bbx proteins are type of zinc finger proteins,

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Om Prakash Patidar
University of agricultural
sciences , dharwad, karnataka
Date of seminar 31 oct 2014

3. Introduction
B-box proteins in animals
B-box proteins in plants
Conclusion
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Structural classification
Functions and related Case studies

4. Zinc finger proteins
• Zinc finger protein contain Zinc finger domains that are stabilized by metal
ions including zinc.
 Characterized by 2 anti parallel b sheets and 1 a helix
 Structure stabilized by binding of Zinc ion
 Zinc binding mediated by specific cysteine (b sheets) and histidine (a
helix) residues
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2 b sheets
1 a helix
Zn

5. zinc finger domains
• Zinc finger domains make multiple contacts on target molecule
• Can bind to DNA, RNA or protein.
• Versatility in binding results in specialized functions including gene
transcription, translation, mRNA trafficking, cytoskeletal organization and
chromatin remodeling.
• There are several classes in zinc finger protein. One of them is BBX
proteins.
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6. B-Box (BBX) Proteins-
are a class of zinc-finger transcription factors.
 Involved in protein-protein interactions.
contain a B-box domain with one or two B-box
motifs, and
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A-box B-box
RING B-1 B-2
COILED COIL

7. B-Box proteins in Animals
• Here BBX often associated with other domains
like RING and coiled coil domains.
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R B CC
(TRIM)

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B-box protein functions in animals

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Genomic organization of TRIM/RBCC family genes in
humans

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TRIM
Role of TRIM/RBCC IN HUMANS
1. In Ubiquitination Cascade

11. Roles of TRIM/RBCC IN HUMANS
3. Retrovirus (HIV) life cycle and TRIMS for
immunity in Humans
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2.Cause of Ovarian and
Breast cancer in humans

12. B-Box in Plants
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13. B-box in plants
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B1 B2 CCT
CCT= CONSTANS, CO-LIKE, TOC1

14. No. of BBX proteins in green plants
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Total 214

15. Phylogenetic tree for 214 BBX proteins from 12
representative species of green plants
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16. Conserved sequences of B1 and B2 in
214 representative green plant
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18. Distribution of the BBX genes on the rice chromosomes.
The segmental duplicated genes are indicated in a different color and are connected by lines 18

19. Roles In Arabidopsis
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20. Roles In Crop Species
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21. Summary of roles of B-box proteins in
plants
• In seedling photomorphogenesis.
• In flowering.
• In shade avoidance Responses.
• In abiotic and biotic stresses.
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22. Plant life cycle & Light receptors
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Photo receptors in
plants

23. 1. Photomorphogenesis
• Skotomorphogenesis-(seed germination).
• Photomorphogenesis- (seedling growth).
-Hypocotyl growth rate reduced
-Cotyledons open
-Accumulation of chlorophyll and
anthocyanin pigments
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24. Role in Photomorphogenesis
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sun

25. Case study
LZF1, a HY5-regulated transcriptional factor, functions in
Arabidopsis de-etiolation (photomorphogenesis)
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Chang et al., 2008

26. Mechanism of regulation of photomorphogenesis
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Phytochrome
HY5
LZF1 (light regulated zinc
finger protein, BBX)
MYB75/PAP1
(Anthocyanin
accumulation)
Ferredoxin-thioredoxin
cascade for
chloroplast
biogenesis
Inhibition of
Hypocotyl
growth rate
sun
Photomorphogenesis

27. LZF1 acts synergistically with HY5 in the light-regulated inhibition of hypocotyl
elongation
Wild genotypes (normal Hy5 and LZF1)
Over expression of LZF1
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1
23
45
6
7
(Double mutants)
1 2 3 4 5 6 7 1 2 3 4 5 6 7
DARK LIGHT
Less hypcotyl length

28. RT-PCR analyses of Lzf1 and MYB75 in Col (wild) and
LZF1-ox plants ( at 5 day old stage)
(Anthocyanin promoting
factor)
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(Control)
(Wild) (overexpressing)

29. LZF1(B-box protein) regulates anthocyanin and chlorophyll accumulation in
seedlings during photomorphogenesis.
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overexp
ressing
overexpr
essing
mutant
mutant

30. Shade Avoidance Response(SAR)
Shade Avoidance:
Morphological changes like Hypocotyl and stem elongation, Acceleration of flowering
to avoid shade and to compete for light in high density plantings.
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Mechanism of shade avoidance response
More far red in
shade
BBX
(red) (Far red)
Jiao et al., Nature review genetics, 2007

32. few examples showing shade avoidance response
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More far red in
shade
Sunlight

33. BBX in Shade Avoidance Response(SAR)
• Inhibitors of SAR- BBX 19, BBX 21, BBX22
• Promoters of SAR-BBX18,BB24,BBX25
• BBX21- act as component of negative
feedback to avoid exaggerated response of
SAR genes(PAR1, HFR1,ATHB2 etc.)
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34. SAR inhibition by BBX21
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Mutants of BBX
No inhibition of
hypocotyl growth
in shade
Mutant gene

35. Role of B-box in Flowering control
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36. case study
Hd1, A Major Photoperiod Sensitivity QTL in Rice, is Closely Related to the
Arabidopsis Flowering Time Gene CONSTANS (Atbbx1)
- Yano et al., 2000 36

37. mapping population & created genetic map of photo sensitive gene loci
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(hd1) mutant
Late flowering
(Hd1) wild
Early flowering
Major QTL for flowering in rice:
a B-box protein.

