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 Objective: 2  First year harvests and analysis completed.  Second year experimental material planted Milestones Feb

Second Year Growth Study Two varieties grown: –Printanor –Messidrome Two growth media –Hydroponic –Pot-grown in a soil based compost

Hydroponic verses Pot-grown Is there a difference?

Which is most like field-grown? Can compare mean maximum number of leaves per Printanor plant –Hydroponic-grown19 –Pot-grown10 –Field-grown (Remi)13 Messidrome similar

Mean Bulb weights Pot-grown Printanor 38g Pot-grown Messidrome 32g Hydroponic-grown Printanor101g Hydroponic-grown Messidrome 53g These differences are significant

Analysis completed Determined Fresh and Dry weights of Root (R), Leaf (L), and Bulb (B) Determined Protein content (R, L, B) Determined Sulphur content (R, L, B) Determined Nitrogen content (R, L, B) Determined Carbon content (R, L, B) Determination of CSO content (R, L, B) Have now got complete data set

Hydroponic Printanor - Sulphur

Hydroponic Printanor - Nitrogen

Hydroponic Printanor - Protein

Hydroponic Printanor - CSOs

Hydroponic Messidrome - CSOs

Hydroponic Printanor - Leaf CSO composition

Hydroponic Messidrome - Leaf CSO composition

Hydroponic Printanor - Clove CSO composition

Hydroponic Printanor - Root CSO composition

Questions on CSO synthesis Is the main Pre-CSO sulphur stored in the root? Do the roots synthesis (the) CSO’s What form is the sulphur in prior to CSO synthesis? Is Isoalliin an intermediate or product? Is there a Propiin to Alliin and Methiin conversion take place?

Hydroponic Printanor - Carbon

Can define 4 stages of growth Stage 1. Early growth phase Stage 2. Late growth phase Stage 3. Bulb initiation Stage 4. Bulb maturity

Stage One Day: 0-40 hydroponic, 0-70 pot- grown Plants rely on stored nutrients Difference between systems, due to higher hydroponic temperature? Does differences in temperature explain variation between hydroponic, pot and field-grown plants?

Stage two Day: hydroponic, pot Root and leaf tissue increase rapidly leaf tissue is twice fresh weight of root Root and leaf S, N, C, protein and CSO’s maximal Carbon accumulates in leaf and is not lost until leaf senescence. Probably structural Protein synthesis mainly in leaves supported by fact that leaves have 40% more N than root S content is same in root and leaf Since root has a third dry matter of the leaf suggesting this is the major S store

Stage three Day: hydroponic & pot-grown Initiation of bulbing. Independent of plant size –temperature and day-length response –garlic plants clove days after planting. S, N, C, protein and CSO's decline in leaf and root S, N, C, protein and CSO's rapidly accumulate in bulb S & N totals do not change implying no root uptake Root death at end of period Dramatic rise in CSO synthesis

Stage four Day: 200-bulb maturity hydroponic & pot-grown Leaves are senescing and S, N, C and protein levels fall Equivalent rise of S, N, C in bulb Leaf turgor lost due to root death The aerial parts of the plant senesce Neck closure Bulb matures

To identify developmental control points for CSO synthesis Objective: 2 Analysis of second-year field experiment completed Whole plant labelling studies completed Milestones Feb

A B C D Hydroponic Printanor A=50% total sulphur (d. 102, end March) B=Start bulbing (d. 130, 20 Apr) C=Start of senescence of root & leaf (d. 170, end May) D=Harvest (d. 230, end Jul)

A B C D Distribution and remobalisation of sulphur taken up early Distribution and remobalisation of sulphur taken up late * * * * * * * * * * * every 14d Hydroponic Printanor 34 S 32 S * * * * * * * * * * * every 14d

What we want to know When does nutrient uptake from the medium stop When does bulb filling start What proportion of S (and N) comes from the roots, leaves and hydroponic medium The form of the S in the root and leaf

To isolate and characterise alliinase cDNA clones Objective: 3b cDNA clones encoding alliinase isolated and sequenced Milestones Feb

l=Leaf Cloneb=Bulb Clone Clustering relationship of Messidrome alliinase PCR fragment sequences

Alliinase cDNA clone isolation Have used PCR fragments to identify alliinase clones in the library Have isolated 56 alliinase containing plaques and re-amplified Have developed a method of identifying near full-length clones Are screening for full-length clone Expect to be sequencing soon

To isolate and characterise alliinase cDNA clones Objective: 3b Analysis of second-year field experiment completed Whole plant labelling studies completed Milestones Feb

Expression analysis Will use Single Nucleotide Primer Externsion Based on detecting single nucleotide differences between alliinase clones –Bulb Alliinase B6 –Bulb Alliinase B5 –Bulb Alliinase B4 –Bulb Alliinase B3 Have successfully used this approach on onion

SNuPE analysis of Sweet Onion RT-PCR Bulb Leaf Root II I III IV E L E L E L E = Early L = Late