Rice sHsp genes: genomic organization and expression profiling under stress and development

Abstract

Background: Heat shock proteins (Hsps) constitute an important component in the heat shock response of all living systems. Among the various plant Hsps (i.e. Hsp100, Hsp90, Hsp70 and Hsp20), Hsp20 or small Hsps (sHsps) are expressed in maximal amounts under high temperature stress. The characteristic feature of the sHsps is the presence of alpha-crystallin domain (ACD) at the C-terminus. sHsps cooperate with Hsp100/Hsp70 and co-chaperones in ATP-dependent manner in preventing aggregation of cellular proteins and in their subsequent refolding. Database search was performed to investigate the sHsp gene family across rice genome sequence followed by comprehensive expression analysis of these genes.

Results: We identified 40 alpha-crystallin domain containing genes in rice. Phylogenetic analysis showed that 23 out of these 40 genes constitute sHsps. The additional 17 genes containing ACD clustered with Acd proteins of Arabidopsis. Detailed scrutiny of 23 sHsp sequences enabled us to categorize these proteins in a revised scheme of classification constituting of 16 cytoplasmic/nuclear, 2 ER, 3 mitochondrial, 1 plastid and 1 peroxisomal genes. In the new classification proposed herein nucleo-cytoplasmic class of sHsps with 9 subfamilies is more complex in rice than in Arabidopsis. Strikingly, 17 of 23 rice sHsp genes were noted to be intronless. Expression analysis based on microarray and RT-PCR showed that 19 sHsp genes were upregulated by high temperature stress. Besides heat stress, expression of sHsp genes was up or downregulated by other abiotic and biotic stresses. In addition to stress regulation, various sHsp genes were differentially upregulated at different developmental stages of the rice plant. Majority of sHsp genes were expressed in seed.

Conclusion: We identified twenty three sHsp genes and seventeen Acd genes in rice. Three nucleocytoplasmic sHsp genes were found only in monocots. Analysis of expression profiling of sHsp genes revealed that these genes are differentially expressed under stress and at different stages in the life cycle of rice plant.

Figure 1

Phylogenetic analysis of rice ACD gene family. The tree was derived by Neighbor- joining method with bootstrap analysis (1000 replicates) from alignment of amino acid sequences of conserved ACD of sHsp and Acd of rice and Arabidopsis using CLUSTAL X1.83. The tree was analyzed with TREEVIEW 1.6.6. The bootstrap values >50% are denoted at the nodes. Rice sHsps and Acds are depicted in bold. Clusters of Acd genes are denoted with a vertical line on the right.

Figure 2

Phylogenetic relationship of sHsps of rice with diverse plant species. The tree was generated by Neighbor- Joining method on amino acid sequences of sHsps aligned using Clustal X1.83 and analyzed by TreeView1.6.6. The bootstrap values (>50%) from 1000 replications are indicated at the nodes. The abbreviations of species are as follows: At- Arabidopsis thaliana, Cd- Cynodon dactylon, Gm – Glycine max, Hv- Hordeum vulgare, Lp – Lycopersicon peruvianum, Os- Oryza sativa, Ps- Pseudoroegneria spicata, Pt- Populus tremula, Sb- Sorghum bicolor, So- Saccharum officinarum, Ta -Triticum aestivum, Vv- Vitis vinifera and Zm- Zea mays. Accession number of sHsps are – Cd16.6 (ES295769), Gm16.2 (BAG09378), Hv17.1 (CB878637), Hv19.3 (BF265056), Lp16.1 (AAK84869), Lp26.2 (AAB07023), Ps19.8 (FF349294), Pt17.6 (BU893632), Sb17.4 (CN143862), So16.5 (CA143009), So17.4 (CA184170), Ta16.9B (CAA45902), Ta20.1 (CV766415), Ta26.6 (AAC96315), Vv15.7 (CAO22716), Vv16.4 (CAO64962), Vv21.7 (EE098997), Vv22.4 (CAO39056), Vv25.0 (CAO48583), Vv26.3 (CAO62740), Zm16.6 (ACG25839), Zm17.2 (CAA46641), Zm17.2 (ACG27403), Zm17.4 (ACG45088), Zm17.9 (ACF78669), Zm21.9 (ACG27568), Zm22.7 (ACG31638), Zm22.8 (ACG38401), Zm26.4 (AAA33477). CI – CXI- cytoplasmic/nuclear, ER- endoplasmic reticulum, M- mitochondria, P- plastid, Px- peroxisome. Rice sHsps are shown in bold.

