N. Richard Knowles

Structural analysis of phosphatidylcholines by post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The utility of post-source decay (PSD) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was investigated for the structural analysis of phosphatidylcholine (PC). PC did not produce detectable negative molecular ion from MALDI, but positive ions were observed as both [PC+H]+ and [PC+Na]+. The PSD spectra of the protonated PC species contained only one fragment corresponding to the head group (m/z 184), while the sodiated precursors produced many fragment ions, including those derived from the loss of fatty acids. The loss of fatty acid from the C-1 position (sn-1) of the glycerol backbone was favored over the loss of fatty acid from the C-2 position (sn-2). Ions emanating from the fragmentation of the head group (phosphocholine) included [PC+Na-59]+, [PC+Na-183]+ and [PC+Na-205]+, which corresponded to the loss of trimethylamine (TMA), non-sodiated choline phosphate and sodiated choline phosphate, respectively. Other fragments reflecting the structure of the head group were observed at m/z 183, 146 and 86. The difference in the fragmentation patterns for the PSD of [PC+Na]+ compared to [PC+H]+ is attributed to difference in the binding of Na+ and H+. While the proton binds to a negatively charged oxygen of the phosphate group, the sodium ion can be associated with several regions of the PC molecule. Hence, in the sodiated PC, intermolecular interaction of the negatively charged oxygen of the phosphate group, along with sodium association at multiple sites, can lead to a complex and characteristic ion fragmentation pattern. The preferential loss of sn-1 fatty acid group could be explained by the formation of an energetically favorable six-member ring intermediate, as apposed to the five-member ring intermediate formed prior to the loss of sn-2 fatty acid group.

Characterization of Solanum Tuberosum Multicystatin and its Structural Comparison with Other Cystatins.

Potato (Solanum tuberosum) multicystatin (PMC) is a crystalline Cys protease inhibitor present in the subphellogen layer of potato tubers. It consists of eight tandem domains of similar size and sequence. Our in vitro results showed that the pH/PO4−-dependent oligomeric behavior of PMC was due to its multidomain nature and was not a characteristic of the individual domains. Using a single domain of PMC, which still maintains inhibitor activity, we identified a target protein of PMC, a putative Cys protease. In addition, our crystal structure of a representative repeating unit of PMC, PMC-2, showed structural similarity to both type I and type II cystatins. The N-terminal trunk, α-helix, and L2 region of PMC-2 were most similar to those of type I cystatins, while the conformation of L1 more closely resembled that of type II cystatins. The structure of PMC-2 was most similar to the intensely sweet protein monellin from Dioscorephyllum cumminisii (serendipity berry), despite a low level of sequence similarity. We present a model for the possible molecular organization of the eight inhibitory domains in crystalline PMC. The unique molecular properties of the oligomeric PMC crystal are discussed in relation to its potential function in regulating the activity of proteases in potato tubers.

Temperature-induced changes in potato processing quality during storage are modulated by tuber maturity

