Plant NAC transcription factors responsive to abiotic stresses

Accepted Manuscript Plant NAC transcription factors responsive to abiotic stresses

Deyvid N. Marques, Sávio P. dos Reis, Cláudia R.B. de Souza PII: DOI: Reference:

S2352-4073(17)30036-7 doi: 10.1016/j.plgene.2017.06.003 PLGENE 112

To appear in:

Plant Gene

Received date: Revised date: Accepted date:

9 January 2017 5 June 2017 8 June 2017

Please cite this article as: Deyvid N. Marques, Sávio P. dos Reis, Cláudia R.B. de Souza , Plant NAC transcription factors responsive to abiotic stresses, Plant Gene (2017), doi: 10.1016/j.plgene.2017.06.003

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ACCEPTED MANUSCRIPT Plant NAC transcription factors responsive to abiotic stresses Deyvid N Marques1, Sávio P dos Reis1,2, Cláudia RB de Souza1* 1

Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, PA, Brazil. 2Universidade do

Estado do Pará, Marabá, PA, Brazil. *

Corresponding author: Cláudia RB de Souza, Instituto de Ciências Biológicas, Universidade Federal

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do Pará, Belém, PA, 66075-110, Brazil. Tel: ++55 91 32057585; E-mail: [email protected]

Abstract

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Plants are subjected to a wide variety of abiotic and biotic stresses, which have been responsible for huge yield losses worldwide. Among abiotic stresses, drought, high

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salinity and extremes of temperature comprise some of the major factors reducing the agricultural production. On the other hand, plants have evolved several mechanisms of

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response and adaptation to adverse conditions. In order to overcome abiotic stress conditions, insights about physiological components that contribute to endogenous

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defense mechanisms of plants are extremely relevant. Genes coding for NAC transcription factors have been identified from various plant species. NAC proteins are related to several roles at cellular level in improvement of plants against abiotic stresses.

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This review aims to summarize current knowledge about the interaction of NAC

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proteins with cis-acting regulatory elements that function in abiotic stress-responsive gene expression, as well as the regulation of these proteins by plant hormones. Also, we present recent progress revealing the importance of differential expression of theses

plants.

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transcriptional regulators in tolerance against abiotic stresses in genetically transformed

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Keywords: Abiotic stress; Gene expression control; Plant breeding; Plant stress tolerance, Transcription factors.

ACCEPTED MANUSCRIPT Introduction Plants are responsible for significant part of food source worldwide, and through agriculture, they play an important socioeconomic role for world population. In this context, to ensure global food demand, an equivalent improvement of agricultural productivity is required. On the other hand, agricultural productivity can be significantly limited by several biotic and abiotic factors that affect growth and development of plants.

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A global yield reduction of over 50% in major crop plants is due to abiotic stresses (Li et al., 2017). Extremes of temperature and pH, drought and high salinity are

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major abiotic stresses for agriculture, since these adverse factors constitute suboptimal

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conditions that prevent plants from realizing their full genetic potential (Rockstrom and Falkenmak, 2000). As example, soil high salinity affects about 20% of arable land in the

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world and about 40% of irrigated land to various degrees (Sahi et al., 2006). In addition, climate change can gradually affect agriculture compromising the food security in the world (Nelson et al., 2014).

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Abiotic stresses can impair plant growth, productivity and yield, by causing various injuries at cellular level. For instance, salinity and drought (Marques et al.,

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2015), cadmium (Tripathi et al., 2012a), chromium (Tripathi et al., 2012b), lead (Singh et al., 2015), aluminium (Singh et al., 2017) and UV-B (Tripathi et al., 2017) stresses

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can induce damage; and temperature stress causes protein dysfunction (Sanghera et al., 2011; Hasanuzzaman et al., 2013). Damaged cell membrane integrity, restrained photosynthesis and dysfunctional metabolism can result from these cellular changes (Li

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et al., 2017). Because these stresses account for a great part of crop yield losses worldwide, understanding the endogenous molecular mechanisms of plants that act in

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response to abiotic stresses is very relevant, aiming, ultimately, to increase plant tolerance through genetic manipulation. In order to cope with these stresses, plants have developed refined biochemical, physiological and molecular mechanisms of response triggered by phytohormones action. Other signalling molecules (phosphatases and protein kinases), as well stressresponsive proteins (e.g. heat shock proteins, molecular chaperones, aquaporins and antioxidante enzymes) are important components of such mechanisms (Reis et al., 2012; Reis et al., 2016). This response is deeply modulated by regulation of gene expression, mediated by regulatory proteins called transcription factors, which activate or repress transcription of target genes. Transcription factors, such as MYC/MYB (Roy, 2016),

ACCEPTED MANUSCRIPT basic leucine zipper (bZIP) (Sornaraj et al., 2016) and WRKY (Phukan et al., 2016) proteins interact with specific and conserved DNA sequences (cis-acting regulatory elements) in promoters of abiotic stress-responsive genes, regulating their expression at transcriptional level. Proteins of plant-specific NAC domain family are among the transcriptional regulators related to plant strategies under conditions of abiotic stresses (Xu et al., 2014; Shao et al., 2015) and several recent studies have ratified the importance of these

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transcription factors in generating tolerant plants (Gunapati et al., 2016; Hong et al., 2016; Reis et al., 2016; Tak et al., 2016). The identification of genes encoding NAC

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proteins with differential expression in response to abiotic stress contributes to

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understanding their roles in mechanisms of plant response and defense; furthermore, they can be used as candidate genes in the production of transgenic plants tolerant to

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abiotic stress.

Therefore, prospecting of NAC genes related to abiotic stress response in plants involves several steps, including differential gene expression studies, isolation and

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molecular cloning of NAC genes, as well as their functional analysis by genetically transformed plants (Fig. 1). In this review, we have focused on relationship of NAC

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transcription factors in plant response to major abiotic stresses, such as drought, high

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salinity and temperature extremes.

