The influence of magnesium deficiency on carbohydrate concentrations in Norway spruce (Picea abies) needles

Both short- and long-term effects of Mg deficiency on carbohydrate metabolism were investigated in 6-year-old clonal Norway spruce (Picea abies (L.) Karst.) trees cultivated in sand culture with an optimal supply of nutrients, except for Mg which was supplied at 0.203, 0.041 and 0.005 mM to provide optimal, moderately deficient and severely deficient Mg supply, respectively. Annual changes in carbohydrate concentrations (starch, sucrose, glucose and fructose) were analyzed and diurnal changes were investigated on a single day during the summer. Older needles of trees in the moderate Mg-deficiency treatment developed tip-yellowing symptoms, whereas current-year needles remained green. The severe Mg-deficiency treatment led to pronounced yellowing symptoms in needles of all ages. Increased carbohydrate concentrations were observed before needle yellowing occurred. Diurnal and annual changes in carbohydrates were similar in all treatments; however, carbohydrate concentrations were influenced by Mg supply. In both Mg-deficiency treatments, starch concentrations increased in needles, especially during summer and autumn. Starch accumulation was more pronounced at the beginning of the Mg-deficiency treatments than at the end of the treatments. Sucrose, and to a minor extent, glucose and fructose concentrations tended to increase in response to Mg deficiency. The consequences of Mg deficiency on carbohydrate metabolism are discussed with respect to reduced plant growth and decreased transport rates of carbohydrates to sink organs.     

Levels of IAA,ABA and carbohydrates in source and sink leaves of Betula pendula Roth.

Young birch (Betula pendula Roth) seedlings were grown in hydroponic cultures to which nutrients were added in amounts that increased exponentially over time. The nutrient additions were adjusted to give three different suboptimal, but stable, relative growth rates (RGR). Levels of glucose, fructose, sucrose and starch and the hormones 3‐Indolyl acetic acid (IAA) and abscisic acid (ABA) were determined in immature (sink) and mature (source) leaves and related to the measured RGR of the seedlings. The results show that ABA increased and IAA decreased in the sink leaves as the RGR of the plants decreased. This occurred in concert with a decrease in soluble sugar levels and starch accumulation in the source leaves. These observations suggest that ABA and IAA may be involved in source‐sink communication in the seedlings, although such causal relationships remain to be proven.     

Photosynthesis down-regulation precedes carbohydrate accumulation under sink limitation in Citrus

Photosynthesis down-regulation due to an imbalance between sources and sinks in Citrus leaves could be mediated by excessive accumulation of carbohydrates. However, there is limited understanding of the physiological role of soluble and insoluble carbohydrates in photosynthesis regulation and the elements triggering the down-regulation process. In this work, the role of non-structural carbohydrates in the regulation of photosynthesis under a broad spectrum of source-sink relationships has been investigated in the Salustiana sweet orange. Soluble sugar and starch accumulation in leaves, induced by girdling experiments, did not induce down-regulation of the photosynthetic rate in the presence of sinks (fruits). The leaf-to-fruit ratio did not modulate photosynthesis but allocation of photoassimilates to the fruits. The lack of strong sink activity led to a decrease in the photosynthetic rate and starch accumulation in leaves. However, photosynthesis down-regulation due to an excess of total soluble sugars or starch was discarded because photosynthesis and stomatal conductance reduction occurred prior to any significant accumulation of these carbohydrates. Gas exchange and fluorescence parameters suggested biochemical limitations to photosynthesis. In addition, the expression of carbon metabolism-related genes was altered within 24 h when strong sinks were removed. Sucrose synthesis and export genes were inhibited, whereas the expression of ADP-glucose pyrophosphorylase was increased to cope with the excess of assimilates. In conclusion, changes in starch and soluble sugar turnover, but not sugar content per se, could provide the signal for photosynthesis regulation. In these conditions, non-stomatal limitations strongly inhibited the photosynthetic rate prior to any significant increase in carbohydrate levels.     

