Methods for improving seeds

The invention provides methods of modulating seed mass and other traits in plants. The methods involve producing transgenic plants comprising a recombinant expression cassette containing an ADC nucleic acid linked to a plant promoter.

FIELD OF THE INVENTION 
The present invention is directed to plant genetic engineering. In 
particular, it relates to new methods for modulating mass and other 
properties of plant seeds. 
BACKGROUND OF THE INVENTION 
The pattern of flower development is controlled by the floral meristem, a 
complex tissue whose cells give rise to the different organ systems of the 
flower. Genetic and molecular studies have defined an evolutionarily 
conserved network of genes that control floral meristem identity and 
floral organ development in Arabidopsis, snapdragon, and other plant 
species (see, e.g., Coen and Carpenter, Plant Cell 5:1175-1181 (1993) and 
Okamuro et al., Plant Cell 5:1183-1193 (1993)). In Arabidopsis, a floral 
homeotic gene APETALA2 (AP2) controls three critical aspects of flower 
ontogeny--the establishment of the floral meristem (Irish and Sussex, 
Plant Cell 2:741-753 (1990); Huala and Sussex, Plant Cell 4:901-913 
(1992); Bowman et al., Development 119:721-743 (1993); Schultz and Haughn, 
Development 119:745-765 (1993); Shannon and Meeks-Wagner, Plant Cell 
5:639-655 (1993)), the specification of floral organ identity (Komaki et 
al., Development 104:195-203 (1988)); Bowman et al., Plant Cell 1:37-52 
(1989); Kunst et al., Plant Cell 1:1195-1208 (1989)), and the temporal and 
spatial regulation of floral homeotic gene expression (Bowman et al., 
Plant Cell 3:749-758 (1991); Drews et al., Cell 65:91-1002 (1991)). 
One early function of AP2 during flower development is to promote the 
establishment of the floral meristem. AP2 performs this function in 
cooperation with at least three other floral meristem genes, APETALA1 
(AP1), LEAFY (LFY), and CAULIFLOWER (CAL) (Irish and Sussex (1990); 
Bowman, Flowering Newsletter 14:7-19 (1992); Huala and Sussex (1992); 
Bowman et al., (1993); Schultz and Haughn, (1993); Shannon and 
Meeks-Wagner, (1993)). A second function of AP2 is to regulate floral 
organ development. In Arabidopsis, the floral meristem produces four 
concentric rings or whorls of floral organs--sepals, petals, stamens, and 
carpels. In weak, partial loss-of-function ap2 mutants, sepals are 
homeotically transformed into leaves, and petals are transformed into 
pollen-producing stamenoid organs (Bowman et al., Development 112:1-20 
(1991)). By contrast, in strong ap2 mutants, sepals are transformed into 
ovule-bearing carpels, petal development is suppressed, the number of 
stamens is reduced, and carpel fusion is often defective (Bowman et al., 
(1991)). Finally, the effects of ap2 on floral organ development are in 
part a result of a third function of AP2, which is to directly or 
indirectly regulate the expression of several flower-specific homeotic 
regulatory genes (Bowman et al., Plant Cell 3:749-758 (1991); Drews et 
al., Cell 65:91-1002 (1991); Jack et al. Cell 68:683-697 (1992); Mandel et 
al. Cell 71: 133-143 (1992)). 
Clearly, Ap2 plays a critical role in the regulation of Arabidopsis flower 
development. Yet, little is known about how it carries out its functions 
at the cellular and molecular levels. A spatial and combinatorial model 
has been proposed to explain the role of AP2 and other floral homeotic 
genes in the specification of floral organ identity(see, e.g., Coen and 
Carpenter, supra). One central premise of this model is that AP2 and a 
second floral homeotic gene AGAMOUS (AG) are mutually antagonistic genes. 
That is, AP2 negatively regulates AG gene expression in sepals and petals, 
and conversely, AG negatively regulates AP2 gene expression in stamens and 
carpels. In situ hybridization analysis of AG gene expression in wild-type 
and ap2 mutant flowers has demonstrated that AP2 is indeed a negative 
regulator of AG expression. However, it is not yet known how AP2 controls 
AG. Nor is it known how AG influences AP2 gene activity. 
