Plants undergo developmental transitions throughout their lifetime. The most obvious of these is the transition from a leaf-producing vegetative phase to a reproductive phase that is often accompanied by a showy display of flowers. During the vegetative phase, many plants undergo a juvenile-to-adult transition, evident as changes in the features of leaves including their shape. This change is referred to as heteroblasty. In some species, transitions in shape are very dramatic. For example, the Australian native Eucalyptus globulus has short broad juvenile leaves and long narrow adult leaves. In many plant species, however, lifetime changes in leaf shape are distinct but less blatant.
In Arabidopsis thaliana, developmental transitions in leaves can be measured by changes in several shape parameters (Tang et al., 2023; Telfer and Poethig, 1994; Tsukaya et al., 2000). The earliest juvenile leaves are relatively small and have a symmetric oval-shaped blade. The blade has a smooth margin and a sharp junction with a narrow petiole. Successive leaves are progressively larger and develop asymmetrically along their proximodistal length. Thus, adult leaves grow relatively long and narrow, with the leaf blade tapering down the petiole. They also develop small tooth-like protrusions, called serrations, along the leaf margin at regular intervals. When serrations start to appear, they are initially confined to the base of the leaf, but as the plant matures the later leaves have more serrations extending along the margin towards the leaf tip (Bilsborough et al., 2011; Kumar et al., 2007; Nikovics et al., 2006). These morphological differences in juvenile and adult leaves have provided a valuable developmental system for Li and colleagues (Li et al., 2024a preprint) to address what might drive leaf proximodistal asymmetry.
One important family of genes involved in the juvenile-to-adult transition are the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, a family of 17 genes, 11 of which are post-transcriptionally regulated by the microRNAs miR156 and miR157 (Rhoades et al., 2002; Schwab et al., 2005; Wang et al., 2009; Wu and Poethig, 2006). miR156 is highly expressed in the earliest leaves of A. thaliana but its levels decline in later leaves, while the targeted SPL genes have the opposite temporal expression (Wang et al., 2008; Wu et al., 2009). The function of SPLs has been dissected using genetic approaches that alter the level of SPL through loss-of-function mutations, changing the level of miR156 and using transgenes with alterations in the SPL sequence so they are no longer regulated by miR156. There are redundancies between members, but several SPLs are associated with leaf developmental transitions (Tang et al., 2023; Wang et al., 2009; Wu et al., 2009; Xu et al., 2016).
SPL9 is of interest because, individually, it promotes most adult leaf shape characteristics (Wu et al., 2009; Xu et al., 2016). Furthermore, a close analysis of the spatial expression pattern shows SPL9 increases expression levels in successive leaves and is more persistent in later leaves (Li et al., 2024b). Expression occurs throughout the developing adult leaf but as the leaf matures, SPL9 becomes limited to its base and lateral regions (Li et al., 2024b; Tang et al., 2023). This pattern is congruent with leaf maturation, whereby cessation of cell division and cell differentiation starts at the leaf tip and progresses toward the leaf base (Donnelly et al., 1999; Li et al., 2024b; Nath et al., 2003). In fact, SPL9 acts through a mitotic cell cycle regulator, cyclin D3, to maintain cell proliferation and retard differentiation (Dewitte et al., 2007; Li et al., 2024b).
Li and colleagues delve further into whether SPL9-regulated growth contributes to the proximodistal asymmetry of adult A. thaliana leaves. The experiments elegantly combine genetics to manipulate where in the leaf SPL9 is expressed with tracing cell growth parameters and time-lapse imaging to analyse growth patterns. The experimental system focuses on comparing growth in juvenile leaf 1 with adult leaf 8, thus readily allowing a comparative analysis of juvenile-state symmetry and adult-state asymmetry.
