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Where are all the small peptides and what might they do?

Roger P. Hellens, QUT, Brisbane, Australia

@rogerhellens

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Micronutrient deficiency – hidden hunger

http://www.fao.org

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DALYs and deaths attributable to deficiencies of key vitamins and minerals

Steur et al. Nature Biotechnology 33, 25–29 (2015)

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Mechanism of Vitamin C-mediated Iron uptake

Free Radical Biology and Medicine (2014) 75, 69–83

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The problem of iron deficiency anemia can be viewed as one of inadequate iron bioavailability; with that in mind, it has been suggested that the problem may be better thought of as one of vitamin C inadequacy. Combs J, Gerald F. The Vitamins. 4 ed. Burlington: Elsevier Science; 2012

Ann N Y Acad Sci. 1980; 355:32-44.

Vitamin C and Iron Deficiency

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Fresh Fruit as a Cure for ScurvyThe Diffusion Of Innovation, Everett M. Rogers

1617 The Surgeon's Mate by John Woodall (p 160-176)

We have in our owne country here many excellent remedies generally knowne, as namely, Scurvy-grasse, Horse-Reddish roots, Nasturtia Aquatica, Wormwood, Sorrell, and many other good meanes... to the cure of those which live at home...they also helpe some Sea-men returned from farre who by the only natural disposition of the fresh aire and amendment of diet, nature herselfe in effect doth the Cure without other helps." At sea”, he states that experience shows that "the Lemmons, Limes, Tamarinds, Oranges, and other choice of good helps in the Indies... do farre exceed any that can be carried tither from England

1747 James Lind, HMS Sailsbury. (130 years later)1

• cider, • elixir of vitriol (sulphuric acid), • vinegar, • sea water, • 2 oranges and • 1 lemons, and a • purgative mixture of spicy paste and barley water

1795 Scurvy eliminated in the British navy (48 years later)

1856 British Board of Trade forms a policy of using lemons and limes to eliminate scurvy in the trading fleet. (61 years later, 239 in total)

1 - Lind J (1753). A Treatise of the Scurvy. London: A. Millar.

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Vitamin C, So Much More Than a Cure For Scurvy

1920 – anti-scurvy factor called vitamin C

1933 - L-ascorbic acid discovered as anti-scorbutic factor (Haworth and Szent-Györgyi)

1970 - Vitamin C and the Common Cold (Linus Pauling)

1980 - Vitamin C and Iron Bioavailability

2000 - Vitamin C and Cognition/Mood (Visser)

2015 – Vitamin C and some Cancer (Kaiser et al, Science 2015: 619)

2016 –Vitamin C and epigenetics

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VitC concentration in kiwifruit

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

Pistil 1WAA fruit 2WAA fruit 4WAA fruit 6WAA fruit 12WAA fruit Mature fruit

Asco

rbat

e(m

g/10

0g F

W)

Vitamin C in kiwifruit

CRICOS No. 00213JLaing et al., PNAS (2004 and 2007)

Biosynthesis of Ascorbate

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Transients Expression of Enzymes in Vitamin C Biosynthesis

# genes expressed

(control) PMM GMP GME GGP GPP

0 32 ± 21 32 ± 1

35 ± 132 ± 1

223 ± 3149 ± 14

2 34 ± 138 ± 3

542 ± 57177 ± 24

3 34 ± 2505 ± 46

539 ± 624 574 ± 31

680 ± 345 515 ± 53

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Over-expression of GGP in plants

wt 10 3 3b 5 6 9 9a0

20406080

100120140

Strawberry

Transgenic lines

Frui

t asc

orba

te (m

g/10

0 g

FW)

wt vector line 1 line 2 line 3 line 4 line 50

2

4

6

8

10

12Tomato

Frui

t asc

orba

te (m

g/10

0 g

FW)

35S-A

tVT2

35S-S

tVTC2A

PAT-StV

TC2A

Empty ve

ctor

WT

35S-A

tVT5

35S-S

tVTC2B

PAT-StV

TC2B

Empty ve

ctor

WT

0

0.5

1

1.5

2 Potato

Tube

r Asc

orba

te (m

g/g

DW)

