DEVELOPMENTAL
BIOLOGY
143,408-417
(19%)
A Kinematic Analysis of Gynoecial Growth in Lilium longiflorum: Surface Growth Patterns in All Floral Organs Are Triphasic
Sequential marking experiments combined with histological and morphological examination indicate that growth of the gynoecium in Liliu~z hng$kmm~ Thunb. (Easter lily) is triphasic. Phase I (gynoecia less than 10 mm in length) is characterized by spatial and temporal variation in local relative growth rate and mitotic index throughout the entire gynoecium. In Phase II (lo-100 mm), a steady growth peak is restricted to the proximal 5 to 15 mm of the style, while the ovary shows uniform growth. In Phase III (loo-135 mm), the ovary ceases growth and the maximal growth zone of the gynoecium migrates from the proximal to the distal end of the style. Comparison of surface growth patterns and differentiation events in the gynoecium to those previously described in the tepal and anther demonstrates the triphasic :a 1991 Academic Press, Inc. nature of growth in the whole lily flower.
ity and actual enlargement of the plant organ. In another approach, Ritterbusch (1980,1989) has used natural features of the petal surface to describe trajectories of organ growth and predict relative timing of developmental events. Although more quantitative than other descriptions and utilizing a surface marking technique, these studies lack an ix ~ivo or real time component. Growth can be considered in several different ways (Hunt, 1982): the total absolute growth of the organ, the relative growth rate of the organ, and the local relative growth rate of regions along a dimension of the organ. Surface marking experiments can test assumptions about the distribution of growth in an organ by using time-lapse photographs of marked organs to highlight variations in growth rates along the length of an organ (Silk, 1984). Segments of an organ can be delineated by applied marks and their relative elongation rates compared over time. These marking experiments document kinematic effects, motion in the abstract, but do not imply any information about the forces driving growth. In purely descriptive terms, steady growth is centered in the same region over time, as seen in the indeterminate shoot or root apices of plants. In contrast, nonsteady growth occurs when growth centers shift during ontogeny, as in determinate organs such as leaves (Silk, 1984). Surface marking studies have only recently been attempted in floral organs (Lord and Gould, 1989). The Easter lily, Lilizrm lo~g~flo~um Thunb. (Liliaceae) is a useful model organism for growth studies (Erickson, 1948). It has a large flower (150-160 mm at anthesis or maturity) that is hardy enough to withstand the dissection necessary to mark internal organs. The first marking experiments with lily flowers demonstrated that anthers (the pollen-containing part of the stamen) show
INTRODIJCTION
Plants produce their organs from permanently “embryonic” regions called apical meristems. Flowers, the reproductive structures of the angiosperms or flowering plants, are produced by vegetative meristems that have been induced to flower. The floral organs are often formed in acropetally produced whorls, with the perianth initiated first and usually composed of sepals and petals. The stamens are initiated next, and then the carpels or pistils (collective term, gynoecium) are initiated, typically in sequential order. Flowers are complex structures, difficult to follow developmentally. As a result, Greyson’s (1972) and other researchers’ calls for quantitative descriptions of floral organ growth have gone unheeded, for the most part. There are few such studies of the gynoecium, the most complex of the floral organs being heterogeneous in its composition and function. The stigma is a site for pollen capture and hydration; the style is a path for pollen tube growth; and the ovary is the site of fertilization and fruit formation. Previous workers have described the ontogeny and histogenesis of the gynoecium, usually with a comparative, systematic focus (e.g., Sattler, 1973; van Heel, 1981). The use of periclinal chimeras has demonstrated the relative contribution of different apical meristematic layers to gynoecial growth (Satina and Blakeslee, 1943; Dermen and Stewart, 1973). Anatomical studies tend to emphasize localized growth through “meristerns,” or histologically separable regions of cell division and expansion (Boke, 1949; Kaplan, 1968; Weberling, 1989). The techniques used are based on the assumptions of steady-state distributions of growth centers and close relationships between the visible “meristematic” activOOlZ-1606/91 Copyright All rights
$3.00
:C’ 1991 by Academic Press, Inc. of reproduction in any form reserved
408
a nonsteady wave pattern of growth throughout ontogeny (Gould and Lord, 1988). Lily tepals (the term used for perianth organs when they are not divided into distinct sepals and petals) show three phases, including both nonsteady and steady growth (Gould and Lord, 1989). These were patterns that would not have been predicted on the basis of histology or scanning electron micrographs (SEMs)l alone. Previous authors have documented the early initiation patterns of lily flowers (Pfeiffer, 1935; Cremer et al., 1974; de Hertogh ef (LI, 1976). In the present study, we describe the growth patterns in the gynoecium after initiation, utilizing surface marking techniques in combination with SEM and histology. In addition, we provide a composite picture of a kinematic analysis for the entire lily flower. MATERIALS
AND
METHODS
Bulbs of L. lor&lwum cv. Nellie White (Dahlstrom and Watt Bulb Farms, Smith River, CA) were planted in pots and grown in a greenhouse in Riverside throughout the year, with temperatures ranging from 5 to 35°C. At least 2 weeks prior to the marking experiments, the pots were transferred to a growth chamber at one of two settings: 25°C with an 1%hr photoperiod (35 plants), or 21°C and a 12-hr photoperiod (9 plants). The growth chamber had a photon fluence rate of 375 PE me2 s-l with both incandescent and fluorescent light.
The method of Erickson (1948) was used to estimate the in sita growth rate of the gynoecium in control buds. Buds grown under the 25°C X 1%hr regimen (N = 74) were measured daily, from tip to base of the tepal. Fresh and fixed buds (N = 119) were used to plot allometric constants of gynoecial versus bud growth. The relative rate of elongation of gynoecia in situ was estimated as the product of the allometric constant k for the gynoecial and tepal lengths and r as the relative elongation rate of the entire bud.
One side of the gynoecium was exposed for its entire length by removing a portion of the tepals plus one to three stamens with a scalpel. The exposed gynoecium was marked with a file of approximately equidistant dots of activated charcoal in a viscous mixture with water or petroleum jelly. The cut tepal edges were coated with petroleum jelly and the entire bud was cov-
1 Ahhreviations useti: DAPI, ~,6-tliamidino-2-~),henYlindolc chloride; FAA, formaldrhydc~acetic acid-alcohol; LRGR, tivr growth rate: SEM, scanning elrctron micrograph.
tlihydrolocal rcla-
ered with polyethylene film to minimize desiccation. The first and last marks were always located at the base and the tip of the gynoecium, respectively. In total, 126 gynoecia varying in length from 2.0 to 133.5 mm were marked. In six buds (gynoecial length 3.4-9.9 mm) a sample of one of each organ was marked along its length simultaneously. Each marked organ was photographed alongside a reference scale using Kodak Technical Pan 2415 black and white film (Eastman Kodak, Rochester, NY) at 24-hr intervals, the minimum time for measurable growth. Additional marks were placed between dots when original ones grew more than 4 to 5 mm apart. Most gynoecia grew for 1 to 3 days after the initial marking. Prints were enlarged up to a final magnification of 20-40X. Recognizable features of the individual marks were used to delineate segments for each 24-hr period. The distance separating each mark from the base of the gynoecium was recorded from the prints using a Summagraphics (Fairfield, CT) digitizer and bit pad data tablet coupled to an IBM (Boca Raton, FL) personal computer. For the smallest gynoecia, both top and bottom features of marks were used to increase the number of data points. Data manipulations were performed using the spreadsheet software SuperCalc 4 (Computer Associates International, San Jose, CA). In order to highlight localized regions of growth and yet smooth the growth curves, a running refit analysis was undertaken for each day’s data (Erickson, 1976; Hunt, 1982). We used overlapping sets of four marks along the organ. Local relative growth rate (LRGR) for a segment was calculated as the difference in the values of the natural logarithms of the four point segment length over time (Erickson, 1976; Silk, 1984). LRGR was plotted versus the distance of the initial midpoint of the four point segment measured from the base of the gynoecium. Our study focused on the ovary and the style, since these two regions make up most of the length of the gynoecium.
