We provide genetic evidence that the production of methanol in tomato fruit is regulated by pectin methylesterase (PME, EC 3.1.1.11), an enzyme that catalyzes demethoxylation of pectins. The role of PME in methanol production in tomato fruit was examined by relating the tissue methanol content to the PME enzymatic activity in wild-type Rutgers and isogenic PME antisense fruits with lowered PME activity. In the wild-type, fruit development and ripening were accompanied by an increase in the abundance of PME protein and activity and a corresponding ripening-related increase in methanol content. In the PME antisense pericarp, the level of methanol was greatly reduced in unripe fruit, and diminished methanol content persisted throughout the ripening process. The close correlation between PME activity and levels of methanol in fruit tissues from wildtype and a PME antisense mutant indicates that PME is the primary biosynthetic pathway for methanol production in tomato fruit. Interestingly, ethanol levels that were low and unchanged during ripening of wild-type tomatoes increased progressively with the ripening of PME antisense fruit. In vitro studies indicate that methanol is a competitive inhibitor of the tomato alcohol dehydrogenase (ADH, EC 1.1.1.1) activity suggesting that ADH-catalyzed production of ethanol may be arrested by methanol accumulation in the wild-type but not in the PME mutant where methanol levels remain low.Methanol is emitted by actively growing plant tissues (1) and ripening fruit (2). A major source of methanol may be pectin methyl esters (3) that are de-esterified to methanol and pectic substances by a PME 1 -catalyzed reaction (4). Although methanol production is correlated with PME activity in germinating seeds or other plant tissues (1), the role of PME in methanol accumulation in plants has not been firmly established (4,5).A developmentally regulated increase in PME gene expression occurs in developing tomato fruit (6) and may be used as a test system to relate PME activity to methanol metabolism. We have created transgenic tomato fruits with severely impaired expression of PME by introducing a fruit-specific PME antisense gene under the control of cauliflower mosaic virus 35S promoter (7). We compared methanol production in the wildtype and the PME antisense tomato fruits to examine whether the arrest of PME gene expression results in diminished production of methanol. Our results show that methanol accumulation in ripening tomato pericarp of either the wild-type or the PME antisense is related to PME activity, suggesting that the PME activity is the primary source of methanol production in tomato fruits. A surprising finding is that the tissue methanol and ethanol content in ripening tomato fruit were inversely related.

A new cold-inducible genetic construct was cloned using a chloroplast-specific omega-3-fatty acid desaturase gene (FAD7) under the control of a cold-inducible promoter (cor15a) from Arabidopsis thaliana. RT-PCR confirmed a marked increase in FAD7 expression, in young Nicotiana tabacum (cv. Havana) plants harboring cor15a-FAD7, after a short-term exposure to cold. When young, cold-induced tobacco seedlings were exposed to low-temperature (0.5, 2 or 3.5 degrees C) for up to 44 days, survival within independent cor15a-FAD7 transgenic lines (40.2-96%) was far superior to the wild type (6.7-10.2%). In addition, the major trienoic fatty acid species remained stable in cold-induced cor15a-FAD7 N. tabacum plants under prolonged cold storage while the levels of hexadecatrienoic acid (16:3) and octadecatrienoic acid (18:3) declined in wild type plants under the same conditions (79 and 20.7% respectively). Electron microscopy showed that chloroplast membrane ultrastructure in cor15a-FAD7 transgenic plants was unaffected by prolonged exposure to cold temperatures. In contrast, wild type plants experienced a loss of granal stacking and disorganization of the thylakoid membrane under the same conditions. Changes in membrane integrity coincided with a precipitous decline in leaf chlorophyll concentration and low survival rates in wild type plants. Cold-induced double transgenic N. alata (cv. Domino Mix) plants, harboring both the cor15a-FAD7 cold-tolerance gene and a cor15a-IPT dark-tolerance gene, exhibited dramatically higher survival rates (89-90%) than wild type plants (2%) under prolonged cold storage under dark conditions (2 degrees C for 50 days).

