7.1 Introduction. Summary

Transcription

1

2 Regulation of Plastid Gene Expression by High Temperature during Light Induced Chloroplast Development in Dark Grown Wheat Seedlings : Study using the Cloned DNA Fragments of Chloroplast Genome Summary 7.1 Introduction 7.2 Results and Discussion Wheat chloroplast DNA Localization of genes on various DNA fragments of wheat chloroplast DNA Total RNA from wheat seedlings Expression of plastid genes encoding proteins involved in photosynthetic process at high temperature Expression of psba gene Expression of psbdic operon Expression of psbb operon Expression of psbe operon Expression of psaa and atpa operon Expression of atpb operon and rbcl gene Expression of ndh genes Expression of plastid genes coding for the components of protein synthesis apparatus Expression of ribosomal genes Expression of genes coding for subunits of RNA polymerase Expression of genes coding for ribosomal proteins Expression of genes coding for trnas Expression of nuclear genes involved in the plastid functions Expression of genes coding for RNA-binding proteins Expression of genes coding for translation elongation factors 72

3 Summary Effect of high temperature on the plastid gene expression during the light induced chloroplast development in dark-grown seedlings was studied by northern hybridization using cloned DNA fragments of wheat chloroplast genome. Based on their response to high temperature, plastid genes can be grouped into three different categories: 1) plastid genes whose expression was not affected by high temperature. These consisted genes mainly for rrna, ribosomal proteins, trnas and some genes coding for the putative NADH dehydrogenase; 2) plastid genes whose expression was increased at high temperature. These consisted gene coding for the subunit of RNA polymerase and some unidentified transcripts; and 3) plastid genes whose expression was decreased at high temperature. These consisted genes coding for proteins involved in the photosynthetic process. Loss of primary transcripts originating from a number of operons consisting of genes coding for proteins involved in the photosynthetic process was observed. Expression of all the light inducible plastid genes was inhibited suggesting that the light inducibility property was lost at high temperature. 7.1 Introduction Chloroplasts are membrane bound intracellular organelles present in plant cells which contain the entire enzymatic machinery required for the photosynthesis. In higher plants, chloroplasts are derived from proplastids/etioplasts. The development of chloroplasts from proplastids/etioplasts is a complex process that requires both activation and co-operative expression of a large number of chloroplast and nuclear genes (Mullet, 1988; Gruissem, 1989; Rochaix, 1992; Gruissem & Tonkyn, 1993). During this phase, a number of events such as accumulation of pigments, assembly of photosynthetic electron transfer components into PSI, PSII, cyt bsf and ATP synthase complex occurs (Mullet, 1988; Gruissem, 1989). The normal developmental program of chloroplasts is regulated by environmental signals such as light and, by other metabolic signals (Mullet, 1988; lgloi & Kassel, 1992; Danon & Mayfield, 1994; Mayfield et al., 1995). Photosynthesis, together with other plastid functions require the products of several hundred genes. The complete nucleotide sequence of ctdna from tobacco (Shinozaki et al., 1986), liverwort (Ohyama et al., 1986) and rice (Hiratsuka et al., 1989) revealed the existence of ca. 130 genes. About half of these are involved in the plastid protein synthesis and about 30 code for subunits of the four supramolecular complexes involved in the photosynthetic process (Ohyama et al., 1986; Shinozaki et al., 1986; Hiratsuka et al., 1989). Information on the regulation of plastid genes expression during the chloroplast development is limited to few genes (Gruissem, 1989; Mullet, 1993). Initial studies were concentrated on the elucidation of control of expression of plastid genes coding for the proteins involved in the photosynthetic process. These studies suggested that the light regulation of plastid genes expression could occur at transcriptional (Mullet, 1993), posttranscriptional (Gruissem, 1989; Rochaix, 1992; Gruissem & Tonkyn, 1993), translational 73

4 (Danon & Mayfield, 1991; Mayfield et al., 1995) and post-translational (Klein & Mullet, 1987; Kim et al., 1994) level depending on plant species. Factors that interfere with the normal development and maintenance of chloroplasts limit the ability of plant cells to carry out photosynthesis (Berry & Bjorkman, 1980; Powles, 1984). Since most of the components required for the chloroplast functions are developed during the development of chloroplast from etioplast, this makes the etiolated seedlings a suitable system to study the effect of environmental factors on the regulation of plastid gene expression. High temperature is one such environmental factors which effect on chloroplast function is well characterized (Smillie et al., 1978; Berry & Bjorkman, 1980). High temperature profoundly affects the structure and function of photosynthetic apparatus. Decline in the rates of photosynthetic activity are well documented and are attributed to disruption in the functional integrity of the photosynthetic apparatus (Smillie et al., 1978; Berry & Bjorkman, 1980; Quinn & Williams, 1985; Yordanov et al., 1987). Loss of chloroplast functions in high temperature-treated rye and euglena is known for long, although the molecular mechanism leading to these effects is not known (Hess et al., 1993; Thomas & Ortiz, 1995). It was shown that high temperature leads to the loss of chloroplast ribosome (Feierabend & Berberich, 1991 ), proteins required for photosynthesis as well as for other plastid functions, a number of mrnas, rrnas and trnas (Feireband & Berberich, 1991) whereas transcripts of some other genes accumulated to high level (Hess et al., 1993). In the euglena, a steady loss of chi was observed due to decrease in the rate of protein synthesis and transcription of chi binding proteins during the high temperature treatment (Thomas & Ortiz, 1995). Thus it is clear that high temperature affects the function of chloroplast in light grown seedlings. The detrimental effect of high temperature on chloroplast functions may be characterized in two parts: in short term, it primarily affects photosynthesis whereas in the long term it also affects the plastid transcription/ translation machinery. In the present work, we have studied the effect of high temperature on the expression of plastid genes during light induced chloroplast development in the dark-grown seedlings utilizing cloned DNA fragments of wheat plastid genome. 7.2 Results and Discussion Wheat chloroplast DNA The recombinant vectors covering approximately 95% of total chloroplast genome were kind gift of Dr. Elizabeth A Kelogg. These clones were constructed in pbr322 after either 7-l

