The document describes experiments to identify genes that regulate miRNA activity in Arabidopsis thaliana plants. The experiments involved mutagenizing plants with T-DNA insertions, screening for mutants with altered responses to ABA and BASTA, identifying locations of T-DNA insertions, and analyzing effects on miRNA target gene expression. Several mutant lines (HOAT2-2, HOAT3-2, HOAT5-2) were identified that showed atypical responses to ABA and altered expression of the miRNA target gene GRF3, suggesting they may have mutations affecting miRNA activity.

1. miRNA activity in
Arabidopsis thaliana plants
Helen Oh
Andrew Tsai

2. Purpose
 To identify potential genes that regulate miRNA
activity, especially in regards to growth and
development.

3. Overview
 To identify those potential genes:
 Mutagenize with T-DNA insertions
 Screen using BASTA
 Screen using ABA
 Screen for typical mutant miRNA phenotypes
 Find location of T-DNA insertion
 Determine whether miRNA target genes are affected
 Germination assay
 BASTA assay

4. either too slow (ABA hypersensitive) or too fast (ABA hyposensitive), these collection of
Mutagenize with T-DNA
mutant plants will be highly enriched in mutations that affect miRNA activity. In sum, we
will direct our studies on screening for mutations that enhance or diminish the response
of Arabidopsis plants to the plant hormone abscisic acid (ABA). This approach has
insertion
already resulted in the identification of a number of interesting genes, and will hopefully
lead to identification of a number more within this semester's class.

5. Overview
 To identify those potential genes:
 Mutagenize with T-DNA insertions
 Screen using BASTA
 Screen using ABA
 Screen for typical mutant miRNA phenotypes
 Find location of T-DNA insertion
 Determine whether miRNA target genes are affected
 Germination assay
 BASTA assay

6. Screen using BASTA
I1. Mutagenesis strategy using T-DNA containing three tandem strong
rview of procedure Brian Gregory used to prepare the mutagenized seed th
screen. B) Cartoon depicting thegerminated is inserted into the plant genom
 Ten plants T-DNA that on BASTA.
ns. Shown is a random gene that is induced by the insertion (green arrow, r
borders of the T-DNA (RB, LB, orange), the herbicide resistance gene exp
r
DNA that we will use to select mutagenized seedlings (BASTA , purple), and
enhancers (35S enhancers, red).

7. Overview
 To identify those potential genes:
 Mutagenize with T-DNA insertions
 Screen using BASTA
 Screen using ABA
 Screen for typical mutant miRNA phenotypes
 Find location of T-DNA insertion
 Determine whether miRNA target genes are affected
 Germination assay
 BASTA assay

8. Screen using ABA
 Six plants displayed mutated responses to ABA ,
specifically they displayed hypersensitive root
growth.

9. Overview
 To identify those potential genes:
 Mutagenize with T-DNA insertions
 Screen using BASTA
 Screen using ABA
 Screen for typical mutant miRNA
phenotypes
 Find location of T-DNA insertion
 Determine whether miRNA target genes are affected
 Germination assay
 BASTA assay

10. Screen for typical mutant
miRNA phenotype
 HOAT2-2

11. Screen for typical mutant
miRNA phenotypes
 HOAT3-2

12. Screen for typical mutant
miRNA phenotype
 HOAT5-2

13. Overview
 To identify those potential genes:
 Mutagenize with T-DNA insertions
 Screen using BASTA
 Screen using ABA
 Screen for typical mutant miRNA phenotypes
 Find location of T-DNA insertion
 Determine whether miRNA target genes are affected
 Germination assay
 BASTA assay

14. Find location of T-DNA
insertion
 TAIL PCR was
performed.
AD1/AD2
 This gel shows
that AD primers
AD2
are about 100 bps.

15. Find location of T-DNA
insertion
 TAIL PCR product for
HOAT2-2AD1
HOAT2-2AD2
HOAT2-2AD3
HOAT2-2AD2 and
HOAT3-2AD1
HOAT3-2AD2
HOAT3-2AD3
HOAT5-2AD1
HOAT3-2AD2 are
about 750 bps.
 Lower bands are AD
primers.

16. Find location of T-DNA
insertion
 Heavy upper bands could be
HOAT5-2AD2
HOAT5-2AD3
due to precipitated DNA.
 Lower bands are AD primers.

17. Find location of T-DNA
insertion
 DNA sequence analysis
performed.
 HOAT3-2:
 Had greater or equal to 200
as alignment score.
 Expected value was 0.0.

18. Find location of T-DNA
insertion
 AT3G09720 codes for a
upstream
protein with a p-loop.
 Binds nucleotides and
upstream
ATP
 Helicase functions
downstream
 AT3G09730 has no
known function.

19. Overview
 To identify those potential genes:
 Mutagenize with T-DNA insertions
 Screen using BASTA
 Screen using ABA
 Screen for typical mutant miRNA phenotypes
 Find location of T-DNA insertion
 Determine whether miRNA target genes
are affected
 Germination assay
 BASTA assay

20. Determine whether miRNA
target genes are affected
 GRF3 is a known target of miRNA.
 Look at amount of mRNA transcribed from GRF3 to
determine if miRNA activity is potentially altered.

21. Determine whether miRNA
target genes are affected
HOAT2-2
HOAT5-2
HOAT3-2
Col-0
 Non-degraded RNA has
the following bands: 28s
rRNA and 18s rRNA.
28s rRNA
 Create cDNA for HOAT3-2 18s rRNA
and HOAT5-2.