The AP2 gene in Arabidopsis has been isolated by T-DNA insertional 
mutagenesis as described in Jofuku et al. The Plant Cell 6:1211-1225 
(1994). AP2 encodes a putative nuclear factor that bears no significant 
similarity to any known fungal, or animal regulatory protein. Evidence 
provided there indicates that AP2 gene activity and function are not 
restricted to developing flowers, suggesting that it may play a broader 
role in the regulation of Arabidopsis development than originally 
proposed. 
In spite of the recent progress in defining the genetic control of plant 
development, little progress has been reported in the identification and 
analysis of genes effecting agronomically important traits such as seed 
size, protein content, oil content and the like. Characterization of such 
genes would allow for the genetic engineering of plants with a variety of 
desirable traits. The present invention addresses these and other needs. 
SUMMARY OF THE INVENTION 
The present invention provides methods of modulating seed mass and other 
traits in plants. The methods involve providing a plant comprising a 
recombinant expression cassette containing an ADC nucleic acid linked to a 
plant promoter. The plant is either selfed or crossed with a second plant 
to produce a plurality of seeds. Seeds with the desired trait (e.g., 
altered mass) are then selected. 
In some embodiments, transcription of the ADC nucleic acid inhibits 
expression of an endogenous ADC gene or activity the encoded protein. In 
these embodiments, the step of selecting includes the step of selecting 
seed with increased mass or another trait. The seed may have, for 
instance, increased protein content, carbohydrate content, or oil content. 
In the case of increased oil content, the types of fatty acids may or may 
not be altered as compared to the parental lines. In these embodiments, 
the ADC nucleic acid may be linked to the plant promoter in the sense or 
the antisense orientation. Alternatively, expression of the ADC nucleic 
acid may enhance expression of an endogenous ADC gene or ADC activity and 
the step of selecting includes the step of selecting seed with decreased 
mass. This embodiment is particularly useful for producing seedless 
varieties of crop plants. 
If the first plant is crossed with a second plant the two plants may be the 
same or different species. The plants may be any higher plants, for 
example, members of the families Brassicaceae or Solanaceae. In making 
seed of the invention, either the female or the male parent plant can 
comprise the expression cassette containing the ADC nucleic acid. In 
preferred embodiments, both parents contain the expression cassette. 
In the expression cassettes, the plant promoter may be a constitutive 
promoter, for example, the CaMV 35S promoter. Alternatively, the promoter 
may be a tissue-specific promoter. Examples of tissue specific expression 
useful in the invention include fruit-specific, seed-specific (e.g., 
ovule-specific, embryo-specific, endosperm-specific, integument-specific, 
or seed coat-specifiic) expression. 
The invention also provides seed produced by the methods described above. 
The seed of the invention comprise a recombinant expression cassette 
containing an ADC nucleic acid. If the expression cassette is used to 
inhibit expression of endogenous ADC expression, the seed will have a mass 
at least about 20% greater than the average mass of seeds of the same 
plant variety which lack the recombinant expression cassette. If the 
expression cassette is used to enhance expression of ADC, the seed will 
have a mass at least about 20% less than the average mass of seeds of the 
same plant variety which lack the recombinant expression cassette. Other 
traits such as protein content, carbohydrate content, and oil content can 
be altered in the same manner. 
Definitions 
The phrase "nucleic acid sequence" refers to a single or double-stranded 
polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to 
the 3' end. It includes chromosomal DNA, self-replicating plasmids, 
infectious polymers of DNA or RNA and DNA or RNA that performs a primarily 
structural role. 
The term "promoter" refers to a region or sequence determinants located 
upstream or downstream from the start of transcription and which are 
involved in recognition and binding of RNA polymerase and other proteins 
to initiate transcription. A "plant promoter" is a promoter capable of 
initiating transcription in plant cells. 
The term "plant" includes whole plants, plant organs (e.g., leaves, stems, 
flowers, roots, etc.), seeds and plant cells and progeny of same. The 
class of plants which can be used in the method of the invention is 
generally as broad as the class of higher plants amenable to 
transformation techniques, including angiosperms (monocotyledonous and 
dicotyledonous plants), as well as gymnosperms. It includes plants of a 
variety of ploidy levels, including polyploid, diploid, haploid and 
hemizygous. 
