Microclimate and ecophysiological significance of the tree-like life-form of Lobelia rhynchopetalum in a tropical alpine environment

The microclimate and the adaptive significance of the tree-like plant life-form for growth in a tropical alpine environment was investigated with the pachycaul arborescent giant rosette plant, Lobelia rhynchopetalum (Hochst. A. Rich.) Hemsl. in the Bale and Simen Mountains, Ethiopia. The microclimate of plants of three height classes was examined with respect to temperature, relative humidity and the effect of wind. Although the total heat gains were rather similar, leaves of young, still stemless (acaulescent) individuals of Lobelia were subjected to a high diurnal temperature fluctuation of up to 29 K compared to a 14-K fluctuation for the leaves of an individual 3.5 m in height. During the cold nights, temperatures of the inner rosette leaves and inside leaf buds of caulescent plants were 4–5 K above air temperature, while corresponding temperatures of acaulescent individuals were 1–2 K below air temperature. The inner temperature of the stem tissue was higher than the surface temperature of the stem by about 5 K for most of the cold night. The annual rates of increment in whole plant, stem and rosette height, and stem diameter of L. rhynchopetalum showed that the young, still acaulescent individuals, with an annual increment of 5.6 cm in plant height, had the lowest growth rate, compared to 12.1 and 22.1 cm for caulescent life-forms. The results show that the most important advantage gained by the tree-like life-form of adult L. rhynchopetalum is probably a more favourable microclimate in which the strong diurnal temperature fluctuations at the ground are mitigated and nocturnal temperatures do not drop below freezing point. Received: 16 August 1996 / Accepted: 9 September 1997     

Biology of afroalpineDendrosenecio (Asteraceae)

The genusDendrosenecio (giant groundsels), encompassing three species and 12 subspecies, is endemic to the high mountains of East and Central Africa where it constitutes the most conspicuous components of the afroalpine vegetation. Two lifeforms, the arborescent and the prostrate rhizomatous, are regarded as the results of evolution from forest-living woody or herbaceous ancestors. Due to the uninterrupted growth period in the tropics, there are no anatomical or morphological features which allow conventional age determination. However, stem elongation rates have been determined (3–5.5 cm per year) and indicate an age of about 250 years for the tallest arborescent Dendrosenecios which may reach a height of 10 m. 30 to 120 large leaves are clustered in an enormous terminal rosette, justifying the term giant rosette plants. A leaf bud, consisting of about as many developing leaves as the rosette contains, is found in the center. During the nocturnal frost period the adult rosette leaves form a so-called night-bud by nyctinastic upwards bending and thus protect the leaf bud from freezing by insulation. The stem is surrounded by a mantle of persistent dead leaves; this ameliorates the microclimate of the pith-cells which greatly contribute to water transport into the leaves. Below the leaf rosette a zone of putrefaction is found, from where the decay of the dead leaves apparently provides nutrients directly to the growing stem. The population dynamics of the arborescentD. keniodendron is characterized by a simultaneous inflorescence development at irregular intervals of up to more than twenty years. Due to sporadic flowering and a seedling survival rate of less than 1%, oscillations of the population size are to be expected.     

Frost resistance of winter rape leaves as related to the changes in water potential and growth capability

Differential thermal analysis indicated that the frost resistance of winter rape leaves ( Brassica napus L. var. oleifera L. cv. Gòrczanski), collected from plants grown in the cold (5/2°C), relies mainly on their ability to supercool to −9 to −11°C, i.e. consists in freezing avoidance. Initiation of ice formation in the cold-acclimated leaves resulted in the death of more than 50% of the cells as determined with a conductivity method. The development of freezing tolerance appeared to be an attribute of the second stage of plant hardening and was induced by the exposure of plants to a slightly subzero temperature (−5°C) for 18 h. Such a treatment brought about a sudden and persistent water potential decrease in the leaves, despite the fact that they had reabsorbed water from the medium prior to water potential measurements. Water potential changes were associated with a higher growth capability of the leaves as checked by determinations of disk area increments. It is suggested that the increased frost tolerance of the cold-grown winter rape leaves, subjected to subfreezing temperature, is related to the decreased water potential of the tissue caused by changes in turgor and/or in osmotic pressures of the cells.     

