„Plant hormone markers for inherently rapid stem

diameter & height growth in balsam poplar and

hybrid poplar genotypes growing in Canada‟s

Prairie Provinces‟

• Richard P. Pharis, Biological Sciences, University of Calgary, Calgary, Alberta

T2N 1N4, Canada

• Co-Authors Alberta-Pacific Forest Industries, Boyle, AB - Dr. Barb R. Thomas

NZ Horticulture & Food Research Institute, Palmerston North - Ms. Sarah Hurst & Dr.

Lindsay Fung

UBC School of Forestry (Ms. Virginie Pointeau, Ms. Faride Unda, Dr. Robert Guy & Dr.

Shawn Mansfield)

AAFC-AESB Agroforestry Research Centre, Indian Head, SK (Dr. Salim Silim & Raju Y

Soolanayakanahally)

Research Branch, B.C. Ministry of Forests (Dr. Chang-Yi Xie)

U. Calgary (Dr. Ruichuan Zhang, Dr. Leonid Kurepin, Mrs. Loeke Janzen).

New Zealand‟s “famous” 80055

genotype of Pinus radiata

A Fast Growing Hybrid Cross in NZ

At age 7 years A Initial Working Hypothesis:

Inherently Fast-growing Poplar Trees

(Families & Individual Genotypes)

Have Higher Levels of „Growth-

effector‟ Gibberellin (GA1) in

Growing Wood (Xylem) & Bark

Tissues & Higher Levels of Both GA1

and GA4 in Elongating Internode

Tissues

Gibberellin A1

One Biosynthesis Pathway for Synthesis of

the Growth Active Gibberellin, GA1

HCO2H

H

CO

O OH

CO2H

HCO2H

H

OH

O

HCO2H

H

CO

OOH

GA19GA44 GA20

GA20oxGA20ox

HCO2H

H

CO

O

HO

OH

HCO2H

H

CO

O

HO

OH

HO

GA1GA8

GA3ox

GA2ox

A 2nd Biosynthesis Pathway for Synthesis of the Growth

Active Gibberellin, GA4 (and Possibly GA1 Also)

HCO2H

HCO

O

CO2H

HCO2H

H

CHO

HCO2H

HCO

O

GA15 GA9GA24

H CO2H

HCO

O

GA9

H CO2H

HCO

O

HO

GA4

H CO2H

HCO

O

HO

GA34

HO

GA3ox GA13ox

HCO2H

H

CO

O

HO

OH

GA1

HCO2H

H

CO

O

GA9

HCO2H

H

CO

O

HO

GA4

Another Hypothesis: Gibberellin A1 is Required

Not Only for Radial & Height Growth of the Tree

Trunk, But Also for Xylem Fibre Length.

3 -hydroxylation of GA20 to GA1 was blocked in Eucalyptus

globulus by use of the acylcyclohexanedione,

trinexapac ethyl (Syngenta)

-- Ridoutt, B.G., R.P. Pharis & R. Sands. 1996. Secondary xylem fibre

development ……. Physiol. Plant. 96:559-566.

Control (XF=xylem fibre) GA1 synthesis blocked

Overexpression of the GA20ox Gene in Hybrid Aspen Gives

Increased

Gibberellin Biosynthesis, Increased Radial Stem Growth &

Longer Xylem Fibres

Eriksson et al., 2000

Control Xylem Fibres

GA20ox Overexpressed

Xylem Fibres

Question: Are Plant Hormones Causal For Radial

Stem Growth In Poplar? Gibberellin A1 - a proven effector of shoot elongation.

Xylem GA1 content is significantly (P=0.0001) correlated

with stem diameter in 4 year-old NZ-grown P. deltoides

Stem diameter (cm) at breast height (DBH)

10 11 12 13 14 15 16 17 18

GA

1 c

on

ten

t (n

an

og

ram

s p

er

gra

m d

ry w

eig

ht)

0

5

10

15

20

25

30

35

40

GA1 Content in Growing Xylem Tissue “Reflects” (is

Significantly (P=0.001) Correlated with) Year 4 Stem Diameters

Across Genotype for 4 year-old NZ-grown P. deltoides

genotypes (seedlings) from a 2 family cross

Answer: “Yes”, GA1 is a highly significant

variable for stem diameter growth across

genotype (n = 11) and the slow-growing family

(Oklahoma parents) has a significantly (P =

0.01) lower concentration of GA1 in growing

xylem tissues than the faster-growing family

(Mississippi parents)

Question: What about other poplar species which

are also adapted to „temperate climates‟, such as P.

trichocarpa? When “across genotype” is set as the

experimental variable, do the same or similar

significant correlations with plant hormone

concentrations also hold true?

