240 Mol Genet Genomics (2015)290:239-255 in Asia.Moreover,Chinese cabbage has become a vegeta- made great progress in elucidating the roles of these genes ble that is grown worldwide due to its high yield and good in plant development.Further genetic and molecular anal- quality.Thus,the growth,development and flowering time yses regarding their biological functions have focused on of this plant are significant for its yield.Recently.the Chi- flower organogenesis,which acts as the major component nese cabbage(Chiifu-401-42)genome has been sequenced, in the well-known ABCDE model:sepals(A +E),petals and this sequence can help us with the analysis of MADS- (A +B+E),stamens (B+C+E),carpels (C E),and box genes from the entire genome (Wang et al.2011).This ovules (D+E)(Zahn et al.2006).Briefly,a previous study genome has undergone triplication events since its diver- of Arabidopsis MIKC genes classified these genes into five gence from Arabidopsis (13-17 mya)(Wang et al.2011); functional classes as follows:Class A includes APETALAl however,a high degree of sequence similarity and con- (API);class B includes PISTILATA (PD)and AP3;class C served genome structure remain between these two species, includes AGAMOUS (AG):class D includes SEEDSTICK/ these traits make B.rapa a good species to use to study the AGAMOUS-LIKEII (STK/AGL11);and class E includes retention and ortholog groups of MADS-box genes dur- SEPALLATA (SEPI,SEP2,SEP3,and SEP4)(Pinyop- ing genome duplication events.Furthermore,plant growth ich et al.2003).Other MIKC genes were later identified and development are influenced greatly by numerous plant as being involved in different regulatory steps,such as: growth regulators and environmental factors. (1)Determination of flowering time genes,which include MADS proteins are characterised by the presence of a Suppressor of Overexpression Of Constansl (SOCI) conserved 58-60 amino acids long DNA-binding domain (Samach et al.2000;Moon et al.2003a,b),AGAMOUS- in the N-terminal region,which is known as the MADS LIKE GENE 24 (AGL24)(Liu et al.2008),Short Vegeta- domain,and which binds to CArG boxes (Yanofsky et al. tive Phase (SVP)(Lee et al.2007),MADS Affecting Flow- 1990).Based on the phylogenetic analysis,the plant MADS ering (MAF1/FLM),Flowering Locus c(FLC)(Michaels gene family is divided into two large lineages,type I and and Amasino 1999;Ratcliffe et al.2003)and AGL/5, type II,which were generated by an ancestral gene duplica- AGL/8(Adamczyk et al.2007);(2)Fruit ripening genes, tion event (Alvarez-Buylla et al.2000;Becker and TheiBen which include SHATTERPROOF 1-2(SHPI.SHP2)and 2003).The type I genes encode SRF-like domain proteins, FUL (Liljegren et al.2000);(3)Seed pigmentation and whereas type II genes encode MEF2-like proteins(De Bodt embryo development genes,which include TRANSPARENT et al.2003).The plant type II proteins are named MIKC due TESTA/6(TT/6)(Nesi et al.2002).Apart from reproduc- to their four domains.In addition to the MADS(M)domain. tive development,MIKC genes also function in vegetative MIKC type proteins contain the I (intervening),K(keratin- development and root development,such as AGL/2 and like)and C(C-terminal)domains (Cho et al.1999).The I AGLI7 genes (Tapia-Lopez et al.2008). domain contributes to dimer formation (Henschel et al. Some MIKCC genes have already been shown to play 2002).The K domain is characterised by a coiled-coil struc- key roles to control flowing time in Brassica,such as ture,which primarily regulates to the dimerisation of MADS BrFLCI,2,3,BcFLC,BrAGL20 and BnAP3 (Pylatuik proteins (Diaz-Riquelme et al.2009).The C domain func- et al.2003;Hong et al.2012;Liu et al.2013).For example, tions in transcriptional activation and in the formation of the overexpression of BrAGL20 can significantly affect the higher order protein complexes (Honma and Goto 2001). flowering time of B.napus,and BrFLC genes act similar to MIKC-type