Knowledge of how potato (Solatium tuberosum L.) cultivars respond to non-conventional storage temperature regimes may expand options for managing immature, overmature, or otherwise stressed potatoes. Here we demonstrate that temperature-dependent changes in the processing quality of tubers are modulated by tuber maturity. ‘Russet Burbank’ (RB), ‘Ranger Russet’ (RR), and ‘Umatilla Russet’ (UR) tubers from early- and late- planted crops (grown for 163 d and 133 d, respectively) were conditioned for a month at 4.5, 6.7, or 9 C, and subsequently stored at 4.5, 6.7 or 9 C (nine temperature regimes) for an additional 182 d before reconditioning at 16 C. Fry color darkened as conditioning temperature (CT) decreased from 9 to 4.5 C, and tubers were most sensitive to low temperatures for loss of processing quality during the initial conditioning period following wound-healing. Conditioning at higher temperatures increased the tolerance of tubers to lower subsequent holding temperatures (HT) later in the storage season. The processing quality of chronologically older (early planting) RR and UR tubers was inferior to the younger tubers (late planting), resulting in fewer CT/HT combinations for managing the former. Regardless of maturity, RR tubers maintained processing quality for up to 230 d when conditioned and stored at higher temperatures; however, tubers from the late planting had a greater tolerance of lower temperatures than those from the early planting. Although reconditioning improved the processing quality for all cultivars and thus expanded the CT/HT management options, the responsiveness of cold-sweetened UR tubers to reconditioning attenuated over the 230 d storage period, reflecting earlier onset of irreversible senescent sweetening in this cultivar. Chronologically younger tubers of the most dominant processing cultivars in the Pacific Northwest stored better than tubers produced over a prolonged growing season. Collectively, these studies suggest strategies to manage tubers of differing maturity to best preserve processing quality during storage.ResumenEl saber de cómo los cultivares de papa (Solanum tuberosum L.) responden a regímenes de temperatura no convencional de almacenamiento puede aumentar las opciones para el manejo de papas inmaduras, sobre maduras o estresadas. Aquí demostramos que los cambios temperatura-dependiente de la calidad del producto son regulados por la madurez del tubérculo. Los tubérculos de ‘Russet Burbank’ (RB), ‘Ranger Russet’ (RR) y ‘Umatilla Russet’ (UR) de cultivos sembrados temprano y tardíamente (crecidos por 163 y 133 días, respectivamente) fueron acondicionados por un mes a 4.5, 6.7 y 9 C y luego almacenados por 182 días adicionales a 4.5, 6.7 o a 9 C (nueve regímenes de temperatura) antes de reacondicionarlos a 16 C. E1 color de la fritura se oscureció a medida que la temperatura de acondicionamiento (CT) disminuyó de 9 a 4.5 C y los tubérculos fueron más sensibles a las temperaturas bajas para pérdida de su calidad de procesamiento durante el periodo inicial de acondicionamiento, después de la cicatrización de las heridas. El acondicionamiento a temperaturas más altas incrementó la tolerancia de los tubérculos a mantenimiento posterior a temperaturas mas bajas (HT) hacia el final del período de almacenaje. La calidad de procesamiento de los tubérculos más viejos (siembra temprana) de RR y UR fue inferior a la de tubérculos más jóvenes (siembra tardía) resultando en pocas combinaciones CT/HT de mane jo para los primeros. Prescindiendo de la madurez, los tubérculos de RR mantuvieron la calidad de procesamiento hasta 230 días cuando se acondicionaron y almacenaron a temperaturas más altas; sin embargo, los tubérculos de siembra tardía tuvieron mayor tolerancia a temperaturas bajas que aquellos de siembra temprana. Aunque el acondicionamiento mejoró la calidad de procesamiento de todos los cultivares y por lo tanto amplió las opciones de manejo CT/HT, la respuesta de los tubérculos de UR endulzados por frío al acondicionamiento se atenuó después de 230 días de almacenamiento, reflejando una temprana aparición de dulzor senescente irreversible en este cultivar. Tubérculos cronológicamente más jóvenes de los cultivares prominentes en el Pacífico noroccidental se almacenaron mejor que los tubérculos de permanencia prolongada en el campo. Colectivamente, estos estudios sugieren estrategias de manejo de tubérculos de diferente estado de madurez para preservar mejor su calidad de procesamiento durante el almacenamiento.

Phosphorus status affects postharvest respiration, membrane permeability and lipid chemistry of European seedless cucumber fruit (Cucumis sativus L.)

