Coleus Blumei Benth Research Articles

Branch development in Lupinus angustifolius L. II. Relationship with endogenous ABA, IAA and cytokinins in axillary and main stem buds

level appeared to play a secondary role, as a growth inhibitor. The concentrations of indole-3-acetic acid (IAA), cytokinins (CK) and abscisic acid (ABA) were measured in Key words: Apical dominance, cytokinin, auxin, abscisic buds of different regions (main stem and lateral acid, Lupinus angustifolius. branches) of Lupinus angustifolius L. (cv. Merrit) and at different stages in the development of branches. In lupin, branching patterns are the result of discrete Introduction regions of axillary branches (upper, middle and basal) which elongate at much different rates. Early in devel- A strongly growing main shoot apex represses the opment only the main shoot elongates, followed usu- growth of lateral buds by exerting ‘apical dominance’. ally by basal branch growth and then rapid upper Sachs and Thimann (1967) proposed that apical dominbranch growth. Branches in the middle of the main ance was hormonally controlled and that auxin played a stem grow only weakly or fail to develop. Levels of IAA dominant role. The model envisages auxin being transwere generally high in the apical buds of slowly grow- ported from its principal site of synthesis, the dominant ing branches and low in buds from strongly growing apical bud, towards the lateral buds where it inhibits branches, whereas CK levels showed the opposite growth either directly or indirectly. Support for this relationship. CK5IAA ratio showed a closer rela- model relies on the fact that exogenous auxin applicationship with the rate of growth of a particular branch tion, either re-establishes apical dominance when applied better than the levels of either CK or IAA alone. During to the stump of decapitated plants, or suppresses lateral early stages of growth ABA concentration did not growth when applied to lateral buds of intact plants follow the rate of branch growth. However, later in (Cline, 1991; Tamas, 1995). development, where growth did not closely match the In such a scenario, quiescent axillary buds are regarded ratio of CK5IAA, ABA level showed a strong negative as ‘replacement apices’; being called upon following loss relationship with growth. A significant decrease in ABA of the strongly growing shoot apex. While this scenario was associated with continued strong growth of the may apply to many plant species, the hypothesis has main stem apex following a decline in CK5IAA ratio. limitations, and cannot readily explain branching patOverall, the best relationship between the level of terns in plants with weak apical dominance, such as growth factors in apical buds and branching pattern Coleus blumei Benth. and Arabidopsis thaliana L., or in in lupin was the ratio of CK5IAA, implying that high those for which decapitation leads to outgrowth of buds which are furthest away from the apex (Cline, 1996).

Amino acid accumulation in sink and source tissues of Coleus blumei Benth. during salinity stress

The purpose of this study was to analyse the accumulation of amino acid in source and sink tissues of variegated Coleus blumei Benth. leaves during an extended exposure to salinity stress. The imposed stress resulted in a reduction in shoot height and leaf size, as well as a reduction in total protein and nitrogen content in both the sink and source tissues. At the same time, accumulation of low molecular weight nitrogen-containing compounds in Coleus leaves was observed, which peaked within the first 10 d of exposure to salinity, and then declined, but remained slightly elevated for the remainder of the study. A number of amino acids were found to accumulate in both the sink and source tissues, including arginine, asparagine, and serine. A larger proportion of asparagine and less arginine was observed in the sink tissue than the source tissue of the salinity-stressed plants. This difference may reflect the mobility of these compounds in the phloem. No proline was found to accumulate in either the source or sink tissue during the exposure to salinity. From the pulse-chase labelling of stressed Coleus leaves it can deduced that some of the observed accumulation of amino acids and amides observed is due to de novo synthesis and not simply the result of protein degradation.

Amino acid accumulation in sink and source tissues of Coleus blumei Benth. during salinity stress

Amino acid accumulation in sink and source tissues of Coleus blumei Benth. during salinity stress

Root-Zone Salinity Alters Raffinose Oligosaccharide Metabolism and Transport in Coleus.

