Research Area: Environmental Stress and AdaptationOct 07, 2014 · promoters to sense heat is largely attributed to heat shock elements (HSE) located in the promoter region (Sugio

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Running head: Promoter analysis of AtClpB-cytoplasmic/Hsp100 gene 1

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Corresponding author 3

Prof. Anil Grover 4

Department of Plant Molecular Biology 5

University of Delhi South Campus 6

New Delhi-110021, India 7

Phone: 91-11-24115097 8

FAX: 91-11-24115270 9

Email: [email protected] 10

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Research Area: Environmental Stress and Adaptation 14

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Plant Physiology Preview. Published on October 7, 2014, as DOI:10.1104/pp.114.250787

Copyright 2014 by the American Society of Plant Biologists

www.plantphysiol.orgon October 19, 2020 - Published by Downloaded from Copyright © 2014 American Society of Plant Biologists. All rights reserved.

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Intergenic Sequence between Arabidopsis ClpB-C/Hsp100 and Choline Kinase Genes 1

Functions as a Heat Inducible Bidirectional Promoter 2

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Ratnesh Chandra Mishra, Anil Grover* 4

Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi-110021, 5

India 6

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Summary of most important finding: 10

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Two Arabidopsis genes, ClpB-C/Hsp100 and choline kinase, with role in thermotolerance are 12

oriented in head to head manner in the genome and the intergenic region between these genes 13

functions as heat inducible bidirectional promoter. 14

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Financial source: The work was supported by financial grant from Center for Advanced 1

Research and Innovation on Plant Stress and Developmental Biology, Department of 2

Biotechnology (DBT), Government of India and J.C. Bose Fellowship award grant from 3

Department of Science and Technology (DST), Government of India to AG. 4

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Corresponding author: 9

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Prof. Anil Grover 11

Phone: 91-11-24115097 12

FAX: 91-11-24115270 13

Email: [email protected] 14

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ABSTRACT 1

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In Arabidopsis thaliana, At1g74310 locus encodes for ClpB-cytoplasmic/Hsp100 protein 3

(AtClpB-C) which is critical for the acquisition of thermotolerance and At1g74320 encodes for 4

choline kinase (AtCK2) that catalyzes the first reaction in the Kennedy pathway for 5

phophatidylcholine biosynthesis. Previous work has established that the knockout mutants of 6

these genes display heat sensitive phenotypes. While analyzing AtClpB-C promoter and 7

upstream genomic regions in this study, we noted that AtClpB-C and AtCK2 genes are head-to-8

head oriented on chromosome 1 of Arabidopsis genome. Expression analysis showed that 9

transcripts of these genes are rapidly induced in response to heat stress treatment. In stably 10

transformed Arabidopsis plants harboring this intergenic sequence between head-to-head 11

oriented GFP and Gus reporter genes, both transcript and protein of the two reporters were up-12

regulated upon heat stress. Four HSE-like elements were noted in the intergenic region by in 13

silico analysis. In homozygous, T-DNA insertion mutant Salk_014505, 4393 bp T-DNA is 14

inserted at position -517 upstream to ATG of AtClpB-C gene. As a result, AtCK2 loses 15

proximity to 3 of the 4 HSEs in the mutant line. Heat inducible expression of AtCK2 transcript 16

was completely lost whereas the expression of AtClpB-C was not affected in the mutant plants. 17

Our results suggest that the 1329 bp intergenic fragment functions as a heat inducible 18

bidirectional promoter and the region governing the heat inducibility is possibly shared between 19

the two genes. We propose a model that AtClpB-C shares its regulatory region with heat-induced 20

choline kinase which has possible role in heat signaling. 21

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INTRODUCTION 1

High temperature stress constitutes a major environmental stress particularly for the 2

sessile plant systems. On experiencing heat stress (HS), plants respond by vigorously 3

transcribing Hsp genes which function largely as molecular chaperones in protecting cells from 4

damage caused by misfolding of proteins (Kotak et al., 2007; Richter et al., 2010; Sarkar et al., 5

2014). Plants harbor several classes of Hsps i.e., Hsp100, Hsp90, Hsp70, Hsp40 and sHsps 6

(Rajan and D'Silva, 2009; Sarkar et al., 2009; Singh et al., 2012; Sarkar et al., 2013; Sarkar et al., 7

2013). Hsp100 class proteins, referred to as Clp (Caseinolytic Protease) family proteins, are 8

exclusively involved in dissolution of toxic protein aggregates (Agarwal et al., 2001; Katiyar-9

Agarwal et al., 2001; Singh and Grover, 2010). This family comprises ClpA, ClpB, ClpC, ClpD, 10

ClpX, ClpM, ClpN and ClpY/HslU subfamilies (Singh et al., 2010). Unlike others, ClpB/Hsp100 11

proteins which are primarily heat inducible, function solely in renaturation of aggregated 12

proteins. Plants comprise three ClpB homologs which are localized to cytoplasm/nucleus (ClpB-13

C), mitochondria (ClpB-M) and chloroplast (ClpB-P). ClpB-C protein is considered critical for 14

survival under conditions of high temperature stress. Maize mutant null for ClpB-C/Hsp100 15

protein are highly sensitive to extreme temperatures as against wild type (WT) plants (Nieto-16

Sotelo et al., 2002). Rice Hsa32 (HS associated 32 kDa protein) knockout mutant in which decay 17

of heat-induced ClpB-C is faster has defective long-term acquired thermotolerance (Lin et al., 18

2014). In addition to heat-induced regulation, ClpB/Hsp100 proteins are developmentally-19

regulated. Uninduced levels of rice ClpB-C/Hsp100 are particularly high in mature grains and 20

decline during the seed germination phase (Singla et al., 1998). Seeds/grains of several other 21

plant genera (i.e. wheat, maize, and mustard) also contain high uninduced levels of ClpB-22

C/Hsp100 proteins. In maize, ClpB-C is expressed at a high level in the pre-meiosis stage of 23

tassel development, the ears, silks, endosperm, and embryo but is present at only a low level in 24

foliar leaves and roots (Young et al., 2001). 25

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Past studies show that at the transcriptional level, the ability of heat shock (HS) 27

promoters to sense heat is largely attributed to heat shock elements (HSE) located in the 28

promoter region (Sugio et al., 2009; Akerfelt et al., 2010). The consensus sequence that 29

demarcates HSE in eukaryotic heat-inducible promoters is defined by altering units of 5’-30

nGAAn-3’, upstream of the TATA box (Mittal et al., 2011; Scharf et al., 2012). The organization 31



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of this consensus sequence separates HSEs into three groups: perfect, gapped and stepped 1

(Yamamoto et al., 2005). HSEs provide the binding targets for the trans-active heat shock factors 2

(Hsfs), which mediate the expression of Hsp genes (Zhu et al., 2006; Mittal et al., 2009). Maize 3

ClpB-C promoter contains five HSEs present within the proximal 289 bp of promoter region 4

