ASCORBIC ACID LOCALIZATION DURING VEGETA-TIVE AND REPRODUCVEGETA-TIVE DIFFERENTIATION IN
CYPERUS ROTUNDUS L.
N. V. MADHAVAN UNNI and C. K. SHAH
Botar~y Department, GuJarat University, A!zmedabad-9
Received on December 8. 1968
SUMMARY
Shoot apices and spikes of different developmental stages of
ASCORBIC ACID LOCALIZATION IN CYPERUS 173
INTRODUCTION
Considerable evidence has accumulated during the past few decades to show that ascorbic acid (AA) plays a vital role in the metabolic activities related to growth and development in plants. High concentration of AA in actively growing zones of root tips (Reid, 1937, 194la, b, c), germinating seeds (Ahlberg, 1935), in sprouting of potato tubers (Pett, 1936) and during active vegetative growth in pea, clover and wheat (Virtanen, 1949) has been reported. Embryos of different varieties of pea seedlings diDered in their ability to sythesise vitamin C in culture medium when cotyledons were removed (Bonner and Bonner, 1938). Mitotic chromosome division in polyploid cells and meiosis in diploid cells of onion roots were induced by treatment with AA (Sharma and Datta, 1956). Chinoy (1962) co-related the metabolism of AA with that of nucleic acids and proteins and postulated 'Ascorbic acid-Nucleic acid-Protein Metabolism Concept of Growth and Development in plants'. This paper deals with the histochemical localization of AA in
Cyperus rotundus during the periods of vegetative and reproductive
differentiation.
MATERIALS AND METHODS
Modified method (Dave et al., 1968) of Barnett and Bourne
174 N. V. MADHAVAN UNNI AND C. K. SHAH
Thus the specificity of the stain was determined from three sets of slides.
RESULTS
The L.S. of the vegetative bud at the tuber level (Fig. 1) shows the concentration of AA in the following order: (1) the extreme tip of the apex, (2) the peripheral meristem on the left, (3) the buttress of leaf primordium on the right, (4) the upper part of the leaf primordium on the left and ( 5) the basal part of the developing leaf on the right. The five zones are indicated by serial numbers in fig. 1.
The ray primordia are formed at an early stage at the tip of the reproductive axis (Fig. 2) which elongates by the activity of an intercalary meristem at the base. Here, two sides of the ray primordia differentiate into ridges forming growth centres for the production of secondary branches. Shoot apices at this stage and the new growth centres register higher concentrations of AA when compared to the previous stage. The storage cells of the tuber below the intercalary meristem (Fig. 3) record higher AA concentration when compared to the storage cells at the same part (Fig. 4) when the apex is at the tuber level only. In these cells AA is more in cytoplasm while the cells of the intercalary meristem (Fig. 5) have big nuclei, densely packed with black grains showing the heavy concentration of AA. Some buds at the same stage show smooth periphery and negligible AA. These are the apices of aerial axes which produce no spikes.
AA content increases in the newly formed growth centres of the shoot apex. This is maintained until the florets are formed. The primordia of florets record very heavy AA concentration. Figure 6 shows florets at different stages of development at the tip of a young spike. The AA concentration is in the following order of the production of florets. (a) The cells towards the left hand side below the apex (1), (b) the cells forming a convex projection on the right hand side (2), (c) further below on the left hand side the cells forming the buttress of a flower primordium (3), (d) still below on the right hand side where the buttress has divided into an upper lobe ( 4) and a lower lobe ( 5).
ASCOkBIC ACID LOCAtlZATION IN crn:1ws 175
FIGs. 1-7. 1. L s. of shoot apex at the tuber level; 2. L s. of differentiated rays; 3. Cells of the tuber during the emergence of reproductive axis; 4. Cells of the tuber during vegetative stage; 5. Intercalary meristem; 6. L. s. of spike with florets at different stages of development; 7. L. s. of differentiated stamen and developing carpel. (an-anther; ant-antipodals; ca-carpel; en-connective; cw-cell wall; e--egg; emc-mdosperm mother cell; ep-epidermis; fil-filament; mic-microspore mother cell;mmc-megaspore mother cell;nu-nucleus; pn-polar nuclei; syn-synergids; t--tapetum; wl-walllayers; zgt-zygote).
