Plant Cell Advance Publication. Published on May 18, 2017, doi:10.1105/tpc.17.00186
RESEARCH ARTICLE
A Regulatory Hierarchy of the Arabidopsis Branched-chain Amino Acid
Metabolic Network
Anqi Xinga and Robert L. Lasta, b,1
a
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing,
Michigan 48824–1319
b
Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824–1319
1
Corresponding Author: [email protected]
Short title: Branched-chain amino acid biosynthesis
One sentence summary: Branched-chain amino acid homeostasis in leaves and seeds is
regulated by the interplay of three committed enzymes in Arabidopsis.
The author responsible for distribution of materials integral to the findings presented in this
article in accordance with the policy described in the Instructions for Authors is: Robert L. Last
([email protected]).
ABSTRACT
The branched-chain amino acids (BCAAs) Ile, Val and Leu are essential nutrients that humans
and other animals obtain from plants. However, total and relative amounts of plant BCAAs
rarely match animal nutritional needs, and improvement requires a better understanding of the
mechanistic basis for BCAA homeostasis. We present an in vivo regulatory model of BCAA
homeostasis derived from analysis of feedback-resistant Arabidopsis thaliana mutants for the
three allosteric committed enzymes in the biosynthetic network: threonine deaminase (also
named L-O-methylthreonine resistant 1, OMR1), acetohydroxyacid synthase small subunit 2
(AHASS2) and isopropylmalate synthase 1 (IPMS1). In this model, OMR1 exerts primary
control on Ile accumulation and functions independently of AHAS and IPMS. AHAS and IPMS
regulate Val and Leu homeostasis, where AHAS affects total Val+Leu and IPMS controls
partitioning between these amino acids. In addition, analysis of feedback-resistant and loss-offunction single and double mutants revealed that each AHAS and IPMS isoenzyme contributes to
homeostasis rather than being functionally redundant. The characterized feedback-resistant
mutations caused increased free BCAA levels in both seedlings and seeds. These results add to
our understanding of the basis of in vivo BCAA homeostasis and inform approaches to improve
the amount and balance of these essential nutrients in crops.
1
2
INTRODUCTION
3
The branched-chain amino acids (BCAAs) Ile, Val and Leu are essential nutrients, which
4
humans and other animals must obtain from their diets (Binder et al., 2007). Besides their roles
5
as building blocks of protein, BCAAs function as signaling molecules in animals, and BCAA
1
©2017 American Society of Plant Biologists. All Rights Reserved
6
supplementation helps to prevent oxidative damage and supports cardiac and skeletal muscle
7
mitochondrial biogenesis (Valerio et al., 2011). In plants, BCAAs and their derivatives
8
contribute to growth, defense and the production of food flavor components (Kang et al., 2006;
9
Yoshikawa et al., 1995; Zeier, 2013; Kimball and Jefferson, 2006; Gonda et al., 2010; Galili et
10
al., 2016). In addition, BCAA catabolism provides an alternative source of energy in plants under
11
long-term dark treatment conditions (Peng et al., 2015; Kochevenko et al., 2012). Moreover,
12
acetohydroxyacid synthase (AHAS), the committed enzyme of Val biosynthesis, is the target of
13
four classes of commercial herbicides, and numerous plant herbicide resistance AHAS alleles
14
have been reported (Jander et al., 2003). In addition to an essential role in protein synthesis,
15
BCAA metabolism provides precursors for specialized metabolism; for example, variants
16
derived from isopropylmalate synthase (IPMS), the committed enzyme in Leu biosynthesis,
17
contribute to specialized metabolism in Arabidopsis thaliana (de Kraker and Gershenzon, 2011)
18
as well as cultivated (Solanum lycopersicum) and wild tomatoes (Solanum pennellii) (Ning et al.,
19
2015). Thus, this metabolic network (Figure 1) contributes directly and indirectly to plant
20
defense, growth and metabolic diversity along with animal nutrition.
21
Despite their importance, BCAAs and other essential amino acids are present in limiting
22
amounts in major crops used for human consumption and animal feed (Ufaz and Galili, 2008).
23
Over accumulation of amino acids leads to various toxic effects on growth and development in A.
24
thaliana, indicating the importance of amino acid homeostasis (Phillips et al., 1981; Angelovici
25
et al., 2009; Zhu and Galili, 2003). Biosynthesis and catabolism both play roles in BCAA
26
regulation in plants (Galili et al., 2016). A. thaliana mutants blocked in BCAA degradation
27
accumulate BCAAs in seeds of plants grown under standard environmental conditions
28
(Angelovici et al., 2013; Gu et al., 2010; Lu et al., 2011) and in leaves of dark-treated plants
29
(Peng et al., 2015; Araújo et al., 2010), implicating BCAA catabolism in seed BCAA
30
homeostasis and as an alternative energy source under energy-deprived conditions. These
31
catabolic enzymes are coregulated at steady-state mRNA level under diel and extended darkness
32
conditions as well as during seed development, consistent with the mutant results (Peng et al.,
33
2015; Uygun et al., 2016). While less is known about the importance of genetic regulation of
34
BCAA biosynthetic enzymes, the activities of committed enzymes of the BCAA network are
35
sensitive to in vitro regulation by amino acid products (Less and Galili, 2008; Pratelli and Pilot,
2
36
2014). Such allosteric mechanisms could play an important role in BCAA homeostasis; however,
37
relatively little is known about their in vivo importance.
38
Plants synthesize BCAAs in the chloroplast through a metabolic network conserved from
39
bacteria to flowering plants (Diebold et al., 2002; Ellerström et al., 1992; Binder, 2010; Curien et
40
al., 2008) (Figure 1). Ile is synthesized from the Asp-derived amino acid biosynthetic pathway
41
with Thr as an intermediate. Val is derived from pyruvate, and this pathway shares four common
42
catalytic enzymes with Ile biosynthesis. Leu biosynthesis branches off before the final
43
transamination step of Val biosynthesis. In A. thaliana, the three committed enzymes in BCAA
44
biosynthesis, threonine deaminase (referred to in this work as OMR1 for L-O-methylthreonine
45
resistance 1), AHAS and IPMS, are feedback regulated, as demonstrated in vitro for A. thaliana
46
(Mourad and King, 1995; de Kraker et al., 2007; Chen et al., 2010; Lee and Duggleby, 2001).
47
OMR1 activity is inhibited by Ile, and Val antagonizes this inhibition (Halgand et al., 2002;
48
Garcia and Mourad, 2004). In addition, Leu is mildly inhibitory to OMR1 in vitro (Mourad and
49
King, 1995). The two IPMS isoenzymes, IPMS1 and IPMS2, are both subject to Leu inhibition
50
in vitro (de Kraker et al., 2007). In contrast to OMR1 and IPMS enzymes, with catalytic and
51
regulatory domains on single proteins, the active plant AHAS enzyme is a α2β2 type
52
heterotetramer formed by the large catalytic subunit AHASL and the small regulatory subunits
53
AHASS1 and AHASS2 (McCourt and Duggleby, 2006). Binding of the small subunits stimulates
54
AHASL activity and confers it with BCAA sensitivity. In vitro assays revealed that the AHASS1
55
isoform is sensitive to all three BCAAs (Lee and Duggleby, 2001), and the AHASS2 isoform is
56
sensitive to Val (Chen et al., 2010); however the effects of Leu and Ile on AHASS2 are not
57
known. These regulatory effects are conferred through evolutionarily conserved ACT (Aspartate
58
kinase, Chorismate mutase and TyrA) domains in the allosteric regions of OMR1, AHASS1 and
59
AHASS2 or ACT-like domains in IPMS1 and IPMS2 (Chipman and Shaanan, 2001).
60
Despite extensive work on in vitro end-product regulation of OMR1, AHAS and IPMS,
61
relatively little is known about the in vivo importance of allosteric regulation in BCAA
62
homeostasis. A feedback-resistant mutation in OMR1 caused up to 20-fold free Ile accumulation
63
in Arabidopsis seedlings (Mourad and King, 1995), whereas a Val-tolerant AHASS2 mutant
64
enzyme led to two- to three-fold increase in free Val and Leu (Chen et al., 2010), suggesting that
65
the biosynthesis of Ile may be regulated independently from that of Val and Leu. These studies
66
demonstrate the power of forward genetic selection for mutants with relaxed allosteric control,
3
67
while leaving important questions to be answered. For example, what is the in vivo role of IPMS
68
in regulating Leu homeostasis and how does allosteric regulation of the three committed
69
enzymes interact to regulate BCAA homeostasis? How does each of the AHASS and IPMS
70
isoforms contribute to allosteric regulation and overall in vivo enzyme activity? What is the
71
relative importance of allosteric regulation in BCAA homeostasis in vegetative and reproductive
72
tissues?
73
To address these questions, we performed a genetic dissection of the committed enzymes
74
of the BCAA metabolic network. Forward genetic screens in Arabidopsis using toxic Ile and Leu
75
analogs led to the identification of dominant inhibitor-resistant mutants of OMR1, IPMS1 and the
76
AHAS small subunit AHASS2 gene. Recessive T-DNA insertion mutations were identified and
77
characterized in each of the AHASS and IPMS genes. Analysis of free amino acids in seedlings of
78
single and double mutants provided evidence that OMR1 primarily regulates Ile accumulation
79
and functions independently of AHAS and IPMS. We demonstrated that the joint action of
80
AHAS and IPMS maintains Val and Leu homeostasis, with the former affecting the overall flux
81
into the two pathways and the latter controlling partitioning between them. Moreover, evidence
82
was obtained that both AHAS isoenzymes contribute to Val+Leu accumulation, while IPMS1
83
appears to be more important for Val/Leu partitioning than IPMS2. In addition to documenting
84
BCAA in vivo regulatory mechanisms, these results inform transgenic, genome editing and
85
breeding approaches to produce crops with balanced essential amino acids.
