Metabolic response of apple fruits (Malus domestica Borkh.) to physiological disorders

V nalogi smo želeli z analizo metabolitov preveriti odziv plodov jablane (Malus domestica Borkh.) na izbrane fiziološke motnje (sončni ožigi, steklavost, grenka pegavost in skald). Pri nekaterih fizioloških motnjah smo določili tudi encimsko aktivnost in izražanje genov fenilpropanoidne/flavonoidne poti ter določili aktivnost polifenol oksidaz (PPO) in peroksidaz (POX). V raziskavah smo za določanje vsebnosti metabolitov uporabili metode UV-VIS, HPLC in HPLC-MS, aktivnost encimov smo določali s pomočjo spektrofotometrije, radiografskega skeniranja TLC in izražanja genov z metodo RT-qPCR. Ugotovili smo, da odziv plodov na fiziološke motnje ni enoten. Pri skoraj vseh fizioloških motnjah se je vsebnost hidroksicimetnih kislin povečala v prizadetem tkivu, razen pri steklavosti. Splošno večjo vsebnost vseh fenolnih spojin smo opazili v pegah grenke pegavosti, medtem ko je bila vsebnost antocianinov in flavonolov v kožici nad pego manjša, je bila vsebnost slednjih pri ožigih močno povečana. Med najbolj zanimivimi in hkrati najmanj raziskanimi odzivi je vsebnost dihidrohalkonov v povezavi s fiziološkimi motnjami. Pri steklavosti, sončnih ožigih in v pegi pri grenki pegavosti je bila njihova vsebnost v prizadetem tkivu večja od vsebnosti v neprizadetem tkivu. Izkazalo se je, da je bila tudi aktivnost encima P2\u27GT večja v plodu s steklavostjo v primerjavi s plodovi brez steklavosti. Pri ostalih določenih encimih fenilpropanoidne/flavonoidne poti (PAL, CHS/CHI, FHT, FLS in F3’GT) v poskusu s steklavostjo nismo ugotovili razlik v aktivnosti med obravnavanji. Razvoj steklavosti se odraža tudi v kožici prizadetih plodov, kar lahko služi v pomoč pri razvijanju cenovno ugodnih metod razvrščanja plodov. Najbolj neenoten odziv na fiziološke motnje je bil v skupini flavanolov. Pri skaldu smo ugotovili povečano izražanje genov fenilpropanoidne/flavonoidne poti (MdCHS, MdCHI, MdFHT, MdANS in MdUFGT) v kožici s skaldom, ki pa se ni odražala v večji aktivnosti encimov (PAL, FHT, CHS/CHI, DFR). Pri nastanku fizioloških motenj smo ugotovili tudi večjo aktivnost POX v steklavem mesu ter na mestu sončnih ožigov, medtem ko je bila aktivnost POX pri skaldu manjša. Aktivnost encima PPO smo določali pri nastanku skalda in steklavosti in v obeh primerih smo ugotovili večjo aktivnost v prizadetem tkivu.The aim of this PhD thesis was to evaluate the response of apple fruits (Malus domestica Borkh.) to selected physiological disorders (sunburn, watercore, bitter pit and scald) through metabolite analysis. Additionally, gene expression and enzyme activity of phenylpropanoid and flavonoid pathway plus polyphenol oxidase (PPO) and peroxidase (POX) activity were determined for certain disorders. Metabolites were analysed using UV-VIS, HPLC and HPLC-MS, gene expression with RT-qPCR and enzyme activities with UV-VIS and radiographic TLC scan. The response of apple fruits to different physiological disorders was not uniform. The affected tissues from all physiological disorders except watercore had higher contents of hydroxycinnamic acids. Generally higher contents of phenolic compounds were observed in the pits of bitter pit, whereas peels above the pit contained lower amounts of anthocyanins and flavonols. The latter were strongly increased in sunburned peels. One of the most notable results and yet least researched responses was the content of dihydrochalcones in connection to physiological disorders. Their content was higher in sunburned peels, flesh with watercore and in the pits of bitter pit in comparison to unaffected tissue. The P2\u27GT enzyme activity was also higher in the flesh of apples with watercore in comparison to flesh of apples without watercore. No statistically significant differences were observed in the activity of the rest of the enzymes of phenylpropanoid and flavonoid pathway (PAL, CHS/CHI, FHT, FLS in F3’GT) between treatments. Watercore incidence is reflected in the apple peel, which could aid in developing cost-efficient methods of fruit sorting. The most unequal response to physiological disorders was in the group of flavanols. Higher gene expression (MdCHS, MdCHI, MdFHT, MdANS and MdUFGT) was observed with scald development, but was not reflected in enzyme activity (PAL, FHT, CHS/CHI and DFR). Higher activity of POX was determined in flesh affected with watercore and sunburned peel, whereas it was lower in peel affected with scald in comparison to unaffected tissues. Comparing to unaffected tissues, PPO activity was higher in flesh with watercore and peel with scald