Gynoecia in control buds grown in both the greenhouse and growth chamber were fixed in formaldehydeacetic acid-alcohol (FAA) (Johansen, 1940), dehydrated in an ethanol series, infiltrated and embedded with glyco1 methacrylate (JB-4, Polysciences, Inc., Warrington, PA), serially sectioned at 3 to 5 pm, and stained with either 1% aqueous toluidine blue-0 or 1% aqueous acid fuchsin (McCully and O’Brien, 1981). For determination of the mitotic index, serial sections at intervals of 9 to 200 pm apart, depending on the total length of the gynoecium, were scored for mitotic figures and total cells present on that section. Since mitoses were infrequent, any cell at mid prophase to late telophase was scored as
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DEVELOPMENTALBIOLOGY
++---I--------
a
0
10 20 30 Time [days)
40
50
b
Length
of bud
(mm)
FIG. 1. Itr situ growth of control buds of Lili/otr Io?~,qiflwrrnr. (a) Bud length vs time (K = 741. The smallest buds that could be measured ir, sitar were 3.5 mm in length. Arrow = anthesis. Bars are t I standard deviation. ? = 0.999. (b) Allometric plot of ggnoecium length versus tepal length (A’ = 119). P = 0.977.
a mitotic figure. The mitotic index for that section was determined by the number of mitoses/total number of cells X 100. These values were plotted against the distance of that section from the base of the gynoecium, with enough sections scored to create a mitotic index profile along the entire length of the gynoecium.
VOLUME 1X3,1991
anthesis, Y’,was calculated by linear regression to be r = 0.09391day. The logarithmic plot of tepal length vs gynoecial length was allometric (Fig. lb). In other words, there was a constant relationship between the elongation of the two organs with an allometric constant of k = 1.253, derived by linear regression. The relative rate of gynoecial elongation was calculated as kr = O.lWday. This allometric plot allowed the estimation of gynoecial size without dissection of the flower. In addition, this iv sit,u known relative growth rate supplied a standard or control to compare with gynoecia treated by dissection and marking. The closeness of the fit (r2 = 0.977) demonstrated that the gynoecium grew as a unit.
After initiation of the three carpel primordia that fuse laterally and form a cylindrical structure, most of the early vertical elongation of the gynoecium was in the region of the ovary (Figs. 2a-2c, 3a, 3b). The stigma was the first to differentiate as evidenced by precocious Other Mitotic Determinations cell enlargement (Fig. 2~). By the time the gynoecium was 4 to 5 mm, the style had begun to intercalate beTwenty-seven gynoecia (2-112 mm in length) from control flowers were fixed overnight in 3:l 95%1 tween the stigma and the ovary (Fig. 3b). A defined ethanokglacial acetic acid. They were then softened in ridge at the junction of the ovary and proximal style was 10% aqueous sodium sulfite for at least an hour, and noticeable after the gynoecium reached a total length of 8 to 10 mm or more (Fig. 3~). From this point until antransferred to 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) aqueous solution, 0.5 pg/ml, for at least thesis, the majority of length increase in the gynoecium was stylar (Figs. 3d-3f). 2 hr (McCully and O’Brien, 1981). DAPI is a fluorescent stain for DNA. The gynoecia were sliced in half, gently squashed onto a slide, and viewed with a Zeiss fluorescence microscope with Zeiss filter set 487702. This technique was used to estimate cell division activity in One-hundred and twenty-six gynoecia on 44 plants various regions of the gynoecium. were marked under the above growth chamber conditions (Figs. 3a-3f). Of these, 101 (80%) had significant Scwnniqy Electron Microscopy und 3-D Computer portions of the LRGR profile at or above O.OG/day for 24 Reconstruction hr or more and so were considered appropriate for furYoung control gynoecia were fixed and dehydrated as ther analysis. Of the 101 gynoecia analyzed, 46 (46%) above, critical point dried with CO,, coated with gold- grew at or faster than the in situ rate of 0.118/day, and palladium, and viewed in a JOEL (Tokyo, Japan) 35C 55 (54%) grew slower. The growth of lily gynoecia from SEM. One entire control bud (length 1.9 mm) was pre- 2.0 mm to maturity could be separated into three dispared and sectioned serially as described above. Camera tinct phases, as determined from the surface marking lucida tracings of sections throughout the bud were di- experiments. These observations were based on a total gitized into an Evans and Sutherland (Salt Lake City, of 199 plots showing the spatial distribution of LRGR in UT) ES 390 computer. The reconstruction program Anthe 101 gynoecia. atograph (Applied Medical Systems, Salt Lake City, UT) Phase I was a period of spatial and temporal variation was used to create a 3-D image. in growth rate, i.e., nonsteady growth (Figs. 4 and 5). This phase encompassed gynoecia 2.0 to 10 mm in length RESULTS (Figs. 3a and 3b). The peaks in the LRGR profile showed rises and falls with no predictable location in space. This Growth Profiles was particularly so for those gynoecia less than 5 mm in Elongation of the floral bud, i.e., tepal length, of Lillength (Figs. 3a and 4). After the start of stylar intercaium was exponential until anthesis, when growth ceased lation at 5 mm (Fig. 3b), the base of the style began to (Fig. la). The relative rate of elongation for growth until predominate as the site of the greatest LRGR, even
CRONE
growth in Lilium lo~z&kw-um. FIG. : Early gynoecial S, stigma; o, ovary; 1, ovary locule. 3.0 mm gnoecium.
AND
LORD
Gynoecial
Growth
(a) SEM of 1.4 mm gynoecium; Scale bars, 250 Frn.
in Liliu
(b) SEM
m
of 2.2 mm gynoecium;
(c) longitudinal
section
of
412
FIG. 3. Dissected buds of Lili~w lo~ry~or~rrc style intercalation; (c) 9.0, arrowhead on joint c); 10 mm (d, e, f).
DEVELOPMENTALBIOLOGY
VOLUME 143, 1991
with marks applied to gynoecia of various lengths (mm): (a) 2.2; (b) 5.2, arrow indicates site of between ovary and style; (d) 20.7; (e) 44.7; (f) 123.5, arrow on mature stigma. Scale bars: 1 mm (a, b,
though peaks of growth could still be found throughout the gynoecium (Fig. 5). Phase II, gynoecia 10 to 100 mm in length, was characterized by predominantly stylar growth, with the LRGR maximum localizing to the basal 5 to 15 mm of the style (Figs. 3c-3e and 6). The magnitude of the LRGR here was greater than that seen in Phase I. In gynoecia 10 to 30 mm in length, growth occurred throughout the Z-10 mm style (Figs. 3c-3d, 6a, 6b). As the style elongated in the 30 to 100 mm gynoecia, the location of the LRGR maximum appeared in a 5 to 15 mm range from the base of the style (Figs. 3e, 6c, 6d). During this phase, the ovary, when studied apart from the whole gynoecium, showed steady, uniform growth, reaching maturity by early Phase III (Figs. 6d and
‘7a). The stigma also reached its mature size by early Phase III. Phase III, in gynoecia 100 to 135 mm in length, was characterized by nonsteady growth, with a shift in the LRGR maximum from the proximal to the distal end of the style (Figs. 3f and 7). This occurred in two ways: the LRGR maximum appeared to be either an acropetally moving single wave during this phase (8 of 14 observations) (Fig. 7), or the base of the style maintained a separate LRGR peak from the apically progressing one (6 of 14 observations). In both cases,by anthesis, almost all growth was at the distal lo-20 mm of the style (Fig. 7~). For the six Phase I buds which had a sample of all types of floral organs marked simultaneously, there was
CRONE
-.I]
, 0
a
.4
1 Illstance
AND
LORD
Distance
, 2 from
3 base
from base
j?rnl
b
Oi-
5
D~smce
lo-
from base
(mm]
4 (mm)
1
FIG. 6. LRGR profiles of two individual Lilirrnl Itmg~&run/ gynoecia over 2 days of growth during early (a, b) and late (c, d) Phase II. Initial gynoecium length, 16.0 mm. (a) Day 1; tb) Day 2. Initial gynoecium length, 52.2 mm. Cc) Day 1; ((11 L)ay 2. Arrow indicates ovary/style junction.
l-
bol
Distance
2
from
3
base
(mm)
4
4-
early Phase I, the lower and upper rz:rions of the ovary are growing maximally, but soon the upper ovary and the style become a more common site for the maximum LRGR in this phase of nonsteady growth throughout the gynoecium. During most of Phase II, the lower style is the predominant site of growth, although by early Phase III, the LRGR maximum migrates up the style to the apex where it remains until anthesis when a small surge of expansion occurs as well in the whole organ.
