Determining the function of ripening associated genes and biochemical changes during tomato (Solanum Lycopersicum L.) fruit maturation

doi:10.21203/rs.3.rs-2873487/v1

Download PDF

Research Article

Determining the function of ripening associated genes and biochemical changes during tomato (Solanum Lycopersicum L.) fruit maturation

https://doi.org/10.21203/rs.3.rs-2873487/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

This article aimed to explore biochemical alterations and gene changes during tomato fruit development and ripening. During fruit ripening, color changes and texture softening was observed, along with an increase in the accumulation of reducing sugar and total carotenoids. Free radical content was elevated during the early stages of ripening and it declined at the later stages. Activity of α-mannosidase,β-N-acetyl hexosaminidase,and polygalacturonase was measured. qPCR experiments revealed that the α-mannosidase gene was upregulated during the breaker (1.2 fold) stages of tomato ripening, the β-N-acetyl Hexosaminidase gene was upregulated throughout the breaker (2 fold) and pink (1.2 fold)stages of tomato ripening, and the β-xylosidase gene was upregulated significantly during the breaker stage(3.9 fold) of tomato ripening. The current findings revealed that the genes α-mannosidase,β-N-acetyl hexosaminidase, ethylene-responsive factors, aminocylco propane carboxylic oxidase, pectin methylesterase, and have significant associations with textural softening which was confirmed through correlation coefficient during tomato fruit ripening.

Ripening

Antioxidants

Tomato

Mannosidase

β -N acetyl Hexosaminidase

Ripening is a protracted form of senescence, and tomato is a climacteric fruit where ripening is triggered by ethylene and its associated transcription factors (Alexander and Grierson 2002; Cara and Giovannoni 2008). Excessive textural softening is a major problem occurring during tomato ripening resulting in post-harvest losses. Many ripening related-enzymes act on cell wall of tomatoes and cause the degradation of cell wall proteins like pectin polysaccharides and N-glycoproteins (Barnes and Patchett 1976; Brummell and Harpster 2001; Meli et al. 2010; Chea et al. 2019), leading to the softening of tomato fruit. Globally, tomato is the second most-consumed vegetable after potato, and discovering a link between ripening and its association with various genes involved in softening, ethylene and ROS pathways fundamental requirement for reducing its post-harvest losses.

Ethylene is the major phytohormone responsible for fruit ripening in climacteric fruit such as tomato, papaya, apple, watermelon, etc. (Alexander and Grierson 2002; Iqbal et al. 2017; Botton et al. 2019). Blocking of ethylene and its receptor extends the shelf life of fruit and vegetables (Serek et al. 1995; Tan et al. 2012; Botton et al. 2019). Amino cyclopropane carboxylic acid oxidase (ACO) and amino cyclopropane carboxylic acid synthase (ACS) are the two biosynthetic genes of ethylene, which have a role in seed germination to fruit ripening process (Lincoln et al. 1993; Polko and Kieber 2019). Ethylene responsive factor, Auxin response factor, and expansin are the ethylene receptor genes essential in fruit ripening, reported in several climacteric fruit (Brummell et al. 1999; Müller and Munné-Bosch 2015; Liu et al. 2016; Gao et al. 2020; Diao et al. 2020).

Polygalactouronase (PG) and pectin methylesterase (PME) are the major pectin cleavage enzymes, which have a specific role in fruit ripening where they cleave the cell wall polysaccharides leading to fruit softening (Jiang et al. 2019). Several studies showed that gene silencing of PG and PME can extend the fruit ripening of different fruits (Liu et al., 2021; Yang et al., 2017). Glycoprotein cleavage enzymes such as α-mannosidase and β-N-acetyl Hexosaminidase cleaves the terminal α mannosidic linkages and hexose amine linkages, which leads to the accumulation of N-glycans in the fruit cell wall and causes the softening of the tomato fruit cell wall (Meli et al. 2010; Ghosh et al. 2011; Irfan et al. 2014; Dorairaj et al. 2020; Bose et al. 2021).

This research work investigated the expression patterns of cell wall-associated ripening genes in tomatoes during ripening, and identified critical texture softening genes that can be targeted to increase the shelf life of tomato fruit. Before sampling, fruit were picked at four different stages of ontogeny; mature, breaker, ripe and overripe. N-glycan processing (α-mannosidase and β-N acetyl Hexosaminidase), ethylene biosynthesis genes (ACS and ACO), pectin cleavage genes (Polygalctouranase and Pectin methylesterase), and antioxidant genes (NADPH oxidase and catalase) were investigated. The main objective was to examine the correlation between cell wall genes and physicochemical parameters such as color, texture, and reducing sugar in tomato fruit. Multiple correlation analysis of physiological changes and expression of multiple pathway genes was also performed to demonstrate the statistically significant fruit-cell wall texture softening genes.

2.1 Physicochemical parameters

Sample collection:

Local variety of tomato were grown in Mysuru, Karnataka, India, and fruit harvested at 70 to 80 days after flowering. The harvested fruit was divided into four categories according to their maturity stage: mature green, breaker, pink and ripen (Wang et al. 2009; Moniruzzaman et al. 2013; Skolik et al. 2019)

2.1.1. Color measurements:

Fruit assessments were performed by measuring the external colour of each biological replication with a Hunter Lab Color Measuring System (Minolta CM3500D) at visible wavelengths of the light instrument. L*, a*, b* (L = lightness *(higher positive values indicate more lightness while negative readings indicate darkness), a = ranging from green to red, a*(negative readings indicate greenness while positive values indicate redness), b = ranging from blue to yellow, b*(negative readings are indicative of blueness and higher positive readings are indicative of yellowness), hue angle (h°), Chroma (C*), colour index, the colour difference with true red (DE), a*/b*, and (a*/b*) (McGuire, 1992.)

2.1.2 Estimation of total chlorophyll and carotenoids

Spectrophotometric analysis was used to determine the chlorophyll and carotenoid content using the previously described methods (Su et al. 2015; Wang et al. 2018). Chlorophyll a, b, total chlorophyll, and total carotenoids contents were estimated and represented in mg/100 g of FW.

2.1.3 Analysis of Texture

The texture of each biological replication of different maturity stages of tomato fruit was measured using a Universal Testing Machine (UTM Lloyds, LR—5 K, Lloyd Instruments Ltd., Fareham, U.K.) (Candir et al. 2017; Li et al. 2017; Cheema et al.2018).

