Manabu Nagao

Cold acclimation in bryophytes: low-temperature-induced freezing tolerance in Physcomitrella patens is associated with increases in expression levels of stress-related genes but not with increase in level of endogenous abscisic acid

Bryophyte species growing in areas in which temperatures fall below zero in winter are likely to have tolerance to freezing stress. It is well established in higher plants that freezing tolerance is acquired by exposure to non-freezing low temperatures, accompanied by expression of various genes and increases in levels of the stress hormone abscisic acid (ABA). However, little is known about the physiological changes induced by cold acclimation in non-vascular plants such as bryophytes. We examined the effects of low temperatures on protonema cells of the moss Physcomitrella patens (Hedw.) Bruch & Schimp. The freezing tolerance of protonema cells was clearly increased by incubation at low temperatures ranging from 10°C to 0°C, with maximum tolerance achieved by incubation at 0°C for several days. The enhancement of freezing tolerance by low temperatures occurred in both light and dark conditions and was accompanied by accumulation of several transcripts for late-embryogenesis-abundant (LEA) proteins and boiling-soluble proteins. By de-acclimation, low-temperature-induced expression of these transcripts and proteins, as well as the freezing tolerance, was reduced. Interestingly, endogenous levels of ABA in tissues or that secreted into the culture medium were not specifically increased by low-temperature treatment. Furthermore, removal of ABA from the medium by addition of activated charcoal did not affect low-temperature-induced freezing tolerance of the protonema cells. Our results provide evidence that bryophytes have an ABA-independent cold-signaling pathway leading to expression of stress-related genes and resultant acquisition of freezing tolerance.

Abscisic acid-induced freezing tolerance in the mossPhyscomitrella patens is accompanied by increased expression of stress-related genes

Abscisic acid (ABA)-induced genes are implicated in the development of freezing tolerance during cold acclimation in higher plants, but their roles in lower land plants have not been determined. We examined ABA- and cold-induced changes in freezing tolerance and gene expression in the moss Physcomitrella patens. Slow equilibrium freezing to -4 degrees C of P. patens protonemata grown under normal growth conditions killed more than 90% of the cells, indicating that the protonema cells are freezing-sensitive. ABA treatment for 24 h dramatically increased the freezing tolerance of the protonemata, while cold treatment only slightly increased the freezing tolerance within the same period. We examined the expressions of fourteen Physcomitrella patens ABA-responsive genes (PPARs), isolated from ABA-treated protonemata. ABA treatment resulted in a remarkable increase in the expression of all the PPAR genes within 24 h. Several of the PPAR genes (PPAR 1 to 8, and 14) were also responsive to cold, but the response was much slower than that to ABA. Treatment with hyperosmotic concentrations of NaCl and mannitol increased freezing tolerance of protonemata and also increased the expression levels of eleven PPAR genes (PPAR2, 3, 5 to 8, and 10 to 14). These results suggest that ABA and environmental stresses positively affect the expression of common genes that participate in protection of protonema cells leading to the development of freezing tolerance.

Klebsormidium flaccidum, a charophycean green alga, exhibits cold acclimation that is closely associated with compatible solute accumulation and ultrastructural changes

To elucidate the fundamental mechanisms and subsequent evolutionary aspects of plant cold acclimation, we examined the effect of cold acclimation on freezing tolerance in Klebsormidium flaccidum, a green alga belonging to Charophyceae, a sister group of land plants. Freezing tolerance of K. flaccidum was significantly enhanced by cold treatment: survival increased from 15% at -10 degrees C when grown at 18 degrees C to 55 and 85% after exposure at 2 degrees C for 2 and 7 d, respectively. Accompanying the development of freezing tolerance, soluble sugars (glucose and sucrose), a putative glycoside and amino acids, including gamma-aminobutyric acid (GABA), accumulated to high levels in the alga, suggesting that these solutes play a crucial role in the cold acclimation of K. flaccidum. Interestingly, the application of abscisic acid (ABA) did not change the freezing tolerance of the alga. We also observed changes in cell structure, including increased numbers and sizes of starch grains in chloroplasts, chloroplast enlargement, vacuole size reduction and cytoplasmic volume increase. These results suggest that K. flaccidum responds well to cold treatment and develops freezing tolerance in a process comparable to that of land plants.

Long- and short-term freezing induce different types of injury in Arabidopsis thaliana leaf cells

In nature, intact plant cells are subjected to freezing and can remain frozen for prolonged periods. We assayed the survival of Arabidopsis thaliana leaf cells following freezing and found that short- and long-term exposures produced different types of cellular injury. To identify the cause of these injuries, we examined the ultrastructure of the cell plasma membranes. Our results demonstrate that ultrastructural changes in the plasma membrane due to short-term freezing are associated with interbilayer events, including close apposition of the membranes. In both acclimated and non-acclimated leaf cells, these interbilayer events resulted in “fracture-jump lesions” in the plasma membrane. On the other hand, long-term freezing was associated with the development of extensive protein-free areas caused by the aggregation of intramembrane proteins with consequent vesiculation of the affected membrane regions; this effect was clearly different from the ultrastructural changes induced by interbilayer events. We also found that prolonged exposure of non-acclimated leaf cells to a concentrated electrolyte solution produced effects that were similar to those caused by long-term freezing, suggesting that the ultrastructural changes observed in the plasma membrane following long-term freezing are produced by exposure of the leaf cells to a concentrated electrolyte solution. This study illustrates multiple causes of freezing-induced injury in plant cells and may provide useful information regarding the functional role of the diverse changes that occur during cold acclimation.

