Erin E. Irish

The maize tasselseed4 microRNA controls sex determination and meristem cell fate by targeting Tasselseed6/indeterminate spikelet1

In maize (Zea mays), sex determination occurs through abortion of female carpels in the tassel and arrest of male stamens in the ear. The Tasselseed6 (Ts6) and tasselseed4 (ts4) mutations permit carpel development in the tassel while increasing meristem branching, showing that sex determination and acquisition of meristem fate share a common pathway. We show that ts4 encodes a mir172 microRNA that targets APETALA2 floral homeotic transcription factors. Three lines of evidence suggest that indeterminate spikelet1 (ids1), an APETALA2 gene required for spikelet meristem determinacy, is a key target of ts4. First, loss of ids1 suppresses the ts4 sex determination and branching defects. Second, Ts6 mutants phenocopy ts4 and possess mutations in the microRNA binding site of ids1. Finally, IDS1 protein is expressed more broadly in ts4 mutants compared to wild type. Our results demonstrate that sexual identity in maize is acquired by limiting floral growth through negative regulation of the floral homeotic pathway.

JOINTLESS is a MADS-box gene controlling tomato flower abscission zone development

Abscission is a universal and dynamic process in plants whereby organssuch as leaves, flowers and fruit are shed, both during normal development,and in response to tissue damage and stress. Shedding occursby separation of cells in anatomically distinct regions of the plant, calledabscission zones (AZs). During abscission, the plant hormone ethylene stimulatescells to produce enzymes that degrade the middle lamella between cells inthe AZ. The physiology and regulation of abscission at fully developed AZsis well known, but the molecular biology underlying theirdevelopment is not. Here we report the first isolation of a gene directlyinvolved in the development of a functional plant AZ. Tomato plants with the jointless mutation fail to develop AZs on their pedicelsand so abscission of flowers or fruit does not occur normally. We identify JOINTLESS as a new MADS-box gene in a distinct phylogenetic clade separatefrom those functioning in floral organs. We propose that a deletion in JOINTLESS accounts for the failure of activation of pedicel AZ developmentin jointless tomato plants.

Interactions between jointless and Wild-Type Tomato Tissues during Development of the Pedicel Abscission Zone and the Inflorescence Meristem

The jointless mutation of tomato results in the formation of flower pedicels that lack an abscission zone and inflorescence meristems that revert to vegetative growth. We have analyzed periclinal chimeras and mericlinal sectors of jointless and wild-type tissue to determine how cells in different meristem layers (L1, L2, and L3) and their derivatives interact during these two developmental processes. Cells in the inner meristem layer, L3, alone determined whether the meristem maintained the inflorescence state or reverted to vegetative growth. Moreover, L3 derivatives determined whether a functional pedicel abscission zone formed. Limited and disorganized autonomous development of wild-type L2-derived cells occurred when they overlay mutant tissue. Adjacent mutant and wild-type L3-derived tissues in pedicels developed autonomously, indicating little or no lateral communication. Only the outermost L3-derived cells within the pedicel were capable of orchestrating normal pedicel development in overlying tissues, revealing the special status of those cells as coordinators of development for L1- and L2-derived cells, whereas the innermost L3-derived cells developed autonomously but did not influence the development of other cells.

JOINTLESS suppresses sympodial identity in inflorescence meristems of tomato.

