Unveiling Conservation in Plant Extracellular Vesicle Proteomes: A Sorghum and Arabidopsis Study

Plant extracellular vesicles (EVs) are pivotal in intercellular communication and defense responses. A recent study by scientists from the University of Delaware, Indiana University and Brookhaven National Laboratory delved into the EV proteomes of Sorghum bicolor and Arabidopsis thaliana, revealing intriguing insights into the partial conservation between these plant species. The isolation of EVs from sorghum leaves showcased a heterogeneous population with distinct lipid bilayers and content. Advanced techniques, including nanoparticle tracking analysis and cryo-electron tomography, contributed to the characterization of sorghum EVs. Proteomic analysis identified 437 proteins enriched in sorghum EVs, with a significant overlap observed in the EV proteome of Arabidopsis. This suggests a partial conservation in both function and contents between the monocot sorghum and the distantly related eudicot Arabidopsis.

Despite the extensive research on mammalian EVs, the field of plant EVs faces challenges, necessitating further exploration into fundamental aspects such as biogenesis, morphology, cargo packaging, and interkingdom movement. The study addressed these challenges by adapting an Arabidopsis EV isolation protocol for sorghum. Optimization techniques, including leaf tip snipping and efficient buffer retention, were introduced to enhance EV capture during vacuum infiltration. Additionally, homologs of the marker proteins like PENETRATION1 (PEN1, AT3G11820.1), PATELLIN 1 (PATL1, AT1G72150.1), the ABC transporter PENETRATION3 (PEN3, AT1G59870.1), and TETRASPANIN8 (TET8, AT2G23810.1), as well as the proposed RNA-binding annexin proteins ANN1 (AT1G35720.1) and ANN2 (AT5G65020.1) were identified, laying the groundwork for future investigations in sorghum and other monocots. Biological function GO term enrichment of sorghum orthologs suggested a core of proteins enriched for vesicle docking/fusion, transport across membranes, exocytosis, carbohydrate metabolism, and translation. Cryo-electron tomography unveiled the diverse population of sorghum EVs, while attempts to stain them with fluorescent dyes provided crucial insights into the limitations of commonly used lipophilic dyes. Overall, this research contributes to the evolving understanding of plant EVs, paving the way for deeper exploration into the intricate world of intercellular communication and defense responses in plants.

The goal of this manuscript was to provide a robust extracellular vesicle isolation protocol for sorghum. With this in hand, we were able to show that 60% of the sorghum EV proteins identified have orthologs in the Arabidopsis EV proteome. We hope that our isolation method and characterization by proteomics and microscopy can be used as a guide for other monocot EV research and further our understanding of the key proteins and functions of EVs in plants. – Chaya

SorghumBase examples: 

AT1G72150 PATL1 was picked up as an example to get sorghum orthologs to explore SorghumBase below: 

Figure 1: A search for orthologs of PATL1 shows sorghum genes on chromosomes 3 and 9 in most genomes with some additional paralogs on chromosome 5.
Figure 2: The location of the one of the ortholog of PATL1 SORBI_3003G373100 (Sb03g041370, Sobic.003G373100 similar to P0648C09.9 protein) is displayed on Chromosome 3 (within 3:68853615:68857222) in SorghumBase.
Figure 3: This figure displays the phylogram of SORBI_3003G373100 (Sb03g041370, Sobic.003G373100 similar to P0648C09.9 protein) based on the Ensembl compara gene tree. There are 5 paralogs and 46 orthologs of this gene.
Figure 4: This figure displays the baseline gene expression of SORBI_3003G373100 (Sb03g041370, Sobic.003G373100 similar to P0648C09.9 protein) in the five published sorghum BTx623 datasets ( Makita et al. 2015; Davidson et al. 2012; Turco et al. 2017; Olson et al. 2014; Thurber et al 2015; Wang et al. 2018) curated and processed by EMBL-EBI atlas in collaboration with Gramene team.
Figure 5: This figure displays the baseline gene expression of SORBI_3003G373100 (Sb03g041370, Sobic.003G373100 similar to P0648C09.9 protein) along with its six paralogs curated and processed by EMBL-EBI atlas in collaboration with Gramene team.


Chaya T, Banerjee A, Rutter BD, Adekanye D, Ross J, Hu G, Innes RW, Caplan JL. The extracellular vesicle proteomes of Sorghum bicolor and Arabidopsis thaliana are partially conserved. Plant Physiol. 2023 Dec 4:kiad644. PMID: 38048422. doi: 10.1093/plphys/kiad644. Read more

Related Project Websites: 

Image 1: Sorghum grown at the University of Delaware. Inset showing cryo-electron tomography of extracellular vesicles isolated from 2 week old sorghum plants. Photo credit Timothy Chaya (field image) and Guobin Hu at the Brookhaven National Lab (CryoET).