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A tumor suppressor homolog, AtPTEN1, is essential for pollen development in Arabidopsis.

Although it is well known that Tyr phosphatases play a critical role in signal transduction in animal cells, little is understood of the functional significance of Tyr phosphatases in higher plants. Here, we describe the functional analysis of an Arabidopsis gene (AtPTEN1) that encodes a Tyr phosphatase closely related to PTEN, a tumor suppressor in animals. The recombinant AtPTEN1 protein, like its homologs in animals, is an active phosphatase that dephosphorylates phosphotyrosine and phosphatidylinositol substrates. RNA gel blot analysis and examination of promoter-reporter constructs in transgenic Arabidopsis plants revealed that the AtPTEN1 gene is expressed exclusively in pollen grains during the late stage of development. Suppression of AtPTEN1 gene expression by RNA interference caused pollen cell death after mitosis. We conclude that AtPTEN1 is a pollen-specific phosphatase and is essential for pollen development.

Amino Acid Sequence↗

A beta-galactosidase-like gene is expressed during tobacco pollen development.

cDNA clone (TP5) with significant homology to ss-galactosidases has been isolated from a mature tobacco pollen cDNA library by differential screening. The predicted protein of 715 aa shows high levels of homology to plant beta-galactosidases expressed during fruit ripening and senescence. Northern analysis shows that the TP5 transcript is expressed exclusively in developing anthers and mature pollen. The transcript is present at very low levels at meiosis and increases dramatically, late in microspore development after mitosis suggesting that the primary role for the protein is during pollen tube growth. beta-galactosidase activity, measured by scanning densitometry of histochemically stained tobacco microspores, is first detectable in the early to mid-vacuolate stage, and reaches a peak at microspore mitosis, thereafter decreasing as the microspores reach maturity. Southern analysis indicates that the TP5 gene is present in two copies, probably corresponding to the two ancestral genomes of N. tabacum.

Amino Acid Sequence↗

Rapid assessment of microspore and pollen development stage in wheat and maize using DAPI and membrane permeabilization.

The use of the DNA-specific fluorochrome DAPI has been extended to stage assessment of fresh pollen in wheat and maize. Membrane permeabilization by Triton X-100 incorporated in the staining solution allows access of the fluorochrome to nuclear DNA. At all stages of gametophytic development, the nuclei can be sharply visualized. Starch does not interfere with the fluorochrome so that it is possible to study the second pollen grain mitosis and sperm differentiation. With its rapidity and reliability, this technique represents an efficient tool for routine staging or investigation of the nuclear status of the pollen grains.

Cell Membrane Permeability↗

The appearance of male gamete-specific histones gH2B and gH3 during pollen development in Lilium longiflorum.

The male gametic (generative) nucleus within the nearly mature pollen of Lilium longiflorum contains specific variants of histone H2B and H3, which have been designated gH2B and gH3, respectively. Using specific antibodies raised against gH2B and gH3, we examined in detail the temporal and spatial aspects of the appearance of these histone variants during male gametogenesis. Neither gH2B nor gH3 was detected in microsporocytes during meiotic division or in microspores before microspore mitosis. However, both gH2B and gH3 were abundantly present in mid bicellular pollen and these histones continued to be detectable in germinated pollen tubes after pollination. Furthermore, immunocytochemistry revealed that these proteins were present not only in generative nuclei during the maturation process, but also in the two sperm nuclei formed by division of the generative nucleus. By contrast, these proteins were not found in the vegetative nuclei. It appears, therefore, that both gH2B and gH3 are specific to male gametic (generative and sperm) nuclei in L. longiflorum. The results also suggest that these male gamete-specific histones might be newly synthesized in the progenitor of sperm cells for the differentiation of male gametes, as occurs in spermatogenesis in animals.

Cell Differentiation↗

The Arabidopsis thaliana ACT4/ACT12 actin gene subclass is strongly expressed throughout pollen development.

Plants contain complex actin gene families composed of several diverse and ancient subclasses of genes. One Arabidopsis actin gene subclass represented by the ACT4 and ACT12 genes has been isolated and characterized. Both actin genes have typical plant actin gene structures, including three small introns interrupting the coding region and an intron within the mRNA leader. Their encoded proteins differ from each other in only one amino acid, whereas they differ in 3-10% of their amino acids from the other five Arabidopsis actin subclasses. They also share a few small blocks of DNA sequence homology in the 5' flanking region near their TATA boxs, but not in their introns, 3' flanking regions, or degenerate positions within codons. Southern analysis with gene-specific probes from 5' flanking sequences showed that both were single copy genes in the genome. Both RNA gel blot analysis with 3' gene-specific probes and reverse transcriptase-mediated polymerase chain reactions (RT-PCR) with gene-specific primers detected low levels of ACT4 and ACT12 mRNAs in flowers and very high levels in pollen. The RT-PCR detected very low levels of these mRNAs in the vegetative organs. The 5' region from both genes, including the promoter region, TATA box, the sequence for the mRNA leader and its intron, and the first 19 actin codons, was fused to a beta-glucuronidase (GUS) reporter gene. Expression of the GUS fusions were examined histochemically in 40 independent transgenic Arabidopsis plants. Expression of the ACT4/GUS fusion was restricted to young vascular tissues, tapetum, and developing and mature pollen. Similar expression patterns in these tissues and cell types were observed for ACT12/GUS fusion, yet unlike ACT4, ACT12 was also strongly expressed in the root cap and in a ring of pericycle tissues during lateral root initiation and early development. The unique expression patterns of the ACT4/ACT12 actin gene subclass are discussed in light of recent data on the other expressed members of the Arabidopsis actin gene family.

