Huiling Xu

Isolation and developmental expression of Bcp1, an anther-specific cDNA clone in Brassica campestris.

Differential screening of a mature Brassica campestris pollen cDNA library has identified five cDNA clones that represent transcripts expressed exclusively, or at elevated levels, in pollen. We show here that the expression of one of these, clone Bcp1, is tissue specific and temporally regulated. The gene is activated during microspore development, as detected by in situ hybridization. Expression is enhanced at the time of pollen maturation and during pollen germination. In situ hybridization has also shown that Bcp 1 is activated in the tapetal cells in early anther development and continues to be expressed until tapetal dissolution. Homologous transcripts are present in pollen of other taxa of Brassicaceae including Arabidopsis, but not in pollen of any other families tested.

Identification of a Ca2+ Binding Protein as a New Bermuda Grass Pollen Allergen Cyn d 7: IgE Cross-Reactivity with Oilseed Rape Pollen Allergen Bra r 1

cDNA clones encoding two isoforms of an allergen from pollen of Bermuda grass (Cynodon dactylon) have been isolated using IgE from allergic patients. Homologous transcripts are present in pollen of 15 other grasses tested. This allergen, tentatively designated as Cyn d 7, contains two calcium binding domains and shows significant sequence similarity with other Ca2+ binding pollen allergens, namely Bet v 4 from birch and Bra r 1 from oilseed rape. Approximately 10% of allergic sera tested showed IgE reactivity to this allergen. IgE cross-reactivity was observed between this allergen and Bra r 1 of oilseed rape. IgE reactivity of this allergen requires protein-bound Ca2+. Using IgE affinity-purified from the recombinant allergen to probe Western blots of pollen extracts Cyn d 7 has been identified as a 12 kDA protein.

A cDNA clone encoding an IgE-binding protein from Brassica anther has significant sequence similarity to Ca2+-binding proteins

Thirteen cDNA clones encoding IgE-binding proteins were isolated from expression libraries of anthers of Brassica rapa L. and B. napus L. using serum IgE from a patient who was specifically allergic to Brassica pollen. These clones were divided into two groups, I and II, based on the sequence similarity. All the group I cDNAs predicted the same protein of 79 amino acids, while the group II predicted a protein of 83 amino acids with microheterogeneity. Both of the deduced amino acid sequences contained two regions with sequence similarity to Ca2+-binding sites of Ca2+-binding proteins such as calmodulin. However flanking sequences were distinct from that of calmodulin or other Ca2+-binding proteins. RNA-gel blot analysis showed the genes of group I and II were preferentially expressed in anthers at the later developmental stage and in mature pollen. The recombinant proteins produced in Escherichia coli was recognized in immunoblot analysis by the IgE of a Brassica pollen allergic patient, but not by the IgE of a non-allergic patient. The cDNA clones reported here, therefore, represent pollen allergens of Brassica species.

Cloning, expression and immunological characterization of Ory s 1, the major allergen of rice pollen.

We have isolated and characterized a cDNA clone, Ory s 1, encoding a group-1 allergen of rice pollen. The Ory s 1 protein shows significant sequence identity to the major allergen of rye-grass pollen, Lol p 1. RNA gel blot analysis shows that the Ory s 1 gene is expressed in mature anthers, but not in vegetative or other floral tissues tested. Southern blot analysis indicates that this clone represents a member of a small gene family in rice. Western blot analyses of total rice pollen proteins with the group-1 allergen-specific monoclonal 3A2 and IgE antibodies from grass pollen-allergic patients, revealed the presence of cross-reactive antigenic and allergenic epitopes in Ory s 1.

Isolation and characterization of a flowering plant male gametic cell-specific promoter1

Flowering plant male gametic cell‐specific gene expression has been reported recently but the regulatory elements controlling specificity of such genes expressed in generative cell and sperm cells have not been identified and studied. Here, we report the 0.8 kb promoter sequence upstream of the start of the transcription site of the generative cell‐specific gene, LGC1, sufficient to regulate the expression of reporter genes in a cell‐specific manner. In addition, the diphtheria toxin A‐chain‐ (DT‐A)‐coding region under the control of the LGC1 promoter sequence confirmed unequivocally the lack of LGC1 expression in vegetative tissues. Transgenic tobacco plants carrying the LGC1‐DT/A construct showed normal phenotype except for anthers of these plants that contained sterile and aborted pollen. Truncation and internal deletion analysis of the LGC1 promoter identified −242 bp as the minimal sequence necessary for male gametic cell‐specific expression. In addition, a regulatory sequence required for determining generative cell‐specific expression of LGC1 was identified. Deletion of this regulatory sequence led to loss of the generative cell specificity resulting in activation of this promoter in other tissues where it is normally repressed. Therefore, male gametic cell specificity of the LGC1 gene seems to be regulated by factors that suppress its activation in other plant cells. This is the first report of a male gametic cell‐specific promoter, hence can be used as a novel tool in molecular analyses and experimental manipulation of flowering plant spermatogenesis and fertilization.

