Identification of the profile of endogenous cytokinin-like compounds during different plant growth stages and their effects on flower bud abscission in pistachio (Pistacia vera L.)

Pistachio (Pistacia vera L.), a member of the Anacardiaceae family, is an important nut crop of Turkey and has a long history in that country. In particular, the Southeastern Anatolia region is well suited for pistachio cultivation due to its suitable climate conditions and thus it holds 95% of the total pistachio growing area which covers 91.5% of pistachio production in Turkey (Yavuz, 2011; Gundesli, 2017). P. vera ‘Uzun’ is the most popular cultivar, contributing more than 60% of the total production in Turkey. One of the main problems in pistachio production is the phenomenon of alternate bearing (irregular crop load) and flower bud abscission. Pistachio trees show some physiological disorders such as abscission of flower buds and fruit, production of empty fruit, unsplit nutshells and deformed nuts. Alternate bearing is identified by a year of bumper nut load, referred to as the ‘On’ year, followed by a year with an extremely light, light or no fruit load, called the ‘Off’ year in pistachio. This phenomenon is a problem not only in pistachio trees but also in several other fruit trees such as mango, olive and citrus (Vemmos, 1999a, 1999b; Baninasab and Rahemi, 2006; Lavee, 2007; Rosenstock et al., 2010; Goldschmidt, 2013; Sharma et al., 2015). Although this phenomenon is still not explained, different hypotheses have been reported. The alternate bearing in pistachio, as in all other fruit plants, is governed by both internal and external factors. The internal factors include the genotype and the endogenous hormone balance, whereas the external factors are the environmental conditions encountered by the pistachio tree (Mok and Mok, 2001; Foster et al., 2003; Achard et al., 2006; Al-Shdiefat and Qrunfleh, 2008; Bangerth, 2006; Dag et al., 2010; Gundesli et al., 2019). There are also other factors, such as planting density, irrigation and fertilisation, which may affect the growth and development of the plant (Weinbaum et al., 1994; Brown et al., 1995; Picchioni et al., 1997; Gundesli, 2017). Thus, floral development involves a large number of interwoven, well-defined growth, senescence and abscission processes. It is highly likely that plant growth regulators (PGRs) play distinct roles in the development of flowers, organs and functions. Accordingly, PGRs are thought to be among the most important causes of the activation of abscission zone and flower abscission (Aziz et al., 2001; Koshita and Takahara, 2004; Gomez-Jimenez et al., 2010; Gundesli et al., 2019).

Since the discovery of PGRs, they have been used to manipulate plant growth and development. PGRs are active at micromolar or lower concentrations in the growth and development of plants. PGRs are classified into five ‘classic’ groups – abscisic acid, auxins, cytokinins (CKs), ethylene and gibberellins (Davies, 2004; George et al., 2008; Bons et al., 2015; Sharma et al., 2019). Among these groups, CKs, N⁶-substituted purine derivatives, are a significant class of PGRs that regulate a large number of physiological and developmental processes in plants. Numerous researchers have proved that the effects of CKs on both major and minor physiological processes are important throughout the development in the plant, from seed germination to leaf senescence, including photosynthesis and respiration (Ahmadi and Baker, 2000; Mok and Mok, 2001; George et al., 2008). The most common endogenous CK-free bases are isopentenyl adenine (iP), cis- and transzeatins (c-Z, t-Z) and dihydrozeatin (DHZ), which may occur in different metabolic forms (Ahmadi and Baker, 2000; Novak et al., 2003; Stirk et al., 2004). CKs control various processes in plant growth and development, such as the delay in senescence, the transmission of feeding signals or control in the balance of root/root growth (Kamada-Nobusada and Sakakiba, 2009). Despite the richness of knowledge on the chemistry and physiology of CKs, the transition to molecular biology appears to be relatively slow and small compared with other plant hormones such as ethylene and gibberellins. In recent years, studies for CK receptors have begun to appear (Stirk et al., 2004; Novak et al., 2003). Although many metabolites have been identified, their importance and the regulation of metabolic transformations are still largely unknown. It has been reported that due to the transport of CK-like compounds to other organs, the species and concentrations of these compounds and also their functions showed differences in tissues between the plant organs (Wagner and Beck, 1993; Baktir et al., 2004; Davies, 2004). Palmer and Wong (1985) found 10 different CKs, including Z, ZR and DHZ. However, since the contribution of individual CKs to specific growth processes is unclear, the usefulness of CK amounts is open to interpretation. In short, CK research is important to solve many of the critical problems. A lot of information on the roles of endogenous CKs in fruit-bud abscission (alternate bearing) and embryo development in pistachio is still unknown. However, in recent years, a new approach to studying CKs has been developed. Therefore, in this study, major free-base endogenous CKs (Z, DHZ, iP) and their corresponding ribosides (ZR, DHZR, iPR) were determined using ultra-performance liquid chromatography–electrospray ionisation–tandem mass spectrometry (UPLC-ESIMS/MS) analysis. The objective of our research was to determine the levels of CKs in pistachio trees 35, 45, 55 and 65 days after full blooming (DAFB) to provide a complete CK profile during the critical stages of flower bud abscission and early embryo development.

