Biology 137 2008 Spring Semester
Meta-report 2: Environmental Modulation of Critical Period for Dahlia Stem Pigmentation

Dahlia 'Smooth Operator', a red cactus type,
with characeristic purple stems,
the anthocyanins of which are diluted during cell elongation


          Critical periods exist for numerous developmental events, including song learning in songbirds, visual cortex wiring in cats, language learning in humans, and anthocyanin production (Mancinelli 1984, Squire et al. 2003). During a critical period, the system in question is responsive to environmental stimuli and may require such stimuli for normal development. Before this critical period, tissues are unresponsive to the signal, and after the critical period elapses tissues are again non-responsive. The critical period is a “window” of developmental time during which the test tissue can respond to a specific stimulus; this is generally termed a “phenocritical period”. Whereas a single critical period typically exists for a given organ or process in animal systems, the iterative growth of plants enables similar, sequentially produced organs to enter and exit the critical period at distinct time points. For example, induced overexpression of the maize R gene in the Arabidopsis ttg1 mutant restores trichome production over the entire surface of leaves that are very young at the time of induction, whereas older leaves show either no response or a response limited to the basal region (Lloyd et al. 1994). By irradiating maize kernels with white light at different developmental stages, anthocyanin production was shown to be contingent on different developmental windows during which the plant was responsive to light, generally (Procissi 1997) or certain light wavelengths (Oelmuller and Mohr 1985). Sorghum bicolor seedlings are only responsive to light-triggered anthocyanin synthesis for a few days after initial photoactivation (Rajasekhar et al. 1988). Previous experiments suggest the existence of a similar critical period in dahlia stem pigmentation, such that anthocyanin production and/or maintenance is strongly dependent on light signals in young internodes, but is largely light-independent in older tissues. Anthocyanins play a number of different roles in plants, from pollinator attraction, antipathogen responses, and protection against a variety of environmental stressors (Lloyd et al. 1992, Weger et al. 1993). The present study examines the effects of greenhouse vs. field conditions on critical period duration and strength of developmental response in light-dependent anthocyanin pigmentation of the dahlia stem. This report synthesizes the work of students in the Spring 2008 quarter of Biological Sciences 137; Dahlias in Plant Genetics: E. Abrash, A. Christian, R. Kamber, J. McCallen, A. Schultink, W. White, and C. Young.



          There were three major objectives of this study:

  1. Determine if stems that appeared to exhibit full anthocyanin expression actually continue light-dependent anthocyanin accumulation as cell elongation occurs.
  2. Determine the differences between the duration of the critical periods for light-dependent stem pigmentation of greenhouse and field conditions.
  3. Determine when during floral development does anthocyanin production require light, throughout flower development from bud to antithesi

Materials and Methods

          Methods of experimentation differed based on experimental objective. For objective (1), internodes were chosen based on age and covered with 0.5 cm bands of aluminum foil. Apical portions of upper internodes were considered to be actively growing, and tissue covered with foil was measured for growth and for anthocyanin development. Alternatively, covering an entire branch in foil and measuring growth as compared to a control branch was attempted to fulfill objective (1). Objective (2) required sets of plants acclimatized to outdoor conditions and greenhouse conditions; these plants were covered at basal and apical regions of young internodes to determine if outdoor conditions accelerated or depressed the production of anthocyanins and the overall growth. The main differences between greenhouse and outdoor conditions are higher fluence (more photons) and the presence UV-A and UV-B photons outdoors. Depending on ambient weather conditions outdoor plants might also experience cooler nights or warmer days than the greenhouse plants. Covering young buds, more mature buds, and open flowers with foil allowed students to determine if flowers or buds require light to synthesis anthocyanins, fulfilling objective (3). Typically treatments were in place for five to seven days prior to data collection, and most groups used replication and control plants that, based on the first lab did not differ in anthocyanin production in the presence or absence of light.

Results and Discussion

          Differences in growth showed younger, more apical internodes to grow faster than basal internodes, and within these still elongating stems to grow faster than basal regions. Pigment reduction in apical tissues was postulated to reflect dilution through cell elongation without further stimulation of anthocyanin production by light in the Dahlia cultivars Smooth Operator (purple stems), Clear Choice (tan stems), and Fire Mountain (tan stems). In answering objective (1), then, the presence of light is required in actively growing tissues in order for anthocyanin production to continue within those tissues; old tissue previously exposed to light did not allow the newly formed tissue to continue anthocyanin production (apparently there is no mobile signal from the older parts of the plant), and dilution of pigment does occur during elongation.

          Results for objective (2) were consistent with a model in which field vs. greenhouse conditions alter duration of the phenocritical period and the requirement for light input in the tan-stemmed cultivar Pinot Noir. The shorter phenocritical period induced by field conditions caused internodes to grow more quickly and thus be less responsive to light deprivation than the corresponding internodes in indoor plants. In addition, the requirement for a light input could be reduced under field conditions (e.g., by non-cell-autonomous compensatory mechanisms dependent on UV), causing light deprivation to have a weaker effect on pigmentation. These changes appear relatively quickly, and indicate that prolonged exposure to field conditions is not necessary for reduction of plasticity.

Figure 1. The experimental set-up: foil covered flower of Dahlia Mish Mash, covered when the bud was just opening.

Figure 1a. Dahlia Mish Mash from Figure 1 seven days after foil covering to exclude all light.

Figure 2. Covering flowers of varieties with brown paper bags, which exclude about 90% of sunlight. Two brown bags were used to cut light intensity to about 1% of solar radiation.

Figure 2a. Dahlia CG Elegance after 7 days.

Figure 2b. Dahlia Devon Paree after 7 days.

Figure 2c. Dahlia Ferncliff Illusion after 7 days.

Figure 2d. Dahlia Moray Susan after 7 days.

Figure 2e. Dahlia My Fritz after 7 days.

Figure 2f. Dahlia Valley Porcupine after 7 days.

          Flower anthocyanin production was significantly impacted only in certain early stages of development. Flowers or portions of flowers covered after antithesis were not affected by reduction in light. Immature (closed) buds failed to develop if covered too early in development; mature buds, however, continued to develop into flowers without high anthocyanin content. These flowers were in some cases simply altered developmentally, in those cases in which the flowers would not have typically had high anthocyanin content as measured by flower color (the cultivars Lakeview Premier and Honka). Likewise not tremendously affected in terms of flower color was the cultivar Clear Choice, which exhibits typically high stem anthocyanin content. The cultivar Kari Fruit Salad, which has a dark blend color flower with reds and yellows was however, highly affected by foil treatment, resulting in a much paler overall flower. Additional experimentation following objective (3) has led to this finding in many dahlias, which are annotated as such in the databases.

          A related question concerns “escape time” – this is the term applied to denote the length of treatment during the critical period that is required for the full response. In the case of dahlia stem and floral pigmentation, results were collected after 5 or 7 days. Would 2 or 3 or 4 days been sufficient?

Figure 3. Buds covered too early never open. Dahlia Pooh young bud (left) and the same bud, now dead, after 7 days inside a bag.

Figure 4. Opening flowers more than half unfurled show little impact of covering. Dahlia Kari Fruit Salad (left) and Dahlia Lavid (right) with flowers half covered yielded no changes in color.


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