# Presentation: 773349

## Slide 1

## Outline of Talk

- Cereal Crops Research Activities
- Avenanthramide biosynthesis
- Field trials effect of crown rust on avenanthramide content
- Tocol biosynthesis
- Eric Jackson: Mapping tocol production in oat

**Notes:**
Want to describe a little bit about the research activities at the CCRU
Provide a background discussion on biosynthesis of avenanthramides
 Then describe some recent work we’ve been doing on the environmental effects and genetics of avenanthramide production
Then talk a little about Tocols (vitamin E) in oats and have Eric describe his work on mapping the genes involved in tocol production

## Cereal Crops Research UnitMadison, WI

- Total: 18,000 Ft2 to 38,000 Ft2
- Lab space: 900 Ft2 to 1200 Ft2
- 5 PIs working on barley and oat

**Cereal Crops Research Unit****Madison, WI**

**Notes:**
Cereal Crops Research Unit was formerly known as the Malt and Barley Lab
Came into being shortly 1933 after prohibition repeal to assist barley breeders develop high quality malting barley
Housed in this building, build ca 1948, addition in 1968
Moved to our new digs in Oct 2006 (across the street)
 5 PIs currently with one vacancy (to be filled in 2010)

## Collaborations and Analyses Over Last Five Years

**Notes:**
1)  Bulk of the work in this unit is directed toward barley, we still have the malting barley quality testing lab that processes ~5-6000 lines of barley each year
2) Of the five PIs I’m most active in oat research
3) My research directed mostly toward biosynthesis of avenanthramides, do some collaborative analysis work on phytonutrient content of oat and barley
4) Have also provided synthetic avenanthramides to investigators interested in their nutritional value

## Health Claim/Phytonutrients

- FDA approved healthy food claim (1997)
- Largely attributed to high soluble fiber (beta-glucan) content
- Oats also rich in tocopherols and tocotrienols
- Folk cure for skin irritations
- Avenanthramides

**Notes:**
Oat have long been recognized as a healthy component of the diet
FDA granted approval of a healthy food claim in 1997 
Cholesterol lowering effect of b-glucan
Worthwhile to note that oats are rich in tocotrienols, also shown to lower cholesterol, inhibits HMG-CoA Reductase

## Health Effects Specific to Avenanthramides

- Mohsen Meydani, Tufts University
- Reduces vascular smooth muscle cell proliferation
- Increased NO production
- Inhibition of ppRb, arrest G1-S1 cell cycle progression
- Stabilized p53
- Li Li Ji, University of Wisconsin
- Elevation in anti-oxidant enzyme system (SOD, Catalase) in exercise stressed rats
- Lowered myeloperoxidase levels (neutrophiles)

**Notes:**
1)  Bottom line is that avenanthramides are proving to be highly beneficial “nutraceuticals” and there are some very sophisticated studies available to demonstrate that.

## Avenanthramide Structure and Function

- Phytoalexins
- Anti-oxidants
- Cell wall component
- Phytohormone??

- R1 - R4 = H, OH, OCH3

**Notes:**
Generalized structure of the avenanthramides
This portion comes from the phenylpropanoid pathway, may recognize as component of lignans (gives structural integrity to plants)
This portion comes from anthranilic acid
Some forms have additional olefin (double bond)
The “R” groups can be any combination of H, OH, or Methoxy group, can see that there are potentially a large number of potential congeners
These natural products are believed to play a number of physiological roles. 

