1. Molecular Factors Influencing phosphorus use efficiency(PUE) in rice
Ph.D.
Semester II
Dept of Plant Mol Biology & Biotech,
Indira Gandhi Krishi Vishwavidyalaya,
Raipur ,
(C.G.),
INDIA
Presented by
Datta P. Kakade Ph.D. Scholar
I.D.:
Dept.: PMBB

2. Contents Introduction Phosphorus Use Efficiency (PUE) ? Mechanism of
P Uptake,
P Transport
P Homeostasis
Physiological changes in response to P deficiency
Genetic studies on
P uptake
P-use efficiency
Genetic regulation of P-starvation induced changes

3. Introduction
Among the macro-nutrients, phosphorus (P) is the least available to the plants as major phosphorus content of the fertilizer is sorbed by soil particles
P is the most important inorganic nutrient after nitrogen (N)
P is one of the most important elements that significantly affect plant growth and metabolism
Constituent of key molecules such as
ATP,
Nucleic acids,
Phospholipids

4. About 80–90% of the P applied as fertilizer is sorbed by soil particles and makes it unavailable for plants that lack specific adaptation to access sorbed P
High P-fixing capacity of the soils results in very low P uptake by the plants

5. What is Phosphorus Use Efficiency (PUE) ?
Total biomass or grain yield produced per unit P accumulated in tissue (g tissue DM/mg tissue P)
Phosphorus Efficiency Ratio (PER)
Grain yield per unit of P accumulated in above ground plant material/ shoot (g grain DM/mg of shoot P)
Critical Tissue P Concentration
P conc. required for the given percentage of maximal grain yield (90%)
Plant PUE is inherently complex, as each step including
P uptake,
Translocation,
Assimilation,
Remobilization

6. In Case of Rice

7. Mechanism of P uptake, transport and P homeostasis in plants

8. P uptake in plants
P is absorbed by plants mainly in the form of phosphate ion
(H2PO4)2− or (HPO4)2−
Several forms of P exist in the soil such as
(H2PO4)−,
(H2PO4)2− and
(PO4)3−
Dihydrogen form of orthophosphates (H2PO4−) is most readily transported into the plant
Uptake of P under non-limiting conditions appears to be regulated by internal levels of P
P uptake is against a steep concentration gradient (100-fold or higher) which is energy mediated co-transport process
P absorption is accompanied by H+ influx with a stoichiometry of 2 to 4 H+ per H2PO4− transported

9. P transport in plants
P acquired by roots is rapidly loaded on to xylem and then transported to different parts of the plant according to the metabolic need
Two categories of transporters
Low-affinity transporters
High-affinity transporters
P transporters are actually proton/phosphate (H+/H2PO4−) symporters
Low-affinity transporters are active in
Vascular loading and unloading
Internal distribution
Remobilization of acquired P
High-affinity P transporters play an important role in acquisition of P
Low-affinity transport system is expressed constitutively, High-affinity transport system is regulated by the availability of P in the plant

10. Conti….
Two major check points of regulation for ion transport across roots
Uptake across the plasma membrane into the symplast of the epidermal and cortical cells
Subsequent release into xylem
Under P-limiting condition
Increase in capacity of roots for P uptake occurs accompanied by increase in capacity of the transfer of absorbed phosphate to shoots by increasing phosphate release into xylem

11. P homeostasis in plants
Strong tendency to maintain constant cytoplasmic concentration of ions such as N, P, and K, irrespective of the large fluctuations in the external concentrations
Higher plants generally store P as polyphosphates in vacuoles
P homeostasis in cytoplasm is maintained by P transport across the tonoplast
Tonoplast H-ATPase or pyrophosphates provide required energy to maintain electrochemical gradient for P transport
The transport mechanism for the tonoplast P transporters is unclear
But vacuoles probably play the dual role of
Sink and
Source for P in plant cells

12. Physiological changes in P deficiency to enhance P acquisition
Plants have developed highly specialized adaptations to acquire and utilize P from the environment
Morphological,
Physiological
Biochemical
Adaptations include
High-affinity transporters
Root morphology & root architecture
Phosphate scavenging and recycling enzymes
Alternative pathways of cytosolic glycolysis
Tonoplast H+-pumping pyrophosphatase
Alternative pathways of respiratory electron transport
Other metabolic pathways
These adaptations include many features like enhanced uptake ability through activation of high-affinity transporters, adaptive root development leading to altered root morphology and root architecture, induction of phosphate scavenging and recycling enzymes, induction of alternative pathways of cytosolic glycolysis, induction of tonoplast H+-pumping pyrophosphatase, alternative pathways of respiratory electron transport, and other metabolic pathways associated with signal transduction and transcription regulation

13. Genetic studies on P uptake and P-use efficiency
Fig. Chromosome wise representation of the QTLs reported for P-deficiency tolerance in rice
Numbers on the left side are the position on the physical map in Mb and indicate the start and end position of the QTL on physical map
Names on the right are the accession IDs of the QTLs

14. Genetic regulation of P-starvation induced changes
P starvation in plants leads to co-ordinated gene expression
Some genes function to acquire and utilize P efficiently
Others are involved in regulating the expression of P-starvation induced genes
Regulatory mechanisms
Escherichia coli
Yeast
E. coli, at least 30 genes are involved in response to P during starvation
PhoB, a regulator, and PhoR, a sensor, act in concert as a two-component system that responds to the cell's need for P
Pi starvation in plants leads to co-ordinated gene expression, which includes induction of many enzymes and genes but the functions of only a few are known

15. Conti…
In yeast, the PHO-regulon responds to changes in P concentration
Acidic and alkaline phosphates (PHO5, PHO8, PHO10, PHO11)
High-affinity P transporters (PHO84)
Negative regulators (PHO80, PHO85)
bHLH transcription factor PHO4 generally interacts with transcription factor PHO2
Induce the expression of the P-starvation induced acid phosphates and high-affinity P transporters (PHO5 and PHO84)

