Phylogenetic analyses of key developmental genes provide insight into the complex evolution of seeds

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Highlights

Abstract

Gene duplication plays a decisive role in organismal diversification and in the appearance of novel structures. In plants the megagametophyte covered by the integuments, which after fertilization becomes the seed constitutes a novel structure: the ovule. In Arabidopsis thaliana, genetic mechanisms regulating ovule development, including the genetics underlying ovule initiation, ovule patterning and integument development, have been identified. Among seed plants, integuments are not only a novelty in evolution, but integuments also present an enormous morphological variation. This study is focused on the evolution of gene families that play a role in the proper morphological development of the integuments, BELL1 (BEL1), KANADIs (KAN1, KAN2, and KAN4/ATS), UNICORN (UCN) and SHORT INTEGUMENTS1 (SIN1). In Arabidopsis, BEL1 establishes the initiation of integument development. KAN1 and 2 act in the proper development of the outer integument. While ABERRANT TESTA SHAPE (ATS), is involved in the correct separation of both integuments. UCN acts in planar growth of the outer integument repressing ATS. SIN1 is involved in cell elongation in the integuments. The results of our analyses show that each of these genes has a different evolutionary history and that while gymnosperms appear to have a simpler ovule morphology, they have more homologues of these candidate genes than angiosperms. In addition, we present the conserved and novel motifs for each of these genes among seed plants and their selection constraints, which may be related to functional changes and to the diversity of ovule morphologies.

Introduction

Ovule development is genetically controlled by a complex molecular circuitry including transcription factors mediating ovule initiation and identity, followed by the initiation, identity, and development of the integuments; and finally, embryo sac development. Functional studies in the model species Arabidopsis thaliana (Arabidopsis; Brassicaceae) have identified more than 12 transcription factors, belonging to different gene families, functioning at different ontogenetic stages of the ovule, allowing its proper development (Baker et al., 1997, Gasser et al., 1998, Colombo et al., 2008, Gasser and Skinner, 2019). Ovule primordia initiation is determined by multiple genes including SEEDSTICK (STK), AGAMOUS (AG), SHATTERPROOF (SHP), APETALA2 (AP2) and AINTEGUMENTA (ANT) in Arabidopsis (Colombo et al., 1997, Dreni et al., 2007, Colombo et al., 2008, Mizzotti et al., 2012) and FLORAL BINDING PROTEIN 7 (FBP7) in Petunia (Angenent et al., 1995, Colombo et al., 1995).

Integument initiation seems to be mainly determined by BELL1 (BEL1). The bel1 mutant shows significant growth in the chalazal region where an amorphous structure develops instead of integuments (Robinson-Beers et al., 1992, Modrusan et al., 1994, Ray et al., 1994, Reiser et al., 1995, Brambilla et al., 2007, Bencivenga et al., 2012; Fig. 1A). BEL1 belongs to the Three Amino acid Loop Extension (TALE) class of Homeodomain proteins with the TALE motif located within the triple helix of the Homeodomain. However, the BELL group (or BELL-like Homeodomain; BLH) is characterized from other TALE-HD proteins by a ZIBEL motif (Bürglin, 1997, Becker et al., 2002, Kumar et al., 2007, Mukherjee et al., 2009). In addition to integument initiation, BEL1 activates INNER NO OUTER (INO; Meister et al., 2004, Colombo et al., 2008) which belongs to the YABBY gene family (Bowman and Smyth, 1999, Bowman, 2000, Floyd and Bowman, 2007). Phylogenetic analyses for the YABBY gene family indicated that INO is angiosperm specific (Bowman, 2000, Floyd and Bowman, 2007, Bartholomes et al., 2012). INO is functionally restricted to the proper development of the outer integument, Arabidopsis ino mutants lack an outer integument (Baker et al., 1997, Siegfried et al., 1999, Villanueva et al., 1999; Fig. 1B). Its expression is conserved in other angiosperms such as Nicotiana benthamiana (Skinner et al., 2016) and in the genus Impatiens (McAbee et al., 2005). In the unitegmic ovule of Solanum lycopersicum, INO is expressed in the single integument (Skinner et al., 2016).

