Chromium Toxicity in Plants: Signaling, Mitigation, and Future Perspectives.

Plants are very often confronted by different heavy metal (HM) stressors that adversely impair their growth and productivity. Among HMs, chromium (Cr) is one of the most prevalent toxic trace metals found in agricultural soils because of anthropogenic activities, lack of efficient treatment, and unregulated disposal. It has a huge detrimental impact on the physiological, biochemical, and molecular traits of crops, in addition to being carcinogenic to humans. In soil, Cr exists in different forms, including Cr (III) "trivalent" and Cr (VI) "hexavalent", but the most pervasive and severely hazardous form to the biota is Cr (VI). Despite extensive research on the effects of Cr stress, the exact molecular mechanisms of Cr sensing, uptake, translocation, phytotoxicity, transcript processing, translation, post-translational protein modifications, as well as plant defensive responses are still largely unknown. Even though plants lack a Cr transporter system, it is efficiently accumulated and transported by other essential ion transporters, hence posing a serious challenge to the development of Cr-tolerant cultivars. In this review, we discuss Cr toxicity in plants, signaling perception, and transduction. Further, we highlight various mitigation processes for Cr toxicity in plants, such as microbial, chemical, and nano-based priming. We also discuss the biotechnological advancements in mitigating Cr toxicity in plants using plant and microbiome engineering approaches. Additionally, we also highlight the role of molecular breeding in mitigating Cr toxicity in sustainable agriculture. Finally, some conclusions are drawn along with potential directions for future research in order to better comprehend Cr signaling pathways and its mitigation in sustainable agriculture.

Endophytic Fungi: Key Insights, Emerging Prospects, and Challenges in Natural Product Drug Discovery.

Plant-associated endophytes define an important symbiotic association in nature and are established bio-reservoirs of plant-derived natural products. Endophytes colonize the internal tissues of a plant without causing any disease symptoms or apparent changes. Recently, there has been a growing interest in endophytes because of their beneficial effects on the production of novel metabolites of pharmacological significance. Studies have highlighted the socio-economic implications of endophytic fungi in agriculture, medicine, and the environment, with considerable success. Endophytic fungi-mediated biosynthesis of well-known metabolites includes taxol from Taxomyces andreanae, azadirachtin A and B from Eupenicillium parvum, vincristine from Fusarium oxysporum, and quinine from Phomopsis sp. The discovery of the billion-dollar anticancer drug taxol was a landmark in endophyte biology/research and established new paradigms for the metabolic potential of plant-associated endophytes. In addition, endophytic fungi have emerged as potential prolific producers of antimicrobials, antiseptics, and antibiotics of plant origin. Although extensively studied as a "production platform" of novel pharmacological metabolites, the molecular mechanisms of plant-endophyte dynamics remain less understood/explored for their efficient utilization in drug discovery. The emerging trends in endophytic fungi-mediated biosynthesis of novel bioactive metabolites, success stories of key pharmacological metabolites, strategies to overcome the existing challenges in endophyte biology, and future direction in endophytic fungi-based drug discovery forms the underlying theme of this article.

Recent Advances in the Metabolic Engineering of Yeasts for Ginsenoside Biosynthesis.

Ginsenosides are a group of glycosylated triterpenes isolated from Panax species. Ginsenosides are promising candidates for the prevention and treatment of cancer as well as food additives. However, owing to a lack of efficient approaches for ginsenoside production from plants and chemical synthesis, ginsenosides may not yet have reached their full potential as medicinal resources. In recent years, an alternative approach for ginsenoside production has been developed using the model yeast Saccharomyces cerevisiae and non-conventional yeasts such as Yarrowia lipolytica and Pichia pastoris. In this review, various metabolic engineering strategies, including heterologous gene expression, balancing, and increasing metabolic flux, and enzyme engineering, have been described as recent advanced engineering techniques for improving ginsenoside production. Furthermore, the usefulness of a systems approach and fermentation strategy has been presented. Finally, the present challenges and future research direction for industrial cell factories have been discussed.

