Maize Harvest

Author(s): Sue Wessler and Sarah Hake

Source: The Plant Cell, Vol. 2, No. 6 (Jun., 1990), pp. 495-499
Published by: American Society of Plant Biologists (ASPB)
Stable URL: https://www.jstor.org/stable/3869110
Accessed: 29-11-2018 22:00 UTC

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The Plant Cell, Vol. 2, 495-499, June 1990 ? 1990 American Society of Plant Physiologists

Maize Harvest

Sue Wessler91 and Sarah Hakeb

a Department of Botany, University of Georgia, Athens, Georgia 30602

b United States Department of Agriculture/Agricultural Research Service, Plant Gene Expression Center, 800 Buchanan
Street, Albany, California 94710

MEETING REPORT

The 32nd Maize Genetics Meeting, held from March with 8 the
to 100 cloned genes and the approximately 660
11 in Delavan, Wisconsin, was very different from the restriction
first fragment length polymorphism (RFLP) loci. In
meeting in 1959. At that time, about 25 geneticists addition,
sat more cytogenetic tools are available in maize
around an oval table at Allerton Park, a small estatethan owned in any other plant. There are 879 A-A reciprocal
by the University of lllinois. The intent of the meeting translocations
was distributed over the whole genome. There
to be informal, informative, and argumentative. are The also
3184 B-A translocations (involving the accessory B
intervening years have witnessed a rapid growth in the
chromosomes), which have the ability to generate deficien-
number of participants, increasing to the currentcies or duplications
total of of defined parts of the genome. All of
more than 350. As the group grew in size, the original the translocations are slated for mapping by Coe and
room with the oval table gave way to a larger library colleagues
and, through a deficiency-generating strategy, which
in 1983 when the number exceeded 100, Allerton Park should produce a comprehensive and unparalleled physical
was reluctantly abandoned for even larger quarters. map that can be mated with the recombinational maps.
Despite this rapid growth, the organizers of the Maize Jeff Bennetzen (Purdue) has used more than 50 of the
Genetics Meetings have attempted to maintain some of maize RFLP markers to generate the first RFLP map of
the intimacy associated with the early days. This is prob? sorghum and finds that the two maps are virtually colinear
ably due to the fact that many of the "Allerton Twenty- (except for a few inversions). His finding that maize repet?
Five" are still active participants, including John Laughnan itive sequences are not homologous with sorghum se?
(University of lllinois), Ed Coe, Jr. (University of Missouri), quences may assist in very limited chromosome walks or
Oliver Nelson, Jr. (University of Wisconsin), Don Robertson in the analysis of jumping libraries.
(lowa State), Peter Peterson (lowa State), Gerald Neuffer The molecular characterization of genes previously
(University of Missouri), and Ellen Dempsey (Indiana Uni? cloned by transposon tagging, especially genes involved
versity). Even Marcus Rhoades (Indiana University) and in signal transduction pathways, represented a major em-
Charles Burnham (University of Minnesota), who were in phasis of this meeting. The ease in identifying mutants in
the now-famous 1929 photo with R.A. Emerson, George the anthocyanin pigment pathway led to the isolation of
Beadle, and Barbara McClintock, attended until the late transposable element alleles for six genes, including four
1980s. Their participation provides a sense of continuity, that encode enzymes (A1, Bz1, Bz2, C2) and two that are
which is such an important component of these meetings. presumed to be transcriptional activators (C1, R). The
This gathering represents a unique opportunity for those proteins encoded by C1 and R are not related; however,
new to the organism, such as first-year graduate students each has acidic and basic domains that are characteristic
and postdoctoral associates from other fields, to rub shoul- features of transcriptional activators. In addition, the basic
ders with geneticists who may have provided both the domains of C1 and R have amino acid similarity to the myb
insight and genetic stocks for their molecular projects. and myc binding domains, respectively (Cone et al., 1986;
The 44 verbal and 70 poster presentations at the 32nd Paz-Ares et al., 1987; Ludwig et al., 1989). Mutants of C1
Meeting clearly demonstrated that the large genome of or R fail to accumulate Bz1 or A1 transcripts, consistent
maize is not an impediment to exciting science. More than with their putative role as transcriptional activators. The
60 years of maize genetic studies have provided a wealth cloning of both the regulators and the regulated genes,
of raw materials for molecular biologists to exploit. With and the use of particle bombardment to introduce these
one foot in the past and one in the future, major progress genes into maize tissues to complement mutant pheno?
on the "Integrated Mapping Project" was reported by Ed types (Klein et al., 1989), provide the tools necessary to
Coe, Jr. (Missouri). This project serves to combine the study the mechanisms underlying correct gene expression.
more than 500 loci represented on the genetic linkage map The R gene family, on chromosome 10, controls where
and when pigment forms in the plant. More than 50 pat?
terns of pigmentation can be attributed to the genetic
1 To whom correspondence should be addressed. constitution of this gene family. Using the particle bom-

