Identification and expression analysis of 11 subtilase genes during natural and induced senescence of barley plants

Subtilases are one of the largest groups of the serine protease family and are involved in many aspects of plant development including senescence. In wheat, previous reports demonstrate an active participation of two senescence-induced subtilases, denominated P1 and P2, in nitrogen remobilization during whole plant senescence. The aim of the present study was to examine the participation of subtilases in senescence-associated proteolysis of barley leaves while comparing different senescence types. With this purpose, subtilase enzymatic activity, immunodetection with a heterologous antiserum and gene expression of 11 subtilase sequences identified in barley databases by homology to P1 were analyzed in barley leaves undergoing dark-induced or natural senescence at the vegetative or reproductive growth phase. Results showed that subtilase specific activity as well as two inmunoreactive bands representing putative subtilases increased in barley leaves submitted to natural and dark-induced senescence. Gene expression analysis showed that two of the eleven subtilase genes analyzed, HvSBT3 and HvSBT6, were up-regulated in all the senescence conditions tested while HvSBT2 was expressed and up-regulated only during dark-induced senescence. On the other hand, HvSBT1, HvSBT4 and HvSBT7 were down-regulated during senescence and two other subtilase genes (HvSBT10 and HvSBT11) showed no significant changes. The remaining subtilase genes were not detected. Results demonstrate an active participation of subtilases in protein degradation during dark-induced and natural leaf senescence of barley plants both at the vegetative and reproductive stage, and, based on their expression profile, postulate HvSBT3 and HvSBT6 as key components of senescence-associated proteolysis.