NAC of fleshy fruit development and stress responses. To

NAC transcription factors are knownregulators of diverse developmental processes and have been associated with carotenoidaccumulation and plant abiotic defense responses, such as drought andtemperature stress tolerance. Tomato fruit (Solanumlycopersicum L.

) is an established model for the study of fleshy fruitdevelopment and stress responses. To explore further the role of NAC, weanalyzed the expression of six NAC family transcription factors (SlNAC1, SlNAC4, SlNAC5, SlNAC6, SlNAC7and SlNAC9) in four hybrid and fournative tomato fruit (Solanum lycopersicumL.) genotypes of different colors. RT-qPCR analysis showed that nativegenotypes, which have been exposed to open-air conditions promoting thedevelopment of defense mechanisms that increase plant survival, had a higherexpression of all NAC genes whencompared to hybrid genotypes.

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The expression of SlNAC1 and SINAC4 wasassociated with fruit pigmentation, since both hybrid and native yellow fruitsexhibited lower expression levels. This report provides new insights into theexpression of NAC family transcription factors in hybrid and native tomatogenotypes of different color.   NAC genesconstitute one of the largest families of plant-specific transcription factorsand influence a diverse set of developmental processes.

The term NAC derives fromthe names of three transcription factors which share the same DNA-bindingdomain: NAM from Petunia, ATAF1-2from Arabidopsis thaliana and CUC2 fromArabidopsis (Souer et al. 1996;Nuruzzaman et al. 2015). Since the activation and expression of NAC family members are crucial for plantsurvival, these genes have been studied in several species, such as bean, rice,maize and tomato (Bhattacharjee et al.2016; Wu et al. 2016).

There are over 117 NAC genes in Arabidopsis,151 in rice and 79 in grape, while more than 20 have been described in tomato (Fanget al. 2008; Rushton et al. 2008; Nuruzzaman et al. 2015; Kou et al.

2016;Tranbarger et al. 2017). In addition to their participation in the regulationof several developmental processes, including senescence, NAC proteins havebeen associated with plant abiotic defense responses such as drought stresstolerance mechanisms and biotic stress response and been used in crop plants toimprove tolerance to several types of stress by genetic engineering (Mao et al.2014; Shao et al.

2015). Thetomato (Solanum lycopersicum L.) isone of the most commonly consumed fleshy fruits in the world due to itsversatility of use (fresh or processed), characteristic flavor and nutritionalproperties, being also considered a good model for the study of fleshy fruits (Sinesio et al.

2010). Native genotypes have been produced on asmall scale, under backyard open-air conditions. It is believed that long-termexposure to continuous environmental stresses and pathogen infections promotethe development of defense mechanisms that increase plant survival, such as biotic and abiotic stress responses(Bonilla-Barrientos et al. 2014).

Although several NAC family transcriptionfactors have been characterized in tomato, only little information concerningstress-related NAC genes isavailable. It is suggested that NACgenes are not only associated with stress responses, but also with fruitdevelopment and carotenoid accumulation (Zhu et al. 2014). The aim of this study was to analyzethe expression of six NAC family transcription factors in eight hybrid andnative tomato (Solanum lycopersicum L.)genotypes, in order to associate its expression with stress-response andpigmentation. Four native and four hybrid tomato (Solanum lycopersicum L.) genotypes ofdifferent colors (Table 1) were cultivated in volcanic sand substrate and aStainer nutrient solution in two nearby greenhouses. The first was located at(19.

463327, -98.907527, 2295 MASL), while the second was located at (19.486792,-98.900117, 2295 MASL).

Allgenotypes used in this study are part of the Mexican Network of Plant GeneticResources (REMEFI). Fruits werecultivated simultaneously under temperature conditions within the range of22-30 °C and a relative humidity of 75%.Fruits were selected when ripe on the basis of a color chartdeveloped specifically for each genotype according to maximum color development and harvested free of mechanical damage and physicaldefects, and transported to the Autonomous Metropolitan University-Iztapalapa.These fruits (three replicates of 27 fruits for each genotype; 81 total) werewashed and deseeded. The whole fruit tissue was chopped, frozen in liquid nitrogen,and stored at -75°C until use.    The RNA extraction protocol wasperformed according to the technique reported by Chang et al. (1993).

Six g oftissue were crushed to a powder in liquid nitrogen. The purity andconcentration of the RNA were determined by spectrophotometry (absorbance260/280 nm) (Nanodrop ND-1000), and the integrity was verified by a 1% w/vagarose gel electrophoresis stained with ethidium bromide.The identity of amplificationproducts was verified before performing the RT-qPCR reactions and theamplification efficiency was corroborated to be between 95 and 100%. 2 µg ofRNA were treated with DNase I, RNase free 1 U/L (Thermo Fisher Scientific). Thetranscripts of interest were amplified by RT-qPCR using the kit Express SYBRGreenER qPCR Supermix (ThermoFisher Scientific, USA). 1 µl of each dilutedsample was loaded per tube, obtaining a final volume of 10.

