Control of leaf and vein development by auxin

Abstract

Leaves are the main photosynthetic organs of vascular plants and show considerable diversity in their geometries, ranging from simple spoon-like forms to complex shapes with individual leaflets, as in compound leaves. Leaf vascular tissues, which act as conduits of both nutrients and signaling information, are organized in networks of different architectures that usually mirror the surrounding leaf shape. Understanding the processes that endow leaves and vein networks with ordered and closely aligned shapes has captured the attention of biologists and mathematicians since antiquity. Recent work has suggested that the growth regulator auxin has a key role in both initiation and elaboration of final morphology of both leaves and vascular networks. A key feature of auxin action is the existence of feedback loops through which auxin regulates its own transport. These feedbacks may facilitate the iterative generation of basic modules that underlies morphogenesis of both leaves and vasculature.

Figure 1.

Axes of leaf asymmetry and diversity of leaf shape. (A) A simple, serrated leaf of the Columbia ecotype of Arabidopsis thaliana. The proximo–distal (P–D) and medio–lateral (M–L) axes are indicated in the image. The asterisk marks one marginal serration. (B) The lobed leaf of the Arabidopsis thaliana relative Arabidopsis lyrata. The asterisk depicts the position of one lobe. Lobes are deep serrations, so the definition of an outgrowth as a serration or lobe is somewhat arbitrary. (C) The dissected leaf of Cardamine hirsuta. The asterisk marks a lateral leaflet. Leaflets are clearly defined as distinct units of the same leaf, which connect with the rachis (R) via a structure called a petiolule (Pu). (D) The dissected leaf of the cultivated tomato. Tomato demonstrates additional orders of dissection with respect to Cardamine hirsuta leaf and produces both primary leaflets (black asterisk) and secondary leaflets (red asterisk). (E) Scanning electron micrograph of the shoot apex of tomato. The white asterisk marks a leaf primordium (1) initiating from the meristem. The adaxial (yellow) and abaxial (orange) domains are marked on the subsequent developing leaf (2). Tomato is a compound leaf plant where leaflets are formed from the leaf blade soon after leaf initiation (a developing leaflet is marked by an arrow in leaf 3). Images in panels A–D are leaf silhouettes. Scale bars: (A–D) 1 cm, (E) 100 µm.

Figure 2.

Stages of leaf development and associated polarities of auxin transport. (A) Leaf initials are specified at the flanks of the SAM (purple) and correspond to sites of elevated auxin activity (red) resulting from convergence points of PIN1 polarity in the epidermal layer (black arrows). From PIN1 convergence points, auxin is transported in internal tissues (white arrow), where it gradually induces formation of a vascular strand. (B) During leaf initiation, a small primordium (green) becomes visible at the flanks of the SAM. Epidermal auxin flow converges to form a maximum of auxin activity at the tip of the primordium. There, auxin is drained through the center of the primordium, marking the position of the midvein (orange, M). (C) In primary morphogenesis, leaves grow predominantly via cell division to acquire their shape and vascular pattern. Auxin activity maxima at the margins of the leaf correlate with sites of lateral vein (orange, LV) formation and positions of serration development. Marginal veins (yellow, MV) emerge in continuity with lateral veins from PIN1 domains initiated within the growing lamina. Open-ended marginal vein precursors form the upper part of each vein loop and display uniform auxin transport polarity toward pre-existing lateral veins, but they switch to bipolarity as they become connected at both ends to give rise to closed vein loops. (D) After primary morphogenesis, the basic leaf and vasculature patterns are already formed. Similar to marginal veins, higher-order veins (yellow) have appeared in continuity with pre-existing vasculature from PIN1 domains initiated within the expanding blade. Higher-order veins can end freely in the lamina (FV) or become connected (CV) on proximity to other PIN1 domains. During secondary morphogenesis, leaves grow primarily through cell expansion. (E) Examples of two basic leaf shapes, simple and dissected. In simple leaves, the leaf blade is composed of one unit (regardless of whether the leaf is smooth, serrated, or lobed), whereas, in dissected leaves, the blade is divided into distinct units called leaflets. The dissected leaf of the cartoon is similar to leaves of C. hirsuta, where the terminal leaflet (TL) is located at the tip of the leaf, whereas lateral leaflets (LL) are borne from the rachis (purple, R). Auxin activity maxima are present in both the lobes and serrations of the simple leaf and in the serrations of the terminal leaflet, but they are also associated with positions of lateral leaflet formation. Arrows within panels depict auxin flow, as inferred by PIN1 localization, whereas arrows between panels temporally connect successive stages of leaf formation.

