An advanced method for the release, enrichment and purification of high-quality Arabidopsis thaliana rosette leaf trichomes enables profound insights into the trichome proteome

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.

Efficient Isolation and Purification of High‐Quality Arabidopsis thaliana Trichomes

“…Metabolomic profiling, however, requires an altered buffer composition during trichome release, as sample preparation for metabolomics requires the quenching of metabolic processes, for instance, by cold methanol (de Jonge, Douma, Heijnen, & van Gulik, 2012). Lastly, we note that the EDTA‐based trichome release protocol described here is applicable to plant species other than A. thaliana , including N. benthamiana (tobacco) and S. lycopersicum (tomato) (Huebbers et al., 2022). However, the exact amount of deployed plant mass and the time frame for agitation in EDTA‐PBS buffer requires optimization for specific plant species.…”

“…However, the exact amount of deployed plant mass and the time frame for agitation in EDTA‐PBS buffer requires optimization for specific plant species. Helianthus annuus (sunflower), for instance, provided a comparably low yield of 3.89 mg g ‐1 trichome fresh mass per harvested leaf mass (Table 1), and microscopy of processed leaves revealed that a considerable amount of trichomes remained attached to the leaf surface during the release step (Huebbers et al., 2022).…”

“…Thus, we recommend a maximum seedling fresh mass of 36 g in 250 ml of EDTA‐PBS buffer. Moreover, prolonged seedling agitation (>1 hr) may cause a sluice of cytosolic trichome material, as indicated by a reduction in trichome density (Huebbers et al., 2022). Accordingly, an agitation time of 30 min before trichome enrichment should not be exceeded.…”

“…This protocol describes the procedure to isolate A. thaliana trichomes at high yield, based on our previous publication (Huebbers et al, 2022). This is achieved by extended incubation of A. thaliana seedlings in EDTA-containing buffer, in combination with mild agitation on a magnetic stirrer.…”

“…Based on the procedure by Marks et al. (2008), we have developed an enhanced method for the release of trichomes that takes advantage of prolonged yet gentle agitation of comparably large amounts (up to 36 g) of A. thaliana seedlings (Huebbers et al., 2022). Besides the larger batch size compared to the original protocol, we observed that prolonged agitation by magnetic stirring boosted trichome yield per harvested plant mass by 60% in comparison to high‐speed mixing.…”