Chapter 5

DISCUSSION

Discussion

The broader aim of the present study was to understand the

mechanism of regulation of transcript levels in response to salinity

stress in rice. While there have been some work on the understanding

the upregulation of genes in response to stress, yet there are very few

reports to check for genes that are downregulated. A detailed work was

undertaken on elucidating the mechanism of downregulation of rpL32

gene and briefly on MSRMK2 promoter. The results of this work

indicated that the four rpL32 genes of rice, especially the rpL32_8.1, is

differentially downregulated and the MSRMK2 gene is differentially

upregulated in the tolerant and sensitive varieties of rice. The outcomes

of these results are broadly discussed in this section.

rpL32 belongs to multigene family

The NCBI and TIGR database mining resulted in the identification of four

60S ribosomal protein rpL32 genes in rice which were named as

rpL32_8.1, rpL32_9.1, rpL32_9.2 and rpL32_9.3 according to their nth

copy number on the chromosomes on which they were found (Figure 1

and 2). The proteins encoded by these genes are almost similar in their

sequences. Multiple genes encoding the same ribosomal protein are not

uncommon in plants. It is estimated that in plants almost all ribosomal

protein are encoded by multiple genes. A study in rice, including 57

ribosomal protein genes, indicated that 90% of the ribosomal proteins

were encoded by small multigene families (Wu et. al., 1995). More

recently, from an in silico genome wide survey done in Arabidopsis, it was

inferred that the 80 different cytoplasmic ribosomal proteins of this

plant, are encoded by 249 genes (Barakat et. al., 2001). Multiple genes

encoding the same ribosomal protein were also inferred by the earlier

studies in maize and Arabidopsis (Larkin et. al., 1989; Williams and

89

Sussex, 1995; Revenkova et. al., 1999). Multiple genes for ribosomal

proteins are also found in other organisms. In yeast, ( S. cereviciae), of the

identified 78 different ribosomal protein genes, 59 are found in multiple

copies (Planta and Mager, 1998). All the above studies predicted that the

multiple genes of ribosomal proteins have arisen by duplication and

fragmentation of chromosomal segments within the genome of these

organisms during the course of evolution. The observation that the three

rpL32 genes on chromosome 9 were very closely placed, raises the

possibility that ribosomal genes could occur in cluster within the plant

genome, but the genome wide studies in Arabidopsis and rice did not

support this view. However, the ribosomal protein gene density was

found to be much higher in some regions of the genome.

The introns within rpl32 gene could contribute in the

transcriptional regulation of these genes

In all the four rpL32 genes identified in rice genome, three introns were

found to be present in each gene (Figure 2 C). The first intron lies in the

5' UTR region of the gene and the other two within the coding region of

the gene. The importance of such conserved gene structure is not well

understood. Intron mediated gene regulation has been reported from

many systems. The rpL32 gene of mouse also contains similar gene

structure. The importance of these introns in mouse was studied by a

series of experiments involving transient transfection of monkey kidney

(COS) cells with recombinant constructs carrying rpL32 gene having

complete or partial deletion of one or more intron (Chung and Perry,

1989). The results of these transfection experiments suggested that the

first intron (which lies in the 5' UTR region) was necessary for the

expression of rpL32 gene. A nuclear run-on assay made it clear that this

intronic regulation of rpL32 gene of mouse operates at the transcriptional

level. It was found that the 1st intron contains an element that could

increase the expression of the gene by 5-10 folds and relocalization of

90

this element in the promoter or the third intron did not bring this effect.

A similar phenomenon might function for the rpL32 genes in rice also.

TIGR database also predicts two alternative spliced form for two of the

rice rpL32 genes present on chromosome 9 which involves the first

intron. This suggests that this intron could also operate at the post­

transcription and translational regulation of these two genes.

The rpL32 genes vary in their expression profile

The northern hybridization and nuclear run-on assay indicated that

rpL32_8.1 is the highest expressing gene of this small family (Figure 4, 5

and 8). The northern studies depict that rpL32_9.3 is faintly expressed.

Although all of these genes down regulate under salt stress, there are

certain fine differences in their expression pattern. In pokkali rice

rpL32_8.1 is downregulated in shoots at a relatively slower rate than

rpl32_9.1 and rpL32_9.2. Moreover, rpL32_9.2 shows a small oscillation

from 30% to 60% of its original level (control conditions) under salt stress

condition. The downregulation of rpL32 was steeper and more in stress

sensitive variety of rice. In roots, however no apparent decrease in

rpL32'-8.1 transcript was observed under salt stress condition. Genes

encoding similar or different ribosomal proteins are known to express

differentially in plants and other systems as we have found for rpL32

genes in rice. The database search forESTs of genes (showing complete

ORF) encoding same ribosomal protein in Arabidopsis have shown varied

number of representative ESTs indicating the members of these small

families may vary greatly in their expression pattern (Barakat et. al,

2001).

