Jen-Chih Chen

Functional activity of sporamin from sweet potato (Ipomoea batatas Lam.) : a tuber storage protein with trypsin inhibitory activity

Sporamin accounts for about 60% to 80% of total soluble protein in sweet potato tubers, and the predicted protein sequence of sporamin shares significant amino acid sequence identity with some Kunitz-type trypsin inhibitors. We constructed three recombinant plasmids with cDNAs that encode preprosporamin, prosporamin, and sporamin, and these three were expressed in Escherichia coli cells as fusion proteins. All three forms of sporamin expressed in E. coli were shown to have strong inhibitory activity to trypsin in vitro, suggesting that post-translational modifications are not essential for trypsin inhibitory activity. Northern blot analysis showed that sporamin transcripts could be systemically induced in leaf tissue of sweet potato by wounding. Therefore, sporamin may have a defense role as a protease inhibitor, in addition to its role as a storage protein.

Chalcone synthase as a reporter in virus-induced gene silencing studies of flower senescence

Agrobacterium-mediatedinfection of petunia (Petunia hybrida) plants with tobacco rattle virus (TRV) bearing fragments of Petuniagenes resulted in systemic infection and virus-induced gene silencing (VIGS) of the homologous host genes. Infection with TRV containing a phytoene desaturase (PDS) fragment resulted in reduced abundance of PDS transcripts and typical photobleaching of photosynthetic tissues. Infection with TRV containing a chalcone synthase (CHS) fragment resulted in silencing of anthocyanin production in infected flowers. The silencing phenotype ranged from scattered white spots on the normal purple background to entirely white flowers. Symptoms in the V26 cultivar were a diffuse mosaic, but infection of some purple-flowered commercial cultivars resulted in large white sectors and even entirely white flowers. Abundance of CHS transcripts in the white flowers was less than 4 of that in purple flowers on the same plant. Infection with TRV containing a tandem construct of PDS and CHS resulted in leaf photobleaching and white patterns on the flowers. Transcripts of CHSand PDSwere reduced both in leaves and in flowers confirming simultaneous silencing of both genes by the tandem construct. We tested the effects of infection with TRV containing CHS and a fragment of a petunia gene encoding for 1-aminocyclopropane-1-carboxylate oxidase (ACO4) Abundance of transcripts encoding ACO4 and ACO1 were reduced (by 5 and 20, respectively) in infected flowers. Whether the flowers were treated with ACC or pollinated, the white (silenced) flowers or flower sectors produced less ethylene and senesced later than purple (non-silenced) tissues. These results indicate the value of VIGS with tandem constructs containing CHS as reporter and a target gene as a tool for examining the function of floral-associated genes.

Recruitment of CRABS CLAW to promote nectary development within the eudicot clade

Nectaries are secretory organs that are widely present in flowering plants that function to attract floral pollinators. Owing to diversity in nectary positions and structures, they are thought to have originated multiple times during angiosperm evolution, with their potential contribution to the diversification of flowering plants and pollinating animals being considerable. We investigated the genetic basis of diverse nectary forms in eudicot angiosperm species using CRABS CLAW (CRC), a gene required for nectaries in Arabidopsis. CRC expression is conserved in morphologically different nectaries from several core eudicot species and is required for nectary development in both rosids and asterids, two major phylogenetic lineages of eudicots. However, in a basal eudicot species, no evidence of CRC expression in nectaries was found. Considering the phylogenetic distribution of nectary positions and CRC expression analyses in eudicots, we propose that diverse nectaries in core eudicots share conserved CRC gene regulation, and that derived nectary positions in eudicots have altered regulation of CRC. As the ancestral function of CRC lies in the regulation of carpel development, it may have been co-opted as a regulator of nectary development within the eudicots, concomitant with the association of nectaries with reproductive organs in derived lineages.

Molecular changes occurring during acquisition of abscission competence following auxin depletion in Mirabilis jalapa.

