The overexpression of OsNAC9 alters the root ... - Wiley Online Library

Plant Biotechnology Journal (2012) 10, pp. 792–805

doi: 10.1111/j.1467-7652.2012.00697.x

The overexpression of OsNAC9 alters the root architecture of rice plants enhancing drought resistance and grain yield under field conditions Mark C.F.R. Redillas1,†, Jin S. Jeong1,†, Youn S. Kim1, Harin Jung1, Seung W. Bang1, Yang D. Choi2, Sun-Hwa Ha3, Christophe Reuzeau4 and Ju-Kon Kim1,* 1

School of Biotechnology and Environmental Engineering, Myongji University, Yongin, Korea

2

School of Agricultural Biotechnology, Seoul National University, Seoul, Korea

3

National Academy of Agricultural Science, RDA, Suwon, Korea

4

CropDesign NV, a BASF Plant Science Company, Gent, Belgium

Received 29 December 2011; revised 14 March 2012; accepted 20 March 2012. *Correspondence (Tel +82 31 330 6197 fax +82 31 335 8249; email [email protected]) † These authors contributed equally to this work.

Keywords: drought, NAC, root architecture, grain yield, root-specific expression.

Summary Drought conditions limit agricultural production by preventing crops from reaching their genetically predetermined maximum yields. Here, we present the results of field evaluations of rice overexpressing OsNAC9, a member of the rice NAC domain family. Root-specific (RCc3) and constitutive (GOS2) promoters were used to overexpress OsNAC9 and produced the transgenic RCc3:OsNAC9 and GOS2:OsNAC9 plants. Field evaluations over two cultivating seasons showed that grain yields of the RCc3:OsNAC9 and the GOS2:OsNAC9 plants were increased by 13%–18% and 13%–32% under normal conditions, respectively. Under drought conditions, RCc3:OsNAC9 plants showed an increased grain yield of 28%–72%, whilst the GOS2:OsNAC9 plants remained unchanged. Both transgenic lines exhibited altered root architecture involving an enlarged stele and aerenchyma. The aerenchyma of RCc3:OsNAC9 roots was enlarged to a greater extent than those of GOS2:OsNAC9 and non-transgenic (NT) roots, suggesting the importance of this phenotype for enhanced drought resistance. Microarray experiments identified 40 up-regulated genes by more than threefold (P < 0.01) in the roots of both transgenic lines. These included 9-cis-epoxycarotenoid dioxygenase, an ABA biosynthesis gene, calcium-transporting ATPase, a component of the Ca2+ signalling pathway involved in cortical cell death and aerenchyma formation, cinnamoyl CoA reductase 1, a gene involved in lignin biosynthesis, and wall-associated kinases¸ genes involved in cell elongation and morphogenesis. Interestingly, O-methyltransferase, a gene necessary for barrier formation, was specifically up-regulated only in the RCc3:OsNAC9 roots. Such up-regulated genes that are commonly and specifically up-regulated in OsNAC9 transgenic roots may account for the altered root architecture conferring increased drought resistance phenotype.

Introduction Drought is a major limiting factor in agricultural production systems because it prevents crops from reaching their genetically determined theoretical maximum yields (Peet and Kramer, 1980). Typical consequences for plants exposed to drought stress include cellular dehydration that reduces the cytosolic and vacuolar volumes as well as production of reactive oxygen species that negatively affects cellular structures and metabolism (Bartels and Sunkar, 2005). Various attempts have been made to produce plants that can withstand drought stress by manipulating stress-responsive genes such as OsCDPK (Saijo et al., 2000), P5CS (Zhu et al., 1998) and TPSP (Jang et al., 2003). Stress-responsive genes are generally regulated by the interaction between transcription factors (TFs) and cis-elements located in the promoter regions of the target genes. Examples of these cis-elements include dehydration-responsive element ⁄ C-repeat (DRE ⁄ CRT), ABA-responsive element (ABRE), NAC recognition sequence (NACRS), ZFHD recognition sequence (ZFHDRS) and MYCRS ⁄ MYBRS (Shinozaki

792

and Yamaguchi-Shinozaki, 2007). In addition, well-characterized TFs that play a role in the regulation of stress response pathways include DREB ⁄ CBF (Liu et al., 1998; Stockinger et al., 1997), AREB ⁄ ABF(Choi et al., 2000; Uno et al., 2000), MYB (Villalobos et al., 2004), bZIP (Zhang et al., 2008), AP2 (Oh et al., 2009), zinc-finger (Sugano et al., 2003) and NAC (Hu et al., 2006). Recently, Yang et al. (2010) classified droughtresponsive genes into three groups based on their biological functions: transcriptional regulation, post-transcriptional RNA or protein phosphorylation, and osmoprotectant metabolism or molecular chaperones. Among the most widely studied, drought-responsive genes are the transcriptional regulators belonging to the NAC (NAM, ATAF and CUC) gene family, which are found only in plants, and many are involved in stress responses. NAC proteins have conserved N-terminus, a DNA-binding domain that can form a b-sheet structure where proteins form either a homodimer or a heterodimer (Ernst et al., 2004; Hegedus et al., 2003; Jeong et al., 2009; Takasaki et al., 2010; Xie et al., 2000), and a highly variable C-terminal region (Zheng et al., 2009). NAM

ª 2012 The Authors Plant Biotechnology Journal ª 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd

OsNAC9 improves drought resistance and grain yield in rice 793 (NO APICAL MERISTEM) was the first reported NAC gene in petunia (Petunia hybria) responsible for the formation of the shoot apical meristem and primordium of the plant (Souer et al., 1996). Genes in the ATAF subfamily of NAC, such as ATAF1 and ATAF2 from Arabidopsis, are induced by pathogen attack and wounding, whilst CUC2 (CUP-SHAPED COTYLEDON) are involved in the development of embryos and flowers (Aida et al., 1997). Members of the NAC family that enhance both abiotic and biotic stress resistance in plants have also been reported. For example, AtNAC1 mediates auxin signalling to promote lateral root development (Xie et al., 2000), whereas the overexpression of AtNAC2 functions downstream of ethylene and auxin signalling pathways, which subsequently enhances salt resistance (He et al., 2005). The STRESS-RESPONSIVE NAC1 and 2 (SNAC1 and SNAC2) genes in rice are NAC genes involved in responses to various environmental stress stimuli. SNAC1 has been found to confer drought resistance in transgenic rice (Hu et al., 2006), whilst SNAC2 significantly increases the germination and growth levels of transgenic plants under high-salinity conditions (Hu et al., 2008). Likewise, transgenic plants overexpressing OsNAC045 show increased resistance to both drought and salt stress (Zheng et al., 2009), while the constitutive overexpression of OsNAC6 resulted in an improved resistance to both abiotic and biotic stress conditions such as drought, high-salt and blast disease (Nakashima et al., 2007). To develop stress-resistant transgenic plants that could be cultivated in agricultural practices, such plants must be evaluated under field conditions. Most of the studies on the overexpression of stress-related genes have been conducted under greenhouse conditions (Dubouzet et al., 2003; Garg et al., 2002; Park et al., 2005; Shen et al., 1997; Xiong and Yang, 2003; Xu et al., 1996) with very few reports to date on longterm field testing (Jeong et al., 2010; Oh et al., 2009; Wang et al., 2005). It is also an important consideration that the new transgenic plant lines with an acquired stress-resistant phenotype do not manifest yield penalty and ⁄ or growth retardation. The constitutive overexpression of stress-related genes often causes reduced growth and thus a loss in productivity. For example, stress-resistant transgenic Arabidopsis expressing 35S:DREB1A displays growth retardation and a severe reduction in seed production (Kasuga et al., 1999; Liu et al., 1998). Similarly, a transgenic tomato expressing 35S:CBF1 (Hsieh et al., 2002a,b), a transgenic rice line expressing 35S:OsNAC6 (Nakashima et al., 2007) and a transgenic tobacco plant expressing 35S:TPS1 (Romero et al., 1997) have been shown to exhibit varying degrees of growth retardation, abnormal development and reduced seed production. The use of a root-specific and ⁄ or stress-inducible promoter provides an alternative approach by which the adverse effects of constitutive overexpression can be potentially bypassed. For example, Jeong et al. (2010) have overexpressed OsNAC10 using both a root-specific (RCc3) and a constitutive (GOS2) promoter to evaluate the grain yields of transgenic rice plants under normal and drought field conditions. The RCc3:OsNAC10 plants showed higher grain yield both under drought and normal field conditions than GOS2:OsNAC10 plants. The growth retardation observed in transgenic Arabidopsis expressing 35S:DREB1A was also found to be diminished when the stressinducible promoter rd29A was used in place of 35S (Kasuga et al., 1999). Furthermore, the rice stress-inducible promoters LIP9 and OsNAC6 have been effectively used to overexpress the

