Bot. Bull. Acad. Sin. (2003) 44: 31-35

Huang et al. — Phase change and photosynthesis in Sequoia sempervirens

Photosynthetic potentials of in vitro-grown juvenile, adult, and rejuvenated Sequoia sempervirens (D. Don) Endl. shoots

Li-Chun Huang, Jui-Hsi Weng, Chiu-Hui Wang, Ching-I Kuo, and Yuh-Jang Shieh*

Institute of Botany, Academia Sinica, Nankang, Taipei, Taiwan 11529

(Received June 7, 2002; Accepted November 7, 2002)

Abstract. In vitro shoot tips of Sequoia sempervirens (D. Don.) Endl.—including a juvenile, two adult, and two rejuvenated adult clones—were examined for differences in basic physiological characteristics. Moisture contents were the same, around 85%, for all tissues regardless of origin. Growth rates, determined by fresh weight increase and shoot elongation, were higher for the juvenile and rejuvenated shoots. They correlated with higher total nitrogen contents. Juvenile and rejuvenated shoots also showed higher rates of photosynthesis and respiration, evidenced by faster O₂ evolution and consumption. The photosynthetic rates were associated with more chlorophyll, especially chlorophyll a, in the juvenile and the rejuvenated shoots. Nevertheless, identical quantum efficiencies of photosystem II indicated the same photosystems were operating and with equal effectiveness in juvenile, adult, and rejuvenated tissues.

Keywords: Phase change; Photosynthesis; Respiration; Sequoia sempervirens; Total nitrogen content.

Introduction

Plant development normally begins with a strictly vegetative juvenile phase and culminates in the sexually reproductive adult phase. Although herbaceous annuals achieve phase change, or maturation, within weeks, in woody perennials (Franclet, 1983), especially trees, this phase change or maturation process can take several years and is associated with changes of different characteristics, e.g., loss of adventitious rooting competence, diminished vigor and growth rate, changes in phyllotaxy, shape and size of leaves, re-orientation of leaves from plagiotropism to orthotropism, and decreased thorniness depending on species. During the past several years we have been employing Sequoia sempervirens (D. Don.) Endl., the coastal redwood, as a model to characterize the juvenile and mature phases of plants through their biochemical and physiological differences, as well as to progress in the understanding of the underlying mechanism controlling phase change in trees. This species was chosen because its excised shoots can be easily cultured in vitro on a chemically defined medium without phytohormonal supplements. Also, a phase reversal resulting in emergence of juvenile shoots from adult shoots is readily achieved (Huang et al., 1992). Furthermore, the shoots of the two phases display distinct morphogenetic characteristics. Repeated grafting in vitro of adult shoot tips onto rooted shoot segments of juvenile seedlings eventually results in scion growths with juvenile characteristics, such as a higher capacity for adventitious rooting, vigorous growth, and plagiotropic stems (Huang et al., 1992). Differences in esterase and peroxidase

isozymes were observed between juvenile or rejuvenated and adult shoots (Huang et al., 1996). The rate of protein phosphorylation was also higher in the juvenile shoots (Kuo et al., 1995), compared to the adult ones. Although juvenile and rejuvenated S. sempervirens produced more ethylene per cultured shoot, the rate of ethylene emission per gram of tissue was found to be the same (Huang et al., 2000). Restriction fragment length polymorphism of mtDNA (mitochondrial DNA) was also pointed out between juvenile and adult phase shoots (Huang et al., 1995). In this report, phase change aspects in tissue-cultured Sequoia sempervirens are investigated with respect to the potential for photosynthesis, and this is complemented by information on respiration rates and nitrogen contents.

