A Protein Hydrolysate Mitigates the Adverse Effect of Chilling Stress on Cucumber Plants

Salicylic acid (SA) has been proven to be a multifunctional signaling molecule that participates in the response of plants to abiotic stresses. In this study, we used cold-sensitive cucumber and cold-tolerant pumpkin as experimental materials to examine the roles of SA in root–shoot communication responses to aerial or/and root-zone chilling stress in own-root and hetero-root grafted cucumber and pumpkin plants. The results showed that pumpkin (Cm) rootstock enhanced the chilling tolerance of grafted cucumber, as evidenced by the observed lower levels of electrolyte leakage (EL), malondialdehyde (MDA), and higher photosynthetic rate (Pn) and gene expression of Rubisco activase (RCA). However, cucumber (Cs) rootstock decreased the chilling tolerance of grafted pumpkins. Cs/Cm plants showed an increase in the mRNA expression of C-repeat-binding factor (CBF1), an inducer of CBF expression (ICE1), and cold-responsive (COR47) genes and CBF1 protein levels in leaves under 5/25 and 5/5°C stresses, or in roots under 25/5 and 5/5°C stresses, respectively, compared with the Cs/Cs. Chilling stress increased the endogenous SA content and the activity of phenylalanine ammonia-lyase (PAL), and the increase in SA content and activity of PAL in Cs/Cm plants was much higher than in Cs/Cs plants. Transcription profiling analysis revealed the key genes of SA biosynthesis, PAL, ICS, and SABP2 were upregulated, while SAMT, the key gene of SA degradation, was downregulated in Cs/Cm leaves, compared with Cs/Cs leaves under chilling stress. The accumulation of SA in the Cs/Cm leaves was mainly attributed to an increase in SA biosynthesis in leaves and that in transport from roots under aerial and root-zone chilling stress, respectively. In addition, exogenous SA significantly upregulated the expression level of cold-responsive (COR) genes, enhanced actual photochemical efficiency (ΦPSII), maximum photochemical efficiency (Fv/Fm), and Pn, while decreased EL, MDA, and CI in grafted cucumber. These results suggest that SA is involved in rootstock–scion communication and grafting-induced chilling tolerance by upregulating the expression of COR genes in cucumber plants under chilling stress.

以谷子(<em>Setaria italica </em>(L.) Beauv.)为对象,从拔节期开始持续给予低氮(1.875 mmol/L)和高氮(15 mmol/L)两种氮供应条件并从抽穗期开始进行26 d两种强度(4.29、7.12 kJ·m^-2·d^-1)的增强UV-B辐射处理,研究了谷子叶中光合色素含量、类黄酮含量和苯丙氨酸解氨酶(PAL)活性的变化。结果表明:与高氮供应条件相比,低氮供应条件明显降低了谷子叶中光合色素含量但提高了类胡萝卜素/叶绿素含量比值;在开花期中段和灌浆期中段,高氮供应条件下谷子叶中光合色素含量对增强UV-B辐射比低氮供应条件下的谷子更敏感。从灌浆期开始到处理结束,两种影响因子对谷子叶中类黄酮含量均有显著影响,增强UV-B辐射导致谷子叶中类黄酮含量逐渐升高,且相同增强UV-B辐射强度下低氮供应条件下的谷子叶中类黄酮含量明显高于高氮供应条件下的谷子。谷子叶中PAL活性对两种影响因子的响应较类黄酮含量更加敏感,低氮供应条件使谷子叶中PAL活性明显提高。结合上述指标的相关性分析结果可知,低氮供应条件加强了处于繁殖期主要阶段的谷子叶中类黄酮的积累,并使谷子叶中的类胡萝卜素/叶绿素含量比值明显提高,进而有助于维持谷子叶中光合色素含量在增强UV-B辐射条件下的相对稳定性,对植株抵抗UV-B辐射伤害有利。;Enhanced UV-B radiation resulting from ozone depletion in stratosphere with the increase of human activities can harm plants greatly, whereas the change of nitrogen supply can affect the sensitivity and resistance to UV-B radiation in plants. Foxtail millet (<em>Setaria italica </em>(L.) Beauv.), a typical C₄ crop plant, was chosen as experimental model to investigate the effects of enhanced UV-B radiation and nitrogen supply on photosynthetic pigments and non-enzymatic protection system in leaves. The plants of foxtail millet were fertilized under two different nitrate-nitrogen conditions (1.875, 15 mmol/L) from the jointing stage to the grouting stage, and were stressed by two different intensities of enhanced UV-B radiation (4.29, 7.12 kJ·m^-2·d^-1) from the heading stage to the grouting stage (26 days). During this period, the photosynthetic pigments and flavonoids content and the phenylalanine ammonia-lyase (PAL) activity in leaves of foxtail millet were measured. The nitrogen supply showed significant effect on the photosynthetic pigments contents in leaves of both UV-B treated and non-treated foxtail millet. The leaves in low nitrogen supply group showed significantly lower photosynthetic pigments content but higher carotenoid/chlorophyll ratio when compared to those of high nitrogen supply group according to most of statistical test results (<em>P</em><0.05, least-significant difference, LSD). However, enhanced UV-B radiation treatment showed significant effects on the photosynthetic pigments contents in leaves of foxtail millet in only the 11th day and the 21st day, and the photosynthetic pigments were more sensitive to enhanced UV-B radiation treatment at the high nitrogen group compared to the low nitrogen group. The nitrogen supply showed significant effect on PAL activity in leaves of foxtail millet during the whole period of this experiment, whereas enhanced UV-B radiation showed significant effect on the PAL activity only after the 11th day. Meanwhile, most LSD results showed that the PAL activity in leaves of foxtail millet increased in the low nitrogen supply group compared to the high nitrogen group, whether treated by enhanced UV-B radiation or not. Furthermore, both the nitrogen supply and enhanced UV-B radiation affected the flavonoids content in leaves of foxtail millet significantly after the 16th day, and the flavonoids content in leaves increased significantly in the low nitrogen supply in the 26th day independent of enhanced UV-B radiation. There was a significant positive correlation between the flavonoids content and the PAL activity, but both the flavonoids content and the PAL activity had a significantly negative correlation with the chlorophyll content. Moreover, there was a significant correlation between the chlorophyll content and the carotenoid content. This study indicated that in leaves of foxtail millet, low nitrogen supply treatment could enhanced accumulation of flavonoids and the carotenoid/chlorophyll ratio, thus caused more stability of photosynthetic pigments contents when foxtail millet was stressed by enhanced UV-B radiation compared to the high nitrogen supply during the breeding period. Therefore, low nitrogen supply treatment may improve the resistance to UV-B radiation in plants.

