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Light and hormonal regulation of the tomato plastidial development and maintenance gene GOLDEN 2-LIKE 2 and its effect on fruit nutritional quality

Plastids are organelles responsible for several essential aspects for plant development, like photosynthesis, nitrogen assimilation and synthesis of several compounds of secondary metabolism. Chloroplasts differentiation and activity are highly regulated by light, and several proteins and mechanisms involved in these processes have been characterized. The GOLDEN 2-LIKE (GLK) transcription factors controls the expression of several genes related to photosynthesis, plastid biogenesis and maintenance. Solanum lycopersicum genome harbors two copies of this gene, SlGLK1 and SlGLK2 and, although they are functionally redundant, their expression pattern is different, once SlGLK1 predominates in leaves, while only SlGLK2 is expressed in fruit, precisely at the pedicel region. During tomato domestication, selection for varieties that ripened evenly resulted in the fixation of uniform ripening mutation (Slglk2) in most cultivated varieties, resulting in alterations in fruit metabolic composition. In this context, the objective of this work was to functionally characterize SlGLK2 gene aiming to understand in which way phytochrome mediated light and phytohormones, particularly auxins and cytokinins, regulates this gene expression, and how SlGLK2 presence affects fruit nutritional quality. To achieve this, a detailed transcriptional profile of SlGLK2 was performed in fruits of wild plants, Slglk2 mutant and plants deficient for light perception or hormonal signaling. The effect of SlGLK2 over nutritional quality was evaluated by characterizing carbon and vitamin E metabolism. Additionally, reporter protein GUS activity was quantified in transgenic plants that express uidA gene under control of promoters responsive to cytokinins or auxins in SlGLK2 or Slglk2 genotypes, to analyze if hormonal activity is affected by SlGLK2 presence. Finally, in order to verify if the presence of SlGLK2 is sufficient to reverse the chlorotic phenotype of the mutant aurea, promoting the differentiation and plastidial maturation even in the absence of functional phytochromes, transgenic lines were generated by overexpressing the SlGLK2 gene on aurea-Slglk2 genetic background. The integrated data analysis allowed us to conclude that the content of soluble sugars and vitamin E correlate with the expression of SlGLK2, that the expression of SlGLK2 is repressed by auxins, that SlGLK2 positively participates in the signaling of cytokinins, and that its overexpression partially reverts the phenotype of the aureaSlglk2 mutant fruits. The results obtained in this work contributes to a better understanding of the regulatory network that interconnects SlGLK2 gene, phytohormones and light, promoting the plastidial activity and consequently, determining the nutritional quality of the tomato fruit, an important component of the human diet.


Abstract
Plastids are organelles responsible for several essential aspects for plant development, like photosynthesis, nitrogen assimilation and synthesis of several compounds of secondary metabolism.
Chloroplasts differentiation and activity are highly regulated by light, and several proteins and mechanisms involved in these processes have been characterized. The GOLDEN 2-LIKE (GLK) transcription factors controls the expression of several genes related to photosynthesis, plastid biogenesis and maintenance. Solanum lycopersicum genome harbors two copies of this gene, SlGLK1 and SlGLK2 and, although they are functionally redundant, their expression pattern is different, once SlGLK1 predominates in leaves, while only SlGLK2 is expressed in fruit, precisely at the pedicel region. During tomato domestication, selection for varieties that ripened evenly resulted in the fixation of uniform ripening mutation (Slglk2) in most cultivated varieties, resulting in alterations in fruit metabolic composition. In this context, the objective of this work was to functionally characterize SlGLK2 gene aiming to understand in which way phytochrome mediated light and phytohormones, particularly auxins and cytokinins, regulates this gene expression, and how SlGLK2 presence affects fruit nutritional quality. To achieve this, a detailed transcriptional profile of SlGLK2 was performed in fruits of wild plants, Slglk2 mutant and plants deficient for light perception or hormonal signaling. The effect of SlGLK2 over nutritional quality was evaluated by characterizing carbon and vitamin E metabolism. Additionally, reporter protein GUS activity was quantified in transgenic plants that express uidA gene under control of promoters responsive to cytokinins or auxins in SlGLK2 or Slglk2 genotypes, to analyze if hormonal activity is affected by Introduction

