Generation of 919 expressed sequence tags from immature flower buds and gene expression analysis using expressed sequence tags in the model plant Lotus japonicus

Lotus japonicus has received increased attention as a potential model legume plant. In order to study gene expression in reproductive organs and to identify genes that play a crucial function in sexual reproduction, we constructed a cDNA library from immature flower buds containing anthers at the stage of developing tapetum cells in L. japonicus, and characterized 919 expressed sequence tags (ESTs) randomly selected from a cDNA library of the immature flower buds. The 919 ESTs analyzed were clustered into 821 non-redundant EST groups. As a result of a database search, 436 groups (53%) out of the 821 groups showed sequence similarity to genes registered in the public database. Out of these 436 groups, 109 groups showed similarity to genes encoding hypothetical proteins whose function had not yet been estimated. Three hundred eighty five groups (47%) showed no significant homology to known sequences and were classified as novel sequences. A comparison of 821 non-redundant EST sequences and EST sequences derived from the whole plant L. japonicus revealed that 474 EST sequences derived from immature flower buds were not found in the EST sequences of the whole plant. In order to confirm the expression pattern of potential reproductive-organ specific EST clones, nine clones, which were not matched to ESTs derived from the whole plant, were selected, and RT-PCR analysis was performed on these clones. As a result of RT-PCR, we found two novel anther specific clones. One clone was homologous to a gene encoding human cleft lip and palate associated transmembrane protein (CLPTM1) like protein, and the other clone did not show a significant similarity to any genes deposited in the public database. These results indicate that ESTs analyzed here represent a valuable resource for finding reproductive-organ specific genes in Lotus japonicus.     

Analysis of expressed sequence tags of flower buds in Lotus japonicus.

In order to study gene expression in a reproductive organ, we constructed a cDNA library of mature flower buds in Lotus japonicus, and characterized expressed sequence tags (ESTs) of 842 clones randomly selected. The EST sequences were clustered into 718 non-redundant groups. From BLAST and FASTA search analyses of both protein and DNA databases, 58.5% of the EST groups showed significant sequence similarities to known genes. Several genes encoding these EST clones were identified as pollen-specific genes, such as pectin methylesterase, ascorbate oxidase, and polygalacturonase, and as homologous genes involved in pollen-pistil interaction. Comparison of these EST sequences with those derived from the whole plant of L. japonicus, revealed that 64.8% of EST sequences from the flower buds were not found in EST sequences of the whole plant. Taken together, the EST data from flower buds generated in this study is useful in dissecting gene expression in floral organ of L. japonicus.     

EST sequencing and time course microarray hybridizations identify more than 700 <Emphasis Type="Italic">Medicago truncatula</Emphasis> genes with developmental expression regulation in flowers and pods

To evaluate the molecular mechanisms during pod and seed formation in legumes, starting with the development of reproductive organs, we constructed two cDNA libraries from developing flowers (MtFLOW) and pods including seeds (MtPOSE) of the model plant Medicago truncatula Gaertner. A total of 2,516 expressed sequence tags (ESTs) clustered into 1,776 nonredundant sequences (2k-set), which were annotated and assigned to functional classes. While about 30% of the ESTs encoded proteins of yet unknown function, typical annotations pointed to seed storage proteins, LTPs and lipoxygenases. The 2k-set was used to upgrade Mt6k-RIT microarrays (Küster et al. in J Biotechnol 108: 95, 2004) to Mt8k versions representing approximately 6,300 nonredundant M. truncatula genes. These were used to perform time course expression profiling studies based on hybridizations of samples that covered eight different developmental stages from flower buds to almost mature pods versus leaves as a common reference. About 180 up- and 70 downregulated genes were typically found for each stage and in total, 782 genes were either twofold up- or downregulated in at least one of the eight stages investigated. Based on this set, a combination of self-organizing map and hierarchical clustering revealed genes displaying expression regulation during characteristic stages of M. truncatula flower and pod development. Amongst those, several genes encoded proteins related to seed metabolism and development including novel regulators and proteins involved in signaling.Electronic supplementary material Supplementary material is available for this article at     

Expressed sequence tags of Chinese cabbage flower bud cDNA.

