Indian Agricultural Research Institute, New Delhi - 12 Influence of high [CO2] and sulphur levels on root membrane stability and organic acid exudation under low phosphorus in wheat Milan Kumar Lal* and Renu Pandey Mineral Nutrition Lab Division of Plant Physiology Indian Agricultural Research Institute New Delhi – 110012, India *Email: milan2925@gmail.com Presented at: National Seminar on Plant Genomics and Biotechnology, OUAT, BBSR

Availability of Phosphate rock by the end of this century Indian Agricultural Research Institute, New Delhi - 12 Availability of Phosphate rock by the end of this century Mining of Phosphate rock – A non-renewable resource Syers et al., 2011

INTRODUCTION Indian Agricultural Research Institute, New Delhi - 12 Phosphorus is vital to plant growth and is found in every living plant cell, involved in several key plant functions; Energy transfer Photosynthesis Structural component of nucleic acid and membranes Mostly taken up as the primary orthophosphate ion (H2PO4-) at (pH < 7.0), but also absorbed as secondary orthophosphate (HPO42 -) at (pH > 8.0). It is incorporated into organic compounds, including nucleic acids (DNA and RNA), phosphoproteins, phospholipids, sugar phosphates, enzymes, and energy-rich phosphate compounds, adenosine triphosphate (ATP) . Activation of enzymes and enhanced production of phosphatases RNases and organic acids, changes in protein phosphorylation Organic acid exudates under low P help to mobilize the fixed phosphorus from the soil to plant Indian Agricultural Research Institute, New Delhi - 12

Objectives of Research Indian Agricultural Research Institute, New Delhi - 12 Objectives of Research To study the interactive effect of high [CO2] and phosphorus starvation on growth with different sulphur level To analyze the interactive effect of high [CO2] and phosphorus on membrane injury and root exudation capacity in wheat grown with different sulphur levels To analyze the expression of candidate genes involved in phosphorus remobilization from membranes in wheat grown under high [CO2] and phosphorus starvation with different sulphur levels Hypotheses: Elevated CO2 would provide extra carbon to maintain the flux and thereby improving the organic acid exudation under low P Since Pi is replaced with S in membrane lipids, the elevated CO2 might also influence membrane permeability and uptake of nutrient.

Materials and Methods Indian Agricultural Research Institute, New Delhi - 12 Crop : Wheat Varieties : a. PDW 233 b. PBW 396 Treatments : P level (sufficient 500 µM and deficient 5 µM) S level (sufficient 2000 µM and deficient 10 µM) Carbon dioxide- Ambient Elevated 700 ppm Replications : Three Design of experiment: Factorial CRD

Results- Biomass accumulation Indian Agricultural Research Institute, New Delhi - 12 Results- Biomass accumulation 0.533 0.422 0.257 0.284 0.062 0.151 0.121 0.075 0.614 0.257 0.296 0.543 0.294 0.265 0.319 0.399 Shoot dry weight averaged over nutrient interaction is increased by 64 and 88% in PDW-233 and PBW-396 respectively Root dry weight also doubled under eCO2 condition The total biomass showed 70% more accumulation in PBW 396 under eCO2 The R/S ratio averaged over CO2 X variety increased under low P (41%) and low P/S (12%)

PBW 396 PDW 233 Control -P -S -P/-S Elevated CO2 Ambient CO2 Indian Agricultural Research Institute, New Delhi - 12 PBW 396 PDW 233 Control -P -S -P/-S Elevated CO2 Ambient CO2

Results- Root architecture Indian Agricultural Research Institute, New Delhi - 12 Results- Root architecture 154.3 325.9 183.1 291.4 17.39 29.92 19.78 32.2 0.16 0.24 0.17 0.28 106.6 102.8 65.7 86.5 The root length increased under eCO2 condition in both varieties and under –P and –P/-S Root surface area was more than 60% at eCO2 in both varieties averaged over nutrient treatment as compared to aCO2 Low P and low P/S has more root volume and low S perform better under eCO2 No. of lateral increased under eCO2 and also increased in –P/S in PDW 233 under eCO2

Root system of PDW 233 seedlings under nutrient treatments Control -P -P/-S -S Ambient CO2 Elevated CO2 Indian Agricultural Research Institute, New Delhi - 12 Root system of PBW 396 seedlings under nutrient treatments

Root hairs of PBW 396 seedlings under nutrient treatments Root hairs of PDW 233 seedlings under nutrient treatments Indian Agricultural Research Institute, New Delhi - 12 Control -P -S -P/-S Ambient CO2 Elevated CO2

Membrane Injury of root Indian Agricultural Research Institute, New Delhi - 12 Membrane Injury of root 53.18 52.78 51.14 54.43 0.10 0.29 0.13 0.12 Membrane injury found to higher in the aCO2 condition compared to eCO2 Overall MII increased by 25 and 27% at low P and low P/S, respectively in comparison to control. The membrane damage of root was significantly less at low P and low S under eCO2 MGDG content was increased under eCO2 condition in PDW 233 and DGDG content was increased under low P in both eCO2 as well as aCO2 SQDG content decreased under –S and increased under –P and –P/S both in elevated and ambient CO2 The amount of PG under aCO2 was reduced by 58 mol% while the reduction in MGDG was marginal at low P over control

