Accumulation of Free Proline in Citrus Leaves during Cold Hardening of Young Trees in Controlled Temperature Regimes

Free proline increased in leaves of orange (Citrus sinensis [L.] Osb. cv. Valencia) and grapefruit (Citrus paradisi Macfad. cv. Star Ruby) trees on a wide range of citrus rootstocks during cold hardening. Increases in sugars accompanied proline accumulation. During cold hardening, the rate of proline accumulation was greater in old than in young leaves. In leaves of grapefruit trees kept in the dark during cold hardening, neither proline nor sugars increased and the degree of cold hardiness was less than in trees exposed to light. Like sugar accumulations, proline accumulation does not reflect specific degrees of cold hardiness in citrus cultivars.     

Causes of variation in cold hardiness among fast-growing willows (Salix spp.) with particular reference to their inherent rates of cold hardening

Causes of variation in cold hardiness in the autumn were assessed among closely related, fast‐growing clones of willow of northern/continental and southern/maritime origins, under controlled regimes and natural conditions. Cold hardiness was assessed by controlled freezing followed by injury analysis, based on measurements of chlorophyll fluorescence (stems) and electrolyte leakage (leaves). During growth at a given temperature, the cold hardiness of the clones' stems was negatively correlated with their rate of growth. This apparently phenotypic variation was independent of temperature and, hence, the absolute growth rate. At later stages, cold hardiness of stems varied mainly with respect to genetic differences in the timing and rate of cold hardening. Cold hardening began up to 7 weeks earlier in northern/continental clones, and their rates of hardening in cool temperature regimes were up to three times higher than in southern/maritime clones. Ranking of clones with respect to rates was essentially the same whether natural or abrupt reductions of day length were used to trigger cold hardening. Results closely agreed with those of a previous field trial. Comparisons of rates at cool and warm temperatures suggest that cold hardening became increasingly dependent on cool temperatures with time. Increasing sucrose‐to‐glucose ratios, and especially dry‐to‐fresh weight ratios, paralleled early cold hardening. Before leaves were shed in the autumn they underwent cold hardening in parallel with stems, eventually allowing them to tolerate temperatures down to ?10 °C.     

Effects of cold hardening on acyl lipids of citrus tissue

Cold hardened and unhardened 8- or 16-month-old citrus plants were examined for differences in fatty acid (FA) content. Unhardened leaves from 8-month-old Valencia scion budded on sour orange rootstock had 29% less FAs than leaves from seedling sour orange. After cold hardening triacylglycerol (TAG) FAs increased 4-fold in Valencia on sour orange and 6-fold in sour orange seedling. The percentage of FAs associated with TAGs for unhardened-hardened 16-month-old Valencia on sour orange tissues were: upper leaves 7–20, lower leaves 6–17, bark 6–9, and roots 57–73%. Cold hardening increased the amount of TAG FAs of 16-month-old Valencia on sour orange in upper leaves by 226% and in lower leaves by 173%. Concentrations of linoleic acid increased by 479% in upper leaves and by 303% in lower leaves. Quantities of lionolenic acid in monogalactosyl diacylglycerols declined by 27% in upper leaves and by 20% in lower leaves.     

Changes in glutathione content during cold acclimation in Cornus sericea and Citrus sinensis

Glutathione content was evaluated in relation to freezing tolerance in red osier dogwood stems and Valencia orange leaves. Exposure of dogwood and citrus to cold-acclimating conditions in controlled environments led to increases in reduced glutathione (GSH) content which were correlated with freezing tolerance. GSH did not accumulate in field-grown dogwood stems during cold acclimation in fall, but did increase in content prior to deacclimation in late winter. Further studies showed that accumulation of GSH in dogwood at low temperatures is dependent on adequate levels of sulfate in the soil. In citrus, modulation of GSH content by infiltration of leaf tissue with various compounds including GSH did not alter freezing tolerance. Root treatment with N,N-diallyl-2,2-dichloroacetamide (R-25788) increased leaf GSH content, but not hardiness. Evidence presented indicates that glutathione accumulates in plant tissues exposed to low temperatures, but that GSH accumulation is not associated with freezing tolerance.     

