Roles of the Arabidopsis KEULE Gene in Postembryonic Development.

Cytokinesis in plant cells begins with the fusion of vesicles that transport cell wall materials to the center of the cell division plane, where the cell plate forms and expands radially until it fuses with the parental cell wall. Vesicle fusion is facilitated by trans-SNARE complexes, with assistance from Sec1/Munc18 (SM) proteins. The SNARE protein KNOLLE and the SM protein KEULE are required for membrane fusion at the cell plate. Due to the crucial function of KEULE, all Arabidopsis (Arabidopsis thaliana) keule mutants identified to date are seedling lethal. Here, we identified the Arabidopsis serrata4-1 (sea4-1) and sea4-2 mutants, which carry recessive, hypomorphic alleles of KEULE. Homozygous sea4-1 and sea4-2 plants are viable and fertile but have smaller rosettes and fewer leaves at bolting than the wild type. Their leaves are serrated, small, and wavy, with a complex venation pattern. The mutant leaves also develop necrotic patches and undergo premature senescence. RNA-seq revealed transcriptome changes likely leading to reduced cell wall integrity and an increase in the unfolded protein response. These findings shed light on the roles of KEULE in postembryonic development, particularly in the patterning of rosette leaves and leaf margins.

Leaf dissection and margin serration are independently regulated by two regulators converging on the CUC2-auxin module in strawberry.

The remarkable diversity of leaf forms allows plants to adapt to their living environment. In general, leaf diversity is shaped by leaf complexity (compound or simple) and leaf margin pattern (entire, serrated, or lobed). Prior studies in multiple species have uncovered a conserved module of CUC2-auxin that regulates both leaf complexity and margin serration. How this module is regulated in different species to contribute to the species-specific leaf form is unclear. Furthermore, the mechanistic connection between leaf complexity and leaf serration regulation is not well studied. Strawberry has trifoliate compound leaves with serrations at the margin. In the wild strawberry Fragaria vesca, a mutant named salad was isolated that showed deeper leaf serrations but normal leaf complexity. SALAD encodes a single-Myb domain protein and is expressed at the leaf margin. Genetic analysis showed that cuc2a is epistatic to salad, indicating that SALAD normally limits leaf serration depth by repressing CUC2a expression. When both Arabidopsis homologs of SALAD were knocked out, deeper serrations were observed in Arabidopsis rosette leaves, supporting a conserved function of SALAD in leaf serration regulation. We incorporated the analysis of a third strawberry mutant simple leaf 1 (sl1) with reduced leaf complexity but normal leaf serration. We showed that SL1 and SALAD independently regulate CUC2a at different stages of leaf development to, respectively, regulate leaf complexity and leaf serration. Our results provide a clear and simple mechanism of how leaf complexity and leaf serration are coordinately as well as independently regulated to achieve diverse leaf forms.

Is auxin enough? Cytokinins and margin patterning in simple leaves.

The interplay between auxin and cytokinins affects facets of plant development as different as ovule formation and lateral root initiation. Moreover, cytokinins favor complexity in the development of Solanum lycopersicum and Cardamine hirsuta compound leaves. Nevertheless, no role has been proposed for cytokinins in patterning the margins of the simple leaves of Arabidopsis thaliana, a process that is assumed to be sufficiently explained by auxin localization. Here, we discuss evidence supporting the hypothesis that cytokinins play a role in simple leaf margin morphogenesis via crosstalk with auxin, as occurs in other plant developmental events. Indeed, mutant or transgenic arabidopsis plants defective in cytokinin biosynthesis or signaling, or with increased cytokinin degradation have leaf margins less serrated than the wild type.

Tannin phenotyping of the Vitaceae reveals a phylogenetic linkage of epigallocatechin in berries and leaves.

BACKGROUND AND AIMS: Condensed tannins, responsible for berry and wine astringency, may have been selected during grapevine domestication. This work examines the phylogenetic distribution of condensed tannins throughout the Vitaceae phylogenetic tree. METHODS: Green berries and mature leaves of representative true-to-type members of the Vitaceae were collected before 'véraison', freeze-dried and pulverized, and condensed tannins were measured following depolymerization by nucleophilic addition of 2-mercaptoethanol to the C4 of the flavan-3-ol units in an organic acidic medium. Reaction products were separated and quantified by ultrahigh pressure liquid chromatography/diode array detection/mass spectrometry. KEY RESULTS AND CONCLUSIONS: The original ability to incorporate epigallocatechin (EGC) into grapevine condensed tannins was lost independently in both the American and Eurasian/Asian branches of the Vitaceae, with exceptional cases of reversion to the ancestral EGC phenotype. This is particularly true in the genus Vitis, where we now find two radically distinct groups differing with respect to EGC content. While Vitis species from Asia are void of EGC, 50 % of the New World Vitis harbour EGC. Interestingly, the presence of EGC is tightly coupled with the degree of leaf margin serration. Noticeably, the rare Asian EGC-forming species are phylogenetically close to Vitis vinifera, the only remnant representative of Vitis in Eurasia. Both the wild ancestral V. vinifera subsp. sylvestris as well as the domesticated V. vinifera subsp. sativa can accumulate EGC and activate galloylation biosynthesis that compete for photoassimilates and reductive power.

