Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2024.06.11.598459

Transcriptomic analysis of Picea abies tissue culture reveals the impact of culture conditions and the presence of glucuronoxylan on extracellular lignin production

Sapouna, I.·Sivan, P.·Vilaplana, F.·Srivastava, V.·McKee, L. S.
View original source ↗Export metadata ↓

Abstract

Tissue cultures are an important study model for woody plant tissue and can be used to study lignin biosynthesis. The greatest disadvantage of protocols based on extraction of lignin from wood biomass is the almost inevitable alteration of the native structure of lignin. Using a Norway spruce tissue culture with the ability to secrete monolignols into a liquid culture medium, fundamental aspects of lignin have been studied in the past, such as its structure, the enzyme activity related to its polymerization, and its interactions with a secondary cell wall hemicellulose. In this study, parameters that can induce monolignol production and secretion in the tissue culture are investigated via gene expression analysis. The impact of the composition of the solid growth medium, which was in some cases supplemented with xylan, was studied in depth through transcriptomic investigation. We find that the state (i.e. liquid or solid) and the xylan content of the medium can impact gene expression, although microscopic analysis suggests that cellular morphology is consistent. Extracellular lignin was collected from a formulation of liquid medium with the same composition as that used for cellular growth, which was previously presumed to be "non-inducing" of lignin biosynthesis. Chemical analysis of this lignin was performed using nuclear magnetic resonance spectroscopy and size exclusion chromatography, which revealed changes in its structure compared to the polymer produced in the previously developed "inducing" liquid medium. These experiments show that there is still much we do not understand about an oft-used tissue culture system, but show the way to a deeper understanding of the genetic control of lignin biosynthesis.

Explore related subjects

plant biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Sapouna, I., Sivan, P., Vilaplana, F., Srivastava, V., McKee, L. S.. 2024-06-12. Transcriptomic analysis of Picea abies tissue culture reveals the impact of culture conditions and the presence of glucuronoxylan on extracellular lignin production. https://doi.org/10.1101/2024.06.11.598459

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↗