Relationships of leaf Fatty acids to cold hardening of citrus seedlings

Three cultivars of citrus with different sensitivities to freezing temperatures (citron, Citrus medica L.; rough lemon, C. limon Burm. F; sour orange, C. aurantium L.) were cold hardened for 4 weeks. Lipids from leaves of hardened and control seedlings were fractionated and analyzed for fatty acids. The absolute amount of triglycerides and phospholipids increased in the leaves upon hardening. With hardening, total linoleic acid also increased 141% in citron, 210% in rough lemon, and 233% in sour orange. Specific increases in linoleic acid were found in triglycerides, in the four phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol), and in neutral lipids more polar than triglycerides. Trans-3-hexadecenoic acid was found only in phosphatidylglycerol.     

Evaluation of Polyamine and Proline Levels during Low Temperature Acclimation of Citrus

The polyamines (PA) putrescine (Put), spermidine (Spd), and spermine (Spm) were measured during 3 weeks exposure to cold hardening (15.6°C day and 4.4°C night) and nonhardening (32.2°C day and 21.1°C night) temperature regimes in three citrus cultivars: sour orange (SO) (Citrus aurantium L.), `valencia' (VAL) (Citrus sinensis L. Osbeck), and rough lemon (RL) (Citrus jambhiri Lush). The changes in PA were compared to the amount of free proline, percent wood kill and percent leaf kill. A 2- to 3-fold increase in Spd concentrations were observed in hardened RL, SO, and VAL leaves compared to nonhardened leaves. Spermidine reached its highest level of approximately 200 nanomoles per gram fresh weight after 1 week of acclimation in both SO and VAL leaves, while RL spermidine content continued to increase up to the third week of acclimation. Spm levels in acclimated VAL and RL leaves increased 1- to 4-fold. However, SO leaves Spm content decreased with acclimation. Putrescine levels in SO and VAL increased 20 to 60% during the first 2 weeks of acclimation then declined after 3 weeks. RL putrescine content was not affected by cold acclimation. The data presented here provided direct relationship between increased Spd concentration and citrus cold hardiness. Free proline was 3- to 6-fold higher in acclimated than in nonacclimated trees. Results also demonstrate that in acclimated versus nonacclimated citrus trees the absolute amount rather than the ratio of increase in free proline is more important in predicting their ability to survive freezing stress.     

Freezing tolerance of citrus, spinach, and petunia leaf tissue : osmotic adjustment and sensitivity to freeze induced cellular dehydration

Seasonal variations in freezing tolerance, water content, water and osmotic potential, and levels of soluble sugars of leaves of field-grown Valencia orange (Citrus sinensis) trees were studied to determine the ability of citrus trees to cold acclimate under natural conditions. Controlled environmental studies of young potted citrus trees, spinach (Spinacia pleracea), and petunia (Petunia hybrids) were carried out to study the water relations during cold acclimation under less variable conditions. During the coolest weeks of the winter, leaf water content and osmotic potential of field-grown trees decreased about 20 to 25%, while soluble sugars increased by 100%. At the same time, freezing tolerance increased from lethal temperature for 50% (LT₅₀) of −2.8 to −3.8°C. In contrast, citrus leaves cold acclimated at a constant 10°C in growth chambers were freezing tolerant to about −6°C. The calculated freezing induced cellular dehydration at the LT₅₀ remained relatively constant for field-grown leaves throughout the year, but increased for leaves of plants cold acclimated at 10°C in a controlled environment. Spinach leaves cold acclimated at 5°C tolerated increased cellular dehydration compared to nonacclimated leaves. Cold acclimated petunia leaves increased in freezing tolerance by decreasing osmotic potential, but had no capacity to change cellular dehydration sensitivity. The result suggest that two cold acclimation mechanisms are involved in both citrus and spinach leaves and only one in petunia leaves. The common mechanism in all three species tested was a minor increase in tolerance (about −1°C) resulting from low temperature induced osmotic adjustment, and the second in citrus and spinach was a noncolligative mechanism that increased the cellular resistance to freeze hydration.     