The four rpl32 genes could also vary in their spatial and temporal

expression pattern. rpL32_9.3, which was found to express very weakly,

might contribute toward the generation of major rpL32 transcripts m

some specific cell type and/ or at a different developmental stage. In

91

Arabidopsis rpL16 is encoded by two genes and the promoter:reporter

assays of these revealed that rpL16B expresses in proliferating tissue

including shoot and root meristem while rpL 16A expression was mainly

found in cells of root stele and in anthers (Williams and Sussex, 1995).

Both these genes were found to be co-expressed in auxin stimulated

lateral root primordial. It was further found that rpL 16 expressiOn

correlates with cell division and the expression of rpL16A is more cell

specific. A similar situation was also observed m case of Arabidopsis

rpS18 gene which has three copies within the genome. Of these three

genes rpS18A, via promoter:reporter assays, was found to be expressed

in meristematic cells at embryonic heart stage, and in cells at the

wounding sites (Lijsebettens et. al., 1994). Such a phenomenon of

differential expression could also account for the expression pattern

differences within these rpL32 genes under control and stress conditions

and for rpL32_8.1 in shoot and root and in different varieties of rice.

As the rpL32_8.1 was found to be the major transcript among all the

rpL32 RNAs in rice system, it was expected to be expressed in majority of

the cell types under the specified growth conditions and at the specified

developmental stage. Due to this reason, the regulatory studies under

salt treatment were carried out for this gene.

rpL32_8.1 could be an indicator of the degree of physiological stress

experienced by a monocot plant

rpL32_8.1 gene was found to get downregulated in shoots of rice plants

when subjected to salt, drought and cold stress (Figure 4 and 10). Few

ribosomal protein genes from different plants are known to get down- or

upregulated on being subjected to stress. rpL25 and rpL34 were found to

get upregulated in wounding stress in tobacco (Gao et. al., 1994). BnC24

ribosomal protein transcripts in Brassica and rpS6 and rpL37 in

soyabean were upregulated when subjected to cold stress (Saez-Vasquez

92

et. al., 2000; Kim et. al., 2004). rpS26 is down regulated in pea by UV­

stress (Brosche and Strid, 1999). rpL2 is transiently downregulated in

soyabean in response to pathogen infection (Ludwiga and Tenhaken,

2001). The analysis of clones of differential display library between

control and stressed plants of barley and rice led to the idea that among

the downregulated genes, rpL32 is one of the gene that could be

commonly downregulated in both these plants. This could be an

indication that rpL32 downregulates atleast in all monocot plants under

stress conditions. From the northern studies done in this work, it seems

that the rate and the degree of down regulation of rpL32_8.1 gene

depends on the degree of physiological stress experienced by the plant. In

Pokkali rice, under drought stress (air dried), the severe most stress a

plant can experience, rpL32_8.1 is downregulated at a faster rate than

other treatments. Under cold stress the level of transcript after 24 hours

of treatment was found to be lower than that in salt stress after the same

time of treatment. Under salt stress, the rate of decrease in sensitive

variety (PB1) was found to be much faster than in the tolerant variety.

The sensitive varieties are supposed to experience higher degree of stress

than the tolerant varieties under the same environmental conditions. All

these inferences indicates that rpL32_8.1 gene can be used as an

indicator of the physiological stress experienced by a plant and can be

used as one of the markers to validate stress tolerant transgenic cereal

crops.

rpL32 gene could be indispensible for proper growth and functioning

of rice plants

Northern analysis m this study has shown that the expressiOn of

rpL32_8.1 returned back to its original level as soon as the stress

condition was removed (Figure 11). This could be an indications that

rpL32 gene is an important component of the rice system. Homologues of

rpL32 are present in all organisms from archea to higher eukaryotes

93

(figure 49' and 48). Protein sequences of rpL32 of rice share more than

70% homology with Drosophila rpL49 (homologue of rpL32). In

Drosophila, of the different mutations in ribosomal protein that result in

minute phenotype, rpL49 is one of them (Lambertsson, 1998) which

shows reduced growth and cell division rate. The minute phenotype is

characterized by a reduced body size and short and thin bristles. Being

very similar to rpL49, rpL32 protein of rice is expected to be an absolute

necessary for proper growth and development of the rice. Thus as soon

as the return of favorable growth conditions is sensed by the rice plants,

the cell system brings back the rpL32 transcript level to its original level.

rpL32 provides direct evidence for transcriptional regulation of

ribosomal proteins under stress conditions

Ribosomal proteins are known to be regulated at various steps of gene

regulation. Interestingly, biasness towards a particular type of regulation

has been encountered in different groups of organisms for the regulation

of ribosomal proteins. In yeast (S.cerevisiae), it has been suggested that

regulation of majority of the ribosomal proteins occur at the

transcriptional level (Planta and Mager, 1998). It was found that

promoter regions of 90 out of the analyzed 137 yeast ribosomal protein

genes contain a double Raplp-binding site, 12 ribosomal protein genes

contains Abflp-binding site and 2 of such genes contain Reblp-binding

site. In mammals, ribosomal proteins are believed to be regulated

predominantly at the translational level (Meyuhas, 2000; Meyuhas and

Hornstein, 2000). A similar kind of translational regulation of ribosomal

proteins has been observed in plants and also operates under abiotic

stress which involves TOR (TARGET OF RAPAMYCIN) kinase, RAPTORl

(REGULATORY ASSOCIATED PROTEIN!), PDKl (PROTEIN DEPENDENT

KINASE!) and S6K (rpS6 Kinase) and probably works in connection with

pathways involving phosphatidylinositol 3 (PI3)-kinase (Aida et. al., 2004;

