Genome-Wide Identification and Role of the bHLH Gene Family in Dendrocalamus latiflorus Flowering Regulation.

The basic helix-loop-helix (bHLH) gene family is a crucial regulator in plants, orchestrating various developmental processes, particularly flower formation, and mediating responses to hormonal signals. The molecular mechanism of bamboo flowering regulation remains unresolved, limiting bamboo breeding efforts. In this study, we identified 309 bHLH genes and divided them into 23 subfamilies. Structural analysis revealed that proteins in specific DlbHLH subfamilies are highly conserved. Collinearity analysis indicates that the amplification of the DlbHLH gene family primarily occurs through segmental duplications. The structural diversity of these duplicated genes may account for their functional variability. Many DlbHLHs are expressed during flower development, indicating the bHLH gene's significant role in this process. In the promoter region of DlbHLHs, different homeopathic elements involved in light response and hormone response co-exist, indicating that DlbHLHs are related to the regulation of the flower development of D. latiflorus.

Computational insights into diverse aspects of glutathione S-transferase gene family in Papaver somniferum.

Plant glutathione S-transferases are an ancient protein superfamily having antioxidant activity. These proteins are primarily involved in diverse plant functions such as plant growth and development, secondary metabolism, signaling pathways and defense against biotic and abiotic stresses. The current study aimed to comprehensively identify and characterize the GST gene family in the medicinally important crop Papaver somniferum. A total of 93 GST proteins were identified belonging to eight GST classes and found to be majorly localized in the cytoplasm. All GST genes were found on eleven opium chromosomes. Gene duplication analysis showed segmental duplication as a key factor for opium GST gene family expansion under strong purifying selection. Phylogenetic analysis with gymnosperm, angiosperm and bryophyte revealed the evolution of GSTs earlier than their division into separate groups and also prior to the divergence of monocot and dicot. The secondary structure prediction showed the dominance of α-helices indicative of PsomGSTs as structurally stable and elastic proteins. Gene architecture showed the conservation of number of exons across the classes. MEME analysis revealed only a few class specific and many across class conserved motifs. Ser was found to be the active site residue of tau, phi, theta and zeta class and Cys was catalytic residue of DHAR, lambda and GHR class. Promoter analyses identified many cis-acting regulatory elements related to hormonal, cellular, stress and light response functions. Ser was the key phosphorylation site. Only three glycosylation sites were found across the 93 PsomGSTs. 3D structure prediction was also performed and was validated. Interactome analyses revealed the correlation of PsomGSTs with glutathione metabolizing proteins. Gene enrichment analysis and KEGG pathway analyzed the involvement of PsomGSTs in three major pathways i.e. glutathione metabolism, tyrosine metabolism and ascorbate metabolism. The outcome revealed high model quality of PsomGSTs. The results of the current study will be of potential significance to understand the functional and structural importance of the GST gene family in opium, a medicinally important crop.

ICE-CBF-COR Signaling Cascade and Its Regulation in Plants Responding to Cold Stress.

Cold stress limits plant geographical distribution and influences plant growth, development, and yields. Plants as sessile organisms have evolved complex biochemical and physiological mechanisms to adapt to cold stress. These mechanisms are regulated by a series of transcription factors and proteins for efficient cold stress acclimation. It has been established that the ICE-CBF-COR signaling pathway in plants regulates how plants acclimatize to cold stress. Cold stress is perceived by receptor proteins, triggering signal transduction, and Inducer of CBF Expression (ICE) genes are activated and regulated, consequently upregulating the transcription and expression of the C-repeat Binding Factor (CBF) genes. The CBF protein binds to the C-repeat/Dehydration Responsive Element (CRT/DRE), a homeopathic element of the Cold Regulated genes (COR gene) promoter, activating their transcription. Transcriptional regulations and post-translational modifications regulate and modify these entities at different response levels by altering their expression or activities in the signaling cascade. These activities then lead to efficient cold stress tolerance. This paper contains a concise summary of the ICE-CBF-COR pathway elucidating on the cross interconnections with other repressors, inhibitors, and activators to induce cold stress acclimation in plants.

Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy.