OsNAC6 gene in transgenic rice and improve stress resistance without inducing growth retardation (Nakashima et al., 2007). In our current study, we evaluated the overexpression of the TF OsNAC9 in T5 and T6 generation transgenic rice in a rice paddy field under both normal and drought conditions. OsNAC9 is identical to the SNAC1 gene that was previously reported to confer drought resistance (Hu et al., 2006). The use of the GOS2 (de Pater et al., 1992) and RCc3 (Xu et al., 1995) promoters to achieve the overexpression of OsNAC9 in a constitutive and root-specific manner, respectively, enabled us to address the effects of this transgene on stress resistance and grain yield more specifically. Under normal conditions, grain yield was found to be improved in both transgenic lines. Under drought conditions, however, RCc3:OsNAC9 plants performed better than GOS2:OsNAC9 plants and showed a significantly enhanced grain yield.

Results Transgenic overexpression of OsNAC9 confers stress resistance in rice at the vegetative stage of growth We performed RNA gel blot analysis using total RNA preparations from the leaves and roots of 14-day-old rice seedlings exposed to drought, high-salinity, low temperature and ABA over a particular time course. The expression of endogenous OsNAC9 in the rice leaves and roots was found to be up-regulated significantly by drought, high-salinity and ABA but only weakly by low-temperature conditions (Figure S1). To overexpress OsNAC9 in transgenic rice plants, the full-length cDNA of this gene was linked to two different promoters, RCc3 for root-specific expression (RCc3:OsNAC9) and GOS2 for constitutive expression (GOS2:OsNAC9). Fifteen to 20 independent transgenic lines per construct were produced through the Agrobacterium-mediated transformation method. T1–6 seeds from transgenic lines that grew normally and without stunting were collected, and three independent T5–6 homozygous lines of both RCc3:OsNAC9 and GOS2:OsNAC9 plants were selected for further analysis. The expression of RCc3:OsNAC9 and GOS2:OsNAC9 was confirmed by RNA gel blot analysis in both the roots and the leaves (Figure 1a). The expression of the transgene OsNAC9 was not detectable in the leaves of RCc3:OsNAC9 plants but the roots showed high levels of transgene expression, validating the root specificity of the RCc3 promoter. The transgene expression levels were increased to similar levels in both the roots and the leaves of the GOS2:OsNAC9 plants. In addition, expression levels of the transgene were higher in the roots of RCc3:OsNAC9 plants than in those of the GOS2:OsNAC9 plants, whereas the expression of the reference tubulin gene was equivalent between these two lines. To evaluate the stress resistance of OsNAC9 overexpressors at the mature vegetative stage of growth, 4-week-old transgenic and NT control plants were subjected to drought stress for up to 5 days (Figure 1b). Transgenic plants showed delayed leaf rolling compared with the NT plants during drought treatments. After re-watering, however, the transgenic plants started to recuperate, whilst the NT plants continuously withered with no signs of recovery. This demonstrated the drought resistance of the transgenic plants at the vegetative stage. Because environmental stresses affect the photosynthetic machinery of plants, the maximum photochemical efficiency of PSII (Fv ⁄ Fm; where Fv is the variable fluorescence and Fm is the

ª 2012 The Authors Plant Biotechnology Journal ª 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 10, 792–805

794 Mark C.F.R. Redillas et al. RCc3:OsNAC9

(a)

NT

10 + –

34 + –

60 + –

NT

GOS2:OsNAC9 2 63 78 + – + – + –

OsNAC9 rbcs

Leaf

EtBr OsNAC9 Root

tubulin EtBr (b)

(c) RCc3:OsNAC9

+2 day

Fv/Fm

0 day

3 day

+5 day

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

Drought

High-Salinity

**** ** ** **

0

0.5

Low temperature NT RCc3:OsNAC9-10 RCc3:OsNAC9-34 RCc3:OsNAC9-60

** ** ** **

**

1

**

**

0

2

6

0

6

12 (h)

GOS2:OsNAC9 5 day

Fv/Fm

+7 day

10 34 60

RCc3: OsNAC9

NT

2 63 78

10 34 60

GOS2: OsNAC9

RCc3: OsNAC9

2 63 78

NT

GOS2: OsNAC9

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

Drought ** **

High-Salinity

Low temperature

**

NT GOS2:OsNAC9-2 GOS2:OsNAC9-63 GOS2:OsNAC9-78

**** ** **

0

0.5

** **

1

** **

0

2

6

**

0

6

12 (h)

Figure 1 Stress resistance of RCc3:OsNAC9 ad GOS2:OsNAC9 plants at the vegetative stage. (a) RNA gel blot analysis of three homozygous T5 lines of RCc3:OsNAC9, GOS2:OsNAC9 and NT plants. Total RNAs were blotted and hybridized with OsNAC9 for gene-specific region. Equal loadings of the RNAs were determined using ethidium bromide (EtBr) staining. ()) and (+) represent null and transgenic lines, respectively. (b) Images of rice plants during drought stress. Three independent homozygous T5 lines of RCc3:OsNAC9 and GOS2:OsNAC9 plants and NT controls were grown for 2 weeks, subjected to 5 days of drought stress followed by 7 days of re-watering in greenhouse as indicated by (+). (c) Comparison of the chlorophyll fluorescence (Fv ⁄ Fm) of rice plants exposed to drought, high-salinity and low-temperature stress conditions. Each data point represents the mean ± SE of triplicate experiments (n = 10). Asterisks (**) indicate a significant level (P < 0.01).

maximum fluorescence) was measured using a pulse amplitude modulation fluorometer (Figure 1c). Fourteen-day-old plants were subjected to a time course of drought, high-salinity and low-temperature stress conditions and their Fv ⁄ Fm values were then determined. Both under drought and high-salinity conditions, RCc3:OsNAC9 and GOS2:OsNAC9 plants showed 10%– 30% higher Fv ⁄ Fm values than NT control plants depending on the extent of the stress and transgenic line. Under low-temperature conditions, in contrast, no differences were found in the Fv ⁄ Fm values between the transgenic and NT control plants. Taken together, these results indicate the enhanced resistance of both RCc3:OsNAC9 and GOS2:OsNAC9 plants to drought stress at the vegetative stage of growth.

