Scott D. Russell

BAK1 and BKK1 Regulate Brassinosteroid-Dependent Growth and Brassinosteroid-Independent Cell-Death Pathways

Brassinosteroids (BRs) are phytosteroid hormones controlling various physiological processes critical for normal growth and development. BRs are perceived by a protein complex containing two transmembrane receptor kinases, BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) [1-3]. BRI1 null mutants exhibit a dwarfed stature with epinastic leaves, delayed senescence, reduced male fertility, and altered light responses. BAK1 null mutants, however, only show a subtle phenotype, suggesting that functionally redundant proteins might be present in the Arabidopsis genome. Here we report that BAK1-LIKE 1 (BKK1) functions redundantly with BAK1 in regulating BR signaling. Surprisingly, rather than the expected bri1-like phenotype, bak1 bkk1 double mutants exhibit a seedling-lethality phenotype due to constitutive defense-gene expression, callose deposition, reactive oxygen species (ROS) accumulation, and spontaneous cell death even under sterile growing conditions. Our detailed analyses demonstrate that BAK1 and BKK1 have dual physiological roles: positively regulating a BR-dependent plant growth pathway, and negatively regulating a BR-independent cell-death pathway. Both BR signaling and developmentally controlled cell death are critical to optimal plant growth and development, but the mechanisms regulating early events in these pathways are poorly understood. This study provides novel insights into the initiation and crosstalk of the two signaling cascades.

Female Germ Unit: Organization, Isolation, and Function

Publisher Summary This chapter focuses on megagametophytes and examines the female germ unit from four new perspectives: (1) the ultrastructure and cytochemistry of megagametophyte development and cellularization, (2) the organization and function of the female germ unit in reproduction, (3) the isolation and characterization of embryo sacs and their component cells, and (4) the involvement of the cytoskeleton in megagametophyte development and function. The development of the megagametophyte is divided into two connected developmental phases: (1) megasporogenesis, which entails the formation and maturation of the initial products of meiosis, followed by (2) megagametogenesis, which begins with the mitotic division of the meiotic products and continues through the cellularization and maturation of the megagametophyte. The female germ unit is comprised of the egg, two synergids, and the central cell. As such, it constitutes the minimum number of cells required to (1) receive the pollen tube, (2) cause the discharge of the sperm into the receptive portion of the female gametophyte, and (3) undergo double fertilization.

A genome-wide survey of imprinted genes in rice seeds reveals imprinting primarily occurs in the endosperm.

Genomic imprinting causes the expression of an allele depending on its parental origin. In plants, most imprinted genes have been identified in Arabidopsis endosperm, a transient structure consumed by the embryo during seed formation. We identified imprinted genes in rice seed where both the endosperm and embryo are present at seed maturity. RNA was extracted from embryos and endosperm of seeds obtained from reciprocal crosses between two subspecies Nipponbare (Japonica rice) and 93-11 (Indica rice). Sequenced reads from cDNA libraries were aligned to their respective parental genomes using single-nucleotide polymorphisms (SNPs). Reads across SNPs enabled derivation of parental expression bias ratios. A continuum of parental expression bias states was observed. Statistical analyses indicated 262 candidate imprinted loci in the endosperm and three in the embryo (168 genic and 97 non-genic). Fifty-six of the 67 loci investigated were confirmed to be imprinted in the seed. Imprinted loci are not clustered in the rice genome as found in mammals. All of these imprinted loci were expressed in the endosperm, and one of these was also imprinted in the embryo, confirming that in both rice and Arabidopsis imprinted expression is primarily confined to the endosperm. Some rice imprinted genes were also expressed in vegetative tissues, indicating that they have additional roles in plant growth. Comparison of candidate imprinted genes found in rice with imprinted candidate loci obtained from genome-wide surveys of imprinted genes in Arabidopsis to date shows a low degree of conservation, suggesting that imprinting has evolved independently in eudicots and monocots.

The Egg Cell: Development and Role in Fertilization and Early Embryogenesis.

