Journal of Experimental Marine Biology and Ecology 185 (1995) 191-202

JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY

Impact of daily photosynthetic period on protein synthesis and carbohydrate stores in Zostera marina L. (eelgrass) roots:

implications for survival in light-limited environments

George P. Kraemer”,‘, Randall S. Alberteb**

aDepartment of Biology. University of California, Los Angeles, CA 90024, USA

bBiologicaI Science and Technology Program, Ojice of Naval Research. 800 N. Quincy Street, Arlington.

VA 22217, USA

Received 21 March 1994; revision received 13 August 1994; accepted 2 September 1994

Abstract

The daily period of light-saturated photosynthesis (H,,,) determines the length of time that Zosteru marzha L. roots must function anaerobically. The cffccts of the duration of H,,, period on leaf growth rates, root protein metabolism and plant carbohydrate stores of eelgrass were investigated. Growth rates declined linearly with decreasing H,,,. Protein synthesis in roots subjected to 2 h H,,, was elevated in the first 10 min of aerobiosis during the transition from anaerobic metabolism. This elevation of protein synthesis capacity was not observed in roots from 4 and 12 h H,,, treatments. Root tissue age had a significant effect on protein metabolism, with rates of protein synthesis in root bundle no. 1 (youngest) 58 y0 higher than those of bundle no. 3. This age dependency was also observed in root protein content. Soluble carbohydrate levels in root bundle no. 1 and leaf no. 2 decreased linearly with H,,, period, while reductions in rhi- zome segment no. 1 were not significant. Levels in all tissues subjected to the 2 h H,,, treatment were 57% of levels following the 12-h treatment. Although protein synthesis in Z. marina root tissue was influenced only slightly by the anoxia associated with reduced irradiance during these short (lo-14 day) periods, longer and/or more severe periods of light limitation may disrupt root protein metabolism due to carbohydrate starvation.

Keywords: Anoxia; Carbohydrate; Protein synthesis; Root; Zostera

* Corresponding author. ’ Present address: Stazione Zoologica di Napoli, Laboratorio di Ecologia del Benthos, Punta S. Pietro, Ischia Porte 80077, Italy.

Elsevier Science B.V. SSDI 0022-0981(94)00145-6

192 G.P. Kraemer, R.S. Aiberte 1 J. Exp. Mar. Bid. Ed. 185 11995) 191-202

1. Introduction

The temperate zone marine angiosperm Zostera marina L. (eelgrass) is a keystone species in many estuarine environments, and shows a wide geographical distribution in temperate zone waters (Phillips & McRoy, 1980). Eelgrass flourishes in organic-rich, anoxic sediments that exclude most other macrophytes. Subterranean tissues (roots and rhizome) are supplied with oxygen via well-developed aerenchyma (Tomlinson, 1980), and function aerobically while shoot photosynthesis is at or near saturation (Smith et al., 1984). Eelgrass roots cannot store O,, and must depend on anaerobic fermen- tation for energy generation when subterranean tissues become anoxic (Pregnall et al., 1987; Smith et al., 1988). Root anoxia ensues 15-30 min after cessation of shoot photosynthesis (e.g. due to increased water column turbidity or darkness; Smith et al., 1984). Since Z. marina often inhabits light-limited environments, its survival depends on adaptations that enable its roots to tolerate anoxia for extended periods each day (Smith et al., 1988; Zimmerman et al., 1991). Tissue death occurs in all plants, including Z. marina, if anoxia is prolonged (Kozlowski, 1984; Smith et al., 1988). However, the time-course over which anoxia depresses metabolism and leads to death differs greatly in anoxia-tolerant and -intolerant species. For example, eelgrass roots can tolerate O2 deprivation for 3 days and fully recover (Smith, 1989), while roots of anoxia-intolerant species such as Pisum sativum survive for only a few hours under anoxia (Crawford, 1982). Although root tissue constitutes a small part of total plant biomass in eelgrass (5-6%; Kraemer & Alberte, 1993), roots can supply more than 50% of the nitrogen required for plant growth (Zimmerman et al., 1987).

The capacity for protein synthesis is a significant indicator of tissue metabolic re- sponse to anoxia, since protein synthesis is required to maintain cellular integrity and metabolic function. In most organisms, protein synthesis is disrupted upon the tran- sition from aerobiosis to anaerobiosis. Large decreases in the rates of protein synthe- sis, and dramatic differences in the products have been documented in response to anoxia in terrestrial plants (e.g. Lin & Key, 1967; Sachs et al., 1980; Kennedy et al., 1992).