5 complete digestion with pst1 and Sal1 or by partial digestion with BamH1. The specific DNA fragments were isolated and purified as described in Material and Methods. Figure 40 shows the restriction digestion of recombinant vectors with restriction enzymes. The size of different fragments obtained after pst1, Sal1 and BamH1 used in the present study are given in table Localization of genes on various DNA fragments of wheat chloroplast DNA Figure 41 shows the location of different fragments used in the present study on the wheat chloroplast genome (after Bowman et al., 1981). Most plastid genes are organized into polycistronic transcription units. The sequence analysis of the entire tobacco (Shinozaki et al., 1986), liverwort (Ohyama et al., 1986) and rice (Hiratsuka et al., 1989) chloroplasts genome together with the partial sequence and mapping data from other plant chloroplasts genome has revealed that the arrangement of genes within these transcription units is highly conserved although transcription units are rearranged in some plant species (Palmer & Thompson, 1982). Comparison of known location of plastid genes in wheat suggest similar arrangement of genes as in rice plastid genome. Besides, plastid genome size of wheat and rice are approximately equal in size (135 kb) and a 20 kbp inversion in maize and wheat has also occurred in rice chloroplast genome (Quigley & Weil, 1985; Hiratsuka et al., 1989). The nucleotide sequence of wheat chloroplast gene coding for the subunit of RNA polymerase (rpoa) is shown to be largely contained within the B20 fragments (Hird et al., 1989). The various genes on different fragments of wheat chloroplast genome have been assigned following walking upstream and downstream of rpoa gene on rice chloroplast genome. This yielded the similar location of genes on the fragments of wheat chloroplast genome which have been characterized separately (See legend of Fig. 42) Total RNA from wheat seedlings Total RNA from dark-grown and light-grown wheat seedlings were isolated as described by Chymozcinki & Sacchi (1987). Equal amount of RNAs from all the four samples were fractionated on denaturing formaldehyde-agarose gel (Fig. 43) of total RNAs was used for the northern analysis. 75

6 Fig.40 Analysis of recombinant vectors carrying ctdna. Recombinant vectors were restriction digested using either pstl, BamHI or Sail and were fractionated on 0.8 /c agarose gel. Lanes 2-9 are recombinant vectors digested with pst1 ; lane 10 is recomibanant vector digested with S3a and lanes are recombinants vectors digested with BamH1. Lane 1 is lambda-hindlll digest and lane 14 is 1 kb ladder (BRL).

7 Table 14: Size of ctdna fragments cloned in pbr322 Recombinant Molecular Recombinant Molecular Vector size Vector Size S3a 14.2 kb kb P kb kb P kb kb P kb kb P6 8.4 kb kb P7 8.1 kb kb P8 5.6 kb kb P9 5.3 kb kb P kb kb kb kb kb kb

8 Fig.41 Map of wheat chloroplast genome. Localization of different fragments of pst1, BamH1 and Sal1 on wheat chloroplast genome.

9 P3 I P4 I PS I P6 I P7 I P8 I pg I P10I 82 I o I o S3a I '!>09 rpoc1 I!'JI0 2 I rn=j GifCJ llllfh OL:J CD I :-;;cj C!!iiCl ["'!ij!c"] m QJ OiL] I t!!a Li!!COJ I IRFHO I 0"20 m OD Li!!D G! 1 G!LJ w LiiiU w OiJ UJ m m LTILJ co LiJ LiU w en m CEL:J I s e I LLJ ~ I psbh OD O!LJ cp;g:=j QiJ w on OFl on w DD CT] on [J" "] lpo!g G!LJ w I "'FE LiLJ QLJ QLJ c;;;cj I polo [ ] I~ I~ I f!!!e pn on ljlj u::::::j c...-=] =rn=j O"iCl ~ w w Li.&J QiLJ LiiLJ noo=j cp;bcl cp;oc== LD lpo!a I ns Fig.42 Localization of various genes on the DNA fragments of chloroplast genome (for detail see text). The plastid genes localized in wheat i.e, 16S, 23S, rbcl (Bowman et al., 1981); atpf (Bird et al., 1985); atph, atpa, atp~. atpc. (Howe et al., 1985); psbe-f-l (Webber et al., 1989); psbh (Hird et al., 1986); psbb, psbc, psbd, psaa, peta, pets, petd (Courtice et al., 1985); psac (Dunn & Gray, 1988); rpoa (Hird et al., 1989); S2 (Hoglund & Gray, 1987); psbg, ndhc (Nixon et al., 1989) and ORF62, tmg, trnm, trng (Quigley & Weil, 1985) have been shaded. Open rectangle at the end of fragments indicates that gene is also present on other fragment. The numerical numbers denotes the number of codon in an open reading frame. trnas carrying different amino acids are represented by assigning the one letter name of amino acid carried by trnas. The genes coding for proteins of small and large subunit of ribosome are given by the polypeptide number preceded by letter L for Large subunit and S for small subunit.

