Expression profiling of OsGLP genes in leaves
To examine the effect of salt and drought stress on the transcriptomic abundance of OsGLP genes in leaves of two rice varieties (KS282 and Super Basmati), the relative expression pattern of 8 OsGLP genes (OsGLP3-6, OsGLP3-7, OsGLP4-1, OsGLP8-4, OsGLP8-7, OsGLP8-10, OsGLP8-11 and OsGLP8-12) was determined by RT-qPCR. Taking the two-way interaction of abiotic stress treatments and rice varieties into account, statistical analysis identified 5 OsGLP genes (OsGLP3-6, OsGLP4-1, OsGLP8-7, OsGLP8-10 and OsGLP8-11) in KS282 (Fig. 1a, 1c, 1e, 1f and 1g) and 2 OsGLP genes (OsGLP3-6 and OsGLP4-1) in Super Basmati (Fig. 1a and 1c) that were differentially expressed by nearly 2-folds or more than 2-folds in response to at least one of the stress treatments. Among these OsGLP genes, OsGLP3-6 was observed with highest up-regulation under salt and drought stress in both rice varieties (Fig. 1a). Moreover, comprehensive overlap analysis demonstrated that, in leaves of both rice varieties, changes in the expression pattern of OsGLP genes under salt and drought stress are both general and specific to a particular variety as well as to a particular stress (Fig. 2a). Apart from this, some of the OsGLP genes exhibited significantly different relative expression in response to both stress treatments however, the relative expression of some OsGLP genes was same under both stresses depending on a particular rice cultivar.
In KS282 leaves, compared to the untreated control, a significant up-regulation of 5 OsGLP genes (OsGLP3-6, OsGLP4-1, OsGLP8-7, OsGLP8-10 and OsGLP8-11) under salt stress (Fig. 1a, 1c, 1e, 1f, 1g) and 4 OsGLP genes (OsGLP3-6, OsGLP4-1, OsGLP8-7 and OsGLP8-11) under drought stress (Fig. 1a, 1c, 1e and 1g) was observed. However, relative expression of 4 OsGLP genes (OsGLP3-6, OsGLP4-1, OsGLP8-10 and OsGLP8-11) under salt stress (Fig. 1a, 1c, 1f and 1g) and 3 OsGLP genes (OsGLP3-6, OsGLP4-1 and OsGLP8-11) under drought stress (Fig. 1a, 1c and 1g) was up-regulated in leaf samples of Super Basmati. The fold change in expression of these up-regulated OsGLP genes in both varieties, except for OsGLP4-1 and OsGLP8-7 in Super Basmati, was significantly different in response to salt and drought stress. In this aspect, higher relative expression of OsGLP3-6 and OsGLP4-1 was observed under drought stress as compared to salt stress (Fig. 1a and 1c) whereas OsGLP8-7, OsGLP8-10 and OsGLP8-11 showed high level of expression in response to salt stress (Fig. 1e-1g) suggesting that these genes from chromosome number 8 might play a significant role in response to salt stress. Further, OsGLP3-7, OsGLP8-4 and OsGLP8-12 showed similar expression patterns by being down-regulated in both varieties in response to at least one of the stress treatments (Fig. 1b, 1d and 1h).
Comparison of differential expression of OsGLP genes in leaves of KS282 and Super Basmati under salt and drought stress
Among the up-regulated OsGLP genes in leaves under salt and drought stress, relative expression of OsGLP3-6, which showed highest up-regulation in both rice verities, was significantly higher in KS282 in comparison with Super Basmati (Fig. 2b and Fig. 2c). Overall, relative change in expression of 5 OsGLP genes (OsGLP3-6, OsGLP4-1, OsGLP8-7 OsGLP8-10 and OsGLP8-11) under salt stress (Fig. 2b) and 3 OsGLP genes (OsGLP3-6, OsGLP4-1 and OsGLP8-7) under drought stress (Fig. 2c) was significantly higher in KS282 than Super Basmati.