We randomly selected and partially sequenced cDNA clones from a library of Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower bud cDNAs. Out of 1216 expressed sequence tags (ESTs), 904 cDNA clones were unique or nonredundant. Five hundred eighty-eight clones (48.4%) had sequence homology to functionally defined genes at the peptide level. Only 5 clones encoded known flower-specific proteins. Among the cDNAs with no similarity to known protein sequences (628), 184 clones had significant similarity to nucleotide sequences registered in the databases. Among these 184 clones, 142 exhibited similarities at the nucleotide level only with plant ESTs. Also, sequence similarities were evident between these 142 ESTs and their matching ESTs when compared using the deduced amino acid sequences. Therefore, it is possible that the anonymous ESTs encode plant-specific ubiquitous proteins. Our extensive EST analysis of genes expressed in floral organs not only contributes to the understanding of the dynamics of genome expression patterns in floral organs but also adds data to the repertoire of all genomic genes.     

Isolation of early genes expressed in reproductive organs of the dioecious white campion (Silene latifolia) by subtraction cloning using an asexual mutant

The dioecious white campion (Silene latifolia) has been chosen as a working model for sexual development. In this species, sexual dimorphism is achieved through two distinct developmental blocks: inhibition of carpel development in male flowers, and early arrest of anther differentiation in female flowers. The combined advantages of the dioecious system and the availability of a sexual mutant lacking both male and female reproductive organs have been exploited in a molecular subtraction approach using male and asexual flower buds. This resulted in the cloning of 22 cDNA clones expressed in stamens at distinct stages of development. Fourteen of these clones corresponded to genes whose expression was detected in pre-meiotic stamens, a stage of development for which very little information is presently available. Furthermore, the absence of similarities with database sequences for ten clones suggests that they represent novel genes. Functional analysis of each clone will enable their positioning within the reproductive organ developmental pathway(s). In parallel, these clones are being exploited as developmental markers of early differentiation within the flower.     

A polygalacturonase inhibitory protein gene (<Emphasis Type="Italic">BcMF19</Emphasis>) expressed during pollen development in Chinese cabbage-pak-choi

The level of polygalacturonase inhibitory protein (PGIP) genes involved in pollen development remains unclear. Characterization of the different PGIP genes that are expressed in pollen is necessary in understanding the similarities and differences of functions between the members of this gene family, as well as the underlying mechanism of pollen development. A gene-encoding putative PGIP, BcMF19 was successfully cloned on a cDNA-amplified fragment length polymorphism fragment after it was found to be up-regulated in the fertile flower buds of Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino) genic male sterile AB line (Bajh97-01A/B). The amino acid sequence of BcMF19 possessed the basic feature of PGIPs, containing an N-terminal signal peptide, several potential N-glycosylation sites, two disulfide bridges flanking both the N- and C-terminal regions, and 10 leucine-rich repeat (LRR) consensus sequences. Real-time RT-PCR verified the higher expression of BcMF19 in the fertile flower buds compared to the sterile flower buds. In situ hybridization showed that BcMF19 was exclusively expressed in the tapetal cells and microspores during anther development. These results indicate that BcMF19 is a novel PGIP gene that might be involved in pollen or tapetum development.     