June 2006 UBC Vertical Whips [Coppiced]. Upper Internode Tissue

GA1 Levels at Age 90 days were Correlated with Tree Stem Volumes at

Age 3 Years (October 2009) in a Field Provenance Trial Near Harrison

Hot Springs for the Same 8 Genotypes

r2=0.557

p=0.033

2009 Harrison site stem volumes (cm3)

1000 2000 3000 4000

UB

C 2

006

Upp

er in

tern

ode

GA

1 leve

ls (

ng/g

DW

)

2

4

6

8

10

12

14

16

18

June 2006 UBC Vertical Whips [Coppiced]. Upper Internode Tissue

GA4 Levels at Age 90 days were Correlated with Tree Stem Volumes at

Age 3 Years (October 2009) in a Field Provenance Trial Near Harrison

Hot Springs for the Same 8 Genotypes

r2=0.686

p=0.011

2009 Harrison site stem volumes (cm3)

1000 2000 3000 4000

UB

C 2

006

Upp

er in

tern

ode

GA

4 le

vels

(ng

/gD

W)

2

4

6

8

10

12

Structures of the Two Growth „Effector‟ Gibberellins Identified and

Quantified in Rapidly Elongating Internode Tissues of Populus

trichocarpa Coppiced “Whips” that were Harvested in Mid-June

2006

Gibberellin A4 Gibberellin A1

A comparison of relative levels of gene expression of GA3ox1 (June 2006

vertical whips produced by coppice cutting – tissue was scraped from the

lower stem phloem side) with July 2007 stem basal diameters of the same 9

Populus trichocarpa genotypes -- Totem Field, UBC

r2=0.472

p=0.041n=9

2007 Mean stem base diameter (mm)

20 30 40 50 60 70 80 90 100

Ph

loe

m G

A3

ox

rela

tive

exp

ress

ion

0

1e-4

2e-4

3e-4

4e-4

5e-4

HCO2H

H

CO

OOH

GA20

HCO2H

H

CO

O

HO

OH

GA1

GA3ox

Relative levels of gene expression of GA3ox1 in mid-June 2006. The

vertical whips had been produced by coppice cutting. Tissue for

analysis was scraped from the phloem side of the lower stems of the

whips at age 3 months. GA3ox1 expression was then correlated with

tree stem volumes [age 3 years in 2009] across genotype in a Field

Provenance Trial near Harrison Hot Springs.

r2=0.617

p=0.021

2009 Harrison site stem volumes (cm3)

1000 2000 3000 4000

UB

C 2

006

Low

er s

tem

phl

oem

GA

3ox1

rel

ativ

e ex

pres

sion

0

1e-4

2e-4

3e-4

4e-4

5e-4

6e-4

Nine randomly selected genotypes from the McMillan Islands

provenance where elongating internode tissue was analyzed

for GA1 at age 30 days and related to (correlated with) stem

volumes of the same genotypes in a provenance field growth

trial near Harrison Hot Springs at age 3 years

r2=0.577

p=0.018

2009 Harrison site McMillan stem volumes (cm3)

1000 1500 2000 2500 3000

20

07

McM

illa

n h

arv

est

01

GA

1 le

ve

ls (

ng/g

DW

)

0

1

2

3

4

5

6

7

8

r2=0.593

p=0.015

2009 Harrison site McMillan stem volumes (cm3)

1000 1500 2000 2500 30002

00

7 M

cM

illan

ha

rve

st

01 G

A1 le

ve

ls (

ng/c

m)

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

Ten randomly selected genotypes from the McMillan Islands

provenance. Elongating internode tissue was analyzed for

GA1 at age 90 days and related to stem volumes of the same

genotypes in a provenance field growth trial near Harrison

Hot Springs at age 3 years

r2=0.550

p=0.014

2009 Harrison site McMillan stem volumes (cm3)

500 1000 1500 2000 2500 3000 3500

2007 M

cM

illa

n h

arv

est

03 G

A1 levels

(ng/g

DW

)

