Young Anthers Research Articles

The Golgi-localized transporter OsPML3 is involved in manganese homeostasis and complex N-glycan synthesis in rice.

Manganese (Mn) is involved in many biochemical pathways as an enzyme cofactor, and is essential for maintaining metabolic processes in various plant cell compartments. Here, we determined the function of a rice (Oryza sativa) Mn transporter, PHOTOSYNTHESIS-AFFECTED MUTANT 71-LIKE 3 (OsPML3), belonging to the UNCHARACTERIZED PROTEIN FAMILY 0016 (UPF0016), in regulating Mn homeostasis and late-stage Golgi N-glycosylation. OsPML3 was highly expressed in rapidly developing tissues such as young leaves, root caps, lateral root primordia, and young anthers. Heterologous expression of OsPML3 restored the growth of Mn uptake-defective yeast strain Δsmf1 under Mn-limited conditions. Sub-cellular localization analysis revealed that OsPML3 localizes in the Golgi apparatus. At the vegetative stage, we observed necrotic root tips and lateral root primordia, and chlorotic young leaves in OsPML3 knockout lines under Mn-deficient conditions. Knocking out OsPML3 reduced the Mn content in the young leaves but did not affect the older leaves. Additionally, knocking out OsPML3 reduced the deposition of cell wall polysaccharides and the content of Lea (Lewis A structure)-containing N-glycan in roots and young leaves. OsPML3 knockout lines grown in the paddy field had reduced pollen fertility. Moreover, we found that the Lewis A structure was reduced in young anthers of OsPML3 knockout lines. Collectively, our results indicate that OsPML3 maintains Mn homeostasis in the Golgi apparatus of the rapidly developing rice tissues, and regulates the deposition of cell wall polysaccharides and late-stage Golgi N-glycosylation, especially biosynthesis of the Lewis A structure.

Meiotic behavior and diploidy in Schinus terebinthifolia (Anacardiaceae)

Abstract The genus Schinus (Anacardiaceae) comprises 30 species found in Peru, Bolivia, Chile, Paraguay, Uruguay, Argentina and Brazil. Schinus terebinthifolia (Brazilian pepper tree) is socioeconomically important in the Brazilian state of Espírito Santo. Dried fruit is used as a pepper-like spice and natural populations are exploited. Studies on the meiotic behavior and pollen stainability from an ecological and evolutionary point of view are essential, since they can promote future sustainable management strategies for this species. The objective was to evaluate meiotic behavior and characteristics of pollen grains of individuals from a natural population of S. terebinthifolia localized in the municipality of Vitória, Espírito Santo state, Brazil. Young anthers were squashed in 1% acetocarmine dye to obtain the pollen mother cells. The pollen stainability with 1% acetocarmine, Lugol’s iodine solution and Sudan IV and pollen shape were used to assess the pollen potential viability, cytochemistry and morphology. All individuals had a gametic number of n = 7, a regular meiotic behavior and so the somatic number is 2n = 2x = 14 chromosomes. Median pollen stainability was 94%. Pollen grains are dispersed as monads, 3 - colporate and subspheroidal. The exine has striate-reticulate ornamentation. Pollen grains are starch positive and lipid negative. This is the first report of the occurrence of diploidy for the species. The high pollen stainability is the result of the occurrence of regular meiosis. The presence of starch as pollen reserve can increase the resistance of pollen grains to hostile environments in this species.

CHROMOSOME COUNT ON YOUNG ANTHER OF BANANA MALE BUD USING EZYMATIC MACERATION AND DAPI STAINING IN SLIDE PREPARATION

Chromosome counting is the basis in describing the chromosomes number of organism that might useful for genetic study and classification. In banana studies, the root tip with a combination of non-fluorescent staining methods such as carmine or orcein and squash is the most common material for chromosome counting. In this study, we presented the usefulness of young anther of banana male bud with enzymatic maceration method for cell spreading and 4,6-diamino-2-phenyl-indole (DAPI) for staining agent to get a satisfying chromosomes image at metaphase for mitotic study of diploid and tetraploid bananas. The principle of this study is fixation using ethanol:acetic acid (3:1), enzymatic digestion, maceration and staining using DAPI. Our result showed that this method can provide well spread cells with intensely contrast of chromosomes images that satisfying for chromosome counting.

