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Department of Biology, University of California, Santa Cruz, California 95064
Received for publication March 27, 1998. Accepted for publication October 29, 1998.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: end-of-day far-red light • Lamiaceae • phytochrome • Satureja • Sequoia • shade avoidance • Taxodiaceae
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Smith, 1995
). Other physiological parameters, such as leaf area, axillary branch growth, biomass, and the amount of chlorophyll and anthocyanins, can also be influenced by the R:FR ratio. These physiological changes to shading can be initiated by irradiating plants with end-of-day far-red light and are among the responses that have been termed a shade-avoidance response or syndrome (Smith, 1982
).
In earlier studies, we examined the influence of phytochrome on monoterpene variation in coastal redwood [Sequoia sempervirens (D. Don) Endl.] and monoterpene chemotypes in the evergreen labiate Satureja douglasii (Benth) Briq., which occur with Sequoia in the coastal redwood forest as well as other plant communities (Peer, 1996
; Peer and Langenheim, 1998
). Because phytochrome differentially affected monoterpene accumulation in Satureja chemotypes and Sequoia, we decided to analyze the shade-avoidance response of these monoterpene-producing plants as part of a larger study comparing angiosperm and gymnosperm responses to light quality.
The coastal redwood forest is mainly characterized by Sequoia sempervirens, but a number of coniferous and angiosperm trees are associated with it, e.g., Douglas fir [Pseudotsuga menzieii (Mirbel) Franco], tan oak [Lithocarpus densiflorus (Hook. & Arn.) Rehder], and the California bay [Umbellularia californica (Hook. & Arn.) Nutt.] (Cooper, 1965
), throughout its range from central California to southern Oregon. These trees can make a closed canopy, forming a forest floor that is dense shade and relatively depleted in red light (Smith, 1982
). However, there are gaps in the shade created by disturbance; the shade is also less dense under the canopy in the drier southern portion of the range, and hence more red light would be expected on the forest floor in both situations. This is also assumed to be the case in the redwood forest border dominated by oak (Quercus agrifolia Nee or Q. wizlizenii A.DC.) and madrone (Arbutus menziesii Pursh), which is common along the central California coast where this study was carried out.
Satureja douglasii occurs throughout the range of the coastal redwood forest and is commonly present in the oak-madrone forest border. It also ranges through other conifer forests into oak woodland and chaparral, thus occupying various different light-quality conditions. Populations have been characterized based on genetically controlled patterns of monoterpenes known as "chemotypes" (Lincoln and Langenheim, 1981
), which are characterized by the predominant monoterpenes pulegone, isomenthone, carvone and the bicyclic compounds camphor and camphene (Lincoln and Langenheim, 1976
). Two of the chemotypes in this study (pulegone and isomenthone types) occur in extremes of light quality habitats, and the other two (carvone and bicyclic types) range across the different light environments. Their distribution may be due in part to herbivory (Rice, Lincoln, and Langenheim, 1978
; Lincoln and Langenheim, 1979
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Collection of Satureja douglasii clones and growing conditions
Collection and propagation of chemotypes of Satureja douglasii (Benth.) Briq. were previously described (Peer and Langenheim, 1998
). Clones were maintained under the same conditions as Sequoia seedlings.
Light sources and end-of-day far-red light treatments
The light sources used were previously described (Peer and Langenheim, 1998
).
Fourteen days after germination the Sequoia seedlings with expanded cotyledons (2 cm) were transplanted and then allowed 4 d to become established. After establishment, the Sequoia seedlings were either given 20 min of 13 µmol·m-2·s-1 far-red (+FR), 20 min of 13 µmol·m-2·s-1 far-red light followed by 20 min 10 µmol·m-2·s-1 red light (+FR/R), or a mock light treatment (-FR) at the end of the light period each day for 3 wk. Each chemotype of Satureja also received the above light treatments.
Chlorophyll extraction and determination
The needles of the Sequoia seedlings and the fifth leaf pair of Satureja clones were collected after the last dark period and weighed for chlorophyll extraction. The fifth leaf pair of Satureja was used because the leaves are mature at this stage. Chlorophyll was extracted using N,N-dimethylformamide (Moran and Porath, 1980
) and measured on a Shimadzu UV-160 spectrophotometer (Shimadzu Scientific Instruments, Columbia, Maryland) at A664, A647, and A603. The amount of chlorophyll and the chlorophyll a/b ratio were calculated using equations from Moran (1982)
.
Anthocyanin extraction and determination
The needles from Sequoia and the fifth leaf pair from Satureja were collected after the last dark period. After weighing the needles and leaves, anthocyanins were extracted after the method described by Peters (1992)
. The absorbance of the aqueous layer was measured on a Shimadzu UV-160 spectrophotometer at A536 for the Sequoia needles and A535 for the leaves of Satureja clones.