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496 The Plant Cell

also reported that two mutant alleles of vpl?vp1-c a

bardment assay, Ludwig et al. (1990) have demonstrated
that pigmentation can be induced in most cellvp1-Mc?have
types by a normal dormancy but no kernel pigmen
cauliflower mosaic virus 35S-ft cDNA construct even tion. The finding that both mutants synthesize a trunc
though the cDNA is encoded by a gene that normally
polypeptide has led to the hypothesis that the Vp1 prot
may be bifunctional with the amino terminus required
pigments only a subset of these cell types. This suggested
that the various patterns of pigmentation are determined dormancy and the carboxy terminus involved in antho
not by what the R genes encode but when the anin genebiosynthesis.
product is expressed. Vicki Chandler (University of Oregon) Unlike vpl, four other viviparous mutants, vp2, vp5, v
used an R probe to clone the B gene, located on chromo? and vp9, are blocked in carotenoid biosynthesis and
not accumulate ABA. The white endosperm phenotyp
some 2 (Chandler et al., 1989). Unlike R, B alleles condition
strong plant pigmentation. The duplicate nature of B andmutants was exploited by Karen Oishi (Universit
these
R was inferred from the existence of a B allele (B-Peru),Arizona), who reported the cloning of vp7 (ps1) usin
which can substitute for a functional R gene to conditiontransposon tagging with Mu elements. Brent Buckne
aleurone pigmentation. Chandler reported that the (lowa
se? State) used Mu tagging to clone y1, a gene that
quence of the B gene showed 80% amino acid similarity has a white endosperm mutant phenotype due to a bl
with R, including 93% identity in the myc homology in carotenoid biosynthesis. However, unlike the vp loci,
region.
Steve Goff (USDA Plant Gene Expression Center), in mutants
col- are not viviparous. Screening for the white end
laboration with Chandler, went on to show that either sperm thephenotype should facilitate the cloning of the ot
B cDNA fused to the 35S promoter, or the intact Bvp loci by similar tagging strategies. The availability
gene,
these genes, coupled with the ability to complement m
can induce pigmentation in maize tissue following particle
bombardment. Thus, the R and B alleles may represent tant phenotypes
a by assaying their expression when in
wonderful collection of tissue-specific promotersduced whoseinto intact tissues or protoplasts, provides powe
patterns of expression are reflected in the pigmentation tools to understand the carotenoid biosynthetic pathw
patterns of the plant. and the involvement of ABA in seed dormancy.
Goff, Chandler, and Karen Cone (University of Missouri) The ease of scoring nonlethal kernel phenotypes h
have used co-transformation of plasmids carrying regula? also facilitated the identification of several genes invo
in regulating the zein (seed storage) proteins of maize. T
tory genes and structural gene promoters fused to reporter
genes to begin to dissect the molecular interactions. zeinBy gene family comprises more than 100 genes tha