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Fine mapping of Hd1 region using markers on chromosome 6 of rice
RFLP
YAC
PAC
4 CAPS markers co-segregated with Hd1
P0038c5

39. Comparison Of rice Hd1, Arabidopsis CO, and Brassica
napus BnCOA1 B-boxes
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40. hd1 NILs(mutant, late flowering) are transformed with Hd1(wild, early flowering) candidate
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gene segment (apal 7.1 kb) isolated from Nipponbare
8
6
4
2
0
52 53 54 55 73 74 75 76
Hd1 Transformed NILs
hd1 NILs.
Days to heading
No. of plants
Hd1(wild)
Transformation of hd1 containing
NILs, late flowering lines
Those lines transformed shown early flowering due to presesnce of Hd1

41. Case study
Expression of the Arabidopsis thaliana BBX32 Gene in
Soybean Increases Grain Yield
- Preuss et al.,2012
(Monsanto Company, USA & Mendel Biotechnology inc., USA)
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42. Micro array to detected that there is higher alteration of gene expression near dawn(6 am)
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Arabidopsis
Atbbx32 Soybean 8 transgenic lines
Multilocation multiseason trial for yield
Out of 8, 4 yielded more than 5% over control
2 lines selected of it & grown in field and in controlled chamber
Observation taken at different stages and Similar results found as earlier in multilocation trials
Studied circadian clock components and found the cause of phenotypic changes
and higher yield
Search for Atbbx32 homolog in soybean by phylogeny
study
Gmbbx52 & Gmbbx53 found homolog,
There over expression gave same results as by Atbbx32

43. Atbbx32 transgenic soybean demonstrate improved grain yield
over control
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N= No. of environments

44. To understand physiological impacts two representative lines
(line1&2) grown in both controlled and field conditions
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45. AtBBX32 extends the reproductive period between R3
and R7developmental stages in soybean resulting in a
delay in final maturity compared to control
Developmental stages
R1(Initiation of
flowering.)
R3 (Onset of pod
development)
R7 (Beginning of
maturation)
R8 Stage where
95% of the pods are
physiologically
mature.
Control 38.1 57.8 112.5 120.4
Event1 39.3 57.7 115.8* 122.8*
Event2 39 57.2 116.7* 123.6*
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AtBBX32 expression- Delays leaf senescence
and brown pod maturity
*at 5%

47. Microarray data from field grown lines
(line1 and line 2)
(3 am) (6 am) (9 am) (12 pm) (3pm)
Total 219 genes show 2–8 fold changes in abundance in both transgenic events relative to the control.
Dark bar- Genes increased in abundance
light bar- Genes decreased in abundance.
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Incr
eas
ed
D
e
cr
e
a
s
e
d
84% of total
genes changes
at 6 am (dark to
light transition)

48. Expression of AtBBX32 in soybean affects the transcript
abundance of central clock components near 6 am(ZT 0)
Gm LCL2
GmTOC1
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(5 am) (2 pm) (2 am)
(5 am) (7 am) (5 pm) (2 am)

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Search for ortholog to Atbbx32 in soybean

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1
2
3
4
5
6
7
8
1
2
3
4
1
2
3
4
5
6
7
8
Avg. 6.1%
higher
Avg. 4.1%
higher
Avg. -11.8%
less

51. Labs working on B-box proteins
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Postal Address:
Mendel Biotechnology lab, Inc.
3935 Point Eden Way
Hayward, California ,
USA
St. Louis --World Headquarters
Monsanto Company lab
800 North Lindbergh Blvd.
St. Louis, Missouri, USA 63167
Signal Transduction lab
National Institute of technology
Durgapur, West Bengal, India
National Institute of Plant
Genome Research
Aruna Asaf Ali Marg,
New Delhi - 110 067
Dr. holm’s laboratory
Box 100, SE-405 30
Gothenburg, SWEDEN

52. Future line of work
• understanding the molecular mechanisms of
each individual BBX protein.
• the complexity and modularity of the system
is to be understand and simplified
• Bringing this knowledge from lab to farmers
field at commercial level in order to increase
food production.
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