In this study, we evaluated the importance of exotherm characters on the response of the freezing-resistance mechanism of flower buds. Our findings showed that a single-peaked exotherm per specimen was detected in the flower buds of cultivars exposed to DTA tests during the bud break stage, and they were considered as the lethal temperature for flower buds (Fig. 1e). Previous studies supporting our results have revealed that a single exothermic peak of flower buds of deciduous fruit crop species was detected using DTA during the bud break stage (Meng et al., 2007; Gil-Albert, 1998; Kaya et al., 2018; 2020; Kaya and Kose, 2019). Indeed, studies evaluating the freezing response of different organs such as herbal parts, flower buds, and flowers of some plant species have reported that single peak determined by DTA is reliable and acceptable in predicting the lethal temperatures of the buds (Fiorino and Mancuso, 2000; Gale and Moyer, 2017; Smith and Redpath, 2018; Kaya et al., 2018; 2020). In fact, these single exotherms in our findings could be explained by the hypothesis that the buds lose their deep supercooling capacity in species belonging to the sub-genus Prunus during the deacclimation stage (Ashworth, 1990; Rodrigo 2000; Kaya et al. 2018; 2020). Basically the number of exotherms depends on the presence or absence of xylem in the flower primordia or whether the vascular continuity of the xylem is associated with the shoot tissues. More clearly, since the continuity of the xylem vessels between the shoot and the flower is established during the bud break phase, the flower buds of many deciduous species lose their deep supercooling capacity, and so that the ice crystals in the bud tissues spread immediately via the vascular vessels (Wisniewski et al., 1997; Kuprian et al. 2016). In cases where xylem vascular connections are established between the shoot and the flower in the spring, freezing occurs simultaneously in the extracellular and intracellular regions of the bud tissues, and thus a single exotherm is obtained from the sample (Fiorino and Mancuso, 2000; Gale and Moyer, 2017; Kaya et al., 2018; 2020; Kaya and Kose, 2019).
In general, frost tolerance of sweet cherry flowers has been previously studied mainly under field and laboratory observation conditions (Miranda et al., 2005; Matzneller et al., 2016). These methods allow the measurement of frost damage of flowers at different phenological stages of bud burst and are performed visually by observing dark discoloration or browning of the pistil after samples exposed to freezing have been thawed at room temperature for 24 or 48h. However, it is not very clear to understand what temperature level causes the death of pistil organs in this method, and it is not practical to research a few samples. On the contrary, the exothermic temperatures (mLTE values) of the flowers of many deciduous fruit species were determined in different phenological stages with DTA, which is an easier and more difficult method (Kaya et al., 2020). In contrast to classic visual evaluation studies after and during bud burst, in this study we used the only DTA method, and flowers were exposed to frost in a temperature control transparent climate chamber, so that the mLTE values of flower buds in our study showed compliance with the results reported by Kaya et al. (2020).
Considering the phenological stages, it was seen that all cultivars were tolerant to frost at the side stage, and a partial decrease in frost tolerance occurred at the green tip stage (except for 'Merton Late' and 'Bigarreau Gaucher' cultivar for 2020), and the significant losses of frost tolerance occur at the open cluster stage (Tab. 1). Previous studies have reported a gradual decrease in the frost tolerance of the buds with the progression of phenological stages after bud burst. Investigations have shown that the tolerance of the buds to low temperatures usually follows the order of side green > green tip > open cluster > first white > full bloom according to phenological stages (Proebsting and Mills 1978; Miranda et al., 2005; Salazar-Gutierrez et al., 2014; Matzneller et al., 2016). In our study consistent with these results, it was found that frost tolerance gradually decreased in the buds from bud burst to the open cluster stage. But interestingly, in contrast to previous findings on frost tolerance of flowers at the first white and full bloom stages (Proebsting and Mills 1978; Andrews et al., 1983; Rodrigo 2000; Miranda et al. 2005; Salazar-Gutierrez et al., 2014; Matzneller et al. 2016; Alhamid et al., 2018), all cultivars in our study showed the highest frost tolerance at the first white and full bloom stage compared to other stages of development in both years. Our findings, however, seem to confirm the preliminary observations of Kaya et al. (2020) at the first white and full bloom stages. Although they did not test the frost tolerance of the buds in the open side green, green tip and open cluster stages, they determined the critical temperature values for the cherry flower buds in the first white (-9.54°C = 50% damage) and full bloom (-9.19°C = 50% damage) stages. In this study, we focused on the hypothesis that flower buds have more cell count and water content at side green, green tip and open cluster stages, and so they may have shown mLTE values at higher temperatures. As is known, there may be one to four or five stalks (flowers) in a dormant cherry bud and these stems are reserved within sepal up to the open cluster stage. In other words, in the first white and full bloom stage, which are the stages after the open cluster stage, the flowers completely separate from the sepals. We, therefore, think that the water content and cell number of the flower buds tested in the first white and full bloom stages were low compared to other stages of development, and thus they exhibited mLTE values at lower temperatures. In studies supporting this hypothesis, it has been reported that high water content and tissue size in flower buds have a negative effect on freezing tolerance (Ashworth et al., 1983; Quamme, 1991). Although we assume that this may be related to the number of cells and water in the flower buds, we argue that the main reason for this may be due to different physiological, morphological and biochemical events and that more research should be carried out. Because, it has been reported that frost tolerance in plant occurs as a result of a complex process involving a series of physiological and biochemical events, such as changes in the accumulation of certain proteins, lipid-membrane compounds (Wisniewski et al., 2003; Chinnusamy et al., 2007).
