Abscisic acid and putrescine synergistically regulate the cold tolerance of melon seedlings

Highlights

• A total of 14 genes related to ABA and polyamines biosynthesis are identified in melon.

• ABA has a positive feedback relation with putrescine in melon seedlings under cold stress.

• ABA and putrescine may act as signals to trigger the antioxidant system and increase melon cold tolerance.

Abstract

Low temperature in early spring severely endangers the growth and development of melon seedlings. Abscisic acid (ABA) and polyamines (PAs) are important signal molecules in plant response to stress. However, the issue of whether they interact to regulate melon cold tolerance remains largely uncharacterized. Here, we identified a total of 14 key genes related to ABA and PAs biosynthesis, including four CmNCEDs, and ten genes in PA pathway (one CmADC, one CmODC, four CmSAMDCs, two CmSPDSs, and two CmSPAMs). Two oriental melon cultivars (IVF571, cold-tolerant; IVF004, cold-sensitive) were selected to explore the difference of ABA and PAs biosynthesis under cold stress (15 °C/6 °C, day/night). Results showed that the expressions of CmNCED3, CmNCED3-2, CmADC, CmSAMDCs, CmSPDS2 and CmSPMS1 were significantly up-regulated. ABA and putrescine levels were significantly increased in IVF571 under cold stress. Inhibiting the biosynthesis of endogenous ABA with nordihydroguaiaretic acid (NDGA) or Put with D-Arginine (D-Arg) dramatically decreased the levels of each other and aggravated the cold injury of melon seedlings. In addition, spraying with exogenous 75 μM ABA or 1 mM Put improved the activities of superoxide dismutase, catalase and ascorbate peroxidase, and reduced the membrane lipid peroxidation damage of melon seedlings under cold stress. In all, the higher cold tolerance of IVF571 seedlings than that of IVF004 seedlings might be related to the increase in ABA and Put levels triggered by cold stress. ABA and Put could regulate the biosynthesis of each other and might act as signals to trigger the antioxidant system, thereby increasing melon cold tolerance.

Introduction

Melon (Cucumis melo L.) is of tropical origin and fails to cold acclimate. It is vulnerable to frost and cold injury at the early cultivation stages due to the anomalous variations in global climate (Li et al., 2020). Low temperature seriously limits crops growth and production (Ding et al., 2019). In the long process of acclimation, plant species have evolved sophisticated adaptations to defend against low temperature, among which, the abscisic acid (ABA) and polyamine (PA) signaling pathways are the important components (Ali et al., 2020; Sah et al., 2016).

ABA plays a crucial role in plant response to various stresses (Chen et al., 2020; Sah et al., 2016), which is one of the central regulators of cold stress signaling (Ciura and Kruk, 2018). As a signal molecule, ABA cooperates with other hormone pathways (Eremina et al., 2015) or signal molecules such as, NO, H₂O₂, and H₂S (Guo et al., 2014; Prakash et al., 2019), to reduce excessive reactive oxygen species (ROS) accumulation regulated by antioxidant systems, such as catalase (CAT), superoxide dismutases (SOD) and the ascorbate-glutathione cycle under abiotic stress (Chen et al., 2020). Transcriptome analysis revealed that 10% of ABA-responsive genes also contribute to cold stress (Hartung et al., 2002). ABA is mainly synthesized via the catalysis by 9-cis-epoxycarotenoid dioxygenase (NCED), which is the key rate-limiting enzyme (Hwang et al., 2010). Since the first NCED gene was identified from maize (Tan et al., 1997), additional NCED genes have been discovered from other plant species, including tomato (Burbidge et al., 1999), Arabidopsis (Iuchi et al., 2001), peach and grape (Zhang et al., 2009b), and kiwifruit (Gan et al., 2020). NCED expression is involved in fruit ripening; for example, AcNCED1, PpNCED1 and VVNCED1 promote fruit softening in postharvest (Gan et al., 2020; Zhang et al., 2009b). Abiotic stress can also induce NCED expression, and the expression level of this gene is directly related to the ABA signal transduction pathway and endogenous ABA accumulation. An NCED gene found in Stylosanthes guianensis, namely, SgNCED1, can be induced by drought, dehydration, salt stress, and cold stress (Yang and Guo, 2007). Another NCED gene found in Malus hupehensis, that is, MhNCED3 can enhance plant tolerance to Cl-stress (Zhang et al., 2015a). However, the NCED gene in the melon genome has not been identified, and the issue of whether it is involved in the cold tolerance of melon seedlings remains unsettled.

