The Plant Cell, Vol. 3, 1141, November 1991
IN THlS ISSUE
Filling Tomorrow’s Shopping Basket: Practical
Applications of Plant Molecular Biology
One of the reasons that plant biology
is so exciting is that discoveries about
how plants function can have profound
practical applications. Many researchers are exploring the molecular
basis of agronomically important plant
traits with the eventual goal of engineering “better” crop plants, ones with
improved nutritional content, that are
better able to withstand pathogen attack, or that are less likely to spoil.
Although the fruits of agricultura1 biotechnology have yet to reach the
market, it is only a matter of time
before recent discoveries are translated into improved crop plants.
Although traditional breeding programs do not allow for the range of
cross-species gene-transfer events
that biotechnology makes possible,
such programs have done a remarkable job at enhancing the food value
(and many other properties) of important crop plants. A case in point is
corn. Corn seed is a staple food for
many of the world’s people, especially
in developing nations, yet it does not
contain nutritionally well-balanced
protein. The storage proteins known
as zeins, which constitute 60% of the
endosperm protein in the seed, are
lysine deficient; consequently, the
average lysine content of corn endosperm is low.
Over 25 years ago, it was found that
a corn mutant known as opaque-2 (02)
has a higher-than-normal lysine content (Mertz et al., 1964). The 02 gene
encodes a protein that is required for
the transcription of a subset of zein
genes, most notably the a-zeins
(Schmidt et al., 1990). 02 corn is more
nutritious than normal corn mainly
because it contains far less a-zein. With
less lysine-poor protein, the relative
content of the lysine-rich, non-zein
proteins that normally comprise 40%
of the endosperm rises. Not surpris- in normal corn; with two doses, the yingly, however, o2 corn kernels are zein content is still higher, and with
less dense and contain less protein three doses, it is higher still. The
than normal corn kernels. Moreover, modifier-mediated increase in y-zein
they are soft and floury (opaque to content appears to be independent of
light, hence their name). o2 kernels the o2 genotype: when the modifiers
also dry out more slowly than normal are crossed into a normal (02)gekernels and, as a result, are especially netic background, the y-zein level also
susceptible to insect and funga1 attack. rises.
The increased amounts of y-zein in
To circumvent these problems, plant
breeders developed 02 varieties that modified o2 mutants raised the quescontain genetic modifiers of the o2 tion of how the extra y-zein is distribuphenotype. The kernels of these QPM ted in the endosperm. In normal en(Quality Protein Maize) varieties main- dosperm, the concentration of p z e i n
tain the relatively high lysine content and y-zein is higher in the outer cell
of 02 corn but are as vitreous (glassy) layers than in the inner cell layers,
and hard as normal corn. It was dis- whereas the concentration of a-zein is
covered recently that the level of a- greater in the inner layers than the
zein in QPM corn seed remains Iow outer layers. (Lending and Larkins,
but that the amount of y-zein has in- 1989). In QPM endosperm, however,
creased to two to three times the y-zein is present at high concentranormal level (Wallace et al., 1990). tions throughout the endosperm, and
One of the challenges ahead is to the level of a-zein is much reduced.
determine the molecular mechanisms RNA gel blot analysis indicates that
by which the o2 modifiers in QPM corn the increase in the amount of y-zein
alter the protein composition and phe- protein reflects an increase in the
steady-state level of y-zein mRNA.
notype of the o2 endosperm.
In this issue, Geetha and cowork- This effect is probably mediated at the
ers (pages 1207-1219) characterize transcriptional or post-transcriptional
the y-zein alterations in QPM corn, level, for the authors find no evidence
analyzing the accumulation of these that the modifiers increase the copy
storage proteins during seed develop- number of the y-zein genes.
Although the increase in y-zein corment. The authors also take advantage of the triploid nature of the en- relates with the hard and glassy
dosperm to study the effects of differ- phenotype of QPM seeds, it does not
ent doses of modifier genes. In crosses explain why QPM retains a high lysine
in which the standard o2 plant is the content: y-zein is as devoid of lysines
female parent, the triploid endosperm as a-zein. QPM corn must also conwill have one dose of the modifier, and tain a significantly increased concenin crosses in which a QPM plant is tration of lysine-rich protein. It is
the female parent, the endosperm will possible that a secondary effect of the
have two doses. These studies indi- increased y-zein synthesis is a n incate that the 02 modifiers in Pool 34 crease in some other, lysine-rich proQPM, the line used in these experi- teins. A more detailed analysis of the
ments, are semidominant. With one modifier genes and the proteins they
dose of the modifiers, y-zein accu- encode as well as of the protein
mulates to slightly higher levels than content of o2 and QPM corn should
1142
The Plant Cell
IN THlS ISSUE
reveal the source of the increased
lysine content of QPM corn.
Nutritional quality is not the only trait
that growers are hoping to improve.
Years of breeding efforts have been
dedicated to increasing the shelf life
of climacteric fruits such as tomatoes,
bananas, and avocados. These fruits
ripen rapidly and uncontrollably, and
often overripen during shipment and
storage, causing enormous losses.
Ethylene has long been supposed to
be a central factor in fruit ripening:
ripening fruit produce ethylene and are
in turn induced to ripen by ethylene.
