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Unraveling the effect of the transcription factor DOF1147 on the in vitro effect of auxin on cell division and endoreduplication in developing seeds of the model
legume Medicago truncatula
Max Wirsich, Rana Muhammad Atif, M.B. Schroeder, Richard Thompson, Sergio Ochatt
To cite this version:
Max Wirsich, Rana Muhammad Atif, M.B. Schroeder, Richard Thompson, Sergio Ochatt. Unraveling
the effect of the transcription factor DOF1147 on the in vitro effect of auxin on cell division and en-
doreduplication in developing seeds of the model legume Medicago truncatula. Meeting of the German
Society of Plant Biotechnology: Plant Biotechnology - Applications in Breeding and Production, Aug
2013, Geisenheim, 2013. �hal-02748513�
Unraveling the effect of the transcription factor DOF1147 on the in vitro effect of auxin on cell division and endoreduplication in developing seeds of the model legume Medicago truncatula
WIRSICH M 1,2 , ATIF RM 1 , SCHROEDER M-B 2 , THOMPSON RD 1 , OCHATT SJ 1
1 , UMR 1347 Agroécologie, INRA, CR Dijon, BP86510, 21065 Dijon Cedex, FRANCE
2 , Hochschule Geisenheim University, Von Lade Strasse1 - D-65366 Geisenheim- GERMANY
We studied the role of auxins in morphogenesis of immature seeds of Medicago truncatula, focusing on the transition from embryo cell division phase to seed maturation comparing wild type plants (WT) with transformants overexpressing gene DOF1147 (TR), involved in embryo development and seed filling (Verdieret al, 2008; Noguero
et al, 2013), by analysing the development of seeds (at 8, 10, 12, 14 and 16 Days After Pollination; DAP ) in vitro without auxin, or with IBA or NAA, by flow cytometry and through the kinetics of seed fresh weight and surface area, every 2 days from the start of in vitro culture.Fig. 2
Fig. 3
Medicago truncatula genotype R108pMDC83_20b_T1
TR genotype transformed by Atif (2012) usingAgrobacterium tumefaciens strain EHA105 and binary vector system pMDC83 (Fig. 2) to prepare the translational GFP reporter fusion constructp35S::dof-gfp was used to monitor transcriptional activity of DOF1147 protein.
The expression construct contains dual CaMV35S promoter, controlling nos terminator and DOF1147 coding sequence, fused to the N-terminus of histidine-tagged GFP6 in expression vector pMDC83 (Fig. 3; Curtis and Grossniklaus, 2003; Atif, 2012).
Transformation was verified by PCR and Southern-Blot (Atif, 2012; Ochatt et al, 2013), Greenhouse-grown plants here belong to generation T1, from pod “b” of transformant 20. The in-vitro cultured immature seeds, hence, are T2,
Labeling of flowers was done the day following pollination when petal size is maximum but pistils not yet apparent (0 DAP; Fig. 4). Each labeling and harvest included ~ 15 pods.
Fig. 4
Pods harvested at 8, 10, 12, 14 and 16 DAP were disinfected (Atif et al, 2013), opened and seeds weighed prior to culture up to 30 DAP, under a 16h/8h (day/night) photoperiod (100 µE/m
2/s) at 23±1 °C, on MS medium (Murashige & Skoog, 1962) with 6 g/l agar, 130 g/l sucrose and without growth regulators or with naphthalene acetic acid (NAA) and indole-3-butyric acid (IBA), respectively, at 1 mg/l each (pH 5,6). Such high sugar concentration creates conditions permitting the comparison of development in vitro to in planta (Gallardo et al, 2006; Ochatt, 2011).
In vitro development of seeds was assessed by their surface area, fresh weight and flow cytometric status, each measurement being carried out on the day of harvest and every 2 days thereafter up to 16 DAP, and at 30 DAP (end of seed development in planta ). Every measurement was repeated three times per developmental stage and culture medium and results were expressed as the means ± SE from ≤ 25 replicates, in mm
2for surface area and in mg for fresh weight.
Determination of surface area of immature seeds was performed on images acquired with ARCHIMEDPRO (Microvision, Evry, France) and treated using HISTOLAB (Microvision), as detailed by Ochatt et al (2008) and Ochatt and Moessner (2010).
