Developmental genetic programs, which are regulated by both endogenous and exogenous stimuli, govern the transition between the distinct growth phases that comprise the life cycle of flowering plants. After germination and prior to attaining reproductive maturity, most plant shoots undergo a period of juvenile vegetative growth. As juvenile annual plants move towards reproductive competence also known as the adult vegetative phase, they show progressive developmental changes in their vegetative organs such as dimension, shape and phyllotaxis of the leaves, changes in internode elongation, or the patterns of trichome distribution (Huijser & Schmid, 2011; Keller, 2020a). When a well-defined set of environmental and endogenous conditions triggers the initiation of reproductive development, such as photoperiod, vernalisation or age, the adult vegetative plants initiate the development of flowers (Poethig, 1990; Poethig, 2010). It remains uncertain if the same variables regulating the juvenile-to-adult phase transition in annual plant species also regulate this transition in woody perennial plants. Shrubs and trees frequently display more pronounced distinctions between their juvenile and adult stages, with woody plants generally revealing these differences more consistently than herbaceous species. Secondly, the juvenile and adult vegetative stages are relatively short in herbaceous plants like Arabidopsis thaliana and maize, whereas they can persist for several years in perennial species (Lawson & Poethig, 1995a; Wang et al., 2011). Understanding this regulation is particularly essential in fruit trees, since an extended juvenile phase severely precludes crop selection and improvement, given that breeding and production features depend on attaining reproductive maturity, which includes flowering and fruiting (Ahsan et al., 2019).

It has been evident in recent years that there are several key regulatory variables shared by the networks that govern the transitions from the juvenile-to-adult phases. Furthermore, several of these factors influence specific heteroblastic characteristics that differentiate between juvenile and adult phases in annual plants (Poethig, 2010). At the transcriptional level, two highly conserved microRNAs (miR156 and miR172) have been widely identified as essential elements of the genetic control mechanisms underlying phase variations in several plant species like A. thaliana (Wu & Poethig, 2006), maize (Chuck et al., 2007), rice, (Xie et al., 2012) and tomato (Silva et al., 2014). The expression of miR156 is highly abundant in seedlings of the model plant A. thaliana, and it declines as growth progresses, whereas miR172 exhibits an opposite expression pattern. The inhibition of miR156 during the transition from the juvenile stage to the adult stage enables overexpression of several SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors that are negatively regulated by miR156 (Wu et al., 2009; Wu & Poethig, 2006; Xu et al., 2016b). Since miR172 is directly targeted by SPL genes, its expression increases gradually during the transition to the adult phase. In turn, miR172 participates in floral induction by directly repressing the expression of six Arabidopsis AP2-like transcriptional repressors including APETALA2 (AP2), TARGET OF EARLY ACTIVATION TAGGED (EAT) 1 (TOE1), TOE2, TOE3, SCHLAFMUTZE (SMZ), and SCHNARCHZAPFEN (SNZ). These proteins are responsible for the suppression of SOC1 and FT, which are activated during floral induction by increasing the activity of miR172 (Aukerman & Sakai, 2003; Chen, 2004). Such expression activation of SOC1 (and various SPLs) allows for the upregulation of different floral meristems to identity genes, such as APETALA1 (AP1), FRUITFULL (FUL), and LEAFY (LFY) promoting flower meristem development (Lee et al., 2008; Liu et al., 2008; Xu et al., 2016b). The conservation of the molecular regulation of these transition from the juvenile-to-adult phases in maize, rice and tomato is mediated by the corresponding orthologous genes and gene families (Chuck et al., 2007; Silva et al., 2014; Xie et al., 2012).

Despite the well-known fact that vegetative development is regulated by a temporal decrease in miR156 levels, how this decreased expression is initiated and then maintained during shoot development remains elusive. Recent research suggests miR159 and MYB33 function as moderators of vegetative phase change. Specifically, miR159 facilitates vegetative phase change by repressing MYB33 expression, targeting MYB33 transcripts for degradation in Arabidopsis. This prevents MYB33 from hyperactivating miR156 expression throughout shoot development, ensuring the correct timing of the juvenile-to-adult transition (Guo et al., 2017). Sugar metabolism was also associated with the promotion of phase transition through the repression of miR156 expression. In this sense, the repression of miR156 induced by glucose was dependent on the signalling activity of HEXOKINASE1 (HXK1) (Yang et al., 2013). Moreover, carbohydrate status was also involved in flowering induction, where TREHALOSE-6-PHOSPHATE SYNTHASE 1 (TPS1) seems to be required for the timely initiation of flowering (Wahl et al., 2013).

Gibberellins (GAs) are involved in the regulation of plant growth and are also known as adult phase promoters in Arabidopsis and maize (Lawson & Poethig, 1995b; Telfer & Poethig, 1998). The gibberellin pathway, which is required for flowering promotion in Arabidopsis, induces LFY expression, independent of photoperiod (Blazquez & Weigel, 2000). Additionally, by using the rice d18-dy mutant, Tanaka (2012) has shown that GAs participate in the change from juvenile to adult phase independent of microRNA regulation, particularly miR156 and miR172.