86
87
88
RESULTS
89
Identification of amino acid analog-resistant mutants
90
Two forward-genetic selections were used to isolate mutants with altered regulation of
91
BCAA biosynthesis. The first used the toxic L-leucine analog, 5,5,5-trifluoro-DL-leucine (TFL),
92
while the other employed the toxic L-isoleucine analog, L-O-methylthreonine (OMT). Ethyl
93
methanesulfonate (EMS) mutagenized seeds from the Columbia-0 accession (Col-0) were sown
94
under the lowest concentrations of compound that completely inhibited the growth of Col-0
95
(Figure 2). Thirty TFL-resistant and nine OMT-resistant putative mutants were identified,
96
rescued to sterile nutrient agar plates lacking inhibitor and then allowed to self-pollinate in soil-
97
less mix. Three classes of mutants were anticipated: enzymes with altered allosteric regulation or
4
98
catalytic function, variants that have modified genetic regulation of one or more enzyme in
99
BCAA biosynthesis or plants defective in inhibitor uptake.
100
We hypothesized that feedback-resistant mutants would carry dominant mutations in
101
allosteric domains of enzymes at committed steps (Mourad and King, 1995; Rognes et al., 1983;
102
Chen et al., 2010; Li and Last, 1996). Three enzymes of Asp-derived and branched-chain amino
103
acid biosynthesis are documented to be inhibited in vitro by Leu (Figure 1). These are IPMS
104
(IPMS1 and IPMS2), the AHAS regulatory small subunit 1 (AHASS1) and the bifunctional
105
aspartate kinase/homoserine dehydrogenase I (AK-HSDH I) (de Kraker et al., 2007; Curien et al.,
106
2005; Lee and Duggleby, 2001). Sequencing the allosteric domains of these four genes as well as
107
the AHASS2 gene revealed single nucleotide mutations in 29 of the 30 TFL-resistant putative
108
mutants. A total of nine different missense mutations were identified; six in AHASS2 and three in
109
IPMS1 (Supplemental Table 1). Ile inhibits Arabidopsis threonine deaminase and AHASS1 in
110
vitro (Mourad and King, 1995; Lee and Duggleby, 2001). The allosteric regions in OMR1,
111
AHASS1 and AHASS2 were sequenced and analyzed from the nine OMT-resistant mutants. We
112
found four different missense changes in the ACT allosteric domains of OMR1 gene in five of
113
these lines (Supplemental Table 1). Surprisingly, the ahass2-1D mutation (in the TFL-resistant
114
mutants tfl101 and tfl106) was re-isolated in the weakly OMT-resistant line omt4 (Supplemental
115
Table 1). No mutation was identified in IPMS2 or AHASS1.
116
Genetic analysis of inhibitor-resistant mutants
117
Genetic analysis was performed on strong OMT- and TFL-resistant alleles to test whether
118
the mutations identified in OMR1, AHASS2 and IPMS1 are heritable and genetically linked to the
119
inhibitor resistance trait. The three strongest OMT-resistant mutants—omr1-11D, omr1-12D and
120
omr1-13D—were chosen along with two TFL-resistant alleles, ahass2-1D (found in tfl101 and
121
tfl106) and ipms1-1D (carried by tfl102 and tfl111) (Table 1). These tfl mutant lines showed
122
stronger TFL resistance than alleles with other mutations in each gene and were found in
123
multiple mutants from independent seed pools, consistent with the hypothesis that these
124
mutations cause the inhibitor resistance phenotype (Supplemental Table 1). Self-crossed progeny
125
were germinated on nutrient plates with varying concentrations of OMT or TFL; the results
126
indicated that all of the mutant phenotypes are heritable (Figure 2). The mutants were then
127
crossed with Col-0 wild type: the F1 plants grew in the presence of 0.4mM OMT or 0.2mM TFL,
128
consistent with the hypothesis that these mutations are dominant. The F1 plants showed
5
129
intermediate inhibitor resistance compared with the homozygous mutant parents (Figure 2), a
130
characteristic observed for other amino acid analog feedback-insensitive mutants (Mourad and
131
King, 1995; Rognes et al., 1983; Li and Last, 1996).
132
To test the hypothesis that the mutations in OMR1, AHASS2 and IPMS1 are genetically
133
linked to the observed inhibitor resistance, the mutants were crossed with Ler and F2 co-
134
segregation analysis performed (Jander et al., 2002). The F2 plants that grew on nutrient agar
135
plates containing 0.6mM OMT or 0.3mM TFL were genotyped using PCR assays that detect
136
OMR1 (CAPS1-1), AHASS2 (CAPS2-1), IPMS1 (CAPS3-1) and marker AMU-4-272, which is
137
unlinked to any of the candidate genes. As expected for linked dominant mutations, only
138
homozygous Col-0 and heterozygotes were identified in the resistant F2 plants using the OMR1-,
139
AHASS2- or IPMS1-linked markers in the corresponding populations (Supplemental Table 2). In
140
contrast, the unlinked AMU-4-272 marker showed a segregation ratio of roughly 1:2:1 for
141
homozygous Col-0, heterozygous and homozygous Ler genotypes in all five F2 populations
142
(Supplemental Table 2). These results demonstrate cosegregation of mutations in candidate
143
genes and the inhibitor resistance traits, consistent with the hypothesis that the sequenced
144
mutations are causal.
145
OMR1D enzyme activities are resistant to Ile in vitro
146
As shown in Figure 3, the amino acid substitutions found in OMT-resistant mutants
147
omr1-11D, omr1-12D and omr1-13D are located in the second ACT domain at sites that are
148
conserved in land plants (OMR1-12D) or across all sequences analyzed (OMR1-11D and OMR1-
149
13D) (Figure 3A and 3B). Homology modeling of the OMR1 C-terminal regulatory region—
150
containing the two ACT domains—revealed that the three amino acid changes reside in regions
151
predicted to link two anti-parallel β-sheets (OMR1-12D and OMR1-13D) or α-helix and β-sheet
152
domains (OMR1-11D) (Figure 3C).
D
153
In vitro enzyme assays revealed that the amino acid substitutions in OMR1-11 , OMR1-
154
12 and OMR1-13 reduced the Ile feedback sensitivity of the OMR1 protein. Escherichia coli
155
(E. coli)-expressed purified recombinant wild-type and mutant OMR1 proteins were tested for
156
Ile inhibition and catalytic efficiency. The three OMR1D protein activities showed increased Ile
157
resistance up to 10mM, the maximum tested (Figure 3D). At 1mM Ile, ~90%, ~70% and ~30%
158
residual activity was observed for OMR1-11D, OMR1-12D and OMR1-13D, respectively—and all
159
were more resistant than the wild-type enzyme, which showed ~20% residual activity in the
D
D
6
160
presence of 1mM Ile (Figure 3D). Kinetic analysis revealed minor differences between the
161
OMR1D enzymes and wild type in the absence of Ile (Supplemental Table 3). Only OMR1-12D
162
had modestly increased catalytic efficiency with a Kcat/Km of 7.7 s-1 mM-1 compared with 5.2 s-1
163
mM-1 for wild-type enzyme. Taken together with the genetics, these results provide strong
164
evidence that the mutations identified in the omr1D mutants cause Ile feedback resistance through
165
alteration of the second ACT protein domain.
166
Altered BCAA sensitivity is associated with ahass2-1 D TFL-resistant mutant
167
We asked whether the ACT2 Ser349Phe ahass2-1D amino acid change influenced the
168
AHAS enzyme catalytic activity or allosteric control. As shown in Figure 4, amino acid Ser349
169
is conserved from E. coli to flowering plants (Figure 4A and 4B), suggesting that it influences
170
enzyme activity or regulation. Because the active AHAS enzyme comprises both large and small
171
subunits, E. coli-expressed recombinant wild-type AHASL (the catalytic large subunit) and
172
AHASS2 (regulatory small subunit 2) variants—expressed in-frame with N-terminal GST tags to
173
improve accumulation of soluble protein—were reconstituted into active enzyme complexes and
174
tested in vitro (McCourt and Duggleby, 2006). The reconstituted wild-type AHASS2 enzyme
175
activity was most strongly inhibited by Val (~30% inhibition at 1mM); in contrast, Ile and Leu
176
had more modest impacts up to 10mM concentration (4.9% and 1.2% inhibition at 1mM,
177
respectively) (Figure 4C). AHASS2-1D had reduced sensitivity to BCAA inhibition (Figure 4C),
178
with minimal changes in Km and catalytic efficiency (Table 2). This mutant showed reduced
179
sensitivity to all BCAAs—1mM Val and Leu caused 2.3% and 3.3% reduction in enzyme
180
activity, respectively, whereas 1mM Ile was not inhibitory (Figure 4C). These results show that
181
Ser349Phe in AHASS2-1D causes reduced sensitivity to the BCAAs without changing catalytic
182
efficiency.
183
IPMS1-1D enzyme is Leu tolerant
184
The ipms1-1D mutation is predicted to cause a Gly to Glu substitution at position 606 in
185
the IPMS C-terminal domain, a region previously demonstrated to be involved in Leu binding
186
and allosteric regulation in plants (de Kraker and Gershenzon, 2011; Ning et al., 2015) (Figure 5).
187
Gly606 is in a region of the allosteric domain of IPMS proteins conserved from bacteria to
188
flowering plants (Figure 5A and 5B). In addition, homology modeling places Gly606 in the α-
189
helix close to the Leu-binding pocket on the IPMS1 dimer, suggesting that this amino acid
190
substitution may affect Leu docking or binding (Figure 5C). The impact of the Gly606Glu
7
191
substitution in IPMS1-1D was investigated in vitro using recombinant IPMS1 proteins. The wild-
192
type and mutant IPMS1 enzymes had no difference in enzyme activity without Leu; in contrast,
193
IPMS1-1D showed higher activity than the wild type at all tested Leu concentrations (Figure 5D).
194
Combined with the genetic mapping results, these data demonstrate a strong link between the
195
Gly606Glu substitution and in vivo TFL-resistance.
