phyA Gene Research Articles

Emerging roles for diguanylate cyclase during the evolution of soma in dictyostelia

BackgroundCyclic di-guanylate (c-di-GMP), synthesized by diguanylate cyclase, is a major second messenger in prokaryotes, where it triggers biofilm formation. The dictyostelid social amoebas acquired diguanylate cyclase (dgcA) by horizontal gene transfer. Dictyostelium discoideum (Ddis) in taxon group 4 uses c-di-GMP as a secreted signal to induce differentiation of stalk cells, the ancestral somatic cell type that supports the propagating spores. We here investigated how this role for c-di-GMP evolved in Dictyostelia by exploring dgcA function in the group 2 species Polysphondylium pallidum (Ppal) and in Polysphondylium violaceum (Pvio), which resides in a small sister clade to group 4.ResultsSimilar to Ddis, dgcA is upregulated after aggregation in Ppal and Pvio and predominantly expressed in the anterior region and stalks of emerging fruiting bodies. DgcA null mutants in Ppal and Pvio made fruiting bodies with very long and thin stalks and only few spores and showed delayed aggregation and larger aggregates, respectively. Ddis dgcAˉ cells cannot form stalks at all, but showed no aggregation defects. The long, thin stalks of Ppal and Pvio dgcAˉ mutants were also observed in acaAˉ mutants in these species. AcaA encodes adenylate cyclase A, which mediates the effects of c-di-GMP on stalk induction in Ddis. Other factors that promote stalk formation in Ddis are DIF-1, produced by the polyketide synthase StlB, low ammonia, facilitated by the ammonia transporter AmtC, and high oxygen, detected by the oxygen sensor PhyA (prolyl 4-hydroxylase). We deleted the single stlB, amtC and phyA genes in Pvio wild-type and dgcAˉ cells. Neither of these interventions affected stalk formation in Pvio wild-type and not or very mildly exacerbated the long thin stalk phenotype of Pvio dgcAˉ cells.ConclusionsThe study reveals a novel role for c-di-GMP in aggregation, while the reduced spore number in Pvio and Ppal dgcAˉ is likely an indirect effect, due to depletion of the cell pool by the extended stalk formation. The results indicate that in addition to c-di-GMP, Dictyostelia ancestrally used an as yet unknown factor for induction of stalk formation. The activation of AcaA by c-di-GMP is likely conserved throughout Dictyostelia.

Apical Dominance and Branching in Plantlets of Colt Cherry Lines Expressing Different Light and Auxin Signalling Sensitivities

The establishment of plant architecture requires coordination of distinct processes including shoot branching and apical dominance (AD). AD involves the bud apical shoot, mainly through indole-3-acetic acid (IAA) synthetized by the cells of the meristem and young leaves. The rootward flow generates an auxin gradient in the stem and buds, regulating lateral bud (LB) outgrowth. Phytochromes and AD are involved in the shade-avoidance syndrome in woody plants. However, the underlying mechanisms remain poorly understood. The aim of this study was to evaluate the sensitivity of cherry rootstocks to light, mediated by the photoreceptor phytochrome, and its effect on the role of auxin in driving branching by AD. Pharmacological treatments using transport inhibitors and a competitor of IAA were applied to transgenic lines of Colt cherry rootstock, which showed different sensitivities to light because of the ectopic expression of a rice phyA gene. Results showed different physiological behaviours among the transgenic lines and between themselves and the Colt-wt line. Exogenous IBA inhibited Colt-wt LB outgrowth, and this inhibition was less intense in transgenic lines. The IAA-inhibitors and IAA-competitor promoted branching. In in vitro phyA-transgenic plantlets, the ectopic gene induced greater branching and a higher number of buds developed in new shoots. This work confirms a positive action of phytochrome on lateral branching in cherry rootstock, playing a role in the regulation of AD. Moreover, we suggest that the confined in vitro system might now be used as a phenotyping screening to test the plasticity of the response, highlighting the behaviour of modified genotypes due to an ectopic insertion event by simple and rapid procedures.

