Expression Pattern of AsFLC Gene and Its Relationship with the AsSOC1, AsAP1 and AsAP2 Genes During Vernalization in Bolting, Semi- and Non-bolting Iranian Garlic (Allium sativum L.)

Document Type : Research Article

Authors

• F. Ghaemizadeh ¹
• F. Dashti ²

¹ Department of Horticultural Sciences, Faculty of Agriculture, Bu-Ali Sina University, Hamedan, Iran.

² Department of Horticultural Sciences, Faculty of Agriculture, Bu-Ali Sina University, Hamadan. Iran.

https://doi.org/10.22067/jhs.2024.88467.1352

Abstract

Introduction
At present, garlic (Allium sativum L.) production is completely dependent on asexual propagation, but a wide diversity of bolting and scape formation is observed in garlic. Based on their ability to produce flowering stem (scape), garlic clones are classified into non-bolting, semi-bolting, and bolting clones. In non-bolting clones, scape is not formed or abort at early stages. In semi-bolting clones, cessation of scape development often results in the formation of very short scape between the leaves and development of the elongated leaf-like bracts in the center of the inflorescence. In bolting clones after exposure to low temperature during autumn and winter (vernalization) and long day during spring, long and thick scape is formed which contains inflorescence with flowers. Transition from vegetative to reproductive phase and formation of scape, inflorescences and flowers in plants includes a series of continuous stages which control by several gene groups. Vernalization reduces the expression of the flowering inhibitors like FLOWERING LOCUC C (FLC), resulting in increased flowering integrators expression like SUPPRESSOR OF OVER EXPRESSON OF CONSTANT 1 (SOC1), APETALA 1 (AP1) and APETALA 1 (AP2). So, a correct understanding of the vernalization control genes expression pattern will improve garlic flowering and breeding programs. The aim of this study was to investigate the relative expression of AsFLC, AsSOC1, AsAP1 and AsAP2 before and after vernalization in Iranian bolting, semi-bolting and non-bolting garlic clones.
 
Materials and Methods
In this study, three garlic clones including, bolting (Mazand Zabol), semi-bolting (Langrud), and non-bolting (Hamedan) garlic clones were selected from vegetable collection of Bu-Ali Sina university (Hamedan, Iran). At first, RNA extracted from meristems of three clones monthly, from 4 to 20 weeks after planting (for AsFLC, AsSOC1, AsAP1 and AsAP2 expression analysis) and from inflorescence of semi-bolting and bolting clones at 24 weeks after planting (for AsSOC1, AsAP1 and AsAP2 expression analysis) at 2 biological replicates. Then, cDNA synthesized using Oligo d(T) primer and relative expression pattern of the mentioned genes were analyzed using quantitative Real time- PCR.
 
Results and discussion
The highest expression of the AsFLC in all three clones were observed at 4 weeks after culture (before vernalization). Its expression in non-bolting clone at 4 weeks after planting was 2.03 and 1.13 times more than bolting and semi-bolting garlic, respectively. After vernalization AsFLC expression decreased in the meristem of the all three clones. The decrease in the relative expression of AsFLC in bolting garlic occurred at a faster rate compared to the other two garlic clones. Then the relative expression of the AsSOC1 was increased in the meristem during vegetative to reproductive transition phase (12 weeks after planting). The highest AsSOC1 expression was observed in the meristem of bolting garlic at 12 weeks after planting which was 10.98 and 18.41 times more than the meristem of semi-bolting and non-bolting garlic, respectively. AsAP1 was expressed in the meristem of three clones in the vegetative to reproductive phase, but its highest expression was in bolting clone at 12 weeks after planting and was 1.22 and 3.64 times more than the meristem of semi-bolting and non-bolting clone respectively. AsAP2 was just expressed in the meristem of semi-bolting and bolting clones and after reproductive transition. The highest expression of the AsAP2 was observed at 16 weeks after planting in the meristem of semi-bolting and bolting garlic, which was higher in bolting garlic (2.33 times) in comparison to semi–bolting garlic. Decreases in the expression of the AsFLC during vernalization and increases in the expression of the AsSOC1 and AsAP1 during vegetative to reproductive phase in the meristem may lead to scape formation in bolting garlic. However, the higher AsFLC and the lower AsSOC1 and AsAP1 expression in the meristems of non- and semi bolting garlics in comparison to bolting garlic inhibit scape formation. In non-bolting garlic scape aborts and in semi-bolting garlic short and thin scape formed in the middle of leaves. According to the results AsSOC1 and AsAP1 were expressed in the inflorescence of semi-bolting and bolting garlic. AsSOC1 and AsAP1 relative expression in the inflorescence of bolting garlic were 4.28 and 11.25 times more than semi-bolting garlic, respectively. AsAP2 was just expressed in the inflorescence of the bolting clone but wasn’t expressed in the inflorescence of semi-bolting garlic. The differences in the expression pattern of AsSOC1, AsAP1 and AsAP2 in the inflorescence of the garlic clones could be the reason of the abnormal inflorescence in semi-bolting garlic.
 