Decoding family-level features for modern and fossil leaves from computer-vision heat maps.

PREMISE: Angiosperm leaves present a classic identification problem due to their morphological complexity. Computer-vision algorithms can identify diagnostic regions in images, and heat map outputs illustrate those regions for identification, providing novel insights through visual feedback. We investigate the potential of analyzing leaf heat maps to reveal novel, human-friendly botanical information with applications for extant- and fossil-leaf identification. METHODS: We developed a manual scoring system for hotspot locations on published computer-vision heat maps of cleared leaves that showed diagnostic regions for family identification. Heat maps of 3114 cleared leaves of 930 genera in 14 angiosperm families were analyzed. The top-5 and top-1 hotspot regions of highest diagnostic value were scored for 21 leaf locations. The resulting data were viewed using box plots and analyzed using cluster and principal component analyses. We manually identified similar features in fossil leaves to informally demonstrate potential fossil applications. RESULTS: The method successfully mapped machine strategy using standard botanical language, and distinctive patterns emerged for each family. Hotspots were concentrated on secondary veins (Salicaceae, Myrtaceae, Anacardiaceae), tooth apices (Betulaceae, Rosaceae), and on the little-studied margins of untoothed leaves (Rubiaceae, Annonaceae, Ericaceae). Similar features drove the results from multivariate analyses. The results echo many traditional observations, while also showing that most diagnostic leaf features remain undescribed. CONCLUSIONS: Machine-derived heat maps that initially appear to be dominated by noise can be translated into human-interpretable knowledge, highlighting paths forward for botanists and paleobotanists to discover new diagnostic botanical characters.

Gene prediction for leaf margin phenotype and fruit flesh color in pineapple (Ananas comosus) using haplotype-resolved genome sequencing.

Pineapple (Ananas comosus (L.) Merr.) is one of the most economically important tropical fruit species. The major aim of the breeding programs in several countries, including Japan, is quality improvement, mainly for the fresh market. 'Yugafu', a Japanese cultivar with distinctive pipe-type leaf margin phenotype and white flesh color, is popular for fresh consumption. Therefore, genome sequencing of 'Yugafu' is expected to assist pineapple breeding. Here, we developed a haplotype-resolved assembly for the heterozygous genome of 'Yugafu' using long-read sequencing technology and obtained a pair of 25 pseudomolecule sequences inherited from the parental accessions 'Cream pineapple' and 'HI101'. The causative genes for leaf margin and fruit flesh color were identified. Fine mapping revealed a 162-kb region on CLG23 for the leaf margin phenotype. In this region, 20 kb of inverted repeat was specifically observed in the 'Cream pineapple' derived allele, and the WUSCHEL-related homeobox 3 (AcWOX3) gene was predicted as the key gene for leaf margin morphogenesis. Dominantly repressed AcWOX3 via RNAi was suggested to be the cause of the pipe-type leaf margin phenotype. Quantitative trait locus (QTL) analysis revealed that the terminal region of CLG08 contributed to white flesh and low carotenoid content. Carotenoid cleaved dioxygenase 4 (AcCCD4), a key gene for carotenoid degradation underlying this QTL, was predicted as the key gene for white flesh color through expression analysis. These findings could assist in modern pineapple breeding and facilitate marker-assisted selection for important traits.

TCP5 controls leaf margin development by regulating KNOX and BEL-like transcription factors in Arabidopsis.

Development of leaf margins is an important process in leaf morphogenesis. CIN-clade TCP (TEOSINTE BRANCHED1/CYCLOIDEA/PCF) transcription factors are known to have redundant roles in specifying leaf margins, but the specific mechanisms through which individual TCP genes function remain elusive. In this study, we report that the CIN-TCP gene TCP5 is involved in repressing the initiation and outgrowth of leaf serrations by activating two key regulators of margin development, the Class II KNOX factor KNAT3 and BEL-like SAW1. Specifically, TCP5 directly promotes the transcription of KNAT3 and indirectly activates the expression of SAW1. We also show that TCP5 regulates KNAT3 and SAW1 in a temporal- and spatial- specific manner that is largely in accordance with the progress of formation of serrations. This regulation might serve as a key mechanism in patterning margin morphogenesis and in sculpting the final form of the leaf.

