Shu-Jen Wang

Regulation of starch granule-bound starch synthase I gene expression by circadian clock and sucrose in the source tissue of sweet potato

Studies on regulations of transitory starch synthesis and degradation in the leaf tissue are important for understanding how carbons could be distributed effectively from the source to sink tissues. Therefore, expressions of starch granule-bound starch synthase I (GBSSI) gene in leaves of sweet potato were studied under different photoperiodic conditions and various sugar treatments. Results indicated that accumulations of GBSSI mRNA and its protein were controlled by an endogenous biological clock. Starch accumulations in leaves also showed a pattern characteristic of circadian rhythm. In addition to circadian clock, sucrose also played an important role in regulating GBSSI mRNA accumulations. Although sucrose stimulated the transcription of GBSSI, it had no effect on the rhythmic pattern of GBSSI gene expressions. Protein phosphorylation/dephosphorylation were involved in the sucrose-related signal transduction for GBSSI gene expressions. However, the sugar sensing for regulation of GBSSI was independent of the hexokinase-mediated pathway. In conclusion, the GBSSI gene expression in leaves of sweet potato appears to be regulated by two independent pathways. First, light is responsible for setting up biological clock(s) that control the circadian expression of GBSSI gene; and second, light plays an indirect signal to enhance GBSSI mRNA accumulations mediated by the photosynthetic product, sucrose.

Simultaneous Detection of Eight Genetically Modified Maize Lines Using a Combination of Event-and Construct-Specific Multiplex-PCR Technique

To fulfill labeling and traceability requirement of genetically modified (GM) maize for trade and regulation, it is essential to develop an event-specific detection method for monitoring the presence of transgenes. In pursuit of this purpose, we systematically optimized and established a combined event- and construct-specific multiplex polymerase chain reaction (mPCR) technique for simultaneous detection of 8 GM maize lines. Altogether 9 sets of primers were designed, including six that were event-specific for Event176, Bt11, TC1507, NK603, MON863, and Mon810; two that were construct-specific for T25 and GA21, and one for an endogenous zein gene. The transgene in each GM maize line and the endogenous zein gene could be clearly detected and distinguished according to the different sizes of PCR amplicons. The limit of detection (LOD) was approximately 0.25% (v/v), although the detection can be as sensitive as 0.1% as demonstrated by the International Seed Testing Association (ISTA) proficiency test. This study further improves the current PCR-based detection method for GM maize. The method can be used in an easy, sensitive, and cost and time effective way for the identification and quality screening of a specific GM maize line.

Nucleotide sequence of a sporamin gene in sweet potato.

Sporamin is tissue specific and may account for more than 80% of the soluble protein found in tuberous root of sweet potato (Ipomoea batatas Lam.) (Maeshima et al., 1985). SDS-PAGE analysis indicates that sporamin may be resolved into two bands with molecular masses in the range of 25 kD. These two protein bands were characterized and classified into two subfamilies, based on the homology of their nucleotide sequences (Murakami et al., 1986; Hattori et al., 1989). Within the subfamilies, the homology may be greater than 90%, whereas the cDNAs between the two subfamilies share only 80% homology. Recent studies of two sporamin genes, gSPO-A1 and gSP0-B1, which belong to the two distinct subfamilies A and B, respectively, have shown that they are intronless genes (Hattori and Nakamura, 1988). Comparison of the 5’ flanking region also revea led t h e presence of two conserved sequence blocks, namely Suc box 2 and box 3 (Hattori and Nakamura, 1988; Tsukaya et al., 1991). In this paper, we present a nove1 sporamin gene, gSPOR5-31, which was identified to be an isogene of the gSPO-A1 within subfamily A. A hEMBL3 genomic library comprising 6 X 105 plaqueforming units was constructed with partially Sau3A-digested genomic DNA of tuberous roots. The library was screened by plaque hybridization using the cDNA probe of SP-B (an antisense strand of sporamin cDNA; K.-W. Yeh, unpublished results). One positive signal was obtained during the primary screening. It was purified to a single plaque, from which the DNA was extracted and characterized by restriction endonuclease and Southern blot analysis (Table I). The results showed that the clone (gSPOR5-31) contained an insert DNA of approximately 7 kb. When the insert DNA was digested by BamHI, resolved by agarose gel electrophoresis, and hybridized with the SP-B cDNA probe, only a 2.2-kb fragment clearly showed a strong signal. Therefore, it was subsequently subcloned into pGEM3Z (Promega) at the BamHI site for further studies. The nucleotide sequence of the 2.2-kb BamHI fragment was determined by nested deletion and primer walking. It included an open reading frame of 660 nucleotides corresponding to 220 amino acid residues. The genomic clone of gSPOR5-31 was similar to those of the gSPO-A1 and gSPO-B1 containing no intron. These observations suggest that sporamin genes are intronless. Analysis of the coding