The overexpression of OsNAC9 increases grain yield under both normal and drought conditions We evaluated the yield components of the transgenic plants under normal and field drought conditions over two cultivating seasons (2009 and 2010). Three independent T5 (2009) and T6

(2010) homozygous lines of both the RCc3:OsNAC9 and GOS2:OsNAC9 plants, together with NT controls, were transplanted to a paddy field and grown to maturity. The yield parameters were scored for 30 plants per transgenic line from three replicates. The data sets for both years of the field test were generally consistent, and the total grain weights of the RCc3:OsNAC9 and the GOS2:OsNAC9 plants were increased by 13%–18% and 13%–32%, respectively. The increase in total grain weight was due mainly to the increased panicle length in RCc3:OsNAC9 plants and to both the increased panicle length and number in GOS2:OsNAC9 plants (Figure 2a and Table S1). To test the growth response of these transgenic plants under drought conditions, three independent T5 and T6 lines of RCc3:OsNAC9 and GOS2:OsNAC9 plants were transplanted to a paddy field with a removable rain-off shelter. Plants were exposed to intermittent drought stress at the panicle heading stage (from 10 days before heading and 10 days after heading). The level of drought stress imposed under the rain-off shelter caused a 50%–60% reduction in the normal grain weight, as


OsNAC9 improves drought resistance and grain yield in rice 795 Normal

(a) 2009

2010

NT

RCc3: OsNAC9

RCc3:OsNAC9-10 RCc3:OsNAC9-34 RCc3:OsNAC9-60

NT

GOS2: OsNAC9

GOS2:OsNAC9-2 GOS2:OsNAC9-63 GOS2:OsNAC9-78

Drought

(b) 2009

Figure 2 Agronomic traits of RCc3:OsNAC9 and GOS2:OsNAC9 plants grown in the field under normal (a) and drought (b) conditions for two cultivating seasons (2009–2010). Agronomic traits of three independent homozygous T5 (2009) and T6 (2010) lines for each transgenic plant together with NT controls were plotted using Microsoft Excel 2007. Each data point represents the percentage of the mean values (n = 30) with the NT plants assigned a reference value of 100%. CL, culm length; PL, panicle length; NP, number of panicles per hill; NSP, number of spikelets per panicle; TNS, total number of spikelets; FR, filling rate; NFG, number of filled grains; TGW, total grain weight; 1000 GW, 1000 grain weight.

RCc3: OsNAC9

2010

NT RCc3:OsNAC9-10 RCc3:OsNAC9-34 RCc3:OsNAC9-60

GOS2: OsNAC9

evidenced by the NT plant yields under both set of conditions (Tables S1 and S2). In addition, we compared the yield parameters of non-stressed NT controls grown between the normal field test site and drought test site (Table S3). We did not find any significant statistical differences (P < 0.05) in the yield parameters of the controls between the two sites, eliminating difference in effects of radiation and temperature. Statistical analysis of the yield parameters scored over two cultivating seasons in 2009 and 2010 showed that the decrease in grain yield under drought conditions was significantly smaller in the RCc3:OsNAC9 plants than that in the NT controls. Specifically, in the drought-treated RCc3:OsNAC9 plants, the filling rate was 18%–36% higher than in the drought-treated NT plants, which resulted in a total grain weight increase of 28%–72%, depending on the transgenic line (Figure 2b and Table S2). In the drought-treated GOS2:OsNAC9 plants, in contrast, the total grain weight was found to be similar to the drought-treated NT controls. Given the similar levels of drought resistance during

NT GOS2:OsNAC9-2 GOS2:OsNAC9-63 GOS2:OsNAC9-78

the vegetative stage in the RCc3:OsNAC9 and GOS2:OsNAC9 plants, the differences observed in total grain weight under field drought conditions between these transgenic lines were unexpected. These seemingly contradictory observations prompted us to examine the root architecture of the transgenic plants. We, therefore, measured the root volume, length, dry weight and diameter of RCc3:OsNAC9, GOS2:OsNAC9 and NT plants grown to the heading stage of reproduction. As shown in Figure 3b, the root diameters of the RCc3:OsNAC9 and GOS2:OsNAC9 plants were thicker than those of the NT control plants by 30% and 7%, respectively. Microscopic analysis of cross-sectioned roots revealed that the increase in root diameter was owing to the enlarged stele, aerenchyma and epidermis in the RCc3:OsNAC9 roots. In particular, the aerenchyma (ae in Figure 3b) was larger in the RCc3:OsNAC9 roots compared with the GOS2:OsNAC9 and NT plants, which may have contributed to the enlargement of the RCc3:OsNAC9 roots along with an enlarged stele. The root-specific overexpression of OsNAC9


796 Mark C.F.R. Redillas et al. Figure 3 Comparison of the root growth of RCc3:OsNAC9, GOS2: OsNAC9 and NT plants grown at the heading stage of reproduction. (a) The upper panel shows representative roots for each plant, whilst the lower panel shows one representative root for each plant. Bars, 10 cm and 2 mm in the upper and lower panels, respectively. (b) Light microscopic images of cross-sectioned transgenic and NT plant roots. The whole-cross-section of the roots (top panel), vascular bundles within the stele (middle panel) and the epidermis and part of the aerenchyma (bottom panel) are shown. xy, xylem; ae, aerenchyma; the epidermis is indicated by an arrowhead. Bars, 500 lm in the top panel, 100 lm in the middle and bottom panels. (c) The volume, length, dry weight and diameter of transgenic plant roots were normalized to NT plants. Values are the means ± SD of 50 roots (ten roots from each of five plants). Asterisks (**) indicate a significant mean difference at 0.01 level (LSD).

(a)

RCc3: OsNAC9

NT

leading to an increased root diameter accompanied by a larger aerenchyma, thus, correlated with the enhanced drought resistance of transgenic plants at the reproductive stage. The volume, length and dry weight of the GOS2:OsNAC9 roots increased by 50%, 20% and 35% relative to the NT roots, respectively, suggesting that these parameters also affected the increase in grain yield under normal growth conditions.

