Phylogenetic relationship and exon-intron structure analysis
The phylogenetic analysis was conducted by comparing predicted AQPs/MIPs of Helianthus annuus with clearly classified MIPs of Arabidopsis thaliana as shown in Fig. 1. this phylogenetic relationship between sunflower and Arabidopsis revealed four major clusters of MIPs. A total of 31 predicted MIPs of sunflower were classified into four different subfamilies, plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic protein (TIPs), nodulin 26-like intrinsic proteins (NIPs) and small intrinsic protein (SIPs) with 11 members of TIPs, 8 members of PIPs, 9 members of NIPs and 3 members of SIP. Among 11 members of TIPs, six distinct members were selected for further spatial and temporal expression analysis based on this phylogenetic analysis. The exon intron structure analysis clearly indicated that HaGTIP1;1, HaTIP1;1 and HaTIP7 contained only one intron while HaTIP-RB7, HaTIP4;1 and HaTIP5;1 contained two introns. The length and insertion positions of introns were different among the six members of HaTIPs as shown in Fig. 2.
Expression analysis of HaTIPs in various tissues of sunflower
The expression of all samples was normalized using Actin-7 as internal control. It expressed constitutively in all samples. The differential expression of selected HaTIPs was observed in leaves, roots, stem and flower of selected sunflower entries as presented in Fig. 3. The expression of these HaTIPs was higher in cross as compared to parents in all the tissues. HaTIP7, HaTIP1;1 and HaGTIP1;1 expressed in all tissues. HaTIP7 expressed strongly in leaf, stem and root of cross and in leaf and flower of Hysun-33 and root of 3447, while a marginal expression was observed in root of 9591, stem of 3447 and flower of cross. A weak expression of this gene was noted in leaf and flower of 3447, leaf, stem and flower of 9591, and in stem and root of Hysun-33. Ha TIP1;1 showed strong expression in stem, root and flower of cross and in leaf of Hysun-33, stems of 3447 and flower of Hysun-33. A marginal expression of this gene was found in leaf and stem of 9591, in leaf of cross, and flower of 3447, while showing weak expression in leaf of 3447, stem of Hysun-33 and flower of 9591. A strong expression of HaGTIP1;1 gene was noted in leaf and flower of cross, while it showed marginal expression in leaf and flower of 9591, shoot of Hysun-33 and root of cross. A weak expression of this gene was noted for leaf and root of Hysun-33 and root of 9591. HaTIP-RB7 expressed in all tissues except flower showing strong expression in root and stem of the cross (C = 9591×3447). A marginal expression was shown by this gene in leaf of Hysun-33, and roots of 3447 (G1), 9591 (G2) and Hysun-33, while it showed weak expression in stem of 3447. HaTIP4;1 expressed in all tissues except root. Its showed strong expression in stem of the cross. It showed marginal expression in leaf of cross and Hysun-33 while weak expression was observed in leaf and flower of 9591, in stem of 3447 and in flowers of cross and Hysun-33. Ha TIP5;1 expressed in leaf and flower. It showed strong expression in flower of cross. A marginal expression of this gene was observed in leaf and flower of 9591 and Hysun-33. While a weak expression was noted in leaves of 9591, cross and Hysun-33.
Temporal expression of HaTIPs in roots of sunflower through semi qPCR
The expressions of HaTIPs analyzed through semi q-PCR in roots of selected sunflower entries at 0, 2, 4 and 12 hours of PEG treatment are presented in Fig. 4. The expression of HaTIP RB7 and HaTIP7was gradually increased in Hysun-33, 9591 and cross from 0 to 12 hours of PEG treatment. Their expression was highest at 12 hours of stress in cross. While the expression of this gene was decreased in 3447 at 2, 4 and 12 hours compared to 0 hour of stress. The expression of HaGTIP 1;1 was decreased in roots of 9591, cross and Hysun-33 at 2, 4 and 12 hours compared to 0 hour of PEG treatment. It did not express at 12 hours in cross and at 4 and 12 hours of PEG treatment in Hysun-33, while in 3447 it did not express at all. The expression of HaTIP 1;1 was induced in 9591 at 2 hours which was increased at 4 hours and then lowered down at 12 hours of PEG treatment. This gene did not express in 3447 and Hysun-33 at all. Under normal condition (at 0 hours of stress), HaTIP 1;1 expressed strongly in root of cross. Its expression lowered down in cross at 2 hours, then upregulated at 4 hours and then again lowered down at 12 hours compared to 0 hour of PEG treatment. HaTIP 4;1 did not express in roots of any accession at all.
Validation of PEG inducible expression of HaTIPs in roots of sunflower through real time PCR
The expression of HaTIP7 and HaTIP-RB7 at 0, 4 and 12 hours and expression of HaTIP1;1 at 0, 2, 4 and 12 hours of PEG treatment was compared with internal control (Actin-7) in parental accessions (3447 and 9591) and cross (9591×3447) as presented in Fig. 5. For the gene HaTIP-RB7, the relative expression was decreased in drought sensitive accession 3447 at 4 and 12 hours compared to 0 hour, while in drought tolerant entries 9591 and cross 9591×3447 it was increased at 4 and 12 hours of PEG treatment compared to 0 hour. Similar pattern was observed for the gene HaTIP7 in parental accessions and cross. The relative expression of both of these genes was highest at 12 hours of stress treatment. However, the relative expression of HaTIP1;1 was increased at 2 and 4 hours but then lowered down at 12 hours of PEG treatment in the parental accession 9591. The relative expression of HaTIP1;1 was highest at 0 hour in cross and gradually lowered down 2 and 4 hours of PEG treatment followed by a rapid decrease at 12 hours of stress. Thus, the expression pattern of these selected HaTIPs was found quite similar as that of the results of semi q-PCR.