Exposure of variegated coleus (Coleus blumei Benth.) plants to a saline root-zone environment (60 mM NaCl:12 mM CaCl2) resulted in a significant decline in elongation growth rate over the 30-d experimental period. During the initial 5 to 10 d of exposure, mature source leaves showed strongly diminished rates of photosynthesis, which gradually recovered to close to the control rates by the end of the experiment. In green leaf tissues, starch levels showed the same transient decline and recovery pattern. Low starch levels were accompanied by the appearance of several novel carbohydrates, including high-molecular-weight raffinose family oligosaccharides (RFOs) with a degree of polymerization (DP) of 5 to 8, and an O-methylated inositol (OMI). New enzyme activities, including galactan:galactan galactosyltransferase, for the synthesis of high-DP RFOs and myo-inositol 6-O-methyltransferase for O-methylation of myo-inositol, were induced by salinity stress. Phloem-sap analysis showed that in the stressed condition substantially more sucrose than RFO was exported, as was the OMI. In white sink tissues these phloem sugars were used to synthesize high-DP RFOs but not OMIs. In sink tissues galactan:galactan galactosyltransferase but not myo-inositol 6-O-methyltransferase was induced by salinity stress. Models reflecting the changes in carbohydrate metabolism in source and sink tissues in response to salinity stress are presented.

わが国のコリウス（Ｃｏｌｅｕｓ ｂｌｕｍｅｉ Ｂｅｎｔｈ．）から検出されたウイロイドの全塩基配列と宿主範囲

A viroid was detected from coleus (Coleus blumei Benth.) in Japan, which was 1 nucleotide larger (249nt) than coleus blumei viroid 1 (CbVd1) and coleus yellow viroid (CYVd), indicating that the viroid was an isolate of CbVd1. Four species of plants in Labiatae (Mentha spicata, Mentha arvensis var. piperascens, Ocimum basilicum and Melissa officinalis), in addition to coleus, were first found to be infected with the CbVd1 without showing any detectable disease symptoms.

Purification of rosmarinic acid synthase from cell cultures of Coleus blumei Benth

Rosmarinic acid synthase from cell cultures of Coleus blumei Benth. was purified to apparent homogeneity by fractionated ammonium sulfate precipitation (60–80% saturation), hydrophobic interaction chromatography, affinity chromatography and gel filtration. This purification procedure resulted in a 225-fold-enriched specific enzyme activity with a yield of 9%. The protein preparation was apparently pure according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis. The apparent molecular mass determined by gel filtration and SDS-PAGE was 77 kDa, indicating that rosmarinic acid synthase is a monomeric enzyme.

Isolation of protoplasts and vacuoles from cell suspension cultures of Coleus blumei Benth.

In order to study the accumulation and transport of rosmarinic acid in suspension cells of Coleus blumei we established an efficient method to isolate protoplasts and vacuoles. Protoplasts were disrupted by an osmotic shock in a medium with basic pH containing ethylenediamine tetraacetic acid. The resulting vacuoles were purified on a two-step Ficoll gradient. The comparison of the rosmarinic acid contents of cells, protoplasts and vacuoles showed that the depside is localized in the vacuole. Data concerning the yield and purity of the vacuoles are presented. In addition we show that at the physiological pH of the cytoplasm rosmarinic acid is present almost exclusively as an anion and cannot pass a membrane by simple diffusion. We therefore propose a carrier system for the transport of rosmarinic acid into the vacuole.

Proposed biosynthetic pathway for rosmarinic acid in cell cultures of Coleus blumei Benth

A biosynthetic pathway for rosmarinic acid is proposed. This pathway is deduced from studies of the enzymes detectable in preparations from suspension cells of Coleus blumei. Phenylalanine is transformed to 4-coumaroyl-CoA by the enzymes of the general phenylpropanoid pathway: phenylalanine ammonia-lyase (EC 4.3.1.5), cinnamic acid 4-hydroxylase (EC 1.14.13.11) and hydroxycinnamic acid:CoA ligase (EC 6.2.1.12). Tyrosine is metabolized to 4-hydroxyphenyllactate by tyrosine aminotransferase (EC 2.6.1.5) and hydroxyphenylpyruvate reductase. The ester can be formed from 4-coumaroyl-CoA and 4-hydroxyphenyllactate by the catalytic activity of rosmarinic acid synthase with concomitant release of CoA. Microsomal hydroxylase activities introduce the hydroxyl groups at positions 3 and 3′ of the aromatic rings of the ester 4-coumaroyl-4′-hydroxyphenyllactate giving rise to rosmarinic acid.

