Glycine betaine improves Listeria monocytogenes tolerance to desiccation on parsley leaves independent of the osmolyte transporters BetL, Gbu and OpuC

Aims: To investigate the effect of glycine betaine (GB) on the survival of Listeria monocytogenes on leaf surfaces under low relative humidity (RH). Methods and Results: The addition of GB (≥25 mmol l^?1) improved the survival of L. monocytogenes under low RH on parsley leaves, thus suggesting that GB can improve the tolerance of L. monocytogenes to desiccation. Ten times less GB was needed to improve L. monocytogenes survival under low RH on nonbiological surfaces compared with parsley leaves, suggesting that, on the leaf surface, L. monocytogenes may have to compete for the available GB with autochthonous bacteria and/or the plant itself. Wild type and mutants carrying deletions in the three GB uptake systems, BetL, Gbu and OpuC, behaved similarly with and without added GB on parsley leaves (P > 0·05). In addition, preaccumulation of GB, triggered by osmotic stress prior to inoculation, failed to improve survival under low RH compared with osmotic stress without GB accumulation. Conclusions: Exogenous GB had a protective effect on L. monocytogenes cells from desiccation during survival on parsley leaves. This effect was independent of intracellular GB accumulation by the known uptake systems. Significance and Impact of the Study: Presence of GB could improve the survival of L. monocytogenes to desiccation on leaf surfaces and nonbiological surfaces.     

Assessment of leaf micromorphology after full desiccation of resurrection plants

Resurrection plants are unique among higher plants because of their ability to withstand long periods of dehydration without damages. In this study, leaf epidermis and palisade mesophyll of three resurrection species, Haberlea rodopensis, Ramonda serbica and Ramonda myconi, grown under full desiccation and benign conditions, were analyzed by differential interference contrast microscopy. Detailed investigation of adaxial and abaxial leaf surfaces revealed species-specific differences in the size and number of epidermal cells and stomatal density. The applied full desiccation did not cause any significant deviations of these parameters from the controls. There were no changes in the size and number of mesophyll cells as well. Analysis of stomatal patterning displayed essentially hypostomatic leaves, having stomata mainly abaxially positioned. The most significant change detected in the leaves of dehydration-treated plants was the increased formation of adaxially positioned trichomes. This increase was very high in R. myconi, where the adaxial leaf surface was fully covered by trichomes. Despite the existence of small species-specific differences, the results showed uniform desiccation-related responses of the studied resurrection species. The quantified leaf epidermal and mesophyll features are discussed with respect to their possible contribution to the desiccation tolerance of resurrection species.     

Protective mechanism of desiccation tolerance in <Emphasis Type="Italic">Reaumuria soongorica</Emphasis>: Leaf abscission and sucrose accumulation in the stem

Reaumuria soongorica (Pall.) Maxim., a perennial semi-shrub, is widely found in semi-arid areas in northwestern China and can survive severe desiccation of its vegetative organs. In order to study the protective mechanism of desiccation tolerance in R. soongorica, diurnal patterns of net photosynthetic rate (Pn), water use efficiency (WUE) and chlorophyll fluorescence parameters of Photosystem II (PSII), and sugar content in the source leaf and stem were investigated in 6-year-old plants during progressive soil drought imposed by the cessation of watering. The results showed that R. soongorica was characterized by very low leaf water potential, high WUE, photosynthesis and high accumulation of sucrose in the stem and leaf abscission under desiccation. The maximum Pn increased at first and then declined during drought, but intrinsic WUE increased remarkably in the morning with increasing drought stress. The maximal photochemical efficiency of PSII (Fv/Fm) and the quantum efficiency of noncyclic electric transport of PSII(Φ_PSII) decreased significantly under water stress and exhibited an obvious phenomenon of photoinhibition at noon. Drought stressed plants maintained a higher capacity of dissipation of the excitation energy (measured as NPQ) with the increasing intensity of stress. Conditions of progressive drought promoted sucrose and starch accumulation in the stems but not in the leaves. However, when leaf water potential was less than −21.3 MPa, the plant leaves died and then abscised. But the stem photosynthesis remained and, afterward the plants entered the dormant state. Upon rewatering, the shoots reactivated and the plants developed new leaves. Therefore, R. soongorica has the ability to reduce water loss through leaf abscission and maintain the vigor of the stem cells to survive desiccation.     

