Phytohormone Quantification Research Articles

Ethylene promotes fruit ripening initiation by downregulating photosynthesis, enhancing abscisic acid and suppressing jasmonic acid in blueberry (Vaccinium ashei).

Blueberry fruit exhibit atypical climacteric ripening with a non-auto-catalytic increase in ethylene coincident with initiation of ripening. Further, application of ethephon, an ethylene-releasing plant growth regulator, accelerates ripening by increasing the proportion of ripe (blue) fruit as compared to the control treatment. To investigate the mechanistic role of ethylene in regulating blueberry ripening, we performed transcriptome analysis on fruit treated with ethephon, an ethylene-releasing plant growth regulator. RNA-Sequencing was performed on two sets of rabbiteye blueberry ('Powderblue') fruit: (1) fruit from divergent developmental stages; and (2) fruit treated with ethephon, an ethylene-releasing compound. Differentially expressed genes (DEGs) from divergent developmental stages clustered into nine groups, among which cluster 1 displayed reduction in expression during ripening initiation and was enriched with photosynthesis related genes, while cluster 7 displayed increased expression during ripening and was enriched with aromatic-amino acid family catabolism genes, suggesting stimulation of anthocyanin biosynthesis. More DEGs were apparent at 1 day after ethephon treatment suggesting its early influence during ripening initiation. Overall, a higher number of genes were downregulated in response to ethylene. Many of these overlapped with cluster 1 genes, indicating that ethylene-mediated downregulation of photosynthesis is an important developmental event during the ripening transition. Analyses of DEGs in response to ethylene also indicated interplay among phytohormones. Ethylene positively regulated abscisic acid (ABA), negatively regulated jasmonates (JAs), and influenced auxin (IAA) metabolism and signaling genes. Phytohormone quantification supported these effects of ethylene, indicating coordination of blueberry fruit ripening by ethylene. This study provides insights into the role of ethylene in blueberry fruit ripening. Ethylene initiates blueberry ripening by downregulating photosynthesis-related genes. Also, ethylene regulates phytohormone-metabolism and signaling related genes, increases ABA, and decreases JA concentrations. Together, these results indicate that interplay among multiple phytohormones regulates the progression of ripening, and that ethylene is an important coordinator of such interactions during blueberry fruit ripening.

Effects of chitin and chitosan on root growth, biochemical defense response and exudate proteome of Cannabis sativa.

Fungal pathogens pose a major threat to Cannabis sativa production, requiring safe and effective management procedures to control disease. Chitin and chitosan are natural molecules that elicit plant defense responses. Investigation of their effects on C. sativa will advance understanding of plant responses towards elicitors and provide a potential pathway to enhance plant resistance against diseases. Plants were grown in the in vitro Root-TRAPR system and treated with colloidal chitin and chitosan. Plant morphology was monitored, then plant tissues and exudates were collected for enzymatic activity assays, phytohormone quantification, qPCR analysis and proteomics profiling. Chitosan treatments showed increased total chitinase activity and expression of pathogenesis-related (PR) genes by 3-5 times in the root tissues. In the exudates, total peroxidase and chitinase activities and levels of defense proteins such as PR protein 1 and endochitinase 2 were increased. Shoot development was unaffected, but root development was inhibited after chitosan exposure. In contrast, chitin treatments had no significant impact on any defense parameters, including enzymatic activities, hormone quantities, gene expression levels and root secreted proteins. These results indicate that colloidal chitosan, significantly enhancing defense responses in C. sativa root system, could be used as a potential elicitor, particularly in hydroponic scenarios to manage crop diseases.

Comparative proteomic analysis between mature and germinating seeds in Paris polyphylla var. yunnanensis.

