Dr. Toshio Yamaguchi (U.C. Davis) Dr. Lianhai Fu (U.C. Davis) Jordan Sottosanto (U.C. Davis) Marina Large (U.C. Davis) Prof. Larry Teuber (U.C. Davis) Dr. Maris Apse (Arcadia BioSciences) Dr. Jos van Boxtel (Arcadia BioSciences) Prof. Juan M. Ruiz (Granada, Spain) Prof. Shimon Gepstein (Technion Institute, Israel) Dr. Amira Gepstein (Technion Institute, Israel) Dr. Abdel Ismail (IRRI, Philipines) Dr. Ju-Kon Kim (Myongji Univ, Korea) Supported by: National Science Foundation, Arcadia BioSciences Inc. California Specialty Crops Program, Will.W. Lester Endowment, U.C.Davis Extension and global distribution of salt-affected soils Pessarakli and Szabolcs, 1999 1
10,000 acres Water Use (cubic Km) 5000 4000 3000 2000 1000 0 1940 1950 1960 1970 1980 1990 2000 Year Agriculture Industry Domestic 2
Ogallala aquifer 3
Salt concentrations Pacific Ocean ppm 32,000 mg/l 32,000 mm 550 EC (ds/m) 50 Colorado River 650/750 650/750 11/13 1/1.2 Chicken Soup 600 600 10.5 0.95 4
ECONOMICAL IMPACT: Decline in productivity and yields Increased input (seeds, fertilizer) to compensate for yield reduction Reduced efficient water use Increased labor requirements, high costs ENVIRONMENTAL IMPACT: Reduction of green cover, leading to soil erosion (crust formation, wind and water erosion) Leaching of nutrients, loss of organic matter, run-off, leading to the contamination of water supplies Increase of carbon dioxide released to the atmosphere What are our options? Management and Rehabilitation of salt-affected soils (1) manipulate the environment by agronomic and irrigation practices so as to minimize the salinity crops are exposed to. Most common method. Very expensive, requires continuos input and large amounts of water. (2) use of halophytes and saline environments for the production of edible seeds, pharmaceuticals, Leaching feedstocks and biomass. Only applicable to coastal regions. Deep ploughing Crops + levelling yielding fruits and pommes are excluded. Drainage Could provide important traits for breeding new salt tolerant crops. (3) generation of genetically modified salt tolerant plants able to grow and produce high yields under high salinity conditions. Relatively fast methodology Can be applied everywhere Regulatory and safety issues are not widely accepted. Spreading of gypsum Mulching Organic manure 5
H + ph out =5.7 ATP ADP ph cyt =7.1 Cl - vacuole ph vac =6.0 ATP Na + sos1 H + vacuole +60 mv H + H + PPi Na + H + nhx1 H + Na + Na + nhx1 cytosol out Cl - -120 mv Salinity tolerance (gain of function) Working hypothesis H 2 O Na + Na + K + Na + Na + K + Na + Na + K + Na + K + 6
Overexpression of AtNHX1 increases salt tolerance Arabidopsis High Canola Low OEX1 X1OE 1 X1OE 2 X1OE 3 WT Tomato WT 0 50 100 150 200 NaCl (mm) (Apse, et al., 1999). Science 285,1256-1258 X1OE 1 X1OE 2 X1OE 3 WT 200 mm NaCl Zhang, et al. (2001) PNAS 98:12832-12836. 200 mm NaCl Zhang & Blumwald.(2001). Nature Biotechnology 19:765-768. Rice Rice [AgNHX1] (Ohta et al., 2002) Maize [AtNHX1] (Yin et al., 2004) Tobacco [GhNHX1] (Wu et al., 2004) Cotton [AtNNHX1] (Zhang et al., 2004) Wheat [AtNHX1] (Xue et al., 2004) T2 lines recovering from exposure to 100 mm NaCl for 35 daysl (van Boxtel et al, 2005). Only ONE gene? DNA arrays from plants exposed to 100 mm NaCl (sublethal!!) for 0.5, 2, 7 and 14 days. The plants were grown in pots, All the plants were at the same developmental stage, The only difference was the time of exposure to NaCl. 7
wt nhx1 NHX1::nhx1 0 0.5 2 7 14 0 0.5 2 7 14 0 0.5 2 7 14 defense growth processing energy signalling 50 genes unclassified structural translation metabolism transcription transport defense growth processing energy signalling 76 genes unclassified structural translation metabolism transport transcription (1) From the lab to the field! Challenges and Opportunities (a) Variability of salt levels in the field (i) naturally salty soils (ii) irrigation with salty water - type of irrigation! (b) Interactions with other factors (i) soil fertility (ii) temperature (iii) soil ph (alkaline soils with ph>8.3) (iv) soil composition -Na 2 SO 4 -MgSO 4 - Selenium - Boron 8
ZhongJin Lu et al, 2004 Challenges and Opportunities (2) Promoters most commonly used are primarily constitutively expressed (CaM35S, ubiquitin, actin, etc.). Stress-induced and/or tissue specific promoters display better phenotype. (3) There have been a number of successes in the production of abiotic-stress tolerant plants using tobacco or Arabidopsis. We have to introduce these tolerance genes in crop plants. (4) We should produce transgenes in specific genetic backgrounds already characterized by breeding practices. 9
Canola (Brassica napus) g/100 g DW 7 6 5 4 3 2 1 0 leaves Na + roots # seed/plant 400 300 200 100 0 Seed production Wt (10 mm NaCl) oe (200 mm NaCl) Canola Wt (10 mm NaCl) oe (10 mm NaCl) oe (200 mm NaCl) FATTY ACID COMPOSITION (mol%) 80 70 60 50 40 30 20 10 0 wild type salt - SEED OIL COMPOSITION 16:0 16:1 18:0 18:1 18:2 18:3 20:0 20:1 FATTY ACID 10
Strategy for soil reclamation (alfalfa) Root zone WATER TABLE Assuming: 1) an average (conservative) alfalfa yield of 20 ton/ha/year, 2) and a NaCl accumulation in the plant foliage of 8% of the dry mass, the total amount of salt collected by the plants should be 1.6 ton/ha/year The plants (containing NaCl) could be used for feed, eliminating the need for the use of salt blocks. Challenges and Opportunities (2) Promoters most commonly used are primarily constitutively expressed (CaM35S, ubiquitin, actin, etc.). Stress-induced and/or tissue specific promoters display better phenotype. (3) There have been a number of successes in the production of abiotic-stress tolerant plants using tobacco or Arabidopsis. We have to introduce these tolerance genes in crop plants. (4) We should produce transgenes in specific genetic backgrounds already characterized by breeding practices. (5) We need to develop rational concepts for combining genes. 11
Wild Type Line T2-36 Before Drought 9 days Drought After 12 Days Re-watering Plants grown under salinity stress (200mM NaCl) WT T2-36 NT03 NT03 T2-36 WT Drought tolerant Salt tolerant T2-36xNT03 WT T4-24 NT15 NT15 T4-24 WT Drought Salt tolerant tolerant T4-24xNT15 12
Challenges and Opportunities (1) Promoters most commonly used are primarily constitutively expressed (CaM35S, ubiquitin, actin, etc.). Stress-induced and/or tissue specific promoters display better phenotype. (2) There have been a number of successes in the production of abiotic-stress tolerant plants using tobacco or Arabidopsis. We have to introduce these tolerance genes in crop plants. (3) We should produce transgenes in specific genetic backgrounds already characterized by breeding practices. (4) We need to develop rational concepts for combining genes. (5) It is the phenotype that counts!! 13