David D. Cass

Ultrastructure of the sperms ofPlumbago zeylanica

SummaryMale gametes ofPlumbago zeylanica were examined in pollen grains and tubes using light and electron microscopy of chemically and physically fixed tissues, and Nomarski interference microscopy of isolated, living sperm cells. Male gametes are elongate, spindleshaped cells containing a nucleus, mitochondria, ER, ribosomes, vesicles, dictyosomes, probable microfilaments, and a variable number of plastids. In mature pollen grains ofP. zeylanica, the two sperm cells are directly linked; they share a transverse cell wall with plasmodesmata and are enclosed together by the inner vegetative cell plasma membrane. One of these two sperms is also associated with the vegetative nucleus as a consistent feature of pollen grain organization. The basis of this association appears to be a long, narrow projection of the sperm cell (averaging < 1 μm wide and about 30 μm long) which wraps around the periphery of the vegetative nucleus and occupies embayments of that nucleus. This association is maintained throughout pollen tube growth but becomes less extensive near the completion of tube growth and is severed following tube discharge. The consistent occurrence of the sperm-vegetative nucleus association in pollen grains, tubes and isolated pollen cytoplasm suggests that the two structures may be directly connected, but attempts to visualize this type of connection were unsuccessful. Possibly, the entwining nature and extent of complementary interfaces between vegetative nucleus and sperm may have a role in stabilizing their association. Functionally, the two sperms and vegetative nucleus appear to travel as a linked unit within the pollen tube, possibly increasing the effectiveness of gamete delivery and helping to ensure nearly simultaneous transmission of sperms into the receptive megagametophyte.

Chloroplast Structure after Water and High-Temperature Stress in Two Lines of Maize that Differ in Endogenous Levels of Abscisic Acid

Chloroplast structure in mesophyll cells from the high-level abscisic acid (ABA) line of maize (Zea mays L.), ZPBL 1304, and the low-level ABA line, ZPL 389, was studied in response to leaf dehydration and high temperature (45 C). Thirteen-day-old seedlings were exposed to 7-d soil drying followed by either 6-h or 24-h heat stress. Seven-day soil drying followed by 6-h high-temperature stress did not affect chloroplasts in line ZPBL 1304 but damaged many chloroplasts of line ZPL 389; chloroplast-envelope membranes were broken and not distinct, and many thylakoids were swollen. Seven-day soil drying followed by 24-h heat stress affected chloroplasts in both lines, but changes in chloroplast structure were greater in ZPL 389 than in ZPBL 1304. The main damage to the chloroplasts in ZPBL 1304 included thylakoid swelling, disruption of the chloroplast envelope, and distortion of chloroplast shape. Chloroplasts in ZPL 389 suffered severe damage: their shape was irregular, envelope membranes were not visible, grana were barely recognizable, and many lipid droplets were visible inside the chloroplast. Modifications in chloroplast structure were reversible in ZPBL 1304. Chloroplasts in ZPL 389 were irreversibly damaged after heating for 24 h. Chloroplast structure differences between lines under stress conditions were likely the result of genotypic differences in leaf dehydration, and possibly the result of genotypic differences in thermal stability of chloroplast membranes. The results support the hypothesis that higher levels of endogenous ABA can indicate drought and heat resistance in maize.

Ultrastractural organization of the egg ofPlumbago zeylanica

SummaryEggs ofPlumbago zeylanica (Plumbaginaceae, Plumbagineae) have been examined by electron microscopy. The major features of this angiosperm egg which are considered to be unique are the possession of an elaborate basal wall structure, known as the filiform apparatus, and microtubules, the latter both in association with the filiform apparatus and elsewhere in the cell adjacent to the plasmalemma. Dictyosomes and mitochondria are abundant throughout the cytoplasm with mitochondria exhibiting higher frequencies near the filiform apparatus. Other aspects of the ultrastructural organization of the egg ofP. zeylanica are presented and discussed. The interpretation of the egg ofP. zeylanica as an egg having relatively high metabolic activity differs from interpretations of the level of metabolic activity of some other angiosperm eggs based on ultrastructural studies. A previous interpretation of the egg ofPlumbago as a cell in which both gametic and synergid functions might reside is substantiated by this ultrastructural study.