In some instances, lignin content may not be significantly correlated with phenylalanine ammonia lyase (PAL) activity because: (1) PAL is not committed exclusively to lignin, and (2) the time of maximum PAL activity may not coincide with maximum lignin deposition. This study evaluates correlations and timing of PAL activity and lignin deposition during legume stem maturation. Three forage legumes, alfalfa (Medicago sativa L.), birdsfoot trefoil (Lotus corniculatus L.), and red clover (Trifolium pratense L.), were established, and basal stem regrowth was sampled, biweekly, for 10 wk, for dry weight (DW), cell wall (CW), lignin, and PAL analyses. Nonlinear regression of lignin content by the Gompertz function indicated that lignin increased sigmoidally, and PAL activity by the third-order quadratic demonstrated rapid initial increases in activity, followed by decreases, as a function of regrowth days. First derivative of the Gompertz function demonstrated that changes in lignin deposition closely resembled changes in PAL activity. Among species, peak deposition of DW and CW content occurred 3–11 d prior to maximum lignin deposition. Time of maximum PAL activity occurred 8 d prior to maximum lignin deposition in birdsfoot trefoil and red clover and 3 days after maximum lignin deposition in alfalfa. Across species, lignin content was not positively correlated with PAL activity on a protein basis. However, lignin deposition was positively correlated with PAL per unit protein (r = 0.76, P &lt; 0.05) and lignin content was positively correlated with PAL on a per plant basis (r = 0.60, P &lt; 0.05). These results indicate that the activity of PAL is related to lignin deposition in a cause-and-effect relationship. Key words: Alfalfa, birdsfoot trefoil, cell wall, lignin, phenylalanine, ammonia lyase, red clover

Phenylalanine ammonia lyase (PAL) activity was measured in p-fluorophenylalanine (PFP)-sensitive and -resistant tobacco (Nicotiana tabacum L.) and carrot (Daucus carota L.) cell lines which are known to oversynthesize phenylalanine. A correlation between phenolic levels and PAL activities was detected. The phenylalanine analog-resistant and -sensitive carrot cells showed no differences in the accumulation of phenolic compounds and PAL activities. The PFP-resistant tobacco cells, however, had 10 times higher levels of phenolics and also 10 to 20 times higher PAL activities than the PFP-sensitive line. The PAL activity in the resistant tobacco line increased dramatically after inoculation of the cells into fresh medium. Conditions affecting this increase were characterized.A comparison of the responses of PAL activity to light treatment and changes in the 2,4-dichlorophenoxyacetic acid concentration in the medium revealed no differences for both tobacco lines. After inoculation of the cells into fresh medium, the rates of nitrate uptake from the medium were similar for both lines as were the rates of protein synthesis. The reason for the increased PAL levels in the resistant tobacco cells remains unknown.