196
Feedback-insensitive mutants reveal an allosteric regulatory hierarchy
197
The in vivo roles OMR1, IPMS and AHAS play in regulating BCAA homeostasis were
198
investigated by analyzing soluble amino acids in the two-week-old vegetative tissues of the
199
feedback-resistant mutants. As shown in Figure 6, the primary change in three threonine
200
deaminase-deregulated omr1D mutants was a 7 to >140 fold increase in Ile accumulation (Figure
201
6A and 6B, Table 3). These increases were accompanied by up to 50% reduction in the OMR1
202
substrate Thr (Table 3). In contrast to the large increases in Ile, the other two BCAAs—Val and
203
Leu—were much less affected in the mutants. For example, the strongest mutant, omr1-11D, had
204
only 1.5- and 3.8-fold increases of Val and Leu compared to the wild type despite a >140-fold
205
increase in Ile, while the weakest mutant, omr1-13D, showed no Val and Leu changes (Figure 6A
206
and 6B, Table 3). These results are consistent with a model in which the allosteric control of
207
OMR1 primarily regulates Ile homeostasis in Arabidopsis seedlings.
208
Consistent with a role for IPMS in feedback regulation of Leu biosynthesis, ipms1-1D
209
mutant seedlings had 2.5-fold higher free seedling Leu compared with the wild type (Figure 6C
210
and 6D, Table 4). This Leu increase was associated with a 60% decrease in Val in the mutants.
211
As a result, the total amount of Val+Leu was the same as in the wild type, consistent with the
212
hypothesis that IPMS feedback regulation did not indirectly influence flux through AHAS
213
(Figure 6C and 6D, Table 4). No change was detected in free Ile in seedlings. The ipms1-1D
214
mutant phenotypes support a role of this enzyme in controlling Val/Leu partitioning.
215
Results with the feedback-insensitive ahass2-1D mutants argue that allosteric regulation
216
of this enzyme primarily controls partitioning into the Val+Leu subnetwork. The ahass2-1D
217
single mutants showed seven- to eight-fold increases in both Val and Leu, respectively, without
218
influencing the 3:1 ratio of Val to Leu seen in the wild type (Figure 6C and 6E, Table 4). These
219
results suggest that AHAS allosteric regulation influences the total amount of Val+Leu without
220
affecting the partitioning between these two amino acids. AHAS regulation also has a minor
221
influence on Ile concentrations, presumably reflecting a secondary role in regulating Ile
8
222
homeostasis compared to that of threonine deaminase. Consistent with its role as the second
223
committed enzyme in Ile biosynthesis, an up to 3.5-fold increase in Ile was observed in ahass2-
224
1D mutants; these changes are subtle compared with the 7-140 fold increases in the omr1D
225
mutants (Figure 6A, 6B, 6C and 6E, Table 3, Table 4).
226
Taken together, the BCAA phenotypes of the feedback-resistant single mutants suggest a
227
simple allosteric regulatory hierarchy. In this model, AHAS regulates the diversion of flux into
228
both Val and Leu, IPMS controls partitioning between Val and Leu, and threonine deaminase
229
feedback regulation exerts primary control on Ile homeostasis.
230
BCAA profiling of the feedback-resistant double mutant F1 seedlings
231
To test this model, double heterozygous dominant feedback-resistant mutant F1 plants
232
were generated and free amino acids analyzed in two-week-old seedlings (Figure 7). The
233
phenotypes of double mutants with omr1D yielded results consistent with threonine deaminase
234
regulation
235
1D/AHASS2;omr1D/OMR1 and ipms1-1D/IPMS1;omr1D/OMR1 double mutants had BCAA
236
profiles similar to those for the single mutant F1 lines (Figure 7A to 7D; Supplemental Table 4).
237
For example, the ahass2-1D/AHASS2;omr1-11D/OMR1 double mutant F1 had 63-fold increase in
238
Ile—which is close to the 54-fold boost seen in the heterozygous omr1-11D/OMR1 F1 single
239
mutant—and 3.8- and 4.1-fold increased Val and Leu—similar to that observed in the ahass2-
240
1D/AHASS2 F1 single heterozygote (Figure 7A; Supplemental Table 4). Similarly, while
241
exhibiting
D
acting
the
Ile
independently
increase
seen
of
IPMS
in
and
omr1D/OMR1
AHAS.
F1
Heterozygous
plants,
the
two
ahass2-
ipms1-
D
242
1 /IPMS1;omr1 /OMR1 heterozygous double mutants had decreased Val comparable to the ~40%
243
decrease seen in the ipms1-1D/IPMS1 single mutant (Figure 7C and 7D; Supplemental Table 4).
244
The double heterozygotes had Leu increases similar to those seen in the heterozygous omr1D
245
single mutants, whereas the ipms1-1D/IPMS1 single heterozygous F1 exhibited no change in Leu
246
(Figure 7C and 7D; Supplemental Table 4). Together, these results support the hypothesis that
247
OMR1 primarily regulates Ile biosynthesis and it functions independently from the Val and Leu
248
pathways.
249
In contrast, results with the ahass2-1D/AHASS2;ipms1-1D/IPMS1 double heterozygous
250
mutant reinforce the idea that both AHAS and IPMS function in regulation of Val and Leu
251
homeostasis in two-week-old seedlings. First, the heterozygous double mutant displayed
252
increased total Val+Leu that is similar to that seen in the ahass2-1D/AHASS2 single heterozygote,
9
253
with ~2.0-fold higher Val+Leu than the wild type in both the single and double heterozygous
254
mutant (Figure 7E; Supplemental Table 5). Second, Val/Leu partitioning was altered in the
255
doubly heterozygous mutant—with roughly equal amounts of Val and Leu observed compared
256
with the ~3:1 wild-type Val/Leu ratio (Supplemental Table 5). This ratio was reminiscent of the
257
heterozygous ipms1-1D/IPMS1 single mutant. These results are consistent with the hypothesis
258
that AHAS and IPMS allostery regulate Val and Leu homeostasis in a coordinated manner in
259
vivo, with AHAS controlling entry into the Val+Leu subnetwork and IPMS affecting partitioning
260
between Val and Leu.
261
IPMS1 loss-of-function mutant BCAA phenotype supports a role in Val/Leu partitioning
262
Arabidopsis has two IPMS genes that encode isoforms with qualitatively similar but
263
quantitatively distinct transcript profiles throughout vegetative development (Supplemental
264
Figure 1). To investigate how each isoenzyme contributes to in vivo BCAA homeostasis, we
265
studied ipms1 and ipms2 loss-of-function mutants. Public microarray analysis revealed that
266
IPMS1 transcript accumulates up to five-fold more than that of IPMS2 in vegetative tissues
267
during development (Supplemental Figure 1). Indeed, T-DNA disruption of the more highly
268
expressed IPMS1 gene provided evidence for a role of this isoenzyme in Val/Leu partitioning
269
(Figure 8; Supplemental Figure 2A and 2B), causing up to a 3.6-fold boost in free seedling Val
270
compared to the wild type, whereas Leu and Ile were unaffected (Figure 8A and 8B;
271
Supplemental Table 6). As a result, the proportion of Leu to total Val+Leu decreased by up to 17%
272
compared to the wild type—opposite to the 44% increase seen in the reduced allosteric
273
regulation ipms1-1D mutants (Figure 8A to 8C; Supplemental Table 6). In contrast, the loss-of-
274
function mutants in the less highly expressed IPMS2 gene showed marginal increases in the
275
proportion of Leu to total Val+Leu compared to wild type (4% and 7% in ipms2-1 and ipms2-2
276
respectively) (Supplemental Table 6 and Supplemental Figure 1 and 2). Collectively, the higher
277
IPMS1 transcript levels and stronger loss-of-function ipms1 mutant phenotype results are
278
consistent with the hypothesis that the IPMS1 isoform plays a more prominent role in regulating
279
vegetative tissue Val/Leu partitioning.
280
Although the ipms2 loss-of-function mutations alone had no significant impact on BCAA
281
homeostasis (Supplemental Table 6), BCAA levels were altered in the ipms1-1D ipms2 double
282
mutants compared with the wild-type or ipms1-1D single mutant plants (Figure 8A, 8C and 8D;
283
Supplemental Table 6 and Supplemental Figure 2B and 2C). We observed an up to 1.9-fold
10
284
increase in Val+Leu in the double mutants compared to the wild type, in contrast to the ipms1-1D
285
and ipms2 homozygous single mutants, which exhibited no change in Val+Leu (Figure 8A, 8C
286
and 8D; Supplemental Table 6). An up to 2.7-fold increase in Ile was also observed in the double
287
mutants, which was not seen in either single mutant line (Figure 8A, 8C and 8D; Supplemental
288
Table 6). In contrast, reduction or absence of IPMS2 did not influence partitioning between Val
289
and Leu in the double mutants—which had the same proportion of Leu to Val+Leu as seen in the
290
ipms1-1D single mutant (Figure 8C and 8D; Supplemental Table 6). Together, results with the
291
ipms1-1D ipms2 double mutants revealed evidence for a role of the IPMS2 isoform in BCAA
292
homeostasis.
293
Evidence for a role of AHASS1 in BCAA homeostasis
294
Transcript analysis and in vitro enzyme assays revealed differential gene expression and
295
allosteric feedback regulation of the AHASS1 and AHASS2 isoenzymes. AHASS1 and
296
AHASS2 transcript levels were anti-correlated in public microarray data, with more AHASS1
297
transcript than AHASS2 in vegetative tissues (Supplemental Figure 3). These results were
298
validated by qRT-PCR analysis, where AHASS1 transcript was 4.5-fold more abundant than
299
AHASS2 in two-week-old Col-0 seedlings. In addition, AHASS1 and AHASS2 protein
300
responded differently to BCAA inhibition in vitro (Figure 9). First, AHASS1 showed sensitivity
301
to both Val and Leu (Figure 9A to 9C), in contrast to AHASS2, which only was sensitive to Val
302
under our assay conditions (Figure 4C). Second, the AHASS1 isoenzyme was subject to
303
synergistic inhibition by Val+Leu, consistent with the report by Lee and Duggleby (Lee and
304
Duggleby, 2001) (Figure 9D). In contrast, we observed a simple additive inhibitory effect of
305
Val+Leu on wild-type AHASS2 activity (Figure 9E). We sought in vivo evidence for an impact
306
of the allosteric regulation of the AHAS isoforms by testing the effects of exogenous applied
307
BCAAs on germination and seedling growth of Col-0 wild type as well as ahass1 and ahass2
308
loss-of-function mutants (Figure 10; Supplemental Figure 4A to 4D). Wild-type seedling growth
309
was strongly inhibited by a mixture of 0.5mM Val plus 0.5mM Leu (Figure 10A; Supplemental
310
Figure 5A), whereas growth inhibition was not seen with any other pairwise mix that included Ile
311
or with individual BCAAs at this concentration (Supplemental Figure 5B to 5F). The ahass1
312
mutants were tolerant of Val+Leu inhibition (Figure 10B and 10C), while the ahass2 lines
313
exhibited wild type-like sensitivity to Val+Leu (Figure 10D and 10E). Taken together, these data
11
314
revealed differences in expression and allosteric regulation of the two AHAS isoforms and
315
evidence for an in vivo role of AHASS1 in Val+Leu allosteric control.