Seasonal variation in biomass and carbohydrate partitioning of understory sugar maple (Acer saccharum) and yellow birch (Betula alleghaniensis) seedlings

We investigated seasonal patterns of biomass and carbohydrate partitioning in relation to shoot growth phenology in two age classes of sugar maple (Acer saccharum Marsh.) and yellow birch (Betula alleghaniensis Britt.) seedlings growing in the understory of a partially harvested forest. The high root:shoot biomass ratio and carbohydrate concentration of sugar maple are characteristic of species with truncated growth patterns (i.e., cessation of aboveground shoot growth early in the growing season), a conservative growth strategy and high shade tolerance. The low root:shoot biomass ratio and carbohydrate concentration of yellow birch are characteristic of species with continuous growth patterns, an opportunistic growth strategy and low shade tolerance. In both species, starch represented up to 95% of total nonstructural carbohydrates and was mainly found in the roots. Contrary to our hypothesis, interspecific differences in shoot growth phenology (i.e., continuous versus truncated) did not result in differences in seasonal patterns of carbohydrate partitioning. Our results help explain the niche differentiation between sugar maple and yellow birch in temperate, deciduous understory forests.     

Boron remobilization at low boron supply in olive (Olea europaea) in relation to leaf and phloem mannitol concentrations

For plant species in which a considerable portion of the photoassimilates are translocated in the phloem as sugar alcohols, boron is freely translocated from mature organs to growing tissues. However, the effects of decreased plant boron status on boron remobilization are poorly understood. We conducted a growth chamber experiment (CE) and a field experiment (FE) to study the effects of low boron supply on boron remobilization in olive (Olea europaea L.), a species that transports considerable amounts of mannitol in the phloem. For the CE, several physiological parameters were compared between control (B+) and boron-deficient olive plants (B-) during the expansion of new leaves. Boron remobilization was assessed by measuring boron content of selected leaves at the beginning and at the end of the CE. As expected, boron was remobilized from mature leaves to young leaves of B+ plants; however, considerable boron remobilization was also observed in B- plants, suggesting a mechanism whereby olive can sustain a minimum boron supply for growth of new tissues despite an insufficient external boron supply. Boron deficiency caused inhibition of new growth but had no effect on photosynthetic capacity per unit leaf surface area of young and mature leaves, thereby altering the carbon utilization pattern and resulting in carbon allocation to structures within the source leaves and accumulation of soluble carbohydrates. Specifically, in mature B- leaves in the CE and in B- leaves in the FE, mannitol concentration on a leaf water content basis increased by 48 and 27% respectively, compared with controls. Carbon export ability (assessed by both phloem anatomy and phloem exudate composition of FE leaves) was enhanced at low boron supply. We conclude that, at low boron supply, increased mannitol concentrations maintain boron remobilization from source leaves to boron-demanding sink leaves.     

Seasonal patterns of carbohydrate reserves in red spruce seedlings

We studied seasonal dynamics of carbohydrate storage in red spruce (Picea rubens Sarg.) seedlings by measuring starch and sugar concentrations of old needles (>/= one year old), new needles (< one year old), stems, and roots in two stands in the Green Mountains of Vermont. Although the two stands differed in many site characteristics including percent slope, aspect, soil type, drainage, and 564 m in elevation, concentrations and seasonal patterns of carbohydrates were similar for the two stands. For all tissues, starch concentrations peaked in late spring, declined through summer, and reached a minimum in winter. Sugar concentrations were greater than starch concentrations in all months except May and June. Sugar concentrations peaked in winter, and old needles showed a significant increase in sugar concentration between February and March. This increase in sugar concentration occurred without any reduction in localized starch concentrations or reductions in sugar or starch concentrations in new needles, stems or roots. Because March measurements were made toward the end of a prolonged thaw, a time when increases in photosynthesis have been documented for red spruce, it is likely that the March increase in sugar concentrations resulted from photosynthesis during the thaw. Compared with stems and roots, needles generally contained the highest concentration of carbohydrates and exhibited the greatest seasonal change in carbohydrate concentration. Needles were also the largest reservoir of carbohydrates throughout the year, especially during winter. Because of the critical roles of needles in photosynthesis and storage of carbohydrates, we conclude that any factors that disrupt the accumulation or availability of carbohydrates in red spruce needles will greatly alter plant carbon relations.     

