Evolution and Functional Differentiation of the C-terminal Motifs of FtsZs during Plant Evolution.

Filamentous temperature-sensitive Z (FtsZ) is a tubulin-like GTPase that is highly conserved in bacteria and plants. It polymerizes into a ring at the division site of bacteria and chloroplasts and serves as the scaffold protein of the division complex. While a single FtsZ is present in bacteria and cyanobacteria, there are two subfamilies, FtsZ1 and FtsZ2 in the green lineage, and FtsZA and FtsZB in red algae. In Arabidopsis thaliana, the C-terminal motifs of AtFtsZ1 (Z1C) and AtFtsZ2-1 (Z2C) display distinct functions in the regulation of chloroplast division. Z1C exhibits weak membrane-binding activity, whereas Z2C engages in the interaction with the membrane protein AtARC6. Here, we provide evidence revealing the distinct traits of the C-terminal motifs of FtsZ1 and FtsZ2 throughout the plant evolutionary process. In a range of plant species, the C-terminal motifs of FtsZ1 exhibit diverse membrane-binding properties critical for regulating chloroplast division. In chlorophytes, the C-terminal motifs of FtsZ1 and FtsZ2 exhibit both membrane-binding and protein interaction functions, which are similar to those of cyanobacterial FtsZ and red algal FtsZA. During the transition from algae to land plants, the functions of the C-terminal motifs of FtsZ1 and FtsZ2 exhibit differentiation. FtsZ1 lost the function of interacting with ARC6 in land plants, and the membrane-binding activity of FtsZ2 was lost in ferns. Our findings reveal the functional differentiation of the C-terminal motifs of FtsZs during plant evolution, which is critical for chloroplast division.

A novel amphiphilic motif at the C-terminus of FtsZ1 facilitates chloroplast division.

In bacteria and chloroplasts, the GTPase filamentous temperature-sensitive Z (FtsZ) is essential for division and polymerizes to form rings that mark the division site. Plants contain two FtsZ subfamilies (FtsZ1 and FtsZ2) with different assembly dynamics. FtsZ1 lacks the C-terminal domain of a typical FtsZ protein. Here, we show that the conserved short motif FtsZ1Carboxyl-terminus (Z1C) (consisting of the amino acids RRLFF) with weak membrane-binding activity is present at the C-terminus of FtsZ1 in angiosperms. For a polymer-forming protein such as FtsZ, this activity is strong enough for membrane tethering. Arabidopsis thaliana plants with mutated Z1C motifs contained heterogeneously sized chloroplasts and parallel FtsZ rings or long FtsZ filaments, suggesting that the Z1C motif plays an important role in regulating FtsZ ring dynamics. Our findings uncover a type of amphiphilic beta-strand motif with weak membrane-binding activity and point to the importance of this motif for the dynamic regulation of protein complex formation.

Immunofluorescence staining of chloroplast proteins with frozen sections of plant tissues.

KEY MESSAGE: Immunofluorescence staining with frozen sections of plant tissues and a nest tube is convenient and effective, and broadens the applicability of immunofluorescence staining. Immunofluorescence staining is an indispensable and extensively employed technique for determining the subcellular localization of chloroplast division proteins. At present, it is difficult to effectively observe the localization of target proteins in leaves that are hard, or very thin, or have epidermal hair or glands with the current immunofluorescence staining methods. Moreover, signals of target proteins were predominantly detected in mesophyll cells, not the cells of other types. Thus, the method of immunofluorescence staining was further explored for improvement in this study. The plant tissue was embedded with 50% PEG4000 at -60℃, which was then cut into sections by a cryomacrotome. The sections were immediately immersed in fixation solution. Then, the sample was transferred into a special nested plastic tube, which facilitated the fixation and immunofluorescence staining procedures. The use of frozen sections in this method enabled a short processing time and reduced material requirements. By optimizing the thickness of the sections, a large proportion of the cells could be well stained. With this method, we observed the localization of a chloroplast division protein FtsZ1 in the wild-type Arabidopsis and various chloroplast division mutants. Meanwhile, the localization of FtsZ1 was also observed not only in mesophyll cells, but also in guard cells and epidermal cells in a lot of other plant species, including many species with hard leaf tissues. This method is not only easy to use, but also expands the scope of applicability for immunofluorescence staining.

Improvements for Tissue-Chopping-Based Immunofluorescence Staining Method of Chloroplast Proteins.

Immunofluorescence staining is a very common method for the subcellular localization study of proteins. A tissue-chopping-based immunofluorescence staining method for chloroplast proteins overcomes the restriction of plant cell wall, makes the operation simpler, and uses less experimental materials. Here we provide some improvements for this method. We found that the stained tissues can be directly observed with a confocal microscope without tissue lysis. Samples maintained at a low temperature (0-4 °C) throughout the process can reduce the intensity of chlorophyll autofluorescence and the background signal. A low temperature is also good for the storage of the sample. Fluorescence signal of the stained samples can be kept for several weeks if they are stored at -20 °C. FtsZ is an essential component of the chloroplast division apparatus. We demonstrated this method with the immunofluorescence staining of FtsZ1 in wildtype Arabidopsis and some chloroplast division mutants. We also successfully tested this method by the immunofluorescence staining of FtsZ1 in many other plants, including woody plants. With these procedures, the performance of tissue-chopping-based immunofluorescence staining method are further improved.

A Tissue-Chopping Based Immunofluorescence Staining Method for Chloroplast Proteins.

Immunofluorescence staining is an important method for detecting the localization of proteins in the cell. It is also frequently used in the localization study of chloroplast-division proteins. Although this method has been improved before by using protoplasts, it still has some limitations. Now we developed a new method to make it much easier. We just broke the plant leaf tissue with a serrated blade, stained the samples directly, and simply lysed the tissue into separatable cells. The localization of the target protein can then be observed with a clear view. Since this method directly uses broken leaf pieces, it is very fast. It can also be applied to the plants in which protoplasts are difficult to prepare. We first used this method to observe the localization of a chloroplast division protein FtsZ1 in the wild-type Arabidopsis. A ring was clearly seen in the middle of chloroplasts. In addition, we used this method to analyze the localization of FtsZ1 in arc3 and pdv2 mutants, as well as in dozens of other species, including some woody plants. This new immunofluorescence staining method is not only easy to use, but also has a wide applicability in various plants.

Visualizing the Integrity of Chloroplast Envelope by Rhodamine and Nile Red Staining.

Chloroplasts are essential organelles in plant cells with many important functions. Chloroplasts isolated by Percoll density gradient centrifugation are widely used in the study of chloroplasts. The intactness of isolated chloroplasts is necessary for many of the experiments. In the past, those isolated chloroplasts were either simply believed to be intact or had to be analyzed by indirect biochemical methods. Here we show a new method to check the intactness of isolated chloroplasts by staining their envelope with fluorescent dyes, Rhodamine or Nile red, and then observing them with a fluorescence microscope. With this method, broken chloroplasts and intact chloroplasts can be distinguished easily and their integrity can be checked in a few minutes. Results of this method agreed well with those of biochemical methods. Moreover, we have also found that sometimes the middle layer chloroplasts from the Percoll gradient centrifugation could be mostly broken, which could cause mistakes in the experiment. With our method, this problem can be easily found. This chloroplast envelope staining method can be used in the preparation of isolated chloroplasts to ensure the intactness.