One of the most significant biosphere contamination problems worldwide is derived from heavy metals. Heavy metals can be highly reactive and toxic according to their oxidation levels. Their toxic effects are associated with the increased production of reactive oxygen species (ROS) and cellular damage induced in plants. The present study focuses on the effects of nickel (Ni), copper (Cu), and zinc (Zn) applied to the soil on the antioxidant response and allergen production in the aromatic plant basil (Ocimum basilicum L.) following a combined physiological, biochemical and analytical approach. The concentrations used for the three heavy metals were based on the 2002 Regulation of the Polish Ministry of the Environment on Soil Quality Standards [(i) agricultural land (group B): Ni 100 ppm, Ni 210 ppm, Cu 200 ppm, Cu 500 ppm, Zn 720 ppm and (ii) industrial land (group C): Ni 500 ppm, Cu 1000 ppm, Zn 1500 ppm, Zn 3000 ppm]. The highest physiological and cellular damage in basil plants was caused by Cu and Zn. Increasing concentrations of Cu resulted in a further increase in cellular damage and nitro-oxidative stress, correlating with an induction in activity of reactive oxygen and nitrogen species metabolism enzymes (SOD, CAT, APX, NR). Treatment with Cu led to increased concentration of the allergenic protein profilin, while increasing concentrations of Cu and Zn led to a decrease in the concentration of total proteins (likely due to proteolysis) and antioxidant capacity. Interestingly, severe Cu stress resulted in the accumulation of specific proteins related to transpiration and photosynthetic processes. On the basis of these findings, Ni stress in basil plants appears to be less damaging and with lower allergenic potential compared with Cu and Zn stress, while Cu-stressed basil plants experience most detrimental effects and display highest allergen production.

Carotenoids are the main colouring substances found in orange-fleshed loquat fruits. The aim of this study was to unravel the carotenoid biosynthetic pathway of loquat fruit (cv. ‘Obusa’) in peel and flesh tissue during distinct on-tree developmental stages through a targeted analytical and molecular approach. Substantial changes regarding colour parameters, both between peel and flesh and among the different developmental stages, were monitored, concomitant with a significant increment in carotenoid content. Key genes and individual compounds that are implicated in the carotenoid biosynthetic pathway were further dissected with the employment of molecular (RT-qPCR) and advanced analytical techniques (LC-MS). Results revealed significant differences in carotenoid composition between peel and flesh. Thirty-two carotenoids were found in the peel, while only eighteen carotenoids were identified in the flesh. Trans-lutein and trans-β-carotene were the major carotenoids in the peel; the content of the former decreased with the progress of ripening, while the latter registered a 7.2-fold increase. However, carotenoid profiling of loquat flesh indicated trans-β-cryptoxanthin, followed by trans-β-carotene and 5,8-epoxy-β-carotene to be the most predominant carotenoids. High amounts of trans-β-carotene in both tissues were supported by significant induction in a chromoplast-specific lycopene β-cyclase (CYCB) transcript levels. PSY1, ZDS, CYCB and BCH were up-regulated and CRTISO, LCYE, ECH and VDE were down-regulated in most of the developmental stages compared with the immature stage in both peel and flesh tissue. Overall, differential regulation of expression levels with the progress of on-tree fruit development was more evident in the middle and downstream genes of carotenoid biosynthetic pathway. Carotenoid composition is greatly affected during on-tree loquat development with striking differences between peel and flesh tissue. A link between gene up- or down-regulation during the developmental stages of the loquat fruit, and how their expression affects carotenoid content per tissue (peel or flesh) was established.

The term vitamin E refers to a group of eight lipophilic compounds known as tocochromanols. The tocochromanols are divided into two groups, that is, tocopherols and tocotrienols, with four forms each, namely α-, β-, γ-, and δ-. In order to explore the temporal biosynthesis of tocochromanols in olive (Olea europaea cv. ‘Koroneiki’) fruit during on-tree development and ripening over successive growing years, a combined array of analytical, molecular, bioinformatic, immunoblotting and antioxidant techniques were employed. Fruits were harvested at eight successive developmental stages (10-30 weeks after flowering (WAF)), over three consecutive years. Intriguingly, climatic conditions affected relative transcription levels of vitamin E biosynthetic enzymes; a general suppression to induction pattern (excluding VTE5) was monitored moving from the 1st to the 3rd growing year, probably correlated to decreasing rainfall levels and higher temperature, particularly at the fruit ripening stage. A gradual diminution of VTE5 protein content was detected during the fruit development of each year, with a marked decrease occurring after 16 WAF. Alpha-tocopherol was the most abundant metabolite with an average percentage of 96.82 ± 0.23%, 91.13 ± 0.95% and 88.53 ± 0.96% (during the 1st, 2nd and 3rd year, respectively) of total vitamin E content in 10–30 WAF. The concentrations of α-tocopherol revealed a generally declining pattern, both during the on-tree ripening of the olive fruit and across the three years, accompanied by a parallel decline of the total antioxidant activity of the drupe. Contrarily, all other tocochromanols demonstrated an inverse pattern with lowest levels being recorded during the 1st year. It is likely that, in a defense attempt against water deficit conditions and increased air temperature, transcription of genes involved in vitamin E biosynthesis (excluding VTE5) is up-regulated in olive fruit, probably leading to the blocking/deactivating of the pathway through a negative feedback regulatory mechanism.