The daily period during which eelgrass shoots can maintain maximal photosynthetic rates (incident irradiance > photosynthetic light saturation point [Zk]), Hsat, strongly influences the maximum depth inhabited by Z. marina (Dennison & Alberte, 1985; Zimmerman et al., 1991). H,,, defines the period of carbon (C) influx and aerobic function by subterranean tissues. Smith et al. (1988) hypothesized that light limitation (either chronic or transient; Zimmerman et al., 1991) leads to C limitation in roots in the long-term, which impacts survival and production. Not only must the roots toler- ate anoxia during periods of light limitation, but the translocation of respiratory sub- strate (sucrose) also ceases during anaerobic periods in eelgrass (Zimmerman & Alberte, in review). Thus, the adverse impacts of root tissue anoxia may result from both C starvation in subterranean tissues and disruption of protein synthesis. Furthermore, because eelgrass plants can possess 12 or more pairs of root bundles of increasing age along the subterranean rhizome, and because tissue age is correlated with metabolic potential (Mazzella & Alberte, 1986; Kraemer & Chapman 1991; Kraemer & Alberte, 1993), the importance of roots to whole plant metabolism varies with root tissue age.

G.P. Kraemer. R.S. Alberte 1 J. Exp. Mar. Biol. Ecol. 185 (1995) 191-202 193

The objective of this study was to examine the effects of a range of daily photosyn- thetic periods (H,,,) on the protein metabolism of Z. marina roots and relate this to carbohydrate stores available to support this and other metabolic requirements. Mea- surements of relative rates of aerobic and anaerobic protein synthesis and tissue car- bohydrate levels allowed the assessment of the influence ofH,,, on root protein synthetic capacity, gross C metabolism in subterranean and leaf tissues, and the partitioning of C between tissue compartments.

2. Materials and methods

Healthy Z. marina plants, containing abundant root carbon reserves (Smith, 1989; Kraemer & Alberte, 1993) were collected from a subtidal population (5-7 m) located off Del Monte Beach, Monterey Bay, CA (36” 37’ 19” N, 121” 54’ 12” W) from Sep- tember to November (1992) using the rhizosphere coring method (Dennison & Alberte, 1982). For evaluation of protein synthetic capacity, individuals (one shoot each) with at least three rhizome segments embedded in a sediment plug were placed into 4 1 pots, and six pots were placed into 80 1 incubation chambers containing filtered seawater, with airstones to provide aeration and mixing. Plants were maintained at 14 ( k 2) “C, and halogen floodlamps supplied light in excess of photosynthetic saturation (1,) throughout the containers (> 125 pmol quanta.m-2.s-‘; Dennison & Alberte, 1985). Illumination was timer-controlled to provide saturating photosynthetic irradiance for daily 1ight:dark periods of 2:22, 4:20, and 12:12, yielding Hsat periods of 2, 4, and 12 h, respectively. Plants were harvested after 10 days and assayed for protein synthesis capacity and shoot growth. The effects of root tissue age on protein synthesis were assessed using both freshly collected tissue and tissue harvested after 10 days of the Hsat treatments. For evaluation of the effects of H,,, on carbohydrates stores, plants were subjected to H,,, periods of 2, 4, 6, or 12 h for 14 days. Experimental treatments were repeated three times to obtain replicates for each Hsat level.

Growth rate. At the beginning of the growth period, a hole was punched through the leaf bundle 4-5 cm above the first root bundle (Zieman & Wetzel, 1980). After 10 days, the amount (cm) of new tissue (root or shoot) was measured and normalized to total tissue.

