Phenotypic Study of Photomorphogenesis in Arabidopsis Seedlings.

Light is one of the most important environmental factors, serving as the energy source of photosynthesis and a cue for plant developmental programs, called photomorphogenesis. Here, we provide a standardized operation to measure physiological parameters of photomorphogenesis, including in hypocotyl length, cotyledon size, and anthocyanin content.

Targeted mutagenesis of EOD3 gene in Brassica napus L. regulates seed production.

Seed size and number are central to the evolutionary fitness of plants and are also crucial for seed production of crops. However, the molecular mechanisms of seed production control are poorly understood in Brassica crops. Here, we report the gene cloning, expression analysis, and functional characterization of the EOD3/CYP78A6 gene in rapeseed. BnaEOD3 has four copies located in two subgenomes, which exhibited a steady higher expression during seed development with differential expression among copies. The targeted mutations of BnaEOD3 gene were efficiently generated by stable transformation of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat) vector. These mutations were stably transmitted to T1 and T2 generations and a large collection of homozygous mutants with combined loss-of-function alleles across four BnaEOD3 copies were created for phenotyping. All mutant T1 lines had shorter siliques, smaller seeds, and an increased number of seeds per silique, in which the quadrable mutants showed the most significant changes in these traits. Consequently, the seed weight per plant in the quadrable mutants increased by 13.9% on average compared with that of wild type, indicating that these BnaEOD3 copies have redundant functions in seed development in rapeseed. The phenotypes of the different allelic combinations of BnaEOD3 copies also revealed gene functional differentiation among the two subgenomes. Cytological observations indicated that the BnaEOD3 could act maternally to promote cotyledon cell expansion and proliferation to regulate seed growth in rapeseed. Collectively, our findings reveal the quantitative involvement of the different BnaEOD3 copies function in seed development, but also provided valuable resources for rapeseed breeding programs.

Method for Phenotypic Chemical Screening to Identify Cryptochrome Inhibitors.

After germination, plants determine their morphogenesis, such as hypocotyl elongation and cotyledon opening, by responding to various wavelengths of light (photomorphogenesis). Cryptochrome is a blue-light photoreceptor that controls de-etiolation, stomatal opening and closing, flowering time, and shade avoidance. Successful incorporation of these phenotypes as indicators into a chemical screening system results in faster selection of candidate compounds. Here, we describe phenotypic screening for the blue-light response of Arabidopsis thaliana seedling and the resulting process that clarifies that the compound obtained in the screening is an inhibitor of cryptochromes.

Characterization of Quantitative Trait Loci for Germination and Coleoptile Length under Low-Temperature Condition Using Introgression Lines Derived from an Interspecific Cross in Rice.

Previously, five putative quantitative trait loci (QTLs) for low-temperature germination (LTG) have been detected using 96 BC3F8 lines derived from an interspecific cross between the Korean japonica cultivar "Hwaseong" and Oryza rufipogon. In the present study, two introgression lines, CR1517 and CR1518, were used as parents to detect additional QTLs and analyze interactions among QTLs for LTG. The F2 population (154 plants) along with parental lines, Hwaseong and O. rufipogon, were evaluated for LTG and coleoptile length under low-temperature conditions (13 °C). Among five QTLs for LTG, two major QTLs, qLTG1 and qLTG3, were consistently detected at 6 and 7 days after incubation. Three minor QTLs were detected on chromosomes 8 and 10. Two QTLs, qLTG10.1 and qLTG10.2, showing linkage on chromosome 10, exerted opposite effects with the Hwaseong allele at qLTG10.2 and the O. rufipogon allele at qLTG10.1 respectively, in turn, increasing LTG. Interactions among QTLs were not significant, implying that the QTLs act in an additive manner. Near-isogenic line plants with the combination of favorable alleles from O. rufipogon and Hwaseong exhibited higher LTG than two introgression lines. With regard to coleoptile length, three QTLs observed on chromosomes 1, 3, and 8 were colocalized with QTLs for LTG, suggesting the pleiotropy of the single gene at each locus. According to the results, the introgression of favorable O. rufipogon alleles could hasten the development of rice with high LTG and high coleoptile elongation in japonica cultivars.

