Plant Science Letters, 16 (1979) 95--99 © Elsevier/North-Holland Scientific Publishers

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CHANGES IN THE CONTENT OF CHLOROPHYLL, PROTEIN AND NUCLEIC ACIDS AND IN THE EFFICIENCY OF PHOTOELECTRON TRANSPORT OF CHLOROPLASTS DURING GROWTH OF MAIZE SEEDLINGS

N.K. CHOUDHURY and U.C. BISWAL*

Laboratory of Biophysics and Biochemistry, School of Life Sciences, Sambalpur University, Burla 768017 (India)

(Received December 5th, 1978) (Accepted March, 27th, 1979)

SUMMARY

The chlorophyll content of developing maize leaves shows a maximum value and remains in a steady state between the fourth and seventh day of seedling growth. During this period, the levels of nucleic acids and of the Hill reaction by isolated chloroplasts, do not exhibit any significant change. On the contrary, a considerable loss in the content of leaf protein during the same period probably suggests that the loss of total leaf protein, which is normally considered to be an index of leaf senescence, may not always reflect the loss of the chloroplast proteins associated with the electron transport system of the organelle.

INTRODUCTION

Extensive studies have been made on leaf development, maturation and senescence [1--6]. Leaf maturation normally corresponds to a state where most of the physiological and biochemical characteristics of leaves are fully developed and in a steady state. This stage is followed by senescence, a deteriorative process leading to death. These sequences of leaf growth are normally characterised by the level of leaf chlorophyll, protein or nucleic acids [1,2,7--10]. Since the levels of pigment and macromolecules show a parallel increase and decrease during leaf development and senescence re- spectively [1,9,10], the status of the leaves is readily determined by moni- toring either of these parameters.

However, our results show that pigment and protein levels do not run in parallel in the developing leaves of maize seedlings. The leaves, in a steady

*To whom any correspondence should be addressed.

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and matured state with reference to the chlorophyll content and photo- synthetic efficiency of isolated chloroplasts, show a considerable loss in the content~of total protein, a symptom of leaf senescence.

MATERIALS AND METHODS

Maize seeds (Zea mays L. Hybrid, Ganga-5) were germinated on water- soaked cotton in petri dishes, and germinated seedlings were kept in 10 ml of distilled water in small beakers at 25°C under continuous il lumination (approx. 2 500 lux).

Estimation of pigments Pigments were extracted in 80% cold acetone and estimated according to

Arnon [ 11]. All the experiments were conducted under a green safety light.

Estimation of macromo lecules Ten to fifteen leaves were homogenised and macromolecules were pre-

cipitated by 10% ice cold trichloroacetic acid [12]. The precipitate was washed with 5 ml of 5% trichloroacetic acid (TCA). Lipids were removed by washing the precipitate with alcohol, alcohol/chloroform ( 1: 2) and finally with ether. Then the precipitate, with 5 ml of 0.3 N NaOH, was incubated for 3.5 h in a shaking water bath maintained at 37°C, for RNA hydrolysis [ 13]. After the hydrolysis, 20% TCA was added for complete DNA precipi- tation and centrifuged. RNA in the supematant was estimated by the orcinol method [14]. The residue with 5 ml of 20% TCA was kept in a water bath maintained at 90°C for 20 rain for DNA hydrolysis and after centrifugation, estimation of DNA in the supernatant was carried out using the diphenyl- amine method of Dische [15]. Protein in residue was dissolved in 5 ml of 1 N NaOH and assayed using the method of Lowry et al. [16].

Isolation of chloroplasts Chloroplasts were isolated from the leaves of light-grown seedlings. Twenty

to thirty leaves were taken and homogenised in a blender with ice cold buffer at maximum speed for 20 s. The homogenising medium contained 0.4 M sucrose, 0.01 M EDTA Na2 and 0.1 M phosphate buffer (pH 7.8). After homogenisation the paste was squeezed through 16 layers of cheese cloth. The filtrate was centrifuged in a controlled-temperature centrifuge at 500 g for I rain. The supernatant was again centrifuged at 1000 g for 10 min. The pelleted chloroplasts were suspended in a small volume of homogenising mediun~ Chlorophyll was assayed according to Arnon [11].