Actins↗

A gene showing sequence similarity to pectin esterase is specifically expressed in developing pollen of Brassica napus. Sequences in its 5' flanking region are conserved in other pollen-specific promoters.

Differential screening of a Brassica napus genomic library led to the isolation of the clone named Bp 19 containing a gene which is highly expressed during microspore development. The accumulation of Bp19 mRNA starts in uninucleate microspores, increases during development reaching a peak in the late stages but declines considerably in mature pollen. The nucleotide sequence of the entire coding region and of extended portions of the 5' and 3' flanking regions was determined. Several homologous cDNA clones were also isolated and sequenced. The Bp 19 gene contains a single intron of 137 bp and gives origin to a mRNA of ca. 1.9 kb which codes for a polypeptide of 584 amino acids. Bp 19 protein has an estimated molecular weight of 63 kilodaltons and has a highly hydrophobic amino terminal region which shows features of a signal peptide. The carboxy half of the Bp 19 protein, starting at amino acid 269, has striking sequence similarity to the pectin esterases of tomato and of the plant pathogen Erwinia chrysanthemi. Four short domains are extremely well conserved in all the three proteins and therefore could represent catalytic sites responsible for enzyme activity. Comparison of the 5' flanking region of the Bp 19 gene with the sequence of other pollen-specific promoters revealed the presence of several conserved regions. These short promoter sequences could correspond to regulatory elements responsible for pollen-specific gene expression.

Amino Acid Sequence↗

[Pollen development in the anthers of several cereal strains and hybrids during cultivation in vitro].

In order to induce androgenesis in vitro anthers of some cereals were cultivated. The highest number of proembryos was obtained in the hybrid Triticale in F3 generation on Blayder's medium supplemented with 3 and 12% sucrose. Proembryos represented multi-nuclear and multicellular formations which stagnated at the globular stage of development. Origin of roots from calluses was not accompanied by formation of buds. There was no formation of embryos from pollen grains in case of lines Triticum aestivum and Secale cereale.

Edible Grain↗

The DELAYED ABAXIAL TRICHOMES Helitron has dual functions in vegetative and pollen development in Arabidopsis thaliana.

Transposons drive genetic diversity and evolution by altering the genomic landscape over time. Here, we describe DELAYED ABAXIAL TRICHOMES (DAB), a Helitron/RC transposable element in Arabidopsis thaliana that has a role in vegetative phase change and gametogenesis. A genome-wide association study (GWAS) for the timing of abaxial trichome development (an adult leaf trait) in A. thaliana revealed a conserved haplotype of polymorphisms within DAB that delays abaxial trichome production. CRISPR-Cas9-induced deletions of DAB are gametophytic pollen-lethal, indicating that this locus is also required for pollen production. DAB produces 24-nucleotide siRNAs with sequence complementarity to genes involved in embryogenesis, gametogenesis, and seed development. DAB also impacts the expression of ARGONAUTE genes, genes involved in RNA-directed DNA methylation (RdDM), as well as genes in several key genetic pathways. This global effect on gene expression suggests that DAB may have functions beyond those identified in this study.

Arabidopsis↗

Expression of antisense SnRK1 protein kinase sequence causes abnormal pollen development and male sterility in transgenic barley.

A chimaeric gene was constructed comprising a wheat high molecular weight glutenin subunit gene promoter, a 304-bp sucrose non-fermenting-1-related (SnRK1) protein kinase sequence in the antisense orientation, and the cauliflower mosaic virus 35S RNA gene terminator. Transgenic barley plants containing the antisense SnRK1 chimaeric gene were produced by particle bombardment of barley immature embryos with the aim of obtaining plants expressing the antisense SnRK1 sequence in the seeds. Despite the fact that the promoter was expected to be active only in seeds, two independent transgenic lines were found to fail to transmit the transgene to the T1 generation. These T0 plants had matured and died before this was discovered, but subsequently four other independent transgenic lines were found to be affected in the same way. Cytological analysis of the pollen grains in these lines showed that about 50% were normal but the rest had arrested at the binucleate stage of development, were small, pear-shaped, contained little or no starch and were non-functional. The presence of antisense SnRK1 transcripts was detected in the anthers of the four lines analyzed and a ubiquitin promoter/UidA (Gus) gene, one of the marker genes codelivered with the antisense gene, was found to be expressed only in the abnormal pollen. Expression analyses confirmed that SnRK1 is expressed in barley anthers and that expression of one class of SnRK1 transcripts (SnRK1b) was reduced in the abnormal lines. All of the abnormal lines showed approximately 50% seed set, and none of the transgenes were detected in the T1 generation.

