Linge Li

Tomato Shade Avoidance: Unraveling Internode Elongation and Hormonal Harmony Linge Li

ISBN: 978-94-6483-864-0 Cover design: Youyang Hu huyyoo@outlook.com Lay-out design: Parntawan | www.ridderprint.nl Print: Ridderprint | www.ridderprint.nl © Copyright 2024: Linge Li, Utrecht, The Netherlands All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, by photocopying, recording, or otherwise, without the prior written permission of the author.

Tomato Shade Avoidance Unraveling Internode Elongation and Hormonal Harmony Het ontwijken van schaduw in tomaten planten: Het ontrafelen van de hormonale regulatie achter de strekking van stengels (met een samenvatting in het Nederlands) 番茄的避阴反应：探索节间伸长和激素协调的奥秘（内附中文摘要） Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof. dr. H.R.B.M. Kummeling, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op woensdag 27 maart 2024 des ochtends te 10.15 uur door Linge Li geboren op 12 november 1993 te Province Liaoning, City Dalian, China

Promotor: Prof. dr. R. Pierik Copromotor: Dr. K. Kajala Beoordelingscommissie: Dr. R.B. Karlova Prof. dr. R. Offringa Prof. dr. ir. C.M.J. Pieterse Prof. dr. I. Rieu Prof. dr. R. Sasidharan Dit proefschrift werd (mede) mogelijk gemaakt met financiële steun van China Scholarship Council (CSC)(No.201807720067).

TABLE OF CONTENTS Chapter 1: General Introduction 7 Chapter 2: Cellular Anatomy of Tomato Stems in Response to Far-Red Light 27 Chapter 3: Transcriptome Changes of Tomato Internode Elongation Induced by Far-Red Light 55 Chapter 4: Hormone Interplay in the Regulation Far-Red-Responsive Stem Elongation in Tomato 107 Chapter 5: Exploring Conservation of Cellular-Level Traits in Shade Avoidance Syndrome among Species 161 Chapter 6: General Discussion 207 Appendix References 224 General Summary 247 General Summary (Dutch) 248 General Summary (Chinese) 250 About the author 251 Acknowledgements 252 EPS Education Statement 255 List of publications 258

GENERAL INTRODUCTION

Chapter 1 8 This PhD thesis reports studies on the effects of supplemental far-red light on tomato growth and development. The background information for the research questions examined in the coming chapters will be provided in this introductory chapter. This introduction provides an overview of the historical significance and cultivation practices of the model plant, tomato. It subsequently draws a comparison between two model tomato cultivars. The narrative then introduces the key phenotypes in the shade avoidance response, shedding light on how these responses are regulated by photoreceptors. The intricate interplay of hormones in shade avoidance is then explored. Lastly, the discussion delves into the involvement of stem structure in the study of shade avoidance. 1.1 TOMATO 1.1.1 History of tomato cultivation Tomato (Solanum lycopersicum) has a unique position in the botanical and culinary realms, tracing its origins to western South America, Mexico, and Central America (Naika et al., 2005). The journey of this versatile fruit, commonly recognized as a vegetable in culinary usage, spans centuries and continents, leaving an indelible mark on global agriculture. With its botanical roots firmly embedded in the Andes region of South America, the tomato found its way into human cultivation and culinary practices through the ingenious efforts of indigenous people in Mexico (Naika et al., 2005). These early cultivators recognized the potential of this fruit, and it became an integral part of their cuisine long before the arrival of Spanish explorers. The transformative encounter between the Spanish conquistadors and the Aztec Empire during the 16th century brought the tomato to Europe as part of the monumental Columbian exchange. The culinary traditions of Europe were forever altered by this novel addition, as tomatoes found their place in a wide array of dishes, sauces, and culinary concoctions (Musselman, 2002; 2005). Despite its fruit classification from a botanical standpoint, the tomato is predominantly utilized as a vegetable in culinary applications. Its rich umami flavour profile, when consumed raw or cooked, lends itself to an extensive range of culinary creations worldwide. From fresh salads to hearty sauces and refreshing beverages, the tomato continues to captivate the global dinning plates (Musselman, 2002). Today, tomato production remains a vital component of global agriculture, with diverse varieties cultivated in temperate climates across continents but still reach a 39.2 million metric Ton (Dillon, 2022). For the reason that tomato is a crop with low maintenance and

General introduction 9 1 high yield, and also a short life cycle, the economic value makes growing it attractive to breeders and farmers (Naika et al., 2005). Advancements in greenhouse technology enable year-round cultivation, making tomatoes an accessible crop regardless of season. The plant’s growth characteristics, such as its sprawling vine-like nature and support requirements, further contribute to its cultivation practices. 1.1.2 Tomato growth habit Tomato (Solanum lycopersicum) is a highly versatile plant that reaches impressive heights exceeding two meters. With careful maintenance, tomato plants can be harvested consecutively for multiple years, offering prolonged productivity. The first harvest typically occurs within a span of 45-55 days after flowering (Naika et al., 2005). In our experimental set-up, tomatoes flower at 35-42 days. Among the diverse tomato cultivars, two main categories can be distinguished: cultivars for fresh consumption and cultivars for processing. We selected a cultivar from each category as our experimental models, Moneymaker (MM) and M82, respectively. Moneymaker stands out as an exceptional greenhouse variety renowned for its abundant yield, delightful bright red fruits, and smooth texture, making it an ideal choice for fresh consumption. This particular cultivar thrives in hot and humid climates, demonstrating its adaptability both outdoors and within greenhouse environments. MM, while not a current choice for large-scale greenhouse farming due to the availability of more modern varieties in recent years, it still remains a greenhouse variety favored by home gardeners. Originating from England, Moneymaker exhibits vigorous vine growth, necessitating proper staking to optimize results (Marks, 2010). M82 is a processing tomato, suited for field growth and machine harvest. M82 plants typically reach a height of 1.5 to 2 meters and have a bushy growth habit. The fruits are typically red when fully ripe, with a smooth skin and a juicy, flavorful flesh but comparing to MM, the fruits are less oval. In our greenhouse set-up, we noticed M82 and Moneymaker (MM) have very similar life cycle and flowering time within 5-6 weeks, similar height with around 1.5 m. To distinguish these two species (Figure 1.1), we can use the first true leaf. The first true leaf of M82 and MM has a distinguishable pattern as shown in Figure 1.2. M82 usually has smaller main leaf lamina more similar to a simple leaf, whereas MM has a leaf pattern more similar to later true leaves with clear separation of each leaflet.