2.1.4 Estimation of reducing sugar

Reducing sugar was determined with the 3, 5-dinitrosalicylic acid (DNS) method (Fu et al. 2010). One gram of fresh fruit pericarp was extracted in 2.0 mL 100 mM Phosphate buffer and centrifuged for 20 minutes at 9000 g. After 5 minutes incubation with the 3, 5-dinitrosalicylic acid (DNS) reagent in a boiling water bath, samples were cooled and mixed with 40% 0.33 mL of sodium potassium tartrate. GENESYS UV-Vis spectrophotometer (Thermo Scientific, USA) was used to detect absorbance at 510 nm, and results were expressed as galactose equivalent.

2.2. Estimation of Phenolics and Flavonoids

The total phenolics were determined spectrophotometrically using the Folin Ciocalteu reagent.

Fresh tomato fruit were weighed and extracted twice with 2 mL of methanol. A rotary evaporator was used to evaporate the solvent (Buchi, Rotavapor R-205, Switzerland), and the extract was dissolved with 3 mL of distilled water. 100 µL extract was diluted with 3 mL of distilled water, and 500 µL of FC reagents was added to each test-tube and the solution was incubated for 3 minutes. After which, 200 µL of 20% sodium carbonate was added to each test tube, vortexed, this was further incubated for 1 minute in a boiling water bath, cooled, and measured the reading at 650 nm against the reagent blank. The standard graph with gallic acid was used to calculate phenolic quantities.

Total flavonoids were determined using the (Marinova et al. 2005; Wang et al. 2018) technique; in brief, 100 µL of methanol-extracted samples were combined with 1 mL of aluminum chloride solution and incubated for 15 minutes in the dark before measuring absorbance at 430 nm. Flavonoids were quantified using the standard graph of quercetin as represented as quercetin equivalent.

2.3 Determination of oxidation status during tomato fruit maturation: Total antioxidants, H₂O₂, Reducing power assay

Sample preparation

One gram of different stages of fresh tomato pericarp was taken and grounded with 2 mL of methanol in ice-cold condition, and the supernatant was collected after centrifugation at 8000 g (MPW- 260 R Poland). The supernatant (100 µL) was used for all the assays.

2.3.1 Total antioxidants:

Total antioxidants was calculated using a previously reported method (Núñez Sellés et al., 2002). Briefly 20 µL of different stages tomato fruit-extracts was taken and 1.8 mL of reagent buffer was added, incubated at 95 ℃ for 90 mins. After incubation, absorbance was taken at 695 nm. phenolic content was calculated using the standard graph of ascorbic acid.

2.3.2 FRAP/RPA:

For analysing the ferric reducing antioxidant power (FRAP), 100 µL of supernatant was mixed with 250 µL of phosphate buffer (pH 6.6), and 1% of potassium ferricyanide and incubated at 50 ℃ for 30 mins. After the incubation, the reaction mixture was mixed with 10% trichloroacetic acid, centrifuged at 8000 g for 1 min. The supernatants were collected, and 250 µL of distilled H₂O was added. The final solution was mixed up with 0.1% ferric chloride, and absorbance was measured at 700 nm. Ascorbic acid was used as standard.

2.3.3 Quantification of Hydrogen peroxide (H₂O₂)

The method by Sergiev et al. (1997) was used to determine the H₂O₂ content. 5 mL of 0.1 percent (w/v) ice-cold trichloroacetic acid was used to homogenise the fresh tomato fruit (1g). After centrifuging the homogenate at 12000 g for 15 minutes at 4°C, 0.5 mL of the supernatant was added to 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) and 1 mL of potassium iodide (1 M). At 390 nm, the absorbance of the supernatant was measured. The H₂O₂ content was compared to the standard curve (Sun et al. 2020).

2.4 Enzyme assays of α-mannosidase, β-N acetyl Hexosaminidase and Polygalactouronase

One gram of fresh tomato fruit was extracted overnight in 0.1 M sodium phosphate buffer 6.8 pH, and enzyme reactions were performed at 37°C for 20 minutes with pNP –N acetyl glucosamine (3mM) and pNP –N acetyl alpha gluocpyranaside acid (3mM) in 0.1 M sodium acetate pH 4.5 and 5.0 for mannosidase and beta N acetyl Hexosaminidase, respectively. The reaction was terminated with sodium carbonate. At 405 nm, the colour formed was recorded, and the amount of pNP was calculated using a standard curve. We used 1% pectic acid as the substrate in 0.1 M sodium acetate (pH 5), and followed the DNS technique for estimating polygalacturonase (Miller 1959).

2.5 Gene expression studies by qPCR:

One gram of fresh tomato pericarp was grounded in liquid nitrogen, and RNA was extracted with the Trizol method. One microgram of total RNA was reverse transcribed to cDNA using verso cDNA synthesis kit (Thermo Fisher). QRT PCR was performed using Quantstudio-5 (applied Biosystems) with SYBR green dye. The analysis was carried out in triplicate. Changes in gene expression data were calculated as fold changes, normalised to the endogenous reference gene actin and set relative to the control treatment by the 2^ΔΔct method (Livak and Schmittgen 2001).

2.6 Fruit cell wall studies by Scanning electron microscopy (SEM)

Different stages of the tomato pericarp were fixed in 2.5% (w/v) glutaraldehyde in 0.1 mM sodium phosphate buffer, pH 7.2, for 2 h at 4°C, then washed extensively with sodium phosphate buffer. The cubes were dehydrated in an ethanol series (0–100%). Pictures of the air-dried samples were taken using SEM (LEO-435VP, Cambridge, UK).

2.7 Statistical analysis:

All experiments were conducted in triplicate. Biological replications were individual fruit from different plants. Experiments values were analysed using Tukey's multiple comparisons test, One way ANOVA. GraphPad Prism 8 statistical software (San Diego, CA, USA). Correlation among them was measured by corrplot package in R-studio. All the experiments were performed twice with triplicate sample size. The data are represented the mean values (mean ± SD) of three biological replicates for physico biochemical, enzyme, and gene-level analysis.

3.1 Physiological parameters of colour, texture, and reducing sugar during tomato ripening

Colour can be measured based on the perception of lightness (L*), chroma (C*), hue angle (h), a*/b, and colour difference (DE*) for tomato ripening (Wang et al. 2018). During tomato ripening, colour changes from green to red and is mainly because of disassembly of chlorophyll pigments and lycopene synthesis, and carotenoid accumulation, which leads to turning tomato colour into the red as shown in Table 1. Lightness decreased during tomato fruit maturation, and the observed changes in the colour coordinates were accompanied by a decrease in L*from 63.28 to 41.35 units from the mature green to red stage. Furthermore, chroma suggested that as the ripening increased from 41.2 to 48.3 units, where the fruit developed red colour due to the synthesis of secondary metabolites such as carotenoids and lycopene.

