Root specific expression of OsNAC10 improves drought tolerance and grain yield in rice under field drought conditions 1

Drought poses a serious threat to the sustainability of rice yields in rainfed agriculture. Here we report the results of a functional genomics approach that identified a rice NAC-domain gene, OsNAC10 , which improved performance of transgenic rice plants under field drought conditions. Of the 140 OsNAC genes predicted in rice, 18 were identified to be induced by stress conditions. Phylogenic analysis of the 18 OsNAC genes revealed the presence of 3 subgroups with distinct signature motifs. A group of OsNAC genes were pre-screened for enhanced stress tolerance when overexpressed in rice. The OsNAC10 , one of the effective members selected from pre-screening, is expressed predominantly in roots and panicles, and induced by drought, high salinity and abscisic acid. Overexpression of OsNAC10 in rice under the control of the constitutive promoter GOS2 and the root-specific promoter RCc3 increased the plant tolerance to drought, high salinity and low temperature at the vegetative stage. More importantly, the RCc3:OsNAC10 plants showed significantly enhanced drought tolerance at the reproductive stage, increasing grain yield by 25-42% and 5-14% over controls in the field under drought and normal conditions, respectively. Grain yield of GOS2:OsNAC10 plants in the field, in contrast, remained similar to that of controls under both normal and drought conditions. These differences in performance under field drought conditions reflect the difference in expression of OsNAC10 -dependent target genes in roots as well as in leaves of the two transgenic plants, as revealed by microarray analyses. Root diameter of the RCc3:OsNAC10 plants was thicker by 1.25-fold than that of the GOS2:OsNAC10 and NT plants due to the enlarged stele, cortex and epidermis. Overall, our results demonstrated that root specific overexpression of OsNAC10 enlarges roots, enhancing drought tolerance of transgenic plants, which increases grain yield significantly under field drought conditions. (2009) homozygous lines of the RCc3:OsNAC10 and GOS2:OsNAC10 plants, together with non-transgenic (NT) controls were transplanted to a paddy field at the Rural Development Administration, Suwon, Korea. A randomized design was employed with two (2008) and three (2009) replicates. At 25 d after sowing, the seedlings were randomly transplanted within a 15 X 30 cm spacing and single seedling per hill. Fertilizer was applied at 70N/40P/70K kg ha -1 after the last paddling and 45 d after transplantation. Yield parameters were scored for 20 (2008) and 30 (2009) plants per Dip1 gene which was used as a marker for up-regulation of key genes following stress treatments. Ethidium bromide staining was used to determine equal loading of RNAs. the