Knowledge about the genetic regulation of phase transition in perennial species is less abundant than in annuals. However, Wang et al. (2011), working with different woody trees, and Ahsan et al. (2019), working with horticultural tree crops, coincided in remarking that miR156 is an evolutionarily conserved regulator of vegetative phase change. However, in both reports, the role of miR172 was less clear than in annual plants, while the expression of flowering transition regulators was not deeply evaluated (Ahsan et al., 2019; Wang et al., 2011).

When grapevine (Vitis vinifera L.) plants are grown from seeds, vegetative development involves a transition from a juvenile to an adult phase after the formation of 6–10 nodes. This phase change is marked by a shift from spiral to alternate phyllotaxis, changes in leaf morphology from round to lobed forms, and the development of lateral meristems, known as uncommitted primordia, which arise opposite to leaf primordia and initially form tendrils. As in other Vitaceae species, the grapevine is characterised by a climbing growth habit supported on tendrils, which share a common ontogenetic origin with inflorescences (Lacroix & Posluszny, 1989; Mullins et al., 1992). In later years, under flowering-inductive conditions, some of those lateral uncommitted primordia will produce inflorescences instead of tendrils, depending on the environmental conditions and the plant status, related to the levels of bioactive gibberellins (Boss & Thomas, 2002; Gerrath, 1992; Keller, 2020a; Srinivasan & Mullins, 1980). While gene expression related to the flowering transition has been studied in grapevine (Carmona et al., 2008; Díaz-Riquelme et al., 2014), no studies have yet analysed the vegetative transition from the juvenile to the adult phase.

To understand the transcriptional regulation of the juvenile-to-adult phase transition during the development of grapevine plantlets grown from seeds, we performed a global gene expression analysis of plants derived from the self-cross of the near-homozygous line PN40024 (Jaillon et al., 2007; Velt et al., 2023), allowing a replicated RNA-seq analysis of different developmental stages. The quasi-homozygous state of this line warrants that all derived selfed-plantlets share a very similar genetic composition and behave similarly regarding the timing of phase transition.

The first targets for studying the existence of distinct juvenile and adult phases of vegetative development in plants were tree species, due to their typically prolonged and stable vegetative growing periods. However, the knowledge about the development and molecular regulation of these processes in perennials remains less studied than in annual plants, where the studies in maize (Poethig, 1988) and Arabidopsis (Poethig, 2010; Wang et al., 2011) provided the foundation in this field. Still, grapevines present various additional particularities regarding the biology of phase transition. In addition to the change in the phyllotaxis of the seedlings (from spiral to alternate) and the morphological change from juvenile to adult leaves, the vegetative phase transition is particularly characterised in grapevines by the appearance of tendrils (Carmona et al., 2007b; Mullins et al., 1992). Moreover, in grapevines (and most perennial crops), the vegetative transition of the plant from the juvenile phase to the adult phase and the induction of flowering occur at different seasons, even after two or three years of the seed germination and the corresponding seedling development (Keller, 2020b; Wang et al., 2011). These facts, together with the very limited existing literature on the biology of the grapevine plant during its first years of development, condition both the study of physiological responses of the plant and their gene regulation under such transition. The knowledge of the molecular basis of phase change in grapevine may be useful to understand the basis of its genetic variation and its possible relationship with the plant's fertility in different traits, like the number of inflorescences per shoot. Moreover, to what extent can the phase state affect the expression of reproductive traits?

Performing a whole-genome transcriptomic analysis of the vegetative phase transition in grapevine plantlets grown from seeds presents several challenges. Given the high heterozygosity of cultivated grape varieties, the seeds generated in a cluster are all genetically different (Keller, 2020a; Martínez-Zapater et al., 2010). This led us to select the PN40024 line, the only quasi-homozygous genotype available in grapevine (Jaillon et al., 2007; Velt et al., 2023), from which we developed a self-cross population for the transcriptomic experiments.