Materials and Methods

Tissues Analyzed

Five different origins of shoots—juvenile (SS), adult from two different trees (AS and AST1), and rejuvenated shoots (RS and RST1) from the two adult S. sempervirens were investigated. Stocks of juvenile shoots were initiated from seedlings germinated in vitro. The adult stocks were established by culturing shoot tips excised from trees that were at least 60 years old. Stock cultures of AS were initiated from shoots excised in 1976, and those of AST1 were established from another mature tree in 1994 (Huang et al., 2000). Stocks of rejuvenated shoots, RS and RST1, were derived from AS and AST1, respectively. Rejuvenated shoots were obtained by 5-times grafting of the shoot tips from the two mature trees onto rooted juvenile seedling segments in vitro. Shoots of all five were available in stock cultures, maintained by monthly subculturing on a medium containing MS (Murashige and Skoog, 1962) salts, 3%

*Corresponding author. E-mail: yjshieh@gate.sinica.edu.tw

Botanical Bulletin of Academia Sinica, Vol. 44, 2003

sucrose, 0.25% gelrite, and, in mg L^-1: i-inositol, 100; thiamine•HCl, 1; nicotinic acid and pyridoxine•HCl, 0.5 each; and glycine, 2. Initial growth measurements were made by re-culturing 2-cm long shoot terminals for 14 days. Moisture and nitrogen contents were determined on pooled samples of 1 g of 0.7 cm tall terminals each, consisting of approximately 66 shoot tips per sample. The samples were oven-dried at 70°C overnight, weighed for moisture determinations, and pulverized for Kjeldahl nitrogen analysis using a Kjeltec 2300 Analyser Unit (Foss Tecator, Sweden). For measuring photosynthetic parameters and respiration rates 0.2 g of terminal 0.6-0.7 cm portions of shoots from newly sub-cultured stocks (10 days following transfer to fresh medium) were used. The samples consisted mainly of leaves with a minimum of stem tissue.

Measurements of Photosynthetic Parameters

Photosynthesis rates were based on the measurements of photosynthetic O₂ evolution from 0.3 g of sequoia shoot tips. Photosynthesis and respiration measurements were made using a Hansatech leaf disc oxygen electrode system to trace the O₂ exchange of samples, as described by Delieu and Walker (1981).

Chlorophyll was determined according to Wintermans and De Mots' procedure (1965) after extraction in 96% ethanol.

Ten shoot terminals were employed for each Chlorophyll fluorescence analysis. The shoot terminals were first pre-cultured individually in 25- × 150-mm test tubes for 10 days. Each tube contained 20 ml of the MS medium described above. Chlorophyll fluorescence was measured by means of a PAM 101 chlorophyll fluorometer (H. Walz, Effeltrich, Germany). Just prior to fluorescence measurements, the cultures were placed in darkness, horizontally, for 40 min. The dark fluorescence yield (F_o) was obtained by exciting a 0.7 cm region of each shoot tip with weak red light (1 µmole m^-2 s^-1, emission peak at 650 nm) and fluorescence was detected at wavelength above 700 nm. Each shoot tip was then given one flash (1 s) of saturated light (250 µmole m^-2 s^-1) to obtain the maximal (F_m) fluorescence value. The light signal was recorded and calculated by the software DA-100, proven by the manufacturer to obtain a (F_m-F_o)/F_m ratio. It is a measure of the quantum efficiency, or the potential quantum yield, of photosystem II (Bilger et al., 1995).

Statistical Analysis

Statistical significance was determined by computing standard errors of means or obtaining 95% confidence limits from tables of binomials (Lentner, 1982).

Results

Growth Rates of Experimental Shoots

As expected (Huang et al., 1992), increases of fresh weights and elongation of shoots were significantly greater

for the juvenile and rejuvenated shoots than adult shoots (Figures 1A, B).

Moisture and Nitrogen Contents

Juvenile, rejuvenated, and adult phase shoots had the same moisture content, approximately 85%. The total nitrogen content, however, was significantly higher in the juvenile and rejuvenated shoots than in the adult ones (Figure 2). They averaged nearly 5% of the dry weight of juvenile phase tissues. The adult tissues, AS and AST1, contained about 4.5% total nitrogen.

