Changing winter climate and snow conditions induce various transcriptional stress responses in Scots pine seedlings.

In northern boreal forests the warming winter climate leads to more frequent snowmelt, rain-on-snow events and freeze-thaw cycles. This may be harmful or even lethal for tree seedlings that spend even a half of the year under snow. We conducted a snow cover manipulation experiment in a natural forest to find out how changing snow conditions affect young Scots pine (Pinus sylvestris L.) seedlings. The ice encasement (IE), absence of snow (NoSNOW) and snow compaction (COMP) treatments affected ground level temperature, ground frost and subnivean gas concentrations compared to the ambient snow cover (AMB) and led to the increased physical damage and mortality of seedlings. The expression responses of 28 genes related to circadian clock, aerobic and anaerobic energy metabolism, carbohydrate metabolism and stress protection revealed that seedlings were exposed to different stresses in a complex way depending on the thickness and quality of the snow cover. The IE treatment caused hypoxic stress and probably affected roots which resulted in reduced water uptake in the beginning of the growing season. Without protective snowpack in NoSNOW seedlings suffered from cold and drought stresses. The combination of hypoxic and cold stresses in COMP evoked unique transcriptional responses including oxidative stress. Snow cover manipulation induced changes in the expression of several circadian clock related genes suggested that photoreceptors and the circadian clock system play an essential role in the adaptation of Scots pine seedlings to stresses under different snow conditions. Our findings show that warming winter climate alters snow conditions and consequently causes Scots pine seedlings various abiotic stresses, whose effects extend from overwintering to the following growing season.

Separating the effects of air and soil temperature on silver birch. Part II. The relation of physiology and leaf anatomy to growth dynamics.

The aboveground parts of boreal forest trees grow earlier in the growing season, the roots mostly later. The idea was to examine whether root growth followed soil temperature, or whether shoot growth also demanded most resources in the early growing season (soil temperature vs internal sink strengths for resources). The linkage between air and soil temperature was broken by switching the soil temperature. We aimed here to identify the direct effects of different soil temperature patterns on physiology, leaf anatomy and their interactions, and how they relate to the control of the growth dynamics of silver birch (Betula pendula Roth). Sixteen 2-year-old seedlings were grown in a controlled environment for two 14-week simulated growing seasons (GS1, GS2). An 8-week dormancy period interposed the GSs. In GS2, soil temperature treatments were applied: constant 10 °C (Cool), constant 18 °C (Warm), early growing season at 10 °C switched to 18 °C later (Early Cool Late Warm) and 18 °C followed by 10 °C (Early Warm Late Cool) were applied during GS2. The switch from cool to warm enhanced the water status, net photosynthesis, chlorophyll content index, effective yield of photosystem II (ΔF/Fm') and leaf expansion of the seedlings. Warm treatment increased the stomatal number per leaf. In contrast, soil cooling increased glandular trichomes. This investment in increasing the chemical defense potential may be associated with the decreased growth in cool soil. Non-structural carbohydrates were accumulated in leaves at a low soil temperature showing that growth was more hindered than net photosynthesis. Leaf anatomy differed between the first and second leaf flush of silver birch, which may promote tree fitness in the prevailing growing conditions. The interaction of birch structure and function changes with soil temperature, which can further reflect to ecosystem functioning.

Host species shape the community structure of culturable endophytes in fruits of wild berry species (Vaccinium myrtillus L., Empetrum nigrum L. and Vaccinium vitis-idaea L.).

Wild berries are interesting research subjects due to their rich sources of health-beneficial phenolic compounds. However, the internal microbial communities, endophytes, associated with the wild berry fruits are currently unknown. Endophytes are bacteria or fungi inhabiting inside plant tissues, and their functions vary depending on the host species and environmental parameters. The present study aimed to examine community composition of fungal and bacterial endophytes in fruits of three wild berry species (bilberry Vaccinium myrtillus L., lingonberry Vaccinium vitis-idaea L. and crowberry Empetrum nigrum L.) and the effects of host plant species and their growth sites on shaping the endophytic communities. We found that the endophytic community structures differed between the berry species, and fungi were predominant over bacteria in the total endophytic taxa. We identified previously unknown endophytic fungal taxa including Angustimassarina, Dothidea, Fellozyma, Pseudohyphozyma, Hannaella coprosmae and Oberwinklerozyma straminea. A role of soluble phenolic compounds, the intracellular components in wild berry fruits, in shaping the endophytic communities is proposed. Overall, our study demonstrates that each berry species harbors a unique endophytic community of microbes.