Proteomic Changes in the Sound Vibration-Treated Arabidopsis thaliana Facilitates Defense Response during Botrytis cinerea Infection.

Sound vibration (SV) treatment can trigger various molecular and physiological changes in plants. Previously, we showed that pre-exposure of Arabidopsis plants to SV boosts its defense response against Botrytis cinerea fungus. The present study was aimed to investigate the changes in the proteome states in the SV-treated Arabidopsis during disease progression. Proteomics analysis identified several upregulated proteins in the SV-infected plants (i.e., SV-treated plants carrying Botrytis infection). These upregulated proteins are involved in a plethora of biological functions, e.g., primary metabolism (i.e., glycolysis, tricarboxylic acid cycle, ATP synthesis, cysteine metabolism, and photosynthesis), redox homeostasis, and defense response. Additionally, our enzyme assays confirmed the enhanced activity of antioxidant enzymes in the SV-infected plants compared to control plants. Broadly, our results suggest that SV pre-treatment evokes a more efficient defense response in the SV-infected plants by modulating the primary metabolism and reactive oxygen species scavenging activity.

Positive regulatory role of sound vibration treatment in Arabidopsis thaliana against Botrytis cinerea infection.

Sound vibration (SV), a mechanical stimulus, can trigger various molecular and physiological changes in plants like gene expression, hormonal modulation, induced antioxidant activity and calcium spiking. It also alters the seed germination and growth of plants. In this study, we investigated the effects of SV on the resistance of Arabidopsis thaliana against Botrytis cinerea infection. The microarray analysis was performed on infected Arabidopsis plants pre-exposed to SV of 1000 Hertz with 100 decibels. Broadly, the transcriptomic analysis revealed up-regulation of several defense and SA-responsive and/or signaling genes. Quantitative real-time PCR (qRT-PCR) analysis of selected genes also validated the induction of SA-mediated response in the infected Arabidopsis plants pre-exposed to SV. Corroboratively, hormonal analysis identified the increased concentration of salicylic acid (SA) in the SV-treated plants after pathogen inoculation. In contrast, jasmonic acid (JA) level in the SV-treated plants remained stable but lower than control plants during the infection. Based on these findings, we propose that SV treatment invigorates the plant defense system by regulating the SA-mediated priming effect, consequently promoting the SV-induced resistance in Arabidopsis against B. cinerea.

Use of Ultrasonication Technology for the Increased Production of Plant Secondary Metabolites.

Plant secondary metabolites (PSMs) provide taste, color, odor, and resistance to plants, and they are also used to treat cancer and cardiovascular diseases. Synthesis of PSMs in plants is stimulated in response to different forms of external stress. Use of ultrasonication (US) to clean or decontaminate fruits and vegetables leads to physical stress that finally results in the accumulation of PSMs. US can stimulate accumulation of taxol, ginsenoside saponins, shikonin, and resveratrol, e.g., up to 319-fold increase of resveratrol synthesis has been observed in grape due to US. US also increases carotenoids, total phenolics, and isoflavonoids accumulation. Furthermore, US shows synergistic effects in PSMs synthesis-when combined with ultraviolet (UV) irradiation, jasmonic acid (JA) or salicylic acid (SA). It has been observed that US stimulates the production of reactive oxygen species (ROS) which then upregulates expression of phenylalanine ammonia lyase (PAL), resulting in the synthesis of PSMs. In this review, we summarize the effects of US, as a physical stress, to maximize the accumulation of PSMs in crop produce and in cell cultures.

Novel Genomic and Evolutionary Perspective of Cyanobacterial tRNAs.