Turck et. al., 2004; Kawaguchi et. al., 2004; Anderson et. al., 2005;

94

I Q

l

~ tXPI1Utd h~o1htficlll pro loin [TrichopiO>< odh .. t1nS] 0 Pt1dicted pro !tin IN•rn•tosttlla vec!tnsiSJ

<:1 111es 115leaves

i

-Q ribosomal proftin L!2 [Branchiostom•belcheri l>ingfiuntse] putative libosomol proftin L32 [5ipunculus nuduSJ

9 libosOI'IIIIi proltin U2 [8os tauruS]

I '~ prim•lts 121tavu Q Rlbosomol proltin L32 [Mus musculuS] 8 PREDICT EO: h-1htficlli prot.in isofonn I [PNl !roglodyftS]

;:;! prim•t.s 121ewes ~ ribosomal prot. in Ll2 1$~1tldc construct

~ .:> PREDICTfO: simHill'to 605 ribosomal proftin Ll2 [h1•o.ca.mui•II>J

~ -o PRfDICTfO:similill'to60 5 ribosomal proftin Ll2 [Conis f>rnilioriS] ribosomal prot. in Ll2 ~Oiho sapienS]

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• ~ frogs a. fiolds 12 lewes \l...;:t bony~shesl71ewts

Oput>.fivelibosomol prot.in Ll2 [Bmnfsilelong•fill

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-4 Wl>PS &IC. Illtavts

0 ..:. pufafivolibosomal prot.in •9 l\3~hoctphlliufropuncfaf>l • O tibosomal prot.in ~9 [llthalilrosle]

o Ribosomlli prot.in L 32 [Pklt.lll w!lo•ftii>J .l'"Q u ribosomlli prol>ein L32 ~oijconius melpomene] '( () libosomal prot.in L 32 (Bomb~"< morlJ

Qo 60~ libosomal prot.in L!2 ribosomol proltin Ll2e [Sph .. tiu.s sp. A.PV.2005J

Opufafive ribosomal proltin L32 [IWI..:onellicoccus hirsutuS) il PREDICT ED: simililT to ribosomlli pro !tin ll2 [Sfrong!loctnfrotus purpur1tuSJ

-v ribosomal proftin ll2, <omponentofcytos .. ~mosses l 5lewts

... 1 conif!rs t21eaues eudicol> 1 ~lewes

~ eudicol> 1 7 lewes eudicofs 1 5lewes

~ mollocol> 1 lleo.ves il 605 ribosomol proftin L~2A [Cucumis meloJ

<t monocols 1 7 lew" 1]1 4 monocoi:s 12lewes

c) 0 0S08g05H600 lOIYZi.SlfiVl(j. unnomed pro !tin product ~ h~othefical prot.in OsL0289 ..

Figure 48: A guide tree showing rpl32 is present in different groups of higher eukaryotes and shares high percentage of identity

Deprost et. al., 2005; Wullschleger et. al., 2006; Otterhag et. al., 2006;

Mahfouz et. al., 2006). Post-transcriptional regulation of ribosomal

proteins 84, 86, L3 and L16 was observed in maize where the translation

of these proteins occurs from the stored mRNA in seeds during early

periods of germination (Beltran-Pena et. al., 1995). Post-transcriptional

regulation of ribosomal protein P2 was discovered in anoxic roots of

maize (Bailey-Serres et. al., 1998). Ribosomal protein 828 in peach was

also supposed to be regulated post-transcriptionally where its pre-mRNA

was detected in cells showing intense metabolic (but not mitotic) activity

(Giannino et. al., 2000). However, no hnRNA was discovered in stress

downregulation of rpS28 in sunflower (Liu and Baird, 2003). BnC24

(rpL13) in Brassica napus was found to be regulated by both

transcriptional and post transcriptional ways where the increase in

mRNA did not correlate with protein level in response to cold stress

(Saez-Vasquez et. al., 2000). The promoter of a large number of

ribosomal proteins in both monocots and dicots have been found to

contain TEOSINTE BRANCHED 1, CYCLOIDEA, PCF (TCP)-domain

protein binding elements (Tremousague et. al., 2003; Tatematsu et. al.,

2005; Maughan et. al., 2006) . These TCP domain proteins has been

shown to be necessary for high expression of PCNA, cyclins and

ribosomal proteins in actively dividing and growing cells (Tremousague

et. al., 2003; Li et. al., 2005). Hence, it is presently belived that majority

of co-ordinate regulation of ribosomal proteins in plants happen at the

transcriptional level. In the present work, nuclear run-on assay,

promoter:reporter assay and in vivo footprinting prove the transcriptional

regulation of rpL32_8.1 under stress conditions (Figure 8, 20, 23 and

24). This provides further concrete evidence in support of the above

hypothesis and indicates that under stress conditions also the ribosomal

proteins are mainly regulated transcriptionally.