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 effect of photoperiod and temperature on growth and frost resistance of winter rye root systems

Root growth, development and frost resistance were examined in winter rye ( Secale cereale L. cv. Puma) plants grown under 6 combinations of temperature and photoperiod (20/16°C or 5/3°C, day/night; 8, 16- or 24-h days). Overall root system growth is influenced by the interaction of temperature and photoperiod. Maximum shoot growth occurs at a 24-h photoperiod in 20°C plants and at a 16-h photoperiod in 5°C plants, and is correlated in both treatments with a high root:shoot ratio. Frost resistance of rye roots is affected by short photoperiods in 2 ways. First, short photoperiod and low temperature delay production of new adventitious roots so that newly developing roots are not exposed to freezing temperatures. Second, short photoperiod alone can induce several degrees of frost tolerance in existing roots during the lag phase of growth. Low temperature alone does not decrease the rate of dry weight accumulation in rye root systems, but cold temperature does retard developmental processes within the roots. Rye roots grown at 5°C develop first order lateral roots, differentiate metaxylem vessels and suberize endodermal cell walls more slowly than roots grown at 20°C.     

Energy budgets and temperatures of nyctinastic leaves on freezing nights

Temperatures of exposed horizontal and vertical soybean leaves (Glycine max [L.] Merr. var. Chippewa) were measured on calm, clear nights with temperatures near freezing. Average leaf-air temperature differences for 5 nights were −1.5 C and −1.0 C for horizontal and vertical leaves respectively. The horizontal leaves were cooler than the vertical leaves. The mean of all observed horizontal-vertical leaf temperature differences was −0.5 C with a maximum average for 1 night of −0.8 C, while maximum differences theoretically attainable in similar leaves were calculated to be −1.7 C. No differences were observed in the extent of frost damage in horizontal and vertical leaves. The apparent reduction in frost damage in vertical leaves observed by Charles Darwin was probably caused by his method of using corks to hold the horizontal leaves and not by leaf orientation. Theoretical considerations and the experimental results indicate that nyctinastic leaf movements probably do not provide significant protection from frost for any plants.     

Adaptations to low temperature in high altitude insects from Mount Kenya

Abstract. 1. The strategies for low temperature survival in insects on Mount Kenya were investigated. The insects were collected from their natural habitats and their supercooling points and low temperature tolerances determined.
2. Most insects showed no special adaptations to low temperature survival and seem to depend on spending the cold nights in protected habitats, such as beneath stones and fallen trunks of plants, as well as within the wet frills of dead leaves of alpine plants, where they are protected by the heat released from freezing water.
3. Some insects, e.g. Collembola, aphids and a curculionid beetle, which live in relatively unprotected habitats, had low supercooling points, allowing them to remain unfrozen when exposed to low night temperatures. A nucleator free diet is apparently essential for the survival of such species.
4. Two species of curculionid beetles were found to withstand freezing down to -7C. These beetles had nucleating agents in their haemolymph and higher supercooling points than most of the other species studied.
5. A moderate freezing tolerance was found in larvae of a midge that lives in the watery liquid between the leaves of Senecio brassica .     

Seasonal changes in frost hardiness in cloudberry (Rubus chamaemorus) in relation to carbohydrate content with special reference to sucrose

Monthly determinations of frost hardiness of cloudberry ( Rubus chamaemorus L.) buds and rhizomes were done from October 1978 to October 1979. For buds LT₅₀ (lethal temperature for 50% of the plant material) was calculated from the percentage of bud breaking and for rhizomes from visual estimations of the degree of coloring by triphenyltetrazolium chloride. The frost hardiness varied from—11.5°C in November to—4°C in May to July for buds and from—16°C in January to—3°C in June— July for rhizomes. Dehardening started in February while the plants were still covered with snow. In connection with the determinations of frost hardiness, carbohydrate analyses were done. There was a good correlation between the degree of frost hardiness and the amount of soluble carbohydrates determined with anthrone. Sucrose, determined by gas chromatography, seemed to be the sugar contributing most in this correlation.     

