Eleven StOSMs Genes in the Potato Genome Response to Water Deficiency  

Linbo Qiao , Huihua Fan , Xiaoping Zhang , Haixia Dai , Xinling Yao
The State Education Ministry Laboratory of West Bioresource Protection and Utilization, Life Science College, Ningxia University, Yinchuan, Ningxia 750021, China
Author    Correspondence author
Bioscience Methods, 2015, Vol. 6, No. 1   doi: 10.5376/bm.2015.06.0001
Received: 08 Nov., 2014    Accepted: 13 Jan., 2015    Published: 20 Jan., 2015
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Qiao et al., 2015, Eleven StOSMs Genes in the Potato Genome Response to Water Deficiency, Bioscience Methods, Vol.6, No.1 1-13 (doi: 10.5376/bm.2015.06.0001)


Osmotin plays an important role in plant response to low temperature and pathogen infection. To date little is known on existence and full function of OSM genes in genome-wide. In this study, based on an OSM expression sequence tag identified from a drought-treated subtractive library, StOSMs in the potato genome were identified and characterized in response to a water deficit gradient. BLAST and bioinformatic analysis revealed that there are at least eleven StOSMs inthe potato genome. Among eleven StOSMs,eight StOSM mRNAs accumulated in leaf at the drought-lethal critical point (DLP) were at least 4-fold higher than in the control. The peak StOSM-8E mRNA accumulation was 49-fold higher than the control. Three StOSM mRNAs in leaf at DLP were 9.2-fold lower than in the control. This result of qRT-PCR quantification was identical with the FPKM values of all eleven StOSMs examined in the the Potato Genome Sequence database (PGSD). In conclusion, water deficiency does induce the regulation of the expression of the eleven StOSMs. Either up or downregulated by water deficiency, StOSMs play a positive role in enhancing potato tolerance to drought stress. Therefore, osmotins can be considered to be drought responsive molecules involved in withstanding water deficits. The result provides an evidence to reveal how potato adapts to drought stress.

OSM; Osmotin; Expression; mRNA; Drought

Osmotin(accession No. M29279), encoding the stress response molecule osmotin, was initially identifiedin cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) induced by low water potential (Singh NK et al 1989). Subsequently, it was showed that abscisic acid (ABA), low temperature and NaCl can induce the accumulation of three StOSM mRNAs (pA13, pA35, and pA81) in S. commersonii grown in vitro (Zhu et al 1995). Later on StOSM wasshown to exhibit antifungal activity against a range of fungal pathogens (Rivero et al. 2012, Mani et al. 2012 and Patade et al. 2013).Recently, StOSM overexpression in transgenic Arabidopsis and pepper results in H2O2 accumulation and the response of hypersensitive cell death in the leaf and reduces susceptibility to pathogen infection (Choi et al. 2013).

