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Auxin levels at different stages of carrot somatic embryogenesis
Phyzockemistry, Vol. 31, No. 4, pp. 1097-l 103, 1992 Printed in Great Britain.
0031~9422/92 SS.oO+O.OO Pergamon Press plc
AUXIN LEVELS AT DIFFERENT STAGES OF CARROT SOMATIC EMBRYOGENESIS
LECH MICHALCZUK,* TODD J. COOKE and JERRY D. COHEN~: Department
of Botany, University of Maryland, College Park, MD 20742, U.S.A.; tPIant Hormone Laboratory, Ag~cuttur~ Research Service, USDA, Beftsville Agricultural Research Center, Beltsvilie, MD 20705,U.S.A. (Received24 July 1991)
Key Word Index-Caucus carota;Umbelliferae; carrot; auxin; 2,4-dichlorophenoxyacetic somatic embryogen~is.
acid; indole-3-acetic acid;
Abstract-The role of auxin in somatic embryogenesis was evaluated by characterizing the changes in the concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), and their conjugates in callus suspension cells and developing embryos of llaucus carota. Both emb~ogenic and non-embryogenic lines exhibited similar growth rates and levels of IAA and 2,4-D on ~4-D-supplements medium. Total endogenous IAA in both lines exposed to 2,4-D reached high levels greater than 600 ng g- 1 fresh weight which suggests that IAA levels in carrot callus are not regulated via auxin feedback mechanisms. After being transferred to 2,4-D-free medium, the embryogenic line exhibited a rapid decline in both free and conjugated 2,4-D metabolites within seven days, while IAA levels remained relatively steady for seven days in the preglobular stage after which the levels declined steadily in all subsequent stages of embryo development. Individual analyses of different embryo fractions collected from asynchronous cultures confirmed that each stage in embryo development had lower IAA levels than the preceding stage. The non-embryogen~c line maintained similar 2,4-D levels but higher IAA levels than the embryogenic line throughout the experiment. The present results suggest that high IAA levels may be necessary but are not sufficient for the initial events in plant embryogenesis, whereas low IAA levels are associated with the later stages of embryo development.
INTRODUCTION
Physiological experiments indicate that both exogenous and endogenous auxins are intimately involved with several stages of carrot somatic embryo~nesis. Various sources of carrot cells, such as petiole explants [l], hypocotyl explants E23, and single cells isolated from established suspension cultures [3], require a minimum exposure of one, two or seven days, respectively, to the exogenous auxin 2,4-D (~~~c~orophenoxya~ti~ acid) in order to acquire the competence to eventually undergo somatic embryo development. In general, competent carrot cells continue to proliferate as an unorganized callus without any observable embryo initiation when cultured in a standard culture medium supplemented with 1 mg 1-l of 2,4-D; however, a few carrot lines are reported to produce pre~obular or even globular embryos in the presence of such typical 2,4-D 1eveIs [4,5]. Finally, exogenous auxins cause carrot embryos at later developmental stages to revert to proliferating callus [6}. These observations have led to the int~guing notion that once 2,4-D has made the cells com~tent to initiate somatic embryogenesis, it will then inhibit subsequent
*Permanent address: Research Institute of Pomology, 96-100 Skierniewice, Potand. $Author to whom correspondence shontd be addressed.
embryo development, with only the permissive condition, i.e. the absence of exogenous auxin, being necessary to complete embryo formation [7]. The endogenous auxin IAA (indole-3-attic acid) in cultured plant cells appears to have very different effects than the exogenous auxins used in most culture media. For instance, even though typical cultured cells accumulate free endogenous IAA at the levels of 2 to 20 ng g - ‘fr. wt [S, 93 which are comparable to those measured in the vegetative tissues of intact plants [lo], plant cell cultures are usually supplemented with exogenous auxin in order to maintain cell proliferation. More importantly, although carrot cells must be transferred to a 2,4-D-free medium before they can develop into mature embryos, it can be concluded from inhibitor experiments that endogenous auxin is, in contrast, required for the successful completion of embryo development. For example, the auxin analogues ~4,6-t~chiorophenoxyacetic acid and pchlorophenoxyisobutyric acid, which seem to act as competitive inhibitors of endogenous auxin activity [ 11, 121, will prefe~ti~ly inhibit embryo initiation, thereby suggesting that high endogenous IAA activity may be necessary for the initial stages of somatic embryogenesis [6, 131. Moreover, as the polar auxin transport inhibitors 2,3,5triiodobenzoic acid and N-( l-naphthyl)-phthalamic acid cause new embryonic axes to form on older heart and torpedo embryos, it appears that polar auxin transport is necessary to maintain the bipolar axis of the developing embryo [6].
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L. MICHALCZUK et al.
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Since our overall objective is to elucidate the underlying mechanisms by which auxin acts to regulate carrot somatic embryogenesis, we decided that it was first necessary to characterize the levels of both 2,4-D and IAA and their conjugates in callus cells and developing embryos. These hormone analyses were performed on two carrot cell lines, one of which has a high embryogenic potential, and the other of which lacks the ability to initiate any embryos. We observed that each stage of somatic embryogenesis is correlated with specific levels of free and conjugated auxin metabolites. Furthermore, the data demonstrate that the application of exogenous auxins like 2,4-D leads to significant changes in endogenous IAA levels of carrot cells, with the apparent consequence that these cells become competent to undergo somatic embryogenesis. An initial report of this work was presented elsewhere [ 141.
RESULTS AND DISCUSSION
clusters from the embryogenic line started to differentiate into globular embryos (Fig. lc) in seven days and heart and torpedo embryos in 21 days (Fig. Id). A rapid increase in fresh weight of the culture accompanied the formation of globular and post-globular embryos which occurred in the low-density conditions (Fig. 2). In contrast, the non-embryogenic line without 2,4-D exhibited no evidence of even globular embryos. This line showed a slight increase in fresh weight in the first week of low density conditions but no additional growth over the next two weeks even though it remained viable. As most lines lacking the potential to even initiate embryogenesis grow at much slower rates than embryogenic lines following the transfer to 2,4-D-free medium, the non-embryogenic line in this study does not represent the ideal control for our observations. The data from the non-embryogenic line are nonetheless informative, and thus the auxin levels of the two lines are compared in the following sections.
General features of the two cell lines
Auxin analyses of callus cells in 2,4-D-supplemented medium
Both the embryogenic and non-embryogenic cell lines grew at similar rates as unorganized callus with no observable embryos in 2,4-D-supplemented MS medium (Fig. la). The fractions of cell clusters 43-109 pm in diameter, which were obtained from both cell lines by sieving through two screens of these mesh sizes, were also composed only of callus cells (Fig. lb). However, when transferred to 2,4-D-free medium at low density, the cell
Although both 2.4-D and IAA undergo conjugation reactions as well as other metabolic transformations in plants, it was decided for the purposes of this work to concentrate on the free acids and the esterified and amidebound conjugates. The free acids are usually considered to act as the active form of the growth regulator while the conjugates seem to function as short-term storage forms that can readily be converted back into the free acids
Fig. 1. Representative fractions of callus cells and developing embryos in carrot suspension culture. (a) Undifferentiated callus cells cultured in MS medium plus 1 mgl- 1of 2,4-D. (b) 43-109 pm fraction collected from suspension cultures maintamed in the same medium as a. (c) Globular embryos isolated from the embryogenic line cultured in 2,4-D-free MS medium. (d) Heart and torpedo embryos isolated as in c The scale bar corresponds to 400 pm in all photographs.
