Physiology of Citrus Flowering and Fruiting

Abstract

The physiology of citrus flowering and fruiting is a crucial and multidirectional component of citrus agriculture. It encircles a web of intricate biological processes and factors determining when and how citrus trees flower and produce fruit. These critical factors are temperature and water stress, which profoundly impact flowering initiation. Hormonal regulation, with gibberellins and auxins as primary actors, is associated with the timing and progression of flowering and fruiting. Understanding floral induction mechanisms is essential for optimizing flowering schedules and fruit production since it is the fastest step in the reproductive process. Successful pollination and fruit set are crucial for productive citrus yield. Moreover, environmental variables like temperature, humidity, and nutrient availability substantially influence citrus physiology. In summation, delving into the physiology of citrus flowering and fruiting is essential for effective orchard management, increased fruit harvests, and the cultivation of premium citrus crops. Understanding these physiological aspects is crucial for optimizing citrus orchard management, enhancing fruit yield, and ensuring higher production with better quality citrus fruits.

1. Introduction

The physiology of the citrus (Citrus spp.) crop’s flowering and fruiting is a complex and dynamic process shaped by an amalgamation of genetic, hormonal, and environmental factors [1, 2]. Citrus trees exhibit a highly regulated reproductive cycle that determines the successful development of flowers and subsequent fruit formation. Central to this process is the concept of floral initiation, a critical stage influenced by environmental and endogenous factors. As temperature fluctuations and daylength vary, the transition from vegetative growth to reproductive development is triggered [3, 4]. Once the floral initiation is underway, the trees undergo a series of developmental phases, including bud differentiation and the emergence of floral organs. Hormones play pivotal roles in coordinating these events, orchestrating the growth and differentiation of cells to form the intricate structures of blossoming flowers [5]. Understanding the citrus flowering and fruiting physiology is paramount in agricultural contexts. Farmers and researchers can leverage this knowledge to optimize cultivation practices, manage crop yields, and address challenges such as irregular fruiting patterns or environmental stressors. This chapter delves into the physiological mechanisms of citrus flowering and fruiting, encompassing environmental cues, hormonal regulation, and developmental stages.

2. Factors influencing flowering in Citrus spp.

The factors influencing citrus flowering can broadly be divided into environmental or exogenous and endogenous factors (Figure 1).

Figure 1.

Factors influencing the flowering in Citrus spp.

2.1 Environmental influences

In temperate climates, the onset of low temperatures triggers floral induction [6, 7]. Webber [8] highlighted a temperature-dependent variation in the onset of flowering in Citrus, with higher temperature zones (such as Florida) experiencing flowering a month earlier than cooler temperature zones (like California). The floral induction in mandarins and sweet oranges occurs during autumn [9] while, in Satsuma mandarins, it occurs later in January [9, 10]. In contrast, flower induction in tropical climates, characterized by minimal temperature fluctuations year-round, is not dependent on low temperatures. Citrus trees in these areas bloom multiple times annually, with spring flowering being the most robust [11]. The higher temperature range at or above 25°C put forth only vegetative growth [12]. In comparison, the lower temperature at 15°C for a period of 1.5 months initiates flowers [13] in the Satsuma mandarin tree, showing a positive correlation with the CiFT3 gene expression [14]. The flower-inducing genes CsLFY, CsAP1, and CsTFL1 express at the 15°C-inductive period due to upregulation at the onset of floral meristem differentiation [14]. At the field conditions of the “Mediterranean basin”, CiFT2 expression in ‘Moncada’ mandarin [C. clementina × (C. unshiu × C. nobilis)] is triggered when the minimum temperature drops below 15°C [15]. The optimal temperature range for flower initiation appears to be between 10 and 15°C [6, 16].

A strong correlation between the hours of low temperatures and flowering intensity has also been demonstrated in Citrus spp. During winter ectodormancy, accumulation of inductive temperatures (712–1474 hours/year) below 20°C explains significant year-to-year variations in flowering intensity [17]. Occasional winter warmth following low-temperature periods induces flower bud differentiation, causing flowering waves. In regions with continuous low temperatures, a thirty-day induction period results in moderate flowering, while 45 days or more with low temperatures typically leads to robust flowering [6]. The optimum low temperature and ideal drought stress induction levels are seldom met in intermediate climates. The impact on flowering is unclear when there’s both drought stress and insufficient duration of low temperatures for flower bud induction.

While the citrus trees can endure low light and continue flowering and bear fruit, optimal flowering occurs in full sun with maximized light availability through the canopy. Hence, effective pruning to prevent overcrowding is crucial for achieving the best flowering outcomes. The Citrus spp., under tropical climates, requires a period of water stress for flowering induction [7, 18]. A minimum of 45–60 days of water stress is necessary for an economically viable level of flowering [18, 19]. However, prolonged water stress of 70 days or more may harm tree health and productivity [18].

2.2 Hormonal regulation

The hormonal regulation of citrus flowering and fruiting is a complex process involving various plant hormones that interact to coordinate the growth and development of reproductive structures. Essential plant hormones such as gibberellins (GAs), auxins (Ax), cytokinins (CK), abscisic acid (ABA), and ethylene play crucial roles in different stages of citrus flowering and fruiting. During floral organogenesis, a critical divergence emerges among the functions of floral meristem and inflorescence meristem [20]. Developmental dynamics within the inflorescence meristem are multifaceted, characterized by the coexistence of both determined and indeterminate shoots, ensuring the polycarpic nature of the plant [21]. Following the formation of the floral meristem, the precise growth of the flower bud is controlled by a complex cascade of genes under hormonal control. Here’s an overview of their roles.

