The management of extracellular ice by petioles of frost-resistant herbaceous plants

BACKGROUND AND AIMS: Some frost-tolerant herbaceous plants droop and wilt during frost events and recover turgor and posture on thawing. It has long been known that when plant tissues freeze, extracellular ice forms. Distributions of ice and water in frost-frozen and recovered petioles of Trifolium repens and Escholschzia californica were visualized. METHODS: Petioles of intact plants were cryo-fixed, planed to smooth transverse faces, and examined in a cryo-SEM. KEY RESULTS: With frost-freezing, parenchyma tissues shrank to approx. one-third of their natural volume with marked cytorrhysis of the cells, and massive blocks of extracellular icicles grew under the epidermis (poppy) or epidermis and subepidermis (clover), leaving these layers intact but widely separated from the parenchyma except at specially structured anchorages overlying vascular bundles. On thawing, the extracellular ice was reabsorbed by the expanding parenchyma, and surface tissues again contacted the internal tissues at weak junctions (termed faults). These movements of water into and from the fault zones occurred repeatedly at each frost/thaw event, and are interpreted to explain the turgor changes that led to wilting and recovery. Ice accumulations at tri-cellular junctions with intercellular spaces distended these spaces into large cylinders, especially large in clover. Xylem vessels of frozen petioles were nearly all free of gas; in thawed petioles up to 20 % of vessels were gas-filled. CONCLUSIONS: The occurrence of faults and anchorages may be expected to be widespread in frost-tolerant herbaceous plants, as a strategy accommodating extracellular ice deposits which prevent intracellular freezing and consequent membrane disruption, as well as preventing gross structural damage to the organs. The developmental processes that lead to this differentiation of separation of sheets of cells firmly cemented at determined regions at their edges, and their physiological consequences, will repay detailed investigation.     

Root growth of subalpine and montane Eucalyptus seedlings at low soil temperatures

 This study examines the effect of different soil temperatures on root growth in seedlings of Eucalyptus pauciflora Sieber ex Sprengel subsp. pauciflora and Eucalyptus nitens (Deane & Maiden) Maiden. Seedlings were grown in a glasshouse in pots containing soil. Pots were held in water baths maintained at 3, 7 or 13°C, whilst shoots were exposed to ambient glasshouse temperatures. The experiments were designed to separate direct effects of soil temperature from effects due to differences in seedling size. In the first experiment, seedlings were grown to constant height (25 cm for both species), in the second to constant time (100 days for E. pauciflora and 64 days for E. nitens) and in the third experiment seedlings were transferred between soil temperatures. The rate of growth of both species increased with increasing soil temperature. E. nitens grew faster than E. pauciflora at 7 and 13°C, but E. pauciflora grew faster than E. nitens at 3°C. The rate of browning of roots increased with decreasing soil temperature and at a faster rate in E. nitens than E. pauciflora. Root length was highly correlated to root mass within diameter and colour classes (r² > 0.7). However, brown roots were heavier than white roots. Consequently, changes in root mass did not reflect changes in root length when the proportion of brown to white root also changed. For example, at a constant height of 25 cm at 3°C, E. nitens had greater root mass but lesser root length than E. pauciflora. E. pauciflora at 3°C grew faster, and had more root length and less brown roots than E. nitens. This supports the argument that E. pauciflora is better adapted than E. nitens to survive and grow at lower soil temperatures. Received: 16 December 1996 / Accepted: 2 April 1997     

毛尖紫萼藓(Grimmia pilifera P.Beauv) PSII光化学效率对脱水和复水的响应

利用快速叶绿素荧光动力学技术研究了毛尖紫萼藓脱水和复水过程中叶绿素荧光变化,结果显示在脱水过程中毛尖紫萼藓PSⅡ的最大光化学效率（Fv/Fm）、光合机构电子传递的量子产额（ETo／ABS）、捕获的激子将电子传递到电子传递链中超过QA的其它电子受体的概率（ETo／TRo）、单位叶面积的反应中心的数量（RC／CSo）以及PSⅡ受体库（Area）对叶片含水量的响应等均存在相对含水量阈值。在阈值范围内脱水,对以上荧光参数影响不大,低于阈值后,各荧光参数值迅速下降,直至PSⅡ反应中心完全关闭以及光化学过程结束。再复水后,毛尖紫萼藓光合机构的最大捕光效率、实际光化学效率、PSⅡ反应中心受体侧的电子传递链以及反应中心均能得到快速而有效的恢复。表明一定时间内脱水不会对毛尖紫萼藓的光合器官造成严重伤害,光合系统仍维持在可恢复状态。     

