Mitochondrial proteins contribute to plastid retrograde signaling
The gun mutants are the best-characterized regulators of plastid retrograde signaling. Whereas GUN1 encodes a chloroplast nucleoid pentatricopeptide repeat protein (Koussevitzky, 2007), the other GUN proteins are involved in the tetrapyrrole biosynthetic pathway.GUN2 encodes a heme oxygenase (Strand et al., 2003), GUN3encodes a photosensitive pigment chromophore synthase (Susek et al., 1993), GUN4 encodes a regulatory protein involved in chlorophyll synthesis (Larkin et al., 2003), and GUN5 encodes the H subunit of magnesium chelatase (Mochizuki et al., 2001). GUN1 might also play a role in the tetrapyrrole pathway (Cheng et al., 2011). Because these GUN proteins are located in the chloroplast, they play a direct role in the retrograde chloroplast signaling pathway (Surpin, Larkin, & Chory, 2002). However, little is known regarding the roles of mitochondrial proteins in regulating plastid retrograde signaling.
The nucleus controls the majority of processes in chloroplasts, including organelle gene expression (OGE) via ‘anterograde signaling’. The nucleus, in turn, depends on the signals originating from the chloroplasts that convey information to the nucleus via ‘retrograde signaling’. This system allows for changes in nuclear gene expression (NGE) in response to the status of the chloroplast. We propose a model from our study in which environmental cues affect the chloroplast and mitochondrial state which in turn gives rise to retrograde signals that alter nuclear gene expression from the transcriptional to the post-translational level and ultimately feedback to plastid function (Figure 10). Initially, the environmental stimulus is perceived by the chloroplast. In this study, environmental factors include LIN, NF, and high light (shown by the yellow arrows) (Figure 10). After LIN treatment, the expression ofGUN4 and GUN5 was decreased to different degrees in the mutants, in which the expression of GUN1 increased relative to the control (Figure 8a). We also analyzed the expression of important mitochondria-related genes like ALTERNATIVE OXIDASE 1D(AOX1D ), AOX1A, AOX1C, and AOX2 in the mitochondria. The expression of AOX1D and AOX1A increased in WT seedlings treated with LIN, and the expression of these genes was decreased in SALK_047877 and SALK_083115 (Figure 8b). The expression of AOX2 was suppressed in all mutants treated with LIN (Figure 8b). Further, analysis of the expression of carbon metabolism genes revealed that 1-Aminocyclopropane-1-Carboxylate Oxidase 1(ACO1 ) was decreased in all groups, except in WT seedlings following LIN treatment; the expression of ACO2 , ACO3 ,hexokinase 1 (HXK1 ), and HXK2 was decreased to different degrees in all samples (Figure 8c-d), and the expression ofmitochondrial Malate Dehydrogenase 1 (mMDH1 ) was significantly inhibited in WT seedlings (Figure 9d). The transcript levels of GUN4 , GUN5 , AOX1C , HXK1 ,HXK2 , and mMDH1 , were higher in gun1 ,SALK_047877 , and SALK_083115 seedlings than in WT seedlings treated with LIN. In contrast, the transcript levels of the LIN-induced genes AOX2 , AOX1D , AOX1A , ACO1 , and ACO2 were lower in gun1 , SALK_047877 , andSALK_083115 , seedlings than in WT seedlings in the presence of LIN. In summary, these findings indicate that GUN1 andAT5G08670 dependent signaling pathways play important roles in regulating the expression of nuclear genes of both chloroplast and mitochondrial proteins in response to LIN (Figure 10).
The level of ATP synthase -subunit and the total ATP synthase activity in the mutants were significantly lower than in WT (Figure 4a and b). It is thus likely that the effects observed are due to changes in cellular ATP indicating that perturbation of ATP homeostasis in mitochondria affects not only mitochondrial but also chloroplast metabolism and retrograde signaling. Interactions between chloroplast and mitochondrial ATP metabolism have also been observed in Chlamydomonas in a suppressed strain of a chloroplast mutant lacking theatpB gene. This suppressed strain was able to grow photoautotrophically in the absence of chloroplast ATP synthase (Lemaire, Wollman, & Bennoun, 1988). In this strain, photosynthesis was sensitive to specific inhibitors of mitochondrial electron transport suggesting that photosynthesis was restored through an unusual interaction between mitochondria and chloroplast involving the export of reduced compounds from the chloroplast to mitochondria to stimulate the synthesis of mitochondrial ATP which in turn would be exported from the mitochondria to the chloroplast. It remains to be determined how ATP levels are sensed in mitochondria and chloroplasts and how perturbations in ATP homeostasis in either organelle are compensated.