Plastids and microsomal membranes were isolated from pericarp tissue of mature green and red-ripe tell pepper fruit harvested from greenhouse and field grown plants. The lipid composition of these membrane fractions changed far more with ripening of field grown than greenhouse grown fruit. Also, the phospholipid (PL), free sterol (FS), steryl glycoside (SG) and acylated steryl glycoside (ASG) content of microsomes and plastids from both green and red fruit were very different under the two growing conditions. Total steryl lipids (TSL = FS + SG + ASG), and the TSL/PL ratio, increased in microsomes and decreased in plastids with ripening. These changes were much greater in field grown fruit. The ASG/SG ratio decreased with ripening in both membrane fractions, under both growing conditions. Ripening and growth conditions affected the phospholipid and sterol composition in plastids much more than in microsomes. Lipid changes associated with the chloroplast – chromoplast transformation were similar in field and greenhouse grown fruit, including an increase in the galactolipid/PL ratio. Future studies will assess how differences in membrane lipid composition affect postharvest storage life of the fruit.
Bruce D. Whitaker
Altered metabolism of membrane lipids has been proposed as a mechanism for the beneficial effects of postharvest calcium treatment on apple quality. A previous study showed that after transfer of apples stored 6 months at 0C to 20C, calcium-treated fruit exhibited slower loss of galactolipid and altered levels of sterol conjugates. The present study of lipids in “control” fruit was conducted as a prelude to further in-depth analyses of the effects of postharvest calcium and heat treatments on lipid metabolism in apples during and after cold storage. Neutral lipid, glycolipid (GL), and phospholipid (PL) fractions were obtained by column chromatography followed by TLC separation of GL and PL classes. The major GL were steryl glycosides (SG), acylated steryl glycosides (ASG), cerebrosides (CB), and mono- and digalactosyl diacylglycerols. Phosphatidylcholine (PC) > P-ethanolamine (PE) > P-irositol (PI) were the major PL. The fatty acids of PC and PE were quite similar, whereas those of PI were more saturated. CB included only 2-hydroxy fatty acids. Among the steryl lipids, free sterols > SG > ASG, with beta-sitosterol >90% of the total sterol in each.
Bruce D. Whitaker
A previous study of lipids from pericarp tissue of tomato fruit ranging from mature-green to red-ripe showed a large increase in total sterols accompanied by dramatic changes in sterol composition and conjugation with ripening. This study was conducted to determine whether similar changes occur in microsomal membranes derived from tomato fruit pericarp. Acylated steryl glycoside (ASG), the predominant steryl lipid, declined during ripening, with increases in steryl glycoside (SG) and free sterol (FS). Only minor changes in fatty acid composition were associated with the drop in ASG. The stigmasterol:sitosterol ratio increased throughout ripening, but much more in Fs than in SG or ASG. The ratio of FS to phospholipid (PL) increased with ripening. However, FS was never greater than 10 percent of the total membrane sterol (TMS), and TMS:PL actually declined over the middle stages of ripening. It is not known why tomato tissues maintain such high levels of ASG and SG, but sterol conjugation is thought to regulate the physical properties of cell membranes.
Bruce D. Whitaker
MG tomato fruit were stored for four or 12 days at chilling (2C) or nonchilling (15C) temperature. Fruits stored 12 days at 15C ripened to the turning stage, whereas fruits at 2C did not ripen. Lipids of microsomes and plastids from pericarp tissue were analyzed at harvest and after four or 12 days of storage. After 12 days at either 15C or 2C, the ratio of phospholipid (PL) to protein in microsomes declined, with a concomitant increase in the ratio of total membrane sterols (TMS) to PL. The TMS/PL ratio also increased in crude plastids. In both microsomes and plastids, free sterols (FS) increased more at 2C than at 15C, and thus accounted for a larger percentage of the TMS. The ratio of stigmasterol to sitosterol in steryl lipids, particularly in FS, increased more at 15C than at 2C. The unsaturation index of fatty acids in PL and galactolipids generally increased slightly during storage at both 15C and 2C. The ratio of phosphatidylethanolamine to P-choline increased in both membrane fractions at both temperatures. In plastids, the ratio of mono- to digalactosyldiacylglycerol declined substantially at 2C but not at 15C.
Bruce D. Whitaker
Bruce D. Whitaker
Lipid composition and pigment content were determined in pericarp of `Pik Red' tomatoes (Lycopersicon esculentum Mill.) that were harvested when mature-green (MG) then ripened for 1 or 14 days at 20C, chilled for 11 or 21 days at 2C, or chilled for 21 days and transferred to 20C for 4 days (rewarmed). During ripening, chlorophyll fell below a detectable level, carotenes increased 100-fold, phospholipids (PLs) dropped ≈20%, and galactolipids (GLs) dropped ≈35%. Fatty-acid unsaturation decreased slightly. Steryl esters (SEs), more than free sterols (FSs) and steryl glycosides (SGs), increased at the expense of acylated steryl glycosides (ASGs), and in all four steryl lipids, the stigmasterol: sitosterol ratio rose dramatically, whereas the level of isofucosterol fell sharply. During chilling, chlorophyll declined ≈40% and carotenes ≈60%. PL content did not change, whereas GL fell ≈15%. Fatty-acid unsaturation increased slightly. FS, much more than SG and SE, increased at the expense of ASG. The stigmasterol: sitosterol ratio changed little in ASG, SG, and SE but declined in FS. Isofucosterol increased in FS and SE. Rewarming had little effect on the levels of chlorophyll, carotenes, or PL levels, but caused GL to fall another ≈15%. Fatty-acid unsaturation decreased slightly in GL and ASG. The distribution of total sterol in ASG, SG, FS, and SE changed dramatically, yielding proportions close to those in unchilled MG fruit. Also, 4 days after rewarming, the stigmasterol: sitosterol ratio had increased sharply, particularly in FS and SE, and there was a further rise in isofucosterol in all four steryl lipids. These results indicate that chloroplast damage occurs during chilling, but PL-rich cell membranes are not degraded, even upon rewarming. Changes in sterol composition and conjugation during chilling and after rewarming could result in membrane dysfunction.