316
The role of AHASS1 in regulating BCAA homeostasis was further investigated by
317
analyzing BCAA profiles of two-week-old ahass1 loss-of-function single mutant seedlings
318
(Figure 11). Both ahass1 mutants exhibited ~2-fold increase in free seedling Val, whereas Ile
319
and Leu remained at wild-type levels (Figure 11A; Supplemental Table 7). The altered Val levels
320
in the ahass1 loss-of-function mutants provided evidence for an in vivo function of the AHASS1
321
isoform in regulating BCAA homeostasis.
322
Double mutants were constructed between the two loss-of-function ahass1 mutations and
323
the feedback-resistant ahass2-1D allele to further explore the role of these two isoforms. The
324
AHASS2-1D mutant enzyme is resistant to all single BCAAs as well as Val+Leu (Figure 4C;
325
Supplemental Figure 6). While ahass1 and ahass2-1D homozygous single mutants each were
326
viable, we were unable to recover ahass1/ahass1;ahass2-1D/ahass2-1D double mutants after
327
screening 96 plants in each ahass1 × ahass2-1D F2 population. In addition, <70% of the F2 seeds
328
from each population germinated on unsupplemented medium. We hypothesize that lethality of
329
the homozygous ahass1/ahass1;ahass2-1D/ahass2-1D double mutants is due to BCAA
330
dyshomeostasis—as proposed for other amino acid metabolic networks (Joshi et al., 2006; Zhu
331
and Galili, 2003). Consistent with the hypothesis, ahass1/ahass1;ahass2-1D/AHASS2 double
332
mutant F3 seedlings exhibited higher BCAA levels compared to the corresponding single mutants:
333
up to 7.3- and 5.2-fold higher Val and Leu than the wild type, respectively, compared to the up to
334
2.4-fold increases of Val and Leu in ahass1/ahass1 and ahass2-1D/AHASS2 single mutants
335
(Figure 11B; Supplemental Table 8). In addition, BCAA accumulation in ahass1/ahass1;ahass2-
336
1D/AHASS2 F3 seedlings was comparable to that seen in the homozygous ahass2-1D;ahass2-1D
337
single mutant, which had 6.1- and 5.0-fold increases in Val and Leu respectively compared to
338
wild type (Figure 11B; Supplemental Table 8). Together, the ahass1 single and double mutant
339
analysis supports the hypothesis that the AHASS1 isoform plays a role in maintaining BCAA
340
homeostasis.
341
A similar approach was taken to analyze the role of AHASS2 isoform in regulating
342
BCAA homeostasis in vivo. Changes in BCAA profiles of the ahass2 loss-of-function mutants
343
were mixed. On the one hand, free Val and Leu were modestly reduced in ahass2-7—to 0.76-
344
and 0.64-fold of the wild type respectively—whereas Ile was unaffected (Figure 11C;
12
345
Supplemental Table 7). In contrast, ahass2-8 had wild-type levels of the BCAAs (Supplemental
346
Table 7). In addition, an up to 1.8-fold upregulation of AHASS1 transcript levels was detected in
347
the ahass2 loss-of-function mutants by qRT-PCR (Supplemental Figure 4E); this is in contrast to
348
the lack of change in AHASS2 transcript level observed in ahass1 loss-of-function mutants
349
(Supplemental Figure 4F). Together, characterization of the ahass single and double mutants
350
reinforces the hypothesis that the AHASS1 isoform has a role in maintaining BCAA homeostasis
351
in vivo.
352
Ile deficiency underlies Val+Leu toxicity on seedling growth
353
We asked how exogenous Val+Leu inhibit the growth of wild type Col-0 seedlings. Two
354
mechanisms were previously documented for in vivo toxicity resulting from AHAS activity
355
inhibition using herbicides. One is toxicity due to accumulation of the AHAS substrate 2-
356
oxobutanoate—documented in the bacterium Salmonella typhimurium (Van Dyk and LaRossa,
357
1986; Primerano and Burns, 1982)—while BCAA starvation was proposed for plants (Shaner
358
and Singh, 1993). The relatively minor inhibitory effect of up to 1mM exogenous 2-
359
oxobutanoate or pyruvate on Col-0 seedling growth argues against toxicity due to intermediate
360
accumulation (Supplemental Figure 7A). In contrast, Ile supplementation reversed the Val+Leu
361
mediated inhibition of Col-0 seedlings (Supplemental Figure 7B). Given that we observed ~20%
362
residual OMR1 enzyme activity at 10mM Ile in vitro (Figure 3D), 2-oxobutanoate could
363
accumulate in plants treated with 0.5mM Ile. The strongest evidence for the Ile starvation
364
toxicity hypothesis is the reversal of Val+Leu inhibition by Ile in the Ile-feedback-insensitive
365
omr1-12D mutant (Supplemental Figure 7C). This is because Ile addition to OMR1-12D is even
366
less prone to reduction of 2-oxobutanoate than the wild-type enzyme (Figure 3D). Collectively,
367
these data support the hypothesis that the in vivo Val+Leu inhibition of Arabidopsis seedling
368
growth is due to Ile starvation rather than inhibitor accumulation.
369
Feedback-resistant mutants accumulate higher seed BCAAs
370
The feedback-resistant mutants show increases in seed BCAAs (Table 5) qualitatively
371
similar to those seen in seedlings (Table 3 and Table 4). Consistent with an in vivo role of OMR1
372
in seed Ile homeostasis, omr1D mutant seeds had up to 8.9-fold increase in Ile compared to wild
373
type (Table 5). However, these Ile increases were subtle compared to the up to 140-fold increase
374
seen in leaf (Table 3). Seed Val and Leu levels were boosted in the omr1-11D and omr1-13D
375
mutants—by up to 3.7- and 3.9-fold, respectively—compared to wild type (Table 5). Results
13
376
with the ahass2-1D and ipms1-1D homozygous single mutants showed that AHAS and IPMS
377
allostery regulates Val and Leu homeostasis in seeds, with ahass2-1D increasing total Val+Leu—
378
by up to 5.5-fold—and ipms1-1D altering Val/Leu partitioning by ~20% (Table 5), similar to that
379
observed for two-week-old mutant seedlings (Table 4). These data provide evidence for a similar
380
regulatory hierarchy of the three committed enzymes in seedling and seed BCAA homeostasis
381
and the impacts of these feedback-insensitive mutations in boosting seed BCAA levels.
382
383
DISCUSSION
384
Although plants are the main source of animal dietary essential amino acids, plant
385
BCAAs are rarely in balance with the nutritional requirements of these primary consumers.
386
Understanding the regulation of BCAA biosynthesis can inform breeding and transgenic
387
approaches to improve plant nutritional quality. Despite the documented in vitro feedback
388
regulation of the BCAA committed enzymes in plants and microbes (Lee and Duggleby, 2001;
389
Chen et al., 2010; de Kraker et al., 2007; Duggleby and Pang, 2000; Pang and Duggleby, 2001;
390
Curien et al., 2008; de Kraker and Gershenzon, 2011), relatively little is known about how these
391
enzymes influence free amino acid homeostasis in vivo. In contrast, altered regulation of IPMS is
392
of documented importance in specialized metabolism. The evolution of methionine-derived
393
glucosinolate biosynthesis in the Brassicaceae (de Kraker and Gershenzon, 2011) and acylsugar
394
production in glandular trichomes of cultivated and wild tomatoes (Ning et al., 2015) included
395
deletion of the IPMS feedback regulatory C-terminal domain combined with altered
396
transcriptional regulation, leading to the production of specialized metabolic enzyme genes.
397
In this study, we characterized Arabidopsis feedback-resistant and loss-of-function
398
mutants of BCAA committed enzymes, both singly and in combination. This work leads us to
399
propose a model for a regulatory hierarchy in which the three committed enzymes—OMR1,
400
AHAS and IPMS—control BCAA levels in leaves and seeds. These results illustrate that
401
paralogous isoforms contribute unequally to regulation. Taken together, our results indicate that
402
the control of Arabidopsis free amino acid abundance results from the interplay between these
403
committed enzymes. The complexities that were observed, and the previously documented
404
differences in plant growth and seed viability associated with increases in different essential
405
amino acids and plant species (Angelovici et al., 2009; Zhu and Galili, 2003), suggest that
14
406
engineering of improved amino acid balance will benefit from detailed understanding of the
407
genetic and biochemical architecture of this network in the target crop.
408
A regulatory model for Arabidopsis BCAA homeostasis
409
The regulatory model that comes from this study includes several key components. The
410
most straightforward is that Ile homeostasis is primarily determined by OMR1 feedback control.
411
This is dramatically illustrated by the >140-fold increase in Ile in omr1-11D mutant seedlings,
412
with only 1.5- and 3.8-fold increases in Val and Leu, respectively (Figure 6A and 6B, Table 3).