Transfer RNA (tRNA) plays a central role in protein synthesis and acts as an adaptor molecule between an mRNA and an amino acid. A tRNA has an L-shaped clover leaf-like structure and contains an acceptor arm, D-arm, D-loop, anti-codon arm, anti-codon loop, variable loop, Ψ-arm and Ψ-loop. All of these arms and loops are important in protein translation. Here, we aimed to delineate the genomic architecture of these arms and loops in cyanobacterial tRNA. Studies from tRNA sequences from 61 cyanobacterial species showed that, except for few tRNAs (tRNAAsn, tRNALeu, tRNAGln, and tRNAMet), all contained a G nucleotide at the 1st position in the acceptor arm. tRNALeu and tRNAMet did not contain any conserved nucleotides at the 1st position whereas tRNAAsn and tRNAGln contained a conserved U1 nucleotide. In several tRNA families, the variable region also contained conserved nucleotides. Except for tRNAMet and tRNAGlu, all other tRNAs contained a conserved A nucleotide at the 1st position in the D-loop. The Ψ-loop contained a conserved U1-U2-C3-x-A5-x-U7 sequence, except for tRNAGly, tRNAAla, tRNAVal, tRNAPhe, tRNAThr, and tRNAGln in which the U7 nucleotide was not conserved. However, in tRNAAsp, the U7 nucleotide was substituted with a C7 nucleotide. Additionally, tRNAArg, tRNAGly, and tRNALys of cyanobacteria contained a group I intron within the anti-codon loop region. Maximum composite likelihood study on the transition/transversion of cyanobacterial tRNA revealed that the rate of transition was higher than the rate of transversion. An evolutionary tree was constructed to understand the evolution of cyanobacterial tRNA and analyses revealed that cyanobacterial tRNA may have evolved polyphyletically with high rate of gene loss.

Pronounced Phenotypic Changes in Transgenic Tobacco Plants Overexpressing Sucrose Synthase May Reveal a Novel Sugar Signaling Pathway.

Soluble sugars not only serve as nutrients, but also act as signals for plant growth and development, but how sugar signals are perceived and translated into physiological responses in plants remains unclear. We manipulated sugar levels in transgenic plants by overexpressing sucrose synthase (SuSy), which is a key enzyme believed to have reversible sucrose synthesis and sucrose degradation functions. The ectopically expressed SuSy protein exhibited sucrose-degrading activity, which may change the flux of sucrose demand from photosynthetic to non-photosynthetic cells, and trigger an unknown sucrose signaling pathway that lead to increased sucrose content in the transgenic plants. An experiment on the transition from heterotrophic to autotrophic growth demonstrated the existence of a novel sucrose signaling pathway, which stimulated photosynthesis, and enhanced photosynthetic synthesis of sucrose, which was the direct cause or the sucrose increase. In addition, a light/dark time treatment experiment, using different day length ranges for photosynthesis/respiration showed the carbohydrate pattern within a 24-h day and consolidated the role of sucrose signaling pathway as a way to maintain sucrose demand, and indicated the relationships between increased sucrose and upregulation of genes controlling development of the shoot apical meristem (SAM). As a result, transgenic plants featured a higher biomass and a shorter time required to switch to reproduction compared to those of control plants, indicating altered phylotaxis and more rapid advancement of developmental stages in the transgenic plants.

Identification of new metabolites of bacterial transformation of indole by gas chromatography-mass spectrometry and high performance liquid chromatography.

Arthrobacter sp. SPG transformed indole completely in the presence of an additional carbon source. High performance liquid chromatography and gas chromatography-mass spectrometry detected indole-3-acetic acid, indole-3-glyoxylic acid, and indole-3-aldehyde as biotransformation products. This is the first report of the formation of indole-3-acetic acid, indole-3-glyoxylic acid, and indole-3-aldehyde from indole by any bacterium.

Selective Interaction Between Chloroplast ß-ATPase and TGB1L88 Retards Severe Symptoms Caused by Alternanthera mosaic virus Infection.

