- Email: [email protected]
Changes in free and conjugated indole-3-acetic acid during early stage of flower bud differentiation in Polianthes tuberosa
Plan? Physiol. Biochem., 1999, 37 (2), 161-165
Short paper Changes in free and conjugated indole-3-acetic acid during early stage of flower bud differentiation in Polianthes tuberosa Shih-Fen
Ding’, Wen-Shaw
’ Department
of Biological
Chen’*,
Sciences,
Chi-Ling
Sul, Bo-Shiun
Du’, Bruce Twitchin3,
National Sun Yat-Sen University,
Kaohsiung
* National Chia-Yi Institute of Technology,
Chia-Yi City, Taiwan, ROC.
’ Research
National University,
School of Chemistry,
Australian
* Author to whom correspondence
should be addressed
(Received June 24, 1998; accepted
December
Vijaya
K. Bhaskar$
City, Taiwan, ROC.
Canberra ACT 2601, Australia.
(fax +886 7 725 1903; e-mail [email protected])
12, 1998)
Abstract - Tuberose (Polianthes tuberosa L. cv. Double) corms at the vegetative, early floral initiation, and flower bud differentiation stages were assayed for free indole-3-acetic acid (IAA), esterified IAA, and peptidyl IAA. The corms in the vegetative stage contained higher free IAA than those from the early floral initiation stage. Free IAA in corm tissues increased 2.7-fold at flower bud differentiation as compared to the vegetative stage. In the vegetative corms, a marked promotion of leaf differentiation was recorded. In contrast, corms from the early floral initiation stage contained less free IAA, whereas esterified IAA and peptidyl IAA increased dramatically. It is concluded that the level of free IAA in vegetative corms is correlated with leaf differ-
entiation, and that the early floral initiation stage is correlated with a reduction in free IAA and an increase in IAA conjugates in the corms. Moreover, increases in free IAA and decreases in IAA conjugates in the floral differentiation stage, as compared to the early floral initiation stage, indicates that free IAA is correlated with flower development. 0 Elsevier, Paris Flowering I free IAA I conjugated
IAA I Polianthes tuberosa
HPLC, high performance liquid chromatography spectrometry I IAA, indole3-acetic acid
1.
/ GC-SIM-MS,
INTRODUCTION
Among the many growth and developmental phenomena that can be influenced or induced by hormones is flower initiation. Flower bud development is attained through transition of the vegetative apex to a reproductive structure in tuberose corms. Consequently, flower induction occurs only when a critical number of leaves already have developed. As a tropical ornamental plant which is believed to have originated in Mexico [14], the tuberose corms grown at high temperature (30 “C) for two weeks or more, have a shortened period between planting and sprouting, and an increased rate of flower bud formation [ 121. Harada and Nitsch [8] reported that indole-3-acetic acid (IAA) decreases in the buds of Chrysanthemum molifolium before the appearance of the flower merPlant Physiol. Biochem.,
098 l-9428/99/2/0
Elsevier, Paris
gas chromatography-selected
ion monitoring-mass
istem. Tsukamoto et al. [14] indicated that floral initiation was inhibited when exogenous auxin was applied just before flowering in the same species. Endogenous auxin appears to have an indirect, but favorable effect on floral initiation at the beginning of the growing period. This is important because the early, fast development of the leaf primordia, and of young leaves during the early stage of growth, is a prerequisite of floral initiation. Auxin conjugates are known to play a major role in the regulation of the content of free IAA in plants. For example, Hangarter and Good [7] showed that conjugated IAA is a storage form of IAA that is slowly and continuously decarboxylated upon application to pea segments. The observed biological effects involve the free IAA that is regulated by the hydrolysis of the conjugates. However, data concerning changes in IAA conjugates and
162
S.-F. Ding et al.
is not known whether all of the IAA made is conjugated and stored when floral initiation occurs. This view is supported by the fact that at the early floral initiation stage, a significant increase in the ratio of ester and amide conjugates to free IAA was found. Free IAA constitutes approximately 4.6 % of the total auxins, the remainder being ester and amide conjugates (95.4 % of total auxins,figure I) at the early floral initiation stage, whereas 9.3 % of free IAA were detected at the vegetative stage (table I>. Corm tissues from vegetative and floral differentiation plants contained lower detectable levels of ester- and amide-conjugates than tissues from floral initiation plants.
free IAA during floral initiation and development in plants is limited, especially in the case of the tropical plant, tuberose. IAA is known to be conjugated either to amino acids or peptides via an amide linkage, or to sugar moieties via an ester linkage [2]. Several destinations of conjugated IAA have been reported: storage, transport, protection from peroxidation and catabolism [9]. Hence, the regulation of IAA conjugate formation is an important process during floral initiation and differentiation. The purpose of this study was to analyze the destinations of free IAA and IAA conjugates in tuberose corms during vegetative, early floral initiation and the flower differentiation stages of tuberose. Our results indicate that the corms of tuberose may be autonomous with respect to the IAA supply during floral initiation. De novo synthesis of amide-linked IAA conjugates in the corms of flowering seedlings suggests a role for these compounds in the regulation of the concentration of endogenous free IAA in tuberose corms.
