THE ASSOCIATION OF DRUSE CRYSTALS WITH THE DEVELOPING STOMIUM OF CAPSICUM ANNUUM (SOLANACEAE) ANTHERS

Each of the two stomiums in the anther of Capsicum annuum (sweet pepper) consists of a single layer of cells immediately below the epidermis between two adjacent locules. Each stomium extends the entire length of the anther and splits open at pollen maturity. Many calcium oxalate druse crystals form within the vacuoles of the stomium cells in association with membrane complexes and paracrystalline bodies. These latter structures are reported here for the first time and each is considered to be a nucleation site for druse crystal formation. Prior to the appearance of membrane complexes and crystals within the vacuoles, plasmalemmasomes are visible next to the stomium cell walls and contain vesicles and fibrous material. We propose that these bodies carry wall materials, including calcium ions and possibly oxalate ions, into the vacuoles. Their presence coincides with crystal formation. Two other types of crystals occur in the connective tissue between stomiums and the single vascular strand. These crystals, along with those in the two stomiums, form at precise times during anther development. Contrary to the more numerous suggestions that crystals protect against predators or are metabolic waste products, we believe their formation aids in degradation and weakening of the cell walls between the locules and, thus, contributes to the release mechanism for the pollen.     

虎掌（天南星科）中草酸钙晶体的形态和分布

在光学显微镜下对虎掌（Pinellia pedatisecta）营养器官和繁殖器官中晶体的类型和分布进行了观察和分析,探讨晶体的功能与作用机制。结果表明：（1）虎掌各个器官中都发现有晶体,且晶体类型有针晶、簇晶、砂晶和柱晶4种形态,其中针晶最为常见。（2）虎掌叶中的晶体大多以针晶状分布在叶片上表皮下的叶肉中,少数分布在叶下表皮下的叶肉中,其次砂晶和星芒状簇晶也在叶中较常见,叶中也有少量的柱晶。（3）虎掌的块茎中分布有大量的针晶束,在输导组织附近还有一些大的簇晶;虎掌的不定根中分布有不整齐的针晶和排列不规则的针晶束以及少量大的簇晶。（4）虎掌的佛焰苞中分布有针晶、簇晶和砂晶,且在佛焰苞中的针晶主要分布于上、下表皮之下的叶肉中,砂晶多分布在佛焰苞的上、下表皮上。（5）虎掌的花药壁中分布有针晶束,其方向和花药壁表面垂直,而花粉囊中只有小的簇晶。（6）虎掌的果皮和种皮上分布有大量的针晶。根据晶体在酸中的溶解性,虎掌体内所有晶体的化学成分都为草酸钙。研究认为,虎掌各个器官中的各种草酸钙晶体对于保护虎掌免受食草动物取食具有重要的作用。     

On the presence of extracellular calcium oxalate crystals on the inflorescences of Araceae

This study presents a survey of the species of the Araceae where extracellular production of calcium oxalate crystals has been observed and discusses the patterns of production of the crystals in different genera. For all Araceae studied using SEM, the oxalate crystals exuding on the epidermal surface correspond to extended aggregate/druses or crystal sand and the oxalate crystals mixed with pollen correspond to raphides or styloids (prismatic crystals). The type of crystals associated with pollen varies among genera. However, the presence of crystals associated with pollen is a specific rather than a generic characteristic. Our results show that the presence of raphides mixed with pollen seems to be a widespread phenomenon in the aroid family.  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 146 , 181–190.     

Calcium crystals in the anther of Petunia: the existence and biological significance in the pollination process

Using an X-ray microanalysis system fitted with variable-pressure scanning electron microscopy, we noted that many calcium crystals accumulated under the stomium in the anther of Petunia. When the anther was dehisced and pollen grains were released from the stomata, the calcium crystals adhered to pollen grains and moved to the stigma together with pollen grains. In contrast, an X-ray microanalysis of the stigma surface before pollination detected no calcium emission on the stigma surface. Furthermore, pollen germination and pollen tube growth in medium without Ca occurred as in complete medium. However, after the pollen grains had been washed with abundant germination medium without calcium, pollen germination in the medium without Ca was inhibited. These results show that the calcium crystals dissolved in the aqueous drop under the exudate on the stigma and supplied calcium ions for pollen germination. In addition, calcium crystals were produced not only in the anther of Petunia but also in Nicotiana, suggesting that calcium crystals supply pollen grains with the calcium ions required for pollen germination and serve to improve reproduction efficiency in Solanaceae.     

