High temperature in the root zone repressed flowering in Lilium × formolongi by disturbing the photoperiodic pathway and reconfiguring hormones and primary metabolism

Highlights

• High temperature in the root zone caused the inhibition of LD-mediated flowering induction in Lilium×formolongi seedlings.

• Through the analysis of gene expression, the key genes in circadian clock and photoperiod pathways were disturbed by root zone heating.

• The metabolome analysis revealed the fluctuation of hormones and primary metabolites caused by temperature were flowering regulators.

• The work can provide deep understanding on flowering induction in complicated natural environment for lily seedlings.

Abstract

Lilium × formolongi, a facultative long-day (LD) plant, has precocious flowering characteristics, as it can flower within one year from seeds without the need for vernalization. Under normal LD conditions, the seedlings entered the flowering induction phase and bolted with 9−10 rosette leaves at 6 months after sowing. High temperature, especially at the root zone, inhibited bolting of the seedlings in LDs, accompanied by changes in plant shape and rapid expansion of underground bulbs. Low temperature in the natural environment could rapidly reverse the inhibition caused by high temperature, and promote bolting even under non-induced short days. The circadian clock and photoperiod pathways were disturbed at the transcriptional level by high temperature in both leaves and bulbs, such as LfCCA1, LfCRY, LfCOP1, LfFKF1, LfCOLs and LfPIFs. Thus, the flowering integrators LfFT1 and LfSOC1 were suppressed by high temperature. To determine the flowering inhibitors induced by high temperature in the root zone, the metabolome was analyzed under different conditions. ABA and ACC accumulation in the leaves and bulbs respectively during high temperature, was crucial to flowering inhibition possibly via interaction with the circadian clock and photoperiod-dependent pathways. Additionally, under high temperature conditions, _D-allose, pentose and glucuronate interconversions and glutathione metabolism in the leaves were significantly activated. After cold exposure, TCA cycle was suppressed and SA, JA and some sugar (_D-mannose, _D-fructose, _D-arabinose and maltotriose) levels were upregulated in the leaves, simultaneously with the vigorous fatty acid metabolism in the bulbs. These metabolic regulations were crucial to the phase transition under different conditions.

Introduction

Light and temperature are the most crucial environmental factors for plants to synchronize their growth and development to coincide with seasons. Unlike the well-controlled conditions in the laboratory, the natural environment is more complicated. Global warming is one of the greatest threats to plant survival. For year-round production, high temperatures during the summer in low latitude regions (temperate and tropical areas) whether in greenhouses or in the field, bring many problems for growers (Wang et al., 2008). This high temperature inevitably causes high temperatures in the root zone. The roots are more sensitive to high temperature than the aboveground parts (Hao et al., 2012; Kawasaki et al., 2014; Sakamoto and Suzuki, 2015). High temperature alters several physiological and metabolic processes in cells and causes a series of stresses in plants, especially during the reproductive stage. The change in the anthesis date caused by high temperature is widespread among many crops and commercial flowers, such as wheat, barley, cauliflower, tomato, chrysanthemum and more (Gonzalo et al., 2020; Hemming et al., 2012; Matschegewski et al., 2015; Nakano et al., 2013; Wang et al., 2008).