Calcium distribution during anther development in Impatiens balsamina

The subcellular localization of loosely bound calcium in developing anthers of Impatiens balsamina (rose balzam) was investigated using potassium antimonate to precipitate the ions. At the sporogenous stage, few calcium precipitates were found in the young anthers. During meiosis of the microspore mother cell, the amount of calcium precipitates increased both inside these cells and in their cell walls. After meiosis, the resulting microspores accumulated abundant calcium precipitates in their cytoplasm and vacuoles. The microspores underwent mitosis to produce a bicellular pollen grain, in which the large vacuoles disintegrated, and the calcium precipitates of the vegetative cell were again localized in the cytoplasm. The amount of precipitates decreased during pollen maturity and finally disappeared from the inside of the pollen grains and accumulated on the pollen walls.

Morphological Studies of Insect-Induced Galls in Flower and Fruit of Alstonia scholaris (L.) R. Br

Morphological changes in insect-induced galls in flowers and fruits of Alstonia scholaris (L.) R. Br. (Apocynaceae) were recorded. The insect Pauropsylla tuberculata Crawford (Homoptera: Psyllidae) penetrates the thalamus of young flowers inducing hypertrophy and hyperplasia in the parenchymatous tissue resulting in the formations of green galls of various sizes and shapes. The reproductive organs, stamens, and pistil of galled flowers are sterile. Pollen sterility was associated with abnormal behaviour of endothecium and the tapetum. In young anthers of severely infested flowers, the sporogenous tissue and tapetum degenerate. In anthers of some infested flowers, the callose wall surrounding pollen mother cells was thick although tapetal cells showed normal signs of degeneration. In anthers of a few infested flowers, the microspores were formed but they degenerated along with tapetal cells. Anthers of some diseased flowers contained thick-walled vacuolated and sterile microspores surrounded by degenerated tapetum layer and radially elongated endothecial cells which lacked the usual fibrous thickenings. The size of gynoecium and the size and number of ovules were reduced in galled flowers. Ovules failed to differentiate and represented by a globular undifferentiated mass of cells. A large number of green, conical galls are formed on fruits by hypertrophy and hyperplasia in the parenchymatous pericarp. Size of severely infested fruits was reduced, and they became thick, circular and seedless. As a consequence, sexual reproduction of the host tree was completely inhibited.

Structural Organization and Functional Activity of the Orthologous TaGLW7 Genes in Bread Wheat (Triticum aestivum L.)

It is reported that GLW7 encoding the transcription factor OsSPL13, positively regulates grain size and shape in rice. We have limited knowledge about its orthologs in wheat. Here, based on the rice OsGLW7 we isolated and identified the TaGLW7 gene in wheat, characterized its nucleotide and protein structures, predicted the cis-elements of its promoter, analyzed its expression patterns. The orthologs in barley (HvGLW7), Brachypodium (BdGLW7), wild emmer (TtGLW7), Aegilops tauschii (AtGLW7) were also used for comparative analysis. As predicated, TaGLW7, HvGLW7, TtGLW7, and AtGLW7 were mapped onto group 2 chromosomes in the respective species. Multiple alignments indicated GLW7 possesses two exons and one intron in the analyzed species. GLW7 contains a conserved domain SBP and two neighboring low complexity regions. GLW7 was highly expressed in spike organs including wheat young spikes, barley inflorescence, and rice anthers. Additionally, biotic stress significantly down-regulated GLW7 in wheat and barley. Significant correlations between the expression patterns of predicted transcription factor ABF2 and TaGLW7 were detected. In conclusion, the conserved structure and expression of GLW7 among the investigated species and the predicted transcription factors significantly related to GLW7 are helpful for further manipulating GLW7 and uncovering its roles in plants.

Morphological studies and meiotic chromosome analysis of Epimedium elatum (Morr & Decne) - Rare endemic medicinal plant of Northwestern Himalayas in India.