Changes in the activity of proteases and trypsin inhibitors in wheat leaves in the initial period of cold hardening and subsequent dehardening

The dynamics of amidase, cysteine protease, and trypsin inhibitor activities were studied in the leaves of wheat (Triticum aestivum L.) seedlings grown under controlled conditions (25°C, illuminance 10 kLx, 14-h photoperiod) and subjected to cold hardening (5°C, 10 kLx, 14-h photoperiod). Changes in the activity of amidases and cysteine proteases proved to precede an increase in cold resistance during cold hardening and a decrease in cold resistance after the end of cold hardening. The activity of trypsin inhibitors changed only during cold hardening. It is suggested that amidases, cysteine proteases, and trypsin inhibitors are involved in the cold adaptation of plants.     

渗透调节参与循环干旱锻炼提高烟草植株抗旱性的形成

对漂浮育苗的烟草幼苗进行控水一半萎焉．复水一恢复的循环干旱锻炼。结果表明,这种干旱锻炼能显著提高烟草幼苗根、茎、叶中的渗透调节物质可溶性糖和脯氨酸的含量,降低细胞渗透势。当干旱锻炼过的烟草植株遭受后续的干旱胁迫时,与未锻炼的对照相比,其根、茎、叶能积累更多的可溶性糖和脯氨酸,从而降低了细胞渗透势,使叶片能维持较高的膨压。这些结果表明渗透调节参与了循环干旱锻炼提高的烟草植株抗旱性的形成过程。此外,干旱锻炼提高了烟草幼苗的根／冠比。循环干旱锻炼过程中烟草植株一方面使其各部位通过渗透调节来对干旱环境进行生理适应,另一方面通过调节光合产物在地上部和地下部的分配以影响根／冠比来对干旱环境进行形态适应,以最终提高其抗旱性。     

Components of Pink Snow Mould Resistance in Winter Wheat are Expressed Prior to Cold Hardening and in Detached Leaves

Resistance to pink snow mould, caused by Microdochium nivale, was investigated in four resistant winter wheat lines from the USDA World Cereal Collection (CI9342, CI14106, PI173440 and PI181268) and three Nordic wheat lines (Bjørke, Rida and V1004). Pink snow mould resistance was tested in non‐hardened and cold‐hardened plants incubated under artificial snow cover and in detached leaf segments mounted on water agar and incubated at either 3°C in darkness or at room temperature with light during the day. The wheat lines CI9342, CI14106 and PI181268 were more resistant than the Nordic lines, both before and after cold hardening. Thus, although cold hardening strongly increases the level of snow mould resistance in all the wheat lines, some resistance mechanisms are also present prior to cold hardening in some of the resistant lines. CI9342, CI14106 and PI181268 also had a higher level of resistance than the other lines in the detached leaf assay, indicating that these lines have some resistance mechanisms acting in the leaves. The resistance of PI173440 was expressed only in intact hardened plants and not in non‐hardened plants or in detached leaves. This indicates that this line relies on cold hardening‐related changes in the crown for its resistance. In the detached leaf assay the rate of lesion development varied greatly between leaves of different order. The highest correlation with the whole plant test was obtained when using secondary leaves and incubation at 3°C in the dark.     