NAC transcription factors

The NAC acronym originated from the detection of a conserved domain present

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in petunia NAM (no apical meristem) and Arabidopsis ATAFI, ATAF2 (Arabidopsis transcription activation factor) and CUC2 (cup-shaped cotyledon) proteins, when a

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CUC2 DNA sequence was used to search sequences significantly homologous to region encoding the N-terminal part of CUC2 protein (Aida et al., 1997). According to PereiraSantana et al. (2015), expansion of NAC family proteins have occurred shortly before the origin and rapid radiation of flowering plants. The family-defining conserved domain named NAC (approximately 150 amino acids in the N-terminal region) is divided into five subdomains. It is responsible for DNA binding in cell nucleus and dimer formation; whereas the diverse C-terminal region is related to transcriptional regulation (Ooka et al., 2003; Olsen et al., 2005). Recent studies have confirmed the subcellular localization of NAC proteins in nucleus important for tolerance of plants to abiotic stresses (Lv et al., 2016a; Yu et al., 2016;

ACCEPTED MANUSCRIPT Zhao et al., 2016; Zhu et al., 2016). NAC genes are expressed in various developmental stages and tissues (Ooka et al., 2003). They have been isolated in Arabidopsis thaliana (Liu et al., 2016) and crop plants, such as banana (Tak et al., 2016), canola (Wang et al., 2015), soybean (Pimenta et al., 2016), chickpea (Yu et al., 2016), maize (Zhu et al., 2016), rice (Huang et al., 2016), wheat (Huang et al., 2015); common bean (Wu et al., 2016a), tomato (Li et al., 2016) and sweet potato (Chen et al., 2016). Several NAC genes were identified in plant kingdom. There are more than 100 genes of this family

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identified in Arabidopsis, rice and soybean (Ooka et al., 2003; Fang et al., 2008; Pinheiro et al., 2009).

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Some NAC proteins are anchored in plasma membrane or in endoplasmic

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reticulum membrane by the presence of an α-helical transmembrane (TM) motif Cterminal, besides conserved N-terminal NAC domain and the diversified middle

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transcription regulatory region (Liang et al., 2015a). Such proteins are merged into transmembrane NAC protein subfamily named NTL (from NTM1-Like) and are transported to nucleus in response to abiotic stresses after proteolytic cleavage (Seo et

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al., 2008). For instance, NTL6 is proteolytically activated by abscisic acid (ABA) and cold (Seo and Park, 2010) and its processing occurs via the regulated intramembrane

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proteolysis mechanism in response to cold (Seo et al., 2010). TM motif is involved at processing step of NTLs and severe phenotypes in plants overexpressing NTL genes are

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typically generated by TM-deleted form production (Tran et al., 2010). Protein activity encoded by members of this large gene family is related to several processes, such as senescence (Christiansen et al., 2016), secondary walls

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formation (Nakano et al., 2015), cell division (Kim et al., 2006), flowering (Ning et al., 2015) and both biotic and abiotic stresses (He et al., 2016; Seo and Park, 2010; Wang et

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al., 2016). However, complete role of NAC proteins in response to abiotic stresses remains to be elucidated.

Relationship of NAC proteins with plant hormones in response to abiotic stresses Transcriptional regulation of NAC genes is related to the presence of stress response regulatory elements in their promoter region, such as DREs (Dehydrationresponsive elements), ABREs (ABA-responsive elements), jasmonic acid responsive element, salicylic acid responsive element, LTREs (Low-temperature responsive elements), MYB (Myeloblastosis) and MYC (Myelocytomatosis) binding sites

ACCEPTED MANUSCRIPT (Nakashima et al., 2012). At post-transcriptional level, in addition to alternative splicing, members of miR164 family regulate the translation of mRNAs encoding NAC proteins, modulating their response to various developmental processes (Mallory et al., 2004), including abiotic stresses (Fang et al., 2014). After NAC protein synthesis, it can be post-translationally regulated and act by performing its activity at cellular level. This regulation may involve ubiquitination (Huang et al., 2012, Miao et al., 2016), phosphorylation for subcellular localization (Kleinow et al., 2009) or phosphorylation

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for activation in response to abiotic stresses (Zhu et al., 2016), as well as dimerization (Welner et al., 2012; Mathew et al., 2016) and interaction with other proteins related to

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defense response (Xu et al., 2013).

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Phytohormones often act as endogenous triggers or intermediates in cascade of plant cell signaling in response to a particular environmental stimulus, such as abiotic

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stresses. Several studies have proposed that NAC proteins action in response to abiotic stresses is downstream action of various plant hormones directly related to response, such as ABA (Mao et al., 2016) and ethylene (He et al., 2005). ABA acts as a central

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regulator in plant response against abiotic stresses, coordinating an array of functions (Sah et al., 2016). Whereas some NAC genes are shown to be ABA responsive (Liu et

2014; Fang et al., 2015).

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al., 2016; Mao et al., 2016), others are independent of this stress hormone (You et al.,

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In addition to ABA-induced NAC genes, some proteins encoded by NAC genes also act in upregulation of genes related to ABA biosynthesis (Hong et al., 2016; Mao et al., 2016). Jensen et al. (2013) verified that ATAF1, an Arabidopsis thaliana NAC

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transcription factor, directly regulates NCED3 gene by binding to its promoter, correlated with increased NCED3 expression and ABA hormone levels. Overexpression

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of several NAC genes were reported to generate transgenic plants more sensitive to exogenous ABA (Hong et al., 2016; Mao et al., 2016). NAC proteins are also related to direct or indirect transcriptional regulation of ABA-induced genes encoding functional proteins. Relationship of ABA-regulated gene expression control mediated by NAC was verified by NAC suppression by signaling repressor proteins of drought tolerance mediated by ABA (Kim et al., 2009). Relationship of NAC transcription factors with other important hormones to better adaptation under abiotic stress conditions was also reported. Overexpression of GmNAC20 is related to tolerance of transgenic Arabidopsis plants against salt and freezing, as well as promotion of root formation by altering auxin signaling-related

