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Disruption of NTPHAN function in
antisense transgenics produces ectopic
leaf blades and hyperelongation of the
leaf petiole in Nicotiana sylvestris
Neil A. McHale1, Ross Koning 2, and
Hsu-Liang Hsieh1.
1The
Connecticut Agricultural Experiment Station,
New Haven, CT 06504
2Eastern Connecticut State University,
Willimantic, CT 06226
MYB Proteins Are:
Transcription Factors
Found in Eukaryotic Organisms
A Small Gene Family in Mammals
A Large Gene Family in Plants
Have 3 Repeat Domains in Mammals:
R1
R2
R3
Have 2 Repeat Domains in Plants…including
Phantastica:
R2
R3
C terminus
Sequence Homology:
MYB Domain
Nicotiana: MRERQRWRSEEDALLRAYVKQYGPKEWHLVSQRMNTALNRDAKSCLERWKNYLKP
MKERQRWRPEEDALLRAYVKEYGPRDWHLVTQRMNKPLNRDAKSCLERWKNYLKP
Arabidopsis:MKERQRWSGEEDALLRAYVRQFGPREWHLVSERMNKPLNRDAKSCLERWKNYLKP
Antirrhinum:
Nicotiana: RIKKGSLTQEEQRSVIHLQAKHGNKWKKIAAEVPGRTAKRLGKWWEVFKEKQHRE
GIKKESLTQEEQISVINLQAKHGNKWKKIAAEVPGRTAKRLGKWWEVFKEKKHRE
Arabidopsis:GIKKGSLTEEEQRSVIRLQEKHGNKWKKIAAEVPGRTAKRLGKWWEVFKEKQHRE
Antirrhinum:
C terminus
Nicotiana: PFVTELVECCKELDEGHRAWAAHKKEAAWRLRRVELQLESEKICKVREKMEEIEA
HIVPELLECCKELEEGQRAWAAHRKEAAWRLKRVELQLESEKACRRREKMEEIEA
Arabidopsis:VFLSELVECCKELEEGHRAWADHKKEAAWRLRRVELQLESEKTCRQREKMEEIEA
Antirrhinum:
Nicotiana: KMKALREEQKATLDRIEAEYKEQLAGLRRDAEAKEQKLAEQWASKHLRLSKFLEQ
KMKALREEQKASLDRIEAEYREQLAGLRREAEVKEQKLAEQWAAKHLRLTKFLEQ
Arabidopsis:KMKALREEQKNAMEKIEAEYREQLVGLRRDAEAKDQKLADQWTSKHIRLTKFLEQ
Antirrhinum:
The PHAN genes of three species have
two conserved domains
Phantastica...
•Is a MYB Protein
•Is involved in leaf dorsoventrality
•Regulates shoot apical meristem function
•Produces bladeless leaves in Antirrhinum
•Produces lobed leaves in Arabidopsis
We cloned the orthologous gene (NTPHAN)
and generated antisense transgenics in
Nicotiana sylvestris
35S enh
(329 bp)
pFF19
4.2 kb
EcoR1
NTPHAN
BamH1
BamH1
Xba1
Sal1
Pst1
Sph1
Nsi1
35S polyA
(203 bp)
Sst1
Nru1
35S prom
(462 bp)
Sst1
The coding sequence of
NTPHAN with its 5’ UTR
was cloned as a Bam/Sst
fragment in antisense
orientation to the
35S CaMV promoter
in pFF19.
Hind III
EcoR1
Sac1
Kpn1
Sma1
BamH1
Xba1
Sal1
Pst1
HindIII
(1092)……………….MYB………….(-348)
RB
LB
pPZP221
The HindIII/EcoR1 fragment
from pFF19 was inserted into
the multiple cloning site of
pPZP221, and introduced
into Agrobacterium for leaf
disc transformation of WT
Nicotiana sylvestris.
Antisense NTPHAN leaves are abnormal, but maintain normal dorsoventrality.
•Juvenile leaves produce ectopic leaf
blades on the dorsal surface on both
sides of the midrib.
•These ectopic blades become
vascularized and have normal leaf
anatomy.
•The dorsal surface always faces
outward toward the leaf margins.
•Adult leaves have an elongated
petiole/midrib similar to PHAN
mutants in Antirrhinum.
•The vascular system is cylindrical
but a dorsal rib is present in a normal
position.
•The blades of adult leaves are
reduced in area, become peltate, and
ultimately bladeless on cauline
leaves.
In wild-type plants,
cauline leaves become
progressively smaller and
the petiole becomes
shorter.
In antisense NTPHAN
plants, cauline leaves
remain long and the
petiole elongates as blade
is reduced…sometimes
disappearing!
Does NTPHAN regulate KNOX genes?
ELK
homeo
Helix 1
Helix 2
turn
Helix 3
Gene expression studies in maize and Arabidopsis suggest that
PHAN functions as a negative transcriptional regulator of class
I KNOX genes. Maize RSI and Arabidopsis KNATI are
expressed ectopically in developing leaves.
Does loss of NTPHAN produce ectopic expression of genes in
the NTH (Nicotiana tabacum homeobox) genes?
We are examining expression of NTH 15 and NTH 20, the
KNOX genes most similar to Arabidopsis STM and KNAT1,
respectively.
RT-PCR = Reverse Transcription Polymerase Chain Reaction
•RNA is extracted from plant tissue of specific age
•Reverse transcriptase, dNTPs, buffer are added
•Incubated to produce DNA from RNA
•Custom oligonucleotide primers for specific gene added
•Taq DNA polymerase, dNTPs, buffer are added
•Thermal Cycling used to amplify specific gene
•Electrophoresis with ethidium bromide staining
RT-PCR Analysis of NTH Expression
PHAN and NTH23 are controls for
comparing “loading” factors
Juvenile leaves express NTH15 and 20
Young leaves stop producing NTH15
Young antisense PHAN leaves
continue to produce NTH20 while
young WT leaves stop.
Maturing leaves stop producing both
NTH 15 and NTH 20
Even in antisense PHAN leaves.
Conclusions:
•PHAN is expressed at all stages of leaf development.
•Loss of PHAN causes ectopic blade formation.
•Loss of PHAN correlates with extended NTH20
expression…but this extension disappears rapidly.
•Because ectopic blade development continues through
all of leaf development, it is unlikely to be caused
directly by ectopic NTH20 expression.
?