Identification of MADS-Box Transcription Factors in Iris laevigata and Functional Assessment of IlSEP3 and IlSVP during Flowering

Abstract

Iris laevigata is ideal for gardening and landscaping in northeast China because of its beautiful flowers and strong cold resistance. However, the short length of flowering time (2 days for individual flowers) greatly limits its applications. Molecular breeding and engineering hold high potential for producing I. laevigata of desirable flowering properties. A prerequisite is to identify and characterize key flowering control genes, the identity of which remains largely unknown in I. laevigata due to the lack of genome information. To fill this knowledge gap, we used sequencing data of the I. laevigata transcriptome to identify MADS-box gene-encoding transcription factors that have been shown to play key roles in developmental processes, including flowering. Our data revealed 41 putative MADS-box genes, which consisted of 8 type I (5 Mα and 3 Mβ, respectively) and 33 type II members (2 MIKC* and 31 MIKC^C, respectively). We then selected IlSEP3 and IlSVP for functional studies and found that both are localized to the nucleus and that they interact physically in vitro. Ectopic expression of IlSEP3 in Arabidopsis resulted in early flowering (32 days) compared to that of control plants (36 days), which could be mediated by modulating the expression of FT, SOC1, AP1, SVP, SPL3, VRN1, and GA20OX. By contrast, plants overexpressing IlSVP were phenotypically similar to that of wild type. Our functional validation of IlSEP3 was consistent with the notion that SEP3 promotes flowering in multiple plant species and indicated that IlSEP3 regulates flowering in I. laevigata. Taken together, this work provided a systematic identification of MADS-box genes in I. laevigata and demonstrated that the flowering time of I. laevigata can be genetically controlled by altering the expression of key MADS-box genes.

Keywords: Arabidopsis; Iris; MADS-box; SEP3; SVP; flowering; transcription factor.

Figure 1

Phylogenetic analysis of MADS-box proteins. Red and black denote MADS-box proteins from I. laevigata and Arabidopsis, respectively.

Figure 2

Sequence alignment of IlSEP3 proteins from different species. IlSEP3: Iris laevigata (ON398430); AoAGL9: Asparagus officinalis (XP_020247630.1); CsSEP3: Crocus sativus (ACB69509.1); AcSEP3: Allium cepa (QCT25556.1); FhSEP3: Freesia hybrid cultivar (QGV23785.1); AcAGL9: Ananas comosus (XP_020107646.1); PdAGL9: Phoenix dactylifera (XP_038986890.1); EgAGL9: Elaeis guineensis (XP_010913017.1); MsAGL9: Musa acuminata subsp. Malaccensis (XP_009415892.1); PaAGL9: Persea americana (AAX15924.1); AhSEP3: Alpinia hainanensis (ALB09087.1); DcAGL9: Dendrobium catenatum (XP_020706096.1); EpSEP3: Euptelea pleiosperma (ADC79706.1); NnAGL9: Nelumbo nucifera (XP_010250667.1); CgSEP3: Cymbidium goeringii (AHJ80843.1).

Figure 3

Sequence alignment of IlSVP proteins from different species. IlSVP: Iris laevigata (ON398431); CsSVP: Crocus sativus (QIH12017.1); NnSVP: Nelumbo nucifera (XP_010254525.1); EgSVP: Elaeis guineensis (XP_010942683.1); CnSVP: Cocos nucifera (EHA8591139.1); HhSVP: Hemerocallis hybrid cultivar (QBX87860.1); VvSVP: Vitis vinifera (XP_019073897.1); MeSVP: Manihot esculenta (XP_021631111.1); HbSVP: Hevea brasiliensis (XP_021662492.1); VrSVP: Vitis riparia (XP_034692566.1); SiSVP: Sesamum indicum (XP_020554864.1); PcSVP: Paphiopedilum callosum (QXO37013.1); QlSVP: Quercus lobata (XP_030923627.1); PdSVP: Phoenix dactylifera (XP_038983281.1); MaSVP: Morus alba var. alba (AYK27567.1).

Figure 4

Subcellular localization of IlSEP3 and IlSVP. (A) Bright-field; (B) GFP; (C) merged signals.

Figure 5

Interaction between IlSEP3 and IlSVP by yeast-two-hybrid. Numbers on top indicate dilution factors. The relative interaction strengths were determined by increasing dilution of yeast colonies. AD, active domain; BD binding domain. For co-transformation, eight groups were assessed: the test group (pGBKT7-IlSEP3 and pGADT7-IlSVP), (pGBKT7-IlSVP and pGADT7-IlSEP3), control for self-activation (pGBKT7-IlSEP3 and empty), (pGBKT7-IlSVP and empty), (empty and pGADT7-IlSVP), (empty and pGADT7-IlSEP3), positive control (pGADT7-T and pGBKT7-53), and negative control (pGADT7-T and pGBKT7-Lam). Transformed cells were diluted and then grown on either SD/-Trp/-Leu or SD/-Trp/-Leu/-Ade/-His/X-α-Gal/ABA at 28 °C for 48 h.

Figure 6

Phenotypes of transgenic Arabidopsis overexpressing IlSEP3. (A) Representative plants grown under long-day conditions. Bar = 10 cm. (B) Days to bolting (inflorescence stalk of 1 cm). (C) Days to flowering (opening of the first flower). (D) The number of rosette leaves at the time of flowering. All data represent mean ± standard error. Statistical difference was denoted by lowercase letters (p < 0.05).

Figure 7

Expression of endogenous flowering time genes in transgenic Arabidopsis overexpressing IlSEP3. Data represent mean ± standard error. Statistical difference was denoted by * (p < 0.05) or ** (p < 0.01).