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Plant Physiol, January 2001, Vol. 125, pp. 464-475 Developmental Regulation of Indole-3-Acetic Acid Turnover in Scots Pine Seedlings1Department of Forest Genetics and Plant Physiology, The Swedish University of Agricultural Sciences, S-901 83 Umeå, Sweden
Indole-3-acetic acid (IAA) homeostasis was investigated during seed germination and early seedling growth in Scots pine (Pinus sylvestris). IAA-ester conjugates were initially hydrolyzed in the seed to yield a peak of free IAA prior to initiation of root elongation. Developmental regulation of IAA synthesis was observed, with tryptophan-dependent synthesis being initiated around 4 d and tryptophan-independent synthesis occurring around 7 d after imbibition. Induction of catabolism to yield 2-oxindole-3-acetic acid and irreversible conjugation to indole-3-acetyl-N-aspartic acid was noticed at the same time as de novo synthesis was first detected. As a part of the homeostatic regulation IAA was further metabolized to two new conjugates: glucopyranosyl-1-N-indole-3-acetyl-N-aspartic acid and glucopyranosyl-1-N-indole-3-acetic acid. The initial supply of IAA thus originates from stored pools of IAA-ester conjugates, mainly localized in the embryo itself rather than in the general nutrient storage tissue, the megagametophyte. We have found that de novo synthesis is first induced when the stored pool of conjugated IAA is used up and additional hormone is needed for elongation growth. It is interesting that when de novo synthesis is induced, a distinct induction of catabolic events occurs, indicating that the seedling needs mechanisms to balance synthesis rates for the homeostatic regulation of the IAA pool.
Indole-3-acetic acid (IAA) is a
well-known regulator of plant growth and development, which is active
in submicromolar amounts and is associated with a variety of
physiological processes, including apical dominance, tropisms, shoot
elongation, induction of cambial cell division, and lateral root
initiation. The IAA content of plant tissues is believed to be
regulated by several processes. De novo synthesis and the hydrolysis of
IAA conjugates represent inputs into the IAA pool, which can be
counterbalanced by a variety of conjugative and catabolic pathways
(Normanly, 1997 The biosynthesis of IAA in plants is still not fully understood and
there are reasons to believe that several pathways exist (Bartel,
1997 The first comprehensive attempt to measure the different components
comprising the IAA homeostasis mechanism, published in 1980 by Epstein
et al., elegantly showed that the maize kernel utilizes stored forms of
IAA for initial seedling growth. We have chosen germinating Scots pine
(Pinus sylvestris) seedlings for our experiments since this
is one of the few species for which we have an extensive overview of
the catabolic and conjugative pathways (Andersson and Sandberg, 1982 In this study we have considered the major components supplying the IAA pool, as well as the metabolic components involved in its regulation. We have investigated the developmental control of these mechanisms and have shown that the induction of key elements of IAA metabolism is strongly correlated with major physiological and morphological events. We have also identified two new IAA conjugates in germinating Scots pine seedlings, two N-linked hexoses of IAA and indole-3-acetyl aspartic acid (IAAsp), probably glucosides. This type of conjugation to an intact indole nucleus has not previously been described in plants.
Seed Development during Germination When Scots pine seeds are germinating (Fig. 1A), the first visible change occurs 2 d after imbibition when the secondary wall in the cortex of the embryo hypocotyl starts to develop. During the 3rd d, the secondary wall continues to develop, and swelling caused by a general cell enlargement starts to separate the two halves of the seed coat. At the same time the first increment in cell length in the hypocotyl occurs, which leads to the root cap penetrating the nucellus and reaching the opening in the seed coat. Between d 3 and 4, the hypocotyl and root elongate rapidly, with the hypocotyl ending its elongation growth around d 8. The main root continues to grow at approximately the same rate for the rest of the period covered in our experiments. The elongation of the cotyledons is somewhat delayed, being induced at around d 5 and continuing for the remainder of the 12 d analyzed, although at a slower rate after d 10. Figure 1B shows the germination percentage of the Scots pine seeds.
Quantification of IAA and IAA Conjugates Free IAA and conjugated IAA were analyzed over a period of 10 d following imbibition (Fig. 1C), and with the use of different strengths of hydrolysis, it was possible to distinguish between the
pools of amide-linked and ester-linked conjugates. A rapid increase in
the content of free IAA was observed in the first few days following
imbibition, reaching a maximum of 100 ng/g of tissue after 48 h.
