Planta I35, 233 240 (1977)
9 by Springer-Verlag 1977
Development of the Quiescent Center in Maturing Embryonic Radicles of Pea (Pisum sativum L. cv. Alaska) P.A. Jones Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA
Abstract. Maturing embryos of pea (Pisum sativum L. cv. Alaska) were treated with an aqueous solution of tritiated thymidine for 1 h, sectioned, and processed for autoradiography. An analysis of the distribution of labelled nuclei and mitotic figures demonstrated the presence of a quiescent center (QC) in the radicles of developing embryos. The QC developed in the radicle during the growth of the embryo. Immature radicles that did not contain a well-formed zone of root-cap initials did not show a QC. In the latter stages of seed ripening, the pattern of arrest of DNA synthesis and mitosis was tissue-specific. Cells within the QC remained inactive. The region lacking labelled nuclei and mitotic figures progressively expanded to include the root cap initials and then the provascular cylinder. Mitosis was arrested before DNA synthesis in the embryonic cortex. Cells within the QC synthesized DNA during the first stages of seed germination. Key words: Cell cycle - DNA synthesis - Embryo - Pisum - Quiescent center - Root (radicle).
The apical meristem of an angiosperm root contains a region, the quiescent center (Clowes, 1956), in which DNA synthesis seldom occurs. The QC is defined as an area whose "cells proliferate more slowly than any of the surrounding cells of the meristem" and is "always situated at the pole of the stelar and cortical complexes of cells adjacent to the initials of the central part of the root cap" (Clowes, 1975). The development of the QC has been examined by Clowes (1958) who found that roots from germinating seeds Abbreviations: [3H]TdR=tritiated thymidine,QC=quiescent cen-
of Sinapis alba initially had no QC but developed one within 3 days of germination. Alfieri and Evert (1968) also reported that in Medicago and Trifolium the QC developed after germination, starting as a small area and increasing in size. However, Byrne and Heimsch (1970) concluded that in M a l v a sylvestris "the quiescent center was established during embryogeny and continued to exist during germination ". There is some evidence that radicles in maturing embryos show a region within the apical meristem which is comparable to the QC in roots. Sterling (1955), studying maturing lima bean embryos, suggested that a "focal zone" of mitotically inactive cells develops in the apical meristem of the radicle. This inactive "focal zone" surrounded by actively dividing cells could correspond to the QC in seedling root tips. Thomas (1975) summarized the results of investigations of several workers (Norreel, 1973 ; Rondet, 1962; Vallade, 1972) that showed that embryonic radicles contained a region within the apical meristem that stained very lightly with methyl-green pyronin or Schiff's reagent. Thomas (1975) labels this region the "centre quiescent radiculaire" and indicates that it is comparable to the QC found in seedling root tips. If the QC does exist in radicles of maturing embryos, it should show a lack of incorporation of [3H]TdR after exposure of the radicle to this radioactive DNA precursor. This paper is the first report of an analysis of the pattern of label incorporation designed to detect the presence of the quiescent center in the radicle of a maturing embryo. Material and Methods Plant Growth
Plants were grown under controlled conditions so that embryos at various stages of maturitywould be available. Pea (Pisum sat#
rum L. cv. Alaska) seeds from W. Atlee Burpee Co., Clinton, Ia., USA, were surface sterilized with undiluted Clorox (5% sodium hypochlorite) for 5 rain, rinsed well with sterile water, and soaked in water overnight. The seeds were planted in a soil mixture (loam, manure, leaf mold; 2:1:1) in 20-cm (8-in.) plastic pots, 15~0 seeds per pot. The plants were grown in an environmental walk-in chamber (Model GC-808, Vestal Modern Design Corp., Vestal, N.Y., USA) on 16-h day, 8-h night cycles. The day temperature was 19~ C, the night temperature 14~ C. Relative humidity was maintained between 40% and 50%. Plants taller than 30-40 cm were supported by vinyl-covered wire mesh. Under these conditions, the plants flowered within 6-7 weeks of planting.
Application of Label In order to apply [3H]TdR to the radicle it was necessary to expose the embryo. First, the distal end of the pod was cut off and the two halves of the pod were separated along the dorsal and ventral sutures. The seed coat covering the embryo was carefully slashed to form a flap that, when lifted, exposed the radicle. [3H]TdR (New England Nuclear, Boston, Mass., USA), spec. act. 50 Ci] mmol, was applied to the radicle as a 3-gl drop of aqueous solution corresponding to a dose of 0.75 gCi. The embryo was then covered with the flap formed by the seed coat, the cut area of the seed was sealed with a melted mixture of vaseline, paraffin and lanolin (1:1:1), and the pod was wrapped in plastic film (Parafilm). The treated embryos were harvested after 1 h.