Here comes the flood! Stress effects of continuous and interval waterlogging periods during the growing season on Scots pine saplings.

Future climate scenarios for the boreal zone project increasing temperatures and precipitation, as well as extreme weather events such as heavy rain during the growing season. This can result in more frequent short-term waterlogging (WL) leading to unfavorable conditions for tree roots. In addition, it is decisive whether short-term WL periods during the growing season occur continuously or periodically. We assessed the effects of short-termed WL on 4-year-old Scots pine (Pinus sylvestris L.) saplings after shoot elongation started. Waterlogging (WL) lasted either continuously for 2.5 weeks (ContWL) or noncontinuously for 5 weeks, consisting of three repeated 1-week-interval WL periods (IntWL). Both treatments resulted in the same duration of soil anoxia. We studied soil gases, root and shoot growth and physiology, and root survival probability and longevity during the experiment. In the final harvest, we determined shoot and root biomass and hydraulic conductance and electrical impedance spectra of the root systems. Soil CO2 and CH4 concentrations increased immediately after WL onset and O2 decreased until anoxia. Waterlogging decreased fine root survival probability, but there was no difference between WL treatments. Shoot growth suffered more from ContWL and root growth more from IntWL. Needle concentrations of pinitol increased in the WL saplings, indicating stress. No WL effects were observed in photosynthesis and chlorophyll fluorescence. Increased starch concentration in needles by WL may be due to damaged roots and thus a missing belowground sink. Electrical impedance indicated suffering of WL saplings, although root hydraulic conductance did not differ between the treatments. Oxidative stress of short-term and interval WL can have long-lasting effects on shoot and root growth and the physiology of Scots pine. We conclude that even short-term WL during the growing season is a stress factor, which will probably increase in the future and can affect carbon allocation and dynamics in boreal forests.

Let it snow! Winter conditions affect growth of birch seedlings during the following growing season.

Air temperatures and precipitation are predicted to increase in the future, especially at high latitudes and particularly so during winter. In contrast to air temperatures, changes in soil temperatures are more difficult to predict, as the fate of the insulating snow cover is crucial in this respect. Soil conditions can also be affected by rain-on-snow events and warm spells during winter, resulting in freeze-thaw cycles, compacted snow, ice encasement and local flooding. These adverse conditions during winter could counteract the otherwise positive effects of climate change on forest growth and productivity. For studying the effects of different winter and snow conditions on young Downy birch (Betula pubescens Ehrh.) seedlings, we carried out a laboratory experiment with birch seedlings subjected to four different winter scenarios: snow covering the seedlings (SNOW), compressed snow and ice encasement (ICE), flooded and frozen soil (FLOOD) and no snow at all (NO SNOW). After the winter treatments we simulated a spring and early summer period of 9.5 weeks, and monitored the growth by measuring shoot and root biomass of the seedlings, and starch and soluble sugar concentrations. We also assessed the stress experienced by the seedlings by measuring leaf chlorophyll fluorescence and gas exchange. Although no difference in mortality was observed between the treatments, the seedlings in the SNOW and ICE treatments had significantly higher shoot and root biomass compared with those in the FLOOD and NO SNOW treatments. We found higher starch concentrations in roots of the seedlings in the SNOW and ICE treatments, compared with those in the FLOOD and NO SNOW treatments, although photosynthesis did not differ. Our results suggest a malfunction of carbohydrate distribution in the seedlings of the FLOOD and NO SNOW treatments, probably resulting from decreased sinks. The results underline the importance of an insulating and protecting snow cover for small tree seedlings, and that future winters with changed snow pattern might affect the growth of tree seedlings and thus possibly species composition and forest productivity.

Bacterial and fungal communities in boreal forest soil are insensitive to changes in snow cover conditions.

