REVIEW

32 GM Crops Volume 1 Issue 1

GM Crops 1:1, 32-39; January/February 2010; 2010 Landes Bioscience
Introduction
Plants grow in a dynamic and uncontrolled environment that can
frequently impose constraints on growth and development, result-
ing in signicant losses to the yields of economically important
crops. Among the adverse abiotic factors commonly encountered
by plants are extreme temperature, water decit, high salinity and
submergence that affect plant growth and productivity. Water
is an increasingly limited resource, and water shortages limit
crop productivity in many parts of the world. By contrast, in the
humid regions of South and Southeast Asia millions of hectares
are technically suited for crop production but are left unculti-
vated or are grown with very low yields because of salinity. To
deal with scarce good quality water resources, agronomic research
activity has focused on the optimization of water use for irriga-
tion and on the use of alternative sources of waters. Breeders take
a relatively traditional approach. They grow and cross varieties,
*Correspondence to: Lam-Son Phan Tran; Email: tran@psc.riken.jp
Submitted: 08/27/09; Revised: 11/09/09; Accepted: 11/10/09
Previously published online:
www.landesbioscience.com/journals/gmcrops/article/10569
Abiotic stresses such as extreme temperature, drought, high
salinity, cold and waterlogging often result in signifcant losses
to the yields of economically important crops. Plants constantly
exposed to capricious conditions have adapted at the molecu-
lar, cellular, physiological and biochemical level, enabling them
to survive and cope with adverse environmental stresses. NAC
(NAM, ATAF and CUC) transcription factors (TFs), which con-
stitute one of the largest families of plant-specifc TFs, have
been reported to enhance tolerance against various stresses,
such as drought, high salinity and cold, in a number of plants. In
this review the NAC TF family will be described and the poten-
tial use of NAC TFs in development of improved stress tolerant
transgenic crops will be discussed.
then evaluate how the progenies vary in their ability to deal with
stresses. The best-adapted plants will then be selected for growing
in elds exposed to stresses. Biotechnologists, meanwhile, have
taken advantage of recent advances in functional genomics and
biotechnology to genetically engineer crops which can give better
yield than the unmodied ones in adverse conditions.
1-5
The genetic basis of stress tolerance is complex and not well
understood. In order to cope with stresses, which vary in timing
and severity from place to place, season to season, plants activate
a number of defense mechanisms that function to increase toler-
ance to the unfavorable conditions imposed by these stresses. This
inducible adaptation or acclimation process has evolved through-
out the plant kingdom and is critical for the survival of all plants.
Molecular and cellular responses to environmental stimuli have
been analyzed extensively at the biochemical, physiological, mor-
phological and metabolic levels. The early events of plant adapta-
tion to environmental stresses are the stress signal perception and
subsequent signal transduction through either ABA-dependent or
ABA-independent pathways, leading to the activation of various
physiological and metabolic responses. Furthermore, a large array
of genes are activated by stress conditions, meaning several pro-
teins are produced to join the pathways which subsequently lead
to synergistic enhancement of stress tolerance.
3-16
These genes are
classied into two groups: regulatory genes and functional genes.
The regulatory group includes genes encoding various transcrip-
tion factors (TFs), which can regulate various stress-inducible
genes cooperatively or separately, and may constitute gene net-
works. The functional group contains genes encoding metabolic
components such as sugar, sugar alcohols and amines, which
play an important role in stress tolerance. Gaining an under-
standing of the mechanisms that regulate the expression of these
genes is a fundamental issue in plant biology and will be neces-
sary for the genetic improvement of plants cultivated in extreme
environments.
