Phytohormones in fruit development and maturation

Phytohormones are integral to the regulation of fruit development and maturation. This review expands upon current understanding of the relationship between hormone signaling and fruit development, emphasizing fleshy fruit and highlighting recent work in the model crop tomato (Solanum lycopersicum) and additional species. Fruit development comprises fruit set initiation, growth, and maturation and ripening. Fruit set transpires after fertilization and is associated with auxin and gibberellic acid (GA) signaling. Interaction between auxin and GAs, as well as other phytohormones, is mediated by auxin-responsive Aux/IAA and ARF proteins. Fruit growth consists of cell division and expansion, the former shown to be influenced by auxin signaling. While regulation of cell expansion is less thoroughly understood, evidence indicates synergistic regulation via both auxin and GAs, with input from additional hormones. Fruit maturation, a transitional phase that precipitates ripening, occurs when auxin and GA levels subside with a concurrent rise in abscisic acid (ABA) and ethylene. During fruit ripening, ethylene plays a clear role in climacteric fruits, whereas non-climacteric ripening is generally associated with ABA. Recent evidence indicates varying requirements for both hormones within both ripening physiologies, suggesting rebalancing and specification of roles for common regulators rather than reliance upon one. Numerous recent discoveries pertaining to the molecular basis of hormonal activity and crosstalk are discussed, while we also note that many questions remain such as the molecular basis of additional hormonal activities, the role of epigenome changes, and how prior discoveries translate to the plethora of angiosperm species.     

Abscisic acid perception and signaling transduction in strawberry: A model for non-climacteric fruit ripening

On basis of fruit differential respiration and ethylene effects, climacteric and non-climacteric fruits have been classically defined. Over the past decades, the molecular mechanisms of climacteric fruit ripening were abundantly described and found to focus on ethylene perception and signaling transduction. In contrast, until our most recent breakthroughs, much progress has been made toward understanding the signaling perception and transduction mechanisms for abscisic acid (ABA) in strawberry, a model for non-climacteric fruit ripening. Our reports not only have provided several lines of strong evidences for ABA-regulated ripening of strawberry fruit, but also have demonstrated that homology proteins of Arabidopsis ABA receptors, including PYR/PYL/RCAR and ABAR/CHLH, act as positive regulators of ripening in response to ABA. These receptors also trigger a set of ABA downstream signaling components, and determine significant changes in the expression levels of both sugar and pigment metabolism-related genes that are closely associated with ripening. Soluble sugars, especially sucrose, may act as a signal molecular to trigger ABA accumulation through an enzymatic action of 9-cis-epoxycarotenoid dioxygenase 1 (FaNCED1). This mini-review offers an overview of these processes and also outlines the possible, molecular mechanisms for ABA in the regulation of strawberry fruit ripening through the ABA receptors.     

Extensive transcriptomic studies on the roles played by abscisic acid and auxins in the development and ripening of strawberry fruits

Strawberry is an ideal model for studying the molecular biology of the development and ripening of non-climacteric fruits. Hormonal regulation of gene expression along all these processes in strawberries is still to be fully elucidated. Although auxins and ABA have been pointed out as the major regulatory hormones, few high-throughput analyses have been carried out to date. The role for ethylene and gibberellins as regulatory hormones during the development and ripening of the strawberry fruit remain still elusive. By using a custom-made and high-quality oligo microarray platform done with over 32,000 probes including all of the genes actually described in the strawberry genome, we have analysed the expression of genes during the development and ripening in the receptacles of these fruits. We classify these genes into two major groups depending upon their temporal and developmental expression. First group are genes induced during the initial development stages. The second group encompasses genes induced during the final maturation and ripening processes. Each of these two groups has been also divided into four sub-groups according their pattern of hormonal regulation. By analyzing gene expression, we clearly show that auxins and ABA are the main and key hormones that combined or independently are responsible of the development and ripening process. Auxins are responsible for the receptacle fruit development and, at the same time¸ prevent ripening by repressing crucial genes. ABA regulates the expression of the vast majority of genes involved in the ripening. The main genes expressed under the control of these hormones are presented and their physiological rule discussed. We also conclude that ethylene and gibberellins do not seem to play a prominent role during these processes.     