Table 1

Physiological responses of surface colour indexes during tomato ripening
	Mature green	Breaker	Pink	Ripe
L*	63.28^a ± 0.47	57.13^b ± 0.48	43.79^c ± 0.23	41.35^d ± 0.92
a*	-4.45^b ± 6.01	-6.38^b ± 0.37	35.63^a ± 1.48	36.76^a ± 0.60
b*	40.33^a ± 0.33	41.26^a ± 3.96	32.83^a ± 1.41	31.64^a ± 2.29
Hue (h)	-1.46^b ± 0.15	-1.4^b ± 0.01	0.74^a ± 0.04	0.71^a ± 0.04
*Chroma(c)**	41.27^a ± 0.31	41.75^a ± 3.97	48.47^a ± 0.13	48.36^a ± 1.27
a/b	-0.21^b ± 0.003	0.15^b ± 0.010	1.08^a ± 0.91	1.24^a ± 0.20
*(a2/b2)*	0.04^b ± 0.001	0.02^b ± 0.001	1.18^a ± 0.20	1.56^a ± 0.52
*Different stages of tomato fruit are represented horizontally and colour parameters represented vertically. Values are represented as mean ± S.D. for n = 3. Different superscripts indicate significant difference between the values according to the one way ANOVA Post hoc test.

Colour is the most critical attribute in determining fruit quality to assess ripeness and postharvest life. Furthermore, various morphological parameters such as fruit colour, size, firmness, flavour, and nutritional aspects influence tomato quality for fresh eating. One of the most significant aspects is the colour of the fruit, as customers prefer tomatoes with a pleasing appearance. Any enzymatic and non-enzymatic changes during ripening lead to fruit colour, texture and flavour (Airianah et al. 2016). We observed that the chroma value increased as the ripening progressed. Similar results also reported previously (López Camelo and Gómez 2004; Wang et al. 2018).

3.2 Total Chlorophyll and Carotenoids

Chlorophyll is the primary pigment present in the early stage of tomatoes. As the tomato fruit's maturation starts, chlorophylls are degraded, and carotenoids accumulate, e.g., lycopene. Both chlorophyll and carotenoid compounds are significant indicators of tomato fruit quality (Lokesh et al. 2014; Wang et al. 2018; Gao et al. 2020). As depicted in Fig. 1, chlorophyll a and b were observed to decrease during ripening. Whereas, carotenoids began to accumulate in the final stages of ripening. As observed during colour parameter measurements, chroma and hue values began to rise as tomato ripening progressed. Changes in colour parameters are associated with total chlorophyll and carotenoid content, according to these data. The carotenoid syntheses were related with ethylene biosynthesis; ethylene considerably increases the destruction of chlorophyll content in tomato fruit during ripening, a phenomenon described by numerous researchers. (Su et al., 2kl,.015; Wang et al., 2018).

3.3 Texture and reducing sugar assay

The texture is another most important parameter to determine the quality of fruit and vegetables. The fruit undergoes many changes during ripening due to enzymatic, environmental, and oxidative stress, involving degradation of cell wall proteins that cause the fruit to soften (Kumar et al. 2016; Corpas et al. 2018). Pre-climacteric tomato fruits have a more rigid texture due to the compact nature of pectin polysaccharides and glycoproteins present in the cell wall. When it undergoes climacteric stages, the ethylene rate increases, and it also influences the enzymatic cleavage of cell wall polysaccharides. We observed accumulation of glycan and other sugar moieties in the fruit resulting in its softening (Fig. 2a).

We observed that fruit loses its firmness after the breaker stages of tomato fruit ripening, and the maximum firmness was observed during the mature green stage of tomato 42 Newton (N) and least was ripe stages 22 N of tomato. Several enzymes are involved in cell wall degradation. Thus N-glycan processing enzymes are implicated in tomato cell softening owing to glycoprotein cleavage in the primary cell wall (Basse and Boller 1992; Meli et al. 2010; Kumar et al. 2016; Bose et al. 2021).

Generally, fruit ripening increases the sugar level in the different stages of tomato fruit. Pectin polysaccharides are a significant component of the tomato fruit cell wall. During the initiation of ripening, starch is altered into simple sugars like glucose, which results in the accumulation of reducing sugars during tomato maturation (Hulme 1971; Giovannoni 2001; Klee and Giovannoni 2011). As previously researchers reported, sugars have been implicated in fruit ripening, such as pigment accumulation, and their regulatory roles in this complicated process is either independent or in conjunction with the hormone signalling (Durán-Soria et al. 2020). We observed that reducing sugar accumulation was higher during the pink and ripe stages of tomato (Fig. 2b).

3.2.1 Phenolics and Flavonoids

Phenolics are major secondary metabolites, which are present in climacteric and non-climacteric fruit. An essential biological function of phenolic compounds is their antioxidant properties. The total phenol content of tomatoes increased gradually as they ripened, until the pink stages, when it declined or remained steady until the red stage (Fig. 3a). Apart from their antioxidant capacity, phenolic compounds may impact the sensory characteristics of fruit due to their taste or odour (Bernalte et al. 1999; Dumas et al. 2003). During the developmental phases of tomato, phenolic chemicals accumulated, reaching peak levels during the pink stage (82mg/100g FW) and at the end it decreased in red ripe stage (41 mg/100h FW) (Fig. 3a). The values found in this study agreed with those found in other investigations (Wang et al. 2018). The variances in total phenolic content were primarily due to differences in cultivars and other factors such as temperature and light.

Flavonoids are common secondary metabolites found in plants with a wide range of biological functions, including colouration, defense against biotic and abiotic stressors, and contributing to plant growth and development (Mou et al. 2015). The largest and most prominent category of polyphenols, the flavonoids and their derivatives offer potent anti-inflammatory, anticancer, hepatoprotective, and antioxidant action due to their capacity to scavenge reactive oxygen species and reduce oxidative stress (Bhandari and Lee 2016). The flavonoid level and composition differed significantly between cultivars and ripening phases. Flavonoids were found lowest during the mature green stage of tomato and increased in the pink and ripe stage (Fig. 3b). The physiological functions of flavonoids during tomato fruit ripening are unknown. However, it is assumed that some specific flavonoids may play important roles in regulating climacteric ethylene biosynthesis, while ethylene may stimulate their biosynthesis in the epicarp of tomato fruit during the ripening stage (Wang et al. 2018).