Transcriptome Analysis Identifies Key Genes Responsible for Red Coleoptiles in Triticum Monococcum.

Red coleoptiles can help crops to cope with adversity and the key genes that are responsible for this trait have previously been isolated from Triticum aestivum, Triticum urartu, and Aegilops tauschii. This report describes the use of transcriptome analysis to determine the candidate gene that controls the trait for white coleoptiles in T. monococcum by screening three cultivars with white coleoptiles and two with red coleoptiles. Fifteen structural genes and two transcription factors that are involved in anthocyanin biosynthesis were identified from the assembled UniGene database through BLAST analysis and their transcript levels were then compared in white and red coleoptiles. The majority of the structural genes reflected lower transcript levels in the white than in the red coleoptiles, which implied that transcription factors related to anthocyanin biosynthesis could be candidate genes. The transcript levels of MYC transcription factor TmMYC-A1 were not significantly different between the white and red coleoptiles and all of the TmMYC-A1s contained complete functional domains. The deduced amino acid sequence of the MYB transcription factor TmMYB-A1 in red coleoptiles was homologous to TuMYB-A1, TaMYB-A1, TaMYB-B1, and TaMYB-D1, which control coleoptile color in corresponding species and contained the complete R2R3 MYB domain and the transactivation domain. TmMYB-a1 lost its two functional domains in white coleoptiles due to a single nucleotide deletion that caused premature termination at 13 bp after the initiation codon. Therefore, TmMYB-A1 is likely to be the candidate gene for the control of the red coleoptile trait, and its loss-of-function mutation leads to the white phenotype in T. monococcum.

Rht8 gene as an alternate dwarfing gene in elite Indian spring wheat cultivars.

Optimizing wheat height to maximize yield has been an important aspect which is evident from a successful example of green revolution. Dwarfing genes (Rht) are known for yield gains due to lodging resistance and partitioning of assimilates into ear. The available and commercially exploited sources of dwarfism in Indian spring wheat are Rht1 and Rht2 genes inspite of availability of over 20 dwarfing genes. Rht8 a Gibberellic acid sensitive dwarfing gene is another reduced height gene commercially exploited in some Mediterranean countries. Two F2 populations segregating for Rht1 and Rht8 genes with each comprising 398 and 379 plants were developed by crossing European winter wheat cultivars Beauchamp and Capitole with Indian spring wheat cultivar PBW 621. Different genotypic combinations for Rht1 and Rht8 genes were selected from these populations through linked molecular markers and selected F3:4 lines were evaluated for various agronomic traits in a replicated trial. Reduction in plant height with Rht8 and Rht1 averaged 2.86% and 13.3% respectively as compared to the group of lines lacking dwarfing gene. Reduction was spread along all the internodes of wheat culm and reduction was lower as progress towards the lower internode. Grain number per spike and highest yield was observed in lines carrying only Rht1 gene. Reduction in plant biomass was observed with either of the dwarfing gene. Longest coleoptile length and seedling shoot length averaged 4.4 ± 0.09 cm and 19.5 ± 0.48, respectively was observed in lines lacking any of the dwarfing gene. Negligible reduction of 6.75% and 2.84% in coleoptile length and seedling shoot length, respectively was observed in lines carrying only Rht8 gene whereas F3:4 lines with Rht1 gene showed 21.64% and 23.35% reduction in coleoptile length and seedling shoot length, respectively. Additive effect of genes was observed as double dwarfs showed 43.31% and 43.34% reduction in coleoptile length and seedling shoot length.

C4 photosynthesis and transition of Kranz anatomy in cotyledons and leaves of Tetraena simplex.