Carbon gain and bud physiology in Populus tremuloides and Betula papyrifera grown under long-term exposure to elevated concentrations of CO2 and O3

Paper birch (Betula papyrifera Marsh.) and three trembling aspen clones (Populus tremuloides Michx.) were studied to determine if alterations in carbon gain in response to an elevated concentration of CO(2) ([CO(2)]) or O(3) ([O(3)]) or a combination of both affected bud size and carbohydrate composition in autumn, and early leaf development in the following spring. The trees were measured for gas exchange, leaf size, date of leaf abscission, size and biochemical characteristics of the overwintering buds and early leaf development during the 8th-9th year of free-air CO(2) and O(3) exposure at the Aspen FACE site located near Rhinelander, WI. Net photosynthesis was enhanced 49-73% by elevated [CO(2)], and decreased 13-30% by elevated [O(3)]. Elevated [CO(2)] delayed, and elevated [O(3)] tended to accelerate, leaf abscission in autumn. Elevated [CO(2)] increased the ratio of monosaccharides to di- and oligosaccharides in aspen buds, which may indicate a lag in cold acclimation. The total carbon concentration in overwintering buds was unaffected by the treatments, although elevated [O(3)] decreased the amount of starch by 16% in birch buds, and reduced the size of aspen buds, which may be related to the delayed leaf development in aspen during the spring. Elevated [CO(2)] generally ameliorated the effects of elevated [O(3)]. Our results show that both elevated [CO(2)] and elevated [O(3)] have the potential to alter carbon metabolism of overwintering buds. These changes may cause carry-over effects during the next growing season.     

Effects of coppicing on the root and stump carbohydrate dynamics in birches

Whole birch stems were cut off in order to determine how coppicing affects root and stump starch, glucose, fructose and sucrose concentrations and their correlation with shoot regeneration capacity. The Betula pubescens Ehrh. and B. pendula Roth studied included intact trees, trees that had been coppiced 8 years earlier, trees coppiced at the beginning of the experimental season, and birches that had been coppiced twice, 8 years earlier and at the beginning of the experimental season. Carbohydrate accumulation differed between 8 years earlier coppiced and intact trees. Recent coppicing clearly decreased the starch and sugar concentrations of the roots, which were often highest in the thin roots. The concentrations of these compounds in the stumps were always low, although the carbohydrate concentrations of stumps, in particular, correlated with shoot regeneration capacity. Starch was the most labile of the carbohydrates measured and most clearly reacted to coppicing. Differences in starch- and sugar-reserve dynamics indicate a difference between these birch species in the use and replenishing of root and stump reserves. This information may also be of help when the effects of other stresses, for example, severe animal damage or burning, on the regrowth of young birch stands are estimated.     

Atmospheric carbon dioxide enrichment reduces carbohydrate and nitrogen reserves in overwintering Picea mariana

Abstract

Elevated levels of atmospheric carbon dioxide (CO₂) can directly affect the cold hardening process in evergreens through their effect on the accumulation of carbon and nitrogen reserves. This study investigated the biochemical responses of black spruce [Picea mariana (Mill.) B.S.P.] seedlings to CO₂ enrichment during growth, cold hardening and dehardening. Seedlings were grown under 350 (ambient) or 710 (elevated) ppm of CO₂ for 12 months in eight mini-greenhouses. Photoperiod and temperature were gradually lowered in autumn to induce cold hardening, and the conditions were reversed in spring to promote dehardening. At regular intervals, cold tolerance was assessed and sugars, starch and amino acid concentrations were measured. The freezing tolerance differed between the two treatments only in early autumn, with seedlings growing under high CO₂ being more tolerant. The northern ecotype was more cold tolerant with concomitant higher concentrations of sucrose, fructose, pinitol, glucose and total soluble sugars. The concentration of soluble sugars increased in needles and roots of black spruce along with cold hardening, and the concentrations of the cryoprotective sugars sucrose and raffinose were lower under elevated CO₂. Amino acid concentrations were also lower under elevated than under ambient CO_2. The lower level of reserve did not translate into a lower level of freezing tolerance.     