Cell Differentiation↗

Control of petal and pollen development by the plant cyclin-dependent kinase inhibitor ICK1 in transgenic Brassica plants.

The cyclin-dependent protein kinases (CDKs) have a central role in cell cycle regulation and can be inhibited by the binding of small protein CDK inhibitors. The first plant CDK inhibitor gene ICK1 was previously identified in Arabidopsis thaliana. In comparison to known animal CDK inhibitors, ICK1 protein exhibits unique structural and functional properties. The expression of ICK1 directed by the constitutive CaMV 35S promoter was shown to inhibit cell division and plant growth. The aim of this study was to determine the effects of ICK1 overexpression on particular organs and cells. ICK1 was expressed in specific tissues or cells of Brassica napus L. plants using two tissue-specific promoters, Arabidopsis AP3 and Brassica Bgp1. Transgenic AP3-ICK1 plants were morphologically normal except for some modified flowers either without petals or with petals of reduced size. Surprisingly, petals of novel shapes such as tubular petals were also observed, indicating a profound effect of cell division inhibition on morphogenesis. The cell size in the smaller modified petals was similar to that in control petals, suggesting that the reduction of petal size is mainly due to the reduction of cell numbers and that the inhibition of cell division does not necessarily lead to an increase in cell size. Transgenic Bgp1-ICK1 plants were normal morphologically; however, dramatic decreases in seed production were observed in some plants. In those plants, the ability of pollen to germinate and pollen nuclear number were affected. These results are discussed in relation to the cell cycle and plant development.

Arabidopsis↗

Isolation and expression during pollen development of a tobacco cDNA clone encoding a protein kinase homologous to shaggy/glycogen synthase kinase-3.

We have isolated a tobacco (Nicotiana tabacum L.) cDNA clone encoding a putative serine/threonine protein kinase, which shows highest homology to previously described families of alfalfa and Arabidopsis protein kinases and to their homologues rat glycogen synthase kinase-3 and Drosophila shaggy kinases. Northern experiments showed that NtK-4 is expressed in all sporophytic tobacco tissues tested, as well as in gametophytic and embryogenic pollen.

Amino Acid Sequence↗

Molecular characterization of one of the maize polygalacturonase gene family members which are expressed during late pollen development.

A gene exhibiting homology to the polygalacturonases of several species, including tomato and Oenothera, has been shown by RNA dot-blot analysis and in situ hybridization experiments to be expressed post-first microspore mitosis in maize. A 2.87 kbp section of the promoter fused to E. coli beta-glucuronidase (uidA) coding sequence conferred the correct spatial and temporal expression in transgenic tobacco plants. However, low levels of expression were detected in other tissues, and in particular in the tissues surrounding the vascular branch points of leaf nodes. The maize polygalacturonase gene is one member of a highly conserved gene family. The lack of detectable expression in sporophytic tissues and the isolation of a number of related cDNAs from maize suggests that all expressed members of this family show the same spatial and temporal regulation.

Base Sequence↗

A Brassica napus gene family which shows sequence similarity to ascorbate oxidase is expressed in developing pollen. Molecular characterization and analysis of promoter activity in transgenic tobacco plants.

The genomic clone named Bp10 contains a member of a small pollen-specific gene family of B. napus. The expression of the Bp10 gene family is maximal in early binucleate microspores and declines considerably in mature trinucleate pollen. Homologues of the Bp10 genes are expressed in the pollen of other plant species. The pollen-specific expression of the gene contained in the genomic clone was confirmed in tobacco plants transformed with a chimeric Bp10 promoter/GUS construct. A promoter fragment of 396 bp is sufficient to direct a strong and correct spatial and temporal expression in transgenic plants. The Bp10 gene family codes for proteins of 62 kDa showing approximately 30% sequence identify to cucumber and pumpkin ascorbate oxidases (AAOs). However, the AAO active centres are not conserved in the Bp10 products, suggesting an evolutionary relationship but a different enzymatic activity for these proteins. Expression of a recombinant Bp10 protein in E. coli inhibits bacterial growth on minimal medium, suggesting the production of an enzymatically active polypeptide in bacteria. No AAO activity could be correlated with the expression of the recombinant protein. Moreover, substances affecting AAO activity do not appear to influence the inhibitory activity of the protein produced in bacteria. However, as indicated by the rescue of bacterial growth in the presence of sodium bicarbonate or gaseous CO2, the Bp10 protein activity could be modulated by CO2 levels.

Amino Acid Sequence↗