The results of the transcriptomic analysis of juvenile to adult phase transition provided some insights into the complex regulatory networks underlying this developmental shift in grapevine. In Figure 8 we summarise the main molecular events identified as involved in the transition from the juvenile to adult phase in grapevine. These results align with previous studies in model herbaceous annual plants such as Arabidopsis, rice, maize or tomato, where the downregulation of miR156 and the consequent upregulation of SPL transcription factors were established as key drivers of the juvenile to adult phase transition (Poethig, 2010; Wu et al., 2009; Wu & Poethig, 2006; Xu et al., 2016b). Likewise, the promotion of phase change, based on the repression of miR156 mediated by sugar (Yang et al., 2013), also seems to be conserved in grapevine. However, grapevine plants display features that might be distinctive. Particularly, Guo et al. (2017) suggested that repression of miR156 by miR159 regulates the timing of the juvenile-to-adult transition in Arabidopsis by repressing MYB33 expression and preventing this R2R3 MYB domain transcription factor from hyperactivating miR156 expression throughout shoot development, but grapevine initiation of phase transition does not seem to be related to a repression of miR156 by miR159 and MYB33. Moreover, the observed lack of miR172 activation in grapevine as miR156 decays, even with significant SPL gene upregulation, underlines a notable difference from the regulatory interactions observed in annual plants. In Arabidopsis, miR172 is consistently activated following miR156 repression, facilitating the transition to flowering (Wu et al., 2009; Xu et al., 2016b). However, in the present work, we observe a significant expression of genes like SOC1, FT, AP1, LFY, and FUL in adult vegetative plants, despite the absence of miR172 activation. This may suggest that these genes may be primed for alternative developmental stages rather than immediately driving floral induction. This observation is particularly relevant given the fact that grapevine, unlike annual species, does not initiate flowering within its first year, but accommodates its vegetative development to a perennial lifecycle with annual reproductive events. The slight but significant induction of SOC1, FT and the strong upregulation of other flowering-related genes in the apex of adult vegetative grapevine plants may thus serve as a priming mechanism, setting the stage for later reproductive development and/or to establish a default developmental program of on the lateral meristems for the development of tendrils (Carmona et al., 2007b; Mullins et al., 1992). In this sense, the extremely high expression of TFL1, the fourth most induced DEG in the apex of adult vegetative plants, seems to agree with its putative role in the maintenance of meristem indetermination in grapevine, resulting in a flowering delay as well as in the development of tendrils (Boss et al., 2006; Carmona et al., 2007a; Fernandez et al., 2014). In grapevine, the transition from the juvenile to adult phase is required early, because the plant needs the tendrils (sterile modified inflorescences) to climb through the riverbank forest to reach the canopy light, but this could take several years (Carmona et al., 2008; Keller, 2020b). The adult plant needs light to grow and accumulate reserves and will not flower until it has fulfilled it. In this sense, the low levels of TPS could indicate that the plants are not yet ready to begin flowering, in agreement with the reports on the Arabidopsis TPS loss of function plants, showing extremely late flowering (Wahl et al., 2013).

Figure 8. Expression interactions between the main genes involved in the floral and tendril development during the juvenile-to-adult vegetative phase transition in grapevine (Modified from Carmona et al., 2007b). The dashed arrows indicate that the floral development process does not occur with the phase change, but subsequently.

In grapevine (Boss & Thomas, 2002; Carmona et al., 2007b; Carmona et al., 2008) and other perennial plants, particularly fruit trees, GAs are generally found to inhibit flowering (Khan et al., 2014; Nishikawa et al., 2009). Under such context, in noninduced adult vegetative grapevine plants, one may expect higher levels of bioactive gibberellins and the plant's increased sensitivity to respond to GAs. Conversely, here we observe a potential increased degradation of GAs (due to GA2OX8 up-regulation), a reduced biosynthesis (due to GA20OX1) and a putative decreased sensitivity to GAs (due to SLY1, GID1A and GID1B down-regulation), factors more expectable for Arabidopsis than for grapevine. A more complete understanding of these phenomena requires references to GA levels and their activity thresholds during the development period under study. An additional experimental limitation is that the apexes may present different levels of GAs in meristems, leaf primordia, and tendril primordia.

In Arabidopsis, flowering is followed by the overexpression of several genes associated with flower induction and development (Blázquez et al., 2001). On the other hand, in grapevine adult apex we found a significant expression of most of the corresponding orthologous genes, but at lower levels than those found in inflorescence meristems (Díaz-Riquelme et al., 2009; Díaz-Riquelme et al., 2012; Díaz-Riquelme et al., 2014), and not inducing the development of inflorescences and flowers but rather of primordia and tendrils. Even though tendrils are considered organs analogous to inflorescences, supported by their shared developmental origins, functional roles, and regulatory mechanisms (Boss & Thomas, 2002; Calonje et al., 2004; Carmona et al., 2007b; Carmona et al., 2008; Srinivasan & Mullins, 1980), it would seem that some missing factors could allow floral induction during the early years of development. In the present work, two main singularities are characterising plant development and transition. First, the absence of miR172 expression, a key regulator of flowering induction in adult vegetative plants (Khan et al., 2014; Wu et al., 2009), and secondly, the downregulation of TPS, highlighting the putative role of carbohydrates-mediated signalling in this step. To what extent these two phenomena are cause and/or effect of the absence of floral induction presumably mediated by other different factors is a question still to be resolved. Deciphering those questions will allow us to include these still-unknown factors in the model described in Figure 8.

This work was supported by European Commission’s Horizon 2020 Marie Sklodowska-Curie-Research and Innovation Staff Exchange programme “(vWISE) Vine and Wine Innovation through Scientific Exchange”, Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT): PICT2018-02381 and PICT-2021-00039; Spanish Agencia Estatal de Investigación PID2020-120183RB-I00. We are grateful to Pablo Carbonell-Bejerano for his helpful comments and discussion, Servicio de Recursos Vegetales (ICVV) for the maintenance of the plants and Miguel Angulo for technical assistance.