Abstract
Key message
MsTFL1A is an important gene involved in flowering repression in alfalfa (Medicago sativa) which conditions not only above-ground plant shoot architecture but also root development and growth.
Abstract
Delayed flowering is an important trait for forage species, as it allows harvesting of high-quality forage for a longer time before nutritional values decline due to plant architecture changes related to flowering onset. Despite the relevance of delayed flowering, this trait has not yet been thoroughly exploited in alfalfa. This is mainly due to its complex genetics, sensitivity to inbreeding and to the fact that delayed flowering would be only advantageous if it allowed increased forage quality without compromising seed production. To develop new delayed-flowering varieties, we have characterized the three TERMINAL FLOWERING 1 (TFL1) family of genes in alfalfa: MsTFL1A, MsTFL1B and MsTFL1C. Constitutive expression of MsTFL1A in Arabidopsis caused late flowering and changes in inflorescence architecture, indicating that MsTFL1A is the ortholog of Arabidopsis TFL1. Overexpression of MsTFL1A in alfalfa consistently led to delayed flowering in both controlled and natural field conditions, coupled to an increase in leaf/stem ratio, a common indicator of forage quality. Additionally, overexpression of MsTFL1A reduced root development, reinforcing the role of MsTFL1A not only as a flowering repressor but also as a regulator of root development.We conclude that the precise manipulation of MsTFL1A gene expression may represent a powerful tool to improve alfalfa forage quality.
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References
Aung B, Gruber MY, Amyot L, Omari K, Bertrand A, Hannoufa A (2015) MicroRNA156 as a promising tool for alfalfa improvement. Plant Biotechnol J 13:779–790
Ball DM, Collins M, Lacefield GD, Martin NP, Mertens DA (2001) Understanding Forage quality. pp. 1–01.
Barros J, Temple S, Dixon R (2019) A, Development and commercialization of reduced lignin alfalfa. Curr Opin Biotechnol 56:48–54
Bosworth SC, Stringer WS (1992) Cutting management of Alfalfa, Red Clover, and Birdsfoot Trefoil. In: Agronomy Facts No. 7 (Cooperative Extension. State College ed). PA: Pennsylvania State University: Pennsilvania State Extension.
Bouché F, D’Aloia M, Tocquin P, Lobet G, Detry N, Périlleux C (2016) Integrating roots into a whole plant network of flowering time genes in Arabidopsis thaliana. Sci Rep 6:29042
Bradley D, Carpenter R, Copsey L, Vincent C, Rothstein S, Coen E (1996) Control of inflorescence architecture in Antirrhinum. Nature 379:791–797
Bradley D, Ratcliffe O, Vincent C, Carpenter R, Coen E (1997) Inflorescence commitment and architecture in Arabidopsis. Science 275:80–83
Corbesier L, Vincent C, Jang S, Fornara F, Fan Q, Searle I, Giakountis A, Farrona S, Gissot L, Turnbull C, Coupland G (2007) FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis. Science 316:1030–1033
Cheng X, Li G, Tang Y, Wen J (2018) Dissection of genetic regulation of compound inflorescence development in Medicago truncatula. Development 145.
Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32
Foucher F, Morin J, Courtiade J, Cadioux S, Ellis N, Banfield MJ, Rameau C (2003) DETERMINATE and LATE FLOWERING are two TERMINAL FLOWER1/CENTRORADIALIS homologs that control two distinct phases of flowering initiation and development in pea. Plant Cell 15:2742–2754
Fernandez-Cornejo J, Seth W, Mike L, Lorraine M (2014) Genetically engineered crops in the United States, ERR-162 U.S. Department of Agriculture. Econom Res Service
Freiman A, Shlizerman L, Golobovitch S, Yablovitz Z, Korchinsky R, Cohen Y, Samach A, Chevreau E, Le Roux PM, Patocchi A, Flaishman MA (2012) Development of a transgenic early flowering pear (Pyrus communis L.) genotype by RNAi silencing of PcTFL1-1 and PcTFL1-2. Planta 235:1239–1251
Gao R, Austin RS, Amyot L, Hannoufa A (2016) Comparative transcriptome investigation of global gene expression changes caused by miR156 overexpression in Medicago sativa. BMC Genom 17:658
Gao R, Gruber MY, Amyot L, Hannoufa A (2018) SPL13 regulates shoot branching and flowering time in Medicago sativa. Plant Mol Biol 96:119–133