3 z' 2 T2
.2.I
E J
O-.1-
& 0
C
1 Distance
2 from
3 base
4 (mm1
FIG. 4. Local relative growth rate (LRGR) profiles of one Li/i/rnl lor/qiflo/xn/ gynoecium over 3 days of growth during early Phast I. Initial length, 3.9 mm. (a) Day 1; 0)) Day 2; (cl Day 3.
no consistent pattern in growth profiles. The locations of LRGR maxima and minima appeared to be random among the organs (data not shown). Definite relationships exist among the different parts of the gynoecium throughout development (Fig. 8). In
a
I ‘3
? Distance
4 from base
6 lmml
bo-ra Distance
from base
$ imml
FIG. 5. LRGR profiles of one Lilirrnl /on~~jflor.~rtc gynoecium over 2 days of growth during late Phase I. Initial length, 7.4 mm. The style has begun to intercalate between the ovary and stigma. (a) Day 1; (b) Day 2. Arrow indicates ovary/style junction.
In Phase I (gynoecia < 10 mm), the peaks of LRGR were interpreted to represent regions of primarily cell expansion and the troughs, regions of primarily cell division. The smallest marked gynoecium in this study was 2.0 mm. Since the nonsteady growth in Phase I may be set up early, serial sections of gynoecia 0.36 to 4 mm (N = 8) were examined for patterns of mitotic activity along the organ. Examination of the profiles of mitotic index confirmed that cell division activity was not uniform nor was it localized consistently to specific regions of the gynoecium (Fig. 9). These data correlate with the growth profiles from the surface marking experiments. The DAPI-stained whole gynoecia gave a grosser picture of mitotic activity for all phases of growth. In gynoecia 2 to 9 mm in length (Phase I), mitotic activity occurred at a high level throughout the gynoecium. In gynoecia 11 to 62 mm in length (Phase II), there was an overall decrease in cell division activity. Cell divisions contributing to the elongation of the gynoecium became restricted to the ovary and base of the style before ceas-
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DEVELOPMENTALBIOLOGY
a
-.l
4 0
b
-.l
C
Distance
from
base
(mm)
40 Illstance
from
80 base
120 (mm)
from
80 base
120 (mm)
t 0
40 Distance
FIG. ‘7. LRGR profiles of one Lilium longi&vum days of growth during Phase III. Initial gynoecium final length, 137 mm. (a) Day 1; (b) Day 2; (c) Day ovary/style junction.
gynoecium over 3 length, 94.1 mm; 3. Arrow indicates
V0~~~~143,1991
terns occur in gynoecia grown in various temperatures. Phase I gynoecia were sensitive to the dissection necessary for the marking experiments and did not grow as well as the controls. In spite of this, the overall patterns of cell division activity correlate with results from the surface marking experiments for Phase I and lend support to their validity. Analogous data on mitotic activity were used to discount the argument that the nonsteady growth patterns in anthers and Phase I tepals are a wound response (Lord and Gould, 1989). Phase II is a maturation phase in which the ovary and stigma achieve their mature lengths. In addition, ovules mature and undergo meiosis in this phase (Bennett and Stern, 1975; Erickson, 1948). One intriguing aspect of Phase II is the localization of the LRGR maximum to the proximal 5 to 15 mm of the style. When the style is less than this length, e.g., for gynoecia less than 30 mm in total length, the appearance is that of overall stylar growth in an intercalary fashion. The LRGR maximum becomes basal in its location as the style elongates by cell expansion alone, since cell division has typically ceased by this time. The analogy to be used is that of a waterfall (Silk, 1984). The same position of activity holds, although materials (cells) move through it. As more style is produced, the same region of growth appears more basal with respect to the entire style. Phase III is an expansion phase. The ovary and stigma, having matured in Phase II, show no further growth. Increase in gynoecial length in this phase occurs almost entirely by stylar cell expansion. The apical progression of stylar expansion could represent differ-
100
ing in gynoecia 40 to 50 mm in length, halfway through Phase II. Computer Reconstruction Computer reconstruction of a 1.9 mm bud (Fig. 10) demonstrated the close packing of organs in the lily flower. This tight packing extended laterally and vertically; little space existed between the apices of the anthers and gynoecia and the recurved inner tepals. Length of gynoeda Phase I
DISCUSSION
There Are Three Distinct Phases of Growth Gynoecium
in
the
Phase I is a time of nonsteady growth, with LRGR maxima shifting throughout the length of the gynoecium. In addition, the peaks of cell division activity vary in time and space along the organ. These growth pat-
Phase II
(mm) Phase Ill
FIG. 8. Distribution of LRGR maxima in gynoecia of increasing size. Histogram bars: black, lower ovary (lo); stippled, upper ovary (uo); white, lower style (Is); striped, upper style (us). Number of observations in each size category: O-4.99 mm (N = 28; lo = 16, uo = 12); 5-9.99 mm (N = 40; lo = 1, uo = 14,ls = 13, us = 12); 10-14.99 mm (N = 15; lo = 1, uo = 3, Is = 7, us = 4); 15-29.99 mm (N = 36; Is = 20, us = 16); 30-99.99 mm (N=49;ls=43,us=6);100-119.99 mm(N=14;ls=8,us = 6); 120-140 mm (N = 17; lo = 1, uo = 1,ls = 1, us = 14).
Time
(days)
,.
stamen
gynoecium I
Phase
1
J
1 I Phase
e
-L
II
ential cell expansion responses to a stimulatory gradient. As seen in the histogram (Fig. X), transitions exist between these phases. In real time though (Fig. la),
FIG. 10. Three-dimensional computer partial reconstruction of a 1.S mm Lilitrw /o,lg/(flor./c IV flower bud. The flattened tops of thr stamens arc an artifact. T, tepal: s, stamen; g, gynoecium. Scale bar. 1 mm.
Phase
III
FIG. 11. (‘orrclative growth in lily flower organs. Time scale drriacd from Fig. la. Phase 1, cell proliferatCon and nonstvady surfaw growth patterns. *Phase I in the stamen ceases hrnrta only with rrspwt to prolifrrativc cell divisions; thr nonsteady growth patttrns charwttlristic of Phase I persist until anther maturity at thr end of Phase II. Phase II, difl’crentiation and maturation. Arrow indicates when cell divisions wasr in each organ. Anther and ovary are mature at the end of this phase. Phase III. expansion of tepal. filament, and style: this phase ends in anthcsis.
these periods of transition are brief those of the three phases described
I’IG. 9. Mitotic index protiles from eight Phase I gynorcia; lengths (mm): (al 0.X; (111 1.11: (CI 1.23; Cdl 1.26: (e) 2.00; (fl Zll; (a) :1.X; (h) 4.03.