Measurement of DCPIP reduction The measurement of DCPIP (2,6-dichlorophenol indophenol) reduction

by isolated chloroplasts was carried out using the procedure described previously [ 10]. The chloroplasts were illuminated for 30 s with saturating

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white light (approx. 7 X 1 0 -3 J/cm 2 Is) from a 300 W projection on lamp. The incident light rays were passed thorugh a water filter to reduce infra-red light. The final volume of the reaction mixture was 3 ml and contained chloroplasts equivalent to 10--15 pg chlorophyll, DCPIP, 15 pM; KCI, 100 raM; MgSO4, 0.1 mM and 10 mM of phosphate buffer, adjusted to pH 6.8. The absorption of DCPIP was measured at 600 nm as described by Mohanty et al. [17] . The concentration of exogenous electron donor like DPC (diphenyl carbazide) in reaction mixture for DCPIP reduction was 0.5 mM. DPC was prepared fresh every day in methanol and the concentration of methanol in the final 3 ml of reaction mixture was 1.6%. Methanol at this concentration had no effect on the DCPIP-Hill reaction. The reaction mixture contained 5 mM phosphate buffer instead of 10 mM when DPC was used as electron donor.

RESULTS

Figure I displays the changes in the content of total chlorophyll of the leaves as a function of age of the seedlings grown in continuous light• The

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Fig. 1. (left) Changes in the content of chlorophyll o! maize leaves as a function of seedling age. Each value in the figure is the average of 4 experiments.

Fig• 2. (right) Changes in the content of chlorophyll and macromolecules of the leaves of 4-day old maize seedlings grown in continuous light for 3 days. The inset shows DCPIP photoreduction with H20 and DPC as electron donors. Each value in the figure is the average of 4 experiments. (m-----m) DPC -~ DCPIP; ( e - - - - e ) H20 -~ DCPIP; ( v - - - - ~ ) protein; (o------a) RNA; (o-----o) DNA; (A-----~) Chlorophyll.

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level of pigments increases almost linearly from the second to fourth day of germination, after which the level remains steady up to the seventh day and declines thereafter.

The steady state of the leaves between the fourth t~) seventh day of seedling germination is further characterised by monitoring the level of various leaf macromolecules. Figure 2 shows the changes in the contents of DNA, RNA and protein of the leaves of the seedlings from the fourth to seventh day of germination in continuous light. The level of RNA does not show any significant change during this period, whereas the content of DNA declines slowly till the end of the seventh day. The loss in the content of protein is very much significant. The rate of loss in the macromolecular cont, ent is rapid during the first day followed by a slow decline thereafter. The final protein loss by the end of seventh day is approx. 37%.

The inset in Fig. 2 shows the DCPIP photoreduction by chloroplasts isolated from the leaves of the seedlings from the fourth to seventh day of gemdnation, the period of steady state of the seedlings with reference to leaf chlorophyll content. The DCPIP reduction, either water or DCP as electron donor, does not exhibit any change in the level of dye reduction during the entire period of 3 days of seedling growth.

DISCUSSION

The matured and fully~xpanded leaves are normally used to study various physiological and biochemical processes particularly the process of leaf senescence [7,8,18,19]. It is reasonably assumed that at full expansion, the leaves were well differentiated both structuraUy and functionally and most of the physiological and biochemical processes are in a steady state.

The content of total chlorophyll of the developing maize leaves, as shown in Fig. 1, shows a maximum value, and the value does not change between the fourth and seventh days of seedling growth. This period may be con- sidered as the steady state of the leaves. The levels of nucleic acids also do not show any significant change during seedling growth between the fourth to seventh day. On the contrary, a considerable loss in the content of leaf protein is observed during the same period (Fig. 2). It has previously been noted that during development and ageing, the leaves show a parallel increase or decrease in the contents of chlorophyll and protein [1,9,10]. There- fore, ageing status of the leaves has normally been characterised by measuring either the content of the pigment or protein. But our data contra- dict these findings, and show that the leaves remain healthy and are in a steady state with reference to chlorophyll, while the loss of protein during the same period (Fig. 2) may suggest the onset of leaf senescence. The loss in protein content may be attributed to the enhancement in the activity of protease or loss in the ability of leaves to synthesise new proteins.

Surprisingly, the considerable loss in total protein during the fourth to seventh day of germination does not affect the electron transport system of chloroplasts. The level of DCPIP photoreduction either with H20 or DPC

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as electron donor does no t show any loss during this period (Fig. 2). Thus, the loss in the total leaf protein may be at tr ibuted to the loss of non~hloro- plastic protein, or the proteins of chloroplasts no t associated with electron transport system of the organelle.

ACKNOWLEDGEMENT

We thank Professor M.C. Dash, School of Life Sciences, for encouragement and providing the facilities.

REFERENCES

1 A.C. Leopold and JP.E. Kriedemann, in Plant Growth and Development, McGraw-Hill Inc., New York, 1975, p. 249.

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