Sugar synthesis and phloem loading in Coleus blumei leaves

Sugar-synthesis and -transport patterns were analyzed in Coleus blumei Benth. leaves to determine where galactinol, raffinose, and stachyose are made and whether phloem loading includes an apoplastic (extracellular) step or occurs entirely within the symplast (plasmodesmata-connected cytoplasm). To clarify the sequence of steps leading to stachyose synthesis, a pulse (15 s) of (14)CO2 was given to attached leaves followed by a 5-s to 20-min chase: sucrose was rapidly labeled while galactinol, raffinose and stachyose were labeled more slowly and, within the first few minutes, to approximately the same degree. Leaf tissue was exposed to either (14)CO2 or [(14)C]glucose to identify the sites of synthesis of the different sugars. A 2-min exposure of peeled leaf tissue to [(14)C]glucose resulted in preferential labeling of the minor veins, as opposed to the mesophyll; galactinol, raffinose and stachyose were more heavily labeled than sucrose in these preparations. In contrast, when leaf tissue was exposed to (14)CO2 for 2 min for preferential labeling of the mesophyll, sucrose was more heavily labeled than galactinol, raffinose or stachyose. We conclude that sucrose is synthesized in mesophyll cells while galactinol, raffinose and stachyose are made in the minorvein phloem. Competition experiments were performed to test the possibility that phloem loading involves monosaccharide uptake from the apoplast. Two saturable monosaccharide carriers were identified, one for glucose, galactose and 3-O-methyl glucose, and the other for fructose. Washing the apoplast of peeled leaf pieces with buffer or saturating levels of 3-O-methyl glucose, after providing a pulse of (14)CO2, did not inhibit vein loading or change the composition of labeled sugars, and less than 0.5% of the assimilated label was recovered in the incubation medium. These and previous results (Turgeon and Gowan, 1991, Plant Physiol. 94, 1244-1249) indicate that the phloem loading pathway in Coleus is probably symplastic.

Hydroxyphenylpyruvate Reductase From Cell Suspension Cultures Of Coleus Blumei Benth.

Cell suspension cultures of Coleus blumei Benth. producing high amounts of rosmarinic acid were used to study the biosynthetic pathway of this caffeic acid ester. One of the involved enzymes, the hydroxyphenylpyruvate reductase (HPPR), is characterized in this paper. HPPR catalyzes the NAD (P)H dependent reduction of p-hydroxyphenylpyruvate to p-hydroxyphenyllactate. The enzyme developed maximal activity at an incubation temperature of 37 °C and at a pH of 6.5 to 7.0. The reaction proceeded linearly for an incubation time of 60 min and up to a protein concentration of 0.2 mg per assay. As electron donor HPPR accepted NADH and NADPH with Km-values of 190 µm and 95 µm respectively. The enzyme reduced differently substituted hydroxyphenylpyruvates but not β-phenylpyruvate. The apparent Km-values for the various substrates were at 10 µM for p-hydroxyphenylpyruvate, at 130 µm for 3,4-dihydroxyphenylpyruvate and at 250 µM for 3-methoxy-4-hydroxyphenylpyruvate. HPPR was competitively inhibited by rosmarinic acid and pyruvate with Ki-values of 210 µM and 200 µM respectively. Caffeic acid, p-coumaric acid and cinnamic acid did not affect the enzyme activity but p-coumaroyl-CoA inhibited HPPR

Phloem Loading in Coleus blumei in the Absence of Carrier-Mediated Uptake of Export Sugar from the Apoplast