INTRODUCTION
Plants respond and adapt to abiotic stresses to survive under adverse conditions. Upon exposure of plants to such stresses, many genes are induced and their products are involved in the protection of cellular machinery from stress-induced damage (Bray, 1993;Thomashow, 1999;Shinozaki et al., 2003). The expression of stress-related genes is largely regulated by specific transcription factors. The overexpression of such transcription factor genes often results in activation of many functional genes related to the particular stress conditions, consequently conferring stress tolerance. For example, the DREB1A/CBF3 gene in transgenic Arabidopsis activates expression of its stress-related downstream genes thereby enhancing stress tolerance (Liu et al., 1998;Kasuga et al., 1999).
The rice and Arabidopsis genomes each encode more than 1300 transcriptional regulators, which account for 6% of the estimated total number of genes in each plant. About 45% of these transcription factors are reported to be from gene families specific to plants (Riechmann et al., 2000;Kikuch et al., 2003). One example of such a plant-specific family of transcription factors is the NACs. The NAC acronym is derived from the names of the three genes that were first described as containing a NAC domain, namely NAM (no apical meristem), ATAF1,2 and CUC2 (cup-shaped cotyledon). The NAC domain is a highly conserved N-terminal DNA-binding domain, and NAC proteins also contain a variable Cterminal domain (Ooka et al., 2003) and appear to be widespread in plants. The genomes of rice and Arabidopsis were initially predicted to contain 105 and 75 NAC genes, respectively (Ooka et al., 2003;Xiong et al., 2005). Later, a total of 140 NAC genes were identified in rice (Fang et al., 2008). However, only a few of these genes have been characterized so far and show diverse functions in both plant development and stress responses. The earliest reported 6 and ANAC102 from Arabidopsis (Fujita et al., 2004;Tran et al., 2004;Christianson et al., 2009), BnNAC from Brassica napus (Hegedus et al., 2003), and SNAC1 and SNAC2 from rice (Hu et al., 2006(Hu et al., , 2008 have been found to be involved in responses to various environmental stresses. AtNAC2, another stress-related NAC gene in Arabidopsis, functions downstream of the ethylene and auxin signal pathways and enhances salt tolerance when overexpressed (He et al., 2005). A wheat NAC gene, NAM-B1, has been reported to be involved in nutrient remobilization from leaves to developing grains (Uaury et al., 2006).
Drought is one of the major constraints to rice production worldwide. In particular, exposure to drought conditions during the panicle development stage results in a delayed flowering time, reduced number of spikelets and poor grain filling. To date, a number of studies have suggested that the overexpression of stress related genes may improve drought tolerance in rice to some extent (Xu et al., 1996;Garg et al., 2002;Jang et al., 2003;Ito et al., 2006;Hu et al., 2006Hu et al., , 2008Nakashima et al., 2007;Oh et al., 2007). However, despite a number of such efforts to develop drought-tolerant rice plants, very few have shown an improvement in grain yields under field conditions. These include transgenic rice plants expressing SNAC1 (Hu et al., 2006), OsLEA3 (Xiao et al., 2007) and AP37 (Oh et al., 2009).
In our current study, a genome-wide analysis of rice NAC transcription factors was conducted to identify genes that improve tolerance to environmental stress. A group of OsNAC genes were pre-screened for enhanced stress tolerance when overexpressed in rice.
Here, we report the role of OsNAC10, one of the effective members selected from the prescreening, in drought tolerance. Overexpression of OsNAC10 under the control of GOS2 (de Pater et al., 1992), a constitutive promoter, and RCc3 (Xu et al., 1995), a root-specific promoter, improved plant tolerance of transgenic rice to drought, high salinity and low temperature during the vegetative stage of growth. In addition, RCc3:OsNAC10 plants showed significantly enhanced drought-tolerance at the reproductive stage, with a grain yield increase of 25-42% over the controls under field drought conditions. Grain yield of GOS2:OsNAC10 plants under the same conditions, in contrast, remained relatively unchanged, demonstrating the potential use of the root-specific expression strategy for improving drought tolerance in rice.

Identification of Stress-Inducible Rice NAC-Domain Genes
Previously, the rice genome was predicted to contain 140 OsNAC genes (Fang et al., 2008).
To identify those that are stress-inducible, we performed expression profiling with the Rice 3'-Tiling microarray (GreenGene Biotech, Yongin, Korea). We obtained RNAs from the leaves of 14-d-old rice seedlings that had been subjected to drought, high-salinity, abscisic acid (ABA), and low-temperature. When three replicates were averaged and compared with untreated leaves, a total of 18 OsNAC genes were found to be up-regulated by 1.9-fold or greater (P<0.05) upon exposure to one or more stress conditions (Table I). Phylogenic analysis of the amino acid sequences of the corresponding 18 OsNAC proteins revealed the presence of 3 subgroups (I to III) ( Fig. 1; Supplemental Fig. S1). Furthermore, comparison of the amino acid sequences spanning the NAC domains in these proteins revealed signature motifs by which these subgroups can be distinguished (Fig. 1). For example, signature motifs Ia-c and IIa-c are specific to subgroups I and II, respectively. In addition to sequence similarities, the members of each subgroup were found to be closely related in terms of their response to stress. For example, the expression of the genes in subgroup II and III is not induced by low temperature.
One of the stress-responsive OsNAC genes, OsNAC10 (AK069257), which is in subgroup I, was functionally characterized in our present study. RT-PCR analysis of this gene in various tissues at different stages of development revealed that it is predominantly expressed in roots and flowers (panicles) ( Fig. 2A). We also performed RNA gel-blot analysis using total RNAs from leaf tissues of 14-d-old seedlings exposed to high salinity, drought, ABA and low temperature (Fig. 2B). Consistent with the results from our microarray experiments, the expression of OsNAC10 was induced by drought, high salinity and ABA, but not by low temperature.

