Drought Stress Alters the Concentration of Wood Terpenoids in Scots Pine and Norway Spruce Seedlings

Drought is known to have an impact on the resistance of conifers to various pests, for example, by affecting resin flow in trees. Little is known, however, about the quantitative and qualitative changes in resin when trees are growing in low moisture conditions. We exposed Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.) seedlings to medium and severe drought stress for two growing seasons and analyzed the monoterpenes and resin acids in the main stem wood after two years of treatment. In addition to secondary chemistry, we measured the level of nutrients in the needles and the growth response of seedlings. After the first year of treatment, drought stress did not affect the growth of seedlings, but in the second year, shoot growth was retarded, especially in Scots pine. In both conifer species, severe drought increased the concentrations of several individual monoterpenes and resin acids. Total monoterpenes and resin acids were 39 and 32% higher in severe drought-treated Scots pine seedlings than in the controls, and 35 and 45% higher in Norway spruce seedlings. In Scots pine needles, the concentrations of nitrogen and phosphorus increased, while magnesium and calcium decreased compared to controls. In Norway spruce needles, nutrient concentrations were not affected. The results suggest that drought stress substantially affects both the growth of conifers and the chemical quality of the wood. We discuss the potential trade-off in growth and defense of small conifer seedlings.     

Engelmann Spruce Chemotypes in Colorado and their Effects on Symbiotic Fungi Associated with the North American Spruce Beetle

Conifer secondary metabolites play a key role in mechanisms of resistance to biotic disturbance, especially by bark beetles and beetle-associated microorganisms. Here, we describe variation in constitutive monoterpenes isolated from Engelmann spruce, Picea engelmannii, phloem across fourteen high-elevation populations in the Rocky Mountains of Colorado, and test interactions between phloem monoterpenes and an endophloedic symbiotic fungus, Leptographium abietinum, associated with the North American spruce beetle, Dendroctonus rufipennis. We consistently identified ten monoterpenes in Engelmann spruce phloem, and the trees in our samples could be classified into two geographically interspersed chemical phenotypes, or ‘chemotypes’: one in which α- and β-pinene were the most abundant monoterpenes, and one in which 3-carene was the most abundant monoterpene. Media amended with low concentrations of α-pinene, β-pinene, 3-carene, myrcene, and terpinolene stimulated growth of L. abietinum. Increasing monoterpene concentrations uniformly retarded fungal growth. Linalool completely suppressed fungal growth at all concentrations, while terpinolene completely suppressed growth at low and intermediate concentrations, indicating relatively high toxicity of these compounds. Tests with monoterpene blends representing the ‘average’ monoterpene composition of each chemotype indicated that representative chemotypes are equivalent in fungistatic activity, with chemotype blends being inhibitory even at low concentrations. Total constitutive monoterpene abundances in Engelmann spruce phloem ranged from 42 to 1796 μg/g. Induction of Engelmann spruce phloem monoterpenes in response to L. abietinum or other biotic agents has yet to be quantified, but is important for further understanding Engelmann spruce resistance to the D. rufipennis-L. abietinum complex.     

Genome-Wide Identification and Expression Patterns of the C2H2-Zinc Finger Gene Family Related to Stress Responses and Catechins Accumulation in Camellia sinensis [L.] O. Kuntze

The C2H2-zinc finger protein (C2H2-ZFP) is essential for the regulation of plant development and widely responsive to diverse stresses including drought, cold and salt stress, further affecting the late flavonoid accumulation in higher plants. Tea is known as a popular beverage worldwide and its quality is greatly dependent on the physiological status and growing environment of the tea plant. To date, the understanding of C2H2-ZFP gene family in Camellia sinensis [L.] O. Kuntze is not yet available. In the present study, 134 CsC2H2-ZFP genes were identified and randomly distributed on 15 chromosomes. The CsC2H2-ZFP gene family was classified into four clades and gene structures and motif compositions of CsC2H2-ZFPs were similar within the same clade. Segmental duplication and negative selection were the main forces driving the expansion of the CsC2H2-ZFP gene family. Expression patterns suggested that CsC2H2-ZFPs were responsive to different stresses including drought, salt, cold and methyl jasmonate (MeJA) treatment. Specially, several C2H2-ZFPs showed a significant correlation with the catechins content and responded to the MeJA treatment, which might contribute to the tea quality and specialized astringent taste. This study will lay the foundations for further research of C2H2-type zinc finger proteins on the stress responses and quality-related metabolites accumulation in C. sinensis.     

