Subtilisin-like serine proteases involved in N remobilization during grain filling in wheat

Abstract

The induction of two subtilisin-like proteases (P1 and P2) associated with stress-induced senescence in young plants was investigated in adult wheat plants during the grain-filling period. Western blot analysis of flag leaf extracts showed that P1 was induced very late in the life cycle of the plants (9 days post-anthesis) and that 7 days later it reached a 2.5-fold increase with respect to the initial value at anthesis. On the other hand, the P2 signal was already detected previous to anthesis and increased soon after anthesis, reaching a fourfold increase by the end of the grain-filling period. The induction of P1 and P2 temporally correlates with the degradation of the Rubisco small and large subunits in the flag leaf, as well as with nitrogen (N) accumulation in the ears. At the same time, a decrease in the endogenous concentration of the cytokinins isopentenyladenine and isopentenyladenosine (iP ? iPA) in the leaves was observed. In detached leaves senescing in the dark, the levels of both proteases were affected by 6-benzylaminopurine application: the induction of P1 was completely prevented, whereas the induction of P2 was reduced. Our findings demonstrate that both P1 and P2 are expressed in leaves of adult plants and are induced during natural senescence. These results enable us to postulate their participation in N remobilization to developing grains during monocarpic senescence and their regulation by a cytokinin-mediated mechanism.

Subtilases – versatile tools for protein turnover, plant development, and interactions with the environment

“…Both the 67 and 54 kDa forms are active and exhibit broad substrate specificity with peptide and protein substrates, accepting a variety of residues in P1 and P1′ positions (Uchikoba et al 1995, Arima et al 2000c, Yonezawa et al 2000). Enzymes with similar characteristics (broad substrate specificity, high level of expression and stability) have been reported from many plant species including cucumisins from other cucurbits (Curotto et al 1989, Uchikoba et al 1998), green malt (germinating barley; Terp et al 2000, Fontanini and Jones 2002), tung fruits (Dyer et al 1999), soybean seeds (Beilinson et al 2002) and senescing wheat leaves (Roberts et al 2003, 2011). Similar properties have been reported for macluralisin from the fruits of Maclura pomifera (Rudenskaya et al 1995), taraxalisin, indicain, benghalensin and carnein in the latex of dandelion roots, mulberry trees, Banyan trees and morning glory (Rudenskaya et al 1998, Patel et al 2007, Singh et al 2008, Sharma et al 2009), as well as several other cucumisin‐like subtilisins in the latices of various Euphorbiaceae (Lynn and Clevette‐Radford 1988, Arima et al 2000a, Shimada et al 2000, Domsalla and Melzig 2008) and in bamboo shoots (Arima et al 2000b).…”

Nitrogen remobilization and conservation, and underlying senescence‐associated gene expression in the perennial switchgrass Panicum virgatum

“…We identified several SAG genes encoding cysteine, serine, or aspartate proteases in both switchgrass leaves and stems (Table S4). In wheat, serine proteases were reported to be responsible for much of the protein degradation that occurs during senescence of flag leaves, which drives N remobilization during grain filling (Chauhan et al, 2009;Roberts et al, 2011). Inhibition of cysteine proteases with inhibitors in wheat and tobacco (Nicotiana tabacum) leaves resulted in an impaired Rubisco degradation, indicating the involvement of cysteine proteases in degradation of plastid proteins (Thoenen et al, 2007;Prins et al, 2008).…”

Senescence‐associated proteases in plants

“…In addition, two subtilisin‐like proteases (P1 and P2) belonging to the family S8 of SPs according to the MEROPS classification (Rawlings et al 2010) were demonstrated to be strongly associated to senescence in wheat. Induction of both proteases was shown in wheat leaves under dark‐ and N starvation‐induced senescence (Roberts et al 2006) and more recently, in the flag leaf of naturally senescing plants, where they were suggested to participate in Rubisco degradation and N remobilization to developing grains (Roberts et al 2011). Also in barley it has been shown that subtilases are highly expressed under natural and steam girdling‐induced senescence (Parrott et al 2007).…”