176 N. V. MADHAVAN UNNl AND C. K. SHAH
FIGs, 3-14. 3. L. S. of anther with microspore mother cells; 9. L. S. of anther showing pollen mother cells; I 0. Pollen grains; I I. Young ovule with megaspore mother cell; 12. Ovule with linear tetrads; 13. Embryo sac before fertilization; 14. Embryo s~c after fertilization. (ad~anther; ant~antipodals; ca~carpel; cn~connective; cw~cell wall; e~egg; emc~endosperm mother cell;
ep~epidermis; fil~fllament; mic~microspore mother cell; mmc~megaspore
mother cell; nu~nucleus; pn~polar nuclei; syn~synergids; t~tapetum; wl~wall
N. V. MADHAVAN UNNI AND C. K. SHAH 177
forming a cleft as seen in the L.S. (Figs. 9 and 1 0). The outer lobe in the figure is the primordium of an anther and the inner one of a carpel. The cells of the stamen and carpel primordia register uniformly heavy concentration of AA. But in a stamen, the filament shows negligibleAAwhile the anther head shows high concentration (Fig. 7). When the differentiation of wall layers, tapetum and sporogenous tissue takes place, the AA concentration shifts towards the periphery. The tapetum and the middle layers have the maximum AA (Fig. 8). Then there is a decline in AA during meiosis and when pollen mother cells are formed, it is comparatively less (Fig. 9). But when pollen grains mature they regain the AA concentration and fully matured pollen grains record very heavy concentration (Fig. 10). The cells of the filament during the later stages of microsporogenesis start recor-ding an increase of AA and when the stamen is fully matured the filament has the maximum concentration. This co-relates with the further elongation of the filament. The connective and the wall layers also show a high AA concentration at this stage. The ovarian wall develops as a collar-like outgrowth around the central ovule primordium (Fig. 7). All the cells of the carpel at this stage have appreciable AA but slightly less than the cells of the anther of the same floret. The development of the anther is faster than that of the carpel. In the carpel at this stage the ovarian wall has more concentration than the ovule primordium. The ovarian wall grows faster, later on converges above the ovule and fuses to form the style.
The AA content of all the cells of the ovule in the initial stages is uniformly high (Fig. 11). But during later stages the occurrence of AA in high concentration is restricted to certain specific regions only. When a linear tetrad is formed (Fig. 12) the integument and the base of the funiculus show highest con-centration while the nucellar tissue and megaspore have less AA. At this stage the ovary wall, especially the innermost layer and the style also have high AA concentration.
173 N. V. MADHAVAN UNNI AND C. K. SHAlt
DISCUSSION
Synthesis of AA in storage tissues and its subsequent translocation to the growing regions has been reported by a number of workers (von Hausen, 1936, 1936a; Bonner and Bonner, 1938; Virtanen and vonHausen,'19"'"9). InC)perus rotur;dus,
the cells of the tuber register high AA with the onset of repro-ductive phase indicated by the shooting up of reprorepro-ductive axis. Chayen et al. ( 1953) found heaviest concentration of precipitated silver in meristematic cells, the greatest abundance being on the chromosome region of interphase nuclei and on the mitotic chromosomes. Sharma and Datta (1956) hold that AA increases nucleic acid production which induces mitosis in permanent polyploid cells and meiosis in diploid cells. The intercalary meristems responsible for the elongation of the reproductive axis of
Cyperus rotundus too have the active nuclei with high
concentra-tion of AA. Moreover, the shoot apices also record higher AA concentration. Hence it seems that the soluble carbohydrates in the tuber are converted into AA at the time of reproductive differentiation and this AA triggers active division in the inter-calary meristem which results in the springing of reproductive axis. This AA translocated to the shoot apex initiated
differen-tiation of organs.
Chinoy (1962, 1964) assigns a regulatory role to AA in growth and development. According to him, the increase of AA content of the shoot apex enhances increased production of nucleic acids and proteins. This induces high metabolic activity of the cells and results in the formation of various organs. In
Cyperus mtundus there is a steady increase of AA in the shoot apex
during the differentiation of reproductive organs. It shows specific accumulation in the tissues destined to produce new growth centres from which new organs arise. During organo-genesis the concentration of AA fluctuates in the tissues showing active cell division and differentiation during the development of essential organs indicating that it participates in metabolic processes. A decrease of AA content in sporogenous tissue may be due to the high AA utilization during the energy consuming process of gametogenesis.
ASCORBIC ACID LOCALIZATION lN crPERUS 179
layers, like integuments, ovarian wall, anther wall, etc., inCyperus rotundus may be interpreted to mean that AA offers protection to
the -SH group (Harrer and King, 1941) as well as to the activity ofsome enzymes (Hellerman, 1937). The high accumulation of AA in mature pollen grains also may be attributed to such a defensive mechanism against injury due to adverse environmental conditions.
The high ascorbic acid content of post-fertilized synergids and antipodals of C:yperus rotundus corroborates the findings of
Poddubnaya-Arnoldi and Zinger ( 1959) who found that embryo sac, egg and antipodals of C:ypreprdium insigne were rich in AA, the
antipodals registering the maximum. This leads one to postulate that by fertilization two growth centres are formed in the embryo sac, the zygote and the fusion nucleus, and the svpply of AA to them is through the synergids and antipodals, respectiYcly. 'I he fact that the cells of the nucellus on either side of the embryo sac show concentrated accumulation of silver grain supports this postulation.
ACKNOWLEDGEMENT
We express our deep felt thanks to Professor
J .
.J.
Chinoy for his generous counsel and guidance at every stage of this work.REFERENCES
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