Functional characterization and expression of GASCL1 and GASCL2, two anther-specific chalcone synthase like enzymes from Gerbera hybrida

Abstract

The chalcone synthase superfamily consists of type III polyketidesynthases (PKSs), enzymes responsible for producing plant secondary metabolites with various biological and pharmacological activities. Anther-specific chalcone synthase-like enzymes (ASCLs) represent an ancient group of type III PKSs involved in the biosynthesis of sporopollenin, the main component of the exine layer of moss spores and mature pollen grains of seed plants. In the latter, ASCL proteins are localized in the tapetal cells of the anther where they participate in sporopollenin biosynthesis and exine formation within the locule. It is thought that the enzymes responsible for sporopollenin biosynthesis are highly conserved, and thus far, each angiosperm species with a genome sequenced has possessed two ASCL genes, which in Arabidopsis thaliana are PKSA and PKSB. The Gerbera hybrida (gerbera) PKS protein family consists of three chalcone synthases (GCHS1, GCHS3 and GCHS4) and three 2-pyrone synthases (G2PS1, G2PS2 and G2PS3). In previous studies we have demonstrated the functions of chalcone synthases in flavonoid biosynthesis, and the involvement of 2-pyrone synthases in the biosynthesis of antimicrobial compounds found in gerbera. In this study we expanded the gerbera PKS-family by functionally characterizing two gerbera ASCL proteins. In vitro enzymatic studies using purified recombinant proteins showed that both GASCL1 and GASCL2 were able to use medium and long-chain acyl-CoA starters and perform two to three condensation reactions of malonyl-CoA to produce tri- and tetraketide 2-pyrones, usually referred to as alpha-pyrones in sporopollenin literature. Both GASCL1 and GASCL2 genes were expressed only in floral organs, with most expression observed in anthers. In the anthers, transcripts of both genes showed strict tapetum-specific localization.

Tetraketide α-pyrone reductases in sporopollenin synthesis pathway in Gerbera hybrida: diversification of the minor function

“…G TKPR1 exhibited a typical tapetum-specific expression pattern as do various reported sporopollenin biosynthesis associated genes (Fig. 3b ), including gerbera GASCL1 and GASCL2 24 . This is highly consistent with AtTKPR1 expression pattern in Arabidopsis in the tapetum of anthers, ranging from the meiosis stage to microspore stage 12 .…”

“…For verification of the expression of transformed genes in Arabidopsis, flower buds at stage 9 from transgenic lines and nontransgenic controls were collected for RNA isolation. The total RNA isolation and cDNA synthesis was performed as described previously 24 . For RT-PCR, gene-specific primers (Table S1 ) were used to amplify the corresponding cDNA sequences under the following PCR program: 95 °C for 3 min, followed by 28 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min, and followed by 72 °C for 8 min.…”

“…For RT-PCR, gene-specific primers (Table S1 ) were used to amplify the corresponding cDNA sequences under the following PCR program: 95 °C for 3 min, followed by 28 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min, and followed by 72 °C for 8 min. qRT-PCR was performed with gene-specific primers (Table S1 ) as described previously 24 and relative expression was calculated by using the ΔΔCt method as described by Pfaffl 40 . The gerbera housekeeping gene GAPDH and Arabidopsis TUBULIN were used as reference genes for gerbera and Arabidopsis, respectively.…”

“…Later, we identified two homologues of G2PS1 (G2PS2 and G2PS3) that use the same substrates but catalyze a longer elongation in the biosynthesis of another defense-related polyketide, 4-hydroxy-5-methylcoumarin 23 . Orthologues of Arabidopsis PKSA and PKSB, the gerbera anther-specific chalcone synthase-like 1 (GASCL1) and GASCL2, display the conserved tapetum-specific expression pattern and catalyze the conserved sporopollenin precursor biosynthesis with medium- to long-chain fatty CoA esters as starters 24 . During the exploration of accessory reductases in gerberin and parasorboside biosynthesis, we identified a reductase gene that displays a similar wide expression pattern as G2PS1, Gerbera reductase 1 ( GRED1 ), and its close paralogue GRED2 .…”

The Toughest Material in the Plant Kingdom: An Update on Sporopollenin

“…In rice, orthologous genes for each Arabidopsis sporopollenin biosynthesis-related gene have been characterized, including DPW (Shi et al, 2011), CYP703A3 (Yang et al, 2014(Yang et al, , 2018, CYP704B2 (Li et al, 2010), ACOS12 (Li et al, 2016;Yang et al, 2017), PKS1/LAP6 (Wang et al, 2013;Zou et al, 2017;Shi et al, 2018), PKS2 (Zhu et al, 2017;Zou et al, 2018), and TKPR1 (Xu et al, 2019), and exhibit largely redundant gene product activities (Table 1). These findings, together with the characterization of orthologs required for sporopollenin biosynthesis in monocots Zea mays (Tian et al, 2017) and Triticum aestivum (Singh et al, 2017), and eudicots Brassica napus (Yi et al, 2010;Qin et al, 2016), Nicotiana tobacum (Wang et al, 2013), Gerbera hybrida (Kontturi et al, 2017), and Hypericum perforatum (Jepson et al, 2014) support strong conservation of a core sporopollenin metabolic pathway in angiosperms (Table 1). Despite a deficiency in the study of sporopollenin-related orthologs in non-flowering species, characterization of MS2 (Wallace et al, 2015), ACOS6 (Li et al, 2019b), and ASCL (Colpitts et al, 2011;Daku et al, 2016) in the early emerging moss Physcomitrella patens supports substantial overlap in gene product activities, lending further support to the conservation of sporopollenin biosynthetic machinery among land plants (Table 1).…”