SAW homeodomain transcription factors regulate initiation of leaf margin serrations.

Plant leaves are the main photosynthetic organ of plants and they occur in an array of different shapes. Leaf shape is determined by morphogenesis whereby patterning of the leaf margin can result in interspaced leaf serrations, lobes, or leaflets, depending on the species, developmental stage, and in some instances the environment. In Arabidopsis, mutations in the homeodomain transcription factors SAW1 and SAW2 result in more prominent leaf margin serrations. Here we show that serrations appear precociously in the saw1 saw2 mutant. The pattern of auxin maxima, and of PIN1 and CUC2 expression, which form a feedback loop that drives serration outgrowth, is altered in saw1 saw2 and correlates with precocious serration initiation. SAW1 is not expressed in the outer epidermal cell layer where PIN1 convergence points generate auxin maxima. Instead, SAW1 is expressed on the adaxial side of the leaf and expression in this domain is sufficient for function. We suggest that SAW1 and SAW2 repress serration initiation and outgrowth by promoting the transition to a determinate fate in the leaf margin.

Two new monobasic thrips genera for a gall-inducing species and its kleptoparasite (Thysanoptera, Phlaeothripinae).

Drypetothrips korykis gen. et sp.n. is described as inducing leaf-margin galls on a small tree in Australia, Drypetes deplanchei [Putranjivaceae]. This thrips is similar in appearance to the smaller species of the genus Kladothrips that induce galls on Acacia species. The galls are invaded by a phytophagous kleptoparasitic thrips, Pharothrips hynnis gen. et sp.n., females of which have a forked plough-like structure protruding ventrally on the frons that is unique amongst Thysanoptera.

Leaf margins in a deciduous lineage from the Greater Cape Floristic Region track climate in unexpected directions.

PREMISE: The functional significance of leaf margins has long been debated. In this study, we explore influences of climate, leaf lobing, woodiness, and shared evolutionary history on two leaf margin traits within the genus Pelargonium. METHODS: Leaves from 454 populations of Pelargonium (161 species) were collected in the Greater Cape Floristic Region and scored for tooth presence/absence and degree of lobing. Tooth density (number of teeth per interior perimeter distance) was calculated for a subset of these. We compared five hypotheses to explain tooth presence and density using mixed effect models. RESULTS: Tooth presence/absence was best predicted by the interaction of leaf lobing and mean annual temperature (MAT), but often in patterns opposite those previously reported: species were more likely to be toothed with warmer temperatures, particularly for unlobed and highly lobed leaves. In contrast, tooth density was best predicted by the interaction of MAT and the season of most rain; density declines with temperature as consistent with expectations, but only in winter-rain dominated areas. Woody and nonwoody species within Pelargonium have similar associations between tooth presence/absence and MAT, contrary to the expectation that patterns within nonwoody species would be insignificant. CONCLUSIONS: We conclude Pelargonium leaf margins show predictable responses to climate, but these responses are complex and can contradict those found for global patterns across plant communities.

Ectopic expression of the transcription factor CUC2 restricts growth by cell cycle inhibition in Arabidopsis leaves.

Plant leaf margins produce small outgrowths or teeth causing serration in a regular arrangement, which is specified by auxin maxima. In Arabidopsis, the spatiotemporal pattern of auxin dependents on both, the transcription factor CUC2 and the signal peptide EPFL2, a ligand of the growth-promoting receptor kinase ERECTA (ER). Ectopic expression of CUC2 can have contrary effects on leaf growth. Ubiquitous expressed CUC2 suppresses growth in the whole leaf, whereas cuc2-1D mutants have enlarged leaves, through ER-dependent cell proliferation in the teeth. Here we investigated the growth dynamics of cuc2-1D leaves and the growth restricting the function of CUC2 using the ubiquitous inducible CUC2-GR transgene. In time courses, we dissected the serration promoting the function of CUC2 in the leaf margin and ectopic growth inhibition by CUC2 in the leaf plate. We found that CUC2 limits growth rather by cell cycle inhibition than by cell size control. Furthermore, endogenous CUC2 was rapidly induced by CUC2-GR indicating a possible auto-inducible feedback. In contrast, EPFL2 was quickly decreased by transient CUC2 induction but increased in cuc2-3 mutant leaves suggesting that CUC2 can also counteract the EPFL2-ER pathway. Therefore, tooth growth promotion and growth inhibition by CUC2 involve partially the same mechanism but in contrary ways.