Higher daytime leaf temperatures contribute to lower freeze tolerance under elevated CO2

Elevated atmospheric CO2 adversely affects freezing tolerance in many evergreens, but the underlying mechanism(s) have been elusive. We compared effects of elevated CO2 with those of daytime warming on acclimation of snow gum (Eucalyptus pauciflora) to freezing temperatures under field conditions. Reduction in stomatal conductance g(c) under elevated CO2 was shown to cause leaf temperature to increase by up to 3 degrees C. In this study, this increase in leaf temperature was simulated under ambient CO2 conditions by using a free air temperature increase (FATI) system to warm snow gum leaves during daytime, thereby increasing the diurnal range in temperature without affecting temperature minima. Acclimation to freezing temperatures was assessed using measures of electrolyte leakage and photosynthetic efficiency of leaf discs exposed to different nadir temperatures. Here, we show that both elevated CO2 and daytime warming delayed acclimation to freezing temperatures for 2-3 weeks after which time freeze tolerance of the treated plants in both the FATI and open top chamber (OTC) experiments did not differ from control plants. Our results support the hypothesis that delayed development of freezing tolerance under elevated CO2 is because of higher daytime leaf temperatures under elevated CO2. Thus, potential gains in productivity in response to increasing atmospheric CO2 and prolonging the growing season may be reduced by an increase in freezing stress in frost-prone area.     

Proteins accumulate in the apoplast of winter rye leaves during cold acclimation

During cold acclimation, winter rye ( Secale cereale L.) plants develop the ability to tolerate freezing temperatures by forming ice in intercellular spaces and xylem vessels. In this study, proteins were extracted from the apoplast of rye leaves to determine their role in controlling extracellular ice formation. Several polypeptides in the 15 to 32 kDa range accumulated in the leaf apoplast during cold acclimation at 5°C and decreased during deacclimation at 20°C. A second group of polypeptides (63, 65 and 68 kDa) appeared only when the leaves were maximally frost tolerant. Ice nucleation activity, as well as the previously reported antifreeze activity, was higher in apoplastic extracts from cold-acclimated than from nonacclimated rye leaves. These results indicate that apoplastic proteins exert a direct influence on the growth of ice. In addition, freezing injury was greater in extracted cold-acclimated leaves than in unextracted cold-acclimated leaves, which suggests that the proteins present in the apoplast are an important component of the mechanism by which winter rye leaves tolerate ice formation     

The behaviour of scribbly gum moth larvae Ogmograptis sp. Meyrick (Lepidoptera: Bucculatricidae) in the Australian Capital Territory

Abstract  Scribbles, the damage caused by mining larvae of the moth Ogmograptis sp. (Bucculatricidae), are a common sight on the trunks of many Eucalyptus species in eastern Australia. They provide a record of larval behaviour that can be easily measured when the trees shed outer bark layers. Measurements were made of scribbles on three species of eucalypt ( Eucalyptus racemosa ssp. rossii , E. pauciflora and E. delegatensis ) in the Australian Capital Territory. Scribble density varied between tree species and study sites but was consistently higher on the southern and eastern aspects of tree trunks. Some characteristics of mining behaviour, such as initial mining direction, were found to be random, but others including mine length and number of direction changes follow distinct patterns but differ between eucalypt species. It is likely that there is more than one species of Ogmograptis Meyrick as currently described ( O. scribula Meyrick, from E. pauciflora ) and each eucalypt species may be host to a different species of Ogmograptis .     