22. Determine whether miRNA
target genes are affected
26 cycles
HOAT3-2
HOAT5-2
 sqPCR was
Col-0
DDL
performed.
• Actin primers (above). 26 cycles
HOAT3-2
HOAT5-2
Col-0
DDL
Col-0
• GRF3 primers (below).

23. Determine whether miRNA
target genes are affected
 qPCR was performed.
qPCR
1200
1000
800
Percentages
600
400
200
0
Col-0 DDL HOAT3-2 HOAT5-2
Plant Name

24. HOAT3-2
HOAT5-2
DDL
26 cycles
Col-0
HOAT3-2
Actin primers
HOAT5-2
DDL 30 cycles
Col-0
HOAT3-2, 30 26 cycles
HOAT3-2, 30 30 cycles
Col-0, 30 26 cycles
Col-0, 30 30 cycles
HOAT3-2, 20 26 cycles
target genes are affected
HOAT3-2, 20 30 cycles
Determine whether miRNA
Col-0, 20 26 cycles
ATG09730 and ATG09720 primers
Col-0, 20 30 cycles

25. Overview
 To identify those potential genes:
 Mutagenize with T-DNA insertions
 Screen using BASTA
 Screen using ABA
 Screen for typical mutant miRNA phenotypes
 Find location of T-DNA insertion
 Determine whether miRNA target genes are affected
 Germination assay
 BASTA assay

26. Germination Assay
 plated onto ABA plates of concentrations:
0 μM
0.5 μM
1 μM
1.5 μM

27. Germination Assay
0 μM ABA • Baseline
100
90
• note 3-2 low
80
70
Percent
60
germination
50
Germination
40
30
20
10
0
wt 2-2 3-2 5-2
Experimental

28. Germination Assay
0.5 μM ABA • Mimics 0 ABA control
100
90 • T-value of 0.5275
80
70
Percent
60 • Don’t see too much
50
Germination
40
30 from this plate
20
10
0
wt 2-2 3-2 5-2
Experimental

29. Germination Assay
1 μM ABA • Start seeing trends
100
90
80
• 3-2, 5-2 hypersensitive
70
Percent
60
50
• 2-2 ambiguous
Germination
40
30
20
10
0
wt 2-2 3-2 5-2
Experimental

30. Germination Assay
1.5 μM ABA • Similar to 1.0 μM ABA
100
90 • 3-2, 5-2
80
70
60
Percent
50
Germination
40
30
20
10
0
wt 2-2 3-2 5-2
Experimental

31. Germination Assay
Seeds from HOAT 2-2 may have a T-DNA
insertion but are not hyper/hyposensitive to ABA!

32. Overview
 To identify those potential genes:
 Mutagenize with T-DNA insertions
 Screen using BASTA
 Screen using ABA
 Screen for typical mutant miRNA phenotypes
 Find location of T-DNA insertion
 Determine whether miRNA target genes are affected
 Germination assay
 BASTA assay

33. BASTA Assay - Theory
HYPOTHESIS: ABA hyper/hyposensitivity
caused by T-DNA insertion.
GIVEN:
 T-DNA insertion confers BASTA resistance
 ABA-hypersensitive plants on BASTA – should all
have T-DNA insertion
Plated surviving plants from ABA treatment onto
BASTA plates

34. BASTA Assay
Survival on BASTA • Data does not confirm
100
80 theory
60
Percent Col-0 • Most surviving plants
Survival 40 HOAT 2-2
HOAT 3-2
20 have BASTA resistance
HOAT 5-2
0
0.5 1 1.5
[ABA] in μM

35. BASTA Assay - Conclusions
Seeds from HOAT 2-2 HAVE a T-DNA insertion
but are not hyper/hyposensitive to ABA!
 Contrary to original screen
 2-2 originally identified as hypersensitive

36. Possible Explanations
 Chance from first hyper/hyposensitivity assay
(n = 1)
 NOT chance from this experiment
n = large (despite contamination)
 Possibly growth conditions
BOTTOM LINE: We have a lot of factors at work
here that we don’t know about!

37. Possible Explanations
Possible interaction
Survival on BASTA
100
between ABA and
80
Percent
60
Col-0
BASTA
Survival 40 HOAT 2-2
HOAT 3-2
20
HOAT 5-2
0
0.5 1 1.5
[ABA] in μM

38. Conclusions
HOAT 2-2, HOAT 3-2, HOAT 5-2
 Root growth assay  potential to have miRNA
mutations
 Observable “miRNA phenotypes”

39. Conclusions
HOAT 3-2, HOAT 5-2
 Increase in GRF3 expression – KNOWN miR396-
REGULATED GENE
 Decrease in ATG09720 gene expression  T-DNA
insertion inside gene.
Future work: How do changes in these gene
expressions explain original phenotype?

40. References
 Earley, et al., An endogenous F-box protein regulates
ARGONAUTE1 in Arabidopsis thaliana Silence 2010, 1:15
 Rodriguez RE, Mecchia MA, Debernardi JM, Schommer
C, Weigel D, Palatnik JF. 2010. Control of cell proliferation in
Arabidopsis thaliana by microRNA miR396. Development
137, 103–112.
 Dr. John Wagner, personal communication.
 Allison Lesher, personal communication.
 NCBI BLAST database: gene identification
 TAIR database: gene information