A polynucleotide sequence is "heterologous to" an organism or a second 
polynucleotide sequence if it originates from a foreign species, or, if 
from the same species, is modified from its original form. For example, a 
promoter operably linked to a heterologous coding sequence refers to a 
coding sequence from a species different from that from which the promoter 
was derived, or, if from the same species, a coding sequence which is 
different from any naturally occurring allelic variants. As defined here, 
a modified ADC coding sequence which is heterologous to an operably linked 
ADC promoter does not include the T-DNA insertional mutants (e.g, ap2-10) 
as described in Jofuku et al. The Plant Cell 6:1211-1225 (1994). 
A polynucleotide "exogenous to" an individual plant is a polynucleotide 
which is introduced into the plant by any means other than by a sexual 
cross. Examples of means by which this can be accomplished are described 
below, and include Agrobacterium-mediated transformation, biolistic 
methods, electroporation, and the like. Such a plant containing the 
exogenous nucleic acid is referred to here as an R.sub.1 generation 
transgenic plant. Transgenic plants which arise from sexual cross or by 
selfing are descendants of such a plant. 
An "ADC (AP2 domain containing) nucleic acid" or "ADC polynucleotide 
sequence" of the invention is a subsequence or full length polynucleotide 
sequence of a gene which, encodes an polypeptide containing an AP2 domain 
and when present in a transgenic plant, can be used to modulate seed 
properties in seed produced by the plant. An exemplary nucleic acid of the 
invention is the Arabidopsis AP2 sequence as disclosed in Jofuku et al. 
The Plant Cell 6:1211-1225 (1994). The GenBank accession number for this 
sequence is U12546. As explained in detail below a family of RAP2 (related 
to AP2) genes have been identified in Arabidopsis. The class of nucleic 
acids claimed here falls into at least two subclasses (AP2-like and 
EREBP-like genes), which are distinguished by, for instance, the number of 
AP2 domains contained within each polypeptide and by sequences within 
certain conserved regions. The differences between these two subclasses 
are described in more detail below. ADC polynucleotides are defined by 
their ability to hybridize under defined conditions to the exemplified 
nucleic acids or PCR products derived from them. An ADC polynucleotide 
(e.g., AP2 or RAP2) is typically at least about 30-40 nucleotides to about 
3000, usually less than about 5000 nucleotides in length. Usually the 
nucleic acids are from about 100 to about 2000 nucleotides, often from 
about 500 to about 1700 nucleotides in length. 
ADC nucleic acids, as explained in more detail below, are a new class of 
plant regulatory genes that encode ADC polypeptides, which are 
distinguished by the presence of one or more of a 56-68 amino acid 
repeated motif, referred to here as the "AP2 domain". The amino acid 
sequence of an exemplary AP2 polypeptide is shown in Jofuku et al., supra. 
One of skill will recognize that in light of the present disclosure 
various modifications (e.g., substitutions, additions, and deletions) can 
be made to the sequences shown there without substantially affecting its 
function. These variations are specifically covered by the terms ADC 
polypeptide or ADC polynucleotide. 
In the case of both expression of transgenes and inhibition of endogenous 
genes (e.g., by antisense, or sense suppression) one of skill will 
recognize that the inserted polynucleotide sequence need not be identical, 
but may be only "substantially identical" to a sequence of the gene from 
which it was derived. As explained below, these substantially identical 
variants are specifically covered by the term ADC nucleic acid. 
In the case where the inserted polynucleotide sequence is transcribed and 
translated to produce a functional polypeptide, one of skill will 
recognize that because of codon degeneracy a number of polynucleotide 
sequences will encode the same polypeptide. These variants are 
specifically covered by the terms "ADC nucleic acid", "AP2 nucleic acid" 
and "RAP2 nucleic acid". In addition, the term specifically includes those 
full length sequences substantially identical (determined as described 
below) with an ADC polynucleotide sequence and that encode proteins that 
retain the function of the ADC polypeptide (e.g., resulting from 
conservative substitutions of amino acids in the AP2 polypeptide). In 
addition, variants can be those that encode dominant negative mutants as 
described below. 
Two nucleic acid sequences or polypeptides are said to be "identical" if 
the sequence of nucleotides or amino acid residues, respectively, in the 
two sequences is the same when aligned for maximum correspondence as 
described below. The term "complementary to" is used herein to mean that 
the complementary sequence is identical to all or a portion of a reference 
polynucleotide sequence. 