Chapter 1 10 1.1.3 Tomato genomic resources Recent advances in genomic research have significantly contributed to our understanding of the tomato species. The initial tomato genome was derived from the Heinz cultivar (Sato et al., 2012). Leveraging the existing S. lycopersicum cv. Heinz genome as a reference, and with extensive sequencing, and incorporating additional single-nucleotide polymorphisms (SNPs), this ultimately culminated in the final M82 genome (Bolger et al., 2014). Notably, the genome of Solanum lycopersicum cv. M82 was meticulously sequenced in conjunction with the Solanum pennellii genome, The 950-Mb tomato genome exhibits a distinct structure, comprising gene-rich euchromatin and gene-poor pericentromeric heterochromatin (Michaelson et al., 1991; Barone et al., 2008). Given the challenge of sequencing the heterochromatic fraction with its repetitive sequences, the initial strategy focused on sequencing the euchromatic portion, estimated to constitute one-quarter (220 Mb) of the tomato genomic sequence, encompassing over 90% of the genes. Various tools have been developed for the Tomato Genome Sequencing Project, (Sherman and Stack, 1995; Todesco et al., 2008; Mueller et al., 2009; Sato et al., 2012; Bolger et al., 2014). S. lycopersicum cv. M82’s genetic information has provided valuable insights into the molecular mechanisms underlying various traits in tomatoes. M82 tomatoes are widely used in comparison to S. pennellii in drought resistance study, fruit development, and genetic comparison in stress (Gong et al., 2010; Ikeda et al., 2017; Liu et al., 2018; Reynoso et al., 2019; Watanabe et al., 2021). For example, the wild tomato relative Solanum pennellii shows a strong response to shade and in contrast, the domestic tomato S. lycopersicum, which has been bred for production in field conditions, shows a milder response (Bush et al., 2015). However, the Shade Avoidance Syndrome (SAS) mechanisms within the different tomato species are not fully characterized, and we set out to explore the significant changes of commercial tomato plants in stem elongation. 1.1.4 Summary Tomato is an economically important crop, renowned as a well-established stress study model and offers distinctive genetic resources. Despite not having received the same level of attention as Arabidopsis, recent research has delved into the molecular and physiological aspects of SAS in tomato. The uniqueness of tomatoes, with their specific growth patterns, hormonal dynamics, and stem structures, using tomato as a model adds an intriguing layer to our understanding of adaptive strategies under varied light conditions.

General introduction 11 1 Figure 1.1. Model plants of M82 (left) vs Moneymaker (right). Figure 1.2. Examples of first true leaves of M82 vs Moneymaker. 1.2 SHADE AVOIDANCE RESPONSE The shade avoidance syndrome (SAS) is a plant’s acclimation reaction to changes in light that signal impending shade. SAS describes a suite of morphological and physiological changes that occur when plants perceive neighbours, typically through a reduction in the ratio of red: far-red (R:FR) light caused by reflection of far-red light from neighbouring leaves (Smith and Whitelam, 1997). Common shade avoidance responses include hyponasty (upward leaf movement), petiole elongation, stem elongation, and accelerated flowering time (Woodward and Bartel, 2005). Studying SAS unveils crucial insights into plant growth strategies with implications for agriculture and horticulture.

Chapter 1 12 1.2.1. Shade avoidance and neighbour detection for farming Light serves as a fundamental driver of plant growth and development, fuelling photosynthesis, through which plants convert light energy into chemical energy. Plants engage in a struggle for light and adapt their growth patterns to secure optimal light capture in the future. The quality and quantity of light received by plants exert a profound influence on various physiological and developmental processes, including seed germination, stem elongation, leaf development, flowering, and tuber formation. SAS enables plants to elongate stems and adjust leaf angles in an attempt to maximize light capture and mitigate the negative effects of shading (Pantazopoulou et al., 2017). Of particular importance is the ratio of red to far-red (R:FR) light (Osborne, 1991; Franklin, 2008; Ballaré and Pierik, 2017), which acts as a crucial signaling mechanism, conveying information about neighbouring plants and redirecting resource availability into growth, for example, at the cost of seed production (Smith and Whitelam, 1997; Hewitt, 1998; Yang and Li, 2017). Smallholder farmers worldwide often confront common challenges, including constraints on resources, food insecurity, and heightened vulnerability to the impacts of climate change. These issues have been reported in various regions, such as Sub-Saharan Africa (Ariom et al., 2022), South Asia (Cerling et al., 1997), Southeast Asia (Nor Diana et al., 2022), and East Africa (Ndoli, 2018). In these areas, the reduction of fallow periods or continuous cropping practices, driven by population pressure, has led to soil erosion, diminished soil organic matter, and nutrient depletion without adequate replenishment. Consequently, agricultural productivity has been negatively affected, exacerbating the problem of food shortages. Addressing these challenges necessitates the adoption of sustainable agricultural practices and innovative solutions that consider the shade avoidance responses of crops to ensure the resilience and well-being of smallholder farmers globally. 1.2.2 Shade signal detection Canopies formed by plants typically have decreased photosynthetically active radiation (PAR) compared to direct sunlight. Therefore, many plants will adjust their photosynthetic activity and growth (Lichtenthaler et al., 1981; Casal, 2013). Plants absorb photosynthetically active wavelengths (380 nm – 750 nm), including red (R) light (750 nm) and blue (B) light (450 nm). They hardly absorb far-red light (FR, >750 nm) light, and hence the reflected and transmitted light is enriched in FR wavelengths (Figure 1.3). In a situation where there are multiple plants in dense vegetation, FR light