2.2.1 Gibberellic acid (GA)

In fruit trees, GA has traditionally been linked to promoting vegetative shoot growth [22] and antagonistic to flowering behavior [7]. In fruiting shoots devoid of flowers, higher GA has been noticed (e.g., Satsuma mandarin and Valencia Sweet orange) compared to vegetative shoots [23, 24]. Flowers in Citrus spp. predominantly emerge from short ‘generative shoots’ with reduced GA levels [25]. During the ectodormancy period (in Autumn-Winter), foliar spraying of GA3 significantly decreases the number of flower Citrus spp. [26]. GA is commercially recommended for the Citrus hybrids exhibiting ‘weak parthenocarpy’ and ‘insufficient cross-pollination,’ aiming to enhance the ‘fruit set’ [27]. Sequential applications of GA3 mitigate flowering and sprouting intensity in ‘Montenegrina’ mandarin trees during the spring [28]. GAs inhibit flower induction during the fall/winter period and can improve flowering quality after “off” years [29, 30]. Several observations support it: (1st) during the fruit’s inhibitory period, there is typically a decrease in GA concentration within the fruit and a simultaneous rise in shoot bark tissue [31]; (2nd) highest GA foliar content aligns with increased sensitivity to externally applied GA for flowering reduction [24] and (3rd) less inductive temperatures resulted in a temporary decrease in concentration of GA in the buds, after floral differentiation [31]. These cumulative findings contribute to the notion that endogenous GA synthesized by the fruit could significantly influence the regulation of flowering in Citrus. Nevertheless, the validity of GA’s regulatory impact has faced scrutiny and skepticism [22, 29, 30, 32]. Several factors contribute to this skepticism: firstly, despite higher GA content in leaves [24] and the phloem sap of fruit-bearing shoots compared to vegetative ones, no discernible differences in GA bud content have been identified between trees without and with fruit at the dormant period [23]. Secondly, the precise timing of GA concentration changes during flower initiation and differentiation. Thirdly, a specific GA threshold quantity enabling flower initiation and differentiation remains unknown. Additionally, paclobutrazol, a GA synthesis inhibitor, counteracts its flowering promotion effect in the presence of fruit. Applying GA₃ reduces flowering without affecting CcMADS19 gene expression in leaves, as observed in fruit [32]. Higher concentrations of GA are recorded endogenously in the plant parts like buds, seeds, or fruit pericarp of Citrus spp. [29, 31, 33]. Seeded fruits demonstrate more potent inhibitory effects on reproductive growth than seedless ones due to greater GA concentration [29]. Seeded varieties display higher GA contents than parthenocarpic or nonseeded [33]. It is essential to state that the gibberellin precursor GA12 [34] is not obtained during the inductive period in citrus buds.

2.2.2 Auxin (Ax)

The well-established role of Ax in the formation of lateral organs involves a transient surge in indole-3-acetic acid (IAA) within vegetative meristematic tissue, facilitated by the PIN protein, due to polar orientation, which ultimately controls the lateral bud formation [35]. Within the floral meristem, Ax distribution’s precise spatial and temporal control plays a pivotal role in organ initiation and development [36]. Polar Ax transport and localized biosynthesis establish distinct concentration gradients, acting as positional cues for organ identity. For example, elevated Ax levels at the gynoecium primordium apex promote the development of the stigma and style, while moderate levels influence ovary development [37]. Ax plays a crucial role in differentiating the gynophore in the basal region, though with exceptions like Citrus spp. [36]. These processes are mediated by AUXIN RESPONSE FACTORS (ARFs), which act downstream of Ax signaling and contribute directly to organ morphogenesis [38]. Interestingly, ARFs seem less effective in governing Ax distribution among flowers, pointing toward onsite biosynthesis within the meristematic regions as a valuable determinant of organ identity [21]. In Citrus spp., specific ARFs (CiARF3/4, CiARF5/6/7/8/10/19) that regulate both the developmental timing of individual organs and overall flower development have been identified. Moreover, Ax plays a critical role in stamen development, controlling pollen’s maturation and anther’s dehiscence [37].

2.2.3 Abscisic acid

Regarding ABA’s involvement in Citrus spp. flowering, a puzzling picture emerges. The CcNCED3 gene, responsible for ABA synthesis, expresses more actively in vegetative shoot buds than in fruit-bearing ones, yet the actual concentration of ABA is lower in the vegetative bud [39, 40]. This paradox suggests that additional factors beyond bud-intrinsic synthesis, possibly fruit-induced stresses, influence ABA levels. Highlighting this inhibitory potential, exogenous ABA applied to Satsuma mandarin buds during flower induction suppressed sprouting and flowering [41]. However, its role in alternate bearing varieties remains ambiguous. This suggests that ABA might not permanently inhibit flowering, potentially even promoting it under specific conditions. Water stress-induced flowering further complicates the picture, leaving the question of ABA’s mediating role unanswered.

2.2.4 Ethylene

Ethylene has been suggested as a potential flowering promoter, although not in citrus. [42] proposed that it might resemble the action of TIBA, an IAA transport inhibitor, by hampering the polar transport of Ax, a putative floral inhibitor. Several GA biosynthesis inhibitors exhibit this effect, moderately reducing Ax export from fruit and shoots [43]. Notably, few of the inhibitors even enhance flowering [32]. This suggests that ethylene can act by suppressing the polar transport of Ax, an inhibitor of floral Induction.

2.2.5 Cytokinin

Cytokinins (CK) are linked to juvenility and likely influence flowering in Citrus spp. ‘Juvenile buds’ show higher CK before breaking dormancy, leading to increased growth. Like GA and Ax, CK likely integrates environmental and internal signals to influence flowering [44]. Numerous studies suggest CK involvement through content and response [45]. CK supplementation in vitro often induces flowering [41, 46]. Optimal CK concentration appears crucial for stimulating meristematic capacity and bud break. While the source of CK during induction remains unclear, synthesis in buds and vicinity likely plays a role [47].