10 Fig.43 Total RNAs isolated from dark-grown seedlings treated at different conditions. Dark-grown seedlings were illuminated at 25 C and 38 C and total RNAs were isolated from these seedlings using Chymoszki and Sachhi (1987) lane 1 total RNAs from darkgrown seedlings; lane 2: total RNAs from seedlings illuminated at 25 C and lane 3 & 4 are total RNAs isolated from seedlings illuminated at 38 C for 4h and 8h respectively.

11 7.2.4 Expression of plastid genes encoding proteins involved in photosynthetic process at high temperature Expression of psba gene psba mrna is expressed monocistronically in higher plants and codes for D1 protein of PSII. Due to the high turnover of D1 protein, psba mrna is present at high level. The high level of psba mrna is suggested due to a strong promoter (Rapp et al., 1992), light induced transcription (Klein & Mullet, 1990) and a very stable RNA (Kim et al., 1993). In wheat, the differential accumulation of psba mrna has been attributed to both enhanced transcription activity and transcript stability (Kawaguchi et al., 1992). The northern blot analysis of psba mrna is shown in figure 44. In etioplasts, a single transcript of 1.3 kb was detected which level increased following illumination of etiolated seedlings to light at 25 C for 8 h. However, when dark-grown seedlings were illuminated at 38 C, psba mrna failed to accumulate as compared to etiolated seedlings. Failure of psba mrna to accumulate at 38 C could be due to the absence of factor(s) required for psba accumulation either during transcription or for transcript stability. The stability of psba mrna has been attributed, in part, to a stem loop structure located at the 3'-end of the transcript (Schuster & Gruissem, 1991). The stability of such mrnas is suggested to be modulated by protein(s) that binds to the stem loop structure. Recently, a 37/38 kda protein was shown to specifically interact with the stem loop structure of psba (Memon et al., 1996). It could be speculated that function of these proteins are disrupted at high temperature. The similar amount of mrna present in the high temperature treated and etiolated seedlings could be due to the high stability of psba mrna. The half-life of psba mrna is known to be more than 40 h (Kim et al., 1993) Expression of psbdic operon Expression of psbdic operon in various plant species is well characterized. psbdic operon consists of six genes in order of psbk-psbl-psbd-psbc-orf62-tmg which encodes four PSII proteins, a conserved open reading frame of 62 amino acids and a trna for glycine (Mullet, 1993). Two tms genes are encoded by the opposite DNA strand of this same region. The genes present on this fragment are transcribed by four distinct promoters producing atleast eight transcripts (Sexton et al., 1990). Three of these operate in dark while the other one is light inducible and produces two transcripts of 4.4 kb and 3.4 kb (Gamble & Mullet, 1989; Kawaguchi et al., 1992). 76

12 1 ".. Fig.44 Effect of elevated temperature on the expression of psba gene. Total RNAs isolated from dar!< grown seedlings for 120 h (lane 1), dar!< grown seedlings exposed to light at 25 C for 8h (lane 2), dark grown seedlings exposed to light at 38 C for 4h (lane 3) and 8h (lane 4) were fractionated on 1.2% formaldehyde-agarose gel. RNAs were transferred on nylon membrane by capillary transfer method and subjected to hybridization with psba gene.