Plastid biogenesis, differentiation and maintenance
Plastids are organelles with a great diversity of shapes and functions, and they are found in all photosynthetic eukaryotes. Besides being responsible for photosynthesis, depending on the plant development stage, these organelles are also responsible for other functions, such as synthesis of amino acids, fatty acids, nitrogenous bases, pigments and hormones. Additionally, they also participate on the assimilation of sulfur and nitrogen (Jarvis & López-Juez 2013).
Land plants have many plastids that play different functions ( Figure 1). Proplastids are found mainly in meristematic tissues, and are the precursors of other plastids. Amyloplasts accumulates starch and are found mainly in storage organs, such as roots, seeds and tubercles, having a key role in energy storage and gravitropism. Gerontoplasts, which are mostly found in senescent tissues, originate from the disassembly of photosynthetic machinery and macromolecule degradation, playing a main role in nutrient recycling and remobilization towards sink organs. Chloroplasts synthetize and accumulate chlorophyll and maintain all the machinery responsible for light capture and photosynthetic activity, allowing atmospheric carbon fixation, on which most forms of life depend. Chromoplasts are found in flowers of different species and also in fleshy fruits, in which they perform various functions, including synthesis and accumulation of a wide spectrum of metabolites, many of them with nutraceutical relevance, i.e. antioxidants. In this regard, great effort has been made to understand minutely the mechanisms responsible for differentiation and maintenance of plastid structure and metabolism, aiming the improvement of nutritional quality of edible fruits (Jarvis & López-Juez 2013).
Among the exogenous factors that influence the differentiation of proplastids into chloroplasts, the light signal has a prominent role ensuring this transition only in appropriate conditions for photosynthetic activity. The main photoreceptors involved in the light perception that regulates this conversion are the PHYTOCHROMES (PHYs). Structurally, PHYs are homodimeric proteins whose unit is composed of an apoprotein bound to a tetrapyrrole chromophore, i.e.
phytochromobilin (Gyula et al. 2003). In the darkness PHYs are found inactive in the cytoplasm while, in the presence of light, phytochromobilin undergoes an isomeric alteration that changes the structure of the apoprotein, directing the active PHYs to the cellular nucleus (Bae & Choi 2008).
Within the nucleus, they promote degradation of PHYTOCHROME INTERACTING FACTORS  (Fitter et al. 2002). Additionally, HY5 positively regulates Arabidopsis thaliana GLK2 (Lee et al. 2007). Together, HY5 and GLKs activate transcription of several proteins related to the photosynthetic machinery and, consequently, chloroplast differentiation (Jarvis & López-Juez 2013 In relation to endogenous factors, plastidial biogenesis and differentiation is also influenced by different phytohormones. Auxins and cytokinins, for example, regulates plastidial differentiation in different plant organs. At the seedling stage, cytokinins play a key role in chloroplast differentiation and division during de-etiolation, whereas auxin has an inhibitory effect, preventing, for example, the development of chloroplasts in the roots (Cortleven & Schmulling 2015;Kobayashi et al. 2017).

Importance of plastid metabolism for nutritional quality of Solanum lycopersicum fruits
Because of its nutritional importance, widespread consumption in the western population and economic importance, the tomato (Solanum lycopersicum) has been configured as the model species for the study of fleshy fruits development and ripening (Giovannoni 2004).
The development of the tomato fruit begins after pollination with a phase of intense cell division comprising the immature green stages (IG), followed by a period of cell expansion until the fruit reaches its final size at mature green stage (MG). During these green stages, the fruits have active photosynthetic machineries (Carrara et al. 2001). Although fruits in general are sink organs, requiring more photoassimilates than they produce, local photosynthesis in tomatoes seems to be responsible for up to 20% of the total carbon in ripe fruits (Cocaliadis et al. 2014).
From MG stage onwards, the fruits become responsive to ethylene, which triggers ripening, turning fruits from MG to breaker stage (Br), when the fruit starts to change color from green to yellow. During this process, a progressive conversion of chloroplasts into chromoplasts is observed involving several biochemical alterations that will contribute to the definition of the color, flavor, aroma and texture of the ripe fruit (five days after Br onwards, Br+5). Gradual chlorophyll degradation leads to loss of green coloration, and the released phytol is recycled and used, at least in part, for tocopherol (vitamin E, VTE) production . Carotenoid biosynthetic route is intensely stimulated during ripening, resulting in accumulation of lycopene, responsible for the intense red color of ripe tomato fruits (Giovannoni 2004). The induction of several aromatic amino acid decarboxylases leads to increased production of volatile compounds, such as phenylethanol and phenylacetaldehyde, which confer the characteristic aroma of tomatoes (Tieman et al. 2006;Tieman et al. 2007). Fruit softening is induced by cell wall hydrolases, changing fruit texture (Fischer & Bennet 1991). Finally, the degradation of starch and cell wall increases the content of soluble sugars and organic acids, determining the texture and density of tomato puree, i.e. the °Brix, a feature of great industrial importance considering that two-thirds of the world's production is consumed as processed tomato (Carrari & Fernie 2006;Sila et al. 2009). All the described changes determine the nutritional and industrial quality of the fruit (Giovannoni 2004;Fraser et al. 1994;Egea et at. 2011).
The most valuable nutraceutical compounds in fleshy fruits, e.g. carotenoids and tocopherols, are synthetized in chloroplast/chromoplast. Therefore, chloroplast abundance and activity in the green stages of the fruits collaborate not only for the synthesis of part of the photoassimilates necessary to the organ development, but also determine future capacity of these fruits to produce nutraceutical compounds in the chromoplasts. In this way, the increment in the number of active chloroplasts, as well as the enhancement of plastidial metabolism, appears as a key target for of fleshy fruit yield and quality improvement (Isaacson et al. 2002;Nashilevitz et al. 2010).
In this work, we focused on tocopherol nutraceutical compound, since it is a subject of special interest in our research group. Moreover, tomato fruit carotenogenesis has been extensively characterized (Liu et al. 2015).   Rastogi et al. 2014;Miret & Munné-Bosch 2015). Beyond photoprotective roles, tocopherol is also involved in seed longevity, seedling germination (Sattler et al. 2004;Mène-Saffrané et al. 2010), and photoassimilate export regulation .
Tocopherol metabolism (Figure 3) is highly linked to chlorophyll and carotenoid metabolic pathways and, in recent years, it has been characterized in tomato (Almeida et al. 2011;Almeida et al. 2016;Quadrana et al. 2013;Guyer et al. 2014;Lira et al. 2016). The precursors for tocopherol biosynthesis are derived from two plastidial secondary metabolism pathways, methylerythritol phosphate (MEP) and shikimate (SK) and the description below highlights the main steps for which the catalyzing enzyme encoding genes have shown to be transcriptional regulated and will be studied in the present work (Quadrana et al. 2013).