GOS2: OsNAC9

(b)

co

500 μm

Identification and real-time PCR analysis of genes up-regulated by the overexpression of OsNAC9

500 μm

500 μm

xy

100 μm

100 μm

100 μm

ae

100 μm

100 μm

RCc3: OsNAC9

(c) 1.80 1.60

**

1.40

100 μm

NT

GOS2: OsNAC9

NT RCc3:OsNAC9 GOS2:OsNAC9

**

**

** **

1.20 1.00 0.80 0.60 0.40 0.20 0.00 Volume

Length

Dry Weight

Diameter

Expression profiling was performed for RCc3:OsNAC9 and GOS2:OsNAC9 roots to identify up-regulated genes following the overexpression of OsNAC9. A rice 3¢-Tiling Microarray was performed on RNA samples extracted from the roots of 14-dayold plants grown under normal conditions. Each data set was obtained from duplicate biological samples. Statistical analysis using one-way ANOVA identified 40 genes that were up-regulated by more than threefold (P < 0.01) in RCc3:OsNAC9 and GOS2:OsNAC9 roots following OsNAC9 overexpression (Table 1). Also, identified were 17 and 28 genes that were specific to RCc3:OsNAC9 and to GOS2:OsNAC9 roots, respectively. The highly up-regulated genes common to both transgenic roots include 9-cis-epoxycarotenoid dioxygenase (NCED), a gene for ABA biosynthesis (Lefebvre et al., 2006), calcium-transporting ATPase (Ca2+-ATPase), a component of the Ca2+ signalling pathway involved in cortical cell death (apoptosis) and aerenchyma formation (Knight, 2000), cinnamoyl CoA reductase 1 (CCR1), a key enzyme for lignin biosynthesis (Tamasloukht et al., 2011), and the wall-associated kinases (WAK) that are involved in cell elongation, morphogenesis and plant development (Lally et al., 2001; Wagner and Kohorn, 2001). Interestingly, three members of the O-methyltransferase (OMT), genes thought to be involved in barrier formation, were specifically up-regulated only in RCc3:OsNAC9 plants. To confirm the expression of these up-regulated genes, we performed qPCR analysis on the roots of 14-day-old transgenic and NT plants grown under normal conditions (Figure 4). From our previous study, we reported that the root-specific overexpression of OsNAC10 enhances drought resistance owing to the alteration on the root structure of transgenic plants (Jeong et al., 2010). We, therefore, compared the expression patterns of the 85 OsNAC9 up-regulated genes with those of OsNAC10 up-regulated genes and found 53 up-regulated genes that were not common to both OsNAC9 and OsNAC10 roots (Figure 4, Table S4). In addition, the expression patterns of some of the up-regulated genes common to both OsNAC9 and OsNAC10 were different. For


OsNAC9 improves drought resistance and grain yield in rice 797 Table 1 Up-regulated root-expressed genes in RCc3:OsNAC9 and GOS2:OsNAC9 transgenic rice plants in comparison with non-transgenic controls

Gene name

Loc no*

RCc3:OsNAC9

GOS2:OsNAC9

(IRGSP)

Mean†

P valueà

Mean†

P valueà

Up-regulated genes in RCc3:OsNAC9 and GOS2:OsNAC9 Protein kinase

Os01g0117600

3.60

6.7E-04

3.04

5.3E-04

ABC transporter

Os01g0609300

3.39

2.3E-03

4.44

4.7E-04

Peptidase aspartic

Os01g0937500

3.40

6.6E-04

3.37

3.3E-04

WAK3

Os02g0807900

5.02

4.1E-04

4.47

2.7E-04

Cinnamoyl CoA Reductase 1

Os02g0811800

10.65

3.1E-05

7.47

6.4E-05

Acyl-activating enzyme

Os03g0130100

3.09

2.1E-03

3.31

6.7E-04

U-box

Os03g0240600

6.16

1.3E-03

7.27

5.4E-04

Aspartyl protease

Os03g0318400

3.59

1.3E-03

3.61

5.4E-04

High affinity K+ transporter 5

Os03g0575200

5.28

9.5E-05

5.03

8.0E-05

Copalyl diphosphate synthetase

Os04g0178300

3.50

2.5E-04

3.56

1.3E-04

RLP (receptor-like protein kinase)

Os04g0202700

4.10

5.1E-04

4.16

2.6E-04

MAPKKK9

Os04g0339800

7.17

6.5E-05

6.91

6.4E-05

WAK

Os04g0365100

4.60

1.2E-04

3.56

1.5E-04

WAK

Os04g0368800

3.87

4.5E-04

3.56

2.9E-04

Glutamate dehydrogenase

Os04g0543900

3.13

4.6E-04

3.00

2.7E-04

Downy mildew resistnant 6

Os04g0581000

4.49

4.2E-04

4.55

2.1E-04

Oxidoreductase, 2OG-Fe(II) oxygenase

Os04g0581100

70.90

3.1E-06

61.65

3.4E-06

Pyruvate kinase

Os04g0677300

3.27

4.6E-04

4.05

1.3E-04

Zinc finger

Os05g0404700

3.11

6.7E-04

5.46

7.8E-05

Early nodulin 93

Os06g0141600

3.06

5.8E-04

3.47

2.0E-04

Haem peroxidase

Os06g0521500

3.01

1.7E-03

3.44

4.9E-04

Pathogenesis-related protein

Os07g0129300

3.08

3.7E-03

3.44

1.0E-03

RLK (receptor lectin kinase)

Os07g0129800

4.87

3.4E-04

3.96

2.8E-04

9-cis-epoxycarotenoid dioxygenase

Os07g0154100

20.06

5.6E-05

25.41

3.5E-05

Cloroplastosos alterados

Os07g0190000

4.09

6.1E-04

4.50

2.5E-04

Leucine-rich repeat transmembrane kinase

Os07g0251900

8.22

7.8E-05

5.96

9.4E-05

Leucine-rich repeat protein kinase

Os08g0203400

7.64

1.4E-05

6.21

2.0E-05

WRKY40

Os09g0417600

7.64

1.2E-04

7.21

9.3E-05

WRKY18

Os09g0417800

6.93

9.5E-05

7.16

6.7E-05

Potassium ion transmembrane transporter

Os09g0448200

7.71

9.0E-05

7.06

7.8E-05

WAK2

Os10g0151100

6.63

2.1E-05

4.06

6.7E-05

Calcium-transporting ATPase

Os10g0418100

26.21

3.9E-06

32.54

3.4E-06

Aspartyl protease

Os10g0537800

4.59

4.5E-04

4.28

2.7E-04

Aspartyl protease

Os10g0538200

4.47

2.8E-04

3.98

2.0E-04

DNA binding ⁄ Homeodomain

Os11g0282700

126.94

3.8E-06

100.82

4.0E-06

Calcium-binding EF hand family protein

Os11g0600500

4.32

7.6E-05

4.02

6.7E-05

Zinc finger

Os11g0687100

5.60

7.9E-04

6.05

3.3E-04

WAK5

Os12g0615100

4.48

3.5E-04

3.24

4.3E-04

Zinc finger

Os11g0702400

3.23

3.5E-03

3.74

8.3E-04

AAA-ATPase 1

Os12g0431100

3.10

4.2E-04

4.24

9.4E-05

Up-regulated genes in RCc3:OsNAC9 Cytochrome P450

Os02g0601500

5.42

1.1E-04

1.86

3.8E-03

MtN3

Os05g0426000

4.03

5.1E-04

1.27

6.8E-02 3.1E-02

Leucine-rich repeat

Os08g0202300

3.34

7.6E-04

1.52

O-methyltransferase

Os09g0344500

3.68

7.4E-04

1.23

4.5E-02

AAA-type ATPase

Os09g0445700

31.09

2.4E-06

1.15

1.1E-01

O-methyltransferase

Os10g0118000

4.39

1.2E-04

1.50

9.3E-03

O-methyltransferase

Os10g0118200

6.36

1.1E-04

1.30

5.6E-02

protein kinase

Os11g0274700

5.00

1.4E-04

1.95

3.7E-03

Disease resistance protein

Os11g0491600

59.47

1.6E-06

1.08

9.1E-01

Cytochrome P450

Os02g0601400

5.35

9.6E-05

2.91

3.8E-04

Phytosulfokine

Os03g0232400

3.69

5.0E-04

2.85

1.9E-04


798 Mark C.F.R. Redillas et al. Table 1 (Continued) Loc no* Gene name

(IRGSP)

