Database for soybean transporter genes

1. Soybean Transporter Database (SoyTD): Genome-
Wide Identification and Exploration of Natural
Variants in Soybean Transporter Genes
Gunvant Patil
(Henry Nguyen Lab)
National Center for Soybean Biotechnology &
Division of Plant Sciences
University of Missouri, Columbia
January 12, 2016

2. Outline
o Introduction
o Methods
o Results
o Genome-wide comparison
o Classification of Soybean transporters
o Insights through – Transcriptome profiling and Whole genome resequencing
o Example: SWEET and SALT transporters
o Database design
o Summary

3. Transport proteins (transporter) are membrane channels/pumps and facilitate
exchange of selective molecules, ions from the external and internal environment.
I n t r o d u c t i o n
Importance
- Transporter are selective barrier and exchange wide variety of molecules and
ions from external or internal environments.
- Nutrient uptake and transport to sink tissue.
- Pump/exclude toxic ions/metals (salt/boron).
- Signal transduction.
- Maintain osmotic balance in different cell types (aquaporin).

4. ATP
Active
Transport
Extracellular space
Intracellular space
Selective
Channels Carriers
Uniport Symport Antiport
Secondary Active TransportFacilitated Diffusion
Facilitated diffusion
Passive transport is powered by the potential
energy of a concentration gradient and does
not require the expenditure of metabolic
energy
Primary active transport
Energy derived from hydrolysis of ATP to
ADP liberating energy from high energy
phosphate bond
Secondary active transport (Co-transporters & Exchange)
No direct coupling of ATP; instead, the electrochemical
potential difference created by pumping ions out of the cell is
used.
Channels
Transport water or specific ions by conc.
Gradients.
Open in response to signal
Milton Saier et al. (http://tcdb.ucsd.edu/tcdb/)
Classed based on driving
energy
o Active Transport (Primary
and Secondary)
o Channel (passive diffusion)
o Carrier-type facilitators
(facilitated diffusion)

5. ATP
Active
Transport
Extracellular space
Intracellular space
Selective
Channels Carriers
Uniport Symport Antiport
Secondary Active TransportFacilitated Diffusion
Facilitated diffusion
Passive transport is powered by the potential
energy of a concentration gradient and does
not require the expenditure of metabolic
energy
Primary active transport
Energy derived from hydrolysis of ATP to
ADP liberating energy from high energy
phosphate bond
Secondary active transport (Co-transporters & Exchange)
No direct coupling of ATP; instead, the electrochemical
potential difference created by pumping ions out of the cell is
used.
Channels
Transport water or specific ions by conc.
Gradients.
Open in response to signal
Milton Saier et al. (http://tcdb.ucsd.edu/tcdb/)
Classed based on driving
energy
o Active Transport (Primary
and Secondary)
o Channel (passive diffusion)
o Carrier-type facilitators
(facilitated diffusion)
Typical transmembrane
protein
Binding Domain

6. Transporter gene resources and database
TCDB (Transporter Classification) Saier et. al. 2014 http://www.tcdb.org/
TransportDB (Relational database) Ren et. al. 2007 http://www.membranetransport.org/
Human Genome wide identification Ye et. al. 2014 http://htd.cbi.pku.edu.cn/
Arabidopsis Genome wide identification Schwacke et. al. 2003 http://aramemnon.botanik.uni-koeln.de/
Medicago Genome wide identification Miao et. al. 2012 http://bioinformatics.cau.edu.cn/MtTransporter/
TOPCON (membrane topology prediction) Tsirigos et. al. 2015 http://topcons.cbr.su.se/
TMHMM (membrane topology prediction) Krogh et. al. 2001 http://www.cbs.dtu.dk/services/TMHMM/
Publically available Resources