This dramatic increase in free IAA was correlated with a decrease in
the pool of ester-linked conjugates during the first 60 h. It is
interesting that the high level of free IAA observed after 48 h
decreased when root elongation was initiated. Amide conjugates were not
a significant component of the stored IAA in the dry seed. However,
this category of IAA conjugates increased when root elongation was
initiated. The pulse of free IAA observed is in agreement with IAA
profiles previously obtained from Scots pine (Sandberg et al., 1987
IAA Biosynthesis from [15N1]Trp and Deuterated Water A study was performed to investigate the stage of development at which IAA biosynthesis is initiated during germination and initial seedling growth. First, to give the tissue a pulse of tracer, seeds and seedlings were incubated with [15N1]Trp for 6 h and the appearance of [15N1]IAA was measured during the following 48 h. A relatively low amount of the added Trp (about 20% of the total) was taken up. Thus the increase in the seeds Trp pool caused by adding label was small (data not shown). Figure 3A shows the relative abundance of synthesized [15N1]IAA. Linked gas chromatography mass spectrometry (GC-MS) analysis of IAA from the incubation starting on d 1 and ending on d 3 showed no significant increase in m/z 203, whereas the experiment that started on d 4 and ending on d 6 showed a significant increase in m/z 203. It can thus be concluded that there is no de novo synthesis of IAA from Trp before d 3, and that this pathway is induced between d 4 and 6. However, there appears to be no further increase in synthesis from this pathway after d 6, according to results from the incubation starting at d 7 and ending at d 9.
Not all synthesis of IAA is believed to involve Trp as a precursor
(Normanly et al., 1993 Turnover of [13C6]IAA Turnover of [13C6]IAA was analyzed 1, 4, and 7 d after imbibition. The [13C6/12C6]IAA ratio at the start of the experiments ranged from 0.13 to 0.39 (Table I). The disappearance of label from the IAA pool followed first order kinetics, which simplified the calculation of IAA half-life. There was almost no turnover of IAA during the first 3 d of imbibition since the half-life was more than 48 h when the experiment started 1 d after imbibition. After 4 d, however, turnover had clearly been initiated, giving a half-life at this point of 16.4 h. This was even more pronounced after 7 d, when the half-life was 7.2 h. Another noteworthy result was the difference between experiments in [13C6/12C6]IAA ratio observed after the initial 5-h incubation period. Clear indications of an induction of IAA uptake was detected between d 1 and 4. This difference is even more pronounced if the increase in the endogenous IAA pool that occurs between d 1 and 4 is taken into account.
Identification of IAA Metabolites Incubation of seedlings with 14C-labeled IAA led to the formation of six metabolites that were designated metabolites 1 through 6 in order of decreasing polarity (Fig. 4). Metabolites 1 and 2 had retention times of 0.06 and 0.25 relative to IAA that changed, after methylation, to 0.77 for both substances. No conclusive identity of these very polar catabolites was obtained by HPLC-MS analysis.
The methylated and acetylated derivative of metabolite 3 had molecular
ions [M + H]+ of m/z 649/655 (Fig.
5A), corresponding to the incorporation of four acetyl groups into the Glc moiety. This was further supported by diagnostic ions of the acetylated Glc observed at m/z 43, 109, 169, 229, and 331. The molecular ions [M + H]+ m/z 649/655 were analyzed by
constant magnetic sector/electric sector (B/E)-linked scanning
(Fig. 5A). Both spectra described the loss of a methyl ester
[M
Fast-atom bombardment (FAB) spectra from the methylated and acetylated
metabolite 4 gave a double-labeled molecular ion [M + H]+/[13C6M + H]+ (m/z 520/526). This corresponds
to the incorporation of four acetyl groups into a
N-linked hexose (Fig. 5B). The loss of an acetylated
hydroxy group or the loss of the methylated carboxylic acid,
[M Spiking experiments with 14C-labeled compounds
indicated that metabolites 5 and 6 were 2-oxindole-3-acetic acid
(OxIAA) and IA-Asp, respectively (Figs. 4 and 7), and these conclusions
were also verified by HPLC-MS analysis (data not shown). Both these compounds have already been identified as endogenous compounds in Scots
pine (Andersson and Sandberg, 1982 Analysis of IAA Metabolism The relative accumulation of different IAA catabolites was determined by HPLC-radioactive counting (RC) after 24 h of incubating 0- to 10-d-old seeds/seedlings with 14C-labeled IAA. Rapid induction of IAA metabolism was observed between d 4 and 6 after imbibition (Fig. 8). The primary metabolites were OxIAA and IAAsp (Fig. 7), which started to accumulate on d 4. IAAsp was further metabolized to IAAsp-N-glucoside. Minor catabolites were also detected one of which (IAA-N-glucoside) appeared simultaneously with IAAsp-N-glucoside 6 d after imbibition.