In the study, mLTE values of sweet cherry flower buds differed depending on different developmental stages of flowering, year and cultivars. These variations can be explained by the differences among cherry cultivars, years and different phenological stages. It has, indeed, been reported that the response to low temperature of plants varies among the flower organs, rootstock and rootstock/cultivar combination, different developmental stages of flowering, among species and cultivars (Proebsting and Mills 1978; Guy, 1990; Köse, 2006). In general, 'Merton Late' 'Noir de Guben' and 'Merton Bigarreau' cultivars had the least hardy frost tolerance at side green, green tip, first white, and full bloom stages, while 'Van', wild genotype and 'Bigarreau Gaucher' were the highest cultivars in those development stages for both years (Fig. 2, 3, 5 and 6). At open cluster stage, on the other hand, there were significant differences in mLTE values of flower buds of the cultivars for both 2019 and 2020, but their mLTE values were obtained at temperatures below -2oC (except for 'Noir de Guben' and 'Bigarreau Gaucher' for 2020). At this stage for 2019, 'Merton Late' was the most sensitive cultivar to low temperatures, while other cultivars were found to be more tolerant to low temperatures. Unlike 2019, in 2020, 'Bigarreau Gaucher' showed the most tolerance to low temperatures, while other cultivars reacted similarly to low temperatures (Fig. 4). The mLTE value differences in among cultivars tested under similar test conditions point out that there is a strong genetic component effectively leaving cultivars with dramatically different safety margins from freezing damage. It has been reported that frost tolerance of plant species, cultivar and organ are mainly related to genetic structure and also frost tolerance in plants is a complex biological process (Wisniewski et al., 2003; Szalay et al., 2019; Chinnusamy et al., 2007). Interestingly, mLTE values of all cultivars in open cluster stage occurred at higher temperatures compared to mLTE values obtained from flower buds of cultivars in other stages. These findings are both the first records in the literature in terms of frost tolerance responses of flower buds according to phenological stages in sweet cherry cultivars and offer a different perspective for subsequent frost tolerance studies. Although we assume that while discussing the above open set stage, the reason for this may be related to the cell number and size and water content, our hypothesis needs to be confirmed. With our current findings, it seems very difficult to explain why sweet cherry cultivars are more susceptible to frost in the open cluster stage compared to the other phenological stage. In this context, we assume that thanks to the physiological, morphological and biochemical studies to be carried out in the following years, the answers to our currently unexplained questions can be found.
On the other hand, mLTE values of flowers differed among cultivars based on ripening at all phenological development stages, and 'Merton Late' cultivar, which has very late ripening characteristics, was injured at higher temperatures than other cultivars, which have very early ripening, middle and late characteristics. Research supporting our results have been reported that frost tolerance is less in late maturing cultivars (Chaplin and Schneider, 1974; Köse, 2006). However, there were significant differences in the mLTE between late ripening cultivars at all phenological development stages, and between these cultivars that mature late, 'Van' cultivar showed mLTE values lower temperatures, usually followed by 'Noir de Guben' and 'Bigarreau Gaucher' cultivars, respectively. Additionally, wild genotype, which has very early ripening, was classified as moderately frost tolerant, whereas 'Merton Bigarreau' cultivar, which has mid-season ripening, was classified as sensitive to frost (Fig. 2, 3, 4, 5 and 6). Although the response of the studied cultivars to low temperatures contradicts with the above literature, we hypothesize that the response of cultivars to spring frost depending on the maturation time may be due to genetic characteristics. On the other hand, it is known that cell walls both thickened and rigid in the flower buds during the acclimation stage (Rajashekar and Lafta, 1996; Mathers, 2004). The opposite occurs during the deacclimation stage, and cell walls typically become thinner, decrease their tensile strength and increase their pore sizes. Perhaps, varieties showing mLTE values at higher temperatures typically have thinned cell walls, reduced tensile strength, and increased pore sizes. However, this assumption needs to be supported both by histological analysis and using different measurement techniques such as microscopic observation, cryomicroscopy and low temperature scanning electron microscopy.