Characterization of the HAK gene and its expression during in vitro germination and seedlings stage of two avocado varieties under salinity stress
Abstract
Soil salinity, a consequence of human and climatic activities, is one of the main challenges facing modern agriculture. Avocado is classified as glycophyte and is very sensitive to salt stress. There are avocado ecological varieties that show different salt tolerance levels. Intracellular potassium (K+) homeostasis is crucial for plant survival in saline environments. K+ channels and some members of the high-affinity K+ transporter HAK/KUP/KT (high-affinity K+) family are believed to function in the regulation of plant salt tolerance. Methods: In vitro seed germination, seedling growth in two ecological avocado varieties; americana and drymifolia were evaluated under different concentrations of NaCl. Physiomorphological variables were evaluated in two seedlings varieties. The concentration of sodium and potassium ions was evaluated by plasma-optical emission spectrometry. The characterization of the PaHAK2 gene was carried out by bioinformatic methods and its expression was evaluated by real-time quantitative PCR. Results: Salinity delay the germination speed of the drymifolia variety with respect to the americana variety. However, both varieties managed to germinate in vitro at 98-100% after 15 days. Salinity affected the physiomorphological variables evaluated in the seedlings after 30 days of growth in the different concentrations of NaCl. The RL/SL index showed significant differences between the two varieties. The K+/Na+ ratio decreased drastically and depended on the NaCl concentration in both varieties. The phylogenetic tree constructed with the sequence of the PaHAK2 gene and its plant orthologs showed 5 well-defined clusters. PaHAK2 gene from avocado is associated with sequences from basal angiosperms. These results helped us to make a computational 3D inference protein model. The most determining factor was the conserved transmembrane domains in the HAK families, observing that for PaHAK2, showed 10 conserved transmembrane domains. During seed germination, no increase in the expression of the PaHAK2 gene was detected and only a drastic increase in its expression was detected in drymifolia variety seedlings at a concentration of 60 mM NaCl. Conclusion: Our data suggest that the drymifolia variety is more sensitive to salinity than the americana variety. The seeds of both varieties can germinate under saline conditions. The americana variety has better physiomorphological variables that allow it to better tolerate salt stress. The PaHAK2 gene showed similarity to orthologs of eudicot plants even though avocado is a basal angiosperm, also presenting conserved domains of the HAK/KUP/KT ion transporters. The expression of the PaHAK2 gene suggests that it does not participate in the germination process in saline conditions of seeds of two avocado varieties and there is only an increase in its expression in the root and aerial part in seedlings of the drymifolia variety at the concentration of 60 mM.