1
compared
with
Gould and Lord (1988, 1989) have described three phases of growth for the tepal and Phase I-type nonsteady growth throughout development in the anther. The LRGR calculation in those studies was different from the one used here, but the fundamental conclusions are not affected, as verified by calculating gynoecial LRGR both ways (data not shown). If one looks at the surface growth patterns for all the organs at once during development of the flower, a pattern of coordination is revealed (Fig. 11). Both the gynoecium and the tepals show shifting, nonsteady growth throughout Phase I, both in terms of surface growth and cell division patterns. This is a phase of cell proliferation or meristematic growth. A phase of proliferative growth occurs as well in the anther and for the same period of time, so it has a Phase I analogous to the tepal and gynoecium. Tinlike the other t,wo organs, the anther shows nonsteady growth throughout its development, though when cell division declines the pattern becomes more wavelike than random. The cessation of proliferative cell division activity in these three organs is staggered by approximately a day. Since these organs are initiated sequentially, it is likely that the duration of this phase is the same in each case. Phase II is the longest in duration and is a differentiation and maturation phase. Here, both the gynoecium and tepal show steady, nonshifting growth with the maximum at the base of the style in the gynoecium and at the base of the tepals in the perianth. Only the anther
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DEVELOPMENTALBIOLOGY
and not the entire stamen was marked in our previous experiments (Gould and Lord, 1988). Of all the organs, only the anther part of the stamen shows nonsteady growth during this period. The filament of the stamen expands rapidly during this phase. Cell division ceases at similar times in the three organs-in the tepal, these cells are at the base; in the stamen, microspore mitosis signals the end of cell division; and in the gynoecium, megagametogenesis in the ovules is the last cell division event. By the end of Phase II, the ovary and the anther are fully extended and differentiated. Erickson (1948) had previously detected such a correlation between growth and maturation events in the lily flower. Phase III is one of expansion for all organs, culminating in anthesis. The gynoecium shows a shift of the growth maximum from the base to the apex of the style and in the tepals, a similar shift occurs along the whole organ up to the tip as it expands in flower opening. In the stamen, the filament alone shows growth in this phase. Many of the studies of correlative effects on floral organ growth concentrate on aspects of organ initiation. Physiological studies have demonstrated correlative effects via hormone production in floral organs after initiation (Kinet et ab, 1985). Our results indicate that there are correlated surface growth patterns among the organs of the flower as well. The coincidence of proliferative, differentiation and maturation, and expansion phases suggests similar underlying mechanisms and a certain amount of coordination in flower development.
Possible Mechanisms
for Growth Patterns in Each Phase
The three phases described for lily organs may represent different modes of morphogenesis. In Phase I, the nonsteady distribution of cell division along the organs may set up the pattern of nonsteady growth, but a mechanism for these shifting mitotic centers is as yet unknown. Field theory, or the concept of positional inhibition of cell division, may be an explanation, Schoute (1913) proposed a generalized field theory to explain phyllotaxy in plants. He proposed that a leaf “center” at the apex produced a substance that inhibited initiation of other leaf centers in the vicinity (Wardlaw, 1952). Since initiation sites for leaves are ones of increased mitotic activity one could expand this proposal to include the patterns of proliferative growth in Phase I organs. Peaks of cell division appear to be centered in particular areas of the primordium with adjoining areas apparently inhibited. These adjoining areas exhibit primarily cell expansion so that the two processes appear to be coordinated to some extent, though the centers appear randomly distributed along the organ. Controls in the cell cycle may determine which cells in a population are dividing and which are not. Recent
VOLUME 143,lW
work indicates that the cd&?cell cycle regulatory system is in plants and may function in much the same way as in animal and yeast models (John et al., 1989). If the field model proposed above holds true, the inhibitory effects could be operating on this gene system. Phase I-type behavior continues in the anther after cell division ceases(Gould and Lord, 1988). In this situation, the waveforms are propagated through cell expansion alone. The question arises whether there is some kind of cytoplasmic clock that either triggers or synchronizes the activity of these early growth centers. Hara et aL. (1980) demonstrated the possibility of a cytoplasmic clock in developingXenopus eggs. More recently, Medina and Bregestovski (1988) and Block and Moody (1990) have shown the presence of membrane ion channels that show a cyclical pattern despite inhibition of mitosis and cytokinesis. Goodwin and Cohen (1969) have postulated the existence of pacemaker centers in developing organisms that help to organize morphogenesis. These are experiments that derive from rapid-cycling chordate eggs and embryos, but may apply as well to other systems. Electric oscillations along the axis of a plant organ may also stimulate elongation, as suggested by recent work in Phaseolus roots (Souda et al., 1990) and stems (Toko et al., 1990). Simultaneous marking of a sample of floral organs in Phase I buds did not show any correlation in terms of growth profiles, positive or negative, among the organs. If there is a physical constraint component that influences the growth patterns of the three organ types, it would be released by the dissection necessary to mark them. The 3-D computer reconstructions suggest that physical constraints are likely, considering the close packing of the organs in the bud. The Phase I pattern of nonsteady growth may be common to all determinate plant organs, although the lily floral organs are the best studied at this early stage. Chen (1963) noted similar growth patterns in leaf primordia of Eupaturiufm. Poethig and Sussex (1985), in one tobacco leaf, noted an early period of spatial variation in LRGR and mitotic index. We plan to perform marking experiments on young lily leaves as a follow-up to these studies of the floral parts of L&urn. Phase II is a steady-state situation that may require a somewhat different mechanism. Phase II patterns are due in part to both growth and cell division being gradually localized to the base of the tepal and style, but these basal patterns are continued well after cell division ceases. Furthermore, the tepal shows basal growth behavior before cell divisions are restricted to the base (Gould and Lord, 1989). Phase III demonstrates an obvious correlation of expansion activity among the floral organs of lily. This expansion in all the organs is presumably mediated by growth substances. Anthers in lily are known to be a rich source of gibberellins (Barendse et al.,
1970), which have been implicated in expansion phenomena in other floral systems (Kinet et al., 1985; Murakami, 19’75;Raab and Koning, 1988). Surface marking experiments provide a dynamic picture of flower organogenesis. In this study, we show that the lily gynoecium grows in three phases. These findings are supported by similar results in other organs of the lily flower and serve to show the coordination of such events in growth of the flower. Proposed mechanisms for the three phases await further testing. \I’e thank advice during Eckard for was supported 02 to E.M.L. vice Award ment of the nia, Riverside.
Dr. Wendy Silk (University of California, Davis) for her this study, We also thank Marie Greene and Kathleen assistance with the computer reconstruction. This work by National Science Foundation Grant DCB-88 28554and National Institute of Health-National Research SerGM13447-01 to W.C. This work represents partial fulfillrequirements for the Ph.D degree, IJniversity of Califorfor W.C. REFERENCES