To understand how auxin regulates sensitivity of abscission zone (AZ) tissues to ethylene, we used a polymerase chain reaction-based subtractive approach to identify gene transcripts in Mirabilis jalapa AZs that changed in abundance during the time the zones became competent to abscise in response to exogenous ethylene. Transcript expression was then examined in leaf and stem AZs over the period they became ethylene competent following indole-3-acetic acid (IAA) depletion either by leaf deblading, treatment with the IAA transport inhibitor naphthylphthalamic acid, or cutting the stem above a node (decapitation). Transcripts down-regulated by deblading/decapitation included Mj-Aux/IAA1 and Mj-Aux/IAA2, encoding Aux/IAA proteins, and three other transcripts showing highest identity to a polygalacturonase inhibitor protein, a β-expansin, and a β-tubulin. Application of IAA to the cut end of petioles or stumps inhibited abscission, and prevented the decline in the levels of transcripts in both AZs. Transcripts up-regulated in the AZ following deblading/decapitation or treatment with naphthylphthalamic acid were isolated from plants pretreated with 1-methylcyclopropene before deblading to help select against ethylene-induced genes. Some of the up-regulated transcripts showed identity to proteins associated with ethylene or stress responses, while others did not show homology to known sequences. Sucrose infiltration of stem stumps enhanced abscission following ethylene treatment and also enhanced the induction of some of the up-regulated genes. Our results demonstrate a correlation between acquisition of competence to respond to ethylene in both leaf and stem AZs, and decline in abundance of auxin regulatory gene transcripts.

A Ferroxidase Encoded by FOX1 Contributes to Iron Assimilation under Conditions of Poor Iron Nutrition in Chlamydomonas

ABSTRACT When the abundance of the FOX1 gene product is reduced, Chlamydomonas cells grow poorly in iron-deficient medium, but not in iron-replete medium, suggesting that FOX1-dependent iron uptake is a high-affinity pathway. Alternative pathways for iron assimilation, such as those involving ZIP family transporters IRT1 and IRT2, may be operational.

EBARDenovo: highly accurate de novo assembly of RNA-Seq with efficient chimera-detection

MOTIVATIONnHigh-accuracy de novo assembly of the short sequencing reads from RNA-Seq technology is very challenging. We introduce a de novo assembly algorithm, EBARDenovo, which stands for Extension, Bridging And Repeat-sensing Denovo. This algorithm uses an efficient chimera-detection function to abrogate the effect of aberrant chimeric reads in RNA-Seq data.nnnRESULTSnEBARDenovo resolves the complications of RNA-Seq assembly arising from sequencing errors, repetitive sequences and aberrant chimeric amplicons. In a series of assembly experiments, our algorithm is the most accurate among the examined programs, including de Bruijn graph assemblers, Trinity and Oases.nnnAVAILABILITY AND IMPLEMENTATIONnEBARDenovo is available at http://ebardenovo.sourceforge.net/. This software package (with patent pending) is free of charge for academic use only.nnnSUPPLEMENTARY INFORMATIONnSupplementary data are available at Bioinformatics online.

Phytoplasma-Induced Floral Abnormalities in Catharanthus roseus Are Associated with Phytoplasma Accumulation and Transcript Repression of Floral Organ Identity Genes

Floral symptoms caused by phytoplasma largely resemble floral reversion in other plants. Periwinkle leaf yellowing (PLY) phytoplasma and peanut witches-broom (PnWB) phytoplasma caused different degrees of floral abnormalities on infected periwinkle plants. The PLY phytoplasma-infected plants exhibited floral discoloration, virescence, small flowers, and only occasionally full floral reversion. In contrast, PnWB phytoplasma frequently induced complete floral reversion and resulted in a witches-broom symptom from the floral reversion. Although different degrees of floral symptoms were induced by these two phytoplasmas, the morphological disorders were similar to those of other plants carrying SEPALLATA mutations or gene silencing. Here, we compared expression levels of organ-identity-related genes and pigmentation genes during floral symptom development. Accumulation of phytoplasmas in malformed flowers and their closely surrounding leaves was also compared. In infected plants, transcript abundance of all examined organ identity genes and pigmentation genes was suppressed. Indeed, CrSEP3, a SEPALLALA3 ortholog, showed the greatest suppression among genes examined. Of the pigmentation genes, transcript reduction of chalcone synthase was most highly correlated with the loss in floral pigmentation. Floral symptom severities were associated with the accumulation of either phytoplasmas. Interestingly, both phytoplasmas accumulated to higher levels in malformed flowers than in their surrounding leaves. Many plant pathogens manipulate host plant development to their advantage. It is intriguing to see whether phytoplasmas alter floral development to increase their population.

