Deciphering the Plant Hormones Cross-Talk during Fruit Development: A Review

Abstract

Horticultural industries are increasingly crucial in providing livelihoods, food quality, profits, and economic growth. In many horticultural plants, extensive studies were conducted to study the roles of hormones, epigenetics, and genes in regulating the development of cell number, cell size, fruit size, fruit weight, and endo-reduplication primarily via a gene-mapping technique known as quantitative trait loci (QTL). In general, these plants encompassed those with full-genomes sequenced, such as the apple, tomato, strawberry, and bananas. However, apart from fully sequenced apple genomes, the genome sequences of many other plants, particularly highly profitable tropical fruits, such as mangoes, pineapples, durians, and coconuts are yet available. This chapter will describe the interplay of plant hormones in determining fruit cell number and cell size, which, in turn, affects the final fruit size in horticultural plants.

1. Introduction

1.1 Origin of apple fruit cultivars

Apple (Malus x domestica Borkh.), also known as M. pumila [1, 2] belongs to the Rosaceae family [2, 3, 4, 5]. Under the Malus genus, between 55 to 79 species have been identified. Each species can be further divided into different cultivars [6]. Based on the heterozygous nature of the genes as well as self-incompatibility, more than 1000 apple cultivars have been developed through crossing [2, 4, 7]. M. domestica is referred to as Malus spp. that had undergone the domestication process, while M. sieversii is referred to as the Central Asian ‘wild apple’, with the ‘wild apple’ term also referring to all apple species other than the domesticated apple. For instance, the most common ‘wild apple’ in Europe is M. sylvestris Miller, while M. coronaria (L.) Miller is a common species in North America [6].

2. Fruit development

2.1 Stages of fruit development

Fruiting bodies of the Angiosperms range from a completely dry fruit, such as Arabidopsis, a species of Brassicaceae, to a fleshy fruit such as the tomato (Solanum lycopersicum). Angiosperm plants include small plants up to large trees, and are the source of many major crops [8]. Dry fruits can either be indehiscent (split down one side to release their seeds) or non-dehiscent. Fleshy fruits can be classified into two categories: either true fruits (fruits derived from the carpel where the ovary wall expands into flesh) or false fruits (fruits derived from carpel and accessory structures) [8]. True fruits consist of a pericarp, which is obtained from the ovary wall, and seeds, which are derived from the fertilised ovules [9, 10]. Arabidopsis produces true fruits which develop from a gynoecium, consisting of two carpels that share a fused tissue called a septum [11], while tomato produces fleshy true fruits. Apple is a fleshy accessory fruit (false fruit). It is comprised of two distinct parts; an expanded ovary that develops as the “core” of the fruit, and a cortex, the edible part of the fruit, which develops from the fused base of the stamens, petals, and sepals called the hypanthium [1]. In Arabidopsis, growth of the embryo, the ovules and gynoecium involve cell differentiation and must be carefully coordinated during fruit development to generate the final product, being the fruit [11]. Most apple cultivars are not self-fruitful, meaning that they cannot produce a consistent yield without being fertilised by pollen from a different cv. [12]. After flowering and pollination, apple fruit development takes place over 20–21 weeks involving stages of fruit setting, cell division, cell expansion, maturation, and ripening which will lead to a crisp fruit with a waxy cuticle [10]. Apple flowers are produced in clusters consisting of five to six flowers; the primary flower, also known as “king flower”, will normally open first and is capable of producing good quality fruit (Figure 1). However, the lateral flowers, which open later, can also produce good quality fruits. Apple trees are considered to reach full bloom after 50% of the king flowers on the tree are open [12].

Figure 1.

The positions of flowers in the cluster. King flower was positioned at the top of the cluster while at the lower positions were the lateral flowers.