Auxin levels during
carrot
embryogenesis
1099
104-
d
i
s
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9
b
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p-=---l //-II’ _~p_____----__ E c
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t---l” ___________d
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99.; c 0 7 Culture time en 2,4-D-free
_.
.:
14
17
21
mediumat low &nsit~Maysl
Fig. 2. The total fresh weight of cultured tissue of the embryogenic (open circles) and nonembryogenic (closed circles) lines at various times after being transferred to 2,4-D-free medium.
Fig. 3. The concentrations of 24-D and its conjugates in the embryogenic line before and after being transferred to 2,4-D-free medium. Open circles = free 2,4-D; closed circles = free + esterified 2,4-D, triangles = total 2,4-D. The concentration data are plotted as the log scale on the ordinate axis. The ‘c’ on the abscissa axis refers to callus cells, the ‘0’ refers to the preglobular stage following seven days in 2,4-D-free medium at the highdensity condition, and all numbers refer to the times for subsequent embryo development at the low-density condition. All data are presented as the treatment mean+ the standard error among three replicates. All symbols without error bars had a standard error less than the size of the symbol.
[15]. The level of total 2,4-D in suspension cells grown in 2,4-D-supplemented medium was quite similar in the two lines (Figs 3 and 4) (3.82f0.26 pgg-‘fr. wt in the embryogenic line vs 4.33 f0.16 pg g- ‘fr. wt in the nonembryogenic line). Assuming that the densities of the callus cells and the culture medium are both close to 1 g ml- ‘, then the callus cells had accumulated total 2,4D at a concentration roughly four times higher than the original concentration in fresh culture medium. However, as only 10% of the 2,4-D in the embryogenic line and 25% in the non-embryogenic line remained as free acid, it is likely that the uptake of the free form occurred by simple diffusion. In both lines, the bulk of the 2,4-D metabolites existed as ester- and amide-bound conjugates. The two cell lines grown in 2,4-D-supplemented medium had very high levels of endogenous total IAA (Figs 5 and 6) (672 + 24 ng g- ‘fr. wt in the embryogenic cell lines vs 603 + 44 ng g- ‘fr. wt in the non-embryogenic
:
:
c
0
:
:
7
Culture age on 2,4-O-fee
14
21
medium at bv denstiy Uoys)
Fig. 4. The concentrations of 24-D and its conjugates in the nonembryogenic line before and after being transferred to 24-D free medium. Open circles = free 2,4-D, closed circles = free + esterified 2,4-D; triangles = total 2,4-D. For additional explanation, see the legend of Fig. 3.
Culture
age on 2,4+free
medwm atlow density (days)
Fig. 5. The concentrations of IAA and its conjugates in the embryogenic line before and after being transferred to 2,4-D-free medium. Open circles = free IAA; closed circles = free + esterified IAA; triangles = total IAA. For additional explanation, see the legend of Fig. 3.
A
0 0
Culture age on 2,4-D-free
Fig. 6. The concentrations
medwm ot low density (days)
of IAA and its conjugates
in the
nonembryogenic line before and after being transferred to 2,4-Dfree medium. Open circles = free IAA; closed circles = free + esterified IAA, triangles = total IAA. For additional explanation, see the legend of Fig 3.
line). Both lines had roughly 80% of the IAA in the form of amide-bound conjugate(s) and similar low amounts of the free and esterified forms. In another experiment, it was also observed that the embryogenic line in the 2,4-
L.
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MICHAIGWK
D-supplemented medium gradually released IAA into the medium so that the concentrations of free and total IAA averaged 13 and 70 pg 1-l of the medium, respectively, with no detectable esterified IAA in one-month-old cultures (data not shown). Since most cell cultures appear to require an exogenous auxin to sustain cell proliferation, it was expected that carrot cells grown in 2,4-D-supplemented medium would have low concentrations of the endogenous auxin IAA; for example, the levels of free IAA in sycamore cells in 2,4D-supplemented medium can be computed (on the basis of a cell density of 1 gml - ‘) to range from 1 to 15 ngg-‘fr. wt over the course of the culture period [16]. However, the IAA levels observed in the callus cells of both carrot lines (Figs 3,4) were markedly higher than those reported for other cell cultures [8, 9, 161 or most vegetative tissues of intact plants [lo]. This observed ability of 2,4-D to mediate a very high IAA concentration in carrot cells reveals a different aspect of hormone metabolism in these cells. Most metaboiic pathways, such as those responsible for amino acid synthesis, are primarily regulated by feedback inhibition where the product and its analogues are able to reduce product synthesis by inhibiting an earlier enzyme in the pathway [17]. However, it has been argued that the measured concentrations of plant hormones are too low for feedback inhibition by itself to serve as an effective mechanism of metabolic regulation [18]. The present work on carrot cell cultures provides direct experimental evidence for this argument: the analogue 2,4-D must instead promote IAA synthesis and/or inhibit its degradation, with other reversible conjugating reactions controlling the concentration of the active free IAA, as has been described for other plant systems [15]. The underlying mechanism of 2,4-D’s action on IAA metabolism in carrot cells is being investigated in our laboratories. Auxin analyses of culrured cells in embryogenic conditions
The transfer to 2,4-D-free medium caused a very sharp decline of all forms of 2,4-D in both lines, with slight differences observed between the two lines (cf. Figs 3 and 4). In the embryogenic line, the level of free 2,4-D dropped to very low levels during the initial week at high density which corresponds to the pre~lobular stage of embryo development. increased dramatically during the first Table 1. Auxm content in ngg-‘frwt
et al.