genes have been further divided into two sub- AtFLC,with lower expression in early-flowering Chinese groups,MIKCC and MIKC*,based on sequence divergence cabbage (Hong et al.2012).Furthermore,plant growth at the I domain (Henschel et al.2002).The MIKC*genes and development are infuenced greatly by numerous plant encode proteins that tend to have longer I domains and have growth regulators and environmental factors.Gibberel- a duplicated K domain.The type I lineage groups genes lin(GA)promotes flower formation and flowering time in with a relatively simple gene structure (only with one or biennial plants.Its involvement in flower initiation in plants two exons)that lack the K domain and that have common is well-established,and there is growing insight into the ancestors.The type I genes are subdivided into three groups, mechanisms by which floral induction is achieved (Mutasa- Ma,MB,My,based on the sequence of the MADS domain Gottgens and Hedden 2009).Salicylic acid(SA)also reg- and on the presence of additional motifs.The function of the ulates flowering time because SA-deficient plants are late type I genes appears to be restricted to female gametophyte flowering (Martinez et al.2004).Abscisic acid (ABA) (AGL80 and AGL61)and seed development (PHEI,PHE2, regulates many aspects of plant growth and development AGL23.AGL28.AGL40.AGL62)(Kohler et al.2003:Bemer (Bezerra et al.2004;Wilmowicz et al.2008).As important et al.2010:Colombo et al.2008:Masiero et al.2011). environmental stress factors,cold and heat also regulate Plant MIKC genes were first identified as floral organ plant growth and development.To learn more about the identity genes in Antirrhinum majus and in Arabidopsis response of B.rapa MADS-box genes to abiotic stresses, (Sommer et al.1990;Yanofsky et al.1990).Biologists have we selected these five treatments to explore in this study Springer240 Mol Genet Genomics (2015) 290:239–255 1 3 in Asia. Moreover, Chinese cabbage has become a vegeta￾ble that is grown worldwide due to its high yield and good quality. Thus, the growth, development and flowering time of this plant are significant for its yield. Recently, the Chi￾nese cabbage (Chiifu-401-42) genome has been sequenced, and this sequence can help us with the analysis of MADS￾box genes from the entire genome (Wang et al. 2011). This genome has undergone triplication events since its diver￾gence from Arabidopsis (13–17 mya) (Wang et al. 2011); however, a high degree of sequence similarity and con￾served genome structure remain between these two species, these traits make B. rapa a good species to use to study the retention and ortholog groups of MADS-box genes dur￾ing genome duplication events. Furthermore, plant growth and development are influenced greatly by numerous plant growth regulators and environmental factors. MADS proteins are characterised by the presence of a conserved 58–60 amino acids long DNA-binding domain in the N-terminal region, which is known as the MADS domain, and which binds to CArG boxes (Yanofsky et al. 1990). Based on the phylogenetic analysis, the plant MADS gene family is divided into two large lineages, type I and type II, which were generated by an ancestral gene duplica￾tion event (Alvarez-Buylla et al. 2000; Becker and Theißen 2003). The type I genes encode SRF-like domain proteins, whereas type II genes encode MEF2-like proteins (De Bodt et al. 2003). The plant type II proteins are named MIKC due to their four domains. In addition to the MADS (M) domain, MIKC type proteins contain the I (intervening), K (keratin￾like) and C (C-terminal) domains (Cho et al. 1999). The I domain contributes to dimer formation (Henschel et al. 2002). The K domain is characterised by a coiled-coil struc￾ture, which primarily regulates to the dimerisation of MADS proteins (Díaz-Riquelme et al. 2009). The C domain func￾tions in transcriptional activation and in the formation of higher