The gateway to morphine biosynthesis in opium poppy (Papaver somniferum) is the stereochemical inversion of (S)-reticuline since the enzyme yielding the first committed intermediate salutaridine is specific for (R)-reticuline. A fusion between a cytochrome P450 (CYP) and an aldo-keto reductase (AKR) catalyzes the S-to-R epimerization of reticuline via 1,2-dehydroreticuline. The reticuline epimerase (REPI) fusion was detected in opium poppy and in Papaver bracteatum, which accumulates thebaine. In contrast, orthologs encoding independent CYP and AKR enzymes catalyzing the respective synthesis and reduction of 1,2-dehydroreticuline were isolated from Papaver rhoeas, which does not accumulate morphinan alkaloids. An ancestral relationship between these enzymes is supported by a conservation of introns in the gene fusions and independent orthologs. Suppression of REPI transcripts using virus-induced gene silencing in opium poppy reduced levels of (R)-reticuline and morphinan alkaloids and increased the overall abundance of (S)-reticuline and its O-methylated derivatives. Discovery of REPI completes the isolation of genes responsible for known steps of morphine biosynthesis.

Benzylisoquinoline alkaloid biosynthesis in opium poppy.

Opium poppy (Papaver somniferum) is one of the world's oldest medicinal plants and remains the only commercial source for the narcotic analgesics morphine, codeine and semi-synthetic derivatives such as oxycodone and naltrexone. The plant also produces several other benzylisoquinoline alkaloids with potent pharmacological properties including the vasodilator papaverine, the cough suppressant and potential anticancer drug noscapine and the antimicrobial agent sanguinarine. Opium poppy has served as a model system to investigate the biosynthesis of benzylisoquinoline alkaloids in plants. The application of biochemical and functional genomics has resulted in a recent surge in the discovery of biosynthetic genes involved in the formation of major benzylisoquinoline alkaloids in opium poppy. The availability of extensive biochemical genetic tools and information pertaining to benzylisoquinoline alkaloid metabolism is facilitating the study of a wide range of phenomena including the structural biology of novel catalysts, the genomic organization of biosynthetic genes, the cellular and sub-cellular localization of biosynthetic enzymes and a variety of biotechnological applications. In this review, we highlight recent developments and summarize the frontiers of knowledge regarding the biochemistry, cellular biology and biotechnology of benzylisoquinoline alkaloid biosynthesis in opium poppy.

Characterization of the GGP gene family in potato (Solanum tuberosum L.) and Pepper (Capsicum annuum L.) and its expression analysis under hormonal and abiotic stresses.

GDP-L-galactose phosphorylase (GGP) is a key rate-limiting enzyme in plant ascorbic acid synthesis, which plays an important role in plant growth and development as well as stress response. However, the presence of GGP and its function in potato and pepper are not known. In this study, we first identified two GGP genes in each potato and pepper genomes using a genome-wide search approach. We then analyzed their physicochemical properties, conserved domains, protein structures and phylogenetic relationships. Phylogenetic tree analysis revealed that members of the potato and pepper GGP gene families are related to eggplant (Solanum melongena L.), Arabidopsis (Arabidopsis thaliana L.), tobacco (Nicotiana tabacum L.) and tomato (Solanum lycopersicum L.), with tomato being the most closely related. The promoter sequences mainly contain homeopathic elements such as light-responsive, hormone-responsive and stress-responsive, with light-responsive elements being the most abundant. By analyzing the structure of the genes, it was found that there is no transmembrane structure or signal peptide in the GGP gene family of potatoes and peppers, and that all of its members are hydrophilic proteins. The expression profiles of different tissues show that StGGP1 has the highest expression levels in leaves, StGGP2 has the highest expression levels in stamens, and CaGGPs have the highest expression levels in the early stages of fruit development (Dev1). It was found that StGGPs and CaGGPs genes showed different response to phytohormones and abiotic stresses. Abscisic acid (ABA) treatment induced the most significant change in the expression of StGGPs, while the expression of CaGGPs showed the most pronounced change under methyl jasmonate (MeJA) treatment. StGGPs responded mainly to dark treatment, whereas CaGGPs responded mainly to NaCl stress. These results provide an important basis for a detailed study about the functions of GGP homologous genes in potato and pepper in response to abiotic stresses.

Characterization of a flavoprotein oxidase from opium poppy catalyzing the final steps in sanguinarine and papaverine biosynthesis.