Root protein synthesis. The oxygenation of root tissue was initiated by illumination of shoots. Prior to illumination, and at intervals during the first 2 (Hsat = 2) to 4 (Hs,, = 4, 12) h of the illuminated period, plants were gently removed from the sediment matrix and rinsed in deoxygenated seawater. The distal 2 cm of root tips from the root-rhizome unit in one pot (100-200 mg) were excised and placed into incubation medium (sterile seawater containing 60 pg.ml-’ streptomycin, 100 pg.rnl-’ penicillin-G, 0.16 M sucrose, and 20 PCi 35S-methionine [specific activity 1100 Ci mmol-‘1, yielding 8.3 PCi ml-‘). Plants from which roots have been removed were discarded. The time course of aerobic recovery of protein synthesis was measured using sequential measurements of 35S-methionine incorporation. To assess the time scales over which the protein

194 G.P. Kraemer, R.S. Alberte / .I. Exp. Mm. Bid. Ed. 185 (1995) 191-202

synthetic metabolism responded to the anaerobic-aerobic transition, root tips were incubated for 5 or 30 min. During the incubations, the medium was bubbled with 320 ppm CO, + N, balance, either with (aerobic) or without (anaerobic) 40% 0, (oxygen may constitute 38% of lacuna1 gas in actively photosynthesizing seagrasses; Roberts & Moriarty, 1987; Carlson et al., 1988). The root tips were then removed, rinsed with sterile seawater containing 1 mM unlabelled methionine, and then with deionized water. Tissue was frozen in liquid N2 and ground in protein extraction buffer (50 mM HEPES, pH 7.5, 10 mM EDTA, 0.5 mM phenylmethanesulfonyl fluoride (PMSF), 1% (w/v) polyvinylpolypyrrolidone (PVPP), 0.1 y/, (v/v) Nonidet-40, and 1 y0 (v/v) 2-mercaptoethanol). The homogenate was centrifuged (20 min, 16 500 x g at 4 “C), and the supernatant collected.

To determine label uptake, a 50 ~1 aliquot of supernatant was added to 5 ml scin- tillation cocktail (Ecolume). Another 50 ~1 aliquot was added to 50 ~1 10% (w/v) trichloroacetic acid (TCA) containing 2 mM methionine, and incubated overnight at 4 “C. The TCA-precipitable fraction was collected on a Whatman GF/F filter, rinsed with 5% TCA containing 1 mM methionine, 95 “/, (v/v) ethanol, and 100% acetone. To solubilize the precipitated polypeptides, the filter was placed in a scintillation vial containing 1 ml 0.5% (v/v) Triton X-100 containing 0.5 N NaOH and left overnight at room temperature. Radioactive decay (CPM) of tissue homogenate and TCA- precipitable materials were measured using a Packard liquid scintillation counter. Quench correction was performed using the channels ratio method. Protein concen- trations were determined by a dye binding assay (Winterbourne, 1986).

To further assess the aerobic recovery of protein synthesis, a high specific activity mixture of tritiated amino acids (Amersham; 86-154 Ci.mmol-‘, 1 pCi.pl~ ‘) was used in lieu of “S-methionine. Root bundle no. 1 (youngest) and no. 3 of plants subjected to H,,, periods of 2, 4, and 12 h were collected during the last 30 min of the anaero- bic (dark) period and after 30 min of aerobic (illuminated) metabolism to evaluate the impact of Hsat period, the availability of O,, and tissue age on protein synthesis capacity (n = 4 per H,,, treatment).

Soluble carbohydrates. Soluble carbohydrates (96% sucrose + fructose; Smith, 1989) in the second leaf (youngest = no. l), and first root bundle and rhizome segment were determined using sucrose standards (Yemm & Willis, 1965). Measurements were rep- licated using tissue from 3 to 4 plants. The aqueous extracts were stabilized against bacterial degradation with sodium azide (0.02T0, w/v).

Data analysis. Several of the protein synthesis data sets violated the assumption of homoscedasticity; consequently, all protein synthesis data were In-transformed prior to statistical analysis. Results in the text are presented as untransformed means (SD). Data were analyzed using t-tests or ANOVA, where appropriate, and Least Significant Difference post-hoc were tests employed to order treatments means when significant effects were revealed. Linear regressions of tissue carbohydrate content and growth rate (untransformed) on H,,, were performed to describe the relationship between light availability and C metabolism.

G.P. Kramer, R.S. Alberte /J. Exp. Mar. Biol. Ecol. 185 (1995) 191-202 195

3 - SHOOT 0

a, + 0

CT- -r 2-

-c-D

p5

G 1 -

0 ’ I , I

2 4 10 12

H sat Periods (h)

Fig. 1. The effect of H,,, (the number of hours of light-saturated photosynthesis) on the biomass-specific growth rates of 2. marina shoots after a 14-day period. Regression highly significant (df= 21, t = 8.08, p<O.OOl, ? = 0.894).

