The authors gratefully acknowledge financial support from th
The authors gratefully acknowledge financial support from the State of São Paulo Research Foundation (FAPESP, Fundação de Amparo à Pesquisa do Estado de São Paulo), grants 2013/07600-3 and 2013/25658-9, and the National Council for Scientific and Technological Development (CNPq, Conselho Nacional de Desenvolvimento Científico e Tecnológico) (465637/2014-0), Brazil.
Introduction Salt stress seriously affects the growth and development of plants. Thus, plants have developed a variety of sophisticated strategies to respond to salt stress. To uncover these resistance mechanisms, the effects of salt stress have been studied in various species, and many genes and proteins involved in salt tolerance, such as OsMYB3R-2, AtSOS1, and H+-Pyrophosphatase have been identified and characterized (Dai et al., 2007, Shi et al., 2003, Yamada et al., 2002). Cysteine proteases, also called thiol proteases, with a cysteine residue at the active center, play essential roles during plant growth and development by degrading endogenous proteins to ensure protein turnover, and are important in senescence and programmed cell death (Wyk et al., 2014). Programmed cell death (PCD), or apoptosis, is the physiological process through which animal GDC-0994 activate an essential self-destructive mechanism to destroy themselves (Vaux and Korsmeyer, 1999). Cysteine proteases have emerged as key proteins in the regulation of animal programmed cell death (PCD). In plants, PCD-associated programs are induced by various biotic and abiotic stresses, e.g., nutrient starvation, oxidative stress, salinity, drought, and heat. Damaged proteins or organelles in the cells are degraded for recycle. Meanwhile, these stresses enhance the accumulation of ROS and oxidized proteins. Cysteine proteases have been thought as crucial participation in relevant aspects of the degradation of oxidized proteins and the regulation of ROS levels (van der Hoorn, 2008). In addition, cysteine proteases are involved in responses to biotic and abiotic stresses. The expression of cysteine protease genes changes in plants suffering from osmotic stress, wounding and temperature extremes. The cysteine protease gene Cyp15a of pea (Pisum sativum L.) shows increased transcription and mRNA levels in plantlets under high salinity stress (Jones and Mullet, 1995). The cysteine protease genes RD21a and RD19a of Arabidopsis thaliana (L.) Heynh., belonging to papain family, are induced by water deficit and are responsive to salt stress (Hayashi et al., 2001, Koizumi et al., 1993, Yamaguchi-Shinozaki et al., 1992). Functional analysis of these proteases has shown that cysteine proteases play an important role in the programmed cell death pathway during stress (Chen et al., 2012, Lim et al., 2007, Yoshida, 2003). The expression of sweet potato papain-like cysteine protease gene SPCP2 is enhanced significantly in natural senescing leaves and in dark-, jasmonic acid-, abscisic acid-, ethephon-induced senescence leaves. The transgenic Arabidopsis of SPCP2 shows higher salt and drought stress tolerance compared to the control (Chen et al., 2010). The sweet potato cysteine protease SPCP3 ectopic expression enhances drought stress sensitivity in transgenic Arabidopsis (Chen et al., 2013). A lot of plant cysteine protease genes have been researched regarding their involvement in physiological defenses against abiotic stresses such as cold and drought, showing that cysteine protease genes function as important components of signaling pathways and are believed to play a key role in defense mechanisms against abiotic stress through some processes associated with programmed cell death. In our previous study, a salt stress-induced cDNA library from the halophyte Salix matsudana Koidz. was constructed using the SMART method (Yang et al., 2009). One of cDNAs cloned from the library shared 77% amino acid identity with thiol protease aleurain-like (Ricinus communis L.), and was named SmCP (S. matsudana cysteine protease; accession number KC715825). Here, the function of this gene was characterized and its role in salt stress was examined. Bioinformatics analysis and expression profiling of SmCP in S. matsudana were also conducted. The results of in vivo functional validation in Escherichia coli and A. thaliana revealed that SmCP is a functional PCD-associated gene and might play an important role in salt stress tolerance. This study provides a foundation for developing candidate gene resources that regulate salt tolerance and resistance in Salix and important theoretical basis for molecular breeding of this important forest tree.