Characterization and Role of an Endogenous Inhibitor in the Induction of Cold Hardiness in Acer negundo

An inhibitor extracted from short day treated Acer negundo leaves was compared to abscisic acid in 4 different solvent systems. The chromatographic properties of abscisic acid and the inhibitor were in very close agreement. Treatment of Acer negundo plants under non-hardening preconditions (long days) with either the inhibitor or abscisic acid increased hardiness after a hardening period of 3 weeks at 40°. A gibberellin-inhibitor relationship was further studied by making comparison of extracts of plants subjected to either 4 weeks of long days, long days + 5° nights, or short days. These tests indicated that gibberellin-like activity was greatest when the treatment included long days. Abscisic acid-like levels were highest when the treatments consisted of short days or long days + 5° nights. Since the latter groups are the most capable of developing hardiness, the hardening process appears to be more closely related to a build-up of abscisic acid levels than a reduction of gibberellin levels.     

The long day leaf as a source of cold hardiness inhibitors

Short photoperiods followed by low temperatures induced cold hardiness in Acer negundo, Viburnum plicatum tomentosum, and Weigela florida. Hardiness was also obtained under long days and natural fall temperatures if the leaves were removed, either manually or by low temperature. Similarly, removal of leaves from plants exposed to long days at 5° brought about an accelerated rate of hardening. These observations suggested the presence of a hardiness inhibitor in the leaves which was counteracted by short days or removal of the leaves.     

Environmental control of cold hardiness in woody plants

The development of cold hardiness in 2 woody plant species (Acer negundo and Viburnum plicatum tomentosum) was shown to be independent of the induction of bud dormancy. Substantial hardiness levels were obtained under controlled conditions with long days and certain low temperatures—without dormancy development as a prerequisite.

Low temperatures given during the dark period with long days induced hardiness to a level not significantly different from that of short days. Giving plants continuous 10° temperatures under long days forced plants to harden as if they were under short days, even though they were not dormant.

Development of hardiness was shown to be a photoperiodic response. Increasing weeks of short days, followed by a low temperature hardening period in darkness, brought about a progressive increase in hardiness. The short day stimulus could be reversed by long days. Following 6 weeks of short days, the rate of hardening in darkness at 5° was over twice that of plants previously exposed to long days.

     

The relative hardening of roots and shoots and the influence of day-length during hardening in perennial ryegrass

The influence of long and short days during the hardening period on the cold hardiness of perennial ryegrass seedlings was studied as was the relative hardiness of roots and shoots. Hardiness was assessed by the electrolyte release method which was a measure of the amount of damage subsequent to low temperature treatment. Long days promoted hardiness in shoots of Pax 0tofte plants and in one case under short days the roots were found to be hardy. Generally roots were less hardy than shoots in Pax 0tofte and S23 plants hardened under long days for 2 wk. When hardened at the fourth leaf stage for 2 wk at + 5 ^oC under long day conditions, Pax 0tofte plants were more hardy than those of S23. The long day effect on hardiness was arrived at more rapidly, there being no difference in hardiness after 3 wk in Pax 0tofte hardened under long or short days, whereas a significant degree of hardening was observed after 1 wk of hardening under long days at – 4 ^oC. The results obtained are discussed in relation to winter kill of grasses in the West of Scotland, and it is considered that root damage is not an important factor in causing winter kill. The promotive effect of long days on hardiness when hardening commences in late autumn is considered an advantage in temperate regions as it may also allow early frosts to be withstood.     

Gibberellins in Relation to Flowering and Stem Elongation in the Long Day Plant Silene armeria

Two long days induced some flowering and 4 or more long days caused 100% flowering in Silene armeria. On long days microscopically detectable flower primordia were first seen after 6 days, which is at least 1 day before the start of stem elongation. Both gibberellin A₃ and A₇ caused flowering on short days, but the results were variable and flowering was never 100%. Three different gibberellins were detected in Silene extracts. The pattern of gibberellins extracted from plants on short and long days was qualitatively the same, but on long days gibberellin content was up to 100% higher than on short days. Only small amounts of diffusible gibberellins were obtained from Silene shoot tips (including very young leaves) on short days. However, on long days the diffusible gibberellins increased by as much as 10-fold after 4 to 6 long days but then declined somewhat after 10 long days. The gibberellins extracted from the shoot tips at the completion of the diffusion period also increased under long days, although the increase was not as large as for the diffusible gibberellins. An A₅-like gibberellin present in extracts was not detected in diffusates.     