Unlike monopodial plants, in which flowering terminates growth of a shoot, plants exhibiting sympodial shoot architecture maintain the potential for indeterminate growth even after converting to floral development. This vegetative indeterminacy is conferred by a special type of axillary meristem, the sympodial meristem, which exhibits precocious but determinate growth. The reiterative formation of sympodial meristems as the plant grows results in a shoot composed of a series of modules, each consisting of a limited number of vegetative nodes and terminated by a flower or inflorescence. To determine how sympodial meristems differ from other shoot meristems, we examined interactions between mutations that affect various shoot meristem types in tomato (Lycopersicon esculentum Mill.). Analysis of double mutant combinations of jointless, lateral suppressor, self-pruning, blind, and anantha showed that sympodial meristems share regulatory features with inflorescence meristems. Genetic studies on the jointless mutation implicated this gene in suppressing sympodial meristem fate in the inflorescence. As this mutation has a second phenotype, the elimination of the pedicel abscission zone, we examined the expression pattern of JOINTLESS to test whether pedicel development is involved in directing shoot architecture. We found that this MADS box gene is expressed in a variety of shoot meristems, including inflorescence, floral, sympodial, and axillary meristems, as well as in early staged floral organs, in sporogenous tissues of anthers, and in ovules. Lack of expression in developing pedicels indicates abscission zone development does not rely on JOINTLESS transcription in the differentiating cells. We conclude that the primary role of JOINTLESS is to suppress sympodial meristem identity in inflorescence meristems.

Class II tassel seed mutations provide evidence for multiple types of inflorescence meristems in maize (Poaceae).

The tassel seed mutations ts4 and Ts6 of maize cause irregular branching in its inflorescences, tassels, and ears, in addition to feminization of the tassel due to the failure to abort pistils. A comparison of the development of mutant and wild-type tassels and ears using scanning electron microscopy reveals that at least four reproductive meristem types can be identified in maize: the inflorescence meristem, the spikelet pair meristem, the spikelet meristem, and the floret meristem. ts4 and Ts6 mutations affect the fate of specific reproductive meristems in both tassels and ears. ts4 mutants fail to form spikelet meristems from spikelet pair meristems. Ts6 mutants are delayed in the conversion of certain spikelet meristems into floret meristems. Once floret meristems are established in both of these mutants, they form florets that appear normal but fail to undergo pistil abortion in the tassel. The abnormal branching associated with each mutant is suppressed at the base of ears, permitting the formation of normal, fertile spikelets. The classification of the different types of reproductive meristems will be useful in interpretation of gene expression patterns in maize. It also provides a framework for understanding meristem functions that can be varied to diversify inflorescence architectures in the Gramineae.

Experimental analysis of Tassel development in the maize mutant Tassel seed 6

The maize (Zea mays L.) mutation Tassel seed 6 (Ts6) disrupts both sex determination in the tassel and the pattern of branching in inflorescences. This results in the formation of supernumerary florets in tassels and ears and in the development of pistils in tassel florets where they are normally aborted. A developmental analysis indicated that extra florets in Ts6 inflorescences are most likely the result of delayed determinacy in spikelet meristems, which then initiate additional floret meristems rather than initiating floral organs as in wild type. I have used culturing experiments to assay whether delayed determinacy of Ts6 mutant tassels is reflected in an altered timing of specific determination events. Length of the tassel was used as a developmental marker. These experiments showed that although Ts6 tassels elongate much more slowly than wild type, both mutant and wild-type tassels gained the ability to form flowers with organs of normal morphology in culture at the same time. In situ hybridization patterns of expression of the maize gene Kn, which is normally expressed in shoot meristems and not in determinate lateral organs, confirmed that additional meristems, rather than lateral organs, are initiated by spikelet meristems in Ts6 tassels.

Apogamy Induction in Ceratopteris richardii

Two distinct generations, the sporophyte and the gametophyte, alternate to complete the life cycle of all land plants. The sporophyte generation undergoes meiosis to form spores, from which gametophytes develop. The gametophyte generation produces gametes, which undergo fertilization to produce the zygote, the first cell of the sporophyte, thus completing the life cycle. As a variation of this life cycle, some fern species can undergo apogamy and apospory, bypassing fertilization or meiosis, respectively, alternating from one generation to the other without changing chromosome number. Both apospory and apogamy occur in nature and can be induced in the laboratory. The apogamy process is particularly amenable to analysis at the molecular level because the sporophytes are generated directly from a single‐layered, free‐living gametophyte whose growing conditions can be easily manipulated and growth synchronized. Here, we report the successful induction of apogamy in the model fern Ceratopteris richardii by culturing gametophytes in the presence of high levels of exogenous sugars. We also have identified the minimum time developing C. richardii gametophytes require sugar to gain competence for apogamous production of sporophytes. At this time, no signs of sporophyte differentiation are detectable. These results establish an experimental system that will allow genes that are important in apogamy to be identified.