OCCURRENCE,TYPE, AND LOCATION OF CALCIUM OXALATE CRYSTALS IN LEAVES AND STEMS OF 16 SPECIES OF POISONOUS PLANTS

Crystals in 16 species of poisonous plants growing naturally in Saudi Arabia were studied with light microscopy. Three types of crystals were observed: druses, prismatics, and crystal sand. Raphides and styloids were not observed in any of the species studied. Druses occur more frequently in the leaf midrib and in the cortex and pith of the stem. In contrast, crystal sand and prismatic crystals are rare and occur in the leaf, intercostal lamina, and in the vascular tissues. The preliminary results show the absence of the three types of calcium oxalate crystals in the stem and leaf of seven species: Ammi majus L., Anagallis arvensis L., Calotropis procera Ait., Citrullus colocynthis (L.) Schard, Euphorbia peplis L., Hyoscyamus muticus L., and Solarium nigrum L., and the presence of druses, prismatic crystals, and crystal sand either in the leaf and stem or in the leaves or stems of nine species: Anabasis articulata (Forssk.) Moq. in DC., Chenopodium album L., Convolvulus arvensis L., Datura stramonium L., Nerium oleander L., Ricinus communis L., Rumex nervosus Vahl., Pergularia tomentosa L., and Withania somnifera (L.) Dun. in DC. These observations indicate that there is no apparent relationship between the distribution of calcium oxalate crystals and the toxic organs of the plants, and supports the view that the presence of calcium oxalate crystals may not be related to plant toxicity.     

Developmentally regulated antigen associated with calcium crystals in tobacco anthers

We have found and characterized an antigen associated with crystal-containing cells in the stomium and connective tissue of the anthers of Nicotiana tabacum L. (tobacco). The antigen, defined by the monoclonal antibody NtF-8B1, localizes to subcellular regions surrounding the crystals. At the light-microscope level, the antigen is detectable just after the first appearance of crystals in the connective tissue of the anther, and at approximately the same time as the appearance of crystals in the stomium. The antigen is not detectable on a Western blot, and gave inconclusive results on a test of periodate sensitivity. It is not the crystals themselves, nor is the presence of the crystals required for antibody recognition. The antigen is sensitive to heat and protease treatment, indicating that it is a protein. The antigen is not tightly membrane-bound, in spite of its localization closely surrounding the crystals. Chemical tests indicate that the druse crystals in the stomium are calcium oxalate.Abbreviations ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate This research was supported by a National Science Foundation postdoctoral fellowship to B.L.H., by National Science Foundation grants DMB-87-15799 and to W.E.F. BSR-88-18035, and by U.S. Department of Agriculture grant GAM-89-01056. The authors thank Phillip T. Evans (Louisiana State University, Baton Rouge, USA), Wilma L. Lingle, Harry T. Horner, Jr. (Iowa State University), and A. Jack Fowler, Jr., for advice and helpful discussions.     

The Occurrence, Type and Location of Calcium Oxalate Crystals in the Leaves of Fourteen Species of Araceae

The occurrence, type and location of calcium oxalate crystalsin the leaves of 14 species belonging to the family Araceaewere studied by light microscopy. The Pizzolato test and theRubeanic acid-silver nitrate test, used to chemically identifyand locate the crystals in cross sections of laminae, showedthe presence of four types of crystals: druses, raphides, prismaticsand crystal sand. Styloids were not observed in any of the species.Crystals identified as calcium oxalate were observed in eachtissue layer of the leaf blade, druses occurring more frequentlyin the palisade mesophyll layers, raphides more often in thespongy mesophyll. Prismatics were sparse, occurring in the mesophyllof only two species. Specialized spindle-shaped crystal idioblasts,located in the spongy mesophyll in all cases, were observedin seven of the 14 aroids. Crystal sand and variations in crystalforms were most frequently observed to be calcium compoundsother than calcium oxalate. Crystals, calcium oxalate, idioblasts, Araceae     

