行政院國家科學委員會專題研究計畫成果報告
期中報告
大型海藻的磷代謝 (2/3)
P
HOSPHORUSM
ETABLOSIM INM
ACROALGAE(2/3)
NSC 90-2311-B-110-003
90 年 8 月 1 日至 91 年 7 月 31 日
李澤民
Tse-Min Lee
國立中山大學海洋生物研究所
Institute of Mar ine Biology, National Sun Yat-sen Univer sity,
Kaohsiung, Taiwan 80424, Republic of China (R.O.C.)
*Tel: 07-5252000 ext. 5110. Fax: 07-5251509. e-mail: [email protected]
中文摘要 本研究為三年計畫之第二年，本年度 目的是探討大型海藻石蓴(Ulva lactuca L.) 及傘房龍鬚菜(Gracilaria coronopifolia J. Agardh) 為材料探討：(1)、缺磷下的 ACP 與 extracellular AP 活性變化之關係。(2)、 缺 磷 對 ACP isozyme 的 影 響 。 (3) 、 extracellular AP 及細胞內的 ACP 酵素特 性。 關鍵詞：大型海藻，石蓴，傘房龍鬚菜， 磷，有機磷分解，細胞外磷酸分解酵素 (extracellular AP)，細胞內磷酸分解酵素 (ACP)。 ABSTRACT
This study is the 2nd-year project of a 3-year
project. The main purpose of this year project was to study the uptake of inorganic phosphate (Pi) and the responses of DOP-degradation enzymes in macroalgae,
Ulva fasciata Delile and Gracilaria
tenuistipitata var. liui Zhang et Xia. In this study, we would like to investigate the following questions: 1. the optimal seawater Pi level for growth; 2.the physiological changes under P deficiency (including photosynthesis 、 respiration 、 nitrogen
metabolism 、 the composition of
phycobiliproteins 、 growth and tissue total carbon (TC)、total phosphorus (TP) levels, the activities of extracellular alkaline phosphatase, intracellular alkaline (AP) and acid (ACP) phosphatase); 3. The comparison the responses to Pi deficiency among different parts of Gracilaria (such as tip and
base); 4. the kinetics of Pi uptake; 5. the ability of P accumulation (storage?) and its formation. Now, we have done the folling items: 1. the optimal seawater Pi level for growth; 2. the kinetics of Pi uptake; 3. The critical and subsistence N and P levels.
前言
磷 是 細 胞 生 化 代 謝 的 必 需 營 養 因 子，缺磷會造成生化代謝及生長發育受阻 (Lynch et al., 1991)，如光合作用 (Lauer et al., 1989)、碳的分配代謝 (Jeschke et al., 1996) 及氮素吸收 (Heuwinkel et al., 1992; Pilbeam et al., 1993)、代謝 (Rufty et al., 1993) 會因缺磷而受阻。
水 體 中 的 磷 種 類 有 無 機 磷 酸 (inorganic phosphate，Pi) 及有機磷 (organic phosphate，P) 兩種。大型海藻可吸收的磷 為無機磷酸，磷的吸收是一需能的主動吸 收過程 (Eppley, 1958); 高等植物的研究指 出 植 物 細 胞 無 機 磷 酸 的 吸 收 是 藉 由 與 plasma membrane H+-ATPases 的 proton 之
co-transport process (Epstein, 1976;
Ullrich-Eberius et al., 1981, 1984; Sakano, 1990)。無機磷的吸收速率因細胞外的磷濃 度增高而增高。
無機磷吸收的 saturation kinetics 則因 海藻種而不同: 紅藻的 Agardhiella subulata 的 Vmax=0.47 µmol/g DW/h ，Ks=0.4 µM
(DeBoer, 1981); 綠藻的Ulva sp.的 Vmax=8.5 µmol/g DW/h ，Ks=3.5 µM (Lavery and
McComb, 1991); 綠藻的Enteromorpha sp.