Elevated atmospheric [CO2] promotes frost damage in evergreen tree seedlings

Growth under elevated [CO₂] promoted spring frost damage in field grown seedlings of snow gum ( Eucalyptus pauciflora Sieb. ex Spreng.), one of the most frost tolerant of eucalypts. Freezing began in the leaf midvein, consistent with it being a major site of frost damage under field conditions. The average ice nucleation temperature was higher in leaves grown under elevated [CO₂] (– 5·7 °C versus – 4·3 °C), consistent with the greater incidence of frost damage in these leaves (34% versus 68% of leaves damaged). These results have major implications for agriculture, forestry and vegetation dynamics, as an increase in frost susceptibility may reduce potential gains in productivity from CO₂ fertilization and may affect predictions of vegetation change based on increasing temperature.     

Differential location of α-expansin proteins during the accommodation of root cells to an arbuscular mycorrhizal fungus

-Expansins are extracellular proteins that increase plant cell-wall extensibility. We analysed their pattern of expression in cucumber roots in the presence and in the absence of the mycorrhizal fungus, Glomus versiforme. The distribution of -expansins was investigated by use of two polyclonal antibodies (anti-EXPA1 and anti-EXPA2, prepared against two different cucumber -expansins) in immunoblotting, immunofluorescence, and immunogold experiments. Immunoblot results indicate the presence of a 30-kDa band specific for mycorrhizal roots. The two antibodies identify antigens with a different distribution in mycorrhizal roots: anti-EXPA1 labels the interface zone, but the plant cell walls only weakly. By contrast, the anti-EXPA2 labels only the plant cell walls. In order to understand the potential role of -expansins during the accommodation of the fungus inside root cells, we prepared semi-thin sections to measure the size of cortical cells and the thickness of cortical cell walls in mycorrhizal and non-mycorrhizal root. Mycorrhizal cortical cells were significantly larger than non-mycorrhizal cells and had thicker cell walls. In double-labelling experiments with cellobiohydrolase–gold complex, we observed that cellulose was co-localized with -expansins. Taken together, the results demonstrate that -expansins are more abundant in the cucumber cell walls upon mycorrhizal infection; we propose that these wall-loosening proteins are directly involved in the accommodation of the fungus by infected cortical cells.     

Periplast development in cryptophyceae I. Changes in periplast arrangement throughout the cell cycle

Summary The cell covering (or periplast) of many cryptomonads consists of discrete plate areas precisely arranged over most of the cell periphery. Developmental changes in periplast arrangement that occur throughout the cell cycle are examined here forKomma caudata andProteomonas sulcata [haplomorph]. In both cryptomonads, pole reversal occurs during cytokinesis, necessitating major realignment of the plate areas. Growth of the periplast occurs by addition of new plate areas to specialized regions (termed anamorphic zones) located around the vestibular margins and along the mid-ventral line of cells. Development of the periplast from these regions enables elongation and lateral expansion of cryptomonads throughout cell growth. Observed differences in cell division and periplast development between these genera are closely associated with variations in the arrangement of anamorphic zones.     

A microscale model for prediction of breast cancer cell damage during cryosurgery

The morphology of cancerous breast tissue is characterized by tightly packed groups of small malignant cells, as found in most duct cell carcinoma. This special structure affects the osmotic responses of the cells to freezing and hence their probability of damage from cellular dehydration or intracellular ice formation. A mathematical model has been developed to study the microscale damage to these breast cancer cells during cryosurgery by accounting for their special structure. The model is based on a spherical unit comprised of an extracellular region that surrounds several layers of cancer cells, as experimentally observed of breast duct cell carcinoma by other researchers. Temperature transients in the breast cancer undergoing cryosurgery are calculated numerically using the Pennes equation. When subjected to various thermal histories, both cellular dehydration and intracellular ice formation in the unit structure are examined by considering the cell-to-cell contact and water transport at the microscale level. It is found that the cells in the inner layers hardly dehydrated while those in the outermost layer do greatly. The results help interpret the previously observed experimental phenomena that breast cancer tissues exhibit intracellular ice formation even at a slow cooling rate of -3 degrees C/min. In the attempt to better define an optimal procedure for breast cancer cryosurgery, various freezing protocols are simulated. The constant heat flux protocol induces greater cellular dehydration and higher intracellular ice formation probability simultaneously compared to the other protocols studied.     

