Abstract
Auxin and polar auxin transport have been implicated in controlling zygotic embryo development, but less is known about their role in the development of somatic embryos. The aim of this study was to determine if indole-3-acetic acid (IAA) and the PIN1 transporter participate in the induction of somatic embryogenesis (SE) and the development of somatic embryos. The results show that IAA levels gradually increase during pre-treatment and accumulate in the chloroplast. During pre-treatment and the globular stage of SE in C. canephora, auxin is distributed uniformly in all of the cells of the somatic embryo. During the subsequent stages of development, auxins are mobilized to the cells that will form the cotyledons and the root meristem. The location of the PIN transporters shifts from the plasmalemma of the protoderm cells during the globular stage to the plasmalemma of the cells that will give rise to the cotyledons and the vascular tissue in the late stages of somatic embryogenesis. The incubation of the explants in the presence of 2,3,5-triiodobenzoic acid (TIBA) produced aberrant somatic embryos, suggesting that PIN1 mediates the transport of IAA.
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Abbreviations
- DAPI:
-
4′,6-diamidino-2-phenylindole
- 2,4-D:
-
2,4-dichlorophenoxyacetic acid
- IAA:
-
indole-3-acetic acid
- NAA:
-
1-naphthaleneacetic acid
- BA:
-
benzyl adenine
- Kin:
-
kinetin
- NPA:
-
N-1-naphthylphthalamic acid
- PAT:
-
polar auxin transport
- PBS:
-
phosphate-buffered saline
- PIN:
-
PIN-FORMED efflux carrier protein
- TIBA:
-
2,3,5-triiodobenzoic acid
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Funding
This work was supported by a grant from the National Council of Science and Technology to VMLV (CONACyT; Grant No. 257436) and a scholarship from CONACyT to REML and CPH.
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VM conceived the idea, proposed the experiments, and drafted the manuscript. RE and AK carried out the confocal observations, CP realized the inhibition experiments, and RM realized the plant tissue culture experiments. All the authors approved the version to be published.
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Fig. S1
Control of autofluorescence. a Transmitted light differential interference images of a transverse section of leaves. b Autofluorescence in the channel of DAPI (405 nm). c Autofluorescence in the IAA channel (488 nm). d Autofluorescence in the channel of chlorophyll (650 nm). Note the very faint autofluorescence for chlorophyll in panel (d). (JPEG 29 kb)
Fig. S2
Control experiment without the IAA antibody. a, d Transmitted light differential interference images of a transverse section of globular embryos. b, c, e, f Confocal images of globular embryos. b, e Confocal images of globular embryos stained with DAPI (channel of 405 nm). c, f Control without the primary Ab against IAA and in the presence of the secondary Ab (channel of 488 nm). e, f close-up of images (b) and (c), respectively. (JPEG 140 kb)
Fig. S3
Control experiment without the PIN1 antibody. a, d Transmitted light differential interference images of a transverse section of the globular embryo. b, c, e, f Confocal images of globular embryos. b, e Confocal images of globular embryos stained with DAPI (channel of 405 nm). c, f Control without the primary Ab against PIM1 and in the presence of the secondary Ab (channel of 488 nm). e, f close-up of images (b) and (c), respectively. (JPEG 165 kb)
Fig. S4
a Phylogenetic tree of C. canephora (Cc) and A. thaliana (At) PIN proteins. The tree was based on the neighbor-joining method with a bootstrap support at each node of 1000. The bar at the bottom of the figure shows the length of the branch that represents the amount of genetic change of 0.2. The protein sequences from A. thaliana were retrieved from Phytozome v7.0 database. C. canephora PIN sequences were obtained from the website providing the genome of C. canephora (http://coffee-genome.org/) using the Ugene platform (http://ugene.net/). b Amino acid sequence alignment of the transmembrane region of A. thaliana PIN1 (AtPIN1) and C. canephora PIN1 (CcPIN1 and CcPINb). The alignment was carried out using the algorithm published by Corpet (1988) (JPEG 967 kb)
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Márquez-López, R.E., Pérez-Hernández, C., Ku-González, Á. et al. Localization and transport of indole-3-acetic acid during somatic embryogenesis in Coffea canephora . Protoplasma 255, 695–708 (2018). https://doi.org/10.1007/s00709-017-1181-1
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DOI: https://doi.org/10.1007/s00709-017-1181-1