Hawaiian flower skirt

Hawaiian flower skirt DEFAULT

Do Hula Dancers Still Use “Grass Skirts”?

Grass Skirts, or Not?

So-called “grass skirts” have always been a misnomer; however, Hawaiians and other Polynesians have traditionally used strips of natural fibers, barks and other materials to create various skirts and adornment. The purpose of such skirts, beyond decoration, was — and is — always to accentuate the dancer’s movements.

For example, strips of the inner wild hibiscus bark — which Hawaiians and Tahitians call hau, and Samoans and Tongans call fau — are still collected today, cleaned and bleached in salt water, dried and then strung together side-by-side to create a dance skirt.

The basic method of doing this is actually easy:

  • Start with an appropriate length of cord for the waist-size of the dancer, leaving enough extra so the two ends can be tied together.
  • Drape one end of a prepared hau strip over the waist cord, dangling a short length on the other side, and tie the two sides of the strip together under the waist cord.
  • Repeat this over and over, cinching the strips of hau next to each other, until the skirt is long enough to encircle the dancer’s waist (again leaving enough cord so it can be tied off).
  • Trim the bottom of the hau strips appropriate to the height of the dancer. Women’s skirts are traditionally longer, as much as down to the ankles, while men’s skirts are generally shorter.

Ti Leaf Skirts

The same or similar technique is used to make a Hawaiian tī-leaf skirt, although the thick end where the leaf attaches to the plant must first be stripped thinner by removing part of the mid-rib. This thin portion is then draped over the waist cord (and tied to itself underneath the cord).

Other Skirt Styles and Materials

Of course, there are many variations of this basic method throughout Polynesia:

  • Tahitians sometimes add tassels to further accentuate the movements, as well as shells and/or flowers for further adornment.
  • New Zealand Māori, for example, generally collected indigenous flax leaves — the wharariki or harakeke,which are not related to linen flax — and scraped most of each one down to the inner fiber. These were then boiled, twisted, and died to make their traditional, swinging piupiu skirts (by the usual method of draping one end over a waist belt).
    • The Māori used wild flax leaves like the other Polynesians living on tropical islands used pandanus leaves (lauhala) to weave into baskets and other various items. The Māori also used their wild flax fibers to weave and/or braid into clothing and cordage.
  • Students in modern hula hālau or hula schools often learn to make their own “grass” and/or tī-leaf skirts, as well as other dance adornments, as part of their training.

Hale Pili

The description of traditional Hawaiian houses as “grass huts” or “grass shacks” is actually more correct than “grass skirts”: Most Hawaiian hale were “roofed” by tying clumps of pili grass in a row (similar to how modern homes are “shingled”), and then tying each succeeding, higher row so that it overlapped the one beneath, causing the rain to run off.


Story and images by Mike Foley

mike_foleyMike Foley, who has worked off-and-on

at the Polynesian Cultural Center since

1968,  has been a full-time freelance

writer and digital media specialist since

2002, and had a long career in marketing

communications and PR before that. He

learned to speak fluent Samoan as a

Mormon missionary before moving to Laie

in 1967 — still does, and he has traveled

extensively over the years throughout

Polynesia and other Pacific islands. Foley

is mostly retired now, but continues to

contribute to various PCC and other media.

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2 Sets Hawaiian Hula Grass Skirt with Flower Leis Hawaiian Costume Set, Elastic Luau Grass and Hawaiian Flower Bracelets, Headband, Necklace for Party Favors (Straw Color, Mixed Color)

2 Sets Hawaiian Hula Grass Skirt with Flower Leis Hawaiian Costume Set, Elastic Luau Grass and Hawaiian Flower Bracelets, Headband, Necklace for Party Favors (Straw Color, Mixed Color)

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Product Details

  • Package includes: 2 sets with 10 pieces items in total; Each set includes 1 pieces grass skirt, 1 piece necklace, 1 piece headband and 2 pieces bracelets
  • Comfortable to wear: flower leis is made of polyester cloth, grass skirt is made of polyester synthetic grass and faux flowers is made of silk, soft and comfortable materials allow for long time wearing
  • Vibrant colors: flower leis are yellow, green, orange, blue, purple and red; 2 Pieces grass skirts, one is pure nature straw color and the other is bright mixed colors, fashion costume makes you stand out in the crowd
  • Adjustable size: with elastic waistband, the 15.7 inch/ 40 cm long hula skirt's waistband extent is 19.7-31.5 inch (50-80 cm); The size of flower leis are also adjustable with elastic rope; The size fits children and little girls
  • Suitable many occasions: good party costume with tropical look, suit for Hawaiian themed parties, dance, show, activity, costume ball, fancy dress parties, Halloween, etc.
Is Discontinued By Manufacturer ‏ : ‎No
Package Dimensions ‏ : ‎9.61 x 6.54 x 2.64 inches (24.4 x 16.6 x 6.7 cm); 4.59 Ounces (130.13 grams)
Manufacturer recommended age ‏ : ‎14 years and up
Department ‏ : ‎Womens
Manufacturer ‏ : ‎Shappy
ASIN ‏ : ‎B074C3HWVM

Description

2 Sets Hawaiian Hula Grass Skirt with Flower Leis Hawaiian Costume Set, Elastic Luau Grass and Hawaiian Flower Bracelets, Headband, Necklace for Party Favors (Straw Color, Mixed Color)

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Grass skirts, Hawaiian grass skirts, Lei package, Red and green dance costume, Best grass skirts for parties, Best hawaiian costumes for kids

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Abstract

A fast neutron-mutagenized population of Arabidopsis (Arabidopsis thaliana) Columbia-0 wild-type plants was screened for floral phenotypes and a novel mutant, termed hawaiian skirt (hws), was identified that failed to shed its reproductive organs. The mutation is the consequence of a 28 bp deletion that introduces a premature amber termination codon into the open reading frame of a putative F-box protein (At3g61590). The most striking anatomical characteristic of hws plants is seen in flowers where individual sepals are fused along the lower part of their margins. Crossing of the abscission marker, ProPGAZAT:β-glucuronidase, into the mutant reveals that while floral organs are retained it is not the consequence of a failure of abscission zone cells to differentiate. Anatomical analysis indicates that the fusion of sepal margins precludes shedding even though abscission, albeit delayed, does occur. Spatial and temporal characterization, using ProHWS:β-glucuronidase or ProHWS:green fluorescent protein fusions, has identified HWS expression to be restricted to the stele and lateral root cap, cotyledonary margins, tip of the stigma, pollen, abscission zones, and developing seeds. Comparative phenotypic analyses performed on the hws mutant, Columbia-0 wild type, and Pro35S:HWS ectopically expressing lines has revealed that loss of HWS results in greater growth of both aerial and below-ground organs while overexpressing the gene brings about a converse effect. These observations are consistent with HWS playing an important role in regulating plant growth and development.

Abscission involves the detachment of an organ from the body of a plant and takes place at a site that is predestined for the purpose (Sexton and Roberts, 1982; González-Carranza et al., 1998). The phenomenon may be triggered by a range of environmental stresses including drought, waterlogging, nutrient deficiency, or pathogen attack (Addicott, 1982; Taylor and Whitelaw, 2001), but is also programmed to occur at discrete stages during plant development such as after leaf senescence, flower fertilization, or fruit ripening have taken place. Although the precise events that bring about shedding are unclear the process is preceded by an increase in the activity of several wall-loosening enzymes including β-1-4 glucanase and polygalacturonase precisely at the site of abscission (Roberts et al., 2002). It is proposed that the action of these enzymes, coupled with an increase in expansin activity (Belfield et al., 2005; Sampedro and Cosgrove, 2005), may lead to the dissolution of the middle lamella that brings about cell separation (González-Carranza et al., 2002; Roberts et al., 2002).