<p><em>Epimedium elatum</em> (Berberidaceace) is a rare endemic medicinal herb of Northwestern Himalayas in India. Recent ethnopharmacological reports have demonstrated its traditional medicinal use against various bone related diseases in the Kashmir Himalayas. It owes its pharmaceutical importance due to high concentration of flavonoid glycosides like Epimedin A, B, C and Icariin which are known mainly for aphrodisiac, antiosteoporosis, anticancer, antioxidant, antiaging, antifatigue and antiviral activities. It is a neglected medicinal plant in Northwestern Himalayan region and may fall in the list of endangered species due to continuous anthropogenic pressures in its native habitats. In this study, we investigated distributional and altitudinal range of this prized species from twenty diverse eco-geographical zones of Kashmir Himalayas for the first time. We also report here its diversity in morphological attributes both in wild and captive cultivation. The species has a very small population size in most of the surveyed habitats with no natural protection. Under cultivation it showed increased plant height (63.09±4.9cm), more number of leaves (95.53±11cm) and flowers (160.76±20cm), indicating importance of high altitude medicinal garden for its immediate <em>ex situ</em> conservation. Further, the acetocarmine staining and squashing of young anthers confirmed it as a diploid species (2n=12) like other <em>Epimedium</em> species. Chromosome number and meiotic abnormalities are also reported for the first time in the species. Finally, constant anthropogenic pressures in Northwestern Himalayas demand immediate <em>in situ</em> and <em>ex situ</em> conservation programmes for <em>E.elatum</em>.<strong></strong></p>

Anther glands in Mimosoideae (Leguminosae) are emergences with a conserved meristematic origin

The six distinct gland types described in Mimosoideae anthers vary from simple projections to robust and vascularized structures. The origin of these glands is attributed to an extension of connective. However, except for an illustrative recording of some developmental stages, nothing is known about the origin and gland development, which prevents their correct classification. This study aimed to elucidate the ontogeny of four of the six gland types detected in the anthers of Mimosoideae. The intention was to understand whether the morphological diversity results from different ontogenetic processes. For this, flower buds of various sizes of four Mimosoideae species with different types of glands were collected: Stryphnodendron adstringens (Piptadenia subtype Piptadenia), Tetrapleura tetraptera (Piptadenia subtype Adenanthera), Adenanthera pavonina (Piptadenia subtype Entada), and Pentaclethra macroloba (Pentaclethra type). The samples were processed for surface (scanning electron microscopy) and anatomical analyses (light microscopy). Despite their morphological differences, the glands share a common meristematic origin. They arise from simultaneous divisions in the first three layers of meristematic cells that cover the apical portion of the young anther. After the formation of the protuberance, the developing glands undergo different specialized processes that include (1) elongation of the epidermal cells in Piptadenia subtype Adenanthera and Piptadenia subtype Entada, (2) degradation of central gland cells in Piptadenia subtype Entada, (3) change from a rounded to a triangular shape in the Pentaclethra type, and (4) cell differentiation into a procambial meristem and subsequently into vascular tissue in the Pentaclethra type, resulting in the diversity encountered in the group. Their origin from protodermal and fundamental meristem cells characterizes the anther glands as emergences. Our data indicate a conserved meristematic origin of glands, favoring the hypothesis of homology of these structures and supporting the monophyly of the Mimosoideae clade.

Dynamics of Polysaccharides and Neutral Lipids during Anther Development in Castor (Ricinus communis)

Anthers contain starch and neutral lipids, which have key roles in microspore ontogeny and gametophyte development. In this study, we observed the dynamic changes in starch and neutral lipids in the anther developmental processes of castor (Ricinus communis) by cytochemical methods. Starch grains and neutral lipids presented a regular dynamic distribution during anther development. In young anthers, some neutral lipids accumulated in sporogenous cells, whereas neutral lipids disappeared with microspore growth. At the late microspore stage, starch grains began to accumulate in microspores, and the starch content of bicellular pollen significantly increased after microspore mitosis. At anthesis, starch grains and neutral lipids accumulated in the mature pollen grains. Visible changes occurred in anther wall cells. The epidermis, middle layer, and tapetum were degenerated, and only a single layer of endothecium remained at anthesis. The dynamic variation of starch grains and neutral lipids in tapetal cells was consistent with the changes in microspores and pollen during anther development. All these findings demonstrated that tapetal cells directly interacted with the developing gametophytes. The tapetal cells play an important role in supplying nutritional substances for microspore absorption. Moreover, the endothecium protects the pollen and contributes to anther dehiscence. The results of this study provide a foundation for the further research on sexual reproduction in angiosperms.