5 µl. The 18S rRNAfragment was used as reference gene since it showed minimal variability in itsexpression between genotypes. The following temperature program was used: RTreaction at 37°C for 10 minutes, initial denaturation at 95 °C for 3 minutes,94 °C for 30 seconds, alignment temperature as indicated below for 20 seconds,and a final temperature of 72 °C for 30 seconds). Thirty cycles were requiredfor all genes (Table 2). The relative mRNA expression wascalculated by 2-??Ct method (Willemset al. 2008; Villa-Hernández et al.

2013). A value of 1 was arbitrarily assignedto the mRNA expression of the red native genotype, and the relative expressionof the other genotypes was calculated using this genotype as reference. Statistical analyzes were carried out usingANOVA + Tukey (significance level ? = 0.05) using Prism 7.

0 (GraphPad SoftwareInc. 2017). Three biological replicates were used for each experiment. Theartwork was created with SigmaPlot 11.0. The expression of all SINAC familyfactors was significantly higher in the native genotypes when compared to thatof the hybrid genotypes (Fig 1). While hybrid genotypes have been selected toimprove postharvest shelf-life, native genotypes have been exposed to open-airconditions, promoting the development of defense mechanisms that increase plantsurvival. Thus, native genotypes exhibit biotic and abiotic stress responses,such as growing in soils with limited moisture availability and tolerance tohigher temperatures.

These results suggest that in native genotypes NAC genes contribute to enhancesurvivability of plantsunder several conditions of environmental stress. Kou et al. (2014) analyzed the expression of NAC genes in multiple tissues duringdevelopment of tomato fruit, and found that the expression of SINAC5, SINAC6, SlNAC7 and SlNAC9 increases during tomato fruitdevelopment. These authors additionally found that the expression of SlNAC4, SlNAC6 and SlNAC7 was higher at the pink and ripe stages, suggesting thatthese genes participate in the same or similar regulatory networks inethylene-stimulated or hormone signaling pathways (Kou et al. 2016). Inagriculture, high or low temperature acts as a major negative factor limitingcrop production.

In this regard, NACgenes have been also associated with responses to both heat and cold stresses(Nuruzzaman et al. 2015; Bonilla-Barrientos et al. 2014). Due to their exposure to extreme environmental conditions,native genotypes analyzed are expected to show greater resistance to various types ofstress, compared to hybrid genotypes that have been selected for higher yieldand pest resistance.

The difference between theexpression in native and hybrid genotypes was greater for SINAC5 and SINAC1. Whilethe expression of SINAC5 has beenfound to be highly induced by NaCl stress (Zhu et al. 2014), SlNAC1 is the most studied NACtranscription factor in tomato, and it is known to also play a crucial role insalt stress tolerance (Golldack et al. 2011).

Additionally, Ma et al. (2014)report that the overexpression of SINAC1resulted in reduced carotenoids by altering carotenoid pathway flux anddecreasing ethylene synthesis, leading to yellow and orange mature fruits. Inthe present study, we found a higher expression of SINAC1 in both native and hybrid yellow fruits, followed by orange,dark coloration and finally red fruits. Red fruits are reported to have highertotal carotenoid levels, while yellow fruits show the lowest levels of lycopeneand ?-carotene (Namitha et al. 2011).

The same trend was observed in bothnative and hybrid genotypes, suggesting that SINAC1 is highly associated with coloration, although it respondsto several stress factors as well.SINAC4 has been reported to play animportant role in response to various abiotic stresses, including wounding,NaCl and dehydration. However, it also plays an important role in carotenoidaccumulation during tomato fruit ripening, acting as a positive regulator bymodulating the hormone ethylene and therefore carotenoid pigmentation. Zhu et al.

(2014) report that RNAi fruits displayed orangecolor in both the pericarp and placenta, which implies decreased accumulationof lycopene and elevated ?-carotene. A reduced expression of the genes PSY1, and the chromoplast andchloroplast lycopene ?-cyclases (CYC-B and LCY-B) were upregulated compared withcontrols in SlNAC4 RNAi fruits. Inthis study, a higher expression of SlNAC4was found in yellow fruits, suggesting that SlNAC4might be associated with the yellow carotenoid lutein, which is regulatedmainly by LCY-B (Namitha etal. 2011).We suggest that the expression of NAC family genes was higher in nativegenotypes due to their exposure to open-air conditions promoting thedevelopment of defense mechanisms that increase plant survival.

The expressionof SlNAC1 and SINAC4 was associated with fruit pigmentation, since both hybridand native yellow fruits exhibited lower expression levels. This reportprovides new insights into the expression of NAC family transcription factorsin hybrid and native tomato genotypes, which could contribute to theelucidation of their regulatory function.