To dissect further the mechanisms responsible for regulating the abscission process, forward genetic strategies have been employed to identify non- or delayed-shedding mutants followed by the mapping and characterization of the mutated genes (Patterson, 2001; Roberts et al., 2002). While a number of such mutants have been documented only a few of the genes responsible have been cloned and characterized. These include jointless, which is the consequence of a mutation in a MADS-box gene involved in the differentiation of abscission cells in the pedicel of tomato (Solanum lycopersicum) flowers (Mao et al., 2000), and inflorescence deficient in abscission where the mutated gene has been shown to encode a novel class of ligand that seems to play a key role both in defining the site of cell separation (Stenvik et al., 2006) and in regulating the final step of floral organ shedding in Arabidopsis (Arabidopsis thaliana; Butenko et al., 2003). Recently, quantitative trait loci analysis has led to the identification of a quantitative trait locus (sh4) that has played a critical role in the domestication of rice (Oryza sativa) by bringing about a reduced shattering phenotype (Li et al., 2006). Although the function of the gene is unknown its characteristics suggest that it acts as a transcription factor. Other genes that have been identified to contribute to the abscission process in Arabidopsis include: HAESA that encodes a Leu-rich receptor-like kinase (Jinn et al., 2000); AGAMOUS-LIKE15, a MADS-domain transcription factor that if nonfunctional results in a delay in the time course of abscission (Fernandez et al., 2000; Harding et al., 2003; Lehti-Shiu et al., 2005); and the redundant BLADE ON PETIOLE1 (BOP1) and BOP2 genes encoding two BTB/POZ domain proteins that when silenced result in a disruption in leaf patterning and floral organ abscission (Hepworth et al., 2005; Norberg et al., 2005).

During the screening of a fast neutron-mutagenized population of Arabidopsis Columbia-0 (Col-0) wild-type plants a mutant, termed hawaiian skirt (hws), was isolated that retained its floral organs even after silique desiccation had taken place. In addition to exhibiting a nonshedding phenotype hws also exhibited sepals that are fused for some distance along their margins. Other mutants from Arabidopsis that show varying degrees of sepal fusion include unusual floral organs (ufo) where the mutated gene has been shown to encode an F-box protein (Levin and Meyerowitz, 1995; Samach et al., 1999); leafy, which is the result of a mutation in a floral meristem identity gene (Schultz and Haughn, 1991; Weigel and Meyerowitz, 1993); and the cup shaped cotyledon (cuc) mutants (cuc1, cuc2, cuc3) that arise as a consequence of mutations in putative NAC-transcription factor genes (Aida et al., 1997; Takada et al., 2001; Vroemen et al., 2003). Interestingly, ectopic expression of the microRNA miR164, which has been shown to induce posttranscriptional down-regulation of CUC1 and CUC2 (Laufs et al., 2004), leads to the generation of flowers with fused sepals that fail to undergo the normal shedding process (Mallory et al., 2004). A number of other genes have been shown to play an important role in the specification of lateral organ boundaries in leaves or flowers including LATERAL ORGAN BOUNDARIES (Shuai et al., 2002), LATERAL ORGAN JUNCTIONS (Prasad et al., 2005), PETAL LOSS (Brewer et al., 2004), the FUSED FLORAL ORGANS (FFO) loci (FFO1, FFO2, FFO3; Levin et al., 1998), HANABA TARANU (Zhao et al., 2004), and RABBIT EARS (Krizek et al., 2006).

In this article, the mapping and identification of a mutation in a putative F-box gene (At3g61590), which is responsible for bringing about the hws-1 phenotype, is described. Although the transcript of HWS accumulates throughout the plant, reporter gene analysis reveals that expression is restricted to only certain tissues. By comparing and contrasting the phenotypic features of hws-1, an overexpressing HWS line driven by the 35S cauliflower mosaic virus (CaMV) promoter wild-type plants, a role for HWS in regulating plant growth and development has been highlighted.

RESULTS

Mutant Isolation and Characterization

The hws-1mutant was isolated, as a result of an inability to shed its floral organs, during a screen of M2progeny grown from a fast neutron-mutagenized population (dose: 55 Gy; Lehle seeds) of M1Arabidopsis seeds in the Col-0 (wild type) background. Sepals, petals, and anther filaments were retained throughout reproductive development and remained in situ even after silique desiccation and dehiscence had taken place. To study flower development in more detail material was harvested from positions throughout the primary inflorescence with the first being where petals were visible. From that position all subsequent flowers were numbered. A scanning electron microscope (SEM) study of hws-1flowers revealed that sepals of the mutant were fused, for a distance along the lower part of their margins, and that characteristically the sepal whorl was broader than that seen in wild-type plants (Fig. 1, A–F

Figure 1.

SEM and light microscope analyses of wild-type and hws-1 flowers. Scanning electron microscopy of flower development stages in Col-0 wild type (A–C) and hws-1 plants (D–F) at positions 2 (A and D), 6 (B and E), and 14 (C and F). Light microscopy of 5 μm longitudinal sections of flower AZs stained with Toluidine blue in Col-0 wild type (G and H) and hws-1 plants (I and J) at position 4 (G and I), when petals (Pe) and sepals (Se) are still attached, and position 12 (H and J), when only sepals are attached to the hws-1 mutant and siliques (Si) are visible at all stages. Sites of sepal (Se-AZ), petal (Pe-AZ), and anther filament (Fil-AZ) abscission are identified. Bars = 500 μm (A–F) and 50 μm (G–J). [See online article for color version of this figure.]

Figure 1.

SEM and light microscope analyses of wild-type and hws-1 flowers. Scanning electron microscopy of flower development stages in Col-0 wild type (A–C) and hws-1 plants (D–F) at positions 2 (A and D), 6 (B and E), and 14 (C and F). Light microscopy of 5 μm longitudinal sections of flower AZs stained with Toluidine blue in Col-0 wild type (G and H) and hws-1 plants (I and J) at position 4 (G and I), when petals (Pe) and sepals (Se) are still attached, and position 12 (H and J), when only sepals are attached to the hws-1 mutant and siliques (Si) are visible at all stages. Sites of sepal (Se-AZ), petal (Pe-AZ), and anther filament (Fil-AZ) abscission are identified. Bars = 500 μm (A–F) and 50 μm (G–J). [See online article for color version of this figure.]

). While the shedding of petals and anther filaments did not take place in the mutant, fine structural analysis of these tissues indicated that abscission of these organs could be detected in both wild-type and hws-1flowers (Fig. 1, G–J). Cell separation at the sepal bases was also apparent in hws-1flowers, however, the timing of this was delayed in comparison to wild-type plants and the sepal whorl was retained even though abscised (Fig. 1, H and J).
Further SEM analysis revealed that no distinction could be made between the wild-type and hws-1plants during early bud development (Fig. 2, A, B, E, and F

Figure 2.

SEM analysis of wild-type and hws-1 buds at developmental stage 6 (A and E), stage 8 (B and F), stage 10 (C and G), and stage 12 (D and H). Buds dissected from Col-0 wild type (A–D) and hws-1 (E–H) plants. Bars = 100 μm.

Figure 2.

