Raffinose Accumulation Research Articles

Metabolite/phytohormone-gene regulatory networks in soybean organs under dehydration conditions revealed by integration analysis.

SummaryMetabolites, phytohormones, and genes involved in dehydration responses/tolerance have been predicted in several plants. However, metabolite/phytohormone–gene regulatory networks in soybean organs under dehydration conditions remain unclear. Here, we analyzed the organ specificity of metabolites, phytohormones, and gene transcripts and revealed the characteristics of their regulatory networks in dehydration‐treated soybeans. Our metabolite/phytohormone analysis revealed the accumulation of raffinose, trehalose, and cis‐zeatin (cZ) specifically in dehydration‐treated roots. In dehydration‐treated soybeans, raffinose, and trehalose might have additional roles not directly involved in protecting the photosynthetic apparatus; cZ might contribute to root elongation for water uptake from the moisture region in soil. Our integration analysis of metabolites–genes indicated that galactinol, raffinose, and trehalose levels were correlated with transcript levels for key enzymes (galactinol synthase, raffinose synthase, trehalose 6‐phosphate synthase, trehalose 6‐phosphate phosphatase) at the level of individual plants but not at the organ level under dehydration. Genes encoding these key enzymes were expressed in mainly the aerial parts of dehydration‐treated soybeans. These results suggested that raffinose and trehalose are transported from aerial plant parts to the roots in dehydration‐treated soybeans. Our integration analysis of phytohormones–genes indicated that cZ and abscisic acid (ABA) levels were correlated with transcript levels for key enzymes (cytokinin nucleoside 5′‐monophosphate phosphoribohydrolase, cytokinin oxidases/dehydrogenases, 9‐cis‐epoxycarotenoid dioxygenase) at the level of individual plants but not at the organ level under dehydration conditions. Therefore, processes such as ABA and cZ transport, among others, are important for the organ specificity of ABA and cZ production under dehydration conditions.

Ectopic over-expression of ABA-responsive Chickpea galactinol synthase (CaGolS) gene results in improved tolerance to dehydration stress by modulating ROS scavenging

Galactinol synthase (EC: 2.4.1.123) is a crucial enzyme of raffinose family oligosaccharides (RFO’s) biosynthesis, essentially catalyzes the first crucial step in the raffinose biosynthetic pathway. Galactinol and raffinose accumulation along with its higher homologs such as stachyose and verbascose has been considered to participate in plant abiotic stress tolerance. Previously, we reported the function of chickpea galactinol synthase (CaGolS) genes in seed vigor and heat and oxidative stress tolerance. Here, we demonstrate the role and regulation of CaGolS in response to dehydration stress in chickpea. Through transcript accumulation and promoter-gus analyses, we demonstrate that the expression of CaGolS1 and CaGolS2 are positively influenced by dehydration stress and ABA treatment. Further, we have shown that the level of galactinol and raffinose are remarkably enhanced in response to dehydration stress and ABA. Utilizing CaGolS1 and CaGolS2 over-expression Arabidopsis lines, we demonstrate the role of CaGolS in dehydration stress tolerance. The biochemical and physiological analysis revealed that CaGolS over-expressing transgenic lines exhibited improved phenotype with respect to higher number of siliques, plant height, and rosette diameter under dehydration stress. The improved dehydration stress tolerance was corelated with higher chlorophyll retention and relative water content of transgenic lines. Further, lower H2O2, MDA content, and ion-leakage in transgenic lines suggest that CaGolS mediates dehydration stress tolerance by protecting the membrane damage from ROS attack. Collectively, our data highlight the prospect of CaGolS genes in improving dehydration stress tolerance in plants.

Metabolic Changes on the Acquisition of Desiccation Tolerance in Seeds of the Brazilian Native Tree Erythrina speciosa.

