Defensin-Like ZmES4 Mediates Pollen Tube Burst in Maize via Opening of the Potassium Channel KZM1

In contrast to animals and lower plant species, sperm cells of flowering plants are non-motile and are transported to the female gametes via the pollen tube, i.e. the male gametophyte. Upon arrival at the female gametophyte two sperm cells are discharged into the receptive synergid cell to execute double fertilization. The first players involved in inter-gametophyte signaling to attract pollen tubes and to arrest their growth have been recently identified. In contrast the physiological mechanisms leading to pollen tube burst and thus sperm discharge remained elusive. Here, we describe the role of polymorphic defensin-like cysteine-rich proteins ZmES1-4 (Zea mays embryo sac) from maize, leading to pollen tube growth arrest, burst, and explosive sperm release. ZmES1-4 genes are exclusively expressed in the cells of the female gametophyte. ZmES4-GFP fusion proteins accumulate in vesicles at the secretory zone of mature synergid cells and are released during the fertilization process. Using RNAi knock-down and synthetic ZmES4 proteins, we found that ZmES4 induces pollen tube burst in a species-preferential manner. Pollen tube plasma membrane depolarization, which occurs immediately after ZmES4 application, as well as channel blocker experiments point to a role of K(+)-influx in the pollen tube rupture mechanism. Finally, we discovered the intrinsic rectifying K(+) channel KZM1 as a direct target of ZmES4. Following ZmES4 application, KZM1 opens at physiological membrane potentials and closes after wash-out. In conclusion, we suggest that vesicles containing ZmES4 are released from the synergid cells upon male-female gametophyte signaling. Subsequent interaction between ZmES4 and KZM1 results in channel opening and K(+) influx. We further suggest that K(+) influx leads to water uptake and culminates in osmotic tube burst. The species-preferential activity of polymorphic ZmES4 indicates that the mechanism described represents a pre-zygotic hybridization barrier and may be a component of reproductive isolation in plants.

FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei

Background: The male germline in flowering plants differentiates by asymmetric division of haploid uninucleated microspores, giving rise to a vegetative cell enclosing a smaller generative cell, which eventually undergoes a second mitosis to originate two sperm cells. The vegetative cell and the sperm cells activate distinct genetic and epigenetic mechanisms to control pollen tube growth and germ cell specification, respectively. Therefore, a comprehensive characterization of these processes relies on efficient methods to isolate each of the different cell types throughout male gametogenesis. Results: We developed stable transgenic Arabidopsis lines and reliable purification tools based on Fluorescence-Activated Cell Sorting (FACS) in order to isolate highly pure and viable fractions of each cell/nuclei type before and after pollen mitosis. In the case of mature pollen, this was accomplished by expressing GFP and RFP in the sperm and vegetative nuclei, respectively, resulting in 99% pure sorted populations. Microspores were also purified by FACS taking advantage of their characteristic small size and autofluorescent properties, and were confirmed to be 98% pure. Conclusions: We provide simple and efficient FACS-based purification protocols for Arabidopsis microspores, vegetative nuclei and sperm cells. This paves the way for subsequent molecular analysis such as transcriptomics, DNA methylation analysis and chromatin immunoprecipitation, in the developmental context of microgametogenesis in Arabidopsis.

Shaping the Dicot Fruit: Molecular and Genomic Approaches to Fruit Development

The fruit is one of the most complex and important structures produced by flowering plants, and understanding the development and maturation process of fruits in different angiosperm species with diverse fruit structures is of immense interest. In the work presented here, molecular genetics and genomic analysis are used to explore the processes that form the fruit in two species: The model organism Arabidopsis and the diploid strawberry Fragaria vesca. One important basic question concerns the molecular genetic basis of fruit patterning. A long-standing model of Arabidopsis fruit (the gynoecium) patterning holds that auxin produced at the apex diffuses downward, forming a gradient that provides apical-basal positional information to specify different tissue types along the gynoecium’s length. The proposed gradient, however, has never been observed and the model appears inconsistent with a number of observations. I present a new, alternative model, wherein auxin acts to establish the adaxial-abaxial domains of the carpel primordia, which then ensures proper development of the final gynoecium. A second project utilizes genomics to identify genes that regulate fruit color by analyzing the genome sequences of Fragaria vesca, a species of wild strawberry. Shared and distinct SNPs among three F. vesca accessions were identified, providing a foundation for locating candidate mutations underlying phenotypic variations among different F. vesca accessions. Through systematic analysis of relevant SNP variants, a candidate SNP in FveMYB10 was identified that may underlie the fruit color in the yellow-fruited accessions, which was subsequently confirmed by functional assays. Our lab has previously generated extensive RNA-sequencing data that depict genome-scale gene expression profiles in F. vesca fruit and flower tissues at different developmental stages. To enhance the accessibility of this dataset, the web-based eFP software was adapted for this dataset, allowing visualization of gene expression in any tissues by user-initiated queries. Together, this thesis work proposes a well-supported new model of fruit patterning in Arabidopsis and provides further resources for F. vesca, including genome-wide variant lists and the ability to visualize gene expression. This work will facilitate future work linking traits of economic importance to specific genes and gaining novel insights into fruit patterning and development.