Changes in Frost Hardiness of Stem Cortical Tissues of Cornus stolonifera Michx. after Recovery from Water Stress

Moderate water stress increases frost hardiness in many woody plants but little attention has been given to changes in hardiness after recovery from water stress. Tests were carried out to examine how much water stress-induced frost hardiness remained when plants were rewatered under different day length regimes. Red osier dogwood plants (Cornus stolonifera Michx.) were water-stressed at normal growing temperatures in long day (LD) or short day (SD) conditions, exposed to 6 nights of freezing temperatures, and then returned to normal growing conditions with full water supply. Water-stressed plants gained an additional 8 to 10 C of hardiness. The amount of freeze-induced hardiness in both stressed and control plants was not significant (approximately 2 C) and was not affected by photoperiod. When plants were kept in or transferred to LD, they lost nearly all of their water stress-induced hardiness within 7 days after rewatering. Water-stressed plants in SD lost the least amount of hardiness (5 C) when rewatered. In dogwood, water stress is an effective way to increase hardiness temporarily, but the photoperiod has a large effect on the retention of the acquired hardiness.     

Free and Bound Gibberellin Activities and Ent-kaurene Synthesis during Induction of Cold Hardiness in Black Locust Seedlings

The role of gibberellin in the development of cold hardiness in black locust (Robinia pseudoacacia L.) seedlings was investigated. Free and bound gibberellin activities were followed during induction of cold hardiness using ethyl acetate partitioning and pH variation, with subsequent paper chromatographic fractionation and gibberellin bioassay. While total gibberellin activity decreased during the induction of cold hardiness in black locust seedlings, no convincing evidence was found to support conversion of free gibberellin to a bound form. However, bound gibberellin activity did appear to be more stable than did free gibberellin activity during the final stages of cold hardening at freezing temperatures. Gibberellin synthesis was followed using ¹⁴C-mevalonate conversion to ent-kaurene in a cell-free extract of the tissue. Ent-kaurene synthesis decreased during cold hardening with no detectable synthesis in fully hardened seedlings. However, since growth cessation precedes development of cold hardiness, decreased gibberellin synthesis and corresponding trends in free and bound fractions might have been expected, and a cause and effect relationship is difficult to establish. Even so, a decline in one step in gibberellin synthesis and a greater stability of bound than free gibberelin activity are associated with induction of cold hardiness in black locust seedlings.     

The relationship between gibberellin and floral stimulus inBryophyllum daigremontianum

Summary The long-short-day plantBryophyllum daigremontianum initiates flower buds both upon change from long to short day and after gibberellin application in short day only at night temperatures of 11° and 15°C, but not at 19°C.Flowering of receptor scions in long day or short day takes place just as easily when the donor stocks have been induced by the shift from long day to short day or by gibberellin treatment in short day. Leaves taken from gibberellin-induced plants can also function as donors, even better so than photoperiodically induced leaves. Receptor scions induced by gibberellin-treated donors can in turn induce other vegetative scions (indirect induction).Flower formation induced by the change from long day to short day as well as by gibberellin treatment in short day is always associated with an increased length of newly formed internodes.It is concluded that gibberellin and the floral stimulus are not identical, but that gibberellin is a factor which normally limits production of the floral stimulus inBryophyllum under short days, and that the shift from long day to short results in an increase of the gibberellin level in the plant.With 5 Figures in the TextThis work was in part supported by the National Science Foundation, grants G-16408 and G-17483.     

Interactions of low temperature, water stress, and short days in the induction of stem frost hardiness in red osier dogwood

The induction of stem frost hardiness by low temperature, water stress, short days, and their combinations in 2- and 4-month-old growing dogwoods (Cornus stolonifera) were investigated. When plants were subjected to more than one factor, the increased hardiness was the sum of the effects of the individual factors involved. No interactions among these factors on hardiness were observed during a 3-week treatment. Results indicate that low temperature, water stress, and short days initially trigger independent frost-hardening mechanisms. Plant ages significantly influenced the change in low temperature-induced frost hardiness, but not the water stress or short day-induced frost hardiness.     