95

A specific repressor element could be responsible for repression of

GUS expression· by a promoter fragment larger than 705 bp

(reference taken from ATG start codon) of rpL32_8.1

The promoter:GUS assays using the full length and its serial deletions

(DFO, DF1, DF2, DF3 and DF4; Figure 16 and 20), in the tobacco (N.

tabecu.m var. xanthii) failed to bring strong GUS expression for DF1 and

DFO fragments (Figure 16 and 20). A genome wide comparison between

Arabidopsis and rice shows that they have comparable number of TF

factors- 1611 in rice vs 1510 in Arabidopsis (Xiong et. al., 2005) It was

found in these studies that about 50% of the TF genes in rice and

Arabidopsis can be classified into orthologous groups. The families of

transcription factors were also found to be same in rice and Arabidopsis.

However, several subfamilies were found to be lineage specific. As

Arabidopsis and rice represent the model for dicots and monocots, it is

expected that such differences in transcription factors do exist between

the monocots and dicot. In this study, as technology for developing

tobacco plants is much easier and less time consuming, this

heterologous dicot system was used for transgenic studies. In silico

analysis predicted that the promoter architecture of ribosomal proteins

are quite similar in dicot and monocot plants, it was expected that stress

specific regulation of rpL32_8.1 should behave similarly in tobacco and

rice. Due to certain differences in the type of transcription factors in

monocot and dicots, it is possible that a particular cis-element could only

be functional in one of the major groups of plants. Hence, some element

the upstream of DF2 fragment could be recognized as repressor site,

specifically in dicots and might be the cause of repression of GUS

expressiOn when driven by DFO and DF1 promoter fragments of

rpL32_8.1 gene. Also, the work was focused on only 1.2 kb fragment

upstream of the ATG codon. Transcriptional regulation can be imparted

on a gene by elements present at much longer distance away (on both

96

sides of the ATG codon of the gene). Thus, under endogenous conditions

in rice, it is possible that such long distance control regions (LCRs) could

nullify the negative impact of any upstream repressor element on the

transcription of this gene. In fact, an in silico analysis did predicted

the€€ presence of a MAR (matrix associated region) at nearly 5kb

upstream of the ATG codon of this gene. This MAR region could behave

as the LCR described above.

Under salt stress conditions transcription factors are prevented

from binding to SITE II motifs

The promoter:GUS assays in the transgenic tobacco plants indicated the

presence of elements for high expression (under control conditions) and

downregulation (under stress conditions) of rpL32_8.1 within the DF3

fragment and more specifically, between 284 bp and 546 bp region,

upstream of the ATG codon. A close analysis revealed that this region

contains many SITE II elements, SORLIP2 sites and UPl binding site (as

determined by PLACE analysis). This region was also found to contain a

G-box (ABRE Element) sequence which has been reported to bind to

repressors in light signaling pathways and two SRE (sugar repressive

element) which is also a binding site of repressors in plant system

(Hudson and Quail, 2003; Tatematsu et. al., 2005). In the present study

exogenous application of sucrose did downregulate the expression of the

gene. However, to find out which of the elements among all these motifs

participate in stress mediated downregulation of rpL32_8.1, the DMS­

LMPCR in vivo footprinting was conducted which is supposed to be the

best way to determine in vivo binding of transcription factor on its cis­

element under defined set of conditions.

The in vivo footprinting experiments of the top and bottom strands of the

mentioned DNA region suggested relatively less binding of transcription

factors on GGCCCA , GGCCCA WWW and AGCCCA sequence motifs

97

(Figure 23, 24 and 25). From PLACE database TGGGCC (reverse

complement of GGCCCA) and TGGGCT (reverse complement of AGCCCA)

sequences were inferred as SITE II elements and GGCCCA WWW was

found to be the UP1 sequence. SITE II elements were first found to be

involved in the expression of PCNA gene in meristematic cells of rice and

two non-E-Box binding bHLH protein PCF1 and PCF2 were found to

interact with two closely related SITE II elements (Kosugi et. al., 1995;

Kosugi and Ohashi, 1997). Later on it was found that SITE II elements

are the binding sites for TCP (TEOSINTE BRANCHED 1, CYCLOIDEA,

PCF)-domain protein and these sequences were found to belong to two

main groups- Class I (GGNCCCAC;) and Class II (GTGGNCC;), the latter

one being the binding site for repressors and the former for activators in

rice system (Kosugi and Ohashi, 2002). It has been observed that for

SITE II binding heterodimerization or homodimerization between TCP

domain protein, (which binds to two separate SITE II elements) is

necessary (Kosugi and Ohashi, 1997;2002). Functional variants of these

sequences have been discovered in Arabidopsis. In this case the

sequences of these elements were found to be TTGGGCC (which is

similar to TGGGCC sequence of rice PCNA promoter) and TGCCCT

(Tremousaygue et. al., 2003). Nearly 70% of the Arabidopsis ribosomal

proteins were found to contain atleast one of these SITE II elements. A

detailed in vivo and in vitro analysis proved the functionality of GCCCR

(R=A or G) in the promoter of CYCB 1; 1, PCNA2 and ribosomal protein

L24B, S15a and S27a (Li et. al., 2005). The SITE II elements which were

found to be functional in the promoter region of rpL32_8.1 gene in the

present work are GGCCCA (reverse complement of TGGGCC) which is

same as found in rice and AGCCCA (reverse complement of TGGGCT)