The long dormancy period of Paris polyphylla var. yunnanensis seeds affects the supply of this scarce plant, which is used as an important traditional Chinese medicine. Mature seeds with a globular embryo and germinating seeds with developed embryo were used to explore the mechanisms of seed germination in this species. The protein profiles between the mature and germinating seeds were compared using the isobaric tags for relative and absolute quantification (iTRAQ) approach. Of the 4,488 proteins identified, a total of 1,305 differentially expressed proteins (DEPs) were detected. A Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of these DEPs indicated that metabolic pathways and the biosynthesis of secondary metabolites were the two top pathways. Additionally, phytohormone quantification shows that the abscisic acid (ABA) level significantly decreased, whereas the GA3 level dramatically increased among nine endogenous gibberellins (GAs), resulting in a significant increase of the GA3/ABA ratio in germinating seeds. The biosynthesis pathways of carotenoid as a precursor for ABA production and GA were further analyzed, and showed that proteinic expressions of the candidate genes in the two pathways did not correlate with the transcriptional abundances. However, 9-cis-epoxycarotenoid dioxygenase (NCED), a rate limited enzyme for ABA biosynthesis, was significantly decreased in mRNA levels in germinating seeds. By contrast, gibberellin 20-oxidase (GA20ox), a key enzyme GA biosynthesis, exhibited the major increase in one copy and a slight decrease in three others at the protentional level in germinating seeds. Gibberellin 2-oxidase (GA2ox), an inactivate enzyme in bioactive GAs, has the tendency to down-regulate in mRNA or at the proteinic level in germinating seeds. Altogether, these results suggested that the analyses of ABA and GA levels, the GA3/ABA ratio, and the expressional patterns of their regulatory genes may provide a novel mechanistic understanding of how phytohormones regulate seed germination in P. polyphylla var. yunnanensis.

Comparative study on Synergistic effect of plant growth promoting microalgae and Panchagavya in reclaiming the wasteland and growth induction of Vigna radiata

Objectives: The present comparison investigation determines the synergistic effects of employing Plant Growth Promoters [Spirulina platensis and organic fertilizer (Panchagavya)] in a waste land soil bag containing Vigna radiata seeds. Method: Soil from the waste landfill were collected and stored for the experiment. The plants were cultivated in experimental pots (Control, A, B, and C groups) using waste land soil supplemented with PGP [Plant growth promoters] in volume separately. Group A was treated with Spirulina platensis. Group B was treated with Panchagavya. Group C were treated with the consortium (Spirulina platensis + Panchagavya). Control Pots were treated only with water. The crop’s growth characteristics were measured after 14 days. Biomass production [Phosphorous], and phytohormone quantification were done. The relationship between various growth and yield were determined using Pearson’s correlation coefficients. Results: Overall growth and physiology of Vigna radiata were significantly influenced by the addition of PGP in Consortium. The available phosphorus status of the soil is 40 kg / ha, after consortium treatment. Significant positive correlations of grain yield were observed with consortium (r = 0.716; p < 0.05) while non consortium showed comparatively weaker correlation, microalgae – Spirulina platensis (r = 0.448; p > 0.05) and panchagavya (r = 0.407; p > 0.05). As a result, using biofertilizer in a consortium mode will be an effective strategy for maintaining biomass production in an alkaline environment or in wasteland management. Keywords: Spirulina platensis; Phyto hormones; Panchagavya; consortium; Vigna radiate; Auxins

Metabolic disturbances in sugar beet (Beta vulgaris) during infection with Beet necrotic yellow vein virus

Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV) is an economically important disease of sugar beet. Management includes the use of single resistance (Rz) genes. However, strains with the ability to overcome the widely used Rz1 resistance gene have already been reported. The mechanisms associated with these genes are unknown, therefore increased understanding of the host defense response may lead to more durable sources of rhizomania resistance. Non-targeted metabolome profiling, coupled with targeted phytohormone quantification, was applied to characterize the metabolic and signaling pathways associated with BNYVV infections in sugar beet. Clear differences in metabolites were identified following pathogen infection independent of plant genotype particularly those associated with host lipid composition or other membrane associated functions.