Data collection and statistical analyses
Hypocotyl and epicotyl extension of the Sequoia seedlings was measured and recorded on days 0, 7, 14, and 21. Aboveground tissue harvested on day 21 was photocopied onto plastic film and area (in square centimetres) was then measured using a LI-COR LI-3000 portable area meter (LI-COR Inc., Lincoln, Nebraska). Tissue collected on day 21 was dried at 65°C to uniform dryness and weighed for dry biomass.
On day 0, the fifth internode from a branch tip for the Satureja clones was marked; then length of each internode were measured from the fifth internode on day 0 to the branch tip and number of axillary branches were counted and recorded on days 0, 7, 14, and 21. The fifth leaf pairs were harvested on day 21 and photocopied onto plastic film; area was then measured using a LI-COR LI-3000 portable area meter. The branches were collected on day 21 and dried at 65°C to uniform dryness and weighed for dry biomass.
A repeated-measures ANOVA was used to analyze the extension growth of the branch data. ANOVA was used to analyze the axillary branch, chlorophyll, anthocyanin, leaf area, dry biomass, and total number of needles data. Significance was determined at the 95 and 99% levels using Student-Newman-Keuls post hoc test. Data were analyzed using Microsoft Excel, Statview, and Superanova.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The amount of anthocyanin was an average of 46% greater than the +FR and +FR/R in the white light only (-FR) plants of the pulegone type. The amount of anthocyanin increased significantly in the carvone type (an average of 53%) and bicyclic type (an average of 39%) with +FR and +FR/R compared to white light alone. However, the light treatments did not change the amount of anthocyanin in the isomenthone type (Table 2).
Leaf area was an average of 26% greater than the controls in the +FR treatment in the bicyclic type, and the increase was photoreversible. However, leaf area was not significantly altered in the other types, although leaf area in the pulegone type was 25% greater than the controls in the +FR treatment. Neither biomass nor number of axillary branches were significantly altered by the light treatments in any of the types (Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). This phytochrome-mediated extension growth is the main characteristic of sun-adapted plants growing in low R:FR and can be triggered by end-of-day far-red light. Sequoia seedlings are sun adapted, exhibiting a photoreversible shade-avoidance response for extension growth of the hypocotyl, epicotyl, and first internode (Table 1). The photoreversal of the far-red light-induced extension by red light demonstrates that phytochrome is the mediator of the response and not chlorophyll. The internode extension response is not advantageous on the densely shaded forest floor of the redwood forest, because it would be difficult for the seedlings to become established. However, it would be advantageous in areas of partial shade in open areas such as forest gaps where seedlings may be in competition from successful sprouts. The significantly greater total amount of chlorophyll in the +FR/R light treatment is unusual and suggests adaptation to an open environment, and the significantly greater anthocyanin accumulation in the +FR/R and -FR light treatments also indicates sun adaptation. This sensitivity to red light suggests that phytochrome in this gymnosperm may have a different response mode from that in angiosperms. However, phytochrome alone may not modulate anthocyanin synthesis; in conifers, it may be controlled by phytochrome and one or more blue light receptors, most likely through a coaction (Fernbach and Mohr, 1990
; Messner, Boll, and Berndt, 1991
). A change in monoterpene variation by light quality may also be a sun-adapted response (Peer, 1996
). Although leaf area and biomass were not influenced by light treatments suggesting shade adaptation for these parameters, overall the Sequoia seedlings respond as sun-adapted plants.
A chemotype-dependent shade-avoidance response occurred in Satureja (Table 2). Types typically occurring in more open, higher light habitats display varying degrees of the shade-avoidance response, and the type that occurs primarily in a shaded habitat lacks the response. A study in Impatiens capensis Meerb. has shown that woodland and open ecotypes have differential sensitivities to light quality. The open ecotype of Impatiens exhibits extension growth under simulated shade conditions whereas the woodland ecotype does not (Dudley and Schmitt, 1995
). This ecotypic observation suggests the possibility that the Satureja chemotypes are adapted to the light environments in which they usually occur.
The pulegone type occurs in the most open and unshaded areas (rich in red light), in woodlands with scattered oaks amid grassland, sometimes extending into chaparral scrub (Lincoln and Langenheim, 1976
); it also occasionally has been found in open areas of the coast redwood forest. Therefore, as anticipated, this type responded like a sun-adapted plant with significantly greater photoreversible internode extension (Fig. 1) and more total chlorophyll (Table 2) with end-of-day far-red light. Although some increase in leaf area and decrease in biomass occurred, these changes were not significant at the 95% level (P < 0.1). The amount of anthocyanin was significantly less with far-red light compared to the white light treatment, but this effect may not be caused by phytochrome alone since the anthocyanin pathway in angiosperms is also regulated by multiple photoreceptors (Beggs, Wellman, and Grisebach, 1986
). In addition, the sun-adapted characteristics of this type are consistent with the influence of a change in light quality on total quantity of leaf monoterpenes and concentration of individual monoterpenes characterizing this type (Peer and Langenheim, 1998
).