PREMISE OF THE STUDY: Tetraena simplex is an independently evolved C4 species in the Zygophylloideae (Zygophyllaceae) and a characteristic forb of saline flats in hot and sandy desert habitats. During early ontogeny, the species had a morphological shift from planar cotyledons (dorsiventral symmetry) to terete, succulent leaves (radial symmetry). We tested whether this shift had a corresponding change in internal Kranz anatomy and tissue patterning. METHODS: For a comprehensive characterization of C4 photosynthesis across early ontogeny in T. simplex, structural and ultrastructural anatomical properties and localization patterns, activities, and immunoblotting of key C4 photosynthetic enzymes were compared in mesophyll and bundle sheath tissues in cotyledons and leaves. KEY RESULTS: Cotyledons and leaves possessed different types of Kranz anatomy (atriplicoid type and a "Tetraena" variant of the kochioid type, respectively), reflecting the change in leaf morphology. In bundle sheath cells, key differences in ultrastructural features included increased organelle numbers and chloroplast thylakoid stacking. C4 enzymes had strict tissue-specific localization patterns within bundle sheath and mesophyll cells in both cotyledons and leaves. The decarboxylase NAD-ME maintained the highest activity, increasing from cotyledons to leaves. This classified T. simplex as fully C4 across ontogeny and a strictly NAD-ME biochemical subtype. CONCLUSIONS: Tetraena simplex cotyledons and leaves showed differences in Kranz type, with associated progression in ultrastructural features, and differing activities/expression levels of C4 enzymes. Furthermore, leaves characterized a new "Tetraena" variation of the kochioid Kranz anatomy.

Identification of monocot flora using pollen features through scanning electron microscopy.

Pollen used to track structural and functional evolution in plants as well as to investigate the problems relative to plant classification. Pollen characters including ornamentation, shape, apertural pattern, pollen symmetry, colpus length, width, and margins used to detect the similarities and dissimilarities between genera and also species of the same genus. In this study pollen features of 20 monocot species belonging to 15 genera of the Amaryllidaceae, Asparagaceae, Iridaceae, Ixioliriaceae, Liliaceae, and Xanthorrhoeaceae were studied using scanning electron microscopy (SEM) and light microscopy (LM). In this study two species that is Zephyranthes citrina and Tulbaghia violacea were reported for the first time from Pakistan. Pollen grains were visualized with LM. Non-acetolyzed and acetolyzed pollen were examined using SEM. A taxonomic key was developed to highlight the variation in pollen features in order to make their systematic application for correct species identification.

Linking hard and soft traits: Physiology, morphology and anatomy interact to determine habitat affinities to soil water availability in herbaceous dicots.

BACKGROUND AND AIMS: Species' habitat affinities along environmental gradients should be determined by a combination of physiological (hard) and morpho-anatomical (soft) traits. Using a gradient of soil water availability, we address three questions: How well can we predict habitat affinities from hard traits, from soft traits, and from a combination of the two? How well can we predict species' physiological responses to drought (hard traits) from their soft traits? Can we model a causal sequence as soft traits → hard traits → species distributions? METHODS: We chose 25 species of herbaceous dicots whose affinities for soil moisture have already been linked to 5 physiological traits (stomatal conductance and net photosynthesis measured at soil field capacity, water use efficiency, stomatal conductance and soil water potential measured when leaves begin to wilt). Under controlled conditions in soils at field capacity, we measured five soft traits (leaf dry matter content, specific leaf area, leaf nitrogen content, stomatal area, specific root length). KEY RESULTS: Soft traits alone were poor predictors (R2 = 0.129) while hard traits explained 48% of species habitat affinities. Moreover, hard traits were significantly related to combinations of soft traits. From a priori biological knowledge and hypothesized ecological links we built a path model showing a sequential pattern soft traits → hard traits → species distributions and accounting for 59.6% (p = 0.782) of habitat wetness. CONCLUSIONS: Both direct and indirect causal relationships existed between soft traits, hard traits and species' habitat preferences. The poor predictive abilities of soft traits alone were due to the existence of antagonistic and synergistic direct and indirect effects of soft traits on habitat preferences mediated by the hard traits. To obtain a more realistic model applicable to a population level, it has to be tested in an experiment including species competition for water supply.

Two-stage patterning dynamics in conifer cotyledon whorl morphogenesis.