week at low density (globular stage) and then slowly declined during the next two weeks (post-globular stages). Conjugated 2,4-D underwent an almost 30-fold drop during the first week and then decreased slowly over the next three weeks. After three weeks in 2,4-D-free medium at low density, the post-globular embryos had ca 20 ngg- ’ fr. wt of total 2,4-D, with approximately equal amounts of the free form and of amide-bound conjugate and almost undetectable levels of ester conjugate(s) (Fig. 3). In the non-embryogeni~ line, the initial drop in the free 2,4-D level was not so pronounced as observed in the embryogenic line. Moreover, the final concentration of total 2,4-D was two times higher in the non-embryogenic line. but the final level of free hormone was very similar in both lines (Fig. 4). As the sole source for the increased free 2.4-D observed in the globular embryos of the embryogenic cultures was the two conjugate pools, metabolic equilibria comparable to those operating in IAA metabolism must also exist between free and conjugated 2,4-D [cf. 193. After the transfer to 2,4-D-free medium at the highdensity condition (preglobular stage), the total IAA in the embryogenic line remained relatively constant but the next transfer to 2,4-D-free medium at low density was followed by a rapid decrease in total IAA (Fig. 5). The formation of globular embryos in this period is apparently accompanied by the dramatic loss of amide conjugate(s) while no significant change was observed in the free and esterified forms. Subsequently, the concentrations of all forms of IAA decreased slowly at similar rates. The post-globular fraction at three weeks had ca 20 ngg _‘fr. wt of total IAA, with 25% in free and 10% in esterified forms. The decrease in IAA levels occurred at much slower rates in the non-embryogenic line as compared to the embryogenic line (Fig. 6). This decrease was primarily attributable to the depletion of IAA conjugates while the free IAA remained relatively constant for the four weeks following the transfer to 2,4-D-free medium. The final concentration of IAA in the non-embryogenic line (37+5 ngg-ifr.wt of free IAA and 186f16 ngg- ‘fr.wt of total IAA) was almost 10 times higher than the levels measured in the embryogenic line, although the relative propo~ions of the three forms are close in the two lines (Fig. 6). The major difference between the two lines was that the free IAA underwent a steady decline for the globular to the torpedo stages in the embryogenie line
m carrot somatic embryos at various developmental stages
Fraction size (pm)
43-109
109180
180-280
280-380
>380
Embryo stage
80% globular 20% unditT.
45% globular 55% oblong
30% oblong 55% heart 15% other
85% heart 15% other
100% torpedo
31 (71 52 (IO) 67 (14) 27 (6) 31 (5) 56 (8)
6 (2) 23 (3) 31 (16) 16 (3) 44 (7) 70 (10)
8 (3) 12 (2) 29 (1) g (3) 36 (6) 33 (3)
8 (I) 7 (3) 20 (9) 5 (2) 7 (2) 23 (8)
5 (1) 6 (2) 18 (1) 4 (1) g (2) 12 (3)
I AA
2,4-D
free free t ester total free free + ester total
The different fractions were isolated by fractionating cultures with different-sized sieves and by collecting individual embryos as described in the Experimental. The data are presented as &hetreatment mean with the standard error of three replicates in parentheses.
Auxin levels during carrot embryogenesis while it was constant throughout the same time period in the non-embryogenic line. The relative changes in the three pools in the embryogenic line may indicate that an equilibrium exists between the free and amide pools, with ester conjugates as possible intermediates. Similar interconversions of other IAA conjugates have been characterized in maize kernels where a specific acyi transferase catalyses the transfer of the IAA moiety from the highenergy l-O-glucosyI-IAA to the more stable inositol ester EW‘ Auxin analyses of digerent stages of embryo development
Since embryo development was somewhat asynchronous in the embryogenic line and since bulk embryonic cultures of this line as measured in the preceding experiments contained a small proportion of undifferentiated cells, 21-day-oldcultures of the embryogenic line were fractionated with different-sized sieves in order to obtain more precise measurements of the IAA and 2,4-D levels in each stage of embryo development (Table 1). The total IAA level was observed to decrease at each successive stage of embryo development from 67 ngg- ‘fr. wt in globular embryos to 18 ng g- ‘fr. wt in torpedo embryos (Table 1). However, the free IAA level was observed to drop more than 80% from the 43-109 pm fraction (80% globular embryos) to the 109-180 pm fraction (globular and oblong embryos) after which it was maintained at the constant low level. Esterifled and amide-bound IAA was measured in globular and oblong embryos, but only the amide conjugate could be detected in heart and torpedo embryos. Despite the extensive period spent in 2,4-D-free medium before embryo isolation, a significant amount of residual 2,4-D remained in all embryonic fractions (Table 1). The highest levels of all three forms were found in the globular and oblong embryonic fraction, with progressively lower levels in more mature embryos. The possible role of auxin metabolism in plant embryogenesis
1101
However, the later stages of embryo development are occasioned by much lower levels of total endogenous IAA, viz. 20-30 ngg- rfr. wt (Table 1). Apparently, these low auxin levels allow the embryos to successfully complete their development, because globular embryos grown in auxin-supplemented medium dedifferentiate into proliferating callus (see Introduction). More importantly, low endogenous auxin activity may permit postglobular embryos to establish internal auxin gradients which are apparently necessary to initiate and maintain polarized growth. In particular, polar auxin transport inhibitors do not affect either the preglobular or globular stages; however, these inhibitors do specifically interfere with the ability of late globular embryos to initiate polarized growth and the ability of heart embryos to maintain a single bipolar axis [6]. Thus, it appears that the postglobular stages are especially sensitive to either exogenous auxins, which may perturb the metabolic homeostasis of endogenous IAA, or transport inhibitors, which may disrupt endogenous auxin gradients. Finally, it is important to address the question of whether the present observations on somatic embryos have any relevance to zygotic embryos, which are much less amenable to hormone analysis than somatic embryos. It turns out that very high levels of endogenous total IAA (even greater than 1000 ngg- ‘fr. wt ) are consistently measured in developing seeds and in developing seed and fruit composites, such as rye kernels [21], maize kernels [22,23], pine seeds [24], peach endocarps with enclosed seeds [25], and bean seeds [26]. Since these structures are principally composed of nutritive tissues, seed coats and other accessory tissues, it could not be determined whether the enclosed zygotic embryos have IAA concentrations comparable to those measured in the entire structures. The present results with free somatic embryos suggest that the early stages of zygotic embryogenesis must also be occasioned by high endogenous IAA, and thus, it appears that somatic embryos represent a realistic model system for studying auxin’s role in plant embryogenesis.
The present results provide further support for the idea that auxins play several roles in carrot somatic embryogenesis. It is repeatedly observed that 2,4-D is much more effective than other synthetic auxins in its ability to make carrot cells competent to undergo somatic embryogenesis [e.g. 1,2]. Such callus may, indeed, be capable of embryo initiation in part because its exposure to 2,4-D has induced very high levels of endogenous IAA (Fig. 5). However, the results with the non-embryogenic line show that a high IAA level is not sufficient by itself to induce the embryogenic potential of all lines (Fig. 6). The early stages of embryo development were also shown to have high to intermediate levels of total endogenous IAA, viz. over 650 ngg- ’ fr. wt in pre~obular fraction and over 100 ngg- ’ fr. wt in globular fraction (Fig. 5). Since the initial development of embryonic structures is similar whether or not auxin is included in the medium and since antiauxins appear to preferentially inhibit embryo initiation (see Introduction), then the high levels of endogenous auxin measured in our analyses suggest that some auxin-regulated process is important in the first stages of embryo development. Perhaps, high auxin activity helps to sustain the rapid rate of cell division required to construct the globular embryo. PHYTC 31:1-B
Cell culture. Carrot callus and embryo cultures were maintained by the routine procedures [27]. In brief, cell lines were started from the hypocotyls of domesticated carrot (Dawus caroraL. cv. Danvers). The callus cultures were maintained in 25 ml of MS medium [28] supplemented with 1 mg 1-r of 2,4-D in 125 ml flasks at 24” on a rotary shaker set at PO rpm. The cells were transferred to fresh medium every 7-10 days. Somatic embryos were initiated by fractionating the cells between stainless steel sieves (mesh size 43 and 109 pm) to obtain the cell clusters of the desired size. The collected frs were washed several times and resuspended in 2,4-D-free MS medium at a density of 10’ cell clusters per ml. After one week, the cell clusters were washed and resuspended in 25 ml of fresh MS medium without 24-D at a density of lo3 clusters per ml in order to initiate embryo formation. Using this procedure, many lines were evaluated for their embryogenic potential in order to select one nonembryogenic line which did not have the ability to form embryos and another embryogenic line which exhibited close to 100% embryogenesis. In the embryogenic line, S@-60% of the cell clusters developed into globular embryos within 7 days following the transfer to low-density conditions, and the vast majority of the embryos reached the heart stage in 21 days.