2.3 Genetic regulation

In Citrus spp., floral induction predominantly occurs during the autumn to winter rest period, with leaves demonstrating their significance in this process [48]. This was substantiated by studies involving the defoliation of trees in autumn, followed by the evaluation of flowering in spring. Insights into the genetic pathways governing flowering have been gleaned from the model plant Arabidopsis thaliana [49]. The intricate web of citrus flowering relies on an ensemble of genes. Key players like FT, SOC1, and LFY act as floral pathway integrators, coordinating the timing and initiation of the process [50]. Once activated, floral meristem identity genes, including AP1 and LFY, shape the basic floral structure [51]. Meanwhile, the SEPALLATA family guides the web of floral patterning, ensuring each petal, sepal, and stamen occupies its rightful place. At the heart of this orchestrated performance lies the protein AtFT. Synthesized in the companion cells of the phloem of induced leaves, it embarks on a journey through the sieve tube, ultimately getting to the meristem. There, it partners with the transcription factor FD, orchestrating the transformation of vegetative cells into a vibrant floral meristem ready to blossom. The resulting AtFD/AtFT heterodimer is a key initiator of floral development by initiating the expression of AtAP1 and AtSOC1 floral genes [52, 53]. Recognized as a principal, if not exclusive, component of florigen, the AtFT protein plays a central role in the intricate process of flowering [54]. AtLFY and AtSOC1 are crucial for precise flower development: AtLFY controls timing and identity, while AtSOC1 integrates internal and external cues [55].

2.4 C:N ratio of the shoot

Carbohydrate levels have been suggested as playing a role in the control of flowering [56]. Fruit negatively correlates to flower number. Buds on young summer shoots and branch tips tend to be more floriferous than older or lateral buds. Notably, fruit load is a potent inhibitor, significantly reducing spring bud sprouting and flowering. Conversely, trees with minimal fruit sets often exhibit a compensatory surge in blooming the following season. This ‘alternate bearing’ pattern underscores the need for agronomic interventions to regulate flower production. Heavy fruit sets likely create a carbon deficit, limiting resources for flower initiation [24, 41, 57]. Additionally, fruit-derived signaling compounds, particularly gibberellins, may suppress flowering [41, 57]. Supporting this notion, girdling can promote flowering, which temporarily disrupts phloem transport and increases sugar availability in stems [58].

3. Floral induction and differentiation

Transitioning to citrus, shoots emerge from initially quiescent buds formed in leaf axils. Upon release from dormancy, buds on adult trees can give rise to either leafy shoots or vegetative shoots devoid of flowers or inflorescences and flowering shoots with or without leaves. The production of leafy vs. leafless inflorescence was also correlated with the timing of sprouting and temperature during shoot development [59]. The inflorescences encompass diverse forms, ranging from single terminal flowers on leafy shoots to inflorescences composed entirely of single or multiple flowers without developed leaves [44]. The springtime distribution of these shoot types, in conjunction with the number of buds bursting, determines the overall flower production. The number of leaves and flowers per new growth in Citrus spp. depends on two factors: the number of ‘primordia’ that exist in the bud and the abscission or lack of development of some organs, mainly leaves [60, 61].

In Citrus spp., floral induction and differentiation transpire 2–3 months before and during bud release, respectively [62]. Initially, as the vegetative meristem transforms into a flower bud (Figure 2), the apex flattens, initiating the development of the flower’s receptacle, stamens, carpels, and petals [63]. Upon dormancy release, flower primordia emerge as fingers curving on the meristem, sequentially producing the floral organs [63]. Reproductive organogenesis proceeds acropetally, with each whorl forming above and within the preceding one. The sepals form first, succeeded by petals, followed internally by stamens in a single whorl (Figure 3). Subsequently, the pistil develops in the innermost zone, featuring an ovary with 8–10 carpels with 2–3 ovules each [64] single style, papillated stigma, and nectariferous disc [60].

Figure 2.

Flowering phenology of Citrus aurantifolia. (a. Plant at pre-bearing phase, b. vegetative shoot, c. newly formed flowering shoot with floral buds, d. flower bud in one season old shoot and e. flower at anthesis).

Figure 3.

Pollinators frequently reported in established Citrus orchard. [1. Honey bee (A. dorsata) collecting both pollen and nectar, 2. Leafcutter bee (Megachile anthracina) side view, 3. Leafcutter bee (M. anthracina) top view showing feeding on the nectar, and (4) red pumpkin beetle (Aulacophora foveicollis) collecting pollen].

Initially, sepals fully cover the bud at the onset of flower development [44]. As the petals become visible, the anthers in the stamens reach the height of the stigma. The continuous growth of the petals displaces the sepals outwards, revealing the androecium and gynoecium. At this stage, the stigma protrudes out from the anthers, and the petals, nearing their final size, overlap to form a globe enveloping the androecium and gynoecium. Anthesis marks the stage when petals open, anthers dehisce, and the stigma becomes receptive to pollen grains [60, 63, 64]. The receptivity of stigma will determine the effective period for pollination [65].

The single terminal flowers surrounded by leaves on the same shoot produce fruits of optimal size, likely due to the additional assimilates supplied by these nearby leaves. Within a single Citrus variety, the number of fruits on an individual tree negatively correlates with the final fruit size as a result of competition for resources [66]. This relationship underlines the tendency of some Citrus spp. to exhibit a ‘biennial bearing’ or ‘alternate bearing’ pattern, characterized by abundant flowering and fruiting in “on” years followed by a pronounced decline in both the next (“off”) season, influencing both flower and fruit numbers [41]. Excessive flower production in “on” years, while often followed by early fruit drop, ultimately leads to smaller final fruit size.