Background and Aims: Conifer embryos, unlike those of monocots or dicots, have variable numbers of cotyledons, even within the same species. Cotyledons form in a single whorl on a dome-shaped embryo. The closely spaced cotyledons are not found outside this ring, indicating a radial control on where they can form. Polar transport of the hormone auxin affects outgrowth of distinct cotyledons, but not the radial aspect of the whorl or the within-whorl spacing between cotyledons. A quantitative model of plant growth regulator patterning is needed to understand the dynamics of this complex morphogenetic process. Methods: A two-stage reaction-diffusion model is developed for the spatial patterning of growth regulators on the embryo surface, with a radial pattern (P1) constraining the shorter-wavelength cotyledon pattern (P2) to a whorl. These patterns drive three-dimensional (3-D) morphogenesis by catalysing local surface growth. Key Results: Growth driven by P2 generates single whorls across the experimentally observed range of two to 11 cotyledons, as well as the circularly symmetric response to auxin transport interference. These computations are the first corroboration of earlier theoretical proposals for hierarchical control of whorl formation. The model generates the linear relationship between cotyledon number and embryo diameter observed experimentally. This accounts for normal integer cotyledon number selection, as well as the less common cotyledon fusings and splittings observed experimentally. Flattening of the embryo during development may affect the upward outgrowth angle of the cotyledons. Conclusions: Cotyledon morphogenesis is more complex geometrically in conifers than in angiosperms, involving 2-D patterning which deforms a surface in three dimensions. This work develops a quantitative framework for understanding the growth and patterning dynamics involved in conifer cotyledon development, and applies more generally to the morphogenesis of whorls with many primordia.

C3 cotyledons are followed by C4 leaves: intra-individual transcriptome analysis of Salsola soda (Chenopodiaceae).

Some species of Salsoleae (Chenopodiaceae) convert from C3 photosynthesis during the seedling stage to the C4 pathway in adult leaves. This unique developmental transition of photosynthetic pathways offers the exceptional opportunity to follow the development of the derived C4 syndrome from the C3 condition within individual plants, avoiding phylogenetic noise. Here we investigate Salsola soda, a little-studied species from tribe Salsoleae, using an ontogenetic approach. Anatomical sections, carbon isotope (δ13C) values, transcriptome analysis by means of mRNA sequencing, and protein levels of the key C4 enzyme phosphoenolpyruvate carboxylase (PEPC) were examined from seed to adult plant stages. Despite a previous report, our results based on δ13C values, anatomy and transcriptomics clearly indicate a C3 phase during the cotyledon stage. During this stage, the entire transcriptional repertoire of the C4 NADP-malic enzyme type is detected at low levels compared to a significant increase in true leaves. In contrast, abundance of transcripts encoding most of the major photorespiratory enzymes is not significantly decreased in leaves compared to cotyledons. PEPC polypeptide was detected only in leaves, correlating with increased PEPC transcript abundance from the cotyledon to leaf stage.

Unique photosynthetic phenotypes in Portulaca (Portulacaceae): C3-C4 intermediates and NAD-ME C4 species with Pilosoid-type Kranz anatomy.

Portulacaceae is a family that has considerable diversity in photosynthetic phenotypes. It is one of 19 families of terrestrial plants where species having C4 photosynthesis have been found. Most species in Portulaca are in the alternate-leaved (AL) lineage, which includes one clade (Cryptopetala) with taxa lacking C4 photosynthesis and three clades having C4 species (Oleracea, Umbraticola and Pilosa). All three species in the Cryptopetala clade lack Kranz anatomy, the leaves have C3-like carbon isotope composition and they have low levels of C4 cycle enzymes. Anatomical, biochemical and physiological analyses show they are all C3-C4 intermediates. They have intermediate CO2 compensation points, enrichment of organelles in the centripetal position in bundle sheath (BS) cells, with selective localization of glycine decarboxylase in BS mitochondria. In the three C4 clades there are differences in Kranz anatomy types and form of malic enzyme (ME) reported to function in C4 (NAD-ME versus NADP-ME): Oleracea (Atriplicoid, NAD-ME), Umbraticola (Atriplicoid, NADP-ME) and Pilosa (Pilosoid, NADP-ME). Structural and biochemical analyses were performed on Pilosa clade representatives having Pilosoid-type leaf anatomy with Kranz tissue enclosing individual peripheral vascular bundles and water storage in the center of the leaf. In this clade, all species except P. elatior are NADP-ME-type C4 species with grana-deficient BS chloroplasts and grana-enriched M chloroplasts. Surprisingly, P. elatior has BS chloroplasts enriched in grana and NAD-ME-type photosynthesis. The results suggest photosynthetic phenotypes were probably derived from an ancestor with NADP-ME-type C4, with two independent switches to NAD-ME type.