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L. MICHALCZUK
Snmple collection. Because embryo development is relatively asynchronous in carrot cultures, two different approaches were used for collecting frs for auxin analysis. In the first approach, the samples were taken as the 43-109 pm fr. directly from 2,4-D-plus medium (callus cells), as the entire flask after 7 days at highdensity conditions (preglobular embryos), and as the entire flask after additional 7 (globular embryos) and 21 days (post-globular embryos) at the low density conditions described above. Each individual flask represented a single replicate for each culture time. These frs were rinsed twice with MS medium and then transferred to cold homogenization buffer for analysis as detailed below. In the second approach, the samples were collected and divided into different stages of embryo development by fractionating 21-day-old embryonic cultures using stainless steel sieves with mesh sizes of 380, 280, 180, 109 and 43 pm. After twice rinsing the frs collected on each sieve with MS medium, the relative content of each stage in the individual frs was assessed using the criteria described in [27]. As the fr. collected on the 380 pm sieve also included some plantlets, torpedo embryos were hand-picked under a dissecting microscope from this fr. with a Pasteur pipette. A single replicate in this collection consisted of the sieved fr. from several flasks which were combined to provide enough material for analysis. All frs were frozen in liquid N, and kept at -70” until being analysed. Alrxin analysis. Auxin determinations were essentially done as described in ref. [29] with slight modifications to facilitate parallel determination of IAA and 2,4-D. All frs were immediately homogenized with cold 65% 2-propanol/35% 0.2 M imidazole buffer, pH 7.0 (4 ml g- 1 fr. wt ) in the presence of the following labelled standards: 100 ng each of [‘3C,]IAA (99 + % isotopic enrichment, synthesized as detailed in ref. [30]) and [“C&2,4-D (99% isotopic enrichment, Cambridge Isotope Laboratories) which serve as the internal standards to calibrate against extraction losses (for calculations, see ref. [30]) and co 1 kBq of C3H]IAA (specific acttvity of 803 GBq mol- ‘, Amersham) as a radioactive tracer for the chromatographic fractionations. After incubation for 1 hr in the homogenization buffer to allow for isotope equilibrium, cellular debris was removed by centrifugation, and the organic phase was evapd from the supematant in uacuo. The aqueous residue was divided into 3 parts: one part was used directly for analysis of free IAA and 2,4-D, the second part was submitted to mild alkaline hydrolysis (1 N NaOH, 1 hr at room temp.) and analysed for free and esterilied IAA and 2,4D, and the third part was subjected to strong alkaline hydrolysis (7 N NaOH, loo” for 3 hr under N2) and analysed for total (free, esterified and amide-bound) IAA and 2,4-D (for details, see ret [30]). The samples were purified on a Fisher PrepSep C,s disposable column and a Fisher PrcpSep amino disposable column that was followed by high performance liquid chromatography on a 4.6 x 125 mm C,, (Partisil ODS-3,5 m, Whatman) column that was eluted for 15 min with 20%MeCN-H,O in 1% HOAc followed by a rapid change to 40% MeCN-H,O containing 1% HOAc for an additional 15 min. The fracttons with IAA and 24-D were separately collected, evapd and methylated with CH,N,. The samples were analysed by 70 eV electron impact GC-MS-SIM with a GC-MS equipped with 15 m x 0.32 mm DB-1701 fused silica capillary (J & W Scientific). The ions at m/z 130 and 136 (unlabelled and i3C-labelled quinolinmm fragment ions) and at 189 and 195 (unlabelled and i3C-labelled molecular ions) were monitored for IAA analysis; and the ions at m/z 175 and 181 (unlabelled and ‘3C-labelled fragment ions) and 234 and 240 (unlabelled and ‘3C-labelled molecular ions) were monitored for 2,4-D analysis. The samples were injected in the splitless mode. Gas chromatography was carried out with an inj. temp. of 250”. The initial oven temp. was set at 140” for 1 min and then the temp. was increased at a rate of 16” mini up to 280”. The R,s for
et al.
the methyl esters of IAA and 24-D were 7.6 and 6.6 min, respectively. Full scan analyses confirmed peak purity and identity for both IAA and 2,4-D isolated from these plant materials (data not shown). A complete analysis of free and conjugated forms required a minimum of 200-300 mg of packed cells. Data analysis. Each treatment, i.e. either culture time or embryo stage, was replicated x 3. The results are presented as free hormone (direct assay of either IAA or 2,4-D), free + esterified hormone (the sum of free hormone and hormone released from ester-bound conjugates by mild alkaline hydrolysis) and the total hormone (the sum of free hormone and hormone released from both ester- and amide-bound conjugates by strong alkaline hydrolysis). The amounts of amide-bound conjugate and esterilied conjugates can be obtained by simple subtraction: amidebound conjugate = total -(free + esterified); esterified conjugate = (free + esterified) -free. Acknowledgements-This paper is dedicated to Aga Schulze on the occasion of her retirement from Michigan State University. We thank Dr H. Lamar Gibble and Marie K. Wykle (Brethren Service Exchange Programs) and Prof. S. W. Zagaja (Research Institute of Pomology, Sktemiewice, Poland), for their help and commitment to scientific personnel exchange. These studies were supported by United States Department of Agriculture competitive research grant 89-37261-4791, National Science Foundation grant DCB-8917378, and the U.S. -Israel Binational Agrtcultural Research and Development fund (BARD US-136287). This work was carried out. in part, under cooperative agreement No. 58-32U4-8-34 of the U. S. Department of Agriculture-Agricultural Research Service and the Universtty of Maryland. Scientific article number A6096 contribution number 8261 of the Maryland Agricultural Experiment Station.
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25. Miller, A. N., Walsh, C. S. and Cohen, J. D. (1987) Plant Physiol. 84,491.