(Nieto-Sotelo et al., 1999). Heat inducible expression of rice ClpB-C is mediated specifically by 5

OsHsfA2C, which binds to the only HSE (with configuration of nnnnnnGAAnnTTC) present in the 6

ClpB-C promoter (Singh et al., 2012). Yeast ClpB/Hsp100 (Hsp104) is induced after exposure to 7

heavy metal like arsenite (Sanchez et al., 1992). Metal-inducibility of Hsp genes has been 8

attributed to stress responsive elements (STREs) and AP-1 cis-elements noted in the HS 9

promoters (Ruis and Schuller, 1995; Grably et al., 2002). At the post-transcriptional level, Hsp 10

mRNAs are preferentially translated while normal cellular mRNAs are translationally suppressed 11

during HS conditions. The 5’UTRs of Hsp mRNAs play a key regulatory role in this process by 12

allowing sustained translation of Hsp transcripts during HS by virtue of the internal ribosome 13

entry site (IRES) element located in this region (Rubtsova et al., 2003; Hernandez et al., 2004; 14

Spriggs et al., 2008). It is indicated that Hsps mRNAs are less dependent on cap-dependent and 15

have alternative, cap-independent mechanism for translation process which involve IRES in their 16

5’UTRs. Drosophila Hsp70, human Hsp70 and Hsp90 (Vivinus et al., 2001; Rubtsova et al., 17

2003) and maize Adh (Mardanova et al., 2008) genes are shown to contain IRES sequences in 18

their 5’UTRs. Further, studies show that IRES is preferably located towards the 3’ end of 5’ 19

UTR, just upstream to AUG codon (Pelletier et al., 1988; Komar and Hatzoglou, 2005). 20

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Plant ClpB-C transcripts are strongly induced by HS in vegetative tissues in Arabidopsis, 22

maize, tobacco, wheat, rice and soybean (Lee et al., 1994; Schirmer et al., 1994; Wells et al., 23

1998; Campbell et al., 2001; Young et al., 2001; Agarwal et al., 2002; Nieto-Sotelo et al., 2002; 24

Singh et al., 2010). By expressing Gus reporter downstream to rice ClpB-C promoter, Singh et 25

al. (2012) showed heat and metal stress-induced expression in vegetative tissues and constitutive 26

expression in anther, style, ovary and embryonal half of the seeds. Presence of IRES in 5’UTR of 27

ClpB-C mRNAs has been shown in maize Hsp100 gene (Dinkova et al., 2005). Most genetic 28

work on plant ClpB-C protein has been carried out on model plant Arabidopsis. Mutant 29

Arabidopsis having non-functional ClpB-C (hot1-1) or null for ClpB-C (hot1-3) is defective in 30

acquisition to high temperature stress (Hong and Vierling, 2000, 2001). Young et al. (2005) 31



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analyzed a 390 bp AtClpB-C promoter fragment, incorporated in a mutator-like element (mule), 1

for its expression pattern. Barring the latter study, AtClpB-C promoter has not been deeply 2

analyzed. In this work, finer details of the genetic regulation of 1kb region upstream to ATG of 3

AtClpB-C gene were aimed at. We show that 1kbAtClpB-C promoter harbors elements for HS 4

and metal stress inducible transcript expression and for constitutive expression of the transcripts 5

in young developing pollens and mature seeds. 1kb AtClpB-C promoter sequence contains 222 6

bp 5’UTR with an intervening intron of 106bp. There was no adverse effect noted on expression 7

of the downstream reporter gene with respect to both HS inducibility and developmental cues 8

when 77 bp (including 22 bp from UTR and 55 bp from intron) were deleted from the 3’ end of 9

the 1kb promoter sequence. 10

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In some studies, the expression of adjacent genes is suggested to be highly correlated. In 12

prokaryotes, it is invariably a case that a single regulatory unit controls two or more functionally-13

related genes (Kruglyak and Tang, 2000). Cho et al. (1998) showed that genes expressed in the 14

same phase of the cell cycle are adjacently-located. Several functionally-related genes are 15

adjacently located and co-expressed in yeast (Zhang and Smith, 1998; Kruglyak and Tang, 16

2000). Expression similarity in clustered genes has also been documented in Caenorhabditis 17

elegans (Lercher et al., 2002), Drosophila melanogaster (Herr and Harris, 2004) and humans 18

(Lercher et al., 2003). William and Bowles (2004), while analyzing the effect of genome 19

organization on expression of genes in Arabidopsis, reported that neighboring genes are co-20

expressed. Bidirectional gene organization is a common architectural feature in human genome 21

where around 10% genes are head-to-head organized (Trinklein et al., 2004). In Arabidopsis, 22

calmodulin methyl transferase (At4g35987) and senescence associated gene (At4g35985) genes 23

are located in a head-to-head orientation with their 1258 bp intergenic sequence serving as a 24

tissue-specific and stress-inducible bidirectional promoter (Banerjee et al., 2013). 25

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While analyzing AtClpB-C promoter in this study, we noted that a choline kinase (CK) 27

gene (At1g74320) is present upstream to AtClpB-C gene in head-to-head orientation on 28

chromosome 1 of Arabidopsis genome. CK enzyme catalyzes the first reaction in the Kennedy 29

pathway for phosphatidyl choline (PC) biosynthesis (Gibellini and Smith, 2010). PC is the major 30

phospholipid in the eukaryotic cell membranes and has a considerable structural and functional 31



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role (Gibellini and Smith, 2010). It is a precursor for the synthesis of glycerolipids like 1

monogalactosyldiacylglycerol, digalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol 2

which constitute the plastid membranes of plants (Ohlrogge and Browse, 1995). Besides, PC 3

serves as a pool for lipid second messengers, e.g. lyso-PC, phosphatidic acid (PA), 4

diacylglycerol and lyso-phosphatidic acid (Mishkind et al., 2009). In higher plants, PC 5

participates in abiotic stress adaptive response (Zheng et al., 2011). As a response to HS, a 6

gradual but significant increase in CK activity was noted in barley aleurone layers (Johnston et 7

al., 2007). The first step of choline phosphorylation in the PC biosynthesis pathway which is 8

catalyzed by CK is considered as a rate-controlling step (Tasseva et al., 2004). Arabidopsis 9

genome contains five CK genes (At1g34100, At1g71697, At1g74320, At2g26830 and 10

At4g09760). At1g71697 (AtCK1) is induced in response to wounding while At1g74320 and 11

At4g09760 are induced by high salt and mannitol (Tasseva et al., 2004). We report that AtClpB-12

C and At1g74320 (hereafter referred to as AtCK2) genes, both of which are critical in governing 13

the phenotype of heat tolerance (Larkindale and Vierling, 2008), possibly share the requisite 14

genetic regulation machinery under cellular conditions. 15

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RESULTS 17

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Expression Characteristics of 1kb AtClpB-C Promoter Region 19