## General Phenylpropaniod Pathway

- Glycolysis

- phosphoenolpyruvate

- erythrose-4-phosphate

- pentose phosphate

**+**

- Shikimate pathway

- Anthranilate

- Tyr + Phe

- Phenylpropanoids

- Protein

- Tryptophan/protein

- phytoalexins

- lignins

- phenolic
- acids

- flavonoids

**PAL**

**Notes:**
First present general overview - link primary metabolism to secondary metabolism of PP
PEP and E4P from glycolysis & pentose phosphate pathways respectively enter termed “Shikimate Pathway” to yield chorismate
Chorismate metabolized to either tyrosine/phenylalanine or to anthranilate and subsequently to tryptophan

## Phenylpropanoid biosynthesis

**Notes:**
1)  The branchpoint from 1o to 2o metabolism occurs with the conversion of phe to cinnamic acid
2)  Catalyzed by phenylalanine ammonia lyase (PAL)
3)  Cinnamate 4 hydroxylase (P450) converts to p-coumaric acid
4)  Some grasses have a TAL activity that can convert tyr to pCA
5)  Phenolic acid is activated as the CoA thioester by 4-CL
6)  Two parallel pathways to decorate the phenyl ring with additional hydroxyl group and subsequent methylation to form caffeic acid and ferulic acid
7)  The free acid of each can be activated to CoA ester by 4-CL
8) This is a simpified view
9) Important role of isozymes of PAL and 4-CL

## Reaction of Hydroxycinnamoyl CoA: Hydroxyanthranilate N-Hydroxycinnamoyl Transferase (HHT)

_**Form        R       phenylpropanoid**_
**A  (Bp)     H         *****p*****-coumaric **
**B  (Bf)    ****OCH****3****   ****ferulate**
**C  (Bc)    ****OH**       **caffeic**

**Notes:**
1) Final biosynthetic step to avns catalyzed by hydroxycinnamoyl CoA:hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT)
2) There are numerous forms of avenanthramides but the three forms found in significant quantities are A, B, C based on p-CA, CA or FA
3) Illustrated here using 5-hydroxy anthranilic acid, this is an assumption we don’t know at what stage the anthranilate is hydroxilated, 
4)  Ishihara has shown HHT Km lower for 5-OH followed by anthranilate and 5-OH.

## HHT Isozymes/Fragments

- Yang et al. Molc. Plant-Microb. Interact. 17, 81 (2004)

**Notes:**
The obligatory sequence alignment
Point to make is that AsHHT 1-4 are cDNA sequences from Atsushi Isihara’s lab and isolated from elicited leaf tissue, suggest that there are at least 4 isozymes of HHT
AsHHT4 is truncated
With the exception of a single threonine for serine substitution in predicted amino acid sequence the callus HHT aligns with HHT 4
Also noteworthy that Ishihara was not able to functionally express 1-3 in e-coli (in contrast to the BCHT from carnation)

## Avenanthramide Biosynthesis Up-regulated by Fungal / Chemical Elicitation

**Notes:**
1) This slide illustrates the typical response of oat to elicitation
2) These two chromatograms are from elicited vs non-elicited oat callus culture using crude crab shell chitin 
3) Yellow trace is the ethyl acetate extract of media from elicited cells; Red trace is non-elicited cultures
4) Ishihara has reported similar responses in oat leaves using a variety of elicitors, including crown rust infection.
5) Main point is that avns are inducible, at least in leaves and callus
6) Not sure the case with the groat; there is HHT activity in immature and mature seed.
7) The rest if this talk will present evidence that grain avn concentrations are effected by fungal infections.

## Grain Avenanthramide Content Shows Strong Genotype X Environment Interaction

- Emmons & Peterson, Crop Sci 41:1676 (2001)

**Notes:**
 The problem is:  Avn production in the oat crop is highly variable!
 David Peterson (CCRU) has done an extensive amount of work documenting the G X E interaction
 Bars are color coded Avn A, B, C bottom up
Three different cultivars (Belle, Gem, Dane) grown in two environments over three different seasons

## Grain Avenanthramide Content Shows Strong Genotype X Environment Interaction

- Emmons & Peterson, Crop Sci 41:1676 (2001)

**Notes:**
1) A different view with cultivars averaged between two locations in Wisconsin (Sturgeon Bay and Arlington).
2) This demonstrates quite dramatically the environmental effect.  There is clearly something about Sturgeon Bay that promotes avenanthramide production

## Oat Cultivars Grown in North Dakota

## Oat Avenanthramide Concentrations in Rust vs Rust Free Environment

**Notes:**
A more recent study in collaboration with Doug Doehlart at ARS Fargo
Examined avn levels in oat grown in three location over two seasons
Two environments (Fargo, Carrington ’05) had rust pressure others did not.