3,4,14,17
Recent efforts have provided vital information for improving
stress tolerance of important crops by increasing root growth and
development as well as enhancing various physiological and met-
abolic responses through genetic engineering of transcriptional
Potential utilization of NAC transcription factors
to enhance abiotic stress tolerance in plants
by biotechnological approach
Lam-Son Phan Tran,
1,
* Rie Nishiyama,
1
Kazuko Yamaguchi-Shinozaki
2,3
and Kazuo Shinozaki
4
1
RIKEN Plant Science Center; Signaling Pathway Research Unit; 1-7-22; Suehiro-cho, Tsurumi, Yokohama, Kanagawa Japan;
2
Graduate School of Agricultural
and Life Science University of Tokyo; Tokyo, Japan;
3
Japan International Center of Agricultural Sciences; Tsukuba, Ibaraki, Japan;
4
RIKEN Plant Science Center;
Gene Discovery Research Group; 1-7-22, Suehiro-cho, Tsurumi; Yokohama, Kanagawa, Japan
Key words: abiotic stress, NAC, transcription factor, transgenic plant, biotechnology, arabidopsis, crops
Abbreviation: ABA, abscisic acid; TF, transcription factor; NAC, NAM, ATAF and CUC transcription factor;
TRR, transcriptional regulation region; TM, transmembrane
www.landesbioscience.com GM Crops 33
REVIEW REVIEW
TFs from both Arabidopsis and rice suggested that NAC family
proteins could be classied into two major groups and 18 sub-
groups by sequence similarity.
33
Interestingly, the NAC TF fam-
ily is widely distributed in plants, but so far has not been found in
other eukaryotes.
20
The NAC TFs are multi-functional proteins
and have been implicated in diverse processes such as abiotic and
biotic stress responses, lateral root and plant development, ow-
ering, secondary wall thickening, anther dehiscence, senescence
and seed quality.
21,23,35-49
Despite intensive studies on the NAC
genes, more coordinated efforts are still needed for comprehen-
sive understanding of this important plant-specic TF family.
The recent acceleration in functional studies of NAC TFs using
both gain- and loss-of-function approaches will rapidly improve
our understanding of the regulatory function of NAC members.
A greater understanding of how NAC TFs operate will be sub-
sequently translated into their potential applications to enhance
plant productivity and seed quality.
Expression of NAC Genes in Response to Abiotic
Stresses
The stress-responsive NAC genes exhibit a large diversity in
stress-induced expression, suggesting that they may participate
in the regulation of a wide spectrum of responses to different abi-
otic stresses. Additionally, stress responsive NAC TFs appeared
to contain a closely homologous NAC domain as indicated by
phylogenetic analyses.
24,27
Large-scale abiotic stress responsive
expression analysis of NAC genes in whole Arabidopsis seed-
lings by microarray analysis using the Agilent 22K Oligo DNA
Microarray revealed that out of 67 ANAC genes, whose deduced
amino acid sequences showed the closest homology to that of
stress inducible RD26/ANAC072, 12 ANAC genes are induced
by ABA and/or at least one type of abiotic stresses.
44
A microar-
ray analysis of the root transcriptome following NaCl exposure
using Array-Ready Oligo Set for Arabidopsis genome of 70-mer
oligonucleotide probes representing 23,686 Arabidopsis genes
(Version 1.0, Qiagen Operon) detected that 23 ANAC genes was
induced and seven genes was reduced, respectively, by a thresh-
old of two-fold.
50
Recently, tiling arrays have become one of the
most powerful tools in genome-wide investigations. Using the
tiling array, Matsui et al.
51
have carried out the whole-genome
expression proling studies in 3-week-old Arabidopsis seedlings
in responses to drought, cold, high-salinity and ABA treatment,
and they discovered that out of 108 ANAC genes, which have an
AGI code (TAIR6 gene model, ftp://ftp.arabidopsis.org/home/
tair/Genes/TAIR6_genome_release), 30 genes were upregu-
lated with the threshold of 1.8 by at least one of the treatments.
Interestingly, no downregulated NAC gene (threshold of 5/9) was
detected in their studies.