An expansin gene expressed in ripening strawberry fruit

Tissue softening accompanies the ripening of many fruit and initiates the processes of irreversible deterioration. Expansins are plant cell wall proteins proposed to disrupt hydrogen bonds within the cell wall polymer matrix. Expression of specific expansin genes has been observed in tomato (Lycopersicon esculentum) meristems, expanding tissues, and ripening fruit. It has been proposed that a tomato ripening-regulated expansin might contribute to cell wall polymer disassembly and fruit softening by increasing the accessibility of specific cell wall polymers to hydrolase action. To assess whether ripening-regulated expansins are present in all ripening fruit, we examined expansin gene expression in strawberry (Fragaria x ananassa Duch.). Strawberry differs significantly from tomato in that the fruit is derived from receptacle rather than ovary tissue and strawberry is non-climacteric. A full-length cDNA encoding a ripening-regulated expansin, FaExp2, was isolated from strawberry fruit. The deduced amino acid sequence of FaExp2 is most closely related to an expansin expressed in early tomato development and to expansins expressed in apricot fruit rather than the previously identified tomato ripening-regulated expansin, LeExp1. Nearly all previously identified ripening-regulated genes in strawberry are negatively regulated by auxin. Surprisingly, FaExp2 expression was largely unaffected by auxin. Overall, our results suggest that expansins are a common component of ripening and that non-climacteric signals other than auxin may coordinate the onset of ripening in strawberry.     

Induced parthenocarpy-a way of manipulating levels of endogenous hormones in tomato fruits (Lycopersicon esculentum Mill.) 2. Diffusible hormones

Indol-3-acetic acid (IAA), gibberellin-like substances (GAs), and abscisic acid (ABA) were measured throughout the first 35 days of fruit development in agar diffusates from seeded and parthenocarpic tomato fruits. Parthenocarpic fruit growth was induced with either an auxin (4-CPA), morphactin (CME) or gibberellic acid (GA₃). IAA and GAs were at their highest levels in diffusates during the early stages of fruit growth, whereas diffusible ABA increased later. Most IAA was found in diffusates from auxin-induced and seeded fruits, whereas GAs were at their lowest levels in seeded fruits. There were only minor differences in ABA concentrations regardlesss of the treatments.Levels of diffusible hormones of tomato fruits may be easily manipulated by inducing parthenocarpic fruit growth. In spite of no obvious relationship between fruit growth and hormone levels in this study, induced parthenocarpy is considered a useful tool to further elucidate the role of hormones in fruit development and sink-source interactions.     

Induced parthenocarpy—a way of changing the levels of endogenous hormones in tomato fruits (Lycopersicon esculentum Mill.) 1. Extractable hormones

Ethylene, indol-3-acetic acid (IAA), gibberellin-like substances (GAs) and abscisic acid (ABA) were analysed in extracts from normal, seed-containing and parthenocarpic tomato fruits throughout fruit development. Parthenocarpic fruit growth was induced with an auxin (4-CPA), morphactin (CME) or gibberellic acid (GA₃) and compared with that of pollinated control fruits. Fruit growth was only affected by the treatment with GA₃, decreasing size and fresh weight by 60%. The peak sequence of hormones during fruit development was ethylene-GAs-IAA-ABA. Seeded fruits contained the highest levels of IAA and ABA but the lowest levels of GAs. Also, in seeded fruits, a high proportion of IAA and ABA was found in the seeds whereas this was not the case for GAs.Hormone levels of tomato fruits may be successfully, easily and reproducibly altered by inducing parthenocarpic fruit growth and thus eliminating development of seeds which are a major source of hormone synthesis. In spite of markedly changed hormone levels, there was no obvious relationship between fruit growth and extractable hormones per se. However, the results indicate that a high ratio of GAs: auxins is unfavourable for growth of tomato fruits.     

Studies on the Active Changes of GA3 and Cytokinins During Development and Ripening in Seabuckthorn Fruits