13 Fragment 815 contains most part of psbdic operon whereas 825 contains most of the psbc coding region and orf62 (Fig. 42). Northern blot analysis of RNAs isolated from etiolated seedlings using these two fragments gave identical pattern of transcripts. Figure 45A shows the transcripts pattern obtained with the 825 fragment. In etiolated seedlings, a total of eight transcripts of 7.5, 5.9, 5.0, 3.9, 3.4, 1.9, 1.5 and 1.3 kb were detected. When the dark-grown seedlings were illuminated at 25 C, a novel transcript of 4.4 kb appeared and the transcript level of 3.4 kb increased. The increased expression of 4.4 and 3.4 kb is due to activation of light-inducible promoter during illumination of dark-grown plants with light at 25 C (Gamble & Mullet, 1989; Kawaguchi et al., 1992). However, when dark-grown seedlings were illuminated at 38 C, the various transcripts originating from psbdic operon showed complex behavior. As such, transcripts of 7.5, 5.9, 3.4, 1.9, 1.5 and 1.3 kb decreased during illumination of dark-grown seedlings at, 38 C. Transcripts of 5.0 kb and 3. 9 kb showed mixed behavior, initially the transcripts disappeared, however longer period of high temperature treatment resulted in the reappearance of these transcripts. Besides, the light accumulated transcripts were also sensitive to high temperature and after four hour of treatment, both the transcripts 4.4 kb and 3.4 kb, were completely lost. It is known that these transcripts are synthesized from a light inducible promoter. A light inducible factor responsible for the synthesis of light induced transcripts has been shown to be present in the chloroplast extract of light grown plants but not in the extracts isolated from etiolated seedlings (Wada et al., 1994; Kim & Mullet, 1995). It is therefore possible that loss of light inducibility at high temperature could be due to the loss of factor(s) required for the light activation Expression of psbb operon psbb operon consists of psbb, psbh, petb and petd genes in this order which encodes two subunits each of PSII and cyt b 6 f complex. Another gene for PSII protein, psbn, is located on the opposite DNA strand in the spacer region between the psbb and psbh genes (Kawaguchi et al., 1992). Both pets and petd gene consist of single intron (Hiratsuka et al., 1989). A total of 20 predominant transcripts are expected to be produced from this operon which are believed to arise from processing of primary transcripts rather than from multiple promoters (Barkan, 1988; Gruissem, 1989; Kawaguchi et al., 1992). In wheat plastids, using psbb and psbh as a probe, a total of seven transcripts were detected in the etiolated seedlings and illumination of dark-grown seedlings at 25 C lead to the accumulation of two more transcripts of 4.2 and 3.8 kb (Kawaguchi et al., 1992). 77

14 A B ~ Q Fig.45 Effect of elevated temperature on expression of psbdic and psbb operon. Blots were probed with (A) B25 fragments and (B) B11 fragments. Experimental details and lane descriptions are similar as described in Fig.44.

15 Genes for psbb, psbh, pets and peto in wheat are located on 811 fragment (Fig. 42). Northern blot analysis of RNAs isolated from dark-grown seedlings using this fragment revealed a total of 10 transcripts of 5.4, 4.7, 3.9, 3.2, 2.5, 1.9, 1.6, 1.0, 0.75 and 0.4 kb in the etiolated seedlings (Fig. 458). Illumination of dark-grown seedlings to light at 25 C caused overall increase in the level of all the transcripts, however no new transcripts were detected in the present condition. When the etiolated seedlings were illuminated at 38 C, transcripts of 5.4, 4.7, 3.9, 3.2 and 2.5 kb disappeared. More drastic disappearance was observed in the case of 4.7, 3.2 and 2.5 kb transcripts. The mechanism of faster disappearance of some transcripts as compared to other transcripts originating from this operon during high temperature treatment is, presently, not known. Two alternative possibilities may be considered. First, spectrum of transcripts originating from this operon are transcribed by two different promoters and one promoter may be sensitive to high temperature. However, several studies have suggested the presence of only one transcription initiation site thus ruling out this possibility (see Kawaguchi et al., 1992). Second, some transcripts may require protein factors for their stability and synthesis/function of these protein factors is sensitive to high temperature. The transcripts of 4. 7, 3.3 and 2.5 kb is known to consist one or two introns (Gruissem, 1989). Thus it could be possible that the instability of these transcripts at high temperature could be related to the presence of introns which makes these transcripts vulnerable to degradation by nucleases in absence of protein factors. It has been shown that transcripts consisting of unprocessed ends are rapidly degraded when incubated with the chloroplast extract (Schuster & Gruissem, 1991). It will be interesting to find out the mechanism(s) leading to the disappearance of some selective transcripts from this operon at high temperature Expression of psbe operon psbe operon consists of psbe, psbf, psbl and psbj genes which are co-transcribed to produce a single transcript of 1.1 kb (Webber et al., 1989). All genes in this operon code for PSII proteins. It has been shown that expression of genes present in this operon are not affected by light (Webber et al., 1989; Kawaguchi et al., 1992). The psbe operon in wheat plastid genome is localized on 812 fragment (Fig. 42). As can be seen, genes for two trnas, ribosomal proteins and several ORF are also present on this fragment. Northern blot analysis of RNAs isolated from etiolated seedlings using this fragment revealed two major transcripts of 1.1 and 1. 7 kb and one minor transcript of 1.5 kb (Fig. 46A). Illumination of dark-grown seedling with light at 25 C lead to 78

16 A B Fig.46 Effect of elevated temperature on expression of psbeif, psaa and atpa operons. Blots were probed with (A) B12 fragments and (B) P4 fragments. Experimental details and lane descriptions are similar as described in Fig.44.

