1. We hypothesize that microorganisms accumulate organic osmolytes in the cytoplasm during soil drying (Osmolyte Accumulation Hypothesis; OAH). Citations Results Metabolite Analysis of Soils and Soil Microorganisms in Response to Water Stress Conclusions Harris, R. F., 1981. Effect of water potential on microbial growth and activity. Pages 23–95 in J. F. Parr, W. R. Gardner, and L. F. Elliott, editors. Water potential relations in soil microbiology. American Society of Agronomy, Madison, Wisconsin, USA. Schimel, J., Scott, W., Killham, K., 1989. Changes in cytoplasmic carbon and nitrogen pools in a soil bacterium and a fungus in response to salt stress. Appl Environ Microbiol 55. 1635–1637. Artz, R.R.E., Chapman,S.J., Robertson,A.H.J., Potts,J.M., Defarge, F.L., GoGo,S., Comont,L., Disnar,J.R., Francez, A.J., 2008. FTIR spectroscopy can be used as a screening tool for organic matter quality in regenerating cutover peatlands. Soil biology &biochemistry 40, 515-527. We are grateful for the advice and technical support of Dr. Kang Xia, Department of Chemistry & Mississippi State Chemical Laboratory. Introduction 1.The dried and salt stressed soils have more extractable sugars than the moist soils. This result is consistent with the osmolyte accumulation hypothesis (OAH). 2.Increase in concentration of sugar alcohols like xylitol and glucitol and mannitol in dried soils is also consistent with OAH, especially fungal responses to water stress in culture. 3.Our data is some of the first to show that microorganisms in soil, insitu, acclimate to soil drying by accumulating compatible solutes, such as sugars and alcohols. 4.We expect that a deeper characterization of microbial & soil pools that are dynamic during soil drying will shed further light on mechanisms of microbial adaptation to water stress. Hypothesis Materials and Methods Results Fig 2 : Phenol sulfuric acid and Ninhydrin-N analysis of moist, dried and salt-affected soils. Figure 6 &7 :Representative FTIR reports of Chloroform+ 0.01 M K 2 SO 4 extracts from moist and dry soil Materials and Methods Acknowledgements Dynamics in the water potential which is common in soil ecosystems pose great physiological stress on the soil microorganisms. It is known from pure culture studies that microbes adapt to the changing water potentials by altering the chemistry and concentration of their intracellular solute pool. The accumulation of organic osmolytes like proline, glycine betaine, ectoine which are collectively called as compatible solutes helps to retain cell water and maintain turgor under hyperosmotic conditions was reported by salt induced water stress studies. Yet, the evidence that this process occur in insitu soil microbes is predominantly anecdotal. Project Goal: To understand the insitu response of microorganisms to the water dynamics in soil ecosystems. Soil Type: Marietta series Top 15-cm soil was collected, sieved and stored at 4 o C until it is used. Approximately 10g of soil by dry weight was weighed into serum cups and all the samples were brought to water potential of -0.003MPa and preincubated for 5 days before starting the treatments. The water potential of the dry and NaCl treatment were gradually brought down to -4.5MPa by air drying and addition of salt to the soils respectively over a period of 3 days. The soils were extracted using 2 methods 1) Quenching 2) Chloroform + 0.01M K 2 SO 4. The extracts were collected, lyophilized and stored at 80 o C for further analysis. As preliminary test, extracts were analyzed for sugars (Phenol sulfuric acid method) and amino acids (Ninhydrin assay). Extracts were derivatized using BSTFA and MTBSTFA solutions and analyzed on Varian 3600 GC-MS for sugars and amino acids respectively. For identifying different functional groups, the extracts were analyzed on Nicolet 6700 FTIR with DTGS KBr detector. Fig 1 : Cellular response to water availability a Department of Plant and Soil Sciences, Mississippi State University, Ms; b National centre for Natural products Research, University of Mississippi, University, MS. The dried and salt stressed soils have significantly greater (20%) amounts of sugars than the moist soils. Ninhydrin (amino-acid) reactive N is low in all soils, but significantly greater in salt stressed compared to moist and dried soils. Sugar alcohols are consistently higher in dried compared to moist soil. Figure 3 and 4 : Gas chromatograms of Moist and dry soils extracts. FTIR reports show the presence of C=N, C=O stretches at 1660- 1535 cm -1 which might be of proteinaceous in origin. The peak between 3600-2800 cm -1 which represents the presence of functional groups like symmetric and assymetric CH2, H bonded OH groups and NH 2 which could possibly assigned to the fats, lipids and nucleic acids which might be microbial origin. Analysis by FTIR indicated few chemical differences between dry and moist soil. Fig 3: Quenching method Fig 4: Chloroform+ 0.01M K 2 SO 4 method 15.382 min 16.531 min 16.608 min 19.019 min 19.549 min20.104 min 17.61 14.653 min 15.612 min 16.531 min 16.772 min 17.610 min 19.013 min 19.544 min 20.101 min 21.413 min Dry Soil Moist soil 18.964 min 19.280 min 19.650 min 20.051 min 21.360 min 15.631 min 18.970 min 19.282 min 19.654 min 20.055 min 21.366 min 16.710 min 18.971 min 19.497 min 20.056 min 21.365 min 15.635 min 16.737 min 18.973 min 19.292 min 19.501 min 19.659 min 20.057 min 21.369 min Moist-R1 Moist-R2 Dry-R1 Dry-R2 Fig 6: Moist soil Fig 7: Dry soil a b b aab *Sugars and amino acids were analyzed separately. Treatments with different letters are significantly different from one another. Madhavi L.Kakumanu a, Mark A. Williams a and Charles Cantrell b C=N, C=O Fats and Nucleic acids C=N, C=O Fats and Nucleic acids i. ii a. ii b. iii. Fig 1 : Cellular response to water availability i.- Normal bacterial cell ii.- Bacterial cells as affected by changing water potentials ii a.- Plasmolyzed bacterial cell when exposed to low water potential. ii b.- Bacterial cell collects excess water when exposed to high water potential. iii.- Bacterial cell accumulating the osmolytes under low water potentials to maintain cell turgor. Glucitol Xylitol Glucitol Xylitol Glucose Myo-Inositol Galactose Mosit Method-1 Dry Method-1 Moist Method-2 Dry Method-2 XylitolNI0.32 NI 0.312 Glucose 0.622.81 6.93 7.81 GalactoseNI 1.250.625 Glucitol0.623.430.7811.78 Myo-inositol0.51.52.181.03 Table 1: Sugars identified in moist and dry soils (Mcounts/gram of soil). Method 1-Extracted by Quenching; Method 2- Extracted by CHCl3+0.01M K 2 SO 4 Sugars Treatments