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. 2021 Jun 4:12:669143.
doi: 10.3389/fpls.2021.669143. eCollection 2021.

PagERF16 of Populus Promotes Lateral Root Proliferation and Sensitizes to Salt Stress

Shengji Wang  1 , Juanjuan Huang  1 , Xingdou Wang  1 , Yan Fan  1 , Qiang Liu  2 , Youzhi Han  1
Affiliations

PagERF16 of Populus Promotes Lateral Root Proliferation and Sensitizes to Salt Stress

Shengji Wang et al. Front Plant Sci. .

Abstract

The aggravation of soil salinization limits the growth and development of plants. The AP2/ERF transcription factors (TFs) have been identified and play essential roles in plant development and stress response processes. In this study, the function of PagERF16 was detected using the overexpressing (OX) and RNAi transgenic poplar 84K hybrids. Plant growth, stomatal conductance, antioxidant enzymes activity, and PagERF16 co-expressed TFs were analyzed using morphological, physiological, and molecular methods. OX showed a more robust lateral root system with a bigger diameter and volume compared to the wild-type plants (WT). Physiological parameters indicated the bigger stomatal aperture and lower stomatal density of OX along with the lower Catalase (CAT) activity and higher malondialdehyde (MDA) content contributed to the salt sensitivity. The plant height and rooting rate of OX and RNAi were significantly worse compared to WT. Other than that, the morphology and physiology of RNAi plants were similar to WTs, suggesting that the function of PagERF16 may be redundant with other TFs. Our results indicate that when PagERF16 expression is either too high or too low, poplar growth and rooting is negatively affected. In addition, a downstream target TF, NAC45, involved in Auxin biosynthesis, was identified and PagERF16 could directly bind to its promoter to negatively regulate its expression. These results shed new light on the function of ERF TFs in plant root growth and salt stress tolerance.

Keywords: PagERF16; lateral root growth; poplar; salt sensitivity; transgenics.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Spatio-temporal expression pattern and sequence alignment of PagERF16. (A) Relative expression of PagERF16 in roots, stems, leaves, and shoots, respectively, under 100 mM NaCl. Different lowercases above the bar chart indicate significant differences among samples (p ≤ 0.05). (B) AP2 conserved domain architecture of PagERF16 of poplar. (C) Phylogenetic analysis of PagERF16 homologs in P. trichocarpa, P. euphratica, Nicotianu tabacum, and Arabidopsis thaliana. (D) Multiple sequence alignment of PagERF16 amino acid sequence of Populus trichocarpa, P. euphratica, Nicotianu tabacum, and Arabidopsis thaliana.
Figure 2
Figure 2
PagERF16 was sensitive to salt stress. (A) Morphology of the OX, RNAi, and WT grown on medium containing 50 mM NaCl for 30 d. Bars, 2 cm. (B) Fresh weight of whole plant. (C) Root fresh weight. (D) Fresh weight of aboveground tissues. (E) Length of primary root. (F) Numbers of leaves per plant. (G) Height of plant. The plot represents the mean ± SD of six plants per line. Three OX and RNAi lines serve as three biological repeats, respectively. Different lowercases indicate significant differences among samples (p ≤ 0.05).
Figure 3
Figure 3
PagERF16 promoted lateral root proliferation by increasing the diameter and volume. (A) Total length of root. (B) Average diameter of root. (C) Total surface area of root. (D) Total volume of root. (E) Tips of root system. (F–H) Represent the length, surface area, and volume of roots with diameter >1.0 mm, respectively. The plot represents the mean ± SD of six plants per line. Three OX or RNAi lines serve as three biological repeats. Different lowercases indicate significant differences among genotypes under the control (blue) or salt stress (red) conditions (p ≤ 0.05). (I) Rooting rate of 60 shoots per genotype (OX, RNAi, and WT) sub-cultured on medium without (black) or with 50 mM NaCl (red).
Figure 4
Figure 4
PagERF16 decreased stomatal density and increased stomatal width. (A–D) Represent area, length, width, and aspect ratio of the second mature leaf. (E) Relative water content of the second mature leaf. (F) Relative electrical conductance of the second leaf. (G) Scanning electron micrograph of the abaxial leaf epidermis. Bars, 20 μm. (H–J) show the stomatal density, width, and length. The plot represents the mean ± SD of six plants per line. Three OX or RNAi lines serve as three biological repeats. Different lowercases indicate significant differences among samples (p ≤ 0.05).
Figure 5
Figure 5
PagERF16 modulated the physiology and gene expression relevant to antioxidant enzymes. (A–D) Superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and MDA contents. The plot represents the mean ± SD of six plants per line. Three OX or RNAi lines serve as three biological repeats. Different lowercases indicate significant differences among samples (p ≤ 0.05). (E–M) Expression of POD1, POD2, POD3, SOD1, SOD2, SOD3, APX1, APX2, and P5CS1 in transgenic and WT plants. The plot represents the mean ± SD of three repeats. Asterisks denote significant differences between transgenic and WT: *p ≤ 0.05.
Figure 6
Figure 6
PagERF16 negatively regulated the expression of NAC45. (A) Expression pattern of differentially expressed TFs related to salt stress and PagERF16. WT_S, WT treated with 100 NaCl for 48 h. Red denotes high expression and blue indicates low expression. (B) Correlation analysis of co-expressed TFs using Spearman methods. Each node (Node) in the Figure represents a gene, and the wiring between nodes represents the correlation of gene expression. The larger the node, the more the number of expression correlations between this gene and other genes. Red line indicates PagERF16 negatively regulated the expression of NAC45. (C) Promoter structure of NAC45. (D) PagERF16 directedly binding to the promoter of NAC45 revealed by yeast one hybrid assay. The pGADT7-Rec-53 and p53-AbAi were used as positive control and pGADT7-ERF16 and pAbAi served as the negative control. The yeast Y1H Gold strains and plated on the SD/-Leu medium containing either 0 or 200 ng/ml AbA. (E) Relative expression level of NAC45 in OX, RNAi, and WT. The plot represents the mean ± SD of three repeats. Asterisks denote significant differences between transgenic and WT: *p ≤ 0.05.
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