Plant Physiology Preview. Published on October 28, 2016, as DOI:10.1104/pp.16.00564
1
Short title: (Gm)AGL15 gene regulation and somatic embryogenesis
2
Corresponding author: Sharyn E. Perry,
[email protected].
3 4
Article title: Gene Regulation by the AGL15 transcription factor Reveals Hormone
5
Interactions in Somatic Embryogenesis
6 7
Authors:
8
Qiaolin Zheng, Yumei Zheng, Huihua Ji, Whitney Burnie and Sharyn E. Perry
9 10
Affiliation:
11
Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky,
12
40546-0312
13
Other authors’ emails:
[email protected];
[email protected];
14
[email protected];
[email protected]
15 16
Author contributions: S.E.P. directed the project, supervised the research and
17
contributed some of the data. Q.Z. designed and performed the majority of the
18
experiments and analyzed the data. Y.Z. performed some of the Arabidopsis work. W.B.
19
contributed to the analysis of the tir1 mutants. H.J. performed the auxin measurements.
20
Q.Z. and S.E.P. contributed to writing the manuscript with comments from Y.Z and H.J.
21
Funding information: This work was supported by the United Soybean Board
22
(#0282/1282/2822), the National Science Foundation (IOS-0922845), and by an award
23
from the University of Kentucky Microarray Pilot Grant Program. This is publication No.
24
16-06-027 of the Kentucky Agricultural Experiment Station and is published with the
25
approval of the Director. This work is supported by the National Institute of Food and
26
Agriculture, U.S. Department of Agriculture, Hatch 0231956.
27 28
Corresponding author email:
[email protected]
29 1 Downloaded from www.plantphysiol.org on February 1, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Copyright 2016 by the American Society of Plant Biologists
30 31
One sentence summary: Embryo and hormonal transcription factors interact in
32
somatic embryogenesis in Arabidopsis and soybean
33 34 35
The author responsible for distribution of materials integral to the findings presented in
36
this article in accordance with the policy described in the Instructions for Authors
37
(www.plantphysiol.org) is: Sharyn E. Perry (
[email protected]).
38 39 40 41 42
2 Downloaded from www.plantphysiol.org on February 1, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
43
ABSTRACT
44
The MADS-box transcription factor Arabidopsis (Arabidopsis thaliana) AGAMOUS-
45
Like15 (AGL15) and a putative ortholog from soybean (Glycine max, GmAGL15) are
46
able to promote somatic embryogenesis (SE) in these plants when ectopically
47
expressed. SE is an important means of plant regeneration, but many plants, or even
48
particular cultivars, are recalcitrant for this process. Understanding how (Gm)AGL15
49
promotes SE by identifying and characterizing direct and indirect downstream regulated
50
genes can provide means to improve regeneration by SE for crop improvement and to
51
perform molecular tests of genes. Conserved transcription factors and the genes they
52
regulate in common between species may provide the most promising avenue to
53
identify targets for SE improvement. We show that (Gm)AGL15 negatively regulates
54
auxin signaling in both Arabidopsis and soybean at many levels of the pathway
55
including repression of AUXIN RESPONSE FACTOR (ARF)6 and ARF8, and
56
TRANSPORT INHIBITOR RESPONSE 1 (TIR1), as well as indirectly controlling
57
components via direct expression of a microRNA encoding gene. We demonstrate
58
interaction between auxin and gibberellic acid (GA) in promotion of SE and document
59
an inverse correlation between bioactive GA and SE in soybean, a difficult crop to
60
transform. Finally, we relate hormone accumulation to transcript accumulation of
61
important soybean embryo regulatory factors such as GmABI3 and GmFUS3, and
62
provide a working model of hormone and transcription factor interaction in control of SE.
