Alternate wiring of a KNOXI genetic network underlies differences in leaf development of A. thaliana and C. hirsuta

Two interrelated problems in biology are understanding the regulatory logic and predictability of morphological evolution. Here, we studied these problems by comparing Arabidopsis thaliana, which has simple leaves, and its relative, Cardamine hirsuta, which has dissected leaves comprising leaflets. By transferring genes between the two species, we provide evidence for an inverse relationship between the pleiotropy of SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP) homeobox genes and their ability to modify leaf form. We further show that cis-regulatory divergence of BP results in two alternative configurations of the genetic networks controlling leaf development. In C. hirsuta, ChBP is repressed by the microRNA164A (MIR164A)/ChCUP-SHAPED COTYLEDON (ChCUC) module and ChASYMMETRIC LEAVES1 (ChAS1), thus creating cross-talk between MIR164A/CUC and AS1 that does not occur in A. thaliana. These different genetic architectures lead to divergent interactions of network components and growth regulation in each species. We suggest that certain regulatory genes with low pleiotropy are predisposed to readily integrate into or disengage from conserved genetic networks influencing organ geometry, thus rapidly altering their properties and contributing to morphological divergence

Leaf margin morphogenesis in crucifer plants

A key question in developmental biology is how form is generated. The model species Arabidopsis thaliana produces simple leaves with marginal outgrowths termed serrations. Serration development in A. thaliana requires both the transcription factor CUP-SHAPED COTYLEDON2 (CUC2) and the auxin efflux facilitator PIN-FORMED1 (PIN1), which regulates polar auxin transport by forming convergence points (Hay et al., 2006; Nikovics et al., 2006; Scarpella et al., 2006). In Chapter 3, I investigate how CUC2, PIN1 and auxin interact to control serration development. I demonstrate that CUC2 promotes PIN1 convergence point and auxin activity foci formation along the margin of the leaf, whilst high auxin activity represses CUC2 expression. Furthermore, interspersed peaks of CUC2 and auxin activity pattern serration development along the proximo-distal axis of the leaf. Thus, auxin, PIN1 and CUC2 form a negative feedback loop that patterns serration development.CUC genes and PIN1 are required for leaflet development in Cardamine hirsuta (Barkoulas et al., 2008; Blein et al., 2008), a close relative of A. thaliana that produces compound leaves subdivided into units termed leaflets. However, it is unclear how CUC and PIN1 interact to control leaflet development. In Chapter 4, I demonstrate that similar to A. thaliana, CUC genes promote PIN1 convergence point and auxin activity foci formation at the C. hirsuta leaf margin, whilst high auxin activity represses CUC2 expression. These genetic interactions likely create interspersed peaks of CUC2 and auxin activity that pattern leaflet development. Thus, the same negative feedback loop between CUC, PIN1 and auxin patterns both leaflet development in C. hirsuta and serration development in A. thaliana.KNOTTED1-LIKE HOMEOBOX (KNOX) genes are expressed in C. hirsuta leaves, and interact with ChCUC and PIN1 in positive and negative feedback loops, respectively, to control leaflet development (Barkoulas et al., 2008; Blein et al., 2008). KNOX genes are not expressed in A. thaliana leaves, but deeply lobed margins reminiscent of leaflets develop in association with ectopic KNOX expression in leaves (Chuck et al., 1996; Hay et al., 2006). However, it is unclear whether regulatory interactions of PIN1, CUC and KNOX which occur in C. hirsuta leaflets are employed during KNOX-induced lobe development in A. thaliana. In Chapter 5, I demonstrate that CUC2 and polar auxin transport are required for ectopic KNOX expression. Conversely, I show that KNOX misexpression up-regulates CUC2 expression in A. thaliana leaves. Thus, interactions between KNOX, CUC and PIN1 that occur in leaflet development in C. hirsuta also occur in association with KNOX-induced lobe development in A. thaliana.In addition to investigating the regulatory interactions between known components of leaf development pathways, I sought to identify novel genes that mediate CUC2-dependent serration development in A. thaliana. In Chapter 6, I identify a suppressor of the smooth margin phenotype of cuc2 leaves that partially restores PIN1 localisation in the absence of functional CUC2. Finally, in the General Discussion I evaluate how interlinking feedback loops between CUC, KNOX and auxin pattern serration and leaflet development. I then discuss why interlinking feedback loops may have been deployed to control outgrowths in both plant and animal systems.</p