Fruit of European seedless cucumber were grown in a greenhouse under low and high phosphorus (P) fertilization regimes. Tissue P concentration of fruit (number one grade) from low-P plants was, on average, 45% of that of fruit from high-P plants. Fruit P status affected membrane lipid chemistry and fruit respiration after harvest. Mesocarp tissue of low-P fruit had a lower concentration of phospholipids, lower level of unsaturation in various pools of fatty acids, and a greater rate of electrolyte leakage than that of high-P fruit. On average, respiration of low-P fruit was 21% higher than that of high-P fruit over a 16-day postharvest interval at 23°C. Moreover, low-P fruit experienced a climacteric in respiration that began about 40 h after harvest, reached a maximum at 72 h, and declined to preclimacteric levels by 90 h. The difference in respiration rate between low- and high-P fruit was as high as 57% during the climacteric. The respiratory climacteric was unique to the low-P fruit and was not associated with an increase in fruit ethylene concentration or ripening. Phosphorus nutrition can thus alter the postharvest physiology of cucumber fruit by affecting membrane lipid chemistry, membrane integrity and respiratory metabolism.

Characterization of Solanum tuberosum Multicystatin and the Significance of Core Domains

This study characterizes the structure and significance of the core of potato multicystatin (PMC), a multidomain cysteine protease inhibitor found in the cortical parenchyma tissue of potato tubers. Papain inhibitory properties of native and recombinant PMC containing core domains are affected by pH. It is likely that pH-mediated regulation imparts unique properties to PMC that modulate proteolysis upon wounding and/or infection via inhibiting cysteine proteases. Potato (Solanum tuberosum) multicystatin (PMC) is a unique cystatin composed of eight repeating units, each capable of inhibiting cysteine proteases. PMC is a composite of several cystatins linked by trypsin-sensitive (serine protease) domains and undergoes transitions between soluble and crystalline forms. However, the significance and the regulatory mechanism or mechanisms governing these transitions are not clearly established. Here, we report the 2.2-Å crystal structure of the trypsin-resistant PMC core consisting of the fifth, sixth, and seventh domains. The observed interdomain interaction explains PMC’s resistance to trypsin and pH-dependent solubility/aggregation. Under acidic pH, weakening of the interdomain interactions exposes individual domains, resulting in not only depolymerization of the crystalline form but also exposure of cystatin domains for inhibition of cysteine proteases. This in turn allows serine protease–mediated fragmentation of PMC, producing ∼10-kD domains with intact inhibitory capacity and faster diffusion, thus enhancing PMC’s inhibitory ability toward cysteine proteases. The crystal structure, light-scattering experiments, isothermal titration calorimetry, and site-directed mutagenesis confirmed the critical role of pH and N-terminal residues in these dynamic transitions between monomer/polymer of PMC. Our data support a notion that the pH-dependent structural regulation of PMC has defense-related implications in tuber physiology via its ability to regulate protein catabolism.

Changes in lipid molecular species and sterols of microsomal membranes during aging of potato (Solanum tuberosum L.) seed-tubers

Changes in sterols and the molecular species composition of polar lipids from microsomal membranes were characterized as a prerequisite to determining how lipid chemistry affects membrane susceptibility to peroxidation during aging of potato tubers. Polar lipid content of the microsomal fraction fell 17% (protein basis) as tubers aged from 2 to 38 mon at 4°C. In younger seed-tubers, PC concentration (protein basis) was the highest, followed by digalactosyldiacylglycerol (DGDG), PE, monogalactosyldiacylglycerol (MGDG), and PI. PC and PE increased 14 and 27%, respectively, whereas glycolipids fell 64 and PI 43% with advancing age. These changes resulted in PC and PE dominating the microsomal membrane lipids of 38-mon-old tubers. Nonpositional analysis of lipid molecular species across lipid pools showed an increase in 16∶0/18∶3, 18∶3/18∶3, and 18∶2/18∶3 (PC and PE only), and a decline in 18∶2/18∶2 and 16∶0/18∶2 (except for MGDG) with advancing tuber age. The increase in 18∶3-bearing species effected a linear increase in double-bond index (DBI) of PC and PE during aging. The DBI of DGDG did not change with age; however, it fell 65% for MGDG, resulting in an overall decrease in average microsomal DBI. In addition, Δ5-avenasterol and stigmasterol concentrations increased 1.6- and 3.3-fold, respectively, effecting a significant increase in the sterol/phospholipid ratio with advancing tuber age. The increase in sterol/phospholipid ratio and the possibility that the increased unsaturation of microsomal membranes reflects a compensatory response to maintain optimal membrane function in light of the age-induced loss of galactolipid and PI are discussed.