General introduction 13 1 will be reflected whereas the red light will be absorbed, increasing the amount of FR light in the canopy relative to other wavelengths, especially red (R). This difference in the light spectrum is detected by the plant, enabling it to perceive their neighbours and (impending) shade (Beall et al., 1996). In short, the shade consists of light spectrum change and lower light level, and in our experiments, we particularly focus on the signals perceived by plants through low R:FR light ratios. In order to outcompete the neighbouring plants, shade-avoiding plants will adopt the SAS (Ballaré and Pierik, 2017), which involves a number of phenotypic changes that generally make the plant taller and more erect. One of the most significant phenotypic SAS changes observed is stem and petiole elongation. This phenomenon was originally identified to be associated with far-red light transmission in tobacco canopies in the 1970s (Kasperbauer, 1971): the stem elongated more with a lower R:FR light ratio in the canopy than in the daylight. This phenomenon has been observed in many species. For example, in Arabidopsis, both hypocotyl and petiole elongate (Reed et al., 1993; Devlin et al., 1996) and the elongation of the petiole can be induced especially by an end-of-day FR light treatment (Reed et al., 1993). In Arabidopsis, petiole elongation can also be induced by far-red treatment to the leaf tip only (Pantazopoulou et al., 2017). In Brassica rapa, FR light sensing in the cotyledon leads to the elongation of the hypocotyl (Procko et al., 2014). In tomato, significant stem elongation and leaf architecture differences have been observed (Chitwood et al., 2015; Maloof, 2015). Suppression of branching was also found in tomato (cv. Amberley Cross) by end-of-day FR-treatment (Tucker, 1975). In soybean (Glycine max), far-red light perceived on nodes can reduce the fruit production on the very same branch (Green-Tracewicz et al., 2011). Plants detect light with various molecules. Photoreceptors are specialized proteins that absorb specific wavelengths of light, enabling plants to sense changes in light quality and quantity. Cryptochromes respond to blue and ultraviolet-A light and are involved in regulating plant growth, circadian rhythms, and photoperiodic flowering (Lin, 2002). Phototropins perceive blue light and are responsible for phototropism (bending toward light) and stomatal opening (Briggs and Christie, 2002). UVR8 is a photoreceptor that detects ultraviolet-B (UV-B) light and mediates UV-B responses, such as stress protection and regulation of pigmentation (Jenkins, 2017). Phytochromes detect red and far-red light and are involved in various processes such as seed germination, shade avoidance, and flowering (Reed et al., 1994). Phytochromes are 120-kD soluble proteins that have a covalently linked linear tetrapyrrole chromophore. They exist in two photointerconvertible forms, Pr and Pfr. Pr is the red light-absorbing form, which can be

Chapter 1 14 converted to Pfr (the far-red light-absorbing form) by the red light. Pfr is the active form of phytochrome, and can be converted back to Pr by far-red light. Consequently, red light-induced positive phototropism mediated by phytochromes are typically reversible by far-red light (Reed et al., 1993; Kiss et al., 2003). Among all the phytochromes, phyB is essential for mediating SAS (Taiz et al., 2010); phyA can be activated by far-red light and therefore can antagonize phyB-meditated responses in light-grown seedlings (Sheerin and Hiltbrunner, 2017). Figure 1.3. Light absorption graph of pigments in plants, adapted from literature (Glenn, 2022; Domenici et al., 2014). The molecular changes accompanying this photoconversion have a significant impact on signal transduction pathways, leading to adjustments in developmental responses based on the prevailing light environment (Bischoff et al., 2001). In the presence of sunlight, phytochrome becomes active and translocates from the cytosol into the nucleus. Within the nucleus, it interacts with PHYTOCHROME INTERACTING FACTORS (PIFs) and facilitates their inactivation and degradation. PIFs are growth-promoting factors involved in various physiological processes (Shin et al., 1997; Ruberti et al., 2012; Yang and Li, 2017). The family of basic helix-loop-helix (bHLH) transcription factors, which PIFs belong to, serves as the central hub in a signaling cascade that promotes cell elongation. These bHLH factors play a crucial role in coordinating and regulating the processes involved in cell elongation (Oh et al., 2014). In response to low R:FR light conditions, the active pool of phytochrome decreases, leading to the

