The Production of Cytokinin, Abscisic Acid and Auxin by CAM Orchid Aerial Roots

J. Plant Physiol.

Vol. 147. pp. 371-377 (1995)

The Production of Cytokinin, Abscisic Acid and Auxin by CAM Orchid Aerial Roots N. G. 1 2

ZHANG!,

J. W. H. YONG2, C. S. HEW2 *, and X. ZHOU 1

Department of Agronomy, Nanjing Agriculture University, Nanjing 210095, China Department of Botany, National University of Singapore, Lower Kent Ridge Road, Singapore 0511, Republic of Singapore

*

Author for correspondence

Received June 6,1995' Accepted July 28,1995

Summary

Aerial roots of two monopodial thick-leaved epiphytic orchids (A randa and Vanda) are a possible source of endogenous plant hormones (abscisic acid, cytokinins, indole-3-acetic acid). Using a monoclonal antibody-based immunoassay, this study also revealed that the levels of plant hormones in root tips of Aranda could vary with root position along the stem, time and growth stages. The levels of plant hormones are usually higher in root tips than elsewhere in the roots. Higher levels of cytokinin (mainly isopentenyladenosine) were detected in root tips of flowering plants than in non-flowering plants of Aranda. Lateral aerial roots(s) could be induced by the removal of root tip. A possible role for these rootderived plant hormones is discussed in relation to orchid growth and development.

Key words: Aranda
Introduction

Aerial roots of epiphytic orchids are rather unique. The entire length of the root, except the tip, is covered by dead and spongy-like velamentous tissues. Lying beneath the velamen is the cortex region containing chloroplasts (Ho et ai., 1983; Hew et al., 1984). In some cases, the orchid roots may form as much as half of the total plant biomass (Benzing and Ott, 1981). The roles of orchid roots in absorption of mineral, water conservation, carbon fixation and anchorage are well established (Hew et al., 1984; Pridgeon, 1987; Benzing, 1991). The respiration of roots in relation to photosynthesis and carbon gain has also been discussed (Avadhani et al., 1982; Hew, 1989; Hew et al., 1991). © 1995 by Gustav Fischer Verlag, Stuttgart

By comparison, less is known about other possible role(s) of aerial roots with reference to the growth and development of epiphytic orchids. It has often been observed by orchid growers that there appears to be a correlation between flower spike production and number of roots for thick-leaved monopodial orchids (Hew and Clifford, 1993; Hew, 1994). These observations suggest that cytokinins synthesized by the aerial roots may influence flower spike production in thick-leaved monopodial orchids. It is noteworthy that thick and thin-leaved orchids have CAM and C) mode of photosynthesis, respectively (Neales and Hew, 1975; Avadhani et al., 1982; Hew, 1989). Roots of most higher plants are a source of endogenous plant hormones that are known to be involved in a number

372

N. G. ZHANG,]. W. H. YONG, C. S. HEW, and X. ZHOU

of physiological processes (Skene, 1975; Torrey, 1976; Davis and Zhang, 1991; Itai and Birnbaum, 1991; Jackson, 1993). Recently, we reported that both CAM and C 3 orchid aerial roots could produce fair amounts of ethylene, particularly for the root tips (Hew et al., 1995). This paper reports the occurrence of the other plant hormones, i.e. ABA, CTKs (mainly iPA) and IAA, in aerial roots of two thick-leaved monopodial orchids and discusses the possible roles of these plant hormones in relation to orchid growth and development.

shade (600 to 7ool1molm-2s-1 at noon), respectively in the field where the environmental conditions were: Temperature with a maximum of 36°C and minimum of 25 °C, relative humidity with a maximum of 99 % and minimum of 63 %, and a photoperiod of 12h. An optimal regime of fertilizer (Foliar Fertilizer 67, Blue Sky Agricultural Supplies, Singapore, 1.6 g in 1 L of water; nitrogen: phosphorous: potassium ratio of 13.5: 27 : 27) treatment was provided two times a week (Yong and Hew, 1995). Plants were watered daily in the evening.

Growth observations Materials and Methods

Plant materials Intact roots of two thick-leaved monopodial orchid hybrids

(Aranda
node of the third leaf (counting from the apex). Both flowering (with a single inflorescence) and non-flowering plants were used for experiments. Experimental plants of Vanda and Aranda were grown under full sunlight (1500 I1mol m- 2 S-I at noon) and under

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The orchid root tip is dark green in colour and is clearly demarcated from the other root region because of the absence of a white velamen layer (Fig. 7). Throughout the 3-month experimental period, the root tip of a new Vanda or Aranda root was marked with Indian ink at 0900 h. Growth of the region adjacent to the rOOt tip (i.e. the part of the root with velamen) was recorded daily. Six roots were used for each measurement.

