Sung Chul Bahn

Identification and Testing of Superior Reference Genes for a Starting Pool of Transcript Normalization in Arabidopsis

Genes that are stably expressed during development or in response to environmental changes are essential for accurate normalization in qRT-PCR experiments. To prevent possible misinterpretation caused by the use of unstable housekeeping genes, such as UBQ10, ACT, TUB and EF-1α, as a reference, the use of 20 stably expressed genes identified from microarray analyses was proposed. Furthermore, it was recommended that at least four genes among them be tested to identify suitable reference genes under different experimental conditions. However, testing the 20 potential reference genes under any condition is inefficient. Furthermore, since their stability still varies, there is a need to identify a subset of genes that are more stable than others, which can be used as a starting pool for testing. Here, we validated the expression stability of the potential candidate genes together with the above-mentioned conventional reference genes under six experimental conditions commonly used in plant developmental biology. To increase fidelity, three independent validation experiments were carried out for each experimental condition. A hypothetical normalization factor, which is the geometric mean of genes that were identified as stably expressed genes in each experiment, was used to exclude unstable genes under a given condition. We identified a subset of genes showing higher expression stability under specific experimental conditions. We recommend the use of these genes as a starting pool for the identification of suitable reference genes under given experimental conditions to ensure accurate normalization in qRT-PCR analysis.

Secretory Low Molecular Weight Phospholipase A2 Plays Important Roles in Cell Elongation and Shoot Gravitropism in Arabidopsis

To elucidate the cellular functions of phospholipase A2 in plants, an Arabidopsis cDNA encoding a secretory low molecular weight phospholipase A2 (AtsPLA2β) was isolated. Phenotype analyses of transgenic plants showed that overexpression of AtsPLA2β promotes cell elongation, resulting in prolonged leaf petioles and inflorescence stems, whereas RNA interference–mediated silencing of AtsPLA2β expression retards cell elongation, resulting in shortened leaf petioles and stems. AtsPLA2β is expressed in the cortical, vascular, and endodermal cells of the actively growing tissues of inflorescence stems and hypocotyls. AtsPLA2β then is secreted into the extracellular spaces, where signaling for cell wall acidification is thought to occur. AtsPLA2β-overexpressing or -silenced transgenic plants showed altered gravitropism in inflorescence stems and hypocotyls. AtsPLA2β expression is induced rapidly by auxin treatment and in the curving regions of inflorescence stems undergoing the gravitropic response. These results suggest that AtsPLA2β regulates the process of cell elongation and plays important roles in shoot gravitropism by mediating auxin-induced cell elongation.

OVEREXPRESSION OF HUMAN ERYTHROPOIETIN (EPO) AFFECTS PLANT MORPHOLOGIES: RETARDED VEGETATIVE GROWTH IN TOBACCO AND MALE STERILITY IN TOBACCO AND ARABIDOPSIS

Erythropoietin (EPO) is a glycoprotein used for curing human anemia by regulating the differentiation of erythroid progenitors and the production of red blood cells. To examine the expression of recombinant EPO in plants, pPEV-EP21, in which human epo cDNA under the control of the CaMV 35S promoter, was introduced into tobacco and Arabidopsisvia Agrobacterium tumefaciens-mediated transformation. The RNA expression level of epo in the transgenic lines was initially estimated by Northern blot analysis. Two transgenic lines, which exhibited a high expression level of epo mRNA determined by Northern analysis, were chosen for Western blot analysis to examine the production of EPO proteins. Those two lines, EP21-12 and EP21-14, revealed detectable bands on the immunoblot. Interestingly, constitutive expression of the human epo gene affected the morphologies in transgenic plants such that vegetative growth of transgenic tobacco was retarded, and male sterility was induced in transgenic tobacco and Arabidopsis

Endoplasmic Reticulum– and Golgi-Localized Phospholipase A2 Plays Critical Roles in Arabidopsis Pollen Development and Germination

This study shows that Arabidopsis PLA2-γ and -δ, which are specifically expressed in pollen, localize to the endoplasmic reticulum and/or Golgi and that the suppression of PLA2s disrupts the endomembrane and induces pollen collapse. The PLA2 product, 18-1:LPE, was found to be required for pollen tube germination. The phospholipase A2 (PLA2) superfamily of lipolytic enzymes is involved in a number of essential biological processes, such as inflammation, development, host defense, and signal transduction. Despite the proven involvement of plant PLA2s in many biological functions, including senescence, wounding, elicitor and stress responses, and pathogen defense, relatively little is known about plant PLA2s, and their genes essentially remain uncharacterized. We characterized three of four Arabidopsis thaliana PLA2 paralogs (PLA2-β, -γ, and -δ) and found that they (1) are expressed during pollen development, (2) localize to the endoplasmic reticulum and/or Golgi, and (3) play critical roles in pollen development and germination and tube growth. The suppression of PLA2 using the RNA interference approach resulted in pollen lethality. The inhibition of pollen germination by pharmacological PLA2 inhibitors was rescued by a lipid signal molecule, lysophosphatidyl ethanolamine. Based on these results, we propose that plant reproduction, in particular, male gametophyte development, requires the activities of the lipid-modifying PLA2s that are conserved in other organisms.

