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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

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(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

2

3

5

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8

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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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