Molecular regulation of starch accumulation in rice seedling leaves in response to salt stress

In this report we show that the starch content decreased in NaCl-stressed rice (Oryza sativa L.) seedling leaves during the daytime. Because photosynthetic efficiency and starch degradation enzyme activity were not significantly affected by the high salt, it is likely that this effect results from repression of starch biosynthesis. To determine the regulatory mechanism, the activities of enzymes such as ADP-glucose pyrophosphorylase (AGPase), granule-bound starch synthase (GBSS), soluble starch synthase (SSS) and starch branching enzyme (SBE) involved in starch synthetic pathway were examined. Data suggest that NaCl-induced repression of GBSS activity was the most significant factor reducing starch accumulation. Based on real-time RT-PCR analysis, the effect of salinity on GBSS expression was primarily controlled on the transcriptional level. Furthermore, the salt-induced decrease of both GBSSI and GBSSII gene expressions could be mostly contributed by ion-specific effect and not by osmotic stress. Although the mRNA accumulation of GBSSI and GBSSII can be down-regulated by exogenous ABA, the negative influence of salt stress on GBSSI and II gene expression could be chiefly mediated via an ABA-independent pathway.

Molecular regulation of sink–source transition in rice leaf sheaths during the heading period

The upper leaf sheath of rice (Oryza sativa L.) serves as a temporary starch sink before heading, subsequently becoming a carbon source tissue to the growing panicle at the post-heading stage. The time of sink–source transition in upper leaf sheaths is highly correlated to the panicle exsertion. Here, we found that the expression profiles of starch synthesis genes such as ADP-glucose pyrophosphorylase large subunit 2, granule-bound starch synthase II, soluble starch synthase I, starch branching enzyme (SBE) I, SBEIII, and SBEIV were highly correlated with starch content changes during the heading period in the second leaf sheath below the flag leaf. In addition, the α-amylase2A and β-amylase were considered as major genes that were in charge of starch degradation at the post-heading period. Of the five sucrose transporter (OsSUT) genes, OsSUT1 and OsSUT4 appeared to play an important role in sucrose loading into the phloem of source leaf sheaths. Moreover, the microarray-based data implied that the dominant processes associated with functional leaf sheath transition from sink to source were carbohydrate metabolism and the translocation of the carbon and nitrogen sources and inorganic phosphate.

Regulations of granule-bound starch synthase I gene expression in rice leaves by temperature and drought stress

Effects of temperature (15/10, 25/20, 30/25, and 35/30 °C) and drought stresses on the expression of granule-bound starch synthase I (GBSSI) gene were examined in rice (Oryza sativa L.) seedlings. The GBSSI expression was higher at the low temperature (15/10 °C), and the transcript level decreased at temperatures higher than 30 °C. Protein phosphorylation was involved in the low temperature-stimulated signal transduction of GBSSI regulation. The expression of GBSSI in rice seedling was reduced under a drought stress. Even though exogenous ABA played a role to reduce the GBSSI transcript accumulation under non-stress condition, the reducing of GBSSI expression by drought stress appeared to be mediated by an ABA-independent pathway.