which was discovered in Arabidopsis and belongs to the GCCCR

category. The GCCCR (including AGCCCA) type of elements had been

found in the promoter regions of ribosomal proteins and genes involved

in cell cycle regulation and translation machinery in rice but were never

98

functionally validated earlier in nee. Interestingly, as mentioned in

results, a GCCCG (which belongs to GCCCR category) element present in

this analyzed region {-269 bp position) did not show any difference in

protein binding between control and stress samples which shows that

the footprinting difference obtained are not artifacts. The two UPl

element {GGCCCAWWW) in the promoter region of rpL32 also showed

difference in transcription binding. This element was originally

discovered in the upregulated genes of the cells of axillary shoots after

main stem decapitation in Arabidopsis (Tatematsu et. al., 2005). Because

this sequence has the exact similarity to SITE II element discovered in

Arabidopsis, this element was suggested to be the binding site of TCP

domain protein. In the same work it was found that disruption of these

elements does not cause the upregulation of ribosomal protein L15 after

main stem decapitation. All these works suggested that SITE II elements

are of immense importance for the high expression of ribosomal proteins.

The present work clearly showed that the ribososmal proteins under

stress conditions are downregulated by preventing these transcription

factor from binding to their sites. However, the possibility of involvement

of a repressor in this downregulation is still not ruled out. Recently, a

genome wide survey has suggested the presence of GGCCC sequences in

the genes that are downregulated in response to stress (Ma and Bohnert,

2007)

It is being hypothesized that the transcription factors that binds to these

SITE II element could be downregulated transcriptionally or post­

transcriptionally upon stress subjection and hence are prevented from

binding to their respective cis-elements. These transcription factors could

also be regulated post-translationally by proteasome mediated

degradation or by modification like phosphorylation. There is another

potent way to regulate these transcription factors though the mechanism

has not been described for plants yet. As mentioned, the SITE II binding

99

TCP-domain proteins are bHLH type proteins which are needed to form

homo- or heterodimer for DNA binding. In animals, a class of non-DNA

binding HLH proteins, the ID-HLH proteins, is known to form

heterotetramer with the DNA binding bHLH proteins and thus preventing

them from binding to their respective sequences (Fairman et. al., 1993).

A recent genome wide analysis has identified 26 HLH proteins in rice

which lacks the basic regions of bHLH and have been considered as non­

DNA binding proteins (Li et. al., 2006). It has been suggested that these

proteins could behave as ID-HLH proteins of animals and hence can

regulate the TCP-binding proteins if present in the cells, under stress

conditions, in an active form.

A element with a sequence TAGGGTTT (reverse complement of

AAACCCTA), known as telo-box (shows similarity to telomeric repeats)

has been found to work synergistically with SITE II elements for strong

expression of the associated genes (Tremousaygue et. al., 1999;

Tatematsu et. al., 2005). This element, however, is not sufficient alone to

initiate the transcription of an associated gene. This element is present

in promoter of rpL32_8.1 gene at -43 bp position and lies in the DF4

fragment. The transgenic experiments showed that plants bearing DF4

promoter fragment does express the downstream uidA gene. Just

upstream of this telo-box is present a SITE II like element at position -51

bp, the sequence of which is AGCCCG. Though GCCCR SITE II motif has

been found to be functional in Arabidopsis but a similar sequence

TGCCCG has not shown any sequence difference and also seems to be

non-functional in this promoter (as inferred from top and bottom strand

footprinting). It is possible that this particular variant (AGCCCG) is

functional. Also there is a SORLIPl site (GCCAC) at -56 bp position.

SORLIPl and SORLIP2 (GGCCC) have been found bioinformatically in

promoters of light regulated genes (Hudson and Quail, 2003). These sites

have been reported to be present in stress mediated downregulated genes

100

of Arabidopsis (Ma and Bonhert, 2007). GGCCC (SORLIP2 site) is a part

of a SITE II element. GCCAC (SORLIP1 site) could also be a SITE II motif

variant (the sequence is also similar to SITE II motif). It is therefore,

possible that the GUS expression driven by DF4 fragment is mediated by

the interaction of the protein binding to telo-box and to any of these

elements. Alternatively, the interacting partner of the telo-box binding

protein could be present in the internal portion of the gene (i.e in the

5'UTR region including the first intron) and the multiple SITE II elements

are needed for higher expression of rpL32_8.1 gene. The repression of

GUS under stress in DF4 transgenics could be due to removal of

transcription factors (less binding) from the mentioned sequences in the

DF4 region.

