Functional Characterization of microRNA171 Family in Tomato

Abstract

Deeply conserved plant microRNAs (miRNAs) function as pivotal regulators of development. Nevertheless, in the model crop Solanum lycopersicum (tomato) several conserved miRNAs are still poorly annotated and knowledge about their functions is lacking. Here, the tomato miR171 family was functionally analyzed. We found that the tomato genome contains at least 11 SlMIR171 genes that are differentially expressed along tomato development. Downregulation of sly-miR171 in tomato was successfully achieved by transgenic expression of a short tandem target mimic construct (STTM171). Consequently, sly-miR171-targeted mRNAs were upregulated in the silenced plants. Target upregulation was associated with irregular compound leaf development and an increase in the number of axillary branches. A prominent phenotype of STTM171 expressing plants was their male sterility due to a production of a low number of malformed and nonviable pollen. We showed that sly-miR171 was expressed in anthers along microsporogenesis and significantly silenced upon STTM171 expression. Sly-miR171-silenced anthers showed delayed tapetum ontogenesis and reduced callose deposition around the tetrads, both of which together or separately can impair pollen development. Collectively, our results show that sly-miR171 is involved in the regulation of anther development as well as shoot branching and compound leaf morphogenesis.

Keywords: STTM; callose; miR171; pollen; tapetum; tomato.

Figure 1

The tomato miR171 family. (A) Nucleotide sequence alignment of Arabidopsis (ath-miR171) and tomato (sly-miR171) miR171 members. Relative abundance in seedlings is indicated for each on the right. (B) Examples of sly-miR171 precursors that encode two sly-miR171 isoforms. The sequence of SlMIR171a,b (SL2.50ch07:60813582..60813672) and SlMIR171d, iso-SlMIR171d (SL2.50ch12:519795..519887) stem and loops. The sequences of sly-miR171a/d, sly-miR171a*/d*, sly-miR171b/ sly-miRiso-d and sly-miR171b*/iso-d* are bold-face, italicized, underlined and double-underlined, respectively. (C) Accumulation of sly-miR171 members in flower and fruit tissues of tomato cv. Microtome (Flower and fruit) and Heinz (leaf) based on small RNA-seq data deposited in the TFGD database. Dpb—days post breaker.

Figure 2

Generation of transgenic STTM171 tomato with reduced sly-miR171 levels. (A) A scheme of the Short Tandem Target Mimic construct used for tomato M82 transformation. The Watson–Crick pairings between group A and B target mimic sites and sly-miR171 representative members are shown in the expanded region. (B) RNA gel blot analysis of sly-miR171 levels in indicated transgenic T0 plants. Total RNA (5 µg) from leaves was probed by sly-miR171a (sly-miR171a-b), sly-miR159 and U6 antisense probes. Sly-miR171 expression levels were determined after normalization to sly-miR159 and U6 snRNA by geometric averaging and are indicated below. (C) RT-qPCR analysis of sly-miR171 target transcripts in RNA from young leaves of one-month old T2 35S:STTM171-17 plants. TIP41 expression values were used for normalization. Error bars indicate ± SD of three biological replicates, each measured in triplicate. Asterisks indicate significant difference relative to 35:GFP control plants (Tukey–Kramer multiple comparison test; p < 0.01). (D) RNA gel blot analysis of sly-miR171 in 5 µg total RNA from the samples analyzed in C. The blots were probed with sly-miR171a (sly-miR171a-b) antisense probe. Sly-miR171 expression levels were determined after normalization to U6 snRNA and are indicated below.

Figure 3

Vegetative phenotypes of 35S:STTM171 plants. (A) Photograph of representative fifth leaf and terminal leaflet from 45 DAG plants of indicated genotypes. (B) Quantitation of primary leaflet area and petiolule angle (indicated in (A)) in leaves (n = 26) similar to those shown in (A). (C) Quantitation of the number of axillary shoots on the main stem (≥0.5 cm) at eight leaf stage plants (n = 13). Error bars indicate ±SD. Asterisks indicate significant difference as determined by Student’s t-test (p ≤ 0.001). Representative plant of each genotype is shown below. Pot diameter = 18.8 cm.

Figure 4

Effect of miR171 family downregulation on pollen quality and quantity. (A) Quantitation of total, aborted, non-aborted, and germinated mature pollen grains per anthesis flower of indicated genotype (n = 30). Asterisks indicate significant difference as determined by Student’s t-test (p ≤ 0.01). (B) Scanning electron micrographs of dehydrated pollen grains from indicated genotypes. Note the high number of collapsed pollen grains in the 35S:STTM171 sample. Inset shows magnified views of few representative pollen grains from each genotype. PW: pollen wall; GA: germinal aperture. (C) Scanning electron micrographs of polar view of representative mature pollen grains from indicated genotypes. The locations of the germinal aperture are indicated by asterisks.

Figure 5

The 35:STTM171 pollen grains contain normal germ unit. DAPI fluorescence micrographs of control and 35:STTM171 tetrads (A), microspores (B), binucleate microspore (C), mature pollen grains (D). Inset in (D) shows magnified view of a representative pollen grain. The vegetative (V) and generative (G) cells are indicated. Note that the pollen surface fluorescence is due to auto-fluorescence at the same wavelength used for DAPI detection.

Figure 6

Histological analysis of control and 35S:STTM171 anthers. Pictures of Toluidine-blue stained cross sections of control and 35:STTM171 anthers at subsequent stages of microspore development as follows: (A,B) microsporocyte stage, (C,D) meiosis stage, (E,F) tetrad stage, (G,H) microspore stage, (I,J) bicellular pollen stage. dT-degenerated tapetum; En-endothecium; Ep-epidermis; ML-middle cell layer; Msp-microspore; MMC-microspore mother cell; T-tapetum; Tds-tetrads; aPollen-aborted pollen. Scale bars = 20 μm.

Figure 7

Callose detection in control and 35S:STTM171 anthers. Pictures of Lacmoid stained cross sections of anthers at the meiosis (A, 4 mm bud) and tetrad (B, 5 mm bud) stages are shown. T-tapetum. Scale bars = 20 μm.