RCc3:OsNAC9

GOS2:OsNAC9

Mean†

P valueà

Mean†

P valueà

Aldo ⁄ keto reductase

Os05g0456100

3.89

1.9E-04

2.37

5.9E-04

Aldo ⁄ keto reductase

Os05g0456200

3.40

2.7E-04

2.57

3.8E-04

Integral membrane protein

Os06g0218900

3.12

4.2E-04

2.37

5.8E-04

WAK1

Os04g0370900

3.03

8.5E-04

2.20

1.6E-03

Leucine-rich repeat protein kinase

Os08g0201700

3.53

3.5E-04

2.26

9.9E-04

Germin-like protein 9

Os12g0154800

3.01

2.8E-04

2.99

1.5E-04

Up-regulated genes in GOS2:OsNAC9 Aminotransferase

Os01g0729600

1.54

1.0E-01

8.41

1.6E-04

Xyloglucosyl transferase

Os02g0280300

)2.09

1.1E-02

4.44

3.2E-04

Cinnamoyl CoA reductase 1

Os02g0808800

)1.23

4.7E-01

9.51

6.4E-05

Downy mildew resistant 6

Os03g0122300

1.78

5.7E-03

3.01

2.5E-04

Proline extensin-like receptor kinase 1

Os03g0269300

1.76

9.8E-03

5.31

6.7E-05

WRKY1

Os03g0335200

1.78

2.9E-02

3.28

6.7E-04

Salt tolerance zinc finger

Os03g0437200

)1.29

7.6E-01

3.65

1.2E-03

AAA-ATPase 1

Os03g0802400

1.44

1.5E-01

3.13

4.6E-03

Hydrolase

Os04g0411800

1.81

9.9E-03

3.30

2.8E-04

Hydrolase

Os04g0412000

1.55

3.0E-02

3.05

3.8E-04

Membrane bound O-acyl transferase

Os04g0481800

1.92

2.4E-02

4.22

5.2E-04

Cinnamyl alcohol dehydrogenase 6

Os04g0612700

)1.33

3.7E-01

5.27

1.4E-04

Leucine-rich repeat

Os04g0621900

)0.05

5.0E-01

4.18

1.9E-04

Phosphofructokinase 3

Os05g0194900

1.73

6.7E-03

4.42

7.8E-05

Pyruvate decarboxylase

Os05g0469600

1.61

1.5E-03

4.65

5.5E-05

L-lactate dehydrogenase

Os06g0104900

1.67

1.1E-02

4.17

1.2E-04

Disease resistance protein

Os06g0279900

)1.18

1.8E-01

4.54

6.2E-05

FAD-binding domain-containing protein

Os06g0548200

2.00

3.4E-03

3.78

1.4E-04

Universal stress protein

Os07g0673400

1.89

6.9E-03

4.39

1.3E-04

Terpene synthase ⁄ cyclase

Os08g0167800

1.89

2.1E-02

3.74

3.2E-04

Acidic endochitinase

Os08g0518900

1.95

4.8E-03

4.03

1.4E-04

Pin-formed 5

Os08g0529000

0.17

4.3E-01

4.10

5.0E-04

Calcium-binding EF

Os09g0483100

)0.08

9.4E-01

6.22

2.0E-04

Calcium-binding EF hand

Os09g0483300

1.39

1.2E-01

3.54

8.5E-04

Purple acid phosphatase 3

Os10g0116800

1.46

2.1E-01

3.96

5.2E-04

Phosphoenolpyruvate carboxykinas 1

Os10g0204400

1.53

5.4E-02

4.42

1.8E-04

HAT dimerization domain-containing protein

Os10g0567900

)0.11

6.0E-01

3.56

2.0E-04

Acidic endochitinase

Os11g0701000

)1.27

6.5E-01

3.03

2.5E-03

*Sequence identification numbers for the full-length cDNA sequences of the corresponding genes. †

The mean of duplicate biological samples.

à

P values were analysed by one-way ANOVA (P < 0.01).

Genes discussed in the text are in boldface. These microarray data sets can be found at http://www.ncbi.nlm.nih.gov/geo/ (Gene Expression Omnibus, accession number GSE31855).

example, the Cinnamoyl CoA reductase was up-regulated in both RCc3:OsNAC9 and GOS2:OsNAC9, whereas it was up-regulated only in RCc3:OsNAC10 (Figure 4 and Table S4).

Discussion In our previous study, 18 NAC domain factors were identified from the expression profiling of stress-treated rice plants (Jeong et al., 2010). An alignment of these factors revealed three closely related subgroups that were suggestive of a common stress response function. OsNAC9 ⁄ SNAC1, a member of subgroup I, was selected for further analysis in our current study and overexpressed under the control of the root-specific promoter RCc3

(RCc3:OsNAC9) and the constitutive promoter GOS2 (GOS2: OsNAC9). Three independent homozygous lines expressing each of these constructs were evaluated in the field for six cultivating seasons and results from the last 2 years are reported here, that is, T5 (2009) and T6 (2010) generations. Under normal growth conditions, both transgenic lines showed higher grain yield compared with NT controls. The improvement in the total grain weight of the RCc3:OsNAC9 plants was found to be due mainly to the increase in the panicle length, whereas that of the GOS2:OsNAC9 plants was found to be due to a number of traits including panicle length, number of panicles and number of spikelets. Under drought conditions, in contrast, RCc3: OsNAC9 plants showed a significantly enhanced total grain


OsNAC9 improves drought resistance and grain yield in rice 799 OsNAC9

OsNAC10 30

60

Relative mRNA level

48.59 50

25

40

20 15

30

10.44

16.50

20

10 5

10

1.00 0.98 1.05

1.91 1.70

1.00

0

0

WAK1

WAK3 12

16

WAK5 16

9.26

12.62

14

10

12

14

8.70

8

8

10

6

6 2.27 1.25

1.00

0

6 2.75

2.07

2 1.00

NCED 131.16

120

25

20

11.00

4.33

1.00 0

O-methyltransferase Os10g0118000 9.66

O-methyltransferase Os10g0118200 31.00

25

15

0

weight (by 28%–72%) due mainly to an increased filling rate, whilst GOS2:OsNAC9 plants showed no significant changes in either trait.

