Characterization of plastidial thioredoxins from Arabidopsis belonging to the new y-type

Abstract

The plant plastidial thioredoxins (Trx) are involved in the light-dependent regulation of many enzymatic activities, owing to their thiol-disulfide interchange activity. Three different types of plastidial Trx have been identified and characterized so far: the m-, f-, and x-types. Recently, a new putative plastidial type, the y-type, was found. In this work the two isoforms of Trx y encoded by the nuclear genome of Arabidopsis (Arabidopsis thaliana) were characterized. The plastidial targeting of Trx y has been established by the expression of a TrxGFP fusion protein. Then both isoforms were produced as recombinant proteins in their putative mature forms and purified to characterize them by a biochemical approach. Their ability to activate two plastidial light-regulated enzymes, NADP-malate dehydrogenase (NADP-MDH) and fructose-1,6-bisphosphatase, was tested. Both Trx y were poor activators of fructose-1,6-bisphosphatase and NADP-MDH; however, a detailed study of the activation of NADP-MDH using site-directed mutants of its regulatory cysteines suggested that Trx y was able to reduce the less negative regulatory disulfide but not the more negative regulatory disulfide. This property probably results from the fact that Trx y has a less negative redox midpoint potential (-337 mV at pH 7.9) than thioredoxins f and m. The y-type Trxs were also the best substrate for the plastidial peroxiredoxin Q. Gene expression analysis showed that Trx y2 was mainly expressed in leaves and induced by light, whereas Trx y1 was mainly expressed in nonphotosynthetic organs, especially in seeds at a stage of major accumulation of storage lipids.

Figure 1.

Intracellular localization of putative cpTrxs. Subcellular localization of premature Trx-fused GFP proteins was monitored by confocal microscopy in Arabidopsis cell protoplasts. Trx types are indicated in the figures. For each Trx type transmission and green fluorescent images are shown side-by-side. GFP, nonaddressed GFP. o1, m2, y2, premature forms of the corresponding Trx isoforms fused to GFP.

Figure 2.

Efficiencies of various plastidial thioredoxins in the activation of a mutant NADP-MDH where only the N-terminal disulfide is left (mutant C207A/C365A/C377A). Mutant NADP-MDH was activated in the presence of either 10 mm DTT (⋄), or DTT plus 20 μm Trxs f1 (□), or m1 (○), or y1 (▿), or y2 (▾). Aliquots (5 μg of enzyme) were withdrawn periodically for activity measurements.

Figure 3.

Activation kinetics of wild-type sorghum NADP-MDH with 1 μm Trx f1, with or without 20 μm Trx y1. Trx y1 alone (▿); Trx f1 alone (•); Trx f1 and Trx y1 added at the beginning of activation (▾); preincubation with DTT-reduced 20 μm Trx y1 for 10 min, then addition of 1 μm Trx f1 (○). Aliquots (5 μg of enzyme) were withdrawn periodically for activity measurements.

Figure 4.

Activity of 2-Cys PRX B with various Trxs as proton donors. The enzyme (20 μm) 2-Cys Prx B was incubated with 400 μm DTT, 400 μm t-BOOH, and 10 μm Trx. The reduction of t-BOOH was measured by colorimetric titration and expressed as the remaining percentage of unreduced reagent. Control without Trx (⋄) or with Trx f1 (□), m1 (○), y1 (▿), y2 (▾), and x (▵). The actual initial rates of reduction, expressed in μmoles t-BOOH reduced min⁻¹ μmol⁻¹ Prx were, respectively, 0.37 (without Trx), 0.78 (Trx y1 or y2), 1.25 (Trx m1 or f1), and 10.00 (Trx x).

Figure 5.

Activity of Prx Q with various Trxs as electron donors. Prx Q (5 μm) was incubated with 400 μm DTT, 400 μm t-BOOH, and 10 μm Trx. The reduction of t-BOOH was measured by colorimetric titration and expressed as the remaining percentage of unreduced reagent. DTT alone or Trx f1 (⋄), m4 (•), y1 (▿), y2 (▾), and x (▵). The actual initial rates of reduction, expressed in μmoles t-BOOH reduced min⁻¹ μmol⁻¹ Prx were, respectively, 2.0 (without Trx), 5.0 (Trx m4), 8.7 (Trx x), and 12.5 (Trx y1 or y2).

Figure 6.

Expression of Trx y genes in different plant organs and in dependence on light conditions. RNA levels were evaluated by semiquantitative RT-PCR using primers specific for Trx genes and reference gene (Actin 2). mRNA levels of Trx f isoforms are shown for comparison. A, RNA from R, roots; L, leaves; S, seeds; YS, young siliques; MS, mature siliques; and Fl, flowers were analyzed. B, After 72 h of dark adaptation, plants were transferred to white light and leaf RNA levels analyzed after 1, 3, and 8 h of exposure to light (180 μE m⁻² s⁻¹). Optimized number of PCR cycles is indicated in brackets.

Figure 7.

Expression of y-type Trxs in Arabidopsis seeds. Semiquantitative RT-PCR with gene-specific primers for tubulin β 9 (constitutive), AtTRXy1, and AtTRXy2 were used to examine y-type-TRX gene expression in Arabidopsis developing seeds: 6 to 8, 12 to 13, or 18 to 20 DAF; in resting seeds (R); and in seeds after 24 and 48 h imbibition with water. A, Agarose gel. B, Densitometric analysis. Black bars, AtTRXy1; gray bars, AtTRXy2. A typical expression profile is presented.