NAC of fleshy fruit development and stress responses. To

NAC transcription factors are known
regulators of diverse developmental processes and have been associated with carotenoid
accumulation and plant abiotic defense responses, such as drought and
temperature stress tolerance. Tomato fruit (Solanum
lycopersicum L.) is an established model for the study of fleshy fruit
development and stress responses. To explore further the role of NAC, we
analyzed the expression of six NAC family transcription factors (SlNAC1, SlNAC4, SlNAC5, SlNAC6, SlNAC7
and SlNAC9) in four hybrid and four
native tomato fruit (Solanum lycopersicum
L.) genotypes of different colors. RT-qPCR analysis showed that native
genotypes, which have been exposed to open-air conditions promoting the
development of defense mechanisms that increase plant survival, had a higher
expression of all NAC genes when
compared to hybrid genotypes. The expression of SlNAC1 and SINAC4 was
associated with fruit pigmentation, since both hybrid and native yellow fruits
exhibited lower expression levels. This report provides new insights into the
expression of NAC family transcription factors in hybrid and native tomato
genotypes of different color.




NAC genes
constitute one of the largest families of plant-specific transcription factors
and influence a diverse set of developmental processes. The term NAC derives from
the names of three transcription factors which share the same DNA-binding
domain: NAM from Petunia, ATAF1-2
from Arabidopsis thaliana and CUC2 from
Arabidopsis (Souer et al. 1996;
Nuruzzaman et al. 2015). Since the activation and expression of NAC family members are crucial for plant
survival, 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 (Fang
et 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 regulation
of several developmental processes, including senescence, NAC proteins have
been associated with plant abiotic defense responses such as drought stress
tolerance mechanisms and biotic stress response and been used in crop plants to
improve tolerance to several types of stress by genetic engineering (Mao et al.
2014; Shao et al. 2015). The
tomato (Solanum lycopersicum L.) is
one of the most commonly consumed fleshy fruits in the world due to its
versatility of use (fresh or processed), characteristic flavor and nutritional
properties, being also considered a good model for the study of fleshy fruits (Sinesio et al. 2010). Native genotypes have been produced on a
small scale, under backyard open-air conditions. It is believed that long-term
exposure to continuous environmental stresses and pathogen infections promote
the development of defense mechanisms that increase plant survival, such as biotic and abiotic stress responses
(Bonilla-Barrientos et al. 2014). Although several NAC family transcription
factors have been characterized in tomato, only little information concerning
stress-related NAC genes is
available. It is suggested that NAC
genes are not only associated with stress responses, but also with fruit
development and carotenoid accumulation (Zhu et al. 2014).

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The aim of this study was to analyze
the expression of six NAC family transcription factors in eight hybrid and
native tomato (Solanum lycopersicum L.)
genotypes, in order to associate its expression with stress-response and

Four native and four hybrid tomato (Solanum lycopersicum L.) genotypes of
different colors (Table 1) were cultivated in volcanic sand substrate and a
Stainer 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). All
genotypes used in this study are part of the Mexican Network of Plant Genetic
Resources (REMEFI). Fruits were
cultivated simultaneously under temperature conditions within the range of
22-30 °C and a relative humidity of 75%.
Fruits were selected when ripe on the basis of a color chart
developed specifically for each genotype according to maximum color development and harvested free of mechanical damage and physical
defects, and transported to the Autonomous Metropolitan University-Iztapalapa.
These fruits (three replicates of 27 fruits for each genotype; 81 total) were
washed and deseeded. The whole fruit tissue was chopped, frozen in liquid nitrogen,
and stored at -75°C until use.





The RNA extraction protocol was
performed according to the technique reported by Chang et al. (1993). Six g of
tissue were crushed to a powder in liquid nitrogen. The purity and
concentration of the RNA were determined by spectrophotometry (absorbance
260/280 nm) (Nanodrop ND-1000), and the integrity was verified by a 1% w/v
agarose gel electrophoresis stained with ethidium bromide.

