https://munin.uit.no/bitstream/handle/10037/5893/article.pdf?sequence=1

New insights into the regulation of anthocyanin biosynthesis in fruits

Background

The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the gene determining fruit flesh and foliage anthocyanin has been termed MYB10. In order to further understand tissue-specific anthocyanin regulation we have isolated orthologous MYB genes from all the commercially important rosaceous species.

Results

We use gene specific primers to show that the three MYB activators of apple anthocyanin (MYB10/MYB1/MYBA) are likely alleles of each other. MYB transcription factors, with high sequence identity to the apple gene were isolated from across the rosaceous family (e.g. apples, pears, plums, cherries, peaches, raspberries, rose, strawberry). Key identifying amino acid residues were found in both the DNA-binding and C-terminal domains of these MYBs. The expression of these MYB10 genes correlates with fruit and flower anthocyanin levels. Their function was tested in tobacco and strawberry. In tobacco, these MYBs were shown to induce the anthocyanin pathway when co-expressed with bHLHs, while over-expression of strawberry and apple genes in the crop of origin elevates anthocyanins.

Conclusions

This family-wide study of rosaceous R2R3 MYBs provides insight into the evolution of this plant trait. It has implications for the development of new coloured fruit and flowers, as well as aiding the understanding of temporal-spatial colour change.

An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae

Identification and validation of promoters and cis-acting regulatory elements.

transformation, co-transformation, linked transgenes, marker gene excision, metabolic engineering, multigene manipulation, polyproteins, pyramiding, retransformation, stacking.

Gene stacking in transgenic plants--the challenge for 21st century plant biotechnology.

Plants offer an alternative to microbial fermentation and animal cell cultures for the production of recombinant proteins. For protein pharmaceuticals, plant systems are inherently safer than native and even recombinant animal sources. In addition, post-translational modifications, such as glycosylation, which cannot be achieved with bacterial fermentation, can be accomplished using plants. The main advantage foreseen for plant systems is reduced production costs. Plants should have a particular advantage for proteins produced in bulk, such as industrial enzymes, for which product pricing is low. In addition, edible plant tissues are well suited to the expression of vaccine antigens and pharmaceuticals for oral delivery. Three approaches have been followed to express recombinant proteins in plants: expression from the plant nuclear genome; expression from the plastid genome; and expression from plant tissues carrying recombinant plant viral sequences. The most important factor in moving plant-produced heterologous proteins from developmental research to commercial products is to ensure competitive production costs, and the best way to achieve this is to boost expression. Thus, considerable research effort has been made to increase the amount of recombinant protein produced in plants. This research includes molecular technologies to increase replication, to boost transcription, to direct transcription in tissues suited for protein accumulation, to stabilize transcripts, to optimize translation, to target proteins to subcellular locations optimal for their accumulation, and to engineer proteins to stabilize them. Other methods include plant breeding to increase transgene copy number and to utilize germplasm suited to protein accumulation. Large-scale commercialization of plant-produced recombinant proteins will require a combination of these technologies.

Approaches to achieve high-level heterologous protein production in plants.

Expression of a bifunctional green fluorescent protein (GFP) fusion marker under the control of three constitutive promoters and enhanced derivatives in transgenic grape (Vitis vinifera).

article i nfo The effects of tillage on the interaction between soil structure and microbial biomass vary spatially and temporally for different soil types and cropping systems. We assessed the relationship between soil structure induced by tillage and soil microbial activity at the level of soil aggregates. To this aim, organic C (OC), microbial biomass C (MBC) and soil respiration were measured in water-stable aggregates (WSA) of different sizes from a subtropical rice soil under two tillage systems: conventional tillage (CT) and a combination of ridge with no-tillage (RNT). Soil (0-20 cm) was fractionated into six different aggregate sizes (N4.76, 4.76- 2.0, 2.0-1.0, 1.0-0.25, 0.25-0.053, and b0.053 mm in diameter). Soil OC, MBC, respiration rate, and metabolic quotient were heterogeneously distributed among soil aggregates while the patterns of aggregate-size distribution were similar among properties, regardless of tillage system. The content of OC within WSA followed the sequence: medium-aggregates (1.0-0.25 mm and 1.0-2.0 mm)Nmacro-aggregates (4.76- 2.0 mm)Nmicro-aggregates (0.25-0.053 mm)Nlarge aggregates (N4.76 mm)Nsilt+clay fractions (b0.053 mm). The highest levels of MBC were associated with the 1.0-2.0 mm aggregate size class. Significant differences in respiration rates were also observed among different sizes of WSA, and the highest respiration rate was associated with 1.0-2.0 mm aggregates. The Cmic/Corg was greatest for the large- macroaggregates regardless of tillage regimes. This ratio decreased with aggregate size to 1.0-0.25 mm. Soil metabolic quotient (qCO2) ranged from 3.6 to 17.7 mg CO2 g −1 MBC h −1 . The distribution pattern of soil microbial biomass and activity was governed by aggregate size, whereas the tillage effect was not significant at the aggregate scale. Tillage regimes that contribute to greater aggregation, such as RNT, also improved soil microbial activity. Soil OC, MBC and respiration rate were at their highest levels for 1.0-2.0 mm aggregates, suggesting a higher biological activity at this aggregate size for the present ecosystem.

https://soils.ifas.ufl.edu/wetlands/publications/PDF-articles/368.Long-term%20tillage%20effects%20on%20the%20distribution%20patterns%20of%20microbial%20biomass%20and%20activities%20within%20soil%20aggregates.pdf

Long-term tillage effects on the distribution patterns of microbial biomass and activities within soil aggregates