HEADLESS Regulates Auxin Response and Compound Leaf Morphogenesis in Medicago truncatula.

WUSCHEL (WUS) is thought to be required for the establishment of the shoot stem cell niche in Arabidopsis thaliana. HEADLESS (HDL), a gene that encodes a WUS-related homeobox family transcription factor, is thought to be the Medicago truncatula ortholog of the WUS gene. HDL plays conserved roles in shoot apical meristem (SAM) and axillary meristem (AM) maintenance. HDL is also involved in compound leaf morphogenesis in M. truncatula; however, its regulatory mechanism has not yet been explored. Here, the significance of HDL in leaf development was investigated. Unlike WUS in A. thaliana, HDL was transcribed not only in the SAM and AM but also in the leaf. Both the patterning of the compound leaves and the shape of the leaf margin in hdl mutant were abnormal. The transcriptional profile of the gene SLM1, which encodes an auxin efflux carrier, was impaired and the plants' auxin response was compromised. Further investigations revealed that HDL positively regulated auxin response likely through the recruitment of MtTPL/MtTPRs into the HDL repressor complex. Its participation in auxin-dependent compound leaf morphogenesis is of interest in the context of the functional conservation and neo-functionalization of the products of WUS orthologs.

Members of the DEAL subfamily of the DUF1218 gene family are required for bilateral symmetry but not for dorsoventrality in Arabidopsis leaves.

Most plant leaves exhibit bilateral symmetry, which has been hypothesized as an inevitable consequence of the existence of the proximodistal and dorsoventral axes. No gene has been described that affects leaf bilateral symmetry but not dorsoventrality in Arabidopsis thaliana. We screened for viable insertional mutations that affect leaf morphology, and out of more than 700 mutants found only one, desigual1-1 (deal1-1), that exhibited bilateral symmetry breaking but no obvious defects in dorsoventrality. We found that deal1-1 is an allele of VASCULATURE COMPLEXITY AND CONNECTIVITY (VCC). Several overlapping regulatory pathways establish the interspersed lobes and indentations along the margin of Arabidopsis thaliana leaves. These pathways involve feedback loops of auxin, the PIN-FORMED1 (PIN1) auxin efflux carrier, and the CUP-SHAPED COTYLEDON2 (CUC2) transcriptional regulator. Early vcc (deal1) leaf primordia fail to acquire bilateral symmetry and instead form ectopic lobes and sinuses. The vcc leaves show aberrant recruitment of marginal cells expressing properly polarized PIN1, resulting in misplaced auxin maxima. Normal PIN1 polarization requires CUC2 expression and CUC2 genetically interacts with VCC; VCC also affects CUC2 expression. VCC has a domain of unknown function, DUF1218, and localizes to the endoplasmic reticulum membrane. VCC acts partially redundantly with its two closest paralogs, DEAL2 and DEAL3, in early leaf margin patterning and is required for bilateral symmetry, but its loss of function does not visibly affect dorsoventrality.

Identification of the suitable leaf margin for liver stereotactic body radiotherapy with flattening filter-free beams.

The purpose of this study is to identify the suitable leaf margin for liver stereotactic body radiotherapy (SBRT) with flattening filter-free (FFF) beams, as compared with that with flattening filter (FF) beams. SBRT treatment planning for 10 patients with liver cancer was performed using 10-MV FFF and FF beams obtained from a Varian TrueBeam (Varian Medical Systems, Palo Alto, CA) linear accelerator. Each plan was generated with the leaf margin to the planning target volume (PTV) ranging from -3 to 5 mm. The prescription dose at D95 (dose covering 95% of the volume) was 48 Gy in 4 fractions to the PTV. The following dosimetric parameters were evaluated quantitatively: homogeneity index (HI), conformity index (CI), gradient index (GI), the normal liver receiving a dose greater than or equal to 20 Gy (V20), and the mean normal liver dose. The HI for FFF and FF beams increased as the leaf margin decreased. The leaf margins that achieved the best CI and GI were 0.1 and -0.3 mm for FFF beams, and 0.1 and -0.9 mm for FF beams. The liver V20 and the mean liver dose reached their minimum values at leaf margins of -0.8 and 0.0 mm for FFF beams, and -0.8 and 0.0 mm for FF beams. The suitable leaf margin for SBRT planning did not differ significantly for FFF and FF beams. Our data showed that, for both FFF and FF beams, a leaf margin of 0 or -1 mm was optimal for liver SBRT planning in terms of both target coverage and normal tissue sparing.

LMI1-like genes involved in leaf margin development of Brassica napus.

