“Beneficial” Metals and Microbes:
Interactions involving soybean and
other plants
PLAYERS at the University of Missouri-Columbia (Each with
links to other MU colleagues and other institutions)
 Dave Emerich
 David Mendoza-Cózatl
 Joe Polacco
 Gary Stacey
 Kristin Bilyeu
 Chung-Ho Lin
 Others: Basic and Applied, Row
Crops and Perennials, including Forestry
Missouri- Ninho de gente de ideas e da
creatividade:
Count Basie
Chuck Berry
Thomas Harte Benton
Sheryl Crowe
Miles Davis
Walt Disney
TS Eliot
Scot Joplin
Charley Parker
Brad Pitt
Mark Twain
Casey Stengel
Lorenzo Pietro (Yogi) Berra
Missouri- atividades ao ar livre
cavernas
ciclismo
rodeios
canoagem
Missouri- Estado Único dos Estados
Unidos
•
•
•
•
•
•
Border State (geographically/psychically)
“Mesopotamia” of the US
Industrial AND Agricultural
cavernas
rodeios
Rural AND Urban (STL, KC)
Southern Flavor
Gateway to Mid-West (Jeffersonian
Expansion)
ciclismo
canoagem
• Northern row crops; Southern forests
“Beneficial” Metals and Microbes:
Interactions involving soybean and
other plants
 Dave Emerich
 David Mendoza-Cózatl
 Joe Polacco
 Gary Stacey
 Kristin Bilyeu
 Chung-Ho Lin
 Others: Basic and Applied, Row
Crops and Perennials, including Forestry
Proteomics and Nodulation
Gary Stacey (Root Hairs: Responses to
Nod factors, ATP, mamps, stresses)
Dave Emerich (Subcompartments of
Infected Nodule Cells– developmental
and subcellular influences)
 Gary Stacey (Root Hairs: Responses to
Nod factors, ATP, mamps, stresses)
Soybean roots and root hairs
Root with root
hairs
Root stripped of
root hairs
Isolated root
hairs
Functional Genomics
of Soybean Root Hair
Infection by
Bradyrhizobium
japonicum
Project description and list of publications can be found at
soyroothair.org
Supported by the National Science Foundation, Plant Genome Program
Science (20 September, 2013) 341: 1384-1387
Nonlegumes Respond to Rhizobial Nod
Factors by Suppressing the Innate
Immune Response
Y Liang, Y Cao, K Tanaka, S Thibivilliers, J Wan, J-M Chang, J Qiu, Gary Stacey
Nodule initiation work that interfaces well with
studies by Dave Emerich on nodule sensescence
Though not part of the above study, this approach
is amenable to proteomics analysis
This just in:
“Identification of a Plant Receptor for Extracellular ATP"
Jeongmin Choi1, Kiwamu Tanaka1, Yangrong Cao1, Yue Qi2,
Jing Qiu2, Yan Liang1, Chang Ho Kang3, Gary Stacey1
1
Divisions of Biochemistry and Plant Sciences, University of
Missouri , Columbia, MO
2
Dept. of Statistics, University of Missouri, Columbia, MO
3
Plant Molecular Biology & Biotechnology Research Center
(PMBBRC), Gyeongsang National University, Bd.6-421, Jinjudaero,
Korea
SECOND SCIENCE ARTICLE IN SIX MONTHS
David W. Emerich (o meu hospedeiro)
Proteomics/Transcriptomics of Nodule
Compartments
 Dave Emerich: (Subcompartments of Infected Nodule Cells–
developmental and subcellular influences)
Infected Plant Cytoplasm
Symbiosome
Membrane
Symbiosome Space
Transcriptomics
Bacteroid
Proteomics
Periplasm
Bacteroid
Inner Membrane
Bacteroid
Outer Membrane
Symbiosome
The Periplasmic Space: The region between the plasma membrane
and the outer membrane of gram negative bacteria. It contains
enzymes and binding proteins for amino acids and sugars.
