CONTINUOUS LIGHT ON TOMATO From Gene to Yield Aarón I. Vélez-Ramírez i Thesis committee Promotor Prof. Dr Harro J. Bouwmeester Professor of Plant Physiology Wageningen University Co-promotors Dr Wim van Ieperen Assistant professor, Horticulture and Product Physiology Wageningen University Dr Dick Vreugdenhil Associate professor, Laboratory of Plant Physiology Wageningen University Other members Prof. Dr Ton Bisseling, Wageningen University Prof. Dr Roberta Croce, VU University Amsterdam, The Netherlands Prof. Dr Gerrit T.S. Beemster, University of Antwerp, Belgium Dr. Ronald Pierik, Utrecht University, The Netherlands This research was conducted under the auspices of the Graduate School of Experimental Plant Sciences (EPS). ii CONTINUOUS LIGHT ON TOMATO From Gene to Yield Aarón I. Vélez-Ramírez Thesis submitted in fulfillment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 19 September 2014 at 11 a.m. in the Aula. iii Aarón I. Vélez Ramírez Continuous Light on Tomato. From Gene to Yield 214 pages PhD thesis, Wageningen University , Wageningen, NL (2014) With references, summaries in English and Dutch ISBN 978-94-6257-078-8 iv Contents Chapter 1 | 1 General Introduction. Continuous-light-induced injury in tomato: An old enigma 13 Chapter 2 | Plants under continuous light Chapter 3 | 29 Continuous light as a way to increase greenhouse tomato production: Expected challenges Chapter 4 | 39 A single locus confers tolerance to continuous light and allows substantial yield increase in tomato Chapter 5 | 81 Continuous-light-tolerance in tomato is graft- transferable Chapter 6 | 91 Circadian asynchrony triggers continuous-light- induced injury in tomato Chapter 7 | 111 Phytochrome A protects tomato plants from injuries induced by continuous light Chapter 8 | Sucrose and starch content negatively correlates 135 with PSII maximum quantum efficiency in tomato plants exposed to injurious light/dark cycles, including continuous light Chapter 9 | 163 General Discussion. On the continuous-light- induced injury in tomato 182 References 198 Summary 201 Samenvatting 204 Acknowledgements 208 Curriculum vitae 209 List of publications 210 EPS education statement v General Introduction General Introduction Con tinuous-light-induced injury in Continuoustomato:-light An- oldinduced enigma injury in tomato: An old… Aaron I. Velez-Ramirez Chapter 1 Why grow tomatoes under continuous light? High-tech greenhouse horticulture is highly efficient in using resources like water and nutrients. In The Netherlands, in 2005, greenhouses occupied an area of 10,500 ha, and about 20% of this area was equipped with supplementary lighting. Supplementary lighting is used to increase both the light intensity during cloudy days and the daily light period during winter. By 2008, when this project was conceived, rose growers in The Netherlands and other Northwest European Countries were already using supplementary lighting for large periods of the day in order to increase yield. In cut and pot roses, continuous light (CL) increases the number of flowers by up to 12 or 34%, respectively, in comparison to an 18-h photoperiod regime (Mortensen & Gislerød, 1999, Pettersen et al., 2007). For tomato (Solanum lycopersicum), more than 160 ha of greenhouse area were equipped with supplementary lighting in 2006 (original report in Dutch, cited by Heuvelink et al. (2006)). This inspired people in the Dutch horticultural industry to pursue the cultivation of tomato under CL in order to increase yield using the infrastructure already in place. Although innovative, this endeavor revived an old scientific enigma — Unlike roses, tomato plants develop potentially lethal injuries if cultivated under CL (Arthur et al., 1930) — and highlighted a huge gap in our understanding of light signaling, photosynthesis and circadian rhythms in tomato. Here, we present the results of a 5-year effort to better understand the physiological basis of the CL-induced injuries in tomato and develop the tools (genetic and conceptual) to cultivate tomatoes under CL. Continuous light induced injuries in tomato Between 1924 and 1928, Arthur et al. (1930) extensively studied the effects of artificial climate on many plant species in an attempt to find the environmental conditions that allowed cultivating plants at their maximum capacity throughout their life cycle. By doing so, they discovered that, unlike most tested species, tomato plants develop a serious disorder when exposed to CL, which can even result in plant death. This seminal work inspired many others; along the decades, each attempt to understand this disorder took advantage of the knowledge and technology of the time. Although important and