NAC conditions promoting the development of defense mechanisms

NAC transcription factors are versatile
regulators of diverse developmental processes and have been associated with carotenoid
accumulation and plant abiotic defense responses, such as temperature stress
tolerance and drought. Tomato fruit (Solanum
lycopersicum L.) is a convenient 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
colors.

 

 

NAC genes
compose one of the largest families of plant-specific transcription factors and
play a crucial role in several developmental processes. The term NAC derives from
the names of three related transcription factors: NAM from Petunia, ATAF1-2 from Arabidopsis
thaliana and CUC2 from Arabidopsis
(Souer et al. 1996; Nuruzzaman et al. 2015). Since the expression and
activation of NAC family members are
crucial for plant survival, these genes have been studied in several plant species,
such as bean, rice, maize and tomato (Bhattacharjee
et al. 2016; Wu et al. 2016). There are over 151 NAC genes in rice, 117 in Arabidopsis
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, such as senescence, NAC proteins have been associated
with plant biotic and abiotic defense responses. Consequently, NAC proteins
have 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 tomato 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 furthermore
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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This study aimed 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 pigmentation.

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 and stored at -75°C.

 

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
the endurance of plants
under several conditions of environmental stress. Kou et al. (2014) analyzed the expression of NAC genes in multiple tissues of tomato
fruit, and found that the expression of SINAC5,
SINAC6, SlNAC7 and SlNAC9 increases
during 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 hormone or ethylene-stimulated
pathways (Kou et al. 2016). In agriculture, 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 a higher expression of SINAC1 decreases ethylene synthesis and
alters carotenoid pathway, leading to yellow and orange mature fruits with
lower levels of carotenoids. In the present study, a higher expression of SINAC1 was found 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 several abiotic stresses, including wounding,
NaCl and dehydration. However, it also plays an important role in carotenoid
accumulation during tomato fruit ripening, acting as a positively regulating the
hormone ethylene and therefore carotenoid pigmentation. Zhu et al. (2014) report that RNAi fruits showed orange color in both placenta
and pericarp, which implies accumulation of ?-carotene. A reduced expression of
the genes PSY1, and the chloroplast and
chromoplast lycopene ?-cyclases (LCY-B and CYC-B) was found compared with SlNAC4 RNAi fruits used as controls. 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.

 

 

 

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