Nucleotide sequence of 1kbAtClpB-Cpro region was obtained from TAIR database 20

(www.arabidopsis.org). Search using in silico tools indicated presence of several stress- and 21

development-related elements in this sequence. Four HSE-like elements were noted with 22

following configurations: TTCCAGATGAATCTCCTTC (-330 to -348 position; appears 23

stepped type with TTCnnnnnGAAnnnnnTTC configuration, GAAAATTC (position -471 to -478; 24

variant of perfect type with configuration of nnnnnnGAAnnTTC), GAAATTC (-500 to -506; 25

GAAnTTC configuration) and GAAATATTTC (-777 to -787 positions; GAAnnnnTTC 26

configuration) (Fig. 1). A single stress-related element (STRE, -942 to -946 position) and one 27

POLLEN1LELAT52 (-555 to -560 position) sequences were noted. Besides, 2 GTGA motifs (-28

464 to -467 and -848 to -851 positions), an E-BOX element (-424 to -429 position) and an AP-1 29

binding element (-164 to -169 position) were marked. A canonical TATA-box was found at -235 30

to -240 position which is 3’ to the predicted transcription start site (TSS), indicating that 31



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transcription initiates without TATA mediation and promoter is TATA-less. All the above 1

elements are marked taking ‘A’ of ATG translation initiation codon as +1 (Fig. 1). 2

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PCR-amplified 1kb AtClpB-Cpro was cloned upstream to Gus gene to make 1kbAtClpB-4

Cpro::Gus construct (Fig. S2A). Arabidopsis (Col-0) plants stably transformed with this binary 5

construct yielded 1kbpro plants. T0 plants were analyzed for integration of T-DNA by PCR using 6

primers specific for Gus and nptII genes. Three PCR-positive, T2 homozygous transgenic lines 7

(1kbpro-1, 1kbpro-2 and 1kbpro-3) were obtained. Gus expression analysis was carried out on 8

homozygous T3 generation plants. 10-d-old seedlings of the 1kbpro lines did not show Gus 9

staining under unstressed control conditions whereas seedlings exposed to 38°C for 2 h showed 10

heat inducible Gus staining in histochemical assays (Fig. S2B). Similar observations were made 11

for the leaves of 1-month-old plants. In quantitative estimation of Gus protein, heat stressed 12

1kbpro-1, 1kbpro-2 and 1kbpro-3 seedlings showed a remarkable increase in Gus activity in 13

contrast to control seedling (Fig. S2C). Gus expression was induced under arsenic and cadmium 14

stress in 1kbpro plants (Fig. S2D). In unstressed (control) 1kbpro plants, Gus expression was 15

prominent in microspores (pollen grains) within the undehisced anthers (Fig. 2A.e). Young 16

developing siliques did not show Gus staining while imbibed mature seeds showed intense Gus 17

staining (Fig. 2A.h). Western blotting using anti-AtClpB-C antibodies showed remarkable level 18

of AtClpB-C protein in young pollens of unstressed WT Arabidopsis plants (Fig. 2B). Mature 19

seeds of 1kbpro plants showed high levels of Gus protein (Fig. 2C). MUG assays showed a 20

remarkable hike in the Gus activity during the first 24 h of seed germination. In subsequent 21

period, Gus activity showed gradual decline with negligible Gus activity post 5 days after 22

germination (DAG; 120 h) stage (Fig. 2C). In western analysis, the level of AtClpB-C protein in 23

24 h germination sample was higher as compared to the imbibed seeds (Fig. 2D). With 24

progressing DAG stages, the levels of AtClpB-C protein declined. 25

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Expression Characteristics of ∆AtClpB-C Promoter 27

1kb AtClpB-C promoter sequence contains 222 bp 5’UTR with an intervening intron of 28

106 bp (Fig. S4A). Promoter fragment with deletion of 77 bp from the 3’ end of the 1kb AtClpB-29

C promoter was PCR amplified. The deleted 77 bp region included 55 bp of the intron and 22 bp 30

of the 3’ end of the 5’UTR sequences (Fig. S4A). Construct ∆AtClpB-Cpro:Gus contained 923 31



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bp AtClpB-C promoter fragment (672 bp sequence of promoter, 200 bp of 5’UTR with 51 bp of 1

intron sequence) upstream of Gus reporter gene. Stable homozygous transgenic Arabidopsis lines 2

(∆pro-1, ∆pro-2 and ∆pro-3 lines) were confirmed for T-DNA insertion by PCR. Gus expression 3

in vegetative tissues (10-d-old seedlings and leaf from 1-month-old plants) of T3 ∆pro plants was 4

strictly heat inducible (Fig. S3A, B). The levels of Gus activity were comparable in ∆pro and 5

1kbpro seedlings. Gus transcript analysis by sqRT-PCR showed similar pattern of induction in 6

∆pro and 1kbpro seedlings (Fig. S4C). Arsenic and cadmium induced Gus expression also 7

appeared comparable in ∆pro and 1kbpro seedlings (Fig. S3C). Unstressed reproductive tissues 8

of ∆pro plants showed constitutive expression of Gus in pollens and mature seeds like in 1kbpro 9

plants (Fig. S3D). From the above work, it is accrued that deletion of 77 bp from 3’ end of this 10

UTR (that includes 22 bp of 5’UTR and 55 bp of intervening intron) has no effect in terms of 11

qualitative and quantitative Gus expression profilings. 12

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Genomic Sequence Further 5’ Upstream of AtClpB-C Gene 14

Analysis of genomic organization of AtClpB-C (At1g74310) gene in Arabidopsis 15

genome database (TAIR; www.arabidopsis.org) showed that a putative choline kinase gene (CK; 16

At1g74320) lies immediately upstream to it. Both the genes are head-to-head oriented on 17

chromosome 1 of Arabidopsis genome (Fig. 3). By in silico analysis, it accrued that the 18

translation initiation sites of the two genes are distanced 1329 bp apart. While AtClpB-C has a 19

222 bp 5’UTR with a 106 bp intervening intron sequence, AtCK2 gene contains 5’UTR of 231 20

bp with 228 bp intervening intron. The core promoter region between the two genes leaving the 21

5’UTRs is 482 bp. All the four HSEs located in 1kb promoter of AtClpB-C gene as mentioned 22

earlier, lie in this core 482 bp fragment (Fig. 3). Transcripts of AtClpB-C and AtCK2 genes were 23

highly HS inducible in semi-quantitative and quantitative PCR (Fig. 4). Induction was relatively 24

rapid and predominant for AtClpB-C as against AtCK2 gene. 25

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To find out whether 1329 bp sequence can drive expression from both the sides, this 27

sequence was cloned upstream to Gus reporter in either orientation and the resulting plasmids 28

were bombarded in onion epidermal peel. Several cells expressing Gus were seen in peels 29

bombarded with the two clones (Fig. 5). Next, the construct containing 1329 bp intergenic 30

sequence between head-to-head oriented GFP and Gus reporter genes (Fig. 6A) was stably 31