Leaf margin morphogenesis in crucifer plants

A key question in developmental biology is how form is generated. The model species Arabidopsis thaliana produces simple leaves with marginal outgrowths termed serrations. Serration development in A. thaliana requires both the transcription factor CUP-SHAPED COTYLEDON2 (CUC2) and the auxin efflux facilitator PIN-FORMED1 (PIN1), which regulates polar auxin transport by forming convergence points (Hay et al., 2006; Nikovics et al., 2006; Scarpella et al., 2006). In Chapter 3, I investigate how CUC2, PIN1 and auxin interact to control serration development. I demonstrate that CUC2 promotes PIN1 convergence point and auxin activity foci formation along the margin of the leaf, whilst high auxin activity represses CUC2 expression. Furthermore, interspersed peaks of CUC2 and auxin activity pattern serration development along the proximo-distal axis of the leaf. Thus, auxin, PIN1 and CUC2 form a negative feedback loop that patterns serration development.CUC genes and PIN1 are required for leaflet development in Cardamine hirsuta (Barkoulas et al., 2008; Blein et al., 2008), a close relative of A. thaliana that produces compound leaves subdivided into units termed leaflets. However, it is unclear how CUC and PIN1 interact to control leaflet development. In Chapter 4, I demonstrate that similar to A. thaliana, CUC genes promote PIN1 convergence point and auxin activity foci formation at the C. hirsuta leaf margin, whilst high auxin activity represses CUC2 expression. These genetic interactions likely create interspersed peaks of CUC2 and auxin activity that pattern leaflet development. Thus, the same negative feedback loop between CUC, PIN1 and auxin patterns both leaflet development in C. hirsuta and serration development in A. thaliana.KNOTTED1-LIKE HOMEOBOX (KNOX) genes are expressed in C. hirsuta leaves, and interact with ChCUC and PIN1 in positive and negative feedback loops, respectively, to control leaflet development (Barkoulas et al., 2008; Blein et al., 2008). KNOX genes are not expressed in A. thaliana leaves, but deeply lobed margins reminiscent of leaflets develop in association with ectopic KNOX expression in leaves (Chuck et al., 1996; Hay et al., 2006). However, it is unclear whether regulatory interactions of PIN1, CUC and KNOX which occur in C. hirsuta leaflets are employed during KNOX-induced lobe development in A. thaliana. In Chapter 5, I demonstrate that CUC2 and polar auxin transport are required for ectopic KNOX expression. Conversely, I show that KNOX misexpression up-regulates CUC2 expression in A. thaliana leaves. Thus, interactions between KNOX, CUC and PIN1 that occur in leaflet development in C. hirsuta also occur in association with KNOX-induced lobe development in A. thaliana.In addition to investigating the regulatory interactions between known components of leaf development pathways, I sought to identify novel genes that mediate CUC2-dependent serration development in A. thaliana. In Chapter 6, I identify a suppressor of the smooth margin phenotype of cuc2 leaves that partially restores PIN1 localisation in the absence of functional CUC2. Finally, in the General Discussion I evaluate how interlinking feedback loops between CUC, KNOX and auxin pattern serration and leaflet development. I then discuss why interlinking feedback loops may have been deployed to control outgrowths in both plant and animal systems

Hormonal input in plant meristems: a balancing act

Plant hormones are a group of chemically diverse molecules that control virtually all aspects of plant development. Classical plant hormones were identified many decades ago in physiology studies that addressed plant growth regulation. In recent years, biochemical and genetic approaches led to the identification of many molecular components that mediate hormone activity, such as hormone receptors and hormone-regulated genes. This has greatly contributed to the understanding of the mechanisms underlying hormone activity and highlighted the intricate crosstalk and integration of hormone signalling and developmental pathways. Here we review and discuss recent findings on how hormones regulate the activity of shoot and root apical meristems