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Transcriptome analysis of Arabidopsis wild-type and gl3-sst sim trichomes identifies four additional genes required for trichome development.

Marks MD, Wenger JP, Gilding E, Jilk R, Dixon RA - Mol Plant (2009)

Bottom Line: Mutations in this gene did not alter trichome expansion, but did alter mature trichome cell walls.Mutations in BLT resulted in a loss of trichome branch formation.Mutations in PEL3, which was previously shown to be required for development of the leaf cuticle, resulted in the occasional tangling of expanding trichomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology, University of Minnesota, St Paul, MN 551108, USA. marks004@umn.edu

ABSTRACT
Transcriptome analyses have been performed on mature trichomes isolated from wild-type Arabidopsis leaves and on leaf trichomes isolated from the gl3-sst sim double mutant, which exhibit many attributes of immature trichomes. The mature trichome profile contained many highly expressed genes involved in cell wall synthesis, protein turnover, and abiotic stress response. The most highly expressed genes in the gl3-sst sim profile encoded ribosomal proteins and other proteins involved in translation. Comparative analyses showed that all but one of the genes encoding transcription factors previously found to be important for trichome formation, and many other trichome-important genes, were preferentially expressed in gl3-sst sim trichomes. The analysis of genes preferentially expressed in gl3-sst sim led to the identification of four additional genes required for normal trichome development. One of these was the HDG2 gene, which is a member of the HD-ZIP IV transcription factor gene family. Mutations in this gene did not alter trichome expansion, but did alter mature trichome cell walls. Mutations in BLT resulted in a loss of trichome branch formation. The relationship between blt and the phenotypically identical mutant, sti, was explored. Mutations in PEL3, which was previously shown to be required for development of the leaf cuticle, resulted in the occasional tangling of expanding trichomes. Mutations in another gene encoding a protein with an unknown function altered trichome branch formation.

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Comparison of blt-1 and sti-ab Mutants.Higher and lower magnification of stem trichomes of (A, E) wild-type, (B, F) blt-1, (C, G) sti-ab, and (D, H) blt-1 sti-ab double mutant.(I–K) Fluorescent, merged and DIC images of Col stage three trichome expressing gfp–BLT fusion protein.(L–N) Fluorescent, merged, and DIC images of sti mutant stage four trichome expressing TdimerRed–cSTI fusion protein. Arrows highlight regions of enhanced fluorescence.Bars in (A–D) and (I–N) represent 10 μm and in (E–H) 100 μm.
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fig8: Comparison of blt-1 and sti-ab Mutants.Higher and lower magnification of stem trichomes of (A, E) wild-type, (B, F) blt-1, (C, G) sti-ab, and (D, H) blt-1 sti-ab double mutant.(I–K) Fluorescent, merged and DIC images of Col stage three trichome expressing gfp–BLT fusion protein.(L–N) Fluorescent, merged, and DIC images of sti mutant stage four trichome expressing TdimerRed–cSTI fusion protein. Arrows highlight regions of enhanced fluorescence.Bars in (A–D) and (I–N) represent 10 μm and in (E–H) 100 μm.

Mentions: The phenotype of blt is very similar to that of sti (Ilgenfritz et al., 2003). To explore the relationship between the two mutants, additional phenotypic and genetic studies were performed. The development of trichomes on both mutants is similar, lacking stage three branch formation (see Supplemental Figure 4A–4C). The unbranched leaf and stem trichomes on these mutants resemble the unbranched trichomes found on the stems of wild-type plants. However, SEM analysis of the tips of the mutant trichomes showed a phenotypic difference. The wild-type unbranched stem trichomes have a sharp point (Figure 8A and 8E), whereas the unbranched trichomes on the leaves and stems of both mutants have blunt tips (compare Figure 5A and 5B, and see Figure 8B, 8C, 8F, and 8G). This indicates that both BLT and STI are required for the initiation of branches, and play a role in branch tip maturation. Double mutants were generated to study the genetic interaction between the two genes (see Methods). As shown in Figure 8D and 8H, the vast majority of the double mutant trichomes resembled those of either of the single mutants. However, an occasional trichome (fewer than 10%) exhibited a more extreme phenotype with stunted growth and a more bloated tip (Figure 8H). Given that the majority of the trichomes on the double mutants had phenotypes no more extreme than those of either of the single mutants, it is likely that the two genes encode products that function in the same developmental pathway. To begin to test for co-localization, GFP-BLT and Tdimer2 RED–STI fusion constructs were moved into the corresponding mutant and wild-type plants (tdimer2 RED is described in more detail in Methods and in Campbell et al., 2002). Both constructs completely rescued the trichome tip phenotype of the corresponding mutants (data not shown), and, as shown in Figure 8I–8N, both fusion proteins localized to the branch tips of stage four trichomes. Images of non-transformed negative control plants for comparison are shown in Supplemental Figure 5.