In most cases, alternate bearing in fruit trees is the result of poor flower initiation, but in pistachio (P. vera L.), unlike in other fruit species, there are plenty of flower buds every year. Abscission of buds occurs during this quiescent period as well as at the stage when vigorous seed growth occurs in fruits in the current year (Monselise and Goldschmidt, 1982). Some investigators have suggested that excessive flower bud abscission in pistachio begins in June in an ‘On’ year when embryo development increases during July and causes it to be an ‘Off’ year in the following year (Cetinkaya, 2004; Okay et al., 2011). However, microscopic examinations performed by us had shown that the rupturing layer in pistachio flower buds began to form in the last week. Evidence in various fruit species has been shown that PGRs have significant effects during the growth and development periods that they particularly control abscission (Lavee, 1989; Aziz et al., 2001; Baktir et al., 2004; Cetinkaya, 2004; Acar et al., 2006; Gomez-Jimenez et al., 2010; Okay et al., 2011; Gundesli et al., 2019). However, the importance of PGRs and the regulation of metabolic transformations are still largely unknown in fruit trees showing alternate bearing. Previous studies had reported that some physiological events, such as flower bud and embryo formation, development, flower bud abscission, fruit set and growth, are regulated by PGRs such as auxins, CKs, many gibberellins, abscisic acid, polyamines and ethylene. According to many studies, different PGRs have been externally applied in pistachio to obtain regular yield, increase flower bud formation and prevent flower bud abscission [Cetinkaya, 2004, Takeda and Crane, 1980 (Abscisic acid); Lin et al., 1984 (Gibberellic acid); Ferguson and Maranto, 1989 (auxin); Lovatt and Ferguson, 1998, 2001; Acar et al., 2006 (GA₃ and CK); Lovatt et al., 2006, Okay et al., 2011 (auxin and CK); Aziz et al., 2001, Gomez-Jimenez et al., 2010, Gundesli et al., 2019 (polyamines)]. However, effective results could not be obtained in most of them because of insufficient data on the change in endogenous PGRs in the species involved. That is why recently, many studies have been carried out to explain the effects of CK-like compounds on plant growth and development. CK also mediates the responses to variable extrinsic factors, such as light conditions in the shoot and the availability of nutrients and water in the root, and it has a role in the response to biotic and abiotic stresses (Baktir et al., 2004; Cetinkaya, 2004; Davies, 2004; George et al., 2008; Al-Shdiefat and Qrunfleh, 2008; Okay et al., 2011; Sharma et al., 2019). The present study has given a fair insight into the status of CK-like compounds during bud abscission in P. vera L.. Using the latest available technology, the CKs in the different organs were identified and quantified. Concentrations of CK-like compounds in different growth periods and organs were determined in the ‘Uzun’ cultivar (Tables 3–7). There were statistically significant differences between the organs and almost all CK ratios in the ‘On’-year and ‘Off’-year trees for most of the periods of the experiment (p < 0.05). The results of the present investigation clearly show the presence of only four types of CK in different tissues, namely zeatin, zeatin riboside, zeatin glucoside and zeatin riboside glucoside (Tables 3–7 and Figures 3–7). This is evident from the results obtained by high-performance liquid chromatography with mass spectrometric detection (HPLC-ESI-MS/MS). In many studies, the physiological responses in flower bud and embryo formation, development, flower bud abscission, fruit set and growth performed by plants are thought to be due to CKs. Recently, many metabolites have been identified for CK-like compounds, but their importance and regulation of metabolic changes are still largely unknown (Ahmadi and Baker, 2000; Baktir et al., 2004; Stirk et al., 2004; Toker et al., 2006; Bajguz and Piotrowska, 2009; Okay et al., 2011). The most biologically active CKs are t-Z, DHZ and 2iP, while c-Z is thought to exhibit poor activity or none. However, it has been reported that cis-isomers are the main forms of CK in different plants (Emery et al., 2000; Sakakibara, 2006; Bajguz and Piotrowska, 2009). In this study, endogenous CK-like compounds were determined in the different organs and growth periods. We detected and quantified three groups of CK components: Z, Ip and DHZ in different tissues during flower bud abscission and kernel development. This is the first report on different types of CKs found before and after flower bud abscission. In particular, iPR and t-ZOG were found to be predominant forms of CK, which may be precursors of the biologically active CK s, and they were present at high concentrations (Table 3–7). These results agree with those of Ahmadi and Baker (2000), Ulvskov et al. (1992), Stirk et al. (2004), Lulsdorf et al. (2013), who found similar CKs, including the CK-like types in different plants. It is known that changes in CK activity are associated with plant growth and vary among plant organs (Palmer and Palni, 1987; Grayling and Hanke, 1992; Nooden and Letham, 1993; Sakakibara, 2006). Some researchers have identified many CKs in different plant species (Sab et al., 1985; Martin et al., 1987; Wagner and Beck, 1993; Komor et al., 1993; Ito et al., 2001; Novak et al., 2003; Stirk et al., 2004; Karady et al., 2011). In fact, more than 80% of the Z-type CK content in Urtica dioica and Rosa hybrida, as reported by Dieleman et al. (1997) and Wagner and Beck (1993), was consistent with our results.

