A Statistical Model of Expansive Growth in Plant and Fungal Cells: The Case of Phycomyces.

Expansive growth is a process by which walled cells of plants, algae, and fungi use turgor pressure to mediate irreversible wall deformation and regulate their shape and volume. The molecular structure of the primary cell wall must therefore perform multiple functions simultaneously, including providing structural support by combining elastic and irreversible deformation and facilitating the deposition of new material during growth. This is accomplished by a network of microfibrils and tethers composed of complex polysaccharides and proteins that can dynamically mediate the network topology via periodic detachment and reattachment events. Lockhart and Ortega have provided crucial macroscopic understanding of the expansive growth process through global biophysical models, but these models lack the connection to molecular processes that trigger network rearrangements in the wall. Interestingly, the helical growth of the fungal sporangiophores of Phycomyces blakesleeanus is attributed to a limited region (called the growth zone) where microfibrils are deposited, followed by reorientation and slip. Based on past evidence of dominant shear strain between microfibrils (slippage), we propose a mechanistic model of a network of sliding fibrils connected by tethers. A statistical approach is introduced to describe the population behavior of tethers that have elastic properties and the ability to break and reform in time. These properties are responsible for global cell wall mechanics such as creep and stress relaxation. Model predictions are compared with experiments from literature on stress relaxation and turgor pressure step up for the growing cells of P. blakesleeanus, which are later extended to incised pea (Pisum sativus L.) and the algae Chara corallina using the unique dimensionless number Πpe for each species. To our knowledge, this research is the first attempt to use a statistical approach to model the cell wall during expansive growth, and we believe it provides critical insights on cell wall dynamics at a molecular level.

Diversity, ecology, and evolution in Phycomyces.

The fungal genus Phycomyces (Mucoromycotina, Mucorales) has been revised by examining 96 strains, received from established collections or newly isolated from different environments. Morphology, sexuality, DNA sequences, and population structure clearly identify the genus and set it apart from other Mucorales. The size of the spores, the sexual interactions, the sequences of genes sexM and sexP that determine sexual identity, and the DNA for ribosomal RNAs validate the species Phycomyces blakesleeanus and Phycomyces nitens and the family Phycomycetaceae. Isolates from the same sample of biomass often differ in spore size, sex, DNA sequences, and restriction-length polymorphisms. The widest diversity was found in similar environments in three of the Canary Islands, implying a failure to disperse rapidly over the sea. All strains have apparently functional sexM and sexP genes and all but some strains of P. nitens complete the sexual cycle in the laboratory. The genetic diversity of P. blakesleeanus strains provides evidence for geographical clustering. Various sequence comparisons, including the newly isolated genes sexM and sexP of P. nitens and Blakeslea trispora, clarify phylogenetic relationships in the Mucorales and recommend the sex genes for the study of speciation.

31P NMR study of polyphosphate levels during different growth phases of Phycomyces blakesleeanus.

The changes in relative polyphosphate content, estimated as the intensity ratio of core polyphosphate signal and intracellular inorganic phosphate signal from 31P NMR spectra, during the growth of Phycomyces blakesleeanus are reported. The ratio increases from 16 h to 28 h of growth, the minimum occurs at 32 h, followed by sharp increase up to 36 h, and a steady decrease afterwards. The changes in the biomass during mycelium growth showed steady increases, with a stagnation period between 32 h and 36 h during which a pronounced increase in the intensity ratio of core polyphosphates to intracellular inorganic phosphate signal occurred. The reduction of growth temperature from 22 degrees C to 18 degrees C significantly decreased the rate and intensity of growth, but the pattern of polyphosphate changes remained unchanged. The changes of the intensity ratio of core polyphosphates to intracellular inorganic phosphate signal are linked to characteristic stages of sporangiophore development. Analysis of core polyphosphates, intracellular inorganic phosphate and beta-ATP signal intensities suggest the role of polyphosphates as an energy and/or a phosphate reserves during Phycomyces development.

Gravity susception by buoyancy: floating lipid globules in sporangiophores of Phycomyces.

