Metagenomic Insights into the Composition and Function of Microbes Associated with the Rootzone of Datura inoxia

The purpose of this paper is to elucidate the roles that microbes may be playing in the rootzone of the medicinal plant Daturainoxia. We hypothesized that the microbes associated with the Datura rootzone would be significantly different than the similar surrounding fields in composition and function. We also hypothesized that rhizospheric and endophytic microbes would be associated with similar metabolic functions to the plant rootzone they inhabited. The methods employed were microbial barcoding, tests of essential oils against antibiotic resistant bacteria and other soil bacterial isolates, 16S Next Generation Sequencing (NGS) metabarcoding, and Whole Genome Shotgun (WGS) taxonomic and functional analyses. A few of the main bacterial genera of interest that were differentially abundant in the Datura root microbiome were Flavobacterium (p = 0.007), Chitinophaga (p = 0.0007), Pedobacter (p = 6 × 10−5), Bradyhizobium (p = 1 × 10−8), and Paenibacillus (p = 1.46 × 10−6). There was significant evidence that the microbes associated with the Datura rootzone had elevated function related to bacterial chalcone synthase (p = 1.49 × 10−3) and permease genes (p < 0.003). There was some evidence that microbial functions in the Datura rootzone provided precursors to important plant bioactive molecules or were beneficial to plant growth. This is important because these compounds are phyto-protective antioxidants and are precursors to many aromatic bioactive compounds that are relevant to human health. In the context of known interactions, and current results, plants and microbes influence the flavonoid biosynthetic pathways of one other, in terms of the regulation of the phenylpropanoid pathway. This is the first study to focus on the microbial ecology of the Datura rootzone. There are possible biopharmaceutical and agricultural applications of the natural interplay that was discovered during this study of the Datura inoxia rhizosphere.

Ocimum sanctum, OscWRKY1, regulates phenylpropanoid pathway genes and promotes resistance to pathogen infection in Arabidopsis

WRKY transcription factor (TF) family regulates various developmental and physiological functions in plants. PAL genes encode enzymes which are involved in plant defense responses, but the direct regulation of PAL genes and phenylpropanoid pathway through WRKY TF is not well characterized. In the present study, we have characterized an OscWRKY1 gene from O. sanctum which shows induced expression after methyl jasmonate (MeJA), salicylic acid (SA), and wounding. Recombinant OscWRKY1 protein binds to the W-box cis-element TTGAC[C/T] and activates the reporter gene in yeast. Overexpression of OscWRKY1 enhances Arabidopsis resistance towards Pseudomonas syringae pv. tomato Pst DC3000. Upstream activator sequences of PAL and C4H have identified the conserved W-box cis-element (TTGACC) in both O. sanctum and Arabidopsis. OscWRKY1 was found to interact with W-box cis-element present in the PAL and C4H promoters. Silencing of OscWRKY1 using VIGS resulted in reduced expression of PAL, C4H, COMT, F5H and 4CL transcripts. OscWRKY1 silenced plants exhibit reduced PAL activity, whereas, the overexpression lines of OscWRKY1 in Arabidopsis exhibit increased PAL activity. These results revealed that OscWRKY1 positively regulates the phenylpropanoid pathway genes and enhances the resistance against bacterial pathogen in Arabidopsis.

Metabolomic Evaluation of Ralstonia solanacearum Cold Shock Protein Peptide (csp22)-Induced Responses in Solanum lycopersicum

Ralstonia solanacearum, the causal agent of bacterial wilt, is one of the most destructive bacterial plant pathogens. This is linked to its evolutionary adaptation to evade host surveillance during the infection process since many of the pathogen’s associated molecular patterns escape recognition. However, a 22-amino acid sequence of R. solanacearum-derived cold shock protein (csp22) was discovered to elicit an immune response in the Solanaceae. Using untargeted metabolomics, the effects of csp22-elicitation on the metabolome of Solanum lycopersicum leaves were investigated. Additionally, the study set out to discover trends that may suggest that csp22 inoculation bestows enhanced resistance on tomato against bacterial wilt. Results revealed the redirection of metabolism toward the phenylpropanoid pathway and sub-branches thereof. Compared to the host response with live bacteria, csp22 induced a subset of the discriminant metabolites, but also metabolites not induced in response to R. solanacearum. Here, a spectrum of hydroxycinnamic acids (especially ferulic acid), their conjugates and derivatives predominated as signatory biomarkers. From a metabolomics perspective, the results support claims that csp22 pre-treatment of tomato plants elicits increased resistance to R. solanacearum infection and contribute to knowledge on plant immune systems operation at an integrative level. The functional significance of these specialized compounds may thus support a heightened state of defense that can be applied to ward off attacking pathogens or toward priming of defense against future infections.