## Oat Avenanthramide Concentrations in Rust vs Rust Free Environment

**Wise, Doehlert and McMullin, Cereal Chemistry, in press 2008**

## Correlation of Avenanthramide Content

## Avenanthramides in Otana/TAM Mapping Population Artificially Infected with Crown Rust 

**Notes:**
1. Preliminary data from a collaborative effort with Eric Jackson (USDA, Aberdeen ID) 
A subsample of a mapping population grown in Aberdeen (Rust Free).  Plants were grown in identical environments with artificial infection with crown rust.
Note the same general trend of higher avenanthramides in infected plants.  BUT, some very high levels expressed even in the non-infected plants (#12 & 13).
Parents (left most) are generally low avenanthramide producers.

## Aberdeen ID Oat Plots

**First Glance: Significant increase in avenanthramide levels resulting from crown rust infection**
***                            ***_**Mean Avn**_
**  ****Infected            70.9  mg/kg**
***  *****Non-infected    45.5  mg/kg**
**  ****P=<0.0001**

## Parent and Selected Crosses Response to Crown Rust

- 24.6% ↓

- 0.47 ↓

- 11.8 ↓

- 28.9 ↓

- 9.5 ↑

- 5.4 ↑

- 41.1 ↑

- 39.7 ↑

- ∆ Avena:

**Parent and Selected Crosses Response to Crown Rust**

## Otana/TAM Crosses: Three Distinct Groups

**1)  Lines where the overall avenanthramide levels decreased under disease pressure (8 lines ranging from 11.8% to 42.8% decrease).**
**2)  Lines where no (significant) change in avenanthramide levels occurred (19 lines).**
**	****	**
**3)  Lines where the overall avenanthramide levels increased under disease pressure (62 lines ranging from 11% to 57.8% increase).**
**	**
**Furthermore:****	**
**	****a)  The amount of disease was not associated with ****	****avenanthramide response**
**	****b)  Appears that avenanthramide content has a ****	****strong ****	****genetic component**

## Avenanthramides in Processed Food

**Notes:**
A quick and dirty look as the levels of avns in some processed food
Not particularly rigorous analyses, used 0-coumaric acid as internal std.  Note the discrepancies in recovery.
Cv Belle was included just as a benchmark for reasonably high avn levels
There does seem to be a trend toward loss of avns in some of the more processed (baked) items
There is also a substantial variation in avn levels even in unprocessed oats.
Unfortunately we did not find ANY avns in any of the oat Stouts we examined.

## Summary

- Strong genetic as well as environmental components to avenanthramide production
- Avenanthramides are elicited by pathogens and molecular mimics in vegetative tissue
- Crown rust infection can result in dramatic elevation of avenanthramides in the grain
- Breeders: Be encouraged but be aware!

## Vitamin E and Tocols

- Vitamin E is a generic term for all eight naturally occurring tocols
-  -Tocopherol preferentially adsorbed in liver
- Tocotrienols have unique physiological effects
- More potent anti-oxidant in artificial membrane assays
- Shown to reduce cholesterol levels
-  and -T3 more effective than -T3
- Inhibits HMG CoA reductase, different mechanism than statins

## Tocol Structure

**Notes:**
Barley also produces significant quantities of tocochromanols (or tocols)
Sometimes referred to (incorrectly) as Vitamin E
Consist of a, relatively polar, chromanol ring with an long hydrocarbon tail
Highly potent anti-oxidants, primarily due to phenolic hydroxyl group.
The phytyl tail anchors molecule in the hydrophobic inner layer of membrane bilayer with chromanol ring exposed to hydrophilic cytosol
Difference between tocopherols and tocotrienols is the poly unsaturated hydrocarbon tail.
Eight form commonly found, differences being the degree of methylation on the phenolic ring.
You might also note that there are three stereogenic carbons in the tocopherols, naturally produced tocopherols are all in the “R” configuration