SEM analysis of wild-type and hws-1 buds at developmental stage 6 (A and E), stage 8 (B and F), stage 10 (C and G), and stage 12 (D and H). Buds dissected from Col-0 wild type (A–D) and hws-1 (E–H) plants. Bars = 100 μm.

). However, by the time the buds have reached stage 10 (Smyth et al., 1990) the line of separation between individual sepals had become demarcated to the bud base in wild-type but not in hws-1material (Fig. 2, C and G). Initially the region of sepal confluence in hws-1is restricted to just a few cells; however, further division must take place so that by the time buds have reached stage 12 sepals are fused for nearly 25% of their distance (Fig. 2, D and H).

The hws-1 mutant was crossed with a wild-type plant and a 3:1 segregation of shedding:nonshedding individuals in the F2 population showed that the phenotype is due to a recessive mutation in a single gene of nuclear origin.

Crossing of an Abscission Zone-Specific Gene Marker into the hws-1 Mutant

To determine whether the nonshedding phenotype of hws-1was due to a failure of abscission zone (AZ) differentiation a cross was made between the mutant and a transgenic marker line (ProPGAZAT:GUS) that expresses the reporter gene GUSspecifically at the site of floral organ separation. PGAZAT(At2g41850) encodes a polygalacturonase that is transcribed immediately prior to organ abscission in Arabidopsis and is thought to contribute to cell wall degradation (González-Carranza et al., 2002). Several homozygous hws-1lines containing the GUSgene were isolated. Figure 3, A and B

Figure 3.

Flower and silique expression from ProPGAZAT:GUS, ProPGAZAT:GUS crossed into hws-1, and ProHWS:GUS. Time course of expression from different developmental positions of flower and siliques of GUS-stained tissue after 12 h incubation in GUS substrate. Flowers are identified from positions 2 to 20. Position 1 is the first flower where petals are visible to the eye. A, ProPGAZAT:GUS. B, ProPGAZAT:GUS crossed into hws-1. C, ProHWS:GUS. Bars = 1 mm.

Figure 3.

Flower and silique expression from ProPGAZAT:GUS, ProPGAZAT:GUS crossed into hws-1, and ProHWS:GUS. Time course of expression from different developmental positions of flower and siliques of GUS-stained tissue after 12 h incubation in GUS substrate. Flowers are identified from positions 2 to 20. Position 1 is the first flower where petals are visible to the eye. A, ProPGAZAT:GUS. B, ProPGAZAT:GUS crossed into hws-1. C, ProHWS:GUS. Bars = 1 mm.

, shows that both wild-type and mutant plants express the ProPGAZAT:GUSgene at the base of the sepals, petals, and anther filaments, indicating that the hwsmutation does not seem to impede AZ differentiation. However, although ProPGAZAT:GUSexpression is apparent in position 8 flowers of wild-type plants (Fig. 3A), it cannot be detected until position 10 in hws-1flowers (Fig. 3B). In addition, ProPGAZAT:GUSexpression is less pronounced in hws-1plants and by position 20, in contrast to wild-type plants, expression of the reporter gene is no longer detectable at the site of floral organ abscission. For comparison, expression of ProHWS:GUSis shown at different positions throughout floral development (Fig. 3C). However, these observations will not be described in detail until a later part of the “Results” section of this article.

Other Phenotypic Features of the hws Mutant

The identification of another allele of HWS (hws-2) from the SALK collection (Alonso et al., 2003), during the course of mapping the gene, lead to the confirmation of additional phenotypic characteristics associated with the hws-1 mutation.

A detailed analysis revealed that 28% of flowers in the hws-1mutant had fused anther filaments to differing extents along their length (Fig. 4A

Figure 4.

Phenotypic characteristics of hws-1. Characteristics shown in C, D, and G appeared consistently. A, Fused anthers (arrow). B, Anther fused to the silique (arrow). C, Top of mature silique from Col-0 wild type (left) or hws-1 (right). Note the difference in width of silique and length of residual papillae. D, Bottom of mature silique from Col-0 wild type (left) or hws-1 (right). Note the difference in longitudinal dimensions of the AZ region. E, Siliques from hws-1 with more than two valves. F, Dissected siliques from Col-0 wild type (left and center) and hws-1 (right) with aberrant septum development. G, First cauline leaves from hws-1 fused to the stem of the inflorescence (indicated by an arrow). Bars = 500 μm (A–F) and 5 mm (G). [See online article for color version of this figure.]

Figure 4.

Phenotypic characteristics of hws-1. Characteristics shown in C, D, and G appeared consistently. A, Fused anthers (arrow). B, Anther fused to the silique (arrow). C, Top of mature silique from Col-0 wild type (left) or hws-1 (right). Note the difference in width of silique and length of residual papillae. D, Bottom of mature silique from Col-0 wild type (left) or hws-1 (right). Note the difference in longitudinal dimensions of the AZ region. E, Siliques from hws-1 with more than two valves. F, Dissected siliques from Col-0 wild type (left and center) and hws-1 (right) with aberrant septum development. G, First cauline leaves from hws-1 fused to the stem of the inflorescence (indicated by an arrow). Bars = 500 μm (A–F) and 5 mm (G). [See online article for color version of this figure.]

), compared with 2% in the wild type. Occasionally, in hws-1plants, these filaments were fused to the side of siliques (Fig. 4B). The top of the hws-1silique was consistently broader than wild type, and the length of the AZ region, exposed by manually removing the floral organs, was longer in the mutant (Fig. 4, C and D). Some hws-1siliques comprised more than two valves (Fig. 4E) and dissection of aberrant pods revealed that this was associated with abnormal development of the septum (Fig. 4F). The lamina tissue of the primary cauline leaves of hws-1plants routinely exhibited fusion to the inflorescence stems (Fig. 4G).

Mapping the hws Locus

Homozygous hws-1(Col-0 ecotype) plants were crossed with the Landsberg erecta(Ler) ecotype and the F2progeny used as a mapping population. An amplified fragment length polymorphism (AFLP)-based genome-wide approach (Peters et al., 2004) was adopted to map the hwsmutation to a 3.28 Mb domain at the bottom of chromosome 3. Using cleaved amplified polymorphic sequences (CAPS), simple sequence length polymorphisms (SSLPs), and insertion/deletion (InDel) markers this interval was reduced to a region of 56 kb containing 18 annotated genes (Fig. 5A

Figure 5.