Biostimulants in Viticulture: A Sustainable Approach against Biotic and Abiotic Stresses

Climate change and disproportionate anthropogenic interventions, such as the excess of phytopharmaceutical products and continuous soil tillage, are jeopardizing viticulture by subjecting plants to continuous abiotic stress. One of the main physiological repercussions of abiotic stress is represented by the unbalanced redox homeostasis due to the overproduction of reactive oxygen species (ROS), ultimately leading to a state of oxidative stress (detrimental to grape quality). To these are added the direct and indirect damages caused by pathogens (biotic stresses). In light of this scenario, it is inevitable that sustainable techniques and sensitivity approaches for environmental and human health have to be applied in viticulture. Sustainable viticulture can only be made with the aid of sustainable products. Biostimulant (PB) applications (including resistance inducers or elicitors) in the vineyard have become interesting maneuvers for counteracting vine diseases and improving grape quality. These also represent a partial alternative to soil fertilization by improving nutrient absorption and avoiding its leaching into the groundwater. Their role as elicitors has important repercussions in the stimulation of the phenylpropanoid pathway by triggering the activation of several enzymes, such as polyphenol oxidase, lipoxygenase, phenylalanine ammonia-lyase, and peroxidase (with the accumulation of phenolic compounds). The present review paper summarizes the PBs’ implications in viticulture, gathering historical, functional, and applicative information. This work aims to highlight the innumerable beneficial effects on vines brought by these products. It also serves to spur the scientific community to a greater contribution in investigating the response mechanisms of the plant to positive inductions.

Phenylalanine Suppresses Cell Death Caused by Loss of Fumarylacetoacetate Hydrolase in Arabidopsis

Fumarylacetoacetate hydrolase (FAH) catalyzes the final step of Tyrosine (Tyr) degradation pathway essential to animals and the deficiency of FAH causes an inborn lethal disease. In plants, a role of this pathway was unknown until we found that mutation of Short-day Sensitive Cell Death1 (SSCD1), encoding Arabidopsis FAH, results in cell death under short day. Phenylalanine (Phe) could be converted to Tyr and then degraded in both animals and plants. Phe ingestion in animals worsens the disease caused by FAH defect. However, in this study we found that Phe represses cell death caused by FAH defect in plants. Phe treatment promoted chlorophyll biosynthesis and suppressed the up‑regulation of reactive oxygen species marker genes in the sscd1 mutant. Furthermore, the repression of sscd1 cell death by Phe could be reduced by α-aminooxi-β-phenylpropionic acid but increased by methyl jasmonate, which inhibits or activates Phe ammonia-lyase catalyzing the first step of phenylpropanoid pathway, respectively. In addition, we found that jasmonate signaling up‑regulates Phe ammonia-lyase 1 and mediates the methyl jasmonate enhanced repression of sscd1 cell death by Phe. These results uncovered the relation between chlorophyll biosynthesis, phenylpropanoid pathway and jasmonate signaling in regulating the cell death resulting from loss of FAH in plants.

Nucleoside 5′-Phosphoramidates Control the Phenylpropanoid Pathway in Vitis vinifera Suspension-Cultured Cells