Genome-Wide Analysis of the Peptidase M24 Superfamily in Triticum aestivum Demonstrates That TaM24-9 Is Involved in Abiotic Stress Response

The peptidase M24 (Metallopeptidase 24, M24) superfamily is essential for plant growth, stress response, and pathogen defense. At present, there are few systematic reports on the identification and classification of members of the peptidase M24 proteins superfamily in wheat. In this work, we identified 53 putative candidate TaM24 genes. According to the protein sequences characteristics, these members can be roughly divided into three subfamilies: I, II, III. Most TaM24 genes are complex with multiple exons, and the motifs are relatively conserved in each sub-group. Through chromosome mapping analysis, we found that the 53 genes were unevenly distributed on 19 wheat chromosomes (except 3A and 3D), of which 68% were in triads. Analysis of gene duplication events showed that 62% of TaM24 genes in wheat came from fragment duplication events, and there were no tandem duplication events to amplify genes. Analysis of the promoter sequences of TaM24 genes revealed that cis-acting elements were rich in response elements to drought, osmotic stress, ABA, and MeJA. We also studied the expression of TaM24 in wheat tissues at developmental stages and abiotic stress. Then we selected TaM24-9 as the target for further analysis. The results showed that TaM24-9 genes strengthened the drought and salt tolerance of plants. Overall, our analysis showed that members of the peptidase M24 genes may participate in the abiotic stress response and provided potential gene resources for improving wheat resistance.     

The E3 Ligase GmPUB21 Negatively Regulates Drought and Salinity Stress Response in Soybean

E3-ubiquitin ligases are known to confer abiotic stress responses in plants. In the present study, GmPUB21, a novel U-box E3-ubiquitin ligase-encoding gene, was isolated from soybean and functionally characterized. The expression of GmPUB21, which possesses E3-ubiquitin ligase activity, was found to be significantly up-regulated by drought, salinity, and ABA treatments. The fusion protein GmPUB21-GFP was localized in the cytoplasm, nucleus, and plasma membrane. Transgenic lines of the Nicotiana benthamiana over-expressing GmPUB21 showed more sensitive to osmotic, salinity stress and ABA in seed germination and inhibited mannitol/NaCl-mediated stomatal closure. Moreover, higher reactive oxygen species accumulation was observed in GmPUB21 overexpressing plants after drought and salinity treatment than in wild-type (WT) plants. Contrarily, silencing of GmPUB21 in soybean plants significantly enhanced the tolerance to drought and salinity stresses. Collectively, our results revealed that GmPUB21 negatively regulates the drought and salinity tolerance by increasing the stomatal density and aperture via the ABA signaling pathway. These findings improved our understanding of the role of GmPUB21 under drought and salinity stresses in soybean.     

Preference of meadow voles (Microtus pennsylvanicus) for conifer seedlings: Chemical components and nutritional quality of bark of damaged and undamaged trees

Meadow voles (Microtus pennsylvanicus Ord.) of the 1987–1988 population density peak left several seedling plantations of Norway spruce (Picea abies) and Norway pine (Pinus resinosa) severely damaged in southern Quebec, Canada, while white spruce (Picea glauca) and white pine (Pinus strobus) suffered no damage. We compared levels of crude protein, total nonstructural carbohydrates (TNC), total phenols, and monoterpenes in the bark to detect differences between damaged and undamaged seedlings within and between plantations. There were no significant differences in levels of nutritional components between both types of seedlings within the same plantation. However, differences were noted among tree plantations for protein, TNC, and phenolics content but these differences could not be assigned to the voles' selection for particular species. Quantitative and qualitative differences also were registered in monoterpene levels between species. -Myrcene and bornyl acetate were found only in seedlings of undamaged species. Moreover, the latter contained higher levels of limonene. We conclude that the presence of specific monoterpenes is more important in defense mechanisms of conifer seedlings than relative levels of nutrients (protein or TNC) or total phenolics.     

