Abstract
In response to challenges caused by climate change, apple (Malus ×domestica) breeding programs must quickly develop more resilient cultivars. One strategy is to breed for various bloom times. Members of the genus Malus, including domesticated apple, wild species, and hybrids, exhibit striking variations in the bloom date. Although bloom time is strongly influenced by chilling requirements, other aspects of floral development in Malus and their contributions to bloom time are less known. The purpose of this study was to investigate potential connections between predormancy flower development and final bloom time in Malus species. We performed a phenological analysis of flower development in wild and domesticated apple with extreme differences in bloom time over the course of one developmental season. We tracked histological changes in the floral apex of representatives of three early-blooming Malus genotypes (M. ×domestica ‘Anna’ PI 280400, M. orthocarpa PI 589392, M. sylvestris PI 633824) and three late-blooming genotypes (M. angustifolia PI 589763, M. angustifolia PI 613880, M. ×domestica ‘Koningszuur’ PI 188517). Our study documented their floral meristem progression and organ development and expanded on current staging systems for apple flower development to describe the changes observed. The developmental trajectories of each genotype did not group according to bloom category, and we observed variations in the floral development stage at the time of dormancy onset.
The domesticated apple (Malus ×domestica) is an economically valuable crop and an important source of nutrients and calories throughout temperate regions of the world. Fruit production is directly influenced by bloom time, which is a highly heritable trait greatly affected by the way a genotype perceives its environment (Celton et al. 2011; Gottschalk and van Nocker 2013; Hauagge and Cummins 1991). Because of climate change, fruit trees are becoming more susceptible to crop loss. First, early spring freezes may damage developing flower buds and reduce crop yield. These erratic weather events are increasing (Campoy et al. 2011; Legave et al. 2008; Rodrigo 2000; Unterberger et al. 2018). Second, climate change has affected chilling accumulation during winter, which disrupts the flower development and synchronicity of bloom (Campoy et al. 2019; Fadón et al. 2020; Luedeling 2012). Related to climate change, a summer drought in Germany affected subsequent chilling requirements and dormancy responses of apple buds (Fernandez et al. 2020). Third, apple flowers are largely self-incompatible, thus requiring growers to select cultivars that bloom synchronously to ensure adequate cross-pollination (Ramírez and Davenport 2013). Finally, environmental conditions that cause later bloom may result in apple trees being more prone to infection by Erwinia amylovora, the bacterial pathogen that causes fire blight (Jones 1992). These production considerations underscore the importance of studying flower development in Malus.
Domesticated apples exhibit a flowering cycle that spans nearly a full year (Goeckeritz and Hollender 2021; Nyéki and Soltész 1996). In summer, the inflorescence is initiated terminally on nascent shoots. The meristem then differentiates into terminal and lateral floral meristems. In Malus, the flower in the terminal position (often called the “king” flower) is dominant, and the usual four to six subtending lateral meristems are increasingly less developed toward the base of the inflorescence (Nyéki and Soltész 1996). Floral meristems continue to develop throughout the summer and rest in an incompletely developed state before winter dormancy. After an extended period of chilling during winter, warm temperatures in early spring of the subsequent season promote final development and bloom (Labuschagné et al. 2002; Powell 1986). Therefore, hypothetically, the ultimate timing of spring bloom could be conditioned by the combined influence of the timing of floral meristem initiation, rate of development before and during winter, amount of chilling required to exit dormancy, and the rate of development in spring (Goeckeritz and Hollender 2021; Gottschalk and van Nocker 2013).
The regulation of apple floral induction/initiation is an area of active research. At the physiological level, crop load, growth dynamics, genotype, architecture, bearing habit (e.g., regular or biennial), and temperature affect both the timing and rate of floral initiation in apple (Belhassine et al. 2022; Gottschalk et al. 2021; Guitton et al. 2012; Haberman et al. 2016; Hanke et al. 2007; Heide et al. 2020; Kofler et al. 2019). At the molecular level, exogenous hormone applications along with transcriptome studies of several M. ×domestica cultivars have highlighted a competitive relationship between cytokinin and gibberellin (GA) pathways during the floral transition (Li et al. 2018, 2019; Zhang et al. 2016). In support of the positive influence cytokinin has on the vegetative-to-floral transition in apple, treatment of spur buds with 6-benzylaminopurine (a synthetic cytokinin) led to upregulation of known flowering-related transcription factors (Li et al. 2019), whereas treatment with exogenous GA3 resulted in the downregulation of some of these same transcription factors and a reduction in flowering (Zhang et al. 2016). Although these broad trends are supported, the types of cytokinin and gibberellin are likely important to their effects on apple floral induction (Belhassine et al. 2022). More controlled, high-resolution experiments at both the cellular and developmental levels should help to clarify the effects of cytokinin and gibberellin on floral induction in Malus.