3. Results

Growth of Z. marina was observed under all H,,, treatments, and H,,, had a sig- nificant effect on the biomass-specific shoot growth rates (Fig. 1). Growth rate de- creased linearly with H,,, period (untransformed data; df = 21, t= 8.08, p<O.OOl, 2 = 0.894) with plants exposed to the 12 h H,,, period adding shoot biomass at 2.5 times the rate of those under the 2 h Hsat treatment. The protein content of the youngest roots (the most metabolically active; Kraemer & Alberte, 1993) also varied significantly with H,,, treatment (Fig. 2). Roots from the 2, 4, and 6 h H,,, treatments had similar protein contents (~4.0 mg.g-’ FW), while roots from the 12-h treatment possessed about 75% more protein than all other treatments.

Roots incorporated 35S-methionine into protein under both anaerobic and aerobic periods, and 12 to 27% of incorporated label was recovered in TCA-precipitable compounds (Figs. 3 and 4). There was substantial variability among individuals in the amount of label assimilated during incubation (data not shown), preventing meaning-

ROOT b c 8 T

2 4 8 8 10 12

H sat Period (h)

Fig. 2. Protein content of the youngest Z. marina roots (bundle no. 1) after a 14 day treatment period as a function of H,,, (the number of hours of light-saturated photosynthesis). Different letters denote statisti- cally different mean values (pi 0.05; LSD post-hoc tests). Error bars k 1 SD.

196 G.P. Kraemer. R.S. Alberte /J. Exp. Mar. Bid. Ed. 185 11995) 191-202

OI’ 0 10 20 30

Aerobic Recovery (min)

Fig. 3. Time courses of the recovery of aerobic protein synthesis by youngest root bundles of Z. marina roots following 10 days under either 2, 4, or 12 h H,,, (the number of hours of light-saturated photosynthesis) periods. Roots incubated with j5S-methionine for 5 min (n = 3-5 root bundles per time point). Error bars are + 1 SD.

ful comparisons of absolute rates of protein synthesis. Variability was reduced when the label incorporated into protein was normalized to label incorporated into whole tissue (data presented as protein synthesis capacity).

When roots were harvested at the end of the dark period (tissue anoxic; anaerobic incubation, t = 0) and at 5-min intervals during the first 30 min of illumination (i.e. during transition from anaerobiosis to aerobiosis; aerobic incubations) and labelled with 35S-methionine for 5 min, there was a transient increase in protein synthesis ca- pacity during the first 10 min of the aerobic recovery of roots from the 2 h H,,, treat- ment (Fig. 3). Protein synthesis capacity during the first and second 5-min period of aerobic recovery was significantly greater than either anaerobic or other aerobic val- ues (F= 4.58, df = 4, ~~0.05). Roots from plants under the 4 and 12 h H,,, periods showed no significant elevation of protein synthesis capacity during aerobic recovery. When root tips were incubated in 35S-methionine for 30-min intervals (roots collected at t = 0, and at 30 min intervals during aerobic period), protein synthesis capacity did not vary significantly during the 2-4 h of aerobic recovery (Fig. 4). Additionally, HsXt did not have a significant effect on anaerobic (t = 0) rates of protein synthesis (F = 1.09, df = 2, p = 0.35).

Tissue age had a strong and significant impact on aerobic protein synthesis capac- ity (Table l), with roots from youngest bundle incorporating, on average, 2.9-times more 35S-methionine into TCA-precipitable compounds than did roots of the third bundle (12.4% vs. 4.3% of incorporated 35S-methionine). Root total protein content showed a similar age dependency, with first bundle roots possessing 2 to 3 times more protein

G.P. Kraemer. R.S. Alberte 1 J. Exp. Mar. Bid. Ecol. 185 (1995) 191-202 197

OI’ 0 60 120 180 240

Aerobic Recovery (min)

Fig. 4. Time courses of the recovery of aerobic protein synthesis by youngest root bundles of Z. marina roots following 10 days under either 2, 4, or 12 h H,,, (the number of hours of light-saturated photosynthesis) period. Roots incubated with “S-methionine for 30 min (n = - 3 5 root bundles per time point). Error bars are + 1 SD.

