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Structure and Development |
2Department of Botany, University of Wyoming, Laramie, Wyoming 82071-3165 USA; 3College of Agriculture, 106 E. Dalton Ave., Coeur d'Alene, Idaho 83815 USA; and 4College of Agriculture, Department of PSES, University of Idaho, Moscow, Idaho 83843 USA
Received for publication March 13, 2001. Accepted for publication July 31, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: bluegrass • grasses • heteroblasty • leaf development • maize • phase change • rice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Sylvester, Smith, and Freeling, 1996
; Kerstetter and Poethig, 1998
). Each phase manifests itself differently in diverse plant species. Some plants display distinct and abrupt changes in leaf shape or anatomy, whereas other plants show a more subtle and gradual transition between juvenile and adult phases (Borchert, 1976
; Hackett, 1985
; Sylvester, Cande, and Freeling, 1990
; Greenwood, 1995
). Regardless of how phase change is displayed in a plant, its regulation is a complex process involving the interplay of environmental, hormonal, and genetic factors (Bernier, 1986
; Poethig, 1988
; Evans and Passas, 1994
; Moose and Sisco, 1994
; Kerstetter and Poethig, 1998
; Araki, 2001
).
Maize has long been a model for evaluating phase change because leaf anatomy changes dramatically as the plant progresses from a juvenile to adult phase (Kerstetter and Poethig, 1998
). Maize mutants that alter the expression of phase-specific traits can be useful for identifying potentially relevant genes. Along with experimental and descriptive studies, genetic analyses have thus provided guiding principles for understanding some aspects of phase change. For example, phase-specific leaf traits are likely acquired gradually during leaf development (Sylvester, Cande, and Freeling, 1990
; Sylvester, Smith, and Freeling, 1996
; Irish and Jegla, 1997
; Orkwiszewski and Poethig, 2000
). Furthermore, these anatomical traits may be separable from the more fundamental physiological control of phase change. In gl15 mutants, for example, the number of leaves bearing juvenile traits is reduced from five to two without affecting the timing of reproduction (Evans and Passas, 1994
; Moose and Sisco, 1994
). Similarly, vp8 mutants may have a prolonged juvenile period, i.e., more leaves with juvenile anatomy than wild-type, but still have normal flowering times (Evans and Poethig, 1997
). These and other mutants suggest that phase-specific anatomy and reproductive timing are not necessarily united. Mutant analysis and experimental studies also show that giberrellin is required for normal progression through phases prior to reproduction (Evans and Poethig, 1995
). Consequently, a model for phase change has to take into account at least three potentially independent networks of genes that are involved in phase change. These genes could include those that regulate phase change itself, those that regulate overall timing of reproduction, and those that mediate leaf development (including timing of leaf initiation and the acquisition of leaf identity).
It is not yet clear whether patterns of phase change in maize provide a set of principles applicable to other plants. Since maize is a cultivated species, it is possible that its phase transitions may be derived and not typical of the family in general. However, comparative analysis of phase change in other grasses could reveal information about the evolution of phase change and could expose patterns of diversification among related taxa. To begin an evolutionary assessment of phase change across species, it is important to document shared as well as unique traits characteristic of each phase. This paper thus provides a starting point for further evolutionary study by documenting the morphology and anatomy of juvenile and adult leaves in divergent grass species.
The grasses are an ideal group to evaluate conceptual models of phase change for several reasons. First, the grasses share a close and recent evolutionary history, as reflected in high synteny and colinearity of grass genomes (Bennetzen and Freeling, 1993
; Kellogg and Birchler, 1993
; Moore et al., 1995
; Gale and Devos, 1998
). The grasses also share similar shoot anatomy and morphology, despite differences in sizes of leaves and minor variations in anatomy (Sharman, 1942
; Evans, 1949
; Etter, 1951
; Kauffman, 1959a
; Clark and Fisher, 1986
; Kellogg and Birchler, 1993
; Watson and Dallwitz, 1999
). A final important reason is that the grasses have diverse life cycles ranging from annual to biennial to perennial (Sylvester and Reynolds, 1999
; Watson and Dallwitz, 1999
). Floral induction requirements also vary considerably, as some grasses are receptive in the embryonic condition, whereas other species require extensive juvenile growth in order to flower (Silsbury, 1965
; Halevy, 1985
; Chastain and Young, 1998
). By comparing phase change in species of divergent reproductive habits, it is possible to determine whether the maize pattern is representative or uniquely derived.
