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Om Prakash PatidarUniversity of agricultural sciences , dharwad, karnatakaDate of seminar 31 oct 2014
Introduction
B-box proteins in animals
Conclusion
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Structural classification
B-box proteins in plants
Functions and related Case studies
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 (ahelix) residues
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2 b sheets
1 a helix
Zn
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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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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B-2B-1
B-boxA-box
RING COILED COIL
B-Box proteins in Animals
• Here BBX often associated with other domains like RING and coiled coil domains.
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(TRIM)
R B CC
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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
3. Retrovirus (HIV) life cycle and TRIMS for immunity in Humans
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2.Cause of Ovarian and Breast cancer in humans
Roles of TRIM/RBCC IN HUMANS
B-Box in Plants
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B-box in plants
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B1 B2 CCT
CCT= CONSTANS, CO-LIKE, TOC1
No. of BBX proteins in green plants
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Total 214
Phylogenetic tree for 214 BBX proteins from 12 representative species of green plants
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Conserved sequences of B1 and B2 in 214 representative green plant
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Distribution of the BBX genes on the rice chromosomes.
18The segmental duplicated genes are indicated in a different color and are connected by lines
Roles In Arabidopsis
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Roles In Crop Species
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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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Plant life cycle & Light receptors
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Photo receptors in plants
1. Photomorphogenesis
• Skotomorphogenesis-(seed germination).
• Photomorphogenesis- (seedling growth).
-Hypocotyl growth rate reduced
-Cotyledons open
-Accumulation of chlorophyll and anthocyanin pigments
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Role in Photomorphogenesis
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sun
Case study
LZF1, a HY5-regulated transcriptional factor, functions in
Arabidopsis de-etiolation (photomorphogenesis)
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Chang et al., 2008
Mechanism of regulation of photomorphogenesis
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HY5
LZF1 (light regulated zinc finger protein, BBX)
MYB75/PAP1(Anthocyaninaccumulation)
Ferredoxin-thioredoxin cascade for
chloroplastbiogenesis
Phytochrome
Inhibition of Hypocotylgrowth rate
sun
Photomorphogenesis
LZF1 acts synergistically with HY5 in the light-regulated inhibition of hypocotyl elongation
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DARK LIGHT
1 2 3 4 5 6 7 1 2 3 4 5 6 7
1234567
(Double mutants)
Over expression of LZF1
Wild genotypes (normal Hy5 and LZF1)
Less hypcotyl length
RT-PCR analyses of Lzf1 and MYB75 in Col (wild) and LZF1-ox plants ( at 5 day old stage)
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(Anthocyanin promoting factor)
(Control)
(Wild) (overexpressing)
LZF1(B-box protein) regulates anthocyanin and chlorophyll accumulation in seedlings during photomorphogenesis.
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overexpressing
overexpressing
mutantmutant
Shade Avoidance Response(SAR)
Shade Avoidance:
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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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BBX
Jiao et al., Nature review genetics, 2007
Mechanism of shade avoidance response
More far red in shade
(Far red)(red)
few examples showing shade avoidance response
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More far red in shade
Sunlight
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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SAR inhibition by BBX21
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Mutants of BBX
Mutant gene
No inhibition of hypocotyl growth in shade
Role of B-box in Flowering control
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case studyHd1, A Major Photoperiod Sensitivity QTL in Rice, is Closely Related to the
Arabidopsis Flowering Time Gene CONSTANS (Atbbx1)
- Yano et al., 2000 36
mapping population & created genetic map of photo sensitive gene loci
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(hd1) mutantLate flowering
(Hd1) wildEarly 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
Comparison Of rice Hd1, Arabidopsis CO, and Brassica napus BnCOA1 B-boxes
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hd1 NILs(mutant, late flowering) are transformed with Hd1(wild, early flowering) candidate gene segment (apal 7.1 kb) isolated from Nipponbare
0
2
4
6
8
52 53 54 55 73 74 75 76
Hd1 Transformed NILs
hd1 NILs.
Days to heading
No
. of
pla
nts
Hd1(wild)
Transformation of hd1 containing NILs, late flowering lines
Those lines transformed shown early flowering due to presesnce of Hd1
Case studyExpression of the Arabidopsis thaliana BBX32 Gene in
Soybean Increases Grain Yield
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- Preuss et al.,2012
(Monsanto Company, USA & Mendel Biotechnology inc., USA)
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ArabidopsisAtbbx32 Soybean 8 transgenic lines
Out of 8, 4 yielded more than 5% over control
Multilocation multiseason trial for yield
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
Search for Atbbx32 homolog in soybean by phylogenystudy
Gmbbx52 & Gmbbx53 found homolog, There over expression gave same results as by Atbbx32
Micro array to detected that there is higher alteration of gene expression near dawn(6 am)
Studied circadian clock components and found the cause of phenotypic changes and higher yield
Atbbx32 transgenic soybean demonstrate improved grain yield over control
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N= No. of environments
To understand physiological impacts two representative lines (line1&2) grown in both controlled and field conditions
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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%
Microarray data from field grown lines
(line1 and line 2)
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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.
(6 am)(3 am) (12 pm)(9 am) (3pm)
Increased
Decreased
84% of total genes changes at 6 am (dark to light transition)
Expression of AtBBX32 in soybean affects the transcript abundance of central clock components near 6 am(ZT 0)
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Gm LCL2
GmTOC1
(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
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3
5
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8
1
2
3
4
1
2
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5
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Avg. 6.1%higher
Avg. -11.8% less
Avg. 4.1% higher
Labs working on B-box proteins
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Postal Address:Mendel Biotechnology lab, Inc.3935 Point Eden WayHayward, California ,USA
St. Louis -- World HeadquartersMonsanto Company lab800 North Lindbergh Blvd.St. Louis, Missouri, USA 63167
Signal Transduction labNational Institute of technology Durgapur, West Bengal, India
National Institute of Plant Genome ResearchAruna Asaf Ali Marg,New Delhi - 110 067
Dr. holm’s laboratoryBox 100, SE-405 30 Gothenburg, SWEDEN
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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