Retrograde signals arise from reciprocal crosstalk within plastids

Abstract

In addition to the cell nucleus, plant cells also possess genomic DNA and gene expression machineries within mitochondria and plastids. In higher plants, retrograde transcriptional regulation of several nuclear genes encoding plastid-located proteins has been observed in response to changes in a wide variety of physiological properties in plastids, including organelle gene expression (OGE) and tetrapyrrole metabolism. This regulation is postulated to be accomplished by plastid-to-nucleus signaling,^1,2 although the overall signal transduction pathway(s) are not well characterized. By applying a specific differentiation system in tobacco Bright Yellow-2 (BY-2) cultured cells,^3,4 we recently reported that the regulatory system of nuclear gene expressions modulated by a plastid signal was also observed during differentiation of plastids into amyloplasts.⁵ While retrograde signaling from plastids was previously speculated to consist of several independent pathways, we found inhibition of OGE and perturbation in the cellular content of one tetrapyrrole intermediate, heme, seemed to interact to regulate amyloplast differentiation. Our results thus highlight the possibility that several sources of retrograde signaling in plastids could be integrated in an intraorganellar manner.

Introduction

In higher plants, plastids can differentiate in response to developmental and environmental cues. Amyloplasts are a subtype of plastid found in non-photosynthetic tissues characterized by starch synthesis and storage. Although many reports have studied plastid differentiation into chloroplasts in photosynthetic tissues, the differentiation steps into non-photosynthetic plastids such as amyloplasts and chromoplasts have not been well characterized. Tobacco BY-2 cells are normally cultured in a medium containing auxin, but exchanging auxin for cytokinin induces differentiation of proplastids into amyloplasts.⁴ Because the whole set of catalytic enzymes involved in starch metabolism are encoded by the nuclear genome, expression of starch biosynthesis genes, including ADP-glucose pyrophosphorylase small subunit (AgpS) and granule-bound starch synthase (GBSS), was actually enhanced during differentiation of plastids into amyloplasts.⁴ However, involvement of plastid gene expression as well as plastid-to-nucleus signaling in the course of amyloplast differentiation remained unclear.

In our recent study,⁵ we demonstrated transcription of nuclear starch biosynthesis genes was affected specifically by supplementation of inhibitors specific to organelle gene expression (OGE) and tetrapyrrole biosynthesis, which represents the presence of a retrograde signaling pathway(s) similar to that observed between the nucleus and chloroplasts as previously reported.^6-8 These two retrograde signals derived from OGE and tetrapyrrole metabolism influenced the efficiency of amyloplast differentiation via positive and negative effects, respectively, on expression of such nuclear genes. Intriguingly, the effects of simultaneous addition of levulinic acid (LA), an inhibitor of early tetrapyrrole biosynthesis, or heme itself, coupled with the OGE inhibitor rifampicin (Rif), appeared to be synergetic rather than independent. In previous studies, plastid signals were believed to be derived from a variety of physiological parameters and transmitted independently from chloroplasts to the nucleus.⁹ However, in a recent model for plastid signaling, the Arabidopsis GUN1 protein was regarded as the key plastid-localized molecule for mediation and integration of several plastid signals.^10-13 Although glutamyl-tRNA, an upstream precursor of the tetrapyrrole biosynthesis pathway, is also necessary for OGE (for a review see ref. 2), it is still unknown whether there is a direct linkage between plastid gene expression activity and tetrapyrrole metabolism. Here we address the question of whether multiple plastid signaling pathways may be interrelated during the signal transduction to the nucleus at amyloplast differentiation step.