BARENDSE, G. W. M., RODRIGUES PEREIRA, A. S., BERKERS, P. A., DRIESSEN, F. M., VAN EYDEN-ENORIS, A., and LINSKENS, H. F. (1970). Growth hormones in pollen, styles, and ovaries of P&u/licr /,&i&l and of Li/iu,r/ species. Actu Bat. Nwrl. 19, 175-186. BENNETT, M. D., and STERN, H. (1975). The time and duration of female meiosis in Lilium. p’r*)c. R. Sot. Lo~clolr B 188, 4511-475. BLOCK, M. L., and MOODY, W. J. (1990). A voltage-dependent chloride current linked to the cell cgcle in ascidian embryos. Scic,riccz 247, 1090- 1092. BOKE, N. H. (1949). Development of the stamens and carpels in \Srlctr rowu L. A ,i,(‘r. eJ. Bat. 36, 535-547. CHEN, J. C. WY. (1963). “Quantitative Analysis of the Growth of Leaf Primordia in Euptrtoritcttr rqqoslrrrl Houtt.” Ph.D. dissertation, 1Jniv. of Pcnnsvlvania. CREMER, M. C., BEIJER, J. J.. and MUNK, W. J. (1974). Developmental stages of flower formation in tulips, narcissi, irises, hyacinths, and I ilies. ,I.k&rtl. Lee tctlbtrrc cc#!oqrsch. Wtrf/c~c itypu 74, No. 15. DE HERTOGH, A. A., RASMUSSEN, H. P., and BLAKELY, N. (1976). Morphological changes and factors influencing shoot apex development of Lilirrnr lon.qiflor/r it, Thunb. during forcing. J. Arrrcr. Sot Horfic. Sri. 101,46:1~471. L~ERMEN, H., and STEWART, R. N. (1973). Ontogenetic study of floral organs of peach (Prxuus ~~rrsictr) utilizing cytochimeral plants. 21 ,Il of Plant Physiologists, Rockville, MD. MCCULLY, M. E., and O’BRIEN, T. P. (1981). “The Study of Plant Structure: Principles and Selected Methods.” Termarcarphi, Melbourne. MEDINA, I. R., and BREGESTOVSKI, P. D. (1988). Stretch-activated ion channels modulate the resting membrane potential during early embryogenesis. E’~oc,. R. Sot. Lor/tlon R 235, 95-102. MURAKAMI, Y. (1975). The role of gibberellins in the growth of Horal organs of &‘irtrhi/is ,jrr/o/j”. I’/(/ trf CPU Ph ysiol. 16, 337-345. PFEIFFER, N. E. (1935). Development of the floral axis and new bud in imported Easter lilies. (‘oufrib. Boycca Z%onr/~so7/ Zvsf. 7, 311-321. POETHIG, R. S., and SUSSEX, I. M. (1985). The developmental morphology and growth dynamics of the tobacco leaf. PIatLfu 165, 158-169. RAAB, M. M., and KONING, R. E. (1988). How is Horal expansion regulated? Bio.‘+ic~nw 38, 67OM74. RITTERBUSCH, A. ( 1980). The modeling of growth and development, a transformational approach to floral ontogenesis. Fltntr 169, 49X509. RI~ERBUSCH, A. (1989). Comparison of temporal patterns in Howtr ontogenesis by mrans of normalized-age sequences. AuI/. Ref. 64, 17!)~183. SATINA, S., and BLAKESLEE, A. F. (1943). Periclinal chimeras in Dtrf/cur in relation to the development of the carpel. Amer. J. Bof. 30, 453-462. SATTLER, R. (1973). “Organogenesis of Flowers. A Photographic TextAtlas.” liniv. of Toronto Press, Toronto. SCHOUTE, J. C. (1913). Beitrage zur Blattstellun~slehre. &cl. Trtrr.. Rol. A-fvd. 10, 153-235. SILK, W. K. (1984). Quantitative descriptions of development. Ant//r. z&Jr. m/r trf Z’h ysiol. 35, 479-518. SOUDA, M., TOKO, Ii., HAYASHI, K., FUJIYOSHI, T., EZAKI, S., and YAMAFUGI, K. (1990). Relationship between growth and electric oscillations in btsan roots. Pltrt/f Ph,pio/. 93, 532-536. TOKO, Ii., TANAKA, C., EZAKI, S., IIYAMA, S., and YAMAFUGI, K. (1990). Growth and electric current Howing at the surface of stems. Proftr j/h/M 154, 71-Z. WARDLAW, (:. W. (1952). “Phylogenq’ and MorphoKenesis.” Macmillan and Co., London. WEBERLING, F. (1989). “Morphology of Flowers and InHorescences” Cambridge TJniv. Press, Cambridge. [Translated by R. J. Pankhurst]
BIOLOGY
143,408-417
(19%)
A Kinematic Analysis of Gynoecial Growth in Lilium longiflorum: Surface Growth Patterns in All Floral Organs Are Triphasic
Sequential marking experiments combined with histological and morphological examination indicate that growth of the gynoecium in Liliu~z hng$kmm~ Thunb. (Easter lily) is triphasic. Phase I (gynoecia less than 10 mm in length) is characterized by spatial and temporal variation in local relative growth rate and mitotic index throughout the entire gynoecium. In Phase II (lo-100 mm), a steady growth peak is restricted to the proximal 5 to 15 mm of the style, while the ovary shows uniform growth. In Phase III (loo-135 mm), the ovary ceases growth and the maximal growth zone of the gynoecium migrates from the proximal to the distal end of the style. Comparison of surface growth patterns and differentiation events in the gynoecium to those previously described in the tepal and anther demonstrates the triphasic :a 1991 Academic Press, Inc. nature of growth in the whole lily flower.
ity and actual enlargement of the plant organ. In another approach, Ritterbusch (1980,1989) has used natural features of the petal surface to describe trajectories of organ growth and predict relative timing of developmental events. Although more quantitative than other descriptions and utilizing a surface marking technique, these studies lack an ix ~ivo or real time component. Growth can be considered in several different ways (Hunt, 1982): the total absolute growth of the organ, the relative growth rate of the organ, and the local relative growth rate of regions along a dimension of the organ. Surface marking experiments can test assumptions about the distribution of growth in an organ by using time-lapse photographs of marked organs to highlight variations in growth rates along the length of an organ (Silk, 1984). Segments of an organ can be delineated by applied marks and their relative elongation rates compared over time. These marking experiments document kinematic effects, motion in the abstract, but do not imply any information about the forces driving growth. In purely descriptive terms, steady growth is centered in the same region over time, as seen in the indeterminate shoot or root apices of plants. In contrast, nonsteady growth occurs when growth centers shift during ontogeny, as in determinate organs such as leaves (Silk, 1984). Surface marking studies have only recently been attempted in floral organs (Lord and Gould, 1989). The Easter lily, Lilizrm lo~g~flo~um Thunb. (Liliaceae) is a useful model organism for growth studies (Erickson, 1948). It has a large flower (150-160 mm at anthesis or maturity) that is hardy enough to withstand the dissection necessary to mark internal organs. The first marking experiments with lily flowers demonstrated that anthers (the pollen-containing part of the stamen) show
INTRODIJCTION
Plants produce their organs from permanently “embryonic” regions called apical meristems. Flowers, the reproductive structures of the angiosperms or flowering plants, are produced by vegetative meristems that have been induced to flower. The floral organs are often formed in acropetally produced whorls, with the perianth initiated first and usually composed of sepals and petals. The stamens are initiated next, and then the carpels or pistils (collective term, gynoecium) are initiated, typically in sequential order. Flowers are complex structures, difficult to follow developmentally. As a result, Greyson’s (1972) and other researchers’ calls for quantitative descriptions of floral organ growth have gone unheeded, for the most part. There are few such studies of the gynoecium, the most complex of the floral organs being heterogeneous in its composition and function. The stigma is a site for pollen capture and hydration; the style is a path for pollen tube growth; and the ovary is the site of fertilization and fruit formation. Previous workers have described the ontogeny and histogenesis of the gynoecium, usually with a comparative, systematic focus (e.g., Sattler, 1973; van Heel, 1981). The use of periclinal chimeras has demonstrated the relative contribution of different apical meristematic layers to gynoecial growth (Satina and Blakeslee, 1943; Dermen and Stewart, 1973). Anatomical studies tend to emphasize localized growth through “meristerns,” or histologically separable regions of cell division and expansion (Boke, 1949; Kaplan, 1968; Weberling, 1989). The techniques used are based on the assumptions of steady-state distributions of growth centers and close relationships between the visible “meristematic” activOOlZ-1606/91 Copyright All rights
$3.00
:C’ 1991 by Academic Press, Inc. of reproduction in any form reserved
408
a nonsteady wave pattern of growth throughout ontogeny (Gould and Lord, 1988). Lily tepals (the term used for perianth organs when they are not divided into distinct sepals and petals) show three phases, including both nonsteady and steady growth (Gould and Lord, 1989). These were patterns that would not have been predicted on the basis of histology or scanning electron micrographs (SEMs)l alone. Previous authors have documented the early initiation patterns of lily flowers (Pfeiffer, 1935; Cremer et al., 1974; de Hertogh ef (LI, 1976). In the present study, we describe the growth patterns in the gynoecium after initiation, utilizing surface marking techniques in combination with SEM and histology. In addition, we provide a composite picture of a kinematic analysis for the entire lily flower. MATERIALS
AND
METHODS
Bulbs of L. lor&lwum cv. Nellie White (Dahlstrom and Watt Bulb Farms, Smith River, CA) were planted in pots and grown in a greenhouse in Riverside throughout the year, with temperatures ranging from 5 to 35°C. At least 2 weeks prior to the marking experiments, the pots were transferred to a growth chamber at one of two settings: 25°C with an 1%hr photoperiod (35 plants), or 21°C and a 12-hr photoperiod (9 plants). The growth chamber had a photon fluence rate of 375 PE me2 s-l with both incandescent and fluorescent light.
The method of Erickson (1948) was used to estimate the in sita growth rate of the gynoecium in control buds. Buds grown under the 25°C X 1%hr regimen (N = 74) were measured daily, from tip to base of the tepal. Fresh and fixed buds (N = 119) were used to plot allometric constants of gynoecial versus bud growth. The relative rate of elongation of gynoecia in situ was estimated as the product of the allometric constant k for the gynoecial and tepal lengths and r as the relative elongation rate of the entire bud.
