Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Jun 14;114(4):72.
doi: 10.1007/s11103-024-01469-2.

A cytosol-tethered YHB variant of phytochrome B retains photomorphogenic signaling activity

Wei Hu  1 , J Clark Lagarias  2
Affiliations

A cytosol-tethered YHB variant of phytochrome B retains photomorphogenic signaling activity

Wei Hu et al. Plant Mol Biol. .

Abstract

The red and far-red light photoreceptor phytochrome B (phyB) transmits light signals following cytosol-to-nuclear translocation to regulate transcriptional networks therein. This necessitates changes in protein-protein interactions of phyB in the cytosol, about which little is presently known. Via introduction of a nucleus-excluding G767R mutation into the dominant, constitutively active phyBY276H (YHB) allele, we explore the functional consequences of expressing a cytosol-localized YHBG767R variant in transgenic Arabidopsis seedlings. We show that YHBG767R elicits selective constitutive photomorphogenic phenotypes in dark-grown phyABCDE null mutants, wild type and other phy-deficient genotypes. These responses include light-independent apical hook opening, cotyledon unfolding, seed germination and agravitropic hypocotyl growth with minimal suppression of hypocotyl elongation. Such phenotypes correlate with reduced PIF3 levels, which implicates cytosolic targeting of PIF3 turnover or PIF3 translational inhibition by YHBG767R. However, as expected for a cytoplasm-tethered phyB, YHBG767R elicits reduced light-mediated signaling activity compared with similarly expressed wild-type phyB in phyABCDE mutant backgrounds. YHBG767R also interferes with wild-type phyB light signaling, presumably by formation of cytosol-retained and/or otherwise inactivated heterodimers. Our results suggest that cytosolic interactions with PIFs play an important role in phyB signaling even under physiological conditions.

Keywords: Cytoplasmic phytochrome signaling; Light-independent phyB signaling; Photomorphogenesis; Plant photoreceptors; Subcellular localization.

PubMed Disclaimer

Conflict of interest statement

There are no conflicts of interest or competing interests.