Figure 3.

Model depicting interactions between auxin function components and key genetic pathways controlling leaf development in Arabidopsis thaliana. Convergence points of auxin flow (arrows) generated via polar PIN1 localization in epidermal cells contribute to establishing an auxin activity maximum (red dot) at the periphery of the SAM (purple). AS1 (together with the LOB domain protein AS2, not shown here) and auxin repress the KNOX gene BP, thereby contributing to leaf outgrowth at the flanks of the SAM. The KNOX gene STM prevents AS1 expression in the meristem, thus establishing a mutually repressive interaction between meristem cells and leaf initials. Expression of STM might be regulated by auxin activity gradients, but this requires further investigation. CUC proteins (gray) are expressed at the boundary between meristem and leaf cells. The coordinated differentiation of abaxial and adaxial cell fates is critical for leaf function because it underpins functional specialization of the upper side (yellow), specialized for light capture, and a lower side (orange), specialized for gas exchange. Members of the HD-ZIP III class, such as PHB, promote adaxial fate and meristem activity, and are regulated by two known pathways. First, miRNA165/166 directly repress HD-ZIP III transcripts, which results in exclusion of HD-ZIP III expression from the abaxial domain and definition of HD-ZIP III expression level in the meristem and adaxial leaf domain. Second, expression of HD-ZIP III genes is repressed by the abaxial fate-promoting KAN proteins. The auxin response factors ARF3/ETT and ARF4 are required for KAN activity. ARF4 is expressed abaxially, whereas ARF3 mRNA may be more broadly distributed throughout the meristem and leaf primordia. Both ARF3 and ARF4 are subject to negative regulation by ta-siRNAs.

Figure 4.

Control of leaf vein formation by polar auxin transport. Stage-specific dynamics of leaf vein patterning and dependency on auxin levels and flow as exemplified for loop formation, but in general equally applicable to all veins. (A) PIN1-labeled auxin transport paths corresponding to preprocambial cell selection zones (green). Note how loops are composed of a lateral PIN1 expression domain (LD) and an initially free-ending marginal PIN1 expression domain (MD). Further, note slightly expanded PIN1 expression domains in a fraction of serration tip-associated third loops during normal development, broad PIN1 domains on the side of local auxin application (arrowhead), and nearly ubiquitous PIN1 expression on systemic auxin flow inhibition. (B) Directions of ATHB8-defined preprocambial strand formation (yellow arrows). Note middle-to-margin progression of preprocambial strand formation during normal loop development. Further, note margin-to-middle preprocambial strand extension in a fraction of third loops during normal development and in all loops forming on the side of auxin application. Finally, note coexistence of middle-to-margin and margin-to-middle polarities of preprocambial strand extension during the formation of individual loops in response to auxin transport inhibition. (C) Gradual appearance of procambial cell identity acquisition (light to dark purple). Note simultaneous differentiation of lateral and marginal procambial strands in normal loop development. Further, note successive formation of lateral and marginal procambial strands in a fraction of third loops during normal development and in all loops formed on the side of auxin application and under conditions of reduced auxin transport. Arrows temporally connect successive stages of vein formation. L1, L2, and L3, first, second, and third loops, respectively. See text for additional details.