Comparative Analysis of Root Proteome in Soybean Genotypes with Contrasting Exudation

Potential under Phosphorus Starvation

Vengavasi Krishnapriya and Renu Pandey*
Division of Plant Physiology, ICAR-Indian Agricultural Research Institute, New Delhi -110012, India
(krishnapriya19@gmail.com, renu.pandey.iari@gmail.com)

pH 4 pH 7

INTRODUCTION
pH 4 pH 7
245 kDa 245 kDa A Carbohydrate
180 kDa 180 kDa metabolic process,
135 kDa Nucleotide binding,
135 kDa 15
21
100 kDa 100 kDa 4 43 11 Catabolic process,

| Root exudation of carbon compounds is one of the

75 kDa 10 75 kDa 13
10
40
11 31
7 22 63 kDa
6
Nucleobase-
63 kDa 29 36 8 9 22
containing
11 30

31
1 16
47 40 4 compound metabolic Cytoplasmic part, 9
21 17 18 50 446 9

mechanisms in plants to adapt to low phosphorus (P) availability

48 kDa 5
process, 22
48 kDa 4
24
27
5

12 30 7 49
28 5 14
39 23
10
Membrane, 8
18
35 kDa 2
35 kDa 27 2

Low-molecular weight exudates such as carboxylic acids,

43
1 3 3

|
32 26

24 Protein metabolic
8 35 20 process, 10
45 Hydrolase activity,
28
13

phenols, free amino acids and sugars mobilise P from inorganic

25 kDa 9 25 kDa 34 15
48

16 13
20
Intracellular
20 kDa 20 kDa membrane-bounded
10
Kinase activity, 8 organelle, 8

sources, while high-molecular weight secretory proteins 17 kDa 17 kDa

2

Biosynthetic
Nucleic acid binding,
7

Single-organism

hydrolyse P from organic pools (Raghothama and Karthikeyan,

35 process, 29 process, 7
34 26 21

11 kDa EC-232019 11 kDa EC-232019 Protein binding, 9

Sufficient P Low P Generation of
precursor Intracellular non-

2005; Neumann and Romheld, 2012)

metabolites and membrane-bounded
245 kDa pH 4 pH 7
245 kDa pH 4 pH 7 energy, 11 organelle, 6
180 kDa 180 kDa
135 kDa 135 kDa

Research efforts till date have led to identification and

100 kDa

|
100 kDa
Nucleolus, 1
75 kDa
9
75 kDa 4 B Cell wall, 1
39
8
11 Cell, 1
63 kDa 4137 33 63 kDa

functional characterisation of efflux transporters for malate and

16
22
25
6
7
22 Plasma Nucleus, 1 Membrane, 7
15 42
30 38 32 1
47 6
9
membrane, 1
3
38
48 kDa 44
19 6
48 kDa 5 Nucleoplasm, 1
5
29
7

citrate (Ryan et al., 2011; Liang et al., 2013)

49
14
19
43 Ribosome, 2
35 kDa 1 35 kDa 1
46 3

36
25 37

Search for the missing players regulating synthesis and efflux of

8
35
Extracellular

|
45 42
12 region, 2 Mitochondrion, 5
25 kDa 25 kDa 10 34

other root-exuded compounds may provide exciting

20 kDa 33
Cytosol, 2
20 kDa

opportunities in enhancing P acquisition efficiency

17 kDa 17 kDa Plastid, 3
Cytoskeleton, 4
41

Cytoplasm, 3

This study aims to explore the physiological and molecular

11 kDa 11 kDa Intracellular, 4

|
EC-113396 EC-113396
Sufficient P Low P

aspects of root exudation in response to low P stress in soybean Fig. 4. 2-DE gels of root proteome in soybean genotypes grown at
sufficient (250 μM) and low (4 μM) P. Differential proteins are
Fig. 6. Functional annotation of differentially
expressed proteins based on (A) role in
denoted in green (down-regulated at low P), red (up-regulated at biological process and (B) cellular localisation
low P) and blue (expressed only at low P)

MATERIALS AND METHODS EC-232019 EC-113396

Fig. 5. Number of differentially expressed proteins on

| Soybean genotypes: EC-232019 & EC-113396 (with contrasting 27 (9) 7 (1) 16 (4)
2-DE gels at low (4 μM) compared to sufficient
(250 μM) P. In green: down-regulated at low P,
root exudation potential, identified from 116 diverse genotypes) 11 (4)
red: up-regulated and blue: expressed only at low P.
| P levels in hydroponic culture 24 (13) 6 (4) 14 (7) Numbers within parenthesis correspond to proteins with
significant MASCOT score.
{ Sufficient P : 250 µM
{ Low P : 4 µM | Staining of 2-DE gels with Coomassie Brilliant Blue revealed ≈325
| P levels in soil culture spots in the root proteome of soybean
{ Sufficient P : 4 mg Olsen P kg-1 soil + SSP at the rate of 25 mg P kg-1 soil | Out of these, 105 (32%) were differentially expressed between
{ Low P : 4 mg Olsen P kg-1 soil sufficient and low P levels (Fig. 4)
| Root exudates were quantified by HPLC (carboxylates) and | A total of 44 proteins were down-regulated by more than two-
spectrophotometry (proteins, phenols, sugars, free amino acids) fold under low P condition, while 50 proteins were up-regulated
| Root morphology was analysed in Root scanner with WinRhizo by more than two-fold and 11 proteins were expressed only at
| Two-dimensional electrophoresis (2-DE) was carried out on the low P (Fig. 5)
total root proteome, followed by gel image analysis (PDQuest), | Proteins down-regulated at low P include
{ Isocitrate dehydrogenase, methionine synthase, glutathione-S-transferase,
in-gel trypsin digestion of differential spots, MALDI-LC MS/MS and glutamine synthase, chalcone isomerase, enolase
MASCOT search for protein identification | Proteins up-regulated/expressed only at low P include
{ Malate dehydrogenase, isoflavone reductase, heat shock-related protein,
adenosine kinase, argininosuccinate lyase, small Arf-related GTPase, nucleoside
RESULTS diphosphate kinase, cysteine synthase, patellin-5
| Differential proteins represented various functional categories
400
A 240 B 12
C 6 D 60 E Sufficient P
including carbohydrate, protein and energy metabolism,
Free amino acids ( µ M g-1 RFW)
Carboxylates (µ M g-1 RFW)

