Comparative transcriptomics of tropical woody plants supports fast and furious strategy along the leaf economics spectrum in lianas

Lianas, climbing woody plants, influence the structure and function of tropical forests. Climbing traits have evolved multiple times, including ancestral groups such as gymnosperms and pteridophytes, but the genetic basis of the liana strategy is largely unknown. Here, we use a comparative transcriptomic approach for 47 tropical plant species, including ten lianas of diverse taxonomic origins, to identify genes that are consistently expressed or downregulated only in lianas. Our comparative analysis of full-length transcripts enabled the identification of a core interactomic network common to lianas. Sets of transcripts identified from our analysis reveal features related to functional traits pertinent to leaf economics spectrum in lianas, including upregulation of genes controlling epidermal cuticular properties, cell wall remodeling, carbon concentrating mechanism, cell cycle progression, DNA repair and a large suit of downregulated transcription factors and enzymes involved in ABA-mediated stress response as well as lignin and suberin synthesis. Altogether, these genes are known to be significant in shaping plant morphologies through responses such as gravitropism, phyllotaxy and shade avoidance.

Methods
Sample Set

The Luquillo transcriptomic set included six lianas (DIO) Dioscorea poligonoides (Dioscoreaceae), (HET) Heteropterys laurifolia (Malpighiaceae), (PAU) Paulinia pinnata (Sapindaceae), (SEC) Securidaca virgata (Polygonaceae), (SMI) Smilax coriaceae (Smilaceae), (DOL) Dolichandra unguis-cati (Bignoniaceae). We also included four more liana species from National Center for Biotechnology Information’s Sequence Read Archive (NCBI-SRA) and One Thousand Plant Genomes (1KP) dataset (Matasci et al. 2014, 1KP 2019) with the following accessions (GNE) Gnetum montanum (Gnetaceae) SRR5908685, (SMISIE) Smilax sieboldii (Smilacae) SRR5134200, (PASCAE) Passiflora caerulea (Passifloraceae) 1KP id:SIZE, (SCHPAR) Schlegelia parasitica (Schlegeliaceae) 1KP id:GAKQ. A full list including non-liana species can be found in the supplementary (SI Appendix Dataset S1, SI Appendix Fig. S1,).

Sample Collection and RNA Library Construction

To analyze the transcriptomes, we chose healthy and fully developed leaves from seedlings of tree and liana species distributed between 350 and 450 m in elevation from the Luquillo Experimental Forest (LEF) in the north eastern part of Puerto Rico. For each species, approximately 5 grams of leaf tissue was collected and placed in a 50 mL polypropylene conical tube with RNAlater (Thermo Fisher Scientific, Waltham, MA, USA). Explants were cut with a razor blade prior to being placed in the tube to allow the RNAlater to penetrate the mesophyll quickly. Samples were then frozen at -80ºC within two days. Rneasy Plant Mini Kit (Qiagen, Valencia, CA, USA) was used for RNA extraction. RNA quantification and quality metrics were carried out using a NanoDrop 2000 spectrophotometer (NanoDrop Products, Wilmington, DE, USA) and an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) RNAseq library preparations and sequencing were performed at the Beijing Genomics Institute, Shenzhen, China on Illumina Hiseq 2000 sequencer generating 100 bp paired-end reads.