The northern regions are experiencing considerable changes in winter climate leading to more frequent warm periods, rain-on-snow events and reduced snow pack diminishing the insulation properties of snow cover and increasing soil frost and freeze-thaw cycles. In this study, we investigated how the lack of snow cover, formation of ice encasement and snow compaction affect the size, structure and activities of soil bacterial and fungal communities. Contrary to our hypotheses, snow manipulation treatments over one winter had limited influence on microbial community structure, bacterial or fungal copy numbers or enzyme activities. However, microbial community structure and activities shifted seasonally among soils sampled before snow melt, in early and late growing season and seemed driven by substrate availability. Bacterial and fungal communities were dominated by stress-resistant taxa such as the orders Acidobacteriales, Chaetothyriales and Helotiales that are likely adapted to adverse winter conditions. This study indicated that microbial communities in acidic northern boreal forest soil may be insensitive to direct effects of changing snow cover. However, in long term, the detrimental effects of increased ice and frost to plant roots may alter plant derived carbon and nutrient pools to the soil likely leading to stronger microbial responses.

Gender Dimorphism Does Not Affect Secondary Compound Composition in Juniperus communis After Shoot Cutting in Northern Boreal Forests.

Due to a difference in plant resource allocation to reproduction, the males of dioecious plants may be more growth-orientated, whereas females may allocate more resources for synthesizing secondary compounds. This mechanism is considered to cause gender-specific differences in the plant responses to the loss of plant biomass. Here, we tested gender dimorphism in the responses of common juniper (Juniperus communis) to shoot cutting in four juniper populations located in northern boreal forests in Finland. We collected shoots from uncut junipers and from junipers subjected to shoot cutting in the previous year, and analyzed them for their shoot growth as well as phenolic and terpenoid concentrations. There were no differences in foliar phenolic or terpenoid concentrations between the males and the females. Shoot cutting increased phenolic but not terpenoid concentrations, similarly, in both males and females. Our study reveals that the nature of gender dimorphism may differ among species and locations, which should be considered in theories on plant gender dimorphism. Given the similar phenolic and terpene concentrations in both genders, the different sexes in the northern juniper populations might experience equal levels of herbivory. This lack of gender dimorphism in biotic interactions could result from the high need of plant secondary metabolites (PSM) against abiotic stresses, which is typical for juniper at high latitudes.

Winter survival of Scots pine seedlings under different snow conditions.

Future climate scenarios predict increased air temperatures and precipitation, particularly at high latitudes, and especially so during winter. Soil temperatures, however, are more difficult to predict, since they depend strongly on the fate of the insulating snow cover. 'Rain-on-snow' events and warm spells during winter can lead to thaw-freeze cycles, compacted snow and ice encasement, as well as local flooding. These adverse conditions could counteract the otherwise positive effects of climatic changes on forest seedling growth. In order to study the effects of different winter and snow conditions on young Scots pine (Pinus sylvestris L.) seedlings, we conducted a laboratory experiment in which 80 1-year-old Scots pine seedlings were distributed between four winter treatments in dasotrons: ambient snow cover (SNOW), compressed snow and ice encasement (ICE), flooded and frozen soil (FLOOD) and no snow (NO SNOW). During the winter treatment period and a 1.5-month simulated spring/early summer phase, we monitored the needle, stem and root biomass of the seedlings, and determined their starch and soluble sugar concentrations. In addition, we assessed the stress experienced by the seedlings by measuring chlorophyll fluorescence, electric impedance and photosynthesis of the previous-year needles. Compared with the SNOW treatment, carbohydrate concentrations were lower in the FLOOD and NO SNOW treatments where the seedlings had almost died before the end of the experiment, presumably due to frost desiccation of aboveground parts during the winter treatments. The seedlings of the ICE treatment showed dead needles and stems only above the snow and ice cover. The results emphasize the importance of an insulating and protecting snow cover for small forest tree seedlings, and that future winters with changed snow patterns might affect the survival of tree seedlings and thus forest productivity.

The Snow Must Go On: Ground Ice Encasement, Snow Compaction and Absence of Snow Differently Cause Soil Hypoxia, CO2 Accumulation and Tree Seedling Damage in Boreal Forest.

