Light regulates ascorbic acid (AsA) synthesis, which increases in the light, presumably reflecting a need for antioxidants to detoxify reactive molecules produced during photosynthesis. Here, we examine this regulation in Arabidopsis thaliana and find that alterations in the protein levels of the AsA biosynthetic enzyme GDP-Man pyrophosphorylase (VTC1) are associated with changes in AsA contents in light and darkness. To find regulatory factors involved in AsA synthesis, we identified VTC1-interacting proteins by yeast two-hybrid screening of a cDNA library from etiolated seedlings. This screen identified the photomorphogenic factor COP9 signalosome subunit 5B (CSN5B), which interacted with the N terminus of VTC1 in yeast and plants. Gel filtration profiling showed that VTC1-CSN5B also associated with the COP9 signalosome complex, and this interaction promotes ubiquitination-dependent VTC1 degradation through the 26S proteasome pathway. Consistent with this, csn5b mutants showed very high AsA levels in both light and darkness. Also, a double mutant of csn5b with the partial loss-offunction mutant vtc1-1 contained AsA levels between those of vtc1-1 and csn5b, showing that CSN5B modulates AsA synthesis by affecting VTC1. In addition, the csn5b mutant showed higher tolerance to salt, indicating that CSN5B regulation of AsA synthesis affects the response to salt stress. Together, our data reveal a regulatory role of CSN5B in light-dark regulation of AsA synthesis.
SUMMARYThe phytohormones abscisic acid (ABA) and ethylene are known to play multiple roles in plant development and stress responses. Ethylene biosynthesis is affected by several factors including drought, cold and the phytohormone auxin, although the role of ABA is unclear. In this work ABA-responsive mutants were screened and a bZIP transcription factor HY5 was identified as a negative regulator of ethylene biosynthesis via modulation of the expression of the ethylene biosynthesis genes ACS2 and ACS5. Members of the ethylene response factor (ERF) family of transcriptional repressors in Arabidopsis have been shown to modulate ABA responses and three ERF members were found to carry putative HY5-binding cis-acting elements. Analyses with biochemical and molecular approaches revealed that HY5 specifically binds to the G-box region of the AtERF11 promoter to activate its transcription. We further demonstrate that AtERF11, which contains a repressor motif at its C-terminal, interacts with the dehydration-responsive element in the ACS2/5 promoters, to repress its expression, resulting in decreased ethylene biosynthesis. Moreover, an AtERF11 knockout mutant showed increased levels of ACS2/5 expression and ethylene emission, while treatment with ABA greatly suppressed ACS5 transcripts but not ACS2 expression and the ethylene content, indicating that AtERF11 is a key negative regulator for ABA-mediated control of ethylene synthesis. In addition, in ethylene over-producer mutants, ABA treatment was shown to suppress ACS5 transcripts and ethylene content, thereby affecting growth and development. Based on these data, in this research we present a model suggesting that the HY5-AtERF11 regulon is a key factor modulating ABA-regulated ethylene biosynthesis.
In the dark, etiolated seedlings display a long hypocotyl, the growth of which is rapidly inhibited when the seedlings are exposed to light. In contrast, the phytohormone ethylene prevents hypocotyl elongation in the dark but enhances its growth in the light. However, the mechanism by which light and ethylene signalling oppositely affect this process at the protein level is unclear. Here, we report that ethylene enhances the movement of CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1) to the nucleus where it mediates the degradation of LONG HYPOCOTYL 5 (HY5), contributing to hypocotyl growth in the light. Our results indicate that HY5 is required for ethylene-promoted hypocotyl growth in the light, but not in the dark. Using genetic and biochemical analyses, we found that HY5 functions downstream of ETHYLENE INSENSITIVE 3 (EIN3) for ethylene-promoted hypocotyl growth. Furthermore, the upstream regulation of HY5 stability by ethylene is COP1-dependent, and COP1 is genetically located downstream of EIN3, indicating that the COP1-HY5 complex integrates light and ethylene signalling downstream of EIN3. Importantly, the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) enriched the nuclear localisation of COP1; however, this effect was dependent on EIN3 only in the presence of light, strongly suggesting that ethylene promotes the effects of light on the movement of COP1 from the cytoplasm to the nucleus. Thus, our investigation demonstrates that the COP1-HY5 complex is a novel integrator that plays an essential role in ethylene-promoted hypocotyl growth in the light.
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