De novo transcriptome analysis and comparative expression profiling of genes associated with the taste-modifying protein neoculin in Curculigo latifolia and Curculigo capitulata fruits.

BACKGROUND: Curculigo latifolia is a perennial plant endogenous to Southeast Asia whose fruits contain the taste-modifying protein neoculin, which binds to sweet receptors and makes sour fruits taste sweet. Although similar to snowdrop (Galanthus nivalis) agglutinin (GNA), which contains mannose-binding sites in its sequence and 3D structure, neoculin lacks such sites and has no lectin activity. Whether the fruits of C. latifolia and other Curculigo plants contain neoculin and/or GNA family members was unclear. RESULTS: Through de novo RNA-seq assembly of the fruits of C. latifolia and the related C. capitulata and detailed analysis of the expression patterns of neoculin and neoculin-like genes in both species, we assembled 85,697 transcripts from C. latifolia and 76,775 from C. capitulata using Trinity and annotated them using public databases. We identified 70,371 unigenes in C. latifolia and 63,704 in C. capitulata. In total, 38.6% of unigenes from C. latifolia and 42.6% from C. capitulata shared high similarity between the two species. We identified ten neoculin-related transcripts in C. latifolia and 15 in C. capitulata, encoding both the basic and acidic subunits of neoculin in both plants. We aligned these 25 transcripts and generated a phylogenetic tree. Many orthologs in the two species shared high similarity, despite the low number of common genes, suggesting that these genes likely existed before the two species diverged. The relative expression levels of these genes differed considerably between the two species: the transcripts per million (TPM) values of neoculin genes were 60 times higher in C. latifolia than in C. capitulata, whereas those of GNA family members were 15,000 times lower in C. latifolia than in C. capitulata. CONCLUSIONS: The genetic diversity of neoculin-related genes strongly suggests that neoculin genes underwent duplication during evolution. The marked differences in their expression profiles between C. latifolia and C. capitulata may be due to mutations in regions involved in transcriptional regulation. Comprehensive analysis of the genes expressed in the fruits of these two Curculigo species helped elucidate the origin of neoculin at the molecular level.

Additional nitrogen fertilization at heading time of rice down-regulates cellulose synthesis in seed endosperm.

The balance between carbon and nitrogen is a key determinant of seed storage components, and thus, is of great importance to rice and other seed-based food crops. To clarify the influence of the rhizosphere carbon/nitrogen balance during the maturation stage of several seed components, transcriptome analysis was performed on the seeds from rice plants that were provided additional nitrogen fertilization at heading time. As a result, it was assessed that genes associated with molecular processes such as photosynthesis, trehalose metabolism, carbon fixation, amino acid metabolism, and cell wall metabolism were differentially expressed. Moreover, cellulose and sucrose synthases, which are involved in cellulose synthesis, were down-regulated. Therefore, we compared cellulose content of mature seeds that were treated with additional nitrogen fertilization with those from control plants using calcofluor staining. In these experiments, cellulose content in endosperm from plants receiving additional nitrogen fertilization was less than that in control endosperm. Other starch synthesis-related genes such as starch synthase 1, starch phosphorylase 2, and branching enzyme 3 were also down-regulated, whereas some α-amylase and ß-amylase genes were up-regulated. On the other hand, mRNA expression of amino acid biosynthesis-related molecules was up-regulated. Moreover, additional nitrogen fertilization caused accumulation of storage proteins and up-regulated Cys-poor prolamin mRNA expression. These data suggest that additional nitrogen fertilization at heading time changes the expression of some storage substance-related genes and reduces cellulose levels in endosperm.

Global gene expression profiles in developing soybean seeds.

The gene expression profiles in soybean (Glycine max L.) seeds at 4 stages of development, namely, pod, 2-mm bean, 5-mm bean, and full-size bean, were examined by DNA microarray analysis. The total genes of each sample were classified into 4 clusters based on stage of development. Gene expression was strictly controlled by seed size, which coincides with the development stage. First, stage specific gene expression was examined. Many transcription factors were expressed in pod, 2-mm bean and 5-mm bean. In contrast, storage proteins were mainly expressed in full-size bean. Next, we extracted the genes that are differentially expressed genes (DEGs) that were extracted using the Rank products method of the Bioconductor software package. These DEGs were sorted into 8 groups using the hclust function according to gene expression patterns. Three of the groups across which the expression levels progressively increased included 100 genes, while 3 groups across which the levels decreased contained 47 genes. Storage proteins, seed-maturation proteins, some protease inhibitors, and the allergen Gly m Bd 28K were classified into the former groups. Lipoxygenase (LOX) family members were present in both the groups, indicating the multi-functionality with different expression patterns.

