Fig 1.
Main phenotypes of the delaying heading (dh) mutant and comparison of the osa-miR171c expression.
(A) A wide-type plant Zhonghua 11 (ZH11) (left) and a dh mutant (right) under ND conditions after flowering of the wide-type plant. ZH11 was shown the plant from the 1st growing season. (B) Statistical analysis of the growth time from sowing to heading of ZH11 at the 1st and the 2nd growing seasons compared with that of dh mutant. (C and D) Statistical comparison of plant height (C) and tiller number (D) between ZH11 and dh mutant. (E and F) dh mutant stems present more nodes than those from ZH11. (G and H) qRT-PCR expression comparison of OsMIR171c gene (G) and mature osa-miR171c and osa-miR171* (H) in different developing organs between ZH11 and dh mutant. Rice plants were grown in a paddy field in Guangzhou, China, during two normal growing seasons (1st growing season from March to July, 2nd growing season, from July to November), with normal fertilizer application. The data were presented from three-year experiments. Error bars indicate SD from at least 20 samples. e-EF-1a was used for OsMiR171c internal control and U6 for osa-miR171c and osa-miR171* internal control. 4L-L and 10L-L, the 4th leaf and the 10th leaf; 4L-SA, shoot apex of 4-leaf stage seedlings; ≤1P, developing panicles with lengths ≤1 cm.
Fig 2.
Four OsHAM transcription factors are targets of osa-miR171c.
(A) Organ-expression profiles of the eight predicted targets of osa-miR171c in ZH11 at the heading stage by semi-RT-PCR. (B) Identification of cleavage sites of the target mRNAs in ZH11 using 5′-RLM-RACE. miRNA binding sites are underlined in black and the 5′ end of the cleaved mRNA in red. The numbers in green refer to the ratio of 5′-RACE clones and the arrows indicate the cleavage sites. The agarose gel images on the left show the bands corresponding to the amplified 3′ cleavage products. (C and D) qRT-PCR expression analysis shows down-regulated (C) and unchanged (D) target genes of osa-miR171c in dh mutant leaves. e-EF-1a was used as internal control in A, C, and D.
Fig 3.
Spatial and temporal parallel expression analyses of osa-miR171c and OsHAMs in different ZH11 organs at vegetative and reproductive stages.
Data were calculated from three replicates. ZH11 plants were grown in a paddy for microRNA and RNA extraction. The shadow showed inverse correlation of expression change between osa-miR171c and 4 OsHAM genes during transit from vegetative to reproductive stage. e-EF-1a and U6 were used as OsHAMs and osa-miR171c internal controls, respectively. Expression level of osa-miR171c and 4 OsHAMs was normalized with their expression at 2L-R stage, respectively. 2L-R, roots of 2-leaf stage seedlings; 2L-S, 2-leaf stage seedlings; 10L-L, the 10th leaf; 2L-SA, shoot apex of 2-leaf stage seedling;4L-SA, shoot apex of 4-leaf stage seedling;10L-SA, shoot apex of 10-leaf stage seedling;≤1P, developing panicles with a length of ≤1 cm; BP, booting panicle.
Fig 4.
Light regulates expression of osa-miR171c and OsHAMs.
(A) Expression level of osa-miR171c under nature day (ND), long day (LD, 14-h light), and short day (SD, 9-h light) conditions. (B) Transcript levels of osa-miR171c and OsHAMs changed during through the 24-h day/night period. (C) Transcript levels of osa-miR171c and OsHAMs changed during the day/night period (48 h). (D) Expression of osa-miR171c and OsHAMs in continued dark and continued light conditions. e-EF-1a and U6 were used for OsHAMs and osa-miR171c internal controls, respectively.
Fig 5.
Expression comparison of nine flowering-related genes in the rice flowering regulation model [57], between ZH11 and dh mutant under SD, ND and LD conditions.
Total RNAs were extracted from leaves, collected from 21-day-old plants grown under short days (SD, 9-h-day/15-h-night), from 21-day-old plants grown in a field under natural day conditions (ND) and from 30-day-old plants grown under long days (LD, 14-h-day/10-h-night). Values are shown as means of two biological replicates. Error bars indicate standard deviation. e-EF-1a was used as internal control.
Fig 6.
Prolonged development of shoot apical meristem (SAM) in dh mutant.
(A and B) Shoot apex of ZH11 (A) and a dh mutant (B) at 40 days after germination (DAG). (C) Expression levels of miR156 and miR172 in the fifth leaf of ZH11 and dh mutant. (D-E and G-H) SAM morphology of ZH11 (D, E) and a dh mutant (G, H) at the 4 leaf-stage and the 10 leaf-stage. (F and I) Shoot apex of ZH11 (F) and a dh mutant (I) at 30 DAG. (J and K) Shoot apex of a dh mutant at 80 DAG (J) and a dh mutant at 100 DAG (K). (L) Statistical analysis of SAM diameters of ZH11 and dh mutant before flowering transition. (M) Expression analysis of SAM identity genes, and floral identity genes in the shoot apex of ZH11 and dh mutant. e-EF-1a was used as internal control. FM, floral meristem; P1, leaf primordial; SAM, shoot apical meristem; B1, first bract; B2, second bract; PB, primary branch. Scale bars = 20 μm. ** represents significant difference compared to ZH11 (p < 0.01). Error bars indicate SD from at least six measurements.
Fig 7.
Reproductive organ abnormalities in dh mutant.
(A) Phenotype of the culms with young panicles. (B—D) Panicle structure of ZH11 (B) and a dh mutant (C, D). (E and F) SEM images showing the formation of primary branches in a ZH11 (E) and a dh mutant (F). PB, primary branch; Bar = 1cm (A—D), 100 μm (D, E).
Fig 8.
A model of osa-miR171c in the phase change pathway.
In the leaf, miR171 delays juvenile—adult phase change mainly by regulating miR156. In the late adult phase, osa-miR171c could affect the expression of florigens RFT1 and Hd3a by altering the expression of OsPHYC. RFT1 and Hd3a proteins move to the SAM, where FD and 14-3-3 is expressed. The module is proposed to activate the transcription of downstream floral promoter genes such as MADS14 and MADS15. At the same time, osa-miR171c and its targets affect SAM maintenance by regulating the expression of FON4, WOX4 genes. Potential influences are indicated by plain arrows (positive associations) and plain T-ended lines (negative associations).