than roots from either second or third nodes. The study of protein synthesis capacity using 3H-amino acids also demonstrated the significant effect of tissue age (Table 2). Age explained almost half of the variation in the incorporation of 3H-amino acids into polypeptides. There was no difference in protein synthesis capacity between the end of the anaerobic period and an early stage (30-60 min post-anaerobic) of the aerobic period. Although the effects of H,,, treatment on root protein synthesis capacity were not statistically significant (p = 0.053), mean values (11.5,8.9, and 8.8% for H,,, periods of 2,4 and 12 h) suggest that reduced daily photosynthetic period may lead to increased

Table 1 Tissue age-related differences in root aerobic protein synthesis and protein content in Zosteru marina

Root bundle Protein content

(mg.g ’ FW)

Protein synthesis

(% label)

1 6.6” (0.3) 12.4” (2.5)

2 3.3b (0.6) ND

3 2.4b (0.7) 4.2b (2.1)

Root bundle No. 1 is the youngest. Protein synthesis measured on plants incubated for 10 days under 12 h H,,, periods, and expressed as y0 of total incorporated label recovered in TCA-precipitable materials.

Protein content determined on freshly-collected plants. Mean values (SD) are untransformed, while statis- tical analyses (ANOVA, LSD post-hoc tests) were performed on In-transformed data. Different superscripts indicate significantly different mean values (~~0.05). ND = not determined.

198

Table 2

G.P. Kraemer, R.S. Alberte 1 J. Exp. Mar. Biol. Ecol. 185 (1995) 191-202

Results of ANOVA of the effects of H,,,, time during the light/dark (aerobic/anaerobic) cycle, and tissue age on the incorporation of 3H-into TCA-precipitable compounds in Zostera matins roots

Effect MS F df P “0 of total variation

explained

H,,, (A) Light/dark (B)

Tissue age (C)

AxB

AxC

BxC

AxBxC

Within

0.141 3.16 2 0.053 12

0.020 0.46 1 0.511 2

0.541 12.18 1 0.002 41

0.108 2.43 2 0.101 9

0.116 2.61 2 0.086 9

0.103 2.31 1 0.134 8

0.088 2.00 2 0.149 7 0.044 36 4

The first (youngest) and the third root bundles of plants subjected to H,,, periods of 2, 4, and 12 h were

collected during the last 30 min of the anaerobic (dark) period and after 30 min of aerobic (illuminated)

metabolism, and incubated with ‘H-amino acids for 30 min (n = 4 for each treatment combination).

short-term protein synthetic rates. This finding is congruent with the data presented in Fig. 1, and the observation by Pregnall et al. (1987) that glutamine synthetase activity increased in roots of plants subjected to decreasing H,,, periods. The soluble carbo- hydrate levels of Z. marina root bundle no. 1 and leaf no. 2 decreased with decreas- ing H,,, periods (Fig. 5). While the linear regressions for root and leaf tissue were significant and paralleled each other, the regression of rhizome carbohydrate content

60 t

SHOOT

40

20

0

20

15

10

5

0

300

200

100

p-T----

t

ROOT

i

RHIZOME

8 ; ii

8 0 0

0

01 2 4 6 a 10 12

H sot Period (h)

Fig. 5. The effect of H,,, (the number of hours of light-saturated photosynthesis) on the soluble carbohydrate contents of leaf no. 2 (no. 1 is youngest), root bundle no. 1, and rhizome segment no. 1 of 2. man‘nu. Re- gressions of shoot and root carbohydrate contents on H,,, period are significant (shoot: df= 17, I= 2.97, ptO.O1, $=0.338; root: df= 14, t=2.98, p<O.Ol, ?=0.424).

G.P. Kraemer, R.S. Alberte /J. Exp. Mar. Biol. Ecol. 18.5 (1995) 191-202 199

on %I, was not different than zero (p = 0.064). Stored carbohydrates of each tissue from the 2 h H,,, treatment was % 57 % of the levels in tissues from the 12-h treatment.

4. Discussion

Results from this and prior studies (Smith et al., 1984; Pregnall et al., 1987; Smith, 1989) demonstrate that eelgrass roots, subjected to anoxia on a die1 cycle, possess adaptations to tolerate and/or minimize metabolic repression or disruption during anoxia. Healthy eelgrass plants tolerated 10 to 1Cday periods during which roots were anoxic most (21-22 h) of each day. However, the long-term impacts of extended an- oxic events on the production ecology and depth distribution of 2. marina are still unclear (Zimmerman et al., 1991).