New gene construction strategy in T-DNA vector to enhance expression level of sweet potato sporamin and insect resistance in transgenic Brassica oleracea

Sporamin, an abundant storage protein in tuberous roots of sweet potato, possesses strong inhibitory activity against trypsin and pest-resistance. To promote consistent high-level expression of sporamin and insect resistance in transgenic Brassica plants, a wound-responsive sporamin promoter (Pspoa) alone or combined with matrix-attached-region-like DNA segment (spoMAR) were constructed for driving sporamin cDNA. The results showed the transgenic plants containing Pspoa-drived sporamin and spoMAR displayed the highest level and low inter-transformant variability of sporamin expression, and the ability of insect resistance of transformants positively correlated with sporamin activity. Furthermore, expressions of Pspoa-drived sporamin especially combined with the spoaMAR retains high and steady levels in the T(1) and T(2) generations, in marked contrast to the variable expression patterns observed in CaMV35S promoter-driven transformants. This study evidently indicates that the Pspoa and spoaMAR would be very efficient for high transgene expression in plants and obtaining inherently stable transformants in consecutive progenies.

Tissue-specific regulation of rice molybdenum cofactor sulfurase gene in response to salt stress and ABA

Molybdenum-containing aldehyde oxidase is a key enzyme for catalyzing the final step of abscisic acid (ABA) biosynthesis in plants. Sulfuration of the molybdenum cofactor (MoCo) is an essential step for activating aldehyde oxidase. The molybdenum cofactor sulfurase (MCSU) that transfers the sulfur ligand to aldehyde oxidase-bound MoCo is thus considered an important factor in regulating the ABA levels in plant tissues. In this study, we identified the rice MCSU cDNA (OsMCSU), which is the first MCSU gene cloned in monocot species. According to the functional domain analysis of the predicted amino acid sequence, the OsMCSU protein contains a Nifs domain at its N-terminus and a MOSC domain at the C-terminus. Expression of the OsMCSU gene was up-regulated by salt stress in root tissues of rice seedlings, but this effect was not observed in leaf tissues. In roots, regulations of OsMCSU expressions could be mediated by both ABA-dependent and ABA-independent signaling pathways under salt stress condition.

Rice develop wavy seminal roots in response to light stimulus

Rice (Oryza sativa L.) seminal roots are the primary roots to emerge from germinated seeds. Here, we demonstrate that the photomorphology of the seminal roots was diverse among rice varieties, and the light-induced wavy roots were found mostly in indica-type rice varieties. The light-induced wavy morphology in rice seminal roots has been different with curling or coiling roots in some other specific conditions, such as high air humidity or high nitrogen nutrient. The efficiency of light-induced root waving was developmental stage dependent. The wavy root phenotype was caused by asymmetric cell growth around the stele. Using the inhibitors to block auxin polar transport and fatty acid oxygenation, the role of auxin and oxylipins in the morphogenesis of light-induced wavy roots was investigated. Expressions of genes encoded in the enzymes involved in fatty acid oxygenation in light-exposed roots were monitored by reverse transcriptase-polymerase chain reaction. Our results suggested that auxin polar transport was essential for inducing wavy seminal roots by light stimulus. In addition, the ketol oxylipins derived from allene oxide synthase (EC 4.2.1.92)-mediated fatty acid oxygenation function as intracellular signals for triggering the light-induced wavy root phenotype.

Circadian control of sweet potato granule-bound starch synthase I gene in Arabidopsis plants

A starch granule-bound starch synthase I (GBSSI) gene is regulated by a circadian clock in sweet potato leaves. In order to examine whether the promoter region is responsible for controlling a circadian expression of the GBSSI gene, the sweet potato GBSSI promoter was isolated and deleted to different lengths for functional analysis with a GUS reporter gene in transgenic Arabidopsis plants. Nuclear run-on transcriptional assays showed that the circadian control was regulated at the transcriptional rate level, and de novo synthesized proteins were necessary for controlling the rhythm. Promoter assays showed that the GBSSI promoter fragments containing six I-boxes, two putative circadian regulation elements (CAANNNNATC) and four circadian clock-associated 1 protein-binding sites (AATCT) maintained the activity to induce the circadian expression of the GUS gene. Similar to the GBSSI in sweet potato, GBSSI, soluble starch synthase and ADP-glucose pyrophosphorylase genes in Arabidopsis leaves also exhibited a circadian rhythm. These results suggested that common signals may exist in dicotyledonous plants to coordinate the circadian expression of genes involved in the transitory starch synthetic pathway.