General introduction 15 1 accumulation of specific phytochrome-interacting factors (PIFs) such as PIF4, PIF5, and PIF7. These PIFs activate growth-promoting genes containing E-box and G-box motifs. This activation stimulates various processes involved in the biosynthesis and transport of important plant hormones like auxin, gibberellins, brassinosteroids, cytokinins, and ethylene (Ueoka-Nakanishi et al., 2011; Casal, 2012; Ruberti et al., 2012; Pantazopoulou et al., 2017; Küpers et al., 2018). Specifically, auxin modulates cell wall remodeling and cell elongation via regulation of expansins and xyloglucan endotransglucosylase/ hydrolases (XTHs) (Fry, 2004; Sasidharan et al., 2010; Sasidharan et al., 2014; de Wit et al., 2015). 1.2.3 Phenotypic response in shade avoidance 1.2.3.1 Hyponasty Hyponasty is the upward bending of leaves or petioles. Hyponastic growth allows plants to reorient their leaves towards available light sources, thereby enhancing light interception and photosynthetic efficiency (Pierik and De Wit, 2014; Pantazopoulou et al., 2017). In recent years, extensive research has shed light on the molecular and physiological mechanisms underlying hyponasty in Arabidopsis in the context of shade avoidance. Studies have revealed the central role of phytohormones, particularly auxins, in regulating hyponasty. Auxins act as key signaling molecules: they are synthesized in the leaf blade in response to FR and transported into the petiole where they induce a hyponastic response specifically localized to the leaf perceiving the FR signal (Michaud et al., 2017). Auxins promote cell elongation and influence the directionality of leaf and petiole bending in response to shade-induced stress (Yang and Li, 2017). The dynamic changes in auxin distribution and transport, mediated by the auxin efflux carriers and signaling pathways, orchestrate the precise adjustments in cell expansion and tissue growth required for hyponasty. The involvement of other phytohormones such as gibberellins and brassinosteroids has been recognized in modulating hyponastic responses. The interplay between these enable plants to fine-tune their growth patterns and optimize resource allocation in response to shade conditions (Bou-Torrent et al., 2014; Pantazopoulou et al., 2017; Yang and Li, 2017; Küpers et al., 2018; Küpers et al., 2023). 1.2.3.2 Elongation When plants are subjected to shading conditions, they exhibit a remarkable ability to elongate their stems, petioles, and hypocotyls to reach and capture more light with their leaves.

Chapter 1 16 In Arabidopsis, shade-induced elongation is regulated by a complex interplay of hormonal signaling pathways. Elongation of the petiole in Arabidopsis involves multiple hormone signaling pathways, including gibberellins, auxins, brassinosteroids, and ethylene. These hormones collectively coordinate cell division, cell elongation, and cell wall modifications to promote elongation in response to shading (Djakovic-Petrovic et al., 2007; MüllerMoulé et al., 2016). Similarly, in tomato, stems elongate in SAS (Figure 1.4) and this process potentially involves key regulators such as gibberellins, auxins, and ethylene, which are have been implicated to play a role (Courbier et al., 2021). Low red/far-red ratios (R:FR) stimulate stem growth-related gene expression and concurrently reduce the expression of genes associated with flavonoid synthesis, isoprenoid metabolism, and photosynthesis. This results in decreased levels of flavonoids and isoprenoid derivatives, alongside a reduction in stem jasmonate levels and photosynthetic capacity. While this model partially explains shade-avoidance responses, further understanding of the complex links between shade and auxin networks is needed. (Cagnola et al., 2012; Iglesias et al., 2018; Schrager-Lavelle et al., 2019; Courbier et al., 2021). However, the intricacies of shade-induced elongation in tomato, particularly the specific molecular mechanisms and signaling components, remain areas that require further investigation. Other dicot plants, such as soybean (Glycine max) and sunflower (Helianthus annuus) also display significant elongation responses in SAS response to shade cues (Green-Tracewicz et al., 2011; Page et al., 2011; Tang and Liesche, 2017; Lyu et al., 2021). The underlying hormonal and molecular mechanisms regulating elongation in these plants are similar to those observed in tomato and Arabidopsis and involve gibberellin and auxin. Figure 1.4. Shade avoidance response leads to a significant elongation response in tomato. Comparison of 21-day-old Moneymaker plants grown in white light (WL) vs far-red light treatment (WL+FR) for 14 days.

General introduction 17 1 1.2.3.3 Leaf morphology In Arabidopsis, research has been done on lamina blade traits in rosette stage of development. Far-red light signals lead to a blade area reduction coupled with curling (Reed et al., 1993; Devlin et al., 1999; Cagnola et al., 2012). Chitwood and colleagues performed a metaanalysis on the morphological consequences of short-term or long-term exposure to shade in tomato (Chitwood et al., 2012; Chitwood et al., 2015). They found that leaf area, stomatal density, and chlorophyll abundance were changed in shade, and that alteration of leaf shape under shade is dictated by the expression of KNOX and other indeterminacy genes in tomato (Chitwood et al., 2012; Chitwood et al., 2015). A SAS study in maize (Zea mays) indicated that decline in biomass and leaf area growth during competition against weeds are likely occurring via low R:FR signaling from weeds to maize (Page et al., 2009). Woody species such as Populus, Acer, and Betula had faster growth rates in response to supplemental FR if they were late succession species in comparison to early succession species (weedy, fugitive) (Gilbert et al., 2001). In densely populated communities, the competition for living space among plants is influenced by leaf morphology and size within the canopy, impacting the generation of proximity signals and the ability to tolerate shade (Gilbert et al., 2001). 1.2.3.4 Root system architecture In shade avoidance response, plants undergo significant alterations in root morphology and architecture to acclimate to low light conditions and optimize their resource acquisition. Several studies have provided insights into the root modifications observed during shade avoidance. Shade has well-documented effects on above-ground plant tissues, yet understanding its impact on root development remains limited (Gundel et al., 2014). Applying FR-enriched light to whole seedlings is known to reduce main root length and lateral roots (LRs) (Salisbury et al., 2007; van Gelderen et al., 2018). However, the influence of shoot R:FR signaling on root development and the underlying mechanisms are unclear. Root growth is traditionally regulated by auxin transport and signalling (Bhalerao et al., 2002), with auxin playing a role in responses to various stimuli (Baster et al., 2013; Galvan-Ampudia et al., 2013; Zhang et al., 2013). It is plausible that auxin dynamics associated with shoot shade avoidance responses impact root auxin levels, thereby influencing root development. Light signalling further influences auxin transport by regulating auxin-transporter proteins (PINs), demonstrating its intricate impact on root development (Van Gelderen et al., 2018). The dynamic changes in root structure and function during shade avoidance demonstrate the plant’s ability to acclimated to shaded conditions and ensure optimal resource acquisition for sustained growth and development.