Recently, researchers compared protein abundances between “on” trees (heavy crop load, associated with lower floral bud initiation in strong biennial bearers) and “off” trees (little to no crop load, associated with higher floral bud initiation in strong biennial bearers) for two M. ×domestica cultivars: Fuji, a biennial bearer, and Gala, a regular bearer. They identified several clusters of proteins that were initially similar in abundance between on and off ‘Fuji’ spur apices but diverged in relative abundance closer to anticipated floral bud initiation (Kofler et al. 2022). These clusters contained proteins with various predicted functions, including flavonoid and phenylpropanoid biosynthesis, response to abiotic stimuli such as light and temperature, and chromatin remodeling. Fan et al. (2018) also implicated extensive chromatin remodeling during the floral transition. Together, these studies highlight the complexity of processes required for the floral transition in apple. Developmental phases are fine-tuned by genotype, environment, and their interactions.
Perhaps as complex as the floral transition and even more studied is the transition of the floral apex into and out of dormancy. As average temperatures decline, apple apices arrest their development irrespective of photoperiod (Heide and Prestrud 2005). To release this growth cessation, individual apices must measure the intensity and duration of cold (termed “chilling”) to resume development when warm temperatures return (termed “heating”) (Abbott et al. 2015). Like other temperate species, a strong genetic component for chilling and heating required to achieve full bloom is well-documented in apple (Abbott et al. 2015; Celton et al. 2011; Hauagge and Cummins 1991; van Dyk et al. 2010). During quantitative trait locus (QTL) studies, usually the limiting requirement (e.g., chill in mild climates and heat in cold climates) has a larger effect on bloom date (Allard et al. 2016; Celton et al. 2011), but QTLs that colocalize for both requirements were identified (Allard et al. 2016), suggesting that general temperature-sensing mechanisms are essential for development. These molecular mechanisms are complex. Researchers have implicated abscisic acid (ABA), GA, and cytokinin pathways (Cattani et al. 2020; Sapkota et al. 2021a; Tylewicz et al. 2018), as well as reactive oxygen species (ROS) (Beauvieux et al. 2018; Sapkota et al. 2021b) and the regulation of the cell cycle (Velappan et al. 2017) as modulators of dormancy transitions in apple and other perennial species.
Although many studies connect M. ×domestica bloom times with dormancy transitions, only a few have explored the influence of predormancy events on bloom time for M. ×domestica, and none that we know of investigated this in other Malus species (Buban and Faust 1982; Dadpour et al. 2011; Foster et al. 2003; Hoover et al. 2004). This lack of data is partly because floral meristem initiation and development are challenging to study. Primordial floral tissues are microscopic, friable, and encased within layers of leaf primordia or bud scales. Additionally, biennial (i.e., alternate) bearing tendencies of some Malus species and domesticated cultivars exacerbate sampling difficulties because of the repression of flowering in response to the crop load of the prior season (Durand et al. 2013; Lauri et al. 1995).
Our research provides insight into the relationship between predormancy events and the timing of spring bloom in apple. We characterized anatomical and structural changes occurring in floral buds from six Malus accessions (representing four Malus species) over the course of one developmental season. These accessions, maintained by the United States Department of Agriculture Plant Genetic Resources Unit (USDA-PGRU, Geneva, NY, USA), include three trees previously characterized as early-blooming (including two extreme early-blooming) and three characterized as extreme late-blooming according to a study documenting bloom time diversity for ∼1800 Malus accessions at the USDA-PGRU (Gottschalk and van Nocker 2013). Between our early-blooming and late-blooming groupings, there is a maximum 3-week difference in bloom time (Supplemental Fig. S1) (Gottschalk and van Nocker 2013). We took advantage of these unique accessions to study the potential relationship of predormancy development and final bloom time in Malus. Note, in the context of this work, we use “bloom time” and “flowering time” to describe flower opening in the spring, whereas “flowering” by itself refers to the rate and frequency of floral induction or initiation.
Materials and Methods
Description of plant material.
The accession species, names, and plant introduction (PI) identifiers used in this study are as follows: M. sylvestris (PI 633824), M. orthocarpa (PI 589392), M. ×domestica ‘Anna’ (PI 280400), M. angustifolia (PI 589763), M. angustifolia (PI 613880), and M. ×domestica ‘Koningszuur’ (PI 188517). Our early-blooming group comprised M. ×domestica cultivar Anna (PI 280400), M. orthocarpa (PI 589392), and M. sylvestris (PI 633824). ‘Anna’ is a commercial cultivar bred in Israel that is known for its low chilling requirement of ∼200 h (Hauagge and Cummins 1991; USDA 2015). For reference, many M. ×domestica cultivars have chilling requirements between 700 and 1200 h (Hauagge and Cummins 1991). The M. orthocarpa accession is a cultivated crabapple from England, and M. sylvestris is a European crabapple and a major progenitor of the domesticated apple (Sun et al. 2020; Velasco et al. 2010). Our M. sylvestris accession was collected in Germany (USDA 2015). The chilling requirements of our early-blooming crabapple accession are unknown.