The role of GOLDEN 2-LIKE genes in chloroplast maintenance and activity.
Several transcriptional factors are involved in chloroplast differentiation. Among them, GLK proteins are one of the key factors, with great importance for both, plastidial biogenesis and activity maintenance (Fitter et al. 2002). In A. thaliana, there are 2 loci that encodes for GLK proteins, AtGLK1 and AtGLK2. Although they have a slightly different transcriptional pattern, they are functionally redundant since only the double mutant, Atglk1Atglk2, is deficient in plastid development. AtGLK1 and AtGLK2 proteins induce expression of several genes involved in the formation of the photosynthetic apparatus, and respond to retrograde signals of chloroplasts, being able to coordinate the expression of nuclear genes related to photosynthesis and optimize this process according to environmental conditions ).
In S. lycopersicum genome there are also two copies of GLKs: SlGLK1 and SlGLK2. While SlGLK1 is mostly expressed in cotyledons, sepals and leaves, SlGLK2 is predominantly expressed in fruits, concentrated at the pedicelar region, resulting in a phenotype called "green shoulder", decreasing in a longitudinal gradient until the base of the fruit (Powell et al. 2012;Nguyen et al. 2014).
Characteristics that facilitate harvesting and shipping, and increase shelf-life had great importance during tomato domestication (Giovannoni 2001). In this context, in order to select fruits that ripen evenly, the uniform ripening mutation was fixed, selecting the mutant allele of SlGLK2 (Slglk2), which is found in most cultivated tomato varieties nowadays. Slglk2 allele has a single base insertion at the coding region, originating a premature stop codon and a truncated protein with only 80 amino acids (Figure 4), while the wild protein encoded by the wild allele has 310 amino acids (Powell et al. 2012).
Fixation of Slglk2 mutation had metabolic consequences, as mutant fruits has lower amount of chlorophyll and soluble sugars (Powell et al. 2012;Nguyen et al. 2014). Also, overexpression of SlGLK2 in mutant background results in fruits with higher content of starch, soluble sugars and carotenoids (Powell et al. 2012;Nguyen et al. 2014). There are no reports of SlGLK2 effect on other nutraceutical compounds, such as VTE. Regarding SlGLKs expression regulation, it has been shown that SlGLK2 expression is higher in fruits that developed in the presence of light when compared to dark grown fruits (Powell et al. 2012 Sagar et al. (2013) demonstrated that the downregulation of SlARF4 expression, an auxin induced repressor of auxin response, leads to the upregulation of SlGLK1, resulting in the increment of chloroplast number and chlorophyll accumulation.
Additionally, senescence delay and maintenance of plastid activity has been largely described in "evergreen" plants with high endogenous cytokinin content, but the role of GLK genes has not been addressed in these studies (Thomas & Ougham 2014).
In this context, the importance of GLK genes for plastid function is evident, however, data on the effect of SlGLKs on fruit quality and their regulatory mechanisms are fragmented, and the studies were mostly restricted to the model species A. thaliana. Thus, the present work intended to improve the knowledge about SlGLK2, specially about their role on tomato fruit development and ripening.
Conclusions 1. GLK genes are a novelty of Embryophyta clade.
2. The duplication that originated SlGLKs occurred in Solanaceae lineage prior to the divergence between Solanum lycopersicum and Solanum tuberosum.
3. The SlGLK2 transcriptional regulation by light is, at least in part, mediated by PHYs.
6. SlGLK2 promotes the differentiation of chloroplasts with highly stacked thylakoids in a PHYindependent manner in fruits.
7. SlGLK2 promotes chlorophyll biosynthesis in immature green stages of fruit development, which is proportional to tocopherol content in ripe fruits.
8. SlGLK2-mediated chlorophyll and tocopherol increment are explained, at least in part, by the transcription upregulation of the biosynthetic enzyme encoding genes.
9. SlGLK2 alters carbon metabolism inhibiting starch accumulation at immature stages of fruit development.
10. SlGLK2 overexpression promotes the accumulation of higher levels of soluble sugar in ripe fruit contributing to higher Brix.
11. SlGLK2 has a negative effect on productivity traits such as fruit weight and aerial biomass.