Effect of Temperature, Photoperiod and Several Growth Substances on the Cold Hardiness of Chrysanthemum morifolium Rhizomes

The effect of 14 combinations of photoperiod, soil and air temperature, and growth substance applications on the cold hardiness of Chrysanthemum morifolium‘Astrid’ rhizomes was evaluated. Both triphenyl tetrazolium chloride and regrowth tests were used to determine the viability of the cold-stressed rhizome tissues. The rhizomes exhibited different degrees of cold hardiness under these environmental conditions. A combination of short photoperiod and low air and soil temperatures induced maximum cold hardiness. Low soil temperature accompanied by long photoperiods and warm aerial temperatures did not induce rhizome hardening, while some hardening in cool soils was evident under either short photoperiods or low aerial temperatures. Warm soils reduced rhizome hardening under the normally inductive short photoperiod-cool aerial conditions. Since the induction of rhizome hardening was dependent on the induction of the aerial organs, the involvement of translocatable hardiness promoters is indicated. Foliar applications of low levels of gibberllic acid (GA₃) or abscisic acid only slightly influenced rhizome hardiness.     

Low night temperature and inhibition of gibberellin biosynthesis override phytochrome action and induce bud set and cold acclimation, but not dormancy in PHYA overexpressors and wild-type of hybrid aspen

Juvenile trees of temperate and boreal regions cease growth and set buds in autumn in response to short day-lengths (SD) detected by phytochrome. Growth cessation and bud set are prerequisites for the development of winter dormancy and full cold hardiness. In this study we show that the SD-requirement for bud set and cold hardening can be overcome in hybrid aspen (Populus tremula L. × tremuloides Michx.) by low night temperature and inhibition of gibberellin (GA) biosynthesis. Bud set and increased cold hardiness were observed under normally non-inductive long day-length (LD) in wild-type plants, when exposed to low night temperature and paclobutrazol. In addition, the effect of PHYA overexpression could be overcome in transgenic plants, producing bud set and cold acclimation by treatment with: SD, low night temperature and paclobutrazol. After cold acclimation, the degree of bud dormancy was lower for wild-type plants prior treated with LD and transgenic plants (overexpressing PHYA), than SD-treated, wild-type plants. Thus, low night temperature in combination with reduced GA content induced bud set and promoted cold hardiness under normally non-inductive photoperiods in hybrid aspen, but was unable to affect development of dormancy. This might suggest separate signalling pathways from phytochrome regulating the induction of cold/cold hardiness and bud dormancy in hybrid aspen or alternatively, there might be one pathway that fails to complete its action in the transgenic and paclobutrazol treated plants.     

FLOWER-PROMOTING EFFECT OF GIBBERELLIN IN WINTER WHEAT AND BARLEY

1. Vernalized and unvernalized seeds of barley were sown in greenhouseunder short day or long day condition. At the second leaf stage,a half of the plants in each plot was sprayed with gibberellin.Plants grown from vernalized seeds headed at about the sametime under long day condition, whether they were treated withgibberellin or not. Under short day condition, however, gibberellinpromoted heading. With plants grown from the unvernalized seeds,gibberellin promoted heading under long day condition whereasit delayed heading under short day condition. Thus the substitutionof gibberellin for long day effect and for vernalization effectwas shown.
2. Immature embryos growing on developing ear of wheatand barleywere vernalized with low temperature combined withor withoutgibberellin application. Plants grown from the seedstreatedat their early ripening stage with low temperature combinedwith gibberellin gave earlier heading, associated with the decreasedleaf number, than the plants from seeds treated with low temperaturealone. This effect of gibberellin lasted for a half-year ofseed storage.
3. Gibberellin was applied to germinating seedsunder vernalizationtreatment. Gibberellin promoted floweringin varieties whichpossess high requirement for chilling vernalization,when thechilling was insufficient to satisfy the high requirement.However,the effect of gibberellin was obscure with varietiespossessingrelatively low requirement for vernalization.