At high latitudes, the climate has warmed at twice the rate of the global average with most changes observed in autumn, winter and spring. Increasing winter temperatures and wide temperature fluctuations are leading to more frequent rain-on-snow events and freeze-thaw cycles causing snow compaction and formation of ice layers in the snowpack, thus creating ice encasement (IE). By decreasing the snowpack insulation capacity and restricting soil-atmosphere gas exchange, modification of the snow properties may lead to colder soil but also to hypoxia and accumulation of trace gases in the subnivean environment. To test the effects of these overwintering conditions changes on plant winter survival and growth, we established a snow manipulation experiment in a coniferous forest in Northern Finland with Norway spruce and Scots pine seedlings. In addition to ambient conditions and prevention of IE, we applied three snow manipulation levels: IE created by artificial rain-on-snow events, snow compaction and complete snow removal. Snow removal led to deeper soil frost during winter, but no clear effect of IE or snow compaction done in early winter was observed on soil temperature. Hypoxia and accumulation of CO2 were highest in the IE plots but, more importantly, the duration of CO2 concentration above 5% was 17 days in IE plots compared to 0 days in ambient plots. IE was the most damaging winter condition for both species, decreasing the proportion of healthy seedlings by 47% for spruce and 76% for pine compared to ambient conditions. Seedlings in all three treatments tended to grow less than seedlings in ambient conditions but only IE had a significant effect on spruce growth. Our results demonstrate a negative impact of winter climate change on boreal forest regeneration and productivity. Changing snow conditions may thus partially mitigate the positive effect of increasing growing season temperatures on boreal forest productivity.

Phenolic responses of mountain crowberry (Empetrum nigrum ssp. hermaphroditum) to global climate change are compound specific and depend on grazing by reindeer (Rangifer tarandus).

Mountain crowberry (Empetrum nigrum ssp. hermaphroditum) is a keystone species in northern ecosystems and exerts important ecosystem-level effects through high concentrations of phenolic metabolites. It has not been investigated how crowberry phenolics will respond to global climate change. In the tundra, grazing by reindeer (Rangifer tarandus) affects vegetation and soil nutrient availability, but almost nothing is known about the interactions between grazing and global climate change on plant phenolics. We performed a factorial warming and fertilization experiment in a tundra ecosystem under light grazing and heavy grazing and analyzed individual foliar phenolics and crowberry abundance. Crowberry was more abundant under light grazing than heavy grazing. Although phenolic concentrations did not differ between grazing intensities, responses of crowberry abundance and phenolic concentrations to warming varied significantly depending on grazing intensity. Under light grazing, warming increased crowberry abundance and the concentration of stilbenes, but decreased e.g., the concentrations of flavonols, condensed tannins, and batatasin-III, resulting in no change in total phenolics. Under heavy grazing, warming did not affect crowberry abundance, and induced a weak but consistent decrease among the different phenolic compound groups, resulting in a net decrease in total phenolics. Our results show that the different phenolic compound groups may show varying or even opposing responses to warming in the tundra at different levels of grazing intensity. Even when plant phenolic concentrations do not directly respond to grazing, grazers may have a key control over plant responses to changes in the abiotic environment, reflecting multiple adaptive purposes of plant phenolics and complex interactions between the biotic and the abiotic factors.

Phenolic composition and antioxidant capacity of bilberry (Vaccinium myrtillus) leaves in Northern Europe following foliar development and along environmental gradients.

Bilberry is a characteristic field layer species in the boreal forests and is an important forage plant for herbivores of the North European ecosystem. Bilberry leaves contain high levels of phenolic compounds, especially hydroxycinnamic acids, flavonols, catechins, and proanthocyanidins. We investigated the phenolic composition of bilberry leaves in two studies, one following foliar development in forest and open areas, and the other along a wide geographical gradient from south to north boreal forests in Finland. An analysis of bilberry leaves collected in open and forest areas showed that major phenolic changes appeared in the first stages of leaf development, but, most importantly, synthesis and accumulation of flavonoids was delayed in the forest compared to the high light sites. Sampling along a geographical gradient in the boreal zone indicated that leaves from higher latitudes and higher altitudes had greater soluble phenolic and flavonol levels, higher antioxidant capacity, and lower contents of chlorogenic acid derivatives. The ecological significance of the results is discussed.

Effect of latitude and altitude on the terpenoid and soluble phenolic composition of juniper (Juniperus communis) needles and evaluation of their antibacterial activity in the boreal zone.