An efficient Agrobacterium-mediated system based on the pyrG auxotrophic marker for recombinant expression in the filamentous fungus Penicillium rubens

This work aimed to construct a versatile, effective, and food-grade Agrobacterium tumefaciens-mediated transformation (ATMT) system for recombinant expression in the filamentous fungus Penicillium rubens (also known as Pencillium chrysogenum). In this study, the wild-type P. chrysogenum VTCC 31172 strain was re-classified as P. rubens by a multilocus sequencing analysis. Further, the pyrG gene required for uridine/uracil biosynthesis was successfully deleted in the VTCC 31172 strain by homologous recombination to generate a stable uridine/uracil auxotrophic mutant (ΔpyrG). The growth of the P. rubens ΔpyrG strain could be restored by uridine/uracil supplementation, and a new ATMT system based on the uridine/uracil auxotrophic mechanism was established for this strain. The optimal ATMT efficiency could reach 1750 transformants for 106 spores (equivalent to 0.18%). In addition, supplementation of uridine/uracil at the concentrations of 0.005-0.02% during the co-cultivation process significantly promoted transformation efficiency. Especially, we demonstrated that the pyrG marker and the amyB promoter from the koji mold Aspergillus oryzae were fully functional in P. rubens ΔpyrG. Expression of the DsRed reporter gene under the regulation of the A. oryzae amyB promoter lighted up the mycelium of P. rubens with a robust red signal under fluorescence microscopy. Furthermore, genomic integration of multiple copies of the Aspergillus fumigatus phyA gene under the control of the amyB promoter significantly enhanced phytase activity in P. rubens. The ATMT system developed in our work provides a safe genetic platform for producing recombinant products in P. rubens without using drug resistance markers.

Moderate salinity improves the availability of soil P by regulating P-cycling microbial communities in coastal wetlands.

Accelerated sea-level rise is expected to cause the salinization of freshwater wetlands, but the responses to salinity of the availability of soil phosphorus (P) and of microbial genes involved in the cycling of P remain unexplored. We conducted a field experiment to investigate the effects of salinity on P cycling by soil microbial communities and their regulatory roles on P availability in coastal freshwater and brackish wetlands. Salinity was positively correlated with P availability, with higher concentrations of labile P but lower concentrations of moderately labile P in the brackish wetland. The diversity and richness of microbial communities involved in P cycling were higher in the brackish wetland than the freshwater wetland. Salinity substantially altered the composition of the P-cycling microbial community, in which those of the brackish wetland were separated from those of the freshwater wetland. Metagenomic sequence analysis indicated that functional genes involved in the solubilization of inorganic P and the subsequent transport and regulation of P were more abundant in coastal soils. The relative abundances of most of the target genes differed between the wetlands, with higher abundances of P-solubilization (gcd and ppa) and -mineralization (phoD, phy, and ugpQ) genes and lower abundances of P-transport genes (pstB, ugpA, ugpB, ugpE, and pit) in the brackish wetland. A significant positive correlation between the concentration of labile P and the abundances of the target genes suggested that salinity may, at least in part, improve P availability by regulating the P-cycling microbial community. Our results suggest that the P-cycling microbial community abundance and P availability respond positively to moderate increases in salinity by promoting the microbial solubilization and mineralization of soil P. Changes in microbial communities and microbially mediated P cycling may represent microbial strategies to adapt to moderate salinity levels, which in turn control soil function and nutrient balance.

Phylogenetic analysis of phytochrome A gene from Lablab purpureus (L.) Sweet

BackgroundPhytochromes are the best characterized photoreceptors that perceive Red (R)/Far-Red (FR) signals and mediate key developmental responses in plants. It is well established that photoperiodic control of flowering is regulated by PHY A (phytochrome A) gene. So far, the members of PHY A gene family remains unexplored in Lablab purpureus, and therefore, their functions are still not deciphered. PHYA3 is the homologue of phytochrome A and known to be involved in dominant suppression of flowering under long day conditions by downregulating florigens in Glycine max. The present study is the first effort to identify and characterize any photoreceptor gene (PHYA3, in this study) in Lablab purpureus and decipher its phylogeny with related legumes. ResultsPHYA3 was amplified in Lablab purpureus cv GNIB-21 (photo-insensitive and determinate) by utilizing primers designed from GmPHYA3 locus of Glycine max. This study was successful in partially characterizing PHYA3 in Lablab purpureus (LprPHYA3) which is 2 kb longer and belongs to exon 1 region of PHYA3 gene. Phylogenetic analysis of the nucleotide and protein sequences of PHYA genes through MEGA X delineated the conservation and evolution of Lablab purpureus PHYA3 (LprPHYA3) probably from PHYA genes of Vigna unguiculata, Glycine max and Vigna angularis. A conserved basic helix-loop-helix motif bHLH69 was predicted having DNA binding property. Domain analysis of GmPHYA protein and predicted partial protein sequence corresponding to exon-1 of LprPHYA3 revealed the presence of conserved domains (GAF and PAS domains) in Lablab purpureus similar to Glycine max. ConclusionPartial characterization of LprPHYA3 would facilitate the identification of complete gene in Lablab purpureus utilizing sequence information from phylogenetically related species of Fabaceae. This would allow screening of allelic variants for LprPHYA3 locus and their role in photoperiod responsive flowering. The present study could aid in modulating photoperiod responsive flowering in Lablab purpureus and other related legumes in near future through genome editing.