17 increase in transcript level of 1. 7 kb, however there was no effect of light on 1.1 and 1.5 kb transcripts. When the illumination was carried at 38 C, the level of all the transcripts decreased as compared to control. The psbe operon is known to be constitutively expressed in light and dark (Webber et al., 1989; Kawaguchi et al., 1992), therefore the decrease in mrna level at high temperature could suggest that the transcription of this operon is affected at high temperature. The slow loss of transcripts could be due to high stability of transcripts similar to psba transcript (Kim et al., 1993) Expression of psaa and atpa operon psaa operon consists of three genes psaa, psab and rps14 in this order. psaa and psab code for the chi binding proteins of PSI and rps14 codes for the ribosomal protein of small subunit. atpa operon consist of four genes atpl-atph-atpf-atpa in this order which encodes proteins for A TP synthase. atpf gene contains a single intron (Hiratsuka et al., 1989). Both atpa and psaa operons are placed near to each oth&r in the plastid genome and are transcribed in opposite polarity. Both these operons in wheat plastid genome are localized on P4 fragment (Fig. 42). The northern blot analysis of RNAs isolated from etiolated seedlings using this fragment is shown in figure 468. A total of six transcripts of 5.2, 2.7, 1.6, 1.2, 0.9 and 0.6 kb were detected in the etiolated seedlings. Besides minor transcripts of 4. 7 and 0. 7 kb could also be detected in the etiolated seedlings. When the dark grown seedlings were transferred to light at 25 C, level of all the six major transcripts slightly increased, however no new transcript appeared from either operons. It could be possible that appearance of a new transcript originating from one operon is compensated by decreased/disappearance of a transcript from another operon. When etiolated seedlings were illuminated at 38 C, transcripts of 5.0 kb and 2.7 kb were most affected as compared to others and after 8 h of illumination at 38 C, almost complete loss of these transcripts occurred. As can be seen, the level of lower transcripts did not changed as compared to control suggesting that stability of these transcripts were not affected by high temperature. Both the operons are expected to produce a primary transcript of 5.2 kb. Therefore the results of figure 46B suggested that the primary transcripts originating from both the operons are affected by high temperature. The transcript of 2.7 kb may belong to spliced RNA that cover atpihf. A 2.6 kb transcript in pea has been suggested to cover atpihf (Hudson et al., 1987). Sensitivity of 5.2 and 2. 7 kb transcripts to high temperature therefore 79

18 suggested that synthesis/stability of these transcripts require some factors whose function is affected by high temperature. Another possibility could be that the transcription of these two operons are sensitive to high temperature leading to the loss of 5.2 and 2. 7 kb transcripts. The size of primary transcript originating from atpl operon is different in spinach (5.5 kb), pea (6.0 kb) and wheat (5.2 kb) (this study, Hudson et al., 1987). In pea, a prominent transcript of 4. 7 kb was detected which was suggested to originate from the intergenic region of atpl and atph (Hudson et al., 1987). As could be seen, this transcript is also present in wheat, although in low amount. Based on the transcripts originating from these operons of Pea and Spinach (Hudson et al., 1987), several transcripts detected in wheat may belong to atphf (1.2 and 1.6 kb), atph (0.6 and 0.9 kb), atph and exon 1 of atpf (0.9 kb). psaa operon is shown to produce a single transcript of 5.5 kb in barley (Baumgartner et al., 1993) and 5.2 kb in tobacco (Meng et al., 1988) Expression of atpb operon and rbcl gene atpb operon consists of two genes atpb and atpe which encode the subunits of A TP synthase. In chloroplast genome these genes are located next to, and of opposite polarity from the rbcl gene which encodes the large subunit of Rubisco (Hiratsuka et al., 1989). In most of the higher plants the coding regions of atpb and atpe genes overlap by four base pairs (Krebbers et al., 1982; Shinozaki et al., 1983; Hiratsuka et al., 1989). In wheat plastid genome, atpb and atpe are located on B2 fragment along with rbcl, peta and several ORFs (Fig. 42). The northern blot analysis of RNAs isolated from etiolated seedlings using B2 fragment revealed the presence of three major transcripts of 2.6, 1.8 and 1.3 kb and several minor transcripts of size greater than 2.6 kb (Fig. 47A). When the etiolated seedlings were illuminated with light at 25 C, level of all the three major transcripts increased. Besides, a novel transcript of 0.8 kb appeared during illumination of etiolated seedlings with light at 25 C. The transcription of the atpb operon is not very well understood. The co-transcription of the atpb and atpe genes has been reported for a number of plants (Krebbers et al., 1982; Zurawski et al., 1982) but several other experiments have suggested the presence of separate transcripts for the atpe gene (Shinozaki et al., 1983). 2.6 kb transcript in figure 47A might have been originated from atpb-e operon. The size of mature transcript of atpb-e are suggested to be kb (Krebbers et al., 1982; Zurawski et al., 1982). 0.8 kb transcript which appeared during illumination of dark-grown seedlings could belong to atpe gene. Transcript of 0.8 kb was 80

19 A. B Fig.47 Effect of elevated temperature on expression of rbcl gene, atpb and ndhc operons. Blots were probed with (A) 82 fragments and (B) P7 fragments. Experimental details and lane descriptions are sim ilar as described in Fig.44.