The identity of amplification
products was verified before performing the RT-qPCR reactions and the
amplification efficiency was corroborated to be between 95 and 100%. 2 µg of
RNA were treated with DNase I, RNase free 1 U/L (Thermo Fisher Scientific). The
transcripts of interest were amplified by RT-qPCR using the kit Express SYBR
GreenER qPCR Supermix (ThermoFisher Scientific, USA). 1 µl of each diluted
sample was loaded per tube, obtaining a final volume of 10.5 µl. The 18S rRNA
fragment was used as reference gene since it showed minimal variability in its
expression between genotypes. The following temperature program was used: RT
reaction 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 required
for all genes (Table 2).


The relative mRNA expression was
calculated by 2-??Ct method (Willems
et al. 2008; Villa-Hernández et al. 2013). A value of 1 was arbitrarily assigned
to the mRNA expression of the red native genotype, and the relative expression
of the other genotypes was calculated using this genotype as reference.

Statistical analyzes were carried out using
ANOVA + Tukey (significance level ? = 0.05) using Prism 7.0 (GraphPad Software
Inc. 2017). Three biological replicates were used for each experiment. The
artwork was created with SigmaPlot 11.0.


The expression of all SINAC family
factors was significantly higher in the native genotypes when compared to that
of the hybrid genotypes (Fig 1). While hybrid genotypes have been selected to
improve postharvest shelf-life, native genotypes have been exposed to open-air
conditions, promoting the development of defense mechanisms that increase plant
survival. Thus, native genotypes exhibit biotic and abiotic stress responses,
such as growing in soils with limited moisture availability and tolerance to
higher temperatures. These results suggest that in native genotypes NAC genes contribute to enhance
survivability of plants
under several conditions of environmental stress. Kou et al. (2014) analyzed the expression of NAC genes in multiple tissues during
development of tomato fruit, and found that the expression of SINAC5, SINAC6, SlNAC7 and SlNAC9 increases during tomato fruit
development. These authors additionally found that the expression of SlNAC4, SlNAC6 and SlNAC7 was higher at the pink and ripe stages, suggesting that
these genes participate in the same or similar regulatory networks in
ethylene-stimulated or hormone signaling pathways (Kou et al. 2016). In
agriculture, high or low temperature acts as a major negative factor limiting
crop production. In this regard, NAC
genes 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 of
stress, compared to hybrid genotypes that have been selected for higher yield
and pest resistance.

The difference between the
expression in native and hybrid genotypes was greater for SINAC5 and SINAC1. While
the expression of SINAC5 has been
found to be highly induced by NaCl stress (Zhu et al. 2014), SlNAC1 is the most studied NAC
transcription factor in tomato, and it is known to also play a crucial role in
salt stress tolerance (Golldack et al. 2011). Additionally, Ma et al. (2014)
report that the overexpression of SINAC1
resulted in reduced carotenoids by altering carotenoid pathway flux and
decreasing ethylene synthesis, leading to yellow and orange mature fruits. In
the 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 higher
total carotenoid levels, while yellow fruits show the lowest levels of lycopene
and ?-carotene (Namitha et al. 2011). The same trend was observed in both
native and hybrid genotypes, suggesting that SINAC1 is highly associated with coloration, although it responds
to several stress factors as well.

SINAC4 has been reported to play an
important role in response to various abiotic stresses, including wounding,
NaCl and dehydration. However, it also plays an important role in carotenoid
accumulation during tomato fruit ripening, acting as a positive regulator by
modulating the hormone ethylene and therefore carotenoid pigmentation. Zhu et al. (2014) report that RNAi fruits displayed orange
color in both the pericarp and placenta, which implies decreased accumulation
of lycopene and elevated ?-carotene. A reduced expression of the genes PSY1, and the chromoplast and
chloroplast lycopene ?-cyclases (CYC-B and LCY-B) were upregulated compared with
controls in SlNAC4 RNAi fruits. In
this study, a higher expression of SlNAC4
was found in yellow fruits, suggesting that SlNAC4
might be associated with the yellow carotenoid lutein, which is regulated
mainly by LCY-B (Namitha et
al. 2011).

We suggest that the expression of NAC family genes was higher in native
genotypes due to their exposure to open-air conditions promoting the
development of defense mechanisms that increase plant survival. The expression
of SlNAC1 and SINAC4 was associated with fruit pigmentation, since both hybrid
and native yellow fruits exhibited lower expression levels. This report
provides new insights into the expression of NAC family transcription factors
in hybrid and native tomato genotypes, which could contribute to the
elucidation of their regulatory function.