7. TCDB
database
G.max
protein
TransporterDB
Mapped Proteins
TMHMM TOPCONs
Data integration and filtering
(>2 TMD)
Classification
(TCDB)
Pfam
annotation
3306 SoyTD
Sequence
Variants (SNPs)
Transcriptome
analysis
blastp blastp
NGS workflow
e.g. Salinity tolerance in soybean e.g. Soybean SWEET gene family
Workflow
Will be available @
www.soykb.org/SoyTD

8. Comparative analysis of primary transporters in 47 plant species
~ 7-12% genes code for
transporter protein in different
plant species

9. Relationship between total genes and transporter gene numbers (>2).
Estimation of all transporter (min_2) in plant genome (47
species). Best-fit linear regression (r2 = 0.89)
y = 0.0831x + 563.65
R² = 0.8948
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
0 20000 40000 60000 80000 100000
T. aestivum
G.max
Arabidopsis
G.max
No. of genes in genome
Primarytransporters

10. GO/InterPro Description Total L.ja M.tr G.ma M.do F.ve M.es R.co P.tr A.th A.ly C.pa T.ca V.vi O.sa
O.Sa
indica
B.di S.bi Z.ma Smo P.pa O.lu O.ta M.rcc V.ca C.re
IPR014047 Chromate transporter 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1
IPR011866
Sulphate ABC transporter,
permease protein
2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1
GO:0046715
Boron transmembrane
transporter activity
58 2 3 4 5 2 2 2 3 6 6 2 2 2 3 4 3 2 5 0 0 0 0 0 0 0
IPR004648
Tetrapeptide transporter,
OPT1/isp4
163 4 12 11 13 8 9 7 12 9 9 6 10 10 7 8 6 8 6 6 2 0 0 0 0 0
GO:0005385
zinc ion transmembrane
transporter activity
267 11 11 21 24 13 12 9 15 22 25 10 14 18 11 12 11 11 10 4 3 0 0 0 0 0
IPR015521
MATE family transporter
related protein
311 27 13 28 10 11 10 16 22 16 17 9 17 18 15 17 15 16 11 15 8 0 0 0 0 0
IPR015606
Cationic amino acid
transporter
252 19 7 19 21 9 15 13 21 9 12 8 8 11 12 11 11 14 11 7 6 1 1 2 2 2
GO:0008271
secondary active sulfate
transmembrane
transporter
257 7 9 27 19 12 16 12 16 12 12 10 10 28 11 14 10 11 9 4 4 0 0 1 1 2
IPR003663 Sugar/inositol transporter 1210 56 64 99 110 55 67 52 70 53 54 42 50 48 65 69 57 63 67 32 20 6 2 6 2 1
IPR000109 Oligopeptide transporter 1484 87 77 125 140 62 73 55 82 53 55 56 62 53 85 99 79 89 78 49 20 1 1 1 1 1
Loss
Gain
Expansion
Lineage specific gain and loss of transporter domains
Dicot Monocot Moss Algae

11. • 3306 (2-TMD) genes in the soybean genome code for transporters or transporter-related
proteins.
• Average 164 transporter gene on 20 chromosomes.
• Transporter genes were classified according to Transporter Classification (TC) system (Saier et. al.
2014)
Major Classes (According to TC system)
• 1:Channels/Pores > Facilitate diffusion (energy independent)
• 2:Electrochemical Potential-driven > Uniporter, symporters and antiporters
• 3:Primary Active Transporters > Energy dependent/drive against concentrations
• 4:Group Translocators > Substrate modified during the transport process
• 5: Transmembrane Electron Carriers > Transport electron flow
• 8: Accessary Factor > Complexes with known transport proteins
• 9: Incompletely Characterized > Unknown classification
• Unclassified in TCDB
Soybean Transporter

12. 693
1709
427
30 47
11
99
290
0
200
400
600
800
1000
1200
1400
1600
1800
1:
Channels/Pores
2:
Electrochemical
Potential-driven
Transporters
3: Primary
Active
Transporters
4:Group
Translocators
5:
Transmembrane
Electron
Carriers
8: Accessary
Factor
9: Incompletely
Characterized
Unclassified in
TCDB
Soybean Transporter classification (TCDB, IUBMB)
Soybean Transporters classification