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transformed in Arabidopsis (Col-0) plants. 10-d-old Bid-1, Bid-2 and Bid-3 seedlings stressed at 1

42°C for 15 min showed HS inducible transcript expression of both Gus and GFP reporters (Fig. 2

6B). The levels of transcript in unstressed control seedlings were negligible. Bid seedlings 3

showed heat induced expression of Gus protein (Fig. 6C, D). Total soluble proteins were isolated 4

from control and stressed Bid seedlings (38°C for 2 h) and analyzed for GFP protein using anti-5

GFP antibodies. Heat inducible increase in the GFP levels were clearly noted in the three Bid 6

lines (Fig. 6E). 7

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Analysis of the Effect of T-DNA Insertion in the Intergenic Region 9

Arabidopsis resource center (ABRC) was searched for polymorphism in the 1329 bp 10

intergenic region. In single T-DNA insertion mutant Salk_014505, a 4393 bp T-DNA (from 11

pROK2 vector) was present in the core promoter region at position -517 upstream to ATG of 12

AtClpB-C gene (Fig. 7A). Insertion of T-DNA divided the core promoter sequences of 482 bp 13

into two regions, one of 239 bp towards the AtCK2 side (containing one of the 4 putative HSEs) 14

and other of 243 bp towards the AtClpB-C side(containing 3 of the 4 putative HSEs). Zygosity 15

of the mutant T3 plants was checked and three homozygous and one heterozygous mutant plant 16

were analyzed. Heat inducible expression of AtCK2 transcript was completely lost whereas the 17

expression of AtClpB-C was not affected in the homozygous mutant lines. In the heterozygous 18

and WT plants, expression of both AtCK2 and AtClpB-C gene was distinctly heat inducible (Fig. 19

7B). 20

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DISCUSSION 22

Previous studies have shown that the transcript expression of Hsp genes by heat and 23

heavy metal stress in vegetative tissues as well as their constitutive expression in pollens and 24

seeds is governed by HS promoters (Haralampidis et al., 2002; Singh et al., 2010; Singh et al., 25

2012). 1kb rice ClpB-C promoter contains a single functional HSE whereas in maize, five HSEs 26

are noted within the proximal 289 bp of the ClpB-C promoter region (Nieto-Sotelo et al., 1999). 27

Through in silico search, we noted four putative HSEs of variable types in the 1kbAtClpB-C 28

promoter. Gus transcript and protein levels were rapidly induced in 1kbPro plants in response to 29

HS conditions. The cooperative binding of the Hsfs to four HSEs may be a reason for the rapid 30

transcript induction noted in this gene. Congruently, Young et al. (2005) noted that the mule 31



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transposon fragment with 390 bp of the AtClpB-C promoter containing only one of the four 1

HSEs retained a relatively lower HS induction. Heat and metal stresses are shown to have an 2

overlap in their signal reception by involving STRE and AP-1 binding elements besides HSEs 3

(Haralampidis et al., 2002). In line with this, we noted that AtClpB-C promoter contains a STRE 4

and an AP-1 binding element and showed As and Cd inducible Gus expression. In the case of 5

ClpB-C promoter driven Gus gene in rice plants, Gus expression was noted under unstressed 6

control conditions in reproductive tissues with a significantly higher expression in embryonal 7

half of the seeds (Singh et al., 2012). Likewise, Gus expression was noted in seeds of 1kbPro 8

plants. In addition to this, constitutive expression of Gus in young developing microspores 9

within the undehisced anthers was also noted in this study. In parallel, detectable levels of 10

AtClpB-C protein were noted in young Arabidopsis pollens. Developmental expression of 11

AtClpB-C so far has been extensively discussed with reference to its accumulation during seed 12

maturation whereas no report specifically comment on expression of this protein in the pollen 13

grains (Boston et al., 1996; Hong and Vierling, 2000; Queitsch et al., 2000; Hong and Vierling, 14

2001; Hong et al., 2003; Kotak et al., 2007). In this study, we report that Arabidopsis pollens 15

contain AtClpB-C protein from the early stages. Flowering and microsporogenesis are the most 16

susceptible stages to high temperatures (Satake and Yoshida, 1978; Farrell et al., 2006; Jagadish 17

et al., 2007; Jagadish et al., 2008; Jagadish et al., 2010). Presence of ClpB-C protein in pollens 18

may have a role in their HS response biology. Pollen related expression of AtClpB-C can 19

possibly be attributed to the presence of GTGANTG10 (GTGA) and POLLEN1LELAT52 20

elements in 1kbAtClpB-C promoter. These elements have been implicated in pollen related gene 21

expression in earlier studies (Rogers et al., 2001; Khurana et al., 2013). In an earlier study, 22

AtClpB-C was detected specifically 4 days after pollination in developing siliques after which it 23

disappeared and then began to re-accumulate during latter stages of seed maturation (Hong and 24

Vierling, 2001). In addition to it, a gradual decline in the levels of this protein was reported 25

during germination. In contrast to this, a hike in the Gus activity in 1kbpro plants during the first 26

24 h of seed germination was noted in this study. There was a parallel increase in AtClpB-C 27

protein levels in WT plants during the first 24 h of seed germination. We thus report that the 28

expression of ClpB-C/Hsp100 increases during the early stages of seed germination, indicating a 29

possible role of ClpB-C in regulation of initial seed germination process in Arabidopsis. The 30



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detailed relationship of various cis-acting sequences in governing heat, metal and developmental 1

stage dependent-inducibility of HS promoters needs to be fully unveiled. 2

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Using a genome-wide approach for understanding the components of heat response in 4

Arabidopsis, Larkindale and Vierling (2008) reported that ClpB-C (Hsp101, At1g74310) and 5

choline kinase (At1g74320) genes are critical for governing high temperature tolerance in 6

Arabidopsis. The latter study showed that AtClpB-C mutant plant is extremely heat sensitive and 7

AtCK2 mutant plant is heat sensitive to moderate levels. While analyzing AtClpB-C genomic 8

region for its promoter analysis, we strikingly noted that AtClpB-C and AtCK2 genes are head-9

to-head oriented on chromosome 1 of Arabidopsis genome. The translation start sites of these 10

genes are 1329 bp apart. We noted that the transcripts of AtClpB-C and AtCK2 genes are 11

coordinately-regulated during high temperature stress (Fig. 4). The 1329 bp intergenic region of 12

the two genes could drive Gus expression in either orientation in transient assays with onion peel 13

epidermal cells (Fig. 5). Further, this region cloned between the Gus and GFP reporters showed 14

heat-inducible expression of both the reporter genes in stably transformed Arabidopsis plants 15

(Fig. 6), indicating that this region acts as a bidirectional promoter. Genome-wide analysis in 16

human indicates that the distance between the two TSSs in a bidirectional promoter is largely 17

within 1000 bp whereas in plants, this distance is generally longer than 1000 bp (Trinklein et al., 18