Understanding the factors regulating citrus flowering is crucial for managing this cyclic trend. In subtropical regions, spring blooms typically coincide with vegetative growth, following winter dormancy and obtaining lower temperatures and shorter days. In tropical climates and areas with defined dry seasons, flower initiation can also be triggered by rehydration after a period of water stress. Techniques like heavy fruit load management, and deliberate leaf removal have been shown to decrease the rate of leafless shoots, typically associated with higher flower numbers [16, 58]. While slight variations in timing may exist across species, floral differentiation typically occurs toward the end of the winter season, coinciding with the bud sprouting stage [9, 63]. At this stage, initial morphological distinctions between vegetative and reproductive buds could be identified [60]. The type of inflorescence significantly impacts the fruit set. Leafless inflorescences, the first to appear, offer lower fruit set potential compared to leafy counterparts, whether terminal or dispersed among leaves [67]. Interestingly, late-opening flowers tend to persist longer, further enhancing fruit set on shoots with a high leaf-to-flower ratio [59].

This positive influence of leaves on fruit sets stems from two primary factors: increased net CO₂ assimilation and the supply of photoassimilates from developing leaves [68]. Flowering control in Citrus spp. has benefited significantly from the application of GA₃, reported to inhibit blossom development [62, 69]. The treatments during the bud sprouting stage (during January-February in the North Hemisphere) significantly decrease flowering by inhibiting the floral bud differentiation [9]. Strategically timed GA₃ applications (25–100 mg/l⁻¹) yield several impactful outcomes: (1) reduced bud sprouting, (2) floral bud conversion to vegetative shoots [9, 41], (3) substantial reductions in flower numbers per tree (45–75% depending on cultivar and treatment), and (4) direct increases in fruit set [70]. This enhanced fruit set then effectively curbs flowering in the following season. Remarkably, despite suppressing flowering, GA3 does not affect the number of leaves or flowers per shoot. Notably, decreased flowering from GA3 enhances other fruit set boosting techniques, like GA3 treatment during the petal fall stage [70] or girdling of branches during the fruitlet abscission stage [15]. Practical application concentrations vary: 25 mg/l⁻¹ in sweet orange and 10 mg/l⁻¹ in ‘Clementine’ mandarin in Spain (mid-November to mid-December) [70]; 25 mg/l⁻¹ in mid-June in New Zealand [69]; 100 mg/l⁻¹ for Satsuma mandarin in Japan (late January) [10].

4. Flowering dynamics

Citrus spp. flowering patterns vary based on species, tree age, and climatic conditions. Flower loads can range up to 250,000 per tree, with only a small percentage maturing into fruit. Some species, like Citrus limon and Citrus aurantifolia, flower regularly throughout the year twice or thrice, while others exhibit alternate bearing. Prior to flower development, buds undergo activation through interactions between external and internal factors. This transition from vegetative to floral meristem, known as floral induction, starts in the leaves, influencing bud meristem identity and facilitating the shift to the reproductive phase and subsequent flower development or differentiation. These phases include induction, differentiation, and organogenesis, which are distinct and independently regulated.

4.1 The flowering processes

In Citrus spp., floral induction precedes floral differentiation by 2–3 months before bud release. The transition from a vegetative to a flower meristem and a flower bud involves apex flattening, initiating sepal, petal, stamen, and carpel [63]. Upon dormancy release, flower primordia emerge, curving on themselves to form the flower bud. ‘Floral organogenesis’ unfolds ‘acropetally’, with sepals forming first, followed by petals, stamens, and, centrally, the pistil. The pistil of a citrus flower comprises an ovary with 8–10 carpels, each with 2–3 ovules, a single style, and have papillated stigma. The nectareous disc, a specialized structure that secretes nectar to attract pollinators, lies between the stamens and carpels [25, 60, 63, 64]. As flower development begins, the sepals initially cover the bud, and later, the petals are visible. Anthers reach the stigma’s height and continuous petal growth results in a globe-like structure enveloping the androecium and gynoecium. Anthesis follows, with open petals, dehiscent anthers, and a receptive stigma [60, 63, 64]. The Stigma and ovule receptivity will determine the effective period of pollination [65].

4.2 Flower distribution

The new shoots developed after dormancy can be determined, evolving into either mixed or fully generative forms. In mixed shoots, the main shoot tip forms a terminal flower bud, while the axillary buds may sprout further in the floral branches [63]. Generative shoots, on the other hand, suppress leaf development at bud break, resulting in leafless floral structures [64]. This interplay between vegetative part and flowers put forth five distinct types of shoots: (1) leafless inflorescences, (2) multiple flowers, (3) leafy inflorescences with a single terminal flower and numerous axillary blooms, (4) entirely floral shoots, and (5) purely vegetative shoots. The number of leaves and flowers on each shoot hinges on two factors: the initial bud primordia and subsequent organ (mainly leaf) abscission or underdevelopment [60, 61].

Interestingly, this shoot diversity pattern remains consistent across cultivated Citrus spp. and varieties, though with quantitative variations. For example, Satsuma mandarins stand out due to the limited sprouting ability of their new shoot axillary buds [61]. Additionally, factors like bud break timing and temperature during shoot development can influence the trajectory of leafless versus leafy inflorescence formation [59]. Remarkably, the bud’s ultimate fate, blossoming into a floral or vegetative shoot, can even be predicted based on the characteristics of its parent shoot, categorized as ‘floral’ or ‘vegetative’ [71, 72].

4.3 Types of inflorescences

In Citrus spp., the journey from floral initiation to fruit set is influenced by several factors, including inflorescence type. Floral differentiation typically occurs toward the last part of the winter season [9, 63], coinciding with the bud sprouting. At this stage, the initial morphological distinctions between reproductive and vegetative buds become evident [60].

