Indirect evidence that eATP regulates growth: apyrases regulate growth

1
Indirect evidence that eATP regulates growth
apyrases regulate growth

• Discovery Lab lecture
• February 1, 2007

2
Ectoapyrase enzymes that control the xATP are
localized in regions critical for growth

• Apyrases are NTPDases (ATP diphosphorylases)

• Ectoapyrases are localized on the PM with
  active site facing into the ECM

• Differences between apyrase and ATPase

• Different substrate specificity
• Apyrase has a broader divalent cation
  requirement
• Apyrase is insensitive to ATPase inhibitors
• Apyrase has a distinct primary structure

3
In situ Expression of Apyrases in Lateral Roots
4
Expression of APsGUS in pollen and pollen tubes
5
Light suppression of APsGUS in wild-type
hypocotyls
6
Light induces loss of apyrase protein sooner
than it induces decrease in growth rate

• Park and Spalding (1999) showed that it took 15
  min before light treatment significantly
  inhibited hypocotyl growth
• Figure above shows that light can induce
  significant loss of apyrase protein within 7 min.

7
KO of both AtApy1 and AtApy2 ( DKO) Results in
Growth Inhibition

• Pollen of DKO plants develops normally, but
  pollen tubes cannot grow out from grain
  (Steinebrunner et al. Plant Physiol. 131 1638
  (2003)
• If DKO plants are complemented with inducible wt
  apyrase, pollen germinates and seeds form. Then,
• if induction withdrawn, the seedling and
  adult plants that grow without apyrase are dwarf.

8
Double KO seedlings
WT vector
leaky DKO
non-leaky DKO
WT
7 d on MS agar

Cotyledons appear to be normal size, but
hypocotyls and roots are shorter.
9
Inhibition of ectoapyrase activity by antibodies
or apyrase inhibitors inhibits pollen tube growth
10
Schedule today

• Transfer seedlings to either normal growth medium
  or to media containing 150 ?M ADP?S and take
  pictures of root hairs at t0 and t1 to assess the
  growth of root hairs transferred on dialysis
  substrate.
• Calculate data and send it to me by 5 PM Friday,
  and arrange with partner a time (ca. 30 min) to
  plant seeds on Monday morning.