Spring patterns of freezing resistance and photosynthesis of two leaf phenotypes of Hedera helix

Subdominant evergreen broad-leaved plants occurring in deciduous forests throughout temperate zones have only a short window of optimum photoassimilation in spring before canopy closure. Yet increasing photosynthetic and metabolic activity occurs concurrently with reductions in freezing resistance, resulting in vulnerability of plant tissues to late spring freezing events. Our goal was to document the temporal patterns of photosynthesis versus freezing resistance during spring in adult and juvenile leaf phenotypes of Hedera helix in Switzerland. Freezing resistances in all leaves were well below long-term minimum temperatures experienced at the study site, with adult leaf phenotypes in the forest canopy being more freezing resistant than juvenile leaves occurring closer to the ground. Reductions in freezing resistance were followed by increases in leaf photosynthetic capacities, which appeared synchronized among leaf phenotypes. Adult canopy leaves maintained a higher freezing resistance but lower photosynthetic capacity than juvenile leaves through the end of winter and into early spring. However, shortly after the cessation of freezing temperatures, adult leaves greatly increased their photosynthetic capacity relative to juvenile leaves, yet maintained freezing resistances sufficient to resist late spring freezing events. These patterns highlight the importance of the tradeoff in H. helix between exposure to potentially damaging cold temperatures in late spring and the need for high photosynthetic carbon gains before full canopy closure.     

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.     

Chilling sensitivity of the frost-tolerant potato Solanum commersonii

Frost tolerance has been reported in the shoots of wild, tuberiferous potato species such as Solanum commersonii when the plants are grown in either field or controlled conditions. However, these plants can survive as underground tubers and avoid unfavorable environmental conditions altogether. As such, leaf growth and photosynthesis at low temperature may not be required for survival of the plants. In order to determine the temperature sensitivity of S. commersonii shoots, we examined leaf growth, development and photosynthesis in plants raised at 20/16°C (day/night). 12/9°C and 5/2°C. S. commersonii leaves grown at 5°C exhibited a marked decrease in leaf area and in total chlorophyll (Chl) content per leaf area when compared with leaves grown at 20°C. Furthermore, leaves grown at 5°C did not exhibit the expected decrease in either water content or susceptibility to low-temperature-induced photoinhibition that normally characterizes cold acclimation in frost-tolerant plants. Measurements of CO₂-saturated O₂ evolution showed that the photosynthetic apparatus of 5°C plants was functional, even though the efficiency of photosystem II photochemistry was reduced by growth at 5°C. A decrease in the resolution of the M-peak in the slow transients for Chl a fluorescence in leaves grown at 12 and 5°C and in all leaves exposed to high light at 5°C indicated that low temperature significantly affected processes on the reducing side of Q_A, the primary quinone electron acceptor in photosystem II. Thus S. commarsonii exhibits the characteristics of a plant that is limited by chilling temperatures. Although S. commersonii can tolerate light frosts, its sensitivity to chilling temperatures may result in shoot dieback in winter in its native habitat. The plants may avoid both chilling and freezing temperatures by overwintering as underground tubers.     

Analysis of the response of leaf extension to chilling temperatures in Lolium temulentum seedlings

Lolium temulentum L. plants were grown at 20°C and transferred to 2°C or 5°C at 21, 28 or 35 days after sowing, when leaves 3, 4 and 5, respectively, were at mid-expansion and leaves 4, 5 and 6 were just emerging. Leaves of plants exposed to 2°C for 7 or 14 days before their date of emergence at 20°C failed to appear at all during the course of the experiment. Transfer to 2°C at emergence resulted in a delay of about 40 days before expansion was detected and subsequent growth was extremely slow. By contrast, although leaves of seedlings exposed to 5°C at or prior to emergence were significantly smaller and slower-growing than the same leaves of plants maintained at 20°C, the difference in vegetative development and tillering between 2°C and 5°C was much less marked than between 5°C and 2°C, implying the existence of a rather sharp threshold for growth between the latter temperatures. Leaves transferred to 2°C at mid-expansion attained a final size not very different from leaves exposed to 5°C at the same time, but expension rates were only 20–30% of those at 5°C, and the time taken to achieve full expansion a corresponding 3 to 5 times longer. These responses were quantified by fitting Richards functions to measurements of leaf extension and determining, from the parameters of the curves, asymptotic maximal lengths, mean relative and absolute extension rates, inflexion points and durations of growth. The potential usefulness of Lolium temulentum as a model species for studying the relationship between temperature and growth in the Granmineae is discussed.     

