Pisatin metabolism in pea ( Pisum sativum L.) cell suspension cultures

ABSTRACT: Cell suspension cultures were established from germinating pea (Pisum sativum L.) seeds. This cell culture, which accumulated pisatin, consisted mostly of single cells containing a few cell aggregates. The cells responded to treatment with a yeast glucan preparation with transient accumulation of pisatin in both cells and culture media. Addition of pisatin to cell cultures resulted in increased synthesis of pisatin. Phenylalanine ammonia-lyase, chalcone synthase and isoflavone reductase activities were present in untreated cells. Upon treatment with an elicitor preparation the activities of the first two enzymes showed a rapid, transient increase up to 20 hours after treatment. Isoflavone reductase showed a major and minor peak at 16 and 36 h, respectively, after elicitor treatment. The time course of the enzyme activity and pisatin accumulation is consistent with an elicitor-mediated respons

Stable expression and phenotypic impact of attacin E transgene in orchard grown apple trees over a 12 year period

<p>Abstract</p> <p>Background</p> <p>Transgenic trees currently are being produced by <it>Agrobacterium</it>-mediated transformation and biolistics. The future use of transformed trees on a commercial basis depends upon thorough evaluation of the potential environmental and public health risk of the modified plants, transgene stability over a prolonged period of time and the effect of the gene on tree and fruit characteristics. We studied the stability of expression and the effect on resistance to the fire blight disease of the lytic protein gene, <it>attacin E</it>, in the apple cultivar 'Galaxy' grown in the field for 12 years.</p> <p>Results</p> <p>Using Southern and western blot analysis, we compared transgene copy number and observed stability of expression of this gene in the leaves and fruit in several transformed lines during a 12 year period. No silenced transgenic plant was detected. Also the expression of this gene resulted in an increase in resistance to fire blight throughout 12 years of orchard trial and did not affect fruit shape, size, acidity, firmness, weight or sugar level, tree morphology, leaf shape or flower morphology or color compared to the control.</p> <p>Conclusion</p> <p>Overall, these results suggest that transgene expression in perennial species, such as fruit trees, remains stable in time and space, over extended periods and in different organs. This report shows that it is possible to improve a desirable trait in apple, such as the resistance to a pathogen, through genetic engineering, without adverse alteration of fruit characteristics and tree shape.</p

Anthocyanin Accumulation Provides Protection against High Light Stress While Reducing Photosynthesis in Apple Leaves

The photoprotective role of anthocyanin remains controversial. In this study, we explored the effects of anthocyanin on photosynthesis and photoprotection using transgenic &lsquo;Galaxy Gala&rsquo; apple plants overexpressing MdMYB10 under high light stress. The overexpression of MdMYB10 dramatically enhanced leaf anthocyanin accumulation, allowing more visible light to be absorbed, particularly in the green region. However, through post-transcriptional regulation, anthocyanin accumulation lowered leaf photosynthesis in both photochemical reaction and CO2 fixation capacities. Anthocyanin accumulation also led to a decreased de-epoxidation state of the xanthophyll cycle and antioxidant capacities, but this is most likely a response to the light-shielding effect of anthocyanin, as indicated by a higher chlorophyll concentration and lower chlorophyll a/b ratio. Under laboratory conditions when detached leaves lost carbon fixation capacity due to the limitation of CO2 supply, the photoinhibition of detached transgenic red leaves was less severe under strong white, green, or blue light, but it became more severe in response to strong red light compared with that of the wild type. In field conditions when photosynthesis was performed normally in both green and transgenic red leaves, the degree of photoinhibition was comparable between transgenic red leaves and wild type leaves, but it was less severe in transgenic young shoot bark compared with the wild type. Taken together, these data show that anthocyanin protects plants from high light stress by absorbing excessive visible light despite reducing photosynthesis

Transgenic Suppression of <i>AGAMOUS</i> Genes in Apple Reduces Fertility and Increases Floral Attractiveness