Conclusions
Finally, it seems that AsFLC is a flowering inhibitor and AsSOC1 and AsAP1 are flowering integrators in bolting garlic. As AsFLC expression decreased after vernalization and AsSOC1 and AsAP2 were expressed highly in the time of vegetative to reproductive transition in the meristem of all clones, and there was difference in the bolting of clones, it is suggested that these genes may influence flower induction but their low relative expression cause incomplete bolting in semi-bolting garlic and forbid bolting in non-bolting garlic.
 

Keywords

• Allium sativum
• Flowering inhibitor gene
• Flowering integrator gene
• Real-Time PCR

Main Subjects

• Growing vegetables

©2024 The author(s). This is an open access article distributed under Creative Commons Attribution 4.0 International License (CC BY 4.0).

References

1- Bao, S., Hua, C., Shen, L., & Yu, H. (2020). New insights into gibberellin signaling in regulating flowering in Arabidopsis. Journal of Integrative Plant Biology, 62(1), 118-131. https://doi.org/10.1111/jipb.12892

2- Brunner, A.M., Rottmann, W.H., Sheppard, L.A., Krutovskii, K., DiFazio, S.P., Leonardi, S.L., & Straus, S.H. (2000). Structure and expression of duplicate AGAMOUS orthologues in poplar. Plant Molecular Biology, 44, 619–634. https://link.springer.com/article/10.1023/A:1026550205851.

3- Chen, M.K., Lin, I.C., & Yang, C.H. (2008). Functional analysis of three lily (Lilium longiflorum) APETALA1-like MADS box genes in regulating floral transition and formation. Plant and Cell Physiology, 49(5), 704-717. https://doi.org/10.1093/pcp/pcn046

4- Chuck, G., Meeley, R., & Hake, S. (2008). Floral meristem initiation and meristem cell fate are regulated by the maize AP2 genes ids1 and sid1. Development, 135(18), 3013-3019. https://doi.org/10.1242/dev.024273

5- Deng, W., Ying, H., Helliwell, C. A., Taylor, J.M., Peacock, W.J., & Dennis, E.S. (2011). FLOWERING LOCUS C (FLC) regulates development pathways throughout the life cycle of Arabidopsis. Proceedings of the National Academy of Sciences of the USA, 108, 6680–6685. https://doi.org/10.1073/pnas.1103175108

6- Ding, L., Wang, Y., & Yu, H. (2013). Overexpression of DOSOC1, an ortholog of Arabidopsis SOC1, promotes flowering in the orchid Dendrobium chao parya smile. Plant Cell Physiology, 54, 595–608. https://doi.org/10.1093/pcp/pct026.

7- Fornara, F., Parenicova, L., Falasca, G., Pelucchi, N., Masiero, S., Ciannamea, S., Lopez-Dee, Z., Altamura, M.M., Colombo, L., & Kater, M.M. (2004). Functional characterization of OsMADS18, a member of the AP1/SQUA subfamily of MADS box genes. Plant Physiology, 135(4), 2207-2219. https://doi.org/10.1104/pp.104.045039.

8-Gocal, G.F., King, R.W., Blundell, C.A., Schwartz, O.M., Andersen, C.H., & Weigel, D. (2001) Evolution of floral meristem identity genes: Analysis of Lolium temulentum genes related to APETALA1 and LEAFY of arabidopsis. Plant Physiology, 125, 1788–1801. https://doi.org/10.1104/pp.125.4.1788.