Effect of spring frost damage on apple fruit (Malus domestica Borkh.) inner quality at harvest

In the last few years, spring frosts have become more of a rule than an exception. There are several prevention measures available for growers and numerous are being testedhowever, fruitlets are often affected to some extent. One of the more common phenomena is the development of frost rings. The aim of our study was to evaluate how the occurrence of frost rings affects sugar, organic acid, and phenol content in the flesh and phenol content in the peel of cv. ‘Gala’ apples. The results show that the frost damage from spring frost affects compounds in the peel, as well as in the flesh of ripe apples. The flesh of fruits with frost rings contained higher content of fructose and in the flesh directly under the rings there was higher sorbitol and malic acid content. Additionally, the hydroxycinnamic acid and dihydrochalcone content in apple flesh was also altered by frost. The frost-affected peel had a completely different ratio of phenolic compounds content in comparison to healthy apples. While there was a significantly higher content of hydroxycinnamic acids and dihydrochalcones in the corky peel in comparison to the unaffected peel, the flavonol and anthocyanin content was considerably lower

Apple (Malus domestica Borkh.) cultivar \u27Majda\u27, a naturally non-browning cultivar

Browning of apple and apple products has been a topic of numerous research and there is a great number of methods available for browning prevention. However, one of the most efficient ways, and the one most acceptable for the consumers, is the selection of a non-browning cultivar. Cultivar ‘Majda’ is a Slovenian cultivar, a cross between ‘Jonatan’ and ‘Golden Noble’. In this study, it was thoroughly examined and compared to the well-known cultivar ‘Golden Delicious’ with the aim to decipher the reason for non-browning. We have determined the content of sugars, organic acids, vitamin C, glutathione and phenolics in apple flesh, with the addition of phenolic content in apple peel and leaves. The change in color in halves and pomace was also measured and the activity of peroxidase (POX) and polyphenol oxidase (PPO) were determined. Additionally, the analyses of flesh were repeated post-storage. The most prominent results were high acidity (malic acid), low phenol content, especially hydroxycinnamic acid and flavan-3-ol content of cultivar ‘Majda’ in comparison to ‘Golden Delicious’, and no difference in PPO activity between cultivars. After the overview of the results, we believe that both low phenol content and high reduced glutathione content impact the non-browning characteristics of cultivar ‘Majda’

Alteration of the phenylpropanoid pathway by watercore disorder in apple (Malus x domestica)

Watercore is a physiological disorder of apple expressed as fluid deposition in intercellular spaces. We analysed the watercore prone cultivar \u27Fuji\u27 to establish a link between enzymatic response and fluid accumulation in the fruit. Individual phenolic content, selected enzyme activities of the phenylpropanoid pathway, sugars and organic acids were quantified in watercore affected flesh and peel. In addition, transcriptome sequencing was performed to obtain a first insight into molecular mechanisms underlying the physiological disorder. Sampled material included: peel (HP) and flesh from healthy fruit (H), peel from watercore affected fruit (WP), healthy flesh from fruit with watercore (HW) and watercore flesh (WW) from fruit with watercore. A sorbitol increase in watercore affected apple was observed not only in the flesh but also in the peel. Moreover, two phenolic groups (hydroxycinnamic acids and flavonols) were significantly affected by watercore and their content was higher in WP in comparison to HP. Dihydrochalcone content was the highest in WW flesh. This was supported by RNA-seq data, which indicated a generally increased expression of genes from the early flavonoid pathway, sorbitol metabolism and polyphenol oxidases. Among the phenylpropanoid pathway enzymes analysed, phloretin 2′-O-glycosyltransferase was the only one exhibiting altered activity