Toxicity and Metabolism of Exogenous α,β-Unsaturated Carbonyls in Potato (Solanum tuberosum L.) Tubers

A group of aliphatic α,β-unsaturated carbonyl compounds was evaluated for their utility as inhibitors of sprout growth in stored potato tubers (Solanum tuberosum L.). Nondormant tubers were treated with vapors of six 8-10-carbon compounds of this chemistry. Subsequent sprout growth at 16 °C (95% relative humidity) over ca. 3 months in storage was suppressed by all compounds in a concentration-dependent manner. The volatile metabolites produced by sprout and associated tuber tissues following treatment with 3-octen-2-one, 3-nonen-2-one, and 3-decen-2-one were the corresponding alkyl ketones and alkyl secondary alcohols. In contrast, (E)-2-octenal, (E)-2-nonenal, and (E)-2-decenal were metabolized by two pathways: (1) parent compound to the corresponding alkyl aldehyde and then to the alkyl primary alcohol and (2) parent compound to the alkenyl primary alcohol. Residues of 3-nonen-2-one and (E)-2-nonenal and their metabolites were analyzed in whole tubers over a 28 day post-treatment period. The concentrations of the parent ketone and aldehyde declined rapidly following application, and the most persistent metabolites were 2-nonanol and (E)-2-nonen-1-ol, respectively. The sequence of reactions leading from the α,β-unsaturated carbonyls to the alcohols was determined by application of each of the 9-carbon compounds individually to tubers. In long-term efficacy studies, a single application of (E)-2-nonenal and 3-nonen-2-one to nondormant tubers terminated sprout growth and prevented regrowth for 2-3 months. A second application suppressed sprouting for at least 4-5 additional months. This efficacy, combined with rapid metabolism and low residue levels, makes the 8-10-carbon α,β-unsaturated ketones and aldehydes worth consideration for use as sprout inhibitors.

Correlative changes in proteases and protease inhibitors during mobilisation of protein from potato (Solanum tuberosum) seed tubers

Potato tubers (Solanum tuberosum L.) contain protease inhibitors that function in plant defence and as storage proteins. A multi-domain cysteine protease inhibitor, potato multicystatin (PMC), has also been implicated in regulating protein accumulation in developing tubers by inhibiting proteases. Unlike developing tubers, sprouting tubers mobilise protein reserves to support growth of developing plants and, therefore, show an increase in protease activity. Using single-eye containing cores (seedcores) from seed tubers, we characterised the relative changes in patatin, PMC, proteases and serine (Ser) protease inhibitors, as a prerequisite to further research on their potential roles in protein mobilisation from tubers during plant establishment. Approximately 63% of seedcore dry matter was mobilised over a 29-day period of plant establishment (1.7 mg seedcore dry matter mobilised for every mg increase in plant dry matter). The gelatinolytic protease isoforms induced in seedcores during plant establishment differed from those characterised previously in developing tubers. Total protease activity increased progressively in seedcores and reached a maximum 23 days after planting. Conversely, seedcore soluble protein content declined, with patatin accounting for the greatest decrease in the soluble protein fraction during plant establishment. PMC also decreased 44% and Ser (trypsin) protease inhibitors decreased to levels barely detectable in seedcores over the 29-day growth interval. Moreover, the temporal changes in PMC, protease activity and patatin content were highly correlated. As PMC decreased from 6 to 4 ng core–1, protease activity increased 9-fold, patatin decreased 2.6-fold and total soluble protein decreased by 58%. These results suggest that catabolism of protease inhibitors may facilitate protein mobilisation from seed tubers. Further work to define unequivocally the role of protease inhibitors in modulating the activity of proteases during protein mobilisation from tubers is warranted.