413
This very high Ile overproduction only resulted in a 50% decrease in the precursor amino acid
414
Thr, suggesting that regulation of the earlier steps in the network responded to increased flux
415
through OMR1 (Table 3). This phenotype, which was not reported for previously published
416
feedback-insensitive OMR1 mutants (Mourad and King, 1995), suggests that engineering
417
increased crop plant Ile can be attempted without changes to aspartate kinase and homoserine
418
dehydrogenase regulation (Figure 1). The up to 3.5-fold increase in Ile in the ahass2-1D mutants
419
suggests that AHAS enzyme activity has a minor role in Ile homeostasis (Figure 6C and 6E,
420
Table 4). This could result from reduced mutant AHAS Ile sensitivity or alternatively from
421
increased soluble Val indirectly causing reduced Ile inhibition of OMR1 (Halgand et al., 2002).
422
Re-isolation of the ahass2-1D allele in the weak OMT-resistant omt4 line is consistent with this
423
hypothesis (Supplemental Table 1). These results support the notion that there is regulatory
424
‘cross talk’, even in the case of Ile regulation by threonine deaminase.
425
Analysis of the ahass2-1D mutant revealed that AHAS allostery mainly affects the
426
Val/Leu subnetwork, with up to a 7.9-fold increase in total seedling Val+Leu (Figure 6C and 6E,
427
Table 4). Furthermore, combining the omr1D mutations with ahass2-1D caused additive effects
428
on free BCAA levels (Figure 7A and 7B; Supplemental Table 4), reinforcing the mutually
429
independent regulation of Ile and Val/Leu subnetworks by OMR1 and AHAS. The observed
430
increase in ipms1-1D seedling Leu and decreased Val argues that IPMS allosteric inhibition
431
regulates partitioning between these amino acids (Figure 6C and 6D, Table 4). Interestingly, Leu
432
levels increased in proportion to Val in ahass2-1D mutants despite the presence of Leu-inhibited
433
wild-type IPMS enzyme activity (Figure 6C and 6E, Table 4). This presumably is a result of
434
the >50% residual in vitro IPMS activity at full Leu inhibition observed in this (Figure 5D) and
435
another study (de Kraker et al., 2007). This proportional Val/Leu increase in ahass2-1D
436
reinforces the in vivo role of IPMS in Val/Leu partitioning. Further supporting evidence came
15
437
from the increased Val/Leu ratio in ipms1 loss-of-function mutant seedlings compared to wild
438
type (Figure 8A and 8B; Supplemental Table 6). Consistent with the single mutant results, the
439
doubly heterozygous ahass2-1D/AHASS2;ipms1-1D/IPMS1 mutant had increased total Val+Leu
440
level and Leu/Val ratio compared to wild type (Figure 7E; Supplemental Table 5). These results
441
demonstrated regulation of Val and Leu homeostasis by both IPMS and AHAS. Taken together
442
with the OMR1 analysis, these findings provide a useful in vivo regulatory starting model of
443
BCAA homeostasis for engineering crops producing high BCAAs.
444
Isoenzymes contribute unequally to maintaining BCAA homeostasis
445
Half of the genes in Arabidopsis are duplicated, a result of whole genome duplications
446
and small scale gene duplications (Jiang et al., 2013; Panchy et al., 2016). Studying the function
447
of paralogous genes is important for understanding the retention mechanism and its impact on
448
physiological homeostasis. We investigated two such gene pairs in this study—AHASS and
449
IPMS—and found evidence that both isoforms of each enzyme play roles in BCAA homeostasis.
450
This reinforces the conclusion of Curien and Bastien (Curien and Bastien, 2009) that isoenzymes
451
contribute unequally to the regulation of flux and are not functionally redundant, and could
452
contribute to future testing and refinement of mathematical models for this network. Our results
453
are consistent with the hypothesis that the two differentially regulated Arabidopsis AHASS
454
isoforms have overlapping function (Figure 11A and 11C; Supplemental Table 7). Despite the
455
lack of genetic redundancy, these genes—which emerged before the divergence of the
456
Arabidopsis-Solanum lineages (Supplemental Figure 8 and Supplemental File 1)—have acquired
457
demonstrably unique characteristics. They are differentially regulated at the mRNA level
458
(Supplemental Figure 3), and loss of the more highly expressed AHASS1 isoform reversed the
459
toxic effect of Val+Leu seen on wild type seedling growth (Figure 10A to 10C). This reveals the
460
potent in vivo inhibitory effect of Val+Leu as well as a role of the AHASS1 isoform in
461
conferring
462
ahass1/ahass1;ahass2-1D/ahass2-1D double mutants provides further evidence for an essential
463
role of AHASS1 in maintaining BCAA homeostasis.
such
feedback
regulation.
Second,
lethality
of
the
homozygous
464
The two IPMS paralogs emerged after the divergence of Arabidopsis-Populus lineages
465
(Supplemental Figure 9 and Supplemental File 2), suggesting a more recent origin than the
466
AHASS gene pair. They are also found in a syntenic block of the A. thaliana α-whole genome
467
duplication (Bowers et al., 2003). While sharing qualitatively similar transcript profiles during
16
468
vegetative development and generally similar enzymatic properties, IPMS1 and IPMS2
469
transcripts display different abundance (Supplemental Figure 1) (de Kraker et al., 2007). Our
470
genetic analysis reveals a clear role for the IPMS1 isoform in Leu homeostasis: T-DNA
471
disruption and feedback-insensitive mutation in the more highly expressed IPMS1 resulted in
472
altered Val/Leu ratios (Figure 8A to 8C; Supplemental Table 6). In contrast, loss of IPMS2 in an
473
otherwise wild type background led to no change in free BCAAs (Supplemental Table 6). This
474
stands in contrast to the surprising result that combining loss-of-function ipms2 alleles with the
475
deregulated ipms1-1D enzyme leads to a modest increase in seedling BCAAs compared to the
476
ipms1-1D single mutant (Figure 8A, 8C and 8D; Supplemental Table 6). This result could reflect
477
a distinct role for IPMS2 or could be an indirect effect of less vigorous growth of the double
478
mutant compared with either single mutant line. Collectively, our findings with AHAS and IPMS
479
provide evidence that each subfunctionalized isoenzyme contributes to BCAA homeostasis in
480
Arabidopsis.
481
Tools for engineering BCAA levels
482
Our results suggest that BCAA levels and ratios can be manipulated in crop plants using
483
a combination of allosteric and loss-of-function mutations. For example, deregulating both
484
OMR1 and AHAS has the potential to increase all three BCAAs, and further modification of
485
IPMS could be used to manipulate the relative abundance of Val and Leu. Increases in BCAAs
486
in both vegetative tissue and mature seeds of the mutants highlight the usefulness of deregulated
487
enzymes in improving nutritional quality in seed (Table 3, Table 4 and Table 5). This adds to our
488
tools for manipulating seed amino acid homeostasis, which was previously shown to be
489
influenced by BCAA catabolism (Peng et al., 2015; Angelovici et al., 2013; Araújo et al., 2010;
490
Gu et al., 2010). These combined results are timely given the rapid development of new genetic
491
and analytical methods including genome editing and facile mass spectrometry approaches,
492
which can be deployed to improve the nutritional quality of target crops.
493
494
METHODS
495
Mutagenesis
496
Mutagenesis of Arabidopsis thaliana was performed using modified published methods
497
(Jander et al., 2003; Kim et al., 2006). All manipulations were done at room temperature in a
17
498
chemical hygiene hood, and the EMS solution was detoxified with a 10× excess of 5M NaOH.
499
Fifty thousand wild-type Col-0 seeds were washed with 0.1% Tween 20 (Sigma-Aldrich) for 15
500
min on a shaker, and then washed with 50mL water. Washed seeds were re-suspended with
501
50mL 0.2% EMS (Sigma) in dH20 and gently rocked on a shaker overnight. The next morning,
502
the EMS solution was decanted and the seeds were washed twice with 50mL water and left to
503
shake gently in 50 mL water for four hours. The water was then poured off and the seeds (M1)
504
were re-suspended in room temperature (22°C) 0.1% agar and planted onto soil-less mix (Redi-
505
earth, Hummert) into 12 flats stratified at 4°C for three-days to promote uniform germination.
506
The M1 plants were grown under 16/8 light/dark cycle (a mix of cool white fluorescent and
507
incandescent bulbs was used; the irradiance was 100 µmol m-2 s-1 photosynthetic photon flux
508
density) for 10 weeks. The M2 progeny from each flat were bulk harvested into independent seed
509
pools. The growth conditions for wild type and mutant Arabidopsis plants in this study were the
510
same as previously described (Lu et al., 2008), except that a 16/8 (light/dark) photoperiod was
511
used.
512
EMS and T-DNA insertional mutant analysis
513
Seeds from this M2 population or a mixed population with M4 and M5 seed (Van
514
Eenennaam et al., 2003; Jander et al., 2004) were surface sterilized and 200-250 seeds were
515
sown on each plate (100 × 25 mm, Thermo Scientific) containing 30mL half-strength Murashige
516
and Skoog (MS) medium (Caisson Labs) with 0.2-0.3mM TFL (Matrix Scientific) or 0.4-0.8mM
517
OMT (Santa Cruz Biotechnology). Two-week-old putatively inhibitor-resistant plants were
518
selected and transplanted onto half-strength MS plates without inhibitor to recover before
519
transplanting onto soil-less mix for M3 seed production. DNA was extracted from the leaves of
520
the putative TFL- and OMT-resistant plants using a DNeasy Plant Mini kit (QIAGEN) and the
521
genomic regions corresponding to the regulatory domains of OMR1, AHASS1, AHASS2, IPMS1,
522
IPMS2 and AK-HSDH I were amplified and Sanger sequenced to identify EMS-induced
523
mutations. Primers used for amplification and sequencing are summarized in Supplemental Table
524
9.
525
T-DNA insertional mutants ipms1-4 (SALK_101771), ipms1-5 (WiscDsLoxHs221_05F),
526
ipms2-1 (WiscDsLox426A07), ipms2-2 (SALK_046876), ahass2-7 (WiscDsLoxHs009_02G),
527
ahass2-8 (WiscDsLoxHs110_12G), ahass1-1 (SALK_096207) and ahass1-2 (SALK_108628),
528
were obtained from the Arabidopsis Biological Resource Center (https://abrc.osu.edu/). The T18
529
DNA insertions in these mutants were validated through PCR as described by Ajjawi and
530
coworkers (Ajjawi et al., 2010). Primers used for genotyping are listed in Supplemental Table 9.