Characterization and Functional Analysis of Transcription Factor LoMYB80 Related to Anther Development in Lily (Lilium Oriental Hybrids)

Anther development is very precise and complicated procedure. In this study, the gene LoMYB80 was isolated from lily (Lilium Oriental Hybrids cv. cultivar ‘Siberia’) anther. Multiple alignment and phylogenetic tree analysis showed that LoMYB80 belongs to the R2R3 MYB family. Furthermore, the protein of LoMYB80 shares a 44 amino acid sequence immediately adjacent to the R3 domain with other MYB80s. Subcellular localization showed that LoMYB80 was predominantly localized in the nucleus. The transcriptional activation experiment revealed that LoMYB80 belongs to the transcription activation factors and its transactivation activity depends on the last 70 amino acids located at the end of the C-terminal region. Real-time quantitative PCR analysis showed that LoMYB80 gene expression was significantly higher in anther than in other organs; the highest level was in anthers of 10-cm flower buds and then was in anthers of 4-cm buds. The relative expression in microspores/pollen of 10-cm buds was much higher than the whole anther in the same stage and also much higher than the microspore/pollen of 8- and 9-cm buds. The gene was mainly expressed in young anthers at the development stages from 6 to 10 by analyzing transgenic Arabidopsis lines which were transformed with LoMYB80 promoter::GUS reporter gene constructs. Complementation experiments revealed that transgenic ms188 mutant lines could produce normal pollen grains and display partial fertility. These results indicated that LoMYB80 was a regulator of lily anther development and pollen formation.

A novel lily anther-specific gene encodes adhesin-like proteins associated with exine formation during anther development

The anther-specific gene LLA1271 isolated from lily (Lilium longiflorum Thunb.) anthers is novel and exists in two forms. The protein encoded by LLA1271 may represent an adhesin-like protein first found in higher plants. The protein contains a typical N-terminal signal peptide followed by a highly conserved repeat domain. The LLA1271 gene is temporally expressed at the phase of microspore development. RNA blot and RNA in situ hybridization analyses demonstrated that the gene was expressed both in the tapetum and in the microspore. The gene is endo- and exogenously induced by gibberellin. Studies with the gibberellin biosynthesis inhibitor uniconazole and an inhibitor of ethylene activity, 2,5-norbornadien (NBD), revealed that LLA1271 is negatively regulated by ethylene, and a cross-talk of regulation between gibberellin and ethylene occurs in young anthers. The treatment with NBD caused the tapetum to become densely cytoplasmic and highly polarized, whereas uniconazole arrested tapetal development in a state close to that of a tapetum without treatment. The LLA1271 protein is heat stable and heterogeneous. An immunoblot of separated protein fractions of the anther revealed that the LLA1271 protein was detected in protein fraction of the microspore released from the cell wall by treatment with either 0.5% or 2% Triton X-100. Ectopic expression of LLA1271 resulted in impaired stamen and low pollen germination. Scanning electron microscopy of TAP::LLA1271 pollen showed distorted exine formation and patterning. The LLA1271 protein once synthesized in both the tapetum and microspore is secreted and deposited on the surface of microspores, moderately affecting exine formation and patterning.

The metabolic incorporation of label from myoinositol-2-3H by the growing young anther of Lilium lorlgiflorum

Fine structural autoradiography involving the incorporation of myo-inositol-2-<sup>3</sup>H (MI-2-<sup>3</sup>H) unto lily anther during development has been used to explore the metabolic processes of microspore development and the interation between the devlaping microspore and degenerating tapetal cells.

A Morphological and Histological Characterization of Male Flower in Chestnut (<em>Castanea</em>) Cultivar 'Yanshanzaofeng'

Chinese chestnut (Castanea mollissima Blume.) is a widely distributed fruit tree and well known for its ecological and economic value. In order to evaluate obstacles to male reproductive in the C. mollissima, a morphological and histological characterization of male flower of chestnut cultivar 'Yanshanzaofeng' were examined by paraffin section technique and scanning electron microscopy. The results showed that male catkins with floral primordia were formed in the buds of one-year olds shoots in later April. Later, a protoderm, ground meristem and a procambium had differentiated in young anthers. Each young anther soon developed to four microsporangia. The anther wall layers developed completely by mid-May and consisted of one-cell-layered epidermis, one-cell-layered endothecium, two or three middle layers and one-cell-layered tapetum. The tapetum was of glandular type. Microspore mother cells underwent meiosis through simultaneous cytokinesis in later May and gave rise to tetrads of microspores, which were tetrahedrally arranged. Mature pollens contained two cells with three germ pores. Anthers were dehiscent and pollen grains shed by early June. Based our results, we did not find the abnormal male flower in the C. molissma cv 'yanshanzaofeng', indicating that male gametes were fertile and thus was considered as pollenizers.