IAA Sources Release of Stored Pools Indole-3 ethanol (IEt) has previously been shown to be an endogenous constituent of Scots pine needles, probably synthesized from Trp and then further converted to IAA. (Sandberg, 1984 ). We have also
earlier demonstrated that the Scots pine seeds contain IEt conjugates
that are released during germination to yield a pulse of free IEt,
peaking 2 d after imbibition, and then decreasing rapidly to
almost undetectable levels 4 d later (Sandberg et al., 1987). In
these previous experiments the content of conjugated IEt and released
IEt was of the same size as the IAA pools, indicating that the stored
IEt pool is almost certainly a significant contributor to the free IAA
pool during the first days of germination. The technique used in the
earlier investigation did not involve stable isotopic dilution
analysis. 14C-IAA was used instead to evaluate
recovery. This procedure gives a method error of ±20% to 25%,
compared with an error of less than ±5% with the techniques used in
this investigation. We therefore decided to repeat the analysis of free
and bound IAA during germination. The data presented in Figure 1C
follow the same general trend we observed earlier (Sandberg et al.,
1987). The majority of the IAA in the dry seed is stored as
ester-linked conjugates and only a minor part occurs as free IAA or as
amide-linked conjugates. The ester-linked pool of IAA is hydrolyzed to
yield free IAA, peaking 2 d after imbibition. We have shown (Fig.
2B) that the IAA conjugates are mainly stored in the embryo itself and
that only a minor proportion is stored in the general nutrient storage tissue, the megagametophyte. This set of data also demonstrates that
the highest concentration of free IAA is localized in the embryo (Fig.
2A). The release of IAA conjugates to yield free IAA in the embryo is a
rapid process, which finishes within 4 to 6 d. It thus makes sense
for the system to store the conjugated hormone in the embryo itself to
allow immediate access and avoid any requirement for long-distance
transport. We propose that homeostatic mechanisms like catabolism and
conjugation that control IAA levels are not needed in the initial stage
of germination, since regulation of the IAA pool size at this stage of
development is probably based on adjustment of the hydrolytic activity,
releasing free IAA from conjugated pools.
De Novo Synthesis IAA synthesis was assayed in such way that Trp-dependent and Trp-independent IAA synthesis could be distinguished. To simplify the analysis, a relative comparison was performed and no attempt was made to quantify the absolute amount of IAA synthesized. Our data clearly show that both pathways of IAA synthesis were inactive during the initial phase of imbibition and that Trp-dependent synthesis was induced first (Fig. 3A) at around d 4, followed by Trp-independent synthesis around d 7 (Fig. 3B). A clear developmental sequence in utilization of the IAA sources is thus part of the homeostatic regulation in this system. This was also demonstrated by Jensen and Bandurski (1996) who fed deuterated water to dark-grown maize
seedlings. Incorporation of deuterium into IAA was not observed in
7-d-old seedlings, although incorporation into Trp could clearly be
demonstrated. In Scots pine seedlings ester-linked conjugates are first
hydrolyzed to give a peak of free IAA 2 d after imbibition, which
coincides with the initial swelling of the seed. This high concentration of free IAA then drops dramatically between d 2 and 5, correlating well with the induction of hypocotyl and root elongation.
It is interesting to note that maximum elongation growth is not
correlated with the highest level of free IAA. After the stored pools
of IAA and IEt have been consumed, Trp-dependent synthesis is induced.
For about 2 d, this pathway is then the only active IAA source in
the seedling. Around d 6, Trp-independent synthesis is initiated. The
differential induction of the two biosynthesis pathways supports the
hypothesis that these processes are developmentally controlled. The
Trp-dependent synthesis is induced at the same time that the
elongation growth of the hypocotyl and root starts, whereas the
Trp-independent pathway is induced concurrently with the major
elongation phase of the cotyledons. The timing of the induction of
Trp-dependent synthesis is consistent with theoretical
expectations, since at that point the stored IAA and IEt has
been used up and major elongation processes are about to start in the
seedling, requiring a direct supply of IAA. This cascade of events is
not obviously linked to the Trp-independent synthesis pathway, but it
is tempting to speculate that this pathway is localized to the
cotyledons, since its induction coincides with the elongation of these organs.