using this approach they have identified a 6-bp region encodeof several classes of zein proteins. opaque-2 (o
the Bz1 promoter that is necessary for gene expression. mutants are characterized by a selective reduction in
This sequence is very similar to a region within the 22-kD
KE2 zein class. Robert Schmidt, as a postdoctoral fel
immunoglobulin enhancer shown to bind an myc-related in Ben Burr's lab, cloned the 02 gene by tagging it w
protein. Goff also demonstrated that he can replace the the
Spm transposable element (Schmidt et al., 1987).
acidic domain of C1 with the acidic region of a known Schmidt (now at San Diego), reported that the 02 protein,
which has a leucine-zipper motif, binds in vitro to a se?
transcriptional activator (yeast Gal4) and still see activation
of Bz1 promoter constructs. quence 300 bp upstream of the start of the 22-kD zein
Don McCarty (University of Florida, Gainesville) and transcript
co- (Schmidt et al., 1990). The binding site is located
workers reported on what may be the next step up in 20 this
bp downstream from the "prolamine box," a conserved
signal transduction pathway. Mutations at the viviparous-element found upstream of all zein genes and even up?
1 (vp1) locus lead to the failure of dormancy and theof the prolamine storage protein genes of wheat
stream
absence of anthocyanin in aleurone and embryo. vp1 is
and barley. The finding that the putative 02 binding site is
unique among the vp loci of maize (vp1, 2, 5, 7, 8, not9)present
in in the promoters of the 19-kD zein genes is
two respects: (1) the other loci do not affect kernel consistent
pig? with the normal levels of the 19-kD class of
mentation and (2) vp1 mutants have normal abscisic zeins
acidseen in o2 mutants. Milo Aukerman (San Diego)
reported that an ethylmethane sulfonate-induced o2 mu?
(ABA) levels. The Vp1 product may control some aspect
tation
of ABA receptor activity or signal transduction within thehas a single base change that results in an Arg-to-
developing seed. The Vp1 gene was cloned by tagging Lys replacement at a highly conserved position near the
with the Mu element (McCarty et al., 1989) and its se?
leucine-zipper motif. Interestingly, the mutant protein is
incapable of binding the target site in vitro even though
quence, as reported by Chris Carson (University of Florida),
shows no similarity with known proteins. However, the harboring this o2 mutation are leaky for 22-kD zein
strains
691-amino acid open reading frame does share certainexpression. Schmidt believes that these and other data
indicate that the 02 protein may interact with another
structural characteristics with known regulatory proteins.
Consistent with a possible regulatory role that may act to control zein transcription initiation. One clue to
protein
before the regulatory C1 gene is the finding thatthe vp1identity of such a protein may come from other genet?
mutant embryos do not accumulate transcripts of ically
many defined mutant loci (such as floury), known to affect
genes in the anthocyanin pathway, including C1. Carson
zein synthesis.