Ultrastructural study of pollen and anther development in Luehea divaricata (Malvaceae, Grewioideae) and its systematic implications: Role of tapetal transfer cells, orbicules and male germ unit

Developmental processes of microsporogenesis and microgametogenesis in Luehea divaricata (Malvaceae, Grewioideae) were analyzed with transmission electron microscopy. The species studied has perfect flowers. The young anthers are bithecal and tetrasporangiate; microspore mother cells undergo simultaneous meiosis, forming tetrads with a tetrahedral arrangement. The development of the anther wall conforms to the basic type and the tapetum is secretory. The results highlight the presence of multinucleate tapetal cells, which acquire ultrastructural features characteristic of transfer cells at the young pollen grain stage, and which are associated with the presence of orbicules. Tapetal transfer cells have not been previously investigated in detail for other species of angiosperms; their function during pollen development is discussed. The present work is the first contribution to the knowledge of ultrastructural pollen development in the genus Luehea, as well as in the subfamily Grewioideae.

Anther structure and pollen development in Melicoccus lepidopetalus (Sapindaceae): An evolutionary approach to dioecy in the family

Anther and pollen development in staminate and pistillate flowers of dioecious Melicoccus lepidopetalus (Sapindaceae) were examined by light and electron microscopy. Young anthers are similar in both types of flowers; they consist of epidermis, endothecium, two to four middle layers and a secretory tapetum. The microspore tetrads are tetrahedral. The mature anther in staminate flowers presents compressed epidermal cells and endothecium cells with fibrillar thickenings. A single locule is formed in the theca by dissolution of the septum and pollen grains are shed at two-celled stage. The mature anthers of pistillate flowers differ anatomically from those of staminate flowers. The epidermis is not compressed, the endothecium does not develop fibrillar thickenings, middle layers and tapetum are generally persisting, and the stomium is nonfunctional. Microspore degeneration begins after meiosis of microspore mother cells. At anthesis, uninucleate microspores and pollen grains with vegetative and generative nuclei with no cytokinesis are observed. Some pollen walls display an abnormal exine deposition, whereas others show a well formed exine, although both are devoid of intine. These results suggest that in the evolution towards unisexuality, the developmental differences of anther wall tissues and pollen grains between pistillate and staminate flowers might become more pronounced in a derived condition, such as dioecy.

Regulation of the Arabidopsis anther transcriptome by DYT1 for pollen development

Several genes encoding transcription factors have been shown to be essential for male fertility in plants, suggesting that transcriptional regulation is a major mechanism controlling anther development in Arabidopsis. DYSFUNCTIONAL TAPETUM 1 (DYT1), a putative bHLH transcription factor, plays a critical role in regulating tapetum function and pollen development. Here, we compare the transcriptomes of young anthers of wild-type and the dyt1 mutant, demonstrating that DYT1 is upstream of at least 22 genes encoding transcription factors and regulates the expression of a large number of genes, including genes involved in specific metabolic pathways. We also show that DYT1 can bind to DNA in a sequence-specific manner in vitro, and induction of DYT1 activity in vivo activated the expression of the downstream transcription factor genes MYB35 and MS1. We generated DYT1-SRDX transgenic plants whose fertility was dramatically reduced, implying that DYT1 probably acts as a transcriptional activator. Furthermore, we used yeast two-hybrid assays to show that DYT1 forms homodimers and heterodimers with other bHLH transcription factors. Our results demonstrate the important role of DYT1 in regulating anther transcriptome and function, and supporting normal pollen development.

Floral ontogeny of Sinojackia xylocarpa (Styracaceae), with special reference to the development of the androecium

Using scanning electron microscopy, we studied the floral ontogeny of Sinojackia xylocarpa. There are 6–7 (–8) sepals. Sepal initiation is staggered; adaxial sepals arise later than abaxial and lateral ones. There are (5–) 6–7 (–8) petals, initiated simultaneously. Petals alternate with the sepals, and occasionally there are two petals instead of one between two sepals. The (10–) 12–14 (–16) stamens are initiated centripetally in two sets (whorls). These floral organ numbers deviate from those of the otherwise mostly pentamerous family Styracaceae. The ovary consists of three (rarely four) locules. In each of the locules, two rows of ovules are differentiated basipetally. Placentation is axile with (5–) 6 (–7) ovules in each locule. Ovules are unitegmic and are ascending with the micropyle directed downwards. Intra-ovarian trichomes are present as in other representatives of Styracaceae and seem to be an apomorphic character of the family as they are absent in the closely related Symplocaceae and Diapensiaceae. Various levels of organ union occur in anthetic S. xylocarpa. The calyx is synsepalous and the ovary syncarpous. Possibly, the basal connation of petals and stamens is postgenital (and not congenital), but this needs further study. The outward curvature of the young anthers of the inner stamen whorl superficially simulates an obdiplostemonous androecium. However, the sequence of stamen initiation shows a diplostemonous pattern.