3.4 Determination of oxidation status during tomato fruit maturation: Antioxidants, Reducing power assay (RPA) and Hydrogen peroxide (H ₂ O ₂₎

Antioxidant capacity, which indicates the ability to block the oxidation process, is an important measure for determining the health benefits of a food product. Tomatoes have significant antioxidant qualities due to natural antioxidants such as lycopene, carotenoids, phenolic compounds, ascorbic acid, and flavonoids. We observed that total antioxidant activity was higher during the later stages of ripening (pink and ripe stages) than in the mature green stage (Fig. 4a). We also measured the antioxidant capacity by a reducing power assay. The highest reducing power activity were found in the breaker and pink stages (Fig. 4b).

Fruit undergo profound changes in colour, aroma, nutrient composition, flavour, and hardness as they mature. Furthermore, the generation of reactive oxygen species is vital during this process, for example, in the biosynthesis of carotenoids and the transformation of chloroplasts to chromoplasts (Li and Yuan 2013). An unexpected metabolic shift was associated with altered thylakoid biogenesis machinery and increased energy production during tomato fruit ripening. Hydrogen peroxide content was highest in the breaker stage, and as ripening progressed, hydrogen peroxide content started declining in the pink and ripe stages (Fig. 4c). We have observed the accumulation of phenolics and flavonoids during the pink and ripe stages, and it can be scavenging the free radicals during the ripening.

3.5 Determining the activity of α-Mannosidase, β-N-Hexosaminidase, and polygalactouronase at different maturity stages of tomato fruit

In many fruit, including tomatoes, cell wall components disintegrate during ripening, resulting in changes in cell wall rheological properties and softening of the fruit (Meli et al. 2010). One approach to understanding fruit softening is identifying ripening-related enzymes such as α-mannosidase and β-N-acetyl hexosaminidase produced during ripening and whose biochemical activity can be linked to the observed changes in the cell wall. During the ripening stages of tomato, we found the maximum specific activity of α-mannosidase in the mature green (0.03 U/mg) and breaker stages (0.075 U/mg) (Fig. 5a) and β-N-acetyl hexosaminidase activity was found highest in the pink (0.032 U/mg) and ripe (0.027 U/mg) stages (Fig. 5b). Similar results were reported previously α-mannosidase and β-N-acetyl hexosaminidase were highest in early and later stages of tomato fruit ripening, respectively (Meli et al. 2010). This shows that α-mannosidase will be cleaving terminal mannosidic bonds, followed by β-N-acetyl hexosaminidase that cleaves terminal hexosaminidase linkages in the later phases of tomato ripening. Polygalactouronase is another class of pectin-cleaving cell wall enzyme, and its specific activity was highest at the pink (1.17 U/mg) and ripe (0.99 U/mg) phases (Fig. 5b). This study found that all ripening-related biochemical changes, such as chlorophyll degradation, synthesis of carotenoids, lycopene, and other secondary metabolites, began at the breaker stages of tomato ripening. Upregulation of ripening-related proteins at the maturity stages indicated these proteins' involvement and ripening-related signals are highly coordinated with each other.

3.6 Gene expression studies by qPCR

3.6.1 Gene expression studies of α-mannosidase, β-N-acetyl hexosaminidase and β-D-xylosidase during different stages of tomato fruit

To investigate the linkage and role of different genes during tomato fruit ripening, gene expression experiments were conducted for various classes of genes related to coordination of ripening, including N-glycan processing genes, ethylene biosynthesis, and receptor genes, pectin cleavage genes, and pectin cleavage cell wall and antioxidant genes.

Excessive softening of fruit during the ripening phase causes them to deteriorate quickly. N-glycan processing enzymes have been shown to play essential roles in fruit ripening-related softening, α-Mannosidase, β-N-acetyl hexosaminidase, β-xylosidase. And α-mannosidase cleaves terminal mannosidic linkages, β-N-acetyl hexosaminidase cleaves the terminal hexose aminidase linkages, and β-D-xylosidase hydrolyses arabinoxylans and xylans, releasing a-L-arabinofuranosyl residues and b-D-xylosyl residues, respectively (Meli et al. 2010; Cao et al. 2014; Irfan et al. 2014; Dorairaj et al. 2020).

Our data indicated that expression of the α-mannosidase gene increased during the breaker (1.2 fold) stage of tomato (Fig. 6a), the expression of the β-N-acetyl hexosaminidase gene was elevated during the breaker (2 fold) and pink (1.2 fold) stages (Fig. 6b), and higher expression of the β-D-xylosidase gene was observed during the breaker (3.9 fold) and ripe (1.2 fold) stages (Fig. 6c). As a result, we hypothesized that these N-glycan processing genes were significantly overexpressed throughout the ripening process, from the early stages of ripening to the ripe phases. These genes are essential during ripening, which involves the destruction of cell wall glycoproteins, resulting in excessive softness. The regulation of these genes by identifying small molecule inhibitors and edible coatings is a promising technology for controlling fruit and vegetable postharvest losses.

3.6.2 Gene expression studies of Ethylene receptor genes and Biosynthetic genes ERF, ARF ACS4 and ACO

Ethylene, as well as its receptors and biosynthetic genes, play an important role in fruit ripening. These genes play an essential role in the development and physiology of climacteric fruit, where ethylene is necessary for fruit development and ripening. There are two ethylene biosynthetic genes, ACS and ACO, and the ethylene-responsive factors (ERF), which stands for auxin-responsive factor (ARF) genes. The regulation of these genes extends the shelf life of fruit and vegetables (Hao et al. 2015; Liu et al. 2016; Polko and Kieber 2019; Gao et al. 2020) indicating the importance of these genes in ripening.

We observed the upregulation of ERF and ARF in the breaker (8.35 and 4.38 fold) stage. After that, it was down-regulated in the later stages; pink and red ripe stages of tomato ripening. ACS4 and ACO are the ethylene biosynthetic gene involved in the developmental and fruit ripening processes (Lincoln et al. 1993). Gene expression data showed higher expression of ACS4 during the later stages of ripening, mainly pink (5.65 fold) and ripe (4.19 fold) stages. ACO was upregulated in the breaker (1.83 fold) and pink (1.82 fold) stages. Previously other researchers also reported that an ethylene receptor and a biosynthetic gene are involved throughout the senescence (Müller and Munné-Bosch 2015; Iqbal et al. 2017; Botton et al. 2019; Gao et al. 2020).