13. 685
425
0
100
200
300
400
500
600
700
800
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Mechanosensitive: Involved in
osmotic stress response
(Hamilton et. al. 2015, Annu.
Rev. Plant Biol.)0 50 100 150 200 250 300 350 400 450
Mechanosensitive Calcium Channel
Metabolite Transporter (DMT)
Major Facilitator (MFS)
ABC Transporter
Amino Acid/Auxin Permease (AAA)
Proton-Dependent Oligopeptide Transporter (POT)
Oligosaccharidyl-Lipid/Polysaccharide (MATE)
P-ATPase
Aquaporins
Voltage-Gated Ion Channel (VIC)
Monovalent Cation:Proton Antiporter-2 (CPA2)
Mitochondrial Carrier (MC)
Sweet Pq-Loop Saliva MtN3 (SWEET)
Auxin Efflux Carrier (AFC)
Sulfate Permease (Sulp)
Soy Transporter
Enrichment:
Top 15 families
Classification based on No. of TMD
No.ofGene
No. of Gene
0
50
100
150
200
250
300
Water Nitrogen Metal Ion Sugar Amino acid Hormones
Classification based on solute transport
No.ofGene

14. Flower
Y_Leaf
1cm_Pd
Pd_shell_10d
Pd_shell_14d
Seed_10
Seed_14
Seed_21
Seed_25
Seed_28
Seed_42
Root
Nodule
Root Specific
Enrichment
Stress responsive, Aquaporin, ABC and AA transporters
Expression profiling of transporters in soybean tissues
 Overall, majority of transporters were expressed in flower, roots,
followed by pod shell and seed tissues.
 339 transporters expressed constitutively in all 14 tissues.
 Highly abundant transporters (top 100) were enriched for intrinsic
proteins (MIPs) and metabolite transporters.
Flower Specific
Enrichment
PQ loop (SWEET), MIFs and metabolite transporters
Nodule
Specific Enrichment
Ammonium transporters, NRT family

15. Example:
Transporter genes
and trait discovery
1. Soybean sugar effluxer
2. Soybean Na+ transporter
3. Maize and wheat boron transporter

16. 1. Soybean SWEET effluxers proteins
• Metabolite transport influences photosynthetic productivity and plant vigor by controlling source/sink relationships
and biomass partitioning.
• SWEETs are uniporter and facilitate diffusion of sucrose across cell membrane
• Involved in sugar efflux, phloem loading, plant-pathogen interaction and reproductive tissue development
Patil et al. 2015 BMC Genomics
Soybean SWEETs are highly expressed during seed
development
FPKM
RNAseq data1 RNAseq data2

17. 2. Soybean salinity tolerance associated with Na+ transporter gene
• GmCHX1 (Glyma03g32900) encodes Na+/H+ exchange protein
• GmCHX1 was identified as the causal gene in the salt tolerant locus (Qi et al 2014 Nat Comm)
GmCHX1 express in roots
and has mechanism to
limits salt transport in
shoot
Analysis of 106
Soybean Lines
Patil et al. 2016 Scientific Reports (In press)

18. GWAS confirmed GmCHX1 as major transporter gene associated with salinity tolerance
5 M SNP (after filtering)
Of 106 lines were used
for GWAS
Patil et al. 2016 Scientific Reports (In press)
Mut (Tolerant)
WT (Sensitive)
Qietal2014
HTP maker development
Str variant identification

19. 3. Boron transporter domain specific to higher plants
• BORON: involve in cross-linking the pectic in primary cell walls, gives physical strength (Miwa 2010 Ann Botany).
• High level are toxic to plant growth.
Pallotta 2014 NatureMach 2014 Plant Cell
Mutant in maize : shows reduced tassel
development and defects in inflorescence.
Natural variants in wheat B transporter (Bot-B5b allele) associated
with B toxicity.