One side of the gynoecium was exposed for its entire length by removing a portion of the tepals plus one to three stamens with a scalpel. The exposed gynoecium was marked with a file of approximately equidistant dots of activated charcoal in a viscous mixture with water or petroleum jelly. The cut tepal edges were coated with petroleum jelly and the entire bud was cov-
1 Ahhreviations useti: DAPI, ~,6-tliamidino-2-~),henYlindolc chloride; FAA, formaldrhydc~acetic acid-alcohol; LRGR, tivr growth rate: SEM, scanning elrctron micrograph.
tlihydrolocal rcla-
ered with polyethylene film to minimize desiccation. The first and last marks were always located at the base and the tip of the gynoecium, respectively. In total, 126 gynoecia varying in length from 2.0 to 133.5 mm were marked. In six buds (gynoecial length 3.4-9.9 mm) a sample of one of each organ was marked along its length simultaneously. Each marked organ was photographed alongside a reference scale using Kodak Technical Pan 2415 black and white film (Eastman Kodak, Rochester, NY) at 24-hr intervals, the minimum time for measurable growth. Additional marks were placed between dots when original ones grew more than 4 to 5 mm apart. Most gynoecia grew for 1 to 3 days after the initial marking. Prints were enlarged up to a final magnification of 20-40X. Recognizable features of the individual marks were used to delineate segments for each 24-hr period. The distance separating each mark from the base of the gynoecium was recorded from the prints using a Summagraphics (Fairfield, CT) digitizer and bit pad data tablet coupled to an IBM (Boca Raton, FL) personal computer. For the smallest gynoecia, both top and bottom features of marks were used to increase the number of data points. Data manipulations were performed using the spreadsheet software SuperCalc 4 (Computer Associates International, San Jose, CA). In order to highlight localized regions of growth and yet smooth the growth curves, a running refit analysis was undertaken for each day’s data (Erickson, 1976; Hunt, 1982). We used overlapping sets of four marks along the organ. Local relative growth rate (LRGR) for a segment was calculated as the difference in the values of the natural logarithms of the four point segment length over time (Erickson, 1976; Silk, 1984). LRGR was plotted versus the distance of the initial midpoint of the four point segment measured from the base of the gynoecium. Our study focused on the ovary and the style, since these two regions make up most of the length of the gynoecium.
Gynoecia in control buds grown in both the greenhouse and growth chamber were fixed in formaldehydeacetic acid-alcohol (FAA) (Johansen, 1940), dehydrated in an ethanol series, infiltrated and embedded with glyco1 methacrylate (JB-4, Polysciences, Inc., Warrington, PA), serially sectioned at 3 to 5 pm, and stained with either 1% aqueous toluidine blue-0 or 1% aqueous acid fuchsin (McCully and O’Brien, 1981). For determination of the mitotic index, serial sections at intervals of 9 to 200 pm apart, depending on the total length of the gynoecium, were scored for mitotic figures and total cells present on that section. Since mitoses were infrequent, any cell at mid prophase to late telophase was scored as
410
DEVELOPMENTALBIOLOGY
++---I--------
a
0
10 20 30 Time [days)
40
50
b
Length
of bud
(mm)
FIG. 1. Itr situ growth of control buds of Lili/otr Io?~,qiflwrrnr. (a) Bud length vs time (K = 741. The smallest buds that could be measured ir, sitar were 3.5 mm in length. Arrow = anthesis. Bars are t I standard deviation. ? = 0.999. (b) Allometric plot of ggnoecium length versus tepal length (A’ = 119). P = 0.977.
a mitotic figure. The mitotic index for that section was determined by the number of mitoses/total number of cells X 100. These values were plotted against the distance of that section from the base of the gynoecium, with enough sections scored to create a mitotic index profile along the entire length of the gynoecium.
VOLUME 1X3,1991
anthesis, Y’,was calculated by linear regression to be r = 0.09391day. The logarithmic plot of tepal length vs gynoecial length was allometric (Fig. lb). In other words, there was a constant relationship between the elongation of the two organs with an allometric constant of k = 1.253, derived by linear regression. The relative rate of gynoecial elongation was calculated as kr = O.lWday. This allometric plot allowed the estimation of gynoecial size without dissection of the flower. In addition, this iv sit,u known relative growth rate supplied a standard or control to compare with gynoecia treated by dissection and marking. The closeness of the fit (r2 = 0.977) demonstrated that the gynoecium grew as a unit.
After initiation of the three carpel primordia that fuse laterally and form a cylindrical structure, most of the early vertical elongation of the gynoecium was in the region of the ovary (Figs. 2a-2c, 3a, 3b). The stigma was the first to differentiate as evidenced by precocious Other Mitotic Determinations cell enlargement (Fig. 2~). By the time the gynoecium was 4 to 5 mm, the style had begun to intercalate beTwenty-seven gynoecia (2-112 mm in length) from control flowers were fixed overnight in 3:l 95%1 tween the stigma and the ovary (Fig. 3b). A defined ethanokglacial acetic acid. They were then softened in ridge at the junction of the ovary and proximal style was 10% aqueous sodium sulfite for at least an hour, and noticeable after the gynoecium reached a total length of 8 to 10 mm or more (Fig. 3~). From this point until antransferred to 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) aqueous solution, 0.5 pg/ml, for at least thesis, the majority of length increase in the gynoecium was stylar (Figs. 3d-3f). 2 hr (McCully and O’Brien, 1981). DAPI is a fluorescent stain for DNA. The gynoecia were sliced in half, gently squashed onto a slide, and viewed with a Zeiss fluorescence microscope with Zeiss filter set 487702. This technique was used to estimate cell division activity in One-hundred and twenty-six gynoecia on 44 plants various regions of the gynoecium. were marked under the above growth chamber conditions (Figs. 3a-3f). Of these, 101 (80%) had significant Scwnniqy Electron Microscopy und 3-D Computer portions of the LRGR profile at or above O.OG/day for 24 Reconstruction hr or more and so were considered appropriate for furYoung control gynoecia were fixed and dehydrated as ther analysis. Of the 101 gynoecia analyzed, 46 (46%) above, critical point dried with CO,, coated with gold- grew at or faster than the in situ rate of 0.118/day, and palladium, and viewed in a JOEL (Tokyo, Japan) 35C 55 (54%) grew slower. The growth of lily gynoecia from SEM. One entire control bud (length 1.9 mm) was pre- 2.0 mm to maturity could be separated into three dispared and sectioned serially as described above. Camera tinct phases, as determined from the surface marking lucida tracings of sections throughout the bud were di- experiments. These observations were based on a total gitized into an Evans and Sutherland (Salt Lake City, of 199 plots showing the spatial distribution of LRGR in UT) ES 390 computer. The reconstruction program Anthe 101 gynoecia. atograph (Applied Medical Systems, Salt Lake City, UT) Phase I was a period of spatial and temporal variation was used to create a 3-D image. in growth rate, i.e., nonsteady growth (Figs. 4 and 5). This phase encompassed gynoecia 2.0 to 10 mm in length RESULTS (Figs. 3a and 3b). The peaks in the LRGR profile showed rises and falls with no predictable location in space. This Growth Profiles was particularly so for those gynoecia less than 5 mm in Elongation of the floral bud, i.e., tepal length, of Lillength (Figs. 3a and 4). After the start of stylar intercaium was exponential until anthesis, when growth ceased lation at 5 mm (Fig. 3b), the base of the style began to (Fig. la). The relative rate of elongation for growth until predominate as the site of the greatest LRGR, even
CRONE
growth in Lilium lo~z&kw-um. FIG. : Early gynoecial S, stigma; o, ovary; 1, ovary locule. 3.0 mm gnoecium.
AND
LORD
Gynoecial
Growth
(a) SEM of 1.4 mm gynoecium; Scale bars, 250 Frn.
in Liliu
(b) SEM
m
of 2.2 mm gynoecium;
(c) longitudinal
section
of
412
FIG. 3. Dissected buds of Lili~w lo~ry~or~rrc style intercalation; (c) 9.0, arrowhead on joint c); 10 mm (d, e, f).
