Lily Cdc42/Rac-interactive binding motif-containing protein, a Rop target, involves calcium influx and phosphoproteins during pollen germination and tube growth

We report unique desiccation-associated ABA signaling transduction through which the Rop (Rho GTPase of plants) and its target LLP12-2 are regulated during the stage of pollen maturation and tube growth. Overexpression of LLP12-2 drastically inhibited pollen germination and tube growth. Studies on the germination inhibitors, Ca2+ influx blocking agents LaCl3 and EGTA and an actin-depolymerizing drug, latrunculin B (LatB), revealed that the LLP12-2-induced inhibition of germination and tube growth is significantly suppressed by LaCl3 and EGTA in the LLP12-2-overexpressing pollen but not by LatB. These results suggested that LLP12-2 is associated with Ca2+ influx in the cytoplasm and may be not with actin assembly. With the addition of LaCl3 and EGTA, LLP12-2-overexpressing pollen increased germination and tube growth compared with the one without addition, whereas pollen expressing GFP decreased germination and tube growth. Thus, an optimum level of [Ca2+]cyt influx is crucial for normal germination and tube growth. Studies on the inhibitors, staurosporine and okadaic acid in the LLP12-2-overexpressing pollen, showed no appreciable increase in germination when compared with the one without addition, suggesting that staurosporine-sensitive protein kinases and dephosphorylation of phosphoproteins may be not involved in the LLP12-2 mediated germination. However, the LLP12-2-induced inhibition of tube length was slightly but significantly suppressed by staurosporine, suggesting that staurosporine-sensitive protein kinases involve in the LLP12-2-induced inhibition of tube growth

Molecular cloning and functional analysis of bergaptol-O-methyltransferase from Angelica dahurica (Bai Zhi) and using it to efficiently produce bergapten in E. coli

Bai Zhi (Angelica dahurica), a Chinese herb, has long been used as a face cream for skin-whitening purposes. One of the known skin-whitening components, 8-hydroxybergapten is a hydroxylated product of bergapten that is converted from bergaptol by bergaptol 5-O-methyltransterase (BMT) in Bai Zhi. The complementary DNA of BMT was cloned from Bai Zhi root using a pair of degenerate primers designed from the highly conserved regions of other plant O-methyltransferases (OMTs). RT-PCR analysis indicated that a single band of DNA fragment corresponding to AdBMT sequence was obtained. The tandem 5'- and 3'-rapid amplification of cDNA ends via polymerase chain reaction was used to obtain the full-length cDNA sequences. The AdBMT cDNA contains an open reading frame of 1,080 bp encoding a BMT polypeptide of 359 amino acids with a calculated molecular mass of 39 kDa and a calculated pI of 5.9. Sequence alignment revealed the considerable sequence similarity of AdBMT to those of other plant OMTs. The AdBMT sequence contains conserved region I-V, similar to other plant OMTs. His-tagged AdBMT was expressed in E. coli and partially purified by ammonium sulfate precipitation. The recombinant AdBMT is most active in potassium phosphate buffer at pH 7.5 and 35 degrees C. The enzyme does not require a divalent cation for activity and the addition of Cu2+, Ni2+, and Co2+ at concentrations even as low as 0.1 mM severely inhibits enzyme activity. A simple and efficient production of bergapten in the E. coli culture overexpressing AdBMT was performed. The bergapten yield is approximately 13-fold higher than that produced by enzymes in the ammonium sulfate-purified fraction. With the supply of bergaptol in the medium, E. con cells can be used as a potential bioreactor to produce bergapten

