Continuous light on tomato : from gene to yield

Light essentially sustains all life on planet earth surface. Plants transform light energy into chemical energy through photosynthesis. Hence, it can be anticipated that extending the daily photoperiod, using artificial light, results in increased plant productivity. Although this premise is true for many plant species, a limit exists. For instance, the seminal work of Arthur <em>et al.</em> (1930) showed that tomato plants develop leaf injuries if exposed to continuous light (CL). Many studies have investigated the physiological mechanism inducing such CL-induced injury. Although important and valuable discoveries were done over the decades, by the time the present project started, a detailed and proven physiological explanation of this disorder was still missing. Here, I present the results of a 5-year effort to better understand the physiological basis of the CL-induced injury in tomato and develop the tools (genetic and conceptual) to cultivate tomatoes under CL. After an exhaustive literature search, it was found that Daskaloff and Ognjanova (1965) reported that wild tomato species are tolerant to CL. Unfortunately, this important finding was ignored by numerous studies done after its publication. Here, we used the CL-tolerance found in wild tomatoes as a fundamental resource. Hence, the specific objectives of this thesis were to (i) better understand the physiological basis of the CL-induced injuries in tomato, (ii) identify the gene(s) responsible for CL-tolerance in wild tomato species, (iii) breed a CL-tolerant tomato line and (iv) use it to cultivate a greenhouse tomato crop under CL. <strong>Chapter 1</strong> describes how innovation efforts encountered the unsolved scientific enigma of the injuries that tomato plants develop when exposed to CL. The term <em>CL-induced injury</em> is defined, and a detailed description of the symptoms observed in this disorder is shown. Additionally, an overview of the most important studies, influencing the hypotheses postulated and/or tested in this dissertation, is presented. Finally, a description and motivation of the main questions that this dissertation pursued to answer is presented alongside a short description of the strategy chosen to answer them. <strong>Chapter 2</strong> reviews the literature, published over the last 80 years, on CL-induced injury using modern knowledge of plant physiology. By doing so, new hypotheses aiming to explain this disorder are postulated in addition to the ones collected from literature. Additionally, we highlight that CL is an essential tool for understanding the plant circadian clock, but using CL in research has its challenges. For instance, most of the circadian-clock-oriented experiments are performed under CL; consequently, interactions between the circadian clock and the light signalling pathway are overlooked. This chapter is published here. <strong>Chapter 3</strong> explores the benefits and challenges of cultivating CL-tolerant tomato under CL. Considering that current commercial tomato varieties need six hours of darkness per day for optimal growth, photosynthesis does not take place during a quarter of the day. Hence, if tomatoes could be grown under CL, a substantial increase in production is anticipated. A simulation study is presented, which shows that if an ideal continuous-light-tolerant tomato genotype is used and no crop adaptations to CL are assumed, greenhouse tomato production could be 26% higher when supplementing light to 24 h day-1 in comparison with a photoperiod (including supplementary lighting) of only 18 h day-1. In addition, the expected changes in greenhouse energy budgets and alterations in crop physiological responses that might arise from cultivating tomatoes under continuous light are discussed. This chapter is published here. <strong>Chapter 4</strong> maps the locus conferring CL-tolerance in wild tomatoes to chromosome seven, and shows that its introgression into modern tomato cultivars enhances yield by 20%, when grown under CL. In addition, genetic evidence, RNAseq data, silencing experiments and sequence analysis all point to the type III Light-Harvesting Chlorophyll a/b Binding protein 13 (CAB-13) gene as a major factor responsible for the tolerance. In <em>Arabidopsis thaliana</em> this protein is thought to have a regulatory role in balancing light harvesting by photosystems I and II. The likely mechanisms that link CAB-13 with CL-tolerance are discussed. This chapter is published here. <strong>Chapter 5</strong> investigates from which part of the plant CL-tolerance originates and whether this trait acts systemically. By exposing grafted plants bearing both tolerant and sensitive shoots to CL, the trait was functionally located to the shoot rather than the roots. Additionally, an increase in continuous-light tolerance was observed in sensitive plants when a continuous-light-tolerant shoot was grafted on it. Our results show that in order to increase yield in greenhouse tomato production by using CL, the trait should be bred into scion rather than rootstock lines. <strong>Chapter 6</strong> discusses the factors that differ between injurious and non-injurious light regimes. Each of these factors may potentially be responsible for triggering the injury in CL-grown tomato and was experimentally tested here. In short, these factors include (i) differences in the light spectral distribution between sunlight and artificial light, (ii) continuous signalling to the photoreceptors, (iii) constant supply of light for photosynthesis, (iv) constant photo-oxidative pressure, and (v) circadian asynchrony — a mismatch between the internal circadian clock frequency and the external light/dark cycles. The evidence presented here suggests that the continuous-light-induced injury does not result from the unnatural spectral distribution of artificial light or the continuity of the light per se. Instead, circadian asynchrony seems to be the factor inducing the injury. As the discovered diurnal fluctuations in photoinhibition sensitivity of tomato seedlings are not under circadian control, it seems that circadian asynchrony does not directly induce injury via photoinhibition as it has been proposed. <strong>Chapter 7</strong> investigates a possible role for phytochromes (PHY) in CL-induced injury in tomato. Mutant and transgenic tomato plants lacking or over-expressing phytochromes were exposed to CL, with and without far-red light enrichment, to test the role of individual phytochromes on the induction and/or prevention of injury. <em>PHYA</em> over-expression confers complete tolerance to CL regardless the light spectrum. Under CL with low far-red content, PHYB1 and PHYB2 diminished and enhanced the injury, respectively, yet the effects were small. These results confirm that phytochrome signaling networks are involved in the injury induction under CL. The link between CAB-13 and PHYA is discussed. <strong>Chapter 8</strong> investigates the role of carbohydrate accumulation in the induction of CL-induced injury in tomato by using untargeted metabolomics and transcriptomics data. These data reveal a clear effect of CL on sugar metabolism and photosynthesis. A strong negative correlation between sucrose and starch with the maximum quantum efficiency of photosystem II (Fv /Fm) was found across several abnormal light/dark cycles, supporting the hypothesis that carbohydrates play an important role in CL-induced injury. I suggest that CL-induced injury in tomato is caused by a photosynthetic down-regulation showing characteristics of both cytokinin-regulated senescence and light-modulated retrograde signaling. Molecular mechanisms linking carbohydrate accumulation with photosynthetic down-regulation are discussed. <strong>Chapter 9</strong> provides a synthesis of the most important findings and proposes a generic model of CL-induced injury in tomato. I propose that CL-induced injury in tomato arises from retrograde signals that counteract signals derived from the cellular developmental program that promote chloroplast development, such that chloroplast development cannot be completed, resulting in the chlorotic phenotype. Finally, perspectives on what future directions to take to further elucidate the physiological basis of this trait and successfully implement it in greenhouses are presented.