Mapping and identification of the HWS gene. A, Mapping strategy used to identify the HWS locus using an AFLP approach to analyze the DNA from 33 F2hws-1 mutant plants. The initial location of the locus was identified to a region of 3.28 MB at the bottom of chromosome 3 between the markers SM148,6 and SM239_119,5. Further use of CAPS and SSLP on DNA from 156 F2hws-1 mutant plants allowed the reduction of the region to a segment of 0.4 MB. With the use of InDels on DNA from 600 F2hws-1 plants, the region was reduced to a 56 kb segment between InDel 470113 and 469675. This region contained 18 candidate genes. Insertional SALK lines of these 18 genes were analyzed for hws-1 characteristics and the DNA from region At3g61590 was amplified by PCR and products were subject to digestion by restriction enzymes. B, Structure of the HWS gene. The gene has a 5′ UTR of 419 bp interrupted by an intron of 532 bp, an ORF of 1,236 bp, and a 3′ UTR of 181 bp. The hws-1 mutation is a consequence of a 28 bp deletion 966 bp downstream from the ATG that introduces a premature amber nonsense codon. Individuals from the SALK_088349 line (hws-2) that phenocopies the hws-1 mutation have two T-DNA insertions inserted in opposite directions 475 and 491 bp downstream from the ATG (shown). C, Patterns of digestion with frequent cutters AluI, RsaI, and TaqI from the region corresponding to the position of gene At3g61590. Arrows indicate different patterns of digestion between the hws-1 mutant and the Col-0 wild type. D, PCR in two progeny plants from the hws-1 mutant and the SALK_088349 line (hws-2) and the Col-0 wild type with insert primers and specific primers; note that two bands were amplified with vector border primer and each one of the specific primers, indicating that this line has two insertions. E, RT-PCR in root, bud/flower, and rosette tissues from the SALK_088349 line (hws-2) and a mix of the same tissues in two progeny plants from the hws-1 mutant and SALK_088349 (hws-2). Ubiquitin (At4g05320) primers were used to demonstrate equal amounts of mRNA amplified. F, Complementation of hws-1 using a 3.5 kb genomic fragment including the promoter, UTRs, intron, and exon of At3g61590. Siliques from position 16: (1) Col-0 wild type, (2) F1 complementation plants, (3) hws-1 mutant, and (4) SALK_088349 KO line (hws-2). [See online article for color version of this figure.]

Figure 5.

Mapping and identification of the HWS gene. A, Mapping strategy used to identify the HWS locus using an AFLP approach to analyze the DNA from 33 F2hws-1 mutant plants. The initial location of the locus was identified to a region of 3.28 MB at the bottom of chromosome 3 between the markers SM148,6 and SM239_119,5. Further use of CAPS and SSLP on DNA from 156 F2hws-1 mutant plants allowed the reduction of the region to a segment of 0.4 MB. With the use of InDels on DNA from 600 F2hws-1 plants, the region was reduced to a 56 kb segment between InDel 470113 and 469675. This region contained 18 candidate genes. Insertional SALK lines of these 18 genes were analyzed for hws-1 characteristics and the DNA from region At3g61590 was amplified by PCR and products were subject to digestion by restriction enzymes. B, Structure of the HWS gene. The gene has a 5′ UTR of 419 bp interrupted by an intron of 532 bp, an ORF of 1,236 bp, and a 3′ UTR of 181 bp. The hws-1 mutation is a consequence of a 28 bp deletion 966 bp downstream from the ATG that introduces a premature amber nonsense codon. Individuals from the SALK_088349 line (hws-2) that phenocopies the hws-1 mutation have two T-DNA insertions inserted in opposite directions 475 and 491 bp downstream from the ATG (shown). C, Patterns of digestion with frequent cutters AluI, RsaI, and TaqI from the region corresponding to the position of gene At3g61590. Arrows indicate different patterns of digestion between the hws-1 mutant and the Col-0 wild type. D, PCR in two progeny plants from the hws-1 mutant and the SALK_088349 line (hws-2) and the Col-0 wild type with insert primers and specific primers; note that two bands were amplified with vector border primer and each one of the specific primers, indicating that this line has two insertions. E, RT-PCR in root, bud/flower, and rosette tissues from the SALK_088349 line (hws-2) and a mix of the same tissues in two progeny plants from the hws-1 mutant and SALK_088349 (hws-2). Ubiquitin (At4g05320) primers were used to demonstrate equal amounts of mRNA amplified. F, Complementation of hws-1 using a 3.5 kb genomic fragment including the promoter, UTRs, intron, and exon of At3g61590. Siliques from position 16: (1) Col-0 wild type, (2) F1 complementation plants, (3) hws-1 mutant, and (4) SALK_088349 KO line (hws-2). [See online article for color version of this figure.]

).

Insertional mutant lines from the SALK collection of these 18 candidate genes were examined and two individuals in a population of 50 plants from line SALK_088349 (located in the gene At3g61590) exhibited phenotypic characteristics reminiscent of hws-1. Further analysis of this line revealed that it contained two T-DNA insertions, arranged in opposing configurations, downstream from the ATG of the At3g61590 gene. These insertions were located 475 and 491 bp from the ATG (Fig. 5B).

PCR amplification of the At3g61590 genomic region of wild type and hws-1 DNA and restriction analyses of the amplified products with the high frequency cutting enzymes AluI, RsaI, and TaqI revealed subtle differences in banding patterns between the two genotypes (Fig. 5C). Sequencing of this region from hws-1 revealed that it contained a 28 bp deletion located 966 bp downstream of the translation start of the open reading frame (ORF) of At3g61590. The consequence of this deletion is to introduce a frame shift resulting in the introduction of a premature termination amber codon in place of an Ile residue and the predicted production of a truncated version of the At3g61590 protein (Fig. 5B).

To determine whether the hws-1 phenotype was a consequence of a mutation in the At3g61590 gene, a cross between the mutant and the SALK_088349 line (knockout [KO]) was performed. All progeny of this cross displayed hws-1 characteristics. To test that these had not arisen by a self-pollination event two plants were analyzed by PCR and reverse transcription (RT)-PCR. The PCR demonstrated that both a gene-specific product (originating from the hws-1 mutant) and an insertion product (originating from the KO) were amplified in each individual but only the former was present in the wild-type control (Fig. 5D). RT-PCR analysis of RNA extracted from various tissues of the SALK_088349 line and from two F1 plants (hws-1 × KO) showed no At3g61590 expression in the KO but the presence of a transcript in both progeny (Fig. 5E).

Proof that HWS is encoded by the At3g61590 gene was obtained by complementing the hws-1 mutant with a 3.513 kb fragment amplified from wild-type DNA containing 1,291 bp upstream of the promoter, plus the 5′ and 3′ untranslated regions (UTRs), and intron and exon of the At3g61590 gene. This segment proved to be of sufficient length to rescue fully the hws-1 mutant (Fig. 5F).

HWS Encodes a Putative F-Box Protein

The likely function of the HWS gene (At3g61590) is that it encodes an F-box protein. These proteins, as part of a SCF complex, are proposed to interact with a substrate leading to their degradation by the 26S proteasome (Ni et al., 2004). HWS encodes a protein of 412 amino acids. It has no introns within the ORF but has an intron of 532 bp within the 5′ UTR of the gene (Fig. 5B). The truncated version of the mutant protein, predicted to be generated in hws-1 plants, contains the intact F-box region. Information retrieved from the Web site PlantsUBQ, which is a functional genomics database for the Ubiquitin/26S proteasome proteolytic pathway in plants (http://plantsubq.genomics.purdue.edu/plantsubq/cgi-bin/detail.cgi?db=plantsubq&id=163137), indicates that the predicted F-box domain of HWS is located between amino acids 40 and 85. The software also proposes the existence of a transmembrane-spanning region between amino acids 120 and 140 and a low similarity (29.3%) to a Kelch_2 motif in the region between amino acids 290 and 338.

Outside the F-box region, the ORF of HWS shows only a low level of sequence similarity with other putative F-box proteins that have been annotated within the Arabidopsis genome. The Arabidopsis gene encoding a protein with highest sequence similarity (approximately 30%) to HWS is UFO. UFO is a protein that has been shown to be required for normal patterning and growth of the floral meristem (Samach et al., 1999).

Spatial and Temporal Expression of the HWS Gene

RT-PCR analysis of RNA isolated from wild-type plants revealed that the HWStranscript is expressed in many different tissues of the plant. Levels of expression were highest in buds and flowers, however, expression could also be detected in roots, leaves, stem, and siliques (Fig. 6

Figure 6.