Differential Root Exudation and Architecture for Improved Growth of Wheat Mediated by Phosphate Solubilizing Bacteria.

Phosphorous (P) deficiency is a major challenge faced by global agriculture. Phosphate-solubilizing bacteria (PSB) provide a sustainable approach to supply available phosphates to plants with improved crop productivity through synergistic interaction with plant roots. The present study demonstrates an insight into this synergistic P-solubilizing mechanism of PSB isolated from rhizosphere soils of major wheat-growing agro-ecological zones of Pakistan. Seven isolates were the efficient P solubilizers based on in vitro P-solubilizing activity (233-365 μg ml–1) with a concomitant decrease in pH (up to 3.5) by the production of organic acids, predominantly acetic acid (∼182 μg ml–1) and gluconic acid (∼117 μg ml–1). Amplification and phylogenetic analysis of gcd, pqqE, and phy genes of Enterobacter sp. ZW32, Ochrobactrum sp. SSR, and Pantoea sp. S1 showed the potential of these PSB to release orthophosphate from recalcitrant forms of phosphorus. Principal component analysis indicates the inoculation response of PSB consortia on the differential composition of root exudation (amino acids, sugars, and organic acids) with subsequently modified root architecture of three wheat varieties grown hydroponically. Rhizoscanning showed a significant increase in root parameters, i.e., root tips, diameter, and surface area of PSB-inoculated plants as compared to uninoculated controls. Efficiency of PSB consortia was validated by significant increase in plant P and oxidative stress management under P-deficient conditions. Reactive oxygen species (ROS)-induced oxidative damages mainly indicated by elevated levels of malondialdehyde (MDA) and H2O2 contents were significantly reduced in inoculated plants by the production of antioxidant enzymes, i.e., superoxide dismutase, catalase, and peroxidase. Furthermore, the inoculation response of these PSB on respective wheat varieties grown in native soils under greenhouse conditions was positively correlated with improved plant growth and soil P contents. Additionally, grain yield (8%) and seed P (14%) were significantly increased in inoculated wheat plants with 20% reduced application of diammonium phosphate (DAP) fertilizer under net house conditions. Thus, PSB capable of such synergistic strategies can confer P biofortification in wheat by modulating root morphophysiology and root exudation and can alleviate oxidative stress under P deficit conditions.

The wheat growth-promoting traits of Ochrobactrum and Pantoea species, responsible for solubilization of different P sources, are ensured by genes encoding enzymes of multiple P-releasing pathways

Production and release of organic acids and phosphatase enzymes by microbes are important for inorganic and organic phosphorus cycling in soil. The presence of microorganisms with corresponding traits in the plant rhizosphere lead to improved plant P uptake and ultimately growth promotion. We studied the potential of two rhizosphere-competent strains, Pantoea sp. MR1 and Ochrobactrum sp. SSR, for solubilization of different organic and inorganic P sources in vitro. In a pot experiment we further revealed the impact of the two strains on wheat seedling performance in soil amended with either phytate, rock phosphate or K2HPO4 as solely P source. To directly link P-solubilizing activity to the strain-specific genetic potential, we designed novel primers for glucose dehydrogenase (gcd), phosphatase (pho) and phytase (phy) genes, which are related to the organic and inorganic P solubilization potential. Quantitative tracing of these functional genes in the inoculated soils of the conducted pot experiment further allowed to compare strain abundances in the soil in dependency on the present P source. We observed strain- and P source-dependent patterns of the P solubilization in vitro as well as in the pot experiment, whereby P release, particularly from phytate, was linked to the strain abundance. We further revealed that the activity of microbial phosphatases is determined by the interplay between functional gene abundance, available soil P, and substrate availability. Moreover, positive impacts of microbial seed inoculation on wheat root architecture and aboveground growth parameters were observed. Our results suggest that screening for rhizosphere-competent strains with gcd, pho and phy genes may help to identify new microbial taxa that are able to solubilize and mineralize inorganic as well as organic bound P. Subsequently, the targeted use of corresponding strains may improve P availability in agricultural soils and consequently reduce fertilizer application.

