Previous studies have shown that proline mutations in the heptad repeat region stabilize the coronavirus spike (S) protein in a pre-fusion state. To understand the impact of proline substitutions on the fusogenicity of the S protein, we engineered the swine acute diarrhea syndrome coronavirus (SADS-CoV) S protein with two proline substitutions (S-PP) and examined its fusogenicity using dual-split-protein based cell-cell fusion assay. Unlike the wild-type S (S-WT), S-PP rarely formed syncytia. Additionally, protein expression of S-PP was impaired compared to S-WT, as previously reported. Our results indicate that pre-fusion stabilized S protein is unable to induce membrane fusion and provide a better understanding of SADS-CoV S and vaccine antigen design.

Ammonium (NH4 +) serves as a nitrogen source, but its elevated levels can hinder plant growth and production. Excess NH4 + with α-ketoglutarate is assimilated into glutamate, a precursor of proline and glutathione (GSH). This study aimed to investigate the effects of excessive NH4 + on the regulation of proline and GSH synthesis. Detached leaves from oilseed rape (Brassica napus L.) were fed with 0, 50, 100, 500, and 1000 mM NH4Cl for 16 h. As the NH4 + concentrations increased, the leaves exhibited progressive wilting and yellowing. Furthermore, total carotenoid and chlorophyll concentrations declined in response to all NH4 + treatments, with the lowest levels observed in 1000 mM NH4 + treatment. Hydrogen peroxide (H2O2) concentration showed a minor increase at low NH4 + concentration (50 and 100 mM) treatments but a significant increase at high NH4 + (500 and 1000 mM), which was consistent with the localization of H2O2. Amino acid concentrations increased with increasing in NH4 + concentration, while the protein concentration displayed the opposite trend. Proline and cysteine concentrations exhibited a gradual increase in response to increasing NH4 + concentrations. However, GSH concentrations rose only in the 50 mM NH4 + treatment and decreased in the 500 and 1000 mM NH4 + treatments. These results indicate that excessive NH4 + is primarily assimilated into proline, while GSH synthesis is adversely affected.

Sulfur is an essential element in plants, including amino acids, vitamin synthesis, and acting as an antioxidant. However, the interaction between endogenous sulfur and proline synthesis has not been yet fully documented. White clover (Trifolium repens L.) is known as a species highly sensitive to sulfate supply. Therefore, this study aimed to elucidate the role of sulfur in regulating proline metabolism in relation to ammonia detoxification and hydrogen peroxide (H2O2) accumulation in white clover. The detached leaves of white clover were immersed in solution containing different concentration of sulfate (0, 10, 100, and 1000 mM MgSO4). As MgSO4 concentrations were increased, the concentration of H2O2 increased up to 2.5-fold compared to control, accompanied with H2O2 detection in leaves. Amino acid concentrations significantly increased only at higher levels (100 and 1000 mM MgSO4). No significant difference was observed in protein concentration. Proline and Δ1-pyrroline-5-carboxylate (P5C) concentrations slightly decreased at 10 and 100 mM MgSO4 treatments, whereas it rapidly increased over 1.9-fold at 1000 mM MgSO4 treatment. Ammonia concentrations gradually increased up to 8.6-fold. These results indicate that exogenous sulfur levels are closely related to H2O2 and ammonia synthesis but affect proline biosynthesis only at a higher level.

