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, 153 (1), 185-97

Root-specific Expression of OsNAC10 Improves Drought Tolerance and Grain Yield in Rice Under Field Drought Conditions

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Root-specific Expression of OsNAC10 Improves Drought Tolerance and Grain Yield in Rice Under Field Drought Conditions

Jin Seo Jeong et al. Plant Physiol.

Abstract

Drought poses a serious threat to the sustainability of rice (Oryza sativa) yields in rain-fed agriculture. Here, we report the results of a functional genomics approach that identified a rice NAC (an acronym for NAM [No Apical Meristem], ATAF1-2, and CUC2 [Cup-Shaped Cotyledon]) 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 three subgroups with distinct signature motifs. A group of OsNAC genes were prescreened for enhanced stress tolerance when overexpressed in rice. OsNAC10, one of the effective members selected from prescreening, 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% to 42% and by 5% to 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 differences 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 nontransgenic 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.

Figures

Figure 1.
Figure 1.
Alignments of NAC domain sequences from 18 stress-inducible rice genes. The deduced amino acid sequences of the NAC domains from these 18 genes (Table I) were aligned using the ClustalW program. Identical and conserved residues are highlighted (gray). Signature motifs are indicated by the boxes: Ia to Ic and IIa to IIc for subgroups I and II, respectively.
Figure 2.
Figure 2.
Expression of OsNAC10 in rice under different stress conditions and in various tissues at different developmental stages. A, Rice seeds were germinated and grown on MS 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 until right after heading (AH). L1C, Coleoptiles from L1 seedlings. RT-PCR analyses were performed using RNAs from the indicated tissues at the indicated stages of development and gene-specific primers. The expression levels of a rice ubiquitin (OsUbi) were used as an internal control. B, Ten micrograms 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 28°C; for high-salinity stress, seedlings were exposed to 400 mm NaCl at 28°C; for low-temperature stress, seedlings were exposed to 4°C; for ABA treatment, seedlings were exposed to a solution containing 100 μm 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 the up-regulation of key genes following stress treatments. Ethidium bromide (EtBr) staining was used to determine equal loading of RNAs.
Figure 3.
Figure 3.
Stress tolerance in vegetative stage RCc3:OsNAC10 and GOS2:OsNAC10 plants. A, RNA gel-blot analyses were performed using total RNA preparations from the roots and leaves of three homozygous T4 lines of RCc3:OsNAC10 and GOS2:OsNAC10 plants, respectively, and of NT control plants. The blots were hybridized with OsNAC10 gene-specific probes and also reprobed for RbcS and Tubulin. Ethidium bromide (EtBr) staining was used to determine equal loading of RNAs. – indicates nullizygous (without transgene) lines, and + indicates transgenic lines. B, The appearance of transgenic plants during drought stress. Three independent homozygous T4 lines of RCc3:OsNAC10 and GOS2:OsNAC10 plants and NT controls were grown for 4 weeks, subjected to 3 d of drought stress, and followed by 7 d of rewatering in the greenhouse. Photographs were taken at the indicated time points. + denotes the number of rewatering days under normal growth conditions. C, Changes in the chlorophyll fluorescence (Fv/Fm) of rice plants under drought, high-salinity, and low-temperature stress conditions. Three independent homozygous T4 lines of RCc3:OsNAC10 and GOS2:OsNAC10 plants and NT controls grown in MS medium for 14 d were subjected to various stress conditions as described in “Materials and Methods.” After these stress treatments, the Fv/Fm values were measured using a pulse modulation fluorometer (mini-PAM; Walz). All plants were grown under continuous light of 150 μmol m−2 s−1 prior to stress treatments. Each data point represents the mean ± se of triplicate experiments (n = 10).
Figure 4.
Figure 4.
Regulated genes in roots and leaves of RCc3:OsNAC10 and GOS2:OsNAC10 plants under normal and stress conditions. The transcript levels of OsNAC10 and six target genes were determined by quantitative RT-PCR (using the primers listed in Supplemental Table S5), and those in RCc3:OsNAC10 and GOS2:OsNAC10 transgenic rice plants are presented as relative to the levels in untreated NT control roots and leaves, respectively. Data were normalized using the rice ubiquitin gene (OsUbi) transcript levels. Values are means ± sd of three independent experiments.
Figure 5.
Figure 5.
Agronomic traits of RCc3:OsNAC10 and GOS2:OsNAC10 plants grown in the field under both normal and drought conditions. Spider plots of the agronomic traits of three independent homozygous T4 and T5 lines of RCc3:OsNAC10 and GOS2:OsNAC10 plants and corresponding NT controls under both normal and drought conditions were drawn using Microsoft Excel. Each data point represents the percentage of the mean values (n = 20 and n = 30) listed in Supplemental 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, 1,000 grain weight.
Figure 6.
Figure 6.
Difference in root growth of RCc3:OsNAC10 and GOS2:OsNAC10 plants. A, RCc3:OsNAC10, GOS2:OsNAC10, and NT control plants were grown to the heading stage of reproduction. Whole plants (left panel) and parts (white box in left panel) of representative roots (10 roots in top right panel and one root in bottom right panel) are shown. Bars = 10 cm in left panel and 1 cm and 2 mm in right panels. B, The root volume, length, dry weight, and diameter of RCc3:OsNAC10 and GOS2:OsNAC10 plants are normalized to those of NT control roots. Asterisks indicate that the mean difference is significant at the 0.01 level (lsd). Values are means ± sd of 50 roots (10 roots from each of five plants). C, Light microscopic images of cross-sectioned RCc3:OsNAC10, GOS2:OsNAC10, and NT roots (top panels) showing the enlarged stele (middle panels), cortex, and epidermis (bottom panels) of RCc3:OsNAC10 roots. Bars = 500 μm in top panels and 100 μm in middle and bottom panels.

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