Fruit development involves fruit set, cell division, cell expansion, and ripening. In accessory fruits, like apples, fruit set is the initiation phase of fruit development, encompassing development of the ovaries and its accessory organs into a rapidly growing young fruit. This happens during and soon after fertilisation has been completed [13]. If fertilisation does not happen, the fruit will be abscised or be small, resulting in very few or no seeds [12]. A successful fertilisation process is influenced by several factors, including adequate pollination, nutrition, and optimum temperature [12]. Fruit produced without fertilisation could be an advantage for improvement of many crops as seedless fruits are easy to consume. Therefore, tomato mutants that exhibit parthenocarpic (seedless) fruit growth in the absence of fertilisation have been extensively studied [14].

For fruit set to take place, pollination and fertilisation must be accomplished or the flowers will be abscised [13, 15, 16]. Pollination is the crucial starting point in fruit development as it is required for fertilisation. During fruit set, rapid cell division occurs and then stops when the fruit reaches its mature size [16, 17, 18]. Levels of plant hormones, such as auxins, CKs (CKs), and gibberellic acids (GAs), increase to promote fruit set [13, 16]. In tomato, after successful fertilisation, ovary development into a fruit starts with cell division until roughly 10–14 days, and during the following six to seven weeks, the fruit grows by cell expansion [14]. During cell division, which starts during fruit setting and continues until three to four weeks after pollination, the concentrations of auxin, CK and GA increase to stimulate and support the process [14, 19, 20].

Cell division ceases to allow cell expansion to then take place. Auxin, gibberellin (GA), brassinosteroid (BR) and abscisic acid (ABA) levels rise during cell expansion [13], which continues until the end of ripening, reaches its peak at 40–60 days after anthesis and becomes constant during ripening [1]. Fruit ripening is a developmental process involving physiological and metabolic changes of colour, texture, aroma, and nutritional status [21]. These changes are helpful in promoting seed dispersal by making it attractive to birds and animals. With the onset of ripening, auxin stimulates ethylene production until reaching its optimum concentration, but it can also delay ripening [22, 23]. ABA and ethylene are produced during ripening to regulate ripening [22].

2.2 Cell differentiation

At the cellular level, fruit cells undergo a differentiation process in order to change into mature cells. The term differentiation is sometimes used in two different manners: 1) it may be used to describe the development of different specialised types of mature cells within an organ or tissue; 2) it can refer to the changes that occur during the development of a meristematic cell into a mature cell, which usually involves cell division and cell expansion [24]. For the purposes of this study, cell differentiation was defined as changes of undifferentiated meristematic cells into differentiated cells that divide, expand, and ripen. The early phase of apple fruit development involves rapid cell division where the cell number is greatly amplified prior to exit from cell division at approximately 24 days after full bloom (DAFB) [25]. Final fruit size is determined by the extent of cell division and cell expansion during growth and development. Therefore, understanding cell cycle regulation and cell expansion is important to the study of fruit development in plants [26].

Once cell proliferation has ceased, cells generally undergo an expansion process that results in water uptake into the central vacuole [24]. Plant cells are large, pressurised, and have a large central vacuole that allows accumulation of water and solutes, along with a strong cell wall [27]. The cell wall is made up of a mixture of carbohydrates and a small amount of proteins, making it rigid yet extensible [28]. During cell division, cells are divided from one cell into two cells while cell expansion occurs because of the expanded vacuole, ensuing in volume growth beyond the size of the mother cell before mitosis [29]. Water uptake and cell turgor are the main factors behind the expansion process. Though the cell size increased, it was generally assumed that little to no increase in cytoplasmic mass occur [30]. Rather, it is the vacuole that grows considerably in volume and the cell wall become thinner as it is stretched and new cell wall material is added to maintain wall thickness. To allow the cell wall to grow, transcripts encoding wall-loosening enzymes, such as polygalacturonase (PG), are up-regulated and newly deposited cell wall materials are hydrated resulting in relaxation and extensive thinning of the wall [31]. Other classes of cell wall-modifying enzymes/proteins are also involved in this process, such as pectin methylesterase (PME), β-galactosidase (β-gal), endo-1,4-β-glucanase (EGAe), xyloglucan endotransglucosylase/hydrolase (XTH), and expansins [32]. These enzymes also interact synergistically by promoting ripening through disassembling the pectin of the cell wall that results in fruit softening. The secondary wall is deposited after expansion has ceased, becoming impermeable and providing strength to cells and tissues [33].