## Tocol Biosynthesis

**Notes:**
In tocol biosynthesis the phenolic moiety originates from the Shikimate pathway via the amino acid tyrosine
The phytyl chain comes from isoprenoid metabolism, similar to cholesterol.
First branch point in tocol biosynthesis is the conjugation of the phytyl chain to homogentisic acid to form 2,3-dimethyl-5-phytyl-1,4-benzoquinone (DMPBQ)

## Selected Tocol Concentrations

| Values expressed as mg/kg |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | α-T* | β-T | γ-T | δ-T | α-T3* | β-T3 | γ-T3 | δ-T3 |
|  |  |  |  |  |  |  |  |  |
| palm | 89 | - | 18 | - | 128 | - | 323 | 72 |
| soybean | 100 | 8 | 1021 | 421 | - | - | - | - |
| maize | 282 | 54 | 1034 | 54 | 49 | 8 | 161 | 6 |
| sunflower | 670 | 27 | 11 | 1 | - | - | - | - |
| rapeseed | 202 | 65 | 490 | 9 | - | - | - | - |
|  |  |  |  |  |  |  |  |  |
| * Abbreviations: T, tocopherol; T3, tocotrienol |  |  |  |  |  |  |  |  |
| Data from: Gunstone, F.D., Harwood, J.L. and Padley, F.B. The Lipid Handbook (Second Edition) (Chapman & Hall, London) (1994). |  |  |  |  |  |  |  |  |
|  |  |  |  |  |  |  |  |  |

**Notes:**
These sources all have very high tocopherol levels
With the exception of palm oil and corn, tocotrienols are quite low or non-exisitant 

## Factors effecting tocopherol levels

- University of Idaho Research and Extension Center, Aberdeen, ID
- 1998-2000

- University of Idaho Research and Extension Center, Tetonia, ID
- 1997 and 1999

- 	Evaluated 136 Ogle/TAM O-301 RILs for:
- 		α-tocotrienol
- 		α-tocopherol
- 		total tocopherol

- Across three location/years:

## Means based on α-tocotrienol, α-tocopherol, and total tocopherols contents in grain

## Environmental Factors

- Water availability:
- 	α-tocotrienol
- 	     Aberdeen, 17.4 mg kg-1
- 	       Tetonia, 11.5 mg kg-1
- 	α-tocopherol
- 	     Aberdeen, 8.8 mg kg-1
- 	     Tetonia, 7.4 mg kg-1
- 	total tocopherol
- 	     Aberdeen, 27.1 mg kg-1
- 	     Tetonia, 18.8 mg kg-1
-           Peterson et al., 2005
- 	     α-tocotrienol
- 	          Aberdeen, 20.3 mg kg-1
- 	          Tetonia, 8.3 mg kg-1
- 	     α-tocopherol
- 	          Aberdeen, 16.7 mg kg-1
- 	          Tetonia, 7.5 mg kg-1

- Soil fertility:
-      Uniform nitrogen:
- 	α-tocotrienol
- 	     H2 Aberdeen, 1998 = 91.9
- 	       H2 Aberdeen, 1999 = 88.6
- 	α-tocopherol
- 	     H2 Aberdeen, 1998 = 92.8
- 	       H2 Aberdeen, 1999 = 90.9
- 	total tocopherol
- 	     H2 Aberdeen, 1998 = 94.2
- 	       H2 Aberdeen, 1999 = 90.6
-       Non-uniform nitrogen:
- 	α-tocotrienol
- 	     H2 Aberdeen, 2000 = 61.1
- 	α-tocopherol
- 	     H2 Aberdeen, 2000 = 23.1
- 	total tocopherol
- 	     H2 Aberdeen, 2000 = 24.1