ACCEPTED MANUSCRIPT genes (Hao et al., 2011). Wang et al. (2016) observed the root growth and development in tobacco transgenic lines overexpressing SINAC35 from tomato along with improved tolerance to drought and salt stress, possibly by regulating NtARF1, NtARF2 and NtARF8 expression, which encode for response factors of auxin signaling. ShahnejatBushehri et al. (2016) verified that Arabidopsis NAC transcription factor JUB1 acts in reducing levels of gibberellins (GAs) and brassinosteroids (BRs), by directly repressing the GA3ox1 and DWARF4 biosynthesis genes, leading to typical GA/BR deficiency

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phenotypes. Also, JUB1 activates the DELLA genes, coding for conserved repressors of GA signaling, and culminating in cell elongation restriction while concomitantly

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enhancing stress tolerance related to reactive oxygen species (ROS) scavenging.

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The role of NAC proteins was also verified regarding antagonistic and synergistic action, which is typical of plant hormones for the maintenance of cellular

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homeostasis. For instance, in transgenic Arabidopsis plants overexpressing GhNAC2 the activation of the ABA/ jasmonic acid (JA) pathways and a suppression of ethylene pathway

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verified,

reducing

expression

of

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ERF6/ERF1/WRKY33/MPK3/MKK9/ACS6 and their targets, related to transpiration reduction and stomatal opening optimization (Gunapati et al., 2016). Ethylene and auxin

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signaling pathways are required for salt response of AtNAC2, but such response does not require ABI2, ABI3 and ABI4, intermediates of the ABA signaling pathway (He et

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al., 2005). In other example of relationship of NAC and JA, the expression of VaNAC26 from Vitis amurensis in transgenic Arabidopsis plants enhanced drought and salt

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tolerance by modulating JA synthesis (Fang et al., 2016).

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Plant tolerance against abiotic stress mediated by NACs

Analysis of gene expression encoding proteins, both at transcription and translation level, is essential to carry out an initial verification of relationship of a gene product with a given response at cellular level. Thus, the monitoring of gene expression patterns allows predicting the relationship of certain gene products to plant response to abiotic stresses. Several studies have reported changes in plant resistance under some kinds of environmental stresses when there is altered expression of NAC genes (Zhu et al., 2015; Lv et al., 2016b). For instance, in two citrus species, using quantitative RTPCR, it was reported that NAC1 was up-regulated by different abiotic stresses: in Citrus limonia by drought stress in leaves, while in Citrus reshni by drought, salt and cold

ACCEPTED MANUSCRIPT stresses in leaves and roots (Oliveira et al., 2011). In Arabidopsis, ANAC092 was upregulated and increased expression of 24 genes associated with salt stress (Balazadeh et al., 2010). Other authors have also reported differential expression of NAC genes under salinity, drought (Rahman et al., 2016; Tak et al., 2016) and temperature stress (Fang et al., 2015; Guo et al., 2015). After nuclear import, NAC proteins can bind to promoters of genes responsive to abiotic stresses and act in their expression regulation (Puranik et al., 2012). Thus, the

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action of NAC transcription factors involves its interaction with regulatory elements that were identified in genes mediating the NAC-response downstream, such as NAC

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recognition site, NACRS, consensus CGT(G/A) and CDBS (core DNA-binding

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sequence, CACG) in promoter region of ERD1 (EARLY RESPONSIVE TO DEHYDRATION 1) gene from Arabidopsis (Tran et al., 2004). However, different

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NAC proteins can bind to different elements, according to NAC protein sequence specificity (Hao et al., 2011). For instance, transcriptional repressor calmodulinbinding NAC of Arabidopsis does not bind to CGT[G/A], but interacts with GCTT

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sequence (Kim et al., 2007). Table 1 shows some genes directly regulated by NACs. In this section, informations obtained from in vivo studies of transgenic plants

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are presented. In addition to the differential expression studies, they contribute to confirm importance of NAC transcription factors in response to major abiotic stresses

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after their interaction with regulatory elements of responsive genes. Also, protein roles

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in tolerance of multiple stresses are often reported (Table 2).

Drought tolerance

Drought tolerance generated by altered expression of NAC family members is

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related to upregulation of genes encoding functional proteins, such as LEA (Hong et al., 2016; Tak et al., 2016), RAB (Hong et al., 2016), P5CS (Hong et al., 2016), ERD10 (Yu et al., 2016), RD29A (Yu et al., 2016; Zhao et al., 2016), COR (Yu et al., 2016; Zhao et al., 2016), KIN1 (Yu et al., 2016); upregulation of genes coding for transcription factors related to abiotic stress response, such as bZIP (Hong et al., 2016), MYB (Hong et al., 2016), DREB (Hong et al., 2016; Yu et al., 2016; Zhao et al., 2016) and WRKY (Tak et al., 2016); downregulation of genes responsive to hydrogen peroxide, oxidative stress and secondary metabolic process (Mao et al., 2016).

ACCEPTED MANUSCRIPT Protein-protein interactions are also important for functions of NAC family proteins. For instance, Arabidopsis ANAC096 interacts with ABF2 (bZIP-type TF family) and cooperate synergistically for full activation of RD29A, which is activated transcriptionally under dehydration conditions (Xu et al., 2013). Zhu et al. (2016) verified that overexpression of maize ZmNAC84 generated tolerance of tobacco plants to drought and ZmCCaMK phosphorylates this protein through a physical interaction between the two proteins. Such interaction is essential for ABA-induced defense and

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related to expression activation of downstream genes related to antioxidant defense (Zhu et al., 2016).