3.6.3 Gene expression studies of cell wall degrading genes: polygalacturonase, pectin methylesterase, expansin, and antioxidant genes catalase and NADPH oxidase

Catalase is an antioxidant gene that mainly scavenges internal reactive oxygen species produced during ripening, protecting fruit from oxidative burst. We discovered that the expression of catalase genes varied with the progression of tomato ripening, with catalase gene expression being elevated throughout the breaker(1.05 fold) and pink (0.59 fold) stages of ripening, followed by a decline phase (Fig. 8c). The primary function of NADPH oxidase is to generate superoxide and oxygen species during oxidative burst during the ripening of both climacteric and non-climacteric fruit. Controlling the expression of these genes increases the shelf life of fruit and vegetables (Chu-Puga et al. 2019; Sun et al. 2019). Expression of the NADPH oxidase gene increased during the breaker (3.62 fold) and pink (2.97 fold) stages of tomato ripening in our study (Fig. 8d). Any biotic or abiotic stimulus that acts directly on these genes causes an oxidative burst during ripening, which causes fruit softness via disintegration of cell-wall polymers (Aghdam et al. 2020).

3.7 Cell wall studies of tomato fruit by scanning electron microscopy (SEM)

The tomato cell wall is a complex structure bounded by pectic-polysaccharides and N-glycoproteins. As the ripening triggers, many cell enzymes and genes activate, acting on polysaccharides, which leads to loosening of the cell wall. As shown in Fig. 8, in the initial mature green stages of ripening, the tomato cell wall is thick and compact. When fruit undergoes ripening, cell walls start gradually loosening in the breaker stage, and it continues until the pink and red ripe stages. Accumulation of glycan and other sugar moieties may be the reason for the fruit to become soften during ripening (Segado et al., 2016)

3.8.1 Correlation between physicochemical parameters of different stages of tomato fruit with ripening associated cell wall genes

Different enzymes and genes act on the fruit cell wall during tomato ripening, causing senescence. N-glycan processing genes are essential in fruit cell softening by cleaving N-glycoproteins and releasing N-glycans from the cell wall. Pectin cleavage genes play a role in the ripening-related softening process. Ethylene and ethylene receptor genes significantly influence both climacteric and non-climacteric fruit. When biotic or abiotic stress occurs, antioxidant genes are activated, protecting the fruit from the cellular oxidative burst. Therefore, we attempted to look at the coordination between these distinct pathway genes and their association with the physicochemical properties of different stages of tomato ripening, such as colour, texture, and sugar content during ripening.

Correlation analysis revealed that the ACS4 gene positively correlated with various colour measurement parameters. Selected N-glycan genes, i.e., α-mannosidase, β-N-acetyl hexosaminidase, and β-D-xylosidase and the cell wall-related gene PME and the ethylene receptor gene, showed a negative correlation with the different tomato fruit colour assessment parameters. Therefore, these genes do not directly influence the colour characteristics of the distinct phases of tomato fruit. We also discovered that the antioxidant genes catalase and NADPH oxidase were negatively associated with colour characteristics (Table.2).

The texture is a significant factor in determining fruit quality because numerous genes affect the texture of different fruit. During the correlation analyses, we discovered that all N-glycan processing genes, namely α-mannosidase, β-N-acetyl hexosaminidase, and β-D-xylosidase, were positively correlated to texture parameters, indicating the involvement of these genes in the textural softening of tomato fruit during ripening.

Many researchers have also reported the significance of postharvest losses of both climacteric and non-climacteric fruit. Ethylene Responsive factors, Auxin responsive factors, and biosynthetic genes of ethylene all positively correlated with the textural characteristics of tomato fruit. However, ethylene is the primary phytohormone involved in the ripening of climacteric fruit such as tomatoes. Many studies have been conducted to investigate the role of ethylene and its receptor genes in the ripening-associated softening process (Müller and Munné-Bosch 2015; Liu et al. 2016; Polko and Kieber 2019). Pectin methylesterase correlated positively with textural metrics of tomato fruit (Table 2). It was discovered that cell wall degrading genes also influence the softening process by cleaving cell wall polysaccharides in the cell wall.

Table 2

Correlation between different cell wall-associated ripening genes with the colour indexes, texture, and reducing sugar properties during tomato fruit ripeningCorrelations between 12 ripening-associated cell wall genes and physicochemical parameters such as color, texture, and reducing sugar content in different stages of tomato fruit. The physicochemical parameters of different stages of tomato correspond to the different classes of ripening genes. *α-mannosidase, β-N-acetyl hexosaminidase, β-D-xylosidase, Ethylene Responsive Factors, Auxin Responsive Factors, Amino Carboxylic Synthase 4* and *Amino Carboxylic Oxidase) Pectin methylesterase, polygalacturonase, Catalase, NADPH oxidase*, and *Expansin*
	Man	Hex	XYL	ERF	ARF	ACO	ACS4	PME	LePG	CAT	NADPH	EXP
L*	0.994	0.900	0.999	0.999	0.984	0.630	-0.918	0.999	-0.334	0.960	0.604	0.081
a*	-0.999	-0.840	-0.987	-0.996	-0.998	-0.531	0.959	-0.993	0.218	-0.919	-0.696	0.039
b*	0.997	0.886	0.997	0.999	0.989	0.606	-0.929	0.999	-0.306	0.951	0.628	0.051
Hue (h)	-0.998	-0.820	-0.980	-0.992	-0.999	0.074	0.968	-0.988	0.183	-0.905	-0.721	0.074
*Chroma (c)**	-0.998	-0.819	-0.980	-0.992	-0.999	-0.499	0.969	-0.988	0.181	-0.904	-0.723	0.076
a/b	-0.998	-0.880	-0.996	-0.999	-0.991	-0.595	0.934	-0.999	0.293	-0.947	-0.638	-0.038
*(a2/b2)*	-0.980	-0.936	-0.998	-0.992	-0.964	-0.698	0.878	-0.995	0.419	-0.982	-0.528	-0.173
Texture	0.773	0.991	0.855	0.816	0.727	0.953	-0.549	0.832	-0.797	0.955	0.066	0.616
Reducing sugar	-0.811	-0.997	-0.886	-0.850	-0.850	-0.850	0.600	-0.865	0.758	-0.971	-0.128	-0.566

Reducing sugar formation occurs by several biochemical changes occurring during fruit ripening, where many pectin cleavage genes get activated leading to the degradation of pectin and starch into glucose and fructose. The correlation analysis showed that the polygalacturonase gene was positively correlated with the amount of reducing sugars during the different phases of tomato fruit development. In tomato ripening, all other genes negatively correlated with sugar formation (Table 2). According to our data, different genes play distinct roles in the textural softening process of tomatoes. Ripening is a highly interconnected process in which many genes’ expression are indirectly related to others, however, directly impacts the physiology of fruit and vegetables.

