Expression analysis of Protease inhibitors in potato plants exposed to different environmental conditions

Abstract

Analysis of the potato genome (SPUD DB) allowed us to identify 142 protease inhibitors (PIs); 42 PI genes are clustered in tandem in chr.3, position 43 Mbp (3.43): 14 encode Kunitz-type PIs (KTIs), 4 encode trypsin type PIs (TPI) and 1 encodes a cystein PI (CPI), and at position 48 MBp (3.48), 10 genes encode KTIs, 8 encode PIN-II and 5 encode serine type PI (PIN-I). On the other hand, 11 genes encoding 5 KTIs, 3 cystatin B, and 3 PIN-I are grouped in Chr. 6, and 12 PIN-I genes are in chr. 9. This multigene family regulates protein turn-over preventing catabolism of essential proteins during metabolic processes and plays a role in the defense against heterologous proteases from pathogens and pests. PIs have been proposed as an alternative to chemical pesticides for the control of herbivorous insects, and as abiotic stress-protective factors. Degenerate primers were designed against five PIs (Soltu.DM.03G018480, -G018520, -G018580, - G018620, -G018650) of 3.43, seven PIs (Soltu.DM.03G023490, -G023500, -G23510, -G23520, -G23530, - G23540, and -G024110) of 3.48, and seven PIs of Chr. 9 (Soltu.DM.09G025840, -G025850 -G025860 - G025880, -G025900, -G025910, and -G025930) that share at least 83.46, 55,2 and 77,53 % of nucleotide identity. According to RNAseq data these three gene clusters are induced upon drought, salt, mannitol and N2 fertilization, and are downregulated during tuber dormancy release. To confirm the RNAseq data, RT-qPCR assays were performed using RNAs from in-vitro plants cultured under control conditions or a) with different NH4+ and NO3- concentrations, b) exposed during 1 week to continuous darkness, and c) to 150 mM NaCl during 24 and 96 h. In addition, RNA was extracted from greenhouse plants exposed to drought conditions. To analyze, the role of PIs during source-sink processes, RNA was obtained from dormant and sprouting tubers, from etiolated and green sprouts, and from young, fully developed and senescent leaves. In the future we aim to characterize one gene of each cluster in overexpressing or knockdown plants to elucidate their potential as biotechnological tools.