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Phosphate Deprivation-Induced Changes in Tomato are Mediated by an Interaction between
Brassinosteroid Signaling and Zinc
Gozde S. Demirer
1,2,*
, Donald J. Gibson
1,*
, Xiaoyan Yue
1,3
, Kelly Pan
1
, Eshel Elishav
4
, Hitaishi Khandal
4
, Guy
Horev
5
,
Danuše
Tarkowská
6
, Alex Cantó-Pastor
1
, Shuyao Kong
1
, Concepcion Manzano
1
, Julin
N.
Maloof
1
,
Sigal
Savaldi-Goldstein
4
, Siobhan
M. Brady
1, #
1
Department
of Plant
Bio
logy and
Genom
e Center, University of
California, Davis, Davis, CA
95616, USA
2
Department
of Chemical
Engineering, California Institut
e of
Technology, Pasadena, CA
91125, USA
3
Department
of Horticultur
e,
Zhejiang
University, Hangzhou, Zhejiang
310058, China
4
Faculty of
Bio
logy, Technion-Israel Institut
e of
Technology, Haifa 3200003, Israel
5
Lorey I. Lokey Interdisciplinar
y Center for Lif
e Scienc
es an
d Engineering
, Technion-Israel Institut
e of
Technology, Haif
a 3200003, Israel
6
Laborator
y of Growt
h Regulators
, Institut
e of Experimental Botan
y,
Czec
h Acad
emy
of Scienc
es and
Palacky Universit
y,
CZ
-78371 Olomouc, Czech Republic
*
Authors
have contributed equally
#
Corresponding
aut
hor:
sbrady@ucdavis.edu
Article acceptanc
e date: 04/28/2023
2
Supplementary Figures
Fig
.
S
1
Statistical analysis of Figure 1. A)
Figure 1 Panel A (primary root length).
B)
Figure 1 Panel B (total
lateral root length).
C)
Figure 1 Panel D (mature root hair length).
D)
Figure 1 Panel G (pNPP assay).
3
4
Fig
.
S
2
Phylogenies for At3g17790 and Solyc03g09810. A) Extended
At3g17790
phylogeny. Phylogenetic
trees generated using protein sequences of several plant species. B) Detailed
At3g17790
phylogeny from
green square with
At3g17790
highlighted in green. C) Extended
Solyc03g098810
phylogeny. Phylogenetic
trees generated using protein sequences of several plant species. D) Detailed
Solyc03g098810
phylogeny
from green square with
Solyc03g098810
highlighted in purple. AmTr:
Amborella trichopoda
, AT:
Arabidopsis thaliana
,
Asparagus:
Asparagus officinalis
, Azfi:
Azolla filiculoides
, Bol:
Brassica oleracea
,
Carub:
Capsella rubella
, CA:
Capsicum annuum
, Cc:
Coffea canephora
, Cp:
Cucurbita pepo
, DCAR:
Daucus
carota
, Gb:
Ginkgo biloba
, HanXRQ:
Helianthus annuus
, MD:
Malus
domestica
, Mapoly:
Marchantia
polymorpha
, Medtr:
Medicago truncatula
, Migut:
Mimulus guttatus
, GSMUA:
Musa acuminata
, OIT:
Nicotiana attenuata
, GWHPAAYW:
Nymphaea colorata
, LOC_Os:
Oryza sativa japonica
, Peaxi:
Petunia
axillaris
, Pp:
Physcomitrella patens
,
MA:
Picea abies
, Potri:
Populus trichocarpa
, Semoe:
Selaginella
moellendorffii
, Seita:
Setaria italica
, Solyc:
Solanum lycopersicum
, PGSC:
Solanum tuberosum
, Sobic:
Sorghum bicolor
, Thecc:
Theobroma cacao
, VIT:
Vitis vinifera
, Zm:
Zea mays
.
Numbers on par
titions
represent the
bootstrap values
;
bootstrap values less than
0.25
were collapsed
.
5
Fig
.
S
3
Statistical analysis of Figure 3. A) Figure 3 Panel A (Root P). B) Figure 3 Panel B (Shoot P). C) Figure
3 Panel C (Root Fe). D) Figure 3 Panel D (Shoot Fe). E) Figure 3 Panel E (Root Zn). F) Figure 3 Panel F (Shoot
Zn).
6
Fig.
S
4
ICP
-
MS results of Cd, Co, and Mn roots and shoots in P sufficiency or deficiency. A) Root and shoot
Cd B)
Root and shoot Co and C) Root and shoot Mn profile of M82 and Penn
in P
i
-
sufficient and P
i
-
limiting
conditions. N= 6 for all plots
.