3.8.2 Correlation matrix studies for different pathway genes

Studies of correlation were conducted to ascertain the impact of various cell wall genes on the physicochemical indicators of tomato fruit ripening. Positive correlation is denoted by the color blue, whereas negative correlation is denoted by the color red (Fig. 10). The correlation matrix revealed that the n-glycan processing genes were connected with physicochemical parameters such as color and texture, as well as with other cell wall genes, specifically the Man gene, which was positively correlated with the CAT and PME genes. The expression of Hex gene was shown to be associated with the Xyl, ERF, and PME genes. The XYL gene expression was found to be significantly associated with the MAN, Hex, ERF, and PME genes. It directly demonstrates that the N-glycan processing genes were clearly involved in tomato fruit ripening and also significantly associated with many other classes of cell wall genes to regulate the tomato fruit ripening process.

However, we have observed that the LePG gene expression was strongly negatively correlated with the other classes of genes, whereas the PME gene was positively associated with MAN, HEX, XYL ERF, and CAT genes. ERF also had a positive correlation with the cell wall genes HEX, Xyl, and PME.

Ethylene biosynthesis genes such as ACS4 were found to be inversely correlated with MAN, Xyl, ERF, ACO, and CAT genes, indicating that later stages of tomato fruit ripening is independent of ethylene biosynthesis. Correlation matrix analysis shown that there was a major participation of ripening linked genes on the physicochemical characteristics of tomato fruit, as well as how a network was formed to regulate the genes in distinct networking pathways.

These experiments examined for the first time the expression patterns of different classes of cell wall-associated softening genes, such as N-glycan processing genes, pectin cleavage genes, ethylene biosynthetic genes, and antioxidant genes how they relate to biochemical changes during different stages of tomato physiology. The findings revealed that physiological, structural, and biochemical changes in tomato ripening were substantially related to the change in sugar content, the content of carotenoids, chlorophyll concentration, and colour parameters. A correlation study revealed that N-glycan processing genes and ethylene biosynthesis and receptor genes were correlated in textural changes during tomato fruit ripening. This study revealed the relevance of N-glycoprotein at the protein and gene levels in tomato ripening. Regulating this class of enzymes during tomato ripening could increase tomato shelf life and reduce post-harvest losses.

Conflicts of interest

The authors declare that there is no conflict of interest of any kind regarding the publication of this article.

Acknowledgments

We thank the Director of the Council for Scientific and Industrial Research, the Central Food Technological Research Institute, for providing all the facilities for the work. We also wish to thank UGC-RGNF for financial support.

Author contributions:

NPS & GP conceived and designed the experiments. DD, SS, BP, and KSM performed the experiments and analysed the data. DD, SS and NPS wrote and edited the manuscript.

