Gene expression data support the hypothesis that Isoetes rootlets are true roots and not modified leaves

Abstract

Rhizomorphic lycopsids are the land plant group that includes the first giant trees to grow on Earth and extant species in the genus Isoetes. Two mutually exclusive hypotheses account for the evolution of terminal rooting axes called rootlets among the rhizomorphic lycopsids. One hypothesis states that rootlets are true roots, like roots in other lycopsids. The other states that rootlets are modified leaves. Here we test predictions of each hypothesis by investigating gene expression in the leaves and rootlets of Isoetes echinospora. We assembled the de-novo transcriptome of axenically cultured I. echinospora. Gene expression signatures of I. echinospora rootlets and leaves were different. Furthermore, gene expression signatures of I. echinospora rootlets were similar to gene expression signatures of true roots of Selaginella moellendorffii and Arabidopsis thaliana. RSL genes which positively regulate cell differentiation in roots were either exclusively or preferentially expressed in the I. echinospora rootlets, S. moellendorffii roots and A. thaliana roots compared to the leaves of each respective species. Taken together, gene expression data from the de-novo transcriptome of I. echinospora are consistent with the hypothesis that Isoetes rootlets are true roots and not modified leaves.

What can lycophytes teach us about plant evolution and development? Modern perspectives on an ancient lineage

“…An annotated S. moellendorffii genome was published by Banks et al, (2011), and there are RNASeq and DNASeq datasets from S. kraussiana (Table 1; Ge et al, 2016). Several lycophyte transcriptomes such Selaginella martensii and Isoëtes echinospora have recently been published (Hetherington, Emms, Kelly, & Dolan, 2019; James et al, 2017; Leebens‐Mack, 2019) and lycophyte transcriptomes from Isoëtes drummondii and Phylloglossum drummondii are also available on request (Table 1; Dixon, Harrison, Hetherington, Mylne, & Zhang, 2016). Such genomic and transcriptomic data are important for analyses of gene copy number and the identification of developmental gene orthologs and will facilitate future evo‐devo studies by providing templates for primer design.…”

“…Genetic studies would determine if lycophyte roots are homologous, or if pre‐existing organs such as leaves acquired root‐like characteristics and associated genetic signatures convergently (Hetherington & Dolan, 2017). A transcriptomic approach recently showed that the short rootlets of Isoëtes echinospora are more transcriptionally similar to Selaginella and Arabidopsis roots than leaves, addressing a longstanding question about the identity of rhizomorphic lycopsid rootlets (Hetherington et al, 2019). It will be important to identify genetic similarities between other root types and the organs on which roots are borne to identify conservation, convergence, and divergence in root evolution.…”

“…While the Lycopodiales are characterised by equally sized spores (homospory) and a lack of leaf ligules, the Selaginellales and Isoëtales have different sized spores (they are heterosporous) and have a ligule on the adaxial side of leaves (Figure 1b; Sporne, 1962). Four-sided strobili with distinct sporangia types and distributions discriminate Selaginellales from Isoëtales, with Isoëtales sporangia being much larger and more productive (Kenrick & Crane, 1997;Sporne, 1962 (Wang et al, 2005;Banks et al, 2011;Weng et al, 2005;Zhu et al, 2017;Mello et al, 2019;Frank et al, 2015;Huang and Schiefelbein, 2015;Ferrari et al, 2020;Leebens-Mack, 2019;James et al, 2017;Yin et al, 2009;Moody et al, 2012;Zhang et al, 2019;Kwantes et al, 2012;Kirkbride et al, 2013;Aya et al, 2011;Zumajo-Cardona et al, 2019;Ge et al, 2016;Hirakawa and Bowman, 2015;Harrison et al, 2005;Ocheretina et al, 2000;Prigge and Clark, 2006;Kawai et al, 2010;Hedman et al, 2009;Zentella et al, 1999;Tanabe et al, 2003;Hetherington et al, 2019;Dixon et al, 2016;Yang et al,2017;Evkaikina et al, 2017;Luo et al, 2010;Floyd et al, 2014;Svensson et al, 2000;Svensson and Engström, 2002).…”

“…Due to the advent of low-cost and high-quality sequencing methods, genomic resources for lycophytes have expanded significantly in the last ten years, lowering the feasibility threshold for reverse genetic approaches. The first lycophyte sequence datasets were a BAC gDNA library and an EST library from S. moellendorffii (W. Wang et al, 2005;Weng, Tanurdzic, & Chapple, 2005 (Hetherington, Emms, Kelly, & Dolan, 2019;James et al, 2017;Leebens-Mack, 2019) and lycophyte transcriptomes from Isoëtes drummondii and Phylloglossum drummondii are also available on request (Table 1; Dixon, Harrison, Hetherington, Mylne, & Zhang, 2016). Such genomic and transcriptomic data are important for analyses of gene copy number and the identification of developmental gene orthologs and will facilitate future evo-devo studies by providing templates for primer design.…”

“… Note: Table showing each molecular and genetic experiment performed in different lycophyte species. The majority of studies have been performed in Selaginella moellendorffii , Selaginella kraussiana , Huperzia selago , and Huperzia lucidula (Wang et al, 2005; Banks et al, 2011; Weng et al, 2005; Zhu et al, 2017; Mello et al, 2019; Frank et al, 2015; Huang and Schiefelbein, 2015; Ferrari et al, 2020; Leebens‐Mack, 2019; James et al, 2017; Yin et al, 2009; Moody et al, 2012; Zhang et al, 2019; Kwantes et al, 2012; Kirkbride et al, 2013; Aya et al, 2011; Zumajo‐Cardona et al, 2019; Ge et al, 2016; Hirakawa and Bowman, 2015; Harrison et al, 2005; Floyd & Bowman, 2006; Ocheretina et al, 2000; Prigge and Clark, 2006; Floyd et al, 2006; Kawai et al, 2010; Hedman et al, 2009; Zentella et al, 1999; Tanabe et al, 2003; Hetherington et al, 2019; Dixon et al, 2016; Yang et al,2017; Evkaikina et al, 2017; Luo et al, 2010; Floyd et al, 2014; Svensson et al, 2000; Svensson and Engström, 2002). …”