3. Hormonal control during early fruit development

After fruit set, fruit undergo cell division and subsequently cell expansion stage. It is difficult to separate fruit set from subsequent stages of fruit development, though auxin and GA are indeed key in the sustained growth of fruit. Plant hormones such as CK and auxins are involved mostly in cell division and cell expansion during fruit development [34]. These hormones have been well-documented in stimulating cell cycle activity [13]. Auxin and GA co-regulate fruit set via auxin activation of GA synthesis [35]. Therefore, auxin and GA have been widely used to increase fruit set by inducing parthenocarpic growth in many crops [36]. For example, GA or auxin treatments to tomatoes can lead to parthenocarpic fruit development [17, 37, 38, 39, 40, 41]. Silencing of SlARF7, an auxin negative regulator, caused production of parthenocarpic fruits as a result of the increasing of auxin and GA concentrations, indicating an interaction between these hormones in regulating fruit set [35, 42]. Early studies showed that auxin concentrations increase during seed development and then GA concentrations increase in the ovaries during fruit set [13, 43, 44], and this is evidenced by the application of GA inhibitors to tomato which resulted in a decreased fruit set. High expression of GA-related genes in parthenocarpic fruit (pat) mutants (a recessive mutation conferring parthenocarpy in tomato) also supports that GA controls or influences tomato fruit set in parthenocarpic fruit [44].

In normal fruit development, successful pollination and fertilisation induces an increase in both auxin and GA concentrations within the ovary [14, 45, 46, 47]. Japanese pears produced larger fruits as a result of hand pollination, indicating that an increased number of fertilised stigma leads to higher levels of GA being produced by the pollen, therefore enhancing cell division and development of larger fruit [48]. This suggests that GA is critical in pollination and fertilisation [43]. Treatment of tomato ovaries with auxin causes the formation of fruits with a higher number of pericarp cells from cell division stimulation by auxin compared to GA-induced fruits that consist of fewer cell numbers but larger cells because of GA cell’s enlargement role during fruit growth [13]. Normal-sized fruits undergo a balance cell division and cell expansion processes as a result of stable concentrations of both auxin and GA [49]. In addition, GA is also involved in the growth of certain fruits without the help of auxin and the outcome are fruit sizes that are twice as large, as shown in a transgenic tomato (S. lycopersicum L.) with low levels of auxin response factor 7 (SlARF7) [17]. Commercially, GA is used to enhance fruit size and fruit cluster in parthenocarpic fruits by increasing carbohydrate import to the fruits, as parthenocarpic fruits are normally smaller and in compact fruit clusters, such as grapes, citrus, and berries [50]. GA has also been used to overcome fruit set problems of apple and pear trees particularly during biennial bearing (a phenomenon where the high production of fruits one year suppresses flower production of the coming year, hence lower yield production) to promote flower production thereby increasing fruit set and yield [50].

Another important function of GA in fruit development is to stimulate organ growth [51]. GA also features in germination, flowering, and fruit set in many plant species [40, 52, 53]. GA concentrations during fruit development increase twice; once during early fruit growth in order to trigger cell division and a second time during cell expansion [18]. During early development, GA is produced by pollen to facilitate pollen tube growth and germination [14]. As the pollen will transfer some GA into the ovary to trigger fruit growth [14, 38], an elevated GA concentration in the ovaries following pollination (which later cause auxin levels to increase [18]) suggests that both are involved together in fruit set and growth of tomato [40]. Studies on GA during cell division have been reported in hypocotyls of cucumber and tomato [48, 54]. GA is also reported to induce and maintain cell expansion [18, 48, 55] when auxin concentrations have decreased [18]. The function of GA in cell expansion is supported by the findings of larger cells in GA₃-induced fruit (parthenocarpic) compared to seeded fruit even though the fruit size was smaller than the seeded [14]. Application of 2,4-D and GA₃ together results in same size and shape of cells of parthenocarpic fruit as seeded fruit [14]. GA maintains cell expansion when it is used during the early stage of cell expansion in Japanese pears, resulting in larger fruits compared with untreated fruits [48, 56, 57, 58]. Meanwhile, in tracheid element differentiation, application of GA₃ in conjunction with auxin results in substantial tracheid expansion as GA causes the cell to expand while application of auxin only results in short tracheid growth [59]. This shows that both auxin and GA play a coordinated role in controlling cell division and cell expansion, probably based on a common response pathway. Early hypotheses speculated that GA may cause an increase in auxin biosynthesis or transport [60].

