The identification and characterization of two dominant r1 haplotype-specific inhibitors of aleurone color in Zea mays.

We report the identification and characterization of two novel dominant inhibitors of aleurone color in Zea mays that interact with specific haplotypes of the r1 locus. One inhibitor locus, inr1 (inhibitor of r1 aleurone color 1), maps to the long arm of chromosome 10, distal to the TB-10L19 breakpoint and tightly linked to dull1, and the second inhibitor locus, inr2 (inhibitor of r1 aleurone color 2), maps to the long arm of chromosome 9. Dominant inhibitory alleles of inr1 and inr2 act by suppressing aleurone color conditioned by certain r1 haplotypes. Two haplotypes, R1-ch:Stadler and R1-Randolph, exhibit nearly complete suppression of aleurone color in the presence of inhibitory alleles of inr1 or inr2. Two members of the R1-d class of haplotypes, R1-d:Catspaw and R1-d:Arapaho, show partial suppression. Other haplotypes tested were not visibly affected. The response of r1 haplotypes to inhibitory inr1 and inr2 alleles provides another means of analyzing the complex behavior of the seed color components of r1 haplotypes. Possible mechanisms of action of inr1 and inr2 are discussed.

Characterization of dull1, a maize gene coding for a novel starch synthase.

The maize dull1 (du1) gene is a determinant of the structure of endosperm starch, and du1- mutations affect the activity of two enzymes involved in starch biosynthesis, starch synthase II (SSII) and starch branching enzyme IIa (SBEIIa). Six novel du1- mutations generated in Mutator-active plants were identified. A portion of the du1 locus was cloned by transposon tagging, and a nearly full-length Du1 cDNA sequence was determined. Du1 codes for a predicted 1674-residue protein, comprising one portion that is similar to SSIII of potato, as well as a large unique region. Du1 transcripts are present in the endosperm during the time of starch biosynthesis, but the mRNA was undetectable in leaf or root tissue. The predicted size of the Du1 gene product and its expression pattern are consistent with those of maize SSII. The Du1 gene product contains two repeated regions in its unique N terminus. One of these contains a sequence identical to a conserved segment of SBEs. We conclude that Du1 codes for a starch synthase, most likely SSII, and that secondary effects of du1- mutations, such as reduction of SBEIIa, result from the primary deficiency in this starch synthase.

CRINKLY4: A TNFR-like receptor kinase involved in maize epidermal differentiation.

The maize crinkly4 (cr4) mutation affects leaf epidermis differentiation such that cell size and morphology are altered, and surface functions are compromised, allowing graft-like fusions between organs. In the seed, loss of cr4 inhibits aleurone formation in a pattern that reflects the normal progression of differentiation over the developing endosperm surface. The cr4 gene was isolated by transposon tagging and found to encode a putative receptor kinase. The extracellular domain contains a cysteine-rich region similar to the ligand binding domain in mammalian tumor necrosis factor receptors (TNFRs) and seven copies of a previously unknown 39-amino acid repeat. The results suggest a role for cr4 in a differentiation signal.

Genetic evidence of Mutator-induced deletions in the short arm of chromosome 9 of maize. II. wd deletions.

Analyses of 113 putative Mutator-induced events involving the yg2 locus of chromosome 9 revealed that 11 of these events were deletions that produce albino seedlings when homozygous. This phenotype is characteristic of wd (white deficiency) deletions. All 11 wd-Mu deletions failed to complement wd1 and pyd1 (pale-yellow deficiency). Nine of the wd-Mu deletions were analyzed cytologically. Two were found to be terminal deletions and seven were internal deletions. Two of the seven had normal pairing throughout the terminal region involved in the pyd1 and wd1 deletions. Because genetic tests established that deletions were present in these two stocks, these deletions were probably too short to disrupt the pairing of the homologous chromosomes. Mechanisms by which the Mutator system might generate these deletions are discussed.

Genetic analysis of 63 mutations affecting maize kernel development isolated from Mutator stocks.

Sixty-three mutations affecting development of the maize kernel were isolated from active Robertson's Mutator (Mu) stocks. At least 14 previously undescribed maize gene loci were defined by mutations in this collection. Genetic mapping located 53 of these defective kernel (dek) mutations to particular chromosome arms, and more precise map determinations were made for 21 of the mutations. Genetic analyses identified 20 instances of allelism between one of the novel mutations and a previously described dek mutation, or between new dek mutations identified in this study; phenotypic variability was observed in three of the allelic series. Viability testing of homozygous mutant kernels identified numerous dek mutations with various pleiotropic effects on seedling and plant development. The mutations described here presumably arose by insertion of a Mu transposon within a dek gene; thus, many of the affected loci are expected to be accessible to molecular cloning via transposon-tagging.