Volatile Emissions from an Odorous Plant in Response to Herbivory and Methyl Jasmonate Exposure

Induced volatile terpenes have been commonly reported among diverse agricultural plant species, but less commonly investigated in odorous plant species. Odorous plants synthesize and constitutively store relatively large amounts of volatiles, and these may play a role in defense against herbivores. We examined the effect of herbivory and methyl jasmonate (MeJA) exposure on the release of volatile organic compounds (VOCs) in the marsh elder, Iva frutescens, which contains numerous constitutive VOCs, mainly mono- and sesquiterpenes. Our specific goal was to test for the presence of inducible VOCs in a naturally occurring plant already armed with VOCs. The abundant, native specialist leaf beetle Paria aterrima was used in herbivore induction trials. VOCs were sampled from herbivore wounded and unwounded, and from MeJA treated and untreated I. frutescens. Total VOC emissions were significantly greater in response to herbivory and MeJA treatment compared to unwounded controls. Herbivore wounding caused a substantial shift in the emission profile (42 VOCs from wounded, compared to 8 VOCs from unwounded I. frutescens), and MeJA had a similar yet less substantial influence on the emission pattern (28 VOCs from MeJA treated compared to 8 VOCs from untreated I. frutescens). Constitutive VOC emissions predominated, but some VOCs were detected only in response to herbivory and MeJA treatment, suggesting de novo synthesis. Several VOCs exhibited a delayed emission profile in contrast to the rapid release of constitutive VOCs, and principal components analysis revealed they were not associated with constitutive emissions. While I. frutescens contains many constitutive VOCs that are released immediately in response to herbivory, it also produces novel VOCs in response to feeding by the specialist P. aterrima and MeJA treatment.     

YdfD,a Lysis Protein of the Qin Prophage,Is a Specific Inhibitor of the IspG-Catalyzed Step in the MEP Pathway of Escherichia coli

Bacterial cryptic prophage (defective prophage) genes are known to drastically influence host physiology, such as causing cell growth arrest or lysis, upon expression. Many phages encode lytic proteins to destroy the cell envelope. As natural antibiotics, only a few lysis target proteins were identified. ydfD is a lytic gene from the Qin cryptic prophage that encodes a 63-amino-acid protein, the ectopic expression of which in Escherichia coli can cause nearly complete cell lysis rapidly. The bacterial 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is responsible for synthesizing the isoprenoids uniquely required for sustaining bacterial growth. In this study, we provide evidence that YdfD can interact with IspG, a key enzyme involved in the MEP pathway, both in vivo and in vitro. We show that intact YdfD is required for the interaction with IspG to perform its lysis function and that the mRNA levels of ydfD increase significantly under certain stress conditions. Crucially, the cell lysis induced by YdfD can be abolished by the overexpression of ispG or the complementation of the IspG enzyme catalysis product methylerythritol 2,4-cyclodiphosphate. We propose that YdfD from the Qin cryptic prophage inhibits IspG to block the MEP pathway, leading to a compromised cell membrane and cell wall biosynthesis and eventual cell lysis.     

TaNBR1, a Novel Wheat NBR1-like Domain Gene Negatively Regulates Drought Stress Tolerance in Transgenic Arabidopsis

Drought stress is an important factor that severely affects crop yield and quality. Autophagy has a crucial role in the responses to abiotic stresses. In this study, we explore TaNBR1 in response to drought stress. Expression of the TaNBR1 gene was strongly induced by NaCl, PEG, and abscisic acid treatments. The TaNBR1 protein is localized in the Golgi apparatus and autophagosome. Transgenic Arabidopsis plants overexpressing TaNBR1 exhibited reduced drought tolerance. When subjected to drought stress, compared to the wild-type (WT) lines, the transgenic overexpressing TaNBR1 plants had a lower seed germination rate, relative water content, proline content, and reduced accumulation of antioxidant enzymes, i.e., superoxide dismutase, peroxidase, and catalase, as well as higher chlorophyll losses, malondialdehyde contents, and water loss. The transgenic plants overexpressing TaNBR1 produced much shorter roots in response to mannitol stress, in comparison to the WT plants, and they exhibited greater sensitivity to abscisic acid treatment. The expression levels of the genes related to stress in the transgenic plants were affected in response to drought stress. Our results indicate that TaNBR1 negatively regulates drought stress responses by affecting the expression of stress-related genes in Arabidopsis.     