The multifunctional triple gene block protein 1 (TGB1) of the Potexvirus Alternanthera mosaic virus (AltMV) has been reported to have silencing suppressor, cell-to-cell movement, and helicase functions. Yeast two hybrid screening using an Arabidopsis thaliana cDNA library with TGB1 as bait, and co-purification with TGB1 inclusion bodies identified several host proteins which interact with AltMV TGB1. Host protein interactions with TGB1 were confirmed by biomolecular fluorescence complementation, which showed positive TGB1 interaction with mitochondrial ATP synthase delta' chain subunit (ATP synthase delta'), light harvesting chlorophyll-protein complex I subunit A4 (LHCA4), chlorophyll a/b binding protein 1 (LHB1B2), chloroplast-localized IscA-like protein (ATCPISCA), and chloroplast ß-ATPase. However, chloroplast ß-ATPase interacts only with TGB1L88, and not with weak silencing suppressor TGB1P88. This selective interaction indicates that chloroplast ß-ATPase is not required for AltMV movement and replication; however, TRV silencing of chloroplast ß-ATPase in Nicotiana benthamiana induced severe tissue necrosis when plants were infected by AltMV TGB1L88 but not AltMV TGB1P88, suggesting that ß-ATPase selectively responded to TGB1L88 to induce defense responses.

The coat protein of Alternanthera mosaic virus is the elicitor of a temperature-sensitive systemic necrosis in Nicotiana benthamiana, and interacts with a host boron transporter protein.

Different isolates of Alternanthera mosaic virus (AltMV; Potexvirus), including four infectious clones derived from AltMV-SP, induce distinct systemic symptoms in Nicotiana benthamiana. Virus accumulation was enhanced at 15 °C compared to 25 °C; severe clone AltMV 3-7 induced systemic necrosis (SN) and plant death at 15 °C. No interaction with potexvirus resistance gene Rx was detected, although SN was ablated by silencing of SGT1, as for other cases of potexvirus-induced necrosis. Substitution of AltMV 3-7 coat protein (CPSP) with that from AltMV-Po (CP(Po)) eliminated SN at 15 °C, and ameliorated symptoms in Alternanthera dentata and soybean. Substitution of only two residues from CP(Po) [either MN(13,14)ID or LA(76,77)IS] efficiently ablated SN in N. benthamiana. CPSP but not CP(Po) interacted with Arabidopsis boron transporter protein AtBOR1 by yeast two-hybrid assay; N. benthamiana homolog NbBOR1 interacted more strongly with CPSP than CP(Po) in bimolecular fluorescence complementation, and may affect recognition of CP as an elicitor of SN.

Bacterial degradation of chlorophenols and their derivatives.

Chlorophenols (CPs) and their derivatives are persistent environmental pollutants which are used in the manufacture of dyes, drugs, pesticides and other industrial products. CPs, which include monochlorophenols, polychlorophenols, chloronitrophenols, chloroaminophenols and chloromethylphenols, are highly toxic to living beings due to their carcinogenic, mutagenic and cytotoxic properties. Several physico-chemical and biological methods have been used for removal of CPs from the environment. Bacterial degradation has been considered a cost-effective and eco-friendly method of removing CPs from the environment. Several bacteria that use CPs as their sole carbon and energy sources have been isolated and characterized. Additionally, the metabolic pathways for degradation of CPs have been studied in bacteria and the genes and enzymes involved in the degradation of various CPs have been identified and characterized. This review describes the biochemical and genetic basis of the degradation of CPs and their derivatives.

Insights into Alternanthera mosaic virus TGB3 Functions: Interactions with Nicotiana benthamiana PsbO Correlate with Chloroplast Vesiculation and Veinal Necrosis Caused by TGB3 Over-Expression.