The type of inflorescence significantly impacts the fruit set. Leafless inflorescences, the first to appear, typically offer lower fruit set potential compared to leafy counterparts, whether terminal or dispersed among leaves [67]. Interestingly, late-opening flowers tend to persist longer, further enhancing fruit set on shoots with a high leaf-to-flower ratio [59]. This positive influence of leaves on fruit set stems from two primary factors: an increase in net CO₂ assimilation and the availability of photoassimilates from the developing leaves [68]. Additionally, hormonal regulation may play a role. Leafy inflorescences often exhibit higher levels of gibberellins compared to leafless ones, potentially contributing to ovary growth and fruit set [73].

4.4 Factors influencing flower bud initiation and fruit set

The flower bud initiation and fruit set in citrus plants are influenced by various factors [16], including both internal and external environmental factors. Here are some key factors:

1. Climate and temperature: many citrus varieties require a period of chilling to induce flower bud initiation. Insufficient chilling hours can lead to delayed or inadequate bud formation [7].

2. Temperature during flowering: extreme temperatures during flowering can affect pollination and fruit set [74]. High temperatures may lead to flower drop, while low temperatures can negatively impact pollen viability and germination.

3. Photoperiod: citrus trees are sensitive to day length, and changes in the photoperiod can influence flower bud initiation. Generally, a period of short days may stimulate flowering in some citrus varieties [75].

4. Hormonal regulation: hormonal balance within the plant is crucial for the transition from vegetative growth to reproductive growth.

5. Pollination: successful pollination is critical for fruit development. Parthenocarpy in self-incompatible Clementine mandarins (Citrus clementina Hort. ex Tan.) is pollination-independent, with fruit set depending on ovary hormone levels. ‘Marisol’ showed greater parthenocarpic ability than ‘Clemenules’, likely due to higher GA1 levels and lower ABA levels [76].

6. Age of the tree: young citrus trees may take some time to reach maturity and start regular flowering while older trees may experience changes in flowering patterns over time.

5. Pollination and fertilization

Understanding floral biology serves as a fundamental prerequisite for analyzing interactions among pollen, stigma, flowers, and pollinators, contributing to the reproductive success of plant species [77]. Floral characteristics such as size, morphology, color, odor, and anthesis play a pivotal role in aiding not only in understanding plant-pollinator relationships but also in unraveling mechanisms behind reproductive success [77, 78]. Another crucial aspect of reproductive biology studies involves floral visitors. Surveying these visitors helps determine native bee species and offers insights into optimal strategies for colony densification and management in crops.

5.1 Anther dehiscence and pollen presentation

In citrus flower development, anther dehiscence, the release of pollen grains, commences between 8.00 and 9.00 am on the first [11] or second day of opening [79]. This process begins with the appearance of a slit across the pollen sacs (thecae) through which pollen gradually oozes out. Within 4–5 hours, all the pollen within each sac is released, coinciding with the arrival of insect visitors. Under natural conditions, pollen foragers, primarily bees, consume nearly all the pollen by the end of the second day. Any remaining pollen within the anthers is minimal and typically gone by the third day. Citrus spp.’s pollen grains are characterized by their bright yellow color, radially symmetrical oblate-spheroidal shape, circular outline, and four to five zonate colporate apertures (elongated pores). The colpi themselves are narrowly elliptic with pointed ends, and the endoaperture (internal opening) is long and oval. The exine, the outer layer of the pollen grain, is 2.5 μm thick with a tegillate texture (covered in small bumps) and equal thickness to the nexine (inner layer). Finally, the surface of the exine exhibits a reticulate pattern [80, 81].

5.2 Floral visitors

A total of 8 insect species have been regularly observed visiting the flowers of Citrus spp. [82]. Apis dorsata is identified as the principal pollinator [82]. Pollinators of Citrus spp. (Figure 4) come from diverse insect orders: Hymenoptera, Diptera, and Lepidoptera. While most, like Apis cerana and Trigona spp., seek both pollen and nectar, some like Halictus spp. focus solely on pollen, and others like Papilio spp. prioritize nectar. The other dipteran visitors include Helophilus spp. and hoverfly (Episyrphus sp.). Lepidopteran spp. plays a minimal role in pollination, acting more as nectar robbers [83]. The peak visitation occurs on the second day of flower opening, mainly between 10:00 am and 11:30 am, when nectar and pollen rewards are maximal [11]. While most nectar visitors return on the second day, fewer visitors, including A. dorsata and Lepidopteran species, visit on the third day for remaining nectar. Pollen visitors are absent on the third day.

Figure 4.

Stigmatic appearance in Citrus aurantifolia [at (a) anthesis, (b) pre-petal fall stage on the 2nd day after anthesis, (c) petal fall stage on the 4th day after anthesis, and (d) fruit set stages on the 7th day after anthesis].

5.3 Floral rewards

The flowers of Citrus spp. offer tempting rewards to attract pollinators (Figure 3), ensuring repeated visits are vital for successful cross-pollination. ‘Pollen grain’ and ‘nectar’ serve as the primary enticements [65] in Citrus spp. Nectar secretion starts early on the second day of flower opening and continues until the third day. This sweet lure attracts a diverse range of pollinators:

1. Pollen gatherers: Halictus spp., Apis dorsata, A. cerana, A. florea, and Trigona spp.

2. Nectar feeders: Apis dorsata, A. cerana, A. florea, Trigona spp., butterflies (Papilio dravidarum and Parantica spp.), and Lepidopteran members (Helophilus spp. and Episyrphus spp.).

3. Additional pollen reward: Tiny, sugar-secreting glandular hairs adorn the stigmatic head, potentially offering another pollen-like reward to incentivize visitation [65].