Stress Tolerance of RCc3:OsNAC10 and GOS2:OsNAC10 Plants at the Vegetative Stage
To enable the overexpression of the OsNAC10 genes in rice, their full-length cDNAs were isolated and linked to the RCc3 promoter (Xu et al., 1995) for root-specific expression, and to the GOS2 promoter (de Pater et al., 1992) for constitutive expression, thereby generating the constructs RCc3:OsNAC10 and GOS2:OsNAC10. These constructs were introduced into rice 8 using Agrobacterium-mediated transformation (Hiei et al., 1994), which yielded 15 to 20 independent transgenic lines per construct. Transgenic T 1-5 seeds were collected and three independent T 4 homozygous lines of both RCc3:OsNAC10 and GOS2:OsNAC10 plants were selected for further analysis. All of the transgenic lines grew normally with no stunting. The transcript levels of OsNAC10 in the RCc3:OsNAC10 and GOS2:OsNAC10 plants were determined by RNA-gel blot analysis. For this purpose, total RNAs were extracted from leaf and root tissues of 14-d-old seedlings grown under normal growth conditions (Fig. 3A).
OsNAC10 expression was clearly detectable in all of the transgenic lines but not in the nontransgenic (NT) controls. The transgenes were expressed at high levels in both the leaves and roots of the GOS2:OsNAC10 plants but in the roots only of the RCc3:OsNAC10 plants, thus verifying the constitutive and root-specific nature of the respective promoters.
To investigate whether the overexpression of OsNAC10 correlated with stress tolerance in rice, four-week-old transgenic plants and NT controls were exposed to drought stress (Fig. 3B). The NT plants started to show visual symptoms of drought-induced damage, such as leaf rolling and wilting with a concomitant loss of chlorophylls, at an earlier stage than the RCc3:OsNAC10 and GOS2:OsNAC10 plants. The transgenic plants also recovered more quickly than the NT plants upon re-watering. Consequently, the NT plants remained severely affected by drought stress at the time at which some of the transgenic lines had partially recovered (Fig. 3B). To further verify this stress-tolerance phenotype, we measured the Fv/Fm values of the transgenic and NT control plants, all at the vegetative stage (Fig. 3C).
These values represent the maximum photochemical efficiency of PS II in a dark-adapted state (Fv, variable fluorescence; Fm, maximum fluorescence) and were found to be 15-30% higher in the RCc3:OsNAC10 and GOS2:OsNAC10 plants compared with the NT plants under drought and low temperature conditions. Under moderate salinity conditions, the Fv/Fm levels were also higher in both transgenic plants by 10% compared with the NT controls. In contrast, under severe salinity conditions, these levels were similar in all plant types. Our results thus indicate that the overexpression of OsNAC10 in transgenic rice primarily increases their tolerance to drought and low temperature stress conditions during the vegetative stage of growth.

Identification of Genes Up-Regulated by Overexpressed OsNAC10
To identify genes that are up-regulated by the overexpression of OsNAC10, we performed 9 expression profiling of the GOS2:OsNAC10 and RCc3:OsNAC10 plants in comparison with NT controls under normal growth conditions. This profiling was conducted using the Rice 3'-Tiling microarray with RNA samples extracted from 14-d-old roots and leaves of each plant, all grown under normal growth conditions. Each data set was obtained from three biological replicates. As listed in Table II, Supplemental Tables S1and S2, statistical analysis of each data set using one-way ANOVA identified a total of 34 root-specific and 40 leaf-specific target genes that are up-regulated following OsNAC10 overexpression with a three-fold or greater induction in the transgenic plants compared with NT plants (P <0.05). More specifically, up-regulation of 34 genes was specific to roots of both RCc3:OsNAC10 and   Table S3). Filling rate and 1000 grain weight of the GOS2:OsNAC10 plants were markedly reduced and the reduction appeared to be balanced by the increase in numbers of panicles and total spikelets, consequently maintaining similar levels of total grain weight to that of the NT controls. In the RCc3:OsNAC10 plants under the same field conditions, however, total grain weight was increased by 5-14% compared with the NT controls, which was due to increased numbers of filled grains and total spikelets. These observations prompted us to examine the yield components of the transgenic rice plants grown under field drought conditions. Three independent T 4 (2008) and T 5 (2009) lines of the RCc3:OsNAC10, GOS2:OsNAC10 and NT plants were transplanted to a paddy field with a removable rain-off shelter, and exposed to drought stress at the panicle heading stage (from 10-d before heading to 20-d after heading). The level of drought stress imposed under the rain-off shelter was equivalent to those that give 40-50% of total grain weight obtained under normal growth conditions, which was evidenced by the difference in levels of total grain weight of NT plants between the normal and drought conditions (Supplemental Tables S3 and   S4). Statistical analysis of the yield parameters scored for two cultivating seasons showed that the decrease in grain yield under drought conditions was significantly smaller in the RCc3:OsNAC10 plants than that observed in the NT controls. Specifically, in the drought-  Table S4). In the drought-treated