In crops, such as rice, Fang and coauthors
24
systematically
analyzed the NAC family and identied 140 putative ONAC
or ONAC-like TFs. Out of these 140 ONAC genes at least 21
genes were identied as drought and/or salt stress-induced and
ve genes as stress-suppressed in four-leaf stage rice seedlings
by microarray analysis using 70mer oligomer microarray, which
covers 36,926 unique genes or gene models.
52
Twenty ONAC
networks.
3-5,17,18
This type of biotechnological approach has
been shown more practical than conventional breeding methods
because stress tolerance is an inherently multigenic trait in nature.
Although the vast majority of stress responsive genes remain to
be identied, in the signal transduction networks involved in
the conversion of stress signal perception to stress-responsive
gene expression, various TFs and cis-acting elements contained
in stress-responsive promoters have been described. These TFs
and cis-motifs function not only as molecular switches for gene
expression, but also as terminal points of signal transduction
in the signaling processes. In plants, approximately 7% of the
genome encodes for putative TFs.
19
Typically, the TFs contain a
distinct type of DNA binding domain and transcriptional regu-
lation region (TRR), and are capable of activating or repressing
transcription of multiple target genes.
14,15,17,18
Individual members
of the same family often respond differently to various stress stim-
uli; on the other hand, some stress-responsive genes may share
the same TF through the conserved cis-acting element in the
promoters of respective target. Several major stress signal trans-
duction pathways, including the major NAC pathway, that are
active in response to abiotic stress have been identied and well
elucidated in Arabidopsis thaliana and rice (Oryza sativa).
14,17,18

Identication and molecular tailoring of novel TFs, such as NAC
TFs, have the potential to overcome a number of important limi-
tations involved in the generation of transgenic crop plants with
superior yield under stress conditions.
5,14,15
The NAC TF Family in Plants
Several members of the NAC family were initially described more
than a decade ago.
20
Since then a great number of NAC TFs have
been identied and functionally characterized in both model and
crop plants such as Arabidopsis, rice, soybean (Glycine max) and
wheat (T. turgidum ssp. durum).
17,20-23
Proper characterization of
particular TFs often requires a detailed study in the biological
context of a whole TF family since functional redundancy is a
common occurrence within TF families.
21,24-27
Furthermore, since
TFs control the expression of the genome, it is not possible to com-
pletely understand their function without performing detailed
functional studies at a genome-wide level.
28,29
In the past decade,
the availability of complete genome sequences and the develop-
ment of high-throughput experimental techniques have enabled
scientists to compile complementary information describing the
function and organization of TF regulatory systems in a number
of organisms. The identication, characterization and classica-
tion of TFs at the genome-wide level will provide an important
resource for researchers who are interested in studying the regula-
tion of gene expression and the functions of the genes. Complete
genomic sequences of several model plants and crops, including
Arabidopsis, rice and soybean are now available. Genome-wide
analyses of their genomes indicated that there are more than 60
reported families of TFs in these plants.
28,30-32
Comprehensive
analyses of the NAC TF family in several plants suggested that
there are at least 105 putative NAC TFs in Arabidopsis, 140 in
rice, 205 in soybean and 152 in tobacco (Nicotiana tabacum),
respectively.
24,32-34
Sequence analysis of NAC domains of NAC
34 GM Crops Volume 1 Issue 1
subdomains has at least two putative motif variants for different
subgroups of NAC members. Furthermore, out of these 15 types,
ve types (J, K, L, M and N) do not have all ve subdomains in
the DNA-binding domain. The sequences of the remaining four
types (F, G, H and I) have only three to four subdomains whose
locations match that of the ve subdomains (A to E subdomains)
of the typical NAC DNA-binding domain but their sequences
are completely different. Several members of rice NAC family
belong to type O, whose sequences are unclassiable based on
motif composition. Together, the authors suggested that the NAC
members, which belong to types F-O, should be called NAC-like
proteins.
24
In addition, the NAC DNA binding domains of stress
responsive NAC TFs appeared to be conserved.