Novel bi-directional duplex promoters (BDDP) were constructed by placing two identical core promoters divergently on both upstream and downstream sides of their duplicated enhancer elements. Estimates of promoter function were obtained by creating versions of CaMV 35S and CsVMV BDDPs that contained reporter marker genes encoding β-glucuronidase (GUS) and enhanced green fluorescent protein (EGFP) interchangeably linked either to the upstream or downstream core promoters. GUS was used for quantitative analysis of promoter function, whereas, EGFP allowed visual qualitative evaluation. In addition, the GUS and EGFP genes placed in downstream positions were modified by translational fusion with neomycin phosphotransferase (NPTII) to allow simultaneous monitoring of promoter activity and selection of stable transformants. These versions of BDDP were compared with each other and with equivalent unidirectional constructs by evaluating their expression in grape and tobacco. For 35S promoter constructs tested in grape somatic embryos (SE), BDDP exhibited transient GUS expression 206- and 300-fold greater in downstream and upstream configurations, respectively, compared to a unidirectional 35S core promoter. Compared with a unidirectional double enhanced 35S promoter, BDDPs exhibited 0.5- and 3-fold increased GUS expression from downstream and upstream core promoters, respectively. The same differences in expression levels determined quantitatively with GUS were distinguished qualitatively with EGFP. Constructs using CsVMV core promoters yielded results relative to those obtained with 35S promoter. For example, the upstream BDDP CsVMV core promoter provided a 200-fold increase in GUS expression compared to a unidirectional core promoter. However, CsVMV promoter was found to have higher promoter activity than 35S promoter in both BDDP and unidirectional constructs. Incorporation of an additional duplicated enhancer element to BDDPs resulted in increased expression. For example, a 35S BDDP with two divergently arranged duplicated enhancer elements resulted in over a 6-fold increase in GUS expression in stably transformed tobacco plants compared to a BDDP with one duplicated enhancer element. Data demonstrate that BDDP composed of divergently-arranged core promoters separated by duplicated enhancers, all derived from a single promoter sequence, can be used to significantly enhance transgene expression and to direct synchronized expression of multiple transgenes.

Bi-directional Duplex Promoters with Duplicated Enhancers Significantly Increase Transgene Expression in Grape and Tobacco

Proembryogenic masses of grapevine (Vitis vinifera L.) `Chardonnay' (clone 02Ch) were exposed to the culture filtrate of Elsinoe ampelina (deBary) Shear, the causal agent of anthracnose disease. After four or five cycles of recurrent in-vitro selection with medium containing 40% fungal culture filtrate, putative resistant lines RC 1 and RC 2 respectively, were established. The selected lines inhibited the growth of E. ampelina and Fusarium oxysporium (Schlecht.) (isolated from watermelon) in a dual-culture assay and reduced the growth of mycelium on a conditioned-medium test, thus suggesting the involvement of extracellular compounds in resistance. Sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis of extracellular proteins from spent suspension-culture medium showed enhanced secretion of new proteins by selected lines. A 36-kDa protein was immunodetected by a chitinase antiserum. This chitinase continued to express constitutively in differentiated somatic embryos and also in the intercellular fluids of plants regenerated from the selected lines. Somatic embryos from selected lines grew uninhibitedly in a medium containing 40% fungal culture filtrate, whereas non-selected (control) somatic embryos became necrotic and died within a few days. Plants regenerated from selected lines exhibited resistance to infection by E. ampelina in both greenhouse tests and detached leaf bioassays. Results suggest that embryogenic cells can be selected for resistance following in-vitro selection, resulting in resistant plants. Whether or not resistant cells pre-existed in the original embryogenic culture or were induced by the selection pressure could not be determined.

In-vitro selection of Vitis vinifera 'Chardonnay' with Elsinoe ampelina culture filtrate is accompanied by fungal resistance and enhanced secretion of chitinase.

Cisgenic engineering involves isolation and modification of genetic elements from the host genome, which are reinserted to develop plant varieties with improved characteristics. As a first step toward production of fungal-disease resistant cisgenic grapevines, the Vitis vinifera thaumatin-like protein (vvtl-1) gene was isolated from “Chardonnay” and reengineered for constitutive expression. Embryogenic cultures of “Thompson Seedless” were initiated from leaves and transformed with Agrobacterium to regenerate cisgenic VVTL-1 plants. Cisgene presence and copy number were confirmed by PCR and quantitative real-time PCR. Protein expression was measured using ELISA. Among the plant lines tested, two exhibited a 7–10 day delay in powdery mildew disease development during greenhouse screening and decreased severity of black rot disease in field tests. Berries exhibited a 42.5% reduction in sour-bunch rot disease incidence compared to non-transformed controls after 3 wk of storage at room temperature. Although plants recovered in this study contain viral promoters and reporter/marker genes, this is the first report of a cisgenic approach to obtain broad-spectrum fungal-disease resistance in genetically engineered grapevine.

Zhijian T. Li

Papers

Expression of a bifunctional green fluorescent protein (GFP) fusion marker under the control of three constitutive promoters and enhanced derivatives in transgenic grape (Vitis vinifera).

Long-term tillage effects on the distribution patterns of microbial biomass and activities within soil aggregates

Bi-directional Duplex Promoters with Duplicated Enhancers Significantly Increase Transgene Expression in Grape and Tobacco

In-vitro selection of Vitis vinifera 'Chardonnay' with Elsinoe ampelina culture filtrate is accompanied by fungal resistance and enhanced secretion of chitinase.

Grapevines engineered to express cisgenic Vitis vinifera thaumatin-like protein exhibit fungal disease resistance