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After almost 45 years of genetic and molecular charac?
terization, the maize transposable elements still have se-
crets to reveal. Following up on the classic studies of
Greenblatt and Brink demonstrating Ac's propensity to
transpose to linked sites, Hugo Dooner (DNA Plant Tech?
nology, Oakland) reported on the distribution of Ac trans-
positions to unlinked sites. By following the transposition
of the Ac element in the bronze-m2 (bz-m2) allele (on the
short arm chromosome 9), he found that approximately
50% of transposed Acs were located within 4 map units
of bronze. One-third of the unlinked Acs were mapped to
the other arm of chromosome 9. The number of trans?
posed Acs that mapped to chromosomes 5 and 7 ex-
ceeded random probability. Interestingly, an independent
study of the nonrandom distribution of maize transloca-
tions (Walden and Jancey, 1972) suggested that chromo?
somes 5 and 9 are close to each other in the interphase
nucleus, thus providing the proximity that is apparently
necessary for transposition. This study, and a previous
study by Dooner demonstrating local transposition of Ac
in tobacco, has important implications with regard to the
design of gene tagging strategies using the Ac element in
maize and several other plants.
Drew Schwartz (Indiana University) has focused on the
methylation of elements in the Ac/Ds family. Previous
studies correlated inactive Ac elements with a high level
of CPG methylation, especially near the Ac promoter region
(Schwartz and Dennis, 1986). In the study reported, he
compared the methylation state of an Ac and a Ds element
on the same chromosome arm and found, surprisingly,
that only the distal element becomes methylated. Schwartz
believes that such long-range cis effects on methylation
may help to explain other unusual genetic phenomena,
such as paramutation.
The solution to the orange pericarp mutant story may
presage a new synergism between two powerful plant
systems, Arabidopsis and maize. Alan Wright and Gerald
Neuffer (University of Missouri) investigated an orange
pericarp mutation that was apparently caused by two
genes because it segregated 15:1 on the ear. These loci,
designated orpl and orp2, were mapped to chromosomes
4 and 10, respectively. Wright analyzed the orange pig?
ment biochemically and found an excess of indole, which
is consistent with a defect in the last step of the tryptophan
pathway, catalyzed by tryptophan synthase. In collabora-
tion with Karen Cone, they used a heterologous hybridi?
zation probe from the Arabidopsis gene (from Robert Last,
Cornell) to clone two maize trpB genes. RFLP mapping
data confirmed the identity of orpl and orp2 as duplicate
structural genes for the B subunit of tryptophan synthase.
Development is another topic in which genetic studies
have paved the way for molecular analysis. Developmental
mutations have always intrigued maize geneticists as well
as serving as useful chromosome markers. The tassel
seed mutations, of which there are six (both dominant and
recessive) are being approached by a number of different
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Maize Harvest 497
methods. These mutations feminize the tassel, which is
normally completely male. Bruce Veit (USDA Plant Gene
Expression Center) presented a double mutant analysis of
the tassel seed mutations in combination with two other
mutations that affect the inflorescence, silkless and
branched silkless. He found that the six tassel seed mu?
tations fell into different epistatic relationships with thes
two mutations, thus permitting the presentation of model
of the gene interactions. With the report of the tagging an
cloning of tassel seed-2 (Ts2) by Alison Delong (Yale), the
mechanisms underlying the genetic interactions can now
be addressed. To tag Ts2, Delong and Steve Dellaport
took advantage of the propensity of Ac to transpose to
nearby sites; Ts2 was about 2 map units from the Ac-
containing P-vv allele in their parental stock. This strategy
of moving the "launching pad" close to the target (or vic
versa) was first used in the cloning of the R locus.
The groups of Zac Cande and Mike Freeling are using
a variety of genetic and cell biological approaches to
address specific developmental questions. Cande and
Chris Staiger (Berkeley) are using maize meiotic mutants
to study microtubule function. Their immunocytological
techniques suggest that abnormal meiosis is correlated
with disruption of the cytoskeleton. It is hoped that their
Mtv-induced mutants will allow the isolation of the respon?
sible genes. The Freeling group (Berkeley) presented sev?
eral preliminary studies on the characterization of ligule
formation. The ligule is a flap of tissue at the junction of
the leaf sheath and blade that is altered in several mutants.
They are using ligule development as a model system to
understand the relationship between timing, position, and
differentiation. In addition to classic approaches of clonal
analysis and scanning electron microscopy, they are at-
tempting to transposon-tag several of the genes already
identified by standard mutations.
One of the highlights of the meeting was the presenta?
tion of the structure and sequence of the Knotted (Kn)
locus, a dominant leaf morphology mutant previously
cloned by tagging with Ds (Hake et al., 1989). Erik Voll-
brecht (USDA Plant Gene Expression Center) reported
that Kn may be the first homeodomain-containing gene in
plants, sharing the greatest conservation with the yeast
homeodomain-containing genes such as MATal. Neelima
Sinha's (USDA Plant Gene Expression Center) clonal
analysis of Kn showed that only certain inner cells of the
leaf need to carry the mutant gene in order to see the
mutant phenotype. Her in situ hybridization experiments
reveal that the transcript is relocalized to a novel cell type
in mutant leaves. These data suggest that the Kn gene
product may be involved in some aspect of maturation and
that its inappropriate expression is, perhaps, responsible
for the mutant phenotype.
In an experimental approach that weds transposons and
developmental phenomena, Rob Martienssen (Cold Spring
Harbor) and Alice Barkan (Berkeley) presented a poster on
the clonal inheritance of the suppression of two Mu-\n-
498 The Plant Cell