9-Ghaemizadeh, F., Dashti, F.A.R.S.H.A.D., & Shafeinia, A.R. (2018). Expression analysis of gaLFY and AsFT during reproductive development in different organs of some Iranian garlic (Allium sativum L.) clones. Iranian Journal of Horticultural Science, 49(1), 269-278. (In Persian with English abstract). https://www.cabidigitallibrary.org/doi/full/10.5555/20203074943

10-Ghaemizadeh, F., Dashti, F., & Shafeinia, A. (2019). Expression pattern of ABCDE model genes in floral organs of bolting garlic clone. Gene Expression Patterns, 34, 119059. https://doi.org/10.1016/j.gep.2019.119059.

11-Ghaemizadeh, F., Dashti, F., & Mosavi, A. (2024). Expression pattern and structural analysis of AGAMOUS-LIKE 6 (AGL6) in Iranian garlic clones (Allium sativum L.). Agricultural Biotechnology Journal, 16(1), 155-174. (in Persian with English abstract). https://www.sid.ir/fileserver/jf/961-279249-fa-1129535.pdf

12-Greenup, A.G., Sasani, S., Oliver, S.N., Talbot, M.J., Dennis, E.S., Hemming, M.N., & Trevaskis, B. (2010). ODDSOC2 is a MADS box floral repressor that is down-regulated by vernalization in temperate cereals. Plant Physiology, 153(3), 1062-1073. https://doi.org/10.1104/pp.109.152488.

13-Hepworth, S.R. Valverde, F., Ravenscroft, D., Mouradov, A., & Coupland, G. (2002) Antagonistic regulation of flowering‐time gene SOC1 by CONSTANS and FLC via separate promoter motifs. The European Molecular Biology Organization Journal, 21(16), 4327-4337. https://doi.org/10.1093/emboj/cdf432

14-Helliwell, C.A., Wood, C.C., Robertson, M., James Peacock, W., & Dennis, E.S. (2006). The arabidopsis FLC protein interacts directly in vivo with SOC1 and FT chromatin and is part of a high‐molecular‐weight protein complex. The Plant Journal, 46(2), 183-192. https://doi.org/10.1111/j.1365-313X.2006.02686.x

15-Hantari, D., Purnomo, D., & Triharyanto, E. (2020). The effects of fertilizer composition and gibberellin on flowering and true shallot seed formation of three shallot varieties at the highlands. Conference Series: Earth and Environmental Science, 423(1), 012032. https://doi.org/10.1088/1755-1315/423/1/012032.

16-Irish, V.F., & Litt, A.T. (2005). Flower development and evolution: Gene duplication, diversification and redeployment. Current Opinion in Genetics and Development, 15(4), 454-460. https://doi.org/10.1016/j.gde.2005.06.001.

17-Kamenetsky, R., Faigenboim, A., Mayer, E.S., Michael, T.B., Gershberg, C., Kimhi, S., & Sherman, A. (2015). Integrated transcriptome catalogue and organ-specific profiling of gene expression in fertile garlic (Allium sativum L.). Biomed Centeral Genomics, 16(1), 12. https://doi.org/10.1186/s12864-015-1212-2.

18-Kennedy, A., & Geuten, K. (2020). The role of FLOWERING LOCUS C relatives in cereals. Frontiers in Plant Science, 11, 617340. https://doi.org/10.3389/fpls.2020.617340.

19-Kim, D.H., & Sung, S. (2014). Genetic and epigenetic mechanisms underlying vernalization. The Arabidopsis Book/American Society of Plant Biologists, 12, 1-15. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3952384/

20-Lee, R., Baldwin, S., Kenel, F., McCallum, J., & Macknight, R. (2013). FLOWERING LOCUS T genes control onion bulb formation and flowering. Nature Communications, 4(1), 1-9. https://www.nature.com/articles/ncomms3884.

21-Pan, R., Xu, L., Wei, Q., Wu, C., Tang, W., Oelmüller, R., & Zhang, W. (2017). Piriformospora indica promotes early flowering in Arabidopsis through regulation of the photoperiod and gibberellin pathways. Plos One, 12(12), e0189791.‏ https://doi.org/10.1371/journal.pone.0189791.