Erythrina speciosa Andrews (Fabaceae) is a native tree of Atlantic forest from Southern and Southeastern Brazil. Although this species is found in flooded areas, it produces highly desiccation tolerant seeds. Here, we investigated the physiological and metabolic events occurring during seed maturation of E. speciosa aiming to better understand of its desiccation tolerance acquisition. Seeds were separated into six stages of maturation by the pigmentation of the seed coat. Water potential (WP) and water content (WC) decreased gradually from the first stage to the last stage of maturation (VI), in which seeds reached the highest accumulation of dry mass and seed coat acquired water impermeability. At stage III (71% WC), although seeds were intolerant to desiccation, they were able to germinate (about 15%). Desiccation tolerance was first observed at stage IV (67% WC), in which 40% of seeds were tolerant. At stage V (24% WC), all seeds were tolerant to desiccation and at stage VI all seeds germinated. Increased deposition of the arabinose-containing polysaccharides, which are known as cell wall plasticizers polymers, was observed up to stage IV of seed maturation. Raffinose and stachyose gradually increased in axes and cotyledons with greater increment in the fourth stage. Metabolic profile analysis showed that levels of sugars, organic, and amino acids decrease drastically in embryonic axes, in agreement with lower respiratory rates during maturation. Moreover, a non-aqueous fractionation revealed a change on the proportions of sugar accumulation among cytosol, plastid, and vacuoles between the active metabolism (stage I) and the dormant seeds (stage VI). The results indicate that the physiological maturity of the seeds of E. speciosa is reached at stage V and that the accumulation of raffinose can be a result of the change in the use of carbon, reducing metabolic activity during maturation. This work confirms that raffinose is involved in desiccation tolerance in seeds of E. speciosa, especially considering the different subcellular compartments and suggests even that the acquisition of desiccation tolerance in this species occurs in stages prior to the major changes in WC.

Genotypic Variation in the Root and Shoot Metabolite Profiles of Wheat (Triticum aestivum L.) Indicate Sustained, Preferential Carbon Allocation as a Potential Mechanism in Phosphorus Efficiency.

Changes in the levels of plant metabolites in response to nutrient deficiency is indicative of how plants utilize scarce resources. In this study, changes in the metabolite profile of roots and shoots of wheat genotypes differing in phosphorus use efficiency (PUE) was investigated. Under low P supply and at 28 days after sowing (DAS), the wheat breeding line, RAC875 (P efficient) produced 42% more shoot biomass than the wheat variety, and Wyalkatchem (P inefficient). Significant changes in the metabolite profile in leaves and roots were observed under low P supply and significant genotypic variation was evident. Under low P supply, an increase in raffinose and 1-kestose was evident in roots of both wheat genotypes, with RAC875 accumulating more when compared to Wyalkatchem. There was no significant increase in raffinose and 1-kestose in leaves when plants were grown under P deficiency. P deficiency had no significant impact on the levels of sucrose, maltose, glucose and fructose in both genotypes, and while phosphorylated sugars (glucose-6-P and fructose-6-P) remained unchanged in RAC875, in Wyalkatchem, glucose-6-P significantly decreased in roots, and fructose-6-P significantly decreased in both leaves and roots. Glycerol-3-P decreased twofold in roots of both wheat genotypes in response to low P. In roots, RAC875 exhibited significantly lower levels of fumarate, malate, maleate and itaconate than Wyalkatchem, while low P enhanced organic acid exudation in RAC875 but not in Wyalkatchem. RAC875 showed greater accumulation of aspartate, glutamine and β-alanine in leaves than Wyalkatchem under low P supply. Greater accumulation of raffinose and 1-kestose in roots and aspartate, glutamine and β-alanine in leaves appears to be associated with enhanced PUE in RAC875. Glucose-6-P and fructose-6-P are important for glycolysis, thus maintaining these metabolites would enable RAC875 to maintain carbohydrate metabolism and shoot biomass under P deficiency. The work presented here provides evidence that differences in metabolite profiles can be observed between wheat varieties that differ in PUE and key metabolic pathways are maintained in the efficient genotype to ensure carbon supply under P deficiency.

Identification of Factors Linked to Higher Water-Deficit Stress Tolerance in Amaranthus hypochondriacus Compared to Other Grain Amaranths and A. hybridus, Their Shared Ancestor.

Water deficit stress (WDS)-tolerance in grain amaranths (Amaranthus hypochondriacus, A. cruentus and A. caudatus), and A. hybridus, their presumed shared ancestor, was examined. A. hypochondriacus was the most WDS-tolerant species, a trait that correlated with an enhanced osmotic adjustment (OA), a stronger expression of abscisic acid (ABA) marker genes and a more robust sugar starvation response (SSR). Superior OA was supported by higher basal hexose (Hex) levels and high Hex/sucrose (Suc) ratios in A. hypochondriacus roots, which were further increased during WDS. This coincided with increased invertase, amylase and sucrose synthase activities and a strong depletion of the starch reserves in leaves and roots. The OA was complemented by the higher accumulation of proline, raffinose, and other probable raffinose-family oligosaccharides of unknown structure in leaves and/or roots. The latter coincided with a stronger expression of Galactinol synthase 1 and Raffinose synthase in leaves. Increased SnRK1 activity and expression levels of the class II AhTPS9 and AhTPS11 trehalose phosphate synthase genes, recognized as part of the SSR network in Arabidopsis, were induced in roots of stressed A. hypochondriacus. It is concluded that these physiological modifications improved WDS in A. hypochondriacus by raising its water use efficiency.