16. Conti… In P-sufficient condition In P-deficient condition
Negative regulator PHO80 in association with PHO85 protein represses the PHO gene transcription probably by hyper-phosphorylation of PHO4
PHO80/PHO85 levels prevent PHO4 and PHO2 from activating PHO transcription
PHO81 is inactivated and its synthesis is reduced
In P-deficient condition
PHO81 is a positive regulator, which inhibits PHO80/PHO85 function
Similarities exist between yeast and higher plants in their response to P starvation

17. Conti…
OsIPS1(Oryza sativa phosphate limitation inducible gene), had sequence similarity with Arabidopsis IPS1
Higher accumulation of OsIPS1 was found in roots compared to shoots
Both OsIPS1/2 are responsive to both systemic and local responses to P starvation
Long-term P starvation
OsPI1, showed most significant increase in transcription in both roots and leaves and was repressed by P re-supply

18. Conti…
Regulatory gene PHR1 plays role in co-ordinate regulation of many late P-starvation genes
The PHR1 encodes a MYB transcription factor with homology to PSR1 from Chlamydomonas
Two types of transcriptional regulations in response to P starvation
Transiently induced genes during early stages of P stress
Highly induced genes during prolonged P stress
OsPHR1 and OsPHR2 are involved in P-signaling pathway by regulating the expression of P-starvation induced genes

19. Conti…
SIZ1 (SUMO/Smt3 ligase) is involved in regulation of phosphate starvation response
SIZ1 encodes a small ubiquitin-like modifier (SUMO) E3 ligase
Sumoylation has many regulatory roles, one of which is to protect proteins from ubiquitin-mediated degradation
Over-expression of transcription factor 1 (OsPTF1) enhances the tolerance to P deficiency
P content are about 20 to 30% higher in P-deficient conditions in the OsPTF1 over-expressing plants
Major associated RNase genes induced under P starvation
RNS1
RNS2

20. P translocation and P transporters and regulation of P homeostasis
In P uptake 13 P transporter genes named as OsPT1–OsPT13 were involved
OsPT11- Induced during arbuscular mycorrhizal symbiosis
OsPT2 - Stored P transport in the plant
OsPT6 - P uptake and translocation across the plant
miR399 is a specific response to deficiency of P
miR399 is a long-distance signal for the regulation of P homeostasis
Induction of miR399-guided cleavage of PHO2 mRNA, inhibits PHO2 and hence transfer of P to shoot is stimulated

21. Fig. Flow chart showing molecular events controlling phosphate uptake and transport during P starvation leading to different responses in rice
Known regulation is represented by solid arrows and
Unknown, predicted regulation is represented by dashed arrows

22. Phosphorus deposition in grain
Most of the phosphate in grain is stored in the form of phytin or inositol hexa phosphate
The enzyme phytase releases the P from the grain during germination
4 QTLs/ genes for phytic acid in seed are known in rice on chromosomes 2, 3, 5 and 12

23. Interaction of P with other micro- and macro-nutrients
Interactions with nitrogen
synergistic interaction between N and P could be more synergistic with application of K in adequate levels
Interactions with iron
iron in the form of ferritin interacts with P in soil or root surface
Fe interacts with P to form precipitates and makes both the nutrients unavailable to the plant

24. Conti… Interactions with silica Interactions with arsenic
Positive silicate effects are usually associated with increase in soil pH
Use of calcium silicate (CaSiO3) or magnesium silicate (MgSiO3) in acid soils enhances phosphorus availability to the plants
Interactions with arsenic
Interactions exist between arsenic and phosphate uptake
Arsenic status in the plant is affected by phosphorus status in the rhizosphere of rice
Arsenate is transported in the plant by the transporters for phosphate
Arsenic can limit the phosphate transport across the plant
Arsenic uptake by rice can be reduced by using more aerobic cultivation practices

25. Case Study Objectives:
Role of transcription factors in the regulation of phosphorus starvation signaling and root architecture in rice

26. Materials and methods Plant Material Growth Conditions
Japonica rice cv Zhonghua 10 was used in experiments
Growth Conditions
Hydroponic experiments were carried out in a growth room with a 16-h-light (300C)/8-h-dark (220C) photoperiod
Relative humidity was controlled at approximately 70%
The solution was refreshed every 3 d

27. Results
Microarray Analysis
Expression profiles of 1-week-old rice seedlings exposed to P-deficient solution for 6, 24, 48, or 72 h were used to extract RNA for microarray studies
Figure:- Isolation of P deprivation-inducible MYB TF from microarray hybridization
A-Signal intensity of Os S1_at in microarray hybridization
B-Real-time PCR to validate Os S1_at microarray results presented in A

28. Figure:- Expression patterns of OsMYB2P-1 in different organs, and effects of the deprivation of P, N, K, and Fe on the expression of OsMYB2P-1
A, OsMYB2P-1 expression in different tissues
B, Time course of OsMYB2P-1 expression in response to P deprivation
C and D, Response of OsMYB2P-1 to Fe, N, and K deprivation

29. Conclusion
Phosphate regulation in rice or other monocots is not fully known
In p-non-limiting condition, the roots of the plant generally acquire phosphate by simple diffusion or mass flow
Low-affinity P transporters are constitutively expressed and take part in the transport of P to different parts of the plant in P-non-limiting condition
In P-limiting condition, highly specific signaling pathways come into play to acquire more P from the soil environment
Signaling events ultimately lead to many morphological adaptive changes in root, shoot structure and growth, which are known as P-deficiency response
P deficiency in cells is sensed by the internal p-use efficiency