Transcriptome analysis of Arabidopsis wild-type and gl3-sst sim trichomes identifies four additional genes required for trichome development.

Marks MD, Wenger JP, Gilding E, Jilk R, Dixon RA - Mol Plant (2009)

Comparison of blt-1 and sti-ab Mutants.Higher and lower magnification of stem trichomes of (A, E) wild-type, (B, F) blt-1, (C, G) sti-ab, and (D, H) blt-1 sti-ab double mutant.(I–K) Fluorescent, merged and DIC images of Col stage three trichome expressing gfp–BLT fusion protein.(L–N) Fluorescent, merged, and DIC images of sti mutant stage four trichome expressing TdimerRed–cSTI fusion protein. Arrows highlight regions of enhanced fluorescence.Bars in (A–D) and (I–N) represent 10 μm and in (E–H) 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2713768&req=5

fig8: Comparison of blt-1 and sti-ab Mutants.Higher and lower magnification of stem trichomes of (A, E) wild-type, (B, F) blt-1, (C, G) sti-ab, and (D, H) blt-1 sti-ab double mutant.(I–K) Fluorescent, merged and DIC images of Col stage three trichome expressing gfp–BLT fusion protein.(L–N) Fluorescent, merged, and DIC images of sti mutant stage four trichome expressing TdimerRed–cSTI fusion protein. Arrows highlight regions of enhanced fluorescence.Bars in (A–D) and (I–N) represent 10 μm and in (E–H) 100 μm.
Mentions: The phenotype of blt is very similar to that of sti (Ilgenfritz et al., 2003). To explore the relationship between the two mutants, additional phenotypic and genetic studies were performed. The development of trichomes on both mutants is similar, lacking stage three branch formation (see Supplemental Figure 4A–4C). The unbranched leaf and stem trichomes on these mutants resemble the unbranched trichomes found on the stems of wild-type plants. However, SEM analysis of the tips of the mutant trichomes showed a phenotypic difference. The wild-type unbranched stem trichomes have a sharp point (Figure 8A and 8E), whereas the unbranched trichomes on the leaves and stems of both mutants have blunt tips (compare Figure 5A and 5B, and see Figure 8B, 8C, 8F, and 8G). This indicates that both BLT and STI are required for the initiation of branches, and play a role in branch tip maturation. Double mutants were generated to study the genetic interaction between the two genes (see Methods). As shown in Figure 8D and 8H, the vast majority of the double mutant trichomes resembled those of either of the single mutants. However, an occasional trichome (fewer than 10%) exhibited a more extreme phenotype with stunted growth and a more bloated tip (Figure 8H). Given that the majority of the trichomes on the double mutants had phenotypes no more extreme than those of either of the single mutants, it is likely that the two genes encode products that function in the same developmental pathway. To begin to test for co-localization, GFP-BLT and Tdimer2 RED–STI fusion constructs were moved into the corresponding mutant and wild-type plants (tdimer2 RED is described in more detail in Methods and in Campbell et al., 2002). Both constructs completely rescued the trichome tip phenotype of the corresponding mutants (data not shown), and, as shown in Figure 8I–8N, both fusion proteins localized to the branch tips of stage four trichomes. Images of non-transformed negative control plants for comparison are shown in Supplemental Figure 5.

Bottom Line: Mutations in this gene did not alter trichome expansion, but did alter mature trichome cell walls.Mutations in BLT resulted in a loss of trichome branch formation.Mutations in PEL3, which was previously shown to be required for development of the leaf cuticle, resulted in the occasional tangling of expanding trichomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology, University of Minnesota, St Paul, MN 551108, USA. marks004@umn.edu

ABSTRACT
Transcriptome analyses have been performed on mature trichomes isolated from wild-type Arabidopsis leaves and on leaf trichomes isolated from the gl3-sst sim double mutant, which exhibit many attributes of immature trichomes. The mature trichome profile contained many highly expressed genes involved in cell wall synthesis, protein turnover, and abiotic stress response. The most highly expressed genes in the gl3-sst sim profile encoded ribosomal proteins and other proteins involved in translation. Comparative analyses showed that all but one of the genes encoding transcription factors previously found to be important for trichome formation, and many other trichome-important genes, were preferentially expressed in gl3-sst sim trichomes. The analysis of genes preferentially expressed in gl3-sst sim led to the identification of four additional genes required for normal trichome development. One of these was the HDG2 gene, which is a member of the HD-ZIP IV transcription factor gene family. Mutations in this gene did not alter trichome expansion, but did alter mature trichome cell walls. Mutations in BLT resulted in a loss of trichome branch formation. The relationship between blt and the phenotypically identical mutant, sti, was explored. Mutations in PEL3, which was previously shown to be required for development of the leaf cuticle, resulted in the occasional tangling of expanding trichomes. Mutations in another gene encoding a protein with an unknown function altered trichome branch formation.

Show MeSH