Expression of the stress-related genes for glutathione S-transferase and ascorbate peroxidase in the most-glycinin-deficient soybean cultivar Tousan205 during seed maturation.

Global analysis of gene expression profiles in most-glycinin-deficient cultivar Tousan205, was performed by DNA microarray analysis. It was confirmed that Tousan205 lacks mRNA expression of three glycinin subunit precursor genes, G1 (A1aB1x), G2 (A2B1a), and G5 (A3B4), and lacks G4 (A5A4B3) protein. Most glycinin subunits were deficient in mature seeds of Tousan205. We compared the gene expression of Tousan205 with those of parent cultivar, Tamahomare, which was used for crossbreeding of Tousan205. As a result, Tousan205 exhibited higher expression of some seed maturation proteins, and stress-related genes such as glutathione S-transferase and ascorbate peroxidase. This result indicates the possibility that the decrease of main storage protein, glycinin causes stress in soybean.

Signal peptide peptidases are expressed in the shoot apex of rice, localized to the endoplasmic reticulum.

Signal peptide peptidase (SPP) is a multi-transmembrane aspartic proteinase involved in regulated intramembrane proteolysis, which is implicated in fundamental life processes such as immunological response, cell signaling, tissue differentiation, and embryogenesis. In this study, we identified two rice SPPs: OsSPP1 and OsSPP2. Green fluorescent protein-fused OsSPP1 and OsSPP2 were localized to the ER in cultured plant cells. In situ hybridization showed that OsSPPs were strongly expressed in vegetative shoot apex, young panicle, developing panicle, and the early developing florets. Undifferentiated cells, which have the potential to differentiate into all of the aerial parts of the plant are presented in the shoot apex. OsSPPs are located in both the undifferentiated cells, and the early differentiated cells at the shoot apex. These results suggest that rice SPPs have an important function in differentiation and development at the shoot apex. The expression of the shoot apex and ER localization is equal to dicot Arabidopsis thaliana, and will have common crucial roles in plant.

Wheat cysteine proteases triticain alpha, beta and gamma exhibit mutually distinct responses to gibberellin in germinating seeds.

We cloned three novel papain-type cysteine proteases (CPs), triticain alpha, beta and gamma, from 1-d-germinating wheat seeds. Triticain alpha, beta and gamma were constituted with 461, 472 and 365 amino acid residues, respectively, and had Cys-His-Asn catalytic triads as well as signal and propeptide sequences. Triticain gamma contained a putative vacuole-sorting sequence. Phylogenetic analysis showed that these CPs were divided into mutually different clusters. Triticain alpha and gamma mRNAs were expressed in seeds at an early stage of maturation and at the stage of germination 2d after imbibition, while triticain beta mRNA appeared shortly after imbibition. The expression of mRNAs for triticain alpha and gamma was suppressed by uniconazol, a gibberellin synthesis inhibitor. All the three CP mRNAs were strongly expressed in both embryo and aleurone layers. These results suggest that triticain alpha, beta and gamma play differential roles in seed maturation as well as in digestion of storage proteins during germination.

Signal peptide peptidase and its homologs in Arabidopsis thaliana--plant tissue-specific expression and distinct subcellular localization.

Signal peptide peptidase (SPP) is an aspartic proteinase that hydrolyses its substrate within the plane of the cellular membrane. In vertebrates, it plays crucial roles in life processes such as differentiation, embryogenesis, cell signaling and immunological response. We first found SPP in plants. An ortholog of human SPP (AtSPP), and its five AtSPP homologs (AtSPPL1-AtSPPL5), were searched for in the Arabidopsis database. These clones were grouped into three different clusters: AtSPP was grouped with human SPP (HsSPP) orthologs, AtSPPL1 with the HsSPPL3 family, and AtSPPL2-AtSPPL5 with the group of SPP-like proteins of plant origin. AtSPP, AtSPPL1 and AtSPPL2 were examined for their expression profiles by in situ hybridization. AtSPP was strongly expressed in both the shoot meristem of germinating seeds and the inflorescence meristem at the reproductive stage. On the other hand, AtSPPL1 and AtSPPL2 were expressed in the shoot meristem of germinating seeds, but at very low levels in the shoot apex at the reproductive stage. The subcellular localization of AtSPP, AtSPPL1 and AtSPPL2 was investigated using green fluorescent protein (GFP) fusion proteins in cultured 'Deep' cells. GFP-AtSPP localized to the endoplasmic reticulum (ER), and GFP-AtSPPL1 and GFP-AtSPPL2 to the endosomes. These results suggest that AtSPP mediates the cleavage of signal peptide in the ER membrane as well as HsSPP does, and also that AtSPPL1 and AtSPPL2 located in the endosomes have distinct roles in cells.

