Matthew M. S. Evans

The indeterminate gametophyte1 Gene of Maize Encodes a LOB Domain Protein Required for Embryo Sac and Leaf Development

Angiosperm embryo sac development begins with a phase of free nuclear division followed by cellularization and differentiation of cell types. The indeterminate gametophyte1 (ig1) gene of maize (Zea mays) restricts the proliferative phase of female gametophyte development. ig1 mutant female gametophytes have a prolonged phase of free nuclear divisions leading to a variety of embryo sac abnormalities, including extra egg cells, extra polar nuclei, and extra synergids. Positional cloning of ig1 was performed based on the genome sequence of the orthologous region in rice. ig1 encodes a LATERAL ORGAN BOUNDARIES domain protein with high similarity to ASYMMETRIC LEAVES2 of Arabidopsis thaliana. A second mutant allele of ig1 was identified in a noncomplementation screen using active Mutator transposable element lines. Homozygous ig1 mutants have abnormal leaf morphology as well as abnormal embryo sac development. Affected leaves have disrupted abaxial–adaxial polarity and fail to repress the expression of meristem-specific knotted-like homeobox (knox) genes in leaf primordia, causing a proliferative, stem cell identity to persist in these cells. Despite the superficial similarity of ig1-O leaves and embryo sacs, ectopic knox gene expression cannot be detected in ig1-O embryo sacs.

A Feedback Regulatory Module Formed by LITTLE ZIPPER and HD-ZIPIII Genes

The Arabidopsis thaliana REVOLUTA (REV) protein is a member of the class III homeodomain-leucine zipper (HD-ZIPIII) proteins. REV is a potent regulator of leaf polarity and vascular development. Here, we report the identification of a gene family that encodes small leucine zipper–containing proteins (LITTLE ZIPPER [ZPR] proteins) where the leucine zipper is similar to that found in REV, PHABULOSA, and PHAVOLUTA proteins. The transcript levels of the ZPR genes increase in response to activation of a steroid-inducible REV protein. We show that the ZPR proteins interact with REV in vitro and that ZPR3 prevents DNA binding by REV in vitro. Overexpression of ZPR proteins in Arabidopsis results in phenotypes similar to those seen when HD-ZIPIII function is reduced. We propose a negative feedback model in which REV promotes transcription of the ZPR genes. The ZPR proteins in turn form heterodimers with the REV protein, preventing it from binding DNA. The HD-ZIPIII/ZPR regulatory module would serve not only to dampen the effect of fluctuations in HD-ZIPIII protein levels but more importantly would provide a potential point of regulation (control over the ratio of inactive heterodimers to active homodimers) that could be influenced by other components of the pathway governing leaf polarity.

Gibberellins Promote Vegetative Phase Change and Reproductive Maturity in Maize

Postembryonic shoot development in maize (Zea mays L.) is divided into a juvenile vegetative phase, an adult vegetative phase, and a reproductive phase that differ in the expression of many morphological traits. A reduction in the endogenous levels of bioactive gibberellins (GAs) conditioned by any one of the dwarf1, dwarf3, dwarf5, or anther ear1 mutations in maize delays the transition from juvenile vegetative to adult vegetative development and from adult vegetative to reproductive development. Mutant plants cease producing juvenile traits (e.g. epicuticular wax) and begin producing adult traits (e.g. epidermal hairs) later than wild-type plants. They also cease producing leaves and begin producing reproductive structures later than wild-type plants. These mutations greatly enhance most aspects of the phenotype of Teopod1 and Teopod2, suggesting that GAs suppress part but not all of the Teopod phenotype. Application of GA3 to Teopod2 mutants and Teopod1,dwarf3 double mutants confirms this result. We conclude that GAs act in conjunction with several other factors to promote both vegetative and reproductive maturation but affect different developmental phases unequally. Furthermore, the GAs that regulate vegetative and reproductive maturation, like those responsible for stem elongation, are downstream of GA20 in the GA biosynthetic pathway.