Chapter 1 18 Overall, phenotypic traits linked with SAS are understood in Arabidopsis while the molecular mechanisms are still being resolved. However, not all plants share the rosette growth habit of Arabidopsis. Understanding the regulation of SAS traits in alternative model plants and other dicots will advance our understanding of the SAS for various traits, and reveal how much of the signalling networks and gene regulatory pathways are shared between different species and growth habits. 1.2.4 Hormonal regulation pathways in shade avoidance Shade avoidance regulation is widely studied in Arabidopsis and various crops, and these studies have identified a network of hormones translating light information into developmental responses (Figure 1.5). In crops, responses to shade involve altered growth patterns and physiological changes, affecting both above-ground and belowground structures. Different tissues, such as stems, leaves, and roots, exhibit specific responses to shade conditions. Hormones, such as auxin, gibberellin and brassinosteroid. The interplay of these hormones in the network of shade avoidance responses varies among crops and tissues, contributing to the adaptability of plants to light availability. Figure 1.5. Arabidopsis SAS hormone regulation scheme adapted from literature (Yang and Li, 2017; Wang et al., 2020). Arabidopsis picture adapted from Biorender.com.

General introduction 19 1 1.2.4.1 Role of Auxins in SAS Auxins are a class of phytohormones that are essential for plant growth and development. Auxin is synthesized in the shoot apical meristem and young leaves, and transported to other parts of the plant through polar transport (Woodward and Bartel, 2005) Auxin biosynthesis is regulated by a family of genes known as YUCCAs (YUC), which encode flavin-containing monooxygenases that catalyze the rate-limiting step in auxin biosynthesis (Zhao, 2010). In Arabidopsis, there are 11 YUC genes, and their expression is regulated by light (Sato et al., 2015). YUCs have also been identified in other species such as tomato (Expósito-Rodríguez et al., 2011) and soybean (Wang et al., 2017). Auxin signaling is mediated by a family of transcription factors, known as AUXIN RESPONSE FACTORs (ARFs), which bind to auxin-responsive elements (AuxREs) in the promoters of target genes (Woodward and Bartel, 2005). Auxin is sensed by a family of F-box proteins known as TIR1/AFB that function as auxin receptors (Kepinski and Leyser, 2005). ARFs have the ability to bind tandem repeat AuxRE sequences either as homodimers, in conjunction with other ARFs, or in combination with repressive Aux/IAA proteins. In the absence of auxin, the function of ARFs is inhibited by Aux/IAA proteins, which form dimers with ARFs to prevent their activity (Woodward and Bartel, 2005; Li et al., 2016). Upon binding of auxin to its receptors, this complex can now interact with AUX/IAA proteins and facilitate their ubiquitination and subsequent degradation. This allows ARFs to homodimerize and regulate auxin-dependent gene expression. Light-dependent interactions of cryptochrome 1 (CRY1) and phytochrome B with AUX/ IAA proteins leads to the stabilization of AUX/IAAs, resulting in the inhibition of auxin signaling (Luo et al., 2018; Xu et al., 2018). When auxin levels increase, auxin binds to TIR1/AFB proteins, causing the degradation of AUX/IAA proteins and the activation of ARF-mediated gene expression (Stacey et al., 2016). As a consequence, hypocotyl elongation is stimulated. This elongation is also regulated by the ARF7 and ARF19 transcription factors, which are induced by low R:FR ratios (Nozue et al., 2015). ARF7 and ARF19 activate the expression of a number of downstream genes, including several encoding cell wall-modifying enzymes, which promote cell elongation (Okushima et al., 2007). It has been shown that auxin primarily acts in the hypocotyl epidermis to regulate low R:FR-induced hypocoyl elongation in Arabidopsis SAS (Keuskamp et al., 2010b; Procko et al., 2016). Extensive investigation into auxin signaling in SAS has been conducted primarily in Arabidopsis, prompting a broader examination to assess the conservation of auxin signaling across different contexts. The role of auxin in the shade avoidance response in

Chapter 1 20 legumes has been studied in several species. In soybean, for example, low R:FR condition has been shown to induce the expression of several auxin biosynthesis and transport genes, including GmYUCCA1, and GmPIN1a, thought to be involved in promoting stem elongation (Wang et al., 2017; Zhang et al., 2022). 1.2.4.2 Role of Gibberellins in SAS Gibberellins (GAs) play a significant role in regulating the shade avoidance response in plants. Under low R:FR conditions, the accumulation of phytochrome-interacting factors (PIFs), modulate the expression of genes involved in GA biosynthesis and signaling pathways, and promote GA levels. Elevated GA levels results in a dynamic distribution of GA in organs, and further stimulate stem and petiole elongation (Djakovic-Petrovic et al., 2007; Casal, 2013; Küpers et al., 2018; Lyu et al., 2021). GA destabilizes DELLA proteins, which are suppressors of PIF action, thus providing a direct SAS modulation mechanism (Djakovic-Petrovic et al., 2007; Feng et al., 2008; De Lucas et al., 2008). Moreover, GAs are also involved in the regulation of other shade-induced responses, such as hyponasty, leaf expansion and reduced branching (Kurepin et al., 2007; UeokaNakanishi et al., 2011; Küpers et al., 2023). 1.2.4.3 Role of Strigolactones in SAS Another aspect of the shade avoidance hormonal network is the role of strigolactones, a class of plant hormones that have been shown to regulate shoot branching and plant architecture in response to environmental cues (Agusti et al., 2011; Siddiqi and Husen, 2017). In Arabidopsis, the transcription factors FAR-RED ELONGATED HYPOCOTYLS3 (FHY3) and FAR-RED IMPAIRED RESPONSE1 (FAR1), integral to phytochrome A-mediated light signaling, act with strigolactone pathway repressors, SUPPRESSORS OF MAX2 1-LIKE (SMXL6/7/8). This alliance directly inhibits SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9, 15 (SPL9 and SPL15), suppressing their activation of BRANCHED 1 (BRC1), a pivotal branching repressor. FHY3 and FAR1 not only modulate SMXL6/7/8 but also directly up-regulate their expression, accentuating the central role of strigolactone in orchestrating branching. Under simulated shade conditions, reduced FHY3 accumulation results in elevated BRC1 expression, limiting branching. This integrated model underscores how light and strigolactone intricately regulate branching through concerted actions on the BRC1 promoter (Xie et al., 2020). Recent studies have suggested that strigolactones may also play a role in the shade avoidance response in legumes by regulating the balance between shoot and root growth (Siddiqi and Husen, 2017). In