22-Liu, X.R., Pan, T., Liang, W.Q., Gao, L., Wang, X.J., Li, H.Q., & Liang, S. (2016) Overexpression of an orchid (Dendrobium nobile) SOC1/TM3-Like ortholog, DnAGL19, in Arabidopsis regulates HOS1-FT expression. Frontiers in Plant Science, 7(99), 1-12. https://doi.org/10.3389/fpls.2016.00099.

23-Livak, K.J., & Schmittgen, T.D. (2001). Analysis of relative gene expression data using Real-Time quantitative PCR and the 2− ΔΔCT methods. Methods, 25, 402-408. https://doi.org/10.1006/meth.2001.1262.

24-Murai, K., Miyamae, M., Kato, H., Takumi, S., & Ogihara, Y. (2003). WAP1, a wheat APETALA1 homolog, plays a central role in the phase transition from vegetative to reproductive growth. Plant and Cell Physiology, 44(12), 1255-1265. https://doi.org/10.1093/pcp/pcg171.

25-Medard, N.G., & Yanofsky, M.F. (2001). Function and evolution of the plant MADS-box gene family. Nature Reviews Genetics, 2(3), 186-195. https://doi.org/10.1038/35056041.

26-Nakamura, T., Song, I., Fukuda, T., Yokoyama, J., Maki, M., & Ochiai, T. (2005) Characterization of TrcMADS1 gene of Trillium camtschatcense (Trilliaceae) reveals functional evolution of the SOC1/TM3-like gene family. Journal of Plant Research, 118, 229–234. https://link.springer.com/article/10.1007/s10265-005-0215-5.

27-Ohto, M.A., Fischer, R.L., Goldberg, R.B., Nakamura, K., & Harada, J.J. (2005). Control of seed mass by APETALA2. Proceedings of the National Academy of Sciences, 102, 3123–3128. https://doi.org/10.1073/pnas.040985810

28-Papaefthimiou, D., Kapazoglou, A., & Tsaftaris, A.S. (2012). Cloning and characterization of SOC1 homologs in barley (Hordeum vulgare) and their expression during seed development and in response to vernalization. Physiologia Plantarum, 146(1), 71-85. https://doi.org/10.1111/j.1399-3054.2012.01610.x

29-Pfaffl, M.W., Horgan, G.W., & Dempfle, L. (2002). Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Research, 30(9), 36-36. https://doi.org/10.1093/nar/30.9.e36.

30-Rotem, N., Einat, Sh., Yuval, P., Fouad, A., Orit, E., Haim. D., Rabinowitch, I., & Rina, K. (2007). Reproductive development and phenotypic differences ingarlic are associated with expression and splicing of LEAFY homologue gaLFY. Journal of Experimental Botany, 58(5), 1133–1141. https://doi.org/10.1093/jxb/erl272 .

31-Rotem, N., David-Schwartz, R., & Peretz, Y. (2011). Flower development in garlic: The ups and downs of gaLFY expression. Planta, 233, 1063–1072. https://link.springer.com/article/10.1007/s00425-011-1361-8.

32-Ruelens, P., De Maagd, R.A., Proost, S., Theißen, G., Geuten, K., & Kaufmann, K. (2013). FLOWERING LOCUS C in monocots and the tandem origin of angiosperm-specific MADS-box genes. Nature Communications, 4(1), 2280. https://doi.org/10.1038/ncomms3280

33-Ryu, C.H., Lee, S., Cho, L.H., Kim, S.L., Lee, Y.S., & Choi, S.C. (2009). OsMADS50 and OsMADS56 function antagonistically in regulating long day (LD)-dependent flowering in rice. Plant Cell Environment, 32, 1412–1427. https://doi.org/10.1111/j.1365-3040.2009.02008.x

34- Searle, I., He, Y., Turck, F., Vincent, C., Fornara, F., Kröber, S., & Coupland, G. (2006). The transcription factor FLC confers a flowering response to vernalization by repressing meristem competence and systemic signaling in Arabidopsis. Genes & Development, 20(7), 898-912. https://doi.org/10.1101/gad.373506   

35-Simon, P.W., & Jenderek, M.M. (2003). Flowering, seed production and the genesis of garlic breeding. Plant Breeding Reviews, 23, 211-244. https://onlinelibrary.wiley.com/doi/book/10.1002/9780470650226#page=424.