Construction and characterization of a cDNA library from floral organs and fruitlets of <Emphasis Type="Italic">Citrus reticulata</Emphasis>

To explore and isolate genes related to flowering and fruit development, we constructed a cDNA library from floral organs and fruitlets of Ponkan mandarin (Citrus reticulata Blanco). A total of 661 high-quality expressed sequence tags (ESTs) were generated and submitted to GenBank with the accession numbers from GO343532 to GO344192. All these ESTs were assembled into 43 contigs and 296 singletons (totally 339 unigenes). The BLAST2GO software was employed to annotate the unigenes, among which 77 ones had no significant homology with the sequences in NCBI non-redundant proteins database by BLASTX analysis. Additionally, gene ontology (GO) analysis revealed an overview of sequences distribution, which implied some specially expressed genes related to flower and fruit development. Furthermore, some abundantly expressed unigenes involved in several crucial metabolic pathways related to fruit quality were highlighted and three types of homologues of miraculin-like protein2 were analyzed by both semiquantitative RT-PCR and real-time PCR. The results showed different expression profiles of these genes, which meant that they contribute distinctly to fruit development.     

Regulation of expression of two novel flower-specific genes from tomato (Solanum lycopersicum) by gibberellin.

Two flower-specific cDNAs have been isolated after differential screening of an anther cDNA library. This library was constructed 48 h after GA(3) treatment of buds of the GA-deficient gib-1 mutant of tomato. Northern blot analysis during flower development in tomato demonstrated that the expression of both genes is regulated by gibberellins (GAs). Application of GA(3) to developmentally arrested gib-1 flower buds induced new expression of tgas100 mRNA 48 h post-treatment, while an increased accumulation of tgas105 mRNA was found after 8 h. In situ analyses showed the spatial distribution of the expression of both genes within the tomato flower. One of the deduced polypeptides (TGAS105) displays similarities to cysteine-rich extensin-like proteins, while the other (TGAS100) shows significant homology with a stamen-specific gene of Antirrhinum majus. Based on the deduced protein sequences, the possible function of the encoded proteins is discussed.     

Structural and expression analysis of prolamin genes in <Emphasis Type="Italic">Oryza sativa</Emphasis> L.

Rice is a staple crop with a small genome of 389 Mb. Rice grain is a source of carbohydrates and proteins and has a relatively low protein content compared to other legume seeds. Glutelin and prolamin are the major storage proteins in rice. Prolamins are characterized by high glutamine and proline content and are generally soluble only in strong alcohol solutions. In this study, we obtained a total of 51,383 expressed sequence tags (ESTs) from Ilpumbyeo (Oryza sativa L.), of which 33,201 and 18,182 clones were obtained from immature and germinating seeds, respectively. From the EST clones, 15,148 unigenes were identified, and 2,590 genes were expressed in both immature and germinating seeds. Gene expression profiling of rice prolamins indicated that prolamin gene expression increased 5 days after heading and reached maximal expression after 30 days, suggesting a high demand for prolamins during seed development and germination. Phylogenetic analysis grouped 33 prolamin genes based on the abundance of sulfur-containing amino acids methionine and cysteine according to the deduced amino acid sequences. Our results enhance the understanding of the regulation of seed maturation and germination, which can result in improved agricultural traits for the seed industry.     

A large scale analysis of cDNA in Arabidopsis thaliana: generation of 12,028 non-redundant expressed sequence tags from normalized and size-selected cDNA libraries.

For comprehensive analysis of genes expressed in the model dicotyledonous plant, Arabidopsis thaliana, expressed sequence tags (ESTs) were accumulated. Normalized and size-selected cDNA libraries were constructed from aboveground organs, flower buds, roots, green siliques and liquid-cultured seedlings, respectively, and a total of 14,026 5'-end ESTs and 39,207 3'-end ESTs were obtained. The 3'-end ESTs could be clustered into 12,028 non-redundant groups. Similarity search of the non-redundant ESTs against the public non-redundant protein database indicated that 4816 groups show similarity to genes of known function, 1864 to hypothetical genes, and the remaining 5348 are novel sequences. Gene coverage by the non-redundant ESTs was analyzed using the annotated genomic sequences of approximately 10 Mb on chromosomes 3 and 5. A total of 923 regions were hit by at least one EST, among which only 499 regions were hit by the ESTs deposited in the public database. The result indicates that the EST source generated in this project complements the EST data in the public database and facilitates new gene discovery.