Cell Wall Reinforcements Accompany Chilling and Freezing Stress in the Streptophyte Green Alga Klebsormidium crenulatum

Abstract

Adaptation strategies in freezing resistance were investigated in Klebsormidium crenulatum, an early branching streptophyte green alga related to higher plants. Klebsormidium grows naturally in unfavorable environments like alpine biological soil crusts, exposed to desiccation, high irradiation and cold stress. Here, chilling and freezing induced alterations of the ultrastructure were investigated. Control samples (kept at 20 • C) were compared to chilled (4 • C) as well as extracellularly frozen algae (−2 and −4 • C). A software-controlled laboratory freezer (AFU, automatic freezing unit) was used for algal exposure to various temperatures and freezing was manually induced. Samples were then high pressure frozen and cryo-substituted for electron microscopy. Control cells had a similar appearance in size and ultrastructure as previously reported. While chilling stressed algae only showed minor ultrastructural alterations, such as small inward facing cell wall plugs and minor alterations of organelles, drastic changes of the cell wall and in organelle distribution were found in extracellularly frozen samples (−2 • C and −4 • C). In frozen samples, the cytoplasm was not retracted from the cell wall, but extensive three-dimensional cell wall layers were formed, most prominently in the corners of the cells, as determined by FIB-SEM and TEM tomography. Similar alterations/adaptations of the cell wall were not reported or visualized in Klebsormidium before, neither in controls, nor during other stress scenarios. This indicates that the cell wall is reinforced by these additional wall layers during freezing stress. Cells allowed to recover from freezing stress (−2 • C) for 5 h at 20 • C lost these additional cell wall layers, suggesting their dynamic formation. The composition of these cell wall reinforcement areas was investigated by immuno-TEM. In addition, alterations of structure and distribution of mitochondria, dictyosomes and a drastically increased endoplasmic reticulum were observed in frozen cells by TEM and TEM tomography. Measurements of the photosynthetic oxygen production showed an acclimation of Klebsormidium to chilling stress, which correlates with our findings on ultrastructural alterations of morphology and distribution of organelles. The cell wall reinforcement

Winter Nights during Summer Time: Stress Physiological Response to Ice and the Facilitation of Freezing Cytorrhysis by Elastic Cell Wall Components in the Leaves of a Nival Species

“…So far, neither the structure nor the chemical composition of cell walls enabling freezing cytorrhysis have been studied. In a recent study of freezing tolerance in a streptophytic green alga, even cell wall reinforcements were established as a consequence of the freezing stress [ 20 ]. Furthermore, it is not known how the plasma membrane behaves during freezing cytorrhysis in intact plant cells, as the few available studies were conducted on isolated protoplasts [ 21 , 22 ].…”

Fusion of Mitochondria to 3-D Networks, Autophagy and Increased Organelle Contacts are Important Subcellular Hallmarks during Cold Stress in Plants

“…It is known from several observations (see references [ 21 , 22 , 23 ] and others), that cell organelles and compartments may alter and interact structurally during stress. In a recent study [ 24 ], structural changes of the cell wall and alterations in organelle distribution were observed in the early branching streptophyte alga Klebsormidium crenulatum (Klebsormidiophyceae) during freezing stress. In the late branching streptophyte alga Micrasterias (Zygnematophyceae, Desmids) for example, the fusion of mitochondria to local networks was observed during ionic stress [ 22 ] and the degeneration of dictyosomes occurred as a consequence of cadmium stress [ 25 ].…”

“…Since 2-dimensional electron microscopy is insufficient for depicting 3-dimensional structural alterations or organelle contacts and fusions [ 22 , 24 , 51 ], we combine 2-D transmission electron microscopic (TEM) methods with 3-D focused ion beam-scanning electron microscopy (FIB-SEM) in the present study. The structural data on cellular responses and rearrangements during low temperature stress obtained by these methods are correlated to functional parameters determined by in-vitro assays (respiration, photosynthesis).…”

3D Dissection of Structural Membrane-Wall Contacts in Filamentous Moss Protonemata

“…It is therefore essential to investigate the form of specific structures in order to understand their function. Modern, computer assisted imaging tools greatly support the visualization of structural details at all levels of complexity [ 13 , 14 ]. 3D presentations of biological structures inspire the imagination of the audience and are a fantastic asset, not only to solve scientific questions but also for teaching [ 2 ].…”

“…Segmentation software like AMIRA ® usually accept also data sets originating from other research fields or imaging techniques like micro computed tomography (µCT) [ 28 ] transmission electron microscopy (TEM; [ 29 ] or focused ion beam scanning electron microscopy (FIB-SEM), as shown for the green alga Micrasterias detenticulata [ 13 ] or Klebsormidium sp. [ 14 ].…”