Hall M, Fales LF, Jung JA (1992) Cutting management of alfalfa, red clover, and birdsfoot trefoil. In: Agronomy Facts 7, The Pennsylvania State University: Pennsilvania State Extension
Hanzawa Y, Money T, Bradley D (2005) A single amino acid converts a repressor to an activator of flowering. Proc Natl Acad Sci USA 102:7748–7753
Iwata H, Gaston A, Remay A, Thouroude T, Jeauffre J, Kawamura K, Oyant LH, Araki T, Denoyes B, Foucher F (2012) The TFL1 homologue KSN is a regulator of continuous flowering in rose and strawberry. Plant J: Cell and Molecular Biol 69:116–125
Kalu BA, Fick GW (1981) Quantifying morphological development of alfalfa for studies of herbage quality1. Crop Sci 21:267–271
Kalu BA, Fick GW (1983) Morphological stage of development as a predictor of alfalfa herbage quality1. Crop Sci 23:1167–1172
Kang J, Zhang T, Guo T, Ding W, Long R, Yang Q, Wang Z (2019) Isolation and functional characterization of MsFTa, a FLOWERING LOCUS T Homolog from Alfalfa (Medicago sativa). Int J Molecular Sci 20
Kotoda N, Hayashi H, Suzuki M, Igarashi M, Hatsuyama Y, Kidou S-I, Igasaki T, Nishiguchi M, Yano K, Shimizu T, Takahashi S, Iwanami H, Moriya S, Abe K (2010) Molecular Characterization of FLOWERING LOCUS T-Like Genes of Apple (Malus × domestica Borkh.). Plant Cell Physiol 51:561–575
Lachowiec J, Shen X, Queitsch C, Carlborg Ö (2015) A genome-wide association analysis reveals epistatic cancellation of additive genetic variance for root length in Arabidopsis thaliana. PLoS Genet 11:e1005541
Lei Y, Hannoufa A, Yu P (2017) The use of gene modification and advanced molecular structure analyses towards improving alfalfa forage. Int J Molecular Sci 18.
Li C, Fu Q, Niu L, Luo L, Chen J, Xu Z-F (2017) Three TFL1 homologues regulate floral initiation in the biofuel plant Jatropha curcas. Sci Rep 7:43090
Lifschitz E, Eviatar T, Rozman A, Shalit A, Goldshmidt A, Amsellem Z, Alvarez JP, Eshed Y (2006) The tomato FT ortholog triggers systemic signals that regulate growth and flowering and substitute for diverse environmental stimuli. Proc Natl Acad Sci USA 103:6398–6403
Liu B, Watanabe S, Uchiyama T, Kong F, Kanazawa A, Xia Z, Nagamatsu A, Arai M, Yamada T, Kitamura K, Masuta C, Harada K, Abe J (2010) The soybean stem growth habit gene Dt1 is an ortholog of Arabidopsis TERMINAL FLOWER1. Plant Physiol 153:198–210
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408
Lorenzo CD, Alonso Iserte J, Sanchez Lamas M, Antonietti MS, Garcia Gagliardi P, Hernando CE, Dezar CAA, Vazquez M, Casal JJ, Yanovsky MJ, Cerdán PD (2019) Shade delays flowering in Medicago sativa. Plant J 99:7–22
Lorenzo CD, Garcia Gagliardi P, Antonietti MS, Lamas MS, Mancini E, Dezar CA, Vazquez M, Watson G, Yanovsky MJ, Cerdan PD (2020) Improvement of alfalfa forage quality and management through the downregulation of MsFTa1. Plant Biotechnol J 18:944–954
Major DJ, Hanna MR, Beasley BW (1991) Photoperiod response characteristics of alfalfa (Medicago sativa L) cultivars. Can J Plant Sci 71:87–93
Menz J, Modrzejewski D, Hartung F, Wilhelm R, Sprink T (2020) Genome edited crops touch the market: a view on the global development and regulatory environment. Front Plant Sci 11:586027. https://doi.org/10.3389/fpls.2020.586027
Mimida N, Goto K, Kobayashi Y, Araki T, Ahn JH, Weigel D, Murata M, Motoyoshi F, Sakamoto W (2001) Functional divergence of the TFL1-like gene family in Arabidopsis revealed by characterization of a novel homologue. Genes to Cells : Devoted to Molecular & Cellular Mech 6:327–336
Mir RR, Kudapa H, Srikanth S, Saxena RK, Sharma A, Azam S, Saxena K, Varma Penmetsa R, Varshney RK (2014) Candidate gene analysis for determinacy in pigeonpea (Cajanus spp.). TAG. Theoretical and applied genetics. Theoretische Und Angewandte Genetik 127:2663–2678
Mohamed R, Wang CT, Ma C, Shevchenko O, Dye SJ, Puzey JR, Etherington E, Sheng X, Meilan R, Strauss SH, Brunner AM (2010) Populus CEN/TFL1 regulates first onset of flowering, axillary meristem identity and dormancy release in populus. Plant J: Cell Molecular Biol 62:674–688
Mueller SC, Fick GW (1989) Converting alfalfa development measurements from mean stage by count to mean stage by weight. Crop Sci 29:821–823
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15.