Down regulation of rpL32_8.1 is associated with alteration in

chromatin architecture

The present work revealed that the DNA region around the TATA-Box of

rpL32_8.1 gene remained nonmethylated under both control and stress

conditions (Figure 29 and 30). An analysis of the 5' boundary of the

nucleosome nearmost to the TATA-Box by Mnase-LMPCR revealed its 13

major boundaries (Figure 37). Occupancy of a nucleosome with diverse 5'

and 3' boundaries had been reported in many other systems (Castanzo

et. al., 1995; Pfaff and Tayor, 1998). Under salt stress conditions, a

relative increase in occupancy of this nucleosome to a position upstream

of the +1 TSS site and TATA-box was observed which otherwise was

confined mainly to a position downstream to the + 1 TSS site under

control conditions. In Arabidopsis, many of Chromatin remodeling

ATPases have been discovered but excepting DDM1, none of them had

been biochemically characterized (Brzesky & Jerzmanowski, 2002).

DDM1 is also linked to the maintenance of methylation status of the

DNA. As the analyzed regions are devoid of any DNA methylation,

chances are more that DDM1 like ATPases are not involved in the

101

regulation. Pickle like ATP dependent chromatin remodeling complex,

could also be involved in the repression of rpL32. Pickle (CHD3 type

remodeling factor) has been found to negatively regulate the activity of

ARF7 and ARF19 in Arabidopsis (Fukaki et. al., 2006). As comparatively

less is known about these remodeling factors in plants, the exact

remodeling complex operating in this promoter fragment cannot be

pointed out presently. The Mnase-LMPCR autoradiogram does suggest

that probably this particular nucleosome might not be repositioned in all

cells under stress and control conditions. This could be due to the

availability of different transcription related factors in different cell types.

When the nucleosome is repositioned over the TSS, it is expected to

hamper the transcript initiation to a greater extent and may be necessary

to severely reduce the transcription of rpL32_8.1 in some cell types.

ChiP-PCR of the 5'UTR region of this gene indicated marked decrease in

histone acetylations (H3ac, H4ac, H3K9ac) under stress condition as the

gene expression goes down (Figure 40) which is consistent with the fact

that DNA acetylation is associated with active genes and has been found

to be true in all the species studied so far (Fuchs et. al., 2006 and the

references there in). The histone acetyltransferases (HATs) and HDACs

could be directly recruited by the transcription factors or could be

associated with a recruited ATP-dependent remodeling factor. The

present work depicts a decrease in H3K4me3 along with an increase in

H3K4me2. Both H3K4me2 and H3K4me3 are markers of euchromatin.

According to a recent ChiP analysis of genes present on two of the

chromosomes of rice, the level of transcription is determined by the ratio

between H3K4me3 and H3K4me2 modification (Li et. al., 2008). The

highly transcribed genes were found to be associated with higher amount

of H3K4me3. In the case of rpL32_8.1, it was found that H3K4me3 mark

was totally abolished by the end of 2 hrs of stress when the gene

expression was found to be nearly 50-60%. In nee, a change from

102

H3K4me2 to H3K4me3 in the 5' and 3' portion of ADH 1 and PDC 1 genes

were also observed during its activation on being subjected to

submergence stress and some amount of H3K9Ac was still associated

with it. A rise in H3K9me2 was found by the end of 24 hrs of stress.

Though H3K9me2 along with H3K27me2 IS considered as

heterochromatic mark in Arabidopsis, but it was also observed 1n

euchromatic regions of the chromosomes in maize (Shi and Dawe, 2006).

This cytological study in maize suggested that the rate of transcription

depends on the ratio of H3K9me2 and H3K4me2. Thus it seems that

transcription of rpL32 is regulated by the levels of H3K4me3, H3K4me2

and H3K4me2. One of the interesting finding in this present work, was

the abundance of H3K9 monomethylation in the 5' upstream region of

the rpL32_8.1 gene. In Arabidopsis H3K9me l was found to be present in

the heterochromatin but was also discovered in the euchromatic region

(Jackson et. al., 2004; Thorstensen et. al., 2006). In maize H3K9me1 was

also found . in some discrete loci in the euchromatic region (Shi and

Dawe, 2006). The ChiP experiments revealed that active rpL32_8.1 gene

is associated with both high levels of H3K9ac and H3K9me 1. It is

possible that these two different signals on same lysine residue could

have riginated from two adjacent nucleosomes. In yeast and mammalian

cells, it has been shown that when the histone modifications H3K9me3

and H3K36me3 were present in the promoter region, it represses

transcription and when present within the gene, it helps is gene

activation (Pfluger and Wagner, 2007). These modificatiqns close

chromatin to prevent transcription initiation. Thus when present within

the gene it prevents transcription initiation from cryptic internal sites,

and helps in generating full length transcripts. Similarly the H3K9me 1

modification could be present in an adjacent nucleosome within the gene

and functions in preventing the initiation of transcripts from some false

sites within the gene of rpL32_8.1. The association of a relatively less

amount of euchromatin specific H3K4me 1 modification with the

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rpL32_8.1 gene cannot be interpreted correctly as much less is known

about this modification in plants. In Chlamydomonas this modification

has been found to be associated with euchromatic gene repression. This

modification along with other modification may operate in some cell types

for a regulated expression of rpL32_8.1 under control conditions.