HETEROBLASTIC FEATURES OF LEAF ANATOMY IN MAIZE AND THEIR GENETIC REGULATION

Heteroblastic features of leaf anatomy in maize were identified by conducting a quantitative analysis of leaf anatomy. Heteroblastic variation in cuticle thickness and epidermal cell shape paralleled changes in previously defined juvenile- and adult-specific traits. The other traits examined in this study (thickness of the leaf blade, epidermal and bundle sheath cell size, vascular area, interveinal distance, mesophyll area : bundle sheath area ratio) varied in a more complex fashion. To determine which of these traits are regulated by genes involved in shoot maturation, we examined the effect of the Teopod2 (Tp2) mutation on their expression. Tp2 increases the number of leaves that express the juvenile form of cuticle thickness, epidermal cell shape, and vascular area and causes all other leaves to produce intermediate (juvenile/adult) forms of these traits. It has little or no effect on any of the other traits we examined. Thus, much of the heteroblastic variation in the internal anatomy of the maize leaf appears to be regulated by factors that are unrelated to the developmental phenomenon of phase change. The effect of Tp2 on leaf anatomy is interesting not only because it provides a criterion for distinguishing between different types of heteroblastic traits, but also because it provides some insight into the nature of the developmental processes involved in shoot maturation. In particular, the observation that Tp2 leaves are quantitatively intermediate between juvenile and adult leaves supports the hypothesis that some phase-specific aspects of leaf identity are regulated in a combinatorial fashion rather than by mutually exclusive patterns of gene expression.

Maternal Gametophytic baseless1 Is Required for Development of the Central Cell and Early Endosperm Patterning in Maize (Zea mays)

In angiosperms, double fertilization of an egg cell and a central cell with two sperm cells results in the formation of a seed containing a diploid embryo and a triploid endosperm. The extent to which the embryo sac controls postfertilization events in the seed is unknown. The novel gametophytic maternal-effect maize mutation, baseless1 (bsl1) affects central cell development within the embryo sac, frequently by altering the position of the two polar nuclei. Despite this irregularity, fertilization is as efficient as in wild type. The spatial expression of basal endosperm-specific transcripts is altered in free-nuclear and cellular mutant endosperms. At later stages of seed development, bsl1 predominantly affects development of the basal endosperm transfer layer (BETL). When bsl1/+ diploid plants were pollinated by wild-type tetraploid plants, the BETL abnormalities observed in bsl1/bsl1/+/+ tetraploid endosperms were diverse and of variable severity. Moreover, the frequency of kernels with severely perturbed BETL development correlated with the percentage of severely affected bsl1 central cells. Therefore, BSL1 is likely required in the central cell before fertilization for correct BETL patterning to occur. These findings provide new genetic evidence that a maternal gametophytic component is necessary for correct endosperm patterning.

Discovery of novel transcripts and gametophytic functions via RNA-seq analysis of maize gametophytic transcriptomes

BackgroundPlant gametophytes play central roles in sexual reproduction. A hallmark of the plant life cycle is that gene expression is required in the haploid gametophytes. Consequently, many mutant phenotypes are expressed in this phase.ResultsWe perform a quantitative RNA-seq analysis of embryo sacs, comparator ovules with the embryo sacs removed, mature pollen, and seedlings to assist the identification of gametophyte functions in maize. Expression levels were determined for annotated genes in both gametophytes, and novel transcripts were identified from de novo assembly of RNA-seq reads. Transposon-related transcripts are present in high levels in both gametophytes, suggesting a connection between gamete production and transposon expression in maize not previously identified in any female gametophytes. Two classes of small signaling proteins and several transcription factor gene families are enriched in gametophyte transcriptomes. Expression patterns of maize genes with duplicates in subgenome 1 and subgenome 2 indicate that pollen-expressed genes in subgenome 2 are retained at a higher rate than subgenome 2 genes with other expression patterns. Analysis of available insertion mutant collections shows a statistically significant deficit in insertions in gametophyte-expressed genes.ConclusionsThis analysis, the first RNA-seq study to compare both gametophytes in a monocot, identifies maize gametophyte functions, gametophyte expression of transposon-related sequences, and unannotated, novel transcripts. Reduced recovery of mutations in gametophyte-expressed genes is supporting evidence for their function in the gametophytes. Expression patterns of extant, duplicated maize genes reveals that selective pressures based on male gametophytic function have likely had a disproportionate effect on plant genomes.

Pollen–pistil barriers to crossing in maize and teosinte result from incongruity rather than active rejection

Many popcorn strains cannot be fertilized by pollen of dent and flint strains although the reciprocal crosses are successful. Similarly, plants of some annual teosinte populations can fertilize maize but do not accept its pollen. Single genes or gene complexes govern these two unilateral barriers to crossing. Failure of fertilization could reflect active rejection by the pistil of pollen containing a contrasting allele (incompatibility). Alternatively, the pistil could require presence of a matching allele in pollen (congruity). To distinguish between these possibilities genetically, the receptivity to pollen having both alleles was determined. If there is active rejection, heteroallelic pollen would not be accepted; if presence of a matching allele is required, heteroallelic pollen would be accepted. In both the popcorn and teosinte crossing barrier systems, heteroallelic pollen functioned, consistent with the congruity model.