Protein Mobilization from Potato Tubers during Long-Term Storage and Daughter Tuber Formation

The role of protease inhibitors in modulating changes in protein content of potato (Solanum tuberosum L.) tubers was investigated using a mother/daughter tuber model system. Changes in patatin, potato multicystatin (PMC), proteases, serine (Ser) protease inhibitors, and their gene expression were temporally coordinated over a 22-mo storage interval in genotypes with short (cv. Ranger Russet) and long (cv. Russet Burbank) dormancy. Daughter tubers were initiated on Ranger Russet tubers at ∼15 mo. PMC (Cys protease inhibitor) declined linearly (∼4.2-fold) in Ranger Russet mother tubers from 4 to 15 mo and then maintained low levels through 22 mo. Conversely, protease activity was low and constant from 4 to 15 mo before increasing 7.4-fold through 22 mo. This increase coincided with the most rapid decline (54%) in patatin and the formation of daughter tubers. The proteases induced during aging of mother tubers were inhibited by PMC. Ser protease inhibitors were maintained in mother tubers throughout storage. In contrast, as daughter tubers developed, PMC and Ser protease inhibitors increased, protease activity declined to 17% of initial levels, and patatin increased threefold. These results implicate a role for protease inhibitors in regulating protein content during mobilization from mother tubers and deposition in daughter tubers.

Lipid metabolism during aging of high-α-linolenate-phenotype potato tubers

Abstract Previous studies demonstrated that high levels of α-linolenate in cell membranes of potato tubers (achieved by overexpressing fatty acid desaturases) enhances lipid peroxidation, oxidative stress, and tuber metabolic rate, effectively accelerating the physiological age of tubers. This study details the changes in lipid molecular species of microsomal and mitochondrial membranes from wild-type (WT) and high-α-linolenate tubers during aging. The microsomal and mitochondrial polar lipids of high-α-linolenate tubers were dominated by 18:3/18:3 and 16:0/18:3 molecular species. Relative to WT tubers, high-α-linolenate tubers had a substantially higher 16:0/18:n to 18:n/18:n molecular species ratio in mitochondria and microsomes, potentially reflecting a compensatory response to maintain membrane biophysical properties in the face of increased unsaturation. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) accounted for 53 and 37% of polar lipids, respectively, in mitochondria from younger WT and high-α-linolenate tubers. The relative proportions of these phospholipids (PL) did not change during aging of WT tubers. In contrast, PE increased to dominate the PL pool of mitochondria during aging of high-α-linolenate tubers. While aging effected an increase in mitochondrial 18:3-bearing PCs and PEs in WT tubers, the concentration of 18:3-bearing PCs fell with a concomitant increase in 18:3-bearing PEs during aging of high-α-linolenate tubers. These age- and high-α-linolenate-induced changes had no effect on the respiration rate and functional integrity of isolated mitochondria. Differential increases in the respiration rates of WT and high-α-linolenate tubers during aging were therefore a consequence of unsaturation-dependent alterations in the microenvironments of cells. Microsomal 18:3-bearing PCs, PEs, digalactosyldiacylglycerols (DGDG), and monogalactosyldiacylglycerols all increased in WT tubers during aging. In contrast, a selective loss of 18:3-bearing PCs and DGDGs from microsomes of high-α-linolenate tubers likely reflects a greater susceptibility of membranes to peroxidative catabolism during aging. Aging resulted in an increase in sterol/PL ratio in microsomes from WT tubers, due primarily to a decline in PL. In high-α-linolenate tubers, the increase in sterol/PL ratio during aging was due to increases in Δ 5-avenasterol and stigmasterol, indicating membrane rigidification and likely contributing to increased membrane permeability. Age-induced changes in 18:3-bearing lipids in membranes of transformed tubers are discussed relative to the development of oxidative stress and accelerated aging.