10

10 0.43

0.82

5 1.00 0

20.14

15

20

1.03

30

20

30

4

1.75

O-methyltransferase Os09g0344500

25

35

6

5 1.00 0

40

8

2 1.00

45

15 10

20

1.00

10

Figure 4 Regulated genes in roots of OsNAC9 and OsNAC10 plants under normal conditions. The transcript levels of OsNAC9, OsNAC10 and nine up-regulated genes were determined by qRT-PCR (using the primers listed in Table S5) and each of transgenic rice plants are presented as a relative concentration to the levels in untreated NT control roots. Data were normalized using the rice ubiquitin gene (OsUbi) transcript levels. Values are the means ± SD of three independent experiments.

37.10

40

0

12

20

60 27.33

27.40

30

80

40

29.55

35

80 60

38.07

40

103.72

100

100

0.87

Cinnamoyl-CoA reductase 45

126.20

107.95

120

2.15

0

Calcium-transporting ATPase 140

140

4 2 1.00

0

160

6.75

8

4

4

13.47

12

8.91

10

2

22.71

1.73

2.22 2.60

5

2.52 1.00

1.96

2.43

0

The potential of using NAC genes for plant yield improvement has received little previous attention owing to roles of these factors in plant fertility, growth and development.


800 Mark C.F.R. Redillas et al. Because of these complex roles, the overexpression of NAC needs to be limited to specific tissues to avoid unwanted pleiotropic effects. In fact, the whole-body overexpression of OsNAC6 has been shown to result in growth retardation and low productivity, although the drought resistance of these plants is enhanced (Nakashima et al., 2007). The results of our current study indicate that the root-specific overexpression of OsNAC9 plays an important role in the improvement of rice yields, particularly under drought conditions. To our knowledge, field tests of up to six generations of transgenic plant lines specifically overexpressing a stress-inducible NAC TF have not been reported previously. This long-term evaluation of transgenic plants for drought resistance provides a higher confidence in the validity of the reported yield traits. This is because the prescreening of T1 through T4 progeny eliminates the contribution of somaclonal variations because the negative effects brought about by tissue culture occur mostly during early generations in transgenic plants (Xiao et al., 2007). The RCc3:OsNAC9 and GOS2:OsNAC9 plants at T5 or later generations did not show any unwanted pleiotropic effects such as growth retardation, abnormal leaf shape and colour, or panicle underdevelopment, which were likely segregated out during the pre-screening at earlier generations if they had arisen. Thus, the changes in the responses exhibited by RCc3:OsNAC9 and GOS2:OsNAC9 plants at T5-6 in comparison with the NT controls were contributed solely by the transgene. The root characteristics of RCc3:OsNAC9 plants at the heading stage of reproduction showed an increase in the root diameter as compared to those of the NT controls and GOS2:OsNAC9 plants. This increase was apparently owing to an enlarged xylem, and larger cortical cells and epidermis. In several reports, the vessel diameter is closely and positively correlated with better water flux and bigger xylem poses a lower risk of cavitation (Vasellati et al., 2001; Yambao et al., 1992). Also, the increased root cortical aerenchyma enhances drought resistance by reducing the root metabolic costs, permitting greater root growth and water acquisition from drying soil. Zhu et al. (2010) reported that the relative water content of mid-day leaf in the high root cortical aerenchyma lines is 10% greater than in the low root cortical aerenchyma lines under water stress. Thus, in this study, the increased root diameter in the RCc3: OsNAC9 plants might have mitigated the effects of drought stress through better water flux, lower risk of cavitation and reduced root metabolic costs, allowing more spikelets to develop and flower normally. In addition, larger roots have a direct impact on improved drought resistance because the large size of root diameter is related to penetration (Clark et al., 2008; Nguyen et al., 1997) and branching (Fitter, 1991; Ingram et al., 1994) ability. Previously, we reported that the rootspecific overexpression of OsNAC10 resulted in an increased root diameter, thereby enhancing drought resistance and increasing the grain yield under drought conditions (Jeong et al., 2010). In addition, the allocation of resources towards the root is high at the vegetative stage and decreases markedly during flowering (Gregory et al., 1997). These observations suggest that the development of larger roots is favourable for drought resistance breeding. Previously, deeper roots and increased root length of upland rice (Kamoshita et al., 2008) and smaller rather than larger root diameter of wheat (Richards and Passioura, 1989) had been implicated in drought resistance, conferring a sharp contrast to our results. Unlike upland rice and wheat, our background cultivar Nipponbare is lowland rice.

Most (69%–94%) of its roots is located in the top 10 cm of soil and very few roots are founded below 30 cm (Gowda et al., 2011). This difference in root growth may explain why the increased root diameter of our transgenic rice plants still account for the increased drought resistance. It is also possible that increased number of filled spikelets in RCc3:OsNAC9 may be due to maintenance of open stomata under drought, hence continuous supply of carbohydrates to spikelets and higher enzyme activities, which might have assisted fertilization thus reducing spikelet sterility (Kamoshita et al., 2008). Results from our microarray analysis revealed 40 up-regulated genes common to the roots of both RCc3:OsNAC9 and GOS2:OsNAC9 (Table 1). In addition, 17 and 28 up-regulated genes were found to be specifically up-regulated in RCc3:OsNAC9 and GOS2:OsNAC9 roots, respectively (Table 1). The common genes in this regard included 9-cis-epoxycarotenoid dioxygenase, calcium-transporting ATPase and cinnamoyl CoA reductase 1. The oxidative cleavage of cis-epoxycarotenoids by 9-cis-epoxycarotenoid dehydrogenase (NCED) to generate xanthoxin is the critical and rate-limiting step in the regulation of ABA biosynthesis (Tan et al., 1997). The NCED gene was found to be up-regulated by more than 20-fold in both the RCc3:OsNAC9 and GOS2:OsNAC9 transgenic plants, which may have contributed to the sensitivity of these plants to drought stress. The Ca2+-transporting ATPase (Ca2+-ATPase) gene was up-regulated by 26- and 32-fold in RCc3:OsNAC9 and GOS2:OsNAC9 plants, respectively. A transient increase in cytosolic Ca2+, derived from either an influx via the apoplastic space or release from internal stores, serves as an early response to low temperature, drought and salinity stress in plant cells (Knight, 2000). Coupled with the increase in cytosolic Ca2+ is the rupture of tonoplasts that also indicate early events preceding the death of root cortical cells, followed by the formation of the aerenchyma—the gas filled spaces in the cortical region of the roots. The aerenchyma serves as an anatomical adaptation in rice that helps to minimize the loss of O2 to the surrounding soil so that it is available for respiration by the apical meristem. These structures include a suberized hypodermis and a layer of lignified cells immediately interior to the hypodermis, both of which are only slightly gas permeable (Drew et al., 2000). Interestingly, cinnamoyl CoA reductase (CCR), a gene encoding a key enzyme (EC 1.2.144) of the lignin biosynthesis pathway, was found to be up-regulated in RCc3:OsNAC9 and GOS2:OsNAC9 plants following OsNAC9 overexpression. CCR is the first enzyme in this biosynthetic pathway that leads to the production of monolignols including P-coumaryl, coniferyl and sinapyl alcohols, and thereby controls the quantity and quality of lignin (Jones et al., 2001; Kawasaki et al., 2006). In maize, a close relative of rice, root developments are inhibited by severe drought stress owing to cessation of root cell wall extension in elongation regions (Yamaguchi and Sharp, 2010). Lignifications were found to increase in drought-stressed roots, decreasing the extensibility of the cell wall. The increased lignifications of epidermis and xylem, in particular, were reported to restrict water loss from the root and also to facilitate longitudinal water transport in soybean (Yamaguchi et al., 2010). Downregulation of the AtCCR1 gene, an Arabidopsis homologue, causes drastic phenotypic alterations (Goujon et al., 2003). In addition, a lossof-function mutation of this gene in maize (Zmccr1) ⁄ )) results in a slight decrease in the lignin content and causes significant changes to the lignin structure (Tamasloukht et al., 2011). The