24,27
Interestingly,
sequence analysis of the NAC family in tobacco revealed that a
NAC subfamily, termed TNACS, represents a major difference in
NAC family members between tobacco and all sequenced plant
genomes. The TNAC subfamily contains the largest number of
tobacco NAC genes with 50 members, an approximate 25% of
NAC genes in tobacco. The TNAC members with their unique
NAC domain appear restricted to the Solanaceae, as they are
absent from currently sequenced plant genomes but present in
tomato (Solanum lycopersicum), pepper (Capsicum annuum) and
potato (Solanum tuberosum).
34
The novel TNAC TFs seem to rep-
resent a previously unknown feature of transcriptional regulatory
circuits, some of which are specic to tobacco or the Solanaceae.
In addition to DNA binding, the NAC domain also possesses
protein binding activity. There is evidence that suggests that the
functions of NAC TFs are dictated, to a large extent, by their
interacting partners. The NAC domain of NAC1 TF interacts
with the RING (really interesting new gene) nger SINAT5,
which promotes ubiquitin-related degradation of NAC1 to atten-
uate auxin signaling in Arabidopsis.
56
Other NAC proteins might
go through ubiquitin-dependent protein degradation, too. For
instance, Arabidopsis ANAC019 can interact with several differ-
ent RING E3 domains.
57
On the other hand, activity of some
NAC proteins could be enhanced by interaction with other pro-
teins through mediation of NAC domain. Recently, it has been
reported that the NAC domain of three stress responsive ANAC
TFsANAC019, ANAC055 and ANAC072interacts with
the zinc-nger homeodomain ZFHD1, a transcriptional activa-
tor which specically binds to the ZFHD recognition sequence
located in the promoter region of ERD1 gene. This interaction
signicantly improved activity of both ANAC and ZFHD pro-
teins in Arabidopsis.
58
The C-terminal transcriptional regulatory region. The
highly variable C-terminal domains of NAC TFs are generally
considered to be the TRR, which can act as either a transcrip-
tional activator or repressor (Fig. 1).
21,22,44,59
In the TRR of rice
ONAC TFs, at least 10 motifs were identied, among which some
motifs are present only in one or two sequences.
24
Interestingly,
the C-terminal domains of relatively large NAC TFs also exhibit
protein binding activity. It has been reported that the C termi-
nal domain of Arabidopsis CBNAC can specically interact with
calmodulin, a ubiquitous calcium-binding protein.
59
The C ter-
minal regions of some NAC TFs also contain transmembrane
(TM) motifs which are responsible for the membrane anchor.
genes with expression level changes higher than two-fold in the
microarray data were checked by real-time PCR analysis using
rice seedlings treated with different stresses, including dehydra-
tion, salt and cold stresses. All of them could be conrmed for
their stress-responsive expressions with ve genes being induced
by drought, 19 genes by salt, and 16 genes by cold, respectively.
24

More recently, in soybean, which is one of the most important
legume crops, computational prediction assumed that there are at
least 205 NAC or NAC-like TFs members in the GmNAC fam-
ily, among which 31 GmNAC members have been recently cloned
and characterized in a comprehensive abiotic stress response
manner.
27,32,53
Nine GmNAC genes were shown to be upregu-
lated by dehydration stress with differential induction levels in
both shoot and root. Six and eight of these nine genes were also
induced by cold stress and high salinity, respectively. Collectively,
it appears that a signicant proportion of NAC genes2025%
according to expression data obtained from genome-wide tran-
scriptome analyses of Arabidopsis and ricefunction in stress
response. The stress-inducible NAC TFs are promising candi-
dates for generation of stress tolerant transgenic plants possessing
traits best suited for survival and/or decreased yield loss under
stressed conditions.
Structural Features of the NAC TFs
The N-terminal DNA-binding NAC domain. The NAC TFs
contain a highly conserved N-terminal DNA-binding NAC
domain and a variable transcriptional regulation C-terminal
domain (Fig. 1).