In general, all flowering responses in plants are considered to be regulated by interactions among phytohormones, mainly auxins, cytokinins, and gibberellins present endogenously in plant tissues as well as those added exogenously. Auxins are thought to influence various aspects of plant development such as cell division, growth, morphogenesis, and response to flowering [2, 51. In the case of IAA, in addition to biosynthesis, catabolism is another way to control the levels of free IAA, and conjugation constitutes an important aspect of IAA catabolism. For example, Tuominen et al. [15] reported that conjugation may also play a role in the catabolism of endogenous IAA in Pop&s hybrids, suggesting that this may be a normal route for the breakdown of endogenous IAA. Also, Campanella et al. [4] showed that IAA conjugates inhibit the growth of Arabidopsis seedlings, probably because the conjugates are hydrolyzed and the IAA released is phytotoxic for plant growth. Although previous works have demonstrated the presence of both ester and amide conjugated IAA in Arubidopsis seedlings [13], to our knowledge, the
2. RESULTS AND DISCUSSION Tuberose corms from early floral initiation contained reduced levels of free IAA compared with corms from the vegetative and floral differentiation stages (table I>. The level of free IAA was higher in the tuberose corms from plants at the flower differentiation stage compared with those from vegetative plants, the increase was about 2.7-fold greater than that in vegetative corms. Free IAA levels were at a high level in corm tissues at the vegetative stage and then decreased at the early floral initiation stage (ruble Z). This value may not equal the corm’s IAA biosynthetic rate because it
Table I. Concentrations of free and bound IAA at different growth stages of tuberose. Although quantities differ significantly between the two replicates (either due to plant-to-plant differences or assay differences), the overall trend with time is similar between the two replicate samples. Free IAA
Growth stage
Ester IAA
Amide IAA
(ng. g-’ fresh mass) Vegetative stage
Early floral initiation
Flower differentiation
stage
stage
Plant Physiol. Biochem.
Sample 1 Sample 2 Average
279 207 243
357 317 337
2 053 1993 2 023
Sample 1 Sample 2 Average
219 167 193
1 127 1 087 1 107
2 954 2 902 2 928
Sample 1 Sample 2 Average
696 628 662
628 558 593
1 269 1 195 1 232
L4A in relation to flowering of tuberose
IAA
163
trast, IAA conjugate accumulation begins when floral initiation has occurred (table I). However, the reason for this accumulation of IAA-conjugates remains unknown. We hypothesized that a decrease in free IAA concentration during the flowering process has a physiological role during the developmental process of new leaves. This view is supported by the fact that free IAA in floral corms would reduce leaf differentiation in tuberose (K.L. Huang, pers. comm.). We also hypothesized that in tuberose, the indole ring is produced in the corm apex and transported to the leaf primordia via polar movement. Bialek et al. [3] reported that excess IAA, which was not utilized by bean seedlings when the growth rate was drastically reduced after the removal of the cotyledons, was conjugated. Simultaneously, they also showed that IAA amide-linked conjugates were hydrolyzed by bean seedlings when they were applied to the stem sections or studied in vitro [3]. Our results suggest a regulatory role for conjugated IAA during early floral initiation. We show that free IAA may be conjugated at this stage due to relatively higher proportion of conjugated IAA to free IAA than those of vegetative and flower differentiation stages (table I>. In summary, it appears that in vegetative tuberose corms, the level of free IAA, normally present in high amounts, is correlated with leaf differentiation, and that a decrease in free IAA levels is required for the initiation of flower buds (table r). The results further show that conjugated peptidyl and esterified IAAs increase significantly during the formation of flower buds in tuberose (table I). These findings indicate that the regulation of particular physiological transformation during flowering processes are really correlated with changes in the levels of endogenous IAA. Our study suggest that conjugated IAA plays an important role during the flowering process in the tuberose. Also, when the level of IAA is reduced during early floral initiation, normal leaf differentiation is affected.
’ ’ CH2CooH 03 :
H
H
<‘HzCOR
IAA amides
<‘H$02R
MA esters
R= amides or esters
Figure 1. Chemical structures of IAA, IAA amides and IAA esters.
