Overexpression of a Novel Apple NAC Transcription Factor Gene, MdNAC1, Confers the Dwarf Phenotype in Transgenic Apple (Malus domestica)

Abstract

Plant height is an important trait for fruit trees. The dwarf characteristic is commonly associated with highly efficient fruit production, a major objective when breeding for apple (Malus domestica). We studied the function of MdNAC1, a novel NAC transcription factor (TF) gene in apple related to plant dwarfing. Localized primarily to the nucleus, MdNAC1 has transcriptional activity in yeast cells. Overexpression of the gene results in a dwarf phenotype in transgenic apple plants. Their reduction in size is manifested by shorter, thinner stems and roots, and a smaller leaf area. The transgenics also have shorter internodes and fewer cells in the stems. Levels of endogenous abscisic acid (ABA) and brassinosteroid (BR) are lower in the transgenic plants, and expression is decreased for genes involved in the biosynthesis of those phytohormones. All of these findings demonstrate that MdNAC1 has a role in plants dwarfism, probably by regulating ABA and BR production.

Keywords: MdNAC1; abscisic acid; apple; brassinosteroid; dwarf.

Figure 1

Gene structure and protein sequence analysis of MdNAC1. (A) Exons/introns of MdNAC1 are displayed. (B) NAM domain detected in the N-terminus region of MdNAC1. (C) Phylogenetic analysis of MdNAC1 (marked by solid-black circle) and homologous proteins from other species. (D) Alignment of MdNAC1 and its homologous proteins. These different colors show the similarity degrees of the amino acids. Homologous protein sequences of MdNAC1 from 18 other species were used for phylogenetic analysis and alignment.

Figure 2

(A) Subcellular localization of MdNAC1. GFP (green fluorescent protein) or MdNAC1-GFP fusion protein were transiently expressed in Nicotiana benthamiana epidermal cells and analyzed by confocal microscopy. The green fluorescent signal in the dark field or merged field shows the localization of GFP or MdNAC1-GFP fusion protein. (B) Transcriptional activity analysis of MdNAC1 in yeast. The transformed yeast strain AH109 with both GAL4 DNA binding domain (BD) and BD-MdNAC1 fusion protein grows well on SD-Trp medium. The yeast with BD-MdNAC1 grows well, but not with BD, on SD-His-Trp-Ade and SD-His-Trp-Ade mediums with X-α-Gal. Growth on SD-Trp means that the protein is expressed in yeast stain AH109; growth on SD-His-Trp-Ade means that there is functional expression of His and Ade biosynthetic genes; and the blue colony is the indicator showing the expression of these genes. SD-Trp medium: synthetic, minimal lacking Trp; SD-His-Trp-Ade: SD medium lacking His, Trp, and Ade; X-α-Gal: SD-His-Trp-Ade medium plus X-α-Gal.

Figure 3

Dwarf phenotype in overexpression lines of MdNAC1. (A) Transgenic plants had smaller plant size and (C) internodes length. (B) They also featured changes in lateral branch architecture, smaller roots (D) and leaves (E), and (F) lower values for plant height, (G) internode length, (H) stem diameter, (L) root length, (M) root diameter, and (N) leaf area. (I) Transgenic plants had lower shoots with lateral branches, (J) lower lateral branches, and (K) shorter lateral branches. The data for Figure 3I–K are presented as means ± standard errors; The data for other Figures are presented as means ± standard deviations. Different letters above the bars indicate the significant difference among these data at a level of p < 0.05 according to Tukey’s multiple comparison test. Any data sharing a same letter means there is no significant difference at this significant level. a: Subset label showing one or more sets of data with the largest mean value(s); b: Subset label showing one or more sets of data with the second-largest mean value(s); c: Subset label showing one or more sets of data with the third-largest mean value(s).

Figure 4

Anatomical structures of stems and leaves from transgenic and wild-type (WT) plants. Longitudinal paraffin sections (A) and transverse paraffin sections (B) of stems from transgenic and WT plants analyzed using a light microscopy. (C) Transverse paraffin sections of leaves from transgenic and WT plants. Genotypes did not differ significantly in the longitudinal lengths of stem epidermis cells (D), or stem cortical parenchyma cells (E), widths of epidermis cells (F), or stem cortical parenchyma cells (G), cell density of cortical parenchyma cells (J) or pith parenchyma cells (K). However, transgenic plants displayed smaller cortical thickness (H) and smaller vascular cylinder diameter (I), but had thicker in leaves (L), upper epidermis (M), palisade tissue (N), spongy tissue (O), and foliar epidermis (P) when compared with WT. Anatomical structures of stems or leaves were analyzed with four biological repeats. The data are presented as means ± standard deviations. Detailed information on statistics, please see Figure 3.

Figure 5

Photosynthetic parameters. Genotypes did not differ significantly in values for net photosynthetic rate (Pn) (A), intercellular CO₂ concentration (Ci) (B), and stomatal conductance (Gs) (C). However, transgenic plants had higher transpiration rate (Tr) (D), but lower instantaneous water-use efficiency (WUEi) (E) when compared with wild-type (WT). Detailed information on statistics, please see Figure 3.

Figure 6

Concentrations of abscisic acid (ABA) (A), brassinosteroid (BR) (B), indole acetic acid (IAA) (C), gibberellin (GA) (D), and cytokinins (CTK) (E) in transgenic and wild-type (WT) plants. The data are presented as means ± standard deviations. Detailed information on statistics, please see Figure 3.

Figure 7

(A) Relative expressions of genes related to biosynthesis and (B) signal pathways for ABA and BR by quantitative real-time PCR (qRT-PCR) analysis. The data are presented as means ± standard deviations. Detailed information on statistics, please see Figure 3.