26. Bialek, K. and Cohen, J. D. (1989) Plant Physiol. 91, 775. 27. Schiavone, F. M. and Cooke, T. J. (1985) Can. J. Botany 63, 1573. 28. Murashige, T. and Skoog, F. (1962) Physiol. Plant 15, 473. 29. Chen, K.-H., Miller, A. N., Patterson, G. W. and Cohen, J. D. (1988) Plant Physiol. 86, 822. 30. Cohen, J. D., Baldi, B. G. and Slovin, J. P. (1986) Plant Physiol. 80, 14.
0031~9422/92 SS.oO+O.OO Pergamon Press plc
AUXIN LEVELS AT DIFFERENT STAGES OF CARROT SOMATIC EMBRYOGENESIS
LECH MICHALCZUK,* TODD J. COOKE and JERRY D. COHEN~: Department
of Botany, University of Maryland, College Park, MD 20742, U.S.A.; tPIant Hormone Laboratory, Ag~cuttur~ Research Service, USDA, Beftsville Agricultural Research Center, Beltsvilie, MD 20705,U.S.A. (Received24 July 1991)
Key Word Index-Caucus carota;Umbelliferae; carrot; auxin; 2,4-dichlorophenoxyacetic somatic embryogen~is.
acid; indole-3-acetic acid;
Abstract-The role of auxin in somatic embryogenesis was evaluated by characterizing the changes in the concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), and their conjugates in callus suspension cells and developing embryos of llaucus carota. Both emb~ogenic and non-embryogenic lines exhibited similar growth rates and levels of IAA and 2,4-D on ~4-D-supplements medium. Total endogenous IAA in both lines exposed to 2,4-D reached high levels greater than 600 ng g- 1 fresh weight which suggests that IAA levels in carrot callus are not regulated via auxin feedback mechanisms. After being transferred to 2,4-D-free medium, the embryogenic line exhibited a rapid decline in both free and conjugated 2,4-D metabolites within seven days, while IAA levels remained relatively steady for seven days in the preglobular stage after which the levels declined steadily in all subsequent stages of embryo development. Individual analyses of different embryo fractions collected from asynchronous cultures confirmed that each stage in embryo development had lower IAA levels than the preceding stage. The non-embryogen~c line maintained similar 2,4-D levels but higher IAA levels than the embryogenic line throughout the experiment. The present results suggest that high IAA levels may be necessary but are not sufficient for the initial events in plant embryogenesis, whereas low IAA levels are associated with the later stages of embryo development.
INTRODUCTION
Physiological experiments indicate that both exogenous and endogenous auxins are intimately involved with several stages of carrot somatic embryo~nesis. Various sources of carrot cells, such as petiole explants [l], hypocotyl explants E23, and single cells isolated from established suspension cultures [3], require a minimum exposure of one, two or seven days, respectively, to the exogenous auxin 2,4-D (~~~c~orophenoxya~ti~ acid) in order to acquire the competence to eventually undergo somatic embryo development. In general, competent carrot cells continue to proliferate as an unorganized callus without any observable embryo initiation when cultured in a standard culture medium supplemented with 1 mg 1-l of 2,4-D; however, a few carrot lines are reported to produce pre~obular or even globular embryos in the presence of such typical 2,4-D 1eveIs [4,5]. Finally, exogenous auxins cause carrot embryos at later developmental stages to revert to proliferating callus [6}. These observations have led to the int~guing notion that once 2,4-D has made the cells com~tent to initiate somatic embryogenesis, it will then inhibit subsequent
*Permanent address: Research Institute of Pomology, 96-100 Skierniewice, Potand. $Author to whom correspondence shontd be addressed.
embryo development, with only the permissive condition, i.e. the absence of exogenous auxin, being necessary to complete embryo formation [7]. The endogenous auxin IAA (indole-3-attic acid) in cultured plant cells appears to have very different effects than the exogenous auxins used in most culture media. For instance, even though typical cultured cells accumulate free endogenous IAA at the levels of 2 to 20 ng g - ‘fr. wt [S, 93 which are comparable to those measured in the vegetative tissues of intact plants [lo], plant cell cultures are usually supplemented with exogenous auxin in order to maintain cell proliferation. More importantly, although carrot cells must be transferred to a 2,4-D-free medium before they can develop into mature embryos, it can be concluded from inhibitor experiments that endogenous auxin is, in contrast, required for the successful completion of embryo development. For example, the auxin analogues ~4,6-t~chiorophenoxyacetic acid and pchlorophenoxyisobutyric acid, which seem to act as competitive inhibitors of endogenous auxin activity [ 11, 121, will prefe~ti~ly inhibit embryo initiation, thereby suggesting that high endogenous IAA activity may be necessary for the initial stages of somatic embryogenesis [6, 131. Moreover, as the polar auxin transport inhibitors 2,3,5triiodobenzoic acid and N-( l-naphthyl)-phthalamic acid cause new embryonic axes to form on older heart and torpedo embryos, it appears that polar auxin transport is necessary to maintain the bipolar axis of the developing embryo [6].
1097
L. MICHALCZUK et al.
1098
Since our overall objective is to elucidate the underlying mechanisms by which auxin acts to regulate carrot somatic embryogenesis, we decided that it was first necessary to characterize the levels of both 2,4-D and IAA and their conjugates in callus cells and developing embryos. These hormone analyses were performed on two carrot cell lines, one of which has a high embryogenic potential, and the other of which lacks the ability to initiate any embryos. We observed that each stage of somatic embryogenesis is correlated with specific levels of free and conjugated auxin metabolites. Furthermore, the data demonstrate that the application of exogenous auxins like 2,4-D leads to significant changes in endogenous IAA levels of carrot cells, with the apparent consequence that these cells become competent to undergo somatic embryogenesis. An initial report of this work was presented elsewhere [ 141.
RESULTS AND DISCUSSION
clusters from the embryogenic line started to differentiate into globular embryos (Fig. lc) in seven days and heart and torpedo embryos in 21 days (Fig. Id). A rapid increase in fresh weight of the culture accompanied the formation of globular and post-globular embryos which occurred in the low-density conditions (Fig. 2). In contrast, the non-embryogenic line without 2,4-D exhibited no evidence of even globular embryos. This line showed a slight increase in fresh weight in the first week of low density conditions but no additional growth over the next two weeks even though it remained viable. As most lines lacking the potential to even initiate embryogenesis grow at much slower rates than embryogenic lines following the transfer to 2,4-D-free medium, the non-embryogenic line in this study does not represent the ideal control for our observations. The data from the non-embryogenic line are nonetheless informative, and thus the auxin levels of the two lines are compared in the following sections.
