low IAA (0.1 μM) with curved growth, whereas the segments grew straight in the presence of high IAA (10 μM). Curvature (measured as the angle subtended at the center of the circle of which the segment formed an arc) induced by BA and low (0.1 μM) IAA was greater than either added separately. Besides, segments induced to curve in BA + low-IAA solution could be made to straighten out by transferring to a solution containing high IAA (10 μM) with or without BA. Thus in vivo patterns of straight and coiling growth could be mimicked reversibly in vitro by adjusting the relative concentrations of cytokinin and auxin; low auxin and cytokinin induced coiling growth, whereas high auxin and cytokinin induced straight growth. p ]Beyond 40 mm, BA had no growth-promoting or curvative-inducing effect.Cuscuta vine segments thus showed sequential sensitivity to applied hormones, the apical region (0–25 mm) to GA3, the subapical (5–40 mm) region to BA and IAA and the region beyond (40–160 mm) to IAA alone.

Auxin-induced ethylene biosynthesis in subapical stem sections of etiolated seedlings of Pisum sativum L.

1-Aminocyclopropane-1-carboxylic acid (ACC) stimulated the production of ethylene in subapical stem sections of etiolated pea (cv. Alaska) seedlings in the presence and absence of indole-3-acetic acid (IAA). No lag period was evident following application of ACC, and the response was saturated at a concentration of 1 mM ACC. Levels of endogenous ACC paralleled the increase in ethylene production in sections treated with different concentrations of IAA and with selenoethionine or selenomethionine plus IAA. The IAA-induced formation of both ACC and ethylene was blocked by the rhizobitoxine analog aminoethoxyvinylglycine (AVG). Labelling studies with L-[U-(14)C]methionine showed an increase in the labelling of ethylene and ACC after treatment with IAA. IAA had no specific effect on the incorporation of label into S-methylmethionine or homoserine. The specific radioactivity of ethylene was similar to the specific radioactivity of carbon atoms 2 and 3 of ACC after treatment with IAA, indicating that all of the ethylene was derived from ACC. The activity of the ACC-forming enzyme was higher in sections incubated with IAA than in sections incubated with water alone. These results support the hypothesis that ACC is the in-vivo precursor of ethylene in etiolated pea tissue and that IAA stimulates ethylene production by increasing the activity of the ACC-forming enzyme.

Rapidly Induced Wound Ethylene from Excised Segments of Etiolated Pisum sativum L., cv. Alaska

Wound-induced ethylene synthesis by subapical stem sections of etiolated Pisum sativum L., cv. Alaska seedlings, as described by Saltveit and Dilley (Plant Physiol 1978 61: 447-450), was half-saturated at 3.6% (v/v) O(2) and saturated at about 10% O(2). Corresponding values for CO(2) production during the same period were 1.1% and 10% O(2), respectively. Anaerobiosis stopped all ethylene evolution and delayed the characteristic pattern of wound ethylene synthesis. Exposing tissue to 3.5% CO(2) in air in a flow-through system reduced wound ethylene synthesis by 30%. Enhancing gas diffusivity by reducing the total pressure to 130 mm Hg almost doubled the rate of wound ethylene synthesis and this effect was negated by exposure to 250 mul liter(-1) propylene. Applied ethylene or propylene stopped wound ethylene synthesis during the period of application, but unlike N(2), no lag period was observed upon flushing with air. It is concluded that the characteristic pattern of wound-induced ethylene synthesis resulted from negative feedback control by endogenous ethylene.No wound ethylene was produced for 2 hours after excision at 10 or 38 C. Low temperatures prolonged the lag period, but did not prevent induction of the wound response, since tissue held for 2 hours at 10 C produced wound ethylene immediately when warmed to 30 C. In contrast, temperatures above 36 C prevented induction of wound ethylene synthesis, since tissue cooled to 30 C after 1 hour at 40 C required 2 hours before ethylene production returned to normal levels. The activation energy between 15 and 36 C was 12.1 mole kilocalories degree(-1).

Änderungen des Chloroplastenpigment-Spiegels bei Sphagnum magellanicum im Verlauf der Synthese von Sphagnorubin und anderer membranochromer Pigmente

A method is described for the determination of the relative content of the red wall pigments synthesized by Sphagnum mag. and other Sphagna cooled to + 0.5°C during a dark period (12 h), when the threshold of light intensity in the following light period (12 h) does not fall short of approximately 2000 lx. The synthesis of sphagnorubin and other wall pigments in the capitulum is linked with characteristic changes in the content of the chloroplast pigments. The content of chlorophyll a decreases comparatively more quickly than that of chlorophyll b; considerably more distinct are the variations in the carotenoid/chlorophyll quotients. It thus becomes evident that the relative content of carotenoids increases during the synthesis of red wall pigments. No significant influence on the content of chloroplast pigments in the 4–5 cm long subapical stem segments cart normally be demonstrated in such an experiment. In agreement with the knowledge that no synthesis of wall pigments takes place without analogous changes in the content of the chloroplast pigments, no sphagnorubin synthesis takes place in this region under the described conditions. That this is merely the consequence of insufficient illumination can be proved by inducing changes in the chloroplast pigment content and synthesis of wall pigments, which then take place when the capitula have been removed prior to the start of the chilling experiment. The colouring produced by a chilling experiment is reversible in so far as it does not involve an especially strong disturbance of metabolic processes. Within a few days after the end of a chilling period which has led to complete colour change of the sphagna capitula, an essential amount of the red pigments in the cell walls will again be mobilized and metabolized. The synthesis of chloroplast pigments is first manifested by the chlorophylls, whereas the synthesis of carotenoids initially retarded. This is especially true of the pigments of the «xanthophyll-cycle».

Altered Cell Microfibrillar Orientation in Ethylene-treated Pisum sativum Stems

Altered Cell Microfibrillar Orientation in Ethylene-treated <i>Pisum sativum</i> Stems