Flowering plant reproduction is unusual in many features, as evidenced by the different topics discussed in this issue of THE PLANT CELL. The egg cell of flowering plants in particular displays several unique features. First, the egg cell is an integral part of the several thousands of cells forming the ovule, and it cannot be released without the aid of enzymes or microdissection. The egg cell appears to be part of a functional assemblage of cells that are fragile in isolation. Surrounding the egg cell in situ are the two synergids (collectively forming the egg apparatus) and the adjacent central cell, as shown in Figure 1. These are almost always derived from the same initial meiotic cell as the egg cell. The surrounding cells appear to provide nutrition for the egg cell and are involved in the positioning of the sperm cells to the precise site where gametic fusion occurs. Different developmental programs are invoked in both the egg and central cells by the fusion of each with a sperm cell in an event characteristic of flowering plants known as “double fertilization.” During this event, one sperm nucleus fuses with the egg nucleus to form the zygote, whereas the other sperm nucleus fuses with the two (or more) central cell nuclei, resulting in the formation of the nutritive endosperm. The developmental potential, biochemical identity, and char

Experimental Analysis of the Fertilization Process

In flowering plants, double fertilization is one of the defining features of reproductive development ([Raghavan, 2003][1]). Double fertilization was first discovered in 1898 by Nawaschin. It involves a complex series of interactions between essentially three plants—the male gametophyte (MG), the

Ultrastructure of the sperm of Plumbago zeylanica : II. Quantitative cytology and three-dimensional organization.

Pollen grains of Plumbago zeylanica L. were serially sectioned and examined using transmission electron microscopy to determine the three-dimensional organization of sperm cells within the microgametophyte and the quantity of membrane-bound organelles occurring within each cell. Sperm cells occur in pairs within each pollen grain, but are dimorphic, differing in size, morphology and organelle content. The larger of the two sperm cells (Svn) is distinguished by the presence of a long (approx. 30 μm) projection, which wraps around and lies within embayments of the vegetative nucleus. This cell contains numerous mitochondria, up to two plastids and, infrequently, microbodies. It is characterized by a larger volume and surface area and contains a larger nucleus than the other sperm cell. The second sperm cell (Sua) is linked by plasmodesmata with the Svn, but is unassociated with the vegetative nucleus. It is smaller and lacks a cellular projection. The Sua contains relatively few mitochondria, but numerous (up to 46) plastids and more microbodies than the other sperm. The degree of dimorphism in their content of heritable cytoplasmic organelles must at fertilization result in nearly unidirectional transmission of sperm plastids into just one of the two female reproductive cells, and preferential transmission of sperm mitochondria into the other.

Calcium function and distribution during fertilization in angiosperms

Calcium has an essential signaling, physiological, and regulatory role during sexual reproduction in flowering plants; elevation of calcium amounts is an accurate predictor of plant fertility. Calcium is present in three forms: (1) covalently bound calcium, (2) loosely bound calcium typically associated with fixed and mobile anions (ionic bonding); and (3) cytosolic free calcium-an important secondary messenger in cell signaling. Pollen often requires calcium for germination. Pollen tube elongation typically relies on external calcium stores in the pistil. Calcium establishes polarity of the pollen tube and forms a basis for pulsatory growth. Applying calcium on the tip may alter the axis; thus calcium may have a role in determining the directionality of tube elongation. In the ovary and ovule, an abundance of calcium signals receptivity, provides essential mineral nutrition, and guides the pollen tube in some plants. Calcium patterns in the embryo sac also correspond to synergid receptivity, reflecting programmed cell death in one synergid cell that triggers degeneration and prepares this cell to receive the pollen tube. Male gametes are released in the synergid, and fusion of the gametes requires calcium, according to in vitro fertilization studies. Fusion of plant gametes in vitro triggers calcium oscillations evident in both the zygote and primary endosperm during double fertilization that are similar to those in animals.