All the above resuts indicates that the expression of rpL32_8.1 is tightly

regulated by alteration 1n nucleosome positioning and histone

modifications.

Transcription of rpl32_8.1 is possibily associated with gene looping

Gene looping or promoter-terminator interaction is a newly discovered

phenomenon in eukaryotes. The promoter and terminator of the genes in

yeast were found interacting with each other (Ansari and Hampsey,

2007). This juxtapositioning of the promoter and terminator was

suggested to be important for reloading of RNA polymerase II on the

promoter from the terminator after one round of transcription. This was

supposed to increase efficiency of transcription of the associated gene.

These transcription-dependent interactions were shown to require the

Ssu72 and Ptal components of CPF 3' end processing complex. The

phosphatase activity of the Ssu72 was also observed to be important for

these interactions. Later on TFIIB was observed to be involved in the

formation of these gene loops (Singh and Hampsey, 2007). Interestingly,

in this work it was found that in mutants of yeast in which the

transcription of the tested genes were not affected, the promoter­

terminator interaction was impaired. This result confused the

interpretation for the need of such interactions. Transcription-dependent

gene-looping was observed in case of human mitochondial rDNA also

(Martin et. al., 2005). In this work, a possibility of existance of this

phenomenon was tested in plants. The results shown in Figure 41

indicated that juxtapositioning of promoter and terminator could also

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exist in plants and operates in a transcription-dependent manner in

rpL32_8.1 gene in rice. A detailed analysis of this phenomenon for

rpL32_8.1 was impaired by the lack of knowledge and limitation of

resources (like the cloned proteins and antibodies agaisnt these proteins

of general transcriptional apparatus) in rice. However, as the frequency

of promoter terminator interaction was found at a relatively low level

under stress conditions, when the gene downregulates, these interaction

seem to be specific. This experiment atleast raises the possibility that

such interaction also happen in plants and under stress such interaction

could be affected.

The need to downregulate rpl32 genes could be attributed to its

ribosomal as well as possible extra-ribosomal functions

Upon exposure to stress, plants exhibit multiple responses at the

molecular, cellular and whole plant levels (Greenway and Munns, 1980;

Zhu et. al., 1997; Yeo, 1998; Bonhert et. al., 1999; Hasegawa et. al.,

2000; Xiong and Zhu, 2002). One of these responses includes inhibition

of growth and cell division in the shoot. This is necessary for either

escaping the period of stress or re-programming the molecular network

in an attempt to adjust to the changed environment. To slow down the

rate of growth, it is necessary to slow down the translational machinery

and hence, the ribosome biogenesis. From this perspective rpL32, which

is a component of ribosome, is needed to be downregulated in response

to stress.

Apart from its ribosomal functions, rpL32 has been reported to possess

extraribosomal functions also. In yeast L32-2 protein has been shown to

have transcriptional transactivation activity also (Wang et. al., 2006).

Owing to the similarity between yeast L32-2 and rpL32 protein of rice

(Figure 49), it can be expected that such functions are also associated

with rpL32 of rice. Thus, the downregulation of rpL32 could be a part of

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rpL32_Tc 62 rpL32 Om 62 rpL32_Ag 62 rpL32 Hs 63 rpL32-Mm 63 rpL32-Xt 63 rpL32-0s 61 rpL32-Zm 61 rpL32 Pt 61 rpL32A At 6 rpL32B=At 61 rpL32 Cr 6 rpL32=Sc 6 0 rpL32_Sp 57 rpL32 Ss 6 rpL32-Pat 63 rpL32e_Pfu 68 rpL32e_Tk 68 rpL32e_Mk

rpL32_Tc 131 rpL32 Om 131 rpL32-Ag 131 rpL32-Hs 135 rpL32-Mm 135 rpL32 =Xt 135 rpL32_0s 133 rpL32 Zm 133 rpL32- Pt 133 rpL32A At 133 rpL32B=At 133 rpL32_Cr 133 rpL32 Sc 13 0 rpL32_Sp 127 rpL32 Ss 131 rpL32 Pat 13 6 rpL32e Pfu 13 d rpL32e Tk 126 rpL32e=Mk 131

Figure 49: A ClustaiW alignment showing similarity between rpl32 genes from different organisms

a broader change which redesigns the transcriptional framework of the

entire plant under stress conditions. Overexpression of L32 in yeast has

been reported to disrupt telomeric silencing (Singer et. al., 1998). It is

possible that L32 downregulation m plants IS associated with

maintenance of the telomeric region of the chromosomes in plants under

stress conditions.

rpL32 belongs to rpL32E superfamily of ribosomal proteins (as detected

by BLAST program of NCBI database) . The 50S rpL32E of Haloarcula

marismortui has been shown to bind to specific RNA secondary structure

known as Kink-turn RNA structures (Klein et. al., 2001). Due to this

interaction rpL32E is believed to stabilize the rRNAs in the ribosomes. A

similar motif has been identified for rice rpL32 protein also. The maize

rpL32 which is almost identical to rice rpL32 gene has been found in a

crosslinked state with RNA under UV stress. This indicate that rpL32 of

rice may be involved in RNA binding. Abiotic stresses induce many types

of secondary structure in mRNA. These RNA structures need to be

disrupted for their further processing. rpL32, due to its RNA binding and

stabilizing effect, can stabilize these structures to a higher degree which

could lead to a more detrimental effect. Thus, downregulation of rpL32

under stress could be a necessity in plants.

