Darleen A. DeMason

Ethylene-Mediated Programmed Cell Death during Maize Endosperm Development of Wild-Type and shrunken2 Genotypes

We characterized the progression of programmed cell death during maize (Zea mays L.) endosperm development of starchy (Su; wild-type) and shrunken2 (sh2) genotypes and tested the involve ment of ethylene in mediating this process. Histological and viability staining demonstrated that endosperm cell death was initiated earlier and progressed more rapidly in sh2 endosperm compared with Su endosperm. Internucleosomal DNA fragmentation accompanied endosperm cell death and occurred more extensively in sh2 endosperm. 1-Aminocyclopropane-1-carboxylic acid levels peaked approximately 16 d after pollination (dap) in Su endosperm and gradually decreased during subsequent development, whereas two large 1-aminocyclopropane-1-carboxylic acid peaks were observed in sh2 endosperm, the first between 16 and 20 dap and the second at 36 dap. Ethylene levels were elevated in sh2 kernels compared with Su kernels, with an initial peak 20 dap approximately 3-fold higher than in Su kernels and a second peak 36 dap approximately 5-fold higher than that in Su kernels. Ethylene treatment of Su kernels resulted in earlier and more extensive endosperm cell death and DNA fragmentation. Aminoethoxyvinylglycine treatment of sh2 kernels reduced the extent of DNA fragmentation. We conclude that ethylene is involved in triggering programmed cell death in developing maize endosperm and is responsible for the aberrant phenotype of sh2 kernels.

Nuclear and cytoplasmic localization of maize embryo and aleurone dehydrin

SummaryThe subcellular location of dehydrins was determined by immunomicroscopy in maize kernels imbibed in the presence of ABA. Antibodies were specific to the consensus sequence that exists near the carboxy terminus of virtually all dehydrins. Aleurone, scutellar epithelium, scutellar provascular strands, and the outermost embryonic leaves contained the highest concentration of dehydrins. In these tissues, and in scutellar parenchyma cells and inner embryonic leaves, dehydrins were present in the cytoplasm and associated with nuclei. In shoot and root apex cells, dehydrins appeared to be present principally in the cytoplasm. This localization pattern of dehydrins, together with the known amino acid sequences and phosphorylation patterns, leads to analogies with other proteins that are present in both the nucleus and cytoplasm of eukaryotic cells.

Histological characterization of root-knot nematode resistance in cowpea and its relation to reactive oxygen species modulation

Root-knot nematodes (Meloidogyne spp.) are sedentary endoparasites with a broad host range which includes economically important crop species. Cowpea (Vigna unguiculata L. Walp) is an important food and fodder legume grown in many regions where root-knot nematodes are a major problem in production fields. Several sources of resistance to root-knot nematode have been identified in cowpea, including the widely used Rk gene. As part of a study to elucidate the mechanism of Rk-mediated resistance, the histological response to avirulent M. incognita feeding of a resistant cowpea cultivar CB46 was compared with a susceptible near-isogenic line (in CB46 background). Most root-knot nematode resistance mechanisms in host plants that have been examined induced a hypersensitive response (HR). However, there was no typical HR in resistant cowpea roots and nematodes were able to develop normal feeding sites similar to those in susceptible roots up to 9-14 d post inoculation (dpi). From 14-21 dpi giant cell deterioration was observed and the female nematodes showed arrested development and deterioration. Nematodes failed to reach maturity and did not initiate egg laying in resistant roots. These results confirmed that the induction of resistance is relatively late in this system. Typically in pathogen resistance HR is closely associated with an oxidative burst (OB) in infected tissue. The level of reactive oxygen species release in both compatible and incompatible reactions during early and late stages of infection was also quantified. Following a basal OB during early infection in both susceptible and resistant roots, which was also observed in mechanically wounded root tissues, no significant OB was detected up to 14 dpi, a profile consistent with the histological observations of a delayed resistance response. These results will be useful to design gene expression experiments to dissect Rk-mediated resistance at the molecular level.

