Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2023.10.30.564862

Cytosolic heme metabolism by alternative localization of heme oxygenase 1 in plant cells

Chen, Y.·Nishimura, K.·Tokizawa, M.·Yamamoto, Y. Y.·Oka, Y.·Matsushita, T.·Hanada, K.·Shirai, K.·Mano, S.·Masuda, T.
View original source ↗Export metadata ↓

Abstract

Heme, an organometallic molecule, is widely engaged in oxygen transport, electron delivery, enzymatic reactions, and signal transduction. Additionally, heme serves as a precursor to phytochromobilin, the chromophore of plant phytochrome. Heme oxygenase (HO) initiates the first committed step in heme metabolism. Our transcription start site-sequencing (TSS-seq) revealed that HO1 in Arabidopsis thaliana and Oryza sativa (rice) has two TSSs, producing long (HO1L) and short (HO1S) transcripts, with or without an intact N-terminal plastid transit peptide. HO1L and HO1S products localize in plastids and the cytosol, respectively. In Arabidopsis, HO1L is prevalent in light-exposed shoots, while HO1S is clearly detected in roots and etiolated seedlings. During de-etiolation and early development, HO1L ratio gradually rises and HO1S ratio decreases. Light perception via phytochrome and cryptochrome elevates HO1L ratio and reduce HO1S ratio through the functioning of HY5 and HYH transcription factors, and the suppression of DET1, E3 ubiquitin ligase COP1, and PIFs transcription factors. As expected, HO1L product was able to complement the HO1-deficient mutant gun2 (hy1), but surprisingly, HO1S expression could also restore the short hypocotyl phenotype and high pigment content, and make the mutant recover from the gun phenotype. This indicates the formation of functional holo-phytochrome within these lines. Our work highlights the presence of a cytosolic pathway for heme metabolism, especially during etiolation and early development. Furthermore, it supports the hypothesis that a mobile heme signal is involved in the mediation of retrograde signaling from the chloroplast. Significance StatementIn this research, through both TSS-seq and CAGE-seq, we discovered that the HO1 (GUN2 or HY1) gene in both Arabidopsis and rice has two TSSs, generating HO1L and HO1S transcripts. We reveal that the HO1 TSS regulation pathway is the same as the light signaling pathway. Significantly, our study identifies that a cytosolic heme metabolism pathway is existent in plant cells.

Explore related subjects

plant biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Chen, Y., Nishimura, K., Tokizawa, M., Yamamoto, Y. Y., Oka, Y., Matsushita, T., Hanada, K., Shirai, K., Mano, S., Masuda, T.. 2023-10-31. Cytosolic heme metabolism by alternative localization of heme oxygenase 1 in plant cells. https://doi.org/10.1101/2023.10.30.564862

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Loss of starch synthase IIa alleviates the negative impact of high temperature on rice starch during grain filling

High temperatures during grain filling stage are becoming increasingly frequent, compromising both grain and eating quality and thereby driving demand for heat-resilient cultivars. Such conditions are known to reduce the expression of granule-bound starch synthase I (GBSSI) and starch branching enzyme IIb (BEIIb), which are involved in starch biosynthesis, resulting in a decrease in amylose content and an increase in long-chain amylopectin. Thus, the present study introduced functional mutation in starch synthase IIa (SSIIa) that increases the proportion of short amylopectin chains to genetically compensate for the high-temperature-induced increase in amylopectin long chain. Rice lines carrying the ss2a mutation were grown at two locations with cooler (Akita) and warmer (Okayama) temperatures. Their grain traits, starch structure, and eating quality were compared. The ss2a mutant lines showed an increased proportion of short amylopectin chains as well as an increased apparent amylose content. Furthermore, these alterations in starch structure varied with the grain-filling temperature of the cultivation sites, ultimately affected eating quality. These results suggest that enriching short amylopectin chain via the ss2a mutation can counteract the increase in long amylopectin chain caused by high temperatures during grain filling, thereby maintaining a desirable starch structure and eating quality.

plant biology↗
bioRxiv2026

Analysis of SpCas9 on- and off-target effects in high efficiency multiplex editing in Arabidopsis

RNA-guided nucleases (RGNs), such as Cas9 from Streptococcus pyogenes (SpCas9), are widely used for plant genome editing. Previous surveys for off-targeting, the modification of unintended targets with similarity to the intended target, indicate high specificity of SpCas9 in plant cells. However, off-targeting has not been assessed for efficiency-optimized editing systems combined with extensive multiplexing, which might increase the likelihood of cleavage at unintended sites. We therefore analyzed Arabidopsis thaliana lines that had been extensively mutagenized using zCas9i and up to 29 gRNAs addressing >45 target sites over several rounds of editing. Genomes were sequenced by short- and long-read technologies, and genome-wide variants were catalogued. Our pipeline for variant calling reliably detected RGN-induced mutations at on-targets. When excluding these on-target modifications, variants were detected in edited lines at frequencies similar to those previously reported for spontaneous mutations. In further analyses, we did not find any evidence for an origin of these variants from RGN activity. Our data are thus consistent with high specificity of SpCas9. In contrast, we detected genomic reorganization events upon editing at two complex loci, RPP1 and RPP7, encompassing multiple homologous genes, and also identified an allele by WGS that had escaped detection by amplicon sequencing. We conclude that, while off-targets may efficiently be avoided by selection of specific gRNAs, on-target modifications may be more extensive than intended, especially at complex loci and/or during multiplexing.

plant biology↗
bioRxiv2026

A cellulose synthase interactome uncovers BAG proteins as regulators of cellulose synthase homeostasis

Cellulose synthase complexes build the load-bearing cellulose microfibrils of plant cell walls, yet how the abundance of their catalytic CELLULOSE SYNTHASE A (CESA) subunits is maintained remains unclear. Here, we used multi-bait TurboID proximity labelling with ten cellulose-synthesis-associated baits and six subcellular controls to define a high-confidence cellulose synthase neighbourhood. Stringent spatial and recurrence-based filtering yielded a core network of 119 interactions among 44 proteins and identified three members of the conserved Bcl-2-associated athanogene (BAG) family as previously unrecognised regulators of cellulose synthase homeostasis. BAG1-3 associated with primary-wall CESAs in reciprocal proximity-labelling experiments. Arabidopsis bag mutants showed reduced cellulose accumulation, hypersensitivity to cellulose-synthesis inhibitors, and markedly decreased CESA protein abundance without corresponding changes in CESA transcript levels. Loss of BAG function also increased the accumulation of CESA6 in vacuolar compartments. These findings identify BAG proteins as previously unrecognised regulators of cellulose synthase homeostasis and link a conserved proteostasis-associated protein family to plant cell wall biosynthesis. More broadly, the study establishes multi-bait proximity labelling, combined with cell location-specific controls, as a strategy for resolving dynamic protein networks whose components traffic through multiple subcellular compartments.

plant biology↗