This correlation has led to the simple
scenario that fruit ripen because they
produce ethylene. If this view is correct, it should be possible to delay or
even inhibit ripening by blocking ethylene production in vivo.
Severa1groups have used antisense
technology to attempt to reduce ethylene production in fruit. From studies
of ethylene biosynthesis in plants, it is
known that 1-aminocyclopropane-1carboxylic acid (ACC), the immediate
precursor of ethylene, is formed from
S-adenosylmethionine by the enzyme
ACC synthase. This is the rate-limiting
step in ethylene biosynthesis in vivo.
ACC oxidase then catalyzes the conversion of ACC to ethylene. Hamilton
et al. (1990) transformed tomato plants
with an antisense ACC oxidase gene
and found that transgenic plants synthesize far less ethylene than normal
plants and produce fruit that ripen
more slowly. Recentiy, Oeller et ai.
(1991) transformed tomato plants with
an antisense ACC synthase gene
specific to an ACC synthase enzyme
that is expressed in ripening fruit. The
antisense gene suppresses ethylene
synthesis in transgenic fruit so strongly
that the fruit remain unripe until they
are exposed to exogenous ethylene,
at which point they ripen and become
indistinguishable in all measured respects from normal ripe tomatoes.
In this issue, Klee and coworkers
(pages 1187-1193) describe a different approach to reducing endogenous
ethylene levels in the plant. They have
transformed tomato plants with a gene
that encodes an enzyme that degrades
ACC rather than blocking its synthesis. To identify ACCdegrading enzymes,
Klee and coworkers gathered a collection of soil bacteria from around the
world and selected for those that could
grow on ACC as a sole nitrogen
source. Two, both Pseudomonas
species, were able to metabolize ACC
into a-ketobutyric acid. By transforming E. coli with a library of DNA
fragments from one of the Pseudomonas strains, Klee and coworkers
were able to clone the responsible
gene, which turns out to encode an
ACC deaminase similar to one that
had previously been isolated (Honma
and Shimomura, 1978).
Like tomatoes from antisense plants,
tomatoes from ACC deaminase-expressing plants ripen far more slowly
than normal tomatoes. Transgenic fruit
produce only one-tenth the ethylene
of normal fruit and remain red and firm
for much longer (over 6 weeks, as
opposed to less than 2 weeks for
normal tomatoes). Together with the
antisense studies, these experiments
confirm that ethylene is not only correlated with but also controls fruit ripening. Ethylene is found in many plant
tissues, and because the constitutively active CaMV 35s promoter
was used to drive ACC deaminase
expression, it was possible that the
transgenic plants would show developmental or other phenotypic abnormalities. This is not the case, however: tomato plants that constitutively
express the ACC deaminase gene are
phenotypically normal in all respects
besides fruit ripening. This result is
consistent with other indications that
ethylene does not play a central regulatory role in normal plant developmental processes outside of ripening.
Ethylene may play a role in certain
stress responses, however, and it will
be interesting to know whether these
responses are attenuated in plants that
express ACC deaminase.
The articles by Geetha and coworkers and Klee and coworkers illustrate
the power of basic research in improving
agriculturally important plants. Knowing the molecular basis of the o2
modifiers in QPM corn will boost future
genetic engineering efforts to produce
even more nutritious corn, just as
elucidation of the route of ethylene
biosynthesis has sparked the many
complementary approaches being
taken to inhibit ethylene production in
tomatoes. The result of both lines of
research should be dramatically enhanced food products that will be of
direct benefit to consumers as well as
to growers.
Rebecca Chasan
REFERENCES
Hamilton, A.J., Lycett, G.W., and
Grierson, D. (1990).Antisense gene that
inhibits synthesis of the hormone
ethylene in transgenic plants. Nature
346, 284-287.
Honma, N., and Shimomura, T. (1978).
Metabolism of 1-amino-cyclopropane-1carboxylic acid. Agric. Biol. Chem. 42,
1825-1831.
Lending, C.R., and Larkins, B.A. (1989).
Changes in the zein composition of
protein bodies during maize endosperm
development. Plant Cell 1, 1O11-1023.
Mertz, E.T., Bates, L.S., and Nelson, O.E.
(1964). Mutant gene that changes protein composition and increases lysine
content of maize endosperm. Science
145, 279-280.
Oelier, P.W., Min-Wong, L., Taylor, L.P.,
Pike, D.A., and Theologis, A. (1991).
Reversible inhibition of tomato fruit se-
nescence by antisense RNA. Science
254, 437-439.
Schmidt, R.J., Burr, F.A., Aukerman,
M.J., and Burr, B. (1990). Maize
regulatory gene opaque-2 encodes a
protein with a "leucine-zipper" motif that
binds to zein DNA. Proc. Natl. Acad. Sci.
USA 87, 46-50.
Wallace, J.C., Lopes, M.A., Paiva, E., and
Larkins, B.A. (1990). New methods for
extraction and quantitation of zeins reveal a high content of y-zein in modified
opaque-2 maize. Plant Physiol. 92,
191-196.
Filling Tomorrow's Shopping Basket: Practical Applications of Plant Molecular Biology
R. Chasan
Plant Cell 1991;3;1141-1142
DOI 10.1105/tpc.3.11.1141
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