Genetic and environmental control of seasonal carbohydrate dynamics in trees of diverse Pinus sylvestris populations

We explored environmental and genetic factors affecting seasonal dynamics of starch and soluble nonstructural carbohydrates in needle and twig cohorts and roots of Scots pine (Pinus sylvestris L.) trees of six populations originating between 49 degrees and 60 degrees N, and grown under common garden conditions in western Poland. Trees of each population were sampled once or twice per month over a 3-year period from age 15 to 17 years. Based on similarity in starch concentration patterns in needles, two distinct groups of populations were identified; one comprised northern populations from Sweden and Russia (59-60 degrees N), and another comprised central European populations from Latvia, Poland, Germany and France (49-56 degrees N). Needle starch concentrations of northern populations started to decline in late spring and reached minimum values earlier than those of central populations. For all populations, starch accumulation in spring started when minimum air temperature permanently exceeded 0 degrees C. Starch accumulation peaked before bud break and was highest in 1-year-old needles, averaging 9-13% of dry mass. Soluble carbohydrate concentrations were lowest in spring and summer and highest in autumn and winter. There were no differences among populations in seasonal pattern of soluble carbohydrate concentrations. Averaged across all populations, needle soluble carbohydrate concentrations increased from about 4% of needle dry mass in developing current-year needles, to about 9% in 1- and 2-year-old needles. Root carbohydrate concentration exhibited a bimodal pattern with peaks in spring and autumn. Northern populations had higher concentrations of fine-root starch in spring and autumn than central populations. Late-summer carbohydrate accumulation in roots started only after depletion of starch in needles and woody shoots. We conclude that Scots pine carbohydrate dynamics depend partially on inherited properties that are probably related to phenology of root and shoot growth.     

Variations in amounts of carbohydrates, amino acids and adenine nucleotides in mulberry tree (Morus alba L.) stems during transitional phases of growth

Quantitative changes in carbohydrates, amino acids and adenine nucleotides in the stems of mulberry trees (Morus alba L., cv. Shin-ichinose) were followed from spring to early summer and from autumn to early spring. Both ATP and ADP content of stems increased before bud break, whereas the content of sucrose, the most abundant sugar among the stem carbohydrates, decreased. The sucrose content fell to its lowest value at the beginning of May, and then increased rapidly, whereas the starch content decreased suggesting consumption of reserve carbohydrate and simultaneous accumulation of current photosynthate. This was confirmed by studies in which reserve carbohydrates were labeled with (14)CO(2). Proline content of stems increased from the time of leaf shedding until early spring. Although it was the most abundant amino acid at the time of bud break, proline rapidly decreased as new shoots developed and was hardly detectable by the beginning of May. The asparagine and arginine contents increased transiently following bud break, and then decreased toward summer. Transient increases in glutamine and arginine were noted at the time of leaf shedding.     

Factors influencing red expression in autumn foliage of sugar maple trees

We evaluated factors influencing the development of autumn red coloration in leaves of sugar maple (Acer saccharum Marsh.) by measuring mineral nutrient and carbohydrate concentrations, water content, and phenology of color development of leaves from 16 mature open-grown trees on 12 dates from June through October 1999. Mean foliar nutrient and carbohydrate concentrations and water content were generally within the range published for healthy sugar maple trees. However, foliar nitrogen (N) concentrations were near deficiency values for some trees. The timing and extent of red leaf coloration was consistently correlated with both foliar N concentrations and starch or sugar concentrations, which also varied with N status. Leaves of trees with low foliar N concentrations turned red earlier and more completely than those of trees with high foliar N concentrations. Low-N trees also had higher foliar starch concentrations than high-N trees. During the autumn development of red leaf coloration, foliar starch, glucose and fructose concentrations were positively correlated with red leaf color expression. At peak red expression, the concentrations of glucose, fructose, sucrose and stachyose were all positively correlated with red color expressed as a percent of total leaf area.     

Early developmental responses of mountain birch (Betula pubescens subsp. czerepanovii) seedlings to different concentrations of phosphorus

Phenolic compounds, identified by high performance liquid chromatography (HPLC), and growth were investigated in the first true leaves, cotyledons, stems and roots of 2.5-week-old seedlings of mountain birch (Betula pubescens subsp. czerepanovii (Orlova) H?metahti) grown with 1.5, 15 and 50 ppm phosphorus (P). Phenolic compounds in seeds and unfertilized 6-day-old seedlings were also studied. Seeds contained few phenolics, but had an abundance of condensed tannins. There were fewer HPLC phenolic compounds in 6-day-old seedlings than in 2.5-week-old seedlings. In 2.5-week-old seedlings, growth of the first true leaves, but not of other plant parts, was affected by P regime. In the first true leaves and stems, the concentration of condensed tannins was significantly affected by P, being higher at 1.5 ppm than at higher concentrations. The concentration of total HPLC phenolics was significantly affected by P only in stems.     

