Ming-Li Wang

Comparative expression analysis of two sugarcane polyubiquitin promoters and flanking sequences in transgenic plants

GUS (uidA) reporter gene expression for two sugarcane polyubiquitin promoters, ubi4 and ubi9, was compared to expression from the maize Ubi-1 promoter in stable transgenic rice (only ubi9) and sugarcane (ubi4 and ubi9). Ubi9 drove high-level GUS expression, comparable to the maize Ubi-1 promoter, in both callus and regenerated plants of rice transformed by Agrobacterium. This high level expression was inherited in R1 plants. Expression from ubi4 and ubi9 was quite high in sugarcane callus transformed via particle bombardment. Expression dropped to very low or undetectable levels in the resulting plants; this drop in expression resulted from PTGS. PTGS in regenerated sugarcane plants also occurred with the maize Ubi-1 promoter. In sugarcane callus, ubi4 was HS inducible, but ubi9 was not. This physiological difference corresponds to a MITE insertion that is present in the putative HSEs of ubi9 but not present in ubi4.

Production of biologically active GM-CSF in sugarcane: a secure biofactory

Over 300 transgenic sugarcane plants representing approx. 200 independent lines producing the human cytokine granulocyte macrophage colony stimulating factor (GM-CSF) were analyzed for recombinant protein accumulation and activity levels. Expression constructs differed in use of the maize polyubiquitin 1, Mubi-1, or the sugarcane polyubiquitin 9, SCubi9, promoters; presence or absence of a C-terminal HDEL tag for ER retention; and presence or absence of a 6X Histidine tag for metal ion affinity purification. Accumulation of GM-CSF protein ranged from undetectable to 0.02 of total soluble protein. No significant difference was observed between the two promoters; however, the ER retention tag was required for higher accumulation levels. Human bone marrow cells (TF-1), which require GM-CSF for cell division, proliferated when growth media was supplemented with transgenic sugarcane extracts. Comparison to purified commercially produced GM-CSF indicated the sugarcane-produced protein had essentially identical activity levels. In a 14-month field trial, accumulation levels remained stable. This is the first report of field production of GM-CSF. During the field trial, no flowering of the trial plants occurred; no pollen or seed was produced. Drying, burning, and burial of the test plants effectively blocked possible routes for the transgenic sugarcane to enter the environment or food supply. Sugarcane may provide a highly secure system for biofactory production of pharmaceutical proteins.

The P0 gene of Sugarcane yellow leaf virus encodes an RNA silencing suppressor with unique activities.

The Sugarcane yellow leaf virus (SCYLV) P0, a member of the highly heterologous proteins of poleroviruses, is a suppressor of posttranscriptional gene silencing (PTGS) and has additional activities not seen in other P0 proteins. The P0 protein in previously tested poleroviruses (Beet western yellows virus and Cucurbit aphid-borne yellows virus), suppresses local, but not systemic, PTGS induced by both sense GFP and inverted repeat GF using its F-box-like domain to mediate destabilization of the Argonaute1 protein. We now report that the SCYLV P0 protein not only suppressed local PTGS induced by sense GFP and inverted repeat GF in Nicotiana benthamiana, but also triggered a dosage dependent cell death phenotype in infiltrated leaves and suppressed systemic sense GFP-PTGS. Deletion of the first 15 N-terminal amino acid residues of SCYLV P0 abolished suppression of both local and systemic PTGS and the induction of cell death. In contrast, only systemic PTGS and cell death were lost when the 15 C-terminal amino acid residues were deleted. We conclude that the 15 C-terminal amino acid residue region of SCYLV P0 is necessary for suppressing systemic PTGS and inducing cell death, but is not required for suppression of local PTGS.

Biosynthesis of chlorogenic acids in growing and ripening fruits of Coffea arabica and Coffea canephora plants.

Chlorogenic acids are major secondary metabolites found in coffee seeds. The accumulation of chlorogenic acids and free quinic acids was studied in Coffea arabica cv. Tall Mokka and Coffea canephora seeds. Growth stages are specified from I to V, corresponding to rapid expansion and pericarp growth (I), endosperm formation (II), mature (green) (III), ripening (pink) (IV), and fully ripened (red) (V) stages. We detected monocaffeoylquinic acids (3CQA, 4CQA and 5CQA), dicaffeoylquinic acids (3,4diCQA, 3,5diCQA and 4,5diCQA) and a monoferuloylquinic acid (5FQA) in whole fruits (stage I), pericarps and seeds. The most abundant chlorogenic acid was 5CQA, which comprised 50-60% of the total of C. arabica and 45-50% of C. canephora seeds. The content of dicaffeoylquinic acid, mainly 3,5d diCQA, was high in C. canephora. A high content of 5FQA was found in seeds of stages III to V, especially in C. canephora. Total chlorogenic acids were accumulated up to 14 mg per fruit in C. arabica and 17 mg in C. canephora, respectively. In contrast, free quinic acid varied from 0.4-2.0 mg (C. arabica) and 0.2-4.0 mg (C. canephora) per fruit during growth. High biosynthetic activity of 5CQA, which was estimated via the incorporation of [U-14C]phenylalanine into chlorogenic acids, was found in young fruits (perisperm and pericarp) in stage I, and in developing seeds (endosperm) in stages II and III. The biosynthetic activity of chlorogenic acids was clearly reduced in ripening and ripe seeds, especially in C. canephora. Transcripts of PAL1, C3′H and CCoAMT, three genes related to the chlorogenic acid biosynthesis, were detected in every stage of growth, although the amounts were significantly less in stage V. Of these genes, CCoAMT, a gene for FQA biosynthesis, was expressed more weakly in stage I. The transcript level of CCoAMT was higher in seeds than in pericarp, but the reverse was found in PAL1. The pattern of expression of genes for the CQA and FQA synthesis is roughly related to the estimated biosynthetic activity, and to the accumulation pattern of chlorogenic acids.