IAA Catabolism The catabolic pathways of IAA in Scots pine have already been
demonstrated to involve oxidation of IAA to OxIAA (Ernstsen et al.,
1987 As shown in Figure 8, there is almost no catabolism of IAA during the
first 3 d following imbibition, but thereafter a progressively stronger induction of catabolism occurs, with turnover reaching rapid
rates after 6 to 7 d. This fits well with our studies of IAA
half-life at d 1, 4, and 7, which also indicate that there is a rapid
induction of turnover between d 4 and 7. The two first-labeled catabolites formed are OxIAA and IAAsp, which are synthesized at
approximately the same rate starting from d 4. Labeled
IAAsp-N-glucoside occurs in significant quantities about
2 d after the first appearance of IAAsp and at the same time
labeled IAA-N-glucoside is observed, but at much lower
levels. This also supports our view that the two new conjugates are
irreversible inactivation forms of IAA in this system. Precautions have
been taken during these experiments to avoid formation of metabolites
caused by feeding with unphysiologically high hormone levels,
microbiological activity, or the preparation itself (Östin et
al., 1998 A Developmental Model for IAA Turnover in Scots Pine Seedlings Figure 9 shows the timing of the different pathways involved in the control of the IAA pool during early seedling growth of Scots pine. A clear difference in function between the two different categories of IAA conjugates is postulated. Ester-linked conjugates of IAA and IEt form a stored pool that is utilized for growth during the initial phase of germination. These conjugates are not involved in a regulatory shunt, i.e. they are not formed at access levels of IAA and, thus after the first 3 d of germination they do not act as sources for release of free IAA. Amide-linked conjugates are only minor constituents in the pool of stored IAA in the seed. The formation of OxIAA and IAAsp is induced around 4 d after germination, together with the other minor catabolites, and IAAsp, as well as the new N-linked sugar conjugates are believed to serve less as a storage form of IAA than as intermediates in irreversible catabolism. There is no catabolism of IAA during the initial growth phase when the predominant IAA sources are IAA and IEt conjugates, but as soon as de novo synthesis begins, catabolism is induced. The close correlation between the induction of de novo synthesis and catabolism indicates that the homeostatic system is dependent on mechanisms such as catabolism and conjugation to control the IAA pool size.
Chemicals and Isotopically Labeled Substrates [1'-14C]IAA with a specific activity of 55 mCi/mmol was purchased from American Radiolabeled Chemicals (St.
Louis). [13C6]IAA and
[15N1]Trp were from Cambridge Isotope
Laboratories (Andover, MA). [2H5]IAA was
supplied by MSD Isotopes (Montreal). Deuterated water came from Norsk
Hydro (Porsgrunn, Norway). Labeled IAAsp, and OxIAA were
synthesized from [1'-14C]IAA according to the methods of
Tuominen et al. (1994) Plant Material and Growth Conditions The Scots pine (Pinus sylvestris) seeds used were
from a local seed orchard (Östteg, Umeå, Sweden; 63°55'N,
19°45'E) and had a germination rate of 95.7%. For anatomical and
metabolic studies, fully developed seeds were selected and germinated
in Petri dishes on moist filter paper at 20°C under 155 µmol
photons m Quantification of IAA and IAA Conjugates For analysis of endogenous IAA levels, 100 mg of seeds were
harvested 0 to 10 d after imbibition and were homogenized in
liquid nitrogen. Subsamples of 10 mg were extracted, purified, and
analyzed by GC-selected reaction monitoring-MS as described in Edlund