The late-blooming group included M. ×domestica cultivar Koningszuur (PI 188517), which was bred in the Netherlands (USDA 2015). Hauagge and Cummins (1991) reported that ‘Koningszuur’ required ∼1450 chilling hours. The remaining trees in the late-blooming group were two different accessions of the southern crabapple M. angustifolia (PI 589763 and PI 613880), which have unknown chilling requirements. M. angustifolia is native to the southeastern United States and the eastern region of Texas, USA. PI 589763 is a triploid accession collected from North Carolina, USA, whereas PI 613880 is a diploid from South Carolina, USA (USDA 2015).
Sample collection.
Shoot apices or buds (Supplemental Fig. S2) were randomly collected from individual representatives of six apple accessions at the USDA-PGRU. All trees were maintained as grafts on semi-dwarfing rootstock (EMLA7 or M.7). To minimize effects of fruit load on early floral development, we verified that accessions were having an off year, removed fruit from trees, and/or collected apices in expected floral positions away from developing fruit whenever possible. Collections for histological analyses were performed on 25 Jul, 28 Aug, 5 Oct, and 8 Nov 2018, and 26 Feb 2019. For each date, at least five apices per genotype were collected and bud scales were removed to ensure proper fixation. Then, they were placed in ice-cold fixative (2.5% glutaraldehyde, 2.5% formaldehyde, 0.1 M sodium cacodylate buffer; pH 7.2) and subjected to vacuum infiltration for 30 min at room temperature. Then, fixative was replaced with fresh solution, and at least one additional vacuum infiltration was performed before storing samples at 4 °C in fixative or 0.1 M sodium cacodylate (pH 7.2).
Sample dehydration and embedding.
Stored tissues were washed three times with 0.1 M sodium cacodylate buffer (pH 7.2) and dehydrated in increasing concentrations of acetone (30%, 50%, 70%, 80%, 90%, 100%, 100%, 100%). For each incubation, samples were subjected to vacuum infiltration for 30 min and then gently shaken for 30 min in fresh solution. During the final 100% acetone step, samples were shaken overnight. Next, the tissues were subjected to gradual infiltration with increasing concentrations of a modified low-viscosity epoxy (Spurr) resin consisting of 10 g vinyl cyclohexene dioxide (ERL-422, cycloaliphatic epoxide resin; Electron Microscopy Sciences, Hatfield, PA, USA), 6 g diglycidyl ether of polypropylene glycol (D.E.R. 736 epoxy resin, Electron Microscopy Sciences), 26 g nonenyl succinic anhydride (epoxy hardener, Electron Microscopy Sciences), and 0.2 g dimethylaminoethanol (curing agent for epoxy resins, Electron Microscopy Sciences) (Spurr 1969). The Spurr-to-acetone solution ratios in sequence were (1:3), (1:3), (1:2), (1:1), (2:1), and (3:1), followed by four 100% Spurr incubations. Samples were vacuum-infiltrated for 1 h in each solution and then shaken in fresh solution two additional times for at least 5 to 12 h each. Then, buds were positioned in gel molds, fresh resin was added, and blocks were polymerized at 60 °C for 48 to 72 h.
Sample sectioning and mounting.
Tissues in resin blocks were sectioned using an ultramicrotome (RMC PTXL; Boeckeler Instruments, Tucson, AZ, USA) at Michigan State University’s Center for Advanced Microscopy (East Lansing, MI, USA). At least three apices were sectioned per sample at a thickness of 1 to 2 μm until the center of the apex was reached. When one or more of the first three apices sectioned from collections on or after 28 Aug 2018 were vegetative, additional ones were sectioned to reach a replicate number of three floral buds (a maximum of five were sectioned). This was based on the expectation that most, if not all, genotypes had undergone the floral transition by this date. This logic could not be applied to the 25 Jul 2018 collection date because it was possible that some genotypes had not made the floral transition; therefore, only three apices per genotype were sectioned on this date. Tissue limitations prevented a few samples from having three floral replicates for some collections. For the earliest time point (25 Jul 2018), the widest part of the apex was deemed the center. For subsequent time points, the approximate middle of the terminal (“king”) floral meristem was deemed the center. Sections were heat-mounted to glass slides, stained with a dilute toluidine blue and basic fuchsin solution (catalog no. 14950, Electron Microscopy Sciences), and imaged using a compound microscope (Eclipse Ni; Nikon, Tokyo, Japan) with a color camera (DS-Fi3, Nikon) and imaging software (NIS-Elements BR 4.60.00, Nikon).