We present here a comparative foundation for understanding patterns of vegetative phase change in the grasses. We used maize anatomy and morphology as a descriptive springboard from which other grasses can be examined. For this purpose, two divergent species in the grass family were compared with maize. The anatomical traits examined were presence or absence of epicuticular waxes, presence or absence of trichomes, and staining pattern of epidermal cell walls. The general size and appearance of epidermal cells was also examined, as previously described elsewhere (Sylvester, Cande, and Freeling, 1990
; Bongard-Pierce, Evans, and Poethig, 1996
). Other traits considered here include the pattern of leaf senescence, changing leaf shape defined by blade width and blade-to-sheath ratio, numbers of embryonic leaves, development pattern of leaf primordia and degree of tillering.
Two species, Poa pratensis L. (bluegrass) and Oryza sativa L. (rice), were compared with maize. These species diverge phylogenetically from one another and from maize, with each belonging in different subfamilies. The three species also represent the range of reproductive habits seen in the grasses. Maize is an annual grass displaying a distinct juvenile phase and with a mild sensitivity to environmental influences on reproduction (Hunter, Tollenauer, and Breuer, 1977
). Bluegrass is a mixed perennial and biennial rhizomatous grass that requires dual daylengths and vernalization for floral induction. Juvenile and adult phases are not characterized explicitly in bluegrass. However, a juvenile growth phase is assumed because bluegrass seedlings must surpass a growth threshold before they will flower, usually in their second season of growth (Peterson and Loomis, 1949
; Evans, 1960
; Canode and Perkins, 1977
; Heide, 1980, 1990
; Meijer, 1984
; Carlson, Ehlke, and Wyse, 1995
; Sylvester and Reynolds, 1999
). Prior characterization of bluegrass development has focused on seedling and tiller growth (Etter, 1951
), but changes in leaf or shoot anatomy during the reproductive cycle have not been reported before.
Rice is an annual warm-season grass that tillers heavily and requires stringent photoperiodic induction of flowering (Summerfield et al., 1992
). Reproductive development of rice has been divided into three phases, similar to those described above for maize (Vergara, 1980
; Vergara and Chang, 1985
). The duration of the rice juvenile phase (termed the "Basal Vegetative Phase"; Vergara and Chang, 1985
) has been characterized only with respect to its lack of sensitivity to photoperiod (Suenaga, 1936
; Chang and Bardenas, 1965
; Vergara and Chang, 1985
). To date, the rice juvenile phase is recognized by counting either the number of days after sowing or leaf number. For most cultivars investigated, a minimum of five leaves must be produced before the juvenile phase is surpassed (Sasamura, 1960
). Anatomy and morphology of embryogenesis and early shoot development in rice has been characterized (Kauffman, 1959a, b
), but specific shoot traits that may correlate with phase have not been investigated before.
Rice, bluegrass, and maize represent three distantly related, physiologically distinct grass species in an otherwise similar family of plants. Anatomical and morphological comparison of phase change in these three grasses provides a starting point for further analysis of the evolution of phase change. We propose that changes in the duration of transitions between phases could account for differences in both phase regulation and reproductive timing among maize, rice, and bluegrass. In this study, we establish that a defined set of anatomical traits are not intrinsic markers of phase change in the different grasses, and we suggest that leaf shape threshold, rather than anatomy, is a more uniform indicator of underlying phase change in diverse grass taxa.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Results were consistent for all four cultivars; therefore, observations and results of experiments using only Huntsville and Glade are presented here, since these two cultivars represent the extremes in flowering induction. Huntsville has the shortest vernalization/induction requirement and thus flowers early in the season, whereas Glade has the longest requirement and flowers later in the season. Bluegrass seed was donated by Jacklin Seed (Post Falls, Idaho, USA). Two inbred lines of maize were selected: the early-flowering ND-101 and the late-flowering B73. Inbred ND101 seeds are maintained by stock originally donated by Dr. William Sheridan (University of North Dakota, Grand Forks, North Dakota, USA). The B73 seeds are maintained from original stock provided by Dr. Michael Freeling (University of California, Berkeley, California, USA). One typical short-day cultivar of rice of the indica genotype, Taichung Native 1 (TN1), was selected for this analysis. The TN1 is maintained by stock originally provided by Dr. Allan Caplan (University of Idaho, Moscow, Idaho, USA).