General features of the two cell lines
Auxin analyses of callus cells in 2,4-D-supplemented medium
Both the embryogenic and non-embryogenic cell lines grew at similar rates as unorganized callus with no observable embryos in 2,4-D-supplemented MS medium (Fig. la). The fractions of cell clusters 43-109 pm in diameter, which were obtained from both cell lines by sieving through two screens of these mesh sizes, were also composed only of callus cells (Fig. lb). However, when transferred to 2,4-D-free medium at low density, the cell
Although both 2.4-D and IAA undergo conjugation reactions as well as other metabolic transformations in plants, it was decided for the purposes of this work to concentrate on the free acids and the esterified and amidebound conjugates. The free acids are usually considered to act as the active form of the growth regulator while the conjugates seem to function as short-term storage forms that can readily be converted back into the free acids
Fig. 1. Representative fractions of callus cells and developing embryos in carrot suspension culture. (a) Undifferentiated callus cells cultured in MS medium plus 1 mgl- 1of 2,4-D. (b) 43-109 pm fraction collected from suspension cultures maintamed in the same medium as a. (c) Globular embryos isolated from the embryogenic line cultured in 2,4-D-free MS medium. (d) Heart and torpedo embryos isolated as in c The scale bar corresponds to 400 pm in all photographs.
Auxin levels during
carrot
embryogenesis
1099
104-
d
i
s
~ICKW--
9
b
A
p-=---l //-II’ _~p_____----__ E c
loo-~
i
,.
\*
t---l” ___________d
dN’
99.; c 0 7 Culture time en 2,4-D-free
_.
.:
14
17
21
mediumat low &nsit~Maysl
Fig. 2. The total fresh weight of cultured tissue of the embryogenic (open circles) and nonembryogenic (closed circles) lines at various times after being transferred to 2,4-D-free medium.
Fig. 3. The concentrations of 24-D and its conjugates in the embryogenic line before and after being transferred to 2,4-D-free medium. Open circles = free 2,4-D; closed circles = free + esterified 2,4-D, triangles = total 2,4-D. The concentration data are plotted as the log scale on the ordinate axis. The ‘c’ on the abscissa axis refers to callus cells, the ‘0’ refers to the preglobular stage following seven days in 2,4-D-free medium at the highdensity condition, and all numbers refer to the times for subsequent embryo development at the low-density condition. All data are presented as the treatment mean+ the standard error among three replicates. All symbols without error bars had a standard error less than the size of the symbol.
[15]. The level of total 2,4-D in suspension cells grown in 2,4-D-supplemented medium was quite similar in the two lines (Figs 3 and 4) (3.82f0.26 pgg-‘fr. wt in the embryogenic line vs 4.33 f0.16 pg g- ‘fr. wt in the nonembryogenic line). Assuming that the densities of the callus cells and the culture medium are both close to 1 g ml- ‘, then the callus cells had accumulated total 2,4D at a concentration roughly four times higher than the original concentration in fresh culture medium. However, as only 10% of the 2,4-D in the embryogenic line and 25% in the non-embryogenic line remained as free acid, it is likely that the uptake of the free form occurred by simple diffusion. In both lines, the bulk of the 2,4-D metabolites existed as ester- and amide-bound conjugates. The two cell lines grown in 2,4-D-supplemented medium had very high levels of endogenous total IAA (Figs 5 and 6) (672 + 24 ng g- ‘fr. wt in the embryogenic cell lines vs 603 + 44 ng g- ‘fr. wt in the non-embryogenic
:
:
c
0
:
:
7
Culture age on 2,4-O-fee
14
21
medium at bv denstiy Uoys)
Fig. 4. The concentrations of 24-D and its conjugates in the nonembryogenic line before and after being transferred to 24-D free medium. Open circles = free 2,4-D, closed circles = free + esterified 2,4-D; triangles = total 2,4-D. For additional explanation, see the legend of Fig. 3.
Culture
age on 2,4+free
medwm atlow density (days)
Fig. 5. The concentrations of IAA and its conjugates in the embryogenic line before and after being transferred to 2,4-D-free medium. Open circles = free IAA; closed circles = free + esterified IAA; triangles = total IAA. For additional explanation, see the legend of Fig. 3.
A
0 0
Culture age on 2,4-D-free
Fig. 6. The concentrations
medwm ot low density (days)
of IAA and its conjugates
in the
nonembryogenic line before and after being transferred to 2,4-Dfree medium. Open circles = free IAA; closed circles = free + esterified IAA, triangles = total IAA. For additional explanation, see the legend of Fig 3.
line). Both lines had roughly 80% of the IAA in the form of amide-bound conjugate(s) and similar low amounts of the free and esterified forms. In another experiment, it was also observed that the embryogenic line in the 2,4-
L.
1100
MICHAIGWK
D-supplemented medium gradually released IAA into the medium so that the concentrations of free and total IAA averaged 13 and 70 pg 1-l of the medium, respectively, with no detectable esterified IAA in one-month-old cultures (data not shown). Since most cell cultures appear to require an exogenous auxin to sustain cell proliferation, it was expected that carrot cells grown in 2,4-D-supplemented medium would have low concentrations of the endogenous auxin IAA; for example, the levels of free IAA in sycamore cells in 2,4D-supplemented medium can be computed (on the basis of a cell density of 1 gml - ‘) to range from 1 to 15 ngg-‘fr. wt over the course of the culture period [16]. However, the IAA levels observed in the callus cells of both carrot lines (Figs 3,4) were markedly higher than those reported for other cell cultures [8, 9, 161 or most vegetative tissues of intact plants [lo]. This observed ability of 2,4-D to mediate a very high IAA concentration in carrot cells reveals a different aspect of hormone metabolism in these cells. Most metaboiic pathways, such as those responsible for amino acid synthesis, are primarily regulated by feedback inhibition where the product and its analogues are able to reduce product synthesis by inhibiting an earlier enzyme in the pathway [17]. However, it has been argued that the measured concentrations of plant hormones are too low for feedback inhibition by itself to serve as an effective mechanism of metabolic regulation [18]. The present work on carrot cell cultures provides direct experimental evidence for this argument: the analogue 2,4-D must instead promote IAA synthesis and/or inhibit its degradation, with other reversible conjugating reactions controlling the concentration of the active free IAA, as has been described for other plant systems [15]. The underlying mechanism of 2,4-D’s action on IAA metabolism in carrot cells is being investigated in our laboratories. Auxin analyses of culrured cells in embryogenic conditions
The transfer to 2,4-D-free medium caused a very sharp decline of all forms of 2,4-D in both lines, with slight differences observed between the two lines (cf. Figs 3 and 4). In the embryogenic line, the level of free 2,4-D dropped to very low levels during the initial week at high density which corresponds to the pre~lobular stage of embryo development. increased dramatically during the first Table 1. Auxm content in ngg-‘frwt
et al.