- ~ 20-40% of the entire
cell volume
- approximately 300 proteins
 Dave Emerich
Proteins Isolated from the B. japonicum
Bacteroid Periplasm
149 Proteins Annotated to
B. japonicum Genome
53 Proteins Annotated to
Plant Genomes
Majority of proteins annotated as unknown,
hypothetical or with tentatively assigned
functions
 Dave Emerich
David W. Emerich
Progression of
Bradyrhizobium japonicum bacteroids
into senescence
(também uma historia humana)
Approaches:
Proteomics
Transcriptomics
Genetics
Expression of nif Genes as a Function of Plant Age
200
nifH
180
Fold Expression
160
Last Day of
Measureable
Acetylene Reduction
Activity
140
120
100
nifD
nifN
80
nifK
60
nifB
40
20
nifW
0
30
40
50
nifE
60
70
Days After Planting
80
90
100
 Dave Emerich
Expression patterns of B. japonicum genes
with homology to Hemibiotroph signature genes
[Ralstonia solanacearum GMI1000, Xanthomonas campestris pv campestris, X. axonopodis
pv citri 306, Pseudomonas syringae pv tomato DC3000 and related strains]
3
Last Day of
Measureable
Acetylene Reduction
Fold Expression
2
1
0
30
-1
-2
-3
-4
50
70
90
Hemibiotroph : An organism
that is parasitic in living tissue
for some time and then
continues to live in dead tissue
Days After Planting
 Dave Emerich
Fold Increase in Selected Pili/Flagellin Genes
Bll 6871 Flagellar
P-ring protein
0
Bll 1436 Pilus
assembly protein
Fold Expression
-5
Bll1877 Flagellar
biosynthetic protein
Bll6862 Probable Flagellar
M-ring protein
-10
-15
Bll6865 Flagellin
-20
Last Day of Measureable Acetylene
Reduction Activity
-25
Bll6876 Flagellar basalbody rod protein
-30
30
50
70
90
110
Days After Planting
 Dave Emerich
 Kristin Bilyeu- Soybean seed composition; Ni and Pi
metabolism
Joe C. Polacco, DL Hyten,
Mônica Medeiros-Silva, DA Sleper
& Kristin D. Bilyeu .2011.
Mutational analysis of the major
soybean UreF paralogue involved in
urease activation
Journal of Experimental Botany, 62: 3599–3608.
Biochemistry Department, Interdisplinary Plant Group
Universidade Federal do Rio Grande do Sul (Bioquímica e Biofísica)
USDA-ARS, Plant Genetics Research Unit, Agronomy Dept., Interdisciplinary Plant
Group
 Joe Polacco
Plant Science 199–200 (2013) 79–90
Opinion –
Nickel and urease in plants:
Still many knowledge gaps
Joe C. Polacco, Paulo Mazzafera, Tiago Tezotto
Generation of NIL lines
• Urease-Positive and Urease-Null lines:
• What is the role of urease in N recycling, in
the senescent nodule, in defense?
 Joe Polacco
(working with Paulo Mazzafera, Tiago & Kristin Bilyeu)
Eu3: UreG, (Glyma08g08970.1
A
B
C
D
E
F
G
H
I
J
K
eu3-a
500 bp
AATCTAATTACTAGTAGACTAAAATATCTTTATTTAATTTCCTCCTATTTTATTTAATTAATTAAAATAAATTTGA
ACT(-50)….........(2,382)AATTGGATTTGTATATGTTGGGTTGCAGTAGCTTTGTCTCCGAGTCAATTTTATTGT
TTTGCTCTATTTTAT
2,383 – (-49) = 2,434 bp deletion
3,266 – 2,434 = 832 nt, expected size of PCR product
L
Generation of NIL lines
e1 x Williams
e1
BC1Fn
Rename, after complementation analysis
eu3-e1
BC1Fn
eu3-e1 x Williams82
BC1Fn
eu3-e1/Eu3
BC2F1
10 generations
single heterozygous
seed selfings (SSD)
eu3-a/eu3-a
BC2F11
and
Eu3/Eu3 NILS
BC2F11
Mol Gen Genet (1987) 209:432 Pleiotropic soybean mutants defective in both urease isozymes. L. Elise Meyer-Bothling,
Joseph C. Polacco, Silvia R. Cianzio
 Joe Polacco
Methylobacterium spp.
(Pink-pigmented facultative methylotrophs: PPFMs)
• Commensal soil and phytoplane bacteria
• A link to some of the pioneering work of
Joanna Doberreiner
• Stimulate germination
• Respond to nickel status of soybean.
tRNA Is the Source of Low-Level
trans-Zeatin Production in
Methylobacterium spp.
Koenig et al. J BACTERIOLOGY
184: 1832 (2002)
Germination of heat-treated soybean,
with and without PPFMs
 David Mendoza-Cózatl:
Ionome dynamics in Arabidopsis and the
transporters behind it
 David Mendoza-Cózatl:
Transition metals are extremely reactive (the good, the
bad, the ugly)
Essential
(Fe, Zn, Mn, Cu)
catalysis, redox centers, structure.
Non essential
(Cd, Hg, Ag)
properties similar to essential metals
Goal:
To understand how plants mobilize and accumulate metals
S2-
Fe2+
thiol
or imidazole
28
Which transporters mobilize metals (and other nutrients)
into seeds?
Companion cells
(phloem loading cells)
Transporters expressed in
companion cell are key to move
molecules into seeds.
