Light signalling pathways regulating the Mg-chelatase branchpoint of chlorophyll synthesis during de-etiolation in Arabidopsis thaliana

Abstract

Precise regulation of tetrapyrrole synthesis is critical for plant survival when seedlings first emerge into the light. At this time there is a massive increase in demand for chlorophyll to drive the assembly of the photosynthetic apparatus. To understand how this demand is met we have followed the expression of genes encoding the chelatase enzymes at the branchpoint between chlorophyll and heme synthesis. Dark-grown Arabidopsis thaliana seedlings were transferred to continuous white, red, far-red or blue light and the expression of eight tetrapyrrole pathway genes was followed using real-time RT-PCR. Our results show that the CHLH gene encoding the H subunit of Mg-chelatase was induced by light under all conditions with an initial peak after 2-4 h light. The other Mg-chelatase subunit genes CHLI and CHLD and the ferrochelatase genes FC1 and FC2 were not strongly regulated at the level of transcript abundance, but the Mg-chelatase regulator GUN4 had an expression profile almost identical to that observed for CHLH. The CHLM gene encoding Mg-protoporphyrin IX methyltransferase, the next enzyme in the pathway, was also light regulated, but showed a very different pattern of expression. Using photoreceptor mutants it was demonstrated that regulation of CHLH and GUN4 is primarily under the control of phytochromes A and B with some input from the cryptochromes. Induction of CHLH and GUN4 under red and far-red light was also compromised in the phytochrome-signalling mutants, fhy1 and fhy3. These results establish GUN4 as a major target of photoreceptor regulation during the earliest stages of de-etiolation.

GLK Transcription Factors Coordinate Expression of the Photosynthetic Apparatus in Arabidopsis

“…Interestingly, expression of the two other subunits of Mgcheletase, CHLD and CHLI, was unchanged, a finding consistent with other work, indicating that transcriptional regulation of this step is achieved through CHLH and GUN4 (Matsumoto et al, 2004;Stephenson and Terry, 2008).…”

“…This is consistent with the well-characterized PIF-dependent pathways and microarray studies showing that both phyA and phyB regulate GLK transcription (Tepperman et al, 2006). Furthermore, photosynthetic gene expression is especially compromised in glk1 glk2 mutants in blue light, which suggests that cryptochromes also regulate GLK activity and is consistent with the role of cryptochromes in regulating chlorophyll biosynthesis (Stephenson and Terry, 2008). Experiments to investigate these interactions more precisely are underway.…”

PIF3 is a repressor of chloroplast development

“…The response in the pif1pif3 double mutant was again equivalent to both single mutants combined. We also analyzed 2 additional genes shown to be key regulatory targets in the tetrapyrrole pathway, CHLH encoding the H subunit of Mg chelatase and the chelatase regulator GUN4 (25)(26)(27). Both genes showed a similar pattern, with the strongest peak 3 d after germination, high expression after 2 d, and little induction if any after 4 d (Fig.…”

“…The rate-limiting step for Pchlide (and chlorophyll) synthesis is the enzyme glutamyl tRNA reductase (24). Light regulation of this step is mediated through changes in expression of the HEMA1 gene (34), and HEMA1 is one of a small group of highly regulated tetrapyrrole genes including CHLH and GUN4 (25,27). The substantial increase in HEMA1 expression and consequent increase in glutamyl tRNA reductase protein can fully account for the observed increase in Pchlide levels in the pif1 and pif3 mutants.…”

“…Chlorophyll was measured as described in ref. 27. Cotyledon samples for transmission electron microscopy were prepared and examined as described in ref.…”