week at low density (globular stage) and then slowly declined during the next two weeks (post-globular stages). Conjugated 2,4-D underwent an almost 30-fold drop during the first week and then decreased slowly over the next three weeks. After three weeks in 2,4-D-free medium at low density, the post-globular embryos had ca 20 ngg- ’ fr. wt of total 2,4-D, with approximately equal amounts of the free form and of amide-bound conjugate and almost undetectable levels of ester conjugate(s) (Fig. 3). In the non-embryogeni~ line, the initial drop in the free 2,4-D level was not so pronounced as observed in the embryogenic line. Moreover, the final concentration of total 2,4-D was two times higher in the non-embryogenic line. but the final level of free hormone was very similar in both lines (Fig. 4). As the sole source for the increased free 2.4-D observed in the globular embryos of the embryogenic cultures was the two conjugate pools, metabolic equilibria comparable to those operating in IAA metabolism must also exist between free and conjugated 2,4-D [cf. 193. After the transfer to 2,4-D-free medium at the highdensity condition (preglobular stage), the total IAA in the embryogenic line remained relatively constant but the next transfer to 2,4-D-free medium at low density was followed by a rapid decrease in total IAA (Fig. 5). The formation of globular embryos in this period is apparently accompanied by the dramatic loss of amide conjugate(s) while no significant change was observed in the free and esterified forms. Subsequently, the concentrations of all forms of IAA decreased slowly at similar rates. The post-globular fraction at three weeks had ca 20 ngg _‘fr. wt of total IAA, with 25% in free and 10% in esterified forms. The decrease in IAA levels occurred at much slower rates in the non-embryogenic line as compared to the embryogenic line (Fig. 6). This decrease was primarily attributable to the depletion of IAA conjugates while the free IAA remained relatively constant for the four weeks following the transfer to 2,4-D-free medium. The final concentration of IAA in the non-embryogenic line (37+5 ngg-ifr.wt of free IAA and 186f16 ngg- ‘fr.wt of total IAA) was almost 10 times higher than the levels measured in the embryogenic line, although the relative propo~ions of the three forms are close in the two lines (Fig. 6). The major difference between the two lines was that the free IAA underwent a steady decline for the globular to the torpedo stages in the embryogenie line
m carrot somatic embryos at various developmental stages
Fraction size (pm)
43-109
109180
180-280
280-380
>380
Embryo stage
80% globular 20% unditT.
45% globular 55% oblong
30% oblong 55% heart 15% other
85% heart 15% other
100% torpedo
31 (71 52 (IO) 67 (14) 27 (6) 31 (5) 56 (8)
6 (2) 23 (3) 31 (16) 16 (3) 44 (7) 70 (10)
8 (3) 12 (2) 29 (1) g (3) 36 (6) 33 (3)
8 (I) 7 (3) 20 (9) 5 (2) 7 (2) 23 (8)
5 (1) 6 (2) 18 (1) 4 (1) g (2) 12 (3)
I AA
2,4-D
free free t ester total free free + ester total
The different fractions were isolated by fractionating cultures with different-sized sieves and by collecting individual embryos as described in the Experimental. The data are presented as &hetreatment mean with the standard error of three replicates in parentheses.
Auxin levels during carrot embryogenesis while it was constant throughout the same time period in the non-embryogenic line. The relative changes in the three pools in the embryogenic line may indicate that an equilibrium exists between the free and amide pools, with ester conjugates as possible intermediates. Similar interconversions of other IAA conjugates have been characterized in maize kernels where a specific acyi transferase catalyses the transfer of the IAA moiety from the highenergy l-O-glucosyI-IAA to the more stable inositol ester EW‘ Auxin analyses of digerent stages of embryo development
Since embryo development was somewhat asynchronous in the embryogenic line and since bulk embryonic cultures of this line as measured in the preceding experiments contained a small proportion of undifferentiated cells, 21-day-oldcultures of the embryogenic line were fractionated with different-sized sieves in order to obtain more precise measurements of the IAA and 2,4-D levels in each stage of embryo development (Table 1). The total IAA level was observed to decrease at each successive stage of embryo development from 67 ngg- ‘fr. wt in globular embryos to 18 ng g- ‘fr. wt in torpedo embryos (Table 1). However, the free IAA level was observed to drop more than 80% from the 43-109 pm fraction (80% globular embryos) to the 109-180 pm fraction (globular and oblong embryos) after which it was maintained at the constant low level. Esterifled and amide-bound IAA was measured in globular and oblong embryos, but only the amide conjugate could be detected in heart and torpedo embryos. Despite the extensive period spent in 2,4-D-free medium before embryo isolation, a significant amount of residual 2,4-D remained in all embryonic fractions (Table 1). The highest levels of all three forms were found in the globular and oblong embryonic fraction, with progressively lower levels in more mature embryos. The possible role of auxin metabolism in plant embryogenesis
1101
However, the later stages of embryo development are occasioned by much lower levels of total endogenous IAA, viz. 20-30 ngg- rfr. wt (Table 1). Apparently, these low auxin levels allow the embryos to successfully complete their development, because globular embryos grown in auxin-supplemented medium dedifferentiate into proliferating callus (see Introduction). More importantly, low endogenous auxin activity may permit postglobular embryos to establish internal auxin gradients which are apparently necessary to initiate and maintain polarized growth. In particular, polar auxin transport inhibitors do not affect either the preglobular or globular stages; however, these inhibitors do specifically interfere with the ability of late globular embryos to initiate polarized growth and the ability of heart embryos to maintain a single bipolar axis [6]. Thus, it appears that the postglobular stages are especially sensitive to either exogenous auxins, which may perturb the metabolic homeostasis of endogenous IAA, or transport inhibitors, which may disrupt endogenous auxin gradients. Finally, it is important to address the question of whether the present observations on somatic embryos have any relevance to zygotic embryos, which are much less amenable to hormone analysis than somatic embryos. It turns out that very high levels of endogenous total IAA (even greater than 1000 ngg- ‘fr. wt ) are consistently measured in developing seeds and in developing seed and fruit composites, such as rye kernels [21], maize kernels [22,23], pine seeds [24], peach endocarps with enclosed seeds [25], and bean seeds [26]. Since these structures are principally composed of nutritive tissues, seed coats and other accessory tissues, it could not be determined whether the enclosed zygotic embryos have IAA concentrations comparable to those measured in the entire structures. The present results with free somatic embryos suggest that the early stages of zygotic embryogenesis must also be occasioned by high endogenous IAA, and thus, it appears that somatic embryos represent a realistic model system for studying auxin’s role in plant embryogenesis.