Remarkably, it is showed that plant osmotin, by binding to the adiponectin receptor as an agonist, activates a pathway concerned with the resistance to animal disease. Osmotin reduces ethanol neurotoxicity via the upregulation of the antiapoptotic Bcl-2 protein, and reverses synaptic dysfunction and neuronal apoptosis. The Bcl-2 protein appears to block a distal step in a common pathway for apoptosis and programmed cell death (Naseer et al. 2014 and Shah et al. 2014).
Osmotin is not only an intercellular component involved in stress tolerance but also an agonist that alternatively binds to the adiponectin receptor involved in animal apoptosis. However, although osmotin was identified in tobacco in 1989, little is known regarding the details of plant osmotin, particularly how many OSM members are present in a plant genome and how these members work together in response to stress.
In potato, nine StOSM genes were identified byscreening a bacterial artificial chromosome (BAC) library. The nine StOSM genes are organised into two loci on chromosomes 08 and 11 (Castillo et al. 2005). Whether there are additional StOSM genes in the potato genome and the functional differences among the potato StOSM genes regarding the response to abiotic stress, such as drought, remain unknown.
With respect to the severity of the water shortage, each plant species is more or less tolerant to a certain drought level. A reliable and comparable level of plant tolerance to drought is known as the drought-lethal critical point (DLP). The availability of water in soil at a level lower than the DLP leads to plant death due to cell dehydration.
In this study, with a full-length StOSM cDNA isolated initially from a drought-treated subtractive library, eleven StOSM genes were identified using BLAST analysis of the Potato Genome Sequence database (PGSD). Leaf mRNA accumulation of StOSMs were assayed using RT-PCR and qRT-PCR and compared with FPKM (Fragments Per Kilobase of exon model per Million mapped reads) value of StOSM genes in the PGSD. The result indicated that expression of eight StOSMs in leafs at DLP was upregulated, three down-regulated. The result revealed that osmotins, as drought responsive molecules involved in withstanding water deficits in potato.
1. Results
1.1 Composition and structure of StOSM family
1.1.1 Cloning and osmotic stress response function of potato StOSM-3B
Through screening a subtractive cDNA library constructed from the young leaves of potato genotype ZHB at 20% ±2% water content in media (WCM) (unpublished work), an expressed sequence tag (EST) was identified. Sequence of the EST shared 99% identity with StOSM-3B belonging toAY737310 from Solanum phureja in the Genbank database (Castillo et al. 2005).
Using StOSM-3B ORF specific primers (O-F and O-R), a cDNA ORF with a length of 744 bp was cloned (Figure 1), which encoded osmotin with 248 amino acids. A sequence alignment showed 97% identity between the cDNA ORF and the StOSM-3B ORF. An alignment with the amino acid sequence showed that the putative amino acid sequences encoded by the isolated cDNA ORF have six residual substitutions compared with the sequence of StOSM-3B (Figure 2).

Figure 1 Cloning of the StOSM-3B ORF from the leaves of potato ZHB under 20% WCM. A: Product of PCR with StOSM-3B specific primer; B: cDNA sequence and putative amino acid sequence of StOSM-3B ORF isolated in this study

Figure 2 Alignment of the cDNA ORF isolated with StOSM-3B by putative amino acid sequence. The sequence alignment was performed using the ClustalW2 online software in EMBL-EBI. The green colour indicates the mutated amino acids (StOSM-3Bm: StOSM-3B isolated in this study; StOSM-3Bc: StOSM-3B isolated by Castillo RA et al [10])

The isolated cDNA in this study was still named after StOSM-3B because the cDNA sequence showed high identity with that of StOSM-3B.
Analysis of the expression of StOSM-3B in E. coli indicated that as the StOSM-3B mRNA accumulates, the tolerance of recombinant E. coli, as described by colony livability under osmotic stress, was significantly improved, even as the osmotic stress generated by PEG6000 increased (Hu et al 2012).
Wounding, abscisic acid (ABA), low temperature, NaCl and salicylic acid (SA) increased the StOSM-3B (pA81) mRNA expression in S. commersonii cell cultures and in plants grown in vitro. The reason for why these conditions induce StOSM-3B mRNA accumulation is unknown yet (Zhu et al. 1995). In addition, the involvement of StOSM-3B in water stress has not yet been reported.

StOSM genes in potato
Thesequence of the cloned StOSM-3B cDNA was retrieved in the PGSC_DM_v3.4_gene.fasta file from the Potato Genome Sequencing Consortium website (http://solanaceae.plantbiology.msu.edu) (Cory et al. 2014). A total of eleven StOSMgenes distributed among five chromosomes in the potato genome were identified from the PGSC database. The sequence structure of the eleven potato StOSM cDNAs is shown in Table 1.

Table 1 Transcript sequence structure of the eleven OSM genes in potato

All potato
StOSM genes were collected from the NCBI GenBank using BLAST analysis and aligned with these eleven StOSM. No one beyond eleven StOSM was found according the sequence alignment. Therefore, seven genes, such as StOSM-3B and -3F, were designated according to names used previously and registered in the database (Castillo et al. 2005). Four new StOSMgenes were identified and named based on the length of the putative encoded peptide.

Overall, eleven StOSM genes were distributed on five of twelve potato chromosomes (Figure 3). Seven of the eleven StOSM genes are near 54277k–54230k on chromosome 08 (Figure 4). The other four are located on chromosomes 01, 03, 06 and 11 (Figure 3 and Table 1).