et al. (1995) IAA Biosynthesis from [15N1]Trp and Deuterated Water Four hundred milligrams of seeds were incubated for 6 h with 2 µg of [15N1]Trp 1, 4, and 7 d after imbibition. The seeds were rinsed in distilled water and left on moist filter paper in darkness for the remainder of the experiment. Approximately 10-mg samples were collected at 6, 12, 24, 36, and 48 h. Four hundred milligrams of seeds were also incubated with deuterated water for 48-h periods starting at d 1, 4, and 7 after imbibition. Samples were collected as described above. The tissue was extracted after the addition of 50 pg [13C6]IAA/1 mg tissue as an internal standard and, after purification, [15N1]IAA, deuterium-labeled IAA, and endogenous IAA were analyzed by GC-MS (operated in the selected ion monitoring mode, R = 5000). No attempts were made to calculate exact synthesis rates. Incorporation of 15N1 from Trp into IAA is expressed as the ratio of the labeled to unlabeled IAA base peaks (m/z 203.102 and 202.105, respectively). Incorporation of 2H from deuterated water into IAA is expressed as the ratio of deuterium-labeled IAA (m/z 203.111 + 204.118) to unlabeled IAA (m/z 202.105). Turnover of [13C6]IAA Four hundred milligrams of seeds was incubated for 5 h with 100 ng of [13C6]IAA dissolved in 5% (v/v) ethanol, 1, 4, and 7 d after imbibition. The seeds were rinsed in distilled water and left on moist filter paper in darkness for the remainder of the experiment. Approximately 10-mg samples were collected at 0, 3, 6, 9, 12, and 24 h for the incubations starting at d 4 and 7, and at 6, 12, 24, and 48 h for the incubation starting at d 1. Free IAA was extracted and purified as described above, except for the addition of 100 ng [2H5]IAA as an internal standard for the quantification of IAA. GC-MS (operated in the selected ion monitoring mode, R = 5000) was performed using m/z 207.137 and 266.149 for measuring [2H5]IAA, m/z 208.125 and 267.139 for [13C6]IAA, and m/z 202.105 and 261.118 for endogenous IAA. The obtained values were corrected for "crosstalk" between the m/z 208.125/207.137 and m/z 267.139/266.149 channels. Identification of IAA Metabolites To produce large amounts of metabolites, pine shoots were placed
in vials with 0.5 mL of incubation media containing a mixture of 100 µM [13C6]IAA, 100 µM IAA, and 10 µM [1'-14C]IAA
in one-half-strength Murashige and Skoog medium. The medium with
tracers was taken up via the transpiration stream and excess one-half-strength Murashige and Skoog medium was thereafter
added to provide the plant with enough liquid for the rest of the
incubation period. Samples were collected for analysis after 24 h.
To verify the existence of the metabolites in the plant system under
study, sterile seedlings with removed roots were immersed in the same incubation media containing tracers for 24 h. Shoots and seedlings were frozen in liquid nitrogen, homogenized, and extracted with methanol containing 0.2% (w/v) antioxidant (diethyldithiocarbamic acid) for 4 h. After filtration, 10 mL of 50 mM sodium
phosphate buffer, pH 7, was added and the sample was reduced to water
phase in a rotary evaporator. Seedling extracts could then be taken directly to the solid phase extraction step (see below), whereas shoot
extracts first had to be subjected to solvent partitioning against
ethyl acetate and water-saturated butanol (Östin et al., 1998 Analysis of IAA Metabolism Seeds (500 mg) were incubated in 5 µM
[1-14C]IAA in one-half-strength Murashige and
Skoog medium, pH 5.6, in the dark for 24 h 0 to 10 d after
imbibition. The plant material was homogenized in liquid nitrogen,
extracted, purified, and analyzed by HPLC-RC (Östin et al.,
1998
The authors are grateful to Gun Lövdahl and Kjell Olofsson for skillful technical assistance.
Received May 1, 2000; modified July 18, 2000; accepted September 18, 2000. 1 This work was supported by The Swedish Council of Forestry and Agricultural Research and The Foundation for Strategic Research.
2 These authors contributed equally to the paper.
3 Present address: National Institute of Working Life, S-907 13 Umeå, Sweden.
* Corresponding author; e-mail goran.sandberg{at}genfys.slu.se; fax 46-90-7865901.
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ABSTRACT
INTRODUCTION
) and ester-linked (
) and
amide-linked (
) conjugates of IAA in Scots pine seeds 0 to 10 d
after imbibition. Vertical bars represent SD. FW, Fresh
weight.
), d 4 (
). Vertical bars represent
SD.
CH3O] (m/z 617/623), loss
of an acetyl group [M
5X0-cleaved Glc
fragment retained on the IAAsp moiety (Domon and Costello, 1988
-methyltryptophan-resistant lines of Lemna gibba showing a rapid rate of indole-3-acetic acid turnover.
Plant Physiol
107: 77-85