Alternanthera mosaic virus (AltMV) triple gene block 3 (TGB3) protein is involved in viral movement. AltMV TGB3 subcellular localization was previously shown to be distinct from that of Potato virus X (PVX) TGB3, and a chloroplast binding domain identified; veinal necrosis and chloroplast vesiculation were observed in Nicotiana benthamiana when AltMV TGB3 was over-expressed from PVX. Plants with over-expressed TGB3 showed more lethal damage under dark conditions than under light. Yeast-two-hybrid analysis and bimolecular fluorescence complementation (BiFC) reveal that Arabidopsis thaliana PsbO1 has strong interactions with TGB3; N. benthamiana PsbO (NbPsbO) also showed obvious interaction signals with TGB3 through BiFC. These results demonstrate an important role for TGB3 in virus cell-to-cell movement and virus-host plant interactions. The Photosystem II oxygen-evolving complex protein PsbO interaction with TGB3 is presumed to have a crucial role in symptom development and lethal damage under dark conditions. In order to further examine interactions between AtPsbO1, NbPsbO, and TGB3, and to identify the binding domain(s) in TGB3 protein, BiFC assays were performed between AtPsbO1 or NbPsbO and various mutants of TGB3. Interactions with C-terminally deleted TGB3 were significantly weaker than those with wild-type TGB3, and both N-terminally deleted TGB3 and a TGB3 mutant previously shown to lose chloroplast interactions failed to interact detectably with PsbO in BiFC. To gain additional information about TGB3 interactions in AltMV-susceptible plants, we cloned 12 natural AltMV TGB3 sequence variants into a PVX expression vector to examine differences in symptom development in N. benthamiana. Symptom differences were observed on PVX over-expression, with all AltMV TGB3 variants showing more severe symptoms than the WT PVX control, but without obvious correlation to sequence differences.

Actin Cytoskeleton and Golgi Involvement in Barley stripe mosaic virus Movement and Cell Wall Localization of Triple Gene Block Proteins.

Barley stripe mosaic virus (BSMV) induces massive actin filament thickening at the infection front of infected Nicotiana benthamiana leaves. To determine the mechanisms leading to actin remodeling, fluorescent protein fusions of the BSMV triple gene block (TGB) proteins were coexpressed in cells with the actin marker DsRed: Talin. TGB ectopic expression experiments revealed that TGB3 is a major elicitor of filament thickening, that TGB2 resulted in formation of intermediate DsRed:Talin filaments, and that TGB1 alone had no obvious effects on actin filament structure. Latrunculin B (LatB) treatments retarded BSMV cell-to-cell movement, disrupted actin filament organization, and dramatically decreased the proportion of paired TGB3 foci appearing at the cell wall (CW). BSMV infection of transgenic plants tagged with GFP-KDEL exhibited membrane proliferation and vesicle formation that were especially evident around the nucleus. Similar membrane proliferation occurred in plants expressing TGB2 and/or TGB3, and DsRed: Talin fluorescence in these plants colocalized with the ER vesicles. TGB3 also associated with the Golgi apparatus and overlapped with cortical vesicles appearing at the cell periphery. Brefeldin A treatments disrupted Golgi and also altered vesicles at the CW, but failed to interfere with TGB CW localization. Our results indicate that actin cytoskeleton interactions are important in BSMV cell-to-cell movement and for CW localization of TGB3.

Influences of four different light-emitting diode lights on flowering and polyphenol variations in the leaves of chrysanthemum (Chrysanthemum morifolium).

Light-emitting diodes (LEDs) are an efficient alternative to traditional lamps for plant growth. To investigate the influence of LEDs on flowering and polyphenol biosynthesis in the leaves of chrysanthemum, the plants were grown under supplemental blue, green, red, and white LEDs. Flower budding was formed even after a longer photoperiod than a critical day length of 13.5 h per day under blue light illumination. The weights of leaves and stems were highest under the white light illumination growth condition, whereas the weight of roots appeared to be independent of light quality. Among nine polyphenols characterized by high-performance liquid chromatography-tandem mass spectroscopy, three polyphenols were identified for the first time in chrysanthemum. A quantitation and principal component analysis biplot demonstrated that luteolin-7-O-glucoside (2), luteolin-7-O-glucuronide (3), and quercetagetin-trimethyl ether (8) were the highest polyphenols yielded under green light, and dicaffeoylquinic acid isomer (4), dicaffeoylquinic acid isomer (5), naringenin (7), and apigenin-7-O-glucuronide (6) were greatest under red light. Chlorogenic acid (1) and 1,2,6-trihydroxy-7,8-dimethoxy-3-methylanthraquinone (9) were produced in similar concentrations under both light types. The white and blue light appeared inefficient for polyphenol production. Taken together, our results suggest that the chrysanthemum flowering and polyphenol production are influenced by light quality composition.