Calculation of chill units and growing degree days.
Hourly temperature data were obtained from the Network for Environment and Weather Applications weather stations near Geneva, NY, USA, where the genotypes of the present study are planted (New York State Integrated Pest Management Program 2022). When possible, data were obtained from the Geneva station. However, occasional missing data warranted supplementation with nearby stations Geneva Agritech North and Geneva Bejo (gaps in hourly measures were less than 10 h in most cases). When data were missing from all three stations, the average of the temperatures flanking the gap was used to fill in the missing hours. Fahrenheit temperatures were converted to Celsius. Chilling accumulation was calculated using chillR’s “chilling” function (Luedeling et al. 2013). This function calculates chill for the dynamic model (Fishman et al. 1987a, 1987b), Utah model (Richardson et al. 1974), and chilling hours model (Bennett 1949; Weinberger 1950). Chill accumulation for select dates between 1 Nov 2018 and 1 Mar 2019 are presented in Supplemental Table S1.
Results
During Summer 2018, we revisited apple trees at the USDA-PGRU previously identified as early-blooming or late-blooming (Gottschalk and van Nocker 2013). Based on tree health, size, and number of floral buds, we selected three early-blooming and three late-blooming genotypes for our phenological study. The six trees we used exhibited bloom time differences in 2019 that were consistent with prior assessments (Fig. 1, Supplemental Fig. S1) (Gottschalk and van Nocker 2013).
Images taken on 17 May 2019, illustrating bloom time differences among Malus genotypes. Each genotype is indicated above the image. The early-blooming genotypes (A–C) were in bloom (A, B) or at petal fall (C). The late-blooming genotypes (D–F) were at tight cluster (D) or approaching pink (E, F). Visible frost damage can be seen on Malus sylvestris (C). Photos were taken by Kevin Maloney.
Citation: Journal of the American Society for Horticultural Science 148, 2; 10.21273/JASHS05236-22
Images taken on 17 May 2019, illustrating bloom time differences among Malus genotypes. Each genotype is indicated above the image. The early-blooming genotypes (A–C) were in bloom (A, B) or at petal fall (C). The late-blooming genotypes (D–F) were at tight cluster (D) or approaching pink (E, F). Visible frost damage can be seen on Malus sylvestris (C). Photos were taken by Kevin Maloney.
Citation: Journal of the American Society for Horticultural Science 148, 2; 10.21273/JASHS05236-22
Images taken on 17 May 2019, illustrating bloom time differences among Malus genotypes. Each genotype is indicated above the image. The early-blooming genotypes (A–C) were in bloom (A, B) or at petal fall (C). The late-blooming genotypes (D–F) were at tight cluster (D) or approaching pink (E, F). Visible frost damage can be seen on Malus sylvestris (C). Photos were taken by Kevin Maloney.
Citation: Journal of the American Society for Horticultural Science 148, 2; 10.21273/JASHS05236-22
Modification and enhancement of the current floral development staging system for apple.
Several staging systems have been developed for apple floral initiation and early differentiation. Hanke et al. (2007) and Milyaev et al. (2017) defined stages 1 (flat, vegetative meristem) to 5 (inflorescence differentiation) for M. ×domestica ‘Fuji’. Dadpour et al. (2011) defined stages 1 (flat, vegetative meristem) to 8 (apex doming) for M. ×domestica ‘Golden Delicious’ based on cell division patterns. Foster et al. (2003) defined eight stages of early floral development (stage 0–7) for the commercial cultivar Royal Gala. They quantified that floral induction is characterized by a widening of the apex and captured the latest stage of the three studies: sepal development in all lateral meristems of the inflorescence.
During the present study, we modified Foster’s staging system by defining each stage based on the development of the terminal floral meristem (which is in the apical position of the inflorescence) and incorporated later stages of development. The terminal meristem was chosen as a focus for comparisons because it was a consistent feature among all accessions. Stages 0 to 5 generally correspond to stages 0 to 5 defined by Foster et al. (2003). However, those authors distinguished stages 0 (vegetative) and 1 (vegetative but committed to floral) by the diameter of the shoot apical meristem (SAM). Because the SAM diameter and appearance varied by genotype in our study, the decision to call a meristem “flat” or “domed” and “wide” or “narrow” was not always clear; therefore, apices with only leaf and bract primordia present were assigned as vegetative and designated as stage 0/1. Stages 6 and 7 were modified based on the progression of the terminal floral meristem. Additionally, we created six new stages (stages 8–13) to describe later developmental events according to our histological analyses. Landmark features of stages and their depictions are shown in Fig. 2. The floral development stages observed for the six accessions at each sampling date are described. Photographs of cross-sections of representative apices from each accession and time point are presented in Fig. 3, and a summary of the staging for each bud is illustrated in Fig. 4.