Analysis of expression of the HWS gene by RT-PCR. RT-PCR of RNA extracted from Arabidopsis Col-0 wild-type tissues using HWS or ubiquitin (At4g05320) primers.

Figure 6.

Analysis of expression of the HWS gene by RT-PCR. RT-PCR of RNA extracted from Arabidopsis Col-0 wild-type tissues using HWS or ubiquitin (At4g05320) primers.

). A more detailed analysis of the spatial and temporal pattern of expression, with the aid of the GUSor green fluorescent protein(GFP) reporter genes fused to the HWSpromoter, showed that the gene is expressed at discrete sites within a range of tissues including the outer margins of cotyledons (Fig. 7A

Figure 7.

Analysis of expression from ProHWS:GUS and ProHWS:GFP transgenic lines. Cleared whole-mount GUS-stained tissue samples after 12 h from ProHWS:GUS plants at different stages of development (A–I) and confocal images from plant ProHWS:GFP (J–L). A, Two-day-old seedling. B, Young flowers (note the GUS expression in sepals including their vascular tissue). C, Close view of youngest floral buds. D, Young buds (note GUS expression becoming more discrete at the top of the silique as the flowers become older). E, Close view of the silique from a flower where petals have not emerged (note that GUS expression is spread along approximately one-third of the top of the silique). F, Separated petal (left) and sepal (right) material from a position 12 flower. G, A flower from position 9 (note how the GUS expression from the top of the silique becomes more localized at the stigma). H, An AZ from a flower of position 10. I, An AZ from a cauline leaf. J, A confocal image of developing seeds isolated from a mature silique. K, A confocal image of an emerging lateral root. L, A confocal image of a root cap. Bars = 300 μm (A, B, and D–K) and 75 μm (C and L).

Figure 7.

Analysis of expression from ProHWS:GUS and ProHWS:GFP transgenic lines. Cleared whole-mount GUS-stained tissue samples after 12 h from ProHWS:GUS plants at different stages of development (A–I) and confocal images from plant ProHWS:GFP (J–L). A, Two-day-old seedling. B, Young flowers (note the GUS expression in sepals including their vascular tissue). C, Close view of youngest floral buds. D, Young buds (note GUS expression becoming more discrete at the top of the silique as the flowers become older). E, Close view of the silique from a flower where petals have not emerged (note that GUS expression is spread along approximately one-third of the top of the silique). F, Separated petal (left) and sepal (right) material from a position 12 flower. G, A flower from position 9 (note how the GUS expression from the top of the silique becomes more localized at the stigma). H, An AZ from a flower of position 10. I, An AZ from a cauline leaf. J, A confocal image of developing seeds isolated from a mature silique. K, A confocal image of an emerging lateral root. L, A confocal image of a root cap. Bars = 300 μm (A, B, and D–K) and 75 μm (C and L).

), the sepals of young buds and flowers (Fig. 7, B–D), the stigmatic papillae and tip of the elongating silique (Fig. 7, E and G), the base and vascular tissue of petals and sepals (Fig. 7F), the anther filaments and pollen (Fig. 7G), the floral and cauline leaf AZs (Fig. 7, H and I), the testa of developing seeds (Fig. 7J), the vascular tissue of the primary root and emerging laterals (Fig. 7K), and lateral root cap (Fig. 7L). A detailed time course of GUS accumulation during flower and silique development revealed that intense HWSexpression could be detected at the site of floral organ abscission in position 8 flowers continuing up to the stage when desiccation of the pods took place (Fig. 3C).

Ectopic Expression of the HWS Gene Produces Smaller Seedlings

Transgenic plants were generated where the ORF from the HWS gene was expressed ectopically using a double 35S CaMV promoter. Homozygous plants from two lines (8.3, A23.3) were examined in detail as RT-PCR analysis had indicated that these lines most strongly expressed both HWS and the transgene (Supplemental Fig. S1) and contained only a single insertion (data not shown).

Line 8.3 exhibited a more severe phenotype than line A23.3 in all the experiments that were undertaken. A growth study, carried out 2 weeks after emergence, revealed that both ectopic expressing lines were substantially smaller than wild type (Fig. 8A

Figure 8.

Comparative phenotypic analysis of young plants. Two-week-old plants were analyzed and compared from: (1) Pro35S:HWS homozygous line 8.3, (2) hws-1, (3) Col-0 wild type, and (4) Pro35S:HWS homozygous line A23.3. A, A comparison of 2-week-old plants. B, Plants from A dissected to show size, shape, and number of leaves and cotyledons. Images of the remaining plantlets are on the far right. Bars = 5 mm. [See online article for color version of this figure.]

Figure 8.

Comparative phenotypic analysis of young plants. Two-week-old plants were analyzed and compared from: (1) Pro35S:HWS homozygous line 8.3, (2) hws-1, (3) Col-0 wild type, and (4) Pro35S:HWS homozygous line A23.3. A, A comparison of 2-week-old plants. B, Plants from A dissected to show size, shape, and number of leaves and cotyledons. Images of the remaining plantlets are on the far right. Bars = 5 mm. [See online article for color version of this figure.]

). However, hws-1plants at the same stage were larger than the control (Fig. 8A). Compared to the wild type, the rosette leaves of the overexpressing lines had shorter petioles, narrower and more rounded lamina with serrated borders, and displayed a greater degree of hyponastic bending (Fig. 8B).

Impact of Ectopic Expression of HWS on Flower Development

A comparison of flower development in wild type, hws-1, and Pro35S:HWSline 8.3 demonstrated that organ shedding took place at an earlier developmental stage in the overexpressing line (position 10) compared to the wild type (position 12). Flowers from line 8.3 also underwent sepal senescence prematurely, as evidenced by a visual decline in chlorophyll, compared to the wild type (Fig. 9

Figure 9.

Comparative phenotypic analysis of flowers. Flowers of 4-week-old plants were analyzed and compared. The flowers are identified from position 1 to 15, considering position 1 as the first flower where petals are visible to the eye, and in the hws-1 mutant a dry silique was included to show the presence of floral organs at that late stage of development. A, Col-0 wild type. B, hws-1 mutant; note the bigger stigmatic papillae in the flowers and wider floral bases. C, Pro35S:HWS line 8.3; note that abscission takes place earlier (position 10) than in the Col-0 wild type (position 12). Bars = 1 mm. [See online article for color version of this figure.]

Figure 9.

Comparative phenotypic analysis of flowers. Flowers of 4-week-old plants were analyzed and compared. The flowers are identified from position 1 to 15, considering position 1 as the first flower where petals are visible to the eye, and in the hws-1 mutant a dry silique was included to show the presence of floral organs at that late stage of development. A, Col-0 wild type. B, hws-1 mutant; note the bigger stigmatic papillae in the flowers and wider floral bases. C, Pro35S:HWS line 8.3; note that abscission takes place earlier (position 10) than in the Col-0 wild type (position 12). Bars = 1 mm. [See online article for color version of this figure.]

; Supplemental Fig. S2). Figure 9shows that hws-1had the longest, while the overexpressing line had the shortest, stigmatic papillae. A close-up view of flowers confirmed these observations (Supplemental Fig. S2).

Measurements of dissected sepals and petals from flowers at position 3 revealed that hws-1 had significantly longer and wider sepals and petals compared to the wild type or Pro35S:HWS A23.3 line. The floral bases of the two overexpressing lines were the narrowest (Fig. 9; Supplemental Fig. S3), while hws-1 was the widest compared to the wild type (see also Fig. 1, B and E).

