Dissection Of Climacteric And Non Climacteric Ripening In Melon

Dissection of Climacteric and Non-Climacteric Ripening in Melon by Using Genetic, Analytic and Genomic Resources

L. Pereira1, S. Pérez1, P. Rios1, K. Alexiou1, M. Pujol*1, M. Phillips2, J. Garcia-Mas1

1 Plant and Animal Genomics, Centre for Research in Agricultural Genomics CSIC-IRTA-UAB-UB, Spain. email: [anonimizat]

2 Plant Metabolism and Metabolic Engineering, Centre for Research in Agricultural Genomics CSIC-IRTA-UAB-UB, Spain

Abstract

Melon (Cucumis melo L.) is a useful plant model to elucidate the mechanism of ripening behaviour given the coexistence of climacteric and non-climacteric cultivars. In addition, useful genetic and genomic resources are available, such as extensive transcriptome data, an annotated genome sequence, resequenced cultivars, and several mapping populations. Among them, a near isogenic line population, obtained by introgressing the exotic PI 161375 (aka SC, non-climacteric) genome into ‘Piel de Sapo’ (PS, non-climacteric) background, facilitated the identification of two quantitative trait loci (QTLs) that confer climacteric ripening to PS: the recently cloned eth6.3 and eth3.5, which has been mapped in a short genomic interval. In order to elucidate additional mechanisms and genes involved in melon ripening behaviour, a new recombinant inbred line (RIL) population has been obtained by crossing ‘Védrantais’ (Ved, a typical climacteric melon) with PS. Phenotypic data related to fruit ripening was collected from the RIL population, such as fruit abscission, external color change, and ethylene production. For the latter, we developed a high throughput gas chromatography-mass spectrometry head space methodology that quantifies the production of ethylene in attached fruit with a limit of quantification near one part per billion (1 nL·L-1). These phenomic and analytical data will be correlated to a genotyping by sequencing analysis of the RIL population in order to identify new genes/QTLs involved in ripening in melon. These combined genetic, phenomic, and analytical approaches will offer significant advances in the characterization of melon fruit ripening.

Keywords: genetic and genomic resources, ethylene, fruit ripening, melon.

Introduction

Plant hormones are signaling molecules that regulate many aspects of growth and development. Ethylene is a hormone involved in many plant processes, among them the regulation of fruit ripening. The presence or absence of a peak of ethylene and a subsequent increase of respiration at the onset of ripening (Lelievre et al. 1997) permits classification of fleshy-fruits into two different categories: climacteric and non-climacteric. While the ripening process includes some structural, physiological and biochemical changes that are common to both ripening types, others are specific of climacteric fruits or even of a particular species. Some of the most important fruit organoleptic properties, as sweetness, color, firmness and aroma production, are usually related to ethylene production (Seymour et al. 2013).

The main plant model to understand climacteric fruit ripening is tomato, a species in which many key genes have been described and cloned. Two enzymes participate in the biosynthesis of ethylene, ACC synthase (ACS) and ACC oxidase (ACO), and only some isoforms are active in fruit tissues (Giovannoni 2004). In the past decades, tomato mutants have been used to figure out the ethylene pathway, including many transcription factors and ethylene receptors as rin (ripening-inhibitor) (Vrebalov et al. 2002), Cnr (colorless non-ripening) (Manning et al. 2006), SlAP2a (APETALA2 transcription factor) (Chung et al. 2010) and Nr (Never-ripe) (Lanahan et al. 1994), among others.

Over the past few years, melon has become an alternative model to study the differences between the two types of ripening given the coexistence of climacteric and non-climacteric varieties (Ezura and Owino 2008) and the large amount of genomic and genetic resources available (Diaz et al. 2011; Garcia-Mas et al. 2012). Previous studies determined which aspects of ripening are ethylene-dependent, as chlorophyll degradation, volatile production, abscission layer formation and part of flesh softening. However, some components of ripening that are a consequence of climacteric ripening in other species, as color flesh and sugar production, are ethylene-independent in melon (Pech et al. 2008).

In this favorable context, some studies have improved the knowledge of melon fruit ripening. The silencing of Aco1 in a climacteric variety using an antisense construction proved the tight relationship between ethylene production and the physiological aspects of fruit ripening (Ayub et al. 1996). Later, the analysis of a RIL population derived from a cross between the cantalupensis melon Ved, a typical climacteric, and the non-climacteric line SC, implicated two major genes and four QTLs in ethylene production and fruit abscission (Périn et al. 2002). Besides, a NIL population constructed using the same exotic parental, SC, and the inodorus non-climacteric PS, revealed two new QTLs that allow recovery of a climacteric phenotype when the SC alleles are introduced into the PS background (Vegas et al. 2013).