Figure 3 Genomic distribution of the StOSM genes throughout the potato chromosomes. The chromosome number is given at the top of each bar. The triangles next to the gene names indicate the direction of transcription

Figure 4 Distribution of the seven StOSMs located on chromosome 08

The seven
StOSM genes on chromosome 08 identified from the PGSC database were not identical with the eight StOSM genes on chromosome 08 distinguished (Castillo et al. 2005). The extra StOSM geneon chromosome 08 identified by Castillo et al.is most likely due to two hybridised signals observed for StOSM-182 (PR-5X) on chromosome 08 because it contains two exons. The StOSM gene on chromosome 11 identified by Castillo et al.is StOSM-251, as shown in Figure 3.
1.1.3 Phylogeny of the putative StOSM amino acid sequences
The eleven putative amino acid sequences encoded by these StOSM genes were used to construct a phylogenetic tree (Figure 5). The eleven osmotins clustered into two main subclusters and further into four main clades. Two new osmotins, StOSM-297 and StOSM-306, were assigned to the same independent branch of the phylogenetic tree. The seven osmotins on chromosome 08 clustered into four subclades.

Figure 5 Phylogenetic relationship of the eleven putative potato osmotin peptides. These data were organised into a phylogenetic tree using the MEGA5.02 package and the neighbour-joining program. The numbers listed at the branching points are boot-strapping values that indicate the percentage significance for the separation of the two branches. The length of the branch line indicates the extent of the difference according to the scale on the lower left-hand side. Putative functions are listed for each gene

According to the phylogenetic relationship, one of the two
main subclusters, composed of StOSM-251, -306 -297 (clade 1), was relatively independent of the other. After separating from StOSM-182 (clade 5),seven of the eight StOSM genes in the other subcluster evolved into three clades: clade 2, containing StOSM-5A, -3Band -3F; clade 3, StOSM-1Gand -8E; and clade 4, StOSM-3Cand -2D.
The alignment of the amino acid sequences of the eleven osmotins revealed four conserved regions: the NNCPYT, RIW and TGDCGG motifs, located in the N-terminal region, and the AYSY motif, located in the C-terminal region (Figure 6A). A Jalview of the alignment of StOSM-5A, -3B, -3F, -1Gand -8E based on their phylogenetic relationship showed that a tandem duplication event occurred in these five genes (Figure 6B).

Figure 6 Amino acid sequence alignments of the putative potato osmotins)

Interestingly, the putative amino acid sequences of all osmotins range from 182 aa for
StOSM-182 to 306 aa for StOSM-306. StOSM-182 and -306 show a greater divergence towards the C-terminal region (Figure 6A). The sequences of four motifs were searched in the UniProt Knowledgebase (UniProtKB) by blastx. However, the result of zero hits from the search showed that the function of these conserved regions is unknown yet.
StOSM-182, with 182 amino acid residuals, is the shortest osmotin protein and does not contain the AYSY motif in its C-terminus (Figure 6A). In addition to the common four motifs, StOSM-182 and -306 share two additional motifs, FCPMKGVKRPN and ILLPFPMLI. Neither an expression sequence tag nor gDNA/cDNA clone of these two genes was registered in the database. Meanwhile, there was no hit for annotated or predicted function of these motifs when these motif sequences BLAST was done in all available protein database. This means that function of these motifs is unknown yet.
1.1.4 Introns and UTRs of StOSMs
The structure of all the StOSM genes was highly conserved. Only three StOSM genes, including StOSM-182 and -306, have two introns (Figure 7). Within the second intron of StOSM-182 is the first exon of a putative gene, which encodes ethylene-responsive proteinase inhibitor 1 (EPI). The second and third EPI exons are located in the 3’ end of the third exon of StOSM-182. In addition, there is one more copy of EPI locating within the promoter region of StOSM-3B (Figure 4).

Figure 7 Diagram of the structure of the eleven StOSM transcripts. The diagram is drawn to scale, according to the alignment of the cDNA sequences with the corresponding genomic sequences. All genomic sequences for StOSM w

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