Maize ROP2 GTPase provides a competitive advantage to the male gametophyte.

Rop GTPases have been implicated in the regulation of plant signal transduction and cell morphogenesis. To explore ROP2 function in maize, we isolated five Mutator transposon insertions (rop2::Mu alleles). Transmission frequency through the male gametophyte, but not the female, was lower than expected in three of the rop2::Mu mutants. These three alleles formed an allelic series on the basis of the relative transmission rate of each when crossed as trans-heterozygotes. A dramatic reduction in the level of ROP2-mRNA in pollen was associated with the three alleles causing a transmission defect, whereas a rop2::Mu allele that did not result in a defect had wild-type transcript levels, thus confirming that mutation of rop2 causes the mutant phenotype. These data strongly support a role for rop2 in male gametophyte function, perhaps surprisingly, given the expression in pollen of the nearly identical duplicate gene rop9. However, the transmission defect was apparent only when a rop2::Mu heterozygote was used as the pollen donor or when a mixture of wild-type and homozygous mutant pollen was used. Thus, mutant pollen is at a competitive disadvantage compared to wild-type pollen, although mutant pollen grains lacked an obvious cellular defect. Our data demonstrate the importance in vivo of a specific Rop, rop2, in the male gametophyte.

Analysis and expression of the alpha-expansin and beta-expansin gene families in maize.

Expansins comprise a multigene family of proteins in maize (Zea mays). We isolated and characterized 13 different maize expansin cDNAs, five of which are alpha-expansins and eight of which are beta-expansins. This paper presents an analysis of these 13 expansins, as well as an expression analysis by northern blotting with materials from young and mature maize plants. Some expansins were expressed in restricted regions, such as the beta-expansins ExpB1 (specifically expressed in maize pollen) and ExpB4 (expressed principally in young husks). Other expansins such as alpha-expansin Exp1 and beta-expansin ExpB2 were expressed in several organs. The expression of yet a third group was not detected in the selected organs and tissues. An analysis of expansin sequences from the maize expressed sequence tag collection is also presented. Our results indicate that expansin genes may have general, overlapping expression in some instances, whereas in other cases the expression may be highly specific and limited to a single organ or cell type. In contrast to the situation in Arabidopsis, beta-expansins in maize seem to be more numerous and more highly expressed than are alpha-expansins. The results support the concept that beta-expansins multiplied and evolved special functions in the grasses.

The control of maize spikelet meristem fate by the APETALA2-like gene indeterminate spikelet1.

The orderly production of meristems with specific fates is crucial for the proper elaboration of plant architecture. The maize inflorescence meristem branches several times to produce lateral meristems with determinate fates. The first meristem formed, the spikelet pair meristem, produces two spikelet meristems, each of which produces two floral meristems. We have identified a gene called indeterminate spikelet1 (ids1) that specifies a determinate spikelet meristem fate and thereby limits the number of floral meristems produced. In the absence of ids1 gene function, the spikelet meristem becomes indeterminate and produces additional florets. Members of the grass family vary in the number of florets within their spikelets, suggesting that ids1 may play a role in inflorescence architecture in other grass species. ids1 is a member of the APETALA2 (AP2) gene family of transcription factors that has been implicated in a wide range of plant development roles. Expression of ids1 was detected in many types of lateral organ primordia as well as spikelet meristems. Our analysis of the ids1 mutant phenotype and expression pattern indicates that ids1 specifies determinate fates by suppressing indeterminate growth within the spikelet meristem.

Plant-pathogen microevolution: molecular basis for the origin of a fungal disease in maize.

A new and severe disease of maize caused by a previously unknown fungal pathogen, Cochliobolus carbonum race 1, was first described in 1938. The molecular events that led to the sudden appearance of this disease are described in this paper. Resistance to C. carbonum race 1 was found to be widespread in maize and is conferred by a pair of unlinked duplicate genes, Hm1 and Hm2. Here, we demonstrate that resistance is the wild-type condition in maize. Two events, a transposon insertion in Hm1 and a deletion in Hm2, led to the loss of resistance, resulting in the origin of a new disease. None of the other plant species tested is susceptible to C. carbonum race 1, and they all possess candidate genes with high homology to Hm1 and Hm2. In sorghum and rice, these homologs map to two chromosomal regions that are syntenic with the maize Hm1 and Hm2 loci, indicating that they are related to the maize genes by vertical descent. These results suggest that the Hm-encoded resistance is of ancient origin and probably is conserved in all grasses.

Analysis of a chemical plant defense mechanism in grasses.

In the Gramineae, the cyclic hydroxamic acids 2,4-dihydroxy-1, 4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7-methoxy-1, 4-benzoxazin-3-one (DIMBOA) form part of the defense against insects and microbial pathogens. Five genes, Bx1 through Bx5, are required for DIBOA biosynthesis in maize. The functions of these five genes, clustered on chromosome 4, were demonstrated in vitro. Bx1 encodes a tryptophan synthase alpha homolog that catalyzes the formation of indole for the production of secondary metabolites rather than tryptophan, thereby defining the branch point from primary to secondary metabolism. Bx2 through Bx5 encode cytochrome P450-dependent monooxygenases that catalyze four consecutive hydroxylations and one ring expansion to form the highly oxidized DIBOA.