Phospholipid Involvement in Frost Tolerance

Changes in frost tolerance and in phospholipid content were studied in the leaves of winter rape plants (Brassica napus L. var. oleifera L. cv. Górczański) grown under natural or artificially controlled conditions. Frost hardening was found to be a three-stage process. During the first stage, occurring at low but above freezing environmental temperatures, phospholipid changes do not seem to be directly related to the leaf frost tolerance. This stage of hardening is possibly related to a metabolic shift caused by the cessation of growth. The achievement of the second level of frost tolerance in the fully turgid leaves depends on the occurrence of sub-freezing temperature and is related to increase in phospholipid level. It was shown that freezing brought about phospholipid degradation which was reversible only in slightly injured leaves with a relatively high phospholipid content. The third stage of hardening is related to frost-induced dehydration of the cells and may overlap the second one.     

Protective mechanism of desiccation tolerance in Reaumuria soongorica: Leaf abscission and sucrose accumulation in the stem

Reaumuria soongorica (Pall.) Maxim., a perennial semi-shrub, is widely found in semi-arid areas in northwestern China and can survive severe desiccation of its vegetative organs. In order to study the protective mechanism of desiccation tolerance in R. soongorica, diurnal patterns of net photosynthetic rate (Pn), water use efficiency (WUE) and chlorophyll fluorescence parameters of Photosystem II (PSII), and sugar content in the source leaf and stem were investigated in 6-year-old plants during progressive soil drought imposed by the cessation of watering. The results showed that R. soongorica was characterized by very low leaf water potential, high WUE, photosynthesis and high accumulation of sucrose in the stem and leaf abscission under desiccation. The maximum Pn increased at first and then declined during drought, but intrinsic WUE increased remarkably in the morning with increasing drought stress. The maximal photochemical efficiency of PSII (Fv/Fm) and the quantum efficiency of noncyclic electric transport of PSII(Φ_PSII) decreased significantly under water stress and exhibited an obvious phenomenon of photoinhibition at noon. Drought stressed plants maintained a higher capacity of dissipation of the excitation energy (measured as NPQ) with the increasing intensity of stress. Conditions of progressive drought promoted sucrose and starch accumulation in the stems but not in the leaves. However, when leaf water potential was less than −21.3 MPa, the plant leaves died and then abscised. But the stem photosynthesis remained and, afterward the plants entered the dormant state. Upon rewatering, the shoots reactivated and the plants developed new leaves. Therefore, R. soongorica has the ability to reduce water loss through leaf abscission and maintain the vigor of the stem cells to survive desiccation. Supported by the Program of the Research of Vegetation Restoration in Arid Areas of Lanzhou (Grant No. 03-2-27) and the National Natural Science Foundation of China (Grant No. 30270243)     

Effect of Whole Plant and Local Heating on the ABA Content in Cucumber Seedling Leaves and Roots and on Their Heat Tolerance

The effects of heating at 38°C of whole cucumber (Cucumis sativus L.) seedlings or local heating of their shoots and roots on ABA content and heat tolerance of leaves and roots were investigated. During the initial period of the high-temperature treatment of whole seedlings, the ABA concentration in leaves and roots increased considerably. Local heating of the shoot or root resulted in an increase in the ABA concentration not only in the heated organ, but also in unheated seedling parts. A high-temperature treatment of the whole seedlings and the local treatment of shoots or roots caused an increase in the heat tolerance of leaf cells. The heat tolerance of root cells virtually did not change after heating of the whole seedlings or shoots, but decreased after heating of roots. The possible role of ABA in changing the heat tolerance of leaf and root cells by local heating of the seedling is discussed.     

Comparative studies on desiccation resistance in leaves ofDactylis glomerata L. andFestuca arundinacea Schreb

Festuca arundinacea Schreb. andDactylis glomerata L., which differ in their ecological requirements toward moisture conditions also express different levels of desiccation tolerance.F. arundinacea was less resistant to severe water stress, showing lower ability to avoid and to tolerate desiccation (according to Levitt’s terminology, 1972). Growth stage of the leaves had no influence on the level of water deficits developed under the field conditions. It is concluded that adaptation ofF. arundinacea to growth in well watered environments might be associated with a decrease of desiccation resistance of its tissue.     