Meanwhile, the function of auxin during fruit set is demonstrated by its presence in pollen, its production in the stalk (style), and during fertilisation [14]. Its role in tomato fruit set regulation has been described by De Jong et al. [42] through the loss-of-function of IAA9 and ARF7, resulting in parthenocarpic fruit growth. This suggests that auxin inhibits fruit growth until fertilisation takes place. IAA9 is a tomato Aux/IAA transcriptional regulator that is linked to plant responses to auxin through the expression of auxin responsive genes [61]. The reduction of IAA9 concentrations in tomato plants elicits pleiotropic phenotypes, indicating that IAA9 acts as a transcriptional repressor of auxin signalling [42, 61]. In addition, another negative regulator of fruit set, ARF8, also had similar effects in Arabidopsis ARF8 mutants [62], where ARF8 caused suppression in ovary growth through a repressive action of the Aux/IAA-ARF complex on auxin responsive genes [63].

Besides fruit set, auxin is also known for promoting cell division and cell expansion [24]. While CKs control cyclin activity during the transition phase between G₁ to S during the cell cycle (which will be discussed further later), auxin’s involvement in the cell cycle occurs much earlier than that by acting as a permissive signal for cell division to start [64]. However, this process is not yet fully understood [64, 65, 66]. Much research has been carried out focusing on identifying auxins` role in regulating cell differentiation in plants. For example, an auxin gene, auxin response factor (ARF106), is expressed during cell division and cell expansion with apple fruit development [19]. Since the gene was also co-localised with a fruit-size QTL, this suggests that auxin is involved in fruit growth control through cell differentiation [67]. Another example would be the auxin receptor, auxin binding protein (ABP1), which modulates ion fluxes in response to the hormone, and is proposed to mediate auxin-dependent cell expansion and is essential for cell division [19, 64]. Evidence has been made available in the form of applying antisense suppression of the ABP1 gene in tobacco BY-2 cell cultures, resulting in slow proliferation, discarding auxin-induced cell expansion and reducing cell division [68]. Moreover, a mutation in the ABP1 gene in Arabidopsis causes a lethal effect to cells [64]. Loss of function of ABP1 in Arabidopsis also resulted in lethal embryo as the result of cell expansion arrest, indicating auxin role, through the function of ABP1, in cell expansion stage during embryogenic development [68].

Along with auxin and GA, CK and ABA are also believed to be involved in fruit set. However, the cross-talk between them during fruit development is only partly understood. ABA is also involved in long-term developmental plant growth processes. While auxins, CKs, and BR are involved in early development, ABA is mainly present in the later stage of development where cell maturation transpires. During pollination of tomato fruit, where auxin and GA are present, ABA concentrations decrease while those of CK increase [69]. ABA concentrations continue to decrease shortly after pollination [69], and validated by a decrease in the mRNA levels of ABA biosynthesis genes after pollination [49] and the diminution of ABA concentrations in tomato pistils after pollination [70]. However, higher concentrations of ABA were observed in pollinated fruits compared to that in parthenocarpic fruits [71]. Despite being suppressed during and shortly after pollination in tomato fruit, ABA levels rose afterwards to support fruit set where they were detected about 5 days after pollination, enhanced in seed and pericarp until 30–50 days after pollination, with concentrations also increasing during cell expansion [20]. In rice (Oryza sativa) grains, low concentrations of ABA are seen in actively dividing endosperm cells [72], indicating its antagonistic effect in cell division. This is supported by higher ABA concentrations in less-dividing of small fruit Japanese pear ‘Shinkou’ compared to large Japanese pear cultivar ‘Atago’ during early fruit development [48]. In tomato, ABA shows a broad peak during cell expansion and cell maturation, showing ABA is involved in the cell expansion phase and reaches peak levels during the cell maturation phase [18]. ABA association in cell expansion has been determined by the reduction of fruit size in ABA-deficient mutants [73]. However, ABA’s exact role in fruit development is not yet known.