<div><p>We investigated the ability of RNA interference (RNAi) directed against two co-orthologs of <i>AGAMOUS</i> (<i>AG</i>) from <i>Malus domestica</i> (domestic apple, <i>MdAG</i>) to reduce the risks of invasiveness and provide genetic containment of transgenes, while also promoting the attractiveness of flowers for ornamental usage. Suppression of two <i>MdAG</i>-like genes, <i>MdMADS15</i> and <i>MdMADS22</i>, led to the production of trees with highly showy, polypetalous flowers. These “double-flowers” had strongly reduced expression of both <i>MdAG-</i>like genes. Members of the two other clades within in the <i>MdAG</i> subfamily showed mild to moderate differences in gene expression, or were unchanged, with the level of suppression approximately proportional to the level of sequence identity between the gene analyzed and the RNAi fragment. The double-flowers also exhibited reduced male and female fertility, had few viable pollen grains, a decreased number of stigmas, and produced few viable seeds after cross-pollination. Despite these floral alterations, RNAi-<i>AG</i> trees with double-flowers set full-sized fruit. Suppression or mutation of apple <i>AG</i>-like genes appears to be a promising method for combining genetic containment with improved floral attractiveness.</p></div

RNAi-<i>AG</i> double-flowers contained a small number of viable pollen grains.

<p>(a) Whole anthers of control flowers, (b) cross sectioned control anthers with (c) pollen grains which (d) stained viable. Viable grains stained dark pink (black arrow) while non-viable grains remained pale (grey arrow). (e) Whole anthers of double-flowers, (f) cross sectioned petaloid anthers of double-flowers with (g) pollen grains, (h) few of which were viable. The average percent viable pollen grains for control and double-flowers are shown. Bar = 200 μm.</p

RNAi-<i>AG</i> trees with double-flowers set fruit with retained petaloid anthers and increased locules.

<p>(a-d) Fruits from control trees shed most of their petals and contained five narrow central compartments. (e-h) Fruits from single-flowered events were similar in overall appearance to control fruits, retained the occasional petal, and contained five internal compartments. (i-l) Fruits formed from double-flowers retained a large cluster of petaloid anthers on their base, had a large hollow in the center of the fruit, and an increased number of locules. (m) Quantification of the average number of locules per fruit showed that events with double-flowers (1600 and 1601) had significantly more locules than control fruits, while fruits from a single flowered event (1609) were similar to controls. All fruits shown were formed in the absence of cross pollination. Bars show standard error of the mean, asterisks indicate significant differences (P < .01).</p

RNAi of apple <i>AG</i>-like genes led to showy, polypetalous flowers with anthers converted to petals.

<p>(a) Control trees developed flowers with a single whorl of five petals (b) as did four RNAi-<i>AG</i> events. (c, d) Four other RNAi-<i>AG</i> events developed flowers with additional whorls of petals and a lack of obvious anthers. Dissection of (e, f) non-transgenic control flowers with 5 sepals, 5 petals and 20 anthers; and (g, h) double-flowers with 5 sepals, 5 outer petals, and 20 inner floral organs with both petal and anther characteristics.</p

RNAi-<i>AG</i> double-flowers had reduced numbers of stigmas and partial conversion of styles to petals.

<p>(a) Quantification of the average numbers of stigmas per flower revealed that double-flower events 1600 and 1612 had significantly fewer stigmas than control flowers. Bars show standard error of the mean, asterisks indicate significant differences (P < .01). (b, e) Microscopy of stigmas and styles from control, (c, f) single-flowered transgenic, and (d, g) double-flowered trees showed that stigmas from double-flowers were smaller than those of control flowers and often had projections of petal-like tissue. Panels b-d and e-g were collected at the same respective magnifications, bar = 500 μm.</p

Fruit of double-flowers had few viable seeds after cross pollination.

<p>(a) Seed formation in the absence of cross pollination. (b) Seed formation with cross-pollination. (c) Germination mature seeds from cross-pollinated flowers. Bars show standard error over individual fruits. Asterisks show significant differences in total seeds as compared to control fruits (P < .01). (d) Examples of control and RNAi-<i>AG</i> fruits (from double-flowers) formed with and without cross-pollination.</p