## 5 Materials and Methods
### Statistical and Bioinformatic Analyses  
Statistical analyses were conducted in R (v4.3.1) using various packages. Two-sample Student's \(t\)-test was used to determine significant differences for protein and metabolite abundance, between treatments and control. A \(p\)-value \(<\) 0.05 with either a fold-change \(<\) 0.67 or a fold-change \(>\) 1.5 was considered significant for proteins, and a \(p\)-value \(<\) 0.05 was considered significant for metabolites. Principal component analysis (factoextra package v1.0.7) was used to cluster GT proteome samples. Heatmaps were created using the pheatmap (v1.0.12) and were hierarchically clustered by Ward clustering using Euclidean distance. Like in [PERSON] et al. [33] and other studies with non-model plants [84], the functional annotation of the identified cannabis GT proteins was determined by aligning sequences to the best BLASTP matches within the National Center for Biotechnology Information (NCBI) and UniprotKB/SwissProt databases, utilising arabidopsis as a reference organism. The threshold for significance was set at an e-value of less than 1.0 \(\times\) 10\({}^{-5}\). Gene ontology enrichment analysis of groups of differentially abundant proteins was conducted using the clusterProfiler (v4.12.6) package [85; 86] with the R package org.At.tair.db: genome-wide annotation package for arabidopsis (v3.19.1). Enriched ontology terms were considered significant when the adjusted (Benjamini-Hochberg) \(p\)-value \(<\) 0.05. Gene ontology semantic similarity analysis was performed with the GOSemSim (v2.30.2) package for R [87]. The following supporting information can be downloaded at [[https://www.mdpi.com/article/10.3990/plants14050694/s1](https://www.mdpi.com/article/10.3990/plants14050694/s1)]([https://www.mdpi.com/article/10.3990/plants14050694/s1](https://www.mdpi.com/article/10.3990/plants14050694/s1)). Supplementary Dataset S1--Statistics results for glandular trichome proteome analysis. Supplementary Figure S1--Enriched GO terms of global increased and decreased DAPs. Supplementary Figure S2--Enriched GO terms and proteins affected by ethephon. Supplementary Figure S3--Abundances of key proteins in controls at 0 h and 24 h. Supplementary Figure S4--Photos of control time course trial. Supplementary Figure S5--Abundances of phytohormone regulatory proteins. Supplementary Figure S6--Abundances of photosystem proteins. Supplementary Figure S7--Photos of high purity trichome disc cells. Supplementary Figure S8--Photos comparing disc cells with and without storage cavities. N.D. and T.K. : conceptualisation; N.D. and Q.G. : formal analysis; T.K. and B.J.B. : funding acquisition; N.D. and L.L. : investigation; N.D., L.L., Q.G., M.N., R.D. and L.G.-d.H.: methodology; T.K. : project administration; J.C.M. : resources; T.K. and B.J.B. : supervision; N.D., L.L. and Q.G. : validation; N.D.: visualisation; N.D.: writing--original draft; all authors: writing--review and editing. All authors have read and agreed to the published version of the manuscript. **Funding:** This project received grant funding from the Australian Government via the Department of Industry, Science, Energy and Resources, as part of the Cooperative Research Centres Project (CRC-P) program--Round 7--Growing the medicinal Cannabis industry--precision farming to pharmaceuticals. The CRC-P program is an industry-led research collaboration and Cann Group Ltd. is the lead partner organisation. **Data Availability Statement:** The primary data supporting this study include the raw mass spectrometry proteomics data and metabolite data. All primary data have been deposited (ID 991013205013802368) to the Southern Cross University research portal ([[https://researchportal.scu.edu.au/](https://researchportal.scu.edu.au/)]([https://researchportal.scu.edu.au/](https://researchportal.scu.edu.au/))) and are openly available (19 December 2024) at [[https://doi.org/10.25918/data.382](https://doi.org/10.25918/data.382)]([https://doi.org/10.25918/data.382](https://doi.org/10.25918/data.382)).