20 also detected with the P7 fragment (Fig. 478). As can be seen in figure 42, _atpe gene is also present on P7 fragment suggesting that 0.8 kb transcript is the mature atpe transcript. Light-dependent accumulation of atpe transcript suggested that either it is synthesized from a light inducible promoter or its transcript is not stable in dark and may require some light regulated factors for its stability. When the illumination of dark-grown seedlings was carried at 38 C, the transcripts of 2.6 and 0.8 kb decreased and after 8h of high temperature treatment, complete loss of these transcript occurred. The sensitivity of 0.8 kb transcript to high temperature further suggested the role of light-inducible factor in the synthesis/stability of atpe transcript. There was no change in the level of two minor transcripts of 4.9 kb and 3.4 kb upon transfer of etiolated seedlings to light either at 25 C or 38 C. rbcl codes for the large subunit of Rubisco. The regulation of rbcl gene expression by light and during chloroplast development have been studied in a number of higher plants and transcriptional, post-transcriptional, and translational control of rbcl gene expression have been proposed (Sasaki et al., 1984). As discussed above, the gene for rbcl is located near to atpb operon and is transcribed in opposite polarity. The transcript level of rbcl (1.8 kb) in the dark grown seedlings is shown in figure 47 A which increased following the illumination of dark grown seedlings at 25 C. Illumination of dark-grown seedlings at 38 C leads to the decrease in the transcript level as compared to dark grown seedlings Expression of ndh genes Evidence for a respiratory chain in the chloroplast of Chlamydomonas has been presented (Bennoun, 1982) and a number of genes coding for the subunits of this complex have been localized on ctdna of tobacco (Shinozaki et al., 1986), liverwort (Ohyama et al., 1986) and rice (Hiratsuka et al., 1989). Matsubayashi et al (1987) showed that all the genes, coding for NADH dehydrogenase, present in the plastid genome of tobacco are transcribed and its transcripts are as abundant as the transcripts of rps7, 3'-rps12. In wheat chloroplast genome, some genes for this complex are localized (Nixon et al., 1989) and based on comparison with rice chloroplast genome, different genes on the wheat ctdna are shown in figure 42. Northam blot analysis of RNAs isolated from etiolated seedlings using P7 fragment revealed the presence of three transcripts of 2.8, 2.4 and

21 kb (Fig. 47B). Illumination of dark-grown seedlings at 25o-C lead to increase in the level of all the transcripts. Besides, a new transcript of 0.8 kb and two minor transcripts of 1.6 and 1.2 kb were detected in the illuminated seedlings. The ndhc, ORF159 and psbg genes, present on P7 fragment, are co-transcribed to produce a single transcript of 2.0 kb (Matsubayashi et al., 1987). In the present condition, 2.0 kb transcript was not observed. However, a 1.8 kb transcript detected with P7 might represent the ndhc operon in wheat. Transcription of all genes originating from this fragment was found to be sensitive to high temperature as all transcripts were absent in seedlings exposed to high temperature. Another ndh operon which consists of ndha, ORF178, ndhg, ndhe are suggested to be co-transcribed (Matsubayashi et al., 1987). In wheat plastid genome, these genes are present on P9 fragment (Fig. 42). The northern hybridization of RNAs isolated from darkgrown seedlings using this fragment revealed one major transcript of kb. Besides, two minor transcripts of 2.6 and 1.5 kb could also be detected with P9 fragment (Fig. 48A). Exposure of etioplast seedling to light either at 25 C or 38 C does not result in any change in the level of these transcripts. Matsubayashi et al. (1987) have shown the presence of a 4.0 kb transcript in tobacco expected to cover the whole ndh operon. However in wheat, using P9 fragment as a probe, no transcript of size greater than 2.6 kb was observed. 0.9 kb transcript detected in figure 48A could be originating from ndhe gene (Matsubayashi et al., 1987). 1.5 kb transcript present in figure 48A might be coming from ORF393 although in tobacco no transcript belonging to ORF393 was found (Matsubayashi et al., 1987). In wheat plastid genome, ndhb gene along with various ORF and genes for ribosomal protein are present on B8 fragment. Northern blot analysis of RNAs isolated from dark-grown seedlings using B8 fragment identified four transcripts of 2.8, 1.6, 1.1 and 0.8 kb (Fig. 48B). Transfer of the etiolated seedlings to light either at 25 C or 38 C did not affected the level of these transcripts. 1.6 kb transcript may represent the mature spliced mrna originating from ndhb (Matsubayashi et al., 1987) Expression of plastid genes coding for the components of protein synthesis apparatus Expression of ribosomal genes All the ribosomal RNAs present in the ribosome are encoded by a single operon located on the chloroplast genome i.e, rm operon. rm operon consists of genes for 16S-tml-tmA-23S- 4.5S-5S rrna in this order. In wheat plastid genome, the rm operon is spread-over PS, P6 82

22 A. e Fig.48 Effect of elevated temperature on expression of genes coding for NADH dehydrogenase. Blots were probed with (A) pg fragment and (B) B8 fragment. Experimental details and lane descriptions are similar as described in Fig.44.