2004; Mitra et al., 2009). The distance between the TSS of OCPI1 and OCPI2 sharing a 19

bidirectional promoter, is 1126 bp (Singh et al., 2009). The distance between the TSS of AtClpB-20

C and AtCK2 is only 482bp. Interestingly, the four putative HSEs present in the intergenic 21

region between these two genes are located within this 482 bp fragment. In Salk_014505 mutant, 22

T-DNA insertion is located at position -517 upstream to ATG of AtClpB-C gene, in almost at the 23

center of the 482 bp core promoter region. The heat inducible expression of AtCK2 gene was 24

lost whereas AtClpB-C gene retained its usual level of expression in this mutant line (Fig. 7). As 25

a result of insertion, AtCK2 loses proximity to 3 of the 4 putative HSEs. This may be the reason 26

for the loss of heat-inducibility of the AtCK2 promoter in the mutant plants. This study suggests 27

that the regulatory elements that govern the HS inducibilty of the 1329 bp bidirectional promoter 28

may lie within the region upstream to ATG of AtClpB-C and -517bp. Combining this finding 29

with the work of Young et al (2005), it can be concluded that the 3 HSEs are crucial and 30

sufficient for sensing the heat stimuli for induction of AtClpB-C and AtCK2 genes. It was 31



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interesting to note that the divergent organization of AtClpB-C and AtCK2 genes is conserved 1

among the homologs of these genes in brassicaceae family of dicots (see Fig. S7; Table S1). 2

Further, we noted that among the 5 AtCK genes, only AtCK2 is HS-induced [GEO microarray 3

data accession no. GSE51879 (Merret et al., 2013)]. In the light of this context, the physical 4

linking of only the AtCK2 gene with AtClpB-C may bear significance. 5

6

Many functionally-related genes are adjacently located and co-expressed in yeast (Zhang 7

and Smith, 1998; Kruglyak and Tang, 2000). Singh et al (2009) showed that the rice protease 8

inhibitor genes OCPI1 and OCPI2 are coordinately regulated and share a bidirectional promoter 9

for their regulation. In rice, two small heat shock proteins, OsHsp17.3 and OsHsp18.0, share a 10

356 bp putative bidirectional promoter (Guan et al., 2004). Xu et al. (2012) performed a 11

comparative study on gene expression and biological functions between mouse and human 12

bidirectional genes. Their analysis indicated that the potential selective constraints for 13

conservation of bidirectional architecture are gene function, not the co-regulation of paired gene 14

expression. We note that a bidirectional promoter is shared between two functionally-dissimilar 15

proteins which are involved in a common phenotype of heat tolerance. AtClpB-C is a chaperone 16

protein whereas AtCK2 encodes for choline kinase enzyme that has a role in plasma membrane 17

synthesis. 18

19

Overall, there are 5763 divergent gene pairs in Arabidopsis (Krom and Ramakrishna, 20

2008). Merret et al. (2013) recently noted that 680 Arabidopsis genes are up-regulated by more 21

than 2 folds under HS. Analyzing these two data sets, we identified that there are 16 divergent 22

gene pairs (with distance between TSSs <1.5 kb) in which both genes show heat up-regulation 23

(see Fig. S8; Table S2). This study has focused on one such case of AtClpB-C and AtCK2 24

among the 16 gene pairs. The above account further reveals that the heat induced fold up-25

regulation for the paired genes differs significantly for members of the divergent gene pairs, 26

suggesting that the bidirectional promoter may generate transcripts in differential amounts. The 27

difference in the heat induced fold up-regulation of AtClpB-C and AtCK2 transcripts as noted in 28

this study is corroborated by the above observation. 29

30



15

Recent studies show the involvement of membrane as the sensor of HS. It is suggested 1

that after the membrane senses the HS, a series of changes are elicited resulting in induction of 2

Hsps (Hofmann, 2009; Horvath et al., 2012). Saidi et al. (2009) showed that HS induced 3

elevation of membrane fluidity activates the expression of Hsp genes in a Ca2+ dependent 4

manner. It has also been noted that HS activates the phopho-lipid based signaling pathways 5

(Mishkind et al., 2009). High temperature triggered phospholipase D (PLD) activity results into 6

the generation of phosphatidic acid (PA) that acts as a second messenger increasing the H2O2 7

levels, inducing Ca2+ influx. The Ca2+ influx in turn regulates the activation of calmodulin 8

binding kinase (CBK), in a calmodulin (CaM)-dependent fashion. Active CBK aids the 9

phosphorylation of Hsfs, which successively modulate the expression of Hsps (Horvath et al., 10

2012). PA is majorly formed by the breakdown of PC and accumulated in plant cell membranes 11

after the onset of HS. However, the increase in PA is not associated with a corresponding 12

decrease in PC levels (Mishkind et al., 2009). The heat inducible CK appears to be involved in 13

generation of PC for maintenance of the membrane integrity during HS and generation of PA 14

molecules (see model in Fig. 8). Clearly, there is a need to understand the relevance of CK in HS 15

biology in greater depths. Our results suggest that a common regulatory module may be involved 16

in AtClpB-C and AtCK2 heat induction. In rice, ClpB-C promoter is specifically driven by 17

OsHsfA2C which is shown to interact with its only HSE (Singh et al., 2012). Arabidopsis 18

genome has 21 Hsfs genes (Fragkostefanakis et al., 2014); however, which Hsf interacts with 19

AtClpB-C promoter is not yet identified. 20

21

The interesting fact emerging from this study is that nature has packed together two genes 22

involved in heat shock response. While AtCK2 is placed at the top of HS signaling, AtClpB-C is 23

placed towards the downstream of the cascade. The possible relevance of this compactness might 24

be the fine tuning of these two genes. We propose that a common regulatory module in heat 25

inducibility of AtCK2 and AtClpB-C genes may provide basis for interdependence of these two 26

processes and prove to be a novel gene regulatory mechanism employed by plants under 27

physiological stress. Finally, the heat inducible bidirectional promoter reported in this study can 28

be used to construct a binary vector for gene stacking where two genes can be expressed in a heat 29

inducible manner in a transgenic system. 30

31



16

MATERIALS AND METHODS 1

2

Plant Material and Growth Conditions 3

Transgenic Arabidopsis lines were raised in Col-0 ecotype background. Seeds were 4

placed in pots containing soil-rite. The pots were kept in cold room (4°C) for 48 h for 5

stratification and subsequently placed in culture room maintained at 22±1°C with 16:8 h light 6

and dark regimes with light intensity of 100-125 µmol m-2s-1 for raising the full-grown plants. 7

For raising seedlings through tissue culture, Arabidopsis seeds were first surface sterilized with 8

70% ethanol for 30 sec followed by a treatment of 2% (v/v) sodium hypochlorite with 1 drop of 9

Tween-20 for 5 min and then washed with sterile water for 3-4 times and finally suspended in 10