红豆草中含晶细胞的形态学研究

红豆草（Ｏｎｏｂｒｙｃｈｉｃｖｉｃｉａｅｆｏｌｉａｓｃｏｐ．）植株的所有器官中都分布有含晶细胞,其结晶的类型主要为棱晶,此外还有砂晶。在营养器官中,含棱晶的细胞主要分布在维管组织之中或外围。横切面上,棱晶则几至几十块纵列成行存在,且常伴生于韧皮纤维旁,但每块棱晶各有一分室隔开;在茎的表皮下偶有与大型粘液细胞伴生的砂晶。花萼的表皮中偶有棱晶,花瓣表皮及雄蕊药隔中有砂晶;子房壁内表皮下一层细胞逐渐发育成含棱晶的连续细胞,同时子房维管组织中也形成大量棱晶。分析表明,结晶成分为草酸钙。     

The Mechanics of the Grass Flower: Anther Dehiscence and Pollen Shedding in Maize

In this paper on the flower mechanics of the grasses, the openingmechanism of the maize anther is studied. Both the septum betweeneach two locules and the stomium of these porate-dehiscing anthersappear to be opened due to lysis of the middle lamellae of theircells. Additional mechanical force of the expanding pollen mightbe necessary to completely dissociate the parenchyma cells ofthe septum. A number of hours before anthesis the anther isstructurally able to dehisce. At anthesis the dehydrating endotheciumcells bend the locule walls bordering the pore in outward direction.Presumably evaporation is not the only cause for this dehydration. Poaceae; Zea mays ; flower; anther; dehiscence; endothecium; pollen     

Mechanism of Anther Dehiscence in Rice (Oryza sativa L.)

This paper presents a new explanation of the mechanism of antherdehiscence in rice during the period from floret opening topollen dispersal. The theca dehisced on the stomium in the apicalpart and the anther wall in the basal part of the large locule.Comparison of the anther dehiscence process under various airhumidity conditions showed that the process, until the splittingat the apical and basal parts, was a moisture-requiring processwhereas the widening of the splits in both parts was a desiccatoryprocess. Observation of the anther transverse section, revealedthe marked development of the U-shaped thick cell wall in theendothecium adjacent to these two splits. From these observations,the anther dehiscence mechanism may be explained as follows.At the time of anthesis, pollen grains swell rapidly in responseto the floret opening and cause the theca to bulge, rupturingthe septum. The pollen pressure combined with the inward bendingof the locule walls adjacent to the stomium causes splittingof the stomium in the apical part of the theca. At the sametime, the septum rupture extends to the bottom of the largelocule supported by the pollen pressure. After these processes,the locule walls adjacent to both splits straighten probablydue to their water loss. This straightening widens the splitsand the swollen pollen grains overflow from the widened splits.Copyright1999 Annals of Botany Company Anther dehiscence, Oryza sativa L., pollen grain swelling, rice, septum, stomium, theca.     

A novel cell ablation strategy blocks tobacco anther dehiscence.

We utilized a new cell ablation strategy to ablate specific anther cell types involved in the dehiscence process. The tobacco TA56 gene promoter is active within the circular cell cluster, stomium, and connective regions of the anther at different developmental stages. We introduced a cytotoxic TA56/barnase gene into tobacco plants together with three different anticytotoxic barstar genes. The anticytotoxic barstar genes were used to protect subsets of anther cell types from the cytotoxic effects of the TA56/barnase gene. The chimeric barstar genes were fused with (1) the tobacco TP12 gene promoter that is active at high levels in most anther cell types; (2) the soybean lectin gene promoter that is active earlier in the connective, and at lower levels in the circular cell cluster and stomium, than is the TA56 promoter; and (3) the tobacco TA20 gene promoter that is active at high levels in most anther cell types but has a different developmental profile than does the TP12 promoter. Normal anther development and dehiscence occurred in plants containing the TA56/barnase and TP12/barstar genes, indicating that barstar protects diverse anther cell types from the cytotoxic effects of barnase. Anthers containing the TA56/barnase and lectin/barstar genes also developed normally but failed to dehisce because of extensive ablation of the circular cell cluster, stomium, and contiguous connective regions. Anthers containing the TA56/barnase and TA20/barstar genes failed to dehisce as well. However, only the stomium region was ablated in these anthers. The connective, circular cell cluster, and adjacent wall regions were protected from ablation by the formation of barnase/barstar complexes. We conclude that anther dehiscence at flower opening depends on the presence of a functional stomium region and that chimeric barnase and barstar genes containing promoters that are active in several overlapping cell types can be used for targeted cell ablation experiments.     