20,54
The presence of NAC domain in the NAC
TFs was rst revealed by database searches with the sequences
of the cloned NAM, ATAF and CUC2.
20,33
Typically, the NAC
domain is ~150 amino acids in length, and crystal structure of
the NAC domain of an Arabidopsis NAC protein, the ANAC
(ABA responsive NAC), was determined.
54,55
Initially, ve sub-
domains, termed A to E, were identied in the DNA-binding
domain of a typical NAC protein.
33
Recently, a comprehensive in
silico analysis of NAC TF family in rice revealed that there are 36
putative motifs within the NAC family, and based on the compo-
sition of these motifs the NAC family of rice can be classied into
15 types (types A-O). Most rice NAC proteins (97 out of 140),
which are classied into types AE, contain a complete NAC
DNA-binding domain with ve major subdomains, termed A to
E as originally identied for typical NAC TFs. Each of these ve
Figure 1. Scheme of NAC TF structure. The N-terminal DNA-binding
NAC domain consists of fve typical subdomains (A to E). The NAC
domain of several NAC TFs also possesses protein binding activity. The
highly variable C-terminal transcriptional regulatory regions (TRRs)
can act as either a transcriptional activator or repressor. The TRR of
several NAC TFs may contain protein binding domain or transmem-
brane motif.
www.landesbioscience.com GM Crops 35
contains a GCTT core binding motif and anking sequenc-
es.
59
Recently, Ogo et al.
65
reported that the iron deciencyre-
sponsive cis-acting element IDE2 containing the core sequence
CA(A/C)G(T/C)(T/C/A)(T/C/A) is recognized and bound to
by IDEF2, which is a NAC TF, in Arabidopsis. Given that the
size of a TF family is inuenced in part by the number of dif-
ferent DNA sequences that they are able to recognize, it is not
surprised that the NAC TF family constitutes one of the largest
TF groups.
Potentials of NAC in Improvement of Plant Stress
Tolerance
NAC TFs enhance stress tolerance in the model plant
Arabidopsis. Signicant progress has indicated that the NAC
TFs regulate a number of biochemical processes that protect
the plants under different stress conditions. Evidence correlat-
ing the involvement of NAC TFs with the regulation of drought
stress response in plants was rst reported in Arabidopsis. Three
Arabidopsis NAC cDNAs encoding the ANAC019, ANAC055
and ANAC072 TFs that bind to the NACRS of the ERD1 gene
were isolated using yeast one-hybrid system.
21
The expression of
ANAC019, ANAC055 and ANAC072 is induced by drought, high
salinity, and ABA, and these ANAC proteins are localized mainly
to the leaves of transgenic Arabidopsis plants. Overexpression of
either ANAC019, ANAC055 or ANAC072 altered the expression
of many stress-inducible genes in the transgenic plants as shown
by genome-wide microarray analysis, and provided plants with
a constitutive increase in drought tolerance. The drought toler-
ance phenotype of the ANAC transgenic plants may be due to the
increased expression of gene encoding glyoxalase I family protein
as glyoxalase enzymes are important for the glutathione-based
detoxication of methylglyoxal, which is formed primarily as a
byproduct of carbohydrate and lipid metabolism. Therefore, it
was postulated that the improved tolerance of the ANAC019,
ANAC055 and ANAC072 transgenic lines against drought stress
is primarily derived from the reduction and, thus, detoxication of
toxic aldehydes through the glyoxalase pathway.
21
More recently,
trangenic Arabidopsis overexpressing a salt inducible rice NAC
gene, the ONAC063, showed enhanced tolerance to high salin-
ity and osmotic pressure by similar mechanisms as ONAC063-
upregulated genes were almost similar to those upregulated by
ANAC019, ANAC055 or ANAC072. The seedlings of transgenic
Arabidopsis showed a higher germination rate than those of wild-
type plants under high salinity and osmotic stress by mannitol
treatment. However, the survival of young seedlings after tran-
sient high salinity was not signicantly improved.