ROLES OF THE AF AND TL GENES IN PEA LEAF MORPHOGENESIS : CHARACTERIZATION OF THE DOUBLE MUTANT (AFAFTLTL)

The pleiofila phenotype (afaftltl double mutant) of Pisum sativum arises from two single-gene, recessive mutations known to affect the identity of leaf pinnae, afila (af), and acacia (tl). The wild-type leaf consists of proximal leaflets and distal tendrils, whereas the pleiofila leaf consists of branched pinnae terminating in small leaflets. Using morphological measurements, histology, and SEM, we characterized the variation in leaf form along the plant axis, in leaflet anatomy, and in leaf development in embryonic, early postembryonic, and late postembryonic leaves of aftl and wild-type plants. Leaves on aftl plants increase in complexity more rapidly during shoot ontogeny than those on wild-type plants. Leaflets of aftl plants have identical histology to wild-type leaflets although they have smaller and fewer cells. Pinna initiation is acropetal in early postembryonic leaves of aftl plants and in all leaves of wild-type plants, whereas in late postembryonic leaves of aftl plants pinna initiation is bidirectional. Most phenotypic differences between these genotypes can be attributed to differential timing (heterochrony) of major developmental events.

ROLES OF THE Uni GENE IN SHOOT AND LEAF DEVELOPMENT OF PEA (PISUM SATIVUM): PHENOTYPIC CHARACTERIZATION AND LEAF DEVELOPMENT IN THE uni AND uni-tac MUTANTS

A number of single‐gene, recessive mutations have been described in Pisum sativum L. that alter the form of the normal pinnately compound leaf and that show promise in elucidating genetic mechanisms of leaf development. Two recessive mutant alleles are known for the Unifoliata gene (the putative Lfy/Flo orthologue): uni and uni‐tac (tendrilled acacia). To better understand the role of Uni in pea, we made observations on shoot development, leaf development, and in situ expression of Uni mRNA in these two mutants in comparison to wild‐type plants. Although uni plants have abnormal, sterile flowers, those of uni‐tac are usually normal and fertile. The uni and uni‐tac plants produce more leaves and flower later than wild type, especially under long days. Some shoot features that are altered under long days are unaffected in uni plants, indicating that Uni may play a role in some photoperiodic responses. Adult uni leaves exceed one lateral leaflet pair only under short days, whereas uni‐tac leaves typically possess two to three lateral leaflet pairs and one lateral tendril pair. Fusions between the ultimate lateral pinnae and the terminal leaflet are common in both mutants. Pinnae are initiated in an acropetal sequence over five plastochrons (P) for wild type, four for uni‐tac, and three for uni. Lateral leaflet initiation and expansion occur earlier on wild‐type leaves than on the mutants. Uni mRNA is expressed in the tips of juvenile leaf primordia through P4 in wild type, through P3 in uni‐tac, and through P2 in uni. Ectopic expression also occurs in the shoot apical meristem of the mutants. We conclude that the Uni gene affects leaf development in pea by prolonging leaf tip growth and the period of pinna initiation and by delaying leaf tip differentiation. Therefore, it allows larger and more complex leaves to be produced by altering the timing of developmental events.

Patterns of DR5::GUS Expression in Organs of Pea (Pisum sativum)

The recent availability of DR5 reporter constructs to visualize regions of auxin response allows us to examine the role of auxin in developmental processes. In Arabidopsis, DR5::GUS is sensitive to auxin pools in a dosage‐dependent manner and has been used extensively to visualize areas of high auxin concentration. We transformed pea (Pisum sativum) with DR5::GUS. Several lines, originating from independent events, have shown stable expression through T5. In pea, GUS expression occurred in embryos, root tips, developing flowers, procambium, and pollen. In root primordia, GUS expression was most abundant in the peripheral root cap cells, protoderm, and protoxylem. In leaf primordia, dark blue staining occurred in the distal tip of the primordium from plastochrons P0 through P4–5. Lateral tendril primordia and tendril tips stained more intensely blue and for more plastochrons than leaflet primordia and tips. Blue (GUS) staining in floral organs during initiation and development also revealed specific, shifting patterns. Quantitative MUG assays showed that DR5 is differentially responsive to different natural and synthetic auxins, with 4‐Cl‐IAA giving the strongest response. The DR5::GUS transgene is being crossed into various mutants, which will allow identification of the role of auxin in different developmental events in pea.