Rice (Oryza sativa L.) cultivars tolerant to high temperature at flowering: anther characteristics

We examined the relationship between morphological characteristics of anthers and fertility in japonica rice cultivars subjected to high temperature (37.5(26 degrees C day/night) at flowering. Percentage fertility was negatively correlated with the number of cell layers that separated the anther locule from the lacuna that formed between the septum and the stomium. The cell layers consisted of the remaining septum and degraded tapetum, and serve to keep the adjacent two locules closed. Anther dehiscence therefore requires the rupture of the cell layers. We conclude that the tight closure of the locules by the cell layers delayed locule opening, and decreased fertility at high temperatures.     

The final split: the regulation of anther dehiscence

Controlling male fertility is an important goal for plant reproduction and selective breeding. Hybrid vigour results in superior growth rates and increased yields of hybrids compared with inbred lines; however, hybrid generation is costly and time consuming. A better understanding of anther development and pollen release will provide effective mechanisms for the control of male fertility and for hybrid generation. Male sterility is associated not only with the lack of viable pollen, but also with the failure of pollen release. In such instances a failure of anther dehiscence has the advantage that viable pollen is produced, which can be used for subsequent rescue of fertility. Anther dehiscence is a multistage process involving localized cellular differentiation and degeneration, combined with changes to the structure and water status of the anther to facilitate complete opening and pollen release. After microspore release the anther endothecium undergoes expansion and deposition of ligno-cellulosic secondary thickening. The septum separating the two locules is then enzymatically lysed and undergoes a programmed cell death-like breakdown. The stomium subsequently splits as a consequence of the stresses associated with pollen swelling and anther dehydration. The physical constraints imposed by the thickening in the endothecium limit expansion, placing additional stress on the anther, so as it dehydrates it opens and the pollen is released. Jasmonic acid has been shown to be a critical signal for dehiscence, although other hormones, particularly auxin, are also involved. The key regulators and physical constraints of anther dehiscence are discussed.     

Receptor-like protein kinase 2 (RPK 2) is a novel factor controlling anther development in Arabidopsis thaliana

Receptor-like kinases (RLK) comprise a large gene family within the Arabidopsis genome and play important roles in plant growth and development as well as in hormone and stress responses. Here we report that a leucine-rich repeat receptor-like kinase (LRR-RLK), RECEPTOR-LIKE PROTEIN KINASE2 (RPK2), is a key regulator of anther development in Arabidopsis. Two RPK2 T-DNA insertional mutants (rpk2-1 and rpk2-2) displayed enhanced shoot growth and male sterility due to defects in anther dehiscence and pollen maturation. The rpk2 anthers only developed three cell layers surrounding the male gametophyte: the middle layer was not differentiated from inner secondary parietal cells. Pollen mother cells in rpk2 anthers could undergo meiosis, but subsequent differentiation of microspores was inhibited by tapetum hypertrophy, with most resulting pollen grains exhibiting highly aggregated morphologies. The presence of tetrads and microspores in individual anthers was observed during microspore formation, indicating that the developmental homeostasis of rpk2 anther locules was disrupted. Anther locules were finally crushed without stomium breakage, a phenomenon that was possibly caused by inadequate thickening and lignification of the endothecium. Microarray analyses revealed that many genes encoding metabolic enzymes, including those involved in cell wall metabolism and lignin biosynthesis, were downregulated throughout anther development in rpk2 mutants. RPK2 mRNA was abundant in the tapetum of wild-type anthers during microspore maturation. These results suggest that RPK2 controls tapetal cell fate by triggering subsequent tapetum degradation, and that mutating RPK2 impairs normal pollen maturation and anther dehiscence due to disruption of key metabolic pathways.     

Effect of Photoperiod on Calcium Distribution in Photoperiod-sensitive Cytoplasmic Male-sterile Wheat During Anther Development

Potassium antimontate was used to locate Ca2+ in fertile and sterile anthers of photoperiod-sensitive cytoplasmic male-sterile wheat (Triticum aestivum L.).The quantity and distribution of calcium precipitates change in microspore and pollen during microsporogenesis and pollen development.The calcium precipitates gradually accumulated on the surface of tetraspore or pollen until pollen mature. They were abundant on the surface of the trinucleate pollen,but not in the cytoplasm.Calcium was transported in anther wall and intervening connective tissue via symplast and apoplast system,meeting the need of microspore and pollen development.Abortive pollens were observed in the anthers developed under long daylight exposure. Abundant calcium precipitates were distributed along the edge of the degradatived pollen plasma and in the locular matrix,but little in the anther wall and intervening connective tissue of the early abortive pollen.Calcium precipitates accumulated on the surface of pollen was less than fertile pollen,but abundant calcium appear in the epiderms,endotheciums,middle layers of anther wall and cells of connective tissue.The results indicate that abnormalities in the distribution of calcium correlate with the failure of pollen development in photoperiod-sensitive cytoplasmic male-sterile wheat.     