Differential expression of wheat aspartic proteinases, WAP1 and WAP2, in germinating and maturing seeds.

Two aspartic proteinase (AP) cDNA clones, WAP1 and WAP2, were obtained from wheat seeds. Proteins encoded by these clones shared 61% amino acid sequence identity. RNA blotting analysis showed that WAP1 and WAP2 were expressed in both germinating and maturing seeds. The level of WAP2 mRNA expression was clearly weaker than that of WAP1 in all tissues of seeds during germination and maturation. APs purified from germinating seeds were enzymatically active and digested the wheat storage protein, gluten. To elucidate the physiological functions of WAP1 and WAP2 in seeds, we investigated the localisation of WAP1 and WAP2 by in situ hybridisation. In germinating seeds investigated 24h after imbibition, both WAP1 and WAP2 were expressed in embryos, especially in radicles and shoots, scutellum, and the aleurone layer. In maturing seeds, WAP1 was expressed in the whole embryo, with slightly stronger expression in radicles and shoots. WAP1 was also expressed in the aleurone layer 3 weeks after flowering. Strong signals of WAP1 mRNA were detected in the whole embryo and aleurone layer 6 weeks after flowering. On the other hand, WAP2 was scarcely detected in seeds 3 weeks after flowering, and thereafter weak signals began to appear in the whole embryo. WAP1 and WAP2 were expressed widely in germinating and maturing seeds. Such diversity in site- and stage-specific expression of the two enzymes suggests their differential functions in wheat seeds.

Plant-specific insertions in the soybean aspartic proteinases, soyAP1 and soyAP2, perform different functions of vacuolar targeting.

Most aspartic proteinases (APs) of plant origin are characterized by the presence of plant-specific insertion (PSI) in their primary structure. PSI has been reported to function as signals for both transport of AP molecules from the endoplasmic reticulum (ER) and for their targeting to the vacuole. To determine the functions of the PSIs in soyAP1 and soyAP2 identified in our previous study, we examined their subcellular localization by transient expression of a green fluorescent protein (GFP) fusion protein in the protoplasts of Arabidopsis suspension-cultured cells. Both soyAP1-GFP and soyAP2-GFP were targeted to the vacuole. To confirm the role of the PSI, we prepared PSI-deleted soyAP1 and soyAP2, and investigated their vacuolar targeting by the same method. While the former deletion mutant was always transported to the vacuole, the latter sometimes remained in the ER and was only sometimes transported to the vacuole. These observations indicated that, in the case of soyAP1, the PSI is not involved in vacuolar targeting, also suggesting that the function of the PSI differs depending on its origin.

Characterization of the genes for two soybean aspartic proteinases and analysis of their different tissue-dependent expression.

We isolated and characterized two cDNAs for aspartic proteinases (APs; EC 3.4.23) in soybean [Glycine max (L.) Merr.]. The encoded enzymes, soyAP1 and soyAP2, share 55% amino acid sequence identity. Northern analysis demonstrated that soyAP1 is expressed specifically in seeds, especially in dry seeds, while the expression of soyAP2 takes place in various tissues such as roots, stems, leaves and flowers, but not in dry seeds. SoyAP1 is highly expressed even at an early stage of germination, with a subsequent decrease in expression intensity. In contrast, the soyAP2 mRNA level increases 48 h after imbibition. To elucidate the physiological functions of soyAPs, we investigated the localization of soyAP expression in seeds germinating for 48 h at 25 degrees C. SoyAP1 shows cell-type-specific expression in sieve tube cells of the hypocotyl. At the root tip, soyAP1 is expressed in immature tracheary elements and sieve tube cells, and its expression pattern changes with distance from the tip; strong signals observed throughout phloem converge gradually to sieve tube cells, whereas those observed in tracheary elements disappear while the elements are still immature. On the other hand, soyAP2 signals were detected broadly in the boundary region between the cortex and the central cylinder. These results suggest that soyAP1 and soyAP2 are functionally different from each other.