identification and quantification of these endogenous IAA conjugates have not been determined during the flowering process, especially in the tropical plant, tuberose. In this study, the most striking result is the finding that both ester& or peptidic IAA occurs in tuberose corm tissues before, during and after floral initiation (ruble I>. It is interesting to note that tuberose corm tissues in the floral initiation stage contain primarily amide-V&, whereas corm tissues in the floral initiation stage contain large amounts of both esterand amide-MA (table I>. Thus, the chemical form of IAA in the vegetative- and reproductive-corms may be different. The IAA content in plants is regulated by the conjugation of excess IAA to amino acids and sugars [ 1, 61. We suggest that synthesis and hydrolysis of the bound IAA is a homeostatic mechanism for maintenance of IAA concentration during the flowering process. After floral initiation, tuberose corms accumulate less conjugated IAA than the vegetative and early floral initiation stages (table I). The assumption is that conjugation of IAA is a means of maintaining free IAA levels [I]. In other words, conjugation leads to a decline in the level of the active hormone, because free IAA is the only biologically active form [6]. Usually, in dicotyledonous plants, most amide conjugates were observed [6, 11, 121, whereas in monocotyledonous plants, the primary conjugates were ester-bound. We were able to detect both ester- and amide-bound IAA in tuberose corms, a monocotyledonous plant (table I). These results also provide evidence that changes in free IAA is correlated with floral initiation in tuberose. The flowering process is most likely attributable to the lowering of free IAA levels in corm tissues. By con-
3. CONCLUSION
In conclusion, the present study demonstrates a about 2-fold greater conjugated IAA concentration in the early floral initiation stage than in vegetative and flower differentiation stages. Free IAA concentration increased 1.3- and 3.4-fold in vegetative and flower differentiation stages, respectively, when compared with early floral initiation stage. In tuberose, we were able to detect both ester- and amide-bound IAA in corm tissues, a monocotyledonous plant. These observol. 37 (2) 1999
164
S.-F. Ding et al.
vations emphasize the need to consider differential distribution of endogenous IAA at different growth stages for an accurate understanding of IAA in the flowering physiology of tuberose. 4. METHODS 4.1. Materials. Tuberose corms (Poliunthes tubema L. cv. Double) 2.0-2.5 cm in diameter were cultured in plastic pots (30 cm x 20 cm, width x depth) filled with an artificial peatmoss/perlite/vermiculite medium (3/1/l, v/v/v). The pots were placed in field conditions during the summer season (30 “Cl25 “C, day/night in average) in Kaohsiung City, Taiwan. After the specified time (21-35 d), the corms were removed fom young seedlings and viewed microscopically. The corms were classified into vegetative, early floral initiation and flower differentiation stages (four to six flower primordia were emerged and small inflorescence observed). Subsequently, their fresh mass were determined. The corms were rinsed with distilled water, and weighed. 4.2. Determination of free and conjugated IAA. The analyses of free and conjugated IAA were as described by Chen et al. [S] with slight modifications. Briefly, fresh samples (1 g) were ground in a mortar with 4 mLg’ of sample in 65 % isopropanol with 0.2 M imidazol (Sigma) buffer pH 7.0. [‘H,]-IAA (enrichment of 84 % deuterium; Cambridge Isotope Lab, Andover, MA, USA) was added as an internal standard. After the known amount of deuterated IAA was equilibrated in the extract for 1 h at 4 “C, the solution was centrifuged at 10 000 x g for 5 min. The isopropanol was then evaporated under vacua, and the aqueous layer was divided into three equal parts: the first for free IAA analysis, the second was hydrolyzed at 25 “C for 1 h with 1 N NaOH to measure free plus ester IAA, and the third was hydrolyzed at 100 “C under N2 for 3 h with 7 N NaOH to measure total IAA (free + esters + amides). For free IAA analysis, a 4-mL extract was applied to a conditioned amino anion exchange column (BAKERBOND speTM). After the sample extract passed through the column, aspiration was applied for 20 s to remove excess water, and the column was washed successively with hexane, ethyl acetate, acetonitrile and methanol (2 mL each). The free IAA was eluted from the column using 3 mL methanol containing 2 % acetic acid. The acidic methanol eluent was evaporated to dryness under vacua, and was taken up in solvent for HPLC purification. The HPLC was equipped with a Cl8 column (30 x 0.4 cm id., pBondapak; Waters Associates) using acetonitrile and water containing 0.1 % acetic acid as the mobile phase. The gradient was programmed to hold for 1 min at water/acetonitrile (9515, v/v), followed by a 5-min linear gradient to water/acetonitrile (80/20, v/v) and held for 20 min. The 1.5-mL fraction at the retention time of IAA was dried in vacua, the sample was then methylated with diazomethane, and analyzed using GCSIM-MS. For the determination of free IAA plus ester form, 2 mL of the extract was base-hydrolyzed. After hydrolyzing
Plant Physiol. Biochem.