Activation of the ABA Signal Pathway Mediated by GABA Improves the Drought Resistance of Apple Seedlings

Drought seriously affects the yield and quality of apples. γ-aminobutyric acid (GABA) plays an important role in the responses of plants to various stresses. However, the role and possible mechanism of GABA in the drought response of apple seedlings remain unknown. To explore the effect of GABA on apple seedlings under drought stress, seedlings of Malus hupehensis were treated with seven concentrations of GABA, and the response of seedlings under 15-day drought stress was observed. The results showed that 0.5 mM GABA was the most effective at relieving drought stress. Treatment with GABA reduced the relative electrical conductivity and MDA content of leaves induced by drought stress and significantly increased the relative water content of leaves. Exogenous GABA significantly decreased the stomatal conductance and intercellular carbon dioxide concentration and transpiration rate, and it significantly increased the photosynthetic rate under drought. GABA also reduced the accumulation of superoxide anions and hydrogen peroxide in leaf tissues under drought and increased the activities of POD, SOD, and CAT and the content of GABA. Exogenous treatment with GABA acted through the accumulation of abscisic acid (ABA) in the leaves to significantly decrease stomatal conductance and increase the stomatal closure rate, and the levels of expression of ABA-related genes PYL4, ABI1, ABI2, HAB1, ABF3, and OST1 changed in response to drought. Taken together, exogenous GABA can enhance the drought tolerance of apple seedlings.     

The BAG2 and BAG6 Genes Are Involved in Multiple Abiotic Stress Tolerances in Arabidopsis Thaliana

The BAG proteins are a family of multi-functional co-chaperones. In plants, BAG proteins were found to play roles both in abiotic and biotic stress tolerance. However, the function of Arabidopsis BAG2 remains largely unknown, whereas BAG6 is required for plants’ defense to pathogens, although it remains unknown whether BAG6 is involved in plants’ tolerance to abiotic stresses. Here, we show that both BAG2 and BAG6 are expressed in various tissues and are upregulated by salt, mannitol, and heat treatments and by stress-related hormones including ABA, ethylene, and SA. Germination of bag2, bag6 and bag2 bag6 seeds is less sensitive to ABA compared to the wild type (WT), whereas BAG2 and BAG6 overexpression lines are hypersensitive to ABA. bag2, bag6, and bag2 bag6 plants show higher survival rates than WT in drought treatment but display lower survival rates in heat-stress treatment. Consistently, these mutants showed differential expression of several stress- and ABA-related genes such as RD29A, RD29B, NCED3 and ABI4 compared to the WT. Furthermore, these mutants exhibit lower levels of ROS after drought and ABA treatment but higher ROS accumulation after heat treatment than the WT. These results suggest that BAG2 and BAG6 are negatively involved in drought stress but play a positive role in heat stress in Arabidopsis.     

Methyl Jasmonate-Induced Monoterpenes in Scots Pine and Norway Spruce Tissues Affect Pine Weevil Orientation