It is not surprising that H,,, had a significant effect on the biomass-specific growth rates of leaf tissue; field studies have amply demonstrated the relationship between light availability and growth (e.g. Dennison & Alberte, 1982, 1985, 1986; Zimmerman et al. 1994). We anticipate that the production of subterranean tissues varies with irradiance in a fashion similar to that of the shoots, since the synthesis of root, shoot, and rhi- zome tissue in Z. marina is coordinated to maintain relatively constant proportions of each tissue type (Dennison et al., 1987; Kraemer & Alberte, 1993). Further support for this conjecture comes from the parallel relationships observed here between Hsat and stored carbohydrate levels in shoots and roots.

A strong age effect was observed on eelgrass root protein metabolism. Young, rap- idly metabolizing roots carried out more protein synthesis and contained more protein than older roots. In fact, protein synthesis capacity appeared correlated with the pro- tein content for the first three root bundles, although the need to analyze separate tissues because of low tissue protein content precluded a statistical evaluation. This age- dependency of protein synthesis capacity is reflected in functional differences between young and old roots, consistent with other age-dependent patterns of metabolism in Z. marina roots (respiration rate, glutamine synthetase and sucrose synthase activities; Kraemer & Alberte, 1993 and in prep.). While an accurate accounting of whole plant N balance in Z. marina requires the consideration of age-dependency in the metabo- lism of subterranean tissues, this is less important for C balance considerations, due in part to the low carbon demand by roots and rhizomes.

The 2 h H,,, treatment mimics an extended light reduction event (e.g. elevation of water column turbidity due to wind-induced mixing), the occurrence of which has been correlated with reduced depth penetration by Z. marina (Zimmerman et al., 1991). It was anticipated that lo-14 days of 2 h Hsat treatment would lead to significant anoxic stress in root tissue, and result in stress to the whole plant in terms of overall C bal- ance (photosynthetic C gain relative to respiratory and fermentative losses). Eelgrass roots and shoots in the present study survived this treatment, and the shoots added biomass at an average rate of 1.2%*day-‘, comparable with measured field growth rates (Dennison & Alberte, 1985). Repeated cycles, however, of extended root anoxia due to short H,,, periods have significant long-term impacts on eelgrass metabolism; plants with marginal root and rhizome C reserves died after 2 to 4 wk of 2 h H,,,

200 G.P. Kraemer. R.S. Alberte 1 J. Exp. Mar. Bid. Ed 185 (1995) 191-202

(Zimmerman & Alberte, unpubl. data). Stored carbohydrate levels in roots are reduced by a combination of (1) reduced C flux from shoots due to the cessation of the trans- location of photosynthate into roots during anoxia (Zimmerman & Alberte, unpubl. data), and, (2) the respiratory and fermentative consumption of reserves (Smith et al., 1988). Assessment of root carbohydrate levels and C consumption rates indicate that young roots, replete with carbohydrates, begin experiencing C-limitation after about 20 h of anoxia (Smith, 1989; Kraemer & Alberte, 1993). Since rates of C consumption do not decline substantially during anoxia as substrate availability decreases (Smith, 1989), translocation of C from either rhizomes or shoots during the short (2-3 h) photosynthetic period must be adequate to replenish C stores in the roots.

Though eelgrass roots under the 2 h H,,, regime were anoxic for about 90% of the lo-14-day treatment, these conditions were not stressful in the short term to Z. marinu