OsNAC9 improves drought resistance and grain yield in rice 801 maize gene ZmCCR2 has been found to be induced by drought conditions and can be detected mainly in roots (Fan et al., 2006). Along with CCR, cinnamyl alcohol dehydrogenase (CAD), another gene encoding a lignin biosynthesis enzyme, was also found to be up-regulated in both the RCc3:OsNAC9 and GOS2:OsNAC9 plants. CAD catalyses the final conversion of hydroxycinnamoyl aldehydes (monolignals) to monolignols in the lignin biosynthesis pathway (Sattler et al., 2009). Furthermore, WAKs that are involved in cell development and morphogenesis were also up-regulated by OsNAC9 overexpression. WAKs contain an extracellular domain within the cell wall and a cytoplasmic serine ⁄ threonine protein kinase domain (Anderson et al., 2001) that serves as the physical link between the cell wall and plasma membrane (Deeks et al., 2002). Antisense experiments show that WAK expression is crucial for cell elongation, morphogenesis and plant development (Lally et al., 2001; Wagner and Kohorn, 2001). Interestingly, O-methyltransferase, a gene encoding an enzyme (EC=2.1.1) involved in lignin and ⁄ or suberin biosynthesis, is specifically up-regulated in RCc3:OsNAC9 plants. In Arabidopsis, transcripts of ZRP4, a gene that encodes an O-methyltransferase, were found to accumulate preferentially in the roots and localize predominantly in the endodermis region with low levels detectable in the leaves, stems and other shoot organs (Held et al., 1993). The up-regulation of three O-methyltransferase genes may have contributed to the enhanced drought resistance of RCc3:OsNAC9 plants over both GOS2:OsNAC9 and NT plants owing to their involvement in lignin and ⁄ or suberin biosynthesis. Lignin and suberin play major roles in impeding radial oxygen loss through lignification and ⁄ or suberization of the walls of the root peripheral layers in a process known as barrier formation. This process on the radial and transverse walls of endo- and exodermal cells forms the water impermeable Casparian strips (CSs). The main function of the endodermal CSs is to inhibit water and salt transport into the stele by blocking the selective apoplastic bypass in the root (Ma and Peterson, 2003), whereas the exodermal CSs blocks apoplastic transport at the root surface (Baxter et al., 2009; Takehisa et al., 2012). Cai et al. (2011) have reported that the development of CSs on the endodermis and exodermis in saltand drought-resistant Liaohan 109 rice plants occurs at an earlier stage than the moderately salt-sensitive Tianfeng 202 or salt-sensitive Nipponbare cultivars. The group also reported that even without salt in the nutrient solution, the development of CSs in Liaohan 109 occurs earlier and at an increased level. Collectively, such up-regulated genes that were commonly or specifically up-regulated in RCc3:OsNAC9 roots contributed to the enhanced drought resistance of the transgenic plants. Because the increase in root diameter of RCc3:OsNAC9 showed similarity to that of RCc3:OsNAC10 roots that we previously reported (Jeong et al., 2010), the up-regulated genes specific to RCc3:OsNAC9 or GOS2OsNAC9 plants were almost entirely different from those of RCc3OsNAC10 or GOS2: OsNAC10 (Figure 4 and Table S4). This suggests that the mechanism involved in altering the root architecture between OsNAC9 and OsNAC10 transgenic plants were different but both conferring drought resistance. Together, our current study provides novel insights into how drought resistance is acquired by transgenic plants following the OsNAC9 overexpression. In summary, we presented the results of a long-term field trial of transgenic rice overexpressing OsNAC9 and the responses of these plants to drought stress. We demonstrated that the root-specific overexpression of OsNAC9 increased the

root diameter owing to an enlarged xylem and augmented cortical cell size relative to that of the NT controls. These changes in the root architecture were also coupled with the up-regulation of genes involved in signal transduction (ABA and Ca2+), barrier formation (lignin and suberin biosynthesis), and cell development and morphogenesis, which aided the plants in surviving drought stress. Our findings revealed that the root-specific overexpression of OsNAC9 enhanced the drought resistance of rice plants at the reproduction stage, resulting in improved grain yield under drought conditions.

Experimental procedures Plasmid construction and transformation of rice The coding region of OsNAC9 (AK067690) was amplified from total RNA preparations using the primer pairs forward 5¢-ATGGGGATGGGGATGAGGAG-3¢ and reverse 5¢-TCAGAACGGGACCATGCCCA-3¢ using the RT-PCR system (Promega, Madison, WI) according to the manufacturer’s instructions. For overexpression in rice, the cDNA for OsNAC9 was linked to the GOS2 promoter for constitutive expression and to the RCc3 promoter for root-specific expression using the Gateway system (Invitrogen, Carlsbad, CA). Plasmids were introduced into Agrobacterium tumefaciens LBA4404 by triparental mating, and embryogenic (Oryza sativa cv Nipponbare) calli from mature seeds were transformed as described by Jang et al. (1999).

Drought treatments of vegetative stage rice plants in a glasshouse Seeds from transgenic and NT rice (O. sativa cv Nipponbare) plants were germinated in half-strength MS solid medium and placed in a dark growth chamber at 28 C for 4 days. Seedlings were subsequently transplanted into soil and grown in a greenhouse (16-h light ⁄ 8-h dark cycles) at 28–30 C. Before undertaking drought stress experiments, 18 seedlings from each transgenic and NT line were grown in pots (3 · 3 · 5 cm; 1 plant per pot) for 4 weeks. Drought stress was simulated by withholding water for 5 days whilst recovery tests were performed by re-watering and observing the drought-stressed plants for 7 days. The numbers of plants that survived or continued to grow were then scored.

Measurement of chlorophyll fluorescence under stress conditions Seeds from transgenic and NT rice (O. sativa cv Nipponbare) plants were germinated and grown in half-strength MS solid medium for 14 days. The light and dark settings of the growth chamber were 16-h light of 150 lmol ⁄ m2 ⁄ s per 8-h dark cycles at 28 C. The green portions of approximately ten seedlings were then cut using a scissors prior to stress treatments in vitro. All stress conditions were conducted under continuous light at 150 lmol ⁄ m2 ⁄ s. For low-temperature stress administration, the seedlings were incubated at 4 C in water for up to 6 h. Highsalinity stress was induced by incubation in 400 mM NaCl for 2 h at 28 C. To simulate drought stress, the plants were airdried for 2 h at 28 C. Fv ⁄ Fm values were then measured as previously described (Oh et al., 2005; Redillas et al., 2011a,b).