Organ boundary NAC-domain transcription factors are implicated in the evolution of petal fusion.

Research rationale: Evolution of fused petals (sympetaly) is considered to be an important innovation that has repeatedly led to increased pollination efficiency, resulting in accelerated rates of plant diversification. Although little is known about the underlying regulation of sympetaly, genetic pathways ancestrally involved in organ boundary establishment (e.g. CUP SHAPED COTYLEDON [CUC] 1-3 genes) are strong candidates. In sympetalous petunia, mutations in the CUC1/2-like orthologue NO APICAL MERISTEM (NAM) inhibit shoot apical meristem formation. Despite this, occasional 'escape shoots' develop flowers with extra petals and fused inter-floral whorl organs. Central methods: To To determine if petunia CUC-like genes regulate additional floral patterning, we used virus-induced silencing (VIGS) following establishment of healthy shoot apices to re-examine the role of NAM in petunia petal development, and uniquely characterise the CUC3 orthologue NH16. KEY RESULTS: Confirming previous results, we found that reduced floral NAM/NH16 expression caused increased petal-stamen and stamen-carpel fusion, and often produced extra petals. However, further to previous results, all VIGS plants infected with NAM or NH16 constructs exhibited reduced fusion in the petal whorl compared to control plants. MAIN CONCLUSIONS: Together with previous data, our results demonstrate conservation of petunia CUC-like genes in establishing inter-floral whorl organ boundaries, as well as functional evolution to affect the fusion of petunia petals.

Node Detection and Internode Length Estimation of Tomato Seedlings Based on Image Analysis and Machine Learning.

Seedling vigor in tomatoes determines the quality and growth of fruits and total plant productivity. It is well known that the salient effects of environmental stresses appear on the internode length; the length between adjoining main stem node (henceforth called node). In this study, we develop a method for internode length estimation using image processing technology. The proposed method consists of three steps: node detection, node order estimation, and internode length estimation. This method has two main advantages: (i) as it uses machine learning approaches for node detection, it does not require adjustment of threshold values even though seedlings are imaged under varying timings and lighting conditions with complex backgrounds; and (ii) as it uses affinity propagation for node order estimation, it can be applied to seedlings with different numbers of nodes without prior provision of the node number as a parameter. Our node detection results show that the proposed method can detect 72% of the 358 nodes in time-series imaging of three seedlings (recall = 0.72, precision = 0.78). In particular, the application of a general object recognition approach, Bag of Visual Words (BoVWs), enabled the elimination of many false positives on leaves occurring in the image segmentation based on pixel color, significantly improving the precision. The internode length estimation results had a relative error of below 15.4%. These results demonstrate that our method has the ability to evaluate the vigor of tomato seedlings quickly and accurately.

Exceptional preservation of tiny embryos documents seed dormancy in early angiosperms.

The rapid diversification of angiosperms through the Early Cretaceous period, between about 130-100 million years ago, initiated fundamental changes in the composition of terrestrial vegetation and is increasingly well understood on the basis of a wealth of palaeobotanical discoveries over the past four decades and their integration with improved knowledge of living angiosperms. Prevailing hypotheses, based on evidence both from living and from fossil plants, emphasize that the earliest angiosperms were plants of small stature with rapid life cycles that exploited disturbed habitats in open, or perhaps understorey, conditions. However, direct palaeontogical data relevant to understanding the seed biology and germination ecology of Early Cretaceous angiosperms are sparse. Here we report the discovery of embryos and their associated nutrient storage tissues in exceptionally well-preserved angiosperm seeds from the Early Cretaceous. Synchrotron radiation X-ray tomographic microscopy of the fossil embryos from many taxa reveals that all were tiny at the time of dispersal. These results support hypotheses based on extant plants that tiny embryos and seed dormancy are basic for angiosperms as a whole. The minute size of the fossil embryos, and the modest nutrient storage tissues dictated by the overall small seed size, is also consistent with the interpretation that many early angiosperms were opportunistic, early successional colonizers of disturbance-prone habitats.