Grape phylloxera (D. vitifoliae) manipulates SA/JA concentrations and signalling pathways in root galls of Vitis spp.

Overcoming host defensive traits is a prerequisite to establish compatible plant-parasite interactions. Following parasite perception, jasmonic (JA) and salicylic acid (SA) signalling pathways mediate biotic stress signals resulting in the activation of host defence responses. Piercing-sucking grape phylloxera (Daktulosphaira vitifoliae) infests Vitis spp. by the formation of organoid root galls. This study aims to investigate whether host defensive SA/JA signalling pathways are affected during D. vitifoliae infestation. We hypothesize that the JA signalling pathway is induced during larval probing (14 hai). Compatible root gall formation (24 hai − 14 dai) involves the reduction of the JA, but the induction of the SA signaling pathway. T5C (V.berlandieri x V.riparia) cuttings are infested with a D. vitifoliae single founder lineage (biotype C). Phytohormone quantification (HPLC-MS) and transcriptional alterations of JA/SA marker genes (qRT-PCR) are determined in root tissues from larval probing (14 hai) until gall formation (>14 dai). Non-infested root tips are considered controls. Our results show a significant induction of all analysed JA marker genes during insect probing (14 hai), but their significant reduction during early gall formation (24 hai). Following gall formation (5–14 dai) SA and JA-Ile increase. However, only the analysed SA marker genes are induced, whereas JA marker gene expression levels are significantly reduced. Based on this data we conclude that the observed suppression of the JA signalling pathway might represent an important step for the compatible D. vitifoliae – Vitis spp. root interaction. We discuss whether the induced SA defences protect nutritive root galls against soil microbes.

Assessment of germination, phytochemicals, and transcriptional responses to ethephon priming in soybean [Glycine max (L.) Merrill

The present work aims to dissect the underlying signaling pathways associated with soybean [Glycine max (L.) Merrill] seed hormo-priming with ethephon (Eth). Our results demonstrated that soybean germination improved significantly upon Eth priming (Ethp). Phytohormone quantification shows relative enhanced endogenous gibberellin A4 (GA4) levels concomitant with impaired biogenesis and signaling of auxin, viz., indole acetic acid (IAA) and abscisic acid (ABA). Phytochemical analysis revealed relative reduced levels of individual and total raffinose family oligosaccharide (RFO) components, starch, soluble sugars, and sucrose concomitant with enhanced levels of reducing sugars, glucose, cellular ATP, and acetyl-CoA pools. Secondary metabolite analysis revealed the activation of the mevalonate (MVA) pathway with a concomitant suppression of the plastidal 2-methyl-d-erythritol-4-phosphate/1-deoxy-d-xylulose-5-phosphate (MEP/DOX) and phenylpropanoid pathways, substantiated by relative reduced levels of total phenolics, tannins, and proanthocyanidin. Ethp also enhances the in vitro antioxidative activity (viz., 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability and ferric reducing antioxidant power (FRAP)) and endogenous antioxidants levels (viz., flavonoids, isoflavones, β-carotene, vitamin C, and vitamin E). Further quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed transcriptional pattern of representative genes in agreement with these metabolic alterations.

Transcriptomic and Phytochemical Analyses Reveal Root-Mediated Resource-Based Defense Response to Leaf Herbivory by Ectropis oblique in Tea Plant ( Camellia sinensis).

Leaf herbivory on tea plants ( Camellia sinensis) by tea geometrids ( Ectropis oblique) severely threaten the yield and quality of tea. In previous work, we found that local defense response was induced in damaged leaves by geometrids at the transcriptome level. Here, we investigated the systemic response triggered in undamaged roots and the potential role of roots in response to leaf herbivory. Comparative transcriptome analysis and carbohydrate dynamics indicated that leaf herbivory activated systemic carbon reallocation to enhance resource investment for local secondary metabolism. The crucial role of jasmonic acid and the involvement of other potential hormone signals for local and systemic signaling networks were supported by phytohormone quantification and dynamic expression analysis of phytohormone-related genes. This work represents a deep understanding of the interaction of tea plants and geometrids from the perspective of systems biology and reveals that tea plants have evolved an intricate root-mediated resource-based resistance strategy to cope with geometrid attack.