Crystal diversity and macropatterns in leaves of Oleaceae

Oleaceae leaves surveyed from herbarium specimens of 240 species from 23 genera were rehydrated, bleached, processed into xylol, mounted unstained, and viewed microscopically between crossed polarizers. Occurrence of five crystal types and two variants (tiny acicular crystals and sphaerites) within one family is unusual. Number of crystal types within a single species was one (108 spp.), two (53 spp.), three (51 spp.), four (15 spp.), and five (6 spp.). Seven species lacked crystals. The tiny acicular crystal variant was most common (167 spp.), followed by prisms (67 spp.), raphides (65 spp.), druses (61 spp.), sphaerites variant (50 spp.), styloids (36 spp.), and crystal sand (21 spp.). Epidermal crystals were common (155 spp.), with epidermal crystals clustering at base of trichomes in 21 species. Jasminum was exceptional in having mostly druses and almost no crystals around vascular bundles. Most Oleaceae crystals are tiny, usually about 5 μm in length, except for larger styloids and raphides.     

The diversity of anther structures and dehiscence patterns among Hamamelididae

HUFFORD, L. D. & ENDRKSS, P. K., 1989. The diversity of anther structures and dehiscence patterns among Hamamelididae. This survey of anther structures and dehiscence patterns focuses on the range of diversity among extant Hamamelididae. The definition and structure of the anther stomium are considered in detail to provide a basis for characterizing dehiscence patterns. We are concerned particularly with the structural basis and distribution of so-called valvate dehiscence, which we define here as occurring only in those anthers that possess stomial bifurcations or markedly eccentric stomia. Valvate dehiscence is restricted to Trochodendrales and Hamamelidales among Hamamelididae, although some Hamamelidaceae possess only linear, not markedly eccentric stomia that lead to longitudinal dehiscence patterns. Anther forms are somewhat variable and do not appear to be highly correlated with stomial patterns, although stomial bifurcations occur most frequently in anthers with broad, thick connectives that extend for the full length (or nearly so) of the thecae. Valvate dehiscence usually occurs in anthers in which the pollen sacs are embedded in bulky superficial tissues. An evolutionarily secondary extension of the stomium around the thecal shoulders seems to have occurred in taxa with a nonextensive connective and may facilitate a broader anther opening in cases of longitudinal dehiscence. An endothecial-like connective hypodermis is a notable characteristic among examined 'Lower Hamamelididae' (except Disanthus) and is also present in Daphnipfiyllum and Eucommia. We hypothesize that this specialized connective hypodermis facilitates a broader opening of the anther.     

Subepidermal idioblasts and crystal macropattern in leaves of Ticodendron (Ticodendraceae)

Previously unknown leaf anatomy and foliar crystal macropattern are described for Ticodendron incognitum Gómez-Laurito and Gómez P. (monotypic eudicot family Ticodendraceae of a Fagales subclade with Betulaceae and Casuarinaceae). Leaf samples of five herbarium specimens were rehydrated, bleached, dehydrated to 100% ethanol, then xylol, mounted in Permount and viewed using polarizing microscopy. Large adaxial and abaxial hypodermal idioblasts occur. Numerous small druses populate palisade mesophyll, and fewer, larger druses populate the spongy mesophyll; this is the opposite of druse distribution in Fagaceae. Druses dominate major veins, many with visible cores, and some prisms occur. Minor veins exhibit isolated groups of 1–8 druses and rare prisms, but most terminal veins lack crystals. Many druses are misshapen or with epitactic crystals; several unusual variants of druses and prisms are common. Hypodermal idioblasts are an apomorphy for Ticodendraceae, but oil and resin cells occur in Myricaceae and Rhoipteleaceae of Fagales. Crystal forms and distribution differ from known Fagalean macropatterns.