Growth conditions
Field-grown bluegrass plants were required for the study to ensure that seedlings would flower in their second year. Bluegrass seeds were planted in a randomized block design under irrigation in fields kindly donated by growers George and Art Thayer near Rathdrum, Idaho, USA, and Wayne Meyer of Rathdrum, Idaho, USA. Seeds were planted in mid-May using a cone-type double disk plot drill with press wheels. The field was fertilized in the fall, according to the growers' suggestions. Recommended herbicide treatments were used in the field prior to planting and occasionally to control weed problems (see Parker-Clark 1997
for details).
Corn seeds were planted in an experimental genetics plot under irrigation and rented from Pat Kirby in a field east of Lewiston, Idaho, USA. Seeds were planted in 20-kernel families, at 0.3-m spacing. Weeds were controlled by hand-hoeing and rototilling. The field was fertilized by standard methods prior to planting. For studying greenhouse-grown plants, corn seeds were planted under long days (16 h day : 8 h night) with supplemental lighting provided by metal halide lamps (Energy Technics, Pennsylvania, USA), with temperatures maintained at 25°C.
Rice plants were grown in hydroponic conditions in a growth chamber following the protocol of Yoshida et al. (1976)
. Briefly, seeds were sterilized and then soaked overnight before sowing in flats of vermiculite. The germinated seedlings were transplanted to light-tight plexiglass containers fitted with foam plugs, each of which held one seedling at a density of 3 plants per 4 L of solution. The solution contained appropriate concentrations of micro- and macronutrients (Yoshida et al., 1976
). Rice plants were grown under short-day photoinductive cycles (10 h day : 14 h night) at 30°C. Supplemental lighting was provided by metal halide lamps (Energy Technics).
Sampling procedures for leaf anatomy
Only fully mature, nonelongating leaf blades were sampled for anatomical analysis. Sampling methods varied due to different growing conditions and sizes of leaves. For bluegrass, individual plants were tagged in the field when leaves 3–4 had emerged, using numbered 1-cm paper tags with strings that were looped around the plants. Surveyors' flags were placed next to the tagged plants. Four to 16 seedling plants of each cultivar were tagged at seedling emergence. Leaf growth was monitored twice weekly by marking the leaf tips as they emerged from the furl with various colors of indelible ink, and the blades were measured when they had stopped growing. For maize, each sequential blade was measured twice weekly as new leaves emerged and then leaf measurements taken only on leaves that had ceased growing. Similarly, for rice, sequential nongrowing leaves of the main seedling plant were measured. The main seedling rice plant was marked to distinguish it from the numerous tillers produced in the TN1 cultivar. For all three species, tiller leaves were not included in anatomical analysis.
Anatomical analysis
For all three species, dental wax impressions were taken of the adaxial epidermis of nongrowing leaf blades using previously described methods (Sylvester, Cande, and Freeling, 1990
; Williams and Sylvester, 1994
). A two-part dental wax (Reprosil, medium body; Patterson Dental Supply, Sunnyvale, California, USA) was applied to the adaxial leaf blade at two positions (one-third and two-thirds distal to the ligule). To insure high magnification view of waxes, a positive impression was made using Epoxy resin and was sputter-coated and viewed with a scanning electron microscope (Hamamatsu, Bridgewater, New Jersey, USA). For lower magnification views of cell shapes, nail polish was applied to the dental wax impression and viewed with a standard light microscope (Carl Zeiss, Thornwood, New York, USA). Epicuticular waxes and hairs were recorded at each position for all blades examined. Thus, transition leaves as well as potentially juvenile or potentially adult leaves could be identified.