Loss of SPL9, through a loss-of-function spl9 mutant and reduction of SPL9 levels via miR156 overexpression, leads to rounder more-symmetrical adult leaves, suggesting that SPL9 is crucial for leaf proximodistal asymmetry. Two approaches were used to test this possibility. First, a domain-specific promoter, pLMI1, was used to drive expression of a miR156-resistant version of SPL9 in the distal and margin regions of leaf 8, such that the gradient of SPL9 expression along the proximodistal axis is evened out. These leaves are more oval-shaped and have serrations that extend along the entire leaf margin. In short, the asymmetric adult leaf becomes more symmetric. Time-lapse imaging and cell-fate tracing show that SPL9 altered cellular growth patterns. Margin cells of adult leaves with ectopic expression of SPL9 grew at the same rate as wild type but there are more and smaller cells, which is indicative of a delay in differentiation. Such delayed differentiation is thought to provide a developmental opportunity for the production of serrations in the distal leaf. Second, in the converse experiment, the domain-specific promoters pCUC2 and pRCO were used to drive expression of a miR156-resistant version of SPL9 in the proximal region of leaf 1. In these plants, the normally symmetric juvenile leaf had an asymmetric shape with a lamina that tapers into a reduced petiole like that of an adult leaf. Ectopic expression of SPL9 in juvenile leaves was found to be associated with repressed growth rate and reduced anisotropy at the base of the leaf, accounting for the less obvious petiole. As such, SPL9 is a key player in leaf proximodistal asymmetry.
Li and colleagues also address whether changes in leaf growth regulation throughout the life of a plant might be conserved in the context of more-complex leaf shapes. This was achieved by comparing the simple undivided leaf of A. thaliana with the dissected compound leaf of Cardamine hirsuta. Despite these extreme shape differences, growth parameters in the two species appear to follow remarkably comparable trajectories. As in A. thaliana, successive leaves in C. hirsuta change shape, which is characterised by a progressive increase in the number of leaflets on each leaf (Cartolano et al., 2015; Rubio-Somoza et al., 2014). At the cellular level, a comparison of C. hirsuta adult leaf 8 with juvenile leaf 1 showed discriminating features of growth that are similar to A. thaliana. In both A. thaliana and C. hirsuta adult leaf 8, the period of cell proliferation was extended, cell expansion and differentiation were slowed, and there was reduced growth anisotropy at the base of the leaf relative to leaf 1. In C. hirsuta, ChSPL9 expression increased with each leaf node from juvenile to adult and, within adult leaves, expression was initially throughout the leaf and declined into proximal regions in a pattern similar to that in A. thaliana. ChSPL9 levels are associated with the heteroblastic progression of leaf development. Increasing ChSPL9 in juvenile leaves increased the number of leaflets, whereas decreasing levels of ChSPL9 decreased the number of leaflets in adult leaves. Accordingly, ChSPL9 is necessary and sufficient for changes in leaflet number. The dissected leaf of C. hirsuta requires KNOXI and RCO homeodomain transcription factors, which do not contribute to leaf shape in A. thaliana. However, ectopic expression of KNOXI genes or RCO in A. thaliana altered the simple leaf to a lobed leaf (Lincoln et al., 1994; Vlad et al., 2014). Despite expression of these transgenes throughout all leaves, the transgene-mediated change in Arabidopsis leaf shape was evident in leaf 8 but not in leaf 1. In fact, the RCO transgene-mediated phenotype was enhanced by SPL9, indicating that SPL9 creates a permissive state for margin serration, lobe and leaflet outgrowths, and the development of adult leaf features.
Both serrations and leaflets form through peaks of auxin, which promotes growth, alternating with regions expressing CUC transcription factors, which suppress growth (Byrne et al., 2024). CUC2 is thought to form active and inactive dimers, depending on its protein partner. SPL9 potentially interferes with inactive protein dimer formation (Rubio-Somoza et al., 2014), which might account for the proximodistal distribution of serrations. Interestingly, a quantitative analysis of A. thaliana naturally occurring accessions from diverse geographical locations has found that individual juvenile and adult leaf traits are poorly correlated, and that the miR156-SPL module might account for some, but not all, differences. As such, the evolution of heteroblasty is likely to be complex (Doody et al., 2022). In any case, the new insights provided by Li and colleagues highlight how one gene can interface with leaf patterning programmes to regulate leaf shape differences over the lifetime of a plant.
Footnotes
Funding
The author declares that no funds, grants or other support were received for the preparation of this Perspective.
References
Competing interests
The author declares no competing or financial interests.