How fall dormancy benefits alfalfa winter-survival? Physiologic and transcriptomic analyses of dormancy process

BackgroundFall dormancy and freezing tolerance characterized as two important phenotypic traits, have great effects on productivity and persistence of alfalfa (Medicago sativa L.). Despite the fact that one of the most limiting traits for alfalfa freezing tolerance in winter is fall dormancy, the interplay between fall dormancy and cold acclimation processes of alfalfa remains largely unknown. We compared the plant regrowth, winter survival, raffinose and amino acids accumulation, and genome-wide differentially expressed genes of fall-dormant cultivar with non-dormant cultivar under cold acclimation.ResultsAveraged over both years, the non-dormant alfalfa exhibited largely rapid regrowth compared with fall dormant alfalfa after last cutting in autumn, but the winter survival rate of fall dormant alfalfa was about 34-fold higher than that of non-dormant alfalfa. The accumulation of raffinose and amino acids were significantly increased in fall dormant alfalfa, whereas were decreased in non-dormant alfalfa under cold acclimation. Expressions of candidate genes encoding raffinose biosynthesis genes were highly up-regulated in fall dormant alfalfa, but down-regulated in non-dormant alfalfa under cold acclimation. In fall dormant alfalfa, there was a significantly down-regulated expression of candidate genes encoding the glutamine synthase, which is indirectly involved in the proline metabolism. A total of eight significantly differentially expressed transcription factors (TFs) related to CBF and ABRE-BFs were identified. The most up-regulated TFs in fall dormant alfalfa cultivar were ABF4 and DREB1C.ConclusionsFall dormant alfalfa drastically increased raffinose and amino acids accumulation under cold acclimation. Raffinose-associated and amino acid-associated genes involved in metabolic pathways were more highly expressed in fall dormant alfalfa than non-dormant alfalfa under cold acclimation. This global survey of transcriptome profiles provides new insights into the interplay between fall dormancy and cold acclimation in alfalfa.

Maize VIVIPAROUS1 Interacts with ABA INSENSITIVE5 to Regulate GALACTINOL SYNTHASE2 Expression Controlling Seed Raffinose Accumulation.

Raffinose, an oligosaccharide found in many seeds, plays an important role in seed vigor; however, the regulatory mechanism governing raffinose biosynthesis remains unclear. We report here that maize W22 wild type (WT) seeds, but not W22 viviparous1 ( zmvp1) mutant seeds, start accumulating galactinol and raffinose 28 days after pollination (DAP). Transcriptome analysis of the zmvp1 embryo showed that the expression of GALACTINOL SYNTHASE2 ( GOLS2) was down-regulated relative to WT. Further experiments showed that the expression of ZmGOLS2 was up-regulated by ZmABI5 but not by ZmVP1, and it was further increased by the coexpression of ZmABI5 and ZmVP1 in maize protoplasts. ZmABI5 interacted with ZmVP1, while ZmABI5, but not ZmVP1, directly binds to the ZmGOLS2 promoter. Together, all of the findings suggest that ZmVP1 interacts with ZmABI5 and regulates ZmGOLS2 expression and raffinose accumulation in maize seeds.

