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 .