23 and P8 fragments (Fig. 42). Northern blot analysis of RNAs isolated from etiolated seedlings using PS and P8 fragments gave identical transcripts pattern and were different from the transcripts pattern obtained with P6 fragment. Although several other protein coding genes are also present on these fragments, transcripts of only rrna genes were detected due to the presence of high amounts of transcripts as compared to transcripts of protein coding genes. As could be seen in figure 49, the transcript level of rrna did not change as compared to etiolated seedlings when the dark grown seedlings were illuminated either at 25 C or 38 C. It has been shown that in heat. treated barley or rye plants which leads to the bleaching of chi and loss of protein biosynthetic machinery, the transcripts of ribosomal genes were not present. The presence of rrna in the heat treated wheat seedlings could be due to the short period of temperature treatment in the present investigation Expression of genes coding for subunits of RNA polymerase Most plastid genome of higher plants contain four genes which code for the proteins homologous to the subunits of RNA polymerase of E. coli (Ohyama et al., 1986; Shinazaki et al., 1986; Hiratsuka et al., 1989). Various experiments lead to suggest that these genes might be coding for the subunits of chloroplast RNA polymerase (little & Hallick, 1988; lgloi & Kessel, 1992). Three of these four genes i.e, rpo8-rpoc-rpoc1 are co-transcribed whereas rpoa, is co-transcribed with the genes coding for ribosomal proteins. Although all the rpo genes are transcribed, their transcript level is present in extremely low amount in normal green plants (Hess et al., 1993). In wheat plastid genome, operon consisting of rpo8-rpoc-rpoc1 genes is present in P3 fragment whereas rpoa is present on 820 fragment (Fig. 42). Northern blot analysis of RNAs isolated from etiolated seedlings using P3 and 820 fragments are shown in figure 50. In etiolated seedlings using P3 fragment as a probe, three transcripts of-2.8, 1.4 and 1.0 kb were detected (Fig. SOA). After illumination with light at 25 C, two new transcript of 2.6 and 1.6 kb appeared while the level of other transcripts slightly increased. Two minor transcripts of 4.5 and 5.5 kb also appeared in the light at 25 C. When the dark grown seedlings were illuminated at 38 C, level of all the transcripts except 1.0 kb decreased. Hess et al. (1993) have shown that transcripts of rpo8 and rpoc1 accumulated to higher level in the bleached barley plants. However, in the present condition, the transcripts of 83

24 Fig.49 Effect of elevated temperature on expression of ribosomal RNA genes. Blots were probed with (A) P5 fragment and (8} P6 fragment. Experimental details and lane descriptions are similar as described in Fig.44.

25 A e Fig.SO Effect of elevated temperature on expression of genes coding the subunits of RNA polymerase. Blots were probed with (A) P3 fragment and (B) B20 fragment. Experimental details and lane descriptions are similar as described in Fig.44.

26 these genes did not increase in temperature treated seedlings. This could be due to the shorter duration of treatment at high temperature in the present study. In contrast to rpob operon, rpoa gene gave complex pattem of transcripts in the etiolated seedlings. The northem blot analysis of RNAs isolated from etiolated seedlings revealed four major transcripts of 2.4, 1.4, 1.2 and 0.6 kb (Fig. 508). Besides few minor transcripts of 3.8, 3.4 and 0.75 kb could also be detected. When the seedlings were transferred to light at 25 C, there was no change in the level of transcripts. However when dark grown seedlings were illuminated with light at 38 C, transcript level of 1.4 kb decreased. There was no change in the level of 2.4, 0.75 and 0.6 kb transcripts at 38 C. The level of 1.2 kb transcript, which is the mature transcript of rpoa gene, increased at 38 C. The increased expression of RNA polymerase gene in the bleached barley seedlings have been shown (Hess et al., 1993). It should be noted, however, that increased level of transcripts was not seen in the case of other three genes coding for the RNA polymerase subunit Expression of genes coding for ribosomal proteins Chloroplast genome of most higher plants codes for approximately 20 ribosomal proteins belonging to small and large subunits (Ohyama et al., 1986; Shinozaki et al., 1986; Hiratsuka et al., 1989). Rest of the ribosomal subunits are synthesized in cytoplasm and transported in the chloroplast. The expression of plastid ribosomal genes are constitutive and does not change much during chloroplast development. However, expression of these genes have been found to increase in the conditions where the chloroplast protein biosynthetic machinery is blocked (Sasaki et al., 1988; Nagano et al., 1991) or ribosome is lost such as in the high temperature treated plants (Feireband & Berberich, 1991 ). In wheat plastid genome, the location of these genes are shown in figure 42. S3a and 83 fragments consists most of the genes coding for ribosomal proteins whereas rest are scattered throughout the plastid genome and are expressed alongwith the genes coding for other plastid proteins. In the S3a and 83 fragments, ribosomal genes are present in operon which consists of rpl23-rpl2-rps19-rpl22-rps3-rp/16-rp/14-rps8-infa-rpl36- rps11-rpoa in this order. All of these genes except rpoa gene are present on S3a and 83 fragments (Fig. 42). Northam blot analysis of RNAs isol~ted from etiolated seedlings using 83 and S3a fragments revealed identical pattem. The northem blot with 83 fragment is shown in figure 51A. A total of four major transcripts of size 2.5, 1.5, 1.2 and 0.8 kb and

27 A B Fig.51 Effect of elevated temperature on expression of genes coding for the ribosomal proteins. Blots were probed with (A) B3 fragment and (B) B 15 fragment. Experimental details and lane descriptions are similar as described in Fig.44.