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Activation of molecular mechanisms by NAC proteins is related to important

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traits of plants related to tolerance against drought, such as seed-setting rate (Hu et al., 2006) and reduced percentage of open stomata (Hong et al., 2016). Hu et al. (2006)

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observed SNAC1-overexpressing plants present acquired enhanced dehydration tolerance and genes encoding proteins related to mechanisms of drought tolerance (osmotic adjustment, cell membrane stability, important macromolecules protection

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from degradation and maintenance of redoxin homeostasis and detoxification) were upregulated in transgenic rice plants, besides SNAC1 induction was reported

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predominantly in guard cells under drought. Xu et al. (2013) reported the cooperation between ANAC096, a NAC protein, and two ABRE transcription factors. Together,

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they increase Arabidopsis tolerance to in vivo drought stress. Thus, many studies in transformed plants have allowed verifying the importance of NAC family members to contribute to several aspects at downstream molecular level to NACs related to drought

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plants tolerance.

Salt tolerance

Several authors have observed that NAC proteins improves tolerance to both salinity and drought and verified increased expression of genes related to tolerance to such stresses (Gunapati et al., 2016; Hong et al., 2016; Tak et al., 2016; Wang et al., 2016; Yu et al., 2016). In studies demonstrating the positive role of NAC proteins to salt tolerance, upregulation of stress-associated genes, such as ERD1 genes (Hu et al., 2006), genes coding to MYB, AP2/ERF and zinc finger proteins (Nakashima et al., 2007), OsLEA3, OsSalT1 and OsPM1 genes (Liu et al., 2016) from rice; AtMYB2 (Han et al., 2015) and genes related to DREB/CBF–COR pathway (Hao et al., 2011) in Arabidopsis were observed. Also, several authors have reported that increased

ACCEPTED MANUSCRIPT expression of NAC genes culminated in increased proline content (related to cell osmotic adjustment against osmotic stress) and reduction of malondialdehyde content (an indicator of membrane damage), with concomitant generation of tolerance against drought and salinity (Liu et al., 2014; Tak et al., 2016; Zhao et al., 2016). Takasaki et al. (2010) overexpressed OsNAC5 in rice and detected increased salinity tolerance as well as upregulation of OsLEA3 gene in transgenic plants and binding of OsNAC5 to OsLEA3 promoter by electrophoretic mobility shift assay. OsLEA3 protein is involved

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in cell structure maintenance in response to salt stress (Chourey et al., 2003). Thus, NAC proteins can also interact directly with promoter region of genes encoding proteins

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related to salt tolerance.

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Different NAC proteins present different functions in plant tolerance against salinity. PeNAC1 overexpression improved tolerance in Arabidopsis to salt stress

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through regulating Na+/K+ homeostasis. This homeostasis is related to AtHKT1 expression regulation by PeNAC1, which could culminate in control of Na+ distribution in different organs of Arabidopsis. The result is root Na+ accumulation, which protects

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the photosynthetic organs from Na+ toxicity (Wang et al., 2013). He et al. (2016) observed the increased tolerance in cotton plants generated by GhATAF1

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overexpression that was related to expression enhancing of ABA response gene GhABI4. Similar results were observed for GhHKT1 transporter gene, involved in

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Na+/K+ homeostasis, as well as for GhAVP1, GhRD22, GhDREB2A, GhLEA3, and GhLEA6, which are stress-related genes. The OsNAC5 silencing in rice plants by RNA interference enhanced

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malondialdehyde and H2O2 accumulation and reduced expression of genes encoding tonoplast Na+/H+ antiporter under both control and salt-stressed conditions (Song et al.,

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2011). On the other hand, Han et al. (2015) detected that genes involved in balancing the ion homeostasis, such as HKT, NHX, SOS1, and SOS2, did not show significant expression changes in Arabidopsis plants overexpressing CiNAC3 and CiNAC4, although the transgenic plants presented enhanced salt tolerance. In rice, a study showed NAC52, when expressed, up-regulate other genes related to salt stress tolerance (Gao et al., 2010).

Temperature stress tolerance NAC role in plant tolerance against heat and cold was also demonstrated. Shahnejat-Bushehri et al. (2012) reported a NAC transcription factor from Arabidopsis

ACCEPTED MANUSCRIPT with thermomemory-related expression that confered heat tolerance on transformed plants. Hu et al. (2008) observed that the transgenic plants overexpressing SNAC2 had higher cell membrane stability than wild type during cold stress. Fang et al. (2015) verified that SNAC3 overexpression led to upregulation of many ROS-associated genes and SNAC3 suppression caused reduced expression of these genes. These authors proposed SNAC3 is a positive NAC regulator of heat resistance through controlling downstream genes expression involved in ROS pathway. Wheat TaNAC2L

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overexpression in transgenic Arabidopsis significantly increased expression of thermotolerance related genes, such as coding for LEA, HSF, RD29A, RD17 and

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DREB2A proteins, in comparison to the wild type (Guo et al., 2015). On the other hand,

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SlNAC1 suppression in tomato plants reduced heat resistance, besides increasing of superoxide dismutase activities and lower accumulation of transcripts of heat-shock

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protein genes (Liang et al., 2015b).

In plants whose NAC protein overexpression generated cold tolerance, it has also been verified upregulation of other thermotolerance-related genes, such as

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COR15A, ERD11, RAB18, DREB1A, ERF5, RD29A (Yang et al., 2015); RD29A/cor78 (Hao et al., 2011); OsTPP1 and OsTPP2 (Song et al., 2011) and HSFA2, HSP18.2,

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HSP21 (Shahnejat-Bushehri et al., 2012); as well as the increased activity of antioxidant enzymes, such as superoxide dismutase and glutathione reductase (Grover et al., 2014).

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In addition, upon exposure to cold, Song et al. (2011) observed increases in proline and soluble sugars contents in transgenic rice plants overexpressing OsNAC5, relatively unchanged Mayetiola destructor susceptibilly (MDS) product amounts in such plants, as

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well as lowest increase in H2O2 content in response to cold, drought and salt stress. SlNAM1 overexpression (a typical NAC gene isolated from tomato) improved

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osmolytes contents and reduced H2O2 and O2•− contents under low temperature, which is associated with alleviating the oxidative damage of cell membrane after chilling stress (Li et al., 2016). Taken together, NAC proteins function in plant strategies to temperature variations.