Expression profiling of OsGLP genes in roots
A comparison of differential expression profiles of 8 OsGLP genes (OsGLP3-6, OsGLP3-7, OsGLP4-1, OsGLP8-4, OsGLP8-7, OsGLP8-10, OsGLP8-11 and OsGLP8-12) was conducted in root samples of KS282 and Super Basmati under salt and drought stress. Statistical analysis revealed that the expression of 7 OsGLP genes (OsGLP3-6, OsGLP3-7, OsGLP4-1, OsGLP8-4, OsGLP8-7, OsGLP8-10 and OsGLP8-11) in KS282 (Fig. 3a- 3g) and 5 OsGLP genes (OsGLP3-6, OsGLP3-7, OsGLP4-1, OsGLP8-4 and OsGLP8-12) in Super Basmati (Fig. 3a-3d and 3h) was increased by nearly 2-folds or more than 2-folds after exposure to at least one of the stress conditions. As in leaves, OsGLP3-6 was observed with highest increase in its expression under salt and drought stress in roots of both rice varieties (Fig. 3a). Furthermore, comparison of regulation pattern of OsGLP genes by overlap analysis suggested that, while not all OsGLP genes, some OsGLP genes were differentially regulated in both rice cultivars after exposure to different stress conditions (Fig. 4a).
Additionally, some OsGLP genes exhibited similar expression pattern in KS282 and Super Basmati depending on a particular stress type. In this aspect, the expression of 6 OsGLP genes (OsGLP3-6, OsGLP3-7, OsGLP4-1, OsGLP8-7, OsGLP8-10 and OsGLP8-11) under salt stress (Fig. 3a, 3b, 3c, 3e, 3f and 3g) and 4 OsGLP genes (OsGLP3-6, OsGLP4-1, OsGLP8-4 and OsGLP8-11) under drought stress (Fig. 3a, 3c, 3d and 3g) was commonly up-regulated in both rice varieties indicating that salt stress positively induced the expression of more OsGLP genes in comparison to drought stress. Further, a significant decrease in expression of OsGLP8-10 was also observed in both cultivars after exposure to drought stress (Fig. 3f). Importantly, expression of OsGLP8-12 was significantly up-regulated in Super Basmati after exposure to both stress treatments while its expression in KS282 was decreased after drought stress and nearly unchanged after salt stress (Fig. 3h).
Comparison of differential expression of OsGLP genes in roots of KS282 and Super Basmati under salt and drought Stress
Although most of the OsGLP genes showed up-regulation in roots of both rice varieties in response to either salt or drought stress, the relative change in the expression of several OsGLP genes was significantly higher in one variety than the other. For example, the expression of 4 OsGLP genes (OsGLP3-6, OsGLP8-7, OsGLP8-10 and OsGLP8-11) under salt stress (Fig. 4b) and 2 OsGLP genes (OsGLP3-6 and OsGLP8-7) under drought stress (Fig. 4c) was significantly higher in KS282 as compared to Super Basmati. Whereas, in comparison with KS282, 2 OsGLP genes (OsGLP3-7 and OsGLP8-12) after salt stress (Fig. 4b) and 2 OsGLP genes (OsGLP8-4 and OsGLP8-12) after drought stress (Fig. 4c) were observed with higher expression in Super Basmati.
Comparative analysis of tissue specific expression pattern of OsGLP genes under control and stress conditions
To get insight into the tissue specific role of OsGLP genes, the expression level of OsGLP genes was analyzed in leaves and roots of both varieties under control and stress (salt and drought) conditions. In general, results indicated same expression pattern of OsGLP genes in both rice varieties, however, the expression level of several OsGLP genes was different in leaves and roots (Fig. 5). For example, in both varieties, 3 OsGLP genes from chromosome number 8 including OsGLP8-4, OsGLP8-7 and OsGLP8-11 were observed with higher expression in roots in comparison with leaves. On the contrary, OsGLP3-6 and OsGLP4-1 were expressed at higher level in leaves as compared to roots. It is noteworthy that OsGLP8-12 which exhibited almost same expression pattern in both tissues was observed with higher expression level in Super Basmati roots as compared to leaves under salt stress. Among all OsGLP genes, OsGLP 8-4 was most prominent with highest level of expression in roots followed by OsGLP8-11 whereas OsGLP3-6 and OsGLP4-1 were observed with highest levels in leaves of both rice varieties.