Phytochrome B control of total leaf area and stomatal density affects drought tolerance in rice

We report that phytochrome B (phyB) mutants exhibit improved drought tolerance compared to wild type (WT) rice (Oryza sativa L. cv. Nipponbare). To understand the underlying mechanism by which phyB regulates drought tolerance, we analyzed root growth and water loss from the leaves of phyB mutants. The root system showed no significant difference between the phyB mutants and WT, suggesting that improved drought tolerance has little relation to root growth. However, phyB mutants exhibited reduced total leaf area per plant, which was probably due to a reduction in the total number of cells per leaf caused by enhanced expression of Orysa;KRP1 and Orysa;KRP4 (encoding inhibitors of cyclin-dependent kinase complex activity) in the phyB mutants. In addition, the developed leaves of phyB mutants displayed larger epidermal cells than WT leaves, resulting in reduced stomatal density. phyB deficiency promoted the expression of both putative ERECTA family genes and EXPANSIN family genes involved in cell expansion in leaves, thus causing greater epidermal cell expansion in the phyB mutants. Reduced stomatal density resulted in reduced transpiration per unit leaf area in the phyB mutants. Considering all these findings, we propose that phyB deficiency causes both reduced total leaf area and reduced transpiration per unit leaf area, which explains the reduced water loss and improved drought tolerance of phyB mutants.     

NaCl胁迫对宁夏枸杞叶和幼根显微及超微结构的影响

以宁夏枸杞为材料,采用超薄切片技术制备样品,应用光学显微镜和透射电镜分析了不同浓度NaCl胁迫条件下宁夏枸杞叶和幼根显微及超微结构的变化。结果表明：随着NaCl胁迫的加重,(1)叶片上表皮细胞增厚,栅栏组织细胞出现缩短现象,排列疏松且紊乱;幼根的初生结构无明显变化。(2)叶片栅栏组织中叶绿体不再紧靠在细胞膜上,叶绿体双层膜破坏,基粒片层松散排列,杂乱无章,出现膨胀和空泡现象,淀粉粒和嗜锇颗粒增多,叶肉细胞中线粒体发生轻微变化;幼根中皮层薄壁细胞线粒体形状发生改变,结构破坏,内膜和外膜模糊甚至破裂,大多数嵴模糊,出现空泡现象;细胞核解体,基质外溢。研究表明, 不同浓度的NaCl胁迫对宁夏枸杞叶片和幼根细胞的显微及超微结构影响不同,NaCl浓度大于200 mmol/L时,宁夏枸杞叶片和幼根细胞的显微及超微结构发生了明显变化,且叶肉细胞中线粒体的变化没有叶绿体的变化显著,推测叶肉细胞中线粒体的耐盐性比叶绿体强。     

The Response of Transpiration Resistance to Leaf Temperature as a Desiccation Resistance Mechanism in Tree Seedlings

Transpiration rate and leaf transfer resistance to water vapor loss were determined under a range of leaf temperatures for Quercus macrocarpa, Q. velutina, Q. alba, Q. rubra, and Acer saccharum. Transfer resistance increased with rising leaf temperatures between 20 and 40°C in all species, but the rate of increase in resistance was greatest in species which normally occupy xeric sites. Increased transfer resistance with rising leaf temperature may be significant in preventing rapid desiccation of leaves under the large evaporative stress imposed by high leaf temperature.     

Do desiccation tolerances control the vertical distribution of intertidal seagrasses?

Photosynthetic processes in Zostera japonica, an upper intertidal species, were found to be more severely affected by desiccation than Z. marina, a lower intertidal and subtidal species, at comparable levels of tissue water content. The data indicate that photosynthetic responses to desiccation at the level of the individual leaf are insufficient to explain observed patterns of intertidal seagrass zonation. Desiccation tolerance in seagrasses is more likely to involve a complex interaction of morphological traits and growth strategies at the level of the whole plant, such as downsizing (e.g. smaller, narrower leaves), reduced structural rigidity and increased rates of leaf abscission and leaf turnover.     

Glycerolipid analysis during desiccation and recovery of the resurrection plant Xerophyta humilis (Bak) Dur and Schinz

Xerophyta humilis is a poikilochlorophyllous monocot resurrection plant used as a model to study vegetative desiccation tolerance. Dehydration imposes tension and ultimate loss of integrity of membranes in desiccation sensitive species. We investigated the predominant molecular species of glycerolipids present in root and leaf tissues, using multiple reaction monitoring mass spectrometry, and then analysed changes therein during dehydration and subsequent rehydration of whole plants. The presence of fatty acids with long carbon chains and with odd numbers of carbons were detected and confirmed by gas chromatography. Dehydration of both leaves and roots resulted in an increase in species containing polyunsaturated fatty acids and a decrease in disaturated species. Upon rehydration, lipid saturation was reversed, with this being initiated immediately upon watering in roots but only 12–24 hr later in leaves. Relative levels of species with short‐chained odd‐numbered saturated fatty acids decreased during dehydration and increased during rehydration, whereas the reverse trend was observed for long‐chained fatty acids. X. humilis has a unique lipid composition, this report being one of the few to demonstrate the presence of odd‐numbered fatty acids in plant phosphoglycerolipids.     