The demand for dry juniper (Juniperus communis) needles as a raw material for the food, pharmaceutical, and cosmetic industries has increased rapidly in recent years. Juniper needles are known to be rich in terpenoids and phenolics, but their chemical composition and antibacterial properties have not been well-characterized. In this study, we describe the soluble phenolic and terpenoid composition of juniper needles collected in Finland (n = 125) and demonstrate that the concentration of these compounds clearly increased with latitude and altitude with, however, a stronger latitudinal effect (a higher content of monoterpenoids, proanthocyanidins, and flavonols in northern latitudes). Analysis of methanolic extracts showed quite good activity against both antibiotic-sensitive and -resistant Staphylococcus aureus strains and suggested an important role of the soluble phenolic fraction. Finally, we demonstrate the relative lack of toxicity of juniper extracts on keratinocytes and fibroblastic cells, raising the possibility of their use in preventing bacterial skin infection.

Effect of the temperature and the exclusion of UVB radiation on the phenolics and iridoids in Menyanthes trifoliata L. leaves in the subarctic.

The long-term effects of UVB exclusion and temperature on the methanol extractable (ME) phenolics (flavonoids, phenolic acids) and iridoids of Menyanthes trifoliata L. (Mt) leaves were studied in northern Finland (68 degrees N) using wooden frames covered with filters for UVB exclusion (polyester filter), control (cellulose acetate filter) and ambient (no filter) conditions. Analysis of ambient plots showed no effect of the daily mean temperature (2sigma = 1.58 degrees C) on the leaf ME compound content and composition, but minimum temperatures decreased the flavonol content. UVB exclusion did not affect the total ME compound content but significantly decreased the proportion of flavonols concomitantly with an increase in iridoids. Due to its high iridoid content, Mt appears as an interesting model plant for studying the iridoid biosynthesis and its regulation under stress conditions.

Influence of solar UV radiation on the nitrogen metabolism in needles of Scots pine (Pinus sylvestris L.).

Needles of 20-year-old Scots pine (Pinus sylvestris L.) saplings were studied in an ultraviolet (UV) exclusion field experiment (from 2000 to 2002) in northern Finland (67 degrees N). The chambers held filters that excluded both UV-B and UV-A, excluded UV-B only, transmitted all UV (control), or lacked filters (ambient). UV-B/UV-A exclusion decreased nitrate reductase (NR) activity of 1-year-old needles of Scots pines compared to the controls. The proportion of free amino acids varied in the range 1.08-1.94% of total proteins, and was significantly higher in needles of saplings grown under UV-B/UV-A exclusion compared to the controls or UV-B exclusion. NR activity correlated with air temperature, indicating a "chamber effect". The study showed that both UV irradiance and increasing temperature are significant modulators of nitrogen (N) metabolism in Scots pine needles.

Changes in freezing tolerance, plasma membrane H+-ATPase activity and fatty acid composition in Pinus resinosa needles during cold acclimation and de-acclimation.

It has previously been suggested that plasma membrane ATPase (PM H+-ATPase, EC 3.6.1.3.) is a site of incipient freezing injury because activity increases following cold acclimation and there are published data indicating that activity of PM H+-ATPase is modulated by changes in lipids associated with the enzyme. To test and extend these findings in a tree species, we analyzed PM H+-ATPase activity and the fatty acid (FA) composition of glycerolipids in purified plasma membranes (PMs) prepared by the two-phase partition method from current-year needles of adult red pine (Pinus resinosa Ait.) trees. Freezing tolerance of the needles decreased from -56 degrees C in March to -9 degrees C in May, and increased from -15 degrees C in September to -148 degrees C in January. Specific activity of vanadate-sensitive PM H+-ATPase increased more than two-fold following cold acclimation, despite a concurrent increase in protein concentration. During de-acclimation, decreases in PM H+-ATPase activity and freezing tolerance were accompanied by decreases in the proportions of oleic (18:1) and linoleic (18:2) acids and increases in the proportions of palmitic (16:0) and linolenic (18:3) acids in total glycerolipids extracted from the plasma membrane fraction. This pattern of changes in PM H+-ATPase activity and the 18:1, 18:2 and 18:3 fatty acids was reversed during cold acclimation. In the PM fractions, changes in FA unsaturation, expressed as the double bond index (1 x 18:1 + 2 x 18:2 + 3 x 18:3), were closely correlated with changes in H+-ATPase specific activity (r2 = 0.995). Changes in freezing tolerance were well correlated with DBI (r2 = 0.877) and ATPase specific activity (r2 = 0.833) in the PM fraction. Total ATPase activity in microsomal fractions also closely followed changes in freezing tolerance (r2 = 0.969). We conclude that, as in herbaceous plants, simultaneous seasonal changes in PM H+-ATPase activity and fatty acid composition occur during cold acclimation and de-acclimation in an extremely winter hardy tree species under natural conditions, lending support to the hypothesis that FA-regulated PM H+-ATPase activity is involved in the cellular response underlying cold acclimation and de-acclimation.

