Coordination of differentiation rate and local patterning in compound-leaf development.

The variability in leaf form in nature is immense. Leaf patterning occurs by differential growth, taking place during a limited window of morphogenetic activity at the leaf marginal meristem. While many regulators have been implicated in the designation of the morphogenetic window and in leaf patterning, how these effectors interact to generate a particular form is still not well understood. We investigated the interaction among different effectors of tomato (Solanum lycopersicum) compound-leaf development, using genetic and molecular analyses. Mutations in the tomato auxin response factor SlARF5/SlMP, which normally promotes leaflet formation, suppressed the increased leaf complexity of mutants with extended morphogenetic window. Impaired activity of the NAC/CUC transcription factor GOBLET (GOB), which specifies leaflet boundaries, also reduced leaf complexity in these backgrounds. Analysis of genetic interactions showed that the patterning factors SlMP, GOB and the MYB transcription factor LYRATE (LYR) coordinately regulate leaf patterning by modulating in parallel different aspects of leaflet formation and shaping. This work places an array of developmental regulators in a morphogenetic context. It reveals how organ-level differentiation rate and local growth are coordinated to sculpture an organ. These concepts are applicable to the coordination of pattering and differentiation in other species and developmental processes.

Chemo-Geographic Variations in Wild Population of Asteriscus graveolens in Israel Based on Volatile Composition Analyses.

Asteriscus graveolens is an aromatic desert shrub which holds medicinal potential. This species belongs to the Asteraceae family and is endemic to the Mediterranean region. In the present study, wild plants were sampled from eleven locations throughout southern Israel and the volatile profiles from leaves and flowers were analyzed using GC/MS. Three methods for volatile sampling were tested for a representative population: solvent extraction (methyl tert-butyl ether), hydrodistillation of the essential oil and headspace solid-phase microextraction. In all methods, the majority of volatiles were characterized as oxygenated mono- and sesquiterpenes. Only solvent extraction was able to detect asteriscunolides that were previously reported as anticancer molecules. Hence, that method was chosen for further analyses. The leaves were dominated by three asteriscunolide isomers, cis-chrysanthenyl acetate and intermedeol. The flowers were dominated by bisabolone, 6-hydroxybisabol-2-en-1-one, cis-chrysanthenyl acetate, epi-α-cadinol, and germacrene-D. k-Means clustering analysis of these data divided the population into four clusters that significantly differ in their volatile composition as was further demonstrated by MANOVA analysis. Geographically, A. graveolens populations growing in Israel were found to be chemically diverse with unique varieties in the Dead Sea basin and the Arava region. This work demonstrates that chemo-geographic variation of volatile composition exists within A. graveolens population growing in Israel, so future research evaluating the medicinal potential of that plant should take this into consideration.

CLAUSA Is a MYB Transcription Factor That Promotes Leaf Differentiation by Attenuating Cytokinin Signaling.

Leaf morphogenesis and differentiation are highly flexible processes, resulting in a large diversity of leaf forms. The development of compound leaves involves an extended morphogenesis stage compared with that of simple leaves, and the tomato (Solanum lycopersicum) mutant clausa (clau) exposes a potential for extended morphogenesis in tomato leaves. Here, we report that the CLAU gene encodes a MYB transcription factor that has evolved a unique role in compound-leaf species to promote an exit from the morphogenetic phase of tomato leaf development. We show that CLAU attenuates cytokinin signaling, and that clau plants have increased cytokinin sensitivity. The results suggest that flexible leaf patterning involves a coordinated interplay between transcription factors and hormones.

Auxin-mediated lamina growth in tomato leaves is restricted by two parallel mechanisms.

In the development of tomato compound leaves, local auxin maxima points, separated by the expression of the Aux/IAA protein SlIAA9/ENTIRE (E), direct the formation of discrete leaflets along the leaf margin. The local auxin maxima promote leaflet initiation, while E acts between leaflets to inhibit auxin response and lamina growth, enabling leaflet separation. Here, we show that a group of auxin response factors (ARFs), which are targeted by miR160, antagonizes auxin response and lamina growth in conjunction with E. In wild-type leaf primordia, the miR160-targeted ARFs SlARF10A and SlARF17 are expressed in leaflets, and SlmiR160 is expressed in provascular tissues. Leaf overexpression of the miR160-targeted ARFs SlARF10A, SlARF10B or SlARF17, led to reduced lamina and increased leaf complexity, and suppressed auxin response in young leaves. In agreement, leaf overexpression of miR160 resulted in simplified leaves due to ectopic lamina growth between leaflets, reminiscent of e leaves. Genetic interactions suggest that E and miR160-targeted ARFs act partially redundantly but are both required for local inhibition of lamina growth between initiating leaflets. These results show that different types of auxin signal antagonists act cooperatively to ensure leaflet separation in tomato leaf margins.