66
One aspect of principal importance in plant adaptation to
abiotic stress, particularly drought stress, is the response of root
growth and development to water-decit conditions.
67
Because
root growth, increased development of lateral roots and prolifera-
tion of root hairs are critical to maintaining function in different
environmental conditions, plasticity for root traits is a critical fac-
tor to acquire resources. For instance, increases in the density and
depth of rooting can help sustain a high rate of water extraction
and may promote substantial improvement in yield in dry years.
These NAC TFs are membrane-associated and designated as NTL
TFs. A genome-wide analysis predicts that out of 105 ANAC
TFs of Arabidopsis there are 13 NTL TFs (NTL1-13).
60
All the
NTL TFs are associated with the plasma membrane, except for
NTM1/NTL12, which is anchored to the endoplasmic reticulum
membrane. The NTL TFs, which have residue numbers rang-
ing from 335 to 652, are larger than non-membrane-associated
NAC TFs, which contain ~320 residues. The TM domains of
NTLs are typically -helical, and many membrane-bound TFs
have a few helix-destabilizing residues within their TM motifs
such as proline and glycine residues that have the lowest tendency
to form helices. At least four NTL (NTL6, NTL8, NTL9 and
NTL12/NTM1) genes have been shown to function in stress
responses. These NTLs are activated by membrane-associated
proteases during stress responses in the endoplasmic reticulum
and when the plants experience environmental stresses, which
liberate the TFs from their TM domain. For instance, NTL8
transcription and proteolytic activation of NTL8 is promoted by
high salinity.
61
Analyses of transgenic plants overexpressing stress
responsive NTL genes, both in full-size and TM-deleted forms,
indicated that the TM domain is involved in regulation of activi-
ties of the NTLs, primarily at the processing step. Transgenic
plants overexpressing full-size NTL genes were indistinguish-
able from control plants, but transgenics producing TM-deleted
forms exhibited severe phenotypes due to the truncated active
forms of NTLs. Evidence also showed that individual NTLs have
both distinct and overlapping roles in a variety of stress responses,
such as cell division, owering initiation, leaf senescence and seed
germination.
60,62
The DNA Binding Sequences of the NAC TFs
In Arabidopsis the DNA binding site of stress-inducible NAC
TFs, the NAC recognition sequence (NACRS) containing the
CACG core motif, has been identied by intensive analysis of
the promoter of the drought inducible EARLY RESPONSE
TO DEHYDRATION1 (ERD1) gene, which encodes a protein
with homology to the ATP-binding subunit of the Clp ATP-
dependent protease from Escherichia coli.
21,63
Molecular analysis
of the ERD1 promoter demonstrated that ERD1 expression dur-
ing dehydration mainly depends upon the integrity of both the
CAC TAA ATT GTC AC ZFHDRS and the ANN NNN
TCN NNN NNN ACA CGC ATG T NACRS sequences.
21,58,63

The CACG core motif is underlined. The rice drought-induc-
ible ONAC TFs bind to a similar NACRS found in rice genome,
demonstrating that the NACRS might be conserved across
the plant kingdom at least for stress-inducible NAC TFs.
22,24

In addition, other sequences have also been reported as NAC
binding sites. The Arabidopsis NAC1 protein has been shown
to bind to a 21-bp segment (CTG ACG TAA GGG ATG ACG
CAC) within the 35S -90 promoter fragment.
39
Independently,
Duval et al.
64
demonstrated that puried AtNAM recombi-
nant protein protected the region of the CaMV 35S promoter
between -70 and -76, which is located in the 21-bp segment
(AGGGATG). On the other hand, CBNAC TF of Arabidopsis
bound specically in vitro and in vivo to CBNACBS, which
36 GM Crops Volume 1 Issue 1
SNAC2 target genes are the same, but comparison of the anking
sequences of the core DNA binding sites in the putative SNAC1
and SNAC2 target genes revealed different conserved anking
sequences of the core binding sites of genes targeted by SNAC1
and SNAC2. Together, these results may suggest that different
stress-responsive NAC TFs may activate the transcription of a
different set of target genes, thus conferring diverse functions
that jointly lead to stress tolerance.