Cold tolerance,water relations and accumulation of osmolytes in <Emphasis Type="Italic">Bituminaria bituminosa</Emphasis>

We determined the cold (freezing) tolerance of five Spanish populations of the perennial shrub Bituminaria bituminosa (L.) C.H. Stirton (Fabaceae), as the temperature at which 50 % of leaf electrolytes are released (LT₅₀) using leaves of field-grown plants, obtained in two winters and one spring. The freezing tolerance was greater in winter and reflected the minimum temperatures at the original sites from which the populations were obtained. Tolerance in vitro was related to osmotic adjustment in the leaves; more negative osmotic potential values and more positive pressure potential values (MPa) were associated with greater tolerance. Tolerance and osmotic potential were not related to leaf cation contents but to leaf amino acids, soluble sugar and proline contents.     

Membrane phospholipid composition during maturation of seeds of Acer platanoides and Acer pseudoplatanus in relation to desiccation tolerance

Membrane phospholipid composition was investigated in seeds of two species from the genus Acer: Norway maple (Acer platanoides L.) — tolerant to desiccation, and sycamore (Acer pseudoplatanus L.) — intolerant to desiccation, during their maturation, from 1 August to 25 September 1995, at weekly intervals. Seeds of Norway maple acquire tolerance to desiccation at the end of August ie. about 125 days after flowering (DAF). Phospholipid composition during development revealed marked differences between studied seeds. Seeds of Norway maple after acquiring tolerance to desiccation contained much more phosphatidylcholine (PC) and phosphatidylethanolamine (PE), compared to sycamore. The ratio of PC/PE in mature Norway maple seeds was evidently higher than those in sycamore. The level of unsaturated fatty acids in the phospholipid fraction substantially increased in Norway maple seeds during development and the saturation of PC and PE was less than in sycamore. The results suggest that phospholipid composition may be involved in desiccation tolerance of Norway maple seeds.     

Response of sun- and shade-adapted plants of Haberlea rhodopensis to desiccation

The differences in some morphological and physiological characteristics of sun- and shade-adapted Haberlea rhodopensis plants were compared. Changes in the photosynthetic activity, electrolyte leakage from leaf tissues, malondialdehyde content (MDA) and leaf anatomy were studied at different degrees of desiccation as well as after rehydration of plants. The MDA content in well-watered sun Haberlea plants was higher compared to shade plants suggesting higher lipid peroxidation, which is commonly regarded as an indicator of oxidative stress, but desiccation of plants at high light did not cause additional oxidative damage as judged by the unaffected MDA content. The electrolyte leakage from dried leaves (8% RWC) from both shade and sun plants increased fourfold indicating similar membrane damage. However, the recovery after rehydration showed that this damage was reversible. Well-watered sun plants had higher photosynthetic activity probably due to the larger thickness of the mesophyll layer in such plants. On the other hand, desiccation at high light reduced CO₂ assimilation which was in accordance with the stronger reduction of stomatal conductance. Stomata were visible only on the abaxial side of sun leaves having also higher abundance of non-glandular trichomes. Increased trichomes density and epicuticular waxes and filaments upon desiccation could help plants to increase reflection, reduce net radiation income, slow down the rate of water loss and survive adverse conditions.     

Determination of leaf heat resistance: comparative investigation of chlorophyll fluorescence changes and tissue necrosis methods

Summary Heat tolerance limits for a variety of vascular plant leaves were determined both with the conventional post-culture necrosis method and by measurements of the heat-induced increase in chlorophyll fluorescence (F-T curves). The reliability of the fluorescence test was improved with the addition of far-red background light which counteracts dark reduction of the Photosystem II acceptor pool by heat-stimulated endogenous electron donors. This was of particular importance in the case of xeromorphic leaves in which the diffusion barrier for oxygen is high. A satisfactory correlation was found between T _L50, the temperature at which a 30 min exposure results in 50% necrotic leaf area following post culture, and the critical temperature, T _c ,the temperature at which the dark fluorescence level begins to increase during slow heating of a leaf sample at a rate of 0.7 K min^-1, in the fluorescence test. The correlation can be described by a linear function, T _L50=1.12 T _c -5.37,with a correlation coefficient, r=0.87. Maximal deviation of the regression line from the line T _L50=T _c was 1.2 K, with 22 determinations for leaves with widely varying heat tolerance limits. This shows that heat-induced fluorescence changes within the thylakoid membrane may be connected with the irreversible leaf tissue damage which occurs following prolonged exposure to high temperature. On the basis of the heat dosage equation of Lepeschkin, a more general expression can be obtained which allows calculation of the accumulated heat dosage under the experimental conditions of the standard fluorescence test (slow heating, 0.7 K min^-1). Such calculations reveal that for a given species the fraction of critical dosage begins to increase, i.e. accumulating heat reaches an injurious level, at a temperature which approximately coincides both with T _L50, obtained with the necrosis method, and with T _c ,the critical temperature derived from the fluorescence test. Hence, the increase in fraction of critical dosage and the rise in chlorophyll fluorescence seem to concur. It is concluded that the fluorescence assay provides a rapid and reliable means of determining the heat tolerance limit of leaf tissue.     

