台灣金線連根部結晶異形細胞之發育及不同鈣離子濃度的供應對其鈣結晶的影響

Abstract

本論文以台灣金線連台東種為實驗材料，觀察T1品系植株根部草酸鈣針束狀結晶異形細胞的分佈以及發育過程中的微細構造變化，並探討其於器官間及物種間的異同；另應用13C/12C穩定性同位素分析，鑑定台灣金線連在套袋與不套袋環境下的光合作用型；並配合不同鈣離子濃度的培養試驗，藉由全鈣、可溶性草酸、不可溶性草酸含量的測定及結晶密度分析，探討在T2品系中，草酸鈣結晶在鈣離子調控上所扮演的角色，並比較品系間的異同。 針束狀結晶異形細胞散生於根部表皮內第3?4層的皮層組織中，其發育過程可區分為六個階段：（1）結晶細胞發育早期、（2）晶體腔出現期、（3）鈣晶體累積期、（4）內質網膨脹期、（5）黏液質堆積期與（6）結晶細胞成熟期。發育初期結晶細胞較周圍皮層細胞早開始形成中央大液胞，接著液胞中大量不定形的電子緻密物質逐漸匯聚形成結晶腔，其內開始累積晶體，待晶體充滿結晶腔後，晶體周圍開始有黏液質產生，之後黏液質漸漸充滿整個液胞，並於晶束外圍及個別針晶之間形成電子緻密的黏液鞘，最終，板狀晶鞘形成，晶體的橫切面由四邊形轉為六至八邊形。整個發育過程中，細胞質明顯較周圍細胞濃稠，內含豐富胞器，顯出高度的生理代謝活性。結晶細胞特有的結晶色素體於發育過程中呈現多種不同的分化形態，其內並常有澱粉粒累積，此與其在葉部及其它物種中所觀察到的不同。另在黏液質開始產生的時期，發現結晶色素體邊緣分化出多個圓盤狀構造，其外具分泌物質而使周圍形成一圈空白區域，並且區域外常有內質網分佈，因此推測結晶色素體可能與黏液質的產生有關。 分化中的結晶色素體含少量不溶性多醣及蛋白質，幾乎不含脂質；而晶束旁的不定形的電子緻密物質及結晶腔，則主要由蛋白質構成；成熟細胞的黏液基質中含少量不溶性多醣及蛋白質，黏液鞘的成分主要為蛋白質。 經13C/12C穩定性同位素分析，並配合前人研究的結果，認為袋外培養的台灣金線連，為典型的CAM植物，生長緩慢；而在套袋栽培時，其可以同時進行C3及CAM型的CO2固定，夜間自介質與空氣中獲取CO2，將之固定於有機酸中累積下來，白晝則分解累積於體內的有機酸，同時自空氣中獲取CO2而進行碳的固定循環，也因此提高了植株的生長速率。 另在不同鈣離子濃度栽培試驗方面，於0Ca（0Mm）、1/8Ca（0.19mM）、1Ca（1.50mM）、8Ca（11.97mM）和16Ca（23.94 mM）五種不同鈣濃度培養基種植的台灣金線連T2品系植株，對高鈣及低鈣的環境都表現出相當強的耐受性，栽培4個月後，其外部形態及生長速度並無明顯差異，直至6個月後，0Ca培養下的植株始出現頂芽褐化凋萎的缺鈣病徵。植株根部及葉部的鈣含量，與培養基內的鈣濃度呈現正相關，但於前2個月的培養期間，葉部可溶性草酸與不可溶性草酸的含量皆不隨著環境中的鈣濃度變動，葉內結晶細胞的密度也維持穩定。至4個月後新葉及不定根中的結晶密度才隨鈣濃度出現顯著的差異，並於16Ca老葉中產生大量另一形態的細小結晶，但所有處理下老葉中的針束狀結晶密度仍維持不變。以上結果顯示此物種在因應環境中鈣離子濃度改變時，初期並不反映在結晶的含量上，然而後期新生組織中針束狀結晶的含量亦會開始隨提供的鈣濃度變動，故於此物種中結晶仍有調控鈣離子濃度的功能。另根據前人的研究，得知T1品系植株於栽培2個月後，新葉的結晶密度即開始產生變動，故可知不同栽培品系間的生理特性會有所差異。