Changes in Tissue Freezing in Senecio vulgaris infected by Rust (Puccinia lagenophorae)

Freezing of healthy and rust (Puccinia lagenophorae) infectedleaves of Senecio vulgaris was compared calorimetrically bythermal analysis. In fully expanded leaves the threshold freezingtemperature was in the range –6.8 to –8.4 °Cin controls but –3.0 to –5.1 °C in leaves withsporulating rust sori. Comparable values in expanding leaveswere –5.0 to –8.9 °C and –3.9 to –6.7°C for healthy and rusted tissues, respectively. The bulktissue freezing point was between –1.0 and –4.0°C in both fully expanded and expanding healthy leaves,and was increased by infection by between +0.2 and 2.5 °C.Whereas healthy leaves supercooled by 3.1–5.8 °C,rusted leaves supercooled by only 1.8–4.9 °C Supercoolingof control leaves was reduced by dusting with aeciospores, particularlywhen leaves were wounded to simulate the rupture of the surfacecaused by sporulation, but wounding alone had no significanteffect. Supercooling of distilled water was also significantlyreduced by aeciospores, suspended at a concentration of 10⁵spores ml^–1. It is concluded that rust-induced changes in leaf freezing inS. vulgaris grown in controlled environments were due to anincrease in the number of sites for ice nucleation, caused bythe presence of the aeciospores, and increased penetration ofice into internal tissues, resulting from damage to the cuticleand epidermis. Although data for frost resistance obtained inthe growth-room are similar to previous field observations,the role of the above mechanisms under field conditions remainsunproven. Senecio vulgaris (groundsel), Puccinia lagenophorae (rust), low temperature, freezing resistance     

The interrelationship of growth and frost tolerance in winter rye

The reduction in growth of winter cereals that occurs in the fall is thought to be required for the development of frost resistance. In the present study, the interrelationship of freezing tolerance and growth was examined by raising winter rye ( Secale cereale cv. Puma) plants at 20/16°C (day/night) and at 5/3°C under 8-, 16- and 24-h daylengths to vary growth rates and frost tolerance. Temperature and irradiance were quantified as thermal time, photothermal time and photosynthetic photon flux and examined by multiple linear regression in order to determine their effects on growth and frost tolerance of rye shoots. At low temperature, both growth and frost tolerance were markedly influenced by daylength and irradiance. Plants grown at 5/3°C with a short daylength accumulated shoot dry weight and increased frost tolerance at a greater rate per unit photothermal time or photon flux than plants grown at longer daylengths. Moreover, 5/3°C plants grown with a 16-h day grew more slowly and were less frost tolerant than plants grown with a 24-h day. We conclude that the interrelationship between growth and frost tolerance is a quantitative one. Frost tolerance is induced only by low temperature, but the development of forst tolerance is dependent upon both irradiance, which affects the amount of photoassimilate available, and daylength, which may affect the partitioning of photoassimilates between growth and frost tolerance.     

Effect of supercooling and freezing on photosynthesis in freezing tolerant leaves of Afroalpine ‘giant rosette’ plants

Summary The effect of supercooling and freezing on the photosynthetic capability of representatives of the permanent frost hardy giant rosette plants Dendrosenecio keniodendron, D. brassica and Lobelia telekii, of the tropical alpine regions was investigated with the non-invasive chlorophyll a fluorescence technique. While supercooling, normal chlorophyll a fluorescence kinetics exhibiting the sequence 0, I, (D), P, S, M, were recorded, however with some retardation of both, the fast and the slow characteristics as compared to those obtained at day-time temperature. As long as the leaves remained unfrozen, the rise of the variable fluorescence F from the level 0 to P was inversely related to a drop of the temperature from about 0°C to-8°C. The increase of F with lower temperature is understood to result from a decrease of the velocity of the quenching reactions while photoreduction of the primary electron acceptor appeared to be unimpeded. The second fluorescence maximum (M), usually interpreted to indicate the commencement of the biochemical reactions of photosynthesis was consistenly to be observed during supercooling. Fluoescence induction kinetics of frozen leaves showed only fast rise to presumably F _max which was not followed by a significant decay for as long as 4 min. The lack of substantial quenching indicates that in the freeze-dehydrated state neither reoxidation of the primary acceptor nor energetization of the thylakoid membrane was accomplished. This effect however was immediately and fully reserved upon thawing of the leaves when the usual fluorescence induction kinetics as well as normal rates of CO₂-uptake were observed. Thus the permanent frost-hardy afroalpine plants do not exhibit any even short-term memory effect of the nocturnal frost on such a delicate process as is photosynthesis.     