For cytochemistry, 1-cm segments of blades from numbered leaves were removed from all three species, fixed in three parts ethanol to two parts acetic acid, allowed to clear for 1–2 wk, and then stained with 0.05% Toluidine Blue-O (TBO) at pH 4.4. For rice, the abaxial surface was slightly abraded with fine silica powder to facilitate staining. Leaves were viewed in either paradermal view and/or hand cross sections, depending on the nature of the leaf. Maize and rice leaves were robust enough to permit observation of the entire stained adaxial surface. The narrow width and delicate nature of the bluegrass leaves prevented adequate viewing of the entire paradermal surface and so only hand sections were observed. Monochromasia was evident when cells stained uniformly blue or purple at low pH. Metachromasia was apparent when the cell walls stained both blue and purple at low pH.
Morphological and developmental analysis
Leaf dimensions were recorded by measuring blade length, sheath length, blade width at the widest point, and by measuring the distance of the widest point from the ligule. To measure width of bluegrass leaves, blades were removed from three of the field-grown plants, which had also been marked and sampled for anatomy. Blade width was measured at the widest point of the fully expanded leaf and compared with the anatomy of the same leaf number. The maximum width was determined by centering a ruler over the blade midrib at the ligule and running the ruler up to the blade tip and noting the first position at which the widest point was reached (at approximately one-third the distance from ligule to blade tip). Once each leaf had fully elongated, maize blades were measured directly on the plant in the greenhouse.
Blade and sheath lengths were measured in dissected plants of all three species. Separate groups of plants were required since the sampling procedure was destructive. Bluegrass plants were grown in the greenhouse in 14 groups of six plants each. Each group was grown to a given leaf stage and all six plants in the group designated for the given leaf stage were dissected. The leaf stage was based on when the outermost leaf had stopped growing. Leaf number was counted from the base, counting the bladeless prophyll as the first leaf. For maize, blade and sheath lengths were recorded for two groups of six plants each, since juvenile leaves senesce early, but adult leaves are retained. Plants in the first group were dissected to the meristem and measured before the fully grown juvenile leaves had begun to senesce. Plants in the second group were allowed to complete tassel formation before all leaves were removed and measured. For rice, four groups of up to ten replicates were dissected to record blade and sheath lengths. As with bluegrass, the rice leaf stage was based on the number of the most recently fully expanded leaf as counted up from the bottom. The bladeless prophyll was counted as leaf one for both rice and bluegrass. To monitor leaf development, plants were fully dissected to the shoot apical meristem under a Zeiss SV-11 stereomicroscope, and all measurements of changing leaf dimensions were recorded.
Assessment of floral induction
For bluegrass, floral induction was monitored by observing changes in the shape of the meristem, using a meristem-staging system established by Canode and Perkins (1977)
. A field of bluegrass was planted adjacent to the plot used for anatomical sampling. Plants were removed periodically from this adjacent plot, transported to the laboratory, and dissected to determine the stage of floral induction. Elongated, stage three meristems (see Canode and Perkins, 1977
) were considered induced. To confirm induction, 10-cm sod strips were removed from middle rows of three of the four replications per cultivar in mid- and late November (when vernalization had begun) and in mid-January. The twelve largest tillers from each sample were dissected, and meristem stages were recorded. Paired samples were placed in the greenhouse under continuous light, provided by metal halide lamps (Energy Technics), and 18°–23°C for a maximum of 3 wk, conditions shown by prior experiments to stimulate growth of panicles from the induced inflorescence (Parker-Clarke, 1997
). After stimulation of floral development, plants from the paired samples were dissected, meristem stages recorded, and floral induction confirmed or refuted. Similar methods were used for rice, but transplant experiments were not conducted because reproductive timing has been reliably established. Dissected rice meristems were considered to be either naked (still vegetative), elongated (early stages of inflorescence development), or ridged (later stages of inflorescence development).
Embryonic sections
Dry seeds of all species were imbibed briefly to soften the seed coat only, fixed using standard protocols, infiltrated with wax under vacuum, and then thick sectioned and stained to confirm numbers of embryonic leaves present in the dormant seed (Sylvester and Ruzin, 1994
).