Rice 3¢-tiling microarray analysis A rice 3¢-tiling microarray was used for expression profiling analysis as previously described (Oh et al., 2009). Transgenic


802 Mark C.F.R. Redillas et al. and NT rice (O. sativa cv Nipponbare) seeds were germinated in soil and grown in a glasshouse (16-h light ⁄ 8-h dark cycle) at 22 C. To identify stress-inducible NAC genes in rice, total RNA (100 lg) was prepared from 14-day-old leaves of plants subjected to drought, high-salinity, ABA and low-temperature stress conditions. For the high-salinity and ABA treatments, the 14-day-old seedlings were transferred to a nutrient solution containing 400 mM NaCl or 100 lM ABA for 2 h in the greenhouse under continuous light of approximately 1000 lmol ⁄ m2 ⁄ s. For drought treatment, 14-day-old seedlings were air-dried for 2 h also under continuous light of approximately 1000 lmol ⁄ m2 ⁄ s. For low-temperature treatments, 14-day-old seedlings were exposed at 4 C in a cold chamber for 6 h under continuous light of 150 lmol ⁄ m2 ⁄ s. For the identification of genes up-regulated in RCc3:OsNAC9 and GOS2:OsNAC9 plants, total RNA (100 lg) was prepared from root and leaf tissues of 14-day-old transgenic and NT rice seedlings (O. sativa cv Nipponbare) grown under normal growth conditions.

Grain yield evaluation of rice plants grown in the field To evaluate the yield components of transgenic rice plants under normal field conditions, three independent T5 (2009) and T6 (2010) homozygous lines of RCc3:OsNAC9 and GOS2: OsNAC9 transgenic plants, together with NT controls, were transplanted to a rice paddy field at the Rural Development Administration, Suwon (127:01E ⁄ 37:16N), Korea (2009), and subsequently at the Kyungpook National University, Gunwi (128:34E ⁄ 36:15N), Korea (2010). A randomized design was employed with three replicates for the two cultivating seasons using three plots each with the size of 5 m2 per plot. A total of 100 plants per genotype were sown on 20 April 2009 in Suwon and on 23 April 2010 in Gunwi. For each plot, 22 seedlings per line were then randomly transplanted with a 15 · 30 cm spacing 25 days after sowing. Fertilizer was applied at 70N ⁄ 40P ⁄ 70K kg ⁄ ha after the last paddling and 45 days after transplantation. Yield parameters were scored for ten plants per plot for a total of 30 plants per line per season. Plants located at the borders were excluded from data scoring. To evaluate the yield components of transgenic plants under drought field conditions, three independent T5 (2009) and T6 (2010) homozygous lines of each of the RCc3:OsNAC9 and GOS2:OsNAC9 plants and NT controls were transplanted to a 1-meter-deep container filled with natural paddy soil under a removable rain-off shelter in Myongji University, Yongin (127:14E ⁄ 37:14N), Korea. A completely randomized design, transplanting distance and use of fertilizer were employed similarly as described previously for normal field conditions. Intermittent type of drought stress was applied during the panicle heading stage (from 10 days before heading to 10 days after heading) by draining the water through the bottom of the container. When the complete leaf rolling had occurred following the first exposure to drought stress, they were irrigated overnight and subjected again to a second round of drought stress until another complete leaf rolling occurred. After exposure to drought stress, the polyvinyl roofs were removed and the plants were irrigated until harvesting. Scoring of agronomic traits was followed as described previously (Oh et al., 2009). The agronomic traits were scored as: panicle length (cm), number of panicles per hill, number of spikelets per panicle, number of spikelets per hill, filling rate (%), number of filled spikelets per hill, total grain weight (g)

and 1000 grain weight (g). The results from three independent lines were analysed by one-way ANOVA and compared with those of the NT controls. ANOVA was used to reject the null hypothesis of equal means of transgenic lines and NT controls (P < 0.05) using SPSS version 16.0 (SPSS Inc., Chicago, IL).

Evaluation of root traits To observe the root traits, RCc3:OsNAC9, GOS2:OsNAC9 plants and NT controls were transplanted to five PVC tubes (1.2 m in length and 0.2 m in diameter) that were filled with natural paddy soil and placed in a 1.5-m-deep container located at Myongji University, Yongin, Korea. Only one seedling was transplanted per tube 25 days after sowing. Fertilizer was employed similarly as described for normal field conditions. Root observations were conducted before heading stage. PVC tubes were taken out from the container and removed the soil carefully. For each plant, only the longest root was used for measuring the length, whilst the total roots were used for measuring the root volume per plant. For the root diameter, ten roots per plant were measured, and the total roots per plant were used for the dry weight. SPSS version 16.0 was used for statistical analysis.

Microscopic examination of roots Roots of transgenic and NT plants at the panicle heading stage were fixed with a modified Karnovsky’s fixative at 4 C overnight and washed with the same buffer three times for 10 min each. They were then post-fixed in the same buffer at 4 C for 2 h and briefly washed twice with distilled water. The post-fixed root tissues were then en bloc stained at 4 C overnight and dehydrated in a graded ethanol series (30, 50, 70, 80, 95 and 100%) followed by three treatments in 100% ethanol for 10 min each. Dehydrated samples were further treated with propylene oxide as a transitional fluid twice for 30 min each and embedded in Spurr’s medium. Ultrathin sections (approximately 1 lm thick) were made with a diamond knife using an ultra-microtome (MT-X; RMC Inc., Tucson, AZ). The sections were stained with 1% toluidine blue, observed and photographed under a light microscope.

qPCR analysis Total RNA was prepared as previously reported (Jung et al., 2011). For quantitative real-time PCR experiments, a SuperScriptTM III Platinum One-Step Quantitative RT-PCR system (Invitrogen) was used. For PCRs, a master mix of reaction components was prepared according to the methods reported by Redillas et al. (2012) and using a Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). Thermocycling and fluorescence detection were performed using a Stratagene Mx3000p Real-Time PCR machine (Stratagene, La Jolla, CA) set at the following, 1 cycle of 95 C for 10 min followed by 40 cycles of 94 C for 30 s, 58 C for 40 s and 68C for 1 min. Analysis was performed in triplicates to validate the qPCR results. The primer pairs are listed in the Table S5.

Acknowledgements This study was supported by the Rural Development Administration under the ‘Cooperative Research Program for Agriculture Science & Technology Development’ (Project No. PJ906910), the Next-Generation BioGreen 21 Program (Project No.


OsNAC9 improves drought resistance and grain yield in rice 803 PJ007971 to J.-K.K., PJ008053 to Y.D.C. and PJ006834 to S.-H.H.) and by the Ministry of Education, Science and Technology under ‘Mid-career Researcher Program’ (Project No. 20100026168 to J.-K.K.).

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Figure S1 Expression of OsNAC9 in response to various stress conditions in rice. Table S1 Analysis of seed production parameters in RCc3: OsNAC9 and GOS2:OsNAC9 transgenic rice plants grown under normal conditions in 2009 and 2010. Table S2 Analysis of seed production parameters in RCc3: OsNAC9 and GOS2:OsNAC9 transgenic rice plants grown under drought stress conditions in 2009 and 2010. Table S3 Seed production parameters of non-stressed NT (Nipponbare) control grown in Yongin and Suwon in 2009. Table S4 Comparison of OsNAC9 up-regulated genes with OsNAC10 up-regulated genes. Table S5 Primers used for qPCR. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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