valuable discoveries were made, by the time this project started, a detailed and substantiated physiological explanation of this disorder was still missing. Tomato is usually grown under natural photoperiods or, if supplementary light is used, under a maximum of 16 to 18h photoperiod. Figure 1.1a shows a healthy leaflet from a tomato plant grown under a 16-h photoperiod. When exposed to CL, tomato plants show a set of characteristic symptoms, of which interveinal mottled chlorosis starting at the leaf/leaflet basis is the most distinctive (Fig. 1.1b-d). Equally distinctive is the fact that such chlorosis gradually extends towards the leaf/leaflet tips/edges in younger leaves until it covers the complete leaf surface (Fig. 1.1f-h). Other symptoms include epinasty (curling of leaf blades) (Fig. 1.1j), necrotic spots (Fig. 1.1k), and smaller leaves/leaflets (Fig. 1.1e-h). Under conditions favoring the development of this disorder (i.e. high light intensity, 2 General introduction: An old enigma exclusion of sunlight from the CL regime and constant temperature), tomato plants eventually die if exposure to CL lasts long enough (Fig. 1.1i). In literature, this disorder has received several names, including photoperiodic chlorosis (Daskaloff & Ognjanova, 1965, Withrow & Withrow, 1949), light-injury (Hillman, 1956), continuous irradiation injury (Tibbitts et al., 1990) and constant-light injury (Cushman & Tibbitts, 1998, Cushman et al., 1995). In this study, we use the term CL-induced injury. Brief chronological summary on key studies As the knowledge in plant physiology increased and new technologies became available, the hypotheses and experiments aiming to explain the physiological mechanism of the CL- induced injuries evolved. One may say that a day in the library is worth years in the laboratory. In Chapter 2, therefore, we critically review the previous research using a modern understanding of plant physiology, and Supplementary Table 4.1 provides an extensive literature list on the topic. Here, therefore, I limit myself to a chronological review of the studies that contributed most to the hypotheses postulated and/or tested in this study. Soon after Arthur et al. (1930) described the phenomenon of CL-induced injury in tomato, Darrow (1933) reported tomato plants growing, vigorously and without injury, under natural CL provided by the Arctic summer in Alaska, less than 2° from the Arctic circle. This posted serious doubts regarding the true nature of the factor inducing the disorder as the important differences between sunlight and artificial light could be the culprit instead of the CL itself. In order to reveal the true nature of the CL-induced injury, in Chapter 6, we used modern light sources, which mimic the spectral distribution of sunlight, to shed light on this concern. Withrow and Withrow (1949) reported that the CL-induced injury is higher at higher temperatures and old leaves developed under 15-h photoperiod did not show marked chlorosis when exposed to CL. Further studying these observations, the work of Hillman (1956) is probably the single most important contribution to the understanding of this disorder. By careful observation of tomato plants transferred to CL, and then back to non-injurious photoperiod regimes, he showed that when a healthy tomato plant is transferred to CL, the first leaves to show injury will show them at the leaf basis, and in progressively younger leaves the injury extends towards the tip. Likewise, when an injured plant is transferred back to a non-injurious photoperiod, the recovery follows an opposite pattern; that is, the leaf tip is injured and the leaf basis remains green. These observations suggest that only young leaves could develop into injured or healthy leaves, depending on the prevalent light regime during a critical developmental stage. Furthermore, he showed that a daily change in temperature prevents CL-induced injury in tomato without affecting plant weight. Then he showed that the light intensity and spectral distribution used to grow the plants under non-injurious photoperiods influenced the injury severity once transferred to CL. Finally, he showed that abnormal light/dark cycles, which are light/dark cycles with a periodicity substantially differing from the terrestrial 24-h periodicity (e.g. 4-h light/4-h dark cycles), induced the same kind of chlorosis in tomato as the one induced by CL. Throughout this dissertation, we are coming back to Hillman’s observations as they offer key clues linking the CL-induced injury with 3 Chapter 1 Figure 1.1 | Continuous-light-induced injuries in tomato. (a) Healthy tomato leaflet grown under 16-h photoperiod provided by a sulfur