Effects of abscisic acid(ABA) and temperature on the anthocyanin accumulation and phenylalanine ammonia lyase(PAL) activity were investigated in seedlings of Arabidopsis thaliana. In time course study, exogenous application of ABA (50-1000 μM) led to a noticeable increase in anthocyanin pigments which persisted over the following 5 days. Anthocyanins increased in concert with the chlorophyll loss. The activity of PAL, a key enzyme in the phenylpropanoid pathway, increased on exposure to ABA and reached maximum on the 4th day. This result shows that anthocyanin synthesis and PAL activity have a close physiological relationships. In the effects of temperatures (10℃, 17℃, 25℃ and 30℃) on anthocyanin accumulation and PAL activity in seedlings, a moderate-low temperatures (17℃) enhanced both anthocyanin content and PAL activity, whereas elevated temperatures (30℃) showed low levels of anthocyanin and PAL activity, suggesting a correlation between temperature-induced anthocyanin synthesis and the accumulation of PAL mRNA. Simultaneous application of ABA with temperatures induced higher anthocyanin synthesis and PAL activity in seedlings than ABA or temperature stress alone. Moderate-low temperature with ABA exposure elicited the maximal induction of anthocyanin synthesis and PAL activity. Therefore, ABA treatment significantly increased thermotolerance in A. thalinan seedlings. Ethephon and ABA showed similar mode of action in physiological effects on anthocyanin accumulation and PAL activity. Our data support that anthocyanins may be protective in preventing damage caused by environmental stresses and play an important role in the acquisition of freezing tolerance.

Secondary metabolites are involved in plant response to salt stress and provide a significant contribution to the antioxidant and anticancer activity of plant tissues. PAL is a key gateway enzyme in the secondary metabolic pathway. The objective of this study is to determine the antioxidant capacity, phenolic acid composition and antiproliferative effect associated with PAL activity in some plants naturally growing under salt stress. Firstly, PAL activity and antioxidant capacity of Cyathobasis fruticulosa (CF), Salsola nitraria and Salvia halophila were evaluated. In addition, phenolic acids in the extracts were screened by LC-MS/MS. Antiproliferative effects of the extracts on HT-29 cells were determined by MTT assay. Among all three plants, CF stood out in terms of all the results. CF had the highest PAL specific activity and antioxidant capacity. The richest plant extract in terms of phenolic acids were found to be CF. CF exhibited a markea antiproliferative activity against HT-29 cells compared to other extracts. In conclusion, high PAL activities of plants that naturally growing under salt stress could contribute to antioxidant and anticancer properties. Therefore, compounds obtained through plants exhibiting high levels of PAL activities could be used in the development of new pharmaceuticals as an antioxidant and anticancer agent.

Phenylalanine ammonia lyase (PAL) activity was studied in pearl millet cultivars with different levels of resistance to the downy mildew disease caused by Sclerospora graminicola, an important oomycete pathogen. PAL activity was elevated in resistant host cultivar and decreased in susceptible cultivars following downy mildew pathogen infection. The enzyme activation varied between cultivars and was correlated with the degree of resistance to downy mildew disease. The induction of PAL as a response to pathogen inoculation was further corroborated by a time-course study in seedlings and cultured cells of pearl millet. The level of PAL activity was highest at 1.5 h in cultured cells and 4 h in seedlings of resistant host cultivar after inoculation with Sclerospora graminicola. Further studies on PAL activity in different tissues of seedlings showed highest enzyme activity in the young growing region of the root of the resistant host cultivars. The accumulation of wall-bound phenolics and lignin was higher in the resistant cultivar seedlings as evidenced by phloroglucinol-HCl staining and p-coumaric acid assay. The temporal changes in lignin concentration and the concentration of soluble phenolics were greater in root tissues of resistant cultivars than in those of susceptible cultivars. Treatment of resistant seedlings with a PAL inhibitor, α-aminooxy-β-phenylpropionic acid, resulted in the enhancement of the enzyme activity, whereas in the presence of 1 mm trans-cinnamic acid the pathogen-induced PAL was completely inhibited. Treatment of pearl millet seedlings with exogenously applied PAL inhibitors induced downy mildew disease susceptibility in the resistant pearl millet cultivar, consistent with direct involvement of PAL in downy mildew resistance. Results are discussed with respect to the presumed importance of host phenolic compounds and lignin accumulation and its relation to PAL activation as a response to the pathogen infection.

