Fig 1.
Inhibited shoot growth and reduced plant size of 35S:PmDAM6 apple plants.
(A) Shoot growth was inhibited in the 2-year-old 35S:PmDAM6 lines, 35S-2 and 35S-4, relative to the growth of wild-type (WT) plants, under long photoperiod (16-h light/8-h dark) conditions. Plant height of 35S-2 and 35S-4 were significantly lower (P < 0.01) than those of WT in all examined periods (Student’s t-test) (B) Photographs of 3-year-old plants taken after they stopped growing under short photoperiod (SP) (8-h light/16-h dark) conditions. 35S:PmDAM6 plants were shorter than the WT plants (right). (C) Photograph of 5-year-old plants taken on 28 August 2015. The 35S:PmDAM6 plants were shortened than the WT plants. Plants were grown in a greenhouse without heating, resulting in exposure to naturally occurring cold conditions in the winter.
Fig 2.
Early bud set observed in 4-year-old 35S:PmDAM6 apple plants.
Photographs of 4-year-old plants taken on 24 July 2014. Early bud set was observed in 35S-2 plants (left).
Fig 3.
Inhibition of lateral bud break in 3-year-old 35S:PmDAM6 apple plants during the spring in a greenhouse without heating (semi-field conditions).
(A) Photo taken on 1 April 2014. (B) Number of lateral buds that opened by 25 March, 1 April, and 8 April 2014 in the 35S:PmDAM6 apple lines, 35S-2 and 35S-4, and wild-type (WT) plants. Significant differences between 35S:PmDAM6 and WT at P < 0.01 and P < 0.05 (Student’s t-test) are indicated with ** and *, respectively.
Table 1.
Accelerated terminal bud break and inhibition of lateral bud break in 3-year-old 35S:PmDAM6 and 35S:PmDAM6-GR apple plants treated with DEX.
Fig 4.
Terminal bud break in the spring was delayed in 5-year-old 35S:PmDAM6 apple plants.
Photograph taken on 15 April 2016. Plants were grown in a greenhouse without heating, resulting in an exposure to naturally occurring cold conditions in the winter.
Table 2.
Delayed bud break timing and repressed bud break competency of dormant terminal buds of 4- to 6-year old 35S:PmDAM6 apple plants.
Fig 5.
Abscisic acid and cytokinins (tZ and iP) contents in the terminal buds were higher and lower, respectively, in the dormancy-enhanced 35S:PmDAM6 apple plants than in the WT plants.
Phytohormone contents in the terminal buds of 4-year-old plants collected on 20 February 2015 (A), and of 6-year-old plants collected on 25 January, 23 February, and 8 and 31 March 2017 (B). Values are presented as the means of three replicates. Bars indicate standard errors. Significant differences in the phytohormone contents between WT and 35S:PmDAM6 apple plants are indicated with ** and * at P < 0.01 and P < 0.05 (Student’s t-test), respectively.
Fig 6.
Dexamethasone (DEX) treatments inhibited bud outgrowth, increased the ABA level, and decreased cytokinin (tZ and iP) levels in the terminal buds of 35S:PmDAM6-GR plants.
The branches collected from 6-year-old 35S:PmDAM6-GR lines, GR21 and GR22, and WT plants on 9 March, 2017 were incubated in DEX- or control solutions under forcing conditions (22°C with a 16-h light/8-h dark photoperiod). The subsequent bud outgrowth and changes in phytohormone contents were analyzed. (A) Branches were photographed at 15 days after initiating the treatment. Control (con) or DEX-treated (DEX) GR21, GR22, and WT plants are presented. (B) Average leaf blade length of the largest leaves on shoots that germinated from buds collected at 15 days after the treatment. (C) Phytohormone contents in the dormant terminal buds of branches at 0, 24, and 96 h after the treatment. Values are presented as the mean of three replicates. Bars indicate standard errors. Significant differences in the phytohormone contents of control and DEX-treated samples at the same time point in each line are indicated with ** and * at P < 0.01 and P < 0.05 (Student’s t-test), respectively.
Fig 7.
Seasonal changes in the phytohormone contents of dormant Japanese apricot ‘Nanko’ buds.
The PmDAM6 expression levels and dormancy status are briefly described at the bottom based on the previous studies. Values are presented as the mean of three replicates. Bars indicate standard deviations.
Fig 8.
Schematic diagram of the proposed model of PmDAM6 functions during dormancy and bud outgrowth in PmDAM6-overexpressing transgenic apple plants and in Japanese apricot.
(A) Proposed model of PmDAM6 functions in 35S:PmDAM6 apple plants. PmDAM6 induces the accumulation of ABA and inhibits the accumulation of cytokinins (CK) in dormant terminal buds, which represses bud break competency and inhibited bud outgrowth. (B) Model of PmDAM6-mediated dormancy regulation in Japanese apricot based on data obtained from transgenic apple plants (this study), seasonal changes in PmDAM6 expression levels [15, 19] and seasonal changes in phytohormone contents (this study) in Japanese apricot leaf buds. The expression of PmDAM6 is up-regulated during endodormancy and down-regulated during the endo- and ecodormancy release stages. Additionally, PmDAM6 expression is undetectable during bud break [15, 19]. Down-regulated PmDAM6 expression coincides with a decrease in ABA during the endo- and ecodormancy release stages and with an increase in CK levels during the ecodormancy release and bud break stages. We hypothesize that PmDAM6 maintains endodormancy by repressing bud break competency and increasing ABA levels and inhibits dormancy release by increasing ABA and decreasing CKs contents in dormant Japanese apricot buds.