Endophytic Trichoderma isolates from tropical environments delay disease onset and induce resistance against Phytophthora capsici in hot pepper using multiple mechanisms.

Endophytic Trichoderma isolates collected in tropical environments were evaluated for biocontrol activity against Phytophthora capsici in hot pepper (Capsicum annuum). Six isolates were tested for parasitic and antimicrobial activity against P. capsici and for endophytic and induced resistance capabilities in pepper. Isolates DIS 70a, DIS 219b, and DIS 376f were P. capsici parasites, while DIS 70a, DIS 259j, DIS 320c, and DIS 376f metabolites inhibited P. capsici. All six isolates colonized roots but were inefficient stem colonizers. DIS 259j, DIS 320c, and DIS 376f induced defense-related expressed sequence tags (EST) in 32-day-old peppers. DIS 70a, DIS 259j, and DIS 376f delayed disease development. Initial colonization of roots by DIS 259j or DIS 376f induced EST with potential to impact Trichoderma endophytic colonization and disease development, including multiple lipid transferase protein (LTP)-like family members. The timing and intensity of induction varied between isolates. Expression of CaLTP-N, encoding a LTP-like protein in pepper, in N. benthamiana leaves reduced disease development in response to P. nicotianae inoculation, suggesting LTP are functional components of resistance induced by Trichoderma species. Trichoderma isolates were endophytic on pepper roots in which, depending on the isolate, they delayed disease development by P. capsici and induced strong and divergent defense reactions.

Expression of thermostable bacterial beta-glucosidase (BglB) in transgenic tobacco plants.

Transgenic tobacco plants expressing the hyperthermostable beta-glucosidase BglB of Thermotoga maritima were generated with the goal of cost-effective production of the enzyme for the application in bioconversion of lignocellulosic biomass. The enzyme was targeted to the cytosol and chloroplasts, where it accumulated to level of 4.5% and 5.8% of total soluble protein, respectively. The optimal temperature and pH of the plant-expressed BglB was 80 degrees C and 4.5, respectively. BglB activity was preserved in leaves after lyophilization, but decreased by over 70% with drying at room temperature. When BglB was synergistically supplied in a 1% (w/v) rice straw with Cel5A for efficient cellulase conversion, a 37% increase in glucose was observed. This report demonstrates the potential of utilizing transgenic tobacco for mass production of BglB.

Mutation of a chloroplast-targeting signal in Alternanthera mosaic virus TGB3 impairs cell-to-cell movement and eliminates long-distance virus movement.

Cell-to-cell movement of potexviruses requires coordinated action of the coat protein and triple gene block (TGB) proteins. The structural properties of Alternanthera mosaic virus (AltMV) TGB3 were examined by methods differentiating between signal peptides and transmembrane domains, and its subcellular localization was studied by Agrobacterium-mediated transient expression and confocal microscopy. Unlike potato virus X (PVX) TGB3, AltMV TGB3 was not associated with the endoplasmic reticulum, and accumulated preferentially in mesophyll cells. Deletion and site-specific mutagenesis revealed an internal signal VL(17,18) of TGB3 essential for chloroplast localization, and either deletion of the TGB3 start codon or alteration of the chloroplast-localization signal limited cell-to-cell movement to the epidermis, yielding a virus that was unable to move into the mesophyll layer. Overexpression of AltMV TGB3 from either AltMV or PVX infectious clones resulted in veinal necrosis and vesiculation at the chloroplast membrane, a cytopathology not observed in wild-type infections. The distinctive mesophyll and chloroplast localization of AltMV TGB3 highlights the critical role played by mesophyll targeting in virus long-distance movement within plants.

Pathogenicity of Alternanthera mosaic virus is affected by determinants in RNA-dependent RNA polymerase and by reduced efficacy of silencing suppression in a movement-competent TGB1.