Differential regulation of phenylalanine ammonia lyase activity and protein level by light in tomato seedlings

Water deficit is one of the limiting factors of plant productions in arid and semi-arid areas. Many adaptive strategies such as accumulation of osmotic adjustments, phenolic compounds and antioxidant enzymes activity have been developed in plants for dealing with drought stress. The use of microorganisms, including probiotic bacteria, is a type of soil management that is effective in reducing the effects of stress. This experiment aimed to determine the effects of some probiotic bacterial strains on proline, sugar, total phenolic compounds (TPC), phenylalanine ammonia lyase (PAL), photosynthesis pigments and antioxidant activities of cucumber plants under drought stress. A completely randomized design was applied with a factorial arrangement of two factors: irrigation levels and bacteria strains with three replications. The results showed that proline, sugar, TPC, PAL and enzymes activity in control and inoculated plants were increased by increasing drought stresses. By contrast photosynthesis pigments significantly decreased under stress. The use of bacterial strains alleviate the harmful effect of stresses by an accumulation of proline, TPC, sugar, PAL activity and enzyme activity. The results also showed that inoculated plants had higher antioxidant activity compared to control plants under drought. It was found that the use of probiotic bacteria is an effective strategy to enhanced drought stress tolerance in plants.

Influence of maturity and bagging on the relationship between anthocyanin accumulation and phenylalanine ammonia-lyase (PAL) activity in ‘Jonathan’ apples

Phenylalanine ammonia lyase (PAL) activity was measured using HPLC in pea leaves following exogenous application of L-phenylalanine and ferulic acid. Treatment with different concentrations (50, 100, 150 ppm) of L-phenylalanine caused increased activity of PAL activity in comparison to control. In pea leaves treated with 50 ppm L-phenylalanine, maximum PAL activity was observed after 72 h of treatment. Application of ferulic acid first reduced PAL activity at lower concentration (50 ppm) but it further increased at higher concentrations of the compound (100 and 150 ppm) in pea leaves compared to control. Minimum PAL activity was 0.19 nM cinnamic acid/min/g fresh wt after 24 h at 50 ppm and then increased with time. Treatment with both compounds significantly increased the accumulation of phenolic acids and salicylic acid and reduced conidial germination of Erysiphe pisi on pea leaves. They were equally effective at 100 and 150 ppm in reducing conidial germination. Conidial germination on L-phenylalanine-treated leaves was 26% after 24 h and that on ferulic acid treated leaves 34% compared to control (46%). Foliar application of different concentrations of L-phenylalanine increased the level of ferulic acid in the leaves of pea plants. Maximum enzyme activity in terms of the accumulation of cinnamic acid (79.3 and 83.5 μg/g fresh wt) was observed following the application of L-phenylalanine after 24 and 48 h respectively. At 50 ppm, cinnamic acid accumulation in pea leaves was 35.6 and 39.4 μg/g fresh wt and 74.3 and 86.5 μg/g fresh wt at 100 ppm.

Russet spotting (RS), consisting of numerous small brown spots on the midrib of head lettuce (Lactuca sativa), is a physiological disorder induced by exposure to ethylene. In leaves suffering RS, the increase in spotting was accompanied by a parallel increase in the amount of phenolic compounds. Of these, chlorogenic acid and isochlorogenic acid were identified. Ethylene induced high phenylalanine ammonia-lyase (PAL) activity and RS formation in the susceptible cultivar Salinas, but not in the resistant cultivar Calmar. In the absence of ethylene neither significant PAL induction nor RS occurred. No correlation was found between the increase in polyphenol oxidase or peroxidase and the development of RS. The increase in PAL activity, however, was closely correlated with the development of RS. The increase in PAL activity preceded the development of RS, and the extent of RS was directly related to the level of PAL. Three temperatures (0.5, 5.5, and 12.5 C) were compared on the basis of their influence on both RS and PAL induction. At the lowest temperature (0.5 C) neither PAL induction nor RS occurred to a significant extent. At the highest temperature (12.5 C) an initial rapid increase in PAL activity and an earlier development of spotting were observed, but subsequently there was a decrease in both PAL activity and the rate of development of RS. At the medium temperature (5.5 C) both PAL activity and RS increased progresively with time. The decline of PAL activity at a higher temperature might be attributed to inactivation of the enzyme. Thus, a temperature favorable for induction of PAL activity by ethylene was also favorable for RS. These observations indicate a close interrelationship between the induction of PAL activity and the development of RS in response to ethylene, and suggest a causal relationship between the two events. PAL serves as a useful biochemical marker for the RS reaction.