The isomenthone type, typically occurring in the red light-depleted dense shade in the redwood forest, responded like shade-adapted plants for all parameters studied, i.e., it did not exhibit internode extension, produced the same amount of anthocyanin and chlorophyll, and maintained the same leaf area in all light treatments. This lack of response to a change in light quality is also observed in monoterpene variation in this type (Peer and Langenheim, 1998
).
The carvone type, which usually occurs in the more open and drier areas of the redwood forest or in the oak and madrone forest bordering it, had a varied response depending on the parameter studied. Since it did not exhibit internode extension nor an increase in leaf area with far-red light, it responded like a shade-adapted plant for those parameters relating to its occasional occurrence in the dense shade of the redwood forest. In contrast, it demonstrated a shade-avoidance response for the total amount of chlorophyll and the amount of anthocyanin (Table 2), corresponding to its occurrence in the oak-madrone forest border and congruent with a change in light quality influencing monoterpene variation (Peer and Langenheim, 1998
). This type is the only one that showed an end-of-day far-red response for the total amount of chlorophyll. This result is supported by a study indicating that the total amount of chlorophyll among species that occur across open, intermediate, and closed-canopied habitats displayed no clear habitat groupings (Morgan and Smith, 1979
).
The bicyclic type has the widest distribution, spanning the northern Douglas-fir forest as well as redwood forests through the open-canopied forests bordering the redwoods and open closed-cone pine (Pinus radiata D. Don) forests in central California (Lincoln and Langenheim, 1976
). As with the carvone type, in the bicyclic type the traits studied are differentially responsive to light quality. This type has typically shade-adapted characteristics, i.e., no extension growth nor increase in chlorophyll amount, but also demonstrated a shade-avoidance response with regard to increased leaf area and the decrease in the amount of anthocyanin with end-of-day far-red light (Table 2). However, its overall response is that of a shade-tolerant plant, consistent with the lack of influence of a change in light quality on monoterpene variation (Peer and Langenheim, 1998
).
Phytochrome responses and growth patterns
Previous studies of phytochrome mutants demonstrated that phytochrome B-like phytochromes play a dominant role in the shade-avoidance response and end-of-day far-red responses (Lopez-Juez et al., 1990
; Robson, Whitelam, and Smith, 1993
; Ballare et al., 1995
; Aukerman et al., 1997
). The influence of phytochrome on the growth pattern of Sequoia seedlings is to initiate a shade-avoidance response that can be induced by end-of-day far-red light. Phylogenetic analysis by Kolukisaoglu et al. (1995)
indicates that conifers posses phytochromes, which are members of the phyB-type phytochrome branch; hence, it is reasonable to suggest that a phyB-type phytochrome regulates these responses in Sequoia. Thus, the role of phytochrome is apparent in the photoreversible responses observed with respect to the parameters studied and monoterpene variation in the seedlings (Peer, 1996
), and the growth pattern corresponds to areas where the seedlings may be established and potentially reach maturity (Table 3).
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Conclusions
There was a range of shade-avoidance responses among the seedlings of dominant trees in the coastal redwood forest and chemotypes of the perennial herbaceous plant that occurs there and in adjacent plant communities (Table 3). Sequoia sempervirens seedlings responded as sun-adapted plants. A chemotype-dependent shade-avoidance response was observed in Satureja douglasii: the pulegone type responded as a sun-adapted plant, whereas the isomenthone type responded as a shade-tolerant plant. The carvone and bicyclic types exhibited variable responses characteristic of both sun-adapted and shade-adapted plants. This range of responses indicates genetic variability for this trait within Satureja douglasii and variability of growth patterns between a conifer and a labiate species that have populations occurring in the same forest type. Phytochrome effects on monoterpene variation fit patterns similar to those found for shade avoidance, i.e., these plants are adapted to the light conditions in which they generally occur (Table 3). Many studies have indicated that phyB-like phytochromes are involved in modulating various parameters of the shade-avoidance and end-of day far-red responses (see Smith, 1995
, for review); we hypothesize that the coastal redwood may have a phyB-type phytochrome and that there may be a phyB-like mutation or a mutation of a component in the shade-avoidance signal transduction pathway in a Satureja population occurring in the coastal redwood forest.
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FOOTNOTES |
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This is Carnegie Institution of Washington, Department of Plant Biology publication number 1399. 