Data analysis
For maize and bluegrass, average blade widths at each sequential leaf were averaged for the replicates. For these samples, anatomical traits and evidence of reproduction were recorded from the same specimens. For all three species, blade-to-sheath ratios were compared in two clusters of leaves from the six to ten replicates. The first cluster was considered to be composed of the earliest formed leaves from the juvenile plant and the second cluster was composed of the latest formed leaves from the adult reproductive plant. Linear regression and r2 were values calculated according to standard methods (Ott, 1993
) and presented using CricketGraph III v1.01 (Computer Associates, Islandia, New York, USA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). These induced meristems overwintered and then grew out as terminal branched panicles in the second season of growth. The 2-yr-old flowering bluegrass plants consisted of a series of leafy tillers and an extensive system of tillered rhizomes. Depending on the time of their emergence, the axillary and rhizomatous tillers were subsequently either annual or biennial in flowering habit (Sylvester and Reynolds, 1999
). The TN1 rice plants were larger and reached an average height of 1.2 m. Branched rice panicles were visible within 3–4 mo, with the primary seedling plant induced to flower around leaf stage 10–12. The TN1 showed extensive tillering beginning at the basal nodes as early as leaf stage three. Tillers also became reproductive in response to inductive photoperiods. Maize plants reached heights of 1.9 m for the early flowering inbred line (ND101) and up to 2.8 m for the late flowering inbred line (B73). Maize plants initiated terminal tassels by 4–5 wk after sowing and tassels had begun to shed pollen by 45–60 d after sowing. Numbers of embryonic leaves were counted in fixed and sectioned seeds of all three grasses (data not shown). Bluegrass embryos consisted of a cotyledon, a coleoptile, a bladeless, nonphotosynthetic leaf called a prophyll, and one incipient embryonic leaf. Rice embryos had a cotyledon (scutellum), followed by a coleoptile, a bladeless but photosynthetic leaf (termed a prophyll or primary leaf), and a single embryonic leaf that was more extensively developed than the incipient leaf in bluegrass. In contrast to these reduced plumules, the maize embryos consisted of the cotyledon (scutellum) and a coleoptile that surrounded five embryonic leaves. Maize lacked a prophyll, but the first embyronic leaf displayed a reduced blade size relative to the blades of the other embryonic leaves.
Each species showed a unique but consistent pattern of overall leaf development. For bluegrass, a new leaf primordium did not initiate until after the blade and sheath of the prior leaf was almost fully extended. Consequently, the bluegrass shoot apex consisted of only two primordia at a given time throughout development. Sheath and ligule began to extend when the blade was nearly fully elongated. Rice leaf primordia initiated only when the prior two leaves had begun to extend, producing a shoot apex with no more than three developing leaves at a given time. This pattern contrasted with maize, which showed an even more rapid production of leaf primordia so that at least five to six developing leaves were always present at the shoot apex. As reported before (Sylvester et al., 1990
), the maize sheath began to elongate before the blade was fully extended, resulting in progressively sized blades and sheaths. In all three species, the pattern of primordium emergence was the same: the basal flank of the meristem protruded circumferentially, then the developing leaf grew up and around to enclose the meristem as a hood-shaped structure. As the leaf margins grew, the primordium appeared as a cone that completely enclosed the younger inner primordia.
For all three species, early formed leaves senesced before flowering was complete. Bluegrass and rice leaves senesced progressively, producing a shoot that always had the same number of photosynthetic leaves (four for bluegrass, approximately six for rice). For maize, only the first five embryonic leaves senesced prior to emergence of the tassel, thus producing a reproductive shoot of given node number (15 for ND101 and 19 for B73). Also, the shoot meristem of all three species gradually increased in diameter and length during vegetative growth, as observed here (data not shown) and also reported before (Abbe and Phinney, 1951
; Kauffman, 1959a, b
; Sylvester and Reynolds, 1999
).