In large parts of Europe, insecticide-free measures for protecting conifer plants are desired to suppress damage by the pine weevil Hylobius abietis (L.). Treatment with methyl jasmonate (MeJA), a chemical elicitor already used in crop production, may enhance expression of chemical defenses in seedlings in conifer regenerations. However, in a previous experiment, MeJA treatment resulted in substantially better field protection for Scots pine (Pinus sylvestris L.) than for Norway spruce (Picea abies (L.) Karst.). Hypothesizing that the variations may be at least due partly to volatiles released by MeJA-treated seedlings and their effects on pine weevil orientation, we examined tissue extracts of seedlings (from the same batches as previously used) by two-dimensional GC-MS. We found that the MeJA treatment increased contents of the monoterpene (?)-β-pinene in phloem (the weevil’s main target tissue) of both tree species, however, the (?)-β-pinene/(?)-α-pinene ratio increased more in the phloem of P. sylvestris. We also tested the attractiveness of individual monoterpenes found in conifer tissues (needles and phloem) for pine weevils using an arena with traps baited with single-substance dispensers and pine twigs. Trap catches were reduced when the pine material was combined with a dispenser releasing (?)-β-pinene, (+)-3-carene, (?)-bornyl acetate or 1,8-cineole. However, (?)-α-pinene did not have this effect. Thus, the greater field protection of MeJA-treated P. sylvestris seedlings may be due to the selective induction of increases in contents of the deterrent (?)-β-pinene, in contrast to strong increases in both non-deterrent (?)-α-pinene and the deterrent (?)-β-pinene in P. abies seedlings.     

Effects of Terpenes and Phenolic and Flavonoid Glycosides from Douglas Fir on Western Spruce Budworm Larval Growth, Pupal Weight, and Adult Weight

Monoterpenes, sesquiterpenes, and phenolic and flavonoid glycosides typical of the current year's foliage of Douglas fir (Pseudotsuga menziesii) were bioassayed using agar diets to determine the effect of these compounds on natural and colony populations of western spruce budworm (Choristoneura occidentalis). Several terpenes adversely affected budworm larval growth, supporting previous field and laboratory studies. However, larvae collected from a population in southeastern Idaho were significantly more tolerant of the monoterpenes than those from a Montana population. For the Montana population, agar diet studies showed that camphene, myrcene, terpinolene, bornyl acetate, and tricyclene adversely affected larval growth rate and pupal weight. For the Idaho population, agar diet studies showed that only terpinolene and bornyl acetate adversely affected larval growth and pupal weights. For both populations bornyl acetate was the most toxic, and this compound in addition to other monoterpenes represent defensive mechanisms in the current year's growth of Douglas fir. Sesquiterpene and phenolic-flavonoid glycoside fractions extracted from Douglas fir current year's growth also were bioassayed using agar diets. The sesquiterpene fraction showed a significant negative effect on budworm larval growth, but phenolics and flavonoid glycosides had no effect. Sesquiterpenes, in combination with tricyclene, camphene, myrcene, limonene, terpinolene, and the acetate fraction appear to represent an effective mixture of defensive compounds against the budworm.     

Root Exposure to 5-Aminolevulinic Acid (ALA) Affects Leaf Element Accumulation,Isoprene Emission,Phytohormonal Balance,and Photosynthesis of Salt-Stressed Arundo donax

Arundo donax has been recognized as a promising crop for biomass production on marginal lands due to its superior productivity and stress tolerance. However, salt stress negatively impacts A. donax growth and photosynthesis. In this study, we tested whether the tolerance of A. donax to salinity stress can be enhanced by the addition of 5-aminolevulinic acid (ALA), a known promoter of plant growth and abiotic stress tolerance. Our results indicated that root exposure to ALA increased the ALA levels in leaves along the A. donax plant profile. ALA enhanced Na⁺ accumulation in the roots of salt-stressed plants and, at the same time, lowered Na⁺ concentration in leaves, while a reduced callose amount was found in the root tissue. ALA also improved the photosynthetic performance of salt-stressed apical leaves by stimulating stomatal opening and preventing an increase in the ratio between abscisic acid (ABA) and indol-3-acetic acid (IAA), without affecting leaf methanol emission and plant growth. Supply of ALA to the roots reduced isoprene fluxes from leaves of non-stressed plants, while it sustained isoprene fluxes along the profile of salt-stressed A. donax. Thus, ALA likely interacted with the methylerythritol 4-phosphate (MEP) pathway and modulate the synthesis of either ABA or isoprene under stressful conditions. Overall, our study highlights the effectiveness of ALA supply through soil fertirrigation in preserving the young apical developing leaves from the detrimental effects of salt stress, thus helping of A. donax to cope with salinity and favoring the recovery of the whole plant once the stress is removed.     