Pectin and the role of the physical properties of the cell wall in pollen tube growth of<Emphasis Type="Italic"> Solanum chacoense</Emphasis>

The cell wall is one of the structural key players regulating pollen tube growth, since plant cell expansion depends on an interplay between intracellular driving forces and the controlled yielding of the cell wall. Pectin is the main cell wall component at the growing pollen tube apex. We therefore assessed its role in pollen tube growth and cytomechanics using the enzymes pectinase and pectin methyl esterase (PME). Pectinase activity was able to stimulate pollen germination and tube growth at moderate concentrations whereas higher concentrations caused apical swelling or bursting in Solanum chacoense Bitt. pollen tubes. This is consistent with a modification of the physical properties of the cell wall affecting its extensibility and thus the growth rate, as well as its capacity to withstand turgor. To prove that the enzyme-induced effects were due to the altered cell wall mechanics, we subjected pollen tubes to micro-indentation experiments. We observed that cellular stiffness was reduced and visco-elasticity increased in the presence of pectinase. These are the first mechanical data that confirm the influence of the amount of pectins in the pollen tube cell wall on the physical parameters characterizing overall cellular architecture. Cytomechanical data were also obtained to analyze the role of the degree of pectin methyl-esterification, which is known to exhibit a gradient along the pollen tube axis. This feature has frequently been suggested to result in a gradient of the physical properties characterizing the cell wall and our data provide, for the first time, mechanical support for this concept. The gradient in cell wall composition from apical esterified to distal de-esterified pectins seems to be correlated with an increase in the degree of cell wall rigidity and a decrease of visco-elasticity. Our mechanical approach provides new insights concerning the mechanics of pollen tube growth and the architecture of living plant cells.     

Feruloylated Arabinoxylans Are Oxidatively Cross- Linked by Extracellular Maize Peroxidase but Not by Horseradish Peroxidase

Covalent cross-linking of soluble extracellular arabinoxylans in living maize cultures, which models the cross- linking of wall-bound arabinoxylans, is due to oxidation of feruloyl esters to oligoferuloyl esters and ethers. The oxidizing system responsible could be H2O2/peroxidase, O2/laccase, or reactive oxygen species acting non-enzymically, To distinguish these possibilities, we studied arabinoxylan cross-linking in vivo and in vitro. In living cultures, exogenous, soluble, extracellular, feruloylated [pentosyl-3H]arabinoxylans underwent cross-linking, beginning abruptly 8 d after sub-culture. Crosslinking was suppressed by iodide, an H2O2 scavenger, indicating dependence on endogenous H2O2. However, exogenous H2O2 did not cause precocious cross-linking, despite the constant presence of endogenous peroxidases, suggesting that younger cultures contained natural cross-linking inhibitors. Dialysed culture-filtrates cross-linked [^3H]arabinoxylans in vitro only if H2O2 was also added, indicating a peroxidase requirement. This cross-linking was highly ionic-strength-dependent. The peroxidases responsible were heat-labile, although relatively heat-stable peroxidases （assayed on o-dianisidine） were also present. Surprisingly, added horseradish peroxidase, even after heat-denaturation, blocked the arabinoxylancross-linking action of maize peroxidases, suggesting that the horseradish protein was a competing substrate for [^3H]arabinoxylan coupling. In conclusion, we show for the first time that cross-linking of extracellular arabinoxylan in living maize cultures is an action of apoplastic peroxidases, some of whose unusual properties we report.     

Effects of ethylene on the orientation of microtubules and cellulose microfibrils of pea epicotyl cells with polylamellate cell walls

Summary Following a 5 hours ethylene treatment, cortical cells of Pea (Pisum sativum L. var Alaska) epicotyl third internode showed a change in the orientation of both microtubules near the plasma membrane and recently deposited cellulose microfibrils. Control cortical cells had mostly transverse microtubules. The ratio of the average frequency of transverse to longitudinal microtubules was 6.0. After 5 hours of ethylene treatment, cortical cells had mostly longitudinal microtubules, with the ratio of transverse to longitudinal microtubules equal to 0.1. Epidermal cells were more variable than cortical cells with regard to the frequency of longitudinal and transverse microtubules. Observation of cortical cell walls in conventionally stained thin sections revealed that recent deposition of microfibrils had been primarily transverse in almost all of the control cortical cells sampled. In contrast, more than half of the ethylene-treated cortical cells had recent deposition oriented primarily longitudinally. This change in microtubule and microfibril orientation may be early enough to constitute the primary effect of ethylene leading to radial cell expansion.Research supported by NSF grant PCM 78-03244, A1, 2 to PBG and by a Research Corporation grant to WRE.     