Comparative anatomy of juvenile and adult leaf epidermis
Anatomical traits were selected from previous reports of phase-specific traits observed in maize (Sylvester, Cande, and Freeling, 1990
; Bongard-Pierce, Evans, and Poethig, 1996
). These traits included epidermal cell shape, chromasia of epidermal cell walls, and the presence or absence of epicuticular waxes, hairs, and bulliform cells. The distribution of hairs, waxes, cell types, and cell shapes for all three grasses (Fig. 1) demonstrate that juvenile and adult leaves are anatomically distinct in maize but not in bluegrass or rice. The juvenile adaxial epidermis of maize is covered with epicuticular wax but lacks hairs and bulliform cells (Fig. 1A), whereas the adult adaxial epidermis is pubescent with bulliform cells but lacks epicuticular waxes (Fig. 1B). In contrast, the adaxial epidermis of all bluegrass leaves is covered with both epicuticular waxes and hairs (Fig. 1C, D). Rice is similar to bluegrass in that all rice leaves are coated with epicuticular waxes and bear hairs (Fig. 1E, F). Rice epidermal cells are also covered with papillae (Fig. 1E, F), which are impregnated with silica, as confirmed by EDAX scanning electron microscopy (data not shown).
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Cytochemistry was used to detect differences in wall compounds characteristic of juvenile vs. adult epidermal cells in maize. The entire intact adaxial surface of leaves could be observed directly for maize and rice (Fig. 2) and in cross sections for bluegrass (Fig. 3). In adaxial surface view, all cells in the juvenile maize leaves stain uniformly purple (Fig. 2A). In adaxial surface view of adult maize leaves, only bulliform cell walls, subsidiary cell walls of the stomatal complex, and cork and silica cells stain purple. The remaining adult cell walls stain blue. Additionally, the abaxial epidermis shifts from uniformly purple to uniformly blue in maize, as is visible in hand cross sections (compare abaxial epidermis in Fig. 3C with that in Fig. 3D). The adaxial surface view of rice leaves revealed metachromatic staining (defined as two different colors of stain visible) present in all sequential leaves (Fig. 2B, C). Cells that stain purple in rice are bulliform cells and intercostal cells, whereas those that stain blue are costal cells (compare Fig. 2C and Fig. 2D with Fig. 2E and Fig. 2F). In bluegrass, bulliform cells are not apparent in the first-formed leaf (Fig. 3A). The next leaf and all subsequent bluegrass blades, however, have bulliform cells and adaxial metachromasia (Fig. 3B). As in maize, the abaxial epidermis shifts to blue in later formed leaves of both bluegrass and rice (Fig. 2E, Fig. 3).
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Comparative leaf morphology
Leaf shape changes are subtle in the grasses due to the uniform lanceolate shape of all leaves and also due to plasiticity of certain leaf shape traits. We found that mature leaf width when compared at sequential nodes represented a common measurement that remained independent of environmental influence. Early- and late-flowering bluegrass (Fig. 4A, B) and also early- and late-flowering maize (Fig. 4C, D) showed progressive increases in width in early formed leaves until a threshold of width was achieved in later formed leaves. For bluegrass, this plateau of leaf width was achieved at leaf six for Glade and leaf seven for Huntsville. Leaf widths plateaued at 3.6 mm for Glade and 3.8 mm for Huntsville, as confirmed by statistical analysis and as reported elsewhere (Parker-Clarke, 1997
). Both epicuticular waxes and hairs were present on all bluegrass leaves measured. Bluegrass plants were receptive to floral induction after five leaves of the maximum width had been produced (asterisks in Fig. 4A, B).