Characterization of Arabidopsis secretory phospholipase A2-γ cDNA and its enzymatic properties1

Plant secretory phospholipases A2 (sPLA2s) probably play important roles in phospholipid signaling based on the data reported from other organisms, but their functions are poorly understood because of the lack of cloned sPLA2 genes. In this study, we cloned and characterized an Arabidopsis secretory phospholipase A2‐γ (AtsPLA2‐γ) cDNA, and examined its enzymatic properties. The recombinant protein of AtsPLA2‐γ showed maximal enzyme activity at pH 8.0, and required Ca2+ for activity. Moreover, AtsPLA2‐γ showed sn‐2 position specificity but no prominent acyl preference, though it showed head group specificity to phosphatidylethanolamine rather than to phosphatidylcholine. AtsPLA2‐γ was found to predominate in the mature flower rather than in other tissues, and subcellular localization analysis confirmed that AtsPLA2‐γ is secreted into the intercellular space.

Identification of differentially expressed genes during flower development in carnation (Dianthus caryophyllus)

Flower development is a complex process mediated by a cascade of transcriptional regulation and signal transduction. Differentially expressed genes in two different developmental stages of carnation flowers were isolated. For this purpose, the suppression subtractive hybridization (SSH) technique followed by the differential hybridization screening was employed to identify rarely transcribed flower maturation-inducible genes. Using the reverse Northern blot analysis we screened 85 positive clones from a total 274 clones obtained by SSH. Among these 85 cDNAs, 60 genes showed obvious distinction of signal intensity. Thirty-five out of 60 clones were analyzed by Northern blot, and 60% (21 of 35) of the clones revealed the true positives of flower maturation-related genes, but others were not certain. Thirteen clones (CFMI-3, 5, 6, 8, 9, 10, 11, 16, 38, 61, 230, 237, 263) were only expressed in mature flowers. None of 13 clones exhibited any visible expression in the driver populations, but were distinctly induced during flower maturation (tester populations). However, the other eight clones (CFMI-7, 14, 49, 66, 203, 205, 243, 388) showed a basal level of expression in the flower bud and increased expression in the mature flower.

Molecular cloning and characterization of a novel low temperature-induced gene, blti2, from barley (Hordeum vulgare L.).

The Suppression Subtractive Hybridization (SSH) was used to isolate large numbers of low temperature-induced genes from the cold-treated winter barley (Hordeum vulgare L. cv. Dongbori). One hundred and sixty blti (barley low temperature-induced) cDNA clones were obtained. Northern blot analyses showed that several blti clones were differentially expressed by treatment of low temperature, NaCl, dehydration and ABA. One of the clones, blti2, was induced from 3 to 72 h after cold treatment while its transcript was detected only at 12 h after ABA treatment, indicating that the expression of blti2 by low temperature was regulated by an ABA-independent pathway. The full-length cDNA sequences were 944 nucleotides long and the open reading frame consisted of 492 nucleotides encoding 164 amino acids. Nucleotide sequences showed no sequence homology with the previously reported low temperature-responsive (LTR) barley genes, and the deduced amino acid sequences revealed that the blti2 contains three membrane-spanning regions. These results suggest that blti2 is a novel transmembrane protein induced by low temperature.

Characterization of PPO (polyphenol oxidase) cDNA in sweet persimmon (Diospyros kaki)

Polyphenol oxidases (PPOs) are copper metalloproteins which play a critical role in producing various derivatives of polyphenolic compounds. These enzymes are encoded in the nuclear genome and subsequently transported into the plastid. A partial cDNA clone encoding polyphenol oxidase was isolated from immature sweet persimmon leaves. Using two degenerate primers, a single band of PCR product was produced by the touchdown PCR method. In sequence analysis, this clone (define SPO; 1381 bp) revealed high similarities to the previously reported plant PPO genes. Moreover, this SPO clone was comprised of copper A and copper B-binding sites and a histidine-rich region, indicating that this clone contained the key conserved elements of PPO. To characterize the spatial and temporal expression of PPO, total RNAs were extracted from leaf and fruit peel tissues at different developmental stages. In northern analysis, transcripts of PPO were detected in all the samples tested, but the strongest expression levels were detected in mature fruit peels and leaves. Expression of PPO was inhibited in the transitional stage from chloroplast to chromoplast. In addition, the expression of PPO was highly induced in wounded immature fruit peel tissues.