Carboxylate exudation Indian Agricultural Research Institute, New Delhi - 12 Carboxylate exudation Sl. No.   Organic acid CO2 level PDW 233 PBW 396 Control Low P Low S Low P/S 1. Oxalic acid AC Nd EC 6.08±0.46 6.71±0.17 4.71±0.28 5.34±0.58 7.86±0.64 9.91±0.36 6.64±0.25 8.47±0.44 2. Citric Acid 2.43±0.15 2.33±.0.07 2.59±0.10 5.80±0.12 2.15±0.09 1.86±0.14 1.74±0.04 2.34±.0.089 3.57±0.16 2.88±0.12 0.81±0.13 3.10±0.32 4.53±0.12 4.09±0.20 2.67±0.18 7.76±0.18 3. Pyruvic acid 5.20±0.42 7.95±0.75 3.60±0.18 6.40±0.15 9.07±0.56 3.34±0.33 3.47±0.17 7.37±0.51 6.02±0.39 9.76±0.43 5.77±0.59 7.29±0.72 7.24±0.27 14.89±0.36 11.09±0.67 10.99±0.52 4. Malic acid 1.31±0.17 1.25±0.21 0.06±0.00 1.14±0.05 1.46±0.20 1.65±0.10 0.51±0.10 1.39±0.31 2.05±0.09 1.82±0.05 0.97±0.02 2.20±0.37 1.07±0.14 3.08±0.10 0.70±0.08 4.88±0.34 5. Succinic acid 3.42±0.06 2.74±0.35 0.017±0.00 2.15±0.18 2.57±0.05 1.53±0.08 1.73±0.03 4.25±0.79 7.59±0.59 8.51±0.35 2.53±0.41 3.86±0.60 4.07±0.42 13.44±0.54 3.86±0.56 13.21±1.00 6. Lactic acid 2.21±0.35 4.30±0.67 3.88±0.25 5.66±1.40 3.89±0.51 6.69±0.31 5.21±0.66 8.39±1.48 3.18±0.15 5.58±0.31 4.05±0.27 7.44±0.26 4.72±0.76 8.23±0.30 6.81±0.64 10.25±0.91 7. Fumaric acid 3.91±0.40 2.38±0.29 2.12±0.09 5.37±0.91 2.66±0.66 5.61±1.39 5.64±0.94 6.98±0.11 4.21±0.20 4.62±0.30 0.63±0.04 4.90±0.50 2.08±0.09 6.00±0.41 2.88±0.66 8.94±0.27 8. Total Organic acid 18.41±0.69 20.98±2.02 12.29± 0.28 26.55±1.22 21.82±0.731 20.94±1.41 18.32±0.46 30.43±2.68 32.72±1.11 39.91±1.36 19.49±0.63 34.16±2.25 37.77±2.26 59.68±2.04 34.50±2.60 64.52±3.47 In aCO2 no Oxalic acid was detected but in eCO2 there was significant exudation The low P/S treatment results in 50% increased in citric acid while other low P and low S showed over control Among varieties, malic acid increased by 87% in PDW-233 and by 2.4-fold in PBW-396 under eCO2 In low P condition have more succinic exudates under eCO2 and similar trend was seen in lactic, fumaric acids. The total carboxylic acid concentration in root exudates doubled in response to eCO2 over aCO2, with more exudation in PBW 396 as compared to PDW 233

Enzymes activity and Nutrient accumulation Indian Agricultural Research Institute, New Delhi - 12 The PEPC activity under low P resulted in dramatic increase by 141% For MDH eCO2 in both varieties have more enzyme activity as compared to its control CS has drastic increase in PBW 396 variety under eCO2 , Increase also seen in –P and –S In response to eCO2, the activity of PK in root averaged over nutrient treatment and varieties increased by 63% over aCO2 In –S under eCO2 condition the total S uptake was increase this show more S uptake under eCO2 The total P accumulation was enhanced in both varieties under eCO2

Conclusion Indian Agricultural Research Institute, New Delhi - 12 The total biomass increases under eCO2 in both varieties as compared to control implying that high CO2 provides more carbon that is accumulated in biomass The root surface area, length, volume and laterals increased under –P, -S and –P/-S indicating that root architecture is altered under nutrient deprived condition Membrane injury was less under eCO2 compared to aCO2 indicating extra CO2 provides membrane stability and sulphur provide membrane stability in form of sulpholipid Carboxylates exudation is higher under eCO2 and also under nutrient deficient condition, eCO2 provides more carbon flux that help in more exudation Under P stress and eCO2 galactolipid like SQDG, DGDG increased suggesting that the phospholipid was replaced with galactolipids thus preventing membrane damage The relative expression of TaPT1 gene and other membrane lipid scavenging genes increased drastically under eCO2 indicating higher synthesis of galactolipids involved in membrane lipid recycling.

Thank You Acknowledgement: Chairperson and member of Mineral Nutrition Lab ICAR for financial support in form of JRF National Seminar of Plant Genomics and Biotechnology-2015