General introduction 21 1 pea (Pisum sativum), exposure to low R:FR conditions has been shown to increase the production of strigolactones, which in turn promote the growth of lateral roots and suppress shoot branching (Dun et al., 2013). In rice, strigolactone has been proven to promote tiller architecture (Zha et al., 2022). 1.3 STEM GROWTH 1.3.1 Comparison of stem and petiole The stem is a support and transport organ in vascular plants. Plant stems have nodes from which they grow leaves, aerial roots, and flowers, and the sections of stem between nodes are called internodes. The conjunctive tissue that extends from the stem to the leaf lamina is the petiole (Mauseth, 2003). Petioles are generated from leaf primordium, and petiole activities are regulated by hormones and light (Sasidharan et al., 2010; Pierik and De Wit, 2014; Bravo et al., 2016). Both petioles and stem show elongation in SAS. Regarding their cellular morphology (Figure 1.6), both of them have epidermis, parenchyma and vascular bundles, and the vascular bundles in the petiole usually show multiple shapes, different from the mostly circular shape in stems (Ergen Akçin et al., 2011; Ragheb et al., 2019). Figure 1.6. Petiole and inflorescence stem cross sections of Arabidopsis. (a) Cross section of the petiole, provided by Sanne Matton. (b) Cross section of the inflorescence stem. 1.3.2 Primary growth and secondary growth Plant growth is categorized into primary growth, which increases the length or height of the plant, and secondary growth, which augments the plant’s diameter. Secondary growth is facilitated by lateral meristems, specifically the vascular cambium and cork cambium (Etchells and Turner, 2010). The cork cambium forms the outer ring of the stem, giving

Chapter 1 22 rise to phellem (cork) externally and phelloderm (secondary cortex) internally. The initiation of fibers and production of xylem indicates the start of secondary growth in hypocotyls (Taiz et al., 2010; Shana Kerr, 2017; Ragni and Greb, 2018). Vascular cambium, a closed ring of lateral meristematic tissue, is present in both roots and shoots, albeit with morphological differences. For instance, interfascicular cambium exists in shoots but is absent in roots, defined as cambium fusing to create a continuous ring in plant stems through a change in cell identity (Ragni and Greb, 2018). In plant development, primary growth, occurring in apical meristems, propels the vertical elongation of shoots and roots, contributing to an increase in height and the development of primary tissues like the epidermis, cortex, and vascular bundles. On the other hand, secondary growth, occurring in lateral meristems such as the vascular cambium and cork cambium, leads to the thickening of stems and roots. This process adds layers of secondary xylem and phloem, contributing to wood formation in dicots and enhancing mechanical support, water transport, and nutrient distribution. While primary growth facilitates upward and downward extension, secondary growth fortifies structural strength and adaptability, collectively shaping the overall form and function of plants. 1.3.3 Tomato stem structure identification in this thesis Tomato stems at four weeks of age are multi-layered structures that serve as a scaffold for plant growth and support (Figure 1.7). The stem consists of outer epidermis (light green layer), hypodermis (purple layer), 1-3 layers of collenchyma cells (blue-green layer), 1-3 layers of parenchyma cells (light orange layers), pith (yellow layer) and a vascular bundle and interfascicular cambium composed of tightly connected tiny cells (red layer). The outer epidermis layer provides a protective barrier against external stresses, while the hypodermis layer provides structural support to the stem. The collenchyma cells offer flexible support for the stem to grow and move, while the parenchyma cells are involved in photosynthesis, storage of water and nutrients, and transport of metabolites. The pith serves as storage tissue for water and nutrients, and the vascular bundle and interfascicular cambium provide the stem with transport tissue for water and nutrients. The tomato stem structure plays a crucial role in the overall growth and development of the plant (Esau, 1953; Evert and Esau, 2006).

General introduction 23 1 Figure 1.7. The cell types of tomato stem. The diagram shows combined cross section and longitudinal section, both taken from the middle of the first internode. In SAS research, some attention has been on how cellular morphology changes in response to shade or low R:FR. The most well-known hypothesis is epidermal growth-control: the epidermal layer is controlled by the tension and stress coming from the expansion of the inner layers of the hypocotyl in the stage of unidirectional growth (Kutschera and Niklas, 2007; Robinson and Kuhlemeier, 2018). Epidermis cell numbers and cell length increase in the petiole in response to shade, as found in Trifolium repens (Huber et al., 2008). In a soybean study, pith cell elongation in particular was related to internode elongation, as well as cell division of all cell types was enhanced to result in internode elongation (Beall et al., 1996). However, there is only limited results from tomato: the cellular morphology changes of stem in SAS have not been characterized previously. 1.4 GENERAL SUMMARY AND OUTLOOKS In this general introduction, it was discussed what are the intricate regulation networks that contribute to the regulation of plant architecture as part of SAS, and the coordination of hormone interplay in shade avoidance in multiple crop species and Arabidopsis was explored. In this thesis, we explore the shade avoidance responses of tomato and other dicots, focusing on stems and the hormonal regulation of internode elongation, involving different cell layers.

