Leaf disks of Rhododendron cv. `English Roseum' were vacuum infiltrated with buffered solutions of L- or D-phenylalanine. Controls were infiltrated with buffer. Following a 24 hour period under lights (23°C, 16 hr light: 8 hr dark), the disks were cold shocked at 0°C for one hour, then held under the same light regime for three days. Disks were then subjected to a series of cold treatments, thawed, and analyzed for damage visually and by phenolic leakage. Disks infiltrated with D-phenylalanine consistently showed greater damage at warmer temperatures than did disks infiltrated with the L isomer. We believe the inhibitory action of D-phenylalanine upon phenylalanine ammonia-lyase is responsible for this decrease in cold hardiness.
P. Lyn Ostolski, Linda Chalker-Scott, and James D. Scott
Phillip N. Miklas, Kenneth F. Grafton, and Phillip E. McClean
We investigated the partial physiological resistance (PPR) of common beans (Phaseolus vulgaris L.) to white mold disease caused by Sclerotinia sclerotiorum (Lib.) deBary. The activity of phenylalanine ammonia-lyase (PAL) was measured in detached stems inoculated with a growing mycelium of the pathogen. Noninoculated detached stems and whole plants were included as controls. Five bean cultivars-Upland, Bunsi, Sierra, UI-114, and Montcalm-and one breeding line-NY 5394-were tested; all varied in PPR to white mold disease. Greater PAL activity in the resistant NY 5394 than in the susceptible `Upland' suggests that PAL activity may be involved in the PPR of common beans to S. sclerotiorum.
Jung-Yoon Yi, Hyo-Won Seo, Kwan-Soo Kim, Hyun-Mook Cho, and Kuen-Woo Park
Antimicrobial peptide gene (shiva) under the promoter of tomato phenylalanine ammonia-lyase (tPAL5) was transformed into potato (Solanum tuberosum L.) plants. Antimicrobial peptide gene was isolated originally from giant silk moth (Hyalophora cecropia) and modified its nucleotide seqnence to increase antimicrobial activity. Phenylalanine ammonia lyase 5 (PAL5) gene was known to express highly by wounding, irradiation, and infection by pathogens. It also expresses specifically on vessel tissues of young roots, stems, and leaves. The vector with shiva and CaMV35S promoter was also introduced into potatoes. The efficiency of regeneration was maximized at the medium containing Zeatin 2 mg/L, NAA 0.01 mg/L, GA3 0.1 mg/L. Putative transgenic potato plants were cultured on the media containing kanamycin 50 mg/L. From the tissue extracts of putative transgenic plants, GUS activity was assayed using 4-methylumbellyferyl glucuronide (MUG) as a substrate for GUS enzyme. In several transformant, GUS activity was 20- to 40-fold higher than non-transformants. Especially, one clone with CaMV35S promoter expressed ≈400-fold higher GUS activity than nontransformants. For histochemical in situ localization of GUS activity, chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide(X-gluc) was used for staining. GUS was highly expressed in the whole tissue of the transformants under CaMV35S promoter, but the other side GUS was expressed especially in the vascular tissues of stems and leaves of transformants with tPAL5 promoter. PCR was carried out at 94 °C for 20 s, 52 °C for 20 s, and 72 °C for 60 s with 45 cycles, using NPTII gene-specific primer set. PCR amplification by NPTII-specific primers confirmed 0.5-kb band in most transformants.
Three years of experiments were carried out with both Delicious fruit on trees and fruit skin discs. There were two peaks of phenylalanine ammonia lyase (PAL) activity during fruit development. One occurred in the fruitlet stage and the other in the fruit enlargement stage. The first peak was coincident with anthocyanin synthesis in fruitlet but the second peak did not correlate with pigment formation during maturation. In fact, PAL activity decreased gradually during fruit maturation and coloration. Treatment with L-α-aminooxy-B-phenylpropionic acid, a specific PAL inhibitor, decreased PAL activity in fruit and in skin discs 57% and 80%, respectively, but did not change anthocyanin content. Cycloheximide inhibited anthocyanin synthesis by 76% in fruit and 85% in skin discs, but did not significantly inhibit PAL activity. On the other hand, PAL activity was positively correlated with concentrations of simple phenols which were direct products of PAL and precursors for synthesis of lignin, anthocyanin and other flavonoid.
W.J. Steyn, D.M. Holcroft, S.J.E. Wand, and G. Jacobs
Changes in activity of phenylalanine ammonia-lyase (PAL) and UDPGalactose: flavonoid-3-o-glycosyltransferase (UFGT) during the development of pear (Pyrus communis L.) fruit and in response to cold fronts were assessed and related to changes in red color. Red and blushed pear cultivars attained maximum redness and highest anthocyanin concentrations in immature fruit. Red color generally faded toward harvest. UFGT activity increased over fruit development and was apparently not limiting to color development. However, the fading of red color and the decreasing level of phenolic compounds toward harvest might relate to decreasing PAL activity. Skin color and enzyme activity in the red pear `Bon Rouge' displayed little responsiveness to low temperatures. In contrast, low temperatures increased red color and activity of both PAL and UFGT in the blushed pear `Rosemarie'. Consistent with the general pigmentation pattern described above, the effect of temperature on enzyme activity was much greater early during fruit development than in the week before harvest.