NAC and senescence The protective role against drought and saline stresses gives NAC factors other importance: senescence control. Several senescence-associated genes are up-regulated

ACCEPTED MANUSCRIPT by NAC factors. In Arabidopsis, Hickman et al. (2013) reported a network between three NAC proteins, showing different roles for each one in environmental stresses and leaf senescence, all related to increased tolerance. Wu et al. (2012) identified a hydrogen peroxide-induced NAC, called JUNGBRUNNEN1 (JUB1), as the main regulating molecule of longevity in Arabidopsis thaliana. When expressed, this protein delays senescence scavenging hydrogen peroxide and at the same time plants improve their resistance to several abiotic stresses. In Medicago trucatula NAC969 expression

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induced by nitrate treatment helps root development adapted to salinity high levels and delays nodule senescence (Zélicourt et al., 2012).

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On the other hand, Lee et al. (2012) proposed that the physiological role of

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NTL4 transcription factor is to promote ROS production in leaves, which triggers programmed cell death to induce leaf senescence under drought conditions. This

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response culminates in remobilizing nutrients and metabolites by plants from senescing leaves to sink organs and newly formed leaves, contributing to the water loss

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minimization from leaves through transpiration.

Different NAC proteins, different effects in abiotic stress tolerance

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Some NAC proteins show function divergence in different species of plants, as well as different NAC proteins can exert distinct effects in same species. For instance,

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Lu et al. (2007) verified Arabidopsis mutants with ATAF1 knocked out presented a recovery rate significantly higher than wild-type plants under drought stress. These authors also proposed that ATAF1 negatively regulates expression of genes responsive

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to drought stress. On the other hand, Wu et al. (2009) observed Arabidopsis plants overexpressing ATAF1 present increased drought tolerance, besides increased

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sensitivity to NaCl and ABA. Liu et al. (2016) detected that the ATAF1 overexpression did not increase rice tolerance against drought, but generated marked tolerance to salt and insensitivity to ABA. Regarding NACs from rice, OsNAC5 and OsNAC6 bind to OsLEA3 promoter, whereas OsNAC6 overexpression generated growth retardation of transgenic plants, transgenic plants overexpressing OsNAC5 showed improved tolerance to high salinity (Takasaki et al., 2010). GmNAC11 and GmNAC20 are NACs from soybean that interact with the core DNA sequence CGT[G/A] and act as transcriptional activator and mild repressor, respectively, although C-terminal end of GmANC20 has transcriptional activation activity. GmNAC20 is involved in tolerance against salt and freezing in

ACCEPTED MANUSCRIPT plants transgenics, whereas GmNAC11 is involved only in salt tolerance (Hao et al., 2011). In some NAC proteins, the presence of a transcriptional repression domain in D subdomain of NAC domains was detected (Hao et al., 2010; Wu et al., 2016b). Such repression activity has already been related to negative effects on tolerance to abiotic stresses. For instance, it was demonstrated PtrNAC72 from Poncirus trifoliata specifically binds to PtADC promoter (coding for an arginine decarboxylase), and

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represses its expression. Tobacco plants overexpressing PtrNAC72 were more sensitive to drought, with concomitant accumulation of ROS. The modulation of putrescine-

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associated ROS homeostasis is involved with the negative regulation of drought stress

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response by PtrNAC72 (Wu et al., 2016b).

ONAC095 acts as a positive regulator of cold response, but as a negative

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regulator of drought response in rice (Huang et al., 2016). Jin et al. (2013) found GmNAC2 overexpression in tobacco generated hypersensitive to drought, high salinity and cold stress. Transgenic leaves also had highest level of malondialdehyde, possibly

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related to cell membrane degradation or dysfunction. Also, downregulated genes downstream to GmNAC2 related to ROS scavenging were identified (Jin et al., 2013).

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Rice plants in which the expression of a NAC-like transcription factor gene was

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abolished by RNA interference gain tolerance to boron toxicity (Ochiai et al., 2011).

Conclusions and perspectives

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Efforts of scientific research to unveil the role of NAC transcription factors have demonstrated an evident participation of these transcriptional regulators as important

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integrants of endogenous defense mechanisms of plants in response to abiotic stresses. Future studies focused on identifying genetic components downstream (such as genes directly or indireclty regulated) or upstream (such as signaling molecules) of NACs that remain to be identified will contribute to the supply of considerable understanding about participation of them in signal transduction pathways triggered by different abiotic stresses. Also, further studies will allow better insights into relationship of NACs with morphological, physiological, biochemical, and molecular changes of plants that culminate in their tolerance to abiotic stresses at field level.

ACCEPTED MANUSCRIPT Acknowledgements The authors thank to: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Amazônia de Amparo a Estudos e Pesquisas do Pará (FAPESPA), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Universidade

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do Estado do Pará (UEPA), and Universidade Federal do Pará (UFPA), Brazil.

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Gao, F., Xiong, A., Peng, R., Jin, X., Xu, J., Chen, J., Yao, Q., Jin, X., 2010. OsNAC52, a rice NAC transcription factor, potentially responds to ABA and confers drought tolerance in transgenic plants. PCTOC. 100, 255–262.

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Overexpression of NAC gene from Lepidium latifolium enhances biomass, shortens life cycle and induces cold stress tolerance in tobacco: potential for engineering fourth generation biofuel crops. Mol. Biol. Rep. 11, 7479–7489.

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Gunapati, S., Naresh, R., Ranjan, S., Nigam, D., Hans, A., Verma, P.C., Gadre, R., Pathre, U.V., Sane, A.P., Sane, V.A., 2016. Expression of GhNAC2 from G.