The faster downregulation of rpL32_8 .1 in sensitive varieties could be a

result of faster rate of removal of transcription factors from the SITE II

elements or due to faster rate of mRNA decay or both. A higher degree of

down regulation of rpL32 in the sensitive varieties could lead to death

under severe stress conditions. Also, as discussed above, many

ribosomal proteins are also induced under stress conditions. Selective

translation of mRNAs under stress conditions are also reported from

plant system (Kawaguchi et. al., 2004). These reports indicate that under

stress many ribosomal proteins could get replaced by a different types of

ribosomal proteins. This might happen with rpL32 also. Thus, a

106

ribosomal protein, homologous to rpL32 but with more specific function

can operate under stress conditions. The accumulation of such a protein

should co-ordinately match with the rate of downregulation of rpL32

under stress condition. A faster down regulation of rpL32 in the sensitive

varieties might not provide a proper chance to the stress specific

ribosomal proteins to accumulate to a minimum threshold level which

may be necessary to cope with the surrounding harsh conditions.

From the results obtained in this work along with some knowledge

gained by various literature, a model for the regulation of rpL32_8.1

under control and stress condition, has been hypothesized and presented

in Figure 50A and SOB

The study on MSRMK2 regulation indicates that the stress tolerant

varieties might be carrying an epigenetic memory of stress

In the present work it was found that MSRMK2 was induced

transcriptionally in the sensitive variety under stress conditions while

this gene was already induced under control conditions in stress tolerant

variety and downregulates upon subjection to stress (Figure 46 and 4 7).

The result indicate that the transcriptional regulation is differently

programmed in tolerant and sensitive varieties of rice. In stress tolerant

variety Pokkali, many early inducible genes have been found to be active

under control conditions. This indicates that probably, the stress tolerant

varieties always senses some degree of virtual stress which might not be

present in reality which provide these plants a better chance of survival

under stress conditions as they are already one step ahead in coping

with harsh environmental conditions. The stress tolerant varieties of rice

should have originated in areas with relative unfavourable conditions

and their generation are possibly carrying a reorganized genetic program

and an epigenetic memory of the stress that were faced by their original

107

The hetero- or homo-dimer fanning SITE II element

specificTCP-Oomain proteins probably causes the

transcription favourable nucleosome positioning an """' modiff~r

s,'t J<r1

''"" ; "' "'>1"'1i:ts~_..,_.,.......

R APII

4>C \\\ rpl32_8.1 1

Gene loop

Figure SOA: A model of transcription regulation of rpl32_8.1 under control conditions.

In absence of any stress the rpl32_8.1 gene expression is very high, The genes probably forms a loop structure where the promoter and terminator could be juxtaposed by TFIIB as found in yeast. The loop may be involved in shifting the RNAPII from terminator to promoter region. The transcription is mediated by the presence of SITE II element binding transcription factor which are probably the TCP domain bHLH proteins. These binding factors are supposed to be involve in maintaining the H3ac,H4ac and H3K4me3 state of the histone in nucleosome present in the S'UTR portions of the gene. This nucleosome is positioned to a region which does not include the TSS site, thus facilitating efficient transcription initiation

TCP-Domal n proteins are not bound to the SITE II e lem!!nts

•• --------------~ .. ~

"~t~ellts

+1 TSS ____ ., I -

H3 K9mo l

Nucleosome is displaced In trans to a position

beyoun the TSS (to the 5' upstream position I

rpl32_8.1 Gene Loop dlssappears

Figure SOB: A model of transcription regulation of rpl32_8.1 under stress conditions.

In presence of any stress the rpL32_8.1 gene expression is very low. The genes loop disappears and it forms a open structure . of SITE II element bind ing transcription factor shows less bind ing. The nucleosome is dominated by H3K9me2 and H3K4me2 which are not favorable for transcription . The nucleosome position changes and it now includes the TSS wh ich might be inhibitory to transcription in it iation .

parental plants (as discussed m detail m the Review & Literature

Chapter).

This work on the regulation of rpL32 and MSRMK2 genes provides

evidence that for the induction of tolerance in sensitive varieties of rice it

is necessary to understand the fine details of transcriptional regulation

in both the tolerant and sensitive varieties of rice. The sincere efforts by

scientists all around the world has helped us in taking a big step in

understanding the regulation of stress responsive genes but a longer

jump is still required.

108