的 Vmax=20.8 µmol/g DW/h ，Ks=10 µM
(Lavery and McComb, 1991)。紅藻的龍鬚菜 (Gracilaria tikvahiae) 的 磷 吸 收 有 三 相 : 0-0.2 µM, 0-2 µM 及 0-11 µM (Friedlander and Dawes, 1985)。 無機磷吸收的 kinetics 因植物細胞內 的磷狀態而不同。高等植物的研究指出在 缺磷下，無機磷的吸收速率是不缺磷植物 細 胞 的 數 倍 (Cartwright, 1972; Clarkson, 1985; Clarkson and Scattergood, 1978)，此一 增加係一植物適應缺磷的策略之一。 材料與方法: 1、材料栽培: 本試驗材料，石蓴 (Ulva lactuca L.) 及傘房龍鬚菜 (Gracilaria coronopifolia J. Agardh) 係取自墾丁南灣。龍鬚菜及石蓴 以滅菌海水 (高雄西子灣抽取天然海水， 取得後高溫高壓 (120°C,1.2 Kgs/cm,20min) 滅菌) 沖洗過後，培養於含 40 公升培養液 ( 含 全 量 ES nutrient solution (Provasoli, 1963) 的滅菌海水) 之養殖箱中，內裝置小 型沉水馬達，使水流保持流通。菊花心種 龍鬚菜的栽培海水鹽度控制為 3%。光照強 度 70~100 µE m-2s-1 ( 光 源 為 日 光 燈 (FL30D， 旭光公司，桃園，台灣) 6 管加 2 個白熱燈泡 (E2760W，東亞公司，新竹， 台灣))，日照時間為 12 小時，溫度為 25°C
(Edelstein et al., 1976)。CO2 來源以 CO2 鋼
瓶提供。每週固定置換二分之一的培養 液。培養四週後即進行試驗。 2、磷酸處理: 龍鬚菜培養於 7.6 x 7.6 x 10.2 cm = W x D x L 的培養盒中，內含 300 mL 含培 養液的人工海水 (35‰ ; 405 mM NaCl，10 mM KCl，30 mM MgSO4，10 mM CaCl2)， 培養密度為 1.2~1.5 g FW/300 mL，培養液 內含全量 ES solution 之 micro-nutrient 及 3 mM NaHCO3，海水磷酸濃度為 0 及 20 µM 的 KH2PO4，氮濃度為 400 µM 的 NaNO3， pH 值以 NaOH 及 HCl 調整為 8.0，光照強 度 70~100 µE m-2s-1 ( 光 源 為 日 光 燈 (FL30D，旭光公司，桃園，台灣) 6 管加 2 個白熱燈泡 (E2760W，東亞公司，新竹， 台灣))，日照時間 12 小時，每天固定更換 全部培養液。溫度以加熱控溫器 (BC-2D， Wisdom Appartus MFG Company，Taiwan， ROC) 及冷卻器（HC-04，Wisdom Appartus MFG Company， Taiwan ， ROC ） 維 持 為 25°C。
3、比生長速率 (specific growth rate) : 經處理之龍鬚菜，以紙把水份吸乾
於 –20°C 冷 凍 乾 燥 機 (SVC-100H ， SAVANT，Farmingdale，N.Y.，USA) 乾 燥 18 小時後取出稱其乾重，以下列式求得 比生長速率 (specific growth rate) (Patwary and van der Meer 1984)。
生長速率=ln(Wt/Wo) t-1 t=生長的天數 Wt=第 t 天的濕重或乾重 Wo=第 0 天的濕重或乾重 4、ACP 及 extracellular AP 活性測定: 取冷凍乾燥後之材料 0.02 g DW 以 液態氮研磨後加入含 0.1 mM PMSF、1 mM MgCl2及 1 mM EDTA 之 Tris-HCl (pH=7.0) 緩衝液 1 mL，於 4o C 下 16000g 離心 20min 後所得之上清液即為 ACP 萃取液。ACP 活 性於 40o C 下以 acetate buffer (pH=4.0) 含 40 mM pNPP 反應 30 min 後以 KOH 終 止，測 A405。 extracellular AP 活性測定則取活體 組織 0.1-0.5 g w. wt.加於含 40 mM pNPP 之 人工海水反應 30 min 後以 KOH 終止，測 A405。 5、磷的測定: 根據 Hernandez et al. (1993) 的方法 將藻體內的磷分為 total P (TP)、soluble reactive P (SRP)、total soluble P (TSP)、
particulate P (PP=TP-TSP) 、 soluble non-reactive P (SNRP=TSP-SRP)。 冷凍乾燥的龍鬚菜後，以液態氮研 磨成粉狀，再加入水振盪 2 h 以抽取細胞內 之無機磷 (SRP)，以 0.45 m filter 過濾後即 為 SRP 待測液。取部份的 SRP 待測液加 acid digestion solution (conc. HNO3 : conc.