root tissues when root carbon reserves were initially full and protein synthesis was used as a litmus for metabolic capacity. The short Hsat conditions had little apparent effect on the steady-state levels of protein, as roots exposed to 2, 4, and 6 h H,,, possessed equal levels of total protein. In fact, the presence/absence of O2 alters only slightly the patterns of protein synthesis in eelgrass roots (Smith, 1989), unlike the situation in most terrestrial plants (Crawford, 1982; Kennedy et al., 1992). The results highlight several points regarding the response of Z. marina to the variable and sometimes stressful submarine light environments characteristic of eelgrass habitats. Z. marina root pro- tein metabolism is resistant and resilient to anoxia stress (at least when C reserves are not depleted). Rates of protein synthesis declined by only 30-45”/, after 3 h of anoxia (Smith, 1989), compared with a reduction of up to 9.5 % in anoxia-intolerant spieces like maize (Freeling & Bennett, 1985). The increase in steady-state protein synthesis observed here during the transition from root anaerobiosis to aerobiosis was transient and had only a small impact on overall protein synthesis. Metabolic adaptations in eelgrass roots sustain metabolism during short episodes of extremely short H,,,. These adaptive strategies include substantial carbohydrate stores, low rates of C consump- tion and the absence of a Pasteur effect in subterranean tissues, and the maintenance of anaerobic protein synthesis at nearly aerobic rates and patterns (Smith et al., 1988; Smith, 1989; Kraemer & Alberte, 1993, and this study). Thus, healthy Z. marina, subjected to a short period of sharply reduced light availability, are able to maintain sufficient metabolic activity to shield subterranean tissues from the deleterious effects of anoxia and, minimizing the impact of root anoxia on shoot processes, the plants continue to grow, although at reduced rates.

These metabolic adaptations, however, are not without restriction. Plants at the light-limited edge of the depth distribution likely do not have replete significant C re- serves in roots or rhizomes due to chronic light limitation. A lo-day period of 2 h H,,,

could constitute a lethal stress to root tissues with little or no mobilizable C reserves. Indeed, recent observations have demonstrated reduced growth of eelgrass exposed to 2 h H,,, periods for 30-45 days when subterranean C reserves were not full at the start of the treatment (Zimmerman & Alberte, unpubl. data). The death of root tissue would have severe consequences since eelgrass rely greatly on roots to supply nitrogen for plant growth (Zimmerman et al., 1987). Longer and/or more severe light attenuation will undoubtedly result in detrimental effects on root protein metabolism and growth

G.P. Kraemer, R.S. Alberte 1 J. Exp. Mar. Biol. Ecol. 185 (1995) 191-202 201

of even healthy, C-replete plants. Recent work has revealed that H,,, periods in 2. marina environments can be less than 2 h for up to 15 days (Zimmerman et al., 1994). Consequently, future metabolic studies must consider both the intensity and duration of the this and other stresses characteristic of the environments inhabited by Z. marina, where spatial and temporal scales of change are dynamic.

Acknowledgements

We thank J. Coyer, G. Suba, and R.C. Zimmerman for SCUBA diving assistance. A. Cabello, C. Dea, and G. Procaccini provided logistical supported by a grant from the National Science Foundation.

References

support. Research was

Carlson, P.R., L.A. Yarbro, W.B. Sargent & H.A. Arnold, 1988. Hypoxic stress in Thalassia festudinum:

Evidence from diurnal changes in rhizome gas composition. Eos, Vol. 69, p. 1115.

Crawford, R.D.M., 1982. The anaerobic retreat as a survival strategy for anaerobic plants and animals. Trans.

Bot. Sot. Edin., Vol. 44, pp. 51-63.

Dennison, W.C. & R.S. Alberte, 1982. Photosynthetic responses of Zostera marina L. (eelgrass) to in situ

manipulations of light intensity. Oecologia, Vol. 55, pp. 137-144.

Dennison, W.C. & R.S. Alberte, 1985. Role of daily light period in the depth distribution of Zostera marina

(eelgrass). Mar. Ecol. Prog. Ser., Vol. 25, pp. 51-61.

Dennison, W.C. & R.S. Alberte, 1986. Photoadaptation and growth of Zostera marina L. (eelgrass) trans-

plants along a depth gradient. J. Exp. Mar. Biol. Ecol., Vol. 98, pp. 265-282.

Dennison, W.C., R.C. Aller & R.S. Alberte, 1987. Sediment ammonium availability and eelgrass (Zosrera

marina) growth. Mar. Bioi., Vol. 94, pp. 469-417.

Freeling, M. & D.C. Bennett, 1985. Adhl in maize. Annu. Rev. Genet., Vol. 19, pp. 297-323.

Kennedy, R.A., M.E. Rumpho & T.C. Fox, 1992. Anaerobic metabolism in plants. Plant Physiol., Vol. 100,

pp. 1-6. Kozlowski, T.T., 1984. Flooding and plant growth, Academic Press, Cambridge, 221 pp.

Kraemer, G.P. & R.S. Alberte, 1993. Age-related patterns of metabolism and biomass in subterranean tis-

sues of Zostera marina L. (eelgrass). Mar. Ecof. Prog. Ser., Vol 95, pp. 193-203.