Four biologically active cDNA clones were derived from the Alternanthera mosaic virus (AltMV; genus Potexvirus) isolate, AltMV-SP, which differ in symptoms in infected Nicotiana benthamiana plants. Two clones induced necrosis and plant death; a mixture of all four clones induced milder symptoms than AltMV-SP. Replication of all clones was enhanced by a minimum of fourfold at 15 degrees C. A mixture of clones 4-7 (severe) and 3-1 (mild) was indistinguishable from AltMV-SP, but the ratio of 4-7 to 3-1 differed at 25 and 15 degrees C. RNA copy numbers of mixed infections were always below those of 4-7 alone. Determinants of symptom severity were identified in both Pol and TGB1; the mildest (4-1) and most severe (3-7) clones differed at three residues in the 'core' Pol domain [R(1110)P, K(1121)R, R(1255)K] and one [S(1535)P] in the C-terminal Pol domain of RNA-dependent RNA polymerase, and one in TGB1 [P(88)L]. Pol [P(1110),R(1121),K(1255)]+TGB1(L(88))] always induced systemic necrosis at 15 degrees C. Gene exchanges of Pol and TGB1 each affected replication and symptom expression, with TGB1(P(88)) significantly reducing silencing suppression. The difference in silencing suppression between TGB1(P(88)) and TGB1(L(88)) was confirmed by an agroinfiltration assay. Further, co-expression of TGB1(P(88)) and TGB1(L(88)) resulted in interference in the suppression of silencing by TGB1(L(88)). Yeast two-hybrid analysis confirmed that TGB1(P(88)) and TGB1(L(88)) interact. These results identify a TGB1 residue that significantly affects replication and silencing suppression, but maintains full movement functions.

The drought response of Theobroma cacao (cacao) and the regulation of genes involved in polyamine biosynthesis by drought and other stresses.

Drought can negatively impact pod production despite the fact that cacao production usually occurs in tropical areas having high rainfall. Polyamines (PAs) have been associated with the response of plants to drought in addition to their roles in responses to many other stresses. The constitutive and drought inducible expression patterns of genes encoding enzymes involved in PA biosynthesis were determined: an ornithine decarboxylase (TcODC), an arginine decarboxylase (TcADC), an S-adenosylmethionine decarboxylase (TcSAMDC), a spermidine synthase (TcSPDS), and a spermine synthase (TcSPMS). Expression analysis using quantitative real-time reverse transcription-PCR (QPCR) results showed that the PA biosynthesis genes were expressed in all plant tissues examined. Constitutive expression of PA biosynthesis genes was generally highest in mature leaves and open flowers. Expression of TcODC, TcADC, and TcSAMDC was induced with the onset of drought and correlated with changes in stomatal conductance, photosynthesis, photosystem II efficiency, leaf water potential and altered emission of blue-green fluorescence from cacao leaves. Induction of TcSAMDC in leaves was most closely correlated with changes in water potential. The earliest measured responses to drought were enhanced expression of TcADC and TcSAMDC in roots along with decreases in stomatal conductance, photosynthesis, and photosystem II efficiency. Elevated levels of putrescine, spermidine, and spermine were detected in cacao leaves 13days after the onset of drought. Expression of all five PA associated transcripts was enhanced (1.5-3-fold) in response to treatment with abscisic acid. TcODC and TcADC, were also responsive to mechanical wounding, infection by Phytophthora megakarya (a causal agent of black pod disease in cacao), the necrosis- and ethylene-inducing protein (Nep1) of Fusarium oxysporum, and flower abscission. TcSAMDC expression was responsive to all stresses except flower abscission. TcODC, although constitutively expressed at much lower levels than TcADC, TcSAMDC, TcSPDS, and TcSPMS, was highly inducible by the fungal protein Nep1 (135-fold) and the cacao pathogen Phytophthora megakarya (671-fold). The full length cDNA for ODC was cloned and characterized. Among the genes studied, TcODC, TcADC, and TcSAMDC were most sensitive to induction by drought in addition to other abiotic and biotic stresses. TcODC, TcADC, and TcSAMDC may share signal transduction pathways and/or the stress induced signal induction pathways may converge at these three genes leading to similar although not identical patterns of expression. It is possible altering PA levels in cacao will result in enhanced tolerance to multiple stresses including drought and disease as has been demonstrated in other crops.