0.1% (w/v) agar. The seeds were plated onto Murashige and Skoog (MS) medium containing 1% 11

sucrose and 0.8% agar (pH 5.8). The plates were kept for stratification (48-72 h) and finally 12

shifted to culture room. For the selection of transformants, surface-sterilized seeds were plated 13

on ½ MS medium plates supplemented with the appropriate antibiotic (kanamycin 50 mgL-1; 14

hygromycin 15 mgL-1) and augmentin (150 mgL-1). Positive seedlings were transferred to pots in 15

culture room for further growth. 16

17

Mutant Analysis 18

T-DNA insertion mutant (Salk_014505; Alonso et al., 2003) seeds were ordered from 19

ABRC seed stock center (www.arabidopsis.org). Individual seeds were grown in pots and plants 20

were analyzed for their zygosity status by PCR (Fig. S6). Information of the primers required for 21

the PCR (Table S3) was obtained from T-DNA express site. Three homozygous and one 22

heterozygous mutant line were selected for analysis. 23

24

Construct Designing 25

For genetic regulation study, two different promoter fragments, 1012 bp (1kbAtClpB-26

Cpro) upstream of ATG codon and 935 bp (∆AtClpB-Cpro; excluding 77 bp region from the 3’ 27

end of the 1012 bp fragment) were PCR amplified from Arabidopsis genomic DNA, sequenced 28

(Fig. S1) and cloned upstream to Gus in pBT2Gus vector. The promoter-Gus fragments were 29

cleaved from the resulting plasmids and introduced in pCAMBIA2300 binary vector. The 30

resulting constructs (1kbAtClpB-Cpro::Gus and ∆AtClpB-Cpro::Gus respectively) were 31



17

transformed in Arabidopsis to raise stable homozygous transgenic plants (referred as 1kbpro 1

plants and ∆pro plants respectively). To analyze the bidirectionality, 1329 bp region upstream to 2

ATG (AtCK2/1329/AtClpB-C) of AtClpB-C was cloned in a bidirectional vector (Singh et al., 3

2009) in between head-to-head oriented Gus and GFP in the background of pCAMBIA1381Z 4

binary vector. The resulting binary plasmid (GFP::AtCK2/1329/AtClpB-C::Gus construct; Fig. 5

6A) in each case was introduced in AGL1 strain of Agrobacterium tumefaciens and stably 6

transformed in Arabidopsis (GFP::AtCK2/1329/AtClpB-C::Gus plants; referred to as Bid plants). 7

Plant transformation was carried out employing floral dip method (Clough and Bent, 1998). 8

Genomic DNA was isolated following protocol described by Kobayashi et al. (Kobayashi et al., 9

1998). For confirmation of transfer DNA (T-DNA) integration, PCR for Gus gene was 10

performed using specific primers. Primers utilized for PCR amplifications are listed in Table S3. 11

12

Protein Isolation from Seed, Seedling and Pollen Tissues 13

Soluble proteins from Arabidopsis seedlings were isolated in HEPES buffer solution [25 14

mM HEPES, pH 7.5, 0.5% Triton X-100, 200 mM NaCl, 0.5 mM EDTA, 10 mM MgCl2 and 1X 15

protease inhibitor cocktail (G-biosciences)]. Seedlings were ground in liquid N2 with a pinch of 16

PVP. HEPES extraction buffer was added to the powdered tissue. The homogenate was 17

centrifuged and supernatant was used as protein extract. Total seed proteins were isolated in Tris 18

buffer (30 mM Tris-Cl pH 8.5, 1 mM EDTA, 1% SDS and 1X protease inhibitor cocktail). Dry, 19

imbibed or germinating Arabidopsis seeds were homogenized with glass pestle in MCTs with 20

required volume of buffer. Homogenate were centrifuged and supernatant was used as seed 21

protein extract. Proteins from Arabidopsis pollens were isolated using HEPES extraction buffer. 22

Young flowers from Arabidopsis inflorescence were taken in the MCT containing required 23

quantity of HEPES extraction buffer. MCT was gently tapped allowing the pollen from the 24

anther to mix in the buffer. Pollens were ground with glass pestle and the homogenate was 25

centrifuged. Clear lysate was used as protein sample. 26

27

Gus Analysis 28

Protocol described by Jefferson et al. (1987) was employed for histochemical Gus 29

staining. Qualitative estimation of Gus activity was performed using 5-bromo-4 -chloro-3-30

indolyl-β-D-glucuronic acid (X-gluc) as substrate. Control tissues (10-d-old seedlings, leaves 31



18

from 1-month-old mature plants, inflorescence, siliques and seeds) were directly stained 1

overnight at 37°C. For HS treatment, seedlings were first exposed to 38°C for 2 h and then 2

stained overnight at 37°C. For each construct, assay was performed with three independent 3

replicates. For metal stress, seedlings were floated in metal solutions of required concentration 4

for 6 h and stained as above. After Gus staining chlorophyll was removed from the tissues with 5

ethanol: acetic acid (3:1). For fluorometric analysis, three transgenic lines positive for 6

histochemical Gus staining were analyzed as described by Jefferson et al. (1987). Total protein 7

from seed and seedlings tissues was extracted in Gus extraction buffer. Reaction was set in 8

replicates of three for each biological sample analyzed. Fluorescence was recorded by a DyNA 9

Quant TM 200 fluorimeter (Hoefer Pharmacia Biotech Inc., California, USA). 10

11

Western Analysis 12

Protein samples were separated on 10% SDS-gel. For immunoblots, gels were processed 13

by electroblotting on nitrocellulose membrane (Hybond C-super membrane; Amersham-14

Pharmacia, USA) as described by Katiyar-Agarwal et al. (2003). For analyzing the levels of 15

AtClpB-C, anti-AtClpB-C primary antibodies (raised against N-terminal region; Agrisera, 16

Sweden) and anti-rabbit secondary antibodies (Sigma-Aldrich) with 1:5000 and 1:10000 17

dilutions, respectively, were used. For GFP protein analysis, anti-GFP primary antibodies 18

(Sigma-Aldrich) and anti-mouse secondary antibodies (Sigma-Aldrich) with 1:10000 and 19

1:10000 dilutions, respectively, were used. 20

21

RNA Isolation, cDNA Synthesis and Transcript Analysis 22

Isolation of total RNA from the control and stressed Arabidopsis seedlings was carried 23

out using GITC extraction buffer (Chomczynski and Sacchi, 1987). For the synthesis of first 24

strand cDNA 2 µg of total RNA was taken and M-MLV reverse transcriptase (MBI Fermentas, 25

Lithuania) was used. Actin was amplified as an internal control for the normalization of mRNA 26

levels in different cDNA preparations. For Q-PCR analysis, primers were designed using 27

PRIMER EXPRESS version 2.0 (PE Applied Biosystems, USA) with default parameters (see list 28

of primers used in Table S3). First strand cDNA was synthesized by reverse transcription using 2 29