It is known that cells contain various uncommon nucleotides such as dinucleoside polyphosphates (NpnN’s) and adenosine 5′-phosphoramidate (NH2-pA) belonging to nucleoside 5′-phosphoramidates (NH2-pNs). Their cellular levels are enzymatically controlled. Some of them are accumulated in cells under stress, and therefore, they could act as signal molecules. Our previous research carried out in Arabidopsis thaliana and grape (Vitis vinifera) showed that NpnN’s induced the expression of genes in the phenylpropanoid pathway and favored the accumulation of their products, which protect plants against stress. Moreover, we found that NH2-pA could play a signaling role in Arabidopsis seedlings. Data presented in this paper show that exogenously applied purine (NH2-pA, NH2-pG) and pyrimidine (NH2-pU, NH2-pC) nucleoside 5′-phosphoramidates can modify the expression of genes that control the biosynthesis of both stilbenes and lignin in Vitis vinifera cv. Monastrell suspension-cultured cells. We investigated the expression of genes encoding for phenylalanine ammonia-lyase (PAL1), cinnamate-4-hydroxylase (C4H1), 4-coumarate:coenzyme A ligase (4CL1), chalcone synthase (CHS1), stilbene synthase (STS1), cinnamoyl-coenzyme A:NADP oxidoreductase (CCR2), and cinnamyl alcohol dehydrogenase (CAD1). Each of the tested NH2-pNs also induced the expression of the trans-resveratrol cell membrane transporter VvABCG44 gene and caused the accumulation of trans-resveratrol and trans-piceid in grape cells as well as in the culture medium. NH2-pC, however, evoked the most effective induction of phenylpropanoid pathway genes such as PAL1, C4H1, 4CL1, and STS1. Moreover, this nucleotide also induced at short times the accumulation of N-benzoylputrescine (BenPut), one of the phenylamides that are derivatives of phenylpropanoid and polyamines. The investigated nucleotides did not change either the lignin content or the cell dry weight, nor did they affect the cell viability throughout the experiment. The results suggest that nucleoside 5′-phosphoramidates could be considered as new signaling molecules.

Satureja montana Essential Oil, Zein Nanoparticles and Their Combination as a Biocontrol Strategy to Reduce Bacterial Spot Disease on Tomato Plants

Tomato bacterial spot (Bs), caused by Xanthomonas spp., including X. euvesicatoria (Xeu) remains a major threat for tomato production. The emergence of copper resistance strains of Xeu calls urgently for eco-friendly phytosanitary treatments as sustainable green alternatives for disease control. Satureja spp. essential oil (EO) has antimicrobial activity against xanthomonads and combined with zein nanoparticles (ZNPs), might offer a viable option for field applications. This study aims to evaluate the effects of S. montana EO, of ZNPs, and their combination in a nanoformulation, on Xeu quantity, and how these compounds modulate molecular and physiological changes in the pathosystem. Uninfected and infected tomato plants (var. Oxheart) were treated with EO; ZNPs and nanoformulation (EO + ZNPs). Treatments reduced Xeu amount by a minimum of 1.6-fold (EO) and a maximum of 202-fold (ZNPs) and improved plants’ health. Nanoformulation and ZNPs increased plants’ phenolic content. ZNPs significantly increased GPX activity and reduced CAT activity. Overall treatments upregulated transcripts of the phenylpropanoid pathway in infected plants, while ZNPs and nanoformulation upregulated those transcripts in uninfected plants. Both sod and aao transcripts were downregulated by treatments in infected plants. These findings demonstrate that S. montana EO, ZNPs and their nanoformulation are suitable to integrate tomato bacterial spot management strategies, mainly due to their antimicrobial activity on Xeu, however further field studies clarifying the long-term action of these products are required. These results also support the prophylactic potential of ZNPs on tomato bacterial spot.

Systematic Analysis and Biochemical Characterization of the Caffeoyl Shikimate Esterase Gene Family in Poplar

Caffeoyl shikimate esterase (CSE) hydrolyzes caffeoyl shikimate into caffeate and shikimate in the phenylpropanoid pathway. In this study, we performed a systematic analysis of the CSE gene family and investigated the possible roles of CSE and CSE-like genes in Populus. We conducted a genome-wide analysis of the CSE gene family, including functional and phylogenetic analyses of CSE and CSE-like genes, using the poplar (Populus trichocarpa) genome. Eighteen CSE and CSE-like genes were identified in the Populus genome, and five phylogenetic groups were identified from phylogenetic analysis. CSEs in Group Ia, which were proposed as bona fide CSEs, have probably been lost in most monocots except Oryza sativa. Primary functional classification showed that PoptrCSE1 and PoptrCSE2 had putative function in lignin biosynthesis. In addition, PoptrCSE2, along with PoptrCSE12, might also respond to stress with a function in cell wall biosynthesis. Enzymatic assay of PoptoCSE1 (Populus tomentosa), -2 and -12 showed that PoptoCSE1 and -2 maintained CSE activity. PoptoCSE1 and 2 had similar biochemical properties, tissue expression patterns and subcellular localization. Most of the PoptrCSE-like genes are homologs of AtMAGL (monoacylglycerol lipase) genes in Arabidopsis and may function as MAG lipase in poplar. Our study provides a systematic understanding of this novel gene family and suggests the function of CSE in monolignol biosynthesis in Populus.