Comparative metabolic profiling of Vitis amurensis and Vitis vinifera during cold acclimation

Vitis amurensis is a wild Vitis plant that can withstand extreme cold temperatures. However, the accumulation of metabolites during cold acclimation (CA) in V. amurensis remains largely unknown. In this study, plantlets of V. amurensis and V. vinifera cv. Muscat of Hamburg were treated at 4 °C for 24 and 72 h, and changes of metabolites in leaves were detected by gas chromatography coupled with time-of-flight mass spectrometry. Most of the identified metabolites, including carbohydrates, amino acids, and organic acids, accumulated in the two types of grape after CA. Galactinol, raffinose, fructose, mannose, glycine, and ascorbate were continuously induced by cold in V. amurensis, but not in Muscat of Hamburg. Twelve metabolites, including isoleucine, valine, proline, 2-oxoglutarate, and putrescine, increased in V. amurensis during CA. More galactinol, ascorbate, 2-oxoglutarate, and putrescine, accumulated in V. amurensis, but not in Muscat of Hamburg, during CA, which may be responsible for the excellent cold tolerance in V. amurensis. The expression levels of the genes encoding β-amylase (BAMY), galactinol synthase (GolS), and raffinose synthase (RafS) were evaluated by quantitative reverse transcription-PCR. The expression BAMY (VIT_02s0012 g00170) and RafS (VIT_05s0077 g00840) were primarily responsible for the accumulation of maltose and raffinose, respectively. The accumulation of galactinol was attributed to different members of GolS in the two grapes. In conclusion, these results show the inherent differences in metabolites between V. amurensis and V. vinifera under CA.

Genome-wide identification and expression analyses of genes involved in raffinose accumulation in sesame

Sesame (Sesamum indicum L.) is an important oilseed crop. However, multiple abiotic stresses severely affect sesame growth and production. Raffinose family oligosaccharides (RFOs), such as raffinose and stachyose, play an important role in desiccation tolerance of plants and developing seeds. In the present study, three types of key enzymes, galactinol synthase (GolS), raffinose synthase (RafS) and stachyose synthase (StaS), responsible for the biosynthesis of RFOs were identified at the genome-wide scale in sesame. A total of 7 SiGolS and 15 SiRS genes were identified in the sesame genome. Transcriptome analyses showed that SiGolS and SiRS genes exhibited distinct expression profiles in different tissues and seed developmental stages. Comparative expression analyses under various abiotic stresses indicated that most of SiGolS and SiRS genes were significantly regulated by drought, osmotic, salt, and waterlogging stresses, but slightly affected by cold stress. The up-regulation of several SiGolS and SiRS genes by multiple abiotic stresses suggested their active implication in sesame abiotic stress responses. Taken together, these results shed light on the RFOs-mediated abiotic stress resistance in sesame and provide a useful framework for improving abiotic stress resistance of sesame through genetic engineering.

Seed desiccation mechanisms co‐opted for vegetative desiccation in the resurrection grass Oropetium thomaeum

Resurrection plants desiccate during periods of prolonged drought stress, then resume normal cellular metabolism upon water availability. Desiccation tolerance has multiple origins in flowering plants, and it likely evolved through rewiring seed desiccation pathways. Oropetium thomaeum is an emerging model for extreme drought tolerance, and its genome, which is the smallest among surveyed grasses, was recently sequenced. Combining RNA-seq, targeted metabolite analysis and comparative genomics, we show evidence for co-option of seed-specific pathways during vegetative desiccation. Desiccation-related gene co-expression clusters are enriched in functions related to seed development including several seed-specific transcription factors. Across the metabolic network, pathways involved in programmed cell death inhibition, ABA signalling and others are activated during dehydration. Oleosins and oil bodies that typically function in seed storage are highly abundant in desiccated leaves and may function for membrane stability and storage. Orthologs to seed-specific LEA proteins from rice and maize have neofunctionalized in Oropetium with high expression during desiccation. Accumulation of sucrose, raffinose and stachyose in drying leaves mirrors sugar accumulation patterns in maturing seeds. Together, these results connect vegetative desiccation with existing seed desiccation and drought responsive pathways and provide some key candidate genes for engineering improved drought tolerance in crop plants.

Overexpression of AtGolS3 and CsRFS in poplar enhances ROS tolerance and represses defense response to leaf rust disease.