Phloem loading in Coleus blumei Benth. leaves cannot be explained by carrier-mediated transport of export sugar from the apoplast into the sieve element-companion cell complex, the mechanism by which sucrose is thought to load in other species that have been studied in detail. Uptake profiles of the export sugars sucrose, raffinose, and stachyose into leaf discs were composed of two components, one saturable and the other not. Saturable (carrier-mediated) uptake of all three sugars was almost completely eliminated by the inhibitor p-chloromercuribenzenesulfonic acid (PCMBS). However, when PCMBS was introduced by transpiration into mature leaves it did not prevent accumulation of (14)C-photosynthate in minor veins or translocation of labeled photosynthate from green to nonchlorophyllous regions of the leaf following exposure to (14)CO(2). The efficacy of introducing inhibitor solutions in the transpiration stream was proven by observing saffranin O and calcofluor white movement in the minor veins and leaf apoplast. PCMBS introduced by transpiration completely inhibited phloem loading in tobacco leaves. Phloem loading in C. blumei was also studied in plasmolysis experiments. The carbohydrate content of leaves was lowered by keeping plants in the dark and then increased by exposing them to light. The solute level of intermediary cells increased in the light (phloem loading) in both PCMBS-treated and control tissues. A mechanism of symplastic phloem loading is proposed for species that translocate the raffinose series of oligosaccharides.

The Effects of Oil-based Fungicides on Water Loss of Coleus blumei Leaves and Pelargonium Hybrid Petals

Effects of oil-based fungicides on plant surfaces were studied by assessing water loss from excised sprayed leaves or petals and by microscopy. Two fungicides (Turbair Zineb Fungicide and Turbair Copper Fungicide) were sprayed, using a Potter tower, onto the adaxial surfaces of excised leaves or petals, and weight loss was determined as a measure of water loss. Doses of approximately 4–9 g m−2 significantly increased water loss from Pelargonium hybrid petals and leaves of Coleus blumei Benth. The fungicides, their supernatants and the carrier oil increased water loss from the petals but effects of the supernatants and the carrier oil on C. blumei leaves were less conclusive. Low Temperature Scanning Electron Microscopy (LTSEM) did not reveal any scoring or marked erosion of the adaxial surface of leaves or petals 24 h after application of the fungicides.

Assimilate Partitioning in the Variegated Coleus Leaf

AbstractMature leaves of a variegated cultivar of Coleus blumei Benth. with a green border and central albino region constitute a source‐sink system suitable for studies on assimilate partitioning. Leaves treated with 14CO2 on a small part of the intact green border export assimilate via the shortest path into the stem. Leaves with all but a small lobe of the green border removed show different partitioning of labeled assimilates when the leaf is exposed to 14CO2 (Fisher and Eschrich, 1985): The whole albino region of the leaf is supplied but no tracer is exported into the stem. When the green border is completely removed, 14CO2‐treatment of the albino region leads to the fixation of CO2, obviously by PEP carboxylase, as indicated by the occurrence of labeled malate. Results show that the albino region of the variegated leaf constitutes a potential sink when deprived of its green border. In addition, CO2‐fixation by PEP carboxylase in albino tissue seems to indicate a common capacity of leaves which is normally masked by photosynthesis. The difference of assimilate partitioning between leaves with intact and leaves with partly removed green borders demonstrates that the unlabeled assimilates control the movement of labeled assimilates.

The Role of Cytokinin in Sieve Tube Regeneration and Callose Production in Wounded Coleus Internodes

Cytokinin proved to be a controlling factor in sieve tube regeneration around wounded collateral bundles in an in vivo system in which the endogenous cytokinin level had been minimized. Both kinetin and zeatin were applied in aqueous solution to the bases of excised, mature internodes of Coleus blumei Benth. that had an active vascular cambium. Each internode also received indoleacetic acid (IAA) in lanolin at its apical end. Under either low (0.1% w/w) or high (1.0% w/w) auxin concentrations, the control internodes (without exogenous cytokinin) exhibited small amounts of sieve tube regeneration. At appropriate concentrations, both kinetin and zeatin induced a significant increase in sieve tube regeneration around the wound. However, the highest concentration of kinetin tested (50 mug/mL) completely inhibited this process. Kinetin was the most effective with high auxin (1.0% IAA), while zeatin was the most effective with low auxin level (0.1% IAA). Kinetin and zeatin showed the strongest promotive effect at 10 mug/mL and 20 mug/mL, respectively. Both cytokinins also induced supplementary phloem regeneration further from the wound surface. In addition to their effects on vascular tissue regeneration, both cytokinins promoted callose production. This was most evident on the sieve plates of the regenerated sieve tube members and on the walls of the parenchyma cells around the wound. The largest deposits of callose were found in both regenerated sieve tube members and parenchyma cells at the highest cytokinin concentration tested (50 mug/mL). The possible role of cytokinin in controlling callose accumulation in the sieve tubes during autumn is discussed.

