Effect of High Temperature on Chloroplast Development during Illumination of Dark Grown Seedlings

Summary 6.1 Introduction 6.2 Results and Discussion

6.2.1 Synthesis of pigments 6.2.2 Absorption spectra of pigments 6.2.3 Development of photochemical activity 6.2.4 Plastid DNA and RNA 6.2.5 Accumulation of chloroplast proteins

6.2.5.1 Thylakoid proteins 6.2.5.2 Stromal proteins

6-l

Summary Effects of high temperature on the light-induced chloroplast development in dark-grown seedlings were studied. High temperature inhibited the accumulation of chi and photosynthetic proteins and development of photochemical activity, however carotenoids continued to accumulate. 2-D gel electrophoresis of stromal proteins showed similar protein patterns in the dark-grown seedlings and seedlings illuminated at high temperature, although some low mol wt proteins accumulated at 3lfC. In contrast, seedlings illuminated at 2S'C showed complex pattern of proteins.

6.1 Introduction

Chloroplasts, the photosynthetic chlorophyll-containing plastids, are derived from small

undifferentiated proplastids which are inherited maternally by the plant zygote (Mullet,

1988). In the dark-grown seedlings, proplastids differentiate into etioplasts. Both

proplastids and etioplastids can develop into a functional chloroplasts. The principal

effector in this process is light (Tobin & Silverthorne, 1985). During the light induced

biogenesis of chloroplast, there is a rapid accumulation of chi, photosynthetic membranes

and its associated proteins, activation of plastid transcription, increase in plastid DNA copy

number, plastid transcript abundance and plastid number per cell (Baumgartner et al.,

1989; Gruissem, 1989).

During the light induced chloroplast development, the most dramatic impact is on

the chi synthesis (Mullet, 1988). Dark-grown plants does not accumulate chi, although its

precursor protochlorophyllide (pchlide) accumulates. Pchlide forms a ternary complex with

NADPH and protochlorophyllide oxidoreductase (POR), enzyme which reduces pchlide to

chlorophyllide (chlide) (Reinbothe & Reinbothe, 1996). This ternary complex is photolabile

and as soon as plants are illuminated, pchlide is reduced to chlide which is subsequently

esterified with geranylgeranyl pyrophosphate in a light-independent step to form chi (Mullet

etal., 1990).

Besides accumulation of chi, the level of some chloroplast proteins can increase

more than 1 0,000-fold during light induced chloroplast development. Although transcription

and post-transcriptional control can influence accumulation of these proteins, the largest

factor affecting this process is protein synthesis (Mayfield et al., 1995). Several studies

have suggested that light-dependent accumulation of proteins may be controlled by

untranslated region of the gene which could provide the binding site for the proteins

encoded by nuclear genes that act as translational activators (Danon & Mayfield, 1991;

Mayfield et al., 1995). Each of these products of nuclear genes affects the translation of

only a single chloroplast mRNA. In some cases, multiple nuclear genes appear to be

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required for the translation of a single chloroplast message. Genetic analysis of chloroplast

mutants has implied that the 5'-untranslated region of the chloroplastic messages, probably

stem-loop RNA structures, are the target for interaction with these nuclear encoded

translational activators (reviewed in Mayfield et al., 1995).

In some other cases, accumulation of chloroplast proteins was linked to

accumulation of its co-factor. For example, the accumulation of D1, D2, CP43, CP47 and

P?OO chi apoproteins depends on the presence of chi. It was suggested that chi is required

either to stabilize the nascent or mature polypeptide chain or to release a translational

arrest. In absence of its co-factors, however, the polypeptides are either degraded or

synthesis is stopped due to translational arrest (Mullet et al., 1990).

The efficient transformation of etioplast to chloroplast during illumination of dark­

grown seedlings with light requires the coordination between chloroplast and nucleus. This

coordination under optimum conditions i.e, normal light intensity and ambient temperature,

leads to acquisition of plastid functions in a time dependent manner. Any changes in the

conditions may delay or stop the chloroplast development. For example, accumulation of a

number of thylakoid proteins is dependent on the accumulation of chi, therefore any

conditions which delays or stops the chi accumulation would be expected to interfere with

the chloroplast development.

In the present investigation, we have studied the effect of high temperature on the

light induced chloroplast development from etioplast in the dark-grown seedlings. Different

parameters including accumulation of pigments and proteins, total plastid DNA and RNA

were determined to compare the chloroplast development at normal and high

temperatures.