It is worthy to note that all the NAC genes used in mak-
ing stress tolerant plants, either Arabidopsis or rice, have been
characterized as transcriptional activators.
21,22,66,72,73,75
Most
importantly, although transgenic plants overexpressing a num-
ber of NAC genes exhibited stress tolerant phenotype, so far the
drought tolerant phenotype of only SNAC1 transgenic plants
was tested in the eld with positive results.
22
Overexpression
of SNAC2/OsNAC6, however, showed no signicant effect on
drought tolerance in eld conditions even though the transgenic
plant showed improved tolerance to either drought tolerance or
osmotic stress by PEG treatment in greenhouse conditions.
72,73

ONAC045 transgenic rice plants have not been tested in eld tri-
als for stress tolerance.
75
Transgenic plants, in which stress responsive TF, such as
DREB1A of AP2/ERF TF family, was constitutively overex-
pressed, although showed an increased tolerance to drought, salt
and freezing in both Arabidopsis and rice, but suffered growth
retardation.
77,78
In contrast, transgenic rice constitutively over-
expressing SNAC1 exhibited a normal phenotype and economic
traits as the wild-type plant under normal growth conditions.
22

Furthermore, there was no signicant difference in plant mor-
phology and yield between either the SNAC2 or ONAC045 trans-
genic lines and wild type under unstressed conditions.
73,75
These
results indicated that transgenic plants overexpressing NAC genes
may not have detrimental effects on the growth and development
of plants under non-stress conditions, suggesting a promising
utility of this gene in genetic improvement of stress tolerance in
economically important crop plants.
Conclusion
Abiotic stress is one of the major factors affecting yield and stabil-
ity of crop production worldwide. As global warming and water
shortage will make arable land in the world less productive or
unusable, the development of stress tolerant plants will become
increasingly important. Transgenic approaches have been shown
to be powerful tools to help manipulate the responses of plants
to stress. There is strong evidence, even in eld trials, that trans-
genic rice plants harboring NAC genes have enhanced stress tol-
erance, suggesting that NAC TFs are promising candidate genes
for genetic engineering of different crops aimed at improving
their productivity under adverse conditions. Moreover, to ensure
the success of the transgenic crops greater emphasis should be
placed on the eld evaluation of the transgenic plants, and the
parameters which will be measured. To facilitate accurate evalu-
ation in the eld for stress tolerance, screening should be carried
out under controllable stressed environments over multiple loca-
tions and years.
There is evidence that soybeans with prolic roots and slower
wilting characteristics can reduce yield losses to plant water de-
cits in the eld.
3,68-70
Therefore inuencing root proliferation by
genetic engineering is a good approach to provide stress toler-
ant plants. A great deal of evidence indicated that overexpression
of NAC genes can promote root development. Overexpressions
of NAC TFs in Arabidopsis, such as NAC1 and AtNAC2 genes
have led to enhanced later root development, suggesting the
useful application of these NAC TFs in improvement of stress
tolerance.
39,71
NAC TFs enhance stress tolerance in crop plants. Following
the discovery of potential use of NAC TFs to improve stress tol-
erance in Arabidopsis, a number of important successes have
reported the application of NAC TFs in genetic engineering of
important crops, such as cultivated rice for enhanced tolerance
against various environmental stresses. Transgenic rice overex-
pressing the stress inducible SNAC1 gene displayed drought and
salt tolerance at the four-leaf stage.