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herbaceum, improves root growth and imparts tolerance to drought in transgenic cotton and Arabidopsis. Sci. Rep. 6, 24978. Guo, W., Zhang, J., Zhang, N., Xin, M., Peng, H., Hu, Z., Ni, Z., Du, J., 2015. The wheat NAC transcription factor TaNAC2L is regulated at the transcriptional and post-translational levels and promotes heat stress tolerance in transgenic Arabidopsis. PLoS ONE 10, e0135667. Han, X., Feng, Z., Xing, D., Yang, Q., Wang, R., Qi, L., Li, G., 2015. Two NAC transcription factors from Caragana intermedia altered salt tolerance of the transgenic Arabidopsis. BMC Plant Biol. 15, 208.

ACCEPTED MANUSCRIPT Hao, Y.J., Song, Q.X., Chen, H.W., Zou, H.F., Wei, W., Kang, X.S., Ma, B., Zhang, W.K., Zhang, J.S., Chen, S.Y., 2010. Plant NAC-type transcription factor proteins contain a NARD domain for repression of transcriptional activation. Planta 232, 1033–1043. Hao, Y., Wei, W., Song, Q., Chen, H., Zhang, Y., Wang, F., Zou, H., Lei, G., Tian, A., Zhang, W., Ma, B., Zhang, J., Chen, S., 2011. Soybean NAC transcription factors promote abiotic stress tolerance and lateral root formation in transgenic plants. Plant

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factor downstream of ethylene and auxin signaling pathways, is involved in salt stress response and lateral root development. Plant J. 44, 903-916. He, X., Zhu, L., Xu, L., Guo, W., Zhang, X., 2016. GhATAF1, a NAC transcription

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Hénanff, G.L., Profizi, C., Courteaux, B., Rabenoelina, F., Gérard, C., Clément, C., Baillieul, F., Cordelier, S., Dhondt-Cordelier, S., 2013. Grapevine NAC1

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around three NAC transcription factors in stress responses and senescence in Arabidopsis leaves. Plant J. 75, 26–39. Hong, Y., Zhang, H., Li, D., Song, F., 2016. Overexpression of a stress-responsive NAC transcription factor gene ONAC022 improves drought and salt tolerance in rice. Front. Plant Sci. 7, 4. http://dx.doi.org/10.3389/fpls.2016.00004. Hu, H., Dai, M., Yao, J., Xiao, B., Li, X., Zhang, Q., Xiong, L., 2006. Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice. Proc. Natl. Acad. Sci. U. S. A. 103, 12987-12992.

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TaNAC29, a NAC transcription factor from wheat, enhances salt and drought tolerance in transgenic Arabidopsis. BMC Plant Biol. 15, 268.

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ONAC095 plays opposite roles in drought and cold stress tolerance. BMC Plant Biol. 16, 203.

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Jin, H., Huang, F., Cheng, H., Song, H., Yu, D., 2013. Overexpression of the GmNAC2 gene, an NAC transcription factor, reduces abiotic stress tolerance in tobacco. Plant Mol. Bio. Rep. 31, 435-442. Kim, Y.S., Kim, S.G., Park, J.E., Park, H.Y., Lim, M.H., Chua, N.H., Park, C.M., 2006. A membrane-bound NAC transcription factor regulates cell division in Arabidopsis. Plant Cell, 18, 3132-3144. Kim, H.S., Park, B.O., Yoo, J.H., Jung, M.S., Lee, S.M., Han, H.J., Kim, K.E., Kim, S.H., Lim, C.O., Yun, D.J., Lee, S.Y., Chung, W.S., 2007. Identification of a calmodulin-binding NAC protein as a transcriptional repressor in Arabidopsis. J. Biol. Chem. 282, 36292–36302.

ACCEPTED MANUSCRIPT Kim, M.J., Shin, R., Schactman, D.P., 2009. A nuclear factor regulates abscisic acid responses in Arabidopsis. Plant Physiol. 151, 1433-1445. Kim, Y.S., Sakuraba, Y., Han, S.H., Yoo, S.C., Paek, N.C., 2013. Mutation of the Arabidopsis NAC016 transcription factor delays leaf senescence. Plant Cell Physiol. 54, 1660–1672. Kleinow, T., Himbert, S., Krenz, B., Jeske, H., Koncz, C. 2009. NAC domain transcription factor ATAF1 interacts with SNF1-related kinases and silencing of its

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ACCEPTED MANUSCRIPT Liu, G., Li, X., Jin, S., Liu, X., Zhu, L., Nie, Y., Zhang, X., 2014. Overexpression of rice NAC gene SNAC1 improves drought and salt tolerance by enhancing root development and reducing transpiration rate in transgenic cotton. PLoS ONE 9, e86895. Liu, Y., Sun, J., Wu, Y., 2016. Arabidopsis ATAF1 enhances the tolerance to salt stress and ABA in transgenic rice. J. Plant Res. 129, 955-962. Lu, P.L., Chen, N.Z., An, R., Su, Z., Qi, B.S., Ren, F., Chen, J., Wang, X.C., 2007. A

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novel drought-inducible gene, ATAF1, encodes a NAC family protein that negatively regulates the expression of stress-responsive genes in Arabidopsis. Plant

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ACCEPTED MANUSCRIPT Table 1. Examples of NAC transcription factors and genes responsive to abiotic stresses directly regulated by NACs in vitro or in vivo.