Flowering regulation is a complex network and under multidirectional control, requiring the synthesis of endogenous signals and environmental factors to ensure flowering at the precise time. Day-length (photoperiod) and temperature are the most crucial environmental factors for plants to synchronize their flowering to coincide with seasons. In Arabidopsis, FLOWERING LOCUS T (FT) is an integrator of several pathways, such as photoperiod, vernalization, ambient temperature, gibberellin (GA), age and autonomous pathways (Blümel et al., 2015). Thus far, the molecular mechanisms of the components involved in these pathways have been well identified under well-controlled laboratory settings. Altering flowering time is a strategy for plants to ensure reproductive success under various stress conditions. Phytohormones, working alone or via cross-talk with each other play important roles in the response to multiple stresses and at the same time act as flowering signaling molecules. It has long been documented that the stress-response hormones abscisic acid (ABA), ethylene (ET), jasmonic acid (JA) and salicylic acid (SA) all regulate the transition to flowering by epigenetic regulation of FLOWERING LOCUS C (FLC), CONSTANS (CO), FT and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) (Campos-Rivero et al., 2017). With the development of large-scale automated analytical platforms, more studies have utilized metabolomics to elucidate the biological processes of environmental and endogenous signals regulation. In Arabidopsis, according to the comparison of the diel metabolic variation between the climate chambers and field conditions, including the levels of amino acids, sugars and anthocyanins, the single varying environmental parameter may fail to capture the complexity of the interactions between multiple simultaneously varying or covarying factors in the natural environment (Annunziata et al., 2018). For many fruits, thermal fluctuation during winter negatively affects endodormancy release (Horikoshi et al., 2018, 2017; Luedeling et al., 2011; Sugiura et al., 2007), and in pear, this unusual fluctuation causes disorders of carbohydrate and organic acid metabolism and the tricarboxylic acid (TCA) cycle (Horikoshi et al., 2018, 2017). In L. longiflorum cv. ‘White Heaven’ bulbs, glycerol has been identified as an inhibitor of sprouting and flowering that is synthesized in the outer scales and can be reduced by vernalization (Lazare et al., 2018). Therefore, the internal metabolites are sensitive to the changing environmental factors, and the fluctuation of metabolites may be crucial to the growth and development of plants.

Lilies are important commercial flowers that are usually used as cut flowers and potted plants and in landscapes with bulb planting. Commercial bulbs are generally propagated by scales and require 2–3 years of cultivation to become flowering bulbs (Ge et al., 2018; Marinangeli and Curvetto, 1997; Robb, 1957). Vernalization is considered obligatory for flowering of commercial bulbs, and the optimum cold treatment temperature and time depend on the variety (Brito De Almeida et al., 2017; Dole and Wilkins, 1994; Holcomb and Berghage, 2001; Roh and Wilkins, 1977). Thus, research on of flowering regulation in lilies focuses on the molecular mechanism of commercial bulbs during the vernalization process (Hamo et al., 2015; Liu et al., 2014; Villacorta-Martin et al., 2015). Lilium × formolongi, obtained from repeated interspecific crosses between L. formosanum Wallace and L. longiflorum Thunb, is an important commercial flower with a precocious flowering ability such that it can flower in 1 year from seeds without vernalization (Okazaki, 1996). L.×formolongi was determined to be a facultative long-day (LD) plant (Li et al., 2017). The seeds are usually sown from late November to January; subsequently, following a period of LD conditions, flower buds appear from July to September in the following year (Roh and Sim, 1996). Based on the morphology, anatomy and expression patterns of CO-LIKE9 (LfCOL9) and LfFT1, Li et al. (2017) identified the phase transition from the rosette leaf stage to the bolting stage as the critical period of flowering induction under LDs, representing the transition from vegetative growth to reproductive growth. However, high temperatures in the summer result in retardation of flowering and a high rate of rosette plants of some L.×formolongi cultivars (Sakamoto, 2006). L. longiflorum seedlings endemic to the northern part of the subtropical Ryukyu Archipelago became dormant at high temperature (≥25 ℃), producing no leaves or flowers until the end of the following January (Hiramatsu et al., 2012; Mojtahedi et al., 2013). High temperature also plays a decisive role in the flowering time regulation of lilies. Thus, analysis of the flowering regulation under actual production conditions is warranted.

Although several reports have been written about research on flowering induction of vernalization and photoperiod pathways, there is little research on the flowering mechanism under temperature stresses in more complicated environments of lily seedlings. Therefore, to determine what occurs under high temperature conditions, the growth and flowering characteristics and molecular and metabolic changes in L.×formolongi seedlings were systematically investigated under different temperature conditions.