The present results provide further support for the idea that auxins play several roles in carrot somatic embryogenesis. It is repeatedly observed that 2,4-D is much more effective than other synthetic auxins in its ability to make carrot cells competent to undergo somatic embryogenesis [e.g. 1,2]. Such callus may, indeed, be capable of embryo initiation in part because its exposure to 2,4-D has induced very high levels of endogenous IAA (Fig. 5). However, the results with the non-embryogenic line show that a high IAA level is not sufficient by itself to induce the embryogenic potential of all lines (Fig. 6). The early stages of embryo development were also shown to have high to intermediate levels of total endogenous IAA, viz. over 650 ngg- ’ fr. wt in pre~obular fraction and over 100 ngg- ’ fr. wt in globular fraction (Fig. 5). Since the initial development of embryonic structures is similar whether or not auxin is included in the medium and since antiauxins appear to preferentially inhibit embryo initiation (see Introduction), then the high levels of endogenous auxin measured in our analyses suggest that some auxin-regulated process is important in the first stages of embryo development. Perhaps, high auxin activity helps to sustain the rapid rate of cell division required to construct the globular embryo. PHYTC 31:1-B
Cell culture. Carrot callus and embryo cultures were maintained by the routine procedures [27]. In brief, cell lines were started from the hypocotyls of domesticated carrot (Dawus caroraL. cv. Danvers). The callus cultures were maintained in 25 ml of MS medium [28] supplemented with 1 mg 1-r of 2,4-D in 125 ml flasks at 24” on a rotary shaker set at PO rpm. The cells were transferred to fresh medium every 7-10 days. Somatic embryos were initiated by fractionating the cells between stainless steel sieves (mesh size 43 and 109 pm) to obtain the cell clusters of the desired size. The collected frs were washed several times and resuspended in 2,4-D-free MS medium at a density of 10’ cell clusters per ml. After one week, the cell clusters were washed and resuspended in 25 ml of fresh MS medium without 24-D at a density of lo3 clusters per ml in order to initiate embryo formation. Using this procedure, many lines were evaluated for their embryogenic potential in order to select one nonembryogenic line which did not have the ability to form embryos and another embryogenic line which exhibited close to 100% embryogenesis. In the embryogenic line, S@-60% of the cell clusters developed into globular embryos within 7 days following the transfer to low-density conditions, and the vast majority of the embryos reached the heart stage in 21 days.
1102
L. MICHALCZUK
Snmple collection. Because embryo development is relatively asynchronous in carrot cultures, two different approaches were used for collecting frs for auxin analysis. In the first approach, the samples were taken as the 43-109 pm fr. directly from 2,4-D-plus medium (callus cells), as the entire flask after 7 days at highdensity conditions (preglobular embryos), and as the entire flask after additional 7 (globular embryos) and 21 days (post-globular embryos) at the low density conditions described above. Each individual flask represented a single replicate for each culture time. These frs were rinsed twice with MS medium and then transferred to cold homogenization buffer for analysis as detailed below. In the second approach, the samples were collected and divided into different stages of embryo development by fractionating 21-day-old embryonic cultures using stainless steel sieves with mesh sizes of 380, 280, 180, 109 and 43 pm. After twice rinsing the frs collected on each sieve with MS medium, the relative content of each stage in the individual frs was assessed using the criteria described in [27]. As the fr. collected on the 380 pm sieve also included some plantlets, torpedo embryos were hand-picked under a dissecting microscope from this fr. with a Pasteur pipette. A single replicate in this collection consisted of the sieved fr. from several flasks which were combined to provide enough material for analysis. All frs were frozen in liquid N, and kept at -70” until being analysed. Alrxin analysis. Auxin determinations were essentially done as described in ref. [29] with slight modifications to facilitate parallel determination of IAA and 2,4-D. All frs were immediately homogenized with cold 65% 2-propanol/35% 0.2 M imidazole buffer, pH 7.0 (4 ml g- 1 fr. wt ) in the presence of the following labelled standards: 100 ng each of [‘3C,]IAA (99 + % isotopic enrichment, synthesized as detailed in ref. [30]) and [“C&2,4-D (99% isotopic enrichment, Cambridge Isotope Laboratories) which serve as the internal standards to calibrate against extraction losses (for calculations, see ref. [30]) and co 1 kBq of C3H]IAA (specific acttvity of 803 GBq mol- ‘, Amersham) as a radioactive tracer for the chromatographic fractionations. After incubation for 1 hr in the homogenization buffer to allow for isotope equilibrium, cellular debris was removed by centrifugation, and the organic phase was evapd from the supematant in uacuo. The aqueous residue was divided into 3 parts: one part was used directly for analysis of free IAA and 2,4-D, the second part was submitted to mild alkaline hydrolysis (1 N NaOH, 1 hr at room temp.) and analysed for free and esterilied IAA and 2,4D, and the third part was subjected to strong alkaline hydrolysis (7 N NaOH, loo” for 3 hr under N2) and analysed for total (free, esterified and amide-bound) IAA and 2,4-D (for details, see ret [30]). The samples were purified on a Fisher PrepSep C,s disposable column and a Fisher PrcpSep amino disposable column that was followed by high performance liquid chromatography on a 4.6 x 125 mm C,, (Partisil ODS-3,5 m, Whatman) column that was eluted for 15 min with 20%MeCN-H,O in 1% HOAc followed by a rapid change to 40% MeCN-H,O containing 1% HOAc for an additional 15 min. The fracttons with IAA and 24-D were separately collected, evapd and methylated with CH,N,. The samples were analysed by 70 eV electron impact GC-MS-SIM with a GC-MS equipped with 15 m x 0.32 mm DB-1701 fused silica capillary (J & W Scientific). The ions at m/z 130 and 136 (unlabelled and i3C-labelled quinolinmm fragment ions) and at 189 and 195 (unlabelled and i3C-labelled molecular ions) were monitored for IAA analysis; and the ions at m/z 175 and 181 (unlabelled and ‘3C-labelled fragment ions) and 234 and 240 (unlabelled and ‘3C-labelled molecular ions) were monitored for 2,4-D analysis. The samples were injected in the splitless mode. Gas chromatography was carried out with an inj. temp. of 250”. The initial oven temp. was set at 140” for 1 min and then the temp. was increased at a rate of 16” mini up to 280”. The R,s for
et al.
the methyl esters of IAA and 24-D were 7.6 and 6.6 min, respectively. Full scan analyses confirmed peak purity and identity for both IAA and 2,4-D isolated from these plant materials (data not shown). A complete analysis of free and conjugated forms required a minimum of 200-300 mg of packed cells. Data analysis. Each treatment, i.e. either culture time or embryo stage, was replicated x 3. The results are presented as free hormone (direct assay of either IAA or 2,4-D), free + esterified hormone (the sum of free hormone and hormone released from ester-bound conjugates by mild alkaline hydrolysis) and the total hormone (the sum of free hormone and hormone released from both ester- and amide-bound conjugates by strong alkaline hydrolysis). The amounts of amide-bound conjugate and esterilied conjugates can be obtained by simple subtraction: amidebound conjugate = total -(free + esterified); esterified conjugate = (free + esterified) -free. Acknowledgements-This paper is dedicated to Aga Schulze on the occasion of her retirement from Michigan State University. We thank Dr H. Lamar Gibble and Marie K. Wykle (Brethren Service Exchange Programs) and Prof. S. W. Zagaja (Research Institute of Pomology, Sktemiewice, Poland), for their help and commitment to scientific personnel exchange. These studies were supported by United States Department of Agriculture competitive research grant 89-37261-4791, National Science Foundation grant DCB-8917378, and the U.S. -Israel Binational Agrtcultural Research and Development fund (BARD US-136287). This work was carried out. in part, under cooperative agreement No. 58-32U4-8-34 of the U. S. Department of Agriculture-Agricultural Research Service and the Universtty of Maryland. Scientific article number A6096 contribution number 8261 of the Maryland Agricultural Experiment Station.
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