Manipulation of HWS Expression Affects Root Growth and Seed Size

Seeds from hws-1, wild type, and the two overexpressing lines were germinated in germination medium (GM) or Murashige and Skoog media and root length was measured after a period of 2 weeks. The mutant exhibited the longest roots while both Pro35S:HWSlines 8.3 and A23.3 had significantly shorter roots than the wild type (Fig. 10A

Figure 10.

Comparative phenotypic analysis of root length and seed size. A, Twenty-six roots from 2-week-old seedlings grown in GM were photographed and their roots measured and statistically analyzed. Root length comparisons among Pro35S:HWS line 8.3, hws-1 mutant, Col-0 wild type, and Pro35S:HWS line A23.3. The significance was analyzed by ANOVA test. *, Different characters (x, y) indicate a significant difference in the mean at P < 0.05, n = 26. B, Seventy dry seeds from five independent plants were photographed, measured, and statistically analyzed. Seed length and width comparisons among Pro35S:HWS line 8.3, hws-1 mutant, Col-0 wild type, and Pro35S:HWS line A23.3. The significance of the differences was analyzed by ANOVA test. *, Different characters (p, q, r and x, y, z) indicate a significant difference in the mean at P < 0.05, n = 70.

Figure 10.

Comparative phenotypic analysis of root length and seed size. A, Twenty-six roots from 2-week-old seedlings grown in GM were photographed and their roots measured and statistically analyzed. Root length comparisons among Pro35S:HWS line 8.3, hws-1 mutant, Col-0 wild type, and Pro35S:HWS line A23.3. The significance was analyzed by ANOVA test. *, Different characters (x, y) indicate a significant difference in the mean at P < 0.05, n = 26. B, Seventy dry seeds from five independent plants were photographed, measured, and statistically analyzed. Seed length and width comparisons among Pro35S:HWS line 8.3, hws-1 mutant, Col-0 wild type, and Pro35S:HWS line A23.3. The significance of the differences was analyzed by ANOVA test. *, Different characters (p, q, r and x, y, z) indicate a significant difference in the mean at P < 0.05, n = 70.

).

The dimensions of mature seeds from the three different genotypes were analyzed. The hws-1 mutant was found to have statistically larger (both in length and width) seeds compared to the wild type while the overexpressing lines produced the smallest seeds (Fig. 10B).

DISCUSSION

We have described the isolation and characterization of a novel Arabidopsis mutant termed hws that fails to shed its floral organs. The mutated gene responsible for bringing about this phenotype (At3g61590) encodes a putative F-box protein. HWS is expressed throughout the plant but reporter gene analysis indicates that expression is restricted to specific tissues. Loss of HWS function results in an elevation of plant size while overexpression of the gene, using the 35S CaMV promoter, generates plants exhibiting a significant reduction in root and vegetative shoot growth. These observations are consistent with HWS playing a role in the regulation of organ development in Arabidopsis.

Phenotype of the hws Mutant

The hws-1 mutant was originally isolated as a consequence of an inability to shed its sepals, petals, and anther filaments. Indeed, these organs are retained throughout silique growth and development and remain at the base of the silique even after desiccation and dehiscence is complete. A close examination of the flowers has revealed that the mutation results in the fusion of the sepals along their basal margins. Ectopic expression of the microRNA miR164 results in the generation of flowers with similar characteristics (Mallory et al., 2004). This miRNA is thought to act by targeting for degradation the mRNA of the genes CUC1 and CUC2 (Laufs et al., 2004). In addition to exhibiting fused sepals, hws-1 has a prevalence to generate fused anthers, multivalved first-formed siliques, and fusion of the cauline leaf lamina to the inflorescence stem. These observations suggest that, like CUC1 and CUC2, the HWS gene might act to regulate lateral boundary development and that the degree of penetrance of some of its phenotypic characteristics could be due, in part, to redundancy between HWS and other peptides.

An examination of the early stages of development in wild-type and hws-1 plants indicates that floral morphology is initially indistinguishable, however, while sepal separation proceeds to the base of the bud in wild type this is terminated prematurely in hws-1 material. Thus the hws phenotype is not the consequence of postgenital fusion of the sepals but due to their failure to undergo complete separation.

To test whether differentiation of the floral AZs was taking place in hws-1 we crossed the gene marker ProPGAZAT:GUS into the mutant. PGAZAT (At2g41850) encodes a polygalacturonase that is expressed specifically within the AZ cells of Arabidopsis and has been proposed to play a role in middle lamella degradation (González-Carranza et al., 2002). The results show that hws-1 is not compromised in terms of AZ differentiation, however, the onset and duration of ProPGAZAT:GUS expression is delayed and reduced, respectively, in the mutant. These revelations, together with our SEM and light microscope observations, indicate that the nonshedding phenotype of hws-1 is not the consequence of a failure of cell separation to take place at the base of petals, anther filaments, and sepals. While the fusion of the sepal margins, encircling the separated petals and anthers, provides a structural barrier to preclude organ shedding in hws-1 our data indicate that the timing of cell separation in the mutant is delayed.

HWS Gene Encodes an F-Box Protein

Mapping and characterization of the hws-1 locus has revealed that the mutant phenotype is a consequence of a 28 bp deletion in the ORF of a gene (At3g61590) encoding a putative F-box protein. The 28 bp deletion in hws-1 introduces a premature translation termination codon, predicted to truncate HWS. The phenotype of hws-1 is indistinguishable from that of a null mutant (SALK_088349; hws-2) caused by the insertion of two T-DNAs into At3g61590, suggesting that the shortened HWS protein, if synthesized, is nonfunctional. Evidence to support the proposal that HWS functions as an F-box protein has come from the demonstration by Takahashi et al. (2004) that in a targeted yeast (Saccharomyces cerevisiae) two-hybrid screen At3g61590 strongly associates with a number of Arabidopsis Skp1-related proteins (ASKs). We have confirmed that HWS interacts with both ASK1 and ASK4 in an anther-specific yeast two-hybrid library (X. Zhang, Z.H. González-Carranza, and J.A. Roberts, unpublished data).

HWS contains a single intron located within the 5′ UTR region of the gene. A bioinformatic analysis of HWS indicates that in addition to having an F-box domain, the protein contains a predicted transmembrane sequence, and a Kelch-like repeat region. The truncated protein produced by the hws-1 mutant would lack an important element of the Kelch_2 motif and it has been proposed that this region, in other F-box proteins, might play a key role in the recognition of the substrate (Jarillo et al., 2001). It is possible therefore that the abbreviated version of HWS might have the capacity to bind ASKs but not the peptide targeted for degradation. Over 700 annotated genes within the genome of Arabidopsis have been classified, on the basis of sequence similarity, to be members of the F-box super family. The ORF of HWS shows only a low level of sequence similarity to other family members with the highest (approximately 30%) being to that encoded by the UFO gene. UFO has been shown to have a role in several aspects of flower development. Mutations in the UFO gene can bring about inappropriately fused floral organs (Levin and Meyerowitz, 1995) through its role in regulating patterning of primordial initiation. In addition, UFO is required for the specification of organ identity through its ability to promote the function of the type B class of floral homeotic genes (Durfee et al., 2003) and, by conditionally restoring UFO function, an additional role for the protein in regulating petal outgrowth has been identified (Laufs et al., 2003). There is no evidence that HWS plays a role in specifying organ identity as neither alleles of the hws mutant, that we have examined, show consistent evidence of abnormalities in floral organ identity. In addition, it is evident that HWS functions within vegetative tissues and that, if silenced, results in fusions of tissues such as the cauline leaf lamina to the stem of the inflorescence.