Plants respond to pathogens through an orchestration of signaling events that coordinate modifications to transcriptional profiles and physiological processes. Resistance to necrotrophic pathogens often requires jasmonic acid, which antagonizes the salicylic acid dependent biotrophic defense response. Recently, myo-inositol has been shown to negatively impact salicylic acid (SA) levels and signaling, while galactinol enhances jasmonic acid (JA)-dependent induced systemic resistance to necrotrophic pathogens. To investigate the function of these compounds in biotrophic pathogen defense, we characterized the defense response of Populus alba × grandidentata overexpressing Arabidopsis GALACTINOL SYNTHASE3 (AtGolS) and Cucumber sativus RAFFINOSE SYNTHASE (CsRFS) challenged with Melampsora aecidiodes, a causative agent of poplar leaf rust disease. Relative to wild-type leaves, the overexpression of AtGolS3 and CsRFS increased accumulation of galactinol and raffinose and led to increased leaf rust infection. During the resistance response, inoculated wild-type leaves displayed reduced levels of galactinol and repressed transcript abundance of two endogenous GolS genes compared to un-inoculated wild-type leaves prior to the up-regulation of NON-EXPRESSOR OF PR1 and PATHOGENESIS-RELATED1. Transcriptome analysis and qRT-PCR validation also revealed the repression of genes participating in calcium influx, phosphatidic acid biosynthesis and signaling, and salicylic acid signaling in the transgenic lines. In contrast, enhanced tolerance to H2O2 and up-regulation of antioxidant biosynthesis genes were exhibited in the overexpression lines. Thus, we conclude that overexpression of AtGolS and CsRFS antagonizes the defense response to poplar leaf rust disease through repressing reactive oxygen species and attenuating calcium and phosphatidic acid signaling events that lead to SA defense.

Trisaccharides isomers, galactinol and osmotic imbalance associated with CO2 stress in strawberries

Treatment with high CO2 atmospheres is effective in preventing strawberry decay and increasing firmness. Nevertheless, CO2 stress generates energy disturbances associated with a high rate of fermentation. This study was designed to measure the impact of high CO2 stress (3 d, 40kPa CO2) and its subsequent removal on fruit quality, osmotic balance and water relations. Fructo-trisaccharides isomers and raffinose were characterized and quantified by mass spectrometry (MS and MS2). CO2 stress removal was marked by both a rapid upsurge in the ratio of unfreezable water to total water and an osmotic adjustment prompted by the accumulation of soluble sugars, 1-kestose, raffinose and galactinol. Due to strong increase in galactinol concentrations, we propose it as a suitable biomarker for fruit having undergone stresses associated with osmotic imbalance.

Differentially expressed galactinol synthase(s) in chickpea are implicated in seed vigor and longevity by limiting the age induced ROS accumulation.

Galactinol synthase (GolS) catalyzes the first and rate limiting step of Raffinose Family Oligosaccharide (RFO) biosynthetic pathway, which is a highly specialized metabolic event in plants. Increased accumulation of galactinol and RFOs in seeds have been reported in few plant species, however their precise role in seed vigor and longevity remain elusive. In present study, we have shown that galactinol synthase activity as well as galactinol and raffinose content progressively increase as seed development proceeds and become highly abundant in pod and mature dry seeds, which gradually decline as seed germination progresses in chickpea (Cicer arietinum). Furthermore, artificial aging also stimulates galactinol synthase activity and consequent galactinol and raffinose accumulation in seed. Molecular analysis revealed that GolS in chickpea are encoded by two divergent genes (CaGolS1 and CaGolS2) which potentially encode five CaGolS isoforms through alternative splicing. Biochemical analysis showed that only two isoforms (CaGolS1 and CaGolS2) are biochemically active with similar yet distinct biochemical properties. CaGolS1 and CaGolS2 are differentially regulated in different organs, during seed development and germination however exhibit similar subcellular localization. Furthermore, seed-specific overexpression of CaGolS1 and CaGolS2 in Arabidopsis results improved seed vigor and longevity through limiting the age induced excess ROS and consequent lipid peroxidation.

Altitude variation in chilling tolerance among natural populations of Elymus nutans in the Tibetan Plateau

Low temperatures limit plant growth, development, and reproductive success. A series of complex adaptive responses in plants evolved to withstand this environmental challenge. Here, eight accessions of Elymus nutans, which originated in Tibet at altitudes between 3720 and 5012 m above sea level, were used to identify heritable adaptations to chilling stress. Dynamic responses of phytohormone, sugar, and gene expression levels related to chilling tolerance were analyzed. During the initial stage of chilling stress (0–24 h), some high-altitude E. nutans accessions exhibited rapid increases in abscisic acid (ABA), jasmonic acid (JA), and zeatin content. This coordinated with decreases in the levels of auxin (IAA), salicylic acid (SA), gibberellins (GA), and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). EnCBF9 and EnCBF14 expression in the high-altitude accessions, Baqing, Xainza, Damxung, and Ali, increased within 1 h of chilling exposure, while chilling induction of EnCOR14a was detected after 3 h of chilling stress. Accessions from high altitudes displayed an increased sucrose and raffinose accumulation and a reduced degradation of chlorophyll under chilling stress. After 24–120 h of chilling exposure, plant adaptation to the chilling treatment was associated with a lower accumulation of ABA and moderate rise of zeatin, IAA, GA, ACC, SA, and JA. EnCBF9, EnCBF14, and EnCOR14a genes were down-regulated during the late stage of chilling stress. Taken together, the dynamic responses of phytohormones and sugars, and the higher expression of the EnCBFs and EnCOR genes play critical roles in the acclimation to chilling in high-altitude accessions of E. nutans, thereby allowing them to achieve higher chilling tolerance.