0

2

4

6

8

10

r2=0.576

p=0.011

2009 Harrison site McMillan stem volumes (cm3)

500 1000 1500 2000 2500 3000 3500

200

7 M

cM

illa

n h

arv

est

03 G

A1 le

ve

ls (

ng/c

m)

-0.1

0.0

0.1

0.2

0.3

0.4

Nine randomly selected genotypes from the McMillan Island, B.C.

provenance. Elongating internode GA1 concentrations were measured at age

30 days and correlated across genotype with stem volumes for the same

genotypes in UBC‟s Totem Field growth trial [which was irrigated] in January

2011 after 3 summers of growth

r2=0.889

p=0.0001

2009 Totem Field stem volumes (cm3)

0 2000 4000 6000 8000 10000 12000

20

07

Ha

rve

st

01

ste

m G

A1 le

ve

ls (

ng/g

DW

)

0

1

2

3

4

5

6

7

8

r2=0.895

p=0.0001

2009 Totem Field stem volumes (cm3)

0 2000 4000 6000 8000 10000 12000

2007 H

arv

est

01 s

tem

GA

1 levels

(ng/c

m)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

Nine randomly selected genotypes from the McMillan Island, B.C.

provenance. Elongating internode GA1 concentrations were measured at

age 30 days and correlated across genotype with stem volumes for the

same genotypes in UBC‟s Totem Field growth trial [which was irrigated]

in January 2011 after 4 summers of growth

r2=0.940

p<0.001

Year 4 - stem volumes (cm3)

0 5000 10000 15000 20000 25000 30000 35000 40000

Age 3

0 d

ays

- G

A1 leve

ls (

ng/c

m)

-0.05

0.00

0.05

0.10

0.15

0.20

CONCLUSIONS: For two poplar species, P. deltoides and P. trichocarpa,

and for their genotypes that are “adapted” to temperate climates, the

concentrations of “growth effector” gibberellins (GA1 and/or GA4)

growing xylem and internode tissues reflects inherent stem growth

capacity across a wide range of genotypes.

That “reflection” may be useful as a “predictive marker” in choosing

inherently fast-growing genotypes for use in afforestation and

agroforestry.

Further, for P. trichocarpa, at least, it appears that the GA biosynthesis

“activation step” - 3 -hydroxylation, catalyzed by GA3 oxidase and

encoded for by GA3ox1 , is a rate-limiting step across genotype for

selections from the natural gene pool.

Question: Are gibberellin & other growth hormone concentrations also

significantly correlated with stem growth of balsam poplar and hybrid

poplar genotypes which are adapted to Canada‟s “interior” provinces, with

their “short summers, very long days & early autumn Arctic cold fronts ?

HCO2H

H

CO

O OH

CO2H

HCO2H

H

OH

O

HCO2H

H

CO

OOH

GA19GA44 GA20

GA20oxGA20ox

HCO2H

H

CO

O

HO

OH

HCO2H

H

CO

O

HO

OH

HO

GA1GA8

GA3ox

GA2ox

Two “N. Alberta-adapted” Hybrid Poplars, cv. „Walker‟ (AP24) and cv.

„Manitou‟ (AP26). Two trees were chosen to represent stem diameter

“rank” within the randomized provenance trial, for each clone. Stem

Diameters and Heights at ca. 12 Years of Age

Tree rank

Small Medium Large

Insid

e b

ark

ste

m d

iam

ete

r at

1 m

(cm

)

0

2

4

6

8

10

12

14

16

AP24

AP26

Tree rank

Small Medium Large

Tre

e h

eig

ht

(m)

0

2

4

6

8

10

12

14

16

AP24

AP26

Two “N. Alberta-adapted” Hybrid Poplars, cv. „Walker‟ (AP24) and cv. „Manitou‟

(AP 26). Comparisons of Growth Hormone Correlations with Stem Diameter

“Rank”. Each „bar‟ represents the mean GA levels for 2 ramets. The GA44

GA19 Biosynthetic Step is catalyzed by GA20 oxidases & encoded for by GA20ox

Tree rank

Small Medium Large

Low

er s

tem

GA

44 levels

(ng/g

DW

)

0

5

10

15

20

25

30

35

AP24

AP26

Tree rank

Small Medium Large

Low

er s

tem

GA

19 levels

(ng/g

DW

)