Diversification of C-function activity in maize flower development.

The Arabidopsis gene AGAMOUS is required for male and female reproductive organ development and for floral determinacy. Reverse genetics allowed the isolation of a transposon-induced mutation in ZAG1, the maize homolog of AGAMOUS. ZAG1 mutants exhibited a loss of determinacy, but the identity of reproductive organs was largely unaffected. This suggested a redundancy in maize sex organ specification that led to the identification and cloning of a second AGAMOUS homolog, ZMM2, that has a pattern of expression distinct from that of ZAG1. C-function organ identity in maize (as defined by the A, B, C model of floral organ development) may therefore be orchestrated by two closely related genes, ZAG1 and ZMM2, with overlapping but nonidentical activities.

Cloning and characterization of the maize An1 gene.

The Anther ear1 (An1) gene product is involved in the synthesis of ent-kaurene, the first tetracyclic intermediate in the gibberellin (GA) biosynthetic pathway. Mutations causing the loss of An1 function result in a GA-responsive phenotype that includes reduced plant height, delayed maturity, and development of perfect flowers on normally pistillate ears. The an1::Mu2-891339 allele was recovered from a Mutator (Mu) F2 family. Using Mu elements as molecular probes, an An1-containing restriction fragment was identified and cloned. The identity of the cloned gene as An1 was confirmed by using a reverse genetics screen for maize families that contain a Mu element inserted into the cloned gene and then by demonstrating that the insertion causes an an1 phenotype. The predicted amino acid sequence of the An1 cDNA shares homology with plant cyclases and contains a basic N-terminal sequence that may target the An1 gene product to the chloroplast. The sequence is consistent with the predicted subcellular localization of AN1 in the chloroplast and with its biochemical role in the cyclization of geranylgeranyl pyrophosphate, a 20-carbon isoprenoid, to ent-kaurene. The semidwarfed stature of an1 mutants is in contrast with the more severely dwarfed stature of GA-responsive mutants at other loci in maize and may be caused by redundancy in this step of the GA biosynthetic pathway. DNA gel blot analysis indicated that An1 is a single-copy gene that lies entirely within the deletion of the an1-bz2-6923 mutant. However, homozygous deletion mutants accumulated ent-kaurene to 20% of the wild-type level, suggesting that the function of An1 is supplemented by an additional activity.

A Biochemical Phenotype for a Disease Resistance Gene of Maize.

In maize, major resistance to the pathogenic fungus Cochliobolus (Helminthosporium) carbonum race 1 is determined by the dominant allele of the nuclear locus hm. The interaction between C. carbonum race 1 and maize is mediated by a pathogen-produced, low molecular weight compound called HC-toxin. We recently described an enzyme from maize, called HC-toxin reductase, that inactivates HC-toxin by pyridine nucleotide-dependent reduction of an essential carbonyl group. We now report that this enzyme activity is detectable only in extracts of maize that are resistant to C. carbonum race 1 (genotype Hm/Hm or Hm/hm). In several genetic analyses, in vitro HC-toxin reductase activity was without exception associated with resistance to C. carbonum race 1. The results indicate that detoxification of HC-toxin is the biochemical basis of Hm-specific resistance of maize to infection by C. carbonum race 1.

Enzymatic Detoxification of HC-toxin, the Host-Selective Cyclic Peptide from Cochliobolus carbonum.

Resistance to the fungal plant pathogen Cochliobolus carbonum race 1 and to its host-selective toxin, HC-toxin, is determined by Hm, a single dominant gene in the host plant maize, (Zea mays L). Radiolabeled HC-toxin of specific activity 70 milliCuries per millimole, prepared by feeding tritiated d,l-alanine to the fungus, was used to study its fate in maize leaf tissues. HC-toxin was converted by resistant leaf segments to a single compound, identified by mass spectrometry and nuclear magnetic resonance as the 8-hydroxy derivative of HC-toxin formed by reduction of the 8-keto group of 2-amino-9, 10-epoxy-8-oxo-decanoic acid, one of the amino acids in HC-toxin. Reduction of HC-toxin occurred in cell-free preparations from etiolated (Hm/hm) maize shoots, and the activity was sensitive to heat and proteolytic digestion, dependent on NADPH, and inhibited by p-hydroxymercuribenzoate and disulfiram. The enzyme (from the Hm/hm genotype) was partially purified by ammonium sulfate precipitation and diethylaminoethyl-ion exchange chromatography. By gel filtration chromatography, the enzyme had a molecular weight of 42,000. NADH was approximately 30% as effective as NADPH as a hydride donor, and flavin-containing cofactors had no effect on activity. When HC-toxin was introduced to maize leaf segments through the transpiration stream, leaf segments from both resistant and susceptible maize inactivated toxin equally well over a time-course of 9 hours. Although these data suggest no relationship between toxin metabolism and host selectivity, we discuss findings in apparent conflict with the current data and describe why the relationship between enzymatic reduction of HC-toxin and Hm remains unresolved.