order protein complexes (Honma and Goto 2001). MIKC-type genes have been further divided into two sub￾groups, MIKCC and MIKC*, based on sequence divergence at the I domain (Henschel et al. 2002). The MIKC* genes encode proteins that tend to have longer I domains and have a duplicated K domain. The type I lineage groups genes with a relatively simple gene structure (only with one or two exons) that lack the K domain and that have common ancestors. The type I genes are subdivided into three groups, Mα, Mβ, Mγ, based on the sequence of the MADS domain and on the presence of additional motifs. The function of the type I genes appears to be restricted to female gametophyte (AGL80 and AGL61) and seed development (PHE1, PHE2, AGL23, AGL28, AGL40, AGL62) (Köhler et al. 2003; Bemer et al. 2010; Colombo et al. 2008; Masiero et al. 2011). Plant MIKC genes were first identified as floral organ identity genes in Antirrhinum majus and in Arabidopsis (Sommer et al. 1990; Yanofsky et al. 1990). Biologists have made great progress in elucidating the roles of these genes in plant development. Further genetic and molecular anal￾yses regarding their biological functions have focused on flower organogenesis, which acts as the major component in the well-known ABCDE model: sepals (A + E), petals (A + B + E), stamens (B + C + E), carpels (C + E), and ovules (D + E) (Zahn et al. 2006). Briefly, a previous study of Arabidopsis MIKC genes classified these genes into five functional classes as follows: Class A includes APETALA1 (AP1); class B includes PISTILATA (PI) and AP3; class C includes AGAMOUS (AG); class D includes SEEDSTICK/ AGAMOUS-LIKE11 (STK/AGL11); and class E includes SEPALLATA (SEP1, SEP2, SEP3, and SEP4) (Pinyop￾ich et al. 2003). Other MIKC genes were later identified as being involved in different regulatory steps, such as: (1) Determination of flowering time genes, which include Suppressor of Overexpression Of Constans1 (SOC1) (Samach et al. 2000; Moon et al. 2003a, b), AGAMOUS￾LIKE GENE 24 (AGL24) (Liu et al. 2008), Short Vegeta￾tive Phase (SVP) (Lee et al. 2007), MADS Affecting Flow￾ering (MAF1/FLM), Flowering Locus c(FLC) (Michaels and Amasino 1999; Ratcliffe et al. 2003) and AGL15, AGL18 (Adamczyk et al. 2007); (2) Fruit ripening genes, which include SHATTERPROOF 1–2 (SHP1, SHP2) and FUL (Liljegren et al. 2000); (3) Seed pigmentation and embryo development genes, which include TRANSPARENT TESTA16 (TT16) (Nesi et al. 2002). Apart from reproduc￾tive development, MIKC genes also function in vegetative development and root development, such as AGL12 and AGL17 genes (Tapia-López et al. 2008). Some MIKCC genes have already been shown to play key roles to control flowing time in Brassica, such as BrFLC1, 2, 3, BcFLC, BrAGL20 and BnAP3 (Pylatuik et al. 2003; Hong et al. 2012; Liu et al. 2013). For example, the overexpression of BrAGL20 can significantly affect the flowering time of B. napus, and BrFLC genes act similar to AtFLC, with lower expression in early-flowering Chinese cabbage (Hong et al. 2012). Furthermore, plant growth and development are influenced greatly by numerous plant growth regulators and environmental factors. Gibberel￾lin (GA) promotes flower formation and flowering time in biennial plants. Its involvement in flower initiation in plants is well-established, and there is growing insight into the mechanisms by which floral induction is achieved (Mutasa￾Göttgens and Hedden 2009). Salicylic acid (SA) also reg￾ulates flowering time because SA-deficient plants are late flowering (Martínez et al. 2004). Abscisic acid (ABA) regulates many aspects of plant growth and development (Bezerra et al. 2004; Wilmowicz et al. 2008). As important environmental stress factors, cold and heat also regulate plant growth and development. To learn more about the response of B. rapa MADS-box genes to abiotic stresses, we selected these five treatments to explore in this study