Figure 3

Diagramatic illustration of sHsp subfamilies of rice. The conserved α- crystallin domain of sHsps is shown in pink. The transit peptide (TP) of the organellar sHsps, the NLS of CII and CIII sHsp, the methionine rich region (Met-bristle) of plastidial sHsp and peroxisomal targeting signal (PTS1; SKL) are marked. The position of the introns is indicated with black arrowhead. Description of sHsp subfamilies is given in the text. Intron- exon structure of subfamilies CVIII, CIX, CX and CXI is similar to CI subfamily.

Figure 4

EST analysis of sHsp and Acd genes of rice. EST based expression of sHsp genes (a) and Acd genes (b). The details of the libraries are given in 'methods'. The library for each tissue is not same for each gene. Expression of sHsps and Acd genes is plotted as percent frequency i.e. (number of ESTs for particular gene/total number of ESTs in the library). γ-irr; gamma irradiated.

Figure 5

Microarray based expression profiles of sHsp genes during developmental stages of rice plant. Expression of sHsp genes during developmental stages are presented as heat maps in blue/white colors generated using meta-analysis tool at Genevestigator . The darker color corresponds to stronger expression.

Figure 6

Expression pattern of sHsp and Acd genes under various stress conditions. The microarray data based expression profiles under stress conditions are presented as heat maps generated using meta-analysis tool at Genevestigator . The transcript levels are depicted by color scale indicating log₂ values. Ubq5 expression is shown as control.

Figure 7

Microarray analysis of expression profiles of sHsps of rice under heat stress. Leaves from one month old plants were given stress at 42°C. Samples were harvested after 10 min and 1 h of HT treatment and 30 min recovery following 1 h HT. Three replicates were performed and the standard error is shown by error bars. 1* represents a probe which cross-reacts with Hsp17.9A-CI, Hsp18.0-CI, Hsp17.4-CI and Hsp17.7-CI. 2* represents a probe on the microarray chip which overlaps with Hsp16.9A-CI, Hsp16.9B-CI, Hsp16.9C-CI, Hsp17.9A-CI, Hsp18.0-CI and Hsp17.4-CI.

Figure 8

Semi-quantitative RT-PCR analysis of sHsps expression under heat, cold, air drying and osmotic stress. Total RNA from the roots and shoots of 7 d old seedlings, dry seeds and panicle was used for RT-PCR analysis. Heat stress (HT): 0.5 h at various temperatures; Osmotic stress: 15% PEG 4000; Dehydration stress: air drying (AD) 20 min; low temperature (LT): 6°C ± 2. Ubq5 was used as internal control for RT reaction.

Figure 9

MEME based consensus sequences in the promoters of sHsp. The motifs obtained by MEME analysis were plotted according to their positions within the sites and their consensus sequences were presented as graphs using MEME LOGO. The occurrences of motif is sorted by P-value and aligned with each other. The E-value of motif is an estimate of the expected number of motifs with the same width and number of occurrences that would be present in a similarly sized set of random sequences. The height of symbols in each stack in the motif indicates the sequence conservation at that position. The sequences are manually highlighted to show recognized consensus HSEs: pink box-nGAAnnTTCnnGAAn or nTTCnnGAAnnTTCn showing perfect HSE, green box-showing imperfect module of HSE. All nGAAn and nTTCn are shown in orange boxes. The 'Start' indicates the distance from translational start site (ATG). Nucleotides in bold indicate the consensus bases present in HSEs.

Figure 10

Overlapping expression of sHsp genes in various abiotic stresses. The numbers in brackets denote the total number of sHsp genes that are upregulated under each stress treatment (H-heat, C- cold, S- salt, A-anoxia and D- dehydration stress). Black circle denotes overlapping expressed genes in H, D, A and S stresses, black triangle denotes genes expressed in H, C, A and S stresses and black square denotes genes expressed in H, C, D and S stresses. Nucleo-cytoplasmic sHsp members are shown in blue font, mitochondrial sHsps are shown in red font, ER sHsps are shown in brown font, peroxisomal sHsps are shown in purple font and chloroplastic sHsps are shown in green font.

Figure 11

Overlapping expressions of sHsps in different developmental stages of rice. The color code of the fonts is same as shown in legend of Figure 10.