20. Database Design

21. www.Soykb.org/soyTD

22. Browse
Solute
Transporter
card

23. Transporter Gene Card
TOPCONs Prediction

24. Transporter Gene Card….
Synonymous
Non_Synonymous

25. Chromosome Distribution

26. Search

27. Blast against transporter proteins
Blast Search

28. Genomic variation data of 106 lines
• SNP (Syn/non-syn)
• Indels
• CNV/PAVs
• Large effect SNPs (STOP/non-STOP)
• Soja specific transporters
Auxin Efflux Amino acid Transport
Line PI
Gm09_22030189_Glyma.
09G251600_+_373:0
Gm09_45198657_Glyma.09G
050600_+_438:2
HN001
PI518664
(Hutcheson)
CAG<->CAG;Q<->Q TAC<->TAC;Y<->Y
HN002
PI548402
(Peking)
CAG<->CAG;Q<->Q TAC<->TAG;Y<->U
HN003 PI089772 CAG<->TAG;Q<->U TAC<->TAC;Y<->Y
HN004 PI090763 CAG<->TAG;Q<->U TAC<->TAC;Y<->Y
HN005 PI404166 CAG<->CAG;Q<->Q TAC<->TAG;Y<->U
HN006 PI407788A CAG<->CAG;Q<->Q TAC<->TAC;Y<->Y
HN007 PI424298 CAG<->CAG;Q<->Q TAC<->TAC;Y<->Y
HN008 PI437655 CAG<->CAG;Q<->Q TAC<->TAC;Y<->Y
HN001 HN002 HN003 HN004 HN005 HN006 HN007 HN008 HN009
HN001 HN002 HN003 HN004 HN005 HN006 HN007 HN008 HN009 HN010 HN011 HN012 HN013 HN014 HN015 HN016
HN017 HN018 HN019 HN020 HN021 HN022 HN023 HN024 HN025 HN026 HN027 HN028 HN029 HN030 HN031 HN032
HN033 HN034 HN035 HN036 HN037 HN038 HN039 HN040 HN041 HN042 HN043 HN044 HN045 HN046 HN047 HN048
HN049 HN050 HN051 HN052 HN053 HN054 HN055 HN056 HN057 HN058 HN059 HN060 HN061 HN062 HN063 HN064
HN065 HN066 HN067 HN068 HN069 HN070 HN071 HN072 HN073 HN074 HN075 HN076 HN077 HN078 HN079 HN080
HN081 HN082 HN083 HN084 HN085 HN086 HN087 HN088 HN089 HN090 HN091 HN092 HN093 HN094 HN095 HN096
HN097 HN098 HN099 HN100 HN101 HN102 HN103 HN104 HN105 HN106 HN107 HN108

29. Summary
• Comparative analysis of 47 plant species showed presence of 8 – 12% transporter genes per
genome.
• Genome size (duplication/ploidy) positively correlated with number of transporter genes.
• Soybean contains 3306 (>2 TMD) transporter genes and majority of soybean transporter encodes
for ‘Electrochemical Potential-driven’ (Uniporter, symporters and antiporters).
• Based on transcriptome profiling, majority of transporter expressed in flower and root tissues.
• Highly abundant transporters were enriched for intrinsic proteins (MIPs) and metabolite
transporters.
• Total 23541 SNPs, 7207 indels and 489 large effect SNPs were identified in transporter gens
across 106 soybean genomes.
• Knowledge-based web resource that integrates information of soybean transporters has been
developed.

30. Acknowledgements
Nguyen Lab (MU)
Babu Valliyodan
Rupesh Deshmukh
Bioinformatics team @ MU
Trupti Joshi
Yang Liu
Shuai Zeng
Dong Xu
ICRISAT, India
Gaurav Agrawal
Rajeev Varshney
D i v i s i o n o f P l a n t S c i e n c e s
Thank you !