Leaf Anatomy of Zea mays L. in Response to Water Shortage and High Temperature: A Comparison of Drought-Resistant and Drought-Sensitive Lines

Leaf anatomical characteristics under normal and reduced water supply in three high-level ABA (abscisic acid) drought-resistant (ZPBL 1304, L-155, and Polj 17) and three low-level ABA drought-sensitive (ZPL 389, B-432, and F-2) lines of Zea mays L. were investigated. The characteristics examined were (1) blade area, (2) thickness of leaf, epidermis, mesophyll, and bulliform cells, (3) stomatal frequency and size of stomatal apparatus, and (4) vessel cross-sectional area. Lines ZPBL 1304 and ZPL 389, L-155 and B-432, and Polj 17 and F-2 were compared. Plant recovery was estimated after exposure to water stress and high-temperature stress. Under normal water supply, the drought-resistant lines were more xeromorphic than the drought-susceptible lines. Water stress affected leaf structures in all lines. Association of xeromorphic features with the ability of the plant to withstand drought varied under dry conditions. Lines ZPL 389, L-155, and Polj 17 were more xeromorphic than lines ZPBL 1304, B-432, and F-2. High temperature induced considerable leaf structural disorganization; however, the changes were not the same in all lines. The greatest differences in leaf structure were observed after the recovery period. The drought-resistant lines showed greater recovery than the drought-sensitive lines. The results, in general, support the hypothesis that cultivars with higher levels of ABA and/or greater drought resistance are more xeromorphic than cultivars with lower levels of ABA and/or lower drought resistance.

Dehydration Avoidance and Damage to the Plasma and Thylakoid Membranes in Lines of Maize Differing in Endogenous Levels of Abscisic Acid

Summary Dehydration avoidance and damage to the plasma and thylakoid membranes were studied in the high-ABA, drought-resistant line of maize ZPBL 1304 and the low-ABA, drought-sensitive line ZPL 389 after exposure to soil drying and high-temperature (45 °C) stress conditions. Thirteen-day-old plants were exposed to 7-d soil drying followed by either 6-h or 24-h high-temperature stress. The high-ABA line ZPBL 1304 was affected by 7-d soil drying followed by 6-h heat stress to a limited extent. The low-ABA line ZPL 389 was affected by 7-d soil drying followed by 6-h heat stress to a great extent, but showed the ability to recover after stress. Extended heat treatment (24 h) affected line ZPBL 1304 reversibly, but was lethal to almost all plants in line ZPL 389. Line ZPBL 1304 showed much greater ability to avoid dehydration and damage to the plasma membrane than line ZPL 389. The stress effects on the stability of thylakoid membranes in lines ZPBL 1304 and ZPL 389 were similar but only when ZPBL 1304 was exposed to high-temperature treatment for a period 4 times longer than ZPL 389. The results support the hypothesis that higher levels of endogenous ABA can indicate drought and heat resistance in maize.

Chloroplast Structure after Water Shortage and High Temperature in Two Lines of Zea mays L. that Differ in Drought Resistance

Chloroplast structure in mesophyll cells from more xeromorphic drought-resistant (Polj 17) and less xeromorphic drought-sensitive (F-2) lines of Zea mays L. was studied in response to water stress and a combination of water and high-temperature stress. In the drought-resistant line water stress partly affected chloroplast structure; membranes of the chloroplast envelope were disrupted only in some chloroplasts, but grana were well preserved. Water stress and high-temperature stress affected chloroplasts to a greater extent but did not severely alter their structure. Grana were still visible, and only in some chloroplasts were they less distinguishable. Water shortage in the drought-sensitive line caused swelling of thylakoids and disappearance of outer membranes of the chloroplast envelope in many chloroplasts. Addition of temperature stress increased chloroplast disruption. The most striking difference was in the structural characteristics of chloroplasts after rehydration. Chloroplasts from the drought-resistant line appeared close to normal. In contrast, chloroplasts from the drought-sensitive line showed signs of additional deterioration in their structure. Differences in chloroplast structure under stress conditions between lines were likely the result of intraspecific differences in leaf dehydration, and possibly the result of intraspecific differences in the sensitivity of chloroplast membranes to high temperature.