DISCUSSION
In our present study, we performed expression profiling using RNAs from stress-treated rice plants and identified 18 NAC-domain factors that are stress-inducible (Table I). Alignment of these stress-inducible factors further revealed three subgroups, within which the members are more closely related, suggesting a common stress response function. Overexpression of one such gene, OsNAC10, under the control of the constitutive promoter GOS2 (GOS2:OsNAC10) and the root-specific promoter RCc3 (RCc3:OsNAC10) was found to increase rice plant tolerance to drought and low temperature at the vegetative stage of growth. Increased tolerance to a moderate level of salinity conditions was also observed in both of these transgenic plants. More importantly, the RCc3:OsNAC10 plants showed significantly enhanced drought tolerance at the reproductive stage, increasing grain yield by 25-42% and 5-14% over controls in the field under drought and normal conditions, respectively. These Recently, the SNAC1 gene, a member of our subgroup I (Table I), was shown to confer tolerance of transgenic rice plants to field drought conditions as well as to drought and high salinity at the vegetative stage (Hu et al., 2006). This is consistent with our current results for RCc3:OsNAC10 plants. In the case of low temperatures at the vegetative stage, however, the effect was more pronounced in our transgenic plants harboring OsNAC10. In addition, SNAC1did not affect grain yield in transgenic plants grown under normal conditions, whilst a 5-14% increase in grain yield was observed in our RCc3:OsNAC10 plants in the normal field conditions. Transgenic rice harboring OsNAC6 (or SNAC2), another member of subgroup I, was shown previously to display enhanced tolerance at the vegetative stage to cold, salt and blast disease as a result of the increased expression of stress-related genes (Nakashima et al., 2007;Hu et al., 2008). Despite their high protein sequence homology (70-73% ) within the NAC domain, the SNAC1 and OsNAC6 (or SNAC2) genes are distinct from OsNAC10 in that their expression is increased upon exposure to low temperature conditions (Table I), which may be responsible in part for the observed functional differences between these genes and OsNAC10.
To date, the potential impact of homeotic genes like the NAC factors upon grain 1 4 yield have received relatively little attention due to their negative effects on fertility, plant growth, and development. Transgenic rice plants overexpressing OsNAC6 in a whole plant body exhibit growth retardation and low reproductive yield (Nakashima et al., 2007). In our current study, we also observed a yield penalty under drought conditions when the OsNAC10 overexpressed in a whole body in the GOS2:OsNAC10 plants; whereas, in RCc3:OsNAC10 plants, significant increase in grain yield was observed. Interestingly, the targeted expression of the prokaryotic Na + /H + antiporter gene in roots of transgenic tobacco plants has been shown to confer a higher tolerance to high-salinity conditions compared with whole plant expression (Hossain et al., 2006). More importantly, Na + content in leaves of transgenic tobacco plants with root-specific expression of the Na + /H + antiporter was lower than that of plants that constitutively expressed this gene, although there was no expression of this transgene in the leaf of the former. These observations together with our results suggest that ectopic expression of a stress response gene in a whole plant may not as effective as rootspecific expression on stress tolerance. It is particularly true for homeotic genes that function in development of reproductive organs.
Arabidopsis HARDY gene (HRD), an AP2 transcription factor, has been previously found to provide enhanced drought tolerance in transgenic Arabidopsis and rice plants (Karaba et al., 2007). HRD was isolated by activation tagging in Arabidopsis; the activationtagged line had a robust root system with increased numbers of secondary and tertiary roots.
We have measured root volume, length, dry weight and diameter of RCc3:OsNAC10, GOS2:OsNAC10 and NT plants after grown to the stage of reproduction (Fig. 6). Root diameter of the RCc3:OsNAC10 plants was thicker than that of the GOS2:OsNAC10 and NT plants. The increase in root diameter of the RCc3:OsNAC10 plants appears to be caused by an increase in cell number rather than cell size, as evidenced by similar size of epidermal and exodermal cells between NT and RCc3:OsNAC10 roots. It was shown that vigorous growth of roots with an increase in length and thickness is correlated with drought tolerance and grain yield of rice (Ekanayake et al., 1985;Price et al., 1997). How the thicker roots of the RCc3:OsNAC10 plants are associated with higher grain yield remains to be investigated.
In summary, we report an analysis of the rice NAC-domain family in their responses to stress treatments. More importantly, we evaluated agronomic traits in transgenic crops throughout the entire stages of plant growth in the field, which allowed us to address the advantages of using such a regulatory gene as OsNAC10, for improving stress tolerance.