6.2 Results and Discussion

6.2.1 Synthesis of pigments

Accumulation of pigments, mainly chi, is the first visible and detectable change observed

during illumination of dark-grown seedlings. Chi accumulation has been found to be

dependent on a number of factors including duration of etiolation and light intensity (Miller

& Zalik, 1965). In normal conditions, chi accumulation can be observed after 1 h of

illumination of dark-grown seedlings

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The chi accumulation during the illumination of dark-grown seedlings at 25°C and

38°C is shown in table 11. As can be seen, illumination of dark-grown seedlings with white

light (100 11E.m-2.s-1) at 25°C leads to the accumulation of chi and subsequently the chi a/b

ratio decreased. The rapid decline of the chla/b ratio is a common indicator of chloroplast

development (Kyle & Zalik, 1982). In contrast, when the dark-grown seedlings were

illuminated at 38°C, chis failed to accumulate and there was no change in the chla/b ratio

as compared to control. Failure of chi to accumulate during illumination of seedlings at

38°C could be due to two reasons; first, the light induced reduction of pchlide is inhibited

and second, the chlide or the synthesized chi is bleached during illumination of dark-grown

seedlings at high temperature. In order to study the involvement of first reason,

experiments were carried out to determine the level of POR protein. It is known that

illumination which causes reduction of pchlide leads to the rapid degradation of POR

enzyme. As shown in Fig.35A, the level of POR was similar in both the seedlings

illuminated either at 25°C or 38°C as compared to control. Furthermore it was shown that

the POR gene is negatively regulated by light and its transcripts decrease very fast during

illumination of seedlings at ambient temperature. As could be seen in figure 358, the

transcript level of POR decreased in the seedlings illuminated at 38°C similar to the

seedlings illuminated at 25°C suggesting that the signal transduction pathway involved in

the regulation of POR transcripts by light is functional and not disrupted by high

temperature. From the above studies, therefore, it is clear that the photochemistry related

to POR involved in the chi synthesis is similar at both the temperatures. We propose that

failure of chi accumulation during illumination of dark-grown seedlings at 38°C is due to

bleaching of chi. The bleaching of chi in plants and in the isolated systems due to exposure

at high light intensity or high temperature is known to occur (Mishra et al., 1992; 1994;

Thomas & Ortiz, 1995).

Carotenoids has been suggested to play an important role in the photoprotection of

PSII during photoinhibitory condition (Demming-Adams & Adams, 1992). The level of

carotenoids was found to increase at 38°C although increase was less as compared to

seedlings illuminated at 25°C. Lesser increase could be due to degradation of carotenoids

at high temperature. The degradation of carotenoids in the photosynthetic apparatus by

high light treatment has been shown (Mishra et al., 1994).

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Table 11: Accumulation of chi and carotenoids in dark-grown seedlings illuminated with light at 25°C (A) and 38°C (B). Values were represented as mg.gfw-1

(A) chi a chlb chla+b chla/b carotenoids

control 0.00 0.00 0.00 - 7.92 4h 0.56 0.06 0.62 9.33 9.22 8h 2.21 0.39 2.60 5.67 15.13

12 h 3.00 0.63 3.63 4.76 17.27 24 h 8.16 1.97 10.13 4.14 27.61

(B) chi a chlb chla+b chla/b carotenoids

control 0.00 0.00 0.00 - 7.55 4h 0.18 0.00 0.18 - 8.80 8h 0.19 0.00 0.19 - 9.01

12 h 0.34 0.00 0.34 - 11.22 24 h 0.37 0.00 0.37 - 12.52

1 2 3 4 1 2 3 4 ......_,

-36koa

-1·4kb

A B

Fig . 35 Effect of high temperature on protochlorophyllide reductase . (A) POR protein and (B) POR transcript . The experimental conditions and lane descriptions were same as described in fig . 44.

6.2.2 Absorption spectra of pigments

The differencial absorption spectra of the pigments isolated from dark grown seedlings and

seedlings illuminated with light at 25°C and 38°C for different periods of time are shown in

figure 36. Illumination of dark-grown seedlings with light at 25°C resulted in the

accumulation of chis and carotenoids. The time course of accumulation indicates that long·

wavelength-absorbing chis (Amax around 672 nm) and carotenoids (490-495 nm) are the

first species to be synthesized during illumination. A significant increase in the absorbance

around 437 nm was also observed which is attributed to the accumulation of chla.