(Received September 15, 1964; )     

光周期对中华通草蛉自然越冬成虫及实验种群幼虫耐寒能力的影响

【目的】滞育诱导期进行短光照处理可影响昆虫耐寒性。为明确光周期对中华通草蛉Chrysoperla sinica (Tjeder)耐寒性的影响, 针对中华通草蛉滞育解除过程及非滞育虫态的耐寒性进行了一系列研究。【方法】测定了中华通草蛉自然越冬成虫的过冷却点（supercooling point, SCP）以及长光周期（15L∶9D）和短光周期（9L∶15D）条件下自然越冬成虫在滞育解除过程中在-12℃下的死亡率, 并测定了室内长、 短两种光周期下实验种群2龄和3龄幼虫的过冷却点（SCP）、 结冰点（freezing point, FP）以及-7℃下的死亡率。【结果】中华通草蛉12月份的自然越冬成虫SCP集中在-10~-14℃之间。SCP低于-12℃的个体占43.70%, 且-12℃处理1 d死亡率为62.00%。-12℃处理1 d条件下的长、 短光周期处理自然越冬成虫, 除处理0 d外, 长光周期处理死亡率均高于短光周期处理的, 且在处理15 （P=0.012）, 20 （P=0.01）和25 d （P=0.001）差异显著。中华通草蛉试验种群相同龄期幼虫在短光周期下的SCP和FP均高于长光周期下, 但差异不显著(P>0.05); 但在-7℃下, 2龄幼虫短光周期下的低温死亡率为67.00%±4.04%, 显著低于长光周期下的低温死亡率（78.00%±1.33%）（P=0.011）, 3龄幼虫短光周期条件下低温死亡率为24.33%±1.33%, 显著低于长光周期下的低温死亡率（53.00%±3.46%）（P=0.002）。【结论】中华通草蛉为结冰敏感型, 诱导滞育的短光照处理可提高其幼虫期及滞育解除过程中成虫的耐寒能力。     

Induction of Frost Hardiness in Stem Cortical Tissues of Cornus stolonifera Michx. by Water Stress: I. Unfrozen Water in Cortical Tissues and Water Status in Plants and Soil

Water supply and day length were varied in cold hardiness studies of red osier dogwood plants (Cornus stolonifera Michx.). The frost killing temperature, the content and freezing of stem cortical tissue water along with soil moisture content and tension were evaluated. Seven days of water stress in long and short day photoperiod regimes caused a rapid decrease in soil moisture content and plant water potential. During the same period, the frost hardiness increased from −3 to −11 C. Further water stress treatment had little effect. Control plants in short days showed only a gradual decrease in plant water potential and only gradually increased in frost hardiness while control plants in long days were unchanged. Freezing studies using nuclear magnetic resonance showed that increased hardiness in water-stressed plants resulted from both an increased tolerance of freezing and an increased avoidance of freezing, the latter resulting from higher solute concentration in the tissue solutions. The short day controls also showed similar changes; however, the changes were smaller over the 21 days of the study.     

Is tissue culture a viable system with which to examine environmental and hormonal regulation of cold acclimation in woody plants?

In vitro-grown saskatoon berry (Amelanchier alnifolia Nutt.) plantlets were exposed to various hormonal treatments, dormancy-inducing and cold acclimation conditions to determine if this in vitro system would be viable for dormancy/hardiness studies in woody plants. Low temperature induced significant hardiness levels in plantlets to ?27°C after 6 weeks at 4°C but did not approach liquid nitrogen levels of fully hardened, field-grown buds. Control plantlets were consistently killed at ?5°C throughout this period. Significant hardiness was attained under both short and long day/low temperature conditions; however, hardiness was reduced under continuous light or dark treatments. A pre-exposure to the typical short photoperiod regime of woody plants did not significantly increase the rate of acclimation in these plantlets. The presence/absence of phytohormones in the media have a pronounced influence on the ability of plantlets to cold acclimate. Hormone-free media increased hardiness to ?10.5°C after 2 weeks in treatment. Addition of abscisic acid (ABA) increased cold hardiness levels (?12°C) while addition of benzylaminopurine (BAP) to this hormone-free media decreased hardiness to ?5.3°C. A combination of BAP and ABA treatments produced LT₅₀ values intermediate between individual applications of either hormone. Conversely, α-naphthaleneacetic acid (NAA) could not counteract the ABA-induced hardiness. ABA treatments alone were not able to harden plantlets to the extent attained under low temperature acclimation conditions. Further, ABA could not maintain the hardiness levels of cold-acclimating treatments and plantlets de-acclimated to ?9°C in BAP + ABA media. Subculturing in itself significantly elevated cold hardiness in plantlets to ?9°C on BAP + NAA media within 3 days after subculture and thereafter plantlets dehardened to ?5°C. While tissue culture has value in specific cases, caution should be taken when using tissue-cultured plantlets as a system to evaluate environmental regulation of cold acclimation in woody plants, in part, due to the influence of phytohormones in the media.     

