OsHsfA2c and OsHsfB4b are involved in the transcriptional regulation of cytoplasmic OsClpB (Hsp100) gene in rice (Oryza sativa L.)

Abstract

ClpB-cytoplasmic (ClpB-cyt)/Hsp100 is an important chaperone protein in rice. Cellular expression of OsClpB-cyt transcript is governed by heat stress, metal stress, and developmental cues. Transgenic rice plants produced with 2 kb OsClpB-cyt promoter driving Gus reporter gene showed heat- and metal-regulated Gus expression in vegetative tissues and constitutive Gus expression in calli, flowering tissues, and embryonal half of seeds. Rice seedlings regenerated with OsClpB-cyt promoter fragment with deletion of its canonical heat shock element sequence (HSE(-273 to -280)) showed not only heat shock inducibility of Gus transcript/protein but also constitutive expression of Gus in vegetative tissues. It thus emerges that the only classical HSE present in OsClpB-cyt promoter is involved in repressing expression of OsClpB-cyt transcript under unstressed control conditions. Yeast one-hybrid assays suggested that OsHsfA2c specifically interacts with OsClpB-cyt promoter. OsHsfA2c also showed binding with OsClpB-cyt and OsHsfB4b showed binding with OsClpB-cyt; notably, interaction of OsHsfB4b was seen for all three OsClpB/Hsp100 protein isoforms (i.e., ClpB-cytoplasmic, ClpB-mitochondrial, and ClpB-chloroplastic). Furthermore, OsHsfB4b showed interaction with OsHsfA2c. This study suggests that OsHsfA2c may play a role as transcriptional activator and that OsHsfB4b is an important part of this heat shock responsive circuitry.

Fig. 1

Linear representation of 2-kb upstream promoter region of OsClpB-cyt gene. ATG as well as selected regulatory elements in this sequence (such as HSE −273 to −280; HSE-like −97 to −107; STRE −578 to −582; AP-1 element −411 to −416; C/EBP element −1,424 to −1,428) are indicated

Fig. 2

Analysis of 2 kb OsClpB-cyt promoter. a Schematic representation of the T-DNA region of 2kbProOsClpB-cyt::Gus construct. b Histochemical expression of Gus in onion epidermal cells shot with 2kbProOsClpB-cyt::Gus construct. Cells showing transient expression of Gus are shown by arrows. c Expression of Gus in heat stressed (42°C, 1 h) calli transformed with 2kbProOsClpB-cyt::Gus construct. d Leaf segments from T1 rice seedlings expressing Gus protein following heat treatment (42°C, 1 h). e Northern analysis showing heat induction of Gus transcript in leaf segments of 2kbProOsClpB-cyt::Gus plants. Probing was done with radiolabeled Gus probe. f Histochemical analysis of Gus expression in 2kbProOsClpB-cyt::Gus plants following heat stress (42°C, 1 h). g Expression of Gus in 2kbProOsClpB-cyt::Gus plants in response to various metals. Treatments were given for 6 h at 28°C in beakers containing 20 μM solutions of arsenic (As), cadmium (Cd), cobalt (Co), copper (Cu), and zinc (Zn). Heat stress was given in a water bath maintained at 42°C for 1 h. Gus expression was analyzed histochemically. h Northern blot showing expression of Gus transcript in metal and heat shocked tissues. Fifteen micrograms of RNA was loaded in each lane. Lower panel depicts rRNA bands as loading control. i Gus expression in the reproductive organs of 2kbProOsClpB-cyt::Gus rice plants. i Whole mount of anther. ii Whole mount of gynoecium tissues. iii Whole mount of seeds. In all the histochemical assays, seven lines were employed; data from one representative transgenic line are shown