5.4 Stigma receptivity

Citrus spp. exhibits a wet-type stigma, becoming receptive early for successful cross-pollination. Bagging experiments revealed peak receptivity from 3.00 am on the second day before anther dehiscence, extending up to the fourth day afternoon with a margin of approximately 58 hours [65]. Notably, under open pollination conditions, the stigma loses receptivity earlier, around the third day afternoon [11]. This extended receptive period in bagging experiments ensures sufficient pollen accumulation on the stigma, exceeding the threshold for successful fertilization. During its receptive phase, the citrus’s stigma boasts a distinctive appearance: shiny, bright yellowish, and juicy (Figure 4). This lustrous and juicy look persists until the end of the second day. However, by the third day afternoon, the stigma begins to brown and lose its luster, signaling the decline in receptivity. This transition in receptivity is confirmed by both the hydrogen peroxide test [84, 85] and in-vivo pollen germination tests on the stigma [65].

5.5 Pollination and parthenocarpy

Among most citrus cultivars, fruit set occurs on successful pollination followed by fertilization, as fertilized ovules trigger fruit development. Lack of pollination leads to ovary arrest, flower senescence, and eventual abscission. Growth arrest in unpollinated ovaries [73]. However, this reliance on pollination varies. Temperature, for example, influences bloom duration: high temperatures shorten it, while low temperatures extend it [59, 60, 86, 87]. Additionally, the temperature impacts bee activity and pollen tube growth rate [65]. Effective pollination periods range from 8 to 9 days for “Clementine” mandarins and sweet oranges to 2–3 days for Satsuma mandarins under optimum temperatures [65]. Beyond optimum temperature, inflorescence type, floral position [59], flower number [16, 88], and nutritional status [65] can influence flower development and, consequently, the final fruit set. Self-incompatible cultivars like “Clementine” mandarins exhibit “facultative parthenocarpy,” setting seedless fruit only in the ‘absence of pollination’. Truly seedless cultivars like Satsuma mandarins and “Navel’ oranges rely on high gametic sterility, producing parthenocarpic fruit without pollination and fertilization.

6. Fruit development and maturation

6.1 Reproductive biology

Citrus fruits offer a unique combination of intriguing reproductive characteristics, making them valuable models for studying diverse biological questions. Citrus spp. undergoes a non-climacteric ripening process [89]. Many citrus cultivars display additional quirks: parthenocarpy, sterility, self-incompatibility, and even pollen defects [90, 91]. In the seeded citrus varieties, fruit development hinges on seed presence, requiring successful pollination and fertilization. Self-pollination often occurs within the enclosed bud or bloom, potentially preceding anthesis. Cross-pollination, mediated by insect vectors, facilitates gene exchange between diverse individuals. However, most modern cultivars have embraced the seedless life, exhibiting high levels of parthenocarpy, often stemming from gametic sterility [90]. This sterility can be partial or complete. Relative sterility can manifest as self-incompatibility (e.g., “Clementine’) or cross-incompatibility, while ‘absolute sterility’ involves complete pollen and/or embryo-sac dysfunction. In Satsuma mandarins and “Washington Navel” oranges very few embryo sacs can occasionally reach maturity.

6.2 Fruit growth and abscission

Citrus fruit development unfolds over a lengthy period, spanning from spring flowering to autumn or even winter harvest, depending on the variety. This journey follows a classic sigmoid growth curve with three distinct phases [92].

1. Phase I (cell division): lasting around 1–2 months, this phase starts after petal fall and culminates in the June drop, characterized by intense cell division and slow growth. During this period, developing fruitlets act primarily as ‘utilization sinks,’ consuming resources for internal construction. This initial phase witnesses two peak abscission waves: one at the phase’s beginning, targeting buds, flowers, and ovaries, and another during the shift to Phase II, focusing on developing fruits [93]. Generally, less than 1.0% of fruits make it past this critical period, known as the ‘fruit set’ [94, 95]. The phase usually ends with cell division proceeding to cell enlargement.

2. Phase II (rapid growth): over 2–6 months, the fruit undergoes a dramatic size increase driven by cell enlargement and water accumulation. This phase transforms the fruitlets into ‘storage sinks,’ accumulating reserves for later ripening. Abscission significantly declines during this phase, though some species and harsh environments can trigger pre-harvest drops of ripe fruits.

3. Phase III (ripening): growth slows significantly in this final phase, and the fruit undergoes a non-climacteric ripening process, with a slow rate of respiration and ethylene production. Interestingly, overripe fruits in many varieties become quite resistant to abscission [73].

While timing varies between early and late-ripening cultivars (some reaching maturity as early as September, others holding on until next summer), a significant feature is the high rate of abscission throughout.

6.3 Regulation of fruit set and growth

Fruit development in Citrus spp. depends on the 3 levels of regulation: environmental, metabolic, and genetic [96]. In seeded cultivars, external stimuli like pollination and bloom quality act as key genetic triggers, while the initiation of these programs in seedless varieties seems more developmentally linked [97]. Despite these distinct control mechanisms, they likely converge on the production and action of shared hormonal messengers. Fruit growth itself depends heavily on resource availability, primarily carbohydrates, water, and mineral elements. While carbohydrate supply can be limiting, and even favorable conditions can turn adverse over time. To cope with such challenges, citrus initiates protective measures triggered by new hormonal signals. These may include temporary closure of stomata and arrest in growth or drastic responses, viz., fruit abscission.

6.4 Hormonal regulators of fruit growth and abscission

Plant growth regulators exert a profound influence on early citrus fruit development, suggesting a crucial hormonal component governing fruit set and abscission [98]. A complex hormonal interaction seems to facilitate this process. GAs and CK typically act as positive growth regulators, while Ax exhibits a dual role, promoting growth and triggering abscission under certain circumstances.

6.4.1 Gibberellin

Gibberellin (GA) treatment stands out as a unique tool for manipulating flowering in citrus. Applying GA during bud development demonstrably suppresses flower production [99], favoring leafy shoots with terminal flowers and ultimately leading to more fruit. Gibberellic acid impairs fertilization in Clementine mandarin under cross-pollination conditions [100, 101].