Figures

Fig. 1
Fig. 1
The G767R mutation strongly, yet incompletely suppresses the constitutive activity of YHB in the phyABCDE mutant background. A Immunoblot analyses of YHBG767R/phyABCDE lines. B Phenotypic comparison of 4-day-old seedlings grown in true darkness or under continuous red light (50 µmol m−2 s−1); # denotes YHBG767R seedlings with etiolated morphology. C Quantification of hypocotyl lengths, cotyledon separation angles and cotyledon areas of two overexpressed YHBG767R/phyABCDE lines (#10, #16) and two less expressed lines (#9, #14) in comparison to other control genotypes; *denotes statistical significance compared to phyABCDE grown under the same condition (p < 0.0001; Student’s t-test), ns, not statistically significant; n =  ~ 30. D Immunoblot analyses of dark-grown seedlings reveal significant, but not complete loss of PIF3 protein by YHBG767R in the overexpressing line #16 and the less expressing line #14. E YHBG767R promotes light-independent seed germination; six transgenic lines were used for YHBG767R and three replicates for other genotypes, each replicate used ~ 100 seeds. F Circular histograms depict seedling growth directions in darkness or under continuous red light (Rc50); YHBG767R data were from two independent lines, n =  ~ 100
Fig. 2
Fig. 2
YHBG767R is localized in cytoplasm. A Confocal fluorescence microscopy reveals the absence of nuclear photobodies in YHBG767R/phyABCDE grown in the dark or under continuous red light, whereas YHB/phyABCDE displays steady nuclear photobodies; Chl., chlorophyll autofluorescence. B Fractionation immunoblotting reveals cytosolic distribution of YHBG767R from light-grown transgenic plants. T, total soluble protein; C, cytoplasmic fraction; N, nuclear fraction. RPII, RNA Polymerase II
Fig. 3
Fig. 3
YHBG767R and PHYBG767R complement the phyB-5 mutant in a dosage-dependent manner. A Immunoblot analysis of nine genetically single-insertion homozygous transgenic lines. B Four-day-old seedlings grown in continuous red light (50 µmol m−2 s−1) or in darkness. * denotes transgenic lines with discernable shorter hypocotyls than phyB-5 mutants; #denote lines with discernable cop phenotype in the dark. C Quantification (mean ± S.D.) of hypocotyl lengths (top) and cotyledon sizes (bottom) of Rc50-grown seedlings; * denotes statistical significance in comparison to phyB-5 (p < 0.01; Student’s t-test), n = 20. D Immunoblot analysis of PIF3 protein levels in dark-grown seedlings. E Immunoblot analysis of red light-induced PIF3 loss in PHYBG767R/phyB-5 lines #2 and #8 with comparable expression levels to endogenous phyB. F Phenotypic comparison of true dark-grown YHBG767R transgenics in the phyB-5 and phyABCDE mutant backgrounds
Fig. 4
Fig. 4
Dosage-dependent effects of YHBG767R in the presence of functional phyB alleles. A Immunoblot analysis of dark-grown seedlings. B Four-day-old seedlings grown in continuous red light (50 µmol m−2 s−1) or in the dark; # denotes lines with discernable cop phenotypes (partially opened cotyledons). C Hypocotyl lengths (mean ± S.D.) of Rc50-grown, 4-d-old seedlings; * denotes statistical significance in comparison to Ler (p < 0.001; Student’s t-test), n = 20. D Dark-grown F1 seedlings of genetic crosses between a weak-expressing YHBg/phyB-5 or an overexpressing 35S::YHB/phyB-5 line with Ler WT, phyB-5 and two cytoplasm-retained PHYB mutation lines
Fig. 5
Fig. 5
Effects of YHBG767R dosage and genetic backgrounds on seedling cotyledon opening in true darkness, mean ± s.e.m., n = 17 ~ 22. YHBG767R/phyABCDE line #16 was used for outcrosses with Ler WT and various phy mutants
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Al-Sady B, Ni W, Kircher S, Schafer E, Quail PH. Photoactivated phytochrome induces rapid PIF3 phosphorylation prior to proteasome-mediated degradation. Mol Cell. 2006;23:439–446. doi: 10.1016/j.molcel.2006.06.011. - DOI - PubMed
    1. Alves FRR, Lira BS, Pikart FC, Monteiro SS, Furlan CM, Purgatto E, Pascoal GB, Andrade S, Demarco D, Rossi M, Freschi L. Beyond the limits of photoperception constitutively active PHYTOCHROME B2 overexpression as a means of improving fruit nutritional quality in tomato. Plant Biotechnol J. 2020;18:2027–2041. doi: 10.1111/pbi.13362. - DOI - PMC - PubMed
    1. Bauer D, Viczian A, Kircher S, Nobis T, Nitschke R, Kunkel T, Panigrahi KC, Adam E, Fejes E, Schafer E, Nagy F. Constitutive photomorphogenesis 1 and multiple photoreceptors control degradation of phytochrome interacting factor 3, a transcription factor required for light signaling in Arabidopsis. Plant Cell. 2004;16:1433–1445. doi: 10.1105/tpc.021568. - DOI - PMC - PubMed
    1. Chen M, Galvao RM, Li M, Burger B, Bugea J, Bolado J, Chory J. Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes. Cell. 2010;141:1230–1240. doi: 10.1016/j.cell.2010.05.007. - DOI - PMC - PubMed
    1. Chen D, Lyu M, Kou X, Li J, Yang Z, Gao L, Li Y, Fan LM, Shi H, Zhong S. Integration of light and temperature sensing by liquid-liquid phase separation of phytochrome B. Mol Cell. 2022;82(3015–3029):e3016. - PubMed

MeSH terms

Substances

LinkOut - more resources

-