Fig. 3

Analysis of mutant forms of OsClpB-cyt promoter. a Schematic representation of ΔPro-HSE_{−273 to −280}::Gus construct. b Northern analysis of ProΔHSE_{−273 to −280}::Gus plants using radiolabeled Gus as probe. T1–T6 represent independent transgenic plants. c Fluorimetry of ProΔHSE_{−273 to −280}::Gus plants using 20 μg of protein. Ratios of Gus activity in unstressed (control) and HS samples were plotted. For both RNA (b) and protein (c) isolation, seedlings given heat stress at 42°C for 1 h were used and unstressed seedlings were taken as control. P1–P7 represent independent transgenic plants. d Schematic representation of ΔUTR-HSE-like_{−97 to −107}::Gus construct. e Northern analysis of ΔUTR-HSE-like_{−97 to −107}::Gus plants, using radiolabeled Gus as probe. T1–T4 represent independent transgenic plants. f Fluorimetric Gus expression in ΔUTR-HSE-like_{−97 to −107}::Gus plants using 20 μg of protein. Ratios of Gus activity in control and HS samples were plotted. For both RNA (e) and protein (f) isolation, seedlings given heat stress at 42°C for 1 h were used. Unstressed seedlings were taken as control. T1–T5 represent independent transgenic plants

Fig. 4

Yeast one-hybrid assay for analyzing binding of OsClpB-cyt promoter with rice heat shock factor proteins. a Schematic representation of the constructs. b Binding assay of OsClp-B-cyt promoter with OsHsfs. Activity of β-galactosidase in yeast cells transformed with different constructs is shown. c Schematic representation of different versions of the construct containing OsClpB-cyt promoter cloned in the vector pLacZi (CYC1, OsClpB-cyt promoter + CYC1, OsClpB-cyt promoter-CYC1 and OsClpB-cyt promoter ΔHSE −273 to −280 + CYC1). d Binding assay of the above OsClp-B-cyt promoter constructs with OsHsfA2c. OsHsfA2c gene was transformed in yeast cells with the above constructs. Liquid β-galactosidase assay is shown in the left panel. Plate assay is shown in the right panel. For plate assays, equal number of cells were taken, spotted, and allowed to grow for 48 h. Thereafter, plates were overlaid with 0.1 M NaPO₄ buffer containing 1% agar and 25 mg/ml X-gal. Plates were kept at 28°C for 24 h

Fig. 5

Yeast two-hybrid assay for analyzing binding between selected OsHsfs and OsClpB proteins. a Schematic representation of the constructs used. b Yeast two hybrid assay showing β-galactosidase activity in YRG2 yeast cells transformed with different constructs. c Growth assay for –His reporter gene. Serial dilutions of YRG2 cells co-transformed with pAD-OsClpB-cyt/chloroplastic/mitochondrial and pBD-OsHsfB4b constructs were spotted on –Leu Trp, –His Leu Trp, and –His Leu Trp containing 1 mM 3AT. PC: positive control (pSE1111-ScSNF1 and pSE1112-ScSNF4 transformed YRG2 strain to yield YRG2-pSE1111-ScSNF1 + pSE1112-ScSNF4 cells). NC Negative control (pAD + pBD vector transformed YRG2 cells). Three independent sets of yeast two hybrid assays were undertaken; data from one representative experiment are shown

Fig. 6

BiFC assays for analyzing OsClpB-cyt:OsHsfB4b, OsClpB-cyt:OsHsfA2c, OsHsfA2c:OsHsfB4b, and OsClpB-cyt:OsClpB-cyt interactions, using onion epidermal cells. a Cells co-transformed with OsClpB-cyt-YFPN and OsHsfB4b-YFPC fusion constructs. b Cells co-transformed with OsHsfA2c-YFPN and OsClpB-cyt-YFPC fusion constructs. c Cells co-transformed OsHsfA2c-YFPN and OsHsfB4b-YFPC fusion constructs. d Cells co-transformed with OsClpB-cyt-YFPN and OsClpB-cyt-YFPC fusion constructs. i YFP signal, ii bright field images, iii DAPI (4′,6′-diamidino-2-phenylindole)-stained images and iv merged images of i, ii, and iii