Agronomic performance and transcriptional analysis of carotenoid biosynthesis in fruits of transgenic HighCaro and control tomato lines under field conditions

Genetic manipulation of carotenoid biosynthesis in higher plants has been the objective of a number of biotechnology programs, e.g. the Golden Rice Program. However, tomato (Solanumlycopersicum L.), which naturally accumulates lycopene in fruits, has attracted the attention of many groups who have manipulated it to increase or diversify carotenoid accumulation. One of the most significant achievements was “HighCaro (HC),” a transgenic tomato plant constitutively expressing the tomato lycopene beta-cyclase (tLcy-b), that produces orange fruits due to the complete conversion of lycopene to β-carotene. In this article we report the results of a field trial conducted in Metaponto (Italy) on HC and on two control genotypes to evaluate the stability of the transgenic trait and their yield performances. Transcriptional regulation of eight genes involved in carotenogenesis was assayed by quantitative real-time PCR (qRT-PCR) analysis on fruits collected at four distinct development stages. Statistical analysis results demonstrated that in field conditions the transgene maintained its ability to induce the conversion of lycopene to β-carotene. Moreover, agronomic performances and fruit quality in the transgenic line were not impaired by this metabolic disturbance. Results of qRT-PCR analysis suggested that transcription of PSY-1, PDS and ZDS genes were developmentally regulated in both genotypes. Unexpectedly, Lcy-b expression in transgenic fruits was also developmentally regulated, despite the fact that the gene was driven by a constitutive promoter. Our data provide evidence that in photosynthetic cells a strict and aspecific mechanism controls the level of transcripts until the onset of chromoplasts differentiation, at which point a gene-specific control on transcription takes place

A xanthophyll-derived apocarotenoid regulates carotenogenesis in tomato chromoplasts

[EN] Carotenoids possess important biological functions that make them essential components of the human diet. 8-Carotene and some other carotenoids have vitamin A activity while lutein and zeaxanthin, typically referred to as the macular pigments, are involved in good vision and in delaying the onset of age-related eye diseases. In order to create a zeaxanthin-producing tomato fruit, two transgenic lines, one with a high 8-carotene cyclase activity and the other with a high 8-carotene hydroxylase activity, have been genetically crossed. Ripe fruits from the resulting progeny contained significant levels of violaxanthin, antheraxanthin, and xanthophyll esters. However, their zeaxanthin content was not as high as expected, and the total level of carotenoids was only 25% of the carotenoids found in ripe fruits of the comparator line. Targeted transcript analysis and apocarotenoids determinations indicated that transcriptional regulation of the pathway or degradation of synthesized carotenoids were not responsible for the low carotenoid content of hybrid fruits which instead appeared to result from a substantial reduction of carotenoid biosynthesis. Notably, the content of an unidentified hydroxylated cyclic (C13) apocarotenoid was 13 times higher in the hybrid fruits than in the control fruits. Furthermore, a GC-MSbased metabolite profiling demonstrated a perturbation of carotenogenesis in ripening hybrid fruits compatible with a block of the pathway. Moreover, carotenoid profiling on leaf, fruit, and petal samples from a set of experimental lines carrying the hp3 mutation, in combination with the two transgenes, indicated that the carotenoid biosynthesis in petal and fruit chromoplasts could be regulated. Altogether the data were consistent with the hypothesis of the regulation of the carotenoid pathway in tomato chromoplasts through a mechanism of feedback inhibition mediated by a xanthophyll-derived apocarotenoid. This chromoplast-specific post-transcriptional mechanism was disclosed in transgenic fruits of HU hybrid owing to the abnormal production of zeaxanthin and antheraxanthin, the more probable precursors of the apocarotenoid signal. A model describing the regulation of carotenoid pathway in tomato chromoplasts is presented.The authors apologize for the inadvertent omission of any pertinent reference in this manuscript. This work was supported in part through the European Union Framework Program FP7 METAPRO Project 244348 and benefit by the activities of the European COST actions CA15136 (EUROCAROTEN) and CA18210 (ROXY) . We are indebted to Prof. Dani Zamir of Hebrew University of Jerusalem for providing us with a seed sample of tomato line e1827m1. JLR acknowledges financiation by the Spanish Ministry of Economy and Competitiveness through a "Juan de la Cierva-Incorporacion" grant (IJC2020-045612-I) . CDA, ALS, and GG would like to thank all Colleagues at the Metapontum Agrobios Research Center of ALSIA (Agenzia Lucana di Sviluppo e di Innovazione in Agricoltura, Matera, Italy) who collaborated or provided help throughout this study.D'ambrosio, C.; Stigliani, AL.; Rambla Nebot, JL.; Frusciante, S.; Diretto, G.; Enfissi, EM.; Granell Richart, A.... (2023). A xanthophyll-derived apocarotenoid regulates carotenogenesis in tomato chromoplasts. Plant Science (Online). 328:1-14. https://doi.org/10.1016/j.plantsci.2022.11157511432