The phenological stages in seabuckthorn (Hippophae rhamnoides L. Subsp. sinensis Rousi and H. thibetana Schlechted) distributed naturally over an area of Wushao ridge were observed. The changes in levels of endogenous phytohormones GA3, iPA, zeatin, IAA and ABA were investigated systematically during development and ripening periods in the two sorts of fruits. The results showed that two peak values of GA3 level in seeds were detected (in 105—109 days and 128--132 days after anthesis). The first peak value of isopentenyl adenine (IPA) in seeds was determined in 92--96 days when the fruit was starting to change its colour and just right now the zeatin first appeared. The iPA content reached the second peak value in 127-136 days after anthesis during fruit full ripening and beginning to deciduous leaf and the maximum of zeatin in seeds appeared near the same time (in 127 days after anthesis). It was remarkable that the zeatin content in fruit stalk and flesh still was kept on high level, in the meantime the zeatin level in seeds dropped down rapidly. Based on the facts that the time of the first peak value in GA3 and iPA level is close to the date when ABA is first detected, and the date of second peak value in GA3, iPA and IAA is coincident with the time of maximum in ABA and zeatin levels make it reasonable to consider that the balance of phytohormones may be more important than absolute amounts of any single hormone during the periods of fruit development and ripening, and meanwhile it also proved that GA3 and CTK play an important regulatory role in controlling fruit ripening and colour changing process of seabuckthorn fruit.     

Downregulation of RdDM during strawberry fruit ripening

Background

Recently, DNA methylation was proposed to regulate fleshy fruit ripening. Fleshy fruits can be distinguished by their ripening process as climacteric fruits, such as tomatoes, or non-climacteric fruits, such as strawberries. Tomatoes undergo a global decrease in DNA methylation during ripening, due to increased expression of a DNA demethylase gene. The dynamics and biological relevance of DNA methylation during the ripening of non-climacteric fruits are unknown.

Results

Here, we generate single-base resolution maps of the DNA methylome in immature and ripe strawberry. We observe an overall loss of DNA methylation during strawberry fruit ripening. Thus, ripening-induced DNA hypomethylation occurs not only in climacteric fruit, but also in non-climacteric fruit. Application of a DNA methylation inhibitor causes an early ripening phenotype, suggesting that DNA hypomethylation is important for strawberry fruit ripening. The mechanisms underlying DNA hypomethylation during the ripening of tomato and strawberry are distinct. Unlike in tomatoes, DNA demethylase genes are not upregulated during the ripening of strawberries. Instead, genes involved in RNA-directed DNA methylation are downregulated during strawberry ripening. Further, ripening-induced DNA hypomethylation is associated with decreased siRNA levels, consistent with reduced RdDM activity. Therefore, we propose that a downregulation of RdDM contributes to DNA hypomethylation during strawberry ripening.

Conclusions

Our findings provide new insight into the DNA methylation dynamics during the ripening of non-climacteric fruit and suggest a novel function of RdDM in regulating an important process in plant development.

     

Changes in gene expression during strawberry fruit ripening and their regulation by auxin

Changes in messenger RNA during the development of the strawberry (Fragaria ananassa Duch.), a non-climacteric fruit, were analysed by extracting total RNA and separating the in-vitro translated products by two-dimensional polyacrylamide gel electrophoresis. Alterations in numerous messenger RNAs accompanied fruit development between the immature green stage and the overripe stage, with prominent changes detected at or before the onset of ripening. A number of messenger RNAs undetectable in immature green fruit increased as the fruit matured and ripened. Others showed a marked decrease in advance of the ripening phase. A further group of messenger RNAs was prominent in immature and ripe fruit but absent just prior to the turning stage. Removing the achenes from a segment of the fruit accelerated anthocyanin accumulation in the de-achened portion and produced a pattern of translated polypeptides similar to normal ripe fruit. Application of the synthetic auxin 1-naphthaleneacetic acid to the de-achened receptacle produced a translation pattern similar to that in mature green fruit. These findings indicate that ripening in strawberry is associated with the expression of specific genes.     

Role of Brassinosteroids, Ethylene, Abscisic Acid, and Indole-3-Acetic Acid in Mango Fruit Ripening