the ester conjugate as above, the sample was diluted, pH adjusted to 2.5 with 1 N HCl and passed through a conditioned (washed with hexane, methanol, water, and 1 % acetic acid successively, 5 mL each) Baker C-18 column to desalt. The C- 18 column was washed with 5 mL distilled water, and the IAA eluted with 2 mL acetonitrile. Imidazole buffer (20 mM, pH 7.0) was added to dilute the eluent to 10 % of the organic phase, and then the sample was applied to the conditioned amino column as above. The solution was then dried in vacua, resuspended in solvent, and purified using HPLC as above. The IAA fraction was evaporated to dryness under vacua and resuspended in 100 pL methanol for methylation. For determination of total IAA, the hydrolysate was filtered, the pH brought to 2.5, and the IAA compounds were purified over a Sep-pak C 18 cartridge equilibrated with H,O. IAA was eluted with 100 % methanol. The methanol was evaporated to dryness and resuspended in 100 pL methanol for HPLC purification. The IAA fraction was taken, and used for methylation. Each analysis was repeated three times. The total IAA determination, appropriate amounts of NaOH were added to the extract after evaporation of the isopropanol. The hydrolysis was performed in a capped PTFE vial purged with water-saturated N2 gas at 100 “C for 3 h. The hydrolysates were diluted, and the pH adjusted to 2.5 with 2 N HCl, then passed through a conditioned C- 18 column, and prepared following the procedures as described above for free and ester IAA. 4.3. GC-NM-MS analysis. GC-SIM-MS analysis of the purified methylated IAA was conducted on a VG Quattro mass selective detector that was interfaced with a HewlettPackard 5890 GC. The GC column was a 30-m DB-5, 0.25 pm film. The temperature program was 100 “C for 1 mitt, followed by an increase of 25 SC.min-’ to 200 “C,
Figure
2. Mass spectrum
IAA isolated from tuherose The molecular ion region of the
of methylated
corm tissues taken during W-MS.
spectrum showing ion at m/z 189 derived from endogenous compound (D,) in addition to corresponding ion at m/z 194 arising from [‘HJlabeled internal standard.
IAA in relation to flowering of tuberose
kept at this temperature for 6 min, increased rate of 25 “C.min-’ and kept at that
to 250 “C at a
165
[6] Cohen J.D., Bandurski R.S., The chemistry and physiology of the bound auxins, Annu. Rev. Plant Physiol. 33 (1982) 403430. [7] Hangarter R.P., Good N.E., Evidence that IAA conjugates are slow-release sources of free IAA in plant tissues, Plant Physiol. 68 (1981) 1424-1427. [8] Harada H., Nitsch J.P., Changes in endogenous growth substances during flower development, Plant Physiol. 34 (1959) 409-415.
Acknowledgements. The authors gratefully acknowledge the help of Jentaie Shiea and Hsiao-Ching Yu in the use of the GC-MS instrument. REFERENCES [l] Bandurski R.S., Schulze A., Concentration of indole-3acetic acid and its derivatives in plants, Plant Physiol. 60 (1977) 211-213. [2] Bandurski R.S., Cohen J.D., Slovin J.P., Reinecke D.M., Auxin biosynthesis and metabolism, in: Davis PJ. (Ed.), Plant Hormones: Physiology, Biochemistry and Molecular Biology, Kluwer Academic Publishers, Dordrecht, 1995, pp. 39-68. [3] Bialek K., Michalczuk L., Cohen J.D., Auxin biosynthesis during seed germination in Phaseolus vulgaris, Plant Physiol. 100 (1992) 509-5 17. [4] Campanella J.J., Ludwig-Muller J., Town CD., Isolation and characterization of mutants of Arabidopsis thaliana with increased resistance to growth inhibition by indoleacetic acid-amino acid conjugates, Plant Physiol. 112 (1996) 735-745. [5] Chen K.H., Miller A.N., Patterson G.W., Cohen J.D., A rapid and simple procedure for purification of indole3-acetic acid prior to GC-SIM-MS analysis, Plant Physiol. 86 (1988) 822-825.
[9] Ludwig-Muller J., Epstein E., Hilgenberg W., Auxinconjugate hydrolysis in Chinese cabbage: Characterization of an amidohydrolase and its role during infection with clubroot disease, Physiol. Plant. 97 (1996) 627634. [lo] Ludwig-Muller J., Sass S., Sutter E.G., Wodner M., acid in Arabidopsis Epstein E., Indole-3-butyric thaliana. I. Identification and quantification, Plant Growth Regul. 13 (1993) 179-187.
[ 1 l] Mori G., Miyake K., Imanishi H., Effect of exposure of corms to high temperature on flowering of Polianthes tuberosa L., J. Jpn. Sot. Hort. Sci. 59 (Suppl. 2) (1990) 538-539. [12] Normanly J., Cohen J.D., Fink G.R., Arabidopsis thaliana auxotrophs reveal a tryptophan-independent biosynthetic pathway for indole-3-acetic acid, Proc. Nat1 Acad. Sci. USA 90 (1993) 10355-10359. [13] Trueblood E.W.E., ‘Omixochitl’- the Tuberose (Polianthes tuberosa), Economic Bot. 27 (1973) 157-173. [14] Tsukamoto Y., Kawashima N., Tanaka T., Retardation of flowering by auxin-gibberellin spray in Chrysanthemum molifolium, Mem. Coll. Agr. Kyoto Univ. 93 (1968) 1-19. [15] Tuominen H., Ostin A., Sandberg G., Sundberg B., A novel metabolic pathway for indole-3-acetic acid in apical shoots of Populus tremula (L.) x Populus tremuZoides (Michx.), Plant Physiol. 106 (1994) 151 l-1520.
vol. 37 (2) 1999
Short paper Changes in free and conjugated indole-3-acetic acid during early stage of flower bud differentiation in Polianthes tuberosa Shih-Fen
Ding’, Wen-Shaw
’ Department
of Biological
Chen’*,
Sciences,
Chi-Ling
Sul, Bo-Shiun
Du’, Bruce Twitchin3,
National Sun Yat-Sen University,
Kaohsiung
* National Chia-Yi Institute of Technology,
Chia-Yi City, Taiwan, ROC.
