Garlic Sulphur Biochemistry

Partner 2:

Horticulture Research International

Laurence Trueman, Brian Thomas, Linda Brown, Brian Smith, & Gareth Griffiths

Workpackage Four

To identify developmental control points for CSO synthesis

Milestones May 2000 - Feb. 2001

• Experimental material planted and harvests begun.

Objective: 2

Strategy: To profile and track sulphur compounds in leaf, leaf

bases, root and clove

• Outline of intended work

• Development of hydroponic system

• Garlic grown to maturity in test system

Report given at Dijon

Experimental material planted...

• Accessions used: Printanor and Messidrome

• Planted first two weeks of December

• Growth media: Hydroponic

• Position: Greenhouse

• Controls: Pot grown garlic – Field grown controls abandoned due to very

wet winter

Harsh Winter conditions in the UK

Growth conditions

• Two full hydroponic systems constructed

• Nutrient medium is a 1/2 strength modified Hewitts solution

Hydroponic verses Pot-grown

Hydroponic verses Pot-grown - Mean (n=3) leaf size (28-3-2001)

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5

10

15

20

25

30

35

40

45

50

1st leaf 2nd leaf 3rd leaf 4th leaf 5th leaf 6th leaf 7th leaf 8th leaf

Leaf number

Len

gth

(cm

)

Printanor hydroponic

Printanor pot-grown

Messidrome hydroponic

Messidrome pot-grown

Hydroponic verses Pot-grown - Mean leaf size (Hydroponic 26-2-01, Pot 28-3-01)

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5

10

15

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25

30

35

40

1st leaf 2nd leaf 3rd leaf 4th leaf 5th leaf

Leaf number

Len

gth

(cm

)

Printanor hydroponic

Printanor pot-grown

Messidrome hydroponic

Messidrome pot-grown

Growth characteristics

• Approximately 2-3 leaves ahead - effectively 31 days

• Little morphological difference although chlorophyl content appears to differ

• Hydroponic garlic sprouted earlier than controls (Printanor 25d, Messidrome 15d)

• Due to higher temperature of hydroponic system?

...and harvests begun Sampling strategy

• At the (visable) emergence of each leaf

• 3 plants (Printanor), 2 plants (Messidrome)

• Equal number pot-grown controls

• Plants divided into leaf base and/or clove, leaf and root material

• Material stored at -40oC whilst awaiting analysis

Sample analysis• Leaf length (ruler)

• Dry weight (freeze drying)

• Soluble protein (bicinchoninic acid assay)

• Chlorophyl content (Arnon assay)

• Total nitrogen (combustion analysis)

• Total sulphur (inductively coupled plasma atomic emission spectrophotometer)

• Cysteine sulphoxide analysis using a (methanol extraction followed by quantification using HPLC)

CSO analysis

Hydroponic garlic Standards

4.271 methiin4.745 ethiin5.531 alliin6.588 propiin

Summary of CSO results

Clove 1 -% of total

Clove 2 -% of total

Clove 3 -% of total

MeanPercentage

Standarderror

Methiin 16.04 18.19 16.21 16.81 ± 1.19

Isoalliin 3.19 2.71 2.93 2.94 ± 0.24

Propiin 2.94 1.89 2.23 2.35 ± 0.54

Alliin 77.83 77.2 78.6 77.88 ± 0.70

Shop bought, hydroponically grown garlic

The two storage organs of garlic

Under extreme conditions garlic plants can form an intermediate bulbing structure

consisting of a swollen leaf base surrounding the meristem

Garlic variety “Solo”Clove 1 -% of total

Clove 2 -% of total

MeanPercentage

Standarderror

Methiin 7.39 4.42 5.91 ± 2.10

Isoalliin 16.60 18.62 17.61 ± 1.43

Propiin 4.63 6.04 5.34 ± 1.00

Alliin 71.38 70.93 71.16 ± 0.32

Comparison of CSO composition hydroponically grown garlic and “Solo”

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20

40

60

80

100

Methiin Isoalliin Propiin Alliin

Cysteine sulphoxide

perc

enta

ge o

f tot

al

cloves

rings

To identify developmental control points for CSO synthesis

Milestones May 2000 - Feb. 2001

• Experimental material planted and harvests begun

Objective: 2

To identify developmental control points for CSO synthesis

Objective: 2

First year harvests and analysis completed.

Second year experimental material planted

Milestones Feb. 2001 - 2002

To isolate and characterise alliinase cDNA clones

Objective: 3b

• To construct a cDNA library from actively synthesizing tissues

Milestones May 2000 - Feb 2001

Thiosulphinates Pyruvic Ammonia acid

Flavour generation in Allium

S-alk(en)yl-L-Cysteine Sulphoxides

Sulphenic acids + Aminoacrylic acid

Other sulphur-containing flavour compounds

Alliinase

SpontaneousSpontaneous

Two cDNA libraries have been constructed from cv Messidrome

from RNA extracted from leaf and bulb tissue from actively growing

plants

cDNA library Statistics

• Range of insert sizes: 400-3000bp

• Number of indepentent clones– bulb 2.22x106 – leaf 2.28x106

• 1x106 clones from each library have been amplified.

• Aliquots stores at -80oC in 7% DMSO

To isolate and characterise alliinase cDNA clones

Objective: 3b

• To construct a cDNA library from actively synthesizing tissues

Milestones May 2000 - Feb 2001

To isolate and characterise alliinase cDNA clones

Objective: 3b

• cDNA clones encoding alliinase isolated and sequenced

Milestones Feb. 2001 - 2002