Abstract
Plant chloroplasts have versatile thioredoxin systems including two thioredoxin reductases and multiple types of thioredoxins. Plastid-localized NADPH-dependent thioredoxin reductase (NTRC) contains both reductase (NTRd) and thioredoxin (TRXd) domains in a single polypeptide and forms homodimers. To study the action of NTRC and NTRC domains in vivo, we have complemented the ntrc knockout line of Arabidopsis with the wild type and full-length NTRC genes, in which 2-Cys motifs either in NTRd, or in TRXd were inactivated. The ntrc line was also transformed either with the truncated NTRd or TRXd alone. Overexpression of wild-type NTRC promoted plant growth by increasing leaf size and biomass yield of the rosettes. Complementation of the ntrc line with the full-length NTRC gene containing an active reductase but an inactive TRXd, or vice versa, recovered wild-type chloroplast phenotype and, partly, rosette biomass production, indicating that the NTRC domains are capable of interacting with other chloroplast thioredoxin systems. Overexpression of truncated NTRd or TRXd in ntrc background did not restore wild-type phenotype. Modeling of the three-dimensional structure of the NTRC dimer indicates extensive interactions between the NTR domains and the TRX domains further stabilize the dimeric structure. The long linker region between the NTRd and TRXd, however, allows flexibility for the position of the TRXd in the dimer. Supplementation of the TRXd in the NTRC homodimer model by free chloroplast thioredoxins indicated that TRXf is the most likely partner to interact with NTRC. We propose that overexpression of NTRC promotes plant biomass yield both directly by stimulation of chloroplast biosynthetic and protective pathways controlled by NTRC and indirectly via free chloroplast thioredoxins. Our data indicate that overexpression of chloroplast thiol redox-regulator has a potential to increase biofuel yield in plant and algal species suitable for sustainable bioenergy production.
Highlights
Thioredoxins (TRX) are crucial components of the regulatory redox networks in all living cells
We propose that overexpression of NADPH-dependent thioredoxin reductase (NTRC) promotes plant biomass yield both directly by stimulation of chloroplast biosynthetic and protective pathways controlled by NTRC and indirectly via free chloroplast thioredoxins
NTRC CONTENT IN TRANSGENIC OVEREXPRESSION LINES To study the function of NTRC domains in vivo, we complemented the ntrc line with a wild type NTRC gene (OE-NTRC lines) and with full-length genes, in which the redox-active 2-Cys motifs of the NTR polypeptide without TRX polypeptide (TRXd) (NTRd) (OE-SAIS lines) or TRXd (OE-SGPS lines) were inactivated by site-directed mutations (Figure 1)
Summary
Thioredoxins (TRX) are crucial components of the regulatory redox networks in all living cells. Plant chloroplasts have versatile thioredoxin systems including two reductases dependent on ferredoxin (FTR) and NADPH (NTR) as reducing power, respectively, and multiple types of TRXs (f, m, x, y, z, CDSP32; Buchanan and Balmer, 2005; Meyer et al, 2008; Jacquot et al, 2009; König et al, 2012). Plastid-localized NADPH-dependent thioredoxin reductase (NTRC) is a unique NTR enzyme constituting a thioredoxin system in a single polypeptide chain (Serrato et al, 2004; Pérez-Ruiz et al, 2006). The protein contains two redox-active 2-Cys motifs, CAIC in its NTR domain (NTRd) and CGPC in the TRX domain (TRXd; Serrato et al, 2004), and it is suggested to function as a dimer (Pérez-Ruiz and Cejudo, 2009; Pérez-Ruiz et al, 2009; Lee et al, 2012). Characterization of knockout lines of NTRC (ntrc) has indicated that NTRC is a crucial redox-regulator of a number of plastidial processes, including biogenesis of chloroplasts, biosynthetic pathways and ROS metabolism in chloroplasts
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