PacBio Long-Read Sequencing Reveals the Transcriptomic Complexity and Aux/IAA Gene Evolution in Gnetum (Gnetales)

Abstract

The genus Gnetum includes pantropical trees, shrubs and lianas, with unresolved phylogenetic relationships with other seed plant groups. Despite the reference genome for this genus being recently published, the molecular mechanisms that regulate the reproductive organ development of Gnetum remain unclear. A previous study showed that indole-3-acetic acid is involved in the regulation of female strobili of Gnetum, while the diversity and evolution of indole-3-acetic acid-related genes—the Aux/IAA genes—have never been investigated in Gnetales. Thus, a pooled sample from different developmental stages of female strobili in Gnetum luofuense C.Y. Cheng was sequenced using PacBio single-molecular long-read technology (SMRT) sequencing. PacBio SMRT sequencing generated a total of 53,057 full-length transcripts, including 2043 novel genes. Besides this, 10,454 alternative splicing (AS) events were detected with intron retention constituting the largest proportion (46%). Moreover, 1196 lncRNAs were identified, and 8128 genes were found to possess at least one poly (A) site. A total of 3179 regulatory proteins, including 1413 transcription factors (e.g., MADS-box and bHLHs), 477 transcription regulators (e.g., SNF2), and 1289 protein kinases (e.g., RLK/Pelles) were detected, and these protein regulators probably participated in the female strobili development of G. luofuense. In addition, this is the first study of the Aux/IAA genes of the Gnetales, and we identified 6, 7 and 12 Aux/IAA genes from Gnetum luofuense, Welwitschia mirabilis, and Ephedra equistina, respectively. Our phylogenetic analysis reveals that Aux/IAA genes from the gymnosperms tended to cluster and possessed gene structures as diverse as those in angiosperms. Moreover, the Aux/IAA genes of the Gnetales might possess higher molecular evolutionary rates than those in other gymnosperms. The sequencing of the full-length transcriptome paves the way to uncovering molecular mechanisms that regulate reproductive organ development in gymnosperms.

A Full-length Transcriptome and Gene Expression Analysis Reveal Genes and Molecular Elements Expressed During Seed Development in Gnetum Luofuense

“…Structural analysis of the full-length transcriptome AS and APA analysis A total of 8,512 genes were identi ed as having at least one poly(A) site, suggesting that alternative polyadenylation may enrich the proteomic complexity and affect the seed ripening process of G. luofuense. Previous studies have suggested that alternative polyadenylation affects the development of G. luofuense leaves and female strobili [29,41]. In addition, we found considerable differences in the number of AS events among different gymnosperm species and among different organs in the same species by comparing AS events in the leaves of G. biloba (12,209) [42] and the leaves (12,998) [41], female strobili (10,454) [29], and seeds of G. luofuense (9,061, the present study).…”

“…Previous studies have suggested that alternative polyadenylation affects the development of G. luofuense leaves and female strobili [29,41]. In addition, we found considerable differences in the number of AS events among different gymnosperm species and among different organs in the same species by comparing AS events in the leaves of G. biloba (12,209) [42] and the leaves (12,998) [41], female strobili (10,454) [29], and seeds of G. luofuense (9,061, the present study). It is noteworthy that intron retention constituted the largest proportion of identi ed AS events in this study.…”

“…Aux/IAA genes also participate in the development of various organs in seed plants by responding to the hormone auxin/indole-3-acetic acid [26][27][28]. In Gnetum, six Aux/IAA genes (GluIAA1-6) were reported to be involved in female strobilus development [29]. Moreover, bHLH and MYB TFs are able to form a complex that regulates stamen development and seed production [30].…”

“…AS generates multiple proteins from a single coding gene [31,32], and APA enhances transcriptome complexity by initiating the expression of multiple 3′ UTR isoforms from the same gene [33,34]. Many examples of AS and APA have been documented in angiosperms [35][36][37][38][39][40], but investigations of AS and APA are rarer in gymnosperms [29,41,42]. In addition, lncRNAs, which are de ned as possessing at least 200 nucleotides, are essential because they take part in transcriptional and post-transcriptional gene regulation [43][44][45].…”

“…In addition, lncRNAs, which are de ned as possessing at least 200 nucleotides, are essential because they take part in transcriptional and post-transcriptional gene regulation [43][44][45]. To date, little attention has been paid to lncRNAs in gymnosperms [29,41,46].…”