The differential absorption spectra of pigments isolated from seedlings illuminated

at 38°C is shown in figure 368. As could be seen, the synthesis of long-wavelength­

absorbing chis (672 nm) was inhibited. As compared to this, significant accumulation of

carotenoids (490-495 nm) and chla (437 nm) occurred. Although this accumulation was

less as compared to seedlings illuminated at 25°C.

6.2.3 Development of photochemical activity

Dark-grown seedlings upon illumination with light develop photochemical activities in a

step-wise manner (Wellburn & Hampp, 1979). Electron transport through PSII in dark­

grown seedlings illuminated at 25°C and 38°C was measured following DCIP

photoreduction in absence or presence of DPC. No oxygen evolving activity could be

detected within first 4 h of illumination at either temperatures (Table 12). Further

illumination of seedlings lead to development of oxygen evolving capacity at 25°C.

However no PSII activity could be detected at 38°C even after 24 h of illumination.

Absence of PSII activity at 38°C was expected as similar treatment of dark-grown seedlings

failed to accumulate chi.

6.2.4 Plastid DNA and RNA

Plastid DNA copy number per chloroplast, transcription activity and number of plastids per

mesophyll cell are known to be regulated by the developmental process (Mullet, 1988;

Baumergtner et al., 1989). Table 13 shows the effect of high temperature on total plastid

DNA and RNA. As can be seen, the total plastid DNA and RNA remained same during

illumination of dark grown seedlings with light either at 25°C or 38°C as compared to dark­

grown seedlings. This is in accordance with the results reported by other workers

(Baumgartner et al., 1989). In the monocots such as in barley and wheat, developmental

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+ 1· 20 A,....-.------------------.

-O~SA~--------~------~------~----~ NM 800.0 350.0

+0.20Ar----------------------------------38•c

-0.0 5 A L...-----'L......------.1.-----~----'-----~ N M 350.0 800.0

Fig.36 Absorption spectra of pigments isolated from dark-grown seedlings illuminated either at 25°C or 38°C.

Table 12: Development of PSI I activity during illumination of dark-grown seedlings at 25°C and 38°C for different time periods. PSII activity was determined following DCIP photoreduction n absence or presence of DPC.

25°C 38°C Control nd nd 4h 4 nd 8h 32 nd 12 h 74 nd 24 h 120 nd 40 h 148 nd Green plants 153

Table 13: Total plastid DNA (A) and RNA (8) in dark-grown seedlin~s and seedlings illuminated at 25°C or 38°C. Equal number of plastids (1 0 ) were collected in an polypropylene tube and plastid DNA and RNA were spectrophotometrically determined as described in Material and Methods. Values (in mg) are the mean of five independent experiments.

(A) I Control 25°C 38°C 1 9.8 ± 1 10.1±0.9 10.0±1.2

28 ± 3 27 ± 2

process leads to the activation of plastid transcription, increase in DNA copy number,

plastid RNA and plastid number per cell even in the absence of light.

6.2.5 Accumulation of chloroplast proteins

As described earlier, illumination of dark-grown seedlings at 38°C inhibits accumulation of

chi and development of photochemical activity, it was interesting to determine the level of

different proteins involved in the photosynthetic process. Furthermore a number of results

have been presented which suggested that various nuclear coded proteins are required for

the gene transcription, mRNA stability and protein synthesis of plastid (Gruissem & Tonkyn,

1993; Mullet, 1993; Mayfield et al., 1995). It could be possible that high temperature affects

the function of these proteins. The effect of high temperature on the accumulation of

chloroplast proteins during illumination of dark-grown seedlings with light were determined

either with western blots or 2-dimensional gel electrophoresis. Soluble and membrane

associated fractions were separated from the intact etiochloroplast as described in

materials and methods. The polypeptide profiles of soluble and membrane associated

fractions isolated are shown in figure 37. High temperature was found to increase the level

of both subunits of Rubisco. However there was no changes in other polypeptides of

soluble fractions isolated from dark-grown seedlings illuminated at either 25°C and 38°C. In

the membrane fractions, a 30 kDa protein band corresponding to LHCII was absent in the

dark-grown seedlings as well as in seedlings illuminated with light at 38°C.

6.2.5.1 Thylakoid proteins

Primary leaf development is largely light-independent in monocots and accumulation of

most plastid proteins occurs in dark-grown seedlings. However, dark-grown plants lack a

number of proteins essential for photosynthetic activity although their transcripts

accumulate in dark. It was suggested that these transcripts/proteins require either co­

factors for their stability (Mullet et al., 1990) or nuclear coded proteins for translation

activation (Danon & Mayfield, 1991; Mayfield et al., 1995).