’ Research
National University,
School of Chemistry,
Australian
* Author to whom correspondence
should be addressed
(Received June 24, 1998; accepted
December
Vijaya
K. Bhaskar$
City, Taiwan, ROC.
Canberra ACT 2601, Australia.
(fax +886 7 725 1903; e-mail [email protected])
12, 1998)
Abstract - Tuberose (Polianthes tuberosa L. cv. Double) corms at the vegetative, early floral initiation, and flower bud differentiation stages were assayed for free indole-3-acetic acid (IAA), esterified IAA, and peptidyl IAA. The corms in the vegetative stage contained higher free IAA than those from the early floral initiation stage. Free IAA in corm tissues increased 2.7-fold at flower bud differentiation as compared to the vegetative stage. In the vegetative corms, a marked promotion of leaf differentiation was recorded. In contrast, corms from the early floral initiation stage contained less free IAA, whereas esterified IAA and peptidyl IAA increased dramatically. It is concluded that the level of free IAA in vegetative corms is correlated with leaf differ-
entiation, and that the early floral initiation stage is correlated with a reduction in free IAA and an increase in IAA conjugates in the corms. Moreover, increases in free IAA and decreases in IAA conjugates in the floral differentiation stage, as compared to the early floral initiation stage, indicates that free IAA is correlated with flower development. 0 Elsevier, Paris Flowering I free IAA I conjugated
IAA I Polianthes tuberosa
HPLC, high performance liquid chromatography spectrometry I IAA, indole3-acetic acid
1.
/ GC-SIM-MS,
INTRODUCTION
Among the many growth and developmental phenomena that can be influenced or induced by hormones is flower initiation. Flower bud development is attained through transition of the vegetative apex to a reproductive structure in tuberose corms. Consequently, flower induction occurs only when a critical number of leaves already have developed. As a tropical ornamental plant which is believed to have originated in Mexico [14], the tuberose corms grown at high temperature (30 “C) for two weeks or more, have a shortened period between planting and sprouting, and an increased rate of flower bud formation [ 121. Harada and Nitsch [8] reported that indole-3-acetic acid (IAA) decreases in the buds of Chrysanthemum molifolium before the appearance of the flower merPlant Physiol. Biochem.,
098 l-9428/99/2/0
Elsevier, Paris
gas chromatography-selected
ion monitoring-mass
istem. Tsukamoto et al. [14] indicated that floral initiation was inhibited when exogenous auxin was applied just before flowering in the same species. Endogenous auxin appears to have an indirect, but favorable effect on floral initiation at the beginning of the growing period. This is important because the early, fast development of the leaf primordia, and of young leaves during the early stage of growth, is a prerequisite of floral initiation. Auxin conjugates are known to play a major role in the regulation of the content of free IAA in plants. For example, Hangarter and Good [7] showed that conjugated IAA is a storage form of IAA that is slowly and continuously decarboxylated upon application to pea segments. The observed biological effects involve the free IAA that is regulated by the hydrolysis of the conjugates. However, data concerning changes in IAA conjugates and
162
S.-F. Ding et al.
is not known whether all of the IAA made is conjugated and stored when floral initiation occurs. This view is supported by the fact that at the early floral initiation stage, a significant increase in the ratio of ester and amide conjugates to free IAA was found. Free IAA constitutes approximately 4.6 % of the total auxins, the remainder being ester and amide conjugates (95.4 % of total auxins,figure I) at the early floral initiation stage, whereas 9.3 % of free IAA were detected at the vegetative stage (table I>. Corm tissues from vegetative and floral differentiation plants contained lower detectable levels of ester- and amide-conjugates than tissues from floral initiation plants.
free IAA during floral initiation and development in plants is limited, especially in the case of the tropical plant, tuberose. IAA is known to be conjugated either to amino acids or peptides via an amide linkage, or to sugar moieties via an ester linkage [2]. Several destinations of conjugated IAA have been reported: storage, transport, protection from peroxidation and catabolism [9]. Hence, the regulation of IAA conjugate formation is an important process during floral initiation and differentiation. The purpose of this study was to analyze the destinations of free IAA and IAA conjugates in tuberose corms during vegetative, early floral initiation and the flower differentiation stages of tuberose. Our results indicate that the corms of tuberose may be autonomous with respect to the IAA supply during floral initiation. De novo synthesis of amide-linked IAA conjugates in the corms of flowering seedlings suggests a role for these compounds in the regulation of the concentration of endogenous free IAA in tuberose corms.