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8 cm at node 12 for B73 (Fig. 4C) and 9 cm at node 12 for ND101 (Fig. 4D). Only four leaves of maximum width were present in ND101, compared with the eight leaves of maximum width in B73. Reduced blade width was also observed in flag leaves of both inbred lines. For both inbred lines, the first five leaves were uniformly waxy, followed by the next two or three transition leaves, which had wax remaining at the leaf tip and hairs near the leaf base. By node 8 for each maize inbred line, waxes were completely absent and adult epidermal features were present. Leaf width continued to increase gradually in all of these anatomically juvenile, transition, and early adult leaves of maize. Leaf width stabilized at ear nodes, which were well past the transition to adult leaf anatomy in both B73 and ND101 (Fig. 4C, D). Blade and sheath lengths were measured in all three species. Two morphological patterns were discerned by comparing blade to sheath in two clusters of fully mature, nongrowing leaves. These leaves were designated as the early formed "juvenile" leaves (Fig. 5A, C) and the later formed "adult" leaves (Fig. 5B, D). For juvenile Glade and Huntsville leaves, blade length increased steadily and in accordance with increasing sheath length (Fig. 5A). In subsequent adult leaves the blade continued to increase gradually, but the sheath increased less rapidly relative to the blade. Leaves that reached a plateau of blade width (Fig. 4) also showed a plateau of sheath length relative to blade length. A similar pattern was observed for B73 leaves of maize. In sequential juvenile maize leaves, blade and sheath lengths also increased uniformly (Fig. 5C), whereas sheaths of fully adult leaves ceased increasing relative to the blade (Fig. 5D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All three grasses show a distinct change in leaf morphology that is independent of anatomy and associated with plant aging. Sequential leaves in all three species cluster into two morphological groups. In terms of maximum blade width, early formed maize and bluegrass leaves show a continuous increase in width until a plateau is reached in the later formed leaves. A similar threshold in leaf shape is observed when blade-to-sheath lengths are compared. This threshold of leaf shape is achieved in nodes that are best characterized as adult vegetative. For maize, the threshold in leaf shape occurs well after a transition in leaf anatomy from typically juvenile to adult and just before ear nodes are established. For bluegrass and rice, the threshold occurs independently from any anatomical indicator but several nodes before flower induction occurs.
Groups of morphologically distinct leaves (as described here) represent a form of heteroblasty, defined as a change in leaf morphology that occurs during shoot development (Ashby, 1948
; Allsopp, 1967
; Kerstetter and Poethig, 1998
). Heteroblasty may be of two types: either leaves change abruptly in shape, as in Pseudopanax (Clearwater and Gould, 1993
) or leaves show progressive and gradual change until a climax leaf shape is acquired, as in Ipomoea (Ashby, 1948
). Heteroblasty in discrete steps may be associated with phase change, whereas progressive changes in leaf shape may correlate with physiological aging of the shoot (Ashby, 1948
). The three grasses studied here show both types of heteroblasty in that a progressive change in leaf shape occurs in the early formed leaves, with each node producing a leaf of gradually increasing blade and sheath lengths. A discrete change then occurs when the blade width plateau is reached and when sheath lengths equalize. For the three grasses, this change precedes and then continues to correlate with the reproductive competence of the plant. Based on the timing of shape change in relation to anatomical phases in maize, we suggest that shape change represents a previously undescribed step in the progression from vegetative to reproductive growth.
These results suggest that a climax leaf shape is attained prior to or during the acquisition of reproductive competence. It has been suggested that leaf shape is not a reliable indicator of developmental phase in maize because growth rates vary due to genetic constitution or environmental conditions (Bongard-Pierce, Evans, and Poethig, 1996
). Given this possibility, we compared two different inbred lines in maize grown under different conditions, greenhouse and field. Additionally, four different bluegrass cultivars grown 2 yr apart were examined. Similar patterns of growth thresholds were achieved in all cases, although final sizes and rates of change varied slightly. We conclude that basic architectural patterns in these grasses are invariant, despite physiological responses that generate differences in final size.
Significant differences in leaf and shoot development were identified in this study. First, embryonic leaf number differs among the three species: maize seeds display five embryonic leaves, regardless of the flowering time, whereas bluegrass and rice seeds display only one embryonic leaf. Interestingly, in maize, the number of anatomically unique "juvenile" leaves in the seedling corresponds to the number of embryonic leaves in the seed. The unique anatomy of the first five leaves of maize could therefore reflect their embryonic legacy rather than a direct relationship to a strictly juvenile phase of growth. In contrast, bluegrass and rice have only one incipient embryonic leaf and an absence of distinctly different juvenile leaves.