Calcium-induced protein phosphorylation and changes in levels of calmodulin and calreticulin in maize sperm cells

Abstract To examine possible calcium (Ca2+)-mediated prefertilization events in male gametes of higher plants, we studied protein phosphorylation and the Ca2+-binding proteins, calmodulin and calreticulin, in sperm cells isolated from maize (Zea mays L.) pollen in the presence and absence of Ca2+. Using immunoblotting, we detected calmodulin and calreticulin and Ca2+-induced variations. Exposure of sperm cells to 1 mM Ca2+ for 1 h increased calmodulin content by 136% compared with the control. Ca2+ had little effect on calreticulin at 1 h, but induced a 34% increase after 3 h. Phosphorylation of proteins was low in 1 h-control and Ca2+-treated cells. However, a 13-fold increase in phosphorylation of a 18-kDa protein was found at 12 h in the presence of Ca2+. Ca2+-induced changes in calmodulin, calreticulin and protein phosphorylation observed in maize sperm cells may reflect prefertilization changes in vivo that facilitate sperm cell fusion with egg and central cells.

RNA and protein synthesis in sperm cells isolated from Zea mays L. pollen

SummarySperm cells are thought to be quiescent in pollen and activated upon pollen germination. To test this hypothesis, protein, RNA and DNA synthesis were assessed in Zea mays sperm cells at different times after isolation from pollen. Protein synthesis changed with time; while some proteins were found to be constitutive in both 0 and 24 h cells, others were synthesized and some disappeared. Overall, the number of proteins detected at 24 h doubled compared with freshly isolated cells. Incorporation of [3H]leucine in 24 h cells was about 50 times that in freshly isolated cells, and that of [5, 6-3H]uridine, about 7 times. Very low incorporation of [6-3H]thymidine into the cells was detected; there was no difference between freshly isolated and 24 h cells. It is possible that the differences in synthetic activity between freshly isolated and 24-h-old cells might correspond to sperm cell activation during pollen tube growth. If so, these metabolic changes may play an important role in fertilization.

Dehydration, Damage to the Plasma Membrane and Thylakoids, and Heat-shock Proteins in Lines of Maize Differing in Endogenous Levels of Abscisic Acid and Drought Resistance

Summary We investigated dehydration avoidance, and damage to the plasma and thylakoid membranes, in the high-level abscisic acid drought-resistant line of Zea mays L., Polj 17, and the low-level abscisic acid drought-sensitive line, F-2, under water shortage, and water shortage and high-temperature stress conditions. The synthesis of heat-shock proteins in Polj 17 and F-2 under water shortage and high-temperature stress conditions was also examined. Under water shortage conditions both lines were mildly dehydrated and suffered damage to the plasma and thylakoid membranes to a similar extent. Exposure to water shortage and high-temperature stress revealed that the high-ABA line Polj 17 had greater capability of withstanding stress conditions than the low-ABA line F-2. Polj 17 showed greater ability to avoid dehydration than F-2. Lines Polj 17 and F-2 showed similar damage to the plasma and thylakoid membranes. However, Polj-17 showed much greater ability to tolerate damage to the cell membranes than F-2. Polj 17 and F-2 did not differ in the pattern of synthesis of heat-shock proteins. Both lines synthesized bands of heat-shock proteins of 43-45 kDa, which appear to be uncommon in maize.

A NOVEL TECHNIQUE FOR THE PARTIAL ISOLATION OF MAIZE EMBRYO SACS AND SUBSEQUENT REGENERATION OF PLANTS

SummaryEmbryo sacs of maize isolated with a few layers of surrounding nucellus or completely isolated with digestive enzymes have resulted in either poorly visible or structurally damaged embryo sacs. We therefore developed a new, more successful method involving mechanical sectioning of maize ovaries using the Vibratome. Sections containing intact embryo sacs are viable and development is normal when sacs are cultured in vitro on semisolid Murashige and Skoog (MS) media. Embryo sacs produce endosperm (90%) and embryos (75%), and mature plants are obtained directly without callus formation or somatic embryogenesis. Immediate applications of this technique may include experimental fertilization and embryogenesis as well as genetic manipulation. Targeting of individual cells was demonstrated with microinjection and confocal microscopy. The methods developed in this study provide a way of studying maize embryo sac development and transformation.