The environmental control of cold acclimation in apple

The role of photoperiod and temperature in the cold acclimation of living Haralson apple (Pyrus malus L.) bark was studied in the autumn under field conditions in Minnesota. Whole trees, or different parts of the same tree, were exposed to either natural conditions, artifically lengthened days, or artificially warmed nights, or they were subjected to manual leaf removal. The results indicate that acclimation occurs in two stages which are induced by short days and frost (or low temperature), respectively. Leaves were stimulated by short days to produce translocatable substance(s) which promoted cold acclimation of the living bark. Leaves of plants grown under long days were the source of a translocatable substance(s) which inhibited acclimation. The second stage of hardiness, induced by frost (or low temperature), did not involve translocatable factors.     

Effects of photoperiod on growth rate and endogenous gibberellins in the long-day rosette plant spinach

The earliest visible responses of spinach plants (Spinacia oleracea L., cv. Savoy Hybrid 612) transferred from short to long days (8 hours of high intensity light supplemented with 16 hours of low intensity illumination from incandescent lamps) were upright leaf orientation and increased elongation of the petioles. The effect of long days on growth rate was direct; i.e., there was no after-effect if the plants were transferred to short days. Gibberellin A₃ applied to plants under short days had an effect similar to that of long days, whereas application of the growth retardant AMO-1618 [2′-isopropyl-4′-(trimethylammonium chloride)-5′-methylphenyl piperidinel-carboxylate] under long days caused a growth habit typical of short-day conditions. Gibberellin A₃ caused more stem growth in plants under long days in which the endogenous gibberellin content had been reduced by AMO-1618 than in plants under short days not treated with the growth retardant.     

Effects of Daylength on Endogenous Gibberellins in Leaves of Solanum andigena

Less gibberellin-like activity was detected in extracts of potato leaves under short day than under long day conditions. Significant decreases in gibberellin levels were detected after two short day cycles. Using thin-layer chromatography coupled with several solvent systems and various bioassays, at least four zones of gibberellin-like activity, A, B, C and D were found in the potato leaf. Under long days only zones C and D were apparently present. However, preliminary purification using polyamide columns revealed that substances A and B were actually present in higher levels under long day than under short day conditions. The chloroplasts of leaves exposed to short days contained less gibberellin-like substances than those from leaves grown under permanent long day conditions.     

Effects of Daylenth on Endogenous Gibberellins in Solanum andigena

When tritiated gibberellin A₁ was fed to intact potato plants growing under different daylength conditions it was converted to a more polar acidic metabolite. The rate of metabolism was the same under both long and short day conditions. Attempts were made to obtain information about the possible sites within the cell where gibberellin metabolism took place.     

Auxin-induced Fruit Set in Capsicum annuum L. Requires Downstream Gibberellin Biosynthesis

A hierarchical scheme for the central role of the plant hormones auxin and gibberellins in fruit set and development has been established for the model plants Arabidopsis and tomato. In the fruit crop Capsicum annuum, the importance of auxin as an early signal in fruit set has also been recognized; however, the effect of gibberellins and their interaction with auxin has not yet been studied. The aim of this study was to determine the role of gibberellin and the hierarchy between auxin and gibberellin. We applied gibberellin alone or in combination with auxin or with the gibberellin biosynthesis inhibitor paclobutrazol on stigmas of emasculated flowers. Gibberellin application enhanced fruit set, whereas application of paclobutrazol reduced fruit set. The effect of paclobutrazol treatment could be counteracted by coapplication of gibberellin but not by auxin_. These results indicate that in C. annuum, like in Arabidopsis and tomato, auxin is the major inducer of fruit set that acts in part by inducing gibberellin biosynthesis. Interestingly, gibberellin does not significantly contribute to the final fruit size but seems to play an important role in preventing flower and fruit abscission, a major determinant of production loss in C. annuum. At the same time, gibberellin together with auxin seems to balance cell division and cell expansion during fruit growth.