Quantitative assessment of mitochondrial DNA copies from whole genome sequencing

BackgroundMitochondrial dysfunction is associated with various aging diseases. The copy number of mtDNA in human cells may therefore be a potential biomarker for diagnostics of aging. Here we propose a new computational method for the accurate assessment of mtDNA copies from whole genome sequencing data.ResultsTwo families of the human whole genome sequencing datasets from the HapMap and the 1000 Genomes projects were used for the accurate counting of mitochondrial DNA copy numbers. The results revealed the parental mitochondrial DNA copy numbers are significantly lower than that of their children in these samples. There are 8%~21% more copies of mtDNA in samples from the children than from their parents. The experiment demonstrated the possible correlations between the quantity of mitochondrial DNA and aging-related diseases.ConclusionsSince the next-generation sequencing technology strives to deliver affordable and non-biased sequencing results, accurate assessment of mtDNA copy numbers can be achieved effectively from the output of whole genome sequencing. We implemented the method as a software package MitoCounter with the source code and users guide available to the public at http://sourceforge.net/projects/mitocounter/.

Virus-Induced Gene Silencing in Ornamental Plants

Virus-induced gene silencing (VIGS) provides an attractive tool for high-throughput analysis of the functional effects of gene knockdown. Virus genomes are engineered to include fragments of target host genes, and the infected plant recognizes and silences the target genes as part of its viral defense mechanism. The consequences of gene inactivation, even of key metabolic, regulatory, or embryo-lethal genes, can thus be readily analyzed. A number of viral vectors have been developed for VIGS; one of the most frequently employed is based on tobacco rattle virus (TRV) due to its wide host range, efficiency, ease of application, and limited disease symptoms. TRV-based VIGS comprises two vectors. One (RNA2) includes a multiple cloning site into which fragments of target genes can be inserted. We have shown that the TRV/VIGS system can simultaneously silence as many as five independent genes. TRV is a mosaic-type virus, and silencing also occurs in a mosaic pattern. It is therefore desirable to have a reporter that can show where target genes have been silenced. The photobleaching induced by silencing phytoene desaturase (PDS) and the loss of purple pigmentation induced by silencing chalcone synthase (CHS) have successfully been used to indicate the location of coordinate silencing of other target genes. In this chapter, we outline our protocols for the use of VIGS for analysis of gene function, focusing particularly on the use of TRV with petunia and tomato.

Virus-Induced Gene Silencing for Functional Characterization of Genes in Petunia

Although functional analysis of genes can be readily carried out in Petunia using standard transformation/regeneration techniques, this process is time- and labor-consuming. High throughput analysis of gene knockouts has been made possible by the use of virus-induced gene silencing (VIGS): fragments of target plant genes are included in the genome of a viral vector, the plant silences them as part of its viral defense mechanism, and the consequences of gene inactivation can be readily analyzed. In Petunia, we use a modified tobacco rattle virus (TRV) vector for VIGS. Infection typically results in chimeric plants, and it is therefore desirable to have a reporter that can show where target genes have been silenced. Inserting a fragment of the gene encoding PHYTOENE DESATURASE (PDS) results in silencing-induced photobleaching of leaves; inserting a fragment of the gene encoding CHALCONE SYNTHASE (CHS) allows us to visualize silencing in floral tissues of purple-flowered Petunia cultivars as white patches, sectors or even entire corollas. We have shown that the VIGS system can silence as many as five independent genes at one time. We describe here the methods that we have found to be efficient and effective for VIGS in Petunia, and describe some results obtained by silencing a range of genes, including some transcription factors.