快速脱水与复水过程中2种藓类植物的活性氧代谢与脂质过氧化特征

以来自不同水分生境的金发藓和湿地匐灯藓为材料,对二者在脱水与复水胁迫条件下的活性氧代谢、脂质过氧化损伤程度及其抗氧化系统应答的差异进行比较研究。结果显示:在脱水与复水过程中,(1)硅胶快速脱水更接近阳光直射条件下藓类植物的水分丧失。(2)随着含水量的变化,湿地匐灯藓虽然能够在复水后迅速修复细胞的完整性,但变化剧烈;金发藓则能够始终维持较低的膜透性。(3)2种藓类植物的丙二醛(MDA)含量变化均呈先升后降趋势,但金发藓的MDA含量明显低于湿地匐灯藓。(4)2种藓类植物的超氧阴离子自由基(O2.-)产生速率和过氧化氢含量(H2O2)的变化均与MDA含量变化相似,且金发藓活性氧水平明显高于湿地匐灯藓。(5)2种藓类植物的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性受活性氧诱导亦呈先升后降的趋势,但金发藓抗氧化酶对活性氧迸发的应答更快,活性更强。(6)2种藓类植物的抗坏血酸(AsA)含量呈先降后升态势,但金发藓的含量低于湿地匐灯藓。研究表明,来自不同生境的2种藓类植物对脱水胁迫所致的氧化胁迫均具有很强的适应能力,尤其是复水过程中的修复能力,但不同藓类可能通过不同途径和机制来适应脱水所致的氧化胁迫;来自易发生水分亏缺生境的金发藓可能因具有更强抗氧化能力,从而获得比来自水分充沛生境的湿地匐灯藓更高的脱水耐性。     

Cold-induced photoinhibition and growth of seedling snow gum (Eucalyptus pauciflora) under differing temperature and radiation regimes in fragmented forests

Fluorescence characteristics and growth of seedling snow gum ( Eucalyptus pauciflora Sieb. ex Spreng.) during autumn and winter were related to variation in radiation and temperature regime in a fragmented forest. Seedlings were planted in four treatments along transects perpendicular to tree island edges to characterize plant responses to microclimates ranging from those of cleared areas to those beneath forest canopies. Three-dimensional mapping of seedling leaf display, in combination with information retrieved from hemispherical photographs about shading from overstory canopies, were used to calculate the intercepted amounts of direct radiation energy for unit area of leaves on clear days (IDRE) . IDRE was highest on the outside, most variable at the edges and lowest well inside the tree islands. Minimum temperature decreased with increasing view of the sky. Photoinhibition, measured as decrease in F _v/ F _m, was correlated with spatial and seasonal differences in weekly minimum temperature and IDRE . Seedlings in the open and under the most canopy cover, with low variability in IDRE in a scale of weeks, exhibited less variability in photoinhibition than those growing along forest edges. Seedlings in the open tended to be most photoinhibited and grew the most. The combination of increased IDRE with reduced minimum temperatures resulted in persistent and strong photoinhibition as the season progressed. Results are discussed in relation to the potential for seedling establishment following forest fragmentation.     

Mesenchymal stem cells: From bench to bedside

Human mesenchymal stem cells (hMSCs) have tremendous promise for use in a variety of clinical applications. The ability of these cells to self-renew and differentiate into multiple tissues makes them an attractive cell source for a new generation of cell-based regenerative therapies. Encouraging results from clinical trials have also generated growing enthusiasm regarding MSC therapy and related treatment, but gaps remain in understanding MSC tissue repair mechanisms and in clinical strategies for efficient cell delivery and consistent therapeutic outcomes. For these reasons, discoveries from basic research and their implementation in clinical trials are essential to advance MSC therapy from the laboratory bench to the patient's bedside.