Physical and nutritional properties of edamame seeds as influenced by stage of development

Changes in physical, chemical and anti-nutritional characteristics of two vegetable soybean varieties (‘Asmara’ and ‘Mooncake’) during seed development were investigated. Pods were sampled weekly for 6 weeks starting from developmental stage 5 (R5) to 8 (R8). Changes over time in measured attributes were similar but the rate of change differed among the two varieties. In both varieties seed moisture content and intensity of green color decreased as seed developed with the most significant decline observed in the last 2 weeks of sampling. Seed weight peaked at R6 then gradually decreased thereafter, while seed hardness increased throughout the sampling period with ‘Asmara’ recording significantly higher seed hardness at R8. For both varieties, protein accumulation occurred mostly in the later stages, while significant lipid accumulation was observed in the early stages of development. Among the sugars, fructose content decreased with seed development, sucrose content increased to R6 before decreasing, and there was rapid accumulation of raffinose and stachyose in the last 2 weeks of sampling. Total phenolic content decreased between R5 and R6 but increased with further seed development. Tannin and phytate content in seed increased throughout the sampling period. Changes in trypsin inhibitory activity varied with variety reaching a maximum at R6 for ‘Asmara, and closer to maturation for “Mooncake’. Our data provide physical, chemical and anti-nutritional basis for harvesting vegetable soybean at R6 namely: peak seed weight and sucrose content, lower oligosaccharide and anti-nutrients values and intense green color.

Phloem transcriptome signatures underpin the physiological differentiation of the pedicel, stalk and fruit of cucumber (Cucumis sativusL.)

Cucumber is one of the most important vegetables grown worldwide due to its important economic and nutritional value. The cucumber fruit consists morphologically of the undesirable stalk and the tasty fruit; however, physiological differentiation of these two parts and the underlying molecular basis remain largely unknown. Here we characterized the physiological differences among the pedicel, stalk and fruit, and compared the respective phloem transcriptomes using laser capture microdissection coupled with RNA sequencing (RNA-Seq). We found that the pedicel was characterized by minor cell expansion and a high concentration of stachyose, the stalk showed rapid cell expansion and high raffinose accumulation, and the fruit featured transition from cell division to cell expansion and high levels of monosaccharides. Analyses of transcriptome data indicated that cell wall- and calcium ion binding-related genes contributed to the cell expansion in the pedicel and stalk, whereas genes implicated in cell cycle and hormone actions regulated the transition from cell division to cell expansion in the fruit. Differential sugar distribution in these three phloem-connected tissues resulted from tissue-specific sugar metabolism and transport. Enrichment of transcription factors in the stalk and fruit may facilitate nutrient accumulation in these sink organs. As such, phloem-located gene expression partially orchestrated physiological differentiation of the pedicel, stalk and fruit in cucumber. In addition, we identified 432 cucumber-unique genes and five phloem markers guiding future functional studies.

Osmotic stress induces genes, enzymes and accumulation of galactinol, raffinose and stachyose in seedlings of pea (Pisum sativum L.)