Phenylalanine Ammonia-Lyase (PAL) Genes Family in Wheat (Triticum aestivum L.): Genome-Wide Characterization and Expression Profiling

Phenylalanine ammonia-lyase (PAL) is the first enzyme in the phenylpropanoid pathway and plays a vital role in adoption, growth, and development in plants but in wheat its characterization is still not very clear. Here, we report a genome-wide identification of TaPAL genes and analysis of their transcriptional expression, duplication, and phylogeny in wheat. A total of 37 TaPAL genes that cluster into three subfamilies have been identified based on phylogenetic analysis. These TaPAL genes are distributed on 1A, 1B, 1D, 2A, 2B, 2D, 4A, 5B, 6A, 6B, and 6D chromosomes. Gene structure, conserved domain analysis, and investigation of cis-regulatory elements were systematically carried out. Chromosomal rearrangements and gene loss were observed by evolutionary analysis of the orthologs among Triticum urartu, Aegilops tauschii, and Triticum aestivum during the origin of bread wheat. Gene ontology analysis revealed that PAL genes play a role in plant growth. We also identified 27 putative miRNAs targeting 37 TaPAL genes. The high expression level of PAL genes was detected in roots of drought-tolerant genotypes compared to drought-sensitive genotypes. However, very low expressions of TaPAL10, TaPAL30, TaPAL32, TaPAL3, and TaPAL28 were recorded in all wheat genotypes. Arogenate dehydratase interacts with TaPAL29 and has higher expression in roots. The analysis of all identified genes in RNA-seq data showed that they are expressed in roots and shoots under normal and abiotic stress. Our study offers valuable data on the functioning of PAL genes in wheat.

Functional Characterization of BrF3'H, Which Determines the Typical Flavonoid Profile of Purple Chinese Cabbage

Flavonols and anthocyanins are the two major classes of flavonoids in Brassica rapa. To elucidate the flavonoid biosynthetic pathway in Chinese cabbage (B. rapa L. subsp. pekinensis), we analyzed flavonoid contents in two varieties of Chinese cabbage with normal green (5546) and purple (8267) leaves. The 8267 variety accumulates significantly higher levels of quercetin, isorhamnetin, and cyanidin than the 5546 variety, indicating that 3′-dihydroxylated flavonoids are more prevalent in the purple than in the green variety. Gene expression analysis showed that the expression patterns of most phenylpropanoid pathway genes did not correspond to the flavonoid accumulation patterns in 5546 and 8267 varieties, except for BrPAL1.2 while most early and late flavonoid biosynthetic genes are highly expressed in 8267 variety. In particular, the flavanone 3′-hydroxylase BrF3′H (Bra009312) is expressed almost exclusively in 8267. We isolated the coding sequences of BrF3′H from the two varieties and found that both sequences encode identical amino acid sequences and are highly conserved with F3'H genes from other species. An in vitro enzymatic assay demonstrated that the recombinant BrF3′H protein catalyzes the 3′-hydroxylation of a wide range of 4′-hydroxylated flavonoid substrates. Kinetic analysis showed that kaempferol is the most preferred substrate and dihydrokaempferol (DHK) is the poorest substrate for recombinant BrF3′H among those tested. Transient expression of BrF3′H in Nicotiana benthamiana followed by infiltration of naringenin and DHK as substrates resulted in eriodictyol and quercetin production in the infiltrated leaves, demonstrating the functionality of BrF3′H in planta. As the first functional characterization of BrF3′H, our study provides insight into the molecular mechanism underlying purple coloration in Chinese cabbage.