Contribution of omega-3 fatty acid desaturase and 3-ketoacyl-ACP synthase II (KASII) genes in the modulation of glycerolipid fatty acid composition during cold acclimation in birch leaves.

Temperate and boreal tree species respond to low positive temperatures (LT) or a shortening of the photoperiod (SD) by inducing cold acclimation. One of the metabolic consequences of cold acclimation is an increase in fatty acid (FA) desaturation in membrane lipids, which allows functional membrane fluidity to be maintained at LT. The molecular mechanisms of FA desaturation were investigated in leaves of birch seedlings (Betula pendula) during cold acclimation. Four genes involved in FA biosynthesis were isolated: a 3-ketoacyl-ACP synthase II gene (BpKASII) involved in the elongation of palmitoyl-ACP to stearoyl-ACP, and three omega-3 FA desaturase genes (BpFAD3, BpFAD7, and BpFAD8) involved in the desaturation of linoleic acid (18:2) to alpha-linolenic acid (18:3). BpFAD7 was the main omega-3 FAD gene expressed in birch leaves, and it was down-regulated by LT under SD conditions. LT induced the expression of BpFAD3 and BpFAD8 and a synchronous increase in 18:3 occurred in glycerolipids. Changes in the photoperiod did not affect the LT-induced increase in 18:3 in chloroplast lipids (MGDG, DGDG, PG), but it modulated the LT response detected in extra-chloroplastic lipids (PC, PE, PI, PS). A decrease in the proportion of the 16-carbon FAs in lipids occurred at LT, possibly in relation to the regulation of BpKASII expression at LT. These results suggest that LT affects the whole FA biosynthesis pathway. They support a co-ordinated action of microsomal (BpFAD3) and chloroplast enzymes (BpFAD7, BpFAD8) in determining the level of 18:3 in extra-chloroplastic membranes, and they highlight the importance of dynamic lipid trafficking.

Factors affecting oligomerization status of UDP-glucose pyrophosphorylase.

UDP-glucose pyrophosphorylase (UGPase) is involved in the production of UDP-glucose, a key precursor to polysaccharide synthesis in all organisms. UGPase activity has recently been proposed to be regulated by oligomerization, with monomer as the active species. In the present study, we investigated factors affecting oligomerization status of the enzyme, using purified recombinant barley UGPase. Incubation of wild-type (wt) UGPase with phosphate or Tris buffers promoted oligomerization, whereas Mops and Hepes completely dissociated the oligomers to monomers (the active form). Similar buffer effects were observed for KK127-128LL and C99S mutants of UGPase; however, the buffers had a relatively small effect on the oligomerization status of the LIV135-137NIN mutant, impaired in deoligomerization ability and showing only 6-9% activity of the wt. Buffer composition had no effect on UGPase activity at UGPase protein concentrations below ca. 20 ng/ml. However, at higher protein concentration the activity in Tris, but not Mops nor Hepes, underestimated the amount of the enzyme. The data suggest that oligomerization status of UGPase can be controlled by subtle changes in an immediate environment (buffers) and by protein dilution. The evidence is discussed in relation to our recent model of UGPase structure/function, and with respect to earlier reports on the oligomeric integrity/activity of UGPases from eukaryotic tissues.

The small subunit ADP-glucose pyrophosphorylase ( ApS) promoter mediates okadaic acid-sensitive uidA expression in starch-synthesizing tissues and cells in Arabidopsis.