The correlation between juvenile leaf appearance and number of embryonic leaves is suggestive and deserves further consideration. It is possible that selective pressures may have similar impact on leaf anatomy and development of the embryonic plumule, but not on reproductive timing (and vice versa). Mutant study has shown that expression of juvenile or adult epidermal anatomy is genetically separable from flowering time in maize (Evans and Passas, 1994
; Moose and Sisco, 1994
). On the other hand, juvenile anatomy and embryonic leaf production can also be separated genetically. We have observed that gl15 mutants retain the normal number of five embryonic leaves even though they have a reduced number of anatomically juvenile leaves (data not shown). Another intriguing experiment shows that phase change is sufficiently flexible that a juvenile maize leaf can be initiated on an isolated adult meristem bearing less than four leaves (Irish and Jegla, 1997
) and that phase identity is acquired late in leaf development (Sylvester, Cande, and Freeling, 1990
; Orkwiszewski and Poethig, 2000
). It is possible that isolated nearly naked meristems are exposed to a hormonal or developmental environment similar to that experienced by developing maize embryos, resulting in the production of a shoot apex that initiates juvenile leaves. An understanding of what triggers these developmental programs deserves continued study.
Another significant developmental difference among the three species concerns the relative rate of leaf initiation compared to leaf growth. These observations are based on the morphology of leaves around the shoot apical meristem. New maize leaf primordia initiate relatively frequently, when compared with the rate of elongation of the blade and sheath of prior formed leaves. Consequently, the maize shoot apex consistently bears at least five to six leaf primordia all at progressive stages of development. In contrast, bluegrass and rice leaves initiate at a slower pace relative to blade and sheath growth. For the bluegrass and rice apex, consequently, no more than two or three primordia reside around the apex at any given time.
There are several implications of the results presented here. First, we suggest that leaf heteroblasty in the grasses, but not anatomy, is a reliable indicator of phase change for comparative purposes in the family. For some species, such as maize, the link between anatomy and phase change is tight enough that the classic anatomical traits remain a useful indicator of at least an early vegetative phase. For other grasses, such as rice and bluegrass, more subtle changes requiring leaf measurement may be needed to evaluate phase change in the absence of defined anatomical indicators. Leaf shape threshold is thus another indicator of developmental progression and is currently the sole shared trait among these divergent grass species. Causal factors associated with this progression remain to be clarified.
The second implication of these results is that comparative anatomy and morphology may help to evaluate how the expression of phase evolved and changed in the grasses. Different grass taxa may show variations in the duration of embryonic and postembryonic development programs superimposed on different rates of shoot apex growth. For maize, these developmental programs could be prolonged, resulting in gradual transitions between phases and long retention of "juvenile" or embryonic traits. Also in maize, architecture of the shoot apex suggests that leaf initiation is juxtaposed against slow overall leaf development, resulting in a protracted production of emerging leaves. In contrast, bluegrass and rice show abbreviated transitions between embryonic and postembryonic development, reflecting the reduced plumule size and anatomically indistinct leaves. Also for these heavy tillering species, reduced rate of leaf initiation combines with accelerated overall leaf development. This could result in a shoot apex that acquires new identities in bluegrass and rice more rapidly than in maize. Abbreviated vs. prolonged developmental phases, as suggested here, may ascribe a role for heterochrony in the evolution of these traits. Heterochrony is likely a significant factor in the evolution of plants (Lord and Hill, 1987
) and has been used to explain shifts in developmental patterns observed in maize before (Bertrand-Garcia and Freeling, 1991
; Evans and Passas, 1994
).
The idea that these grasses show variations in timing of embryonic vs. post-embryonic development programs can be tested by more detailed phylogenetic and morphological analysis of the family. The analysis presented here only considers three distantly related species. Carefully selected taxa from within subfamilies will help to clarify trends in specialization of phases among the grasses. A precise separation of growth phases into distinct juvenile vegetative phase, adult vegetative phase, and adult reproductive phase may not have universal applicability. We show here that change in leaf shape marks a previously undescribed step in the progression through postembryonic growth. For practical purposes, anatomy is still a useful indicator of phase for maize, as long as it is accurately perceived as downstream from the primary genetic regulation of phase change. Maize has both prolonged phase transitions and protracted early leaf development, whereas bluegrass and rice both show abrupt transitions and more abbreviated growth habits. The next step is to evaluate these patterns of phase change in relation to shoot development in a more finely tuned phylogenetic comparison of the grasses.
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FOOTNOTES |
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5 Author for reprint requests (annesyl{at}uwyo.edu
).
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in A and B) is compared with Glade (
in A and B). Only late flowering B73 (
) is shown in (C) and (D). Six individuals at sequential nodes are shown in each case. Overlapping points are not distinct