28 two minor transcripts of size 3.6 kb and 3.9 kb were detected. Illumination of etiolated seedlings either at 25 C or 38 C did not affect the level of any of these transcripts. 815 is another fragment which carry genes for three ribosomal proteins (Fig. 42). Northern blot analysis of RNAs isolated from etiolated seedlings revealed the presence of six transcripts of 2.6, 1.8, 1.6, 1.4, 1.0 and 0.8 kb (Fig. 51 B). Transfer of etiolated seedlings to light at 25 C lead to increase in the level of all the transcripts. However, there was no change in the transcripts level as compared to control when illumination of dark-grown seedlings was carried at 38 C. 821 is another fragment which carry genes for ribosomal proteins (Fig. 42). Northern blot analysis of RNAs isolated from etiolated seedlings revealed four major transcripts of 3.7, 3.5, 1.8, 1.4 kb and two minor transcripts of 2.1 and 1.1 kb (Fig. 528). The level of all the transcripts slightly increased following transfer of dark-grown seedlings to light at 25 C but there was no change at 38 C as compared to control. As pointed above, the increased expression of ribosomal genes in the high temperature treated plants, which lead to the loss of. ribosome in barley and rye plants, were not observed in temperature treated wheat seedlings in our condition. Similar results were also obtained with rpob/c/c1 operon where increased expression of these genes at high temperature was not observed Expression of genes coding for trnas All the required trna for the chloroplast protein biosynthesis are present in plastid genome (Ohyama et al., 1986; Shinozaki et al., 1986; Hiratsuka et al., 1989). Some of the trnas are clustered and expressed together while others are expressed along with the protein coding genes. The clustered trnas are present on P?, P10 and 818 fragments. The pattern of transcripts observed with P10 and 818 were identical. Northern blot of RNAs isolated from etioplated seedlings using P1 0 fragment revealed two transcripts of 1.4 and 0.9 kb (Fig. 52A). After transfer of etioplated seedlings to light at 25 C, two transcripts of 2.4 and 1.8 kb appeared while the transcript level of 0.9 kb increased drastically. When the illumination of dark-grown seedling was carried at 38 C, the transcript level of 0.9 kb did not increased as compared to dark-grown seedlings. It is interesting to note that the accumulation of 0.9 kb is both light dependent and temperature sensitive, a result also found with the expression of various light-inducible genes. The regulation of its expression by light suggest the importance of the product encoded by 0.9 kb transcript in the plastid 85

29 A Fig.52 Effect of elevated temperature on expression of genes coding for subunits of trnas and ribosomal proteins. Blots were probed with (A) P1 0 fragment and (B) B21 fragment. Experimental details and lane descriptions ~re similar as described in Fig.44.

30 functions. The level of other transcripts did not change in response to high temperature. However, a transcript of 4.0 kb appeared at 38 C. The transcripts of trnas discussed here represents those of the primary transcripts synthesized together with other genes. The effect of high temperature on the individual trnas transcripts were not studied in the present investigation Expression of nuclear genes involved in the plastid functions Expression of genes coding for RNA-binding proteins The regulation of plastid gene expression is suggested to involve a number of different RNA-binding proteins which expression is also regulated by light and other metabolic processes (Gruissem, 1989; Rochaix, 1992; Gruissem & Tonkyn, 1993). Unfortunately very few RNA-binding proteins have been identified and characterized. These RNA-binding proteins could be involved in the splicing, processing and stabilization of the plastid transcripts. Li & Sugiura (1990) have identified three such RNA-binding proteins which have strong affinity for ssdna. However, the function of these proteins remain unknown. We have utilize the edna clones of these RNA-binding proteins to study its expression during illumination of dark-grown seedlings at high temperature. The northern blot of RNAs isolated from dark-grown seedlings revealed single transcripts of 1.3 kb for CP29 and 1.4 kb for CP31 (Fig. 53). The expression of both genes was not affected by light at either temperatures Expression of genes coding for translation elongation factors The expression and characteristics of EF-Tu, translation elongation factor, have been widely studied in a number of bacterial species. During protein elongation in bacteria, EF Tu is part of a ternary complex that includes aminoacyl-trna, the ribosome and GTP. EF Tu is the site of GTP hydrolysis during elongation (Kaziro, 1978). Chloroplast ribosomes are similar to bacterial ribosomes in many respects (Hoober, 1984) and EF-Tu is probably essential for chloroplast protein synthesis. Two genes encoding nearly identical EF-Tu proteins, similar to those of E. coli, has been identified in CtDNA in Chlamydomonas (Silk & Wu, 1988) and in nuclear DNA of higher plants (Palmer, 1985) As described earlier, high temperature has led to the loss of plastid translation machinery function. Since function of EF-Tu is essential for the plastid protein synthesis, the high temperature would be expected to affect its expression. Northern blot analysis 86

31 kb -1-4kb Fig.53 Effect of elevated temperature on the expression of RNA-binding proteins. Experimental details and lane descriptions are similar as described in fig.44.

32 was carne a out to stuay the effect of high temperature on the expression of EF-Tu gene. As shown in figure 54, high temperature had no effect on the expression of TufA gene as compared to control, however expression of TufB was found to increase at 38 C. This result is in accordance to those identified in the case of ribosomal RNA thus suggesting that the protein synthesis in chloroplasts is not affected by high temperature treatment in the present conditions. 87

33 1 A kb B Fig.54 Effect of elevated temperature on the expression of plastid translation factors. Experimental details and lane descriptions are similar as described in fig.44.