miRNA and Degradome Sequencing Reveal miRNA and Their Target Genes That May Mediate Shoot Growth in Spur Type Mutant "Yanfu 6"

Abstract

The spur-type growth habit in apple trees is characterized by short internodes, increased number of fruiting spurs, and compact growth that promotes flowering and facilitates management practices, such as pruning. The molecular mechanisms responsible for regulating spur-type growth have not been elucidated. In the present study, miRNAs and the expression of their potential target genes were evaluated in shoot tips of "Nagafu 2" (CF) and spur-type bud mutation "Yanfu 6" (YF). A total of 700 mature miRNAs were identified, including 202 known apple miRNAs and 498 potential novel miRNA candidates. A comparison of miRNA expression in CF and YF revealed 135 differentially expressed genes, most of which were downregulated in YF. YF also had lower levels of GA, ZR, IAA, and ABA hormones, relative to CF. Exogenous applications of GA promoted YF shoot growth. Based on the obtained results, a regulatory network involving plant hormones, miRNA, and their potential target genes is proposed for the molecular mechanism regulating the growth of YF. miRNA164, miRNA166, miRNA171, and their potential targets, and associated plant hormones, appear to regulate shoot apical meristem (SAM) growth. miRNA159, miRNA167, miRNA396, and their potential targets, and associated plant hormones appear to regulate cell division and internode length. This study provides a foundation for further studies designed to elucidate the mechanism underlying spur-type apple architecture.

Keywords: SAM; apple; internode; miRNAs; shoot.

Figure 1

Growth characteristics of CF and YF. (A) Shoot length. (B) Internode number. (C) Internode length. (D) Shoot of Nagafu 2 (CF) and Yanfu 6 (YF). (E) Four-year-old branch of YF and CF. ^* indicates P < 0.05, Student's t-test; Error bars indicate SE; Scale bars in (D,E), 1 cm.

Figure 2

Hormone content of CF and YF. (A) IAA content. (B) GA content. (C) ABA content. (D) ZR content. Approximately 0.5 g leaves near shoot tips were used for plant hormone IAA, GA, ZR, and ABA measurements. ^*Indicates P < 0.05, Student's t-test; Error bars indicate SE.

Figure 3

Summary of miRNA in CF and YF shoot tips. (A) The length distribution of small RNAs. (B) Venn diagrams of known and novel miRNAs in CF and YF shoot tips. (C) Known miRNA family members in CF and YF shoot tips. (D) miRNA reads counts frequency in CF and YF shoot tips. (E) Reads numbers of high expression miRNAs in shoot tips.

Figure 4

Clustering of conserved and novel miRNAs into families.

Figure 5

Expression of miRNA in CF and YF shoot tips. (A) Differential expression of known miRNAs in CF and YF shoot tips. (B) Differential expression of novel miRNAs in CF and YF shoot tips.

Figure 6

Complementary targeting by miRNAs. (A) miRNA397a/b targets the MDP0000237946 at two different sites. (B) miRNA828 and miRNA858 co-target the MYB conserved sequence. (C) miRNA159 and miRNA319 co-target MDP0000147309.

Figure 7

Differentially expressed miRNAs and their targets involved in SAM development between CF and YF. ^*Indicates P < 0.05, Student's t-test; Error bars indicate SE.

Figure 8

Differentially expressed miRNAs and their targets involved in internode elongation between CF and YF. ^*Indicates P < 0.05, Student's t-test; Error bars indicate SE.

Figure 9

Expression of cell cycle and cell elongation related genes. ^*Indicates P < 0.05, Student's t-test; Error bars indicate SE.

Figure 10

Expression of hormone related genes. ^*Indicates P < 0.05, Student's t-test; Error bars indicate SE.

Figure 11

Effect of GA treatment on shoot and internode growth. (A) Effect of GA treatment on internode length. (B) Effect of GA treatment on shoot growth. ^*Indicates P < 0.05, Student's t-test; Error bars indicate SE.

Figure 12

Expression of key miRNA after GA spraying. ^*Indicates P < 0.05, Student's t-test; Error bars indicate SE.

Figure 13

Tentative model for the association of miRNAs with the phytohormone crosstalk regulation the spur type apple tree architecture. Diagram represents a schematic drawing of an apple shoot apex. Arrows and inhibition lines linking the gene names represent positive and negative interactions, respectively. Up and down arrows behind the gene names represent up and down regulation, respectively.