Deirdre M. Holcroft, Maria I. Gil, and Adel A. Kader
`Wonderful' Pomegranates (Punica granatum L.) were placed in jars ventilated continuously with air or air enriched with 10 or 20 kPa CO2 at 10 °C for 6 weeks. Samples were taken initially and after 1, 2, 4, and 6 weeks, and postharvest quality attributes were measured. The arils of the pomegranates stored in air were deeper red than the initial controls and than those stored in CO2-enriched atmospheres. This increased color was associated with increased anthocyanin concentration. Arils from fruit stored in air enriched with 10 kPa CO2 had a lower anthocyanin concentration than air-stored fruit, and atmospheres enriched with 20 kPa CO2 had even lower levels, possibly from suppressed anthocyanin biosynthesis. Anthocyanin concentration correlated well with the activity of phenylalanine ammonia lyase but not with glucosyltransferase activity. Moderate CO2 atmospheres (10 kPa) prolong the storage life and maintain quality of pomegranates, including adequate red color intensity of the arils.
Mark A. Ritenour, Ellen G. Sutter, David M. Williams, and Mikal E. Saltveit
This study was undertaken to determine if endogenous IAA content and axillary bud development correlate with phenylalanine ammonia-lyase (PAL) induction and russet spotting (RS) susceptibility among RS susceptible and resistant cultivars of Iceberg lettuce (Lactuca sativa L.). Final levels of ethylene-induced PAL activity and RS development were highly correlated among cultivars, field conditions, and harvest dates. Harvested Iceberg lettuce midribs contained relatively low amounts of free IAA (maximum of 5.2 ng·g-1 fresh weight). There was poor correlation between free IAA content in lettuce leaf midribs and final RS development among all cultivars, growing conditions, and harvest dates. Axillary bud development, as measured by the number of visible buds per head, bud weight, or bud length, were not significantly correlated with final RS development or midrib IAA content. Cultivars with higher initial free IAA content lost much of their IAA after 8 days storage at 5C in air ± ethylene.
Galen Peiser, Gloria López-Gálvez, Marita Cantwell, and Mikal E. Saltveit
Russet spotting is a physiological disorder of lettuce (Lactuca sativa L.) caused by exposure to hormonal levels (<1 μL·L-1) of ethylene in air at ≈5 °C. Enhanced phenolic metabolism and the accumulation of phenolic compounds accompany the appearance of brown, oval lesions on the leaf midrib. Phenylalanine ammonia-lyase (PAL) is the first committed enzyme in the phenylpropanoid pathway. Three inhibitors of PAL activity [2-aminoindan-2-phosphonic acid (AIP), α-aminooxyacetic acid (AOA), and α-aminooxi-β-phenylpropionic acid (AOPP)] greatly reduced the accumulation of phenolic compounds and browning of lesions. At a concentration of 50 μm, AIP inhibited the formation of chlorogenic and dicaffeoyl tartaric acids in cut midribs of iceberg lettuce by 92% and 98%, respectively. AIP competitively inhibited PAL activity from a lettuce midrib homogenate with an apparent Ki of 22 nm. While the formation of phenolic compounds was strongly inhibited by AIP, the number of lesions associated with russet spotting was not affected. Only the color of the lesions was affected by AIP. In control midribs the russet spotting lesions were brown while those in the AIP-treated midribs were initially olive green and after 3 to 7 days these lesions turned the characteristic brown color. No tyrosine ammonia-lyase activity was detected in a homogenate of lettuce midrib tissue. These results indicate that the early development of russet spotting lesions is independent of the increase in PAL activity and phenolic compounds rather than an effect of these increases as previously suggested. However, accumulation of phenolic compounds does contribute to the subsequent browning symptoms indicative of russet spotting.
M.A. Ritenour, M.E. Saltveit, and M.J. Ahrens
Russet spotting (RS) is an important postharvest disorder in Iceberg lettuce (Lactuca sativa L.). Previous studies showed that RS is induced by exposure to ∼5 ppm (ul/l) ethylene at ∼5C for 3 days and is characterized by the appearance of 1 to 2 mm diam. oval, brown sunken spots along the midrib. Increases in phenylalanine ammonia-lyase (PAL) activity and phenolic content are highly correlated with RS development. Ethylene-induced PAL activity is much less at higher (12C) or lower (0C) temperatures. In this study isolated whole leaves were exposed to a log series of ethylene concentrations from 0.1 to 10 ppm at temperatures from 0.0C to 20C for up to 8 days. Tissue was transferred among these various treatments to investigate the kinetics of PAL induction, activity and deactivation, phenolic accumulation, and RS development. A subjective evaluation was then made of RS development using a 1 to 9 scoring system in which 1 was no RS, and then PAL activity and phenolic content were measured. Preliminary results indicate that ethylene-induced PAL activity was decreased more rapidly upon transfer to temperatures above 10C than to 0.0C. Accumulation of phenolic compounds and development of RS paralleled each other, and were positively related to PAL activity. Practical implications of these results will be discussed.
Mark Ritenour and Mikal E. Saltveit Jr.
Activity of phenylalanine ammonia-lyase (PAL) is critical in the induction of russet spotting (RS) in leaves of Iceberg lettuce (Lactuca sativa L.). RS is a major postharvest disorder of lettuce caused by exposure to ppm levels of ethylene at = 5C. Both PAL and RS are decreased when lettuce tissue previously exposed to ethylene is stored at = 15C or is transferred from = 5C to = 15C. To study the induction and inactivation of PAL, we exposed lettuce leaves to air ± 10 ppm ethylene at 5C for four days to initially induce high PAL levels. After four days, leaves were treated with water ± 2 mg/L cycloheximide, and transferred to air at 5 or 15 C. In leaves previously exposed to ethylene, PAL activity decreased rapidly to baseline levels within two days in non-cycloheximide treated leaves transferred to 15C. PAL activity remain elevated in the same treatment held at 5C. In leaves treated with cycloheximide and transferred to 15C, PAL did not begin to decrease until after four days. Cycloheximide treated leaves held at 5C showed increased PAL activity both two and four days after treatment.