Respiration

Oxygen consumption, or index of respiration rate, was noticeably higher in the juvenile and rejuvenated shoots than in the adult ones. These shoots consumed about 0.3 µmole O₂ per gram tissue per min (Figure 3B). Both adult shoots, AS and AST1, consumed less than 0.25 µmole min^-1 g^-1 fwt.

Figure 1. Fresh weight increases (A) and elongation (B) of juvenile (SS), adult (AS and AST1) and rejuvenated (RS and RST1) S. sempervirens shoots in vitro. In each case, rejuvenation was achieved by five successive grafts of adult shoot tips onto rooted SS segments in vitro. Values are the mean ± SE (n=3). Bars denote standard errors of means.

Huang et al. — Phase change and photosynthesis in Sequoia sempervirens

Photosynthesis

The rate of photosynthetic oxygen evolution was also noticeably higher in the juvenile (SS) and rejuvenated (RS, RST1) tissues than in the adult (AS, AST1) ones (Figure 3A). The O₂ evolution rates in these tissues ranged from ca. 1.2 to 1.4 µmoles min^-1 g^-1 fwt. Those of adult tissues were about 1 µmole min^-1 g^-1 fwt.

Chlorophyll Contents

Shown in Figure 4 are the contents of chlorophylls a and b. Concentrations of both chlorophylls were significantly higher in the juvenile and rejuvenated shoots than in the adult ones although the difference from adult shoots was substantially greater for chlorophyll a. The chlorophyll a/b ratios were significantly lower for adult tissues (2.41 in AS and 2.11 in AST1) compared to the juvenile (2.86 in SS) and rejuvenated ones (2.79 in RS; 3.19 in RST1). Therefore, total chlorophyll was significantly higher in the juvenile and rejuvenated shoots than in the adult ones, which contained less than 600 µg total chlorophyll per gram of tissue. Juvenile and rejuvenated shoots contained nearly 800 µg per gram, in other words, 33% more.

Quantum Efficiency of Photosystem II

Although differing in photosynthetic rates, quantum efficiency was the same for all tissues, juvenile, adult, and rejuvenated (Figure 5).

Discussion

We previously reported on differences between juvenile and adult S. sempervirens in protein phosphorylation (Kuo et al., 1995) and esterase and peroxidase isozymes

Figure 3. Photosynthetic (O₂ evolution) (A) and respiration (O₂ consumption) (B) rates of juvenile (SS), adult (AS and AST1), and rejuvenated (RS and RST1) S. sempervirens shoots. Values are the mean ± SE (n=3). Bars denote standard errors of means.

Figure 2. Total nitrogen contents in shoots of juvenile (SS), adult (AS and AST1), and rejuvenated (RS and RST1) S. sempervirens. Values are the mean ± SE (n=3). Bars denote standard errors of means.

Figure 4. The chlorophyll a and chlorophyll b contents of juvenile (SS), adult (AS and AST1), and rejuvenated (RS and RST1) S. sempervirens shoots. Values are the mean ± SE (n=3). Bars denote standard errors of means.

Botanical Bulletin of Academia Sinica, Vol. 44, 2003

gen evolution and higher chlorophyll content of juvenile or rejuvenated shoots were not suggestive of a more efficient photosynthetic system. The virtual identity among all tissues, i.e., juvenile, adult and rejuvenated, of the (F_m - F_o)/F_m ratios signified no differences in the physiological state of photosynthetic apparatus in intact tissues. Under optimal physiological conditions this parameter was found to have the value of 0.83 (Demmig and Björkman, 1987). The value for sequoia was about 0.76. Differences in the observed rates of photosynthesis are not reflective of differences in basic mechanisms.

Acknowledgements. The investigation was supported by grants from the National Science Council (NSC 89-2313-B-001-017) and the Institute of Botany, Academia Sinica.

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Figure 5. Quantum efficiency of photosystem II in vitro juvenile (SS), adult (AS and AST1) and rejuvenated (RS and RST1) S. sempervirens shoots. Values are the mean ± SE (n=10). Bars denote standard errors of means.

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Huang et al. — Phase change and photosynthesis in Sequoia sempervirens

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