Low P

biosynthetic and catabolic processes, signal transduction and

180
Proteins (µ M g-1 RFW)

Phenols (µ M g-1 RFW)

Sugars (µ M g-1 RFW)

300 9 45
120 4

200
60
6
30
cell cycle regulation (Fig. 6A), and were localised to different
100
8
3
2
8
cellular components (Fig. 6B)
| 14 proteins not characterised or function unknown
4 4

0 0 0 0 0
EC-232019 EC-113396 EC-232019 EC-113396 EC-232019 EC-113396 EC-232019 EC-113396 EC-232019 EC-113396
| RT-qPCR of 42 genes encoding differential proteins revealed that
Fig. 1. Genotypic variation in root-exuded (A) carboxylates, (B) proteins, (C) phenols, (D) sugars and (E) free amino acids of 34 genes displayed trends at the transcript level consistent to
soybean grown at sufficient (250 µM) and low (4 µM) P
Sufficient P
that observed in the proteome profile
A B

| Low P increased efflux of carboxylates,

Low P
500 50
Root surface area (cm 2 )

400 40

proteins and phenols in EC-232019,

Root length (cm)

300 30

CONCLUSIONS
200 20
while sugar and amino acid exudation
100 10 | Higher P acquisition and seed yield in EC-232019 under low soil P
0 0 reduced in both the genotypes (Fig. 1) availability might be attributed to greater efflux of carboxylates,
| Averaged over P, EC-232019 exuded
0.5 C 2500 D

0.4 2000
proteins and phenols, as well as a favourable root morphology
Number of root tips
Root volume (cm 3 )

more carbon compounds compared

| P starvation regulates gene expression at the transcript- and
0.3 1500

to EC-113396, hence the former might

0.2 1000

0.1 500
protein-level, in turn influencing metabolism and the differential
be more efficient in P acquisition
responses of contrasting soybean genotypes to low P stress
0.0 0
EC-232019 EC-113396 EC-232019 EC-113396

Fig. 2. Genotypic variation in (A) root length, Sufficient P

A B
(B) root surface area, (C) root volume and
Photosynthesis ( µ mol CO2 m-2 s-1)

900 25 Low P
(D) number of root tips of soybean grown at
REFERENCES
Le af are a (cm2 plant -1)

20
sufficient (250 µM) and low (4 µM) P 600
15

| At low P, root length, surface area and 10 Liang C, Pineros MA, Tian J, Yao Z, Sun L, Liu J, Shaff J, Coluccio A, Kochian
LV and Liao H (2013) Low pH, aluminium, and phosphorus coordinately
300

volume was approximately 30% higher 5

regulate malate exudation through GmALMT1 to improve soybean

in EC-232019 than EC-113396, but more
0 0

adaptation to acid soils. Plant Physiology. 161: 1347-1361.

18 C 1.5 D
Total biomass (g plant -1)

root tips noted in EC-113396 (Fig. 2)

15

Neumann G and Romheld V (2012) Rhizosphere chemistry in relation to

Root-to-shoot ratio

12 1.0

| Low soil P availability reduced leaf area 9

plant nutrition. In Marschner’s Mineral Nutrition of Higher Plants
(33%), photosynthesis (40%) & biomass
6

3
0.5
(ed. P Marschner) pp: 347-368.
Ryan PR, Tyerman SD, Sasaki T, Furuichi T, Yamamoto Y, Zhang WH and
(28%) in both genotypes, but per cent
0 0.0

Delhaize E (2011) The identification of aluminium-resistance genes

E F
12 12
Total P uptake (mg P plant -1)

reduction lesser in EC-232019 (Fig 3A-C) provides opportunities for enhancing crop production on acid soils.
Se e d yie ld (g plant-1)

9 9

| Root-to-shoot ratio increased by 6 6 Journal of Experimental Botany. 62: 9-20.

five-fold in EC-113396 at low P (Fig. 3D) 3 3 Raghothama KG and Karthikeyan AS (2005) Phosphate acquisition. Plant
and Soil. 274: 37-49.
| Total P uptake and seed yield reduced 0
EC-232019 EC-113396
0
EC-232019 EC-113396

by 67 and 44% at low P compared to Fig. 3. Genotypic variation in (A) leaf area,
ACKNOWLEDGEMENTS
sufficient P, reduction was augmented (B) photosynthetic rate, (C) total biomass,
(D) root-to-shoot ratio, (E) total P uptake and VK acknowledges the senior research fellowship from ICAR-IARI for pursuing doctoral
in EC-113396 (Fig. 3E-F) (F) seed yield of soybean grown at sufficient
(4 mg P kg-1 soil + SSP @ 25 mg P kg-1 soil)
research. Authors acknowledge the Extra Mural Research Division [38(1354)/13/EMR-II]
and low (4 mg P kg-1 soil) P of Council of Scientific and Industrial Research, New Delhi for funding.

Presented in the 3rd International Plant Physiology Congress (Challenges and Strategies in Plant Biology Research) December 11 – 14, 2015, New Delhi.