Temperature and Hormones Associated with Bacterial Etiolation Symptoms of Creeping Bentgrass and Annual Bluegrass

Bacterial etiolation, caused by Acidovorax avenae or Xanthomonas translucens pv. poae, is a problematic disease of creeping bentgrass (Agrostis stolonifera) and annual bluegrass (Poa annua) turfgrass stands. The objective of this study was to determine whether temperature may play a role in phytohormone production by these bacteria and to determine if there is any change in phytohormones specifically in etiolated plant tissues. In vitro, A. avenae and X. translucens were cultured at 25, 30, 35 and 40 °C for 14 days. Bacterial cultures were sampled for gibberellic acid isoforms (GA1, GA3, GA4, and GA20), jasmonic acid, salicylic acid (SA), indole-3-acetic acid (IAA), zeatin riboside, and abscisic acid (ABA). No phytohormones were detected in pure cultures of X. translucens, whereas A. avenae produced GA1, GA3, GA4, and IAA. Acidovorax avenae isolate MSU-13 produced significantly higher GA1 and GA3 at 35 and 40 °C on most days measured. In the field study, etiolated stem and leaf tissues of creeping bentgrass and annual bluegrass were evaluated for bacterial identification, etiolation ratings, and phytohormone quantification. Significantly more etiolation symptoms were observed in both annual bluegrass and creeping bentgrass on days with higher temperatures. In annual bluegrass, etiolated stems and leaves showed significantly higher GA1, GA3, etiolated leaves showed significantly higher GA20, ABA, and lower IAA compared to normal turf tissues on most experimental dates. In creeping bentgrass, higher GA1, GA4, SA, ABA, and lower IAA were detected in etiolated turf tissues than normal tissues on most experimental dates. Plant hormone regulation due to the presence of bacteria may be altered differentially in response to different bacteria species eliciting similar plant symptoms.

GhWIP2, a WIP zinc finger protein, suppresses cell expansion in Gerbera hybrida by mediating crosstalk between gibberellin, abscisic acid, and auxin.

Cell expansion is a key determinant for the final size and shape of plant organ, and is regulated by various phytohormones. Zinc finger proteins (ZFPs) consist of a superfamily involved in multiple aspects of organ morphogenesis. However, little is known about WIP-type ZFP function in phytohormone-mediated organ growth. Using reverse genetics, RNA-seq and phytohormone quantification, we elucidated the role of a new WIP-type ZFP from Gerbera hybrida, GhWIP2, in controlling organ growth via regulation of cell expansion. GhWIP2 localizes to the nucleus and acts as a transcriptional repressor. Constitutive overexpression of GhWIP2 (GhWIP2OE) in both Gerbera and Arabidopsis thaliana caused major developmental defects associated with cell expansion, including dwarfism, short petals, scapes, and petioles. Furthermore, GhWIP2OE plants were hypersensitive to GA, but not to ABA, and showed a reduction in endogenous GA and auxin, but not ABA concentrations. Consistent with these observations, RNA-seq analysis revealed that genes involved in GA and auxin signaling were down-regulated, while those involved in ABA signaling were up-regulated in GhWIP2OE plants. Our findings suggest that GhWIP2 acts as a transcriptional repressor, suppressing cell expansion during organ growth by modulating crosstalk between GA, ABA, and auxin.