Descriptions and corresponding drawings for Malus flower developmental stages. Stages 0 to 5 correspond to stages described by Foster et al. (2003). Stages 6 and 7 were modified from Foster et al. (2003) to describe the development of the terminal meristem (“king” flower). Stages 8 through 13 were created to describe further terminal flower development according to histological sections.
Citation: Journal of the American Society for Horticultural Science 148, 2; 10.21273/JASHS05236-22
Descriptions and corresponding drawings for Malus flower developmental stages. Stages 0 to 5 correspond to stages described by Foster et al. (2003). Stages 6 and 7 were modified from Foster et al. (2003) to describe the development of the terminal meristem (“king” flower). Stages 8 through 13 were created to describe further terminal flower development according to histological sections.
Citation: Journal of the American Society for Horticultural Science 148, 2; 10.21273/JASHS05236-22
Descriptions and corresponding drawings for Malus flower developmental stages. Stages 0 to 5 correspond to stages described by Foster et al. (2003). Stages 6 and 7 were modified from Foster et al. (2003) to describe the development of the terminal meristem (“king” flower). Stages 8 through 13 were created to describe further terminal flower development according to histological sections.
Citation: Journal of the American Society for Horticultural Science 148, 2; 10.21273/JASHS05236-22
Representative longitudinal sections of floral apices from three early-blooming and three late-blooming Malus genotypes. Floral apices were collected at the five indicated time points over the course of the 2018 to 2019 growing season. Dates are organized by column and genotypes are organized by row. Corresponding developmental stages are indicated in each image. (A–C) are early-blooming genotypes, and (D–F) are late-blooming genotypes (approximate 3-week bloom time difference). Sections are 1- to 2-μm thick and stained in a dilute toluidine blue and basic fuchsin solution; scale bar = 200 µm.
Citation: Journal of the American Society for Horticultural Science 148, 2; 10.21273/JASHS05236-22
Representative longitudinal sections of floral apices from three early-blooming and three late-blooming Malus genotypes. Floral apices were collected at the five indicated time points over the course of the 2018 to 2019 growing season. Dates are organized by column and genotypes are organized by row. Corresponding developmental stages are indicated in each image. (A–C) are early-blooming genotypes, and (D–F) are late-blooming genotypes (approximate 3-week bloom time difference). Sections are 1- to 2-μm thick and stained in a dilute toluidine blue and basic fuchsin solution; scale bar = 200 µm.
Citation: Journal of the American Society for Horticultural Science 148, 2; 10.21273/JASHS05236-22
Representative longitudinal sections of floral apices from three early-blooming and three late-blooming Malus genotypes. Floral apices were collected at the five indicated time points over the course of the 2018 to 2019 growing season. Dates are organized by column and genotypes are organized by row. Corresponding developmental stages are indicated in each image. (A–C) are early-blooming genotypes, and (D–F) are late-blooming genotypes (approximate 3-week bloom time difference). Sections are 1- to 2-μm thick and stained in a dilute toluidine blue and basic fuchsin solution; scale bar = 200 µm.
Citation: Journal of the American Society for Horticultural Science 148, 2; 10.21273/JASHS05236-22
Graphical representation of the developmental stage of individual flower buds from three early-blooming Malus genotypes and three late-blooming genotypes. The qualitative staging system is indicated on the y-axis, and the date (in days since 25 Jul 2018, with each collection labeled) is indicated on the x-axis. Individual genotypes are represented with different shapes and colors. This figure was made using ggplot2 (R Core Team 2022; Wickham 2016).
Citation: Journal of the American Society for Horticultural Science 148, 2; 10.21273/JASHS05236-22
Graphical representation of the developmental stage of individual flower buds from three early-blooming Malus genotypes and three late-blooming genotypes. The qualitative staging system is indicated on the y-axis, and the date (in days since 25 Jul 2018, with each collection labeled) is indicated on the x-axis. Individual genotypes are represented with different shapes and colors. This figure was made using ggplot2 (R Core Team 2022; Wickham 2016).
Citation: Journal of the American Society for Horticultural Science 148, 2; 10.21273/JASHS05236-22
Graphical representation of the developmental stage of individual flower buds from three early-blooming Malus genotypes and three late-blooming genotypes. The qualitative staging system is indicated on the y-axis, and the date (in days since 25 Jul 2018, with each collection labeled) is indicated on the x-axis. Individual genotypes are represented with different shapes and colors. This figure was made using ggplot2 (R Core Team 2022; Wickham 2016).
