Plastid-to-Nucleus Retrograde Signals Are Essential for the Expression of Nuclear Starch Biosynthesis Genes during Amyloplast Differentiation in Tobacco BY-2 Cultured Cells    

Abstract

Amyloplasts, a subtype of plastid, are found in nonphotosynthetic tissues responsible for starch synthesis and storage. When tobacco (Nicotiana tabacum) Bright Yellow-2 cells are cultured in the presence of cytokinin instead of auxin, their plastids differentiate from proplastids to amyloplasts. In this program, it is well known that the expression of nucleus-encoded starch biosynthesis genes, such as ADP-Glucose Pyrophosphorylase (AgpS) and Granule-Bound Starch Synthase (GBSS), is specifically induced. In this study, we investigated the roles of plastid gene expression in amyloplast differentiation. Microarray analysis of plastid genes revealed that no specific transcripts were induced in amyloplasts. Nevertheless, amyloplast development accompanied with starch biosynthesis was drastically inhibited in the presence of plastid transcription/translation inhibitors. Surprisingly, the expression of nuclear AgpS and GBSS was significantly repressed by the addition of these inhibitors, suggesting that a plastid-derived signal(s) that reflects normal plastid gene expression was essential for nuclear gene expression. A series of experiments was performed to examine the effects of intermediates and inhibitors of tetrapyrrole biosynthesis, since some of the intermediates have been characterized as candidates for plastid-to-nucleus retrograde signals. Addition of levulinic acid, an inhibitor of tetrapyrrole biosynthesis, resulted in the up-regulation of nuclear AgpS and GBSS gene expression as well as starch accumulation, while the addition of heme showed opposite effects. Thus, these results indicate that plastid transcription and/or translation are required for normal amyloplast differentiation, regulating the expression of specific nuclear genes by unknown signaling mechanisms that can be partly mediated by tetrapyrrole intermediates.Plastids are plant-specific, semiautonomous organelles that have important metabolic functions, such as photosynthesis, carbon and nitrogen assimilation, and biosynthesis of lipids and amino acids. In higher plants, plastids are known to differentiate between different subtypes in response to several developmental and environmental conditions. Each differentiated form of plastid plays a specific role in maintaining a variety of metabolic and physiological functions as a component of plant cells or tissues (for review, see Lopez-Juez and Pyke, 2005). For instance, chloroplasts, the most characterized form of plastid, are found in the majority of green tissues and are involved in photosynthesis. Chromoplasts occur in limited organs, such as fruit and floral petals, or root tissues in carrot (Daucus carota), and are engaged in the synthesis and storage of carotenoid pigments. Leucoplasts are colorless plastids mainly existing in root tissues. Several reactions for terpene biosynthesis take place in leucoplasts as well as in chloroplasts. In meristematic tissues, plastids can be identified as an undifferentiated form, termed proplastids, which are specialized for proliferation instead of any m...

Retrograde signals arise from reciprocal crosstalk within plastids

“…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][7][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.…”

“…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.…”

“…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).…”

“…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.…”

“…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 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.…”