Benzylisoquinoline alkaloids are a diverse class of plant specialized metabolites that includes the analgesic morphine, the antimicrobials sanguinarine and berberine, and the vasodilator papaverine. The two-electron oxidation of dihydrosanguinarine catalyzed by dihydrobenzophenanthridine oxidase (DBOX) is the final step in sanguinarine biosynthesis. The formation of the fully conjugated ring system in sanguinarine is similar to the four-electron oxidations of (S)-canadine to berberine and (S)-tetrahydropapaverine to papaverine. We report the isolation and functional characterization of an opium poppy (Papaver somniferum) cDNA encoding DBOX, a flavoprotein oxidase with homology to (S)-tetrahydroprotoberberine oxidase and the berberine bridge enzyme. A query of translated opium poppy stem transcriptome databases using berberine bridge enzyme yielded several candidate genes, including an (S)-tetrahydroprotoberberine oxidase-like sequence selected for heterologous expression in Pichia pastoris. The recombinant enzyme preferentially catalyzed the oxidation of dihydrosanguinarine to sanguinarine but also converted (RS)-tetrahydropapaverine to papaverine and several protoberberine alkaloids to oxidized forms, including (RS)-canadine to berberine. The K(m) values of 201 and 146 µm for dihydrosanguinarine and the protoberberine alkaloid (S)-scoulerine, respectively, suggested high concentrations of these substrates in the plant. Virus-induced gene silencing to reduce DBOX transcript levels resulted in a corresponding reduction in sanguinarine, dihydrosanguinarine, and papaverine accumulation in opium poppy roots in support of DBOX as a multifunctional oxidative enzyme in BIA metabolism.

Poppy APETALA1/FRUITFULL orthologs control flowering time, branching, perianth identity, and fruit development.

Several MADS box gene lineages involved in flower development have undergone duplications that correlate with the diversification of large groups of flowering plants. In the APETALA1 gene lineage, a major duplication coincides with the origin of the core eudicots, resulting in the euFUL and the euAP1 clades. Arabidopsis FRUITFULL (FUL) and APETALA1 (AP1) function redundantly in specifying floral meristem identity but function independently in sepal and petal identity (AP1) and in proper fruit development and determinacy (FUL). Many of these functions are largely conserved in other core eudicot euAP1 and euFUL genes, but notably, the role of APETALA1 as an "A-function" (sepal and petal identity) gene is thought to be Brassicaceae specific. Understanding how functional divergence of the core eudicot duplicates occurred requires a careful examination of the function of preduplication (FUL-like) genes. Using virus-induced gene silencing, we show that FUL-like genes in opium poppy (Papaver somniferum) and California poppy (Eschscholzia californica) function in axillary meristem growth and in floral meristem and sepal identity and that they also play a key role in fruit development. Interestingly, in opium poppy, these genes also control flowering time and petal identity, suggesting that AP1/FUL homologs might have been independently recruited in petal identity. Because the FUL-like gene functional repertoire encompasses all roles previously described for the core eudicot euAP1 and euFUL genes, we postulate subfunctionalization as the functional outcome after the major AP1/FUL gene lineage duplication event.

Tyrosine aminotransferase contributes to benzylisoquinoline alkaloid biosynthesis in opium poppy.

Tyrosine aminotransferase (TyrAT) catalyzes the transamination of L-Tyr and α-ketoglutarate, yielding 4-hydroxyphenylpyruvic acid and L-glutamate. The decarboxylation product of 4-hydroxyphenylpyruvic acid, 4-hydroxyphenylacetaldehyde, is a precursor to a large and diverse group of natural products known collectively as benzylisoquinoline alkaloids (BIAs). We have isolated and characterized a TyrAT cDNA from opium poppy (Papaver somniferum), which remains the only commercial source for several pharmaceutical BIAs, including codeine, morphine, and noscapine. TyrAT belongs to group I pyridoxal 5'-phosphate (PLP)-dependent enzymes wherein Schiff base formation occurs between PLP and a specific Lys residue. The amino acid sequence of TyrAT showed considerable homology to other putative plant TyrATs, although few of these have been functionally characterized. Purified, recombinant TyrAT displayed a molecular mass of approximately 46 kD and a substrate preference for L-Tyr and α-ketoglutarate, with apparent K(m) values of 1.82 and 0.35 mm, respectively. No specific requirement for PLP was detected in vitro. Liquid chromatography-tandem mass spectrometry confirmed the conversion of L-Tyr to 4-hydroxyphenylpyruvate. TyrAT gene transcripts were most abundant in roots and stems of mature opium poppy plants. Virus-induced gene silencing was used to evaluate the contribution of TyrAT to BIA metabolism in opium poppy. TyrAT transcript levels were reduced by at least 80% in silenced plants compared with controls and showed a moderate reduction in total alkaloid content. The modest correlation between transcript levels and BIA accumulation in opium poppy supports a role for TyrAT in the generation of alkaloid precursors, but it also suggests the occurrence of other sources for 4-hydroxyphenylacetaldehyde.