H2SO4 : conc. HClO4 = 5:1:2) 於室溫下分 解 1 h 後逐漸加溫至 95o C 分解 2 h，降溫後 加入 ddH2O 至 3 mL，為 TSP 待測液。TP 則將藻體直接至於同上的 acid digestion solution 進行酸解及隨後的加水，即為 TP 待測液。SRP、TSP、TP 以 Malachite green 方法測定 (Lanzetta et al., 1979)。分解率以 glycerophosphate 加以估算。 6、總氮測定:
總氮以經 conc. H2SO4 (含 HgO and
K2S2O3) 分解的分解液以 Kjedahl 方法以
0.5 N HCl 滴定 (謝, 1978) 及本研究改良 之 phenol-hypochlorite 方法 (Smith, 1980) 加以測定。
7、藥品與統計分析：
藥品買自 Sigma (USA)，Serva or Merck (Germany). 統計分析以 SAS (Version 6.03, SAS Ltd., NC, USA) 進行之。. 結果與討論 1、石蓴 ACP 及 extracellular AP 利用海水磷酸濃度為 1 及 100 µM 的 KH2PO4形成缺磷與不缺磷之生長，缺磷會 抑制石蓴的生長速率 (Figure 1)。 Extracellular AP 活性則在缺磷之第一 天及快速增高後維持不變，而 ACP 則在缺 磷之第三天才有增高之現象 (Figure 2)。所 以，石蓴缺磷下的 extracellular AP 較 ACP 活性上升為快。可能是因為本研究所栽培 之石蓴的儲藏性磷含量尚未達最高量或不 足為分解用(Figures 3 and 4)，所以對於儲 藏性磷含量不高之石蓴於缺磷環境的策略 是先以 extracellular AP 活性增高來分解獲 取細胞外的儲藏性磷以獲取生長所須的無 機磷營養鹽。 ACP 活性染後的 isozyme 圖譜指出缺 磷會誘導兩個 ACP bands (Figure 5)。
2、傘房龍鬚菜 ACP 及 extracellular AP 傘房龍鬚菜處理之海水磷酸濃度為 0 及 20 µM 的 KH2PO4形成缺磷與不缺磷之 生長，與石蓴處理缺磷之狀態不同。缺磷 會明顯抑制傘房龍鬚菜的生長速率 (data not shown)。 Extracellular AP 活性則在缺磷之第八
天增高後，於第十二天達最高值，接著稍 下降 (Figure 6)。ACP 則在缺磷之第二天即 增高後維持在對照組的十倍左右 (Figure 6)。所以，傘房龍鬚菜在缺磷下的 ACP 較 extracellular AP 活性上升為快。傘房龍鬚菜 與石蓴之情形恰好不同，可能是細胞內磷 之型態不同所致。 缺磷下，傘房龍鬚菜的總磷含量有下 降 (Figure 7) 且 SNRP (soluble non-reactive phosphorus) 含量下降最明顯 (Figure 8)。 傘房龍鬚菜儲藏性磷含量已達 最高量，所以對於儲藏性磷含量高之傘房 龍鬚菜於缺磷環境的策略是先以 ACP 活性 增高來分解獲取細胞內的儲藏性磷以獲取 生長所須的無機磷營養鹽。 傘 房 龍 鬚 菜 的 ACP 活 性 染 後 的 isozyme 圖 譜 指 出 缺 磷 會 誘導兩個 ACP bands (Figure 9)。 目 前 ， 正 待 純 化 藻 體 的 ACP 及 extracellular AP。 REFERENCES
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Days 0 1 2 3 G row th rat e ( % ) 0 70 140 210 280 350 100 µM 1 µM
Figure 1 Growth rate in Ulva lactuca L. in response to phosphorus deficiency. Data are presented as means± 95% confidence interval (n=3). A P (n m o l/m in /g .w . wt .) 0 200 400 600 800 1000 1 µM Days 0 1 2 3 A CP (n m o l/ h /m g p ro te in ) 0 100 200 300 400 100 µM
Figure 2 Changes in extracellular AP and intracellular ACP activities in Ulva lactuca L. in response to P deficiency. Data are
presented as means± 95% confidence interval (n=3). N (% DW) 0 4 8 12 1 µM Days 0 1 2 3 P ( % DW) 0.0 0.1 0.2 0.3 100 µM A B
Figure 3. Changes in N and P contents in Ulva lactuca L. in response to P deficiency. Data are presented as means± 95%
confidence interval (n=3). SR P ( µ mol /g) 0 10 20 30 40 1 µM SN R P ( µ mol /g) 0 10 20 Days 0 1 2 3 T SP ( µ mol /g) 0 20 40 100 µM A B C
Figure 4. Changes in SRP, SNRP and TSP contents in Ulva lactuca L. in response to P deficiency. Data are presented as means± 95% confidence interval (n=3).
1 2 3
Figure 5. ACP zymogram in Ulva lactuca L. in response to P deficiency. 1, initial; 2, P sufficient materials; 3, P deficient materials. Soluble protein of 50 µg was loaded in each lane. AP ( nmo l/ h /g. w . w t. ) 0 5 10 15 20 25 0 µM Days 0 4 8 12 16 ACP (n m ol /h/ mg pro tei n) 0 20 40 60 80 20 µM
Figure 6. Changes in extracellularAP and intracellular ACP activities in Gracilaria coronpifolia in response to P deficiency. Data are presented as means± 95% confidence interval (n=3). N (% D W ) 0 1 2 3 0 µM Days 0 4 8 12 16 P (% D W) 0.00 0.02 0.04 0.06 20 µM A B
Figure 7. Changes in N and P contents in Gracilaria coronpifolia in response to P deficiency. Data are presented as means± ←
95% confidence interval (n=3). SR P ( µ mo l/ g ) 0 30 60 90 120 0 µM SNR P ( µ mo l/ g ) 0 20 40 60 80 Days 0 4 8 12 16 TSP ( µ mo l/ g ) 0 30 60 90 120 20 µM A B C
Figure 8. Changes in SRP, SNRP and TSP contents in Gracilaria coronpifolia in response to P deficiency. Data are
presented as means± 95% confidence interval (n=3).
Figure 9. ACP zymogram in Gracilaria coronpifolia in response to P deficiency. 1, initial; 2, +P for 2 days; 3, -P for 2 days; 4, +P for 4 days; 5, -P for 4 days; 6, +P for 8 days; 7, -P for 8 days; 8, +P for 12 days; 9, -P for 12 days; 10, +P for 16 days; 11, -P for 16 days. Soluble protein of 60 µg was loaded in each lane.