Genetic Isolation, Cloning, and Analysis of a Mutator-Induced, Dominant Antimorph of the Maize amylose extender1 Locus.

We report the genetic identification, molecular cloning, and characterization of a dominant mutant at the amylose extender1 locus, Ae1-5180. The identities of our clones are corroborated by their ability to reveal DNA polymorphisms between seven wild-type revertants from Ae1-5180 relative to the Ae1-5180 mutant allele and between four of five independently derived, Mutator (Mu)-induced recessive ae1 alleles relative to their respective wild-type progenitor alleles. The Ae1-5180 mutation is associated with two Mu1 insertions flanked by complex rearrangements of ae1-related sequences. One of the Mu1 elements is flanked by inverted repeats of ae1-related DNA of at least 5.0 kb in length. This Mu1 element and at least some of this flanking inverted repeat DNA are absent or hypermethylated in six of seven wild-type revertants of Ae1-5180 that were analyzed. The second Mu1 element is flanked on one side by the 5.0-kb ae1-specific repeat and on the other side by a sequence that does not hybridize to the ae1-related repeat sequence. This second Mu1 element is present in revertants to the wild type and does not, therefore, appear to affect ae1 gene function. A 2.7-kb ae1 transcript can be detected in wild-type and homozygous ae1-Ref endosperms 20 days after pollination. This transcript is absent in endosperms containing one, two, or three doses of Ae1-5180. This result is consistent with a suppression model to explain the dominant gene action of Ae1-5180 and establishes Ae1-5180 as an antimorphic allele. Homozygous wild-type seedlings produce no detectable transcript, indicating some degree of tissue specificity for ae1 expression. Sequence analyses establish that ae1 encodes starch branching enzyme II.

Genetic regulation of somatic mutability of two Mu-induced a1 mutants of maize.

Previous studies of stocks of two Mutator-induced mutable a1 alleles (a1-Mum2 and al-Mum3) gave results consistent with the presence of one or more autonomous elements regulating the expression of mutability. This article reports on the results of studies designed to map these autonomous elements by using a series of waxy marked translocations. Linkage of waxy with autonomous elements was found for a1-Mum2 by using the translocations wx T2-9d, wx T4-9e and wx T4-9b. Several different linkage values were found in crosses involving wx T2-9d, suggesting that autonomous elements have transposed to different locations on chromosome 2. Linkage of autonomous elements with waxy was found for a1-Mum3 using translocation wx T2-9d. Again, several different linkage values were found. Some of these values were the same as those observed for a1-Mum2, but some were unique. In some crosses, the number of autonomous elements increased by one or two unlinked elements in addition to the linked element in one generation (i. e. the generation of the cross to the translocation series). Such an increase in number is probably the result of transposition of the original autonomous element to an independent locus while retaining the autonomous element at the original locus. Reduction in the number of autonomous elements is probably the result of the independent assortment in crosses of plants with two or more autonomous elements.

Molecular Analysis of viviparous-1: An Abscisic Acid-Insensitive Mutant of Maize.

The viviparous-1 (vp1) gene in maize controls multiple developmental responses associated with the maturation phase of seed formation. Most notably, mutant embryos have reduced sensitivity to the hormone abscisic acid, resulting in precocious germination, and blocked anthocyanin synthesis in aleurone and embryo tissues. The Vp1 locus was cloned by transposon tagging, using the Robertson's Mutator element present in the vp1-mum1 mutant allele. Detection of DNA rearrangements in several spontaneous and transposable element-induced mutant vp1 alleles, including a partial deletion of the locus, confirmed the identity of the clone. The Vp1 gene encodes a 2500-nucleotide mRNA that is expressed specifically in embryo and endosperm tissues of the developing seed. This transcript is absent in seed tissues of vp1 mutant stocks. Expression of C1, a regulatory gene for the anthocyanin pathway, is selectively blocked at the mRNA level in vp1 mutant seed tissues, indicating the Vp1 may control the anthocyanin pathway by regulating C1. We suggest that the Vp1 gene product functions to potentiate multiple signal transduction pathways in specific seed tissues.

Genetic evidence of mutator-induced deletions in the short arm of chromosome 9 of maize.

Evidence is presented that at least 12 of the Mu-induced yg2 mutants found in an extensive mutation analysis of this locus are the result of deletions in the region of the yg2 locus on the short arm of chromosome 9. Twelve of these putative deletions were characterized genetically, and in every instance, they were confirmed to be deletions involving chromosomal segments that include the yg2 and wd loci as well as additional portions of the short arm of chromosome 9.