(R,S)-Reticuline 7-O-methyltransferase and (R,S)-norcoclaurine 6-O-methyltransferase of Papaver somniferum - cDNA cloning and characterization of methyl transfer enzymes of alkaloid biosynthesis in opium poppy.

S-Adenosyl-L-methionine:(R,S)-reticuline 7-O-methyltransferase converts reticuline to laudanine in tetrahydrobenzylisoquinoline biosynthesis in the opium poppy Papaver somniferum. This enzyme activity has not yet been detected in plants. A proteomic analysis of P. somniferum latex identified a gel spot that contained a protein(s) whose partial amino acid sequences were homologous to those of plant O-methyltransferases. cDNA was amplified from P. somniferum RNA by reverse transcription PCR using primers based on these internal amino acid sequences. Recombinant protein was then expressed in Spodoptera frugiperda Sf9 cells in a baculovirus expression vector. Steady-state kinetic measurements with one heterologously expressed enzyme and mass spectrometric analysis of the enzymatic products suggested that this unusual enzyme is capable of carrying through sequential O-methylations on the isoquinoline and on the benzyl moiety of several substrates. The tetrahydrobenzylisoquinolines (R)-reticuline (4.2 sec(-1) mm(-1)), (S)-reticuline (4.5 sec(-1) mm(-1)), (R)-protosinomenine (1.7 sec(-1) mm(-1)), and (R,S)-isoorientaline (1.4 sec(-1) mm(-1)) as well as guaiacol (5.9 sec(-1) mm(-1)) and isovanillic acid (1.2 sec(-1) mm(-1)) are O-methylated by the enzyme with the ratio kcat/K m shown in parentheses. A P. somniferum cDNA encoding (R,S)-norcoclaurine 6-O-methyltransferase was similarly isolated and characterized. This enzyme was less permissive, methylating only (R,S)-norcoclaurine (7.4 sec(-1) mm(-1)), (R)-norprotosinomenine (4.1 sec(-1) mm(-1)), (S)-norprotosinomenine (4.0 sec(-1) mm(-1)) and (R,S)-isoorientaline (1.0 sec(-1) mm(-1)). A phylogenetic comparison of the amino acid sequences of these O-methyltransferases to those from 28 other plant species suggests that these enzymes group more closely to isoquinoline biosynthetic O-methyltransferases from Coptis japonica than to those from Thalictrum tuberosum that can O-methylate both alkaloid and phenylpropanoid substrates.

Uncoupled defense gene expression and antimicrobial alkaloid accumulation in elicited opium poppy cell cultures.

Treatment of opium poppy (Papaver somniferum L.) cell cultures with autoclaved mycelial homogenates of Botrytis sp. resulted in the accumulation of sanguinarine. Elicitor treatment also caused a rapid and transient induction in the activity of tyrosine/dopa decarboxylase (TYDC, EC 4.1.1.25), which catalyzes the conversion of L-tyrosine and L-dopa to tyramine and dopamine, respectively, the first steps in sanguinarine biosynthesis. TYDC genes were differentially expressed in response to elicitor treatment. TYDC1-like mRNA levels were induced rapidly but declined to near baseline levels within 5 h. In contrast, TYDC2-like transcript levels increased more slowly but were sustained for an extended period. Induction of TYDC mRNAs preceded that of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) mRNAs. An elicitor preparation from Pythium aphanidermatum was less effective in the induction of TYDC mRNA levels and alkaloid accumulation; however, both elicitors equally induced accumulation of PAL transcripts. In contrast, treatment with methyl jasmonate resulted in an induction of TYDC but not PAL mRNAs. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and the protein kinase inhibitor staurosporine partially blocked the fungal elicitor-induced accumulation of sanguinarine. However, only staurosporine and okadaic acid, an inhibitor of protein phosphatases 1 and 2A, blocked the induction of TYDC1-like transcript levels, but they did not block the induction of TYDC2-like or PAL transcript levels. These data suggest that activation mechanisms for PAL, TYDC, and some later sanguinarine biosynthetic enzymes are uncoupled.