duced mutations, the photosynthetic mutationegies, thusand

hcf106 ensuring a continuous supply of interesting
a disease lesion mimic mutation (les). The mutant
genes. plants
To assist in the analysis of these genes, we now
have powerful
contained lesions, characteristic of les, superimposed on transient assay systems that permit the
the pale-green phenotype of the hcf106 mutationcomplementation
and wild- of mutant phenotypes and the analysis
type sectors in which both of these mutant of complex gene interactions. The historical connections
phenotypes
represented
were suppressed. From DNA analysis of the mutant tissueby those attending the meeting may now be
matched by of
and wild-type sectors, they found that the inactivation experiments that permit the newest molecular
Mu correlated perfectly with the suppression oftechniques
the mutant to tap into the vast genetic resources of maize
phenotypes.
With all of the genetic and cytogenetic advantages of-
fered by maize, we often forget that it is also a major crop
ACKNOWLEDGMENTS
plant. In this regard, the meeting brought the first report
of the cloning of what may prove to be the first disease-
resistance locus in a higher plant. Susceptibility We
to thank
Northern
Ed Coe for valuable information and Cliff Weil, Steve
leaf spot, a disease caused by a toxin (hc toxin) produced
Ludwig, Michael Purugganan, and Gary Kochert for critical readin
by the fungus Helminthosporium carbonum, is controlled
of the manuscript.
by two recessive genes, hm1 (chromosome 1) and hm2
(chromosome 9). Strains containing a functional Hm1 locus
are 100-fold more tolerant of the toxin. Guri Johal, in
collaboration with Steve Briggs (Cold Spring Harbor/Pi-
oneer), used the Mu transposable element to tag Hm1 and Received April 6, 1990.
has isolated two independent but tightly linked restriction
fragments containing Mu insertions. Their plans are to use
a tissue culture bioassay to test whether homologous DNA
REFERENCES
fragments, isolated from Hm 1 -containing strains, can con?
fer toxin resistance when introduced into protoplasts by
electroporation. Chandler, V.L., Radicella, J.P., Robbins, T.P., Chen, J., and
Finally, maize transformation is almost in hand. Several Turks, D. (1989). Two regulatory genes of the maize anthocy?
examples were presented of transformed plants resulting anin pathway are homologous: Isolation of B utilizing R genomic
from particle bombardment of immature embryos and em- sequences. Plant Cell 1, 1175-1183.
brogenic callus. This was dramatically illustrated by Ben Cone, K., Burr, F., and Burr, B. (1986). Molecular analysis of the
Bowen (Pioneer) who unveiled "Erika," a plant with large, maize anthocyanin regulatory locus C1. Proc. Natl. Acad. Sci.
red leaf sectors resulting from the introduction of a con? USA 83, 9631-9635.
struct containing the anthocyanin regulatory R gene into Hake, S., Vollbrecht, E.V., and Freeling, M. (1989). Cloning
immature embryos. Although the red sector extended into knotted, the dominant morphological mutant in maize using Ds2
the tassel, the progeny of pollen from red anthers did not as a transposon tag. EMBO J. 8, 15-22.
contain the R construct. The results presented by Kelly Klein, T.M., Roth, B.A., Sanford, J.C, and Fromm, M.E. (1989).
Dawes (Berkeley) on clonal analysis of anther development Regulation of anthocyanin biosynthetic genes introduced into
intact maize tissues by microprojectiles. Proc. Natl. Acad. Sci.
are particularly relevant in this regard. He showed that two
USA 86, 6681-6685.
cell layers of the anther can give colored sectors, but only
Ludwig, S.R., Habera, L.F., Dellaporta, S.L., and Wessler, S.R.
the inner layer gives rise to gametes. Thus, clonal sectors
(1989). Lc, a member of the maize R gene family responsible
of introduced genes need to reside in the L2 to be heritable.
for tissue-specific anthocyanin production, encodes a protein
Michael Fromm (USDA Plant Gene Expression Center) also
similar to transcriptional activators and contains the myc-ho-
used particle bombardment to introduce herbicide resist? mology region. Proc. Natl. Acad. Sci. USA 86, 7092-7096.
ance genes into embryogenic calli and obtained several Ludwig, S.R., Bowen, B., Beach, L., and Wessler, S.R. (1990).
transformed plants that may yet produce transformed A regulatory gene as a novel visible marker for maize transfor?
progeny. Most of us went home feeling that maize trans? mation. Science 247, 449-450.
formation will be reported at the next maize meeting, to McCarty, D.R., Carson, C.B., Stinard, P.S., and Robertson, D.S.
be held at Delavan from March 21 to 24, 1991. (1989). Molecular analysis of viviparous^: An abscisic acid-
This meeting marked a major turning point in maize insensitive mutant of maize. Plant Cell 1, 523-532.
molecular genetics. The first genes involved in develop? Paz-Ares, J., Ghosal, D., Wienand, U., Peterson, P.A., and
mental decisions and disease resistance have been cloned. Saedler, H. (1987). The regulatory d locus of Zea mays
Regulators of some regulatory genes may also be in hand. encodes a protein with homology to myb protooncogene prod?
Increased knowledge of the biology of the maize transpos? ucts and with structural similarities to transcriptional activators.
able elements has led to more sophisticated tagging strat- EMBO J. 6, 3553-3558.

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 Maize Harvest 499

Schwartz,
Schmidt, R.J., Burr, F.A., and Burr, B. (1987). Transposon tag? D., and Dennis, E. (1986). Transposase activity of
ging and molecular analysis of the maize regulatory locusAc controlling element in maize is regulated by its degre
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and Drosophila melanogaster. Can. J. Genet. Cytol. 1
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