Aphid (Myzus persicae) feeding on the parasitic plant dodder (Cuscuta australis) activates defense responses in both the parasite and soybean host

Dodders (Cuscuta spp.) are shoot holoparasites, whose haustoria penetrate host tissues to enable fusion between the parasite and host vascular systems, allowing Cuscuta to extract water, nutrients and other molecules from hosts. Aphids are piercing-sucking herbivores that use specialized stylets to feed on phloem sap. Aphids are known to feed on Cuscuta, but how Cuscuta and its host plant respond to aphids attacking the parasite was unknown. Phytohormone quantification, transcriptomic analysis and bioassays were performed to determine the responses of Cuscuta australis and its soybean (Glycine max) hosts to the feeding of green peach aphid (GPA; Myzus persicae) on C.australis. Decreased salicylic acid levels and 172 differentially expressed genes (DEGs) were found in GPA-attacked C.australis, and the soybean hosts exhibited increased jasmonic acid contents and 1015 DEGs, including >100 transcription factor genes. Importantly, GPA feeding on C.australis increased the resistance of the soybean host to subsequent feeding by the leafworm Spodoptera litura and soybean aphid Aphis glycines, resulting in 21% decreased leafworm mass and 41% reduced aphid survival rate. These data strongly suggest that GPA feeding on Cuscuta induces a systemic signal, which is translocated to hosts and activates defense against herbivores.

Exogenous auxin represses soybean seed germination through decreasing the gibberellin/abscisic acid (GA/ABA) ratio

Auxin is an important phytohormone which mediates diverse development processes in plants. Published research has demonstrated that auxin induces seed dormancy. However, the precise mechanisms underlying the effect of auxin on seed germination need further investigation, especially the relationship between auxins and both abscisic acid (ABA) and gibberellins (GAs), the latter two phytohormones being the key regulators of seed germination. Here we report that exogenous auxin treatment represses soybean seed germination by enhancing ABA biosynthesis, while impairing GA biogenesis, and finally decreasing GA1/ABA and GA4/ABA ratios. Microscope observation showed that auxin treatment delayed rupture of the soybean seed coat and radicle protrusion. qPCR assay revealed that transcription of the genes involved in ABA biosynthetic pathway was up-regulated by application of auxin, while expression of genes involved in GA biosynthetic pathway was down-regulated. Accordingly, further phytohormone quantification shows that auxin significantly increased ABA content, whereas the active GA1 and GA4 levels were decreased, resulting insignificant decreases in the ratiosGA1/ABA and GA4/ABA.Consistent with this, ABA biosynthesis inhibitor fluridone reversed the delayed-germination phenotype associated with auxin treatment, while paclobutrazol, a GA biosynthesis inhibitor, inhibited soybean seed germination. Altogether, exogenous auxin represses soybean seed germination by mediating ABA and GA biosynthesis.

Karrikins delay soybean seed germination by mediating abscisic acid and gibberellin biogenesis under shaded conditions.

Karrikins (KAR) are a class of signal compounds, discovered in wildfire smoke, which affect seed germination. Currently, numerous studies have focused on the model plant Arabidopsis in the KAR research field, rather than on crops. Thus the regulatory mechanisms underlying KAR regulation of crop seed germination are largely unknown. Here, we report that KAR delayed soybean seed germination through enhancing abscisic acid (ABA) biosynthesis, while impairing gibberellin (GA) biogenesis. Interestingly, KAR only retarded soybean seed germination under shaded conditions, rather than under dark and white light conditions, which differs from in Arabidopsis. Phytohormone quantification showed that KAR enhanced ABA biogenesis while impairing GA biosynthesis during the seed imbibition process, and subsequently, the ratio of active GA4 to ABA was significantly reduced. Further qRT-PCR analysis showed that the transcription pattern of genes involved in ABA and GA metabolic pathways are consistent with the hormonal measurements. Finally, fluridone, an ABA biogenesis inhibitor, remarkably rescued the delayed-germination phenotype of KAR-treatment; and paclobutrazol, a GA biosynthesis inhibitor, inhibited soybean seed germination. Taken together, these evidences suggest that KAR inhibit soybean seed germination by mediating the ratio between GA and ABA biogenesis.