DEVELOPMENTALBIOLOGY
VOLUME 143, 1991
with marks applied to gynoecia of various lengths (mm): (a) 2.2; (b) 5.2, arrow indicates site of between ovary and style; (d) 20.7; (e) 44.7; (f) 123.5, arrow on mature stigma. Scale bars: 1 mm (a, b,
though peaks of growth could still be found throughout the gynoecium (Fig. 5). Phase II, gynoecia 10 to 100 mm in length, was characterized by predominantly stylar growth, with the LRGR maximum localizing to the basal 5 to 15 mm of the style (Figs. 3c-3e and 6). The magnitude of the LRGR here was greater than that seen in Phase I. In gynoecia 10 to 30 mm in length, growth occurred throughout the Z-10 mm style (Figs. 3c-3d, 6a, 6b). As the style elongated in the 30 to 100 mm gynoecia, the location of the LRGR maximum appeared in a 5 to 15 mm range from the base of the style (Figs. 3e, 6c, 6d). During this phase, the ovary, when studied apart from the whole gynoecium, showed steady, uniform growth, reaching maturity by early Phase III (Figs. 6d and
‘7a). The stigma also reached its mature size by early Phase III. Phase III, in gynoecia 100 to 135 mm in length, was characterized by nonsteady growth, with a shift in the LRGR maximum from the proximal to the distal end of the style (Figs. 3f and 7). This occurred in two ways: the LRGR maximum appeared to be either an acropetally moving single wave during this phase (8 of 14 observations) (Fig. 7), or the base of the style maintained a separate LRGR peak from the apically progressing one (6 of 14 observations). In both cases,by anthesis, almost all growth was at the distal lo-20 mm of the style (Fig. 7~). For the six Phase I buds which had a sample of all types of floral organs marked simultaneously, there was
CRONE
-.I]
, 0
a
.4
1 Illstance
AND
LORD
Distance
, 2 from
3 base
from base
j?rnl
b
Oi-
5
D~smce
lo-
from base
(mm]
4 (mm)
1
FIG. 6. LRGR profiles of two individual Lilirrnl Itmg~&run/ gynoecia over 2 days of growth during early (a, b) and late (c, d) Phase II. Initial gynoecium length, 16.0 mm. (a) Day 1; tb) Day 2. Initial gynoecium length, 52.2 mm. Cc) Day 1; ((11 L)ay 2. Arrow indicates ovary/style junction.
l-
bol
Distance
2
from
3
base
(mm)
4
4-
early Phase I, the lower and upper rz:rions of the ovary are growing maximally, but soon the upper ovary and the style become a more common site for the maximum LRGR in this phase of nonsteady growth throughout the gynoecium. During most of Phase II, the lower style is the predominant site of growth, although by early Phase III, the LRGR maximum migrates up the style to the apex where it remains until anthesis when a small surge of expansion occurs as well in the whole organ.
3 z' 2 T2
.2.I
E J
O-.1-
& 0
C
1 Distance
2 from
3 base
4 (mm1
FIG. 4. Local relative growth rate (LRGR) profiles of one Li/i/rnl lor/qiflo/xn/ gynoecium over 3 days of growth during early Phast I. Initial length, 3.9 mm. (a) Day 1; 0)) Day 2; (cl Day 3.
no consistent pattern in growth profiles. The locations of LRGR maxima and minima appeared to be random among the organs (data not shown). Definite relationships exist among the different parts of the gynoecium throughout development (Fig. 8). In
a
I ‘3
? Distance
4 from base
6 lmml
bo-ra Distance
from base
$ imml
FIG. 5. LRGR profiles of one Lilirrnl /on~~jflor.~rtc gynoecium over 2 days of growth during late Phase I. Initial length, 7.4 mm. The style has begun to intercalate between the ovary and stigma. (a) Day 1; (b) Day 2. Arrow indicates ovary/style junction.
In Phase I (gynoecia < 10 mm), the peaks of LRGR were interpreted to represent regions of primarily cell expansion and the troughs, regions of primarily cell division. The smallest marked gynoecium in this study was 2.0 mm. Since the nonsteady growth in Phase I may be set up early, serial sections of gynoecia 0.36 to 4 mm (N = 8) were examined for patterns of mitotic activity along the organ. Examination of the profiles of mitotic index confirmed that cell division activity was not uniform nor was it localized consistently to specific regions of the gynoecium (Fig. 9). These data correlate with the growth profiles from the surface marking experiments. The DAPI-stained whole gynoecia gave a grosser picture of mitotic activity for all phases of growth. In gynoecia 2 to 9 mm in length (Phase I), mitotic activity occurred at a high level throughout the gynoecium. In gynoecia 11 to 62 mm in length (Phase II), there was an overall decrease in cell division activity. Cell divisions contributing to the elongation of the gynoecium became restricted to the ovary and base of the style before ceas-
414
DEVELOPMENTALBIOLOGY
a
-.l
4 0
b
-.l
C
Distance
from
base
(mm)
40 Illstance
from
80 base
120 (mm)
from
80 base
120 (mm)
t 0
40 Distance
FIG. ‘7. LRGR profiles of one Lilium longi&vum days of growth during Phase III. Initial gynoecium final length, 137 mm. (a) Day 1; (b) Day 2; (c) Day ovary/style junction.
gynoecium over 3 length, 94.1 mm; 3. Arrow indicates
V0~~~~143,1991
terns occur in gynoecia grown in various temperatures. Phase I gynoecia were sensitive to the dissection necessary for the marking experiments and did not grow as well as the controls. In spite of this, the overall patterns of cell division activity correlate with results from the surface marking experiments for Phase I and lend support to their validity. Analogous data on mitotic activity were used to discount the argument that the nonsteady growth patterns in anthers and Phase I tepals are a wound response (Lord and Gould, 1989). Phase II is a maturation phase in which the ovary and stigma achieve their mature lengths. In addition, ovules mature and undergo meiosis in this phase (Bennett and Stern, 1975; Erickson, 1948). One intriguing aspect of Phase II is the localization of the LRGR maximum to the proximal 5 to 15 mm of the style. When the style is less than this length, e.g., for gynoecia less than 30 mm in total length, the appearance is that of overall stylar growth in an intercalary fashion. The LRGR maximum becomes basal in its location as the style elongates by cell expansion alone, since cell division has typically ceased by this time. The analogy to be used is that of a waterfall (Silk, 1984). The same position of activity holds, although materials (cells) move through it. As more style is produced, the same region of growth appears more basal with respect to the entire style. Phase III is an expansion phase. The ovary and stigma, having matured in Phase II, show no further growth. Increase in gynoecial length in this phase occurs almost entirely by stylar cell expansion. The apical progression of stylar expansion could represent differ-
100
ing in gynoecia 40 to 50 mm in length, halfway through Phase II. Computer Reconstruction Computer reconstruction of a 1.9 mm bud (Fig. 10) demonstrated the close packing of organs in the lily flower. This tight packing extended laterally and vertically; little space existed between the apices of the anthers and gynoecia and the recurved inner tepals. Length of gynoeda Phase I
DISCUSSION
There Are Three Distinct Phases of Growth Gynoecium
in
the
Phase I is a time of nonsteady growth, with LRGR maxima shifting throughout the length of the gynoecium. In addition, the peaks of cell division activity vary in time and space along the organ. These growth pat-
Phase II
(mm) Phase Ill
FIG. 8. Distribution of LRGR maxima in gynoecia of increasing size. Histogram bars: black, lower ovary (lo); stippled, upper ovary (uo); white, lower style (Is); striped, upper style (us). Number of observations in each size category: O-4.99 mm (N = 28; lo = 16, uo = 12); 5-9.99 mm (N = 40; lo = 1, uo = 14,ls = 13, us = 12); 10-14.99 mm (N = 15; lo = 1, uo = 3, Is = 7, us = 4); 15-29.99 mm (N = 36; Is = 20, us = 16); 30-99.99 mm (N=49;ls=43,us=6);100-119.99 mm(N=14;ls=8,us = 6); 120-140 mm (N = 17; lo = 1, uo = 1,ls = 1, us = 14).