Letters represent statistically significant differences as determined by a two
-
way ANOVA and a post
-
hoc Tukey test, p<0.01.
7
Fig
.
S
5
RNA
-
seq and qPCR data of Zn transporter expression levels in M82, Penn, and
Slbzr1a
-
D
in P
sufficient and limiting conditions. A) Expression of HMA4 in M82 and Penn under P
i
sufficient and deficient
conditions, obtained from RNA
-
seq and B) Expression of Z
IP4 in M82 and Penn under P
i
sufficient and
deficient conditions, obtained from RNA
-
seq. C) Expression of HMA4 in M82 and
Slbzr1a
-
D
under P
i
sufficient and deficient conditions, obtained via qPCR and D) Expression of ZIP4 in M82 and
Slbzr1a
-
D
under P
i
suff
icient and deficient conditions, obtained via qPCR. N= 3 for all plots
.
Letters represent
statistically significant differences as determined by a two
-
way ANOVA and a post
-
hoc Tukey test, p<0.05.
8
Fig
.
S
6
Statistical analysis of Fig 4. A) Primary root responses of M82 (left) and
S. pennellii
(right) B) Total
lateral root length responses of M82 (left) and
S. pennellii
(right) C) Mature root hair length responses of
M82 (left) and
S. pennellii
(right) in P
i
sufficiency or deficiency when Zn is absent or present.
9
Fig
.
S
7
Statistical analysis of BR levels in M82 and
S. pennellii
(Figure 5C). P
-
values are obtained from a two
-
way
ANOVA analysis. BL is highlighted as one of my main types of BRs.
The samples were clustered as follows: z
-
scores were calculated for each BR level, Euclidean distances between samples were calculated and
h
ierarchical clustering was performed using the complete method. Hypothesis testing: Two
-
way ANOVA was
performed for each BR with P
i
and Species as main factors and their interaction. For each BR with significant
effect, post hoc comparisons were performed
using Tukey’s HSD test.
Fig. 5A,B For each species one way
analysis of variance was performed with hormone
concentration as the independent variable. When significant
the ANOVA was followed by Tukey’s HSD test to compare between the different concentration pairs.
Fig 5 D,E
-
For M82, two independent experiments were analyzed. Thus, to determine the effect of P
i
and BRZ on LR,
mixed model analysis of variance (ANOVA) was performed with P, BRZ and their interaction as fixed factors
and the batch as random factor. Post
-
hoc analysis was performed using pairwise differences of LS
-
mean as
implemented in the lmerTest R
package. P
-
values are based on the t
-
distribution using degrees of freedom
according to Satterthwaites method.
P
-
values that passed correction for multiple comparisons using the False
Discovery Rate procedure with alpha<0 are indicated by letters.
For Pen
n
t
wo
-
way ANOVA was performed with
P
i
and BRZ treatments as main factors and their interaction.
10
Fig
.
S
8
Statistical analysis of Figure 5 for A) M82 and Penn
primary root length measurement upon BL and
B) BRZ
treatment. C) LR number and D) LR length of mock and BRZ
-
treated (3 μM) M82 and Penn plants.
11
Fig
.
S
9
BZR1/BES1 phylogeny in tomato. A)
Solyc12g089040
and
Solyc04g079980
genes were mutated in
tomato hairy root lines. B) qPCR validation of overexpressio
n of
Solyc12g089040
and
Solyc04g079980
in
mutant hairy root lines. Soly12
-
8 and Soly12
-
10 lines showing promising results for
Solyc12g089040,
and
Soly4
-
1 and Soly4
-
5 showing promising results for
Solyc04g079980.
C) Soly12
-
10 and Soly4
-
5 lines were
used to
check expression of downstream BZR1 target genes using qPCR, and stable mutants of these lines
were generated.
Numbers on partitions represent the
bootstrap values
;
bootstrap values less than
2
0
%
were collapsed
.
12
Fig
.
S
1
0
Statistical analysis of Figure 6. A) Figure 6 Panel A (hypocotyl length). B) Figure 6 Panel C (primary
root length). C) Figure 6 Panel D (total lateral root length). D) Figure 6 Panel E (mature root hair length).
E) Figure 6 Panel F (BCIP staining intensit
y).
13
Fig.
S
1
1
BRZ treatment of second
Slbzr1a
-
D
independent line and
Slbzr1b
-
D
lines. A) Hypocotyl length of
M82 WT and
Slbzr1a
-
D
line a10 upon BRZ treatment grown in dark for 7 days. B) Statistical analysis results
of Panel A. C) Hypocotyl length of M82 WT and
Slbzr1b
-
D
lines b22 and b6 upon BRZ treatment grown in
dark for 7 days. D) Statistical analysis
results of Panel C.