36-Shalom, S.R., Gillett, D., & Zemach, H. (2015). Storage temperature controls the timing of garlic bulb formation via shoot apical meristem termination. Planta, 242(4), 951-962. https://link.springer.com/article/10.1007/s00425-015-2334-0.

37-Sharma, N., Ruelens, P., D'hauw, M., Maggen, T., Dochy, N., Torfs, S., & Geuten, K. (2017). A flowering locus C homolog is a vernalization-regulated repressor in Brachypodium and is cold regulated in wheat. Plant Physiology, 173(2), 1301-1315. https://doi.org/10.1104/pp.16.01161

38- Sharma, N., Geuten, K., Giri, B.S., & Varma, A. (2020). The molecular mechanism of vernalization in Arabidopsis and cereals: Role of Flowering Locus C and its homologs. Physiologia Plantarum, 170(3), 373-383. https://doi.org/10.1111/ppl.13163

39- Schilling, S., Kennedy, A., Pan, S., Jermiin, L.S., & Melzer, R. (2020). Genome‐wide analysis of MIKC‐type MADS‐box genes in wheat: Pervasive duplications, functional conservation and putative neo functionalization. New Phycologist Foundation, 225(1), 511-529. https://doi.org/10.1111/nph.16122

40- Tang, M., Li, G., & Chen, M. (2007). The phylogeny and expression pattern of APETALA2-like genes in rice. Journal of Genetics and Genomics, 34(10), 930-938. https://doi.org/10.1016/s1673-8527(07)60104-0.

41- Takagi, H. (1990). Biochemistry, Food Science, and Minor Crops. In: Onions and Allied Crops. J.L. Brewster H.D. Rabinowitch (eds). CRC Press, BocaRaton, Florida, 3, 109-146. https://cir.nii.ac.jp/crid/1130282270522244736.

42- Winfield, M.O., Lu, C., Wilson, I.D., Coghill, J.A., & Edwards, K. (2009). Cold- and light-induced changes in the transcriptome of wheat leading to phase transition from vegetative to reproductive growth. BMC Plant Biology, 9, 55. https://doi.org/10.1186/1471-2229-9-55

43- Yant, L., Mathieu, J., Dinh, T.T., Ott, F., Lanz, C., Wollmann, H., Chen, X., & Schmid, M. (2010). Orchestration of the floral transition and floral development in Arabidopsis by the bifunctional transcription factor APETALA2. The Plant Cell, 22(7), 2156-2170. https://doi.org/10.1105/tpc.110.075606.

44- Yalovsky, S., Rodríguez-Concepción, M., Bracha, K., Toledo-Ortiz, G., & Gruissem, W. (2000). Prenylation of the floral transcription factor APETALA1 modulates its function. The Plant Cell, 12(8), 1257-1266. https://doi.org/10.1105/tpc.12.8.1257

45-Yu, H., & Goh, C.J. (2000). Identification and characterization of three orchid MADS-box genes of the AP1/AGL9 subfamily during floral transition. Plant Physiology, 123, 1325–1336. https://doi.org/10.1104/pp.123.4.1325

46-Yan, X., Wang, L.J., Zhao, Y.Q., & Jia, G.X. (2022). Expression patterns of key genes in the photoperiod and vernalization flowering pathways in Lilium longiflorum with different bulb sizes. International Journal of Molecular Sciences, 23(15), 8341. https://doi.org/10.3390/ijms23158341

47-Yant, L., Mathieu, J., Dinh, T.T., Ott, F., Lanz, C., Wollmann, H., Chen, X., & Schmid, M. (2010). Orchestration of the floral transition and floral development in Arabidopsis by the functional transcription factor APETALA2. The Plant Cell, 22(7), 2156-2170. https://doi.org/10.1105/tpc.110.075606.

48-Yoo, S.K., Chung, K.S., Kim, J., Lee, J.H., Hong, S.M., Yoo, S.J., & Ahn, J.H. (2005) CONSTANTS activates SUPPRESSORS of OCEREXPRESSION of CONSTANS 1 through FLOWERING LOCUS T to promote flowering in Arabidopsis. Plant Physiology, 139(2), 770-778. https://doi.org/10.1104/pp.105.066928