Aghdam MS, Palma JM, Corpas FJ (2020) NADPH as a quality footprinting in horticultural crops marketability. Trends Food Sci Technol 103:152–161. https://doi.org/10.1016/j.tifs.2020.07.002
Airianah OB, Vreeburg RA, Fry SC (2016) Pectic polysaccharides are attacked by hydroxyl radicals in ripening fruit: evidence from a fluorescent fingerprinting method. Ann Botany 117:441–455
Alexander L, Grierson D (2002) Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripening. J Exp Bot 53:2039–2055. https://doi.org/10.1093/jxb/erf072
Barnes MF, Patchett BJ (1976) Cell Wall Degrading Enzymes and the Softening of Senescent Strawberry Fruit. J Food Sci 41:1392–1395. https://doi.org/10.1111/j.1365-2621.1976.tb01179.x
Basse CW, Boller T (1992) Glycopeptide elicitors of stress responses in tomato cells: N-linked glycans are essential for activity but act as suppressors of the same activity when released from the glycopeptides. Plant Physiol 98:1239–1247
Bernalte MJ, Hernández MT, Vidal-Aragón MC, Sabio E (1999) Physical, Chemical, Flavor and Sensory Characteristics of Two Sweet Cherry Varieties Grown in ‘Valle Del Jerte’ (spain). J Food Qual 22:403–416. https://doi.org/10.1111/j.1745-4557.1999.tb00173.x
Bhandari SR, Lee JG (2016) Ripening-Dependent Changes in Antioxidants, Color Attributes, and Antioxidant Activity of Seven Tomato (Solanum lycopersicum L.) Cultivars. J Anal Methods Chem 2016:5498618. https://doi.org/10.1155/2016/5498618
Bose SK, He Y, Howlader P et al (2021) The N-glycan processing enzymes β-D-N-acetylhexosaminidase are involved in ripening-associated softening in strawberry fruit. J Food Sci Technol 58:621–631. https://doi.org/10.1007/s13197-020-04576-2
Botton A, Tonutti P, Ruperti B (2019) Chap. 5 - Biology and Biochemistry of Ethylene. In: Yahia EM (ed) Postharvest Physiology and Biochemistry of Fruits and Vegetables. Woodhead Publishing, pp 93–112
Brummell DA, Harpster MH (2001) Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants. Plant cell walls. Springer, Dordrecht., pp 311–340
Brummell DA, Harpster MH, Dunsmuir P (1999) Differential expression of expansin gene family members during growth and ripening of tomato fruit. Plant Mol Biol 39:161–169. https://doi.org/10.1023/A:1006130018931
Cao L, Zhao C, Su S et al (2014) The role of β-hexosaminidase in peach (Prunus persica) fruit softening. Sci Hort 169:226–233. https://doi.org/10.1016/j.scienta.2014.02.015
Cara B, Giovannoni JJ (2008) Molecular biology of ethylene during tomato fruit development and maturation. Plant Sci 175:106–113
Chea S, Yu DJ, Park J et al (2019) Fruit softening correlates with enzymatic and compositional changes in fruit cell wall during ripening in ‘Bluecrop’ highbush blueberries. Sci Hort 245:163–170. https://doi.org/10.1016/j.scienta.2018.10.019
Chu-Puga Á, González-Gordo S, Rodríguez-Ruiz M et al (2019) NADPH Oxidase (Rboh) Activity is Up Regulated during Sweet Pepper (Capsicum annuum L.) Fruit Ripening. Antioxid (Basel) 8:E9. https://doi.org/10.3390/antiox8010009
Corpas FJ, Freschi L, Rodríguez-Ruiz M et al (2018) Nitro-oxidative metabolism during fruit ripening. J Exp Bot 69:3449–3463. https://doi.org/10.1093/jxb/erx453
Diao D, Hu X, Guan D et al (2020) Genome-wide identification of the ARF (auxin response factor) gene family in peach and their expression analysis. Mol Biol Rep 47:4331–4344. https://doi.org/10.1007/s11033-020-05525-0
Dorairaj D, Puthusseri B, Shetty NP (2020) Suppression of N-glycan processing enzymes by deoxynojirimycin in tomato (Solanum lycopersicum) fruit. 3 Biotech 10:218. https://doi.org/10.1007/s13205-020-02196-3
Dumas Y, Dadomo M, Lucca GD, Grolier P (2003) Effects of environmental factors and agricultural techniques on antioxidantcontent of tomatoes. https://doi.org/10.1002/JSFA.1370
Durán-Soria S, Pott DM, Osorio S, Vallarino JG (2020) Sugar Signaling During Fruit Ripening.Frontiers in Plant Science11
Gao J, Zhang Y, Li Z, Liu M (2020) Role of ethylene response factors (ERFs) in fruit ripening. Food Qual Saf 4:15–20. https://doi.org/10.1093/fqsafe/fyz042
Ghosh S, Meli VS, Kumar A et al (2011) The N-glycan processing enzymes α-mannosidase and β-D-N-acetylhexosaminidase are involved in ripening-associated softening in the non-climacteric fruits of capsicum. J Exp Bot 62:571–582. https://doi.org/10.1093/jxb/erq289
Giovannoni J (2001) Molecular biology of fruit maturation and ripening. Annu Rev Plant Physiol Plant Mol Biol 52:725–749. https://doi.org/10.1146/annurev.arplant.52.1.725
Hao Y, Hu G, Breitel D et al (2015) Auxin Response Factor SlARF2 Is an Essential Component of the Regulatory Mechanism Controlling Fruit Ripening in Tomato. PLoS Genet 11:e1005649. https://doi.org/10.1371/journal.pgen.1005649
Hulme AC (1971) The biochemistry of fruits and their products, vol 2. Academic Press, London; New York, p 788
Iqbal N, Khan NA, Ferrante A et al (2017) Ethylene Role in Plant Growth, Development and Senescence: Interaction with Other Phytohormones. Front Plant Sci 8:475. https://doi.org/10.3389/fpls.2017.00475
Irfan M, Ghosh S, Kumar V et al (2014) Insights into transcriptional regulation of β-D-N-acetylhexosaminidase, an N-glycan-processing enzyme involved in ripening-associated fruit softening. J Exp Bot 65:5835–5848. https://doi.org/10.1093/jxb/eru324
Jiang F, Lopez A, Jeon S et al (2019) Disassembly of the fruit cell wall by the ripening-associated polygalacturonase and expansin influences tomato cracking. Hortic Res 6:1–15. https://doi.org/10.1038/s41438-018-0105-3
Klee HJ, Giovannoni JJ (2011) Genetics and control of tomato fruit ripening and quality attributes. Annu Rev Genet 45:41–59. https://doi.org/10.1146/annurev-genet-110410-132507
Kumar V, Irfan M, Ghosh S et al (2016) Fruit ripening mutants reveal cell metabolism and redox state during ripening. Protoplasma 253:581–594. https://doi.org/10.1007/s00709-015-0836-z
Li L, Yuan H (2013) Chromoplast biogenesis and carotenoid accumulation. Arch Biochem Biophys 539:102–109. https://doi.org/10.1016/j.abb.2013.07.002
Lincoln JE, Campbell AD, Oetiker J et al (1993) LE-ACS4, a fruit ripening and wound-induced 1-aminocyclopropane-1-carboxylate synthase gene of tomato (Lycopersicon esculentum). Expression in Escherichia coli, structural characterization, expression characteristics, and phylogenetic analysis. J Biol Chem 268:19422–19430
Liu H, Liu L, Liang D et al (2021) SlBES1 promotes tomato fruit softening through transcriptional inhibition of PMEU1. iScience 24:102926. https://doi.org/10.1016/j.isci.2021.102926
Liu M, Gomes BL, Mila I et al (2016) Comprehensive Profiling of Ethylene Response Factor Expression Identifies Ripening-Associated ERF Genes and Their Link to Key Regulators of Fruit Ripening in Tomato. Plant Physiol 170:1732–1744. https://doi.org/10.1104/pp.15.01859
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using Real-Time quantitative PCR and the 2 – ∆∆CT Method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Lokesh V, Divya P, Puthusseri B et al (2014) Profiles of carotenoids during post-climacteric ripening of some important cultivars of banana and development of a dry product from a high carotenoid yielding variety. LWT - Food Science and Technology 55:59–66. https://doi.org/10.1016/j.lwt.2013.09.005
López Camelo AF, Gómez PA (2004) Comparison of color indexes for tomato ripening. Hortic Bras 22:534–537. https://doi.org/10.1590/S0102-05362004000300006
Marinova D, Ribarova F, Atanassova M (2005) Total phenolics and total flavonoids in Bulgarian fruits and vegetables. J Univ Chem Technol Metall 40:255–260
Meli VS, Ghosh S, Prabha TN et al (2010) Enhancement of fruit shelf life by suppressing N-glycan processing enzymes. Proc Natl Acad Sci U S A 107:2413–2418. https://doi.org/10.1073/pnas.0909329107
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428. https://doi.org/10.1021/ac60147a030
Moniruzzaman M, Khatoon R, MFB H, MM R (2013) Ripening of tomato at different stages of maturity influenced by the post harvest application of ethrel. Bull Inst Trop Agric Kyushu Univ 36:017–030
Mou W, Li D, Luo Z et al (2015) Transcriptomic Analysis Reveals Possible Influences of ABA on Secondary Metabolism of Pigments, Flavonoids and Antioxidants in Tomato Fruit during Ripening. PLoS ONE 10:e0129598. https://doi.org/10.1371/journal.pone.0129598
Müller M, Munné-Bosch S (2015) Ethylene Response Factors: A Key Regulatory Hub in Hormone and Stress Signaling1. Plant Physiol 169:32–41. https://doi.org/10.1104/pp.15.00677
Núñez Sellés AJ, Vélez Castro HT, Agüero-Agüero J et al (2002) Isolation and quantitative analysis of phenolic antioxidants, free sugars, and polyols from mango (Mangifera indica L.) stem bark aqueous decoction used in Cuba as a nutritional supplement. J Agric Food Chem 50:762–766
Polko JK, Kieber JJ (2019) 1-Aminocyclopropane 1-Carboxylic Acid and Its Emerging Role as an Ethylene-Independent Growth Regulator. Front Plant Sci 10:1602. https://doi.org/10.3389/fpls.2019.01602
Segado P, Domínguez E, Heredia A (2016) Ultrastructure of the Epidermal Cell Wall and Cuticle of Tomato Fruit (Solanum lycopersicum L.) during Development1[OPEN]. Plant Physiol 170:935–946. https://doi.org/10.1104/pp.15.01725
Serek M, Sisler EC, Reid MS (1995) Effects of 1-MCP on the vase life and ethylene response of cut flowers. Plant Growth Regul 16:93–97. https://doi.org/10.1007/BF00040512
Skolik P, Morais CLM, Martin FL, McAinsh MR (2019) Determination of developmental and ripening stages of whole tomato fruit using portable infrared spectroscopy and Chemometrics. BMC Plant Biol 19. https://doi.org/10.1186/s12870-019-1852-5
Smith CJS, Watson CF, Morris PC et al (1990) Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes. Plant Mol Biol 14:369–379. https://doi.org/10.1007/BF00028773
Su L, Diretto G, Purgatto E et al (2015) Carotenoid accumulation during tomato fruit ripening is modulated by the auxin-ethylene balance. BMC Plant Biol 15:114. https://doi.org/10.1186/s12870-015-0495-4
Sun LR, Zhao ZJ, Hao FS (2019) NADPH oxidases, essential players of hormone signalings in plant development and response to stresses. Plant Signal Behav 14:1657343. https://doi.org/10.1080/15592324.2019.1657343
Sun M, Jiang F, Zhou R et al (2020) NADPH-H2O2 shows different functions in regulating thermotolerance under different high temperatures in Solanum pimpinellifolium L. Sci Hort 261:108997. https://doi.org/10.1016/j.scienta.2019.108997
Tan CK, Ali ZM, Zainal Z (2012) Changes in ethylene production, carbohydrase activity and antioxidant status in pepper fruits during ripening. Sci Hort 142:23–31. https://doi.org/10.1016/j.scienta.2012.04.030
Tieman DM, Handa AK (1994) Reduction in Pectin Methylesterase Activity Modifies Tissue Integrity and Cation Levels in Ripening Tomato (Lycopersicon esculentum Mill.) Fruits. Plant Physiol 106:429–436. https://doi.org/10.1104/pp.106.2.429
Wang H-M, Yin W-C, Wang C-K, To K-Y (2009) Isolation of functional RNA from different tissues of tomato suitable for developmental profiling by microarray analysis. Bot stud 50:115–125
Wang L, Luo W, Sun X, Qian C (2018) Changes in flavor-relevant compounds during vine ripening of tomato fruit and their relationship with ethylene production. Hortic Environ Biotechnol 59:787–804. https://doi.org/10.1007/s13580-018-0067-2
Yang L, Huang W, Xiong F et al (2017) Silencing of SlPL, which encodes a pectate lyase in tomato, confers enhanced fruit firmness, prolonged shelf-life and reduced susceptibility to grey mould. Plant Biotechnol J 15:1544–1555. https://doi.org/10.1111/pbi.12737