The Zea mays Sexual Compatibility Gene ga2: Naturally Occurring Alleles, Their Distribution, and Role in Reproductive Isolation

Major genes govern the fertilization of teosinte ovules by maize pollen. A pollen-pistil compatibility system different from the previously described systems, Ga1-s and Tcb1-s, was identified among maize lines introgressed with chromosome segments from 2 teosinte populations. The pistil barrier is dominant, and pollen competence is determined by genotype of the individual pollen grain. A major gene governing this incompatibility behaves as a strong allele of ga2, a locus identified previously among maize genetic stocks on the basis of transmission ratio distortion. Additionally, pollen simultaneously carrying both ga2 and Ga2 was functional on Ga2 silks, which have the pistil barrier, indicating that Ga2 conditions acceptance of the pollen grain rather than ga2 conditioning rejection of the pollen grain by Ga2 silks. The strong allele (Ga2-s), a weaker one such as reported among maize genetic stocks (Ga2-w), and an allele having only pollen competence (Ga2-m), or some combination of these, was found in all 13 of the teosinte populations sampled. Sympatric and parapatric maize landraces carried Ga2-m or the presumed null allele ga2, but Ga2-s or Ga2-w was not found. The combination of exclusively Ga2-s teosinte with ga2 maize, which could provide strong reproductive isolation, was not characteristic of the 5, paired populations tested.

Analysis of stunter1, a maize mutant with reduced gametophyte size and maternal effects on seed development.

Many higher eukaryotes have evolved strategies for the maternal control of growth and development of their offspring. In higher plants this is achieved in part by postmeiotic gene activity controlling the development of the haploid female gametophyte. stunter1 (stt1) is a novel, recessive, maternal effect mutant in maize that displays viable, miniature kernels. Maternal inheritance of stt1 results in seeds with reduced but otherwise normal endosperms and embryos. The stt1 mutation displays reduced transmission through the male and female parents and causes significant changes in the sizes of both male and female gametophytes. stt1 pollen grains are smaller than wild type, have reduced germination efficiency, and reduced pollen tube growth. stt1 embryo sacs have smaller central cells and abnormal antipodal cells that are larger, more vacuolated, and fewer in number than wild type. Embryos and endosperms produced by fertilization of stt1 embryo sacs develop and grow more slowly than wild type. The data suggest that the morphology of mutant embryo sacs influences endosperm development, leading to the production of miniature kernels in stt1. Analysis of seeds carrying a mutant maternal allele of stt1 over a deletion of the paternal allele demonstrates that both parental alleles are active after fertilization in both the endosperm and embryo. This analysis also indicates that embryo development until the globular stage in maize can proceed without endosperm development and is likely supported directly by the diploid mother plant.

Correlation between a loss of auxin signaling and a loss of proliferation in maize antipodal cells.

The plant life cycle alternates between two genetically active generations: the diploid sporophyte and the haploid gametophyte. In angiosperms the gametophytes are sexually dimorphic and consist of only a few cells. The female gametophyte, or embryo sac, is comprised of four cell types: two synergids, an egg cell, a central cell, and a variable number of antipodal cells. In some species the antipodal cells are indistinct and fail to proliferate, so many aspects of antipodal cell function and development have been unclear. In maize and many other grasses, the antipodal cells proliferate to produce a highly distinct cluster at the chalazal end of the embryo sac that persists at the apex of the endosperm after fertilization. The antipodal cells are a site of auxin accumulation in the maize embryo sac. Analysis of different families of genes involved in auxin biosynthesis, distribution, and signaling for expression in the embryo sac demonstrates that all steps are expressed within the embryo sac. In contrast to auxin signaling, cytokinin signaling is absent in the embryo sac and instead occurs adjacent to but outside of the antipodal cells. Mutant analysis shows a correlation between a loss of auxin signaling and a loss of proliferation of the antipodal cells. The leaf polarity mutant Laxmidrib1 causes a lack of antipodal cell proliferation coupled with a loss of DR5 and PIN1a expression in the antipodal cells.