Genome-Wide Identification and Transcriptional Expression Analysis of Annexin Genes in Capsicum annuum and Characterization of CaAnn9 in Salt Tolerance

Annexin (Ann) is a polygenic, evolutionarily conserved, calcium-dependent and phospholipid-binding protein family, which plays key roles in plant growth, development, and stress response. However, a comprehensive understanding of CaAnn genes of pepper (Capsicum annuum) at the genome-wide level is limited. Based on the available pepper genomic information, we identified 15 members of the CaAnn gene family. Phylogenetic analysis showed that CaAnn proteins could be categorized into four different orthologous groups. Real time quantitative RT-PCR analysis showed that the CaAnn genes were tissue-specific and were widely expressed in pepper leaves after treatments with cold, salt, and drought, as well as exogenously applied MeJA and ABA. In addition, the function of CaAnn9 was further explored using the virus-induced gene silencing (VIGS) technique. CaAnn9-silenced pepper seedlings were more sensitive to salt stress, reflected by the degradation of chlorophyll, the accumulation of reactive oxygen species (ROS), and the decrease of antioxidant defense capacity. This study provides important information for further study of the role of pepper CaAnn genes and their coding proteins in growth, development, and environmental responses.     

Nutrient Supply and Simulated Herbivory Differentially Alter the Metabolite Pools and the Efficacy of the Glucosinolate-Based Defense System in <Emphasis Type="Italic">Brassica</Emphasis> Species

Environmental stress hinders growth of plants and commonly results in the accumulation of carbon-based defense compounds. However, the dynamics of nitrogen (N)-containing defense compounds are less predictable under environmental stress. The impact of nutrient deficiency on plant defenses that require the metabolic conversion of a less toxic compound to a more potent toxin is even more poorly understood. We evaluated the effects of nitrogen (N) and potassium (K) deficiency and simulated herbivory on the concentration of metabolites including glucosinolates (GSLs), on the conversion of GSLs to more toxic isothiocyanates (ITCs), and on the activity of myrosinase (MYR) in leaves of Brassica juncea and Brassica nigra. Both species contained GSLs, predominantly sinigrin, but also derivatives of glucobrassicin. Compared to the control, N deficiency increased the sinigrin concentration in both species. Methyl jasmonate (MeJA) application increased sinigrin production in B. junceae, whereas in B. nigra MeJA increased sinigrin only under K-deficiency. Compared to the aliphatic-glucosinolates, MeJA application produced a greater compositional change in the profiles of indolic-glucosinolates. In both species the increase in sinigrin content of the tissue was associated with a decrease in its overall nutritive value as assessed by the content of sugars and amino acids. In B. juncea, application of MeJA decreased the conversion of sinigrin to allyl isothiocyanate (AITC) under both N and K deficiency. The potential activity of MYR decreased in both species under N deficiency. The reduced conversion of sinigrin to AITC and the lower activity of MYR suggest that the GSL-ITC defense system might have a limited efficiency in deterring generalist herbivores under environmental stress.     

Genome-Wide Analysis and Profile of UDP-Glycosyltransferases Family in Alfalfa (Medicago sativa L.) under Drought Stress

Drought stress is one of the major constraints that decreases global crop productivity. Alfalfa, planted mainly in arid and semi-arid areas, is of crucial importance in sustaining the agricultural system. The family 1 UDP-glycosyltransferases (UGT) is indispensable because it takes part in the regulation of plant growth and stress resistance. However, a comprehensive insight into the participation of the UGT family in adaptation of alfalfa to drought environments is lacking. In the present study, a genome-wide analysis and profiling of the UGT in alfalfa were carried out. A total of 409 UGT genes in alfalfa (MsUGT) were identified and they are clustered into 13 groups. The expression pattern of MsUGT genes were analyzed by RNA-seq data in six tissues and under different stresses. The quantitative real-time PCR verification genes suggested the distinct role of the MsUGT genes under different drought stresses and abscisic acid (ABA) treatment. Furthermore, the function of MsUGT003 and MsUGT024, which were upregulated under drought stress and ABA treatment, were characterized by heterologous expression in yeast. Taken together, this study comprehensively analyzed the UGT gene family in alfalfa for the first time and provided useful information for improving drought tolerance and in molecular breeding of alfalfa.