Citation: Journal of the American Society for Horticultural Science 148, 2; 10.21273/JASHS05236-22
25 Jul 2018: Floral initiation was evident for only one genotype.
On 25 Jul 2018 (1406.9 GDD), all apices sampled from five of the six accessions were vegetative (stage 0/1), with only leaf and bract primordia present (Fig. 3). These could represent purely vegetative apices or apices committed to being floral before differentiation. For the early-blooming M. orthocarpa, all three apices examined were markedly more advanced: one apex initiated floral bracts and lateral floral meristems (stage 3), another had a distinct terminal meristem with a bract and bractlets (stage 4), and the third initiated sepal primordia (stage 5) (Figs. 3, 4).
28 Aug 2018: A wide range of floral development was observed.
On 28 Aug 2018 (2001.6 GDD), two apices from M. ×domestica ‘Anna’ (early-blooming) were at stage 4, and a third was at stage 5 (Figs. 3, 4). For M. orthocarpa (early-blooming), all four floral whorls were clearly distinguishable in all apices (stage 9) (Figs. 3, 4). The single available floral bud from the M. sylvestris (early-blooming) apices had developing sepals and petal primordia (stage 7) (Fig. 3). Two apices of the late-blooming accession M. ×domestica ‘Koningszuur’ were also at stage 7 (Figs. 3, 4). In contrast, apices from the two accessions of M. angustifolia (late-blooming) were much less developed. For M. angustifolia PI 589763, two of the apices were domed (stage 2), and the third was at stage 3 (Figs. 3, 4). For M. angustifolia PI 613880, apices were either vegetative or at stage 2 (Figs. 3, 4). ‘Koningszuur’ (late-blooming) was now more advanced than M. ×domestica ‘Anna’ (Fig. 3A vs. Fig. 3D, second column). This suggests that the floral meristems from ‘Koningszuur’ initiated earlier and/or developed more quickly.
5 Oct 2018: Carpel development had initiated in all but the M. angustifolia genotypes.
Flower bud sections from the 5 Oct 2018 collection (2515.8 GDD) indicated that all buds from the early-blooming accessions (M. ×domestica ‘Anna’, M. orthocarpa, and M. sylvestris) as well as one late-blooming accession (M. ×domestica ‘Koningszuur’) had initiated carpels and reached or passed stage 9 (Figs. 2–4). Furthermore, placental cavities (a biological marker for stage 11) were present in one M. sylvestris bud (Figs. 2–4). Floral buds from the two M. angustifolia accessions continued to be less developed than the other accessions. The more advanced M. angustifolia PI 589763 exhibited sepal elongation (stage 6) (Figs. 3E, 4), whereas all M. angustifolia PI 613880 flowers were at stage 4 and had not yet initiated sepal primordia (Figs. 3F, 4).
8 Nov 2018: Floral buds from M. sylvestris were more developed, whereas those from M. angustifolia PI 613880 were the least developed.
On 8 Nov 2018 (2673.8 GDD), flowers from five of the six genotypes reached a more advanced developmental stage (Fig. 4). Sepals and petals continued to elongate for early-blooming ‘Anna’ and M. sylvestris as well as late-blooming ‘Koningszuur’ (Fig. 3). Carpels were also more developed in ‘Anna’ and ‘Koningszuur’ and slightly more enlarged in M. sylvestris (Fig. 3). All ‘Anna’ carpels exhibited placental cavities (a stage 11 marker), and their anthers were developing a characteristic butterfly shape, indicating the locules were clearly defined (Fig. 3A). In all M. sylvestris anthers, the tapetum surrounding sporogenous tissue (one of the stage 12 indicators) was apparent (Fig. 3C). Interestingly, Malus orthocarpa did not noticeably advance since the last collection (Figs. 3B, 4). Compared with the other accessions, floral development in both M. angustifolia accessions was significantly delayed. Sepal, petal, and anther primordia were visible in M. angustifolia PI 589763 buds, and the flower sections of M. angustifolia PI 613880 showed only sepal primordia (Figs. 3, 4).
26 Feb 2019: Floral development had slightly progressed.
On 26 Feb 2019 (974 chill hours, 15.1 GDD since 1 Jan 2019), all trees had no visible signs of budbreak and were considered dormant. However, compared with the 8 Nov collection, each accession exhibited a slight developmental advancement of one to two stages (Figs. 3, 4). Carpels of the terminal meristem from one ‘Anna’ and one M. sylvestris apex were elongating, marking the initiation of the partially fused style characteristic of Malus (stage 13) (Fig. 3C) (Rohrer et al. 1994). The other apices for both genotypes were at stage 12. Floral buds from the late-blooming ‘Koningszuur’ ranged from stages 11 to 12 (Figs. 3, 4), whereas early-blooming M. orthocarpa flower buds were at stages 10 to 11 (Figs. 3, 4). Consistent with prior collections, the two M. angustifolia accessions remained the least developed. The flower stages for M. angustifolia PI 589763 were between stages 8 and 9, whereas those from PI 613880 were between stages 7 and 8 (Figs. 3, 4).