Once the development of both integuments is initiated, ABERRANT TESTA SHAPE (ATS, also known as KANADI 4) is involved in the development of the inner integument and the separation layer between the two integuments. (Leon-Kloosterziel et al., 1994, McAbee et al., 2006). ATS belongs to the GARP gene family (McAbee et al., 2006, Zhang et al., 2009). In Arabidopsis, there are four KANADI (KAN) paralogues, KAN1 to 4. KAN1 and KAN2, also play a role in integument development, determining proper polarity of the outer integument (Kerstetter et al., 2001, Bowman et al., 2002, McAbee et al., 2006; Fig. 1C, D). Phylogenetic analyses assessing the evolution of the KANADI gene lineage have shown that homologues are present across vascular plants, but the exact origin of the ATS clade is still unclear (Zumajo-Cardona et al., 2019). UNICORN (UCN) encodes an AGC VIII protein kinase, which maintains planar growth of the integuments by directly repressing ATS (Enugutti et al., 2012, Enuguttii and Schneitz, 2013). Phylogenetic analyses have shown that these proteins are present across land plants (Galván-Ampudia and Offringa, 2007; Fig. 1E). Once the identity of the integument is established, SHORT INTEGUMENTS1 (SIN1), also known as DICER LIKE-1, play a role in the proper cell elongation of the integuments and in plant growth generally (Lang et al., 1994, Ray et al., 1996). sin1 mutants have integuments that fail to elongate leaving the nucellus exposed (Golden et al., 2002; Fig. 1F). Genes involved in the identity of integument development also seem to have an impact on the proper development of the embryo sac (Reiser et al., 1995, Elliott et al., 1996, Klucher et al., 1996). It has been shown that if the integuments fail to form so does the female gametophyte (Modrusan et al., 1994, Yang et al., 1999). This cross-talk appears to be maintained during seed development when seed coat growth also regulates endosperm formation (Figueredo and Köhler, 2014).

The molecular genetics that underlies ovule development has been extensively described in Arabidopsis and a few other angiosperms, even the most recent phylogenies have limited sampling (Becker et al., 2002, McAbee et al., 2006, Galván-Ampudia and Offringa, 2007). However, there is an enormous morphological variation of ovules across all seed plants particularly in the number and morphology of the integuments (Bouman, 1984, Endress and Igersheim, 2000, McAbee et al., 2005, Brown et al., 2010, Endress, 2011, Brenner and Stevenson, 2006). Therefore, to better understand the evolution and development of ovules it is necessary to study the genes involved in integument initiation and its proper development, such as BEL1, ATS, UCN and SIN1. Thus, here we report: (1) expanded sampling for each of these gene lineages across vascular plants; (2) the analysis of coding sequences across seed plant homologues to identify conserved regions between the pre- and post-duplication homologues that may help predict putative shifts in protein function; (3) changes in the selection constraints in the different BEL1 and KAN lineages that may suggest functional differences among clades. These results make it possible to formulate hypotheses about their impact on the morphological evolution of ovules among seed plants. This study provides the starting point for further molecular analyses in non-model organisms.

Section snippets

Homologue characterization of genes involved during integument development

For each of the gene families, the initial search was done using the Arabidopsis sequences as query to perform a BLAST search (Altschul et al., 1990). Phylogenetic analyses using only Arabidopsis homologues belonging to the BLH family of transcription factors, show that BEL1 (At5g41410) is sister to BLH2 (At4g36870) and BLH4 (At2g23760; Becker et al. 2002); using those three paralogs as queries, we extended the search across the major vascular plants lineages (angiosperms, gymnosperms, ferns

BEL1 gene lineage

The Maximum Likelihood analysis performed with 193 homologues found, provided a better understanding of the evolutionary history of BEL1 genes. In addition to detecting that BEL1 is angiosperm specific as the result of a duplication event that gave rise also to its sister lineage BLH2/BLH4, no major duplication events were identified in BEL1 (Fig. 2). Prior to this duplication are the gymnosperm homologues, which have undergone two specific duplication events, giving rise to three gymnosperm

Discussion

Most studies have focused on identifying the ovule developmental genes in Arabidopsis and phylogenetic studies have been done for some genes with restricted sampling to model species (i.e., Oryza sativa, Malus domestica, Solanum tuberosum, Solanum lycopersicum and Arabidopsis) in KAN, BEL1 and SIN1 (Becker et al., 2002, Zhang et al., 2009, Mizumoto et al., 2011) while UCN lacks any phylogenetic analyses in plants. Given the enormous diversity of ovule morphologies across seed plants, it is

Conclusion

Our results show that the proteins involved in the ovule regulatory network of Arabidopsis have undergone independent and distinct duplication events during plant evolution. As a result, the number of sequences of the main regulators has changed (Fig. 6; Table 1). SIN1 is the only gene that has been maintained without duplications and with a sequence almost identical to the Arabidopsis homologue across seed plants. All the other gene lineages have undergone duplication events specific to

CRediT authorship contribution statement

Cecilia Zumajo-Cardona: Conceptualization, Methodology, Investigation, Data curation, Writing - original draft, Writing - review & editing, Visualization. Barbara A. Ambrose: Conceptualization, Methodology, Investigation, Funding acquisition, Writing - review & editing.

Acknowledgements

We thank Favio Gonzalez and Natalia Pabón-Mora who helped improve the discussion of this manuscript. Martha Cardona for her help with the final edits of the manuscript. MPE associate editor Fay-Wei Li and the two anonymous reviewers for helping to improve the final version of the manuscript. Beverly Kukiel, our summer 2018 intern who helped with the supplementary table 1. This work was funded by The Eppley Foundation for Research, Inc. (New York, NY, United States).

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