Studies of Spinacia oleracea L. were undertaken to characterize further how Mg2`external to the isolated intact chloroplast interacts with stromal K+, pH, and photosynthetic capacity. Data presented in this report were consistent with the previously developed that Mg" impairment of stromal alkalization is likely the result of altered H+ fluxes across the chloroplast envelope; altered H+ pumping across the thylakoid membrane is thought not to be involved.Mg2+ effects on H+ movement across the chloroplast envelope are known to be linked to K+ counterflux (2,11,14). However, the specific molecular mechanisms that facilitate fluxes of those monovalent cations and allow for Mg2> regulation of the system are not well characterized. Work from this laboratory (21) has established that it is not Mg2+ external to the chloroplast but rather Mg> bound or associated with the chloroplast envelope that exerts the regulatory effect. It is also clear from several different lines of evidence ( 19, 21 ) that millimolar levels of free external Mg> can greatly affect chloroplast metabolism even though Mg2+ movement across the envelope into the stroma does not occur under these conditions.Two hypotheses have been presented in the literature regarding the specific mechanisms that allow for Mg2+ regulation of H+/K+ counterfluxes. The results of work by Huber and Maury ( 11) and Maury et al. (14) suggest that H+ and K+ movement across the chloroplast envelope both occur via a specific antiport enzyme. They postulated that this transport protein, when activated, can facilitate either K+ influx/H+ efflux or K+ efflux directly linked to H+ influx. These researchers speculated that Mg2+ external to chloroplast activates this antiport enzyme and impairs stromal alkalization in the presence of low external K+ due to net K+ efflux from the stroma (coupled directly to H+ influx). Demmig and Gimmler (2,3) speculated that other mechanisms facilitate K+ and H+ fluxes across the envelope and that Mg2+ may influence this system in a different manner. They proposed that a Donnan system (fixed negative charges) develops in the stroma ofthe illuminated chloroplast, increasing the Em2 (inside negative) between the stroma and extrachloroplast medium. They concluded that this Donnan systemgenerated Em drives monovalent cation influx. At high external K+, influx of K+ occurs. At low external K+, impaired stromal alkalization was thought to occur due to H+ influx. Mg2+ was thought to influence this system by altering external solution ionic strength; the Em was proposed to be dependent on ionic strength of the medium. This model, however, does 2 Abbreviations: Em, membrane potential; dimethonium, ethane-1,2-bis-trimethylammonium; TPP, tetraphenylphosphonium.

A K+-conducting protein of the chloroplast inner envelope was characterized as a K+ channel. Studies of this transport protein in the native membrane documented its sensitivity to K+ channel blockers. Further studies of native membranes demonstrated a sensitivity of K+ conductance to divalent cations such as MgZ+, which modulate ion conduction through interaction with negative surface charges on the inner-envelope membrane. Purified chloroplast inner-envelope vesicles were fused into an artificial planar lipid bilayer to facilitate recording of single-channel K+ currents. These single-channel K+ currents had a slope conductance of 160 picosiemens. Antibodies generated against the conserved amino acid sequence that serves as a selectivity filter in the pore of K+ channels immunoreacted with a 62-kD polypeptide derived from the chloroplast inner envelope. This polypeptide was fractionated using density gradient centrifugation. Comigration of this immunoreactive polypeptide and K+ channel activity in sucrose density gradients further suggested that this polypeptide is the protein facilitating K+ conductance across the chloroplast inner envelope.Studies with intact chloroplasts (Wu and Berkowitz, 1992) indicate that K+ flux across the inner envelope occurs through a well-regulated transport pathway that is likely a K+-conducting ion channel. K' flux into or out of the chloroplast stroma is indirectly linked to H+ counterexchange (Maury et al., 1981; Wu and Berkowitz, 1992). Movement of K+ through this channel protein, therefore, has a profound effect on stromal pH and, hence, photosynthesis (Werdan et al., 1975;Maury et al., 1981; Wu and Berkowitz, 1992). Previous work from this laboratory has demonstrated that a K'-conducting protein can be successfully detergent-solubilized from preparations of purified inner-envelope membrane vesicles and functionally reconstituted into artificial liposomes (Wang et al., 1993). This putative K+ channel protein has not previously been characterized. In the work reported here, we used severa1 approaches to characterize the nature of this K+ transport protein. Results of these experiments are consistent with the presence of a K+ channel in the inner envelope that shares some structural, functional, and regulatory properties with K' channels from other membrane systems ( e g Rudy et al., 1991; Anderson et al., 1992). 955 MATERIALS AND METHODS Preparation of Chloroplast Inner-Envelope Membrane VesiclesInner-envelope vesicles were prepared from spinach (Spinacia oleracea L.) as described previously (Berkowitz and Peters, 1993). Briefly, intact chloroplasts (isolated from 5-10 kg of spinach leaves using Perco11 step gradients) were exposed to freeze/thaw cycles in hyperosmotic medium, and the thylakoids were removed before the crude membrane fraction was loaded on a discontinuous SUC step gradient. Inner-envelope vesicles were removed from the 0.8 ~/ 0 . 4 6 M Suc interface of the gradient, washed in envelope medium (0.2 M SUC, 2 mM Na2EDTA, 2 mM DTT, and 10 m~ TricineNaOH, pH 7.5), a...

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