본 연구는 세계채소센터의 고추 유전자원들에 대한 침수조건에서 생육특성을 조사해 침수 저항성 자원을 선발하기 위해 수행하였다. 세계채소센터 유전자원센터에서 보유중인 4종의 고추 8자원을 선발하여 유리온실에서 종자를 파종하여 2개월 간 육묘한 후 본 실험을 위해 비닐하우스로 이동 후 침수시험을 수행하였다. 시험에 이용된 고추는 고온인 여름철에 시험이 수행되어 침수에 대한 반응이 매우 빠르게 나타났다. 1. 침수 처리구에서 생육이 전체적으로 감소 되었으나 저항성 자원들은 대조구와 비교하여 초장에 있어 유의성 있는 차이를 보이지 않았다. 2. 침수 처리 후 고추자원의 엽온 조사에서는 No. 29 (9656-13) 자원을 제외한 모든 침수구에서 엽온이 대조구에 비해 높게 유지되었다. 3. 감수성 자원들은 위조지수가 1.5 이상을 나타냈으나 내침 수성 자원들은 0.5로 낮게 유지되었다. 4. SPAD 값을 조사한 결과에 있어서는 침수로 인해 전체적으로 대조구에 비해 10% 낮은 수치를 나타냈다. 5. Proline 함량에 있어서는 침수구에서 전체적으로 Proline 함량이 높게 나타났으나 내침수성이 강했던 No. 8과 22는 대조구와 유의차가 없이 낮았다. 6. 침수자원들에 대한 침수와 고온에 따른 화분활력 조사에서는 침수에서 일부 자원의 화분활력 감소를 확인하였으며, 고온처리에서 처리구 대부분의 화분 활력이 유의하게 감소하는 결과를 나타냈다. 7. 본 시험을 통해 이번 시험의 고추자원 중 Capsicum bacatum 종인 No. 8 (VI028788), No. 12 (VI044312) 자원 이 침수저항성이 강한 자원으로 판단되어 1차 선발하였다.

In the previous molecular cloning study from human salivary gland cDNA library novel clones (C75‐014, C76‐022) were known as candidate genes for proline rich proteins by GenBank data base search and RNA in situ hybridization. C75‐014 and C76‐022 genes were characterized as those expressing excretory basic proteins primarily composed of alanine, proline, and leucine residues, mimicking basic proline‐rich proteins (bPRPs) with helical structures and multiple consensus sequences of phosphorylation sites. In the immunohistochemical stainings using polyclonal antisera against each C75‐014 and C76‐022 peptide showed strong reaction in the secretory granules of striated and excretory ducts. And in Western blot for the different salivary specimens relatively distinctive bands appeared at lower molecular weight, ranging about 15‐50 kDa. This study was aimed to identify the molecular characteristics of C75‐014 and C76‐022 proteins, which showed properties of basic proline rich protein. These data suggest that C75‐014 and C76‐022 are candidate genes for proline rich proteins in human salivary gland, which may play a role for protecting and stabilizing the mucosal epithelium against numerous proteolytic damages and stresses.

A patient complaining of severe pain in the right submandibular area showed a huge sialolith in radiogram. During the operation, the submandibular gland was much indurated, and large amount of pus was discharged out at an incision of the salivary gland. The removed salivary gland contained a huge sialolith in the major excretory duct of submandibular gland, which had an intact grayish-white surface in ovoid shape. In the histological examination its excretory ducts were extensively dilated without extravasation of saliva, and the involved salivary gland was almost destroyed by the granulomatous r eaction. Most of a cinar cells were d isappeared and r eplaced by ductal cells filled with exudative materials. The microsections of sialolith showed typical laminar structures of calcification containing amorphous basophilic material in the center, in which a lot of Gram positive and Gram negative microorganisms were found. In the center of sialolith numerous microorganisms were admixed with mucinous materials which were strongly positive for the antibody of mucin-1, and formed multiple colonies. In the periphery of the bacterial colonies proline rich proteins (PRPs) were condensely localized, and followed by the consistent positive reaction of transglutaminase 4 (TGase-4). These data suggest that the sialolith of this study is formed from the primary nidus of bacterial colony aggregated with salivary mucin-1 and PRPs by the crosslinking reaction of TGase-4.