Multiplex CRISPR-Cas9 mutagenesis of the phytochrome gene family in Physcomitrium (Physcomitrella) patens

Key messageWe mutated all seven Physcomitrium (Physcomitrella) patens phytochrome genes using highly-efficient CRISPR-Cas9 procedures. We thereby identified phy5a as the phytochrome primarily responsible for inhibiting gravitropism, proving the utility of the mutant library.The CRISPR-Cas9 system is a powerful tool for genome editing. Here we report highly-efficient multiplex CRISPR-Cas9 editing of the seven-member phytochrome gene family in the model bryophyte Physcomitrium (Physcomitrella) patens. Based on the co-delivery of an improved Cas9 plasmid with multiple sgRNA plasmids and an efficient screening procedure to identify high-order multiple mutants prior to sequencing, we demonstrate successful targeting of all seven PHY genes in a single transfection. We investigated further aspects of the CRISPR methodology in Physcomitrella, including the significance of spacing between paired sgRNA targets and the efficacy of NHEJ and HDR in repairing the chromosome when excising a complete locus. As proof-of-principle, we show that the septuple phy− mutant remains gravitropic in light, in line with expectations, and on the basis of data from lower order multiplex knockouts conclude that phy5a is the principal phytochrome responsible for inhibiting gravitropism in light. We expect, therefore, that this mutant collection will be valuable for further studies of phytochrome function and that the methods we describe will allow similar approaches to revealing specific functions in other gene families.

The SNL-HDA19 histone deacetylase complex antagonizes HY5 activity to repress photomorphogenesis in Arabidopsis.

Reprogramming of the transcriptome during photomorphogenesis requires dynamic changes in chromatin and distribution of histone modifications. However, the chromatin-based regulation of this process remains to be elucidated. Here, we identify the conserved SWI-INDEPENDENT3 LIKE (SNL)-HISTONE DEACETYLASE19 (HDA19) deacetylase complex, including HDA19 and SNL1-SNL6, as a negative regulator of the light signaling pathway. Light-repression of HDA19 and SNLs expression is mediated by photoreceptors. HDA19 and SNLs are required for histone deacetylation and chromatin inactivation of PHYA gene. We further examined the interaction between SNL-HDA19 complex and ELONGATED HYPOCOTYL5 (HY5), and their antagonistic regulation on the expressions of target genes. The HDA19 deacetylase complex is recruited by HY5 to the chromatin regions of two positive light signaling genes, HY5 and B-BOX CONTAINING PROTEIN 22 (BBX22), thereby reduces the accessibility and histone acetylation and represses their expression. HDA19, SNL1, and HY5 associate with the same regulatory regions of HY5 and BBX22, and HY5 binding to these loci is enhanced upon SNL-HDA19 dysfunction. Our study reveals a crucial role for the HDA19 deacetylase complex in light signaling and demonstrates that the functional interplay between chromatin regulators and transcription factors regulates photomorphogenetic responses to the changing light environments.

Comparative analysis of two phytochrome mutants of tomato (Micro-Tom cv.) reveals specific physiological, biochemical, and molecular responses under chilling stress