Expression of HWS

As the phenotypic characteristics of the hws-1 mutant were primarily restricted to the shoot and reproductive tissues it was a surprise to discover that the transcript of the HWS gene could be detected by RT-PCR at high levels throughout the plant. Reporter gene analysis, using either GUS or GFP, revealed that although expression is evident in many tissues HWS promoter activity is restricted to specific cell types or regions of an organ. In roots, the pattern of expression is limited to the vascular tissues and the cells that comprise the lateral root cap. In leaves and stems expression is rarely detected in the vasculature but is associated with the margins of cotyledons. Floral tissues strongly express HWS with GUS activity being detected throughout development in sepals, the distal end of the stigma, anther filaments, and pollen. Expression is also evident in the AZs of cauline leaves and floral organs. Aspects of the root and floral organ expression pattern are reminiscent of that exhibited by the F-box protein TIR1 (Gray et al., 1999) that has been shown to encode a receptor for indole-3-acetic acid (Kepinski and Leyser, 2005). Mutations in the TIR1 gene confer a reduced sensitivity to indole-3-acetic acid, however, the responses of hws-1 to auxin are indistinguishable from wild-type plants (Z.H. González-Carranza and J.A. Roberts, unpublished data) and while ectopic expressing lines of HWS exhibit reduced root growth, elongation of laterals root is not promoted.

GUS expression in ProHWS:GUS plants is intense at both the site of abscission of floral organs and cauline leaves. Expression precedes sepal, petal, and anther filament shedding and is maintained throughout silique development and maturation. This spatial and temporal pattern of expression is similar to that observed by genes that have been proposed to contribute to the process of cell separation (González-Carranza et al., 2002). Although anatomical analysis indicates that floral organ shedding is precluded by fusion of the sepals in hws plants, rather than as a consequence of a failure to undergo cell separation, a comparison between the mutant, overexpressing lines and wild-type plants demonstrates that HWS does, in addition, have a direct influence on the timing of abscission. A study of the mutant material crossed with the ProPGAZAT:GUS gene also supports the assertion that HWS is necessary for cell separation to proceed at the normal rate.

Role of HWS in Plant Development

Although the principal characteristic of hws-1 plants is their nonshedding phenotype, the isolation of a null allele (hws-2) of the gene has enabled us to dissect other consistent phenotypic features. A key difference from wild-type plants is that hws-1 plants grown under the same environmental conditions have more elongated leaves and larger seeds. Plants ectopically expressing HWS exhibit a reduced overall stature compared to wild type with the degree of reduction being associated with the intensity of the up-regulation of the gene. These observations indicate that the target for HWS degradation plays a key role in the maintenance of organ growth. Indeed the impact of losing HWS can be seen in individual cells such as the stigmatic papillae where elongation is substantially elevated in the mutant and reduced in the overexpressing lines. A role for UFO in regulating organ growth has been previously identified (Laufs et al., 2003) and a number of F-box proteins have been linked to the regulation of the cell cycle (Taylor et al., 2001). Like UFO, HWS may have more than one direct role or perhaps may target more than one protein for degradation, alternatively, the consequence of HWS on sepal fusion might be mediated through its effects on the lateral expansion of sepal primordia, resulting in the generation of overlapping margins and a failure in organ separation. Intriguingly, silencing of AUXIN RESPONSE FACTOR 2, a transcription factor that mediates gene expression in response to auxin, also results in delayed floral organ abscission (Ellis et al., 2005) and increased growth of aerial organs and seeds (Schruff et al., 2006). Further work is ongoing to identify the mechanism by which HWS exerts its effect on growth and ascertain whether this may be mediated through an auxin-signaling pathway.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Arabidopsis (Arabidopsis thaliana) ecotypes Col-0 and Ler, the hws-1 mutant, SALK lines for the 18 genes in the 56 kb segment that were obtained from the Nottingham Arabidopsis Stock Centre, diverse crosses, and the mapping population were grown in a glasshouse with a temperature of 23°C ± 2°C in plastic pots containing Levington M3 (The Scotts Company) compost and Vermiculite (2.0–5.0 mm, Sinclair) in a 3:1 ratio, respectively. To grow plants under sterile conditions, petri plates were prepared with 4.33 g L−1 of Murashige and Skoog basal salt mixture, pH 5.9 (Sigma; Murashige and Skoog, 1962), and 0.8% agar or GM (Sedbrook et al., 2002); seeds were sterilized in a solution containing 50% (v/v) sodium hypochlorite then rinsed with a solution containing 0.01% (v/v) Triton X-100 for 5 min followed by ethanol (70% v/v). Seeds were then rinsed five times in sterile water before being placed, using a pipette tip, on top of the Murashige and Skoog agar petri dishes. Plates were sealed with micropore tape (3 m) and maintained at 4°C for 72 h to stratify them and to facilitate uniform germination. Plates were then transferred to a growth room with a temperature of 23°C ± 1°C under 16 h light/8 h dark.

The position of the flower was determined from the first site where petals were visible. From that position all subsequent flowers were numbered.

Scanning Electron Microscopy

Inflorescence apices and flowers were taken from Col-0 (wild type) and hws-1 plants. Buds were staged in accord with Smyth et al. (1990) and flowers were harvested at inflorescence position 2, position 4, and position 14. The apex of the floral inflorescence was excised and placed directly into fixative (4% glutaraldehyde [w/v] in 0.5 m potassium phosphate buffer, pH 7.5) for 4 h prior to dehydration through a graded ethanol series and critical point drying in liquid CO2. Specimens were then mounted on steel stubs with double-sided tape and dissected to reveal the floral primorida. They were sputter coated with gold palladium before viewing. Flowers were viewed immediately without fixation or sputter coating at 20 kV accelerating voltage in variable pressure mode under low vacuum (70 Pa). All samples were viewed under a Hitachi S-3000 SEM. A minimum of five buds/flower were examined from each position.

Light Microscopy

Unopened and open flowers were collected from wild type and the hws-1 mutant when the plants were 1 month old. Young buds, mature buds, and flowers from positions 4, 6, 8, 12, 16, or 20 were fixed in 4% (v/v) paraformaldehyde: phosphate-buffered saline (PBS) buffer (1.3 mm NaCl, 0.03 m Na2HPO4, 0.03 m NaH2PO4, pH 7.2, 0.1% [v/v] Triton X-100, and 0.1% [v/v] Tween 20) overnight at 4°C. Tissues were washed with PBS at 4°C for 1 h, then brought to room temperature and washed with PBS for 1.5 h. The tissues were postfixed with 2% (w/v) OsO4 in PBS at room temperature for 3 h, then rinsed twice in PBS for 15 min and dehydrated with an ethanol series (30%, 50%, 70%, 90%, and 100% [v/v], twice, for 1 h in each). Specimens were processed with 100% ethanol/propylene oxide (1:1) for 20 min, washed three times with propylene oxide for 10 min, and propylene oxide/Spurrs resin (1:1; TAAB) for 1.5 h. Specimens were infiltrated with pure Spurrs resin at 4°C for 10 to 12 h. Individual samples were placed in appropriate flat-labeled silicone embedding molds filled with Spurrs resin and placed at 72°C for 10 to 12 h.