To elucidate the mechanisms of gravity susception that operate in the sporangiophore of Phycomyces blakesleeanus, we characterized the function and topography of a large apical complex of lipid globules. Stage-1 sporangiophores (without sporangium) possess a roughly spherical complex of 100-200 large lipid globules whose center is localized 110 microm below the apex. The complex of lipid globules (CLG) is rather stable and is kept in place by positioning forces that resist centrifugal accelerations of up to 150 g. The lipid globules possess an average diameter of 2 to 2.5 microm and a density of 0.791 g cm(-3), which is below that of typical plant oleosomes. The potential energy which is generated by the buoyancy of a CLG of 100 globules is in the order of 10(-17) to 10(-16) J, which is 4 to 5 orders of magnitude above thermal noise. The formation of lipid globules can be suppressed by raising stage-1 sporangiophores for 24 hs at 5 degrees C. Sporangiophores with a reduced number of lipid globules display gravitropic bending angles that are 3 to 4 times smaller than those of sporangiophores with the normal number of lipid globules. The results suggest that the lipid globules function as gravisusceptors of Phycomyces and that buoyancy is the physical principle for their mode of action. The globules contain beta-carotene and two distinct fluorescing pigments that are, however, dispensable for graviperception.

Cycloheximide-induced inhibition of chitin synthesis, and decay of apical vesicles and chitosomes in Phycomyces blakesleeanus.

Addition of cycloheximide rapidly inhibited protein synthesis in Phycomyces blakesleeanus. In contrast, chitin biosynthesis decreased with biphasic kinetics displaying a slow and a rapid decay phases. Electron microscopic studies revealed a decrease in the number of apical vesicles and chitosomes after cycloheximide addition; and no change in wall thickness. It is proposed that the slow phase of decay in chitin biosynthesis represents the exhaustion of the pool of chitosomes which transport the chitin synthase necessary to maintain apical wall growth; whereas the second one corresponds to inactivation of the enzyme, which is short lived in vivo. Data also rule out a change in the polarization of wall synthesis induced by cycloheximide, as suggested in other systems.

Factors responsible for inhibiting the motility of zoospores of the phytopathogenic fungus Aphanomyces cochlioides isolated from the non-host plant Portulaca oleracea.

In a survey of plant secondary metabolites regulating the behaviour of Aphanomyces cochlioides zoospores, we found that root extracts of Portulaca oleracea inhibited zoospore motility. Bioassay-directed fractionation of Portulaca constituents revealed that the inhibitory activity was dependent on the interaction of two chemically different factors. These were identified as a phenolic compound, N-trans-feruloyltyramine, which by itself was active as a zoospore stimulant, and an acidic compound, 1-linoleoyl-2-lysophosphatidic acid monomethyl ester, which had zoospore-repellent activity. When Chromosorb W AW particles coated with a mixture of these pure compounds were bioassayed in Petri dishes, the inhibitory effect on zoospore motility was identical with that caused by root tip or root extracts of P. oleracea. Inhibited zoospores rapidly settled to the bottom of the Petri dishes where they initially encysted, and then germinated within 1-2 h. This is the first report of factors which inhibit zoospore motility without killing or bursting the zoospores.

Cell wall extension behavior of Phycomyces sporangiophores during the pressure response.

The cylindrical, single-celled sporangiophore of Phycomyces blakesleeanus grows (enlarges) predominantly in the longitudinal direction during two stages of development; stage I and stage IVb. Cell enlargement (cell wall extension) occurs in a distinct region termed the "growing zone." It was previously reported that a large step-up or pulse-up in turgor pressure, greater than approximately 0.02 MPa, will elicit a transient decrease in longitudinal growth rate of the stage I and stage IVb sporangiophore. This transient decrease in longitudinal growth rate is termed the "pressure response." Both the magnitude and duration of the pressure response depend on the magnitude of the turgor pressure step-up or pulse-up. Qualitatively, the pressure response is similar to the stretch response, which is produced with the application of a longitudinal force (load) on the sporangiophore. In this investigation, the growth (extension) behavior of the cell wall in the growing zone is studied during the pressure response. It is found that both the extension rate of the cell wall in the growing zone and the length of the growing zone decrease during the pressure response, and that together they account for the observed decrease in longitudinal growth rate.

