Characterization of ScORK28, a transmembrane functional protein receptor kinase predominantly expressed in ovaries from the wild potato species Solanum chacoense

AbstractSolanum chacoense ovule receptor kinase 28 (ScORK28) was found among 30 receptor kinases from an ovule cDNA library enriched for weakly expressed mRNAs. This LRR-RLK displayed high level of tissue specificity at the RNA and protein levels and was predominantly expressed in female reproductive tissues. Protein expression analyses in planta revealed that ScORK28 was N-glycosylated and ScORK28::GFP fusion analyses showed that it was localized at the plasma membrane. Bacterial expression of ScORK28 catalytic domain followed by kinase activity assays revealed that ScORK28 is an active Mg2+-dependent protein kinase and that the juxtamembrane domain is necessary for kinase activity

Identification of genes preferentially expressed in wheat egg cells and zygotes

Wheat genes differentially expressed in the egg cell before and after fertilization were identified. The data support zygotic gene activation before the first cell division in wheat. To have an insight into fertilization-induced gene expression, cDNA libraries have been prepared from isolated wheat egg cells and one-celled zygotes. Two-hundred and twenty-six egg cell and 253 zygote-expressed EST sequences were determined. Most of the represented transcripts were detected in the wheat egg cell or zygote transcriptome at the first time. Expression analysis of fourteen of the identified genes and three controls was carried out by real-time quantitative PCR. The preferential expression of all investigated genes in the female gametophyte-derived samples (egg cells, zygotes, two-celled proembryos, and basal ovule parts with synergids) in comparison to the anthers, and the leaves were verified. Three genes with putative signaling/regulatory functions were expressed at a low level in the egg cell but exhibited increased (2-to-33-fold) relative expression in the zygote and the proembryo. Genes with high EST abundance in cDNA libraries exhibited strong expression in the egg cell and the zygote, while the ones coding for unknown or hypothetical proteins exhibited differential expression patterns with preferential transcript accumulation in egg cells and/or zygotes. The obtained data support the activation of the zygotic genome before the first cell division in wheat

Downregulation of RWA genes in hybrid aspen affects xylan acetylation and wood saccharification

High acetylation of angiosperm wood hinders its conversion to sugars by glycoside hydrolases, subsequent ethanol fermentation and (hence) its use for biofuel production. We studied the REDUCED WALL ACETYLATION (RWA) gene family of the hardwood model Populus to evaluate its potential for improving saccharification. The family has two clades, AB and CD, containing two genes each. All four genes are expressed in developing wood but only RWA-A and -B are activated by master switches of the secondary cell wall PtNST1 and PtMYB21. Histochemical analysis of promoter:: GUS lines in hybrid aspen (Populus tremula x tremuloides) showed activation of RWA-A and -B promoters in the secondary wall formation zone, while RWA-C and -D promoter activity was diffuse. Ectopic downregulation of either clade reduced wood xylan and xyloglucan acetylation. Suppressing both clades simultaneously using the wood-specific promoter reduced wood acetylation by 25% and decreased acetylation at position 2 of Xylp in the dimethyl sulfoxide-extracted xylan. This did not affect plant growth but decreased xylose and increased glucose contents in the noncellulosic monosaccharide fraction, and increased glucose and xylose yields of wood enzymatic hydrolysis without pretreatment. Both RWA clades regulate wood xylan acetylation in aspen and are promising targets to improve wood saccharification.Peer reviewe

Towards genetic modification of the lignin biosynthetic pathway in interior spruce (Picea glauca x engelmanni complex)

