**1. Introduction**

Rice is a staple food for more than half of the world's population and the demand is steadily increasing with the growing human population [1]. Climate change is a significant limiting factor for enhancing food production, because increasing abiotic and biotic stresses negatively affect the yield of all major crops [2–4]. During the past century, the global surface temperature has increased by an average of 0.85 ◦C, and a further increase of up to 3.7 ◦C has been predicted by 2100 [3]. This temperature increase develops asymmetrically, with a faster rise in daily minimum compared to daily maximum temperatures [5–9], leading to "high night temperature" (HNT) conditions. Asymmetric warming causes a reduction in the temperature difference between daily maximum and minimum temperatures, i.e., the diurnal temperature range (DTR), with a negative influence on both wild and

crop plant species [10]. In particular, the main rice-growing countries in Asia, including China [11], the Philippines [12,13] and India [14,15], are affected.

Several studies have reported a strong decrease in yield and grain quality, such as increased chalk formation, and altered grain growth dynamics in rice under HNT [16–21]. HNT can have a stronger impact on grain weight than high day temperatures in rice and wheat [22–24]. Field studies at the International Rice Research Institute (IRRI) in the Philippines showed that rice grain yield was reduced by 10% per 1 ◦C increase in night temperatures during the dry season (DS), whereas the effect of increasing day temperatures was not significant within the investigated time period [12].

Differences in HNT sensitivity among various rice cultivars based on grain yield [25–27], yield-related parameters, or phenotypes in the vegetative stage [28] have been reported, indicating natural variation in HNT tolerance. In addition, HNT reduces the starch content in panicles and negatively affects grain yield and quality (chalk and amylose content) in the sensitive cultivars Gharib and IR64, but not in the tolerant cultivar N22 [29].

Different factors may cause HNT sensitivity. Physiological effects reported under HNT include higher rates of respiration in leaves [28,30,31] and panicles [29], whereas photosynthesis is not affected [28] or may be decreased as well [32]. A reduction in nitrogen and carbohydrate translocation after flowering as a possible cause of yield reduction in HNT sensitive cultivars was also discussed [25]. Reduced grain weight and quality may be caused by lower sink strength due to lower cell wall invertase and sucrose synthase activity in sensitive cultivars, accompanied by higher sugar accumulation in the rachis [29].

Despite the increasing knowledge of the physiological responses to HNT, only little is known about the metabolomic responses of rice under these conditions. The metabolic status is important for growth, development and stress tolerance, and additionally influences important traits such as flavor, biomass, yield and nutritional quality [33–35]. Therefore, the assessment of the metabolomic status of wild and crop species can help to evaluate natural variation [33]. Additionally, the metabolome integrates molecular and environmental effects as endpoints of biological processes [36]. Moreover, metabolites constitute potential markers for the selection of tolerant crop genotypes in breeding programs. Several studies investigated metabolic changes in rice in response to abiotic stress conditions, such as salinity [37–41], osmotic stress [42], drought [43–47], heat [44,48], and combined drought and heat stress conditions [49,50].

In a corresponding study on rice under HNT conditions, sucrose and pyruvate/oxaloacetatederived amino acids were shown to accumulate while sugar phosphates and organic acids involved in glycolysis/gluconeogenesis and the tricarboxylic acid (TCA) cycle decreased in developing caryopses [48]. A dysregulation of central metabolism and an increase in polyamine biosynthesis was described for sensitive cultivars, whereas existing metabolic pre-adaptation under control conditions was found for tolerant cultivars [51,52]. Furthermore, in sensitive cultivars, 4-amino butanoic acid (GABA) signaling—and in tolerant cultivars, the jasmonate precursor *myo*-inositol—were linked to the HNT responses [52]. A metabolomics study investigating early seed development and the early grain-filling stage in six rice cultivars reported a sugar accumulation peak seven days after flowering and 19 significantly different metabolites under HNT compared to under control conditions, with a special focus on the generally higher abundance of sugars and sugar alcohols under HNT [53].

The goal of this study was to investigate the seasonal effects of HNT responses by assessing the metabolic responses to HNT stress in flag leaves and panicles during the DS and wet season (WS) in contrasting rice cultivars under field conditions. Previous studies of the comparison of HNT's effects during the WS and DS were limited to agronomic traits [13,14,20,26,54], while the influence of HNT on the rice metabolome has not been reported yet. The present study sheds new light on the responses of rice to an important climatic stress factor that may severely limit grain yield and quality, and therefore the global food supply.
