Expression Analysis of Sugarcane Aquaporin Genes under Water Deficit

Abstract

The present work is a pioneer study specifically addressing the aquaporin transcripts in sugarcane transcriptomes. Representatives of the four aquaporin subfamilies (PIP, TIP, SIP, and NIP), already described for higher plants, were identified. Forty-two distinct aquaporin isoforms were expressed in four HT-SuperSAGE libraries from sugarcane roots of drought-tolerant and -sensitive genotypes, respectively. At least 10 different potential aquaporin isoform targets and their respective unitags were considered to be promising for future studies and especially for the development of molecular markers for plant breeding. From those 10 isoforms, four (SoPIP2-4, SoPIP2-6, OsPIP2-4, and SsPIP1-1) showed distinct responses towards drought, with divergent expressions between the bulks from tolerant and sensitive genotypes, when they were compared under normal and stress conditions. Two targets (SsPIP1-1 and SoPIP1-3/PIP1-4) were selected for validation via RT-qPCR and their expression patterns as detected by HT-SuperSAGE were confirmed. The employed validation strategy revealed that different genotypes share the same tolerant or sensitive phenotype, respectively, but may use different routes for stress acclimation, indicating the aquaporin transcription in sugarcane to be potentially genotype-specific.

Figure 1

Percentage of HT-SuperSAGE unitags from sugarcane plants (24 h of continuous dehydration or regular daily irrigation) identified as potential aquaporins after keyword searches in the EST annotation (“aquaporin,” “tonoplast intrinsic protein,” “PIP,” “TIP,” “NIP,” “SIP”) or in the GO terms (“water transport”). Total of unitags: 1,579.

Figure 2

Neighbor Joining dendrogram (MEGA v.5.2.1 software [27]) and integrated heat map (bootstrap values of 1,000 replications), showing the phenetic grouping of 24 potential aquaporin amino acid sequences identified by HT-SuperSAGE unitags from sugarcane accessions after 24 h of continuous dehydration (with their respective EST and unitag identifiers), and aquaporins sequences of Arabidopsis thaliana, human and Yersinia pseudotuberculosis (all these labeled by an asterisk). Nomenclature: Isoforms are preceded by the abbreviated species name (At-Arabidopsis thaliana; Hs-Homo sapiens; Hv-Hordeum vulgare; Os-Oryza sativa; Pv-Panicum virgatum; Sb-Sorghum bicolor; So-Saccharum officinarum; Yp-Yersinia pseudotuberculosis; Zm-Zea mays).

Figure 3

Representation of aquaporin subfamilies expressed in sugarcane after 24 h of continuous dehydration ((a), (b), and (c)) and during normal irrigation (d), involving bulks of genotypes tolerant and sensitive to stress. HT-SuperSAGE libraries: SD24T (bulk of tolerant genotypes under stress; SD24S (bulk of sensitive genotypes under stress); SDTC (control bulk of tolerant genotypes); SDSC (control bulk of sensitive genotypes).

Figure 4

Relative quantification of SsPIP1 aquaporin-1 (unitag SD264077) and SoPIP1-3/PIP1-4 (unitag SD231548) with sugarcane cDNAs [tolerant genotypes: (a) CTC6, (b) CTC15, (c) SP83-2847, (d) SP83-5073; sensitive genotypes [(e) CTC9, (f) CTC13, (g) SP90-1638, (h) SP90-3414] under stress (24 h of continuous dehydration). The expression ratios of the same genotypes under control condition were normalized by reference genes GAPDH and 25S rRNA. ^#Relative expression (REST^© v. 2.0.13 software) with the mean value (horizontally dotted line) and range of the observations (100%, horizontal bars); in red: confidence interval at 95%.