Auxin and LANCEOLATE affect leaf shape in tomato via different developmental processes.

Elaboration of a complex leaves depends on the morphogenetic activity of a specific tissue at the leaf margin termed marginal-blastozon (MB). In tomato (Solanum lycopersicym), prolonged activity of the MB leads to the development of compound leaves. The activity of the MB is restricted by the TCP transcription factor LANCEOLATE (LA). Plants harboring the dominant LA mutant allele La-2 have simple leaves with a uniform blade. Conversely, leaves of pFIL > > miR319 are compound and grow indeterminately in their margins due to leaf overexpression of miR319, a negative regulator of LA and additional miR319-sensitive genes. We have recently shown that the auxin-response sensor DR5::VENUS marks and precedes leaflet initiation events in the MB. Mutations in ENTIRE (E), an auxin signal inhibitor from the Aux/IAA family, lead to the expansion of the DR5::VENUS signal to throughout the leaf-primordia margin, and to a simplified leaf phenotype. Here, we examined the interaction between auxin, E, and LA in tomato leaf development. In La-2 leaf primordia, the auxin signal is very weak and is diffused to throughout the leaf margin, suggesting that auxin acts within the developmental-context of MB activity, which is controlled by LA. e La-2 double mutants showed an enhanced simple leaf phenotype and e pFIL > > miR319 leaves initiated less leaflets than wild-type, but their margins showed continuous growth. These results suggest that E and auxin affect leaflet initiation within the context of the extended MB activity, but their influence on the extent of indeterminate growth of the leaf is minor.

ENTIRE and GOBLET promote leaflet development in tomato by modulating auxin response.

Compound leaves produce leaflets in a highly controlled yet flexible pattern. Here, we investigate the interaction between auxin, the putative auxin response inhibitor ENTIRE (E, SlIAA9) and the CUC transcription factor GOBLET (GOB) in compound-leaf development in tomato (Solanum lycopersicum). Auxin maxima, monitored by the auxin response sensor DR5, marked and preceded leaflet and lobe initiation. The DR5 signal increased, but maxima were partially retained in response to the external or internal elevation of auxin levels. E directly interacted with the auxin receptors SlTIR1 and SlAFB6. Furthermore, E was stabilized by a mutation in domain II of the protein and by the inhibition of auxin or proteasome activity, implying that E is subjected to auxin-mediated degradation. In e mutants the DR5 signal expanded to include the complete leaf margin, and leaf-specific overexpression of a stabilized form of E inhibited the DR5 signal and lamina expansion. Genetic manipulation of GOB activity altered the distribution of the DR5 signal, and the inhibition of auxin transport or activity suppressed the GOB overexpression phenotype, suggesting that auxin mediates GOB-regulated leaf patterning. Whereas leaves of single e or gob mutants developed only primary leaflets, the downregulation of both E and GOB resulted in the complete abolishment of leaflet initiation, and in a strong DR5 signal throughout the leaf margin. These results suggest that E and GOB modulate auxin response and leaflet morphogenesis via partly redundant pathways, and that proper leaflet initiation and separation requires distinct boundaries between regions of lamina growth and adjacent regions in which growth is inhibited.

Cytokinin regulates compound leaf development in tomato.

Leaf shape diversity relies on transient morphogenetic activity in leaf margins. However, how this morphogenetic capacity is maintained is still poorly understood. Here, we uncover a role for the hormone cytokinin (CK) in the regulation of morphogenetic activity of compound leaves in tomato (Solanum lycopersicum). Manipulation of CK levels led to alterations in leaf complexity and revealed a unique potential for prolonged growth and morphogenesis in tomato leaves. We further demonstrate that the effect of CK on leaf complexity depends on proper localization of auxin signaling. Genetic analysis showed that reduction of CK levels suppresses the effect of Knotted1 like homeobox (KNOXI) proteins on leaf shape and that CK can substitute for KNOXI activity at the leaf margin, suggesting that CK mediates the activity of KNOXI proteins in the regulation of leaf shape. These results imply that CK regulates flexible leaf patterning by dynamic interaction with additional hormones and transcription factors.