BackgroundPhytochromes are plant photoreceptors that have long been associated with photomorphogenesis in plants; however, more recently, their crucial role in the regulation of variety of abiotic stresses has been explored. Chilling stress is one of the abiotic factors that severely affect growth, development, and productivity of crops. In the present work, we have analyzed and compared physiological, biochemical, and molecular responses in two contrasting phytochrome mutants of tomato, namely aurea (aur) and high pigment1 (hp1), along with wild-type cultivar Micro-Tom (MT) under chilling stress. In tomato, aur is phytochrome-deficient mutant while hp1 is a phytochrome-sensitive mutant. The genotype-specific physiological, biochemical, and molecular responses under chilling stress in tomato mutants strongly validated phytochrome-mediated regulation of abiotic stress. ResultsHere, we demonstrate that phytochrome-sensitive mutant hp1 show improved performance compared to phytochrome-deficient mutant aur and wild-type MT plants under chilling stress. Interestingly, we noticed significant increase in several photosynthetic-related parameters in hp1 under chilling stress that include photosynthetic rate, stomatal conductance, stomatal aperture, transpiration rate, chlorophyll a and carotenoids. Whereas most parameters were negatively affected in aur and MT except a slight increase in carotenoids in MT plants under chilling stress. Further, we found that PSII quantum efficiency (Fv/Fm), PSII operating efficiency (Fq′/Fm′), and non-photochemical quenching (NPQ) were all positively regulated in hp1, which demonstrate enhanced photosynthetic performance of hp1 under stress. On the other hand, Fv/Fm and Fq′/Fm′ were decreased significantly in aur and wild-type plants. In addition, NPQ was not affected in MT but declined in aur mutant after chilling stress. Noticeably, the transcript analysis show that PHY genes which were previously reported to act as molecular switches in response to several abiotic stresses were mainly induced in hp1 and repressed in aur and MT in response to stress. As expected, we also found reduced levels of malondialdehyde (MDA), enhanced activities of antioxidant enzymes, and higher accumulation of protecting osmolytes (soluble sugars, proline, glycine betaine) which further elaborate the underlying tolerance mechanism of hp1 genotype under chilling stress. ConclusionOur findings clearly demonstrate that phytochrome-sensitive and phytochrome-deficient tomato mutants respond differently under chilling stress thereby regulating physiological, biochemical, and molecular responses and thus establish a strong link between phytochromes and their role in stress tolerance.

Heterologous phytase expression in the food filamentous fungus <i> Aspergillus oryzae </i>using the added rice husk cultivation model

Aspergillus oryzae, a safe filamentous fungus, is widely used in food and enzyme production. In this study, we examined a cultivation model using rice husks as carrier to assess the capacity of recombinant protein production in A. oryzae. The model was first tested with the A. oryzae strain expressing the DsRed reporter gene. Expression of DsRed was easily detected by the pink color of the fungal mycelium on culture media and under a fluorescence microscope. The model was then evaluated with the phyA gene encoding a phytase from the fungus Aspergillus fumigatus. The phyA expression cassette regulated by the amyB promoter was permanently integrated into the genome of A. oryzae via Agrobacterium tumefaciens-mediated transformation with the pyrG nutritional marker. Results showed that A. oryzae transgenic strains carrying 2−3 copies of the phyA gene in their genomes exhibited a significant increase in phytase activity on agar medium supplemented with phytate. With rice husks added, these transgenic strains could secrete the recombinant phytase into the culture and phytase activity of the crude enzyme solution increased by 4.3 times compared to the unstransgenic A. oryzae. The established cultivation model and the transgenic approach in this study represent a potential for being used in production of secreted recombinant enzymes for animal feeds.

The Transcription Factor HY5 Is Involved in the Cytokinin-Dependent Regulation of the Expression of Genes Encoding Proteins Associated with Bacterial Plastid RNA-Polymerase during De-Etiolation of Arabidopsis thaliana.

HY5 (ELONGATED HYPOCOTYL5), a bZIP transcription factor, is one of the main regulators of light and hormonal signaling. Among the targets of this gene, the genes for the transcriptional complex of chloroplasts whose coordinated expression ensures the initial stages of photomorphogenesis are particularly significant. In this study, we showed that, during de-etiolation, HY5 functions as a positive CK-dependent regulator of the expression of genes encoding proteins associated with plastid RNA polymerase (PAP), which functions below the primary chain of sensing the cytokinin signal. The absence of blocking effect of mutations of the CRY1, CRY2, PHYA, and PHYB photoreceptor genes on the CK-dependent content of PAP gene transcripts indicates the parallel action of the hormone and light in their regulation.