531
OMT-
532
(https://abrc.osu.edu/). Refer to Supplemental Table 10 for stock numbers.
and
TFL-resistant
single
and
double
mutants
were
donated
to
ABRC
533
534
Segregation analysis of feedback-resistant mutants
535
Feedback-insensitive mutants in the Col-0 genetic background were crossed with
536
Landsberg erecta (Ler) wild type and the F1 plants were sown on half-strength MS plates
537
containing 0.4mM OMT or 0.2mM TFL. Resistant F1 plants were genotyped and transferred to
538
half-strength MS plates for recovery and moved to soil-less mix to generate F2 seeds. 150-200 F2
539
seeds of each mutant were sown on half-strength MS plates containing 0.6mM OMT or 0.3mM
540
TFL, and resistant plants were transplanted onto half-strength MS plates for recovery. Leaf
541
samples were archived onto FTA PlantSaver cards (GE Healthcare) and prepared for PCR
542
following the manufacturer’s instructions. Cleaved amplified polymorphic sequences (CAPS)
543
markers CAPS1-1, CAPS2-1 and CAPS3-1—which are linked to OMR1, AHASS2 and IPMS1
544
respectively—were used to genotype the resistant F2 plants. The marker AMU-4-272, which is
545
not linked to any of the candidate genes, was used as a control. Primers used to amplify these
546
genetic markers are listed in Supplemental Table 9.
547
Vector construction and protein expression
548
Total RNA was isolated from two-week-old Arabidopsis seedlings with an RNeasy plant
549
mini kit (QIAGEN) and first strand cDNA synthesis was carried out with SuperScript III reverse
550
transcription system (Invitrogen) with Oligo(dT) primer (Invitrogen). For the expression of His-
551
tagged proteins, the coding regions of OMR1, AHASL and IPMS1 without their predicted target
552
peptides (TargetP 1.1 Server, http://www.cbs.dtu.dk/services/TargetP/) were amplified using
553
Phusion high-fidelity DNA polymerase (NEB) with primers 42 and 43 (OMR1) (Niehaus et al.,
554
2014), 44 and 45 (AHASL) and 46 and 47 (IPMS1) (Supplemental Table 9). The resulting PCR
555
products were purified (QIAquick PCR purification kit, QIAGEN), double digested with NcoI
556
and NotI (OMR1) or NcoI and XhoI (AHASL and IPMS1) and ligated into pET28b. The two
557
AHAS small subunits, AHASS1 and AHASS2, have low solubility and were expressed in-frame
558
with an N-terminal GST tag. A 6×His tag was attached to the C-termini of AHASS1 and
559
AHASS2 recombinant proteins to facilitate purification. The sequence encoding the predicted
19
560
mature AHASS1 and AHASS2 proteins was amplified with primers 48 to 51 (Supplemental
561
Table 9). Purified PCR products were double digested with BamHI and XhoI and ligated into
562
pGEX-4T-1 vector to express the GST fusion proteins with C-terminal His tags. All the
563
constructs were validated by DNA sequencing and used to transform E. coli strain BL21 (DE3)
564
Rosetta.
565
E. coli strains harboring the pET28b constructs were used to inoculate 1L (OMR1
566
constructs) or 300mL (AHASL and IPMS1 constructs) Luria-Bertani (LB) medium containing
567
100mg/L kanamycin and 34mg/L chloramphenicol. E. coli strains carrying the AHASS1 and
568
AHASS2 constructs were grown in LB medium containing 100mg/L ampicillin and 34mg/L
569
chloramphenicol, and AHASS recombinant proteins were routinely purified from 1L cell culture.
570
Cells were incubated at 37°C until OD600 of 0.7-0.8 and were cooled on ice; isopropyl β-D-1-
571
thiogalactopyranoside (IPTG, Denville Scientific) was added to a final concentration of 0.5mM
572
and cell cultures were incubated overnight at 22°C (for OMR1 expression), 16°C (for the
573
expression of AHASL, AHASS1 and AHASS2) or room temperature (21-25°C, for IPMS1
574
expression). Cells were harvested by centrifugation the next morning and the cell pellets were
575
stored at -80°C until use.
576
His-tagged protein purification
577
All proteins in this study were purified with Ni-NTA agarose (QIAGEN) following the
578
manufacturer’s instructions. Purification steps were carried out at 4°C. Ni-NTA agarose was
579
equilibrated with the corresponding lysis buffer before binding to the proteins. To purify His-
580
tagged OMR1 proteins, the cell pellet was homogenized in 45mL lysis buffer A (pH=8.0, 50mM
581
potassium phosphate, 10mM imidazole, 300mM NaCl and 1mM Ile) and disrupted with a
582
sonicator (W225, Heat systems-Ultrasonics, Inc) using eight 30s pulses at 50% power. Cell
583
lysates were cooled on ice for 2 min between pulses. Insoluble cell debris was removed by a 15
584
min centrifugation at 12,500g at 4°C and the supernatant was incubated with Ni-NTA agarose
585
resin on a shaker at 4°C for ~1h. The protein-bond resin was then transferred to a Poly-Prep
586
chromatography column (2mL bed volume, 10mL reservoir, Bio-Rad) and washed with 30mL of
587
wash buffer (lysis buffer A containing 20mM imidazole). His-tagged OMR1 protein was eluted
588
with 3mL of elution buffer (lysis buffer A containing 200mM imidazole). Protein eluates were
589
desalted into 4mL buffer containing 50mM HEPES, pH=7.5, 1mM EDTA, 1mM DTT, 1mM Ile
590
and 10% (v/v) glycerol using an Econo-Pac 10 DG column (Bio-Rad). OMR1 proteins were
20
591
concentrated to ~20mg/mL with Amicon Ultra-15 10,000 NMWL centrifugal filters (Millipore),
592
aliquoted, snap frozen with liquid nitrogen and stored at -80°C. Protein concentration was
593
determined using the Bio-Rad protein assay system with bovine serum albumin (BSA, Sigma) as
594
a standard. For the purification of the feedback-resistant OMR1D enzymes, 5mM Ile was added
595
to the purification buffers.
596
Purification of His-tagged AHASL was carried out with 12mL lysis buffer B (50mM
597
Tris·HCl buffer, PH=7.5, 300mM NaCl, 5mM of MgCl2, 10µM flavin adenine dinucleotide
598
(FAD, Sigma) and 15% glycerol, v/v), 8mL wash buffer (lysis buffer B containing 30mM
599
imidazole but no glycerol) and 3mL elution buffer (buffer B with 200mM imidazole but no
600
glycerol). AHASL protein eluate was then desalted into 4mL desalting buffer (25mM potassium
601
phosphate, pH=7.5, 5mM MgCl2, 10µM FAD and 15% glycerol, v/v) and stored at -80°C.
602
AHASS1 and AHASS2 recombinant proteins were purified with 45mL lysis buffer,
603
30mL wash buffer, 3mL elution buffer and then desalted into 4mL desalting buffer. The
604
compositions of the buffers were the same as that used for AHASL except that FAD was not
605
added.
606
To purify His-tagged IPMS1, the cell pellet was homogenized with 12mL lysis buffer C
607
(50mM Tris·HCl buffer, pH=8.0, 300mM NaCl, 10mM imidazole, 10% glycerol, v/v and 10mM
608
of MgCl2), and resin-bound protein was washed with 8mL wash buffer (lysis buffer C with
609
30mM imidazole but no glycerol) and eluted with 3mL elution buffer (lysis buffer C with
610
250mM imidazole but no glycerol). IPMS1 proteins were desalted immediately into 4mL buffer
611
containing 50mM Tris·HCl (pH=8.0), 1mM MgCl2 and 10% glycerol (v/v). Due to the instability
612
of the proteins, IPMS1 enzyme assays were carried out immediately after protein purification.
613
Protein purification, desalting and determination procedures for AHASL, AHASS1,
614
AHASS2 and IPMS1 were the same as those for OMR1.
615
Enzyme assays
616
OMR1 enzyme activity assays were performed by monitoring the formation of 2-
617
oxobutanoate (Wessel et al., 2000; Niehaus et al., 2014). 100µl reaction mix containing 100mM
618
potassium phosphate buffer (pH=8.0), 40mM L-Thr (Sigma), 0.05µM enzyme and varying
619
concentrations of L-Ile (Sigma) (For testing Ile inhibition) or 100mM potassium phosphate
620
buffer (pH=8.0), 0.05µM enzyme and various concentrations of L-Thr (for kinetic measurements)
621
was incubated at room temperature and 2-oxobutanoate was measured by absorbance at 230nm
21
622
using a multilabel plate reader (2104 EnVision) at 20min. Pre-experiments indicated that the
623
enzyme reactions of wild-type and mutant OMR1 proteins were linear for the first 22 min under
624
these assay conditions. A no enzyme reaction was used as a control. Kinetic analysis was
625
performed with Prism (GraphPad Software).
626
AHAS activity was assayed using the method of Lee and Duggleby (Lee and Duggleby,
627
2001) with some modifications. 12nM catalytic subunit AHASL was incubated with ~300nM
628
AHASS1 or ~150nM AHASS2 at 30°C for 10min to reconstitute active enzyme. 200mM
629
potassium phosphate buffer (pH=7.5), 1mM thiamine diphosphate (ThDp), 10µM FAD and
630
10mM MgCl2 were added and the reaction mixture was incubated at 30°C for another 10 min to
631
allow co-factor binding. To measure the Km and catalytic parameters of the reconstituted
632
enzymes, various concentrations of sodium pyruvate were added to initiate the enzyme reaction.
633
BCAA inhibition of enzyme activity was assayed by simultaneously adding 200mM sodium
634
pyruvate and amino acid inhibitors of varying concentrations to initiate the enzyme reaction.