In the present study, a high CK concentration was detected at a very early stage, which increased progressively and reached a peak as the flower bud abscission started and then decreased to a low level in the flower bud abscission period (55 DAFB – June) (Table 3–7). Other reports of seasonal variations in CK content in different plants have shown high levels of CKs in the flowering and development stages and then low levels in later periods. The patterns of changes in CK activity in different plants are in agreement with the findings of some researches (Doumas and Zaerr, 1988; Komor et al. 1993; Novak et al., 2003; Cetinkaya, 2004; Okay et al., 2011; Lulsdorf et al., 2013). Leaf and shoot CK contents were higher in ‘On’ than in ‘Off’ trees, while flower buds had a higher amount in ‘Off’ than ‘On’ trees. Cetinkaya (2004), Okay et al. (2011) (pistachio) and Baktir et al. (2004) (olive) found similar results. t-ZOG, t-ZR, c-ZR and iPR were found to be higher in the shoots than in the leaves. Although DHZR was found in nut samples, it was not detected in the other organs. t-Z was detected in shoot samples but not in leaf samples (Tables 3 and 4). CK contents were found to be higher in the panicles compared with the other organs (shoot, leaf and flower bud). The t-Z content of panicles was higher before fruit-bud abscission, but iP was low; however, the t-Z content decreased during fruit-bud abscission and kernel development periods, while the iP content increased (Tables 6 and 7 and Figures 3–7). The pattern of changes in CK activity was in agreement with the findings of Niimi and Torikata (1978). This suggests that there are different changes between the panicle and fruit in terms of CK activity. As a result, it is thought that different CKs are active in different organs of pistachio trees. Results presented elsewhere (Davies, 1995; Ito et al., 2001; Baktir et al., 2004) indicate that at least some of the CK supply entering into the flower passes into the leaves, and some may also pass into other developing organs, similar to our findings. As can be understood from this research, in addition to the amounts of CK that are synthesised in different parts of pistachio trees, flower bud abscission and fruit development periods reveal the activity of CKs. As a result, CK contents were high in the period just prior to flower abscission and low in subsequent stages. Indeed, Lovatt and Ferguson (1998, 2001) and Lovatt et al. (2006) had reported that the levels of CK in pistachio flower buds were low as a result of the effects of ageing, and especially during flower bud abscission, which was similar to our results. In many studies, PGRs have been applied exogenously to achieve regular yields of pistachio nuts to increase the formation of flower buds and to prevent flower bud and fruit abscission (Lovatt and Ferguson, 1998; Acar et al., 2006; Talaie et al., 2006; Lovatt et al., 2006; Vemmos, 2010). However, this is still not very effective. For this reason, it is very important to know the CKs in pistachio in different growth periods to explain the physiological mechanisms such as flower bud formation, and flower and flower bud abscission (alternate bearing). The data obtained in this study show a general relationship between CK-like compounds and flower bud abscission in pistachio trees. Due to this research, it can be concluded that the effect of fruit growth can be influenced by successful exogenous growth regulator applications in periods of slow and low synthesis of CK-like compounds, especially in the determination of effective application periods. Interestingly, the relatively high CK activity was recorded just before flower bud abscission in the fractions corresponding to iP and ZR.

In the present investigation, an experiment was conducted to record the changes in CK-like compounds during flower bud abscission in P. vera ‘Uzun’. In contrast to the studies previously reported in pistachio, CK-like compounds were identified in different organs and growth periods. To the best of our knowledge, this is the first report on CK-like compounds in pistachio. CK activity has been shown to have important effects during flower bud abscission and kernel development. Four CK types, namely zeatin, zeatin riboside, zeatin glucoside and zeatin riboside glucoside were detected. iPR and t-ZOG were the main and dominant CK-like compounds in all the organs analysed, and t-Z, t-ZR, c-ZR and DHZR followed, respectively. According to the results, especially iPR and t-ZOG declined during the heavy bud abscission period in ‘On’-year trees. In most cases, the concentrations of CK-like compounds in organs from ‘On’-year trees were lower than from those of ‘Off’-year trees. The strong negative correlation between CK-like compounds and bud abscission could be the first indication of the role of CK-like compounds in this unique phenomenon in the ‘Uzun’ pistachio cultivar. Thus, exogenous application of CK-like compounds in different concentrations is suggested for further studies to mitigate alternate bearing in pistachio. In conclusion, the relative amount of a specific form of CK may differ not only from one form to another but also in one particular plant tissue under different physiological conditions. This study clearly demonstrated that CK-like compounds play an important role in bud abscission and cause alternate bearing in pistachio.