Chapter 1 24 Chapter 2 of this thesis is a comprehensive phenotyping analysis of two tomato cultivars under different lighting- white light control and white light that is supplemented with far-red light as the treatment. We report various phenotypic traits, including stem and petiole length, leaf area, leaf thickness, chlorophyll content, and cellular level analysis of stem morphology using microscopy. These detailed phenotyping data form the basis for further investigations at the molecular level, focusing specifically on internode 1. Building upon the phenotypic findings, in Chapter 3 we delve into a molecular analysis of internode 1 using time series transcriptomics. This approach allowed for a deeper understanding of the regulatory pathways involved in early-stage elongation, preceding visible changes in plant growth. By examining gene expression patterns over time, we identified several intriguing transcription factors (TFs) including a bZIP finger TF, a GATA TF, and a TCP (TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING) TF for further exploration. In Chapter 4, we unravel the roles of auxin, gibberellin and brassinosteroids in the tomato SAS. Manipulating hormone levels, we aimed to further understand the elongation and cellular changes observed under far-red enrichment. A better understanding of their contribution to SAS was obtained, shedding light on the molecular mechanisms underlying tomato responses to shading. Expanding the investigation outwards in Chapter 5, our study broadened its scope to include multiple dicot species. We aimed to investigate the conservation of pith cell responses in SAS and the conservation of FR-responsiveness of our target TFs across different plants, providing insights into the evolution of this adaptive response. Chapter 6 then brings the discoveries in the experimental chapters together for a general discussion. Through a multidisciplinary approach encompassing phenotyping, transcriptomic analysis, hormone manipulation, and cross-species comparisons, this research thesis unravels the intricate mechanisms of shade avoidance responses in dicots beyond the usual model plant Arabidopsis. Here, we have discussed the rationale behind choosing tomato as the focal point of investigation. This choice may be underscored by the unique characteristics of tomato growth patterns and responses that deviate from established models, thus warranting dedicated exploration. Moreover, the experimental design should be contextualized within the existing gaps in knowledge, emphasizing what remains unknown, particularly in the context of the role of hormones in SAS tomato shade avoidance.

General introduction 25 1

CELLULAR ANATOMY OF TOMATO STEMS IN RESPONSE TO FAR-RED LIGHT Linge Li, Ticho Helming, Ronald Pierik, Kaisa Kajala

Chapter 2 28 ABSTRACT Food scarcity is a pressing global concern, particularly as the world’s population is projected to increase. With limited agricultural land available, the demand for higher crop yields forces dense planting and intensifies competition for limited resources. One crucial resource that becomes scarce in dense canopies is light, prompting some plants to elongate to search for light and to counter shading. This adaptive behavior is known as the shade avoidance syndrome (SAS). This study focuses on two tomato (Solanum lycopersicum) cultivars, M82 and Moneymaker, serving as representative models. Our investigation delves into how M82 and Moneymaker, cultivated in the field and greenhouse respectively, acclimate to light cues of shade. Our objective was to characterize the tomatoes’ cellular developmental plasticity in response to far-red light, a common cue for detecting neighboring plants. We evaluated cellular phenotypic traits in white light versus far-red supplemented white light conditions that simulate shading or light reflection by green leaves. Using statistical analyses, we identified the most notable responses among the measured traits. Furthermore, we conducted microscopy-based quantification of cell types of the stem in the first internode where we had observed increased elongation under far-red treatment compared to white light. We found significant cellular anatomy responses in pith and interfascicular cambium, paving the way for an in-depth exploration of these cell types in subsequent chapters (3-5).

Cellular anatomy of tomato stems in response to far-red light 2 29 2.1 INTRODUCTION With the increasing population and shortage of land area, the huge need for food, fuel, and fiber from field crops cannot be fully satisfied by current agricultural production. Hence, plants are farmed in dense vegetation to increase the yield from the available land area. However, this will not necessarily lead to an increase in yield per plant. On the contrary, dense vegetation increases plant competition for limited resources, one of which is light. When the light captured by the plant is not able to meet the growth demand, plant growth will decrease. The competition for light between neighboring plants is usually triggered by impending shade. Shade from vegetation is a light condition that is characterized by the relative enrichment of far-red (FR) light and overall low light density. FR light is already increased in canopies before true shading occurs by the reflection of light from neighboring plants, and FR enrichment alone can trigger a set of developmental responses known as the shade avoidance syndrome (SAS). Plants that face shade stress can either survive through tolerance to low light, or avoid future shade by SAS; a set of morphological changes to grow away from unfavorable growth condition (Smith and Whitelam, 1997). Plants adapt their growth and photosynthetic activity in response to changes in light conditions, particularly the ratio of red (R) to far-red (FR) light, but also the depletion of blue light. This shift in the light spectrum, which occurs in dense plant canopies, triggers SAS in plants, most commonly making them taller and more erect. Key photoreceptors, such as phytochromes and cryptochromes, play pivotal roles in perceiving these light signals. Phytochromes exist in two forms, Pr (red-absorbing) and Pfr (far-red-absorbing), with Pfr being the active form responsible for mediating SAS. The shade-induced changes, including stem elongation and altered leaf architecture, have been observed across various plant species, such as Arabidopsis, Brassica rapa, and tomato (Osborne, 1991; Pierik and De Wit, 2014; Ballaré and Pierik, 2017; Courbier et al., 2021). While stem elongation is a prominent feature of SAS, the cellular mechanisms underlying these changes have not been extensively studied in tomato. The response to shade varies among tomato species, with some showing a stronger SAS than others. Notably, Solanum pennellii exhibits a robust SAS, while cultivated tomato species like Solanum lycopersicum display a milder reaction. However, the specific SAS mechanisms within different tomato species remain less understood (Bush et al., 2015) In SAS, both stems and petioles undergo elongation, and these tissues share similarities in function and cellular morphology. Cellular changes in response to shade, particularly in the epidermal layer, have been documented in other plant species. For instance,