Long-term exposure to enhanced ultraviolet-B radiation in the sub-arctic does not cause oxidative stress in Vaccinium myrtillus

The aim of this work was to assess whether or not oxidative stress had developed in a dwarf shrub bilberry ( Vaccinium myrtillus L.) under long-term exposure to enhanced levels of ultraviolet-B (u.v.-B) radiation. The bilberry plants were exposed to increased u.v.-B representing a 15% stratospheric ozone depletion for seven full growing seasons (1991–1997) at Abisko, Swedish Lapland (68°N). The oxidative stress was assessed on leaves and stems by analysing ascorbate and glutathione concentrations, and activities of the closely related enzymes ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2). The affects of autumnal leaf senescence and stem cold hardening on these variables were also considered. The results showed that the treatment caused scarcely any response in the studied variables, indicating that u.v.-B flux representing a 15% ozone depletion under clear sky conditions is not sufficient to cause oxidative stress in the bilberry. It is suggested that no strain was evoked since adaptation was possible under such u.v.-B increases. The studied variables did, however, respond significantly to leaf senescence and especially to stem cold hardening.     

Comparison of Barley Response to Short-Term Cold or Dehydration

Abscisic acid (ABA) content and relative water content (RWC) in second fully expanded leaves of cold hardened plants and in dehydrated leaves of freezing tolerant barley (Hordeum vulgare L. cv. Lunet) were compared. ABA content and RWC in leaves did not change during the first day of cold hardening. On the contrary, dehydration of leaves led to a decrease of RWC and to an increase of ABA content.     

Localization and Distribution of 'Candidatus Liberibacter asiaticus’ in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody

‘Candidatus Liberibacter asiaticus’ (CaLas), a non-cultured member of the α-proteobacteria, is the causal agent of citrus Huanglongbing (HLB). Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA polyclonal antibody based direct tissue blot immunoassay to localize CaLas in different citrus tissues and in periwinkle leaves. In citrus petioles, CaLas was unevenly distributed in the phloem sieve tubes, and tended to colonize in phloem sieve tubes on the underside of petioles in preference to the upper side of petioles. Both the leaf abscission zone and the junction of the petiole and leaf midrib had fewer CaLas bacteria compared to the main portions of the petiole and the midribs. Colonies of CaLas in phloem sieve tubes were more frequently found in stems with symptomatic leaves than in stems with asymptomatic leaves with an uneven distribution pattern. In serial sections taken from the receptacle to the peduncle, more CaLas were observed in the peduncle sections adjacent to the stem. In seed, CaLas was located in the seed coat. Many fewer CaLas were found in the roots, as compared to the seeds and petioles when samples were collected from trees with obvious foliar symptoms. The direct tissue blot immuno assay was adapted to whole periwinkle leaves infected by CaLas. The pathogen was distributed throughout the lateral veins and the results were correlated with results of qPCR. Our data provide direct spatial and anatomical information for CaLas in planta. This simple and scalable method may facilitate the future research on the interaction of CaLas and host plant.     

NOCTURNAL THERMAL EXCHANGE OF CITRUS LEAVES

Turrell , F. M., S. W. Austin , and R. L. Perry . (U. California, Riverside & Los Angeles.) Nocturnal thermal exchange of citrus leaves. Amer. Jour. Bot. 49(2) : 97–109. Illus. 1962.—Cooling rates of leaves were measured with fine thermocouples inserted within the leaf laminae. From these rates, total thermal conductances were calculated for leaves of intact greenhouse-grown lemon cuttings, in the dark, in still air and moving air, and in open laboratory rooms of warm to freezing temperatures. Thermal conductances were also calculated for leaves of 4 commercial varieties of citrus picked from mature trees in the grove and measured in low light, in still air, in a microcosm at warm, constant temperatures. The total conductances were fractionated, first, by determining transpiration rates of detached leaves from both sources, in darkness and in still or moving air, through similar temperature ranges and humidities. From transpiration rates, transpiration conductances were calculated. Second fractions (radiation conductances) were calculated for lemon leaves from far-infrared reflectances; and the third fractions (free-convection conductances) were calculated by subtraction of the sum of the radiation and transpiration conductances from the total. A free-convection-conductance coefficient was calculated for lemon, and then applied to 3 other varieties of citrus for which infrared reflectances were unavailable, to obtain their free-convection conductances. These together with experimentally determined transpiration and total conductances permitted calculation of their radiation fractions. The conductances have been tested for 6 different measured microclimates in which the calculated leaf temperatures averaged ± 0.6 C of the measured temperatures, an error compatible with the precision of field temperature measurements. Total thermal conductances of lemon leaves were higher in both warm, still and warm moving air than in cold, whereas the radiation and free-convection fractions were about equal in still air. The transpiration fractions were very small in warm, still or warm moving air but negligible in cold. In cold still and cold moving air, all the conductances were larger for orange fruit than for lemon leaves. Leaves of plants native to tropical rain forests were more efficient in heat transfer than were leaves from the temperate zone.     

