Translation of chloroplast psbA mRNA is regulated by signals initiated by both photosystems II and I

Abstract

Light controls the translation of several mRNAs in fully developed chloroplasts via at least two regulatory pathways. In the first, the light signal is transduced as a thiol-mediated signal that modulates translation in parallel to light intensity. The second light-controlled pathway, termed priming, is a prerequisite to the thiol-mediated regulatory pathway. Light regulation is rapid and requires intrachloroplast photoreceptor(s). To delineate the signaling pathways controlling each of these regulatory events, we assayed the effect of photosynthetic inhibitors and electron donors on the translation of chloroplastic psbA mRNA. We show that the thiol-mediated signal is generated by photosystem I and transduced by vicinal dithiol-containing proteins. We also found that the priming signal probably initiates on reduction of plastoquinone. These findings suggest that translation of chloroplast psbA mRNA is controlled by both linear photosynthetic electron transport, exerted by the reduction of the ferredoxin-thioredoxin system, and the relative activities of photosystems I and II, signaled by the redox state of the plastoquinone pool. These data underscore the function of the light-capturing reactions of photosynthesis as chloroplast photoreceptors.

Figure 1

DTT restores DBMIB-inhibited light-regulated translation of chloroplast mRNAs but not activity of PS II. (A) Dark-adapted intact chloroplasts were treated in the dark with or without 2.5 μM DBMIB, then an aliquot was transferred for 5 min to the light (150 μmol m⁻²⋅sec⁻¹). The translational activities of the dark- and light-incubated chloroplasts were then assayed by labeling newly synthesized proteins for 5 min with [³⁵S]methionine, in the presence or absence of 5 mM DTT. Translation reactions were chased by incubating for an additional 5 min in the presence of 5 mM nonradioactive methionine and stopped by placing tubes in liquid N₂. Chloroplasts were lysed, and extracted proteins were fractionated by SDS/PAGE, blotted onto nitrocellulose membranes, and visualized by autoradiography. (B) Net oxygen exchange was measured in C. reinhardtii cw15 cells with an oxygen electrode. Measurements were performed for 5 min in the dark or light (150 μmol m⁻²⋅sec⁻¹) in the presence or absence of 2.5 μM DBMIB, and then for an additional 5 min with or without 5 mM DTT. (C) Initiation of translation is required for light-regulated translation of chloroplast mRNAs. Dark-adapted intact chloroplasts were treated with or without 15 μM lincomycin (Lync.), then an aliquot was transferred for 5 min to the light (150 μmol m⁻²⋅sec⁻¹), and treatment followed as in A.

Figure 2

Reversal of DBMIB inhibition of chloroplast proteins synthesis by PS I electron transport. Translation reactions of intact chloroplasts were performed in the dark or light, in the presence or absence of 5 μM DBMIB. The inhibition of PS I electron transport by the photosynthetic inhibitor DBMIB was bypassed by incubating with increasing micromolar concentrations of the PS I electron donor DCPIP, as indicated above each lane. Intact chloroplasts translation reactions were performed as in Fig. 1A.

Figure 3

Vicinal dithiol inhibitor blocks the stimulatory effect of PS I electron transport on chloroplast mRNA translation. Translation reactions of intact chloroplasts were performed as in Fig. 1A, in the dark or light, (A) in the presence or absence of micromolar concentrations of PAO, as indicated above each lane, and (B) in the presence or absence of 5 μM DBMIB, 100 μM DCPIP, or 25 μM PAO.

Figure 4

DCMU inhibition of chloroplast proteins synthesis is not reversed by PS I electron transport. Translation reactions of intact chloroplasts were performed, as in Fig. 1A, in the dark or light, (A) in the presence or absence of 5 mM DTT and 5 μM DCMU, and (B) in the presence or absence of 4 μM DCMU or 100 μM of the PS I electron donor DCPIP.

Figure 5

Model of light-signaling control of translation. Translation of several chloroplast mRNAs is regulated in response to light via at least two major pathways. In the first, the reduced PQ pool activates a signal-transduction pathway that leads to the priming of the translational factors required for thiol-mediated modulation of translation. In the second, a thiol-mediated signal, proportional to the reducing potential generated by PS I, is transduced by Fd, Fd–thioredoxin reductase (FTR), and thioredoxin (Trx) to stimulate translation. DCMU binds to the Q_B site of PS II, resulting in oxidation of the PQ pool. DBMIB inhibits cytochrome b₆f (Cyt bf), leading to increased reduction of the PQ pool. DCPIP is an artificial electron donor of PS I that can be used to allow electron transport through PS I under conditions of inhibited PS II to I electron transport.