Studies on a maize mutant sensitive to low temperature I. Influence of temperature and light on the production of chloroplast pigments

A mutant inbred line of Zea mays L. (M11) in which chlorophyll accumulation is particularly sensitive to low temperature is described. Under natural light conditions the chlorophyll content of seedlings is negligible below 17° but is normal at high temperature. Seedlings of M11 can synthesize chloroplast pigments at 16° but at a rate slower than normal. When photo-oxidation is minimized, chlorophyll accumulates, and seedlings can photosynthesize efficiently at low temperature. The primary site of low temperature sensitivity in M11 is the shoot apex where new leaves are developing and undergoing rapid cell expansion. It seems that there is impaired development and associated impaired function of chloroplasts in M11 grown at low temperatures which sensitizes them to rapid photo-oxidation in the light.     

A chloroplast membrane lacking Photosystem I. Changes in unstacked membrane regions

The structure and polypeptide composition of the photosynthetic membrane of a mutant of maize has been investigated. The thylakoid membranes of the mutant plants are deficient in Photosystem I activity, although Photosystem II is at near normal levels. SDS polyacrylamide gel electrophoresis of thylakoid membranes from the mutant shows them to be deficient in two polypeptide bands which have been associated with Photosystem I. Freeze-fracture studies of the membrane show that the absence of these polypeptides is associated with a measurable reduction in particle diameter on the unstacked protoplasmic fracture face. This fracture face is derived from the splitting of membranes in unstacked regions of the thylakoid membrane system. It is suggested that in membranes stacked by salts in vitro, Photosystem I activity may be confined to this region.     

Novel light-regulated chloroplast thylakoid membrane protein

A 64 kilodalton chloroplast membrane polypeptide was dependent on growth irradiance with 10-fold greater quantities of the protein present in barley (Hordeum vulgare) grown under 500 micromoles of photons per square meter per second compared with growth at 50 micromoles per square meter per second. The concentration of the protein was sensitive to changes in irradiance, with a slow time course for the response (days) similar to other reported light acclimation processes. The polypeptide also was observed in maize (Zea mays), oats (Avena sativa), and wheat (Triticum aestivum), but not in soybean (Glycine max Merr). The 64 kilodalton polypeptide did not correspond to any thylakoid membrane protein with an assigned function, so its structural or regulatory role is not known.     

Role of lamellar membrane structure in tether formation from bilayer vesicles.

A theoretical analysis is presented of the formation of membrane tethers from micropipette-aspirated phospholipid vesicles. In particular, it is taken into account that the phospholipid membrane is composed of two layers which are in contact but unconnected. The elastic energy of the bilayer is taken to be the sum of contributions from area expansivity, relative expansivity of the two monolayers, and bending. The vesicle is aspirated into a pipette and a constant point force is applied at the opposite side in the direction away from the pipette. The shape of the vesicle in approximated as a cylindrical projection into the pipette with a hemispherical cap, a spherical section, and a cylindrical tether with a hemispherical cap. The dimensions of the different regions of the vesicle are obtained by minimizing its elastic energy subject to the condition that the volume of the vesicle is fixed. The range of values for the parameters of the system is determined at which the existence of a tether is possible. Stability analysis is performed showing which of these configurations are stable. The importance of the relative expansion and compression of the constituent monolayers is established by recognizing that local bending energy by itself does not stabilize the vesicle geometry, and that in the limit as the relative expansivity modulus becomes infinitely large, a tether cannot be formed. Predictions are made for the functional relationships among experimentally observable quantities. In a companion report, the results of this analysis are applied to experimental measurements of tether formation, and used to calculate values for the membrane material coefficients.     

Association of TMV coat protein with chloroplast membranes in virus-infected leaves

Summary The polypeptide composition of extracts of chloroplasts from tobacco leaves systemically infected with different strains of Tobacco Mosaic Virus (TMV) was analyzed by one- and two-dimensional gel electrophoresis. There were no changes in the protein profiles of chloroplasts from infected leaves when compared to control leaves except for the presence of coat protein (CP) of TMV, identified by immunoblotting. When protease-treated intact chloroplasts isolated on Percoll gradients were osmotically disrupted the CP could be detected in both stroma and membrane fractions. The majority of the CP associated with the thylakoid membranes (about 1–5% of the total thylakoid proteins) was in the form of free molecules while stroma contained aggregated or assembled CP (about 0.1% of the soluble proteins). Thylakoid-associated CP was insensitive to protease digestion unless the membranes were first treated with a detergent, indicating that the CP was embedded inside or otherwise complexed with the thylakoid membranes.Chloroplasts isolated from leaves infected with TMV-PV42, a symptomless strain, contained approximately 10–50 times less CP than did chloroplasts isolated from leaves bearing mosaic symptoms induced by other strains of TMV (U1, PV230 or PV39). A possible role of CP in symptom development is discussed.     

Mapping membrane protein structure with fluorescence

Membrane proteins regulate many cellular processes including signaling cascades, ion transport, membrane fusion, and cell-to-cell communications. Understanding the architecture and conformational fluctuations of these proteins is critical to understanding their regulation and functions. Fluorescence methods including intensity mapping, fluorescence resonance energy transfer (FRET), and photo-induced electron transfer, allow for targeted measurements of domains within membrane proteins. These methods can reveal how a protein is structured and how it transitions between different conformational states. Here, I will review recent work done using fluorescence to map the structures of membrane proteins, focusing on how each of these methods can be applied to understanding the dynamic nature of individual membrane proteins and protein complexes.