HIGH FREQUENCY GENETIC TRANSFORMATION OF CICHORIUM INTYBUS L. USING nptII GENE AS A SELECTIVE MARKER.

Cichorium intybus L. is an important vegetable crop used as salad (leaf form) and for the production of coffee substitutes (root form). At the same time these plants can also be used in biotechnologies for synthesis of pharmaceutical proteins. Here we report the possibility of high frequency Agrobacterium rhizogenes- or A. tumefaciens-mediated transformation of C. intybus L. for construction of transgenic "hairy" roots and plants. The used plasmids contained target human interferonifn-α2b gene, Mycobacterium tuberculosis ESAT6:Ag85B antigene esxA::fbpB(ΔTMD) fused gene and human telomerase reverse transcriptase h Tert gene. Using of nptII gene as a selective one was preferable to the bar gene for chicory. In this case the frequency of transgenic plants or "hairy" roots formation was significantly higher. Cultivation of explants on the medium with Basta in concentration 1-2 mg/l have led to plants death or to significant reduction of number of shoots formed. Frequency of "hairy" roots formation varied from 5.9 to 42.3% after A. rhizogenes-mediated transformation. Frequency of regeneration of transgenic plants varied from 10 to 86% after A. tumefaciens-mediated transformation. Both A. rhizogenes- and A. tumefaciens-mediated transformation frequency depended on the type of explants, roots or cotyledons, and vector used. Usage of A. tumefaciens carrying pCB064 plasmid (target esxA:fbpB(ΔTMD) fused gene and nptII selective gene) resulted in the most effective regeneration of transgenic plants with regeneration frequency up to 86%. In the case of chicory A. rhizogenes-mediated transformation the highest regeneration frequency up to 42.3% was demonstrated using p CB161 vector with ifn-α2b target gene and nptII selective gene.

The tarani mutation alters surface curvature in Arabidopsis leaves by perturbing the patterns of surface expansion and cell division.

The leaf surface usually stays flat, maintained by coordinated growth. Growth perturbation can introduce overall surface curvature, which can be negative, giving a saddle-shaped leaf, or positive, giving a cup-like leaf. Little is known about the molecular mechanisms that underlie leaf flatness, primarily because only a few mutants with altered surface curvature have been isolated and studied. Characterization of mutants of the CINCINNATA-like TCP genes in Antirrhinum and Arabidopsis have revealed that their products help maintain flatness by balancing the pattern of cell proliferation and surface expansion between the margin and the central zone during leaf morphogenesis. On the other hand, deletion of two homologous PEAPOD genes causes cup-shaped leaves in Arabidopsis due to excess division of dispersed meristemoid cells. Here, we report the isolation and characterization of an Arabidopsis mutant, tarani (tni), with enlarged, cup-shaped leaves. Morphometric analyses showed that the positive curvature of the tni leaf is linked to excess growth at the centre compared to the margin. By monitoring the dynamic pattern of CYCLIN D3;2 expression, we show that the shape of the primary arrest front is strongly convex in growing tni leaves, leading to excess mitotic expansion synchronized with excess cell proliferation at the centre. Reduction of cell proliferation and of endogenous gibberellic acid levels rescued the tni phenotype. Genetic interactions demonstrated that TNI maintains leaf flatness independent of TCPs and PEAPODs.

Acorn cotyledons are larger than their seedlings' need: evidence from artificial cutting experiments.