## α-tocotrienol

**Amy2d**

**CDO455a**

**CDO516a**

**RZ892b**

**UMN97c**

**ISU47b**

**CDO412**

**CDO57d**

**RZ596**

**ISU37b**

**OISU563**

**OISU2203**

**OISU5628**

**OT-15**

- QTL Statistics:
- QTL was detected in all five location/years
- QTL was associated with TAM O-301 loci
- 21.6 *R**2*

- α-tocotrienol

## QTL statistics:

-      Intervals:
-          OT-8 (Ogle loci)
-       	*R**2* = 8.5
- 	2 Locations
-         OT-20 (TAM loci)
-       	*R**2* = 5.7
- 	2 Locations
-          OT-32 (TAM loci)
-       	*R**2* = 10.3
- 	*R**2* = 8.4
- 	2 Locations
-          OT-33 (Ogle loci)
*          **	**R**2* = 8.7
- 	3 Locations

## α-tocopherol

**OT-34**

**ISU136b**

**RZ404c**

**RZ446a**

**RZ395b**

**CDO405b**

**RZ69a**

**CDO1502a**

**CDO572b**

**KSUe8a**

**CDO572b**

**CDO57a**

**e40m48-195.o**

**e35m61-091.t**

**e40m48-132.t**

**ISU77c**

- QTL statistics:
- QTL was detected in all five location/years
- QTL was associated with Ogle loci
-  *R**2 = *9.7

- α-tocopherol

## α-tocopherol/Oil 

- α-tocopherol/Oil

## QTL statistics:

-      Intervals:
-          OT-14 (Ogle loci)
-       	*R**2* = 6.8
- 	2 Locations
-         OT-16 (Ogle loci)
-       	*R**2* = 11.9
- 	*R**2** = *7.9
- 	2 Locations
-          OT-30 (TAM loci)
-       	*R**2* = 8.2
- 	3 Locations
-          OT-8 (Ogle loci)
*          **	**R**2* = 8.4
- 	3 Locations

## total-tocopherol

**OT-31**

**ISU35b**

**CDO1326a**

**aa04.810**

**CDO122**

**BCD1643a**

**e35m61-178.t**

**e35m61-120.o**

**OISU1877a**

**PSR115**

**e35m61-110.t**

**RZ2a**

**e35m61-128.o**

**ISU59b**

**CDO795b**

- QTL statistics:
- QTL was detected in Aberdeen, 1999-2000
- QTL was associated with Ogle loci
-  *R**2 = *5.9

## total-tocopherol

- Water and nitrogen availability seem to effect tocopherol content in whole grain oat.
- α-tocotrienol is more abundant in whole grain oat than other tocopherols and is under strong genetic control.
- The QTL on OT-15 accounting for a good portion of the α-tocotrienol content was associated with the STS marker Amy2d.
- Linkage groups OT-8, 15, and 32-33 are genetic areas of high importance in oat based on regions associated with crown rust resistance and tocopherol levels.
- Further work needs to be done to dissect the QTLs for α-tocopherol (associated with Ogle alleles) and the QTL for high oil (associated with TAM O-301 alleles) on linkage group OT-34.

## 	Evaluations of two mapping populations segregating for crown rust resistance and avenanthramide levels are being screened: 

- Ongoing work:
- 	Evaluations of two mapping populations segregating for crown rust resistance and avenanthramide levels are being screened:
- 		i.  Under disease pressure with and without fungicide 			application.
- 		ii.  Under no disease pressure with and without fungicide 			application.
- Results of disease assessment and avenanthramide content will be used to study the genetics of avenanthramide production and the interactions between this quality trait and crown rust disease and fungicide application.

## Acknowlegements

**Fargo Study**
**	****Doug Doehlert, USDA, ARS Fargo ND**
**	****Mike McMullen, NDSU, Dept of Plant Science**
**Madison**
**	****Lauri Herrin, USDA, ARS**
**Aberdeen**
**    ****Mike Bonman and Don Obert, USDA, ARS Aberdeen ID**
**    ****Irene Shakelford, Kathy Satterfield, and Rebecca Caldera, USDA, ARS Aberdeen ID**
***Funding From USDA, ARS***