In general, all flowering responses in plants are considered to be regulated by interactions among phytohormones, mainly auxins, cytokinins, and gibberellins present endogenously in plant tissues as well as those added exogenously. Auxins are thought to influence various aspects of plant development such as cell division, growth, morphogenesis, and response to flowering [2, 51. In the case of IAA, in addition to biosynthesis, catabolism is another way to control the levels of free IAA, and conjugation constitutes an important aspect of IAA catabolism. For example, Tuominen et al. [15] reported that conjugation may also play a role in the catabolism of endogenous IAA in Pop&s hybrids, suggesting that this may be a normal route for the breakdown of endogenous IAA. Also, Campanella et al. [4] showed that IAA conjugates inhibit the growth of Arabidopsis seedlings, probably because the conjugates are hydrolyzed and the IAA released is phytotoxic for plant growth. Although previous works have demonstrated the presence of both ester and amide conjugated IAA in Arubidopsis seedlings [13], to our knowledge, the
2. RESULTS AND DISCUSSION Tuberose corms from early floral initiation contained reduced levels of free IAA compared with corms from the vegetative and floral differentiation stages (table I>. The level of free IAA was higher in the tuberose corms from plants at the flower differentiation stage compared with those from vegetative plants, the increase was about 2.7-fold greater than that in vegetative corms. Free IAA levels were at a high level in corm tissues at the vegetative stage and then decreased at the early floral initiation stage (ruble Z). This value may not equal the corm’s IAA biosynthetic rate because it
Table I. Concentrations of free and bound IAA at different growth stages of tuberose. Although quantities differ significantly between the two replicates (either due to plant-to-plant differences or assay differences), the overall trend with time is similar between the two replicate samples. Free IAA
Growth stage
Ester IAA
Amide IAA
(ng. g-’ fresh mass) Vegetative stage
Early floral initiation
Flower differentiation
stage
stage
Plant Physiol. Biochem.
Sample 1 Sample 2 Average
279 207 243
357 317 337
2 053 1993 2 023
Sample 1 Sample 2 Average
219 167 193
1 127 1 087 1 107
2 954 2 902 2 928
Sample 1 Sample 2 Average
696 628 662
628 558 593
1 269 1 195 1 232
L4A in relation to flowering of tuberose
IAA
163
trast, IAA conjugate accumulation begins when floral initiation has occurred (table I). However, the reason for this accumulation of IAA-conjugates remains unknown. We hypothesized that a decrease in free IAA concentration during the flowering process has a physiological role during the developmental process of new leaves. This view is supported by the fact that free IAA in floral corms would reduce leaf differentiation in tuberose (K.L. Huang, pers. comm.). We also hypothesized that in tuberose, the indole ring is produced in the corm apex and transported to the leaf primordia via polar movement. Bialek et al. [3] reported that excess IAA, which was not utilized by bean seedlings when the growth rate was drastically reduced after the removal of the cotyledons, was conjugated. Simultaneously, they also showed that IAA amide-linked conjugates were hydrolyzed by bean seedlings when they were applied to the stem sections or studied in vitro [3]. Our results suggest a regulatory role for conjugated IAA during early floral initiation. We show that free IAA may be conjugated at this stage due to relatively higher proportion of conjugated IAA to free IAA than those of vegetative and flower differentiation stages (table I>. In summary, it appears that in vegetative tuberose corms, the level of free IAA, normally present in high amounts, is correlated with leaf differentiation, and that a decrease in free IAA levels is required for the initiation of flower buds (table r). The results further show that conjugated peptidyl and esterified IAAs increase significantly during the formation of flower buds in tuberose (table I). These findings indicate that the regulation of particular physiological transformation during flowering processes are really correlated with changes in the levels of endogenous IAA. Our study suggest that conjugated IAA plays an important role during the flowering process in the tuberose. Also, when the level of IAA is reduced during early floral initiation, normal leaf differentiation is affected.
’ ’ CH2CooH 03 :
H
H
<‘HzCOR
IAA amides
<‘H$02R
MA esters
R= amides or esters
Figure 1. Chemical structures of IAA, IAA amides and IAA esters.
