Cultivated and Wild Rices Do Not Accumulate Glycinebetaine Due to Deficiencies in Two Biosynthetic Steps Bala Rathinasabapathi, Douglas A. Gage, David J. Mackill, and Andrew D. Hanson* ABSTRACT Many cereals, grasses and other plants accumulate glycinebetaine in response to drought or salinity. Glycinebetaine can act as a nontoxic or protective cytoplasmic osmolyte, and is therefore a biochemical component of drought- and salt-stress resistance. It is synthesized by a two-step pathway: choline —> betaine aldehyde -» glycinebetaine. There are contradictory literature reports as to whether cultivars of rice (Oryza saliva L.) accumulate glycinebetaine. To resolve this con- flict, we used sensitive and specific fast atom bombardment mass spec- trometry methods to analyze 35 diverse rice cultivars, together with accessions of nine wild rices. No glycinebetaine accumulators were found; levels of glycinebetaine were in all cases < 1 /unol g~' dry wt., and no other betaines were detected. We then applied fast atom bom- bardment mass spectrometry to determine why rice, unlike other cer- eals, does not accumulate glycinebetaine. Rice leaves supplied with deuterium-labeled betaine aldehyde did not convert it to glycinebe- taine, and they lacked an endogenous betaine aldehyde pool. To- gether, these results indicate that rice lacks both the steps in the glycinebetaine biosynthesis pathway. Levels of the precursor choline, however, were comparable to those found in betaine-accumulating cereals. Rice cultivars are therefore rational candidates for the intro- duction of heterologous genes for glycinebetaine biosynthesis, with the aim of improving resistance to drought and salinity. U NDER DRY or saline conditions, the accumulation of inorganic and organic solutes (osmotic adjustment) is essential for growth and survival of many crops (23,26). Since high concentrations of inorganic ions and of many organic solutes are toxic to metabolism, it has been hy- pothesized that special nontoxic organic solutes termed B. Rathinasabapathi and A.D. Hanson, Institut de recherche en biologic vegetate, Univ. de Montreal, 4101 est rue Sherbrooke, Montreal, Quebec, H1X2B2, Canada; D.A. Gage, Biochemistry Dep., Michigan State Univ., East Lansing, MI 48824; and D.J. Mackill, USDA-ARS, Dep. of Agronomy & Range Science, Univ. of California, Davis, CA 95616. Received 24 Aug., 1992. "Corresponding author. Published in Crop Sci. 33:534-538 (1993). compatible osmolytes are accumulated in the cytoplasm, while inorganic ions and other solutes are sequestered in the vacuole (35). Much evidence now supports this hy- pothesis (24). Compatible osmolytes may also mitigate the inhibitory effects of ions on proteins (29). Among the compatible osmolytes found in higher plants, glycinebetaine is probably the most effective (21) and is widely distributed (30,33). Accumulation of glycinebe- taine has therefore drawn attention as a potential osmotic stress resistance trait, for manipulation by conventional breeding methods (10,19) or by genetic engineering (21,24). Glycinebetaine is synthesized via a two-step pathway from its precursor, choline: (CH 3 ) 3 + N-CH 2 -CH 2 OH -* (CH 3 ) 3 + N-CH 2 -CHO Choline Betaine aldehyde -* (CH 3 ) 3 + N-CH ? -COOH Glycinebetaine The first step is catalyzed by choline monooxygenase, the second by betaine aldehyde dehydrogenase (2,32). Three cDNA clones comprising the protein coding re- gion of betaine aldehyde dehydrogenase have been iso- lated from spinach (Spinacia oleracea L.) (34) and sugarbeet (Beta vulgaris L.) (25). Glycinebetaine accumulation has been reported in leaves of cereals and grasses from >20 genera, representing most tribes of the Gramineae (30). The relatively drought- and/or salt-tolerant cereals, sorghum [Sorghum bicolor (L.) Moench], barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.), can accumulate up to ~75 to 100 ju,mol g- 1 dry wt. (9,14,18). Maize (Zea mays L.), which is more susceptible to salt and drought stress, typ- ically shows moderate glycinebetaine accumulation, with levels for stressed plants from 15 to 50 /unol g" 1 dry Abbreviations: ASW, artificial sea water; FABMS, fast atom bombardment mass spectrometry; M + , the molecular ion which represents the molecular weight of a compound; PPFD, photo- synthetic photon flux density. Published May, 1993