Prioritization of potato genes involved in the formation of agronomically valuable traits using the SOLANUM TUBEROSUM knowledge base

The development of highly efficient technologies in genomics, transcriptomics, proteomics and metabolomics, as well as new technologies in agriculture has led to an “information explosion” in plant biology and crop production, including potato production. Only a small part of the information reaches formalized databases (for example, Uniprot, NCBI Gene, BioGRID, IntAct, etc.). One of the main sources of reliable biological data is the scientific literature. The well-known PubMed database contains more than 18 thousand abstracts of articles on potato. The effective use of knowledge presented in such a number of non-formalized documents in natural language requires the use of modern intellectual methods of analysis. However, in the literature, there is no evidence of a widespread use of intelligent methods for automatically extracting knowledge from scientific publications on cultures such as potatoes. Earlier we developed the SOLANUM TUBEROSUM knowledge base (http://www-bionet.sysbio.cytogen. ru/and/plant/). Integrated into the knowledge base information about the molecular genetic mechanisms underlying the selection of significant traits helps to accelerate the identification of candidate genes for the breeding characteristics of potatoes and the development of diagnostic markers for breeding. The article searches for new potential participants of the molecular genetic mechanisms of resistance to adverse factors in plants. Prioritizing candidate genes has shown that the PHYA, GF14, CNIH1, RCI1A, ABI5, CPK1, RGS1, NHL3, GRF8, and CYP21-4 genes are the most promising for further testing of their relationships with resistance to adverse factors. As a result of the analysis, it was shown that the molecular genetic relationships responsible for the formation of significant agricultural traits are complex and include many direct and indirect interactions. The construction of associative gene networks and their analysis using the SOLANUM TUBEROSUM knowledge base is the basis for searching for target genes for targeted mutagenesis and marker-oriented selection of potato varieties with valuable agricultural characteristics.

Genetic transformation of tropical maize (Zea mays L.) inbred line with a phytase gene from Aspergillus niger.

A full-length cDNA of phyA gene of Aspergillus niger, encoding phytase enzyme, was cloned and expressed in E. coli BL21 cells and assayed for its activity. The phyA cDNA consisted of 1404bp, which encoded 467 amino acid residues. The phytase activity of purified phytase was 826.33U/mL. The phyA gene under the control of endosperm-specific promoters was transformed into an Indian maize inbred line, UMI29, using particle bombardment-mediated transformation method to generate transgenic maize plants over-expressing phytase in seeds. PCR and GUS analyses demonstrated the presence of transgenes in T0 transgenic plants and their stable inheritance in the T1 progenies. Three transgenic events expressing detectable level of A. niger phytase were characterized by western blot analysis. Phytase activity of 463.158U/kg of seed was observed in one of the events, JB-UMI29-Z17/2. The phytase activity of transgenic maize seeds was 5.5- to 7-fold higher than the wild-type UMI29 seeds and, consequently, the seeds had 0.6- to 5-fold higher inorganic phosphorus content.

Parallel origins of photoperiod adaptation following dual domestications of common bean.

Common bean (Phaseolus vulgaris L.) is an important grain legume domesticated independently in Mexico and Andean South America approximately 8000 years ago. Wild forms are obligate short-day plants, and relaxation of photoperiod sensitivity was important for expansion to higher latitudes and subsequent global spread. To better understand the nature and origin of this key adaptation, we examined its genetic control in progeny of a wide cross between a wild accession and a photoperiod-insensitive cultivar. We found that photoperiod sensitivity is under oligogenic control, and confirm a major effect of the Ppd locus on chromosome 1. The red/far-red photoreceptor gene PHYTOCHROME A3 (PHYA3) was identified as a strong positional candidate for Ppd, and sequencing revealed distinct deleterious PHYA3 mutations in photoperiod-insensitive Andean and Mesoamerican accessions. These results reveal the independent origins of photoperiod insensitivity within the two major common bean gene pools and demonstrate the conserved importance of PHYA genes in photoperiod adaptation of short-day legume species.

Testing the Hypothesis of Multiple Origins of Holoparasitism in Orobanchaceae: Phylogenetic Evidence from the Last Two Unplaced Holoparasitic Genera, Gleadovia and Phacellanthus.

Orobanchaceae is the largest family among the parasitic angiosperms. It comprises non-parasites, hemi- and holoparasites, making this family an ideal test case for studying the evolution of parasitism. Previous phylogenetic analyses showed that holoparasitism had arisen at least three times from the hemiparasitic taxa in Orobanchaceae. Until now, however, not all known genera of Orobanchaceae were investigated in detail. Among them, the unknown phylogenetic positions of the holoparasites Gleadovia and Phacellanthus are the key to testing how many times holoparasitism evolved. Here, we provide clear evidence for the first time that they are members of the tribe Orobancheae, using sequence data from multiple loci (nuclear genes ITS, PHYA, PHYB, and plastid genes rps2, matK). Gleadovia is an independent lineage whereas Phacellanthus should be merged into genus Orobanche section Orobanche. Our results unambiguously support the hypothesis that there are only three origins of holoparasitism in Orobanchaceae. Divergence dating reveals for the first time that the three origins of holoparasitism were not synchronous. Our findings suggest that holoparasitism can persist in specific clades for a long time and holoparasitism may evolve independently as an adaptation to certain hosts.