Longitudinal semithin sections (0.5 μm) were cut on a Reichert-Jung Ultracut ultramicrotome using glass knives. The glass knives were made from a glass bar (TAAB) on a Leica EM KMR2 knife maker machine. Sections were stained with Toluidine blue (TBO) 0.25% to 1% (w/v), mounted with DePex (Sigma), and observed under a Nikon Optiphot-2 microscope equipped with a Leica DFC320 camera using the IM50 Leica software (Leica Microsystems Imaging Solutions).

Phenotypic and Statistical Analysis

Six plants from the hws-1 mutant, wild type, and 35S CaMV ectopic expression lines 8.3 or A23.3 were grown under the same conditions and 25 flowers were dissected and analyzed under a Zeiss Stemi SV6 Stereo microscope. Organ fusion and other phenotypic characteristics were recorded either with a Kodak MDS290 digital camera attached to the microscope or with a Fujifilm FinePix A205S. Images were analyzed with Adobe photoshop software.

Seedlings of 2 weeks old were grown in Murashige and Skoog media or in soil. Rosette leaves were dissected, and pictures taken with a Fujifilm FinePix A205S. For measurements of roots (26 plants grown in Murashige and Skoog media or GM media), the sepals, petals (12 flowers from different plants at position 3), and seeds (70 seeds from five plants) pictures were taken and measurements were performed with the image processor and analyzer in Java software ImageJ Version 1.37h (Abramoff et al., 2004). Values were then used to perform statistical analysis and create graphics in Microsoft Excel 2002. Statistical analysis of variance (one-way ANOVA) using lsd and Duncan Analysis were performed using the software SPSS 14.0.1.

Mapping of the hws Gene

A mapping population was generated by crossing the hws-1 mutant in Col-0 background with the Ler ecotype and the F1 progeny was allowed to self. DNA was extracted, following manufacturer instructions (Qiagen, DNAeasy Plant Mini kit), from a small population of 33 F2hws-1 mutant plants and used to map the HWS locus to a region of 3.28 MB between markers SM148,6 and SM239_119,5 at the bottom of chromosome 3 using an AFLP strategy as described by Peters et al. (2004).

With a further analysis of 156 hws-1 mutant plants from the original F2 population, using a combination of CAPS and SSLP, the region was reduced to a 0.404 MB between the markers FUS6.2 and NGA6. From 600 F2hws-1 mutant plants, and the use of InDels that were identified from the Cereon Arabidopsis Polymorphism collection (Cereon Genomics; http://www.arabidopsis.org/Cereon/index.html), the region was further reduced to a segment of 56 kb between markers 470113 and 469675. This region contained 18 annotated genes (Fig. 5A). The InDel flanking primers designed for fine mapping hws-1 are summarized in Supplemental Table S1. PCRs were performed in a reaction of 20 μL following red taq (ABgene) manufacturer instructions; the PCR conditions for amplification were: 3 min at 94°C, 30 cycles of 94°C for 30 s, 55°C to 60°C for 30 s (depending on primer combination), 72°C for 30 s, and 7 min at 72°C, 4°C ∞. PCR products were run in a 3% low melting point agarose gel (MetaPhor Agarose, Cambrex).

Salk KO lines were identified for the 18 annotated genes within this region and grown in the glasshouse. Individuals from one of these KO lines (Salk_088349), documented to contain a T-DNA insertion in At3g61590, proved to exhibit a similar phenotype to the hws-1 mutant.

Primers from the region corresponding to the At3g61590 gene that amplified a genomic PCR segment of 1,271 bp are At361590forcDNA: 5′GCTCTTGAGAATGGAAGCAGAAAC 3′ and At3g61590Rev: 5′CAGACCCATTTGCTTCTTCATTGC 3′. PCR reactions were performed in a 50 μL reaction following red taq (ABgene) manufacturer instructions. Conditions for amplification were: 3 min at 94°C, 30 cycles of 94°C for 15 s, 61°C for 1 min, 72°C for 2 min, 7 min at 72°C, 4°C, and the PCR products were run in a 1% agarose gel. The digestion of PCR products was performed in 20 μL reactions containing: 500 ng of PCR product, 10× reaction buffer, 2 μg of bovine serum albumin, and 0.5 μL of each enzyme, incubated at 37°C (RsaI and AluI) or 65°C (TaqI) and run in a 3% agarose gel. The expected restriction sizes for the amplified PCR segment corresponding to the At3g61590 ORF genomic region are shown in Supplemental Table S2.

A plant from the Salk_088349 KO line was used to cross with the hws-1 line and PCR analyses were performed to identify the presence of the two T-DNA insertions and the genomic fragment from the hws-1 mutant from the two parental lines; information about primers is described previously. To test the T-DNA insertion the LBb1 of pBIN-pROK2 for SALK lines primer was used: 5′ GCGTGGACCGCTTGCTGCAACT 3′ (http://signal.salk.edu/tdnaprimers.2.html; Alonso et al., 2003). PCR conditions were the same as for the PCR amplification of the At3g61590 segment described earlier.

RT-PCR Analysis of Expression

Total RNA from roots, buds, flowers, rosette leaves, stem, young siliques, and old siliques from wild type, and a mix of tissues from progeny plants 1 and 2 from the cross between the hws-1 mutant with Salk_088349 line (hws-2) and from a mix of flowers from several developmental positions from F1

Sours: https://academic.oup.com/plphys/article/144/3/1370/6106743
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  • Hula leaf skirt designed with adjustable elastic waistband and with brightly-painted cloth flowers around.
  • Skirt total length: about 14.9inch/38cm; Waistline: from 29.5inch/75cm to 51.1inch/130cm for adult; from 23.6inch/60cm to 35.4inch/90cm for kids. Large and dense petals for the flower bracelets, headband and necklace.
  • The hula skirt is use of environmentally friendly high-quality silk cloth; Connect by Button, easy for put on or put off.
  • It is a good choice for Hawaiian Party, Luau Party, Children's Day, Dance Performance, Halloween,Birthday, and other different kinds of Dance Party.
  • Include one Hula leaf skirt, a pair of flower bracelets, one flower headband, and one flower necklace.
Product Dimensions ‏ : ‎14.9 x 0.7 x 13.5 inches (37.8 x 1.8 x 34.3 cm)
Department ‏ : ‎Unisex-adult
ASIN ‏ : ‎B07S4DZ5F8

Description

Still worried about having no Hawaiian accessory for tropical theme event? Now, we will recommend you a 5-in-1 Hawaii Tropical Leaf Hawaiian Hula Grass skirt set, which includes one leaf skirt, a pair of flower bracelets, one flower headband, and one flower necklace. The leaf skirt is decorated with some brightly-painted cloth flowers around, and equipped with an adjustable elastic waistband for comfortable to wear.

Specification
Material:Silk and cloth
Headband: 50cm,can stretch 100cm
Bracelet:20cm, 40cm stretchable
Neck ring:100cm,no stretch
Adult Leaf Skirt: Length 14.9inch/38cm; Tile Waist 29.5inch/75cm,waist stretch to 51.1inch/130cm
Kids Leaf Skirt: Length 14.9inch/38cm; Tile Waist 23.6inch/60cm,waist stretch to 35.4inch/90cm
Purpose:performances,activities,spoof,anniversary,games,etc.
Packaging:
1*leaf skirt+1*head ring+1*neck ring+2*bracelet

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Sours: https://janetrangi.com/zwwl4522485h5-adult-hawaiian-grass-skirt/lower-.html
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Sours: https://www.kyifi.org/skhula-skirts

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