Calcium Distribution in Developing Anthers of Lettuce (Lactuca sativa)

Potassium antimonite was used to locate calcium in the anthers of lettuce (Lactuca sativa). There are no calcium precipitates in young anthers. After meiosis of microspore mother cells, calcium precipitates first appear in the tapetal cells, from which some small secretive vesicles containing many calcium precipitates are secreted into the locule. At a late stage of the microspore, tapetal cells completely degenerate and their protoplasts move into the locule with many calcium precipitates. The calcium precipitates increase in the early microspores, and in the exine. When the microspores form some small vacuoles containing calcium precipitates, and those vacuoles then fuse to form a large one, the calcium precipitates evidently decrease. The large vacuole of bicellular pollen grain discomposes and calcium precipitates again appear in the cytoplasm and then decrease. When the pollen matures, most calcium precipitates are located in its exine with only a few in the cytoplasm.

Characterization of Raphanus sativus Pentatricopeptide Repeat Proteins Encoded by the Fertility Restorer Locus for Ogura Cytoplasmic Male Sterility

Cytoplasmic male sterility is a maternally inherited trait in higher plants that prevents the production of functional pollen. Ogura cytoplasmic male sterility in radish (Raphanus sativus) is regulated by the orf138 mitochondrial locus. Male fertility can be restored when orf138 accumulation is suppressed by the nuclear Rfo locus, which consists of three genes putatively encoding highly similar pentatricopeptide repeat proteins (PPR-A, -B, and -C). We produced transgenic rapeseed (Brassica napus) plants separately expressing PPR-A and PPR-B and demonstrated that both encoded proteins accumulated preferentially in the anthers of young flower buds. Immunodetection of ORF138 showed that, unlike PPR-B, PPR-A had no effect on the synthesis of the sterility protein. Moreover, immunolocalization experiments indicated that complete elimination of ORF138 from the tapetum of anthers correlated with the restoration of fertility. Thus, the primary role of PPR-B in restoring fertility is to inhibit ORF138 synthesis in the tapetum of young anthers. In situ hybridization experiments confirmed, at the cellular level, that PPR-B has no effect on the accumulation of orf138 mRNA. Lastly, immunoprecipitation experiments demonstrated that PPR-B, but not PPR-A, is associated with the orf138 RNA in vivo, linking restoration activity with the ability to directly or indirectly interact with the orf138 RNA. Together, our data support a role for PPR-B in the translational regulation of orf138 mRNA.

Expression and regulation of two novel anther-specific genes in Lilium longiflorum

Two stage-specific genes have been isolated from a subtractive cDNA library constructed from developing anthers of lily (Lilium longiflorum). The proteins encoded by the two genes have a strong hydrophobic region at the N-terminus, indicating the presence of a signal peptide. The deduced LLA-67 is a new type of small cysteine-rich protein whose sequence exhibits four consecutive CX(3)CX(6-10) repeats that could form signal-receiving finger motifs, while the deduced LLA-115 protein shows significant similarities to a rice unknown protein, and putative cell wall proteins of Medicago truncatula and Arabidopsis. The transcripts of LLA-67 and LLA-115 were anther specific and differentially detected at the phase of microspore development. In situ hybridization with antisense riboprobes of the two genes in the anther showed strong signals localized to the tapetal layer of the anther wall. The LLA-67 mRNA was also detected in the microspore at the phase of microspore development but the LLA-115 mRNA was not. The LLA-115 gene can be exogenously induced by gibberellin (GA), whereas the LLA-67 gene cannot be induced. Studies with the GA biosynthesis inhibitor uniconazole and an inhibitor of ethylene activity, 2,5-norbornadien (NBD), revealed that the two genes were negatively regulated by ethylene and a cross-talk between GA and ethylene was involved in the regulation of the two genes occurring in young anthers. The treatment of NBD caused the tapetum to become densely cytoplasmic and highly polarized, whereas uniconazole arrested tapetal development to a status close to that of control. DNA blots of lily genomic DNA indicated that the two genes were encoded by a small gene family. The different actions of hormones on gene expression and the possible function of the gene products in young anthers are discussed.