Spatial patterning of pigmentation in evergreen leaves in response to freezing stress

Evergreen leaves of temperate climate plants are often subject to frosts. Changes in carbon gain patterns arise from freezing‐related tissue damage, and from interactions between light and temperature stress. We examined relationships between spatial patterns in freezing and concentrations of chlorophyll. Spatial patterns in pigmentation in leaves that had or had not been exposed to naturally occurring frosts were determined by conventional extraction techniques combined with high‐resolution hyperspectral imaging of reflectance from intact leaves. Predictive indices were developed to relate reflectance to chlorophyll content and chlorophyll a/b ratios within intact leaves. Leaves exposed to frosts had lower chlorophyll contents and more variable a/b ratios than protected leaves. In frost‐affected leaves, chlorophyll content was highest near leaf centres and decreased toward leaf tips and margins. Decline in chlorophyll content was associated with shifts in chlorophyll a/b ratios and increases in red pigmentation due to anthocyanin, with effects being greater on leaf sides exposed directly to the sun. These altered pigmentation patterns were consistent with patterns in freezing. The present results illustrate the fine scale of spatial variation in leaf response to freezing, and raise important questions about impacts of freezing on photosynthetic function in over‐wintering evergreens.     

Bacterial ice nucleation: a factor in frost injury to plants

Heterogeneous ice nuclei are necessary, and the common epiphytic ice nucleation active (INA) bacteria Pseudomonas syringae van Hall and Erwinia herbicola (Löhnis) Dye are sufficient to incite frost injury to sensitive plants at −5°C. The ice nucleation activity of the bacteria occurs at the same temperatures at which frost injury to sensitive plants occurs in nature. Bacterial ice nucleation on leaves can be detected at about −2°C, whereas the leaves themselves, i.e. without INA bacteria, contain nuclei active only at much lower temperatures. The temperature at which injury to plants occurs is predictable on the basis of the ice nucleation activity of leaf discs, which in turn depends on the number and ice nucleation activity of their resident bacteria. Bacterial isolates which are able to incite injury to corn at −5°C are always active as ice nuclei at −5°C. INA bacteria incited frost injury to all of the species of sensitive plants tested.     

Resident population of Xanthomonas campestris pv. malvacearum on cotton leaves: a source of inoculum for bacterial blight

Xanthomonas campestris pv. malvacearum was transmitted from infested seed to the cotyledons of cotton cv. Deltapine 61 seedlings at 28°C and relative humidities (RH) of 90% or 73%. A resident population was present on the first and second true leaves but not on the third true leaf of plants at either RH. There were smaller numbers of resident bacteria on fewer leaves of plants at the lower RH than on plants at the higher RH. Cotton plants grown from infested seed at 25°C and 30°C and incubated at 100% RH at different stages of growth developed bacterial blight on leaves that were in bud or partly expanded when incubated. Resident cells of this pathogen can thus invade susceptible leaves when conditions are favourable for infection. Bacterial blight developed on more plants at 30°C than at 25°C. In a field trial, X. campestris pv. malvacearum transmitted from seed was present as resident bacteria on the third leaf from the growing point during the vegetative development of the plant. Resident bacteria, which infected young leaves during rainy periods, were isolated from the bacterial blight lesions which subsequently developed.     

Phytochrome action and frost hardening in black spruce seedlings

Black spruce [ Picea mariana (Mill.) B.S.P.] development is sensitive to photoperiod. To date the implication of photoperiod, and especially phytochrome, in the frost hardening process of black spruce has not been fully tested. Two light fluence rates, night interruption of darkness, and red vs far-red radiation treatments were applied to black spruce seedlings, followed by freezing at –6°C. Parallel to the freezing test, growth measurements, bud formation and the xylem water potential estimates of the seedlings were done. While dry weight accumulation depends on the irradiation energy level, bud formation and freezing tolerance are photoperiodically sensitive. Furthermore, bud formation and frost hardening are dependent upon whether phytochrome is in the active form or inactive form, as demonstrated by the positive effect of short days, far-red radiation and the reversal of the red effect by far-red radiation. Also, xylem water potential appears to be influenced by short day and far-red conditioning.