Transgenic plants of Arabidopsis thaliana Heynh., transformed with a bacterial beta-glucuronidase (GUS) gene under the control of the promoter of the small subunit (ApS) of ADP-glucose pyrophosphorylase (AGPase), exhibited GUS staining in leaves (including stomata), stems, roots and flowers. Cross-sections of stems revealed GUS staining in protoxylem parenchyma, primary phloem and cortex. In young roots, the staining was found in the root tips, including the root cap, and in vascular tissue, while the older root-hypocotyl axis showed prominent staining in the secondary phloem and paratracheary parenchyma of secondary xylem. The GUS staining co-localized with ApS protein, as found by tissue printing using antibodies against ApS. Starch was found only in cell and tissue types exhibiting GUS staining and ApS labelling, but not in all of them. For example, starch was lacking in the xylem parenchyma and secondary phloem of the root-hypocotyl axis. Sucrose potently activated ApS gene expression in leaves of wild-type (wt) plants, and in transgenic seedlings grown on sucrose medium where GUS activity was quantified with 4-methylumbelliferyl-beta-glucuronide as substrate. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, completely blocked expression of ApS in mature leaves of wt plants and prevented GUS staining in root tips and flowers of the transgenic plants, suggesting a similar signal transduction mechanism for ApS expression in various tissues. The data support the key role of AGPase in starch synthesis, but they also underlie the ubiquitous importance of the ApS gene for AGPase function in all organs/tissues of Arabidopsis.

Purification, cloning, and properties of an acyltransferase controlling shikimate and quinate ester intermediates in phenylpropanoid metabolism.

A protein hydrolyzing hydroxycinnamoyl-CoA esters has been purified from tobacco stem extracts by a series of high pressure liquid chromatography steps. The determination of its N-terminal amino acid sequence allowed design of primers permitting the corresponding cDNA to be cloned by PCR. Sequence analysis revealed that the tobacco gene belongs to a plant acyltransferase gene family, the members of which have various functions. The tobacco cDNA was expressed in bacterial cells as a recombinant protein fused to glutathione S-transferase. The fusion protein was affinity-purified and cleaved to yield the recombinant enzyme for use in the study of catalytic properties. The enzyme catalyzed the synthesis of shikimate and quinate esters shown recently to be substrates of the cytochrome P450 3-hydroxylase involved in phenylpropanoid biosynthesis. The enzyme has been named hydroxycinnamoyl-CoA: shikimate/quinate hydroxycinnamoyltransferase. We show that p-coumaroyl-CoA and caffeoyl-CoA are the best acyl group donors and that the acyl group is transferred more efficiently to shikimate than to quinate. The enzyme also catalyzed the reverse reaction, i.e. the formation of caffeoyl-CoA from chlorogenate (5-O-caffeoyl quinate ester). Thus, hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase appears to control the biosynthesis and turnover of major plant phenolic compounds such as lignin and chlorogenic acid.

Oligomerization status, with the monomer as active species, defines catalytic efficiency of UDP-glucose pyrophosphorylase.

Barley UDP-glucose pyrophosphorylase (UGPase), a key enzyme for the synthesis of sucrose, cellulose and other saccharides, was expressed in Escherichia coli and purified. Using both native electrophoresis and gel filtration, the recombinant and crude leaf UGPase proteins were found to exist as a mixture of monomers, dimers and higher-order polymers. In order to understand the molecular basis for the oligomerization of UGPase, a conserved Cys residue was replaced (C99S mutant) and several amino acids were substituted (LIV to NIN, KK to LL and LLL to NNN) in a conserved hydrophobic domain (amino acids 117-138). The C99S mutant had about half the V (max) of the wild-type and a 12-fold higher K (m) for PP(i), whereas NIN and LL mutations lowered the V (max) by 12- and 2-fold, respectively, with relatively small effects on substrate K (m) values (the NNN mutant was insoluble/inactive). The NIN mutation resulted in a low-activity oligomerized enzyme form, with very little monomer formation. Activity staining on native PAGE gels as well as gel-filtration studies demonstrated that the monomer was the sole enzymically active form. Possible implications of the oligomerization status of UGPase for post-translational regulation of the enzyme are discussed.