PAs are low molecular weight aliphatic nitrogen bases, that are widely involved in various biological processes, such as plant cell differentiation, morphogenesis, and programmed cell death (Galston and Sawhney, 1990; Moschou et al., 2012). In plants, Put is mainly formed by arginine, which is catalyzed by arginine decarboxylase (ADC) (Akiyama and Jin, 2007; Fuell et al., 2010). Put can continuously add aminopropyl groups to form spermidine (Spd) and spermine (Spm) via the catalysis of spermidine synthetase (SPDS) and spermine synthetase (SPMS). S-adenosylmethionine decarboxylation can also simultaneously form Spd and Spm by the catalysis of S-adenosylmethionine decarboxylase (SAMDC) (Fuell et al., 2010).

PAs are also involved in plant response to various abiotic stresses, including mineral deficiencies, salt, chilling, drought stress, wounding, heavy metal contamination, UV exposure, ozone, and pesticides (Alcázar et al., 2010; Ali et al., 2020; Minocha et al., 2014; Shen et al., 2019). Free PAs can be used as osmotic regulators and ROS scavengers in response to osmotic stress or cold stress, and they also act as signal molecules in response to stress (Alcázar et al., 2010; Zhang et al., 2009c). PAs have been reported to activate changes in Ca²⁺ efflux and membrane potential, enhance Ca²⁺ signal transduction, and regulate transmembrane transport under stress (Pottosin et al., 2014). Moreover, PAs can improve the cold tolerance of tomato by triggering NO, H₂O₂, and other signaling pathways (Ali et al., 2020; Diao et al., 2016; Song et al., 2014; Xu et al., 2019). In Arabidopsis and tomato, eight and 14 genes involved in PA biosynthesis have been identified, respectively (Liu et al., 2018; Urano et al., 2003). These genes show different profiles of expression not only during plant development but also in response to abiotic stress (Liu et al., 2018; Urano et al., 2003). Some PAs biosynthesis genes have been reported to involve in melon salt stress response; for example, CmADC, CmSAMDC, CmSPDS and CmSPMS can be induced by salt stress (Shen et al., 2019; Xu et al., 2019). To date, the genes of PA synthesis in melon have not been fully identified, and how they respond to cold stress in melon seedlings is unknown.

Accumulating evidence suggests that ABA and PAs are interrelated in stress response (Toumi et al., 2010; Wimalasekera et al., 2011). ABA signaling pathway integrates PAs and apoplastic amine oxidases to regulate H₂O₂ generation, which recruits further stress responses (Toumi et al., 2010). Copper amine oxidase 1 (CuAO1) contributes to enhanced ABA- and polyamine-induced NO biosynthesis (Wimalasekera et al., 2011). ABA-responsive element (ABRE)-binding factor, has been proved to function positively in dehydration tolerance by maintaining ROS homeostasis via the modulation of antioxidant enzymes and PAs through relevant target genes (Zhang et al., 2015b).

Previous studies have demonstrated that exogenous ABA can improve the cold tolerance of melon seedlings by up-regulating endogenous gibberellic acid and salicylic acid (Kim et al., 2016), whereas PAs are involved in γ-aminobutyric acid (GABA)-regulated melon salinity-alkalinity stress tolerance (Xu et al., 2019). However, the role and relationship of the PA and ABA signaling pathways in melon cold stress response is unclear. Moreover, the key genes involved in the two pathways have not been fully identified and elucidated in melon. In this study, we tried to explore whether ABA and PAs were related to the formation of cold tolerance in melon varieties with different cold tolerance, and further clarified if these two metabolites interacted to regulate cold tolerance in melon.