Gene expression associated with apogamy commitment in Ceratopteris richardii

Apogamy is a phenomenon in which a sporophyte develops asexually, directly from a cell or cells of a gametophyte. It is a phenomenon described mainly in lower plants, but shares certain aspects with apomixis in angiosperms. The genes involved in apogamy commitment in ferns are unknown. We hypothesize that the mechanism of asexual reproduction is controlled in lower and higher plants by overlapping sets of genes. To this end, we created a normalized subtracted cDNA library that represents genes with increased expression during apogamy commitment in the fern Ceratopteris richardii. The cDNA library consists of 170 unique sequences. Compared to the mature gametophyte transcriptome of the fern Pteridium aquilinum, the apogamy library is enriched in plant GO-Slim terms that are associated with stress and metabolism. In silico expression analyses of the closest Arabidopsis homologs of the apogamy library revealed many genes that display preferential expression in seed and flower tissues, structures that are absent in ferns. This apogamy library provides a rich resource for investigations into the genetic control of apogamy in ferns and comparisons with the asexual processes of higher plants.

Rejuvenation by Shoot Apex Culture Recapitulates the Developmental Increase of Methylation at the Maize Gene Pl-Blotched

Cytosine methylation provides an attractive epigenetic modification for the global maintenance of phases in plant development; however, there are few known examples of specific genes whose methylation status changes in a developmentally regulated manner. Pl-Blotched, an allele of purple plant1 (pl1), which encodes a myb-like transcription factor that regulates anthocyanin production in maize, is one such gene: certain cytosines at the 3′ end of this allele are hypomethylated in seedlings, become hypermethylated in organs formed in the adult phase, and are hypomethylated again in the next generation. We tested whether this developmental pattern of low juvenile cytosine methylation followed by higher methylation in adult tissues could also be observed in plants “rejuvenated” via shoot apex culture. We found that cytosine methylation patterns at Pl-Blotched were indeed recapitulated in culture-rejuvenated plants, showing hypomethylation in leaves with juvenile patterns of differentiation (even though they were made by an old meristem) followed by hypermethylation in later-formed leaves. Our results show that methylation status at that locus is determined by the developmental phase of the shoot, rather than by the age of the meristem forming it. These results support the hypothesis that DNA methylation is employed by the plant to maintain an epigenetic state.

Transient and stable transformation of Ceratopteris richardii gametophytes

BackgroundFerns, being vascular yet seedless, present unparalleled opportunities to investigate important questions regarding the evolution and development of land plants. Ceratopteris richardii, a diploid, homosporous fern has been advanced as a model fern system; however, the tenuous ability to transform the genome of this fern greatly limited its usefulness as a model organism. Here we report a simple and reliable Agrobacterium-mediated method for generating transient and stable transformants of mature C. richardii gametophytes.ResultsTransformation success was achieved by enzyme treatment that partially digested the cell walls of mature gametophytes to facilitate Agrobacteria infection. Co-incubation of Agrobacteria with enzymatically treated gametophytes was sufficient to generate transient transformants at a frequency of nearly 90% under optimal conditions. Stable transformation was achieved at a rate of nearly 3% by regenerating entire gametophytes from single transformed cells from T0 gametophytes on selective media.ConclusionsThis transformation method will allow for the immediate observation of phenotypes in the haploid gametophytes of transformed plants, as well as the generation of stably transformed C. richardii lines for further analysis. Transformation capability will greatly facilitate gene functional studies in C. richardii, more fully realizing the potential of this model fern species. These protocols may be adapted to other plant species that are recalcitrant to Agrobacterium-mediated transformation.