Discussion
Our data suggest that the individual early-blooming and late-blooming genotypes we examined exhibited variations in floral initiation timing as well as rates of development. Furthermore, these differences did not always correspond to bloom time differences. For early-blooming M. orthocarpa, floral meristem initiation occurred before 25 Jul, as evident by its buds reaching or passing stage 3 (Fig. 4). Apices collected from all other genotypes did not contain buds that were obviously floral until 28 Aug, indicating a later date of initiation. In support of developmental rate differences, flower development in M. orthocarpa progressed only incrementally between 28 Aug and 5 Oct, and then it paused between 5 Oct and 8 Nov. It is possible this was caused by insufficient sampling; however, variability of the floral bud stage was low within genotype. In contrast to M. orthocarpa, appreciable and consistent development was observed for buds from the other five accessions during this timeframe (Fig. 4). Interestingly, during the anticipated winter “dormant” period (8 Nov 2018–26 Feb 2019), an advancement of floral development was observed for at least one bud from all genotypes (Fig. 4). However, without having assessed bud dormancy status (using bloom forcing experiments) and/or assessing developmental stages at more time points during winter, we cannot confidently know when these developmental changes occurred.
A variety of techniques have been used to characterize floral transition and development in M. ×domestica (Dadpour et al. 2011; Foster et al. 2003; Hoover et al. 2004; Kofler et al. 2022; Kotoda et al. 2000; Li et al. 2018, 2019; Mimida et al. 2011), but most did not evaluate the connection between initiation, floral development, and final bloom time. Hoover et al. (2004) noted genotypic variations between floral development timing of four M. ×domestica cultivars was not correlated with spring bloom time. Depending on the contrast in question, our histological analyses support these conclusions. For example, initiation times and development rates of early-blooming M. ×domestica ‘Anna’ and late-blooming ‘Koningszuur’ did not follow a pattern consistent with their final bloom times at all sampling dates (Fig. 4, Supplemental Fig. S1). However, the exaggerated shift in phenology of the M. angustifolia accessions compared with the other genotypes suggests timing of floral initiation and/or rates of flower development may condition the final bloom time for some Malus species. The developmental trajectory of late-blooming ‘Koningszuur’ grouping more closely with that of early-blooming until dormancy demonstrates this genotype probably differs in its chilling and/or heating perception. This result is supported by the high chill requirements of ‘Koningszuur’ compared with the low requirements of ‘Anna’ (Hauagge and Cummins 1991). These results underscore the need to study bloom time for different genotypes holistically, especially in perennial species in which floral progression occurs during all four seasons. This may enable the combination of multiple regulatory mechanisms to manipulate bloom time.
The majority of bloom time research of temperate fruit trees focused on the transition in and out of dormancy (Allard et al. 2016; Cattani et al. 2020; Celton et al. 2011; Fadón et al. 2020; Falavigna et al. 2014; Gottschalk and van Nocker 2013; Hillmann et al. 2021; Sapkota et al. 2021a, 2021b; Wu et al. 2017; Zhang et al. 2018). However, because dormancy describes the growth state of the meristem, it is logical to consider that developmental (and thus, meristematic) differences at the start of dormancy may be related to the amount of chill and heat needed to complete development. In the present study, we showed there is variation in the developmental stage at the time of dormancy onset (near or after 8 Nov). The samples ranged from stage 5 (M. angustifolia PI 613880) to stage 12 (M. sylvestris). These findings are interesting because little research has focused on the flower development stage as the tree enters dormancy and how this could affect final bloom time. It is unknown whether the stage at dormancy is regulated by intrinsic factors (e.g., genetics), environmental factors (e.g., temperature, photoperiod, drought), or their interaction. The stagnation in development for M. orthocarpa between 5 Oct and 8 Nov suggests that there is some genetic regulatory component because all trees were cultivated in the same orchard. If floral stage during dormancy influences bloom time, then it should be accounted for when considering connections between dormancy transitions and bloom time variations. In our study, for example, the M. angustifolia accessions may flower later because of delayed initiation, slower organogenesis rates, dormancy transitions, or any combination thereof. In contrast, because ‘Koningszuur’ flowered later than ‘Anna’ despite being at similar stages at dormancy, we may confidently speculate these cultivars must diverge in development from then until bloom.
Our research was a preliminary investigation of whether timing of floral initiation and organogenesis in Malus correlate with final bloom time. We documented flower development in early-blooming and late-blooming Malus species over the course of the 2018–19 season. Both genotypic differences in rates of floral bud development and differences in developmental stages before dormancy were identified. Furthermore, the rate of floral development did not strictly correlate with early and late bloom categories. Although our study would be stronger if we sampled a larger number of buds or extended collections over multiple seasons, our results and conclusions are reasonable because floral development was fairly synchronized within the genotypes at each time point. Our results provide valuable comparative phenological data from wild and domesticated apple accessions. This research will inform future studies of flowering in fruit trees and may influence breeding strategies targeting bloom time.