Bidirectional Interaction between Heme Concentration and OGE Might Occur Prior to Transduction of Retrograde Signal

Rif selectively inhibits transcriptional activity of PEP, a plastid-encoded plastid RNA polymerase that shows a similar subunit composition to eubacterial RNA polymerase.¹⁴ In fact, mRNA levels of the PEP-dependent plastid-encoded genes psbA, atpA and rbcL decreased in BY-2 cells cultured for 24 h in the presence of 100 mg/L Rif (Fig. 1). Because another type of plastid RNA polymerase encoded in the nuclear genome, termed NEP (nuclear-encoded plastid RNA polymerase), is a bacteriophage-type single subunit enzyme and is insensitive to Rif, transcripts of the NEP-dependent rpoB gene accumulated under the same conditions. Interestingly, supplementation with 40 μM heme instead of Rif resulted in a more drastic reduction of total transcripts of both PEP- and NEP-dependent genes with the only exception for accumulation of the shortest processing product of psbA transcripts (Fig. 1). This result indicated that excess heme might perturb OGE by causing instability of the majority of, but not all, plastid mRNAs. Considering our previous data that decline of mRNA amounts in the presence of heme was not observed overall in nuclear genes,⁵ effects derived from increment in heme content seemed to be restricted to plastid transcription.

Figure 1. Heme supplementation affected expression status of both nuclear- and plastid-encoded genes. Total RNAs were extracted from tobacco BY-2 cells 24 h after inoculation into fresh medium. In the respective lanes, 10 μg (for AgpS), 5 μg (for psbA, atpA, rbcL and rpoB) or 2 μg (for EF-1α) was loaded. Lane 1, mock control (DMSO and methanol); lanes 2 and 3, supplementation with 100 mg/L Rif and 40 μM heme, respectively. Primer sets for probes are listed below or described previously⁵: 5′-GGCTCCCTATTCAGTGCTATGC-3′ and 5′-GAGGGAAGTTGTGAGCATTACG-3′ for psbA; 5′-TGTGGGTATCTCCGTTTCCAG-3′ and 5′-GGGCTTCTGCTTCCTCGGTAA-3′ for atpA; 5′-GTGTTCTACCCGTGGCTTCAG-3′ and 5′-CTTATCCAAAACGTCCACTGCTGC-3′ for rbcL; 5′-ATTTGATGGAAGGACGGGGA-3′ and 5′-GATCGTAGTTCTCGAACGAG-3′ for rpoB.

To test whether OGE inhibition might affect tetrapyrrole metabolism, we monitored changes in heme amounts in response to Rif supplementation. Quantification of the cellular heme level was performed as reported previously.^5,15 A significant increase in subcellular heme content was observed when Rif was added to the cytokinin-containing medium, compared with that in the mock control (Table 1). Rif-dependent accumulation of cellular heme was canceled or even repressed by combined supplementation of Rif and LA, whereas no obvious change was detected in the presence of LA alone (Table 1). These results were consistent with our previous data that indicated moderate and synergetic effects of these two inhibitors on amyloplast differentiation via regulation of AgpS and GBSS gene expression.⁵

Table 1. Quantification of heme contents.

B mock	B+Rif	B+LA	B+Rif+LA
589.11 ± 89.6	865.64 ± 125.66	568.57 ± 72.41	452.21 ± 93.4
			pmol g-1 FW

Tobacco BY-2 cells cultured for 24 h in medium supplemented individually or simultaneously with 100 mg/L Rif and 500 μM LA were used for quantitative analysis of heme content. Extraction and measurement of cellular heme in the cells were performed as described previously.^5,15 The data represent the mean ± SD of three independent experiments.

Based on all of the above analyses, the following model can be proposed: the degree in downregulation of nuclear starch biosynthesis genes and resulting inhibition of amyloplast development was highly correlated with the concurrent plastidic profiles of low OGE activity and high heme levels. Woodson et al.¹⁶ recently reported the heme level within plastids was also important in regulation of photosynthesis-associated nuclear genes that promote chloroplast development. Considering these data and with regard to steps of plastid differentiation into amyloplasts and chloroplasts, regulation of the heme content in plastids seems to be essential for plastid-to-nucleus signaling to monitor overall intraplastid processes including tetrapyrrole metabolism and even plastid gene expression.

Enami K, Ozawa T, Motohashi N, Nakamura M, Tanaka K, Hanaoka M. Plastid-to-nucleus retrograde signals are essential for the expression of nuclear starch biosynthesis genes during amyloplast differentiation in tobacco BY-2 cultured cells. Plant Physiol. 2011;157:518–30. doi: 10.1104/pp.111.178897.