Overexpression of SEPALLATA3-like Gene SnMADS37 Generates Green Petal-Tip Flowers in Solanum nigrum

Abstract

The SEPALLATA3 (SEP3)-like MADS-box genes play crucial roles in determining petal identity and development in the petunia and tomato of Solanaceae. Solanum nigrum is a self-pollinating plant in the Solanaceae family, and produces white flowers. However, the mechanisms controlling the transition from green to white petals during flower development remain poorly understood. In this study, we isolated a flower-specific SEP3-like gene, SnMADS37, from S. nigrum, and investigated its potential role in chlorophyll metabolism during petal development. Our results show that quantitative RT-PCR analysis demonstrates that SnMADS37 is exclusively expressed in petals and stamens during early floral bud development. Overexpression of SnMADS37 clearly enhanced the number of petals, promoting the formation of additional petal-like tissues in stamens and extra organs in some fruits. Moreover, fully opened transformed petals exhibited notable chlorophyll accumulation at their tips and veins, whereas silencing of Snmads37 clearly inhibited petal expansion and reduced green pigmentation in early flower buds. Additionally, the transformed green petals exhibited distinct conical epidermal cells in the green regions, similar to wild type (WT) petals. Our results demonstrate that SnMADS37 plays a critical role in regulating petal identity, expansion, and chlorophyll metabolism during petal development. These findings provide new insights into the functional diversification of SEP3-like MADS-box genes in angiosperms.

Keywords: MADS-box; SEPALLATA3-like gene SnMADS37; Solanaceae; Solanum nigrum; chlorophyll accumulation; green petal-tip flower; overexpression; petal development.

Figure 1

Sequence comparison between SnMADS37 and related MADS-box proteins. The deduced amino acid sequence aligned with the sequences of AtSEP3 (Arabidopsis) and LeSEP3 (tomato). The MADS-box and K-box domains are indicated by lines above, and the three sub-domains in the K-box, namely K1, K2, and K3, are underlined.

Figure 2

Phylogenetic analysis and quantitative RT-PCR analysis of SnMADS37. (A) SnMADS37 identified in this study with selected SEP-like genes. The tree was constructed using the neighbor-joining method using MEGA 11 software and the bootstrap values for 1000 replicates. (B) Relative expression analysis of SnMADS37 in the roots, leaves, stem, and early small inflorescence (Inflo); (C) Relative expression analysis of SnMADS37 in the sepal, petal, stamen, and carpel of earlier stage 1 in supplemental Figure S2. Asterisks indicate statistically significant differences (n = 3; ** p < 0.01).

Figure 3

Quantitative RT-PCR analysis of SnMADS37 in four developmental stages of wild type (WT) flowers. (A) Comparison of the shapes and chlorophyll colorations of the petals among four flower developmental stages. se: sepal; pe: petal. (B) Analysis of the relative expression of SnMADS37 in four flower developmental stages. (C) Chlorophyll content comparison among four flower developmental stages. Asterisks indicate statistically significant differences (n = 3; * p < 0.05; ** p < 0.01).

Figure 4

Flower phenotype comparison between WT and transformed S. nigrum plants grown in a climate chamber (25 °C, 16 h light condition). (A) Inflorescence of WT; (B) inflorescence of OE37-5; (C) inflorescence of OE37-8; (D) inflorescence of OE37-10; (E) flower of WT, showing five petals and stamens; (F) flower, showing seven petals and stamens; (G) flower, showing six petals and stamens; (H) flower, showing notable greenish petals; (I) five stamens of WT plant from panel (E); (J) seven stamens of the case in panel (F); (K) six stamens of the case in panel (G); (L) five stamens of the case in panel (H); (M) magnified petaloid anther of the case in panel (F); (N) WT fruit; (O) Extra organ developing transformed fruits of OE37-8; (P) Extra organ developing transformed fruits of OE37-10. The increased petal-like tissue in panels (F–H,J,K), and (L) formed an additional organ on the fruit in panels (O,P), which is indicated by the white arrow (se: sepal; pe: petal; st: stamen; fil: filament; an: anther; fr: fruit; plt: petal-like tissue, and es: extra shoot like organ).

Figure 5

Petal-tip comparison between the WT and OE37-10 flower. (A) WT flower; (B) OE37-10 flower; (C) SEM image of adaxial surface of petal-tip in panel (A); (D) SEM image of adaxial surface of petal-tip in panel (B); (E) transection of petal-tip in panel (A); (F) transection of petal-tip in panel (B) (Ad: adaxial of petal; Ab: abaxial of petal; Tri: trichome; Cc: conical cell).

Figure 6

Comparison of chlorophyll content and relative expression analysis of B-function genes in fully opened stage 4 petals among a WT and four transgenic S. nigrum plants. (A) Analysis of the total chlorophyll content from petals of a WT and four transgenic plants; (B) SnMADS37 expression analysis from petals of a WT and four transgenic plants; (C) B-function gene SnGLO expression analysis from petals of a WT and four transgenic plants; (D) B-function gene SnDEF expression analysis from petals of a WT and four transgenic plants. OE37-1, 5, 8, and 10: four transformed plants with pBI-35S::SnMADS37. Asterisks indicate statistically significant differences between the WT and transgenic lines (n = 3; * p < 0.05; ** p < 0.01). Chl a: chlorophyll a; Chl b: chlorophyll b.

Figure 7

Comparison of phenotype between a control and an SnMADS37-silenced stage 1 floral bud of S. nigrum by VIGS treatment. (A) a control stage 1 floral bud treated with GV3101/TRV2; (B) an Snmads37-silenced stage 1 floral bud treated with GV3101/TRV2::Snmads37; (C) total RNA was extracted from stage 1 floral bud in (A) and (B), respectively, and relative expression of SnMADS37 was detected by qRT-PCR analysis. Asterisks indicate statistically significant differences (n = 3; ** p < 0.01). Se, sepal; Pe. petal; Sti, stigma.