水稻幼苗冷锻炼过程中钙的效应

冷锻炼处理提高了水稻（Ｏｒｙｚａ ｓａｔｉｖａ Ｌ．）幼苗叶片中抗氧化剂（还原型谷胱甘肽,ＧＳＨ;抗坏血酸,ＡｓＡ）含量和膜保护酶（超氧化物歧化酶,ＳＯＤ）的活性,同时也提高了可溶性蛋白质中热稳定蛋白的含量。ＣａＣｌ２ 浸种处理对上述冷锻炼的作用有加强的效果,且明显地提高了过氧化氢酶（ＣＡＴ）和过氧化物酶（ＰＯＤ）的活性。有无ＣａＣｌ２ 处理的冷锻炼处理均减轻冷胁迫引起的ＧＳＨ 及ＡｓＡ 含量、ＳＯＤ 活性及热稳定蛋白质含量的下降程度,有利于幼苗在恢复过程中ＧＳＨ、ＡｓＡ、ＣＡＴ、ＳＯＤ、ＰＯＤ及热稳定蛋白质水平迅速回升。结合ＣａＣｌ２ 处理的冷锻炼苗在冷胁迫恢复生长时增长迅速,且苗健壮浓绿,说明ＣａＣｌ２浸种对冷锻炼处理提高水稻幼苗的抗冷力有明显的促进作用,这与ＣａＣｌ２ 浸种结合冷锻炼能更有效的提高细胞膜保护能力有关     

Ca2+对冷胁迫柑橘离体叶片的相关生理生化指标的影响

研究了不同浓度的外源Ca^2＋对柑橘离体叶片抗寒性的影响。结果表明：10-20mmol／L Ca^2＋处理能够增加柑橘可溶性糖和可溶性蛋白质的含量,提高SOD和POD的活性,同时减少膜质过氧化物丙二醛（MDA）的积累,从而提高柑橘的抗寒性。     

Adenine Nucleotide Changes during Cold Acclimation of Winter Rape Plants

During the first stage of hardening of winter rape plants (Brassica napus L. var. oleifera L., cv. Górczański), marked increase of ATP content in leaves was observed. Lowering the temperature from 5 to 0 C (the second stage of hardening) had no further effect on ATP content. In roots, not capable of hardening, pronounced decrease of ATP content was noted after prolonged exposure to cold. It was found that increased ATP content and higher energy charge in cold-treated leaves were due to light and dark processes.     

Endogenous abscisic acid content in cucumber leaves under the influence of unfavourable temperatures and salinity

Endogenous abscisic acid (ABA) content was measured in leavesof Cucumis sativus L. under the influence of hardening (lowand high) temperatures and salinity. The rise in cold and heatresistance of the seedlings was accompanied by a considerableincrease in the ABA level in the leaves. Chloride salinity alsobrought about a rise in the ABA content. The data indicate thatABA may induce resistance when the plants are exposed to severalstresses. Key words: Cucumis sativus, ABA, cold and heat hardening, salinity