SOME FACTS AND THOUGHTS ABOUT AMYLOPLAST ENZYME COMPARTMENTATION

PNL Volume 11 1979 COMMENTS 36

Williams, A. F. Williams Laboratories, Williams., Indiana, U.S.A.

1. Of enzymes so far implicated in starch synthesis, only two -- starch

synthetase (ADP-gJucose —►starch + ADP) and phosphorylase (glucose-1-

phosphate -► starch + iP) — sediment with normal pea and maize amyloplasts

from developing seeds.

2. Substrate affinity (for starch) alone fails to account for amyloplast

binding of these enzymes (cf. Tsai, Bchm. Gen. 11:83-95, 1974, for starch

synthetase and item 7, below, for phosphorylase).

3. Observation of a significant difference in R/R vs r/r peas with regard

to soluble phosphorylase level dates back at least to 1957 (NAS-NRC, Laboratory

and Field Studies in Biology: A Sourcebook for Secondary Schools, pp. 681-688,

1957). Matheson and Richardson (Phytochem. 15:887-892, 1976, and 16:1875-1879,

1977), using extraction procedures which are inadequate to release much

amyloplast bound phosphorylase (see PNL 10:78), recently quantified this

difference in developing, mature, and germinating R/R and r/r peas.

4. Matheson and Richardson (1977) mention that mixtures of phosphorylase II

(the larger of two phosphorylase isozymes) extracted from bananas and R/R

peas can be resolved electrophoretically, while similar mixtures from bananas

and r/r peas cannot. I find that phosphorylase II released from R/R amyloplasts

has a slightly slower disc electrophoretic migration rate than soluble

phosphorylase II from r/r seeds. Phosphorylase II from R/r seeds is resolved

by disc electrophoresis into three bands, confirming Matheson and Richardson's

finding that this pea enzyme is dimeric.

5. In the light of 2, 3, and 4, above, I suggest that phosphorylase II

has an "amyloplast binding site" which is separate and distinct from its

"catalytically active site", and that the former is defective or missing in

r phosphorylase II.

6. Among maize, potatoes, and peas, I find that only developing pea

amyloplasts can be ruptured osmotically by pelleting from concentrated sucrose

solution and resuspending in water. Microscopic examination reveals significant

ballooning of the amyloplast membrane even among similarly treated mature pea

amyloplasts. Other workers have indicated unusual difficulty in leaching

soluble starch from pea amyloplasts (e.g., Potter, et al., J. Am. Chem. Soc.

75:1335-1338, 1953) and in deproteinizing pea starch (Senti and Dimler, Food

lech. 63:663-666, 1959).

7. Immature amyloplast fragments from r/r peas bind about 3 times as much

R/R as r/r phosphorylase II when exposed under uniform conditions. Pretreatment

of the ruptured amyloplasts with papain tends to nullify this difference,

indicating that protein(s) plays a role in the specificity of phosphorylase

"receptors" in the amyloplast (membrane?). Use of the r gene "probe" adds

an interesting dimension to amyloplast-phosphorylase binding studies such as

those of Fekete (Arch. Bchein. Bphys. 116:368-374, 1966). I am presently using

starch conjugate (-active site?) bound phosphorylase (see Matheson and Richardson

1977) to search for specific amyloplast proteins with phosphorylase affinities.

I'm assuming that these "receptors" are probably bound into the amyloplast

membrane via hydrophobic regions as discussed by Tanford (Science 200:1012-1018,

1978).

37 COMMENTS

PNL Volume 11

1979

8. The R/R variety 'Alaska' (used by Matheson and Richardson) has a soluble

phosphorylase level late in development which is about twice as high as that

of at least three other R/R_ varieties I have tested. R/R varietal hybrid seeds

from both 'Alaska' x 'Mammoth Melting Sugar' and 'Alaska' x 'Dwarf Grey Sugar'

have higher soluble phosphorylase levels when 'Alaska' is used as the female

parent than when 'Alaska' is used as the pollen parent. According to Marshall

(PNL 2:18-19), 'Alaska' carries the gene di_ (dimpled) which is expressed as

a maternal character; di_ may therefore be involved in organization of amyloplast

receptors for phosphorylase alluded to in 7, above.

9. For possible future research, I suggest that saturation of amyloplast

receptors with phosphorylase may somehow signal a "shutdown" of phosphorylase

synthesis. Such a system of regulation could be essential in the developing

seed since phosphorylase may act both in starch synthesis (when bound??) and

in starch digestion (when unbound??-hence the phenotype of r/r peas and starch??).

10. Suggestions 5 and 9, above, may also hold true for starch synthetase

in maize. Tsai (Maize Genetics Newsletter 39:153, 1965) has shown that the

wx (waxy) gene decreases amyloplast bound starch synthetase level in a dose

dependent' manner. No concomitant increase in soluble enzyme is found, indicating

that wx probably disrupts the catalytic site of starch synthetase. The striking

dose effect of wx and the lack of increase in soluble enzyme by wx may be due

to competitive binding of limited supplies of catalytic (Wx) and non-catalytic

(wx) enzyme proteins by amyloplast receptors (presumably, the binding site

within the mutant "enzyme" and the system of regulation could be intact) in

heterozygotes. Nelson (Maize Genetics Newsletter 50:109-113, 197b) has con-

structed a fine structure map containing over 30 wx alleles, but very little

is known about the actual nature of the w_x gene product (s). If antibodies

can be raised to maize starch synthetase, I plan a quantitative search for

cross reacting material in wx homo- and heterozygotes.

11. I have so far been unable to associate any lesion in the starch

synthetase or phosphorylase pathways to starch nor any differences in starch

synthetase or phosphorylase solubilities with the rb gene in peas. Nor have

I been able to find the solute primarily responsible for higher osmotic poten-

tial of rb cytoplasm. Some of my assays need refinement, however.

12. Isolation of new mutants affecting catalytic activity of pea phosphory-

lases or electrophoretic mobility of both pea and maize phosphorylases and

starch synthetases should provide excellent raw material for further study

of enzyme compartmentation in the amyloplast. Help in identifying possible

candidates is hereby solicited from pea geneticists, especially those who

regularly engage in mutagenesis. If, as suggested here, separate sites for

organelle binding and catalytic activity exist within the structure of an enzyme

(and therefore may be coded within the same locus), they could be expected

to mutate separately. Whether or not such neighboring mutants would result

in identical phenotype and/or be detected as alleles in classic tests of

allelism remains to be seen. Since seed "lethals" may be of value in my

studies, I maintain pod and plant sib identification records in my own muta-

genic work.