0

20

40

60

80

100

120

AP24

AP26

Two “N. Alberta-adapted” Hybrid Poplars, cv. „Walker‟ (6 ramets with open

circles) and cv. „Manitou‟ (6 ramets with closed circles). Comparisons of Growth

Hormone (GA44, left & GA19, right) with Stump Diameters. The GA44 GA19

Biosynthetic Step is catalyzed by GA20 oxidases & encoded for by GA20ox

r2=0.627

p=0.002

Stump diameter (cm)

8 10 12 14 16 18 20

Low

er

ste

m G

A1

9 levels

(ng/g

DW

)

0

20

40

60

80

100

120r2=0.508

p=0.009

Stump diameter (cm)

8 10 12 14 16 18 20

Low

er

ste

m G

A4

4 levels

(ng/g

DW

)

0

5

10

15

20

25

30

35

An Alberta-adapted” Hybrid Poplar, cv. „Walker‟ (6 ramets with closed circles).

Comparisons of Growth Hormone Concentration (GA19, left & the auxin, IAA,

right) with Tree Heights Across Ramet within Clone. The GA44 GA19

Biosynthetic Step is encoded for by GA20ox. IAA is known to regulate GA

metabolism (reviewed in Thomas & Hedden, 2006, page 154).

r2=0.803

p=0.016

Tree height (m)

10 11 12 13 14 15

Low

er

ste

m G

A19 levels

(ng/g

DW

)

20

40

60

80

100

120

r2=0.947

p=0.001

Tree height (m)

10 11 12 13 14 15

Lo

we

r s

tem

IA

A le

ve

ls (

ng

/gD

W)

0

500

1000

1500

2000

2500

3000

Within Genotype Variation in Stump Diameters Across Ramet – The

relationship of indole-3-acetic acid (IAA) concentrations in lower stem

xylem tissues (harvested in mid-July 2005) to stem diameters across 6

ramets of Al-Pac hybrid poplar cv. Walker trees. A similar correlation

occurs for GA19 (data not shown).

r2=0.940

p=0.001

Stump diameter (cm)

10 12 14 16 18 20

Lo

we

r ste

m I

AA

le

ve

ls (

ng/g

DW

)

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

NH

CH2COOH

IAA

Within Genotype Variation in Stem Diameters (DBH) Across Ramet – The sum

of GA44 + GA19 + GA20 concentrations in upper “twig” tissues („elongating

internodes‟ harvested in mid-July), when correlated across ramet, “reflects” tree

DBH for cv. Walker, an Alberta-adapted hybrid poplar. Each closed circle is the

mean of two ramets for both DBH & the GA concentration.

r2=0.999

p=0.024

Mean outer bark DBH (cm)

8 10 12 14 16 18

Mea

n tw

ig G

A20

+19+

44 le

vels

(ng

/gD

W)

40

50

60

70

80

90

100

110

HCO2H

H

CO

O OH

CO2H

HCO2H

H

OH

O

HCO2H

H

CO

OOH

GA19GA44 GA20

Indian Head SK 2006 Hybrids: 6 genotypes X 3 ramets. Xylem tissue was

harvested in early August for hormone analysis – The ratio of „precursor‟ GA19 to

„product‟ GA20 was correlated with tree stump diameter

WP-69 Tristis (white) Walker Hill (yellow) CanAm Max (black)

r2=0.480

p=0.001

Stump diameter (mm)

20 30 40 50

Lo

wer

ste

m G

A1

9:G

A2

0 r

atio

0

20

40

60

80

100

120

r2=0.884

p=0.005

Mean stump diameter (mm)

20 25 30 35 40 45 50M

ea

n lo

we

r ste

m G

A1

9:G

A2

0 r

atio

10

20

30

40

50

60

70

80

CO2H

HCO2H

H

OH

O

HCO2H

H

CO

OOH

GA19GA20

GA20ox

Indian Head SK 2006 Hybrids – Growing xylem tissue GA44 & IAA

concentrations, each expressed as the mean of 3 ramets, were correlated, across 6

genotypes, with mean (of 3 ramets) tree stump diameters of those same genotypes

WP-69 Tristis (white) Walker Hill (yellow) CanAm Max (black)

r2=0.614

p=0.065

Mean stump diameter (mm)

20 25 30 35 40 45 50

Me

an low

er

ste

m G

A4

4 levels

(ng g

DW

-1)

4

5

6

7

8

9

10

r2=0.613

p=0.065

Mean stump diameter (mm)

20 25 30 35 40 45 50

Me

an

lo

we

r ste

m I

AA

le

ve

ls (

ng g

DW

-1)