In this study the distribution and the ultrastructural changes of raphide crystal idioblasts in roots of Anoectochilus formosanus Hay. T1 cultivars were investigated. The raphide idioblasts sporadically occurred in the third or fourth layer of cortex inside the epidermis. The development of the crystal idioblasts can be divided into six stages: (1) crystal idioblast initiation, (2) crystal chamber formation, (3) crystal growth, (4) ER expanded, (5) mucilage accumulation, and (6) crystal idioblast maturation. The crystal initials formed central vacuole earlier than the neighboring ordinary cortex cells. In the central vacuole the electron-dense materials gradually formulated the crystal chambers and then the crystals accumulated in these chambers. When the chambers were saturated with crystals, mucilage appeared around the crystals. The mucilage gradually permeated into the central vacuole and formed an electron-dense mucilage sheath outside the crystal needles. Eventually, lamellaed crystal sheaths came into existent, and the transverse view of the crystals turned from tetragon to hexagon or octagon. During the entire process, abundant organells were found in the cytoplasm of the crystal cells indicating an higher cellular physiological activities. The crystalloplastids showed diverse morphology. In the stage of mucilage accumulation, multiple lobes were found along the margin of the crystalloplastid. Certain secretory materials surrounded the crstalloplastid and formed an unfilled region. There were some ER spreaded outside this region, thus the crystalloplastid may be related to the formation of the mucilage. In the early and late stages, the crystalloplastids contain a few starch grains which were not found in the crystal idioblasts of leaves and that in the other species. The results of histochemical tests showed that the differentiating crystalloplastids contained little polysaccharide and protein, excluded lipid. The amorphous electron-dense materials and crystal chambers mainly contained protein. In the mature crystal cells, the mucilage matrix contained little polysaccharide and protein. The components of mucilage sheaths were mainly proteins. Using the stable isotope ratios (δ13C) analysis for consulting the results of previous studies, it is confirmed that when the plants of Anoectochilus formosanus Hay. cultivated without plastic bags covered, they are typically CAM plants and were obviously grown slowly. However, those cultivated with plastic bags covered were carrying out both the C3 and CAM CO2 fixation pathways. The plants might obtain from CO2 from the air and the culture substratum, and in the daytime they decomposed the organic acids stored in the vacuoles and meanwhile captured CO2 from the air, thus increased the growth rate. With supplying five different calcium ion concentration medium: 0 Ca (0 Mm), 1/8 Ca (0.19 mM), 1 Ca (1.50 mM), 8 Ca (11.97 mM) and 16 Ca (23.94 mM), the T2 cultivars showed strong endurance in both high Ca and low Ca conditions. After four months of cultured period, little difference in morphology and growth rate have been observed. Until the sixth month, the apical buds of those plants grown under 0 Ca condition turned to brown and died. Total Ca contents of the roots and leaves were correlated to the calcium ion concentrations in the medium. However, during the first two months, the soluble and insoluble oxalate contents and the crystal density of the leaves of all the cultured conditions remained stable, which meant they didn't vary with the calcium ion concentrations in the medium. After four months, the raphide crystal density of the newly formed leaves and of the roots began to vary with the calcium ion concentration. And the 16 Ca cultured old leaves produced a large number of tiny crystals with various morphology. The results showed that when the calcium ion concentration in the environment varied, at the beginning, the crystal density remained stable. However, in the newly formed organs, it changed. Therefore, for Anoectochilus formosanus Hay. T2 cultivars, the formation of calcium oxalate crystals in the cells may still have the function of regulating cellular calcium ion concentration. Comparing to the previous study on the T1 cultivars, it is interested to note that somewhat different regulating strategy in calcium crystal formation in these two cultivars.