Comprehensive Expression Profiling of Rice Tetraspanin Genes Reveals Diverse Roles During Development and Abiotic Stress

Abstract

Tetraspanin family is comprised of evolutionarily conserved integral membrane proteins. The incredible ability of tetraspanins to form 'micro domain complexes' and their preferential targeting to membranes emphasizes their active association with signal recognition and communication with neighboring cells, thus acting as key modulators of signaling cascades. In animals, tetraspanins are associated with multitude of cellular processes. Unlike animals, the biological relevance of tetraspanins in plants has not been well investigated. In Arabidopsis tetraspanins are known to contribute in important plant development processes such as leaf morphogenesis, root, and floral organ formation. In the present study we investigated the genomic organization, chromosomal distribution, phylogeny and domain structure of 15 rice tetraspanin proteins (OsTETs). OsTET proteins had similar domain structure and signature 'GCCK/R' motif as reported in Arabidopsis. Comprehensive expression profiling of OsTET genes suggested their possible involvement during rice development. While OsTET9 and 10 accumulated predominantly in flowers, OsTET5, 8, and 12 were preferentially expressed in root tissues. Noticeably, seven OsTETs exhibited more than twofold up regulation at early stages of flag leaf senescence in rice. Furthermore, several OsTETs were differentially regulated in rice seedlings exposed to abiotic stresses, exogenous treatment of hormones and nutrient deprivation. Transient subcellular localization studies of eight OsTET proteins in tobacco epidermal cells showed that these proteins localized in plasma membrane. The present study provides valuable insights into the possible roles of tetraspanins in regulating development and defining response to abiotic stresses in rice. Targeted proteomic studies would be useful in identification of their interacting partners under different conditions and ultimately their biological function in plants.

Keywords: abiotic stress; gene expression; hormone; nutrient deprivation; rice; tetraspanin.

FIGURE 1

Structure of rice tetraspanin genes and phylogenetic tree of predicted proteins. An online tool, Gene Structure Display Server (GSDS; http://gsds.cbi.pku.edu.cn/), was used to draw tetraspanin gene structure. Green boxes indicate exons, black lines depict introns, upstream/downstream sequences are shown by blue boxes. Intron phases are indicated at exon-intron junctions. An unrooted neighbor-joining phylogenetic tree of rice tetraspanin proteins is shown on the left side. Numbers above branches indicate bootstrap percentage values. Clade numbers are indicated by salmon colored boxes. OsTET proteins were clustered based on significant bootstrap value (≥50%).

FIGURE 2

Multiple sequence alignment of predicted rice tetraspanin proteins. Protein sequences of rice tetraspanin proteins were retrieved from RGAP and were aligned using ClustalX 2.1. Red amino acids indicate transmembrane (TM) domains. Predicted palmitoylation and glycosylation sites are highlighted in yellow and green color, respectively. Highly conserved cysteine residues are shaded by blue color. Tetraspanin signature ‘SGCC(K/R)PP’ motif is shaded in pink color. Intron positions are shaded by black color. Sequences were edited with Genedoc software (http://www.nrbsc.org/gfx/genedoc/).

FIGURE 3

Expression profiling of rice tetraspanin genes in different tissues of rice. (A) Quantitative PCR analysis of transcript levels of rice tetraspanins in tissues namely shoot, root, young leaf (YL), active tillering phase (AT-Phase), stem elongation phase (SE-Phase), spikelets (Spks), young flag leaf (YFL), and mature flag leaf (MFL). For root tissue normalized fold change (log₂ scale) was calculated relative to that in shoot tissue of 7-day-old seedlings. For tissues obtained from field grown plants fold change (log₂ scale) was calculated relative to that in young leaf. (B) Quantitative PCR analysis of transcript levels of rice tetraspanins during progression of senescence in flag leaf of field-grown rice plants. CON: fully expanded MFL; 100% chlorophyll, S1: 80–90% chlorophyll; S2: 60–80% chlorophyll; S3: 40–60% chlorophyll. Normalized fold change (log₂ scale) was calculated relative to that in MFL. For normalization eEF-1α was used as internal control. Three biological replicates and two technical replicates were included in the study. Error bars represent standard error (SE) of three independent biological replicates.

FIGURE 4

Heat map representing expression profile of rice tetraspanin genes in rice seedlings exposed to different abiotic stresses. Seven-day-old rice seedlings were exposed to different abiotic stresses such as heat stress at 42°C; salinity stress with 200 mM NaCl; water deficit stress (WDS) imposed by 15% PEG; cold stress at 4°C; oxidative stress with 10 mM H₂O₂ for different time durations as indicated on the left (duration in h). Expression levels of tetraspanin genes were determined by quantitative PCR and normalized fold change (log₂ scale) was calculated relative to that in unstressed seedlings. For normalization eEF-1α was used as internal control. Three biological replicates and two technical replicates were included in the study. Hierarchical clustering analysis of relative fold change was performed to prepare a dendrogram and a heat map using Hierarchical Clustering Explorer v3.5 software.

FIGURE 5

Heat map representing expression profile of rice tetraspanin genes in rice seedlings exposed to nutrient deprivation. Seven-day-old rice seedlings were grown in different nutrient deprivation media such as nitrogen deprivation (N), phosphorous deprivation (P), potassium deprivation (K), sulfur deprivation (S) for different time durations as indicated on the left (in h). Expression levels of tetraspanin genes were determined by quantitative PCR and normalized fold change (log₂ scale) was calculated relative to that in unstressed seedlings. For normalization eEF-1α was used as internal control. Three biological replicates and two technical replicates were included in the study. Hierarchical clustering analysis of relative fold change was performed to prepare a dendrogram and a heat map using Hierarchical Clustering Explorer v3.5 software.

FIGURE 6

Heat map representing expression profile of rice tetraspanin genes in rice seedlings exposed to different hormones. Seven-day-old seedlings were exposed to exogenous hormones namely abscisic acid (ABA), brassinosteroids (BS), gibberellic acid (GA), and methyl jasmonate (MeJA) for different time durations (in h) as indicated on the left. Expression levels of tetraspanin genes were determined by quantitative PCR and normalized fold change (log₂ scale) was calculated relative to that in untreated seedlings. For normalization eEF-1α was used as internal control. Three biological replicates and two technical replicates were included in the study. Hierarchical clustering analysis of relative fold change was performed to prepare a dendrogram and a heat map using Hierarchical Clustering Explorer v3.5 software.

FIGURE 7

Overlap in expression pattern of rice tetraspanin genes in various abiotic stresses. Venn diagram representing overlap of OsTET genes exhibiting significant change (≥2-fold change in log₂ scale) in expression among different abiotic stresses. Both upregulated and downregulated genes are shown. Five abiotic stresses: heat stress (HS), salinity stress (SS), cold stress (CS), water deficit stress (WDS), and oxidative stress (OS) were included in this study.

FIGURE 8

Subcellular localization of rice tetraspanin proteins in tobacco epidermal cells. The OsTETs fused to YFP were transiently expressed in tobacco (Nicotiana benthamiana) leaves. Localization in plasma membrane was confirmed by coexpression of PIP2A-CFP, a plasma membrane (PM) marker. (A) Colocalization of pGWB542 (empty vector) with PM marker. (B) Colocalization of OsTET-YFP proteins with PM marker. The merged fluorescence of marker and OsTET-YFP is shown at the right. Scale bar = 20 μm.