Although the consequences of cotyledon removal have been widely studied in oaks producing large acorns, we have little knowledge of at what level cotyledons can be removed without affecting acorn survival and seedling development. In this study, we aimed to test the hypothesis that the amount of energy reserves in cotyledons is more than the demands of seedlings and that large acorns can tolerate seed predation and/or attract seed predators for seed dispersal. Acorn germination rates were not affected even when 60% of cotyledons were cut off at the basal end, suggesting that the energy reserves contained in cotyledons are not essential for acorn survival. Post-cut acorn mass, more than initial acorn mass, appear to be a better predictor of seedling performance, indicating that the energy reserves in cotyledons are sufficient for seedlings. Acorns with large masses sustained cotyledon damage better than small ones with respect to seedling performance. Large acorns were more likely to be dispersed and cached by animals, implying that producing large acorns is more important for oaks to manipulate seed predators and dispersers rather than provide a seedling with cotyledonary reserves.

Arabidopsis UNHINGED encodes a VPS51 homolog and reveals a role for the GARP complex in leaf shape and vein patterning.

Asymmetric localization of PIN proteins controls directionality of auxin transport and many aspects of plant development. Directionality of PIN1 within the marginal epidermis and the presumptive veins of developing leaf primordia is crucial for establishing leaf vein pattern. One mechanism that controls PIN protein distribution within the cell membranes is endocytosis and subsequent transport to the vacuole for degradation. The Arabidopsis mutant unhinged-1 (unh-1) has simpler leaf venation with distal non-meeting of the secondary veins and fewer higher order veins, a narrower leaf with prominent serrations, and reduced root and shoot growth. We identify UNH as the Arabidopsis vacuolar protein sorting 51 (VPS51) homolog, a member of the Arabidopsis Golgi-associated retrograde protein (GARP) complex, and show that UNH interacts with VPS52, another member of the complex and colocalizes with trans Golgi network and pre-vacuolar complex markers. The GARP complex in yeast and metazoans retrieves vacuolar sorting receptors to the trans-Golgi network and is important in sorting proteins for lysosomal degradation. We show that vacuolar targeting is reduced in unh-1. In the epidermal cells of unh-1 leaf margins, PIN1 expression is expanded. The unh-1 leaf phenotype is partially suppressed by pin1 and cuc2-3 mutations, supporting the idea that the phenotype results from expanded PIN1 expression in the marginal epidermis. Our results suggest that UNH is important for reducing expression of PIN1 within margin cells, possibly by targeting PIN1 to the lytic vacuole.

Leaf fossils of Luzuriaga and a monocot flower with in situ pollen of Liliacidites contortus Mildenh. & Bannister sp. nov. (Alstroemeriaceae) from the Early Miocene.

PREMISE OF THE STUDY: The Foulden Maar lake sediments in Otago, South Island, New Zealand, date to the earliest Miocene and provide an important picture of the diversity of the Australasian biota, paleoecology, and climate at a time when New Zealand had a smaller land area than today. The diverse rainforest contains many taxa now restricted to Australia, New Caledonia, or South America. The presence of Luzuriaga-like fossils in these deposits is important for understanding Alstroemeriaceae evolution and the biogeography of genera shared between New Zealand and South America. METHODS: Leaves and a flower with in situ pollen that resemble extant Luzuriaga are described and placed phylogenetically. Geographic range information and a molecular clock model for the Alstroemeriaceae were used to investigate possible biogeographic scenarios and the influence of the new fossil on inferred divergence times. KEY RESULTS: Luzuriaga peterbannisteri Conran, Bannister, Mildenh., & D.E.Lee sp. nov. represents the first macrofossil record for Alstroemeriaceae. An associated Luzuriaga-like flower with in situ fossil pollen of Liliacidites contortus Mildenh. & Bannister sp. nov. is also described. The biogeographic analysis suggests that there have been several dispersal events across the Southern Ocean for the genus, with the fossil representing a now-extinct New Zealand lineage. CONCLUSIONS: Luzuriaga was present in Early Miocene New Zealand, indicating a long paleogeographic history for the genus, and L. peterbannisteri strengthens biogeographic connections between South America and Australasia during the Oligocene and earliest Miocene.