Rapid ripening of mango fruit limits its distribution to distant markets. To better understand and perhaps manipulate this process, we investigated the role of plant hormones in modulating climacteric ripening of ??Kensington Pride?? mango fruits. Changes in endogenous levels of brassinosteroids (BRs), abscisic acid (ABA), indole-3-acetic acid (IAA), and ethylene and the respiration rate, pulp firmness, and skin color were determined at 2-day intervals during an 8-day ripening period at ambient temperature (21?±?1°C). We also investigated the effects of exogenously applied epibrassinolide (Epi-BL), (+)-cis, trans-abscisic acid (ABA), and an inhibitor of ABA biosynthesis, nordihydroguaiaretic acid (NDGA), on fruit-ripening parameters such as respiration, ethylene production, fruit softening, and color. Climacteric ethylene production and the respiration peak occurred on the fourth day of ripening. Castasterone and brassinolide were present in only trace amounts in fruit pulp throughout the ripening period. However, the exogenous application of Epi-BL (45 and 60?ng?g^?1 FW) advanced the onset of the climacteric peaks of ethylene production and respiration rate by 2 and 1?day, respectively, and accelerated fruit color development and softening during the fruit-ripening period. The endogenous level of ABA rose during the climacteric rise stage on the second day of ripening and peaked on the fourth day of ripening. Exogenous ABA promoted fruit color development and softening during ripening compared with the control and the trend was reversed in NDGA-treated fruit. The endogenous IAA level in the fruit pulp was higher during the preclimacteric minimum stage and declined during the climacteric and postclimacteric stages. We speculate that higher levels of endogenous IAA in fruit pulp during the preclimacteric stage and the accumulation of ABA prior to the climacteric stage might switch on ethylene production that triggers fruit ripening. Whilst exogenous Epi-BL promoted fruit ripening, endogenous measurements suggest that changes in BRs levels are unlikely to modulate mango fruit ripening.     

Gibberellin and auxin signaling genes RGA1 and ARF8 repress accessory fruit initiation in diploid strawberry

Unlike ovary-derived botanical fruits, strawberry (Fragaria x ananassa) is an accessory fruit derived from the receptacle, the stem tip subtending floral organs. Although both botanical and accessory fruits initiate development in response to auxin and gibberellic acid (GA) released from seeds, the downstream auxin and GA signaling mechanisms underlying accessory fruit development are presently unknown. We characterized GA and auxin signaling mutants in wild strawberry (Fragaria vesca) during early stage fruit development. While mutations in FveRGA1 and FveARF8 both led to the development of larger fruit, only mutations in FveRGA1 caused parthenocarpic fruit formation, suggesting FveRGA1 is a key regulator of fruit set. FveRGA1 mediated fertilization-induced GA signaling during accessory fruit initiation by repressing the expression of cell division and expansion genes and showed direct protein–protein interaction with FveARF8. Further, fvearf8 mutant fruits exhibited an enhanced response to auxin or GA application, and the increased response to GA was due to increased expression of FveGID1c coding for a putative GA receptor. The work reveals a crosstalk mechanism between FveARF8 in auxin signaling and FveGID1c in GA signaling. Together, our work provides functional insights into hormone signaling in an accessory fruit, broadens our understanding of fruit initiation in different fruit types, and lays the groundwork for future improvement of strawberry fruit productivity and quality.

An investigation of the mechanism of accessory fruit initiation in diploid strawberry, identifying the function of two hormone signaling genes in fruit initiation.     

Abscisic acid plays an important role in the regulation of strawberry fruit ripening

The plant hormone abscisic acid (ABA) has been suggested to play a role in fruit development, but supporting genetic evidence has been lacking. Here, we report that ABA promotes strawberry (Fragaria ananassa) fruit ripening. Using a newly established Tobacco rattle virus-induced gene silencing technique in strawberry fruit, the expression of a 9-cis-epoxycarotenoid dioxygenase gene (FaNCED1), which is key to ABA biosynthesis, was down-regulated, resulting in a significant decrease in ABA levels and uncolored fruits. Interestingly, a similar uncolored phenotype was observed in the transgenic RNA interference (RNAi) fruits, in which the expression of a putative ABA receptor gene encoding the magnesium chelatase H subunit (FaCHLH/ABAR) was down-regulated by virus-induced gene silencing. More importantly, the uncolored phenotype of the FaNCED1-down-regulated RNAi fruits could be rescued by exogenous ABA, but the ABA treatment could not reverse the uncolored phenotype of the FaCHLH/ABAR-down-regulated RNAi fruits. We observed that down-regulation of the FaCHLH/ABAR gene in the RNAi fruit altered both ABA levels and sugar content as well as a set of ABA- and/or sugar-responsive genes. Additionally, we showed that exogenous sugars, particularly sucrose, can significantly promote ripening while stimulating ABA accumulation. These data provide evidence that ABA is a signal molecule that promotes strawberry ripening and that the putative ABA receptor, FaCHLH/ABAR, is a positive regulator of ripening in response to ABA.     