Bullet et al. Plant Biotechnology Journal (2012) 10, 390–397

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0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0.045

0.05

LUC/REN

0

0.05

0.1

0.15

0.2

0.25

LUC/REN

0

0.5

1

1.5

2

2.5

3

LUC/REN

0

0.1

0.2

0.3

0.4

0.5

0.6

LUC/REN

GGP-LUC TT8-LUC GGP-TT8-LUC TT8-GGP-LUC

+ - + - + - + -VitC

Promoter

Transcriptional or Post-Transcriptional Regulation?

GGP-promoter GGP-UTR LUC

TT8-promoter TT8-UTR LUC

LUC

LUC

TT8-UTR

TT8-promoter

GGP-promoter

GGP-UTR

LUC

:REN

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5’ UTR Alignment of GGP

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RNA fold – 5’UTR region of kiwifruit GGP

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  G  G A  A T GT C AC   T

    C

Non-ATG Start Codons

CRICOS No. 00213JLaing et al. Plant Cell (2015) 27:772-786

A uORF with non-ATG Start (ACG [Thr]) is Highly Conserved in 5’UTR of GGP

dicotyledonous

algae to angiosperms

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sORFs – Where are they in the annotation?

Hellens et.al TIPS (2015) 21:317-328

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Short open reading frames (sORF) and small peptides

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Predicted small open reading frames

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Actual small open reading frames

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sORF - How do you spot the real ones?

• Gene families of small peptides

• Sequence homology between difference species

• sORFfinder – Ka/Ks

• ORF vs. CDS – regulatory role

25-residue peptide (Poneratoxin) from Bullet Ant Paraponera clavata

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sORFs could be everywhere

Hellens et.al TIPS (2015) 21:317-328

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Non-ATG start codons may be common

Ingolia NT, et al. Cell 2011, 147:789–802.

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0

50

100

150

A

Num

ber o

f mat

chin

g se

quen

ces

B

0 500 1000 1500 20000

20

40

Distance along GGP in base pairs

RN

Ase

qR

ibos

ome

prof

iling

Using data from Plant Cell. 2013 25:3699-710

Ribosome Profiling of GGP

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Runway traffic jam. Sunday after the F1 race was the busiest time for departures of private aircraft. At one point, 24 planes were waiting for take off (AUSTIN - Bergstrom International Airport).

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A Model for Vitamin C Regulation

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Observed vs. expected peptide size distribution

0:24 25-50 50:74 75:99 100:124 125:149 150:174 175:1990

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

all ATG

all ATG + ncATG

Annotated

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A role of non-ATG

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Accumulation GGP transcript

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So What - Improve Genome Annotation

ENCODE - Nature 489, 52–55 (2012) . doi:10.1038/489052a

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So What – biofortification in commodity cropsPlant source Amount

(mg / 100g)

Camu Camu 2800

Acerola 1677

Seabuckthorn 695

Mica Muro 500

Indian gooseberry 445

Rose hip 426

Baobab 400

Chili pepper (green) 244

Red pepper 190

Chili pepper (red) 144

Kiwifruit 90

Broccoli 90

Brussels sprouts 80

Kale 41

Cauliflower 40

Garlic 31

Spinach 30

Cabbage raw green 30

Potato 20

Cassava 21

Tomato 10

Banana 10

Carrot 9

Apple 8

Onion 7

Asparagus 6

Beetroot 5

Lettuce 4

Cucumber 3

Eggplant 2

Sweet potato 2

Rice 0

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Summary

• Lots of unannotated small ORFs

• Not all peptides start with an ATG

• The GGP uORF is responsible for the translational regulation of GGP

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Acknowledgements

Willaim A. LaingSean BulleyRoy StoryMarcela Martinez-Sanchez

Richard C. Macknight Matthew A. W. Chisnall Chris M. Brown

Sally RodenMeredith Crisp