A simple and efficient gene functional analysis method for studying the growth and development of peach seedlings

Abstract

Stable genetic transformation of peach [Prunus persica (L.) Batsch] still faces many technical challenges, and existing transient expression methods are limited by tissue type or developmental stage, making it difficult to conduct functional analysis of genes regulating shoot growth. To overcome this dilemma, we developed a three-step method for efficient analysis of gene functions during peach seedling growth and development. This method resulted in transformation frequencies ranging from 48 to 87%, depending on the gene. From transformation of germinating seeds to phenotyping of young saplings took just 1.5 months and can be carried out any time of year. To test the applicability of this method, the function of three tree architecture-related genes, namely PpPDS, PpMAX4, and PpWEEP, and two lateral root-related genes, PpIAA14-1 and -2, were confirmed. Since functional redundancy can challenge gene functional analyses, tests were undertaken with the growth-repressor DELLA, which has three homologous genes, PpDGYLA (DG), PpDELLA1 (D1), and -2 (D2), in peach that are functionally redundant. Silencing using a triple-target vector (TRV2-DG-D1-D2) resulted in transgenic plants taller than those carrying just TRV2-DG or TRV2. Simultaneously silencing the three DELLA genes also attenuated the stature of two dwarf genotypes, 'FHSXT' and 'HSX', which normally accumulate DELLA proteins. Our study provides a method for the functional analysis of genes in peach and can be used for the study of root, stem, and leaf development. We believe this method can be replicated in other woody plants.

Figure 1

Silencing of PpPDS resulted in photo-bleached leaves in peach seedlings. A Seedlings infected with TRV2 (left) and TRV2-PpPDS (middle and right). The photos were taken 15 days post-infiltration. B Expression level of PpPDS in leaves exhibiting mild and severe bleaching. The values represent the average of three biological replicates The error bars indicate means ± standard deviation (SD) (*P < 0.05; ***P < 0.001).

Figure 2

Silencing of PpMAX4 increased the number of lateral branches and lateral roots. A Lateral branching in wild-type seedlings (left) and seedlings infected with TRV2 (middle) and TRV2-PpMAX4 (right). The photos were taken 28 days post-infiltration (DPI). B Analyse the number of lateral branches at each node of the peach seedling 28 DPI (by comparing the branch number to the internode number). C Expression levels of PpMAX4 in shoot tips collected 10, 15, 20, and 25 DPI. The values represent the average of three biological replicates (^****P < 0.0001). D Lateral roots of seedlings 10 DPI. E Fresh roots weight of seedlings 10 DPI. B and E Error bars indicate means ± standard deviation (SD) from three biological replicates. ^**** indicates significant differences at P < 0.0001 between TRV2 and TRV2-PpMAX4.

Figure 3

Silencing of PpWEEP resulted in a phenotype of pendulous branch growth in peach seedlings. A Upright and pendulous growth of dark-grown seedlings. Uninfected wild-type seeds and seeds inoculated with Agrobacterium harboring the empty TRV2 vector and TRV2-PpWEEP were cultured in pots under dark conditions. The photos were taken at 15DPI. B Measure of the angle of the main stem to the vertical. Error bars indicate means ± standard deviation (SD) from three biological replicates. ^**** indicate significant differences at P < 0.0001 between TRV2 and TRV2-PpWEEP.C Expression level of PpWEEP in shoot tips collected at 10, 15, 20, and 25 days post infiltration. The values represent the average of three biological replicates (^*P < 0.05; ^***P < 0.0001).

Figure 4

Silencing of DELLA genes increased plant height. Seedlings were cultured in the dark (A) or treated with PBZ (B). The photos were taken 10 DPI (A) and 15DPI (B). D and E Analysis of plant height of dark-grown (D) and PBZ-treated (E) seedlings. C and F Expression levels of PpDG, PpD1, and PpD2 in shoot tips collected 5, 15 days (C, dark-grown), and 25 days (F, PBZ-treated) post-infiltration (^*P < 0.05; ^****P < 0.0001). The values represent the average of three biological replicates. D and E Error bars indicate means ± standard deviation (SD) from three biological replicates. ^*** and ^**** indicate significant difference at P < 0.001 and P < 0.0001 between TRV2 and TRV2-PpDGYLA or TRV2-DG-D1-D2.

Figure 5

The dwarf phenotypes of ‘FHSXT’ and ‘HSX’ were attenuated by silencing of peach DELLA genes. A The plant height of seedlings. Photos were taken 15 DPI. B Analysis of plant height of the ‘FHSXT’ and ‘HSX’ dwarf genotypes. Error bars indicate means ± standard deviation (SD) from three biological replicates. ^*** and ^**** indicate significant differences at P < 0.001 and P < 0.0001 between TRV2 and TRV2-PpDGYLA or TRV2-DG-D1-D2. Expression levels of PpDGYLA, PpDELLA1, and PpDELLA2 in shoot tips from ‘HSX’ (C) and ‘FHSXT’ (D) collected 25 DPI. The values represent the average of three biological replicates (^**P < 0.01; ^***P < 0.001; ^****P < 0.0001).

Figure 6

Overexpressing PpIAA14–1^P102S and PpIAA14–2^P108S inhibited the number of lateral roots in peach. A Whole seedlings removed from soil 14 days post-infection. B Analysis of lateral root numbers at 14 DPI. In these seedlings, the minimum number of lateral roots was 0, while the maximum was 56. The frequency for each range indicated by color are shown. C Transcript levels of IAA14–1 and IAA14–2 were analysed in the main root 10 days post-infection. The values represent the average of three biological replicates. (^****P < 0.0001) (EV represent pSAK277-GFP).

Figure 7

A simple procedure for transformation of peach seedlings.