Time
(days)
,.
stamen
gynoecium I
Phase
1
J
1 I Phase
e
-L
II
ential cell expansion responses to a stimulatory gradient. As seen in the histogram (Fig. X), transitions exist between these phases. In real time though (Fig. la),
FIG. 10. Three-dimensional computer partial reconstruction of a 1.S mm Lilitrw /o,lg/(flor./c IV flower bud. The flattened tops of thr stamens arc an artifact. T, tepal: s, stamen; g, gynoecium. Scale bar. 1 mm.
Phase
III
FIG. 11. (‘orrclative growth in lily flower organs. Time scale drriacd from Fig. la. Phase 1, cell proliferatCon and nonstvady surfaw growth patterns. *Phase I in the stamen ceases hrnrta only with rrspwt to prolifrrativc cell divisions; thr nonsteady growth patttrns charwttlristic of Phase I persist until anther maturity at thr end of Phase II. Phase II, difl’crentiation and maturation. Arrow indicates when cell divisions wasr in each organ. Anther and ovary are mature at the end of this phase. Phase III. expansion of tepal. filament, and style: this phase ends in anthcsis.
these periods of transition are brief those of the three phases described
I’IG. 9. Mitotic index protiles from eight Phase I gynorcia; lengths (mm): (al 0.X; (111 1.11: (CI 1.23; Cdl 1.26: (e) 2.00; (fl Zll; (a) :1.X; (h) 4.03.
1
compared
with
Gould and Lord (1988, 1989) have described three phases of growth for the tepal and Phase I-type nonsteady growth throughout development in the anther. The LRGR calculation in those studies was different from the one used here, but the fundamental conclusions are not affected, as verified by calculating gynoecial LRGR both ways (data not shown). If one looks at the surface growth patterns for all the organs at once during development of the flower, a pattern of coordination is revealed (Fig. 11). Both the gynoecium and the tepals show shifting, nonsteady growth throughout Phase I, both in terms of surface growth and cell division patterns. This is a phase of cell proliferation or meristematic growth. A phase of proliferative growth occurs as well in the anther and for the same period of time, so it has a Phase I analogous to the tepal and gynoecium. Tinlike the other t,wo organs, the anther shows nonsteady growth throughout its development, though when cell division declines the pattern becomes more wavelike than random. The cessation of proliferative cell division activity in these three organs is staggered by approximately a day. Since these organs are initiated sequentially, it is likely that the duration of this phase is the same in each case. Phase II is the longest in duration and is a differentiation and maturation phase. Here, both the gynoecium and tepal show steady, nonshifting growth with the maximum at the base of the style in the gynoecium and at the base of the tepals in the perianth. Only the anther
416
DEVELOPMENTALBIOLOGY
and not the entire stamen was marked in our previous experiments (Gould and Lord, 1988). Of all the organs, only the anther part of the stamen shows nonsteady growth during this period. The filament of the stamen expands rapidly during this phase. Cell division ceases at similar times in the three organs-in the tepal, these cells are at the base; in the stamen, microspore mitosis signals the end of cell division; and in the gynoecium, megagametogenesis in the ovules is the last cell division event. By the end of Phase II, the ovary and the anther are fully extended and differentiated. Erickson (1948) had previously detected such a correlation between growth and maturation events in the lily flower. Phase III is one of expansion for all organs, culminating in anthesis. The gynoecium shows a shift of the growth maximum from the base to the apex of the style and in the tepals, a similar shift occurs along the whole organ up to the tip as it expands in flower opening. In the stamen, the filament alone shows growth in this phase. Many of the studies of correlative effects on floral organ growth concentrate on aspects of organ initiation. Physiological studies have demonstrated correlative effects via hormone production in floral organs after initiation (Kinet et ab, 1985). Our results indicate that there are correlated surface growth patterns among the organs of the flower as well. The coincidence of proliferative, differentiation and maturation, and expansion phases suggests similar underlying mechanisms and a certain amount of coordination in flower development.
Possible Mechanisms
for Growth Patterns in Each Phase
The three phases described for lily organs may represent different modes of morphogenesis. In Phase I, the nonsteady distribution of cell division along the organs may set up the pattern of nonsteady growth, but a mechanism for these shifting mitotic centers is as yet unknown. Field theory, or the concept of positional inhibition of cell division, may be an explanation, Schoute (1913) proposed a generalized field theory to explain phyllotaxy in plants. He proposed that a leaf “center” at the apex produced a substance that inhibited initiation of other leaf centers in the vicinity (Wardlaw, 1952). Since initiation sites for leaves are ones of increased mitotic activity one could expand this proposal to include the patterns of proliferative growth in Phase I organs. Peaks of cell division appear to be centered in particular areas of the primordium with adjoining areas apparently inhibited. These adjoining areas exhibit primarily cell expansion so that the two processes appear to be coordinated to some extent, though the centers appear randomly distributed along the organ. Controls in the cell cycle may determine which cells in a population are dividing and which are not. Recent
VOLUME 143,lW
work indicates that the cd&?cell cycle regulatory system is in plants and may function in much the same way as in animal and yeast models (John et al., 1989). If the field model proposed above holds true, the inhibitory effects could be operating on this gene system. Phase I-type behavior continues in the anther after cell division ceases(Gould and Lord, 1988). In this situation, the waveforms are propagated through cell expansion alone. The question arises whether there is some kind of cytoplasmic clock that either triggers or synchronizes the activity of these early growth centers. Hara et aL. (1980) demonstrated the possibility of a cytoplasmic clock in developingXenopus eggs. More recently, Medina and Bregestovski (1988) and Block and Moody (1990) have shown the presence of membrane ion channels that show a cyclical pattern despite inhibition of mitosis and cytokinesis. Goodwin and Cohen (1969) have postulated the existence of pacemaker centers in developing organisms that help to organize morphogenesis. These are experiments that derive from rapid-cycling chordate eggs and embryos, but may apply as well to other systems. Electric oscillations along the axis of a plant organ may also stimulate elongation, as suggested by recent work in Phaseolus roots (Souda et al., 1990) and stems (Toko et al., 1990). Simultaneous marking of a sample of floral organs in Phase I buds did not show any correlation in terms of growth profiles, positive or negative, among the organs. If there is a physical constraint component that influences the growth patterns of the three organ types, it would be released by the dissection necessary to mark them. The 3-D computer reconstructions suggest that physical constraints are likely, considering the close packing of the organs in the bud. The Phase I pattern of nonsteady growth may be common to all determinate plant organs, although the lily floral organs are the best studied at this early stage. Chen (1963) noted similar growth patterns in leaf primordia of Eupaturiufm. Poethig and Sussex (1985), in one tobacco leaf, noted an early period of spatial variation in LRGR and mitotic index. We plan to perform marking experiments on young lily leaves as a follow-up to these studies of the floral parts of L&urn. Phase II is a steady-state situation that may require a somewhat different mechanism. Phase II patterns are due in part to both growth and cell division being gradually localized to the base of the tepal and style, but these basal patterns are continued well after cell division ceases. Furthermore, the tepal shows basal growth behavior before cell divisions are restricted to the base (Gould and Lord, 1989). Phase III demonstrates an obvious correlation of expansion activity among the floral organs of lily. This expansion in all the organs is presumably mediated by growth substances. Anthers in lily are known to be a rich source of gibberellins (Barendse et al.,
1970), which have been implicated in expansion phenomena in other floral systems (Kinet et al., 1985; Murakami, 19’75;Raab and Koning, 1988). Surface marking experiments provide a dynamic picture of flower organogenesis. In this study, we show that the lily gynoecium grows in three phases. These findings are supported by similar results in other organs of the lily flower and serve to show the coordination of such events in growth of the flower. Proposed mechanisms for the three phases await further testing. \I’e thank advice during Eckard for was supported 02 to E.M.L. vice Award ment of the nia, Riverside.
Dr. Wendy Silk (University of California, Davis) for her this study, We also thank Marie Greene and Kathleen assistance with the computer reconstruction. This work by National Science Foundation Grant DCB-88 28554and National Institute of Health-National Research SerGM13447-01 to W.C. This work represents partial fulfillrequirements for the Ph.D degree, IJniversity of Califorfor W.C. REFERENCES
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