The Effects of Root Treatment with Various Stress Agents on Plant Cold- and Heat-Tolerance

The root systems of cucumber (Cucumis sativus L.), wheat (Triticum aestivum L.), and barley (Hordeum vulgare L.) plants were subjected to a short-term (6–7-h-long) treatment with low and high temperatures, sodium chloride, and lead nitrate, and the effect of these treatments on the changes in the cold- and heat-tolerance of leaf cells was determined. It was established that chilling of cucumber and wheat seedling roots at 10 and 2°C, respectively, or their heating at 38°C and 40°C, respectively, induced an increase in both cold- and heat-tolerance of leaf cells. An increase in the cold- and heat-tolerance was also observed in roots treated with sodium chloride at concentrations of 0.15 M (cucumber) and 0.2 M (wheat), as well as with lead nitrate at a concentration of 0.1 mM (barley, wheat). The tolerance increase induced by these stress agents was accompanied by a considerable increase in the ABA concentration in leaves. The effect of physical and chemical stress agents is suggested to induce the same nonspecific changes in the aboveground organs. These changes bring about, directly or indirectly, an increase in the cold- and heat-tolerance and are related to an increase in the ABA content.     

Ecophysiological responses of salt cedar (Tamarix spp. L.) to the northern tamarisk beetle (Diorhabdacarinulata Desbrochers) in a controlled environment

The northern tamarisk beetle (Diorhabda carinulata Desbrochers) was released in several western states as a biocontrol agent to suppress Tamarix spp. L. which has invaded riparian ecosystems; however, effects of beetle herbivory on Tamarix physiology are largely undocumented and may have ecosystem ramifications. Herbivory by this insect produces discoloration of leaves and premature leaf drop in these ecosystems, yet the cause of premature leaf drop and the effects of this leaf drop are still unknown. Insect herbivory may change leaf photosynthesis and respiration and may affect a plant’s ability to regulate water loss and increase water stress. Premature leaf drop may affect plant tissue chemistry and belowground carbon allocation. We conducted a greenhouse experiment to understand how Tamarix responds physiologically to adult beetle and larvae herbivory and to determine the proximate cause of premature leaf drop. We hypothesized that plants experiencing beetle herbivory would have greater leaf and root respiration rates, greater photosynthesis, increased water stress, inefficient leaf nitrogen retranslocation, lower root biomass and lower total non-structural carbohydrates in roots. Insect herbivory reduced photosynthesis rates, minimally affected respiration rates, but significantly increased water loss during daytime and nighttime hours and this produced increased water stress. The proximate cause for premature leaf drop appears to be desiccation. Plants exposed to herbivory were inefficient in their retranslocation of nitrogen before premature leaf drop. Root biomass showed a decreasing trend in plants subjected to herbivory. Stress induced by herbivory may render these trees less competitive in future growing seasons.     

The isolation of genes from the resurrection grass Sporobolus stapfianus which are induced during severe drought stress

A modification of the ‘cold plaque’ screening technique (Hodge et al., Plant Journal1992, 2, 257–260) was used to screen a cDNA library constructed from drought‐stressed leaf tissue of the desiccation tolerant (‘resurrection’) grass Sporobolus stapfianus. This technique allowed a large number of clones representing genes expressed at low abundance to be isolated. An examination of expression profiles revealed that several of these genes are induced in desiccation‐tolerant tissue experiencing severe drought stress. Further characterization indicated that the gene products encoded include an eIF1 protein translation initiation factor and a glycine‐ and proline‐rich protein which have not previously been associated with drought stress. In addition, genes encoding a serine/threonine phosphatase type 2C, a tonoplast‐intrinsic protein (TIP) and an early light‐inducible protein (ELIP) were isolated. A number of these genes are expressed differentially in desiccation‐tolerant and desiccation‐sensitive tissues, suggesting that they may be associated with the desiccation tolerance response of S. stapfianus. The results indicate that there may be unique gene regulation processes occurring during induction of desiccation tolerance in resurrection plants which allow different drought‐responsive genes to be selectively expressed at successive levels of water loss.