22
More importantly, SNAC1
transgenic plants had 2234% higher seed setting in the eld
than the negative control under severe drought stress conditions
at the reproductive stage. The SNAC1 plants are drought tolerant
not only due to increased expression of genes encoding proteins
functioned in production of osmolytes, in detoxication and
redox homeostasis and in protection of macromolecules but also
in increasing stomatal closure in transgenic leaves, which pre-
vents water loss from the plant and improves water use efcien-
cy.
22
More recently, overexpression of SNAC2/OsNAC6 in rice
improved drought and salt tolerance of transgenic plants because
of the enhanced expression of a large number of genes encoding
proteins with predicted stress tolerance functions such as detoxi-
cation, redox homeostasis and proteolytic degradation as well.
72

Expression of SNAC2/OsNAC6 is induced by drought, salinity,
cold, wounding and ABA treatment, but decreased by methyla-
tion and deacetylation.
72-74
In another independent study, Hu et
al.
73
found that SNAC2/OsNAC6 transgenic rice plants had sig-
nicantly higher germination and growth rate than WT under
high salinity conditions. These same authors also discovered that
SNAC2/OsNAC6 rice plants confer cold tolerance perhaps due
to higher cell membrane stability in transgenic plants than in
wild type during the cold stress. Overexpression of another rice
NAC gene, the ONAC045 gene, whose expression is induced by
drought, high salinity, low temperature stresses and ABA treat-
ment in both leaves and roots, showed signicantly enhanced
tolerance to drought and salt at the seedling stage. At least,
expression levels of two stress-responsive genes, OsLEA3-1 and
OsPM1, were upregulated in ONAC045 transgenic lines.
75
It is
evident that overexpressing OsLEA3-1 in rice showed signi-
cantly increased drought tolerance under the eld condition.
76

Expressions of OsLEA3-1 and OsPM1 were not affected in either
SNAC1 or SNAC2 transgenic rice, suggesting that ONAC45 TF
improves stress tolerance of transgenic plants in different path-
way than SNAC1 and SNAC2/OsNAC6.
75
Moreover, the poten-
tial transcriptional target genes of SNAC1 and SNAC2 are also
different. There is no overlapping between the two sets of genes
up or downregulated in the two overexpression plants, respec-
tively.
73
The core DNA binding sites for the putative SNAC1 and
www.landesbioscience.com GM Crops 37
industry. Furthermore, integration of genetic and molecular
approaches and advanced high throughput technologies, such as
microarray, proteomic and metabolomic analyses, will undoubt-
edly lead to the discovery of a diverse set of novel candidate genes
that in addition to the NAC TFs, will constitute a novel bio-
resource for the development of improved stress tolerant plants
by biotechnology.
Acknowledgements
Funding support from Grants-in-Aid (Start-up) for
Scientic Research, Ministry of Education, Culture, Sports,
Science and Technology of Japan (No. 21870046) is gratefully
appreciated.
With the recent availability of the entire genome sequence of
a number of crops and genome-wide expression proling data,
comparative analyses of rice and other crops, such as soybean and
maize (Zea mays), will enable scientists to quickly identify those
NAC genes, which can be exploited to engineer the plants for
enhanced stress tolerance. Figure 2 schematically introduces the
process of identication, characterization and selection of NAC
genes for the purpose of improvement of abiotic stress tolerance
in crops by biotechnological approach. Considering their diverse
functions, the NAC TFs possess a number of potentials in broad
aspect of plant biotechnology, ranging from their use in genetic
engineering of abiotic and/or biotic stress tolerance to improv-
ing seed quality and wood as well as its derivatives for woody
Figure 2. Process of identifcation, characterization and selection of NAC candidate genes for improvement of abiotic stress resistance in crops by
biotechnological approach. Potential NAC transcription factors/transcriptional activators can be selected by stress responsive expression profling
and characterization of their transcriptional activity. The selected NAC candidate genes will be then overexpressed in crops, and the transgenic plants
tested in green house and feld for better performance in stressed conditions. Alternatively, the NAC genes can be tested in model plants such as Ara-
bidopsis frst to fasten the screening process.
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