Kraemer, G.P. & D.J. Chapman, 1991. Effects of tensile force and nutrient availability on carbon uptake

and cell wall synthesis in blades of juvenile Egregia menziesii (Turn.) Aresch. (Phaeophyta). J. Exp. Mar.

Biol. Ecol., Vol. 149, pp. 267-271.

Lin, C.Y. & J.L. Key, 1967. Dissociation and reassembly of polyribosomes in relation to protein synthesis

in the soybean root. J. Mol. Biol., Vol. 26, pp. 231-241.

Mazzella, L. & R.S. Alberte, 1986. Light adaptation and the role of autotrophic epiphytes in primary pro-

duction of the temperate seagrass, Zostera marina L. J. Exp. Mar. Biol. Ecol., Vol. 100, pp. 165-180.

Phillips, R.C. & C.P. McRoy, C.P. (eds.), 1980. Handbook of seagrass biology: An ecosystem perspective,

Garland STPM Press, New York, 353 pp.

Pregnall, A.M., R.D. Smith & R.S. Alberte, 1987. Glutamine synthetase activity and free amino acid pools

of eelgrass (Zosrera marina L.) roots. J. Exp. Mar. Biol. Ecol., Vol. 106, pp. 211-228.

Roberts. D.G. & D.J.W. Moriarty, 1987. Lacunal gas discharge as a measure of productivity in the seagrasses Zostera capricorni, Cymodocea nodosa, and Syringodium isoetifolium. Aquat. Bot., Vol. 28, pp. 143-160.

Sachs, M.M., M. Freeling & R. Okimoto, 1980. The anaerobic proteins of maize. Cell, Vol. 20, pp. 761-

167. Smith, R.D., 1989. Anaerobic metabolism in the roots of the seagrass Zostera marina L. Ph.D. Disserta-

tion, University of Chicago, Chicago, IL, 241 pp.

202 G.P. Kraemer, R.S. Alberte 1 J. Exp. Mar. Biol. Ecol. 185 (1995) 191-202

Smith, R.D., W.C. Dennison & R.S. Alberte, 1984. Role of seagrass photosynthesis in root aerobic pro-

cesses. Plant Physiol., Vol. 74, pp. 1055-1058.

Smith, R.D., A.M. Pregnall & R.S. Alberte, 1988. Effects of anaerobiosis on root metabolism of Zostera

marina (eelgrass): implications for survival in reducing sediments. Mar. Biol., Vol. 98, pp. 131-141.

Tomlinson, P.B., 1980. Leaf morphology and anatomy in seagrasses. In, Handbook of seagrass biology: An

ecosystem perspective, edited by R.C. Phillips & C.P. McRoy, Garland STPM Press, New York, pp. 7-

28.

Winterbourne, D.J., 1986. Cell growth determined by dye-binding protein assay. Biochem. Sot. Trans., Vol.

14, p. 1179.

Yemm, E.C. & A.J. Willis, 1965. The estimation of carbohydrates in plant extracts by anthrone. Biochem.

J., Vol. 57, pp. 508-514.

Zieman, J.C. & R.G. Wetzel, 1980. Productivity in seagrasses: methods and rates. In, Handbook of seagrass

biology: An ecosystemperspective, edited by R.C. Phillips & C.P. McRoy, Garland STPM Press, New York,

pp. 87-116.

Zimmerman, R.C., R.D. Smith & R.S. Alberte, 1987. Is growth of eelgrass nitrogen limited? A numerical

simulation of the effects of light and nitrogen on the growth dynamics of Zostera marina. Mar. Ecol. Prog.

Ser., Vol. 41, pp. 167-176.

Zimmerman, R.C., J.L. Reguzzoni, S. Wylie-Echeverria, M. Josselyn & R.S. Alberte, 1991. Assessment of

environmental suitability for growth of Zostera marina L. (eelgrass) in San Francisco Bay. Aquar. Bat.,

Vol. 39, pp. 353-366.

Zimmerman, R.C., A. Cabello-Pasini & R.S. Alberte, 1994. Modeling daily photosynthetic production of

aquatic macrophytes from irradiance measurements. A comparative analysis. Mar. Ecol. Prog. Ser.,

Vol. 114, pp. 185-196.