Genes responsive to abiotic stresses directly regulated by NAC

ANAC019, ANAC055, ANAC072

ERD1

RD26

Methods

References

Transactivation experiments with Arabidopsis protoplasts Tran et al. (2004) Transactivation experiments with Arabidopsis protoplasts

GLY

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NAC protein

COR15A, COR15B, RD29A, RD29B

Electrophoretic mobility shift assays

GmNAC11, GmNAC20

DREB1A

Arabidopsis protoplast transient expression system

SNAC1, SNAC2

OsERD1

Yeast one-hybrid assays

Transactivation experiments with rice protoplasts

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Gene coding for a DUF26-like protein containing a Ser/Thr protein kinase motif

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OsNAC6

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VNI2

OsLEA3

TaNAC69, ONAC131

Gene coding for class I chitinase, glyoxalase I family and ZIM family protein

Yang et al. (2011)

Hao et al. (2011)

Hu et al. (2006); Hu et al. (2008)

Nakashima et al. (2007)

Electrophoretic mobility shift assays

Takasaki et al. (2010)

DNA-binding activity assays according to Xue (2002); Electrophoretic mobility shift assays

Xue et al. (2011)

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OsNAC5

Fujita et al. (2004)

SNAC1

OsSRO1c; OsPP18

NCED3; ABCG40

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ATAF1

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NTL4

Atrboh

Yeast one-hybrid assays

Chromatin immunoprecipitation assays Chromatin immunoprecipitation assays

You et al. (2013); You et al. (2014)

Jensen et al. (2013); Garapati et al. (2015) Lee et al. (2012)

RhNAC2

RhEXPA4

Electrophoretic mobility shift assays

Dai et al. (2012)

IDEF2

OsYSL2

Electrophoretic mobility shift assays

Ogo et al. (2008)

ANAC096

RD29A

Electrophoretic mobility shift assays

Xu et al. (2013)

SNAC3

CATA, APX8, RbohF

Yeast one-hybrid assays

Fang et al. (2015)

NAC016

AREB1, NAP, ORS1

Chromatin immunoprecipitation assays; Yeast one-hybrid assays

Kim et al. (2013); Sakuraba et al. (2015)

OsNAC2

OsLEA3, OsSAPK1

Chromatin immunoprecipitation assays; Yeast one-hybrid assays

Shen et al. (2017)

ACCEPTED MANUSCRIPT Table 2. Examples of genes coding for NAC transcription factors and their action in abiotic stress tolerance. Improved physiological traits in response to abiotic stress

Species transformed

Some genes directly or indirectly regulated

ANAC096

Dehydration and osmotic stress tolerance, sensitivity to ABA, stomatal control

Arabidopsis thaliana

RD29A, RD29B, COR47

GhNAC2

Drought and salt tolerance, leaf abscission, root growth, stomatal control, transpiration rate

Arabidopsis thaliana, Gossypium hirsutum

ERF11, WRKY33

ONAC022

Drought and salt tolerance, contents of proline and soluble sugars, sensitivity to ABA, stomatal control, transpiration rate Drought tolerance, root growth, water loss rate

AaNAC1 ZmNAC55

Arabidopsis thaliana, Artemisia annua Arabidopsis thaliana

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Drought tolerance, stomatal control, water loss rate Drought and salt tolerance, revival ability, relative water content, root and shoot biomass Drought and salt tolerance, contents of proline and malondialdehyde, photosynthetic efficiency, relative water content Drought and salt tolerance, root growth, ROS accumulation in roots

Oryza sativa

EcNAC67

Oryza sativa

References

Xu et al. (2013)

Gunapati et al. (2016)

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Cold tolerance

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Oryza sativa

ONAC095

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NAC genes overexpressed or silenced

OsPP2C30/49/68, OsbZIP23, OsAP37

Huang et al. (2016)

OsNCEDs, OsPSY, OsRAB21, OsLEA3

Hong et al. (2016)

RAB18, RD29A, COR47 RD17, PP2CA, RAB18 -

Lv et al. (2016a) Mao et al. (2016) Rahman et al. (2016)

Musa acuminate

CBF/DREB, LEA, WRKY family

Nicotiana tabacum

NtARF1, NtARF2, NtARF8

Wang et al. (2016)

Drought and salt tolerance, contents Arabidopsis thaliana of proline and malondialdehyde, water loss rate Arabidopsis thaliana Cold, drought and salt tolerance, activities of antioxidant enzymes, content of malondialdehyde, root growth, seed germination rate, sensitivity to ABA Drought tolerance, oxidative damage, Nicotiana tabacum content of malondialdehyde, relative water content Drought and heat tolerance, content of Oryza sativa malondialdehyde, modulation of ROS homeostasis, relative electrolyte leakage

RD29A, ERD10, COR15A, COR47, KIN1, DREB2A COR47, DREB1A, NCED3, RD29A

Yu et al. (2016)

Drought and salt tolerance, oxidative and salt stress tolerance, contents of proline and malondialdehyde, root growth, sensitivity to ABA, seed-setting rate, stomatal control, transpiration rate Cold, drought and salt tolerance, plant cell membrane stabilities, water loss rate

Oryza sativa, Gossypium hirsutum

OsERD1; OsPP18;

VvNAC1

Osmotic, salt and cold stress tolerance

Arabidopsis thaliana

-

Hénanff et al. (2013)

CiNAC3, CiNAC4

Salt tolerance

Arabidopsis thaliana

AtMYB2

Han et al. (2015)

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NAC042

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SINAC35

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CarNAC4

MlNAC9

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ZmNAC84

SNAC1

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SNAC3

TaNAC2

GmNAC11, GmNAC20 DgNAC1

Salt and/or freezing tolerance, lateral root formation, root growth Salt tolerance

Arabidopsis thaliana, Glycine Max Nicotiana tabacum

Zhao et al. (2016)

-

Zhu et al. (2016)

APX3, APX8, PrxIIE2

Fang et al. (2015)

OsSRO1c

Arabidopsis thaliana

Tak et al. (2016)

DREB1A, DREB2A, ABI1, ABI2, ABI5

RD29A/cor78 -

Hu et al. (2006); Liu et al. (2014); You et al. (2013) You et al. (2014) Mao et al. (2012)

Hao et al. (2011) Liu et al. (2011a); Wang et al. (2017)

ACCEPTED MANUSCRIPT ATAF1

Drought and salt tolerance, germination rate, root growth, sensitivity to ABA

OsNAC6

Dehydration and salt tolerance

OsNAC5

Cold, drought and salt tolerance, contents of proline and soluble sugars, reduced accumulation of malondialdehyde and H2O2, Na+/K+ ratio, relative electrolyte leakage, seed germination,