Nowadays, the increase of big data, the decrease in the prizing of sequence technologies and the enormous advances in bioinformatics methods facilitate the use of massive genotyping. Genotyping-by-sequencing (GBS) is a robust and relatively simple technique to genotype an entire population with thousands of SNPs well-distributed across the genome, without a significant cost in terms of economic and bioinformatic resources (Elshire et al. 2011).

The aim of the work presented here is to contribute to identifying new major genes or QTLs involved in climacteric ripening, including ethylene production and its physiological consequences. A RIL population segegating for many traits, including climacteric ripening, was developed by crossing Ved and PS, two popular sweet-fleshed cultivars. A new method, based on gas chromatography-mass spectrometry (GC-MS), to measure fruit ethylene production in planta was tested, obtaining promising results. The present study will significantly contribute to the knowledge of climacteric ripening and ethylene production in melon.

Materials & Methods

Plant material

The parental accessions used to develop the RIL population were Ved, a typical climacteric cantaloupe cultivar, with sweet aroma, presence of abscission layer and change of skin color from white to cream, and PS, a typical non-climacteric cultivar, without any of the above-mentioned properties. The two lines differ also in many interesting traits related to fruit quality: Ved has depressed vein tracts, orange flesh and medium sugar content whereas PS has green skin, white-cream flesh and high sugar content.

A RIL population of 91 individuals was developed in collaboration with Semillas Fitó. After the initial cross, the hybrid was self-pollinated by hand. The F2 generation was entered in a single seed descent scheme, until F7-F8 generations.

Three replicates of the RIL population were grown under greenhouse conditions during the summer of 2015, each replicate separated by three weeks. The plants were hand-pollinated in order to obtain one melon per plant.

Ethylene production

To register the ethylene production with the fruit attached to the plant, a novel system was tested. We used a plastic bag (polyethylene-polyamide) to enclose the fruit, producing a hermetic seal around the pedicel of the fruit (Figure 1 A and B). A PVC tube with a two-point valve was added to the system, to enable a flux of air between the inside and the outside of the bag. The bag was filled with air using a vacuum pump and closed with the valve to accumulate the ethylene that the fruit was producing (Figure 1 C). After one hour, 60 mL of the inner air was extracted with a syringe and collected in a headspace vial of 10 mL (Figure 1 D).

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Figure 1. Method of collecting fruit ethylene production in planta for GC-MS analysis. A. Thermo-sealing of the bag in the area next to the pedicel. B. Hermetic seal with Teflon film around the pedicel. C. Bag is filled with air with a pump. D. Collection of the air with a syringe.

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Once all the samples were collected, the ethylene content was measured with GC-MS; 500 µL were injected in a gas chromatograph (Agilent Technologies 7890A) coupled with a mass spectrometer detector (Agilent 5975C inert MSD). Two standards (5 and 20 ppm) were collected in the greenhouse and utilized to produce a calibration curve. The air volume inside the bag was estimated to calculate the absolute ethylene production per fruit.

The ethylene peak was characterized in 72 RILs in one replicate, using the parents and the hybrid as controls. For each melon fruit, the measurements started before the beginning of ethylene production and were recorded every 1–2 days until the ethylene peak was profiled. The non-climacteric lines were also monitored over ripening to confirm that there was no ethylene production.

Phenotypic evaluation

Phenotypic traits related with climacteric ripening were evaluated in 91 RILs in three replicates. During the season, formation of the abscission layer, aroma and external color change were recorded as qualitative: presence or absence, and as quantitative: days after pollination (DAP) when the trait appears. Abscission and flesh softening were registered at harvest. Abscission was evaluated visually on a scale from 0 (no scar) to 3 (scar totally formed/abscission). Flesh softening was measured with a penetrometer.

Results

Estimation of ethylene production

To characterize the ethylene peak, we defined four different parameters related to ethylene production: maximum ethylene production, DAP to initial ethylene production, DAP to maximum ethylene production and width of the increase of ethylene production, which is measured by counting the number of days between the DAP at which ethylene is detected and the DAP at which the maximum level is reached.

In order to study the suitability of the new method, we calculated the limit of detection (0,4 nL·L-1) and the limit of quantification (1,3 nL·L-1). We also tested the range of linearity, which was maintained between 0 – 20 nL (data not shown).

The results obtained for the parental lines, the hybrid (F1) and the RILs are presented in Table 1. The experiment confirmed the climacteric and non-climacteric behavior of the parental lines, as Ved showed an initial production of ethylene of 0,71 µL kg-1 h-1 at 34 DAP, reaching a maximum ethylene production of 72,6 µL kg-1 h-1 at 36 DAP, whereas PS did not show ethylene production during ripening.

The climacteric parent Ved started to produce the hormone at 34 DAP, while the hybrid did it earlier, at 28 DAP. The RIL population showed a large range of DAP of ethylene production, from 28 to 51 DAP, and the peak appeared between 32 and 53 DAP.