63 64
3 Downloaded from www.plantphysiol.org on February 1, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
65 66
INTRODUCTION AGAMOUS-Like15 (AGL15, Arabidopsis Genome Initiative Identifier At5g13790)
67
encodes a MADS domain transcription factor that is expressed at its highest level in
68
zygotic embryos, although it is not unique to embryo development and has roles after
69
completion of germination (Adamczyk et al., 2007). When ectopically expressed via a
70
35S promoter, AGL15 can enhance somatic embryogenesis (SE) in two different
71
systems in Arabidopsis and can lead to long-term maintenance (over 19 years to date)
72
of development in embryo mode (Harding et al., 2003; Thakare et al., 2008). Loss-of-
73
function alleles of agl15, especially when present with a loss-of-function allele in
74
AGAMOUS-Like18 (agl18), the closest family member to AGL15, show significantly
75
reduced SE (Thakare et al., 2008). Like AGL15, AGL18 can promote SE when
76
overexpressed in Arabidopsis (Adamczyk et al., 2007). Genes encoding putative
77
orthologs of AGL15 and AGL18 were isolated from soybean (Glycine max, referred to
78
as GmAGL15; Glyma12g17721, Glyma11g16105 and GmAGL18; Glyma02g33040) and
79
were able to enhance SE in this species when expressed via a 35S promoter (Zheng
80
and Perry, 2014). Recently three AGL15 orthologs from cotton (Gossypium hirsutum L)
81
were shown to be preferentially expressed during SE especially the induction phase in
82
response to the synthetic auxin 2,4-D, and when overexpressed, all three orthologs led
83
to more rapid production of embryogenic callus and better quality of callus (Yang et al.,
84
2014). In addition, expression of an AGL15-like gene was correlated with early
85
embryogenic culture initiation in maize (Salvo et al., 2014).
86
Somatic embryo development provides an accessible system that has been used
87
as a model for zygotic embryogenesis (Vogel, 2005; Rose and Nolan, 2006), but how
88
this process occurs is not well understood, even though this phenomenon of totipotency
89
has been known since 1958 (Steward et al., 1958). Understanding SE is important
90
because it is one means of regeneration of plants to meet agricultural challenges. In
91
soybean, transformability of different varieties is directly related to competence for SE
92
(Ko et al., 2004; Kita et al., 2007; Klink et al., 2008) suggesting that improvement of
93
regeneration will aid in transformation competence. In addition, testing functions of
94
genes generally includes studying results of increased/ectopic expression as well as 4 Downloaded from www.plantphysiol.org on February 1, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
95
loss-of-function. Both of these approaches include technologies that involve
96
transformation and regeneration of plants. However, little is known about what makes
97
cells competent to respond to induction for SE, a trait that varies depending on plant,
98
tissue, age or even particular cultivar of a species. To better understand how
99
(Gm)AGL15 promotes SE, we assessed transcriptomes in response to (Gm)AGL15
100
accumulation in Arabidopsis and soybean (Zheng et al., 2009; Zheng and Perry, 2014).
101
Comparison of the results in these two species led to the discovery that ethylene has a
102
role in SE (Zheng et al., 2013) and key embryo transcription factors are directly
103
controlled by (Gm)AGL15 in both species (Zheng and Perry, 2014). Here we report on
104
(Gm)AGL15 regulation of auxin signaling and integration with gibberellic acid (GA)
105
metabolism.
106
5 Downloaded from www.plantphysiol.org on February 1, 2017 - Published by www.plantphysiol.org Copyright © 2016 American Society of Plant Biologists. All rights reserved.
107
RESULTS
108
TRANSPORT INHIBITOR RESPONSE 1 (TIR1) is a Direct Target of AGL15
109
Repression
110
Expression of AGL15 via a 35S promoter (35Spro) enhances somatic
111
embryogenesis in both Arabidopsis and soybean (Harding et al., 2003; Thakare et al.,
112
2008; Zheng and Perry, 2014). In one Arabidopsis SE system, seeds are allowed to
113
complete germination in liquid medium containing the synthetic auxin 2,4-
114
dichlorophenoxyacetic acid (2,4-D, Mordhorst et al., 1998). By 21 days after culture
115
(dac) a fraction of the callused seedlings will have SE development from the region of
116
the shoot apical meristem (SAM SE), and a positive correlation between SAM SE with
117
AGL15 accumulation has been reported (Harding et al., 2003; Thakare et al., 2008). To
118
understand how (Gm)AGL15 promotes SE, transcriptome analysis in response to
119
35Spro:(Gm)AGL15 was performed in both species (Zheng et al., 2009; Zheng and
120
Perry, 2014). Because of a central role for auxin, generally added as 2,4-D, in inducing
121
somatic embryogenesis, genes responsive to (Gm)AGL15 and involved in auxin
122
biosynthesis or response were of particular interest. In both the Arabidopsis microarray
123
experiment to assess gene regulation in response to AGL15 (Zheng et al., 2009), and
124
the similar soybean experiment (Zheng and Perry, 2014, discussed below), a gene
125
encoding TRANSPORT INHIBITOR RESPONSE 1 (TIR1, At3g62980), an auxin
126
receptor (Ruegger et al., 1998), was found to be repressed in response to (Gm)AGL15.
127
For the Arabidopsis microarray experiment 10 dac SAM SE tissue was used for RNA
128
extraction and this was before any obvious SE development. The ratio of TIR1 transcript
129
was 2.02 (significant at P