The objective of the present study was to recognize the molecular background of the accumulation of raffinose family oligosaccharides (RFOs) in pea (Pisum sativum L.) seedlings under osmotic stress conditions. The exposure of 5-day-old pea seedlings to osmotic stress for 48 h created by immersing roots in PEG8000 solution (−1.5 MPa) induced synthesis of galactinol and RFOs (raffinose and stachyose) in the epicotyl and root tissues, but not in cotyledons. After 24 h of recovery, galactinol completely disappeared, raffinose decreased fourfold and stachyose decreased twofold in roots, but increased in epicotyls. The temporary accumulation of RFOs resulted from a dramatic increase in the enzymatic activity and changes in expression of galactinol synthase (PsGolS), raffinose synthase (PsRS) and stachyose synthase (PsSTS) genes. PsGolS was induced by osmotic stress in both epicotyls as well as in roots. PsRS and PsSTS were induced only in epicotyls, but repressed or remained unaffected in roots, respectively. During recovery, the expression and activity of PsGolS, PsRS and PsSTS dramatically decreased. The expression of PsGolS gene, that level of mRNA transcript significantly decreased during recovery and whose promoter region was identified to contain some stress-related regulating elements, seems to play a crucial role in the biosynthesis of RFOs under osmotic stress. Possible signals that may trigger the induction of expression of PsGolS, PsRS and PsSTS genes and accumulation of RFOs in pea seedlings are discussed.

Physiological and Biochemical Seasonal Changes in Vitis Genotypes with Contrasting Freezing Tolerance

In this study, we characterized the influence of genotype and bud position on seasonal changes in freezing tolerance (FT, LT50) and soluble sugars, especially raffinose, and the affect of artificial deacclimation in buds of three field-grown cultivars over two years. Generally, basal buds were more freeze tolerant, and the cold-hardy cultivars Couderc 3309 (3309C; <i>Vitis riparia</i> x <i>V. rupestris</i>) and Concord (CD; <i>V. labruscana</i>) had the lowest LT50 compared to the cold-sensitive Cabernet franc (CF; <i>V. vinifera</i>). Among all sugars, fructose, glucose, sucrose, raffinose, and stachyose had strong correlation with LT50, but distinctive responses associated with bud position and cultivar were related to raffinose. Basal buds accumulated the most raffinose, and raffinose concentrations were two- to three-fold higher in the cold-hardy CD and 3309C than those in the cold-sensitive CF. Furthermore, CD and 3309C started accumulating raffinose with decreasing photoperiod prior to exposure to cold temperature, resulting in an early increase of FT. These results suggest that raffinose accumulation might be an early step in the process of cold acclimation that coincides with early development of FT. We concluded that bud raffinose concentration might be a useful detection tool and a metabolic marker during the early stages of cold acclimation to distinguish various <i>Vitis</i> genotypes with contrasting FT.

Postharvest needle abscission resistance of balsam fir (Abies balsamea) is modified by harvest date

Earlier harvest dates have become necessary for Canadian Christmas tree producers to meet international demand, though by harvesting these trees early they may experience poor needle retention. The objective of this study is to understand the effect of harvest date on needle retention and link those changes to cold acclimation. In one experiment, balsam fir trees with varied needle abscission resistance (NAR) were collected in October and in January and monitored for needle retention. This was repeated for 3 years. In a second experiment, 45 branches were collected each month from September to January and monitored for needle retention, xylem pressure, membrane injury, capacitance, and accumulation of galactose, raffinose, and abscisic acid. High-NAR trees had little improvement in needle retention from October to January, whereas low-NAR trees had significantly improved needle retention from October to January. Between September and January, there was an 85% increase in raffinose, 147% increase in galactose, 80% increase in abscisic acid, and 62% decrease in stem capacitance. Early harvest was not detrimental for all trees, and it appears that cold acclimation is linked to postharvest needle abscission, though cold acclimation does not adequately explain differences between NAR classes.

Dehydration induces expression of GALACTINOL SYNTHASE and RAFFINOSE SYNTHASE in seedlings of pea (Pisum sativum L.)

The exposition of 7-day-old pea seedlings to dehydration induced sudden changes in the concentration of monosaccharides and sucrose in epicotyl and roots tissues. During 24h of dehydration, the concentration of glucose and, to a lesser extent, fructose in seedling tissues decreased. The accumulation of sucrose was observed in roots after 4h and in epicotyls after 8h of stress. Epicotyls and roots also began to accumulate galactinol and raffinose after 8h of stress, when small changes in the water content of tissues occurred. The accumulation of galactinol and raffinose progressed parallel to water withdrawal from tissues, but after seedling rehydration both galactosides disappeared. The synthesis of galactinol and raffinose by an early induction (during the first hour of treatment) of galactinol synthase (PsGolS) and raffinose synthase (PsRS) gene expression as well as a later increase in the activity of both enzymes was noted. Signals possibly triggering the induction of PsGolS and PsRS gene expression and accumulation of galactinol and raffinose in seedlings are discussed.