Immobilized mixed microbial cells for wastewater treatment

Abstract Both mono-carrier (cellulose triacetate) and bi-carrier (combined calcium alginate and cellulose triacetate) were used to treat synthetic organic wastewater (glucose and phenol) in an aerobic system. It was found, by means of scanning electron microscopy, that the mono-carrier entrapping process offers a better treatment efficiency (based on COD removal), and more holding capacity of microbial cells. Greater than 90% of COD removal efficiency could be achieved with loading rates of less than 9·0 g COD liter −1 day −1 . Immobilized mixed microbial cells via entrapping method maintains higher SRT which is essential for the operation of a stable and effective biological treatment process.

Analysis of pineapple mealybug wilt associated virus -1 and -2 for potential RNA silencing suppressors and pathogenicity factors.

Higher plants use RNA silencing to defend against viral infections. As a counter defense, plant viruses have evolved proteins that suppress RNA silencing. Mealybug wilt of pineapple (MWP), an important disease of pineapple, has been associated with at least three distinct viruses, Pineapple mealybug wilt associated virus -1, -2, and -3 (PMWaV-1, -2, and -3). Selected open reading frames (ORFs) of PMWaV-1 and PMWaV-2 were screened for their local and systemic suppressor activities in Agrobacterium-mediated transient assays using green fluorescent protein (GFP) in Nicotiana benthamiana. Results indicate that PMWaV-2 utilizes a multiple-component RNA silencing suppression mechanism. Two proteins, p20 and CP, target both local and systemic silencing in N. benthamiana, while the p22 and CPd proteins target only systemic silencing. In the related virus PMWaV-1, we found that only one of the encoded proteins, p61, had only systemic suppressor activity. Of all the proteins tested from both viruses, only the PMWaV-2 p20 protein suppressed local silencing induced by double-stranded RNA (dsRNA), but only when low levels of inducing dsRNA were used. None of the proteins analyzed could interfere with the short distance spread of silencing. We examined the mechanism of systemic suppression activity by investigating the effect of PMWaV-2-encoded p20 and CP proteins on secondary siRNAs. Our results suggest that the PMWaV-2 p20 and CP proteins block the systemic silencing signal by repressing production of secondary siRNAs. We also demonstrate that the PMWaV-2 p20 and p22 proteins enhanced the pathogenicity of Potato virus X in N. benthamiana.

Characterization of Prolyl Oligopeptidase Genes Differentially Expressed Between Two Cultivars of Coffea arabica L.

Two closely related, Coffea arabica cultivars Tall Mokka and Typica, yield coffee with excellent flavor, but they differ distinctively in the size of aerial organs including the fruit and in the degree of branching. Using potato cross-species microarray hybridization, we identified prolyl oligopeptidase (CaPOP) gene(s) as differentially expressed between the shoot tips of ‘Tall Mokka’ and ‘Typica’. Isolation and sequencing of these POP genes identified three paralogs, CaPOP1, CaPOP2 and CaPOP3. All three genes were present in both cultivars, which suggest that differences in the expression of CaPOP are caused by factor(s) regulating their transcription. CaPOP1 differs in sequence from CaPOP2 primarily in having two large deletions in the promoter region. CaPOP3 is probably a recombination product of CaPOP1 and CaPOP2 and encodes a protein identical to CaPOP2. Coffee’s POP (CaPOP) genes are homologous to the arabidopsis gene At1g20380, encoding a post-proline cleaving enzyme that acts on substrates shorter than 30 amino acids. Ectopic expression of CaPOP1 under its native promoter in transgenic arabidopsis resulted in more secondary branches than in the wild type. This is the first study to successfully isolate CaPOP genes and characterize their expression in the developing tissues of coffee. This study also reports a novel role for prolyl oligopeptidase in control of lateral shoot branching.

Analyzing the Papaya Genome

Papaya is a major fruit crop in tropical and subtropical regions worldwide. It has long been recognized as a nutritious fruit with medicinal properties. It has a small genomes and highly homozygous because hermaphrodite varieties are self-pollinated. The novel features of nascent sex chromosomes in papaya and its agricultural importance are additional justifications for sequencing the genome. A female plant of the transgenic variety SunUp was selected for sequencing to avoid the complication of assembling the XY chromosomes in a male or hermaphrodite plant. The draft genome revealed fewer genes than sequenced genomes of flowering plants, partly due to its lack of genome-wide duplication since the ancient triplication event shared by eudicots. Most gene families have fewer members in papaya, including significantly fewer disease resistance genes. However, striking amplifications in gene number were found in some functional groups, including MADS-box genes, starch synthases, and volatiles that might affect the speciation and adaptation of papaya. The draft genome was used to accelerate the construction of physical maps of sex chromosomes in papaya and to clone a gene controlling fruit flesh color. The papaya draft genome, integrated genetic and physical maps, the EST database, and other genomic resources built for the genome sequencing project will expedite papaya improvement and the exploration of its nutritional and medicinal applications in developing countries.