Sequence comparisons between two (or more) polynucleotides or polypeptides 
are typically performed by comparing sequences of the two sequences over a 
"comparison window" to identify and compare local regions of sequence 
similarity. A "comparison window", as used herein, refers to a segment of 
at least about 20 contiguous positions, usually about 50 to about 200, 
more usually about 100 to about 150 in which a sequence may be compared to 
a reference sequence of the same number of contiguous positions after the 
two sequences are optimally aligned. 
Optimal alignment of sequences for comparison may be conducted by the local 
homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by 
the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 
48:443 (1970), by the search for similarity method of Pearson and Lipman 
Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized 
implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and 
TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group 
(GCG), 575 Science Dr., Madison, Wis.), or by inspection. 
"Percentage of sequence identity" is determined by comparing two optimally 
aligned sequences over a comparison window, wherein the portion of the 
polynucleotide sequence in the comparison window may comprise additions or 
deletions (i.e., gaps) as compared to the reference sequence (which does 
not comprise additions or deletions) for optimal alignment of the two 
sequences. The percentage is calculated by determining the number of 
positions at which the identical nucleic acid base or amino acid residue 
occurs in both sequences to yield the number of matched positions, 
dividing the number of matched positions by the total number of positions 
in the window of comparison and multiplying the result by 100 to yield the 
percentage of sequence identity. 
The term "substantial identity" of polynucleotide sequences means that a 
polynucleotide comprises a sequence that has at least 60% sequence 
identity, preferably at least 80%, more preferably at least 90% and most 
preferably at least 95%, compared to a reference sequence using the 
programs described above (preferably BLAST) using standard parameters. One 
of skill will recognize that these values can be appropriately adjusted to 
determine corresponding identity of proteins encoded by two nucleotide 
sequences by taking into account codon degeneracy, amino acid similarity, 
reading frame positioning and the like. Substantial identity of amino acid 
sequences for these purposes normally means sequence identity of at least 
35%, preferably at least 60%, more preferably at least 90%, and most 
preferably at least 95 %. Polypeptides which are "substantially similar" 
share sequences as noted above except that residue positions which are not 
identical may differ by conservative amino acid changes. Conservative 
amino acid substitutions refer to the interchangeability of residues 
having similar side chains. For example, a group of amino acids having 
aliphatic side chains is glycine, alanine, valine, leucine, and 
isoleucine; a group of amino acids having aliphatic-hydroxyl side chains 
is serine and threonine; a group of amino acids having amide-containing 
side chains is asparagine and glutamine; a group of amino acids having 
aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group 
of amino acids having basic side chains is lysine, arginine, and 
histidine; and a group of amino acids having sulfur-containing side chains 
is cysteine and methionine. Preferred conservative amino acids 
substitution groups are: valine-leucine-isoleucine, 
phenylalanine-tyrosine, lysine-arginine, alanine-valine, and 
asparagine-glutamine. 
Another indication that nucleotide sequences are substantially identical is 
if two molecules hybridize to each other, or a third nucleic acid, under 
stringent conditions. Stringent conditions are sequence dependent and will 
be different in different circumstances. Generally, stringent conditions 
are selected to be about 5.degree. C. lower than the thermal melting point 
(Tm) for the specific sequence at a defined ionic strength and pH. The Tm 
is the temperature (under defined ionic strength and pH) at which 50% of 
the target sequence hybridizes to a perfectly matched probe. Typically, 
stringent conditions will be those in which the salt concentration is 
about 0.02 molar at pH 7 and the temperature is at least about 
60.degree..degree.C. 
In the present invention, genomic DNA or cDNA comprising ADC nucleic acids 
of the invention can be identified in standard Southern blots under 
stringent conditions using the nucleic acid sequences disclosed here. For 
the purposes of this disclosure, stringent conditions for such 
hybridizations are those which include at least one wash in 0.2.times. SSC 
at a temperature of at least about 50.degree. C., usually about 55.degree. 
C. to about 60.degree. C., for 20 minutes, or equivalent conditions. Other 
means by which nucleic acids of the invention can be identified are 
described in more detail below.