Disruption of NTPHAN function in antisense transgenics produces ectopic leaf blades and hyperelongat

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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

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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
Have 2 Repeat Domains in Plantsincluding Phantast
ica
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Sequence Homology
MYB Domain
Nicotiana MRERQRWRSEEDALLRAYVKQYGPKEWHLVSQRMNTALN
RDAKSCLERWKNYLKP Antirrhinum MKERQRWRPEEDALLRAYVK
EYGPRDWHLVTQRMNKPLNRDAKSCLERWKNYLKP Arabidopsis M
KERQRWSGEEDALLRAYVRQFGPREWHLVSERMNKPLNRDAKSCLERWKN
YLKP
Nicotiana RIKKGSLTQEEQRSVIHLQAKHGNKWKKIAAEVPGRTAK
RLGKWWEVFKEKQHRE Antirrhinum GIKKESLTQEEQISVINLQA
KHGNKWKKIAAEVPGRTAKRLGKWWEVFKEKKHRE Arabidopsis G
IKKGSLTEEEQRSVIRLQEKHGNKWKKIAAEVPGRTAKRLGKWWEVFKEK
QHRE
C terminus
Nicotiana PFVTELVECCKELDEGHRAWAAHKKEAAWRLRRVELQLE
SEKICKVREKMEEIEA Antirrhinum HIVPELLECCKELEEGQRAW
AAHRKEAAWRLKRVELQLESEKACRRREKMEEIEA Arabidopsis V
FLSELVECCKELEEGHRAWADHKKEAAWRLRRVELQLESEKTCRQREKME
EIEA
Nicotiana KMKALREEQKATLDRIEAEYKEQLAGLRRDAEAKEQKLA
EQWASKHLRLSKFLEQ Antirrhinum KMKALREEQKASLDRIEAEY
REQLAGLRREAEVKEQKLAEQWAAKHLRLTKFLEQ Arabidopsis K
MKALREEQKNAMEKIEAEYREQLVGLRRDAEAKDQKLADQWTSKHIRLTK
FLEQ
The PHAN genes of three species have two
conserved domains
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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
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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.
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.
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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.

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In antisense NTPHAN plants, cauline leaves remain
long and the petiole elongates as blade is
reducedsometimes disappearing!
In wild-type plants, cauline leaves become
progressively smaller and the petiole becomes
shorter.
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Does NTPHAN regulate KNOX genes?
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.
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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

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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.
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Conclusions

• PHAN is expressed at all stages of leaf
  development.
• Loss of PHAN causes ectopic blade formation.
• Loss of PHAN correlates with extended NTH20
  expressionbut 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.

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