Pearson CJ, Hunt LA (1972) EFFECTS OF TEMPERATURE ON PRIMARY GROWTH OF ALFALFA. Can J Plant Sci 52:1007–1015
Pnueli L, Carmel-Goren L, Hareven D, Gutfinger T, Alvarez J, Ganal M, Zamir D, Lifschitz E (1998) The SELF-PRUNING gene of tomato regulates vegetative to reproductive switching of sympodial meristems and is the ortholog of CEN and TFL1. Development 125:1979–1989
Putnam D, Ruselle M, Orloff S, Kuhn J, Fitzhugh L, Godfrey L, Kiess A (2001) Alfalfa Wildlife and the Environment - The importance and benefits of alfalfa in the 21st century:California Alfalfa and Forage Association.
Radovic J, Sokolovic D, Markovic J (2009) Alfalfa-most important perennial forage legume in animal husbandry. Biotechnol Animal Husbandry 25:465–475
Repinski SL, Kwak M, Gepts P (2012) The common bean growth habit gene PvTFL1y is a functional homolog of Arabidopsis TFL1. TAG. Theoretical and applied genetics. Theoretische Und Angewandte Genetik 124:1539–1547
Rodríguez-Leal D, Lemmon ZH, Man J, Bartlett ME, Lippman ZB (2017) Engineering quantitative trait variation for crop improvement by genome editing. Cell 171(2):470-480.e8. https://doi.org/10.1016/j.cell.2017.08.030
Ruan YL, Patrick JW, Bouzayen M, Osorio S, Fernie AR (2012) Molecular regulation of seed and fruit set. Trends Plant Sci 17:656–665
Sachs T (1999) ‘Node counting’: an internal control of balanced vegetative and reproductive development. Plant, Cell Environ 22:757–766
Samac DA, Austin-Phillips S (2006) Alfalfa (Medicago sativa L.). Methods Mol Biol 343:301–311
Serrano-Mislata A, Fernández-Nohales P, Doménech MJ, Hanzawa Y, Bradley D, Madueño F (2016) Separate elements of the TERMINAL FLOWER 1 cis-regulatory region integrate pathways to control flowering time and shoot meristem identity. Development 143:3315–3327
Singer SD, Hannoufa A, Acharya S (2018) Molecular improvement of alfalfa for enhanced productivity and adaptability in a changing environment. Plant, Cell Environ 41:1955–1971
Tamaki S, Matsuo S, Wong HL, Yokoi S, Shimamoto K (2007) Hd3a protein is a mobile flowering signal in rice. Science 316:1033–1036
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28.
Van Deynze A, Putnam D, Orloff S, Lanini T, Canevari M, Vargas R, Hembree K, Mueller S, Teuber L (2004) Roundup ready alfalfa: an emerging technology. agricultural biotechnology in California Series. Publication 8153
Wang X, Fu Y, Ban L, Wang Z, Feng G, Li J, Gao H (2015) Selection of reliable reference genes for quantitative real-time RT-PCR in alfalfa. Genes Genet Syst 90:175–180
Wang Y, Chantreau M, Sibout R, Hawkins S (2013) Plant cell wall lignification and monolignol metabolism. Front Plant Sci 4:220
Weaver JW (1926) Root development of field crops. McGraw-Hill Book Company, Inc., New York
Wenden B, Dun EA, Hanan J, Andrieu B, Weller JL, Beveridge CA, Rameau C (2009) Computational analysis of flowering in pea (Pisum sativum). New Phytol 184:153–167
Wolabu TW, Mahmood K, Jerez IT, Cong L, Yun J, Udvardi M, Tadege M, Wang Z, Wen J (2023) Multiplex CRISPR/Cas9-mediated mutagenesis of alfalfa FLOWERING LOCUS Ta1 (MsFTa1) leads to delayed flowering time with improved forage biomass yield and quality. Plant Biotechnol J. https://doi.org/10.1111/pbi.14042
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This work was supported by the Argentine Agencia Nacional de Promoción Científica y Tecnológica. Grant numbers PICT‐2017‐1626 and PICT-2017–4660 to P.D.C.
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CDL, PGG, CAD, GW, MJY and PDC conceived and designed the experiments. CDL, PGG, SF, LG and MA performed the experiments. CDL, EM and PDC analyzed the data. CDL and PDC wrote the paper.
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Authors Geronimo Watson and Carlos Dezar were employees of INDEAR S.A.. All other authors state that they do not have any conflict of interest.
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Lorenzo, C.D., García-Gagliardi, P., Gobbini, M.L. et al. MsTFL1A delays flowering and regulates shoot architecture and root development in Medicago sativa. Plant Reprod (2023). https://doi.org/10.1007/s00497-023-00466-7
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DOI: https://doi.org/10.1007/s00497-023-00466-7