3 Current address: Dept. of Plant Biology, Carnegie Institution of Washington, 260 Panama St., Stanford, CA 94305.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Ballare, C., A. L. Scopel, M. L. Roush, and S. R. Radosevich. 1995 How plants find patchy canopies: a comparison between wild-type and phytochrome-B-deficient mutant plants of cucumber. Functional Ecology 9: 859–868.[CrossRef][ISI]
Beggs, C. J., E. Wellman, and H. Grisebach. 1986 Photocontrol of flavanoid biosynthesis. In R. E. Kendrick and G. H. M. Kronenberg [eds.], Photomorphogenesis in plants, 467–499, Martinus Nijhoff Publishers, Dordrecht.
Cooper, D. W. 1965 Coast redwood (Sequoia sempervirens) and its ecology. Agriculture Extension Service, Eureka California.
Dudley, S. A., and J. Schmitt. 1995 Genetic differentiation in morphological responses to simulated foliage shade between populations of Impatiens capensis from open and woodland sites. Functional Ecology 9: 655–666.[CrossRef][ISI]
Fernbach, E., and H. Mohr. 1990 Coaction of blue/ultraviolet-A light and light absorbed by phytochrome in controlling growth of pine (Pinus sylvestris L.) seedlings. Planta 180: 212–216.[CrossRef][ISI]
Kolukisaoglu, H. U., S. Marx, C. Wiegmann, S. Hanelt, and H. A. W. Schneider-Poetsch. 1995 Divergence of the phytochrome gene family predates angiosperm evolution and suggests that Selaginella and Equisetum arose prior to Psilotum. Journal of Molecular Evolution 41: 329–337.
Lincoln, D. E., and J. H. Langenheim. 1976 Geographic patterns of monoterpenoid composition in Satureja douglasii. Biochemical Systematics and Ecology 4: 237–248.[CrossRef]
———, and ———. 1979 Variation of Satureja douglasii monoterpenoids in relation to light intensity and herbivory. Biochemical Systematics and Ecology 7: 289–298.
———, and ———. 1981 A genetic approach to monoterpenoid compositional variation in Satureja douglasii. Biochemical Systematics and Ecology 9: 153–160.[CrossRef]
Lopez-Juez, E., W. F. Buurmeijer, G. H. Heeringa, R. E. Kendrick, and J. C. Wesselius. 1990 Response of light-grown wild-type and long hypocotyl mutant cucumber plants to end-of-day far-red light. Photochemistry and Photobiology 52: 143–149.
Messner, B., M. Boll, and J. Berndt. 1991 L-phenylalanine ammonia-lyase in suspension culture cells of spruce (Picea abies). Plant Cell, Tissue and Organ Culture 27: 267–274.[CrossRef][ISI]
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Moran, R. 1982 Formulae for determination of chlorophyllous pigments extracted with N,N-dimethylformamide. Plant Physiology 69: 1376–1381.
———, and D. Porath. 1980 Chlorophyll determination in intact tissues using N,N-dimethylformamide. Plant Physiology 65: 477–479.
Peer, W. A. 1996 The influence of phytochrome on gene regulation of Lhcb expression in Pinus palustris and on ecological responses of monoterpene variation and shade avoidance in Sequoia sempervirens and Satureja douglasii. Ph.D. dissertation. University of California, Santa Cruz, CA.
———, and J. H. Langenheim. 1998 Influence of phytochrome on leaf monoterpene variation in Satureja douglasii. Biochemical Systematics and Ecology 26: 25–34.
Peters, J. L. 1992 Photomorphogenic mutants of higher plants. Ph.D. dissertation. Wageningen Agricultural University, The Netherlands.
Rice, R. L., D. E. Lincoln, and J. H. Langenheim. 1978 Palatability of monoterpenoid compositional types of Satureja douglasii to a generalist molluscan herbivore, Ariolimax dolichophallus. Biochemical Systematics and Ecology 6: 45–53.
Robson, P. R. H., G. C. Whitelam, and H. Smith. 1993 Selected components of the shade-avoidance syndrome are displayed in a normal manner in mutants of Arabidopsis thaliana and Brassica rapa deficient in phytochrome B. Plant Physiology 102: 1179–1184.[Abstract]
Schmitt, J., and R. D. Wulff. 1993 Light spectral quality, phytochrome and plant competition. Trends in Ecology and Evolution 8: 47–51.[CrossRef]
Smith, H. 1982 Light quality, photoperception and plant strategy. Annual Review of Plant Physiolgy 33: 481–518.
———. 1995 Physiological and ecological function within the phytochrome family. Annual Review of Plant Physiology and Molecular Biology 46: 289–315.[CrossRef][ISI]
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