635
After incubating at 30°C for 30min, the reaction was quenched with 25µl 3M H2SO4, followed
636
by incubation at 60°C for 15min to convert acetolactate to acetoin. 200µl of freshly prepared 1:1
637
mix of 5% α-naphthol solution in 10M NaOH (Sigma) and 0.5% creatine (Chem Service) was
638
added and incubated at 60°C for another 15min to convert acetoin to a product with an
639
absorbance peak at 525nm. OD525 was measured with a spectrophotometer (Thermo Spectronic
640
Biomate 3) and kinetic data acquired by subtracting the OD525 of a no enzyme control. Product
641
production was determined with an acetoin (Sigma) standard curve.
642
IPMS1 activity was assayed with a previously described spectrophotometric end point
643
assay using 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB, Sigma) (de Kraker et al., 2007). All steps
644
of the assay followed the same procedures used by de Kraker and coworkers except for an
645
extended incubation time at 30 °C—20 min instead of 10 min.
646
qRT-PCR and transcript analysis
647
Total RNA was isolated from the aerial tissue of two-week-old wild-type Col-0 and the
648
mutants as described above. Aerial tissue was harvested individually from four plants (biological
649
replicates) of the wild-type and each mutant line. Reverse transcription was performed using M-
650
MLV reverse transcriptase (Invitrogen) following the manufacturer’s instructions. qRT-PCR was
651
performed on a 7500 Fast real-time PCR system (Applied Biosystems) with Fast SYBR Green
652
PCR master mix (Applied Biosystems). EIF4A1 was used as the endogenous control. Relative
22
653
changes in the transcript levels of the target genes in the mutants compared to wild-type plants
654
were evaluated using the 2-∆∆Ct method (Livak and Schmittgen, 2001). To compare the transcript
655
levels of AHASS1 and AHASS2 in two-week-old wild-type Col-0, absolute quantification was
656
carried out with the standard curve method. Primers used for qRT-PCR are listed in
657
Supplemental Table 9.
658
Transcript analysis was performed on ipms1-4, ipms1-5, ipms2-1 and ipms1-1D ipms2-1
659
with ACTIN2 as a control. Primers used to amplify IPMS1, IPMS2 and ACTIN2 transcripts are
660
summarized in Supplemental Table 9.
661
Public microarray data used in this study was adapted from AtGenExpress
662
(http://jsp.weigelworld.org/expviz/expviz.jsp) (Schmid et al., 2005).
663
Amino acid extraction and LC-MS/MS analysis
664
Wild-type Col-0 and mutants for amino acid profiling were grown at 22°C, 16/8
665
light/dark cycle (a mix of cool white fluorescent and incandescent bulbs was used; the irradiance
666
was 100 µmol m-2 s-1 photosynthetic photon flux density). omr1D, ahass2-1D, ipms1-1D, ahass1,
667
ahass2, ipms1, ipms2 single mutants and ipms1-1D ipms2 double mutants were planted in soil-
668
less mix (Redi-earth, Hummert). Aerial parts of the two-week-old seedlings (10-20 mg) or dry
669
seeds (5-7 mg) from six to eight individual plants (biological replicates) of wild-type Col-0 and
670
each mutant line were extracted and measured individually for free amino acid levels.
671
For amino acid profiling of the leaves of ahass2-1D/AHASS2;omr1D/OMR1, ipms1-
672
1D/IPMS1;omr1D/OMR1 and ahass2-1D/AHASS2;ipms1-1D/IPMS1 double heterozygous mutants,
673
crosses were made between the homozygous feedback-resistant single mutants and between
674
wild-type Col-0 and the homozygous single mutants. F1 seeds of the resulting single and double
675
mutants as well as wild-type Col-0 were sown on half-strength MS plates and aerial tissue of the
676
two-week-old seedlings was harvested for the amino acid assay.
677
To test free amino acid levels in ahass1/ahass1;ahass2-1D/AHAS2 double mutants, the
678
homozygous ahass2-1D mutant tfl106 was crossed with homozygous ahass1-1 and ahass1-2,
679
respectively. The resulting F1 plants were allowed to self-pollinate to produce F2 seeds. F2 seeds
680
from the two populations were sown on MS plates containing 0.2mM TFL and
681
ahass1/ahass1;ahass2-1D/AHASS2—homozygous for the T-DNA insertion on AHASS1 and
682
heterozygous for the feedback-resistant mutation on AHASS2—and ahass2-1D/AHASS2—has
683
heterozygous ahass2-1D and wild-type AHASS1—F2 individuals were selected from the resistant
23
684
plants via genotyping. These F2 plants were allowed to self-pollinate to generate F3 seeds. F3
685
seeds were sown on MS plates containing 0.2mM TFL and one-week-old resistant plants were
686
transplanted onto unsupplemented MS plates and grown for another week. A small portion of the
687
expanded leaf from each two-week-old F3 seedling was archived onto FTA PlantSaver cards (GE
688
Healthcare) for genotyping and the remaining aerial tissue was harvested and stored at -80°C.
689
The harvested F3 seedlings at -80°C with desired genotypes were analyzed for free amino acids.
690
Wild-type Col-0, ahass2-1D/AHASS2 F1, and homozygous ahass1 and ahass2-1D mutants were
691
planted, harvested and assayed using the same procedure, except that wild-type Col-0 and
692
homozygous ahass1 mutants were grown on MS plates without TFL before transplanting onto
693
unsupplemented MS plates. To be consistent, a small portion of the expanded leaf was removed
694
from all plants that were subjected to the amino acid assay.
695
Leaf and seed amino acid extractions for LC-MS/MS analysis followed a previously
696
documented method (Angelovici et al., 2013) with some modifications. The extraction buffer
697
was prepared with 12 isotope-labeled amino acid standards (Cambridge Isotope Laboratories,
698
Andover, MA); it contains 12µM Gly-D2-15N1, 8µM
699
Gln-U-13C5, L-Leu-D10, L-Lys:2HCl-U-13C615N2, L-His-D3, L-Met-D3, L-Phe-D8, Ser-D3, L-Trp-
700
D5, and L-Val-D8. 1,4-Dithiothreitol (DTT) (Roche) was added to the extraction buffer to a final
701
concentration of 19µM. Less than 10-20mg fresh leaf tissues or 5-7 mg dry seeds were disrupted
702
with a TissueLyser (QIAGEN). Dry seeds were homogenized in 400µl extraction buffer and
703
frozen leaf tissues were homogenized first and then suspended in 400µl extraction buffer. The
704
mixture was incubated at 90°C for 5min and then centrifuged at 3200g for 30 min; the
705
supernatant was transferred on to a 0.45-µm low binding hydrophilic polytetrafluoroethylene
706
filter plate (Millipore) and centrifuged at 2000g for 30 min. Flow through was collected and
707
stored at -80°C until analysis. A series of samples that contained the 12 heavy-labeled AA
708
standards at the same concentrations as that in the extraction buffer as well as the 20 standard
709
amino acids (Sigma) at various concentrations (from 0.1µM to 100µM) were prepared to
710
generate the calibration curve.
711
Leaf and seed extracts as well as the standard curve samples were analyzed by LC-MS/MS with
712
the published 5.6min, 3-function method with some modifications (Angelovici et al., 2013). In
713
addition to the 18 AAs Angelovici and coworkers measured, we analyzed Asn in seeds and both
714
Asn and Cys in seedlings.
24
DL-Ala-D4
and
DL-Asp-D3,
and 4µM of L-
715
Statistical analysis
716
Statistically significant groups in this study were determined by Student’s t-tests or ANOVA
717
analysis followed by multiple comparisons based on Duncan’s multiple range test (Duncan,
718
1955). See Supplemental File 3 for ANOVA Tables.
719
Accession numbers
720
GenBank/EMBL accession numbers for sequences used to build the AHASS and IPMS
721
phylogenetic trees are listed in Supplemental Figure 8 and 9, respectively. The AGI gene
722
identifiers for the genes used in this study are: At3g10050 (OMR1), At2g31810 (AHASS1),
723
At5g16290 (AHASS2), At3g48560 (AHASL), At1g18500 (IPMS1), At1g74040 (IPMS2),
724
At1g31230 (AK-HSDH I), At3g13920 (EIF4A1) and At3g18780 (ACTIN2).
725
726
Supplemental Data
727
Supplemental Figure 1. Transcript profiles of Arabidopsis IPMS1 and IPMS2 in different
728
tissues and developmental stages.
729
Supplemental Figure 2. Transcript analysis of ipms loss-of-function single and double mutants.
730
Supplemental Figure 3. Comparison of AHASS1 and AHASS2 transcript levels during
731
development.
732
Supplemental Figure 4. Transcript analysis of ahass loss-of-function single mutants.
733
Supplemental Figure 5. Individual and combined effects of BCAAs on the growth of wild-type
734
Col-0.
735
Supplemental Figure 6. The AHASS2-1D reconstituted enzyme is resistant to Val+Leu in vitro.
736
Supplemental Figure 7. The inhibition of wild-type Col-0 and omr1-12D by Val+Leu can be
737
reversed by Ile supplementation.
738
Supplemental Figure 8. Phylogenetic relationships among AHASS sequences from different
739
species.
740
Supplemental Figure 9. Phylogenetic relationships among IPMS sequences from different
741
species.
742
Supplemental Table 1. Summary of OMT- and TFL-resistant mutants.
743
Supplemental Table 2. Genetic linkage analysis of the OMT and TFL resistance traits.
744
Supplemental Table 3. OMR1 enzyme kinetic analysis.
25
745
Supplemental Table 4. Levels of free BCAAs in doubly heterozygous ahass2-
746
1D/AHASS2;omr1D/OMR1 and ipms1-1D/IPMS1;omr1D/OMR1 mutant seedlings.
747
Supplemental Table 5. Levels of free BCAAs in doubly heterozygous ahass2-
748
1D/AHASS2;ipms1-1D/IPMS1 mutant seedlings.
749
Supplemental Table 6. Levels of free BCAAs in ipms feedback-resistant and loss-of-function
750
single and double mutant seedlings.
751
Supplemental Table 7. Levels of free BCAAs in ahass1 and ahass2 loss-of-function single
752
mutant seedlings.
753
Supplemental Table 8. Levels of free BCAAs in ahass1/ahass1;ahass2-1D/AHASS2 double
754
mutant seedlings.
755
Supplemental Table 9. Primers used in this study.