In rapeseed (Brassica napus L.), leaf margins are variable and can be entire, serrate, or lobed. In our previous study, the lobed-leaf gene (LOBED-LEAF 1, BnLL1) was mapped to a 32.1 kb section of B. napus A10. Two LMI1-like genes, BnaA10g26320D and BnaA10g26330D, were considered the potential genes that controlled the lobed-leaf trait in rapeseed. In the present study, these two genes and another homologous gene (BnaC04g00850D) were transformed into Arabidopsis thaliana (L.) Heynh. plants to identify their functions. All three LMI1-like genes of B. napus produced serrate leaf margins. The expression analysis indicated that the expression level of BnaA10g26320D determined the difference between lobed- and entire-leaved lines in rapeseed. Therefore, it is likely that BnaA10g26320D corresponds to BnLL1.

Leaf margin phenotype-specific restriction-site-associated DNA-derived markers for pineapple (Ananas comosus L.).

To explore genome-wide DNA polymorphisms and identify DNA markers for leaf margin phenotypes, a restriction-site-associated DNA sequencing analysis was employed to analyze three bulked DNAs of F1 progeny from a cross between a 'piping-leaf-type' cultivar, 'Yugafu', and a 'spiny-tip-leaf-type' variety, 'Yonekura'. The parents were both Ananas comosus var. comosus. From the analysis, piping-leaf and spiny-tip-leaf gene-specific restriction-site-associated DNA sequencing tags were obtained and designated as PLSTs and STLSTs, respectively. The five PLSTs and two STSLTs were successfully converted to cleaved amplified polymorphic sequence (CAPS) or simple sequence repeat (SSR) markers using the sequence differences between alleles. Based on the genotyping of the F1 with two SSR and three CAPS markers, the five PLST markers were mapped in the vicinity of the P locus, with the closest marker, PLST1_SSR, being located 1.5 cM from the P locus. The two CAPS markers from STLST1 and STLST3 perfectly assessed the 'spiny-leaf type' as homozygotes of the recessive s allele of the S gene. The recombination value between the S locus and STLST loci was 2.4, and STLSTs were located 2.2 cM from the S locus. SSR and CAPS markers are applicable to marker-assisted selection of leaf margin phenotypes in pineapple breeding.

Ontogeny of the sheathing leaf base in maize (Zea mays).

Leaves develop from the shoot apical meristem (SAM) via recruitment of leaf founder cells. Unlike eudicots, most monocot leaves display parallel venation and sheathing bases wherein the margins overlap the stem. Here we utilized computed tomography (CT) imaging, localization of PIN-FORMED1 (PIN1) auxin transport proteins, and in situ hybridization of leaf developmental transcripts to analyze the ontogeny of monocot leaf morphology in maize (Zea mays). CT imaging of whole-mounted shoot apices illustrates the plastochron-specific stages during initiation of the basal sheath margins from the tubular disc of insertion (DOI). PIN1 localizations identify basipetal auxin transport in the SAM L1 layer at the site of leaf initiation, a process that continues reiteratively during later recruitment of lateral leaf domains. Refinement of these auxin transport domains results in multiple, parallel provascular strands within the initiating primordium. By contrast, auxin is transported from the L2 toward the L1 at the developing margins of the leaf sheath. Transcripts involved in organ boundary formation and dorsiventral patterning accumulate within the DOI, preceding the outgrowth of the overlapping margins of the sheathing leaf base. We suggest a model wherein sheathing bases and parallel veins are both patterned via the extended recruitment of lateral maize leaf domains from the SAM.

The Leaf Adaxial-Abaxial Boundary and Lamina Growth.

In multicellular organisms, boundaries have a role in preventing the intermingling of two different cell populations and in organizing the morphogenesis of organs and the entire organism. Plant leaves have two different cell populations, the adaxial (or upper) and abaxial (or lower) cell populations, and the boundary is considered to be important for lamina growth. At the boundary between the adaxial and abaxial epidermis, corresponding to the margin, margin-specific structures are developed and structurally separate the adaxial and abaxial epidermis from each other. The adaxial and abaxial cells are determined by the adaxial and abaxial regulatory genes (including transcription factors and small RNAs), respectively. Among many lamina-growth regulators identified by recent genetic analyses, it has been revealed that the phytohormone, auxin, and the WOX family transcription factors act at the adaxial-abaxial boundary downstream of the adaxial-abaxial pattern. Furthermore, mutant analyses of the WOX genes shed light on the role of the adaxial-abaxial boundary in preventing the mixing of the adaxial and abaxial features during lamina growth. In this review, we highlight the recent studies on the dual role of the adaxial-abaxial boundary.