Once ethylene was detected, the levels of the hormone increased progressively over the next few days, until reaching maximum ethylene production. This parameter, defined as width of the increase of ethylene production, varied in the population between zero and nine days, showing a tendency to be higher for lower ethylene peaks (Figure 2). All of the traits described showed transgressive segregation, the maximum ethylene production being the most striking with the highest value of 239.6 µL kg-1 h-1, more than three times the climacteric parental line value.

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Table 1. Values of the controls (parental lines Ved and PS, and hybrid F1) and range observed in the RIL population. The width of the increase of ethylene production is expressed in days, and the maximum ethylene production in µL·kg-1·h-1.

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Figure 2. Ethylene production during fruit ripening in the parents (Ved and PS) and some representative examples from the RIL population.

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Climacteric behavior

Various components of climacteric behavior, including external color change, presence of characteristic aroma and abscission layer formation, were registered in the 91-RIL population in three replicates, with the parents and the hybrid as controls. The qualitative data is presented as a consensus (Table 2) and the quantitative data as a mean of three replicates (Table 3).

In the RIL population, each trait presented a particular segregation; 72 lines of the population showed the characteristic aroma (segregation 3:1, χ2 = 1.56), the external color change was observed in 48 lines (segregation 1:1, χ2 = 1.06) and the abscission layer, with more or less intensity, appeared in 27 lines (segregation 1:3, χ2 = 1.77).

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Table 2. Phenotypic evaluation of qualitative traits related to ripening in the controls (Ved, PS, F1) and the segregation in the RIL population.

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*Population (Yes:No)

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In addition to the most typical qualitative phenotypes, we evaluated other quantitative parameters to characterize the climacteric behavior in the RIL population (Table 3). A wide range of DAP was observed for aroma production, external color change and abscission layer formation. In Ved, the three first effects are almost simultaneous. In the hybrid, the aroma production was the initial symptom, followed by external color change and, later, abscission layer formation. In both cases, fruit was harvested around one week later and the abscission was not total. PS did not show any of the traits related with climacteric ripening and the fruit was harvested at 61 DAP. The RIL population revealed transgressive segregation in all traits, obtaining both extreme early and delayed climacteric lines in comparison with Ved. The penetrometer values did not show a big difference between the parental lines, even though in the population we observed there were values almost four times higher than that of PS.

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Table 3. Means of the quantitative traits related to ripening in the controls and the range in the RIL population.

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Discussion

The molecular control of fruit ripening, discerning between the ethylene-dependent and independent components, is an important topic in plant research (Gapper et al. 2014) and melon is considered as an alternative model to study its genetic regulation (Ezura and Owino 2008). The analysis of the current RIL melon population derived from a cross between two commercial cultivars with opposite and extreme ripening behavior may reveal new genes involved in this trait.

Over the past few decades, the ethylene fruit production has mostly been estimated by cutting the immature fruit and putting it in jars, using either static or dynamic methods (Bassi and Spencer 1982; Trebitsh et al. 1987; Vegas et al. 2013). The analysis of ethylene fruit production with the fruit in planta developed in this work offers some advantages. Traits other than ethylene can be evaluated on the same day, the fruit is conserved in optimal conditions, and the physiological responses are more accurate. The quantity of ethylene registered in our assay for Ved is much greater, around 10 times more, than the one described before with detached fruits (Saladié et al. 2015), probably due to the different methodology (detached fruits and GC-FID measurement). This new approach, with higher sensitivity, allowed us to detect some lines that produced very low levels of ethylene during the last days of the ripening period. Besides, due to the continuous and non-destructive sampling, we confirmed that most of the lines showed the external color change and the production of aromas almost simultaneously with the ethylene production, and sometimes even preceding the peak (Figure 3). Depending on the genetic background of the RILs, low levels of the hormone produced before the peak can be enough to trigger the climacteric ripening-associated phenotypes, including external color change, aroma and abscission layer formation.

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Figure 3. Ethylene production and climacteric symptoms: circles represent the presence of aromas; squares, the external color change, and arrows the abscission layer formation. *Line 191 did not show external color change.

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The RILs showed differences in ethylene levels, precocity, and in the correlation between the physiological consequences of climacteric ripening. Ethylene is the signal that activates secondary pathways determining chlorophyll degradation, abscission layer formation, aroma production and flesh softening (Pech et al. 2008). Genes implicated in these secondary pathways, but not connected with ethylene, could be segregating in our population. In this way, the qualitative and quantitative differences between climacteric lines could be due to quantitative segregation in ethylene production or to differences in other regulators implicated in only one of the traits.

The results of a GBS experiment that is being performed will allow the characterization of QTLs or genes implicated in climacteric ripening, contributing substantially to our current knowledge of ripening in melon.

Acknowledgements

We thank Sergio Martínez (CRAG) for technical assistance with the plant material. The work was funded by the Spanish Ministry of Economy and Competitiveness grants AGL2012-40130-C02-01 and AGL2015-64625-C2-1-R.

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