µg of total RNA of 10-d-old Arabidopsis seedlings in 50 µl of reaction volume using high-30

capacity cDNA archive kit (Applied Biosystems, USA). The reaction was put in 96-well optical 31



19

reaction plates (Applied Biosystems, USA), using ABI Prism 7000 sequence detection system 1

and software (PE Applied Biosystems, USA). For normalization, actin was used as an internal 2

control. Relative expression values were calculated after normalizing against the maximum 3

expression value. For final analysis, two biological replicates and three technical replicates were 4

used. 5

6

ACKNOWLEDGEMENT 7

RCM acknowledges Council of Scientific and Industrial Research (CSIR), Government of India, 8

New Delhi for the Fellowship award. We are grateful to Dr. Ramakrishna Wusirika, Michigan 9

Technological University, USA, for providing us the list of divergent gene pairs in Arabidopsis. 10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31



20

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Zhu B, Ye C, Lü H, Chen X, Chai G, Chen J, Wang C (2006) Identification and 4

characterization of a novel heat shock transcription factor gene, GmHsfA1, in soybeans 5

(Glycine max). Journal of Plant Research 119: 247-256 6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30



28

FIGURE LEGENDS 1

2

Figure 1. Linear representation of 1kb upstream promoter region of AtClpB-C gene. ATG, HSE 3

(-330 to -348, -471 to -478, -500 to -506, -777 to -787), STRE (−942 to −946), AP-1 element 4

(−164 to −169), E-BOX element (−424 to −429), GTGA element (-464 to -467, -848 to -851) 5

and POLLEN1LELAT52 (-551 to -555) sequences are indicated. All elements are marked taking 6

A of ATG as +1. 7

8

Figure 2. Histochemical staining for Gus expression analysis at different developmental stages 9

of 1kbpro plants under unstressed (control) conditions: (A) 10-d-old seedlings (a), mature leaf 10

from 1-month-old plant (b), inflorescence (c), single flower (d), anther (e), pollen grains (f), 11

young silique (g) and mature imbibed seeds (h). (B) Western showing the levels of AtClpB-C 12

proteins in pollens of Arabidopsis. Control (C) and heat stressed (HS, 38°C/2h) protein samples 13

from seedlings were loaded as controls. 2 µg of total protein from seedling and 10 µg of total 14

protein from pollen were loaded. (C) Gus activity at different stages of seed germination. Graph 15

shows the quantitative estimation through MUG assay while photograph shows the 16

histochemical Gus staining. (D) Western analysis showing the levels of AtClpB-C protein during 17

seed germination. 5 µg of protein was analyzed, lower panel represents the Coomassie stained 18

protein bands showing equal loading. 19

20

Figure 3. Schematic representation showing genomic organization of AtClpB-C (At1g74310) 21

and AtCK2 (At1g74320) genes on chromosome number 1 of Arabidopsis. 1329 bp of intergenic 22

region with the core 482 bp promoter has been marked. 519 bp upstream to AtCK2 gene contains 23

231 bp of 5’UTR with an intervening 228 bp intron whereas the 328 bp upstream of AtClpB-C 24

gene represents 222 bp of 5’UTR with an intervening 106 bp intron. 25

26

Figure 4. (A) sqRT-PCR data showing the transcript levels of AtClpB-C and AtCK2 genes 27

under 0, 5, 10, 15, 20, 25 and 30 min HS treatment (42°C). Lower panel shows the 28

standardization with actin primers. (B) Real time PCR data showing the relative transcript 29

abundance of both the genes under similar stress regimes. 30



29

1

2

Figure 5. Transient expression of Gus reporter in onion epidermal cells for confirmation of 3

promoter activity of 1329 fragment in either directions. (A) Construct where Gus reporter is 4

driven by AtClpB-C gene. (B) Construct where the promoter is oppositely oriented and Gus 5

reporter is driven by AtCK2 gene. Respective onion peels expressing Gus are shown towards the 6

right hand side. 7

8

Figure 6. Analysis of Gus and GFP protein expressions in Bid-1, Bid-2 and Bid-3 lines under 9

control and high temperature stress conditions. (A) Diagrammatic representation of the 10

bidirectional GFP::AtCK2/1329/AtClpB-C::Gus construct. (B) sqRT-PCR picture showing heat 11

inducible expression of GFP and Gus reporter transcripts in three Bid lines. (C) Qualitative 12

analysis of Gus protein. Photograph shows the Gus stained seedlings under both C and HS 13

(38°C/2h) conditions. (D) Graph showing the quantification of Gus expression level through 14

MUG assay. (E) Western showing the levels of GFP protein under C and HS conditions. 5 µg of 15

protein was analyzed. 16

17

Figure 7. (A) Schematic representation of 1329 bp intergenic region between AtClpB-C and 18

AtCK2 genes with point of T-DNA insertion (in SALK_014505 mutant) and HSE distribution. 19

4.393kb T-DNA is inserted within the core promoter region. Out of the four HSEs the distal one 20

(considering upstream of AtClpB-C gene) is separated from the rest three by T-DNA insertion. 21

Configuration of the one HSE present within the 1kb promoter region of AtCK2 gene and three 22

HSEs within the 1kb promoter region of AtClpB-C gene is shown. (B) sqRT-PCR analysis of the 23

4 mutant plants for expression of AtCK2 and AtClpB-C gene under HS of 38°C for 15 and 30 24

min. 1, 2 and 3-homozygous lines, 4-heterozygous line, WT-wild type plants. 25

26

Figure 8. Model highlighting the possible function and position of heat-induced CK among the 27

key players of PA mediated heat-signaling pathway, elicited by the plasma-membrane during 28

HS. It is previously shown that heat induced PA accumulation causes a series of reactions 29

ultimately resulting in induction of Hsps (see text for details). The step-wise components 30



30

involved in this cascade are shown in the orange box. Heat induced CK might compensate for 1

the loss of PC by actively phosphorylating choline generated by PC breakdown thereby pacing 2

up PC biosynthesis. In AtClpB-C:AtCK2 divergent pair, CK induction during HS by Hsf may 3

help in generation of the required PC for uninterrupted signaling. The steps in this model 4

indicated by solid arrows are based on published evidence. The dotted arrows represents the 5

hypothesis forwarded in this study. Cycle shown with blue arrows represents the Kennedy 6

pathway for PC biosynthesis. 7

8

9

10



Fig. 1. Linear representation of 1kb upstream promoter region of AtClpB-C gene. ATG, HSE (-330 to -348, -471 to -478, -

500 to -506, -777 to -787), STRE (−942 to −946), AP-1 element (−164 to −169), E-BOX element (−424 to −429), GTGA

element (-464 to -467, -848 to -851) and POLLEN1LELAT52 (-551 to -555) sequences are indicated. All elements are

marked taking A of ATG as +1.