Root Tips Alaska pea seeds were soaked in water for 6 h and placed on wet vermiculite. Twenty-eight h alter the onset of imbibition some seeds were transferred to filter paper saturated with [3H]TdR, spec. act. 50 Ci/mmol, 1 gCi/ml in 1/4-strength Hoagland's solution, pH 4.7. Root tips from these seeds were harvested 48 h after the onset of imbibition. Another group of seeds was exposed to label 72 h after the onset of imbibition by immersing their root tips in aerated 1/4strength Hoagland's solution, pH 4.7, containing 1 ~tC/ml [3H]Tdr (as above). These root tips were harvested after 1 h or 24h of exposure to the label.
Processing the Tissue Embryos and root tips were fixed for 10 rain in acetic acid-ethanol (1:3, v/v), rinsed twice with water, and dehydrated in a series of water, ethanol, t-butanol mixtures (Jensen, 1962). The material was embedded in 56~ nap Paraplast and sectioned at 10 gm. The sections were mounted on slides with gelatin adhesive, deparaffinized in xylene, and hydrated in an ethanol-water series. After hydrolysis in 5 N HCI at 20~ C for 45 min, they were washed briefly in water and stained in Schiff's reagent for 2 h at 20~ C. The sections were bleached (5 g potassium m-bisulfite, 50 ml of 1 N HC1 in 1 I distilled water) for 2 x 10 min each and the slides were dipped in Kodak NTB-2 liquid emulsion. After 5 days exposure, the slides were developed in Dektol (Kodak, Rochester, N.Y., USA), dehydrated through xylene, and permanent mounts were made with Canada balsam.
Evaluation of the Pattern of DNA Synthesis and Mitosis In this investigation the duration of exposme of the embryo to [3H]TdR was deliberately kept short (1 h) to minimize disturbance
P.A. Jones: Embryonic Quiescent Center in Pea Radicles of the normal processes of development caused by the required surgical procedures or by radiation damage resulting from the decay of incorporated tritium. Stetka and Webster (1975) found that prolonged exposure of pea root tips to [3H]TdR results in DNA synthesis in the cells of the quiescent center. However, Phillips and Torrey (1971) commented that short exposures of root tips to [3H]TdR resulted in autoradiographs in which the QC was not sharply delimited in any given medial section. In order to evaluate the pattern of DNA synthesis in pea radicles exposed to [3H]TdR for I h, it was necessary to examine the distribution of labelled nuclei in several sections of the serially sectioned embryo. For this, the median section was first selected for each embryo. Then the location of labelled nuclei in the five central sections (the median section plus 2 sections on each side) were plotted to form a composite drawing representing the location of labelled nuclei in a 50-1am-thick section. Similar techniques for making composite drawings were used by Sachs et al. (1959). The locations of labelled nuclei were determined using a Leitz Orthoplan microscope, the stage of which was fitted with strain gauges accurate to 3 gm (Jackson, 1967). First the outline of the radicle was drawn using the x, y coordinates for 15-20 points along the periphery of the radicle. The locations of all labelled nuclei within the terminal portion of the radicle were plotted. The outlines of each of the 5 sections from a single radicle were superimposed to give the composite. Although the QC is usually detected by its failure to incorporate [aH]TdR, this region also shows fewer mitotic figures than the surrounding meristematic tissue. Haigh and Guard (1963) demonstrated that the QC could be delimited by plotting the location of mitotic figures from the median sections of 100 corn root tips on a drawing of a representative median section. In order to analyze the distribution of mitotic nuclei in pea radicles, the locations of all mitotic figures in the terminal portions of the embryonic radicle were plotted on a composite drawing using the same techniques as described for labelled nuclei. Composite drawings were also made for seedling root tips that were exposed to [alq]TdR for 1 h. This was done as a control to insure that this method of analysis of the pattern of labelled or mitotic nuclei would detect the QC in a root tip that was known to contain such a center.
Measurement of Embryo Development Seed weight was used as a measure of embryo development. Eeuwens and Schwabe (1975) have shown that in Alaska peas seed mass and embryo mass are closely related throughout embryo development. Developing pea seeds were divided into seven weight classes: Stage 1 (150-199 mg), Stage 2 (200 249mg), Stage 3 (250-299 mg), Stage 4 (300-349 rag), Stage 5 (350-399 rag), Stage 6 (400-449 rag), Stage 7 (450 mg or more). Four or five embryos within each weight class were exposed to [3H]TdR.