Download PDF

Editorial decision: Major revisions
07 Apr, 2024
Reviewers agreed at journal
07 Jul, 2023
Reviewers invited by journal
03 Jul, 2023
Editor assigned by journal
13 May, 2023
First submitted to journal
12 May, 2023

You are reading this latest preprint version

Determining the function of ripening associated genes and biochemical changes during tomato (Solanum Lycopersicum L.) fruit maturation

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Physicochemical parameters

2.1.1. Color measurements:

2.1.2 Estimation of total chlorophyll and carotenoids

2.1.3 Analysis of Texture

2.1.4 Estimation of reducing sugar

2.2. Estimation of Phenolics and Flavonoids

2.3 Determination of oxidation status during tomato fruit maturation: Total antioxidants, H₂O₂, Reducing power assay

2.3.1 Total antioxidants:

2.3.2 FRAP/RPA:

2.3.3 Quantification of Hydrogen peroxide (H₂O₂)

2.4 Enzyme assays of α-mannosidase, β-N acetyl Hexosaminidase and Polygalactouronase

2.5 Gene expression studies by qPCR:

2.6 Fruit cell wall studies by Scanning electron microscopy (SEM)

2.7 Statistical analysis:

3. Results and Discussion

3.1 Physiological parameters of colour, texture, and reducing sugar during tomato ripening

3.2 Total Chlorophyll and Carotenoids

3.3 Texture and reducing sugar assay

3.2.1 Phenolics and Flavonoids

3.5 Determining the activity of α-Mannosidase, β-N-Hexosaminidase, and polygalactouronase at different maturity stages of tomato fruit

3.6 Gene expression studies by qPCR

3.6.1 Gene expression studies of α-mannosidase, β-N-acetyl hexosaminidase and β-D-xylosidase during different stages of tomato fruit

3.6.2 Gene expression studies of Ethylene receptor genes and Biosynthetic genes ERF, ARF ACS4 and ACO

3.7 Cell wall studies of tomato fruit by scanning electron microscopy (SEM)

3.8.2 Correlation matrix studies for different pathway genes

4. Conclusion

Declarations

References

Status:

Version 1

Determining the function of ripening associated genes and biochemical changes during tomato (Solanum Lycopersicum L.) fruit maturation

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Physicochemical parameters

2.1.1. Color measurements:

2.1.2 Estimation of total chlorophyll and carotenoids

2.1.3 Analysis of Texture

2.1.4 Estimation of reducing sugar

2.2. Estimation of Phenolics and Flavonoids

2.3 Determination of oxidation status during tomato fruit maturation: Total antioxidants, H2O2, Reducing power assay

2.3.1 Total antioxidants:

2.3.2 FRAP/RPA:

2.3.3 Quantification of Hydrogen peroxide (H2O2)

2.4 Enzyme assays of α-mannosidase, β-N acetyl Hexosaminidase and Polygalactouronase

2.5 Gene expression studies by qPCR:

2.6 Fruit cell wall studies by Scanning electron microscopy (SEM)

2.7 Statistical analysis:

3. Results and Discussion

3.1 Physiological parameters of colour, texture, and reducing sugar during tomato ripening

3.2 Total Chlorophyll and Carotenoids

3.3 Texture and reducing sugar assay

3.2.1 Phenolics and Flavonoids

3.5 Determining the activity of α-Mannosidase, β-N-Hexosaminidase, and polygalactouronase at different maturity stages of tomato fruit

3.6 Gene expression studies by qPCR

3.6.1 Gene expression studies of α-mannosidase, β-N-acetyl hexosaminidase and β-D-xylosidase during different stages of tomato fruit

3.6.2 Gene expression studies of Ethylene receptor genes and Biosynthetic genes ERF, ARF ACS4 and ACO

3.7 Cell wall studies of tomato fruit by scanning electron microscopy (SEM)

3.8.2 Correlation matrix studies for different pathway genes

4. Conclusion

Declarations

References

Status:

Version 1

2.3 Determination of oxidation status during tomato fruit maturation: Total antioxidants, H₂O₂, Reducing power assay

2.3.3 Quantification of Hydrogen peroxide (H₂O₂)