Bruce D. Whitaker
Plastids and microsomal membranes were isolated from pericarp tissue of mature-green and red-ripe bell pepper (Capsicum annuum L.) fruit harvested from greenhouse- and field-grown plants. The lipid composition of these membrane fractions changed much more with ripening of field-grown than greenhouse-grown fruit. Also, the phospholipid (PL), free sterol (FS), steryl glycoside (SG), and acylated steryl glycoside (ASG) content of microsomes and plastids from green and red fruit were very different under the two growing conditions. Total steryl lipids (TSL = FS + SG + ASG) and the TSL: PL ratio increased in microsomes and decreased in plastids with ripening. These changes were much greater in field-grown fruit. The ASG: SG ratio decreased with ripening in both membrane fractions under both growing conditions. Ripening and growth conditions affected the phospholipid and sterol composition in plastids much more than in microsomes. Lipid changes associated with the chloroplast to chromoplast transformation were similar in field- and greenhouse-grown fruit, including an increase in the galactolipid: PL ratio.
Bruce D. Whitaker and Theophanes Solomos
Farnesene and its conjugated triene oxidation products in apple peel are positively correlated with, and thought to be involved in, the storage disorder superficial scald. Levels of these compounds are often estimated by dipping fruit in hexane and measuring the absorbance of the crude extracts at 232 nm (farnesene) and 269 or 281 minus 290 nm (trienes). We have devised a C18 HPLC method with UV detection at 232 and 269 nm that allows the simultaneous quantitation of 80 ng of farnesene and trienes. Using this method we have confirmed the recent report that one conjugated trien-6-ol comprises 90% of the stable oxidation products of farnesene. It was also found that crude hexane extracts of apple peel can give spuriously high values for farnesene and/or trienes when levels of these compounds are low and other UV-absorbing components are present. A group of compounds unrelated to farnesene, with an absorbance maximum at ≈259 nm, were noted in the peel of cv. Gala apples, which produced little farnesene or trienol. This may explain the report that fruit with a high ratio of A258nm/A281nm in peel extracts have a low incidence of scald. The new HPLC method will be applied in subsequent studies of postharvest factors involved in regulation of farnesene synthesis and oxidation.
Gene E. Lester and Bruce D. Whitaker
Postharvest gamma-irradiation of melons at low dosage has been reported to extend shelf life. This study assessed how irradiation alters the structure and function of plasma membrane (PM) from hypodermal-mesocarp tissue. Administration of gamma rays (1 kGy at 0.017 kGy/min) to mature melon (Cucumis melo L.) fruit caused a 14% drop in H+-ATPase activity within 4 h. Total protein content did not differ in PM from non-irradiated (NIR) vs. irradiated (IR) fruits. Following storage (7 days at 7C then 3 days at 21C), H+-ATPase activity was ≈10% to 20% lower in PM from both groups of fruit, with no difference between the two. Total PM protein had declined by 34% and 49% in IR and NIR fruits, respectively. After irradiation, the phospholipid to protein ratio (PL:protein) was substantially higher in PM from IR fruit (0.67 vs. 0.58 in NIR). With storage, PL:protein dropped to 0.52 in NIR fruit PM, but changed little (0.65) in IR fruit PM. These results may indicate that irradiation stimulates PL synthesis or inhibits PL catabolism. Further analyses of PM lipid content and composition are underway.
Bruce D. Whitaker and Gene E. Lester
Increases in phospholipase D (PLD) and lipoxygenase (LOX) activities are thought to play a key role in senescence of mesocarp tissues in muskmelon fruit. We have cloned and characterized two full-length cDNAs, CmPLDα and CmLOX1, encoding PLDα and LOX proteins in honeydew melon (Cucumis melo L. Inodorus Group). Levels of expression of the corresponding genes were determined by semi-quantitative RT-PCR in developing and mature fruit mesocarp tissues (20–60 d after pollination; DAP), and in roots, leaves, and stems from 4-week-old and flowers from 6-week-old plants. The coding regions of CmPLDα1 and CmLOX1 cDNAs are, respectively, 2427 and 2634 nucleotides long, encoding proteins 808 and 877 amino acids in length. CmPLDα1 is most similar to PLDα genes in castor bean, cowpea, strawberry, and tomato (77% nucleotide identity), and is the first cucurbit PLD gene cloned. CmLOX1 has 94% nucleotide identity to a cucumber LOX gene expressed in roots and 80% identity to cucumber cotyledon lipid body LOX. Transcript of CmPLDα1 was much more abundant than that of CmLOX1, but relative levels of transcript in the various organs and tissues were similar for the two genes. Expression was highest in roots, flowers, and fruit mesocarp tissues. CmPLDα1 expression in fruit was high throughout development, although maximum levels occurred at 50 and 55 DAP, respectively, in middle and hypodermal mesocarp. CmLOX1 expression was generally higher in middle than in hypodermal mesocarp with maximum transcript levels at 55 and 50 DAP, respectively. Overall, the patterns of expression of CmPLDα1 and CmLOX1 are consistent with a model in which their encoded enzymes act in tandem to promote or accelerate senescence in fruit mesocarp tissues.