Flow cytometry assessments were done using a Partec PAII flow cytometer, with leaf and seed material prepared as described (Ochatt 2006, 2011; Atif et al, 2013), parallel to surface area and fresh weight determination, i.e . at the same developmental stages. Cytometer calibration was done using leaves of Pisum sativum cv. Frisson and analyzed tissues were compared with leaves of M. truncatula R-108, cultured on MS0 medium or glasshouse-grown.
WT TR
Fig. 1
Genotype R-108 of superior in vitro regeneration (Hoffmann et al, 1997) used for transformation and transgene expression (Trinh et al., 1998;Atif, 2012; Ochatt et al, 2013), was the WT control compared with a transformed genotype (Fig. 1). Plants were kept under a 16h light photoperiod, at 18 °C overnight and 23 °C during daytime and daily fertigated by drip irrigation (N:P:K 10:10:10 + MgSO
4+ NaCl).
Introduction
Conclusion
Results and Discussion Materials and Methods
References
Atif RM (2012) Dissecting the factors controlling seed development in the model legume Medicago truncatula. PhD-Thesis.Univ Dijon, France
Ochatt SJ (2006) Flow cytometry (ploidy determination, cell cycle analysis, DNA content per nucleus).Retrieved Mai 22, 2013, from Medicago truncatula handbook, Chapter 2.2.7: https://www.noble.org/Global/medicagohandbook/pdf/FlowCytometry.pdf Atif RM, Boulisset F, Conreux C, Thompson R, Ochatt SJ (2013) Physiologia Plantarum in press, doi:10.1111/j.1399-3054.2012.01719.x Ochatt SJ (2011) Methods in Molecular Biology 710: 39-52.
Curtis MD, Grossnikolaus U (2003)Plant Physiology 133: 462-469. Ochatt S, Muilu R, Ribalta F (2008) Plant Biosystems142: 480-486.
Gallardo K, Kurt C, Thompson R, Ochatt S (2006) Plant Science 170:1052-1058. Ochatt S, Moessner A (2010) International Journal of Plant Biology 1-e8: 40-42 Hoffmann B, Trinh TH, Leung J, Kondorosi A, Kondorosi E (1997)Molecular Plant-Microbe Interactions 10:307-315. Ochatt S, Jacas L, Patat-Ochatt EM, Djenanne S (2013) Plant Cell Tissue Organ Culture113: 237-244, Murashige T, Skoog F (1962)Physiologia Plantarum 15:473-497. Trinh TH, Ratet P, Kondorosi E, Durand P, Kamate K, Bauer P, et al (1998) Plant Cell Reports17: 345-355.
Noguero M, Atif RM, Ochatt S, Thompson RD (2013) Plant Science 209: 32-45. Verdier J, Kakar K, Gallardo K, Le Signor C, Aubert G, Schlereth A, Town CD, et al.(2008) Plant Mol Biol67:567-80.
Seeds of WT and TR increased in weight and surface area both in planta (Fig. 4) and in vitro under the influence of auxin (Fig. 5), NAA having a stronger effect than IBA, and more pronounced in DOF1147 transformants than in WT. In auxin-free medium, WT seeds exhibited a smaller increase in weight and surface area than TR seeds (Fig. 6).
These results confirm previous data for developing seeds of WT (Atif et al. 2013), but also allow a better comprehension of the involvement of transcription factor DOF1147 in the genetic determinism of seed filling, and may help us understand the fundamental mechanisms underlying the control of yield components for a sustainable agriculture.
Fig. 5
Fig. 6
Flow cytometry results in Figure 7 show that auxin modulated the transition between mitotic cycles and endocycles in developing seed by favoring sustained cell divisions while prolonging endoreduplication (cytogenetic imprint of transition from cell division to storage protein accumulation phase). Differences were apparent between WT and TR, the latter having an earlier onset of endoreduplication on all media, but more marked on NAA than on IBA.
Fig. 7
Flow cytometry profiles shown correspond to immature seeds of TR only
0 1 2 3 4 5 6 7 8
8 DAP in planta
10 DAP in planta
12 DAP in planta
14 DAP in planta
16 DAP in planta
30 DAP in planta
Seed fresh weight (mg)
Developmental stage of seeds
Time course growth of WT and TR seeds in planta
WT TR
