Betaine is an important osmoprotectant in many plants, but its transport activity has only been demonstrated using a proline transporter from tomato, a betaine-nonaccumulating plant. In this study, two fulllength and one partial transporter genes were isolated from betaine-accumulating mangrove Avicennia marina. Their homologies to betaine transporters from bacteria and betaine/4-aminobutyrate transporters from mammalian cells were low but were high to proline transporters from Arabidopsis and tomato. Two fulllength transporters could complement the Na ؉ -sensitive phenotype of the Escherichia coli mutant deficient in betT, putPA, proP, and proU. Both transporters could efficiently take up betaine and proline with similar affinities (K m , 0.32-0.43 mM) and maximum velocities (1.9 -3.6 nmol/min/mg of protein). The uptakes of betaine and proline were significantly inhibited by mono-and dimethylglycine but only partially inhibited by betaine aldehyde, choline, and 4-aminobutyrate. Sodium and potassium chloride markedly enhanced betaine uptake rates with optimum concentrations at 0.5 M, whereas sucrose showed only modest activation. The change of amino acids Thr 290 -Thr-Ser 292 in a putative periplasmic loop to Arg 290 -Gly-Arg 292 yielded the active transporter independent of salts, suggesting the positive charge induced a conformational change to the active form. These data clearly indicate that the betaine-accumulating mangrove contains betaine/proline transporters whose properties are distinct from betaine transporters of bacteria and mammalian cells.

The responses of carbon exchange rate (CER), stomatal conductance (gs), activities of phosphoenolpyruvate carboxylase (PEPcase), NADP malic enzyme (NADP-ME), ribulose-1,5- bisphosphate carboxylase (Rubisco), fructose-1,6-bisphosphatase (FBPase) and pyruvate, orthophosphate dikinase (PPDK), and contents of chlorophyll (Chl) and total soluble protein (Tsp) in leaves of sugar cane (Saccharum sp. cv. NiF4) to gradually developed water stress were investigated. The initial inhibitions of CER, gs, activities of the photosynthetic enzymes and contents of Chl and Tsp were observed from leaf water potentials (Ψw) of -0.37 MPa. During water stress, CER and gs, decreased in a non-linear way, activities of the five enzymes and contents of Chl and Tsp decreased linearly with decreasing leaf Ψw. The changes of gs the photosynthetic enzymes, Chl and Tsp were highly related to the changes of CER. The decline in CER during water stress was caused by both stomatal and non- stomatal limitations. Above leaf �w of -0.85 MPa, the decline in CER was caused by stomatal closure, below -0.85 MPa, the decline in CER was caused by non-stornatal limitation. Among non-stomatal components, PPDK activities decreased 9.1 times during water stress, much more than other enzymes which decreased from 2 to 4 times. Measured PPDK activities were only a little higher than the corresponding CER values at various leaf Ψw suggesting that PPDK is very likely to be the limiting enzyme to photosynthesis under water stress.

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Glycinebetaine is an important osmoprotectant in bacteria, plants, and animals, but only little information is available on the synthesis of glycinebetaine in tree plants. Among four mangrove species, glycinebetaine could be detected only in Avicennia marina. Pinitol was the main osmoprotectant in the other three species. The level of glycinebetaine in A. marina increased under high salinity. Betaine-aldehyde dehydrogenase (BADH) was detected in all four species, but choline monooxygenase could not be detected. A cDNA library was constructed from the leaves of A. marina. Two kinds of BADH cDNAs were isolated, one homologous to the spinach chloroplast BADH, and the other with unique residues SKL at the end of C-terminus. The BADH transcription levels of the former were higher than those of the latter. The levels of the former BADH increased at high salinity whereas those of the latter were independent of salinity. BADHs were expressed in Escherichia coli and purified. Two kinds of A. marina BADHs exhibited similar kinetic and stability properties, but were significantly different from those of spinach BADH. A. marina BADHs efficiently catalyzed the oxidation of betainealdehyde, but not the oxidation of omega-aminoaldehydes and were more stable at high temperature than the spinach BADH.

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Analyzing the assimilation rate (A) relative to the CO(2) concentration inside leaves (C(i)) has been a useful approach for investigating plant responses to various environments. Nevertheless, there are uncertainties in calculating C(i) when stomata close, restricting the application. Here, A-C(i) curves were traced in sunflower (Helianthus annuus L.) leaves using a method for directly measuring C(i). The method was incorporated into an LI-6400 open gas exchange system, and stomata were closed by feeding 10 µM ABA through petioles. The conductance to CO(2) was derived from the directly measured C(i) and compared with the conductance from the water vapor flux (i.e. the standard calculation). When stomata were open, measured and calculated C(i) gave similar A-C(i) curves. When stomata were closed, the curves differed because measured C(i) departed from the calculated value. This difference caused the calculation to trace an artifactual limitation of photosynthesis. The direct measurement avoided this problem and followed the curve for leaves with open stomata. Largely because of the cuticle, the calculation overestimated CO(2) entry into the leaf because the cuticle transmitted more water vapor than CO(2), and the calculation relied on water vapor. Consequently, the standard calculation gave conductances larger than those from directly measured C(i). Although the cuticle conductance to water vapor remained constant as stomata closed, it increasingly contributed to the overestimation of C(i). The system provided here is not affected by these cuticle properties and thus is expected to open up the opportunity for A-C(i) analysis in plant physiology.