Haploid and diploid expression of a Brassica campestris anther-specific gene promoter in Arabidopsis and tobacco

The anther-specific cDNA clone Bcp1 from Brassica campestris is expressed in both the haploid pollen and diploid tapetum, as shown by in situ hybridization. We have isolated Bgpl, a genomic clone homologous to Bcpl. The coding region and extensive 5′ flanking sequences of Bgp1 have been sequenced, and the coding region shows 88% identity with Bcp1. RNA gel blot analysis confirmed the expression of Bgp1-specific transcripts in B. campestris pollen. A 767 by 5′ DNA fragment was fused to the reporter gene β-glucuronidase (gus) and introduced into both Arabidopsis thaliana and Nicotiana tabacum by transformation. This 5′ fragment directed high-level expression in the pollen and tapetum of transgenic Arabidopsis. In transgenic tobacco however, the same construct was expressed only in pollen. A series of 5′ deletion constructs has been created and used to transform A. thaliana to analyse the 5′ region of Bgp1. The results indicate that Bgp1 expression in the tapetum and pollen of Arabidopsis requires the presence of different 5′ DNA sequences.

Developmental expression of polyubiquitin genes and distribution of ubiquitinated proteins in generative and sperm cells

Abstract. Polyubiquitin-encoding cDNA clones were isolated from the generative cells of lily (Lilium longiflorum) and the sperm cells of Plumbago zeylanica. The described genes encode identical amino acid sequences, with no homology outside the coding regions. This gene participates in ubiquitination of proteins, presumably enhancing protein turnover in the germline during male reproductive differentiation. In this paper we show that the gene encoding polyubiquitin is highly up-regulated in both Lilium generative cells and one of the Plumbago sperm cell types in particular.

Isolation and collection of two populations of viable sperm cells from the pollen of Plumbago zeylanica

A protocol is described for individually collecting two populations of sperm cells, Svn and Sua, from pollen of Plumbago zeylanica. Pollen grains were burst in 10 mM MOPS buffer containing 0.8 M mannitol (pH 4.6). Paired sperm cells released from pollen were separated using a microinjector. Svn and Sua were then collected individually with a microinjector, based upon known size differences. Collected sperm cells were washed with isolation medium and transferred to liquid nitrogen until use. Fluorochromatic reaction (FCR) test of isolated sperm cells showed a positive reaction, indicating that the isolated sperm cells are viable; most of the sperm cells retain viability for at least 2 h.

Identification of pronp1, a tobacco profilin gene activated in tip-growing cells

In plant cells, several cellular processes depend on rapid reorganization of a dynamic network of actin cytoskeletal elements in response to internal and environmental stimuli. Profilins, ubiqitous eukaryotic actin monomer-binding proteins with highly conserved three-dimensional structures, regulate the actin cytoskeleton and are considered to link the microfilament system with signal transduction pathways. Plant profilins have been grouped into two distinct classes, gametophytic (pollen-specific) and sporophytic. Here we report the isolation of a profilin gene that seems to be activated during tip growth of specialized cells of gametophytic as well as sporophytic origin. Identification of a genomic DNA clone containing a tobacco profilin gene, pronp1, and analysis of the pronp1 promoter-uidA fusion gene in transgenic Nicotiana tabacum plants revealed a prominent expression of pronp1 in mature pollen and elongating pollen tubes and significant activity in root hairs of developing seedlings. This expression pattern was distinct from that of any other profilin gene isolated so far. Pronp1 thus represents a unique profilin gene that is activated at the transcriptional level in two kinds of tip-growing cells, pollen tubes and root hairs, both of which require rapid organization of the actin cytoskeleton. The isolation of such a gene has fundamental importance for our understanding of modulation of the actin cytoskeleton at the molecular level.

Isolation of a gene preferentially expressed in mature anthers of rice (Oryza sativa L.)

SummaryUsing monoclonal antibodies raised against pollen-specific proteins, we have isolated a cDNA clone, designatedOry-Cl from a rice anther cDNA expression library. A transcript corresponding to theOry-Cl gene showed preferential expression in anthers. This transcript was not detected in any vegetative tissues analysed. RNA gel blot analysis of different developmental stages of anthers showed that theOry-Cl gene is expressed at later stages of pollen development. In situ hybridisation showed that theOry-Cl transcript is only present in mature pollen.