Chapter 2 30 epidermal cell numbers and lengths increase in response to shade, contributing to stem and petiole elongation. While these cellular responses have been observed in Trifolium repens and soybean (Weijschedé et al., 2008; Lyu et al., 2021), similar investigations in tomato are scarce (Courbier et al., 2021). In this chapter, we measured various architectural traits in response to low R:FR in two commercial tomato cultivars: M82 and Moneymaker. M82 is a model for field-grown processing tomatoes. M82 has medium-sized oval fruit, originates from the USA, and has been used for many gene function studies (Brooks et al., 2014; Xu et al., 2015; Gupta and Van Eck, 2016; Kajala et al., 2021). Moneymaker is a greenhouse crop from the UK, cultivated for more than 80 years worldwide, and is a typical model for greenhouse tomatoes in research (https://www.ufseeds.com/product/moneymaker-tomato-seeds/ TOMO.html; Courbier et al., 2021).As these two cultivars have differences in usage and growth environment, we chose to compare them in this study to provide a more comprehensive understanding of the SAS among tomato cultivars. After the architectural traits, we used microscopy to characterize the cellular anatomy SAS of the first internode and identified the most significantly responding cell types, 2.2 RESULTS 2.2.1 FR promotes hypocotyl length, but has no effect on root growth in tomato seedlings In SAS studies of Arabidopsis, many responses at seedling stages are of interest, including hypocotyl elongation, hyponasty (increased leaf angle), and changes in root system architecture (Kasperbauer, 1971; Pierik et al., 2004; Bou-Torrent et al., 2014; Pantazopoulou et al., 2017; van Gelderen et al., 2018). Arabidopsis seedlings root system responds to FR enrichment with decreased lateral root density (van Gelderen et al., 2018; Kang, 2018). We wanted to see how Arabidopsis knowledge translates to a crop species, so we set out to characterize phenotypes of tomato seedlings in white light supplemented with far-red (WL+FR) compared to white light (WL) control. First, we conducted seedling characterization. We grew S. lycopersicum cv. Moneymaker seedlings on plates for 5 dag (days after germination) and then treated them with supplementary FR or continued with control WL for 7 days. At this point, the cotyledons had emerged but the seedlings did not yet have true leaves (Figure 2.1). We measured hypocotyl length (Figure 2.2a), primary root length (Figure 2.2b), lateral root length (Figure 2.2c), total lateral root length (Figure 2.2d), lateral root number (Figure 2.2e), and calculated the lateral root density as lateral root number divided by primary root length (Figure 2.2f).

Cellular anatomy of tomato stems in response to far-red light 2 31 Moneymaker hypocotyls were two times longer (1.9cm compared to 3.9 cm) in WL+FR treatment compared to WL control (Figure 2.4a). However, unlike hypocotyl elongation, main root length, lateral root length, lateral root number and density did not respond to WL+FR treatment (Figure 2.4). Figure 2.1. Seedling stage of Moneymaker grown on a plate for 10 days. WL WL+FR 0 2 4 6 *** Hypocotyl length(cm) WL WL+FR 0 1 2 3 No of lateral roots/length(cm-1) WL WL+FR 0 10 20 30 40 Lateral root number WL WL+FR 0 5 10 15 20 Primary root length(cm) WL WL+FR 0 2 4 6 Lateral root length (cm) WL WL+FR 0 10 20 30 Total lateral root length(cm) (a) (b) (c) (d) (e) (f) Figure 2.2. Hypocotyl and root traits in WL and WL+FR for tomato cultivar Moneymaker. Two independent biological experiments were conducted (n=15). The data includes measurements of (a) hypocotyl length, (b) primary root length, (c) lateral root length, (d) total lateral root length, (e) lateral root number and (f) lateral root density (e divided by b). Asterisks indicate significance between WL and WL+FR as follows: *** p≤0.001.

Chapter 2 32 2.2.2 Stem elongates in response to FR in M82 and Moneymaker Based on the seedling experiment, we chose to focus on shoot responses, with the intention of expanding our research to encompass tomatoes that were bred for two distinct cultivation systems, M82 and Moneymaker (MM). We wanted to conduct phenotyping on young plants to investigate more aspects of stem and leaf responses and zoom in also at the cellular level. We transplanted germinated seedlings into soil and let them recover for a week before 7-day light treatments (WL+FR or WL). We collected phenotypic data of MM and M82 at 21 dag (see Figure 2.3). In general, the stems elongated more and leaves were paler green in both cultivars when they were grown in WL+FR light as compared to WL. Figure 2.3. Comparison of S. lycopersicum cultivars M82 and Moneymaker 24-day-old young plants in WL (left) vs FR+WL (right). This picture was taken at 10 days of treatment to illustrate the different phenotypes between WL and FR+WL. We conducted shoot architecture phenotyping which included measuring the following traits: stem length, hypocotyl length, internode lengths, stem diameters, petiole lengths, rachis lengths, and leaf areas. Length and diameter measurements were taken by caliper and measured traits are shown in Figure 2.4. Leaf area was measured from scanned images using ImageJ. The major finding from the phenotyping is that FR significantly enhances stem and internode length in both varieties (Figure 2.5).

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