1000

2000

3000

4000

5000

6000

HCO2H

H

CO

O OH

CO2H

HCO2H

H

OH

O

HCO2H

H

CO

OOH

GA19GA44 GA20

NH

CH2COOH

IAA

Indian Head SK 2006 Hybrids – Growing xylem tissue GA44 to IAA ratios, each

ratio expressed as the mean of 3 ramets, were correlated, across 6 genotypes, with

mean (of 3 ramets) tree stump diameters of those same genotypes

WP-69 Tristis (white) Walker Hill (yellow) CanAm Max (black)

HCO2H

H

CO

O OH

CO2H

HCO2H

H

OH

O

HCO2H

H

CO

OOH

GA19GA44 GA20

NH

CH2COOH

IAA

r2=0.779

p=0.020

Mean stump diameter (mm)

20 25 30 35 40 45 50

Mea

n lo

wer

ste

m IA

A:G

A44

rat

io

0

200

400

600

800

1000

1200

1400

Balsam Poplar. Both GA1 and its catabolite, GA8 “failed” as markers of field

performance across genotype. However, there were NEW correlations that

appeared. Specifically, stem tissue IAA concentration (ng per g DW tissue, left &

ng per cm lgth of internode tissue extracted, right).

r2=0.293

p=0.132n=9

Carnduff Indian Head year 4 field stem volumes (cm3)

1200 1400 1600 1800 2000 2200 2400

Gla

sshouse u

pper s

tem

IA

A levels

(ng/g

DW

)

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

r2=0.379

p=0.078n=9

Carnduff Indian Head year 4 field stem volumes (cm3)

1200 1400 1600 1800 2000 2200 2400

Gla

sshou

se u

pper

ste

m I

AA

leve

ls (

ng/c

m)

20

40

60

80

100

120

140

160

Balsam Poplar. Further, one of the NEW correlations (with field

performance) that appeared was GA19, and especially, the ratio of IAA to

GA19. . Thus, IAA:GA19 ratio, across genotype, showed a positive and

near-significant correlation with tree stem volumes at Year 4 (2009) in a

field provenance trial at Indian Head, SK.

HCO2H

H

CO

O OH

CO2H

HCO2H

H

OH

O

HCO2H

H

CO

OOH

GA19GA44 GA20

r2=0.429

p=0.055n=9

Carnduff Indian Head year 4 field stem volumes (cm3)

1200 1400 1600 1800 2000 2200 2400

Gla

ssh

ou

se

up

pe

r ste

m I

AA

:GA

19 r

atio

0

10

20

30

40

50

60

NH

CH2COOH

IAA

Conclusions: For poplar hybrids adapted to growing in Alberta and

Saskatchewan, and also for balsam poplar, it is apparent that:

Growing tissue concentrations of GA1 and GA4 do NOT reflect inherent

stem growth capacity “in the field” in the interior of Canada‟s prairie

provinces.

Rather, it appears that “upstream” C20 gibberellins, i.e. GA44, GA19 &

possibly GA20, whose biosynthesis is controlled by GA20 oxidases (&

encoded for by GA20ox) may provide the “markers” for inherent stem

growth capacity. Additionally, the auxin, IAA, which may regulate the

activity of GA20 oxidase, or expression of GA20ox, may also be a useful

“marker”.

Finally, it should be noted that gibberellin biosynthetic steps controlled by

GA20 oxidase (i.e. GA44 GA19 GA20) are under photoperiod

control, with Short Days down-regulating the expression of pttGA20ox, as

was shown for late expanding leaves of the aspen hybrid, P. tremula x P.

tremuloides (Eriksson, M.E. & T. Moritz. 2002. Planta 214:920-930).

Both Tree Height and Diameter of the Slower-

growing Hybrid Poplar cv. Prairie Sky Can Be

Increased – Almost 3-fold by Applied Gibberellin

30 15 0 ppm

of soil-applied

GA3

cv Prairie Sky

cv Tristis

In Contrast, for the Exceptionally Fast-growing

Hybrid Poplar cv. WP-69, the Height & Diameter

Increases that Result from Applied Gibberellin Are

Much Less, ca. 1.3-fold

30 15 0 ppm

of soil-applied GA3

Two-Year Old Willow (Salix aquatica) on

Peat soil in Finland – 30 Tonnes of Dry

Biomass per Hectare