As shown in the figure 38, none of the PSII proteins like 01, 02 and CP47 encoded

by ctDNA were present in the dark-grown seedlings. Illumination of dark-grown seedlings at

25°C lead to the accumulation of all these proteins. However these proteins failed to

accumulate when illumination of dark-grown seedlings was carried out at 38°C.

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LS-

ss-.

Fig.37 Profiles of thylakoid proteins and stromal proteins isolated from dark-grown seedlings and seedlings illuminated either at 25°C or 38°C . Wheat seedlings were grown for 5-days in dark. and were illuminated either at 25°C for 8 h or 38°C for 8 h. Intact etioplast from these seedlings were isolated on percoll gradients. Lane 1 & 4 : proteins isolated from dark-grown seedlings; lane 2 & 5 are proteins isolated from seedlings illuminated at 25°C for 8 h; lane 3 & 6 are proteins isolated from seedlings illuminated at 38°C for 8h . Lane 1-3 and 4-6 are the corresponding stromal and thylakoid fractions respectively.

1 2 3 4 1 2 3 4

32kOa- -34kDa

12 345 6 78 1 2345 678

-47kDa -- -60kDa

1 2 3 4 5 6 7 8

- 33kOa

Fig .38 Effect of high temperature on the accumulation of thylakoid proteins during chloroplast development. Experimental conditions and lane descriptions are same as described in fig . 44.

The 33 kDa extrinsic protein associated with the OEC of PSII is encode_d by nuclear

gene and plays an important role in water photo-oxidation. The 33 kDa protein was present

in dark-grown seedlings and was found to be distributed equally in the thylakoid

membranes and soluble fractions (Fig. 38). Illumination, however. causes its increase in

the membrane fractions and level in the soluble fraction decreased as compared to control.

In contrast, illumination of dark-grown seedlings at 38°C leads to the loss of 33 kDa protein

in both the fractions as compared to control. High temperature induced loss of 33 kDa

protein could be due to either increased turnover or inhibition of its synthesis. It was shown

that synthesis of 33 kDa protein is not affected in a mutant which lacks entire thylakoid

membrane (Palomares et al., 1993), suggesting that it was increased turnover of proteins

which resulted in the loss of 33 kDa protein. It could be possible that loss of 33 kDa protein

in seedlings illuminated with light at 38°C is caused due to increase turnover of 33 kDa

protein .

Similar to the accumulation of 33 kDa protein, CF1cx subunit was also present in

both the fractions of dark-grown seedlings similar to 33 kDa protein and illumination causes

an increase in the level of CF1 ex in the membrane fractions (Fig. 38). Illumination of dark­

grown seedlings at 38°C leads to the loss of CF1cx protein in both the fractions, however,

loss was slower as compared to 33 kDa protein.

6.2.5.2 Stromal proteins

Chloroplast functions require a number of proteins encoded by nuclear genes. These

proteins are involved in splicing, regulation of gene expression, photosynthesis and protein

synthesis (Rochaix, 1992; Mayfield et al., 1995). Recently a number of proteins have been

identified which directly regulates transcription, mRNA stability and translation (Memon et

al.. 1996). Since a number of plastid proteins failed to accumulate during illumination of

dark-grown seedlings at 38°C, we were interested to see whether there is any change in

the profiles of soluble proteins.

Soluble fractions were separated on iso-efectric focussing gel followed by second­

dimensional SDS-PAGE. The protein patterns of stromal fractions of dark-grown seedlings

and seedlings illuminated either at 25°C or 38°C are shown in figure 39. Protein patterns

were similar in dark-grown seedlings and seedlings illuminated at 38°C. However, some

low mol wt proteins specifically appeared in seedlings illuminated at 38°C. Identity of these

proteins remain unknown. In contrast, the protein patterns of seedlings illuminated with

70

A

•

c . -

Fig.39 2-D gel electrophoresis of stromal proteins . Stromal proteins were isolated as described in fig . 37 and were separated on IEF in one dimension followed by second dimension on SDS-PAGE as described in Materials and Methods.

light at 25°C showed the appearance of a number of proteins. It is interesting to note that

some proteins which appeared at 38°C are not present in seedlings illuminated at 25°C

suggesting that these proteins are induced by high temperature stress.

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