5
Finally, we demonstrated that a root specific rather than whole body expression of OsNAC10 increases rice grain yield under drought conditions without yield penalty, providing the potential use of this strategy for improving drought tolerance in other crops.

Plasmid Construction and Transformation of Rice
The coding region of OsNAC10 was amplified from rice total RNA using an RT-PCR system (Promega, WI), according to the manufacturer's instructions. Primer pairs were as follows: forward (5'-ATGCCGAGCAGCGGCGGCGC-3') and reverse (5'-CTACTGCATCTGCAGATGAT-3'). To enable the overexpression of the OsNAC10 gene in rice, the cDNA for this gene was linked to the GOS2 promoter for constitutive expression, and 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 previously described (Jang et al., 1999).

Protein sequence analysis
Of 140 NAC factors predicted from the rice genome (Fang et al., 2008), we selected 87 NAC protein sequences that have full-length EST information from NCBI database search using the tBLAST N program and previously reported annotation of NAC family (Ooka et al., 2003;Xiong et al., 2005). Amino acid sequences of 87 NAC domains were aligned using CLUSTAL W followed by construction of a neighbor-joining phylogenic tree using the MEGA program (B = 1000 bootstrap replications).

Northern blot analysis
Rice (Oryza sativa cv Nipponbare) seeds were germinated in soil and grown in a glasshouse μ mol/m 2 /s. For lowtemperature treatments, 14-day-old seedlings were exposed at 4 °C in a cold chamber for the indicated time course under continuous light of 150 μ mol/m 2 /s. The preparation of total RNA and RNA gel-blot analysis was performed as reported previously (Jang et al., 2002).

Drought Treatments of Rice Plants at Vegetative Stage
Transgenic and non-transgenic (NT) rice (Oryza sativa cv Nipponbare) seeds were germinated in half-strength MS solid medium in a growth chamber in the dark at 28ºC for 4 d, transplanted into soil and then grown in a greenhouse (16-h-light/8-h-dark cycles) at 28-30ºC.
Eighteen seedlings from each transgenic and non-transgenic line were grown in pots (3 x 3 x 5 cm; 1 plant per pot) for four weeks before undertaking the drought-stress experiments. To induce drought stress, 4-week-old transgenic and NT seedlings were unwatered for 3 d followed by 7 d of watering. The numbers of plants that survived or continued to grow were then scored.

Temperature Conditions
Transgenic and non-transgenic rice (Oryza sativa cv Nipponbare) seeds were germinated and grown in half-strength MS solid medium for 14 d in a growth chamber (16-h-light of 150 μ mol m -2 s -1 /8-h-dark cycles at 28ºC). The green portions of approximately 10 seedlings were then cut using a scissors prior to stress treatments in vitro. All stress conditions were conducted under continuous light at 150 μmol m -2 s -1 . To induce low-temperature stress, the seedlings were incubated at 4ºC in water for up to 6 h. High-salinity stress was induced by incubation in 400 mM NaCl for 2 h at 28ºC. To simulate drought stress, the plants were airdried for 2 h at 28ºC. Fv/Fm values were then measured as previously described (Oh et al., 2008).

Rice 3'-Tiling Microarray Analysis
Expression profiling was conducted using the Rice 3'-Tiling Microarray as previously described (Oh et al., 2009). Transgenic and non-transgenic rice (Oryza 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 μ g) was prepared from 14-d-  Supplemental Table S5.

Drought Treatments and Grain Yield Analysis of Rice Plants in the Field for Two (2008 and 2009) Years
To evaluate yield components of transgenic plants under normal field conditions, three independent T 4 (2008) and T 5 (2009)  per panicle, filling rate (%), total grain weight (g), and 1,000 grain weight (g). The results from three independent lines were separately analyzed by one way ANOVA and compared with those of the NT controls. The ANOVA was used to reject the null hypothesis of equal means of transgenic lines and NT controls (P<0.05). SPSS version 16.0 was used to perform these statistical analyses.

Microscopic Examination of Roots
Roots of transgenic and non-transgenic plants at the panicle heading stage were fixed with modified Karnovsky's fixative consisting of 2% (vol/vol) glutaraldehyde and 2% (vol/vol) paraformaldehyde in 0.05 M sodium cacodylate buffer (pH 7.2) at 4°C overnight and washed with the same buffer three times for 10 min each. They were post-fixed with 1% (wt/vol) osmium tetroxide in the same buffer at 4°C for 2 h and washed with distilled water two times briefly. The post-fixed root tissues were enbloc stained with 0.5% (wt/vol) uranyl acetate at 4°C overnight. They were dehydrated in a graded ethanol series (30, 50, 70, 80, 95, and 100%) and three times in 100% ethanol for 10 min each.