Fertilization in Nicotiana tabacum: Cytoskeletal modifications in the embryo sac during synergid degeneration

The cytoskeletal organization of the embryo sac of tobacco (Nicotiana tabacum L.) was examined at maturity and during synergid degeneration, pollen-tube delivery and gamete transfer using rapid-frozen, freeze-substituted and chemically fixed material in combination with immunofluorescence and immunogold electron microscopy. Before fertilization, the synergid is a highly polarized cell with dense longitudinally aligned arrays of microtubules adjacent to the filiform apparatus at the micropylar end of the cell associated with major organelles. The cytoskeleton of the central cell is less polarized, with dense cortical microtubules in the micropylar and chalazal regions and looser, longitudinally oriented cortical microtubules in the lateral region. In the synergid and central cell, F-actin is frequently found at the surface of the organelles and co-localizes with either single microtubules or microtubule bundles. Egg cell microtubules are frequently cortical, randomly oriented and more abundant at the chalazal end of the cell; actin filaments are associated with microtubules and the cortex of the egg cell. At 48 h after pollination and before the pollen tube arrives, the onset of degeneration is evident in one of the two synergids: the electron density of cytoplasmic organelles and the ground cytoplasm increases and the nucleus becomes distorted. Although synergids otherwise remain intact, the vacuole collapses and organelles degenerate rapidly after pollen-tube entry. Abundant electron-dense material extends from the degenerated synergid into intercellular spaces at the chalazal end of the synergid and between the synergids, egg and central cell. Rhodamine-phalloidin and anti-actin immunogold labeling reveal that electron-dense aggregates in this region contain abundant actin forming two distinct bands termed “coronas”. This actin is part of a mechanism in the egg apparatus which appears to precisely position and facilitate the access of male gametes to the egg and central cell for fusion.

Calcium distribution in fertilized and unfertilized ovules and embryo sacs of Nicotiana tabacum L

Abstract. Potassium antimonate was used to localize Ca2+ in tobacco ovules from 0 to 7 d after anthesis in pollinated and emasculated flowers. Antimonate binds “loosely bound” Ca2+ into calcium antimonate; less-soluble forms are unavailable and free calcium usually escapes. Ovules are immature at anthesis. Abundant calcium precipitates in nucellar cells surrounding the micropylar canal. A difference between calcium in the two synergids emerges at 1 d, which is enhanced in pollinated flowers. The future receptive synergid accumulates more precipitates in the nucleus, cytoplasm and cell walls. After fertilization, micropyle precipitates diminish, and the ovule is unreceptive to further tube entry. In emasculated flowers 6 d after anthesis, ovular precipitates essentially disappear; however, flowers pollinated at 4–5 d and collected 2 d later largely restore their prior concentration of precipitates. Ovular precipitates occur initially in the nucellus, then the embryo sac, and finally the synergid and micropylar filiform apparatus. Possibility, calcium is released from the embryo sac, although no structural evidence of exudate formation was observed. Calcium precipitates in the ovule correlate with the ability of the ovule to be fertilized, suggesting that successful pollen tube entry and later development may require calcium of the class precipitated by antimonate.

Response of an allergenic species, Ambrosia psilostachya (Asteraceae), to experimental warming and clipping: Implications for public health

We examined the responses of an allergenic species, western ragweed (Ambrosia psilostachya DC.), to experimental warming and clipping. The experiment was conducted in a tallgrass prairie in Oklahoma, USA, between 1999 and 2001. Warming increased ragweed stems by 88% when not clipped and 46% when clipped. Clipping increased ragweed stems by 75% and 36% in the control and warmed plots, respectively. In 2001, warming resulted in a 105% increase in ragweed aboveground biomass (AGB), and the ratio of ragweed AGB to total AGB increased by 79%. Dry mass per ragweed stem in the warmed plots was 37% and 38% greater than that in the control plots in 2000 and 2001, respectively. Although warming caused no difference in pollen production per stem, total pollen production increased by 84% (P < 0.05) because there were more ragweed stems. Experimental warming significantly increased pollen diameter from 21.2 μm in the control plots to 23.9 μm in the warmed plots (a 13% increase). The results from our experiment suggest that global warming could aggravate allergic hazards and thereby jeopardize public health.