ATG

-1 -1000

100 bp



Fig. 2. Histochemical staining for Gus expression analysis at different developmental stages of 1kbpro plants under unstressed

(control) conditions: (A) 10-d-old seedlings (a), mature leaf from 1-month-old plant (b), inflorescence (c), single flower (d),

anther (e), pollen grains (f), young silique (g) and mature imbibed seeds (h). (B) Western showing the levels of AtClpB-C

proteins in pollens of Arabidopsis. Control (C) and heat stressed (HS, 38°C/2h) protein samples from seedlings were loaded as

controls. 2 µg of total protein from seedling and 10 µg of total protein from pollen were loaded. (C) Gus activity at different

stages of seed germination. Graph shows the quantitative estimation through MUG assay while photograph shows the

histochemical Gus staining. (D) Western analysis showing the levels of AtClpB-C protein during seed germination. 5 µg of

protein was analyzed, lower panel represents the Coomassie stained protein bands showing equal loading.

a b

c

d e

f g h

A

Seedling Pollen

HS C

B

C

D

0

500

1000

1500

2000

2500

3000

n m

ole

s 4

MU

/mg

pro

tein

/ho

ur

1kbPro-1

1kbPro-2

1kbPro-3



1329bp

482bp 519bp 328bp

AtCK2 5’UTR 5’UTR 5’UTR 5’UTR Promoter element Intron Intron AtClpB-C

Chromosome no. 1

Fig. 3. Schematic representation showing genomic organization of AtClpB-C (At1g74310) and AtCK2 (At1g74320) genes

on chromosome number 1 of Arabidopsis. 1329bp of intergenic region with the core 482bp promoter has been marked.

519bp upstream to AtCK2 gene contains 231bp of 5’UTR with an intervening 228bp intron whereas the 328bp upstream of

AtClpB-C gene represents 222bp of 5’UTR with an intervening 106bp intron.



Fig. 4. (A) sqRT-PCR data showing the transcript levels of AtClpB-C and AtCK2 genes under 0, 5, 10, 15, 20, 25 and 30 min

HS treatment (42°C). Lower panel shows the standardization with actin primers. (B) Real time PCR data showing the relative

transcript abundance of both the genes under similar stress regimes.

A

B

0

2

4

6

8

10

12

14

Re

lati

ve t

ran

scri

pt

exp

ress

ion

min

AtCK2

0

200

400

600

800

1000

1200

Re

lati

ve t

ran

scri

pt

exp

ress

ion

min

AtClpB-C

AtCK2

AtClpB-C

Actin

42°C/HS (min)



Fig. 5. Transient expression of Gus reporter in onion epidermal cells for confirmation of promoter activity of 1329bp

fragment in either directions. (A) Construct where Gus reporter is driven by AtClpB-C gene. (B) Construct where the

promoter is oppositely oriented and Gus reporter is driven by AtCK2 gene. Respective onion peels expressing Gus are shown

towards the right hand side.

GUS LB RB

NosT AtCK2----1329bp---AtClpB-C

GUS LB RB

NosT

A

B

AtClpB-C----1329bp---AtCK2



Fig. 6. Analysis of Gus and GFP protein expressions in Bid-1, Bid-2 and Bid-3 lines under control and high temperature stress

conditions. (A) Diagrammatic representation of the bidirectional GFP::AtCK2/1329/AtClpB-C::Gus construct. (B) sqRT-PCR

picture showing heat inducible expression of GFP and Gus reporter transcripts in three Bid lines. (C) Qualitative analysis of

Gus protein. Photograph shows the Gus stained seedlings under both C and HS (38°C/2h) conditions. (D) Graph showing the

quantification of Gus expression level through MUG assay. (E) Western showing the levels of GFP protein under C and HS

conditions. 5 µg of protein was analyzed.

Bid-1 Bid-2 Bid-3

C HS C HS C HS

C HS C HS C HS

Bid-1 Bid-2 Bid-3

0

100

200

300

400

500

600

700

800

900

1000

C HS C HS C HS

n m

ole

s 4

MU

/mg

pro

tein

/ho

ur

Bid-1 Bid-2 Bid-3

D

1329 bp

GFP NosT

GUS NosT Hygromycin CaMV35S

LB RB

A

C

B

E

C HS C HS C HS Bid-1 Bid-2 Bid-3

Gus

GFP

Actin



Fig. 7. (A) Schematic representation of 1329bp intergenic region between AtClpB-C and AtCK2 genes with point of T-DNA

insertion (in SALK_014505 mutant) and HSE distribution. 4.393kb T-DNA is inserted within the core promoter region. Out of the

four HSEs the distal one (considering upstream of AtClpB-C gene) is separated from the rest three by T-DNA insertion.

Configuration of the one HSE present within the 1kb promoter region of AtCK2 gene and three HSEs within the 1kb promoter

region of AtClpB-C gene is shown. (B) sqRT-PCR analysis of the 4 mutant plants for expression of AtCK2 and AtClpB-C gene

under HS of 38°C for 15 and 30 min. 1, 2 and 3-homozygous lines, 4-heterozygous line, WT-wild type plants.

482bp 519bp 328bp

AtCK2

AtClpB-C

- 787 HSE (GAAATATTTC) - 777 - 506 HSE (GAAATTC) - 500 - 348 HSE (TTCn(5)GAAn(5)TTC) - 330

- 478 HSE (GAAAATTC) - 471

T-DNA 4393bp

- 571

5’UTR Intron Core promoter region

A

B

15 30 15 30 15 30 15 30 15 30

HS HS HS HS HS

AtCK2

AtClpB-C

Actin

WT Heterozygous mutant line; 4

1 2 3

Homozygous mutant lines



Plasma membrane

CK

PC

Choline

PLD

PA

Ca2+

Hsf

ClpB-C (Hsps)

CaM/CBK

Heat tolerance

AtCK2

AtClpB-C

Inte

rgen

ic r

egio

n

HS

H2O2

Rboh

He

at-i

nd

uce

d

Fig. 8. Model highlighting the possible function and position of heat-induced CK among the key players of PA mediated heat-

signaling pathway, elicited by the plasma-membrane during HS. It is previously shown that heat induced PA accumulation causes

a series of reactions ultimately resulting in induction of Hsps (see text for details). The step-wise components involved in this

cascade are shown in the orange box. Heat induced CK might compensate for the loss of PC by actively phosphorylating choline

generated by PC breakdown thereby pacing up PC biosynthesis. In AtClpB-C:AtCK2 divergent pair, CK induction during HS by

Hsf may help in generation of the required PC for uninterrupted signaling. The steps in this model indicated by solid arrows are

based on published evidence. The dotted arrows represents the hypothesis forwarded in this study. Cycle shown with blue arrows

represents the Kennedy pathway for PC biosynthesis. www.plantphysiol.orgon October 19, 2020 - Published by Downloaded from

Copyright © 2014 American Society of Plant Biologists. All rights reserved.


Documents

Research Area: Environmental Stress and AdaptationOct 07, 2014 · promoters to sense heat is largely attributed to heat shock elements (HSE) located in the promoter region (Sugio