Auxin role in regulating cell division by stimulating cell cycle progression has been discussed earlier. As for CKs, reducing CKs concentrations by overexpression of an inactivating enzyme [74, 75] and insensitivity to the hormone cause dwarfism because of reduced cell numbers in Arabidopsis [76]. CK role in cell expansion was reported in Arabidopsis leaf expansion which resulted from cell expansion [77]. A point to note, CK function in cell division and cell expansion might require auxin. For example, in Zinnia elegans cell cultures, combination of auxin and CK was required to induce cell division and cell differentiation [33, 78]. Even though CKs are generally considered vital in the stimulation of cell division during fruit development [71], very little experimental data supports their involvement in the initial cell division phase of fruit growth [20]. However, it is known that cyclin D activity in plants is influenced by hormones and carbohydrate levels [79]. In tobacco, it has been shown that overexpression of the cell cycle stimulator CyclinD2 accelerated plant growth [80]. Riou-Khamlichi et al. [81] concluded that cell division can be induced and maintained in the absence of exogenous CK in transgenic plants over-expressing CycD3 (CDK4 to CDK6). Specifically, with overexpression of CycD3, calli may be cultured without the presence of CK [24]. This is further underscored by the transcription of CycD3 in Arabidopsis suspension culture after it is added with CK and sucrose while expression of cyclin D2 and D4 were induced in the presence of sucrose only [79]. Overexpressing CycD3 also reduces endoreduplication [82, 83] while loss of CycD3 function induces endoreduplication in Arabidopsis [26, 84].

BRs play an important role in early fruit development by promoting cell division, cell expansion in the stem, inhibiting root growth, promote xylem differentiation, ripening, and abscission [85, 86, 87, 88] such as in tomato [89, 90], grape berry [91], and cucumber [36, 92]. In tomato fruit, treatment with BR restores the dry mass content, sugar, and amino acid levels in dwarf tomato mutants, showing that BR is required for tomato fruit development [89, 92]. In cucumber, the application of exogenous BR to a cultivar without parthenocarpic capacity induces parthenocarpic growth and increases cell division via cell cycle-related gene expression [36]. In contrast, application of a BR biosynthesis inhibitor (brassinazole (Brz)) to a cucumber cultivar with parthenocarpic growth blocks fruit set, whereas this inhibitory effect was subsequently reversed by applying exogenous BR [36].

4. Fruit ripening, ethylene biosynthesis, and other related hormones

In general, there are two types of fruits based on the rate of respiration. Non-climacteric fruits do not increase ethylene production when they ripen [93]. Climacteric fruits require synthesis, perception, and signal transduction of the plant hormone ethylene in order to fully ripen [94]. In climacteric fruits, when ethylene is detected, they undergo autocatalytic ethylene biosynthesis and increased cell respiration, denoted by increased CO₂ production, which leads to fruit ripening [93]. Non-climacteric fruit, such as strawberries, grapes and citrus, require ethylene for certain ripening processes, such as skin de-greening, and do not undergo the autocatalytic response observed in climacteric fruit [94].