Trehalose serves as a stress protectant and/or reserve carbohydrate in a variety of organisms including bacteria, yeast, and invertebrates. Recently, trace amounts of trehalose have been detected in higher plants, although the function of trehalose in plants remains unknown. A cDNA clone (OsTPP1) encoding a putative trehalose-6-phosphate phosphatase (TPP) for trehalose biosynthesis was isolated from rice. Functionality of the clone was demonstrated by complementation of a yeast mutant and enzymatic activity of the recombinant protein. Northern blots revealed that the OsTPP1 transcript levels were fairly low or under detectable limits in most of the tissues under ambient conditions but were highly induced within 1-2 h of chilling stress (12 degrees C) in both root and shoot tissues of seedlings. This induction was transient and disappeared after 6 h of the chilling stress. Transient expression of OsTPP1 was also induced under severe chilling stress (4 degrees C) as well as salinity and drought stresses at ambient temperatures. Application of exogenous ABA (50 microM) resulted in a transient increase of OsTPP1 expression within 20 min of the treatment, thereby suggesting involvement of ABA in OsTPP1 gene regulation. Measurements of total cellular TPP activity and trehalose content in roots indicated that both TPP activity and trehalose levels were transiently increased after chilling (12 degrees C) stress. Collectively, the data indicate that transient activation of trehalose biosynthesis is involved in early chilling stress response in rice. Possible functions of trehalose in the early stages of chilling stress response are discussed.

Grafting can promote the growth and nitrogen use efficiency (NUE) of cucumber seedlings under reduced nitrogen (N) application, however, its underlying mechanisms and effects on mature plants remain unknown. For this purpose, self-grafted and rootstock-grafted cucumber plants were treated with two N levels (7 and 4 mM) throughout the entire growth period. The long-term reduced-N treatment significantly limited the growth, root morphology, nitrate (NO3 -) uptake, NUE traits, photosynthesis, phenylalanine ammonia-lyase (PAL) activity, yield, and fruit quality of self-grafted plants but had no influence on rootstock-grafted plants, it even improved their NUE traits, total phenolic and flavonoid contents, and PAL activity. Furthermore, the expression of the NRT1.2, NRT1.5, NRT2.2, and NRT2.5 genes were significantly down-regulated in self-grafted plant roots, while they and the transcription factors NLP6 and LBD38 were up-regulated in rootstock-grafted plant roots under reduced-N environments. Correlation analysis showed that plant growth, root surface area, N-accumulation, N-uptake efficiency (NUpE), NUE, photosynthesis, PAL activity, yield, and fruit quality were all positively correlated with each other; meanwhile, the root morphology, NRT1.2 and NRT2.1 gene expression were all positively correlated with NUpE and NUE. The results demonstrate that under reduced-N application, rootstock grafting can enhance NO3 - uptake and N accumulation to improve the NUE of cucumber plants and resist reduced-N environment through secondary metabolism, maintaining growth, photosynthesis, yield, and fruit quality without adverse effects. The up-regulation of NRT genes and related transcription factors regulates the NO3 - uptake in rootstock-grafted plants. Rootstock grafting will be beneficial for fertilizer conservation and efficient cucumber production. yield and fruit quality.

Gibberellic acid (GA 3 ) treatments (30 and 60 μg litre -1 ) were applied to young plants (Fragaria ananassa cv Chandler). Fruits were harvested at various developmental stages (14, 21, 28 and 35 days from fruit set). Weight and size, phenolic compounds (total polyphenols and anthocyanins) and enzyme activities, phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) were determined. Our aim was to obtain detailed information about PAL and TAL activities related to the strawberry colour during development and ripening processes and to determine the effects of exogenous treatments of GA 3 on PAL and TAL activities. Exogenous treatments of GA 3 improve weight, size and colour of strawberry fruits, and affect PAL and TAL activities. We found that the anthocyanin content and PAL activity are enhanced by the exogenous treatment of GA 3 in the range of 30 μg litre -1 . However, with the higher GA 3 treatment, only the anthocyanin content is affected in that way. These findings suggest that gibberellic acid effect on PAL, TAL and ultimately anthocyanin enhancement is dosage related and saturation of the response occurs at 30 μg litre -1 .