병원균 감염에 의한 식물체내 프롤린과 암모니아의 농도 변화와 그것의 스트레스 생리학적 의미를 구명하기 위하여 Rhizoctonia spp.를 처리 후 라지 펫치에 감염된 잔디의 생육 및 관련 화학적 성분을 감염이 되지 않은 대조구와 비교하였다. 라지 펫치에 감염된 잔디의 뿌리의 건사율은 대조구에 비해 약 30% 증가하였다. 가용성 단백질 농도는 병원균 처리후 6일째 잎의 경우를 제외하고는 라지 펫치 감염에 따른 유의적인 영향이 없었다. 암모니아 농도 역

Chinese cabbage grown during autumn season is confronted with drought conditions for a certain period, especially during the early growth stage. In this study, we investigated the effects of drought stress on plant growth characteristics, as well as free amino acid, carotenoid, and proline in Chinese cabbage. Chinese cabbage seeds (Bulam Plus) were germinated, and all the seedlings were transplanted into plastic containers (28 ㎝ diameter × 22 ㎝ high) containing a commercial growth medium. The soil water content was measured and maintained at 10% for the drought-stressed plants and at 30% for the control plants, for three weeks. The results revealed that plant growth parameters were lower in the drought-stressed plants than in the control plants. The total free amino acid content tended to decrease in both drought-stressed and control plants with time. The total free amino acid content was found to be lower in the drought-stressed plants than in the control plants and the proline content was unaffected. Moreover, at three weeks after treatment, carotenoid content in drought stressed plants was significantly higher than that in the untreated plants. We believe that our study makes a significant contribution to the literature because the effects of drought stress on plant growth parameters, free amino acid, carotenoids, and proline accumulation in autumn growing cultivar of Chinese cabbage have not been widely studied in Korea, and our study provides valuable information in this regard, as Chinese cabbage is consumed throughout the year in Korea.

Proline has been shown to accumulate in plant under various type of stresses. In our previous study, changes in cold hardiness and proline content showed contrasting patterns during a constant deacclimation. This study was performed to investigate the proline accumulation and related gene expression in response to repeated deacclimation and reacclimation in peach cultivar ‘Daewol’. Proline content was analyzed using the ninhydrin method and related gene expressions were examined using quantitative real-time RT-PCR. Proline contents of ‘Daewol’ increased during the repeated deacclimation treatments. Interestingly, during the twice deacclimation, expressions of P5CS (Δ1-pyrroline-5-carboxylatesynthase) constantly decreased, whereas expressions of P5CR (Δ1-pyrroline-5-carboxylatereductase) increased. Expressions of OAT (ornithine-δ-aminotransferase) indicated up- and down- pattern in response to repeated deacclimation and reacclimation. Our results indicated that proline responds positively to higher temperature in the shoots of peach cultivar ‘Daewol’ and expressions of both P5CS and P5CR genes could show contrasting patterns during the deacclimation. Moreover, our results suggest that ornithine pathway could serve as an alternative pathway in proline synthesis process during deacclimation in peach.

Proline (Pro) accumulation is a common physiological reaction in response to abiotic stresses in many plants. Accumulation of Pro is believed to play the important role in protecting cellular components from dehydrating effects due to such stresses. The study was performed to investigate the relationship between cold hardiness and Pro content or expression of related genes in peach cultivars during a constant experimental deacclimation. Changes in cold hardiness were determined using electrolyte leakage method in the shoots of 10 peach cultivars (Prunus persica ‘Aikawanakajima’, ‘Chiyomaru’, ‘Daewol’, ‘Janghowon Hwangdo’, ‘Kiraranokiwami’, ‘Mihong’, ‘Misshong’, ‘Soomee’, ‘Suhong’, and ‘Sun Gold’). Pro content was analyzed using the ninhydrin method and related gene expressions were examined using quantitative real-time RT-PCR. While cold hardiness of 10 peach cultivars decreased, Pro contents of those increased during the deacclimation. Notably, at the same time, expression of P5CS (Δ1-pyrroline-5-carboxylatesynthase) decreased in 10 peach cultivars, whereas expressions of P5CR (Δ1-pyrroline-5-carboxylatereductase) and OAT (ornithine-δ-aminotransferase) increased. Our results demonstrate that Pro responds positively to higher temperature in the shoots of 10 peach cultivars and expression of both P5CS and P5CR genes could show contrasting patterns during the deacclimation. Furthermore, our results suggest that ornithine pathway, which has been suggested to be important during seedling development, could serve as an alternative pathway in Pro synthesis process during the deacclimation in peach.