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The processing of plant extract prior to immunoassay was as described by Weiler et al. (1986) with some modifications (Zhang et al., 1991; Zhou et al., in press). Freshly harvested plant material was immersed in liquid nitrogen, ground and extracted overnight with 5mLg- 1 original fresh weight of 80% methanol containing 10 mgL -I BHT at 4 °C-l0 0c. The crude extract was purified (removal of pigments and lipids) using a C\8 Sep-Pak cartridge. The filtrate was collected and an aliquot of 600 ILL of this extract was dried with a stream of nitrogen. The residue was redissolved in 600 ILL TBS for iPA ELISA. Another 600 ILL aliquot of the filtrate was taken and dried using a stream of nitrogen. The residue was redissolved in ethyl acetate and the pH was adjusted to 2.5 with 0.1 N HC!. The solution was extracted 3 times with equal volumes of ethyl acetate. The organic phase was combined and evaporated to dryness with nitrogen. After treatment with an excess of ethereal diazomethane for 1- 5 min on ice (excess diazomethane was subsequently destroyed with a drop of 0.2 N acetic acid in methanol), the sample was dried under nitrogen and redissolved in 50 ILL of methanol and diluted with 550 ILL TBS for IAA and ABA ELiSAs. The iPA Mab and IAA Mab were kindly provided by Dr. Elmar W. Weiler of Germany. The characterization of the two Mabs was described, respectively, by Eberle et al. (1986) and Mertens et al. (1985). The immunogen for the ABA Mab was ABA-C1-BSA. The linear measuring ranges were between 0.15 and 5 pmo!. For the immunoassay, polystyrene microtiter plates were precoated overnight at 4°C with rabbit anti-mouse immunoglobulin [lool1gmL-t in 50mM NaHC03 (pH 9.6) buffer]. The solution was decanted and the wells were coated with suitable amounts of Mab in TBS overnight at 4°C. Samples were added 1 h before the addition of enzyme labeled hormones. After 2 h incubation at 25°C, the substrate (p-nitrophenyl phosphate) for the enzyme was added. The enzyme reaction was carried out in dark at 25°C for 1 h. The reaction was terminated using 0.05 mL 5 M KOH and the absorbance was read at 405nm.

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Results

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The distribution patterns of IAA, iPA and ABA along the axis of the first root (harvested at 12 noon) of Aranda (Fig. 3 C, 3 D, 3 E) and Yanda (Fig. 4 C, 4 D, 4 E) were fairly similar. The levels of IAA peak at the region adjacent to the root tip for both orchids and the levels decreased with distance from the root tip (Figs. 3 C, 4 C). Our preliminary study has shown that iPA appeared to be the major type of CTK present in Aranda roots. The other CTK detected within the aerial roots at very low levels was ZR (data not shown). For the two orchids, the highest level of iPA was observed in root tips, which decreased with distance from the root tip (Figs. 3 D, 4 D). At the 8- 10th cm root sections, the iPA level was between 10 % and 30 % of the levels at the root tips for Aranda and Yanda, respectively. The level of ABA was also highest at the root tips for both orchids. For Yanda roots, it decreased with increasing distance from the tip (Fig. 4E). Conversely, similar levels of ABA within the Aranda

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Aerial roots usually appeared at the node of the 11th or 12th leaf (counting from the apex) for Aranda (Fig. 1). In this experiment, we screened for IAA, iPA and ABA in the root tips of flowering and nonflowering plants of Aranda along different positions on the stem at 1700 h (Fig. 5 A, 5 B, 5 C). The levels of IAA (Fig. SA) and iPA (Fig. 5B) in the root tips were consistently higher for the flowering plants than the nonflowering plants for all six positions along the stem except for ABA (Fig. 5 C). For iPA, the levels in flowering

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ZHANG,].