29
Mendoza-Cozatl et al., 2011 Curr Opin Plant Biol
Ionomics, a high-throughput screening to
understand metal homeostasis
• Take advantage of the different Arabidopsis mutant collections.
(≈ 5,000 indep. lines have been analyzed www.ionomicshub.org)
• Compare the metal content in seeds using wild type as reference.
Ig # 200 (Seeds from A. thaliana, nramp1)
25
2,5
20
1
Z value
Z value
Ig # 3 (Seeds from Arabidopsis var ts-1)
15
10
-0,5
-3,5
0
-5
Ba Ca Cd Fe
K
Mg Mn Na
P
S
K
Mg Mn Na
P
S
Zn
-2
5
-5
Ba Ca Cd Fe
Zn
-6,5
30
Allocation of HM within the plant depends on
ligands and transporters
Fe in the xylem
Fe in the phloem
Citrate-Fe
NA-Fe
NA-Fe
X-Fe (?)
Cadmium in the xylem
Cadmium in the phloem
Histidine-Cd
Maybe citrate (?)
GS2-Cd
PC2-Cd
pH xylem sap ≈ 5.5
Diff from Fe
(HMA4)
pH phloem sap ≈ 7.4
Same as Fe
(IRT1)
31
Reviewed in Mendoza-Cozatl et al., 2011 Curr Opin Plant Biol
Phloem-expressed
transporters
Ribo-seq of phloem-loading cells was used to
identify phloem-specific transporters
SUC2
YSL3
YSL1
OPT3
gene1…n
Transporters (raw signal)
Mather A. Khan, Andrew Riga and Mendoza-Cozatl, unpublished
Organized in 96-well plates
for high throughput screening
 David Mendoza-Cózatl:
Phloem transporters individually
expressed in yeast.
Cd content in yeast
Normalized to
empty vector
1.2
1
0.8
0.6
0.4
0.2
0
-0.2
-0.4
F
1
2
3
4
C
5
6
7
8
9
10
A
11
12
-0.6
No Cd
+Cd
Galactose-inducible promoters have helped identify Ni transporters;
work of Tiago Tezotto
Leaf-to-leaf transport of GSH using radiotracer
experiments
[35S]-GSH was applied to one leaf
(source) and removed before
imaging the rest (sink).
Loaded and imaged in parallel
In collaboration with Jurisson lab, Chem Dept MU
Plants and Associated Microbes)
 Chung-Ho Lin
II. Symbiotic natural systems to remediate contaminated
soils and waters
• Pseudomonas/ADP in riparian buffer to remediate atrazine (to CO2 and
NH3).
•
Pseudomonas putida and Rhodococcus rhodochrous, as above to
remediate TNT and RDX.
•
Forest floor fungal species for remediation of TNT and benzene (in
progress).
•
Plants that stimulate the microbial/biochemical degradation of organic
pollutants, (herbicides, antibiotics, munitions, personal care products
[PPCPs]).
II. Biocatalysts for environmental, energy and biomedical
applications (bioremediation, biofuels, biofilters)
• Recombinant enzymes immobilized (e.g. on biochar and silica) for
industrial applications
• Bacillus thuringiensis spore-based surface biocatalysts (self-expressed
recombinant enzymes).
OBRIGADO
Cheguei ao Brasil, já seis anos atrás
Professor aposentado, mas ainda capaz
de curtir a ciência
e, sem pedir licença
Irritar a gente como obstinado rapaz
Mas acho que juntos algo aprendemos
E conseguir tecer os nossos destinos
e, como fico em Missouri
o espinhoso H. Pylori
Já não é pa’ meus colegas, tão elefantino
Contudo, que agoura somos afastados
Seguemos colegas mutuamente apreciados
e mantenho as lembranças
em forma de alianças
Com os gaúchos (carnívoros disparatados)
Apesar de morar nos Estados Unidos
As lembranças não ficam adormecidas
com sotaque bem gaúcho
preencho cada bucho
Com historias da selva e das garotas floridas*
*e, atendidas, protegidas, lánguidas, envolvidas, não-fugidas, agradecidas
(claro), vestidas (também claro)
Mas, sinceramente, que tenham todo sucesso
E que desculpem minha “poesia” em excesso
a sua acolhida calorosa
e hospitalidade generosa
Me dão saudade, pra vocês, sim, o confesso
Host Plant Infection, Nodule Formation and
Function
B. japonicum
Isoflavones and
Flavanoids
Attachment and
Root hair curling
Soybean Root
Post-Symbiotic Stage Nod Factor
Return to Soil
Bacteroids in
infected plant cell
1 mm
Nodule Formation
Infection thread
invasion
TS of soybean
root nodule
Infection thread development,
induced cortical cell division
 Dave Emerich