Arabidopsis thaliana, Oryza sativa Oryza sativa Oryza sativa

OsLEA3, OsSalT1,OsPM1 MYB, AP2/ERF family OsLEA3, OsTPP1, OsTPP2

Wu et al. (2009); Liu et al. (2016) Nakashima et al. (2007) Takasaki et al. (2010); Song et al. (2011)

Improved physiological traits in response to abiotic stress

Salt tolerance, Na+/K+ ratio

Arabidopsis thaliana

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PeNAC1

Species transformed

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NAC genes overexpressed or silenced

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Table 2 | Continued References

AtHKT1

Wang et al. (2013)

AMY1

Yokotani et al. (2009)

Osmotic and salt stress tolerance

TaNAC2L

Heat tolerance via acquired thermotolerance

SNAC2

Cold and salt tolerance, membrane stability, sensitivity to ABA

Oryza sativa

Zinc finger, C2H2 type family

Hu et al. (2008)

MINAC5

Cold and drought tolerance, sensitivity to ABA

Arabidopsis thaliana

RD29, COR47, NCED3

Yang et al. (2015)

JUB1; ANAC042

Heat tolerance

Arabidopsis thaliana

HSFA2, HSP18.2, HSP21

ShahnejatBushehri et al. (2012)

VaNAC26

Drought and salt tolerance, activities of antioxidant enzymes

TaNAC47

MuNAC4

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Cold tolerance, enhanced biomass

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SINAM1

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ONAC063

LlaNAC

Arabidopsis thaliana

Some genes directly or indirectly regulated

Arabidopsis thaliana

Arabidopsis thaliana

Nicotiana tabacum

LEA, HSF family

Guo et al. (2015)

LTI30, COR15A

Fang et al. (2016)

-

Grover et al. (2014); Singh et al. (2016)

Chilling tolerance, higher germination rates, minor wilting, higher photosynthetic rates

Nicotiana tabacum

NtDREB1,NtP5S, NtERD10s

Li et al. (2016)

Drought, freezing and salt tolerance, sensitivity to ABA

Arabidopsis thaliana

AtRD29A, AtCOR47, AtGSTF6, AtP5CS1

Zhang et al. (2016)

Drought tolerance, proliferated lateral root growth, leaf relative water content, cell membrane stability, total chlorophyll content, contents of proline and soluble sugars

Arachis hypogaea

-

Pandurangaiah et al. (2014)

ACCEPTED MANUSCRIPT CarNAC3, CarNAC6

Drought and salt tolerance, osmotic adjustment, increased antioxidant enzyme activity

Populus deltoides×Populus euramericana

-

Movahedi et al. (2015)

TaNAC67

Drought, freezing and salt tolerance,

Arabidopsis thaliana

DREB2A, COR15, ABI1, ABI2

Mao et al. (2014)

-

Li et al. (2014)

Hsp70, Hsp90, sHsp17.4, sHsp17.6

Liang et al. (2015b)

strengthened cell membrane stability, retention of higher chlorophyll contents, Na+ efflux rates, improved photosynthetic potential, enhanced water retention capability Cold, drought and salt tolerance, higher germination ratio, lower root inhibition rate, sensitivity to ABA

Arabidopsis thaliana

SlNAC1(from Solanum lycopersicum)

Heat tolerance, photochemical efficiency of photosystem II, ion leakage, contents of malondialdehyde content and proline

Solanum lycopersicum

RhNAC3

Drought and dehydration tolerance, cell expansion

Arabidopsis thaliana, Rosa hybrid

RhNAC2

Drought and dehydration tolerance, cell expansion

Arabidopsis thaliana, Rosa hybrid

RhEXPA4

Dai et al. (2012)

TaNAC29

Dehydration and salt tolerance, ABAhypersensitive response, content of malondialdehyde, higher superoxide dismutase and catalase activities

Arabidopsis thaliana

RD29b

Huang et al. (2015)

ZmSNAC1

Dehydration tolerance, sensitivity to ABA

Arabidopsis thaliana

-

Lu et al. (2012)

TaNAC2a

Drought tolerance,

Nicotiana tabacum

-

Tang et al. (2012)

EcNAC1

Drought, osmotic and salt stress tolerance

Nicotiana tabacum

PP2C, ERD1

Ramegowda et al. (2012)

OsNAC10

Cold, drought and tolerance,

Oryza sativa

Improved physiological traits in response to abiotic stress

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NAC genes overexpressed or silenced

PP2C

Jiang et al. (2014)

P450, HAK5

Jeong et al. (2010)

Table 2 | Continued

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SlNAC1(from Suaeda liaotungensis)

Species transformed

Some genes directly or indirectly regulated

References

ANAC019, ANAC055, ANAC072

Drought tolerance

Arabidopsis thaliana

ERD1

Tran et al. (2004)

ONAC045

Drought and salt tolerance

Oryza sativa

OsLEA3-1 WPM-1

Zheng et al. (2009)

TaNAC69

Dehydration tolerance, root and shoot biomass

Triticum aestivum

AhNAC2

Drought and salt tolerance, sensitivity to ABA

Arabidopsis thaliana

RD29A, AtMYB2, AREB1

Liu et al. (2011b)

IDEF2

Iron homeostasis

Oryza sativa

OsYSL2

Ogo et al. (2008)

ZIM gene family

Xue et al. (2011)

ACCEPTED MANUSCRIPT

GhATAF1

Salt tolerance

Gossypium hirsutum

OsNAC52

Drought tolerance, sensitivity to ABA

Arabidopsis thaliana

GhABI4, GhHKT1

He et al. (2016)

RD29B, KIN1

Gao et al. (2010)

DgNAC1

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Wang et al. (2017)

ACCEPTED MANUSCRIPT

Highlights

NAC genes are differentially expressed under abiotic stress conditions.

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NAC proteins act on plant tolerance modulation against abiotic stresses.

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Several genes directly or indirectly regulated by NACs were identified.