Control of GL2 expression in Arabidopsis leaves and trichomes

1. Control of GL2 expression in Arabidopsis leaves and trichomes Daniel B. Szymanski, Ross A. Jilk, Susan M. Pollock and M. David Marks Bjarni Már Óskarsson, Magnús Eysteinsson Sævar Ingþórsson

2. Arabidopsis thaliana • A small flowering plant • A part of the mustard family along with species such as cabbage and radish • Discovered by Johannes Thal, in the Harz mountains in the sixteenth century • Has many advantages for being a model organism in the study of plant biology/genetics

3. Arabidopsis as a model organism • Many Benefits • Small genome (125mb) • Rapid life cycle (6 weeks) • Easy cultivation • Prolific seed production • Efficient transformation • Using Agrobacterium tumefaciens • Large numer of mutant lines • A very large research community

4. Trichomes • Definition: A hairlike or bristlelike outgrowth, as from the epidermis of a plant • Many shapes and sizes that depend on the plant species • Unicellular trichomes • Multicellular trichomes • Secretory trichomes • Root hairs are specialized trichomes found on roots

5. The development of Arabidopsis unicellular trichomes • radial expansion of the trichome precursor in the plane of the leaf • stalk emergence and expansion • formation of branch structures • expansion of the stalk and branches • continued expansion of the stalk and branches, which develop pointed tips • mature trichome with papillate surface

6. What Genes control the development of trichomes? • More than 20 genes are required • The initial selection (stage 1-2) of trichomal precursors depends on 2 genes. • GLABROUS1 (GL1) a myb-class transcription factor • TRANSPARENT TESTA GLABROUS (TTG) WT gl1 ttg

7. What Genes control the development of trichomes? WT, early stages • GLABROUS2 (GL2) is required for subsequent development of trichomes gl2 Later stages WT later stages

8. The expression pattern of the GL2 gene • In order to look at where the GL2 gene was being translated, the researchers made a special T-DNA construct • It was composed of a 5’UTR 2,1kb fragment of the GL2 gene and the b-glucuronidase gene (GUS) • GL2::GUS, (essentially the GUS gene under the control of the GL2 promoter region) • This construct was then used to transform Wild type Arabidopsis plants • The plants were then treated for GUS staining at different developmental stages and GUS activity was analyzed

9. Developing Leaves • Transverse Section through a developing leaf primorida • Apaxial surface of a developing leaf • Cross section through the apical third of a developing leaf • Emerging trichomes at the bse of a mature leaf • Transverse section through leaf base with developing trichomes • Developing leaf with trichomes at several developmental stages • Mature leaf

10. Immunolocalization of the GL2 protein • To confirm the expression pattern of GL2, anti-GL2 antibodies were used to localize the GL2 protein • WT and gl2-2 plants were used and prepared for immunocytochemical staining using a polyclonal antibody against an epitope in the C terminus of GL2. • gl2-2 was used as a negative control as it contains a T-DNA unit insertion consisting of randomly linked T-DNA units in the coding sequence upstream from the epitope.

11. TS through gl2-2 developing leaf • TS through WT developing leaf • TS through a mature WT leaf

12. Dependence of GL2::GUS activity on genes that affect trichome development • Other researchers have shown that GL2 dependant on the presence of GL1 and TTG • Analyzed by looking at GL2::GUS activity in different mutation backgrounds • gl1, ttg, and gl1 ttg plants

13. Dependence of GL2::GUS activity on genes that affect trichome development • In order to quantify the relative levels of GL2 transcription, the total GUS activity of leaves and cotyledons at the four leaf stage was measured in vitro

14. Deletion analysis of the GL2 promoter • To look at the promoter regions of the GL2 gene several deletion constructs using restriction enzymes were made, and fused to GUS and used to transform plants

15. Relationship between GL1 and GL2 • Both the GL1 and TTG loci are required for trichome initiation, while GL2 is required for the earliest morphogenetic events of trichome growth • The Promoter region of GL2 contains a myb-class binding site • Remember that GL1 is a myb-class transcription factor... • A comparison between GL1::GUS and GL2::GUS plants was made

16. GL1::GUS • GL2::GUS • TS of apical section of a GL1::GUS plant leaf • TS of apical section of a GL2::GUS plant leaf

17. Ectopic expression analysis • The Cauliflower mosaic virus promoter 35S can be used to induce ectopic expression of genes • Plants that ectopically express GL1 and the Maize R Gene were analyzed for ectopic GL2 expression • The R gene can compensate for the lack of TTG (ttg plants) • However, the combination of 35S::GL1 35S::R is lethal, unless the R gene contains an N-terminal fusion of the vertebrate glucocorticoid receptor (35S::R-GR) • The plants must also have the genotype ttg/ttg in order to survive • Gene expression can then be controlled by adding the Glucocorticoid homolog Dexamethasone to the growth medium

18. Ectopic expression analysis • To rule out the possibility that the ectopic activation of the GL2 promoter in the 35S::GL1 and 35S::R-GR background represents a nonspecific transcriptional activation, two additional GL2 reporter constructs were analyzed in that genetic background • The DXHp construct (3), which lacks the GL2 promoter regions required for detectable trichome expression • The DRI construct(5), which contains both putative shoot transcription domains

19. A Complex Question... • Because both GL1 and R are required to ectopically activate GL2, it is possible that GL1 and R function as a complex • A construct containing the GL1 gene with a myc epitope as an N-terminal fusion was generated • This same epitope fusion was made to a truncated version of the GL1 protein that expressed only the N-terminal 122 amino acids, which contains the entire myb homologous domain • These two protein fusions, as well as the full length untagged R protein, were cloned into expression vectors for the in vitro production of mRNA and subsequent reticulocyte translations in the presence of [35S]methionine.

20. SDS Page gel containing the results of in vitro translations • GL1mycfull length protein • N terminalGL1 myc protein • mock translation • Full length R • Anti myc immunoprecipitations • tagged GL1 • Tagged N terminal GL1 • R • Co-immunoprecipitations • Full length tagged GL1 and R • N terminal tagged GL1 and R

21. Conclusions • In Arabidopsis the TTG and GL1 genes are required for trichome initiation. • GL2 function does not appear to be required for initiation since gl2 leaves have approximately normal numbers of trichomes. • However gl2 trichome morphology is variable, and shows defects from stage 2 to stage 6 • In leaf primordia, the GL2 gene is expressed in all cell layers. • Because the number of cell layers and leaf morphology of developing gl2 leaves are not obviously different from wild-type, the nontrichome function of GL2 in developing leaves is unknown. • GL2 protein appears to show celltype specific subcellular localization in developing leaves, and is localized in the nucleus in developing trichomes • Both GL1 and TTG may directly bind to the GL2 promoter, either alone or as part of a larger complex, but the interaction between GL1 and R somehow eliminates the requirement for TTG • Further studies [Since 1998] of the mechanisms of GL2 regulation will aid in the understanding of how complex and integrated gene networks control plant development.