W. H. YONG, C. S. HEW, and X.

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and nonflowering plants increased with increasing distance down the stem (Fig. 5 B). Conversely, the levels of IAA in the root tips for either flowering or nonflowering plants were found to be similar (Fig. 5 A). No distinct pattern could be obtained for ABA in root tips except that the levels in flowering plants were either similar or higher than those in nonflowering plants for all six positions along the stem (Fig. 5 C). Diurnal fluctuation ofplant hormones within the aerial roots For this experiment, we examined the levels of IAA, iPA and ABA within the tip of the first root (counting from the apex) of Aranda over a period of 24 h. Within these root tips, the peak for IAA was at 1600 h and this was followed by a

gradual decline throughout the night and until the next day at noon (Fig. 6 A). For ABA, a pattern similar to that for IAA was observed (Fig. 6 C). In contrast, the peak for iPA was at 1200 h, declined to a minimum at 0800 h and returned to similar levels at the next 1200 h (Fig. 6 B). Lateral root formation Lateral root growth was observed after decapitation of the Aranda root tip. New lateral roots emerged from the cut end (Fig. 7 A) and from various positions behind the cut end (Fig. 7B). The number of lateral roots produced (1 to 5) and timing of lateral root appearance (10 days - 30 days) were variable (Data not shown). Discussion

Generally, the growth patterns of aerial roots for the two monopodial thick-leaved orchids (Vanda
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pattern of plant hormones along a root axis in orchid roots is consistent with the fact that they are synthesized in the root apex and transported to the shoot (Torrey, 1976; Itai and Birnbaum, 1991). The presence of IAA, iPA and ABA in roots located at different positions along the stem strongly suggests that all aerial roots are active in the biosynthesis of these three plant hormones. This may account for the substantial amounts of lAA, iPA and ABA available for the control of orchid growth by root-derived plant hormones. The observation that flowering plants of A randa have consistently higher levels of iPA in the root tips than in non-flowering plants substantiates the hypothesis that more CTKs are synthesized by the aerial roots during flower spike production. The difference in levels of plant hormones in roots at different stem positions could possibly account for the flowering gradient reported earlier in thick-leaved monopodial orchids (Goh, 1975). It is therefore possible to increase flower spike production in thick-leaved monopodial orchids by improving the nitrogen status of the orchids since recent studies on a perennial herb, Urtica diocia, indicated that the total root-derived cytokinin gain by the shoot is dependent on the nitrogen status of the plant (Wagner and Beck 1993; Beck and Wagner, 1994). The demonstration of ABA in roots was first reported in the mid 1970s (see Itai and Birnbaum, 1991). As in the case of eTK, the levels of ABA within the roots are clearly affected by root environment. Roots respond more quickly and with greater sensitivity by root environment. Roots respond more quickly and with greater sensitivity to water stress

Fig. 7: The development of lateral roots from the cut end (A) and from various positions behind the cut end (B) after the removal of root tips in aerial roots of Aranda .

than leaves by increasing their ABA levels (Cornish and Zeevart, 1985; Zhang and Davis, 1989). Recently, the importance of roots acting as a sensor for water stress has received considerable attention (Davis and Zhang, 1991). In this regard, the demonstration of orchid aerial root tips as the likely site for ABA synthesis is important and this observation may have ecophysiological significance since both Vanda and Aranda are epiphytic in nature. Studies on CAM plants have demonstrated the possible roles of root-derived ABA and CTK in regulating CAM (Thomas et al., 1992; Schmitt and Piepenbrock, 1994; Dai et al., 1994). In particular, the expression of CAM in Mesem· bryanthemurn crystal/inurn could be induced by feeding ABA, farnesol(an antitranspirant and analog of ABA) and benzylaminopurine to the roots (Dai et al., 1994). As for the epiphytic orchids (especially the thick-leaved orchids with CAM), the physiological role(s) of root-derived plant hormones remains unknown. In this study, the demonstration of diurnal changes in levels of IAA, iPA and ABA (expressed in terms of per g dry weight) within the CAM orchid root tips is interesting. At present, we cannot suggest a plausible explanation for this physiological observation. Nonetheless, this study reveals a possible relationship in the hypothesis that the diurnal rhythm of CAM may be controlled or influenced by root-derived plant hormones; however, more work is needed to substantiate this hypothesis.

376

N. G. ZHANG,J. W. H. YONG, C. S. HEW, and X. ZHOU

The production of lateral roots after removal of root tips clearly demonstrated the presence of apical dominance in orchid aerial roots. It is possible that the growth of lateral root(s) is controlled by certain substances synthesi~ed in the root apex (Wightman and Thimann, 1980; Torrey, 1986). Wightman and Thiman (1980) have shown that the root tip is a possible source of inhibitor, which acts against the formation of lateral roots. Under normal culture conditions, lateral roots of orchids are formed when the tips are at a certain distance away or when the tips are damaged. In addition, we have also observed the development of lateral root primordia (from the region next to the tip) when root tips of Vanda
We thank Mr. Ong Tang Kwee and Miss Gouk Sok Siam for their technical assistance. The financial support from Lee Foundation (Singapore) and the National University of Singapore to N. G. Zhang and J. W. H. Yong is much appreciated.

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