Carbohydrate Metabolism in Photosynthetic and Nonphotosynthetic Tissues of Variegated Leaves of Coleus blumei Benth.

Mature, variegated leaves of Coleus blumei Benth. contained stachyose and other raffinose series sugars in both green, photosynthetic and white, nonphotosynthetic tissues. However, unlike the green tissues, white tissues had no detectable level of galactinol synthase activity and a low level of sucrose phosphate synthase indicating that stachyose and possibly sucrose present in white tissues may have originated in green tissues. Uptake of exogenously supplied [(14)C]stachyose or [(14)C]sucrose into either tissue type showed conventional kinetic profiles indicating combined operation of linear first-order and saturable systems. Autoradiographs of white discs showed no detectable minor vein labelling with [(14)C]stachyose, but some degree of vein labeling with [(14)C]sucrose. Autoradiographs of green discs showed substantial vein loading with either sugar. In both tissues, p-chloromercuribenzenesulfonic acid had no effect on the linear component of sucrose or stachyose uptake but inhibited the saturable component. Both tissues contained high levels of invertase, sucrose synthase and alpha-galactosidase and extensively metabolized exogenously supplied (14)C-sugars. In green tissues, label from exogenous sugars was recovered as raffinose-series sugars. In white tissues, exogenous sugars were hydrolysed and converted to amino acids and organic acids. The results indicate that variegated Coleus leaves may be useful for studies on both phloem loading and phloem unloading processes in stachyose-transporting species.

Abscission inColeus: Light and Phytohormone Control

Light control of leaf abscission in Coleus (Coleus blumei Benth cv. Ball 2719 Red) appears to be regulated by the quantity of endogenous auxin transported from the leaf blade to the abscission zone. Gas chromatographic-mass spectrophotometry analysis indicated that diflfusate collected from leaf tissue treated with red light contained significantly higher levels of auxin than dark and far-red light-treated leaf tissue. In addition, diffusate from red light-treated tissue inhibited abscission of leafless petioles while diffusate from far-red light-treated tissue promoted abcission when compared with diffusate from dark-treated tissue. The effect of red light on abscission could be mimicked by IAA, but not by other phytohormones. An auxin transport inhibitor, 2,3,5 triiodobenzoic acid (TIBA), applied either as a lanolin ring around the petiole or vacuum infiltrated into tissue, could completely eliminate any red light effect on abscission. The data are consistent with a phytochrome-mediated light regulation of endogenous auxin level in the leaf which then controls abscission.

Cryopreservation of Coleus blumei Suspension and Callus Cultures

Callus and suspension cultures of Coleus blumei Benth. were cryopreserved and stored for various periods of time in liquid nitrogen. Sorbitol was the only additive used during preculture and during cryoprotection treatments. The best results were obtained using cells from the early growth period. The duration of storage was 3, 6, 12 and 15 months, respectively. Cell viability could be improved considerably either by thawing the samples rapidly in a 60 °C water bath (instead of 45 °C) or by thawing them in a microwave oven. After a short period of recovery growth on semi-solid agar medium, the cells were cultivated under standard conditions. The growth characteristics and the accumulation of rosmarinic acid, a compound of pharmaceutical value, were found to be in the range of untreated controls.

