Introduction
Mast seeding (or masting) is synchronous highly variable seed production among years by a population of perennial plants (Kelly, 1994; Kelly, Turnbull, Pharis, & Sarfati, 2008; Schauber et al., 2002). This results in irregular heavy flowering and seeding events, which occur in a range of taxa globally, including in various woody and herbaceous endemic species in the New Zealand flora (Kelly et al., 2008; Schauber et al., 2002; Webb & Kelly, 1993). A key question is what external and internal factors allow the plants to synchronously trigger heavy reproduction in only some years. A better understanding of those factors, to allow prediction of changes under global climate change (Kelly et al., 2013; McKone, Kelly, & Lee, 1998; Rees, Kelly, & Bjornstad, 2002) requires clarification of the underlying genetic mechanisms which control masting (Samarth, Kelly, Turnbull, & Jameson, 2020).
Although masting imposes costs, such as missed opportunities for reproduction, it is selectively favoured in plants that gain benefits from one or more Economies of Scale (EOS) (Kelly, 1994; Kelly & Sork, 2002). The two most common EOSs are predator satiation (where seed predators are not able to consume all the seed produced, ensuring higher survival of the offspring) or more efficient wind pollination (Kelly & Sork, 2002). In order for masting to occur, plants need some synchronising factor, typically a weather cue. Several reports have suggested that a likely cue for masting comes from seasonal changes in summer temperature (Kelly et al., 2013; Schauber et al., 2002) so it has been speculated that increases in global temperatures may alter masting behaviour, although the nature of this effect is uncertain (Bogdziewicz, Kelly, Thomas, Lageard, & Hacket-Pain, 2020; Monks, Monks, & Tanentzap, 2016; Pearse, LaMontagne, & Koenig, 2017; Shibata, Masaki, Yagihashi, Shimada, & Saitoh, 2020). Changes in masting would affect the wider community, potentially impacting on food availability for indigenous seed predators and the rest of the food chain (Touzot et al., 2020).
In recent times, the use of ecological genomics tools has enabled us to determine the molecular nature of ecologically important traits including disease resistance, stress-responsive genes, and agro-economic traits, and their variability among individuals or populations (Richards et al., 2017). Molecular studies, such as those of Miyazaki et al. (2014) and Satake et al. (2019), have shown the potential to improve our understanding of the mechanisms that underpin mast flowering behaviour. Both these papers showed nitrogen levels as proximate drivers of masting in Fagus crenata using resource manipulation and gene expression studies. Similar studies can help show how changes in natural conditions may lead to the evolution of flowering-time genes and associated regulatory mechanisms. However, there is currently little molecular evidence on the mechanisms for temperature-driven mast flowering in plants (Samarth et al., 2021).
Information from model plant species provides useful background to the special case of mast seeding species. Molecular and genetic approaches have revealed that various external cues interact with the developmental processes to regulate the floral transition in perennial plants (Khan, Ai, & Zhang, 2014; Kobayashi et al., 2013). Genetic pathways controlling flowering time in model crops and temperate grasses, including Arabidopsis (Arabidopsis thaliana ), tomato, apple, rice, barley, wheat and Brachypodium distachyon (purple false brome), show a high degree of conservation between dicot and monocot species (Shrestha, Gomez-Ariza, Brambilla, & Fornara, 2014). Both dicots and monocots share common floral integrator genes including homologues of florigen, the universal flowering hormone. Florigen, or FLOWERING LOCUS T (FT), is a 175 amino acid long protein belonging to the p hosphatidyl e thanolamine b indingp rotein (PEBP) family, an evolutionarily conserved protein family found in all taxa of organisms from bacteria to animals and plants (Karlgren et al., 2011). Phylogenetic analysis of different homologues of the PEBP gene sequences across the plant kingdom has revealed three sub-families. These are MOTHER OF FT AND TFL (MFT), FT and TERMINAL FLOWER 1 (TFL1) (Karlgren et al., 2011). FT and TFL1 protein sequences share 60% homology with highly conserved amino acid sequences across diverse species. However, these genes act antagonistically to each other: FT promotes flowering whereas TFL1 represses it (Liu, Yang, Wei, & Wang, 2016).
Most of the core components of the regulatory pathways controlling flowering in temperate grasses are similar to Arabidopsis (Hill & Li, 2016). However, in contrast to the role of FLC in the vernalisation response in Arabidopsis, the vernalisation response which mediates flowering-time control in temperate monocots is controlled by the VERNALISATION (VRN) loci (Trevaskis, Hemming, Dennis, & Peacock, 2007). During early growth stages, a floral repressor named VERNALISATION 2 (VRN2), a CCT-domain protein, blocks the floral transition. Exposure to cold temperatures during the winter increases the expression of VERNALISATION 1 (VRN1) , a repressor of VRN2 (Yan et al., 2004). During the winter season, VRN1 protein binds to the promoter of VRN2 and blocks its expression (Woods, McKeown, Dong, Preston, & Amasino, 2016). Post-vernalisation, VRN1 maintains the repressed state ofVRN2 through epigenetic changes and releases the VRN2mediated suppression of VERNALISATION 3 (VRN3 ) (Ream et al. , 2014; Shimada et al. , 2009). VRN3 is an orthologue of FT and Hd3a - the mobile florigen in plants (Shimada et al., 2009). VRN3, expressed in the leaves, then travels to the shoot apical meristem and activates VRN1 to initiate flowering in warm spring conditions (Distelfeld, Li, & Dubcovsky, 2009).
In the current study, molecular tools were used to investigate the regulation of flowering of the alpine snow tussock, Chionochloa pallens (Poaceae), a temperate non-model grass species. This species is one of the most strongly masting species globally (Kelly et al., 2000), which gives the plant selective benefits through predator satiation (Rees et al., 2002). The possible impacts of global warming on masting in this species have been discussed in the literature (Kelly et al., 2013; McKone et al., 1998; Monks et al., 2016; Rees et al., 2002), but information on the molecular mechanisms inducing flowering is lacking. Understanding the molecular regulation of flowering in C. pallenscan provide not only a more accurate prediction of masting years in the face of climate change, but also aid in designing appropriate conservation strategies to save endangered New Zealand fauna (Samarth et al., 2020). From various plants, including some manipulated to induce or prevent flowering, we took leaf samples from tillers (shoots), some of which subsequently flowered and some remained vegetative. We later classified each leaf sample as coming from a plant that subsequently flowered or one from a plant that remained vegetative (Appendix S1). We then used ecological transcriptomics (Samarth et al., 2021, Samarth, Lee, Song, Macknight, & Jameson, 2019; Todd, Black, & Gemmell, 2016) to identify the potential homologues of PEBP sequences involved in the onset of flowering. Subsequent structural, functional and expression analysis of the PEBP sequences led to the identification of an orthologous TFL1 gene with a novel function. In addition, the global transcriptomic analysis revealed crucial transcription factors including thermosensory and floral epigenetic genes involved in the initiation of flowering in C. pallens .