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. 2014 Jan 23;9(1):e84886.
doi: 10.1371/journal.pone.0084886. eCollection 2014.

Functional Analysis of Water Stress-Responsive Soybean GmNAC003 and GmNAC004 Transcription Factors in Lateral Root Development in Arabidopsis

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Functional Analysis of Water Stress-Responsive Soybean GmNAC003 and GmNAC004 Transcription Factors in Lateral Root Development in Arabidopsis

Truyen N Quach et al. PLoS One. .
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Abstract

In Arabidopsis, NAC (NAM, ATAF and CUC) transcription factors have been found to promote lateral root number through the auxin signaling pathway. In the present study, the role of water stress-inducible soybean GmNAC003 and GmNAC004 genes in the enhancement of lateral root development under water deficit conditions was investigated. Both genes were highly expressed in roots, leaves and flowers of soybean and were strongly induced by water stress and moderately induced by a treatment with abscisic acid (ABA). They showed a slight response to treatment with 2,4-dichlorophenoxyacetic acid (2,4-D). The transgenic Arabidopsis plants overexpressing GmNAC004 showed an increase in lateral root number and length under non-stress conditions and maintained higher lateral root number and length under mild water stress conditions compared to the wild-type (WT), while the transgenic plants overexpressing GmNAC003 did not show any response. However, LR development of GmNAC004 transgenic Arabidopsis plants was not enhanced in the water-stressed compared to the well-watered treatment. In the treatment with ABA, LR density of the GmNAC004 transgenic Arabidopsis was less suppressed than that of the WT, suggesting that GmNAC004 counteracts ABA-induced inhibition of lateral root development. In the treatment with 2,4-D, lateral root density was enhanced in both GmNAC004 transgenic Arabidopsis and WT plants but the promotion was higher in the transgenic plants. Conversely, in the treatment with naphthylphthalamic acid (NPA), lateral root density was inhibited and there was no difference in the phenotype of the GmNAC004 transgenic Arabidopsis and WT plants, indicating that auxin is required for the action of GmNAC004. Transcript analysis for a number of known auxin and ABA related genes showed that GmNAC004's role may suppress ABA signaling but promote auxin signaling to increase lateral root development in the Arabidopsis heterologous system.

Conflict of interest statement

Competing Interests: LPT currently works for RIKEN and SKG currently works for Dow AgroSciences LLC. LPT and SKG contributed to this study while they were employed at the University of Missouri. Therefore, this does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Relative transcript abundance of GmNAC003 and GmNAC004 in response to water stress.
Transcript abundance was quantified using qRT-PCR and the data were normalized to the four best internal control genes (CYP2, IDE, UNK1 and UNK2) based on the M stability of GeNorm analysis. Mean relative expression levels were transformed to a value of 1 for the sample having lowest expression. Error bars are standard errors of the means from three replications. (A) Response to water deficit stress. Plants at the 2-leaf (V2) growth stage were allowed to grow without supply of additional water until desired stem water potentials were achieved. V2, V3 and V6 are the growth stages of the control plants corresponding to the time when water stressed-plants were sampled. (B) Response to dehydration stress. V2 growth stage plants were harvested and allowed to dehydrate in a growth chamber for the designated times.
Figure 2
Figure 2. Expression of GmNAC003 and GmNAC004 in response to various stresses and hormonal treatments.
V2 growth-stage soybean plants were harvested and transferred to stress conditions. All treatments of control (Water), 20 nM 2,4-D (2,4-D), 100 µM ABA (ABA), 4°C (Cold) and 250 mM NaCl (NaCl) were under hydroponic conditions at 28°C unless otherwise indicated. Transcript abundance was quantified by qRT-PCR and the data were normalized to the four most stable internal reference genes (CYP2, IDE, UNK1 and UNK2) using GeNorm. Data were normalized using water treatments, and transformed to a value of 1 for the unstressed plant (0 h). The error bars are standard errors of the means (n = 3 plants).
Figure 3
Figure 3. Transgenic Arabidopsis plants overexpressing soybean GmNAC003 and GmNAC004.
(A) Expression of the transgenes in 3-week-old transgenic plants quantified by qRT-PCR using Arabidopsis ubiquitin as the reference gene. The tissues were sampled from homozygous transgenic plants having a single insertion. Error bars are the standard errors of the means from three samples of ten plants. (B) Growth of the 4-week old and T4 generation GmNAC003 (events N3.09 and N3.10) and GmNAC004 transgenic (events N4.01 and N4.03) and WT plants.
Figure 4
Figure 4. Lateral root numbers, lateral root length and primary root length of transgenic Arabidopsis plants overexpressing GmNAC003 and GmNAC004 in response to mild water stress.
(A) Water potentials of PEG-diffused agar plates. Water potentials were measured at the end of the experiment, 8 days from the date of stress exposure. (B) Lateral root number, (C) total lateral root length, and (D) primary root length of transgenic plants. Transgenic GmNAC003 (events N3.09 and N3.10), GmNAC004 (events N4.01 and N4.03), and WT Arabidopsis lines were grown in nutrient agar plates diffused with different concentrations of PEG. The transgenic plants were at the T4 generation and homozygous for the transgenes. Data were recorded at 12 days after sowing (or 8 days of stress exposure). (*) denotes significant difference at 95% confidence level using Duncan's multiple range test from six replications. Error bars are standard errors of the means from 6 replications.
Figure 5
Figure 5. Representative root growth of transgenic Arabidopsis plants overexpressing GmNAC004 in response to water deficit conditions.
Two T4 homozygous transgenic Arabidopsis (N-4.01 and N-4.03) and WT plants were grown on nutrient agar plates diffused with different concentrations of PEG. The plants were 12 days old (8 days after stress exposure).
Figure 6
Figure 6. Germination of GmNAC003 and GmNAC004 transgenic Arabidopsis seeds in response to ABA treatment.
Fifty to seventy seeds of the homozygous GmNAC003 and GmNAC004 transgenic and WT plants were sown on agar plates without ABA (MS) or with 1 µM ABA (ABA). (A & B) Plants were 7 and 14 days old, respectively. (C) Quantification of germination rates 5 days after sowing. Different letters denote significant differences at the 95% confidence level using Duncan's multiple range test from four replications.
Figure 7
Figure 7. Role of GmNAC004 in regulation of lateral root density of transgenic Arabidopsis plants.
(A) Transgenic Arabidopsis overexpressing GmNAC004 in response to ABA and 2,4-D treatments. Four-day-old seedlings of WT and GmNAC004 transgenic plants (T4 generation) were exposed to 5 µM ABA, 20 nM 2,4-D, 2 µM NPA or their combinations for 7 days. Control treatment was not treated with hormones. Error bars are the standard errors of the means from six replications. Asterisks denote significant differences at 95% between the WT and transgenic plants. (B) Expression of representative ABA and auxin signaling genes in transgenic GmNAC003 and GmNAC004 Arabidopsis plants. Duncan multiple-mean comparisons were used and different letters indicates differences of the means.

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Grant support

This work was funded by a grant from the Missouri Soybean Merchandising Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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