Different Patterns of Vein Loading of Exogenous [14C]Sucrose in Leaves of Pisum sativum and Coleus blumei

Vein loading of exogenous [(14)C]sucrose was studied using short uptake and wash periods to distinguish between direct loading into veins and loading via mesophyll tissue. Mature leaf tissue of Pisum sativum L. cv Little Marvel, or Coleus blumei Benth. cv Candidum, was abraded and leaf discs were floated on [(14)C]sucrose solution for 1 or 2 minutes. Discs were then washed for 1 to 30 min either at room temperature or in the cold and were frozen, lyophilized, and autoradiographed. In P. sativum, veins were clearly labeled after 1 minute uptake and 1 minute wash periods. Autoradiographic images did not change appreciably with longer times of uptake or wash. Vein loading was inhibited by p-chloromercuribenzenesulfonic acid. These results indicate that uptake of exogenous sucrose occurs directly into the veins in this species. When C. blumei leaf discs were floated on [(14)C]sucrose for 2 minutes and washed in the cold, the mesophyll was labeled but little, if any, minor vein loading occurred. When discs were labeled for 2 minutes and washed at room temperature, label was transferred from the mesophyll to the veins within minutes. These results indicate that there may be different patterns of phloem loading of photosynthetically derived sucrose in these two species.

Genotypic variation in coleus in the ability to accumulate abscisic acid in response to water deficit

Terry, P. H., Krizek, D. T. and Mirecki, R. M. 1988. Genotypic variation in coleus in the ability to accumulate abscisic acid in response to water deficit. ‐ Physiol. Plant. 72: 441–449.Abscisic acid (ABA) concentration and plant and soil water potentials were determined in leaves of three cultivars of Coleus blumei Benth. cvs. Marty, Buckley Supreme and PI354190, chosen for their differences in sensitivity to drought, SO2 and/or chilling stress. Plants were subjected to ‘gradual’ soil moisture stress (SMS) for 0–6 days, during which time the soil dried out slowly and to more ‘rapid’ SMS for 0–9 days where the soil dried out at a faster rate. Plants were propagated from clonal stock in the greenhouse and transferred to the growth chamber, where they were maintained for 1 week prior to beginning water stress treatment. ABA concentration was determined as the methyl ester using a gas chromatography ‐ electron capture detector method. Samples for ABA determinations were taken from the third pair of leaves from the apex at the same time each day (1430 h). Measurements of stomatal conductance (C5) and leaf water potential (φl) were made on the fourth pair of leaves from the apex, using the same plants as those sampled for ABA. During the more rapid stress portion of the study soil matric potentials (φm) were monitored on a daily basis. Despite large cultivar differences in ABA concentration at 0–6 days, by 7–9 days these differences had largely disappeared. Except for drought‐insensitive cv. Marty, there was generally little correlation between ABA levels and measurements of plant and soil water status at 7–9 days.

Photoassimilate-transport characteristics of nonchlorophyllous and green tissue in variegated leaves of Coleus blumei Benth

The nonchlorophyllous (albino) tissue of mature C. blumei leaves is a sink for photoassimilate. Transport from the green to the albino region of the same leaf was inhibited by cold and anoxia. When the green tissue of mature leaves was removed, the remaining albino portion imported labeled translocate from other mature leaves in the phloem. Photoassimilate unloading in the albino region of mature leaves was studied by quantitative autoradiography. The unloading was inhibited by cold but not by anoxia. No labeled photoassimilate could be detected in the free space of mature albino tissue by compartmental efflux analysis as phloem unloading proceeded in a N2 atmosphere, indicating that unloading, may occur by a symplastic pathway as it apparently does in sink leaves of other species. The minor veins of mature albino leaf tissue did not accumulate exogenous [(14)C]sucrose. Minor veins of green tissue in the same leaves accumulated [(14)C]sucrose but, in contrast to other species studied to date, this accumulation was insensitive to the inhibitor p-chloromercuribenzensulfonic acid (PCMBS).In its capacity to import and unload photoassimilate, and in the inability, of the minor veins to accumulate exogenous sucrose, the albino region of the mature C. blumei lamina differs from mature albino tobacco leaves and darkened mature leaves of other species. This, together with evidence indicating that phloem loading in C. blumei and other species may occur by different routes and with different sensitivity to PCMBS, indicates that the mechanism of transfer of photoassimilates between veins and surrounding tissues, and the mechanism of the sink-source transition, may not be the same in the leaves of all species. It is speculated that the unusual properties of the C. blumei leaf may be a consequence of the presence, in the minor veins, of "intermediary cells", large companion cells connected to the bundle sheath by abundant plasmodesmata.