ClSnRK2.3 negatively regulates watermelon fruit ripening and sugar accumulation

Watermelon(Citrullus lanatus) as non-climacteric fruit is domesticated from the ancestors with inedible fruits. We previously revealed that the abscisic acid(ABA) signaling pathway gene ClSnRK2.3 might infuence watermelon fruit ripening. However,the molecular mechanisms are unclear. Here,we found that the selective variation of ClSnRK2.3 resulted in lower promoter activity and gene expression level in cultivated watermelons than ancestors, which indicated ClSnRK2.3 might be a negative regulator ...     

Transcriptomes of Fruit Cavity Revealed by De Novo Sequence Analysis in Nai Plum (<Emphasis Type="Italic">Prunus salicina</Emphasis>)

Nai plum (Prunus salicina) is an important fruit crop in China having good taste and flavour. Cavity formation occurring during fruit development affects fruit quality. However, the molecular mechanism underlying cavity formation is unclear. To obtain differential expression profiles of cavity fruit (CF) and non-cavity fruit (nCF) in P. salicina, we sequenced the fruits at different time intervals of 7 days after anthesis (DAA), 21 and 28 DAA, respectively, and 83,869 unigenes, 3811 differentially expressed genes, 22,971 simple sequence repeats and over 14,000 single nucleotide polymorphisms were obtained. Twenty-three differentially expressed genes were selected for verification by qRT-PCR. The contents of phytohormones during fruit development showed that there was a positive relevance between phytohormone contents (IAA, ZR and GA), fruit size and ABA contents in the fruits, whereas there was a negative correlation with ZR, GA and IAA. Lower GA content in fruit before 14 DAA and higher IAA and ZR levels during later developmental stages resulted in cavity appearance. Further studies showed that differential expression of phytohormone-related genes IPT, CKX, YUCCA, GA20ox, GID1, CCS1 was determined at key fruit development stages, which is consistent with content changes of IAA, GA, ABA and ZR. Our results suggest that ABA might inhibit the synthesis of IAA, ZR and GA and cause fruit cavity formation in Nai plum.     

香梨果实成熟衰老过程中4种内源激素的变化

以库尔勒香梨[白梨(PyrusbretschneideriRehd.)的变种]为材料,在果实生长发育、成熟衰老期间检测内源IAA、GA3、ABA、乙烯含量变化规律及其相互关系。结果表明果实发育初期IAA、GA3、ABA含量最高,有利于幼果坐果;CA3与ABA的比值变化对果实迅速膨大起关键作用;高浓度GA3对阻抑叶绿素分解起明显作用;果实成熟衰老期间,IAA含量与乙烯释放速率呈方向相同的变化;在此期间GA3含量变化与乙烯释放变化相反。     

Indole-3-acetic acid, ethylene, and abscisic acid metabolism in developing muskmelon (Cucumis melo L.) fruit

Hormonal metabolism associated with fruit development in muskmelon was investigated by measuring IAA, ABA, and ACC levels in several tissues at various stages of development. In addition, levels of conjugated IAA and ABA were determined in the same tissues. Ethylene production, which is believed to signal the ripening and senescence of mature fruit, was also measured. Ethylene production was highest in the outer tissue near the rind and gradually declined during maturation, except for a dramatic increase in all fruit tissues at the climacteric. In contrast to ethylene production, ACC levels increased during maturation and remained equal throughout the fruit until the climacteric, when levels in the outer tissues increased nearly 5-fold over levels in the inner tissues. The consistent presence of ACC indicates that ACC oxidase rather than the availability of ACC regulates ethylene production in developing fruits. ABA and ABA esters generally declined during maturation, however an increase in ABA esters associated with the outer mesocarp tissue was observed in fully mature, climacteric fruit. IAA and IAA conjugates were only found in the outer tissue near the rind, and their levels remained low until the fruit was fully mature and entering the climacteric. At that time, increased levels of conjugates were detected. The late burst of hormonal metabolism in the outer mesocarp tissue appeared to signal its degeneration and the deterioration that typically occurs in ripening fruit. The tissue-specific conjugation of IAA and ABA, in addition to the production of climacteric ethylene, may represent part of the signaling mechanism initiating ripening and eventual deterioration of tissues in muskmelon fruits.Abbreviations ABA abscisic acid - ACC 1-aminocylopropane-1-carboxylic acid - DAP days after pollination - IAA indole-3-acetic acid