Changes in cold hardiness were confirmed with relative electrolyte leakage (REL) method in the shoots of two peach cultivars (Prunus persica Janghowon Hwangdo and Odoroki) during cold acclimation and deacclimation. Changes in proline (Pro) content and related gene expressions were also analyzed. Particularly, transcript accumulations of P5CS and P5CR were examined using quantitative real-time RT-PCR. REL in the shoots of two peach cultivars was significantly different during the entire experimental period. Cold hardiness of two cultivars increased gradually to December 2012, and then decreased to April 2013, whereas Pro contents of ‘Janghowon Hwangdo’ and ‘Odoroki’ were reduced from the beginning of the experiment to February 2012 and then increased in the spring. Interestingly, P5CS gene encoding an enzyme, which catalyzes conversion from glutamic acid (Glu) into glutamic-γ-semialdehyde (GSA) in the first step of Pro pathway, showed the contrasting patterns with Pro contents of two cultivars. On the other hand, P5CR gene encoding an enzyme, which catalyzes conversion from Δ1-pyrroline-5-carboxylate(P5C)intoPro in the final step of Pro pathway, showed the similar patterns to Pro contents in two cultivars. Our results demonstrate that Pro responds negatively to low temperatures in the shoot of different peach cultivars, including the supplemental 10 peach cultivars, and expression of both P5CS and P5CR genes could show contrasting patterns from each other. Our results suggest that identification of both P5CS and P5CR genes are required necessarily for accurate analysis of Pro biosynthesis because Pro accumulation is affected more by expression of P5CR gene.

A cDNA clone encoding CBL-interacting protein kinase 1 (CIPK1) was isolated from Chinese cabbage seedlings. The gene, BrCIPK1 consisted of 1,982 bp long with 216 bp of the 5’-untranslated region (UTR), 1,509 bp of the coding region and 257 bp of the 3’-UTR. It is highly conserved CBL-interacting module with absolutely conserved domain among the 15 amino acid NAF domain of the 15 related genes. Southern blot analysis showed a single copy number. BrCIPK1 gene was localized in the cytoplasm and peripheral region in the plant cell which is highly expressed in seedling of rice and in the shoot and pistil of Arabidopsis. Analyses of gene expression on Ubi-1::BrCIPK1 rice lines was differentially accumulated by cold, salinity and drought, indicating its biological roles in the multiple stress response pathways in plants. Further, the expression of BrCIPK1 is hijacked by rice calcineurin-B-like protein (OsCBL5). Moreover, mRNA expression of P5CS1, a gene responsible for proline biosynthesis is regulated by the BrCIPK1 during abiotic stresses resulting to improved accumulation of proline. The interaction of BrCIPK1 with OsCBL5 along with the regulation of P5CS1 explained the enhanced tolerance of transgenic rice. This gene could be used in the development of rice varieties with enhanced tolerance to abiotic stresses.

A cDNA clone encoding CBL-interacting protein kinase 1 (CIPK1) was isolated from Chinese cabbage seedlings. The gene, BrCIPK1 consisted of 1,982 bp long with 216 bp of the 5’-untranslated region (UTR), 1,509 bp of the coding region and 257 bp of the 3’-UTR. It is highly conserved CBL-interacting module with absolutely conserved domain among the 15 amino acid NAF domain of the 15 related genes. Southern blot analysis showed a single copy number. BrCIPK1 gene was localized in the cytoplasm and peripheral region in the plant cell which is highly expressed in seedling of rice and in the shoot and pistil of Arabidopsis. Analyses of gene expression on Ubi-1::BrCIPK1 rice lines was differentially accumulated by cold, salinity and drought, indicating its biological roles in the multiple stress response pathways in plants. Further, the expression of BrCIPK1 is hijacked by rice calcineurin-B-like protein (OsCBL5). Moreover, mRNA expression of P5CS1, a gene responsible for proline biosynthesis is regulated by the BrCIPK1 during abiotic stresses resulting to improved accumulation of proline. The interaction of BrCIPK1 with OsCBL5 along with the regulation of P5CS1 explained the enhanced tolerance of transgenic rice. This gene could be used in the development of rice varieties with enhanced tolerance to abiotic stresses.