Gibberellins interfere with symbiosis signaling and gene expression and alter colonization by arbuscular mycorrhizal fungi in Lotus japonicus.

Arbuscular mycorrhiza is a mutualistic plant-fungus interaction that confers great advantages for plant growth. Arbuscular mycorrhizal (AM) fungi enter the host root and form symbiotic structures that facilitate nutrient supplies between the symbionts. The gibberellins (GAs) are phytohormones known to inhibit AM fungal infection. However, our transcriptome analysis and phytohormone quantification revealed GA accumulation in the roots of Lotus japonicus infected with AM fungi, suggesting that de novo GA synthesis plays a role in arbuscular mycorrhiza development. We found pleiotropic effects of GAs on the AM fungal infection. In particular, the morphology of AM fungal colonization was drastically altered by the status of GA signaling in the host root. Exogenous GA treatment inhibited AM hyphal entry into the host root and suppressed the expression of Reduced Arbuscular Mycorrhization1 (RAM1) and RAM2 homologs that function in hyphal entry and arbuscule formation. On the other hand, inhibition of GA biosynthesis or suppression of GA signaling also affected arbuscular mycorrhiza development in the host root. Low-GA conditions suppressed arbuscular mycorrhiza-induced subtilisin-like serine protease1 (SbtM1) expression that is required for AM fungal colonization and reduced hyphal branching in the host root. The reduced hyphal branching and SbtM1 expression caused by the inhibition of GA biosynthesis were recovered by GA treatment, supporting the theory that insufficient GA signaling causes the inhibitory effects on arbuscular mycorrhiza development. Most studies have focused on the negative role of GA signaling, whereas our study demonstrates that GA signaling also positively interacts with symbiotic responses and promotes AM colonization of the host root.

Validated method for phytohormone quantification in plants.

Phytohormones are long time known as important components of signaling cascades in plant development and plant responses to various abiotic and biotic challenges. Quantifications of phytohormone levels in plants are typically carried out using GC or LC-MS/MS systems, due to their high sensitivity, specificity, and the fact that not much sample preparation is needed. However, mass spectrometer-based analyses are often affected by the particular sample type (different matrices), extraction procedure, and experimental setups, i.e., the chromatographic separation system and/or mass spectrometer analyser (Triple-quadrupole, Iontrap, TOF, Orbitrap). For these reasons, a validated method is required in order to enable comparison of data that are generated in different laboratories, under different experimental set-ups, and in different matrices. So far, many phytohormone quantification studies were done using either QTRAP or Triple-quadrupole mass spectrometers. None of them was performed under the regime of a fully-validated method. Therefore, we developed and established such validated method for quantification of stress-related phytohormones such as jasmonates, abscisic acid, salicylic acid, IAA, in the model plant Arabidopsis thaliana and the fruit crop Citrus sinensis, using an Iontrap mass spectrometer. All parameters recommended by FDA (US Food and Drug Administration) or EMEA (European Medicines Evaluation Agency) for validation of analytical methods were evaluated: sensitivity, selectivity, repeatability and reproducibility (accuracy and precision).

Biosensors for phytohormone quantification: challenges, solutions, and opportunities

Fluorescent reporters are valuable tools for plant science research, particularly as sensors to monitor biological signals and developmental processes. Such biosensors are particularly useful to monitor the spatial and temporal distribution of small signalling molecules such as phytohormones. Knowledge of perception and signalling pathways can be exploited to design biosensors for estimating intracellular abundance of hormones. However, the nonlinear relationship between target molecule and reporter necessitates the development of parameterised mathematical models to quantitatively relate sensor fluorescence to hormone abundance. In this opinion article, we will discuss the use of transcriptional reporters, sensor design strategy, and the importance of mathematical modelling approaches and technological advances in the development of new techniques to allow truly quantitative analyses of hormone regulated processes.