identification and quantification of these endogenous IAA conjugates have not been determined during the flowering process, especially in the tropical plant, tuberose. In this study, the most striking result is the finding that both ester& or peptidic IAA occurs in tuberose corm tissues before, during and after floral initiation (ruble I>. It is interesting to note that tuberose corm tissues in the floral initiation stage contain primarily amide-V&, whereas corm tissues in the floral initiation stage contain large amounts of both esterand amide-MA (table I>. Thus, the chemical form of IAA in the vegetative- and reproductive-corms may be different. The IAA content in plants is regulated by the conjugation of excess IAA to amino acids and sugars [ 1, 61. We suggest that synthesis and hydrolysis of the bound IAA is a homeostatic mechanism for maintenance of IAA concentration during the flowering process. After floral initiation, tuberose corms accumulate less conjugated IAA than the vegetative and early floral initiation stages (table I). The assumption is that conjugation of IAA is a means of maintaining free IAA levels [I]. In other words, conjugation leads to a decline in the level of the active hormone, because free IAA is the only biologically active form [6]. Usually, in dicotyledonous plants, most amide conjugates were observed [6, 11, 121, whereas in monocotyledonous plants, the primary conjugates were ester-bound. We were able to detect both ester- and amide-bound IAA in tuberose corms, a monocotyledonous plant (table I). These results also provide evidence that changes in free IAA is correlated with floral initiation in tuberose. The flowering process is most likely attributable to the lowering of free IAA levels in corm tissues. By con-
3. CONCLUSION
In conclusion, the present study demonstrates a about 2-fold greater conjugated IAA concentration in the early floral initiation stage than in vegetative and flower differentiation stages. Free IAA concentration increased 1.3- and 3.4-fold in vegetative and flower differentiation stages, respectively, when compared with early floral initiation stage. In tuberose, we were able to detect both ester- and amide-bound IAA in corm tissues, a monocotyledonous plant. These observol. 37 (2) 1999
164
S.-F. Ding et al.
vations emphasize the need to consider differential distribution of endogenous IAA at different growth stages for an accurate understanding of IAA in the flowering physiology of tuberose. 4. METHODS 4.1. Materials. Tuberose corms (Poliunthes tubema L. cv. Double) 2.0-2.5 cm in diameter were cultured in plastic pots (30 cm x 20 cm, width x depth) filled with an artificial peatmoss/perlite/vermiculite medium (3/1/l, v/v/v). The pots were placed in field conditions during the summer season (30 “Cl25 “C, day/night in average) in Kaohsiung City, Taiwan. After the specified time (21-35 d), the corms were removed fom young seedlings and viewed microscopically. The corms were classified into vegetative, early floral initiation and flower differentiation stages (four to six flower primordia were emerged and small inflorescence observed). Subsequently, their fresh mass were determined. The corms were rinsed with distilled water, and weighed. 4.2. Determination of free and conjugated IAA. The analyses of free and conjugated IAA were as described by Chen et al. [S] with slight modifications. Briefly, fresh samples (1 g) were ground in a mortar with 4 mLg’ of sample in 65 % isopropanol with 0.2 M imidazol (Sigma) buffer pH 7.0. [‘H,]-IAA (enrichment of 84 % deuterium; Cambridge Isotope Lab, Andover, MA, USA) was added as an internal standard. After the known amount of deuterated IAA was equilibrated in the extract for 1 h at 4 “C, the solution was centrifuged at 10 000 x g for 5 min. The isopropanol was then evaporated under vacua, and the aqueous layer was divided into three equal parts: the first for free IAA analysis, the second was hydrolyzed at 25 “C for 1 h with 1 N NaOH to measure free plus ester IAA, and the third was hydrolyzed at 100 “C under N2 for 3 h with 7 N NaOH to measure total IAA (free + esters + amides). For free IAA analysis, a 4-mL extract was applied to a conditioned amino anion exchange column (BAKERBOND speTM). After the sample extract passed through the column, aspiration was applied for 20 s to remove excess water, and the column was washed successively with hexane, ethyl acetate, acetonitrile and methanol (2 mL each). The free IAA was eluted from the column using 3 mL methanol containing 2 % acetic acid. The acidic methanol eluent was evaporated to dryness under vacua, and was taken up in solvent for HPLC purification. The HPLC was equipped with a Cl8 column (30 x 0.4 cm id., pBondapak; Waters Associates) using acetonitrile and water containing 0.1 % acetic acid as the mobile phase. The gradient was programmed to hold for 1 min at water/acetonitrile (9515, v/v), followed by a 5-min linear gradient to water/acetonitrile (80/20, v/v) and held for 20 min. The 1.5-mL fraction at the retention time of IAA was dried in vacua, the sample was then methylated with diazomethane, and analyzed using GCSIM-MS. For the determination of free IAA plus ester form, 2 mL of the extract was base-hydrolyzed. After hydrolyzing
Plant Physiol. Biochem.