Structure and biochemistry of endosperm breakdown in date palm (Phoenix dactylifera L.) seeds

SummaryThe zone of endosperm breakdown in the germinated date seed (Phoenix dactylifera L.) is a narrow area immediately adjacent to the surface of the enlarging cotyledon, or haustorium. The zone width is correlated with the amount of cell division in the adjacent region of the haustorium. The sequence of endosperm breakdown is: 1. protein bodies vacuolate, 2. storage cell walls become electron-transparent immediately adjacent to the protoplast of each endosperm cell, 3. all remaining cytoplasm and lipid bodies disappear, and 4. the remaining cell walls become electron-transparent and collapse against the haustorium surface. Two cell wall hydrolases are present—endo-Βmannanase (EC3.2.1.78) and Β-mannosidase (EC3.2.1.25). Β-mannosidase is detectable in the endosperm before germination. At germination, the major portion of activity is found in the softened endosperm. Β-mannanase is only detectable from germination and there is always hundreds of fold greater activity in the softened endosperm than elsewhere. Proteinase is detectable in trace amounts at germination in the softened endosperm but is also found in the haustorium at later stages. Isolated haustoria, incubated in extracted ivory nut (Phytelephas macrocarpa) mannan in buffer, cause no mannan breakdown. Haustoria, incubated in a solution of locust bean galactomannan, cause no decrease in galactomannan viscosity. Our observations suggest that although haustoria probably regulate mannan breakdown in the endosperm, they do not seem to secrete the hydrolytic enzymes concerned.

Genetic Control of Leaf Development in Pea (Pisum sativum)

Three well‐defined genes affect the morphological and anatomical features of the pea (Pisum sativum) compound leaf. Either singly or in combination, they specify five distinct pinna types. Using simple genetics, classical criteria for establishing homology, SEM of leaf development, and pinna histology, the phenotypes of the afila (af), tendril‐less (tl), and tendrilled acacia (uni‐tac)/unifoliata (uni) mutants are compared with that of wild‐type plants, and the roles of the Af, Tl, and Uni genes are deduced. Marx’s concept of inherent regions within the pea leaf is upheld. The leaf blade consists of three genetically/developmentally determined regions: proximal, distal, and terminal. All three genes modify leaf blade form by altering the timing of events during leaf development. In addition, these genes affect most aspects of leaf morphology (pinna pair number, pinna, petiole and leaf lengths, pinna branching) and histology (cell arrangement and size) as well as characteristics of shoot ontogeny (number of leaves, first node to flower, leaf heteroblasty).

Freeze-fracture observations on membranes of dry and hydrated pollen from Collomia, Phoenix and Zea.

Pollen from Collomia grandiflora Dougl. ex Lindl., Phoenix dactylifera L. and Zea mays L. was examined by freeze-fracture electron microscopy. Particular attention was paid to the organization of the cell membranes in the naturally dehydrated, as compared to the fully hydrated, state. All membranes examined had a normal bilayer organization similar to that seen in the hydrated cells of these and other plants. This organization of dry pollen membranes is discussed as it relates to physiological studies (e.g., leakage of ions during hydration), and to biophysical properties of biological and model membranes under various conditions of hydration and dehydration.

Histochemical and ultrastructural changes in the haustorium of date (Phoenix dactylifera L.)

SummaryDuring imbibition ofPhoenix dactylifera embryos, all cotyledon cells show the same changes: protein and lipid bodies degrade, smooth endoplasmic reticulum (ER) increases in amount, and dictyosomes appear. At germination, the distal portion of the cotyledon expands to form the haustorium. At this time, epithelial cells have a dense cytoplasm with many extremely small vacuoles. Many ribosomes are present along with ER, dictyosomes, and mitochondria. The parenchyma cells have large vacuoles and a small amount of peripheral cytoplasm. Between 2 and 6 weeks after germination, epithelial cells still retain the dense cytoplasm and many organelles appear: glyoxysomes, large lipid bodies, amyloplasts, large osmiophilic bodies, and abundant rough and smooth ER which appear to merge into the plasmalemma. A thin electron-transparent inner wall layer with many small internal projections is added to the cell walls. Starch grains appear first in the subsurface and internal parenchyma and subsequently in the epithelium. Lipid bodies, glyoxysomes, protein, and osmiophilic bodies occur in the epithelial and subepithelial cell layers but not in the internal parenchyma. At 8 weeks after germination, the cytoplasm becomes electron transparent, vacuolation occurs, lipid bodies and osmiophilic bodies degrade, and the endomembranes disassemble. After 10 weeks, the cells are empty. These data support the hypothesis that the major functions of the haustorium are absorption and storage.