Accumulation of flavonoids and related compounds in birch induced by UV-B irradiance

A growth chamber experiment was conducted to examine the effects of UV-B exposure (4.9 kJ m(-2) day(-1) of biologically effective UV-B, 280-320 nm) on shoot growth and secondary metabolite production in Betula pendula (Roth) and B. resinifera (Britt.) seedlings originating from environments in Finland, Germany and Alaska differing in solar UV-B radiation and climate. Neither shoot growth nor the composition of secondary metabolites was affected by UV-B irradiance, but the treatment induced significant changes in the amounts of individual secondary metabolites in leaves. Leaves of seedlings exposed to UV-B radiation contained higher concentrations of several flavonoids, condensed tannins and some hydroxycinnamic acids than leaves of control seedlings that received no UV-B radiation. At the population level, there was considerable variation in secondary metabolite responses to UV-B radiation: among populations, the induced response was most prominent in Alaskan populations, which were adapted to the lowest ambient UV-B radiation environment. I conclude that solar UV-B radiation plays an important role in the formation of secondary chemical characteristics in birch trees.     

Growth and nutrition of birch seedlings at varied relative addition rates of magnesium

Growth and nutrition of hydroponically cultivated birch seedlings (Betula pendula Roth.) were investigated at various magnesium (Mg) availabilities. Suboptimum Mg conditions were created by adding Mg once per hour in exponentially increasing amounts at one of four relative addition rates (R(Mg)): 0.05, 0.10, 0.15 or 0.20 day(-1). Seedlings given free access to Mg were used as controls. After an acclimation period, the relative growth rate of the seedlings attained the same value as the corresponding relative rate of Mg addition. In all suboptimum Mg treatments, deficiency symptoms in the form of chloroses and necroses developed in the older leaves, both during and after the phase of growth acclimation. The severity of these symptoms was correlated with the availability of Mg. The relative growth rate of seedlings was linearly correlated with plant Mg status. The root fraction of the total biomass decreased from 22% in control plants to 8% in plants receiving the lowest rate of Mg addition. A shift in Mg availability from free access to R(Mg) = 0.05 day(-1) decreased the photosynthetically active leaf area per plant weight, despite a concomitant increase in the leaf weight ratio (leaf dry weight/plant dry weight) from 0.61 to 0.75. The loss in assimilating leaf area was mainly a consequence of enhanced leaf mortality and formation of necroses, and to a minor extent attributable to increased carbon costs for leaf area production. A decrease in starch concentration was observed in leaves showing Mg-deficiency symptoms, whereas the starch concentration in healthy leaves was unaffected by Mg availability. It was concluded that shortage of carbohydrates constituted the major growth constraint, particularly for roots, under Mg-limiting conditions.     

Root Egress and Field Performance of Actively Growing Betula pendula Container Seedlings

The objective of this study was to evaluate the possibility of enlarging the planting window of container-grown silver birch seedlings from spring and autumn to summer by planting young, actively growing seedlings. For over 3 yrs the study investigated the growth, survival and damage of silver birch seedlings grown in containers and planted at different times during the growing season on 18 sites in fields and forest sites in central Finland. In a pot experiment in the greenhouse, the root-egress ability of seedlings planted at different times during the growing season was monitored. Root egress of seedlings was rapid from the beginning of July to the middle of August. Actively growing seedlings planted in summer on a site suitable for silver birch grew and survived at least as well as seedlings planted in spring during the dormant stage. The risk of drought stress increased when seedlings were planted on easily drying, coarse sandy soils. The risk was also high in soils with high levels of fine soil fractions that become hard when dry and when the dry period continued for several weeks before planting. In conclusion, it is possible to enlarge the planting window of birch seedlings to July and the beginning of August by using actively growing container seedlings.     

Leaf responses of micropropagated apple plants to water stress: nonstructural carbohydrate composition and regulatory role of metabolic enzymes