APPLICATION OF LLA23 GENE THAT CONFERS IMPROVED HEAT AND FREEZING RESISTANCE IN PLANTS

本發明係一種基因片段在使植物具有增加的耐熱和/或抗凍能力上的用途。本發明亦提供一種用於使植物具有增加的耐熱和/或抗凍能力的組成物，其包含：一載劑以及一重組載體，其中該重組載體具有一基因片段以及一與該基因片段可操作地相連結的控制序列，其中該基因片段係具有下列之核苷酸序列：(i)SEQ ID NO.1之核苷酸序列；(ii)具有與SEQ ID NO.l之核苷酸序列相似性大於90%以上的核苷酸序列；(iii)編碼SEQ ID NO.2之胺基酸序列的核苷酸序列；或(iiii)編碼和SEQ ID NO.2實質上相同之胺基酸序列的核苷酸序列。本發明亦提供一種利用前述載體來製備具有增加的耐熱和/或抗凍能力的重組細胞或轉殖植物之方法。本發明亦提供一種利用前述載體所製得的重組細胞

Identification of the tapetum/microspore-specific promoter of the pathogenesis-related 10 gene and its regulation in the anther of Lilium longiflorum

A tapetum/microspore-specific pathogenesis-related (PR) 10 gene was previously identified in lily (Lilium longiflorum Thunb.) anthers. In situ hybridization and RNA blot analysis indicated that the lily PR10 genes are expressed specifically and differentially in the tapetum of the anther wall and in microspores during anther development. The accumulation of PR10 transcripts was exogenously induced by gibberellic acid (GA) and was suppressed by ethylene. Studies using inhibitors of GA and ethylene revealed that the lily PR10 is modulated by an antagonistic interaction between GA and ethylene. The treatment of norbornadien, an ethylene inhibitor, caused the tapetum to become densely cytoplasmic and highly polarized, whereas uniconazole, an inhibitor of GA biosynthesis, arrested tapetal development to a status close to that of control. The expression of the lily PR10g promoter in transgenic Arabidopsis was determined using the β-glucuronidase (GUS) reporter gene indicated that the decisive fragment required for anther specificity is located -1183 bp to -880 bp upstream of the transcription start site. The PR10gPro::barnase transgenic lines exhibited complete male sterility because of the disruption of the tapetum and the deformation of microspore/pollen. The anther specificity of lily PR10 highlights the importance of the tapetum/microspore-specific PR10g promoter for future biotechnological and agricultural applications

AtRH57, a DEAD-box RNA helicase, is involved in feedback inhibition of glucose-mediated abscisic acid accumulation during seedling development and additively affects pre-ribosomal RNA processing with high glucose

The Arabidopsis thaliana T-DNA insertion mutant rh57-1 exhibited hypersensitivity to glucose (Glc) and abscisic acid (ABA). The other two rh57 mutants also showed Glc hypersensitivity similar to rh57-1, strongly suggesting that the Glc-hypersensitive feature of these mutants results from mutation of AtRH57. rh57-1 and rh57-3 displayed severely impaired seedling growth when grown in Glc concentrations higher than 3%. The gene, AtRH57 (At3g09720), was expressed in all Arabidopsis organs and its transcript was significantly induced by ABA, high Glc and salt. The new AtRH57 belongs to class II DEAD-box RNA helicase gene family. Transient expression of AtRH57-EGFP (enhanced green fluorescent protein) in onion cells indicated that AtRH57 was localized in the nucleus and nucleolus. Purified AtRH57-His protein was shown to unwind double-stranded RNA independent of ATP in vitro. The ABA biosynthesis inhibitor fluridone profoundly redeemed seedling growth arrest mediated by sugar. rh57-1 showed increased ABA levels when exposed to high Glc. Quantitative real time polymerase chain reaction analysis showed that AtRH57 acts in a signaling network downstream of HXK1. A feedback inhibition of ABA accumulation mediated by AtRH57 exists within the sugar-mediated ABA signaling. AtRH57 mutation and high Glc conditions additively caused a severe defect in small ribosomal subunit formation. The accumulation of abnormal pre-rRNA and resistance to protein synthesis-related antibiotics were observed in rh57 mutants and in the wild-type Col-0 under high Glc conditions. These results suggested that AtRH57 plays an important role in rRNA biogenesis in Arabidopsis and participates in response to sugar involving Glc- and ABA signaling during germination and seedling growth