Akinete Formation in Tribonema bombycinum Derbes et Solier (Xanthophyceae) in Relation to Freezing Tolerance

Tribonema bombycinum (Xanthophyceae), was examined. T. bombycinum shifted from vegetative cells to akinetes with starving by a prolonged batch culture, by culture with a diluted medium, or by culture with a single nutrient-deficient medium. In addition, akinetes developed by desiccation, but cold treatment at 4 C did not facilitate akinete formation. During starving, the vegetative cells, which had a large central vacuole in the protoplasm and thin cell walls, finally changed to akinetes, which had many small vacuoles and oil droplets in the protoplasm and thick cell walls. During akinete formation by starving, the freezing tolerance (LT50) increased gradually from −3 C in vegetative cells to far below −30 C in akinetes. When vegetative cells were subjected to equilibrium freezing, their size shrank greatly and aparticulate domains accompanied by fracture-jump lesions developed in the plasma membranes. Akinetes subjected to equilibrium freezing showed little shrinkage, and freezing-induced ultrastructural changes did not occur in the plasma membranes. The morphological changes in the process of akinete formation and the responses to equilibrium freezing resembled those of cold-acclimated terrestrial plants.

Sucrose phosphate phosphatase in the green alga Klebsormidium flaccidum (Streptophyta) lacks an extensive C-terminal domain and differs from that of land plants

Previously, it was reported that like land plants, the green alga Klebsormidium flaccidum (Streptophyta) accumulates sucrose during cold acclimation (Nagao et al. Plant Cell Environ 31:872–885, 2008), suggesting that synthesis of sucrose could enhance the freezing tolerance of this alga. Because sucrose phosphate phosphatase (SPP; EC 3.1.3.24) is a key enzyme in the sucrose synthesis pathway in plants, we analyzed the SPP gene in K. flaccidum (KfSPP, GenBank accession number AB669024) to clarify its role in sucrose accumulation. As determined from its deduced amino acid sequence, KfSPP contains the N-terminal domain that is characteristic of the L-2-haloacid-dehalogenase family of phosphatases/hydrolases (the HAD phosphatase domain). However, it lacks the extensive C-terminal domain found in SPPs of land plants. Database searches revealed that the SPPs in cyanobacteria also lack the C-terminal domain. In addition, the green alga Coccomyxa (Chlorophyta) and K. flaccidum, which are closely related to land plants, have cyanobacterial-type SPPs, while Chlorella (Chlorophyta) has a land plant-type SPP. These results demonstrate that even K. flaccidum (Streptophyta), as a recent ancestor of land plants, has the cyanobacterial-type SPP lacking the C-terminal domain. Because SPP and sucrose phosphate synthase (SPS) catalyze sequential reactions in sucrose synthesis in green plant cells and the lack of the C-terminal domain in KfSPP is predicted to decrease its activity, the interaction between decreased KfSPP activity and SPS activity may alter sucrose synthesis during cold acclimation in K. flaccidum.

Accumulation of Nitrate and Nitrite in Chilled Leaves of Rice Seedlings is Induced by High Root Temperature

We previously found a novel type of chilling injury in the leaves of rice seedlings (Oryza sativa L. cv. Akitakomachi). The damage was only observed when the roots were not chilled (10 °C/25 °C, shoots/roots), but not when the whole seedling was chilled (10 °C/10 °C). In this report, we show that the chilling injury induced by high root temperature required nitrate and potassium together with a trace amount of iron, manganese or both in the nutrient solution during the treatment, and that the injury was increased by nitrogen starvation before chilling. Both nitrate and nitrite accumulated in the 10 °C/25 °C leaves when the nutrient solution contained nitrate. The nitrate accumulation in the 10 °C/25 °C leaves was highest at the end of the first light period, and was followed by a decrease with a concomitant increase in nitrite during the first dark period. The photosynthetic electron transport was completely lost in both PSII and PSI in the 10 °C/25 °C leaves when the nutrient solution contained nitrate. However, the activities in the leaves of the 10 °C/25 °C plants treated with the nutrient solution lacking nitrate remained at approximately half those in the 10 °C/10°C leaves. The photochemical quenching of Chl fluorescence and the P700 oxidation state were also intermediate between those in the 10 °C/25 °C and 10 °C/10°C leaves of plants supplied with the complete nutrients. Thus, the chilling injury was closely linked to the accumulation of nitrate and nitrite, as well as to a malfunction of photosynthesis in the 10 °C/25 °C leaves.

Ultrastructure of chloroplasts in‘Marimo’(Cladophora aegagropila, Chlorophyta), and changes after exposure to light

This study compares the ultrastructure of the inner, dark‐habituated cells of the green ‘Cladophora‐ball’, or Marimo, to that of similar cells at the surface. Cells not exposed to light possess fewer, but larger and more irregular, chloroplasts than do the outer cells. Unexposed chloroplasts have a pyrenoid matrix lacking starch sheaths and containing unusually thick granal stacks. Despite prolonged exposure to darkness, the chloroplasts contain small starch grains. After exposure to light, such chloroplasts divide, become smaller and take on the appearance of those in the outer layer cells. Within 48 h, all of the chloroplasts develop substantial starch grains and the pyrenoids are surrounded by starch sheaths. This response is consistent with previous reports of the recovery of photosynthetic activity in inner cells exposed to light.