Identification and expression analysis of photoreceptor genes in kiwifruit leaves under natural daylength conditions and their relationship with other genes that regulate photoperiodic flowering

Kiwifruit (Actinidia chinensis var. deliciosa (A. Chev) A. Chev.) is a dioecious vine highly dependent on pollination, which is limited by a lack of synchrony of flowering time between male and female plants. In many plant species, the regulation of the timing of flowering depends largely on seasonal cues such as photoperiod, which is detected by photoreceptors. In this report, we determined the full sequences of the PHYB (AcPHYB) and PHYA (AcPHYA) genes and a partial sequence of the CRY2 (AcCRY2) gene in kiwifruit. Next, we monitored the expression patterns of these photoreceptor genes (AcPHYA, AcPHYB and AcCRY2) as well as other genes involved in flowering regulation (AcCO-like and AcFT) in the leaves of kiwifruit plants grown under natural photoperiods in the field. The annual expression patterns of AcPHYB, AcPHYA and AcCRY2 genes showed that they were significantly highly expressed from late flower development until full bloom and fitting with floral evocation, closely matching the peaks of expression detected for the AcFT and AcCO-like genes. In addition, the daily expression patterns of AcPHYB, AcPHYA and AcCRY2 were analyzed in leaves collected under different daylength conditions. Under long-day (LD) conditions, maximum expression levels were detected in the middle of the day in April (before full bloom), while their expression lost their daily rhythmic patterns in June (after full bloom) and were consistently expressed at low levels. Under short-day (SD) conditions, AcPHYB, AcPHYA and AcCRY2 gene expression patterns were the opposite of those observed in April. With respect to AcFT, no expression was detected in SD conditions. In contrast, the AcCO-like gene oscillated for all daylength conditions with the same daily rhythm. Our results seem to indicate the involvement of photoreceptor genes in kiwifruit flowering regulation. The different daily expression patterns detected for AcPHYA, AcPHYB, AcCRY2 and AcFT under different daylength conditions suggest that photoperiod regulates their expression, while the uniform expression of the AcCO-like gene is in agreement with its reported regulation by the circadian clock.

Cloning and Sequence Comparison of Phytase (Phy) Genes from Aspergillus niger and Bacillus atrophaeus

Cloning and Sequence Comparison of Phytase (Phy) Genes from Aspergillus niger and Bacillus atrophaeus

Codon Optimization and Expression of phyA Gene in Soybean ( Glycine max Merr.)

Codon Optimization and Expression of <i>phyA</i> Gene in Soybean ( <i>Glycine max</i> Merr.)

FTL2 expression preceding bud set corresponds with timing of bud set in Norway spruce under different light quality treatments

Shoot elongation and bud set in temperate zone trees is under photoperiodic control, but light quality also plays a role with far-red (FR) light sustaining growth, and blue (B) affecting timing of bud set. This may suggest a role of B light receptors in addition to the photoperiod- and red (R)-FR-sensing phytochrome (phy) system. However, there is limited information on effect of light quality on genes thought to be involved in growth-dormancy cycling. In this study we have examined effect of light quality on expression of genes known to be affected by photoperiod as well as CRY and PHY genes, which were also characterised in this study. In contrast to growth-sustaining FR and reference LD treatment, bud-inducing R and B light and reference SD exposure resulted in accumulation of transcripts of PaFTL2, and reduction in PaCOL1, PaCOL2 and PaSOC1 before visible morphological changes associated with bud set. The light receptor transcripts (CRY1, CRY2, PHYN, PHYO, PHYP) also accumulated, but with the largest changes after growth cessation. Reduced or slightly delayed bud set under R compared to B light and SD, was in most cases reflected in changes in transcript levels. Of the studied genes, FTL2 expression preceding bud set correlated the most closely with timing of bud set in in the studied provenances under the different light quality treatments. These results demonstrate action of light quality and photoperiod through a common pathway in control of bud set in Norway spruce, with FTL2 as a key factor.