The functional analysis of SlNCED1 in tomato pollen development

Abstract

Abscisic acid (ABA) regulates plant growth and development, but the role of ABA in the development of reproductive organs in tomato has rarely been addressed. In the present study, the role of ABA in the regulation of male and female gametogenesis as well as pollen development and germination is tested in tomato. qRT-PCR and in situ hybridization analysis of 9-cis-epoxycarotenoid dioxygenase (SlNCED1), a key enzyme in the ABA biosynthetic pathway, showed high expression of SlNCED1 primarily in the meristem during gametogenesis and mainly in ovule, stigma, anther/pollen and vascular tissues during floral organ development. SlNCED1 expression and ABA accumulation in anther peak at stages 13-14, suggesting that ABA plays a role in the primary formation of pollen grains. Over expression and suppression of SlNCED1 led to the abnormal development of anther/pollen, especially in SlNCED1-OE lines, which have serious pollen deterioration. The percentage of pollen germination in wild type is 91.47%, whereas it is 6.85% in OE transgenic lines and 38.4% at anthesis in RNAi lines. RNA-Seq of anthers shows that SlNCED1-OE can significantly enhance the expression of SlPP2Cs and down-regulate the expression of SlMYB108 and SlMYB21, which are anther/flower-specific transcriptional factors in tomato. Finally, anther transcriptome data indicate that SlNCED1 is involved in ABA-mediated regulation in pollen/anther metabolism, cell wall modification, and transcription levels. These results support an important role for ABA in the development of reproductive organs in tomato and contribute to the elucidation of the underlying regulatory mechanisms.

Keywords: ABA signaling; Anther development; Gametogenesis; MYBs; Pollen germination; SlNCED1-RNAi/OE.

Fig. 1

Changes in the expressions of SlNCED1/2, and ABA content and ethylene release in tomato anther and whole flower (except sepals) during floral development. SAND was employed as an internal control (Fig. S8). Three biological replicates (n = 3) are used for each analysis. Error bars are SE

Fig. 2

Temporo-spatial expression patterns of SlNCED1 in tomato flower from in situ hybridization detection. Longitudinal sections of flower bud at different developmental stages (1–20). (1–3) The expression of SlNCED1 in floral meristem (FM) (indicated by the arrow). (4, 5) Presence of SlNCED1 transcripts in the primordia of petals and FM. (6) The expression of SlNCED1 in stamen and pistil primordia. (S) Sense control. (7–14) Expression of SlNCED1 in stamen, pistil and vascular bundle located on the base of bud. (16) Expression of SlNCED1 in pistil stigma and ovule. (18–20) Expression of SlNCED1 in style and ovule. a Enlarged view of stage 9 at 40× magnifying power; b enlarged view of stage 13–14 at 40× magnifying power; c enlarged view of stage 16. Scale bar (a–c) was 50 μm; other scale bar was 500 μm. FM flower meristem, SeP sepal primordium, Se sepal, PeP petal primordium, P petal, St stamen, SP stamen primordia, Pis pistil, PP pistil primordia, C carp

Fig. 3

The relative expression levels of genes related to ABA signaling and transcriptional factors in WT and SlNCED1-OE/RNAi transgenic anther. Gene expressions were examined at the 13–14 stage through qRT-PCR, with SAND as the internal control. *P value t test < 0.05; **P value t test < 0.01

Fig. 4

The phenotypic characterization of flowers at anthesis in wild type and SlNCED1-RNAi and OE lines. a A representative flower at anthesis in WT. The stamen and pistil phenotypes of WT at full bloom. b, c The representative flower at anthesis in SlNCED1-RNAi 3/4. d, e The representative flower at anthesis in SlNCED1-OE 1/2. The stamens and pistil after sepals and petals were removed in SlNCED1-OE 1/2 at full bloom. f Changes in transcription levels of SlNCED1 and ABA accumulation in anthers of SlNCED1-RNAi, SlNCED1-OE and wild-type plants at stages 13–14. SAND was employed as an internal control. Three biological replicates (n = 3) were used for each analysis. Error bars are SE. *P value t test < 0.05; **P value t test < 0.01

Fig. 5

Anther development in SlNCED1-OE 2 plants is abnormal. a Light micrographs of the nine stages of SlNCED1-OE 2 anther development. The cross-sections of anthers at different development stages (6–20). Scale bar was 200 μm. b Comparison of normal pollen rate in WT and SlNCED1-RNAi 3 or OE 2 during different development stages. c Percentage of pollen germination in WT and SlNCED1-RNAi 3 or OE 2. Fresh pollen grains were scattered onto pollen germination medium with or without 10 µM ABA or 10 µM NDGA. The pollen grains were then germinated in dark at 25 °C for 3 h incubation. Three biological replicates (n = 3) were used for each analysis. *P value t test < 0.05; **P value t test < 0.01. d Electron microscope observation of pollen in WT and SlNCED1-RNAi/OE transgenic lines during flower full bloom

Fig. 6

Phylogenetic relationship of transcription factors in the MYB family and transcript patterns of SlMYB21 and SlMYB108. a Analysis of anther transcriptome data of SlNCED1-OE 2 at stage 13–14 of floral development. b Transcript patterns of SlMYB21 and SlMYB108 in different organs and tissues of tomato. c, e Expression patterns of SlMYB21 and SlMYB108 in SlNCED1-OE 2 flowers. The 22 on the horizontal axis indicates 3 days after full bloom. d, f Transcript patterns of SlMYB21 and SlMYB108 in different organs and tissues of SlNCED1-OE 2 tomato. SAND was employed as an internal control. Three biological replicates (n = 3) were used for each analysis. Error bars are SE. *P value t test < 0.05; **P value t test < 0.01

Fig. 7

The expression of genes involved in anther/pollen development and germination. a–c The expression of genes involved in receptor-like protein kinases (PRK) signaling, small GTPase signaling and Ca²⁺ in the anther of WT and SlNCED1-OE 2 at stage 13–14. d, e SlNCED1-OE 2 alters the expression of genes involved in carbohydrate and lipid metabolism in anthers. f, g SlNCED1-OE 2 alters the expression of genes involved in cell wall metabolism in anthers in WT and transgenic lines at stage 13–14. f Expression of genes related to cell wall-degrading enzymes. g Expression of genes related to cytoskeleton components