The inability of transgenic tomato plants to ripen where ethylene biosynthesis has been disrupted shows that the autocatalytic burst of ethylene biosynthesis is essential for climacteric fruit ripening [95]. Nevertheless, the distinction between climacteric and non-climacteric is not clearly delimited; closely related species of melon can be either climacteric, such as the cantaloupes, or non-climacteric like the ‘honey dew’ melon [94]. Climacteric fruit spans an evolutionarily wide range of angiosperm taxa, from eudicotyledons, such as tomato and apple, to monocotyledons like the banana [96]. Climacteric fruits are advantageous from an economic perspective as they are able to continue ripening even when removed from the plant [97]. This allows the fruit to be harvested and transported before they are fully ripe, resulting in reduced loss through fruit spoilage in transit [97]. Many non-climacteric fruit lack this ability and must ripen on the plant [97].

In the ethylene biosynthetic pathway, L-methionine is the main precursor which is then transformed into ethylene by the enzymes 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) and ACC oxidase (ACO). ACS and ACO are encoded by multigene families in various plant species [98]. In addition, ethylene production can be brought about by using propylene with climacteric fruit (but not in non-climacteric type fruit). In apple fruit, the level of ACS and ACO transcripts increase at the onset of ripening after treatment with ethylene [99]. However, these genes are not expressed in non-climacteric fruits as these fruits produce ethylene in very low concentrations [100]. Nevertheless, ethylene production is induced in non-climacteric fruits by various external stimuli, such as physical wounding, auxin treatments, chilling injury, drought, water logging, and pathogen infections [101]. These observations show that non-climacteric fruits have the capability to produce ethylene but are not able to produce ripening-associated ethylene [101, 102, 103].

A number of investigations have been carried out on the ripening process at both the biochemical and genetic levels in many fruits, with tomato being the classical model of choice for fleshy fruit ripening [94, 96]. As tomato ripens, it undergoes a colour change from green to red through the transformation of chloroplasts to chromoplasts [104]. Then, the fruit softens and its texture changes as the fruit cell wall is modified and partially disassembled by enzymes [104]. Alteration of specific volatiles concentrations and the sugar-acid balance will lead to the development of flavours associated with a ripened tomato [104]. Similar changes occur during ethylene-induced ripening in apples and a number of other fruits [104]. When fruits ripen, cellular turgor pressure is decreased, the cell wall is dissembled, and cell adhesion is reduced, resulting in fruit softening; a process which is facilitated by many classes of cell wall-modifying enzymes such as PG, PME, β-gal, XTH, and expansin [32, 99].

ABA concentrations are very low in unripe fruit but increase when the fruit ripens [105]. This happens in both climacteric fruits, such as mango [93], pear, avocado, and apple [106], and non-climacteric fruits like citrus [107], cherry [108], and grapes [109]. In climacteric fruits like apples, ABA concentrations increase from maturation to harvest, while in non-climacteric sweet cherries, ABA concentrations increase before maturation and decrease until harvest [105, 110]. ABA appears to have a similar function to ethylene in regulating the changes of fruit during ripening by supporting the colour changes, cell wall metabolism, fruit softening, and sugar and acid metabolism [105].

In climacteric tomato fruit, BR has been reported to promote the ripening process through increasing ethylene production and lowering chlorophyll levels [90, 92, 93]. However, in mango, low concentrations of castasterone and brassinolide are present throughout the ripening period indicating that BR is unlikely to modulate ripening [93]. Chai et al. [92] demonstrated that BRs are involved in strawberry fruit ripening through downregulating the BR receptor gene (FaBRI1) transcript, producing plants that lack the red colouring traditionally associated with ‘Akihime’ strawberry fruit. As a result of these inconsistent findings, definitive evidence for action of this hormone in fruit ripening is still inadequate.

5. Conclusions

The molecular mechanisms controlling fruit growth and fruit size in apples have yet to be fully understood [26]. The recent publication of the genome sequence of apple provides a powerful tool to reveal the underlying mechanisms during fruit development [5].

Acknowledgments

I would like to express appreciation to the University of Auckland, New Zealand, for my doctorate studies opportunity. An appreciation also goes to Malaysia Ministry of Education and Universiti Teknologi MARA (UiTM) for my doctorate financial support.

Conflict of interest

I declare no conflict of interest.