Although the lignin biosynthetic pathway has been altered successfully in angiosperm species via genetic engineering approach, this has not yet been achieved in gymnosperm species. Therefore, the goal of my thesis research was to apply the transgenic approach to economically important interior spruce (Picea glauca x engelmanni complex). In the first half of my thesis, the poplar PAL2-GUS fusion gene was introduced into hybrid poplar (Populus tremula x P. alba) and interior spruce in order to evaluate the potential use of this promoter for directing xylem-specific gene expression. In transgenic poplar, the poplar PAL2 promoter directed the expression of the GUS gene in the tissues associated with synthesis of phenolic compounds (epidermal/subepidermal cells) and in the tissues associated with lignin synthesis (xylem and phloem cells). In contrast, in transgenic spruce, the activity of the poplar PAL2 promoter was detected only in the tissues associated with lignin and suberin synthesis. The differences in the activity of the poplar PAL2 promoter between the two hosts suggest that the gene regulation system that leads to the synthesis of essential structural components such as lignin and suberin is more likely to be conserved than that leading to the synthesis of specialized phenolic compounds. The second half of this thesis investigated a contribution of coniferin (β-glucosidase (CG) in lignin synthesis in spruce. CG is believed to be involved in the last steps of the lignin biosynthetic pathway by releasing the lignin precursor, coniferyl alcohol, from its glucosides form, coniferin, before polymerization in the cell wall. An antisense construct against the CG gene was prepared using the lodgepole pine CG cDNA sequence and was introduced into interior spruce. Among 45 antisense lines examined, no transgenic lines contained reduced levels of endogenous CG mRNA levels. The failure of the antisense CG gene to cause inhibitory effects on the endogenous CG expression levels in transgenic spruce could be attributed to low expression of the antisense gene, or to insufficient sequence homology between the antisense and target CG sequences. To my knowledge, this is the first study to employ an antisense approach in a gymnosperm speciesLand and Food Systems, Faculty ofGraduat

160 students, 20 groups, 1 TA: transforming a large classroom into a small classroom-like environment.

Class size limits the way students learn. In small classes, students receive plenty of feedbacks from instructors. Nurturing interaction with instructors and peers are critical components of the small classroom environment. This dynamic is completely changed in a large classroom, where individual feedback is almost non-existent. This presentation explores group projects as a way to manufacture a small-classroom like environment in a large classroom in non-major introductory biology. With a class of 160, students are divided into 20 groups, each consisting of 8 students. The final goal is to create collaborative Wiki pages by presenting health information from scientific research articles. For many, this is their first exposure to scientific literature. Students are forced to interact with other group members throughout the term via a number of online and face-to-face group activities. Feedback is given to groups rather than individual students. This is analogous to having 20 students in a class. Each group receives plenty of feedback from the instructor, TA and students from other groups. Projects are evaluated entirely by peer-evaluation. This group project has transformed the way students interact with the instructor and peers. Through this project, students learn key components of evaluating scientific literature in a safe nurturing environment, while learning the art of successful collaboration. Although benefits of group and project-based learning have been well documented, it is rarely adapted to intro-level science courses, possibly due to perceived challenges. How can we overcome the challenges? I invite participants to engage in problem-solving discussions on group projects

Loss of ovule identity induced by overexpression of the fertilization-related kinase 2 (ScFRK2), a MAPKKK from Solanum chacoense

In order to gain information about protein kinases acting during plant fertilization and embryogenesis, a reverse genetic approach was used to determine the role of protein kinases expressed in reproductive tissues. Two cDNA clones named ScFRK1 and ScFRK2 (Solanum chacoense fertilization-related kinase 1 and 2) were isolated from an expressed sequence tag (EST) library normalized for weakly expressed genes in fertilized ovaries. These showed significant sequence similarities to members of the mitogen-activated protein kinase kinase kinase (MAPKKK) family. RNA gel blot and RNA in situ hybridization analyses confirmed the strong up-regulation of ScFRK2 in ovules after fertilization. In addition, ScFRK2 mRNAs accumulate during early ovule development in the megasporocyte and in the integument of developing ovules. Overexpression of ScFRK2 led to the production of fruits with a severely reduced number of seeds. The seeds that were produced also exhibited developmental retardation. Analysis of ovaries prior to fertilization showed that the seedless phenotype was caused by a homeotic conversion of ovules into carpel-like structures. The present observations are consistent with the role of ScFRK2 in pre- and post-fertilization events. Furthermore, overexpression of ScFRK2 led to changes in the expression of the class D floral homeotic gene ScFBP11, suggesting that the ScFRK2 kinase may interact, directly or indirectly, with the FBP7/11 pathway that directs establishment of ovule identity.NRC publication: Ye