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Floral progression of three early-blooming Malus genotypes and three late-blooming genotypes on four dates in Spring 2019. M. ×domestica ‘Anna’, M. orthocarpa PI 589392, and M. sylvestris PI 633824 represent early-bloomers, and M. ×domestica ‘Koningszuur’, M. angustifolia PI 589763, and M. angustifolia PI 613880 represent late-bloomers. Dates are given for each column. Genotypes are organized by row. Photos were taken by Kevin Maloney.
Citation: Journal of the American Society for Horticultural Science 148, 2; 10.21273/JASHS05236-22
Floral progression of three early-blooming Malus genotypes and three late-blooming genotypes on four dates in Spring 2019. M. ×domestica ‘Anna’, M. orthocarpa PI 589392, and M. sylvestris PI 633824 represent early-bloomers, and M. ×domestica ‘Koningszuur’, M. angustifolia PI 589763, and M. angustifolia PI 613880 represent late-bloomers. Dates are given for each column. Genotypes are organized by row. Photos were taken by Kevin Maloney.
Citation: Journal of the American Society for Horticultural Science 148, 2; 10.21273/JASHS05236-22
Floral progression of three early-blooming Malus genotypes and three late-blooming genotypes on four dates in Spring 2019. M. ×domestica ‘Anna’, M. orthocarpa PI 589392, and M. sylvestris PI 633824 represent early-bloomers, and M. ×domestica ‘Koningszuur’, M. angustifolia PI 589763, and M. angustifolia PI 613880 represent late-bloomers. Dates are given for each column. Genotypes are organized by row. Photos were taken by Kevin Maloney.
Citation: Journal of the American Society for Horticultural Science 148, 2; 10.21273/JASHS05236-22
Images capturing the external appearance of shoot apices from the three early-blooming and three late-blooming Malus genotypes on the same days that these tissues were harvested and fixed for sectioning. Early-blooming genotypes are M. ×domestica ‘Anna’, M. orthocarpa PI 589392, and M. sylvestris PI 633824, and late-blooming genotypes are M. ×domestica ‘Koningszuur’, M. angustifolia PI 589763, and M. angustifolia PI 613880. The date of collection is indicated above the column. Genotypes are organized by row.
Citation: Journal of the American Society for Horticultural Science 148, 2; 10.21273/JASHS05236-22
Images capturing the external appearance of shoot apices from the three early-blooming and three late-blooming Malus genotypes on the same days that these tissues were harvested and fixed for sectioning. Early-blooming genotypes are M. ×domestica ‘Anna’, M. orthocarpa PI 589392, and M. sylvestris PI 633824, and late-blooming genotypes are M. ×domestica ‘Koningszuur’, M. angustifolia PI 589763, and M. angustifolia PI 613880. The date of collection is indicated above the column. Genotypes are organized by row.
Citation: Journal of the American Society for Horticultural Science 148, 2; 10.21273/JASHS05236-22
Images capturing the external appearance of shoot apices from the three early-blooming and three late-blooming Malus genotypes on the same days that these tissues were harvested and fixed for sectioning. Early-blooming genotypes are M. ×domestica ‘Anna’, M. orthocarpa PI 589392, and M. sylvestris PI 633824, and late-blooming genotypes are M. ×domestica ‘Koningszuur’, M. angustifolia PI 589763, and M. angustifolia PI 613880. The date of collection is indicated above the column. Genotypes are organized by row.
Citation: Journal of the American Society for Horticultural Science 148, 2; 10.21273/JASHS05236-22
Chill accumulation from 1 Nov 2018. The R package “chillR” from Luedeling et al. (2013) was used to calculate chilling accumulation from 1 Nov to select ending dates with three different models. Calculations were performed using the function “chilling”. The dynamic model (measured in chill portions), the Utah model (measured in hours using differential temperature effects within a certain temperature range), and the chilling hours model (measured in hours between 0 and 7.2°C) were derived from Fishman et al. (1987a, 1987b), Richardson et al. (1974), and Bennett (1949) and Weinberger (1950), respectively. Dates in bold indicate floral bud sampling dates.
Growing degree day (GDD) accumulation from 1 Jan 2018. GDD = sum (hourly_temp − base_temp)/24) when hourly_temp was greater than 4.5 °C. Dates in bold indicate floral bud sampling dates.
Growing degree day (GDD) accumulation from 1 Jan 2019. GDD = sum (hourly_temp − base_temp)/24) when hourly_temp was greater than 4.5 °C. Dates in bold indicate floral bud sampling dates. Dates in green have a corresponding photo in Supplemental Figure S1.