the ester conjugate as above, the sample was diluted, pH adjusted to 2.5 with 1 N HCl and passed through a conditioned (washed with hexane, methanol, water, and 1 % acetic acid successively, 5 mL each) Baker C-18 column to desalt. The C- 18 column was washed with 5 mL distilled water, and the IAA eluted with 2 mL acetonitrile. Imidazole buffer (20 mM, pH 7.0) was added to dilute the eluent to 10 % of the organic phase, and then the sample was applied to the conditioned amino column as above. The solution was then dried in vacua, resuspended in solvent, and purified using HPLC as above. The IAA fraction was evaporated to dryness under vacua and resuspended in 100 pL methanol for methylation. For determination of total IAA, the hydrolysate was filtered, the pH brought to 2.5, and the IAA compounds were purified over a Sep-pak C 18 cartridge equilibrated with H,O. IAA was eluted with 100 % methanol. The methanol was evaporated to dryness and resuspended in 100 pL methanol for HPLC purification. The IAA fraction was taken, and used for methylation. Each analysis was repeated three times. The total IAA determination, appropriate amounts of NaOH were added to the extract after evaporation of the isopropanol. The hydrolysis was performed in a capped PTFE vial purged with water-saturated N2 gas at 100 “C for 3 h. The hydrolysates were diluted, and the pH adjusted to 2.5 with 2 N HCl, then passed through a conditioned C- 18 column, and prepared following the procedures as described above for free and ester IAA. 4.3. GC-NM-MS analysis. GC-SIM-MS analysis of the purified methylated IAA was conducted on a VG Quattro mass selective detector that was interfaced with a HewlettPackard 5890 GC. The GC column was a 30-m DB-5, 0.25 pm film. The temperature program was 100 “C for 1 mitt, followed by an increase of 25 SC.min-’ to 200 “C,
Figure
2. Mass spectrum
IAA isolated from tuherose The molecular ion region of the
of methylated
corm tissues taken during W-MS.
spectrum showing ion at m/z 189 derived from endogenous compound (D,) in addition to corresponding ion at m/z 194 arising from [‘HJlabeled internal standard.
IAA in relation to flowering of tuberose
kept at this temperature for 6 min, increased rate of 25 “C.min-’ and kept at that
to 250 “C at a
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[6] Cohen J.D., Bandurski R.S., The chemistry and physiology of the bound auxins, Annu. Rev. Plant Physiol. 33 (1982) 403430. [7] Hangarter R.P., Good N.E., Evidence that IAA conjugates are slow-release sources of free IAA in plant tissues, Plant Physiol. 68 (1981) 1424-1427. [8] Harada H., Nitsch J.P., Changes in endogenous growth substances during flower development, Plant Physiol. 34 (1959) 409-415.
Acknowledgements. The authors gratefully acknowledge the help of Jentaie Shiea and Hsiao-Ching Yu in the use of the GC-MS instrument. REFERENCES [l] Bandurski R.S., Schulze A., Concentration of indole-3acetic acid and its derivatives in plants, Plant Physiol. 60 (1977) 211-213. [2] Bandurski R.S., Cohen J.D., Slovin J.P., Reinecke D.M., Auxin biosynthesis and metabolism, in: Davis PJ. (Ed.), Plant Hormones: Physiology, Biochemistry and Molecular Biology, Kluwer Academic Publishers, Dordrecht, 1995, pp. 39-68. [3] Bialek K., Michalczuk L., Cohen J.D., Auxin biosynthesis during seed germination in Phaseolus vulgaris, Plant Physiol. 100 (1992) 509-5 17. [4] Campanella J.J., Ludwig-Muller J., Town CD., Isolation and characterization of mutants of Arabidopsis thaliana with increased resistance to growth inhibition by indoleacetic acid-amino acid conjugates, Plant Physiol. 112 (1996) 735-745. [5] Chen K.H., Miller A.N., Patterson G.W., Cohen J.D., A rapid and simple procedure for purification of indole3-acetic acid prior to GC-SIM-MS analysis, Plant Physiol. 86 (1988) 822-825.
[9] Ludwig-Muller J., Epstein E., Hilgenberg W., Auxinconjugate hydrolysis in Chinese cabbage: Characterization of an amidohydrolase and its role during infection with clubroot disease, Physiol. Plant. 97 (1996) 627634. [lo] Ludwig-Muller J., Sass S., Sutter E.G., Wodner M., acid in Arabidopsis Epstein E., Indole-3-butyric thaliana. I. Identification and quantification, Plant Growth Regul. 13 (1993) 179-187.
[ 1 l] Mori G., Miyake K., Imanishi H., Effect of exposure of corms to high temperature on flowering of Polianthes tuberosa L., J. Jpn. Sot. Hort. Sci. 59 (Suppl. 2) (1990) 538-539. [12] Normanly J., Cohen J.D., Fink G.R., Arabidopsis thaliana auxotrophs reveal a tryptophan-independent biosynthetic pathway for indole-3-acetic acid, Proc. Nat1 Acad. Sci. USA 90 (1993) 10355-10359. [13] Trueblood E.W.E., ‘Omixochitl’- the Tuberose (Polianthes tuberosa), Economic Bot. 27 (1973) 157-173. [14] Tsukamoto Y., Kawashima N., Tanaka T., Retardation of flowering by auxin-gibberellin spray in Chrysanthemum molifolium, Mem. Coll. Agr. Kyoto Univ. 93 (1968) 1-19. [15] Tuominen H., Ostin A., Sandberg G., Sundberg B., A novel metabolic pathway for indole-3-acetic acid in apical shoots of Populus tremula (L.) x Populus tremuZoides (Michx.), Plant Physiol. 106 (1994) 151 l-1520.
vol. 37 (2) 1999