6.4.2 Cytokinins

Similar to GAs, CK also promotes cell division and exhibits increased levels in developing ovaries at anthesis. Interestingly, exogenous CK can enhance the parthenocarpic fruit development and boost the sink strength for some cultivars. Benzyladenine, for instance, seems to specifically influence flower differentiation [102] and promote bud sprouting [103, 104, 105]. However, despite these promising effects, CK has not been adopted commercially for improving fruit set in citrus [106].

6.4.3 Auxins

Plant regulators like Ax exhibit a complex, context-dependent relationship with fruit abscission, acting as both ‘growth hormones’ and ‘abscission agents’ [107]. However, Ax also demonstrates a contrasting role in abscission, both delaying and accelerating it. In the initial stages, Ax act as inhibitors, but once abscission begins, they can surprisingly promote it, potentially through enhanced ethylene synthesis [93]. In citrus, synthetic Ax displays a nuanced influence on fruitlets depending on their developmental stages. During the cell division (Phase I) stage, they trigger abscission instead of inhibiting it [107]. However, during early cell enlargement (Phase II), Ax application can offer mild protection against abscission and even increase fruit size [107]. At the next stages, particularly near the end of Phase II and the beginning of Phase III, synthetic Ax become economically valuable tools for preventing or delaying preharvest fruit drop [108]. NAA, plays a crucial role in commercial fruit thinning, especially when excessive fruit set threatens size and quality. Applied around 6–8 weeks after bloom, during the “1/2-inch fruit diameter stage,” NAA effectively triggers the abscission of unwanted fruitlets. This thinning practice, proven effective in Tangerines like “Murcott” and “Sunburst”, ultimately improves overall fruit yield [108] and quality attributes. In addition to increased ethylene synthesis, there is also a transient reduction in photosynthesis when Ax are applied at june drop [109].

6.4.4 Abscisic acid

Despite field experiments showing no abscission induction by aerial ABA application, various observations suggest its involvement alongside ethylene [93]. Notably, ABA levels rise in developing ovaries in the petal fall stage and during the June drop, coinciding with cell division-to-enlargement transition and abscission waves in phases I and II [94, 95]. Elevated ABA also accompanies the duration of low humidity or dehydration, salt stress, and drought stress [110, 111].

6.4.5 Ethylene

Since the early twentieth century, ethylene has been implicated in various detrimental effects on plant growth, notably abscission induction [112]. In citrus specifically, its role in abscission has been long established [93]. Notably, ethylene is also the key hormonal regulator of the abscission of leaves in citrus [113].

6.5 Regulation of fruit set

Low GA levels at anthesis cause ovary and fruitlet drop. Conversely, cultivars with low abscission rates show high GA and low ABA shortly after flowering [114, 115, 116]. Exogenous ABA promotes abscission while GA suppresses it, both in explants [93, 117] and emasculated ovaries [73]. Similarly, pollination elevates GA and reduces abscission in seeded varieties. Thus, ‘GA appears crucial for fruit set’, countering ABA’s abscission-promoting effects [73]. GA deficiency, a ‘growth arrest trigger,’ sparks a hormonal cascade: “rise in ABA, release in ethylene, leading to, abscission of the ovary.” It will be activated under diverse stresses like carbon shortage and water deficit. GAs plays a key role in citrus fruit sets, even for seeded varieties. Exogenous GAs induces parthenocarpic fruit development in emasculated “Sweet” oranges, mimicking the hormonal signals from seeds, leading to initial growth that rivals pollinated fruit until the June drop [73]. However, GAs alone cannot overcome June drop and complete fruit maturation, suggesting additional factors are crucial in seeded varieties. Despite this limitation, GAs stimulates fruit sets in both seedless and parthenocarpic types (“facultative” and “obligate”). Beyond fruit set, GAs strategically applied during citrus maturation improve peel firmness and delay senescence [73].

6.5.1 The role of carbohydrates

Citrus spp. shower blossoms, but the competition for limited nutrients, particularly sugars, sparks significant fruitlet abscission-a harsh reality exceeding their support capacity. These sugars act as potent fuel, driving initial cell division and subsequent fruit enlargement. Carbohydrate availability plays a pivotal role, as evidenced by various studies on source-sink imbalances and defoliation’s impact on growth and abscission [92, 118], and the positive correlation between sugar levels and fruit set observed in girdling, shading, and sucrose supplementation experiments [119, 120]. Phase I is particularly vulnerable to sugar fluctuations. Defoliation during this phase cripples carbohydrate supply, stunting fruit growth and triggering substantial fruitlet drop [92]. However, post-June drop, defoliation’s impact shifts-growth slows, but abscission rates remain unaffected, suggesting a reduced reliance on sugars [118]. This crucial link between sugars and fruit growth is further corroborated by 14C metabolite translocation studies and CO₂-enrichment experiments demonstrating a strong correlation between available carbohydrates and abscission [88, 95, 120]. Photosynthesis plays a critical role, as surging fruit sugar demand during fruit set to development drives up the photosynthetic rate. Conversely, reduced net CO₂ assimilation translates to lower sugar production and compromised fruit set. Girdling, known to enhance fruit set and carbon availability, paradoxically represses overall photosynthetic activity [120]. Fruit set improvement after girdling is driven by a dual mechanism: delayed fruitlet abscission and enhanced photosynthetic efficiency (ΦPSII) in young leaves of leafy flowering shoots [121]. This seemingly contradictory observation highlights the complexity of the carbohydrate-fruit growth relationship. While girdling limits photosynthesis in the developing vegetative stems, it simultaneously stimulates it in the leafy fruiting branches, demonstrating the imbalanced allocation of photosynthetic resources toward fruit development. Pollination and exogenous GAs further bolster fruit set and growth by promoting stronger mobilization of 14C metabolites to ovaries [95]. GAs also stimulates vegetative growth and enhance carbon supply to the vegetative tissues, underscoring the profound influence of sugars on fruitlet growth regulation and highlighting the vital role of adequate resources in preventing early fruit drop and optimizing citrus fruit yield [122]. The scarcity of carbohydrates triggers abscission, while sustained availability fuels growth and yield. Plant nutrition also plays a role, with higher bud and leaf nitrogen (ammonia) content, often achieved through winter urea application, demonstrably increasing flower quantity [19, 123]. Similarly, [124] observed sustained nitrogen fertilization over 3 years improves canopy width and flower yield in Citrus spp.