HydroxyprolineO-arabinosyltransferase mutants oppositely alter tip growth inArabidopsis thalianaandPhyscomitrella patens

Hydroxyproline O-arabinosyltransferases (HPATs) are members of a small, deeply conserved family of plant-specific glycosyltransferases that add arabinose sugars to diverse proteins including cell wall-associated extensins and small signaling peptides. Recent genetic studies in flowering plants suggest that different HPAT homologs have been co-opted to function in diverse species-specific developmental contexts. However, nothing is known about the roles of HPATs in basal plants. We show that complete loss of HPAT function in Arabidopsis thaliana and the moss Physcomitrella patens results in a shared defect in gametophytic tip cell growth. Arabidopsis hpat1/2/3 triple knockout mutants suffer from a strong male sterility defect as a consequence of pollen tubes that fail to fully elongate following pollination. Knocking out the two HPAT genes of Physcomitrella results in larger multicellular filamentous networks due to increased elongation of protonemal tip cells. Physcomitrella hpat mutants lack cell-wall associated hydroxyproline arabinosides and can be rescued with exogenous cellulose, while global expression profiling shows that cell wall-associated genes are severely misexpressed, implicating a defect in cell wall formation during tip growth. Our findings point to a major role for HPATs in influencing cell elongation during tip growth in plants.

Effects of plant regulators on the growth and flowering of Saint Augustine grass plants

The objective of this work was to evaluate the effects of three plant growth inhibitors on the development and emission of floral rachis of Saint Augustine grass [Stenotaphrum secundatum (Walt.) Kuntzel] plants. The study was carried out in a 15 month old lawn with the experimental plots being distributed in accordance with a complete randomized block design with four replications. The treatments consisted of sole application of trinexapac-ethyl (113, 226, 452, 678, and 904 g ai ha(-1)), prohexadione-calcium (100 and 200 g ai ha(-1)), and bispyribac-sodium (40 and 60 g ai ha(-1)) plus a check treatment in which the plants were not submitted to any of the plant growth inhibitors. The effects of those products were evaluated in terms of visual signs of plant intoxication, plant height, emission and height of floral rachises, and chip total dry matter production. All the plant growth inhibitors resulted in visible injury to the plants but these intoxication signs practically disappeared 28 days after the application. Trinexapac-ethyl at the dose of 904 g ai ha(-1) reduced plant height by 59.7%, the emission of floral rachis by 96.4%, and the amount of chip dry matter production by 87.7%. Plant growth inhibitors may reduce the number of times of lawn plants cutting up to 119 days after their application with no harmful effects on the plants visual aspect.

How to grow rare greenhouse plants; 260 flowering varieties for amateur and florist.

Mode of access: Internet.

The orchid-grower's manual : containing descriptions of upwards of nine hundred and thirty species and varieties of orchaceous plants, together with notices of their times of flowering, and most approved modes of treatment ...

Mode of access: Internet.

The home florist: a treatise on the cultivation, management and adaptability of flowering and ornamental plants, designed for the use of amateur florists.

Mode of access: Internet.

Florula Bostoniensis : A collection of plants of Boston and its environs, with their generic and specific characters, synonyms, descriptions, places of growth, and time of flowering, and occasional remarks.

Bound with Dartmouth College. Statutes of Dartmouth University relating to medical graduation. [Hanover? N.H., 1812?]; Dartmouth College. Catalogus eorum, qui adhuc in Universitate Dartmuthensi ... Andoverii, Mass., 1814; Dartmouth College. Catalogus praesidum, senatus academici ... Leuphanae, 1816; and Williams College. Catalogus senatus academici ... Stockbridge, 1814.

Bristol nursery. A catalogue of forest trees, evergreen and flowering shrubs, fruit trees, herbaceous, green-house, and hot-house plants.

Mode of access: Internet.

Flora cestrica : an attempt to enumerate and describe the flowering and filicoid plants of Chester County, in the state of Pennsylvania : with brief notices of their properties, and uses, in medicine, domestic and rural economy, and the arts /

Bookplate of Herbert McLean Evans.

A catalogue of the indigenous flowering and filicoid plants growing within 20 miles of Bridgewater, (Oneida co.) New York.

From Annual Report of the Regents of the University of the state of New York, made to the Legislature Feb. 28, 1832.

Florula bostoniensis. A collection of plants of Boston and its vicinity, with their generic and specific characters, principal synonyms, descriptions, places of growth, and time of flowering, and occasional remarks.

Mode of access: Internet.

Florula bostoniensis. A collection of plants of Boston and its vicinity, with their generic and specific characters, principal synonyms, descriptions, places of growth, and time of flowering, and occasional remarks.

Microfilm. Ann Arbor, Mich., University Microfilms [n.d.] (American culture series, Reel 443.9)