We examined changes in nonstructural carbohydrate biosynthesis and activities of related enzymes in leaves of micropropagated apple plants (Malus domestica Borkh. cv. 'NaganoFuji') in response to water stress, with particular emphasis on the enzymes associated with sorbitol, sucrose and starch metabolism. Water stress resulted in the accumulation of photosynthates in leaves, mainly sorbitol, sucrose, glucose and fructose, accompanied by a reduction in starch concentration. Correlation and path analysis indicated that water stress affected the partitioning of newly fixed carbon among terminal products. In response to water stress, ADP-glucose-pyrophosphorylase (ADPGPPase) activity decreased, becoming a critical and limiting step in shifting partitioning of photosynthetically fixed carbon. Amylase and ADPGPPase affected sucrose and sorbitol metabolism, mainly by regulating substrate supply; however, competition for limited substrate had a greater effect on the biosynthesis of sorbitol than of sucrose. Starch metabolism was also strictly regulated by ADPGPPase and amylase, whereas other related enzymes were downstream of the pathway for synthesis and degradation of carbohydrates and thus had relatively little effect on starch metabolism. Sorbitol dehydrogenase and sucrose phosphate synthase were critical regulators of sorbitol and sucrose metabolism, respectively.     

Trophic control of bud break in peach (Prunus persica) trees: a possible role of hexoses

Vegetative buds of peach (Prunus persica L. Batsch.) trees act as strong sinks and their bud break capacity can be profoundly affected by carbohydrate availability during the rest period (November-February). Analysis of xylem sap revealed seasonal changes in concentrations of sorbitol and hexoses (glucose and fructose). Sorbitol concentrations decreased and hexose concentrations increased with increasing bud break capacity. Sucrose concentration in xylem sap increased significantly but remained low. To clarify their respective roles in the early events of bud break, carbohydrate concentrations and uptake rates, and activities of NAD-dependent sorbitol dehydrogenase (SDH), sorbitol oxidase (SOX) and cell wall invertase (CWI) were determined in meristematic tissues, cushion tissues and stem segments. Only CWI activity increased in meristematic tissues shortly before bud break. In buds displaying high bud break capacity (during January and February), concentrations of sorbitol and sucrose in meristematic tissues were almost unchanged, paralleling their low rates of uptake and utilization by meristematic tissues, and indicating that sorbitol and sucrose play a negligible role in the bud break process. Hexose concentrations in meristematic tissues and glucose imported by meristematic tissues correlated positively with bud break capacity, suggesting that hexoses are involved in the early events of bud break. These findings were confirmed by data for buds that were unable to break because they had been collected from trees deprived of cold. We therefore conclude that hexoses are of greater importance than sorbitol or sucrose in the early events of bud break in peach trees.     

Comparison of the photosynthetic capacity of Siberian and Japanese birch seedlings grown in elevated CO(2) and temperature

The effects of increased CO(2) and temperature on the photosynthetic capacity of Siberian white birch and Japanese white birch (Betula platyphylla Sukatch. and B. platyphylla Sukatch. var. japonica Hara) were measured. Birch seedlings were raised with a CO(2) partial pressure of 36 +/- 0.3 Pa (i.e., ambient) or 70 +/- 0.6 Pa at day/night temperatures of either 30/16 degrees C or 26/12 degrees C. Siberian birch leaves were smaller and thicker than Japanese birch leaves. Water use efficiency and nitrogen use efficiency of Siberian birch grown in the CO(2)-enriched air were higher than those of Japanese birch. Both species showed a physiological adjustment to the growth CO(2) partial pressure. Carboxylation efficiency and quantum yield of both species grown in CO(2)-enriched air were lower than those of seedlings grown in ambient CO(2). The adaptation of Siberian and Japanese birch to elevated CO(2) and temperature are discussed in relation to predicted climate change.     

Frost hardening of Pinus radiata seedlings: effects of temperature on relative growth rate,carbon balance and carbohydrate concentration

Pinus radiata (D. Don) seedlings were grown for 100 days at day/night temperatures of 10/1, 15/1, 20/1 and 25/1 degrees C, to determine whether temperatures above a threshold of 5 degrees C influence frost hardiness development. Relationships between hardening and relative growth rate, carbohydrate concentration and net carbon balance were also investigated. Seedlings hardened at a nearly constant rate in each treatment, although the rate of hardening was strongly temperature dependent. It increased as the temperature declined, but in a curvilinear fashion. Temperatures below 9.5 degrees C were effective in hardening the seedlings. During the daily temperature cycle, dehardening occurred at temperatures above the threshold, whereas hardening occurred at temperatures below the threshold. The net difference between the two processes determined the development of frost hardiness. The development of frost hardiness was negatively correlated with relative growth rate and positively correlated with the accumulation of starch and sugars. We conclude that frost hardening is a complex process that is causally linked to carbohydrate concentrations.