Pressure probe study of the water relations of Phycomyces blakesleeanus sporangiophores.

The physical characteristics which govern the water relations of the giant-celled sporangiophore of Phycomyces blakesleeanus were measured with the pressure probe technique and with nanoliter osmometry. These properties are important because they govern water uptake associated with cell growth and because they may influence expansion of the sporangiophore wall. Turgor pressure ranged from 1.1 to 6.6 bars (mean = 4.1 bars), and was the same for stage I and stage IV sporangiophores. Sporangiophore osmotic pressure averaged 11.5 bars. From the difference between cell osmotic pressure and turgor pressure, the average water potential of the sporangiophore was calculated to be about -7.4 bars. When sporangiophores were submerged under water, turgor remained nearly constant. We propose that the low cell turgor pressure is due to solutes in the cell wall solution, i.e., between the cuticle and the plasma membrane. Membrane hydraulic conductivity averaged 4.6 x 10(-6) cm s-1 bar-1, and was significantly greater in stage I sporangiophores than in stage IV sporangiophores. Contrary to previous reports, the sporangiophore is separated from the supporting mycelium by septa which prevent bulk volume flow between the two regions. The presence of a wall compartment between the cuticle and the plasma membrane results in anomalous osmosis during pressure clamp measurements. This behavior arises because of changes in solute concentration as water moves into or out of the wall compartment surrounding the sporangiophore. Theoretical analysis shows how the equations governing transient water flow are altered by the characteristics of the cell wall compartment.

Absence of significant light-induced changes in cAMP levels in sporangiophores of Phycomyces blakesleeanus.

We were unable to find transient changes in the amount of cAMP or cGMP that had been proposed to mediate the light-growth response in sporangiophores of Phycomyces blakesleeanus.

Avoidance and rheotropic responses in phycomyces. Evidence for an 'avoidance gas" mechanism.

If a mature sporangiophore is placed next to a barrier that is moving in a clockwise direction, it grows both away from the barrier and into the wind; the wind is generated by the moving barrier itself. When the barrier is moving in a counterclockwise direction, the sporangiophore grows towards both the barrier and the wind. The net direction of growth appears to be the vector sum of the rheotropic response and the avoidance aiming error and does not involve the classic stationary-barrier avoidance response. Our experiments all support the suggestion that the avoidance response, the rheotropic response and the variety of reported wind responses can be explained by the presence of a self-emitted, growth-simulating avoidance gas. We present data that suggest that it is the direction of the net flux (mass transfer) of this gas that determines both the direction and the magnitude of the sporangiophore growth. We further suggest that the region of the cell wall showing maximum mass transfer will show a minimum growth rate, i.e., the direction of growth will always be in the direction of maximum transfer. If water is the avoidance gas, then it would follow that the total hydration of the cell wall in an aqueous salt solution should result in cell wall softening; cell wall softening has been correlated directly to cell wall growth. Using the Instron technique, we now show that submerging the entire sporangiophore in an aqueous salt solution for 4 min causes an increase in cell wall extensibility.

Regulation of carotene synthesis in Phycomyces.

Three independent mutations of Phycomyces blakesleeanus resulting in overaccumulation of beta-carotene are recessive and belong to the same complementation group. The corresponding gene has been named carS. Evidence is presented that gene carS is not the same as gene carA, previously defined by mutations blocking carotene production. Vitamin A increases carotenogenesis in wild types and in carS mutants to about the same extent. Intersexual heterokaryosis increases carotenogenesis most prominently in carS genetic backgrounds (up to 300 times the production of the wild type in the same conditions). Vitamin A, intersexual heterokaryosis and carS mutations are thought to stimulate carotenogenesis through different mechanisms. It is suggested that the carS gene product participates in end-product regulation of the pathway.