6.5.2 Regulation of June drop

The carbon deficit, an early fruit drop factor, increased ABA and ethylene, promoting abscission [95]. Defoliating citrus alters the nutrient supply, leaving water status unchanged in developing fruits [92]. June drop’s nutrient imbalance, marked by a rise in nitrogen and a drop in carbon, fuels abscission intensity. This carbon shortage directly correlates with abscission rates, mirroring the rise in ABA and ACC seen in defoliated fruitlets [95]. This suggests ABA acts as a carbon deficiency sensor, modulating ACC and ethylene production, the final abscission trigger.

6.6 Citrus fruit ripening

Citrus fruits, categorized as hesperidia, feature two distinct parts: the endocarp and the pericarp. The pericarp includes the exocarp ‘flavedo,’ an external-colored part, and the mesocarp ‘albedo,’ an inner-cushioned-white peel layer. The pulp contains segments, ovarian locules within a locular membrane, filled with juice vesicles-the primary storage organ in Citrus spp. During non-climacteric ripening, citrus fruits undergo biochemical and physiological changes, slowing active growth. Ripe fruits exhibit low ethylene production and sensitivity, significantly reduced respiration, and gradual texture and composition changes [125]. Additionally, there is no evidence indicating that a particular hormone controls the entire ripening process. Fruit quality characteristics develop during stages II and III, encompassing physical properties like color, shape, texture, size, peel ability, seed number, etc., and chemical components like TSS, acid, flavor compounds, sugars, volatiles, and vitamin C. These properties, dictated by the regulation of physiological and biochemical processes, hold economic significance, influencing consumer perception and impacting the success of the citrus industry.

6.6.1 External ripening

Citrus spp.’s rind ripening relies on chloroplast-to-chromoplast conversion [126]. This resembles leaf senescence but is influenced by the environment, nutrients, and hormones [56]. “Color turning” stage, driven by chlorophyll degradation and carotenoid build-up, typically occurs in mid-autumn as temperature drops and days shorten [127]. Notably, citrus chromoplasts show unique flexibility, reversing back to chloroplasts even after full differentiation [56]. This reversible conversion sets citrus ripening apart from many other fruits, with significant agronomic implications.

6.6.2 Internal ripening

Mature citrus pulp, with a water content of 85–90%, contains various constituents, including carbohydrates, organic acids, amino acids, vitamin C, minerals, and small quantities of lipids, proteins, and secondary metabolites like carotenoids, flavonoids, and volatiles [128]. Total soluble solids make up 10–20% of the fresh weight, primarily composed of carbohydrates (70–80%), with minor amounts of organic acids, proteins, lipids, and minerals. Ripening fruit exhibits decreased titratable acidity (citric acid catabolism) and increased sugars (TSS) [57].

6.6.3 Effect in fruit quality

Climate plays a pivotal role in determining fruit quality in commercial citrus production. In tropical climates with consistently high temperatures, oranges and tangerines maintain green peel color due to stable fruit peel chlorophyll [129]. Conversely, during winter months in subtropical climates, these fruits develop a more vibrant peel color in orange, enhancing their visual appeal at maturity. Lowland tropical areas experience rapid fruit maturation, driven by high respiration rates, leading to insufficient time for significant total soluble solids (TSS) accumulation. In contrast, cool coastal areas witness slower TSS accumulation, with maximum levels typically achieved in humid subtropical and mid-tropical regions featuring warmer winters. Total titratable acid levels reach their peak in arid or semiarid coastal and subtropical areas, declining more gradually during maturation. The quantity of fruit set plays a significant role in determining overall fruit quality, demonstrating a negative correlation among the fruits per tree and the resulting quality [130]. Individual fruit quality varies, with interior fruit seen in the heavily shaded areas exhibiting lower total soluble solids (TSS) due to insufficient light and having more acidity. Shoots with fruit usually do not flower the next year, and having too many fruits in a crop uses up carbohydrates, resulting in poor crops and more growth in the next year. The pruning operation influences crop dynamics: after a heavy crop, it stimulates vegetative growth and reduces fruit yield; conversely, after a light crop, it enhances fruit size and mitigates alternate bearing [131].

7. Conclusion

The physiology of Citrus spp.’s flowering and fruiting is a complex interplay of environmental factors, including genetic mechanisms, temperature, water stress, and plant nutrient status. The response of citrus trees to varying climatic conditions, such as low temperatures (<15°C) and water stress (45 days), highlights the adaptability of these plants to diverse environments. The intricate balance between temperature-induced flower initiation and subsequent flowering waves, as observed in temperate climates like Florida, underscores the need for precise quantification and management of inductive temperatures. Similarly, in tropical climates, the dependency on water stress for flowering necessitates careful consideration of the duration and intensity of this stress to achieve optimal yields without compromising tree health. Moreover, the integration of genetic insights, such as the expression patterns of key genes like CiFT2 and CiFT3, adds another layer of complexity to our understanding of citrus flowering physiology. The ongoing research, both in field conditions and controlled environments, provides valuable insights into the combined effects of climatic stressors and their impact on flowering intensity. As the citrus industry continues to expand and face new challenges, ongoing studies and a comprehensive understanding of the physiological processes involved in citrus flowering and fruiting will be essential for developing sustainable and efficient cultivation practices. Through continued research, we can enhance our ability to manipulate these physiological processes, ultimately leading to improved citrus crop management and increased agricultural productivity.