SUPPLEMENTAL DATA
The following materials are available in the online version of this article Supplemental    The deduced amino acid sequences of the NAC domains from these 18 genes (listed in Table   I) were aligned using the CLUSTAL W program. Identical and conserved residues are highlighted (gray). Signature motifs are indicated by the boxes: Ia-e and IIa-c for subgroups I-III, respectively.

Various Tissues at Different Developmental Stages
A, Rice seeds were germinated and grown on Murashige and Skoog agar medium in the dark for 2 d (D2) and then in the light for 1 d at 28°C (L1). The seedlings were transplanted into soil pots and grown in the greenhouse for 14 d (14d), until meiosis (M), until just before heading (BH) and right after heading (AH). L1C, coleoptiles from L1 seedlings. RT-PCR analyses were performed using RNAs from the indicated tissues at indicated stages of development and gene-specific primers. The expression levels of a rice ubiquitin (OsUbi) were used as an internal control. B, Ten μg of total RNA was prepared from the leaf and root tissues of 14 d-old seedlings exposed to drought, high salinity, ABA or low temperature for the indicated time periods. For drought stress, the seedlings were air-dried at 28ºC; for highsalinity stress, seedlings were exposed to 400 mM NaCl at 28ºC; for low-temperature stress, seedlings were exposed to 4ºC; for ABA treatment, seedlings were exposed to a solution containing 100 μ M ABA. Total RNAs were blotted and hybridized with OsNAC10 genespecific probes. The blots were then reprobed with the Dip1 gene which was used as a marker for up-regulation of key genes following stress treatments. Ethidium bromide staining was used to determine equal loading of RNAs.    Tables S3 and   S4. The mean measurements from the NT controls were assigned a 100% reference value. 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; 1,000GW, thousand grain weight.

Conditions.
Numbers in boldface indicate up-regulation by more than 1.9-fold (P<0.05) in plants grown under stress conditions. a Sequence identification numbers for the full-length cDNA sequences of the corresponding genes. b The mean of three independent biological replicates. These microarray data sets can be found at http://www.ncbi.nlm.nih.gov/geo/ (Gene Expression Omnibus, GEO). c P values were analyzed by one-way ANOVA. Accession numbers: SNAC1,  Subgroups Ⅰ Ⅱ Ⅲ  Table I) were aligned using the CLUSTAL W program. Identical and conserved residues are highlighted (gray). Signature motifs are indicated by the boxes: Ia-e and IIa-c for subgroups I-III, respectively. . The seedlings were transplanted into soil pots and grown in the greenhouse for 14 d (14d), until meiosis (M), until just before heading (BH) and right after heading (AH). L1C, coleoptiles from L1 seedlings. RT-PCR analyses were performed using RNAs from the indicated tissues at indicated stages of development and gene-specific primers. The expression levels of a rice ubiquitin (OsUbi) were used as an internal control. B, Ten mg of total RNA was prepared from the leaf and root tissues of 14 d-old seedlings exposed to drought, high salinity, ABA or low temperature for the indicated time periods. For drought stress, the seedlings were air-dried at 28ºC; for high-salinity stress, seedlings were exposed to 400 mM NaCl at 28ºC; for low-temperature stress, seedlings were exposed to 4ºC; for ABA treatment, seedlings were exposed to a solution containing 100 μ M ABA. Total RNAs were blotted and hybridized with OsNAC10 gene-specific probes. The blots were then reprobed with the Dip1 gene which was used as a marker for up-regulation of key genes following stress treatments. Ethidium bromide staining was used to determine equal loading of RNAs.  The transcript levels of OsNAC10 and six target genes were determined by qRT-PCR (using the primers listed in Supplemental Table S5) and in RCc3:OsNAC10 and GOS2:OsNAC10 transgenic rice plants are presented as a relative concentration to the levels in untreated NT control roots and leaves, respectively. Data were normalized using the rice ubiquitin gene (OsUbi) transcript levels. Values are the means ± SD of three independent experiments.

Subgroups
www.plantphysiol.org on August 28, 2017 -Published by Downloaded from Copyright © 2010 American Society of Plant Biologists. All rights reserved.  Tables S3 and S4. The mean measurements from the NT controls were assigned a 100% reference value. 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; 1,000GW, thousand grain weight.