Results C o m p o s i t e d r a w i n g s s h o w i n g the location o f labelled n u c l e i a n d m i t o t i c f i g u r e s in t h e 5 c e n t r a l s e c t i o n s of pea r o o t tip e x p o s e d to [3H]TdR for 1 h d e m o n s t r a t e the presence of the QC. Figure 1A and 1 C show the location of labelled nuclei a n d mitotic figures, r e s p e c t i v e l y . F i g u r e 1 B is a p h o t o m i c r o g r a p h o f t h e m e d i a n 10 m i c r o n s e c t i o n o f t h e s a m e r o o t tip. T h e Q C , l o c a t e d p r o x i m a l t o t h e r o o t c a p initials, is a
P.A. Jones: EmbryonicQuiescentCenter in Pea Radicles region with few labelled nuclei (Fig. 1A) and few mitotic nuclei (Fig. 1C). Feldman (1975) has used the terms distal and proximal meristems to name the meristematic regions surrounding the QC. In his terminology, the distal meristem would be equivalent to the root-cap initials, and the proximal meristem to the stelar and cortical initials. These regions contain labelled and mitotic nuclei in pea seedling root tips. A QC was present in radicles of Stage 5 and larger. Figure 2A shows the pattern of labelled nuclei in a Stage-5 radicle that was exposed to [3H]TdR for 1 h. Figure 2B is a photomicrograph of the median section of this radicle and Figure 2C shows the location of mitotic divisions in the radicle. There is a group of cells proximal to the root cap initials that contains few labelled nuclei. Numerous nuclei of the distal and proximal meristems surrounding this region are labelled. The region contains no mitotic figures (Fig. 2 C) even though the surrounding meristem contains many mitotic cells. This inactive region within the apical meristem of the radicle clearly corresponds to the QC of the seedling root tip. As mentioned before, the duration of exposure of the radicle to [3H]TdR was intentionally quite short (1 h). During the period of incubation with label, only those nuclei undergoing DNA synthesis would be labelled. Meristematic cells in other phases of the cell cycle (G2 or G1) would not be detected when the patterns of labelled and mitotic nuclei were analyzed. Thus, it could be argued that if the cells in the region of the QC of the radicle were synchronized and by chance were in G 1 or G 2 during the labelling period, they would be unlabelled. In order to demonstrate that the cells in the QC were, in fact, cycling more slowly than the surrounding cells, some radicles were incubated with [3H]TdR for 24 h. Figures 8 and 9 are autoradiographs of the median section of Stage 5 embryos fixed after 24 h of labelling. The QC is evident as a region with few labelled nuclei. The QC develops during embryo growth (Table 1). Embryos of Stages 1 and 2 did not contain a QC. Figure 3A is a composite showing labelled nuclei in an embryo of Stage 2. The region proximal to the root cap initials contained labelled nuclei and mitotic nuclei (Fig. 3 C). The QC develops during Stages 3 and 4 of embryo growth. Its development is gradual, with the number of labelled and mitotic nuclei in the region of the QC decreasing as the embryo matures. As the seed ripens, the cells in the distal and proximal meristems are arrested in the cell cycle. In Stage 6 (Fig. 4A-C) the central root cap initials (distal meristern) no longer contain S phase or mitotic nuclei. As the process of seed ripening nears completion in
235 Stage 7 (Fig. 5A) DNA synthesis is restricted to the embryonic cortex and the periphery of the root cap. Mitoses (Fig. 5C) occur in the provascular cylinder and the cortex, but the apical meristem no longer contains dividing cells. During the first stages of germination, the cells in the QC incorporate [3H]TdR, indicating that they have synthesized DNA. Figure 6 is an autoradiograph of a seedling root tip exposed to label 28 h after the onset of germination. Nuclei in the region of the QC are labelled heavily. In contrast, Figure 7 is an autoradiograph of a seedling root tip exposed to label 72 h after the onset of germination. The cells in the QC do not incorporate [3H]TdR. In seedling root tips (Fig. 1A-C), nuclei within all regions of the root cap incorporate label. Mitotic divisions within the root cap are restricted to the region of the cap initials. This is consistent with Barlow's (1973) report that cells in the root cap of Zea show endoreduplication of DNA. During development of the embryo (Stages 1 4 ; Figs. 2-4), DNA synthesis and mitosis occur in various regions of the cap of the radicle. Only in the last stage of embryo development (Fig. 5) does the pattern of DNA synthesis and mitosis in the cap resemble that in seedling root tips.
The results of this investigation provide information on several aspects of the QC: (1) its presence in the embryonic radicle, (2) its development during embryo maturation, (3) its response to the onset of seed ripening, and (4) its response to seed germination. The QC is present during the later stages of embryogenesis in pea radicles. It is comparable in appearance and location to the QC of seedling root tips. The QC in pea radicles corresponds to Sterling's (1955) "focal zone" of mitotically inactive cells in the radicle of maturing lima-bean embryos. Vallade (1972), in a study of embryogenesis in Petunia, found in the globular embryo a region containing 4 cells ("le centre quiescent") that remained mitotically inactive throughout the entire duration of embryogenesis. In the pea embryo, in contrast, the QC was not present during early stages of the development. Feldman (1976) excised the root cap, the distal meristem and the QC in corn root tips and then studied the regeneration of the QC. He found that the onset of quiescence preceded the development of the histological patterns of tissue formation found in normal seedling roots. In pea embryos, the histological zonation is well established before the onset of quiescence within the apical meristem. The
P.A. Jones: Embryonic Quiescent Center in Pea Radicles
P A Jones: Embryonic Quiescent Center in Pea Radicles
.. ..% ~: