Universität zu Köln

Huebbers, Jan W., Buttgen, Kim, Leissing, Franz ORCID: 0000-0002-9746-7162, Mantz, Melissa ORCID: 0000-0001-7254-5331, Pauly, Markus ORCID: 0000-0002-3116-2198, Huesgen, Pitter F. and Panstruga, Ralph ORCID: 0000-0002-3756-8957 (2022). An advanced method for the release, enrichment and purification of high-quality Arabidopsis thaliana rosette leaf trichomes enables profound insights into the trichome proteome. Plant Methods, 18 (1). LONDON: BMC. ISSN 1746-4811

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Abstract

Background Rosette leaf trichomes of Arabidopsis thaliana have been broadly used to study cell development, cell differentiation and, more recently, cell wall biogenesis. However, trichome-specific biochemical or -omics analyses require a proper separation of trichomes from residual plant tissue. Thus, different strategies were proposed in the past for trichome isolation, which mostly rely on harsh conditions and suffer from low yield, thereby limiting the spectrum of downstream analyses. Results To take trichome-leaf separation to the next level, we revised a previously proposed method for isolating A. thaliana trichomes by optimizing the mechanical and biochemical specifications for trichome release. We additionally introduced a density gradient centrifugation step to remove residual plant debris. We found that prolonged, yet mild seedling agitation increases the overall trichome yield by more than 60% compared to the original protocol. We noticed that subsequent density gradient centrifugation further visually enhances trichome purity, which may be advantageous for downstream analyses. Gene expression analysis by quantitative reverse transcriptase-polymerase chain reaction validated a substantial enrichment upon purification of trichomes by density gradient centrifugation. Histochemical and biochemical investigation of trichome cell wall composition indicated that unlike the original protocol gentle agitation during trichome release largely preserves trichome integrity. We used enriched and density gradient-purified trichomes for proteomic analysis in comparison to trichome-depleted leaf samples and present a comprehensive reference data set of trichome-resident and -enriched proteins. Collectively we identified 223 proteins that are highly enriched in trichomes as compared to trichome-depleted leaves. We further demonstrate that the procedure can be applied to retrieve diverse glandular and non-glandular trichome types from other plant species. Conclusions We provide an advanced method for the isolation of A. thaliana leaf trichomes that outcompetes previous procedures regarding yield and purity. Due to the large amount of high-quality trichomes our method enabled profound insights into the so far largely unexplored A. thaliana trichome proteome. We anticipate that our protocol will be of use for a variety of downstream analyses, which are expected to shed further light on the biology of leaf trichomes in A. thaliana and possibly other plant species.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Huebbers, Jan W. UNSPECIFIED UNSPECIFIED UNSPECIFIED Buttgen, Kim UNSPECIFIED UNSPECIFIED UNSPECIFIED Leissing, Franz UNSPECIFIED orcid.org/0000-0002-9746-7162 UNSPECIFIED Mantz, Melissa UNSPECIFIED orcid.org/0000-0001-7254-5331 UNSPECIFIED Pauly, Markus UNSPECIFIED orcid.org/0000-0002-3116-2198 UNSPECIFIED Huesgen, Pitter F. UNSPECIFIED UNSPECIFIED UNSPECIFIED Panstruga, Ralph UNSPECIFIED orcid.org/0000-0002-3756-8957 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-677363
DOI:	10.1186/s13007-021-00836-0
Journal or Publication Title:	Plant Methods
Volume:	18
Number:	1
Date:	2022
Publisher:	BMC
Place of Publication:	LONDON
ISSN:	1746-4811
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language PLANT GLANDULAR TRICHOMES; EXOCYST SUBUNIT EXO70H4; GENE-EXPRESSION; CELL-WALL; COMPUTATIONAL PLATFORM; REVEALS; METABOLISM; ENCODES; DIFFERENTIATION; TRANSCRIPTOME Multiple languages Biochemical Research Methods; Plant Sciences Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/67736