756
Supplemental Table 10. ABRC stock numbers of the mutants.
757
Supplemental File 1. AHASS alignment used to construct the phylogenetic tree in Supplemental
758
Figure 8.
759
Supplemental File 2. IPMS alignment used to construct the phylogenetic tree in Supplemental
760
Figure 9.
761
Supplemental File 3. ANOVA Tables.
762
763
ACKNOWLEDGMENTS
764
We thank Dean DellaPenna for providing labeled amino acid standards, Ruthie Angelovici, Jan-
765
Willem de Kraker, Thomas Niehaus, Padmanabhan Kaillathe, members of the Yan Lu and Last
766
groups for helpful discussions and Kathryn Harmer for plant care and general lab work. LC-
767
MS/MS assays were performed in the MSU Mass Spectrometry and Metabolomics Core Facility.
768
This work was supported by the National Science Foundation (grants no. MCB-119778 and
769
MCB-124400).
770
771
AUTHOR CONTRIBUTIONS
772
A.X and R.L.L conceived the research and designed the experiments; A.X performed the
773
experiments and analyzed the data; A.X. and R.L.L wrote the article.
774
775
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950
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953
TABLES
Table 1. OMT- and TFL-resistant mutants selected for detailed characterization.
AA
# of mutants
# of seed Domain
Mutant ID Mutation
substitution carrying mutation
pools
affected
D
C to T
Pro519Leu
2
2
ACT2
omr1-11
omr1-12
D
C to T
Ala551Val
1
1
ACT2
D
G to A
Arg544His
1
1
ACT2
C to T
G to A
Ser349Phe
Gly606Glu
3
2
2
2
ACT2
C-term
omr1-13
D
ahass2-1
ipms1-1
954
955
956
957
D
Table 2. AHAS enzyme kinetic analysis.
32
Enzyme
Km
Vmax
Kcat
-1
33
-1
Kcat/Km
(mM)
(nmol s ) ×10
(s )
(s-1 mM-1)
AHASS2-wt
11.4 ± 1.0
16.7 ± 0.9
5.6 ± 0.3
0.49 ± 0.02
D
AHASS2-1
13.1 ± 0.6
19.1 ± 0.1**
6.4 ± 0.04**
0.48 ± 0.02
AHASS2-wt
8.9 ± 0.6
20.6 ± 0.3
6.9 ± 0.1
0.77 ± 0.05
D
AHASS2-1
7.9 ± 0.9
20.9 ± 1.0
7.0 ± 0.3
0.89 ± 0.07
AHASS2 variants were reconstituted with the large subunit AHASL and kinetic parameters
measured. Data are expressed as mean ± SD of three technical replicates (using the same
enzyme preparation). The asterisks indicate significant differences compared to the wild-type
AHASS2 enzyme (** P < 0.01, Student’s t-test). The results are from two experiments with
enzymes prepared on different dates.
958
959
3
960
Table 3. omr1D mutant seedlings accumulate increased free Ile.
Ile
Mutant
Ile
Val
Val Leu
a
Leu Thr
a
(nmol/mg fw) (FC ) (nmol/mg fw) (FC ) (nmol/mg fw)
Col-0
0.021 ± 0.002
0.13 ± 0.02
0.047 ± 0.01
143 0.20 ± 0.02* 1.5
0.18 ± 0.01**
D
3.0 ± 0.2**
D
0.88 ± 0.05** 42
0.12 ± 0.01
D
0.16 ± 0.02** 7.6
0.14 ± 0.01
omr1-11
omr1-12
omr1-13
Thr Val+Leu
a
Val/(Val+Leu) Leu/(Val+Leu)
a
(FC ) (nmol/mg fw) (FC ) (nmol/mg fw) (ratio×100)
1.6 ± 0.2
74 ± 1
26 ± 1
0.80 ± 0.05** 0.50 0.38 ± 0.02** 52 ± 3**
48 ± 3**
0.92 0.095 ± 0.003** 2.0
0.97 ± 0.1*
0.61 0.22 ± 0.01
55 ± 1**
45 ± 1**
1.1
1.3 ± 0.1
0.81 0.20 ± 0.01
71 ± 1
0.060 ± 0.01
3.8
0.18 ± 0.02
(ratio×100)
1.3
a
29 ± 1
D
FC = fold change compared to wild type. Levels of free BCAAs were analyzed in the two-week-old seedlings of omr1 mutants. Data
are expressed as mean ± SE of six to eight biological replicates (extracts from different individual plants). The asterisks indicate
significant differences compared to wild type (* P < 0.05, ** P < 0.01, Student’s t-test)
961
962
963
964
Table 4. ipms1-1D and ahass2-1D mutant seedlings have altered Val and Leu accumulation.
Ile
Ile Val
Val Leu
Leu Val+Leu
Val+Leu Val/(Val+Leu) Leu/(Val+Leu)
Category Mutant
a
a
a
a
(nmol/mg fw) (FC ) (nmol/mg fw) (FC ) (nmol/mg fw) (FC ) (nmol/mg fw) (FC )
(ratio×100)
(ratio×100)
0.019 ± 0.003
0.14 ± 0.02
tfl102
0.022 ± 0.001 1.2
0.058 ± 0.01** 0.41 0.13 ± 0.01** 2.5
0.19 ± 0.01
NC
tfl111
0.019 ± 0.001 NC
0.056 ± 0.002** 0.40 0.13 ± 0.01** 2.5
0.19 ± 0.01
tfl101
0.045 ± 0.01** 2.4
1.1 ± 0.1**
7.9
0.39 ± 0.04** 7.6
tfl106
0.066 ± 0.01** 3.5
1.1 ± 0.04**
7.9
0.35 ± 0.01** 6.9
Wild type Col-0
ipms1-1
D
D
ahass2-1
a
b
0.051 ± 0.01
b
0.19 ± 0.02
74 ± 1
26 ± 1
b
30 ± 2**
70 ± 2**
NC
b
30 ± 1**
70 ± 1**
1.5 ± 0.1**
7.9
73 ± 0.4
27 ± 0.4
1.4 ± 0.1**
7.4
75 ± 0.3
25 ± 0.3
FC = fold change compared to wild type. NC = No change compared to Col-0 wild type. Levels of free BCAAs in two-week-old
D
D
ipms1-1 and ahass2-1 mutants are represented as mean ± SE of six to eight biological replicates (extracts from different individual
plants). The asterisks indicate significant differences compared to Col-0 wild type (** P < 0.01, Student’s t-test). Wild-type Col-0 and
ipms1-1D mutant samples are the same as in Supplemental Table 6.
34
965
966
Table 5. Feedback-resistant mutations affect seed free BCAA levels.
Ile
Category Mutant
D
Val Leu
a
0.18 ± 0.005
69 ± 1
31 ± 1
0.54 ± 0.03
8.9
2.0 ± 0.2**
3.7
b
0.24 ± 0.01
0.71 ± 0.04
0.78 ± 0.04
0.93 ± 0.1** 3.9
2.4 ± 0.3**
3.4
3.0 ± 0.2**
3.8
69 ± 1
31 ± 1
0.27 ± 0.01
0.63 ± 0.1
0.89 0.83 ± 0.03
1.1
67 ± 2
33 ± 2
D
0.56 ± 0.03
D
0.77 ± 0.1** 1.4
0.60 ± 0.04
0.35 ± 0.03** 1.5
0.28 ± 0.02
0.93 ± 0.1*
0.81 ± 0.1
1.3
1.1 ± 0.1**
0.88 ± 0.05
1.4
69 ± 2
68 ± 1
31 ± 2
32 ± 1
1.4 ± 0.3**
5.0
2.5 ± 0.2**
3.1
4.8 ± 0.7**
5.5
72 ± 2
28 ± 2
1.1 ± 0.1** 3.9
0.31 ± 0.02
0.91 0.56 ± 0.04** 1.8
2.0 ± 0.1**
0.67 ± 0.1
1.0 ± 0.2
2.5
3.2
1.5
2.8 ± 0.2**
0.87 ± 0.05
1.1 ± 0.06*
1.3
62 ± 1**
64 ± 1
48 ± 1**
38 ± 1**
36 ± 1
52 ± 1**
1.4 ± 0.4
2.1
1.5 ± 0.04** 1.7
43 ± 1**
57 ± 1**
NC
tfl101
0.86 ± 0.1** 3.9
3.4 ± 0.4**
5.7
tfl106
Wild type Col-0
D tfl102
ipms1-1
tfl111
0.60 ± 0.05** 2.7
0.20 ± 0.01
0.31 ± 0.02** 1.6
1.7 ± 0.1**
0.56 ± 0.03
0.51 ± 0.02
2.8
0.44 ± 0.02** 2.2
0.65 ± 0.03
1.2
a
Val+Leu Val/(Val+Leu) Leu/(Val+Leu)
a
(ratio×100)
omr1-12 0.20 ± 0.01* 1.1
D
Thr Val+Leu
a
(ratio×100)
omr1-13 0.34 ± 0.04** 1.9
Wild type Col-0
0.22 ± 0.01
ahass2-1
Leu Thr
a
(nmol/mg dw) (FC ) (nmol/mg dw) (FC ) (nmol/mg dw) (FC ) (nmol/mg dw) (FC ) (nmol/mg dw) (FC )
omr1-11 1.6 ± 0.2**
omr1
Val
a
Wild type Col-0
D
Ile
1.1
0.85 ± 0.02** 2.7
b
D
FC = fold change compared to wild type. NC = No change compared to Col-0 wild type. Levels of free BCAAs in dry seeds of omr1 ,
D
D
ahass2-1 and ipms1-1 mutants are expressed as mean ± SE of six to eight biological replicates (extracts from different individual
plants). The asterisks indicate significant differences compared to Col-0 wild type (* P < 0.05, ** P < 0.01, Student’s t-test).
967
968$
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A Regulatory Hierarchy of the Arabidopsis Branched-chain Amino Acid Metabolic Network
Anqi Xing and Robert L. Last
Plant Cell; originally published online May 18, 2017;
DOI 10.1105/tpc.17.00186
This information is current as of June 17, 2017
Supplemental Data
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