Phytoglobins in the nuclei, cytoplasm and chloroplasts modulate nitric oxide signaling and interact with abscisic acid.

Symbiotic hemoglobins provide O2 to N2 -fixing bacteria within legume nodules, but the functions of non-symbiotic hemoglobins or phytoglobins (Glbs) are much less defined. Immunolabeling combined with confocal microscopy of the Glbs tagged at the C-terminus with green fluorescent protein was used to determine their subcellular localizations in Arabidopsis and Lotus japonicus. Recombinant proteins were used to examine nitric oxide (NO) scavenging in vitro and transgenic plants to show S-nitrosylation and other in vivo interactions with NO and abscisic acid (ABA) responses. We found that Glbs occur in the nuclei, chloroplasts and amyloplasts of both model plants, and also in the cytoplasm of Arabidopsis cells. The proteins show similar NO dioxygenase activities in vitro, are nitrosylated in Cys residues in vivo, and scavenge NO in the stomatal cells. The Cys/Ser mutation does not affect NO dioxygenase activity, and S-nitrosylation does not significantly consume NO. We demonstrate an interaction between Glbs and ABA on several grounds: Glb1 and Glb2 scavenge NO produced in stomatal guard cells following ABA supply; plants overexpressing Glb1 show higher constitutive expression of the ABA responsive genes Responsive to ABA (RAB18), Responsive to Dehydration (RD29A) and Highly ABA-Induced 2 (HAI2), and are more tolerant to dehydration; and ABA strongly upregulates class 1 Glbs. We conclude that Glbs modulate NO and interact with ABA in crucial physiological processes such as the plant's response to dessication.

Jasmonic acid is a downstream component in the modulation of somatic embryogenesis by Arabidopsis Class 2 phytoglobin.

Previous studies have shown that the beneficial effect of suppression of the Arabidopsis phytoglobin 2 gene, PGB2, on somatic embryogenesis occurs through the accumulation of nitric oxide (NO) within the embryogenic cells originating from the cultured explant. NO activates the expression of Allene oxide synthase (AOS) and Lipoxygenase 2 (LOX2), genes encoding two key enzymes of the jasmonic acid (JA) biosynthetic pathway, elevating JA content within the embryogenic tissue. The number of embryos in the single aos1-1 mutant and pgb2-aos1-1 double mutant declined, and was not rescued by increasing levels of NO stimulating embryogenesis in wild-type tissue. NO also influenced JA responses by up-regulating PLANT DEFENSIN 1 (PDF1) and JASMONATE-ZIM-PROTEIN (JAZ1), as well as down-regulating MYC2. The NO and JA modulation of MYC2 and JAZ1 controlled embryogenesis. Ectopic expression of JAZ1 or suppression of MYC2 promoted the formation of somatic embryos, while repression of JAZ1 and up-regulation of MYC2 reduced the embryogenic performance. Sustained expression of JAZ1 induced the transcription of several indole acetic acid (IAA) biosynthetic genes, resulting in higher IAA levels in the embryogenic cells. Collectively these data fit a model integrating JA in the PGB2 regulation of Arabidopsis embryogenesis. Suppression of PGB2 increases JA through NO. Elevated levels of JA repress MYC2 and induce JAZ1, favoring the accumulation of IAA in the explants and the subsequent production of somatic embryos.

Function of type-2 Arabidopsis hemoglobin in the auxin-mediated formation of embryogenic cells during morphogenesis.

Suppression of Arabidopsis GLB2, a type-2 nonsymbiotic hemoglobin, enhances somatic embryogenesis by increasing auxin production. In the glb2 knock-out line (GLB2-/-), polarization of PIN1 proteins and auxin maxima occurred at the base of the cotyledons of the zygotic explants, which are the sites of embryogenic tissue formation. These changes were also accompanied by a transcriptional upregulation of WUSCHEL (WUS) and SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK1), which are markers of embryogenic competence. The increased auxin levels in the GLB2-/- line were ascribed to the induction of several key enzymes of the tryptophan and IAA biosynthetic pathways, including ANTHRANILATE SYNTHASE (α subunit; ASA1), CYTOCHROME P79B2 (CYP79B2) and AMIDASE1 (AMI1). The effects of GLB2 suppression on somatic embryogenesis and IAA synthesis are mediated by increasing levels of nitric oxide (NO) within the embryogenic cells, which repress the expression of the transcription factor MYC2, a well-characterized repressor of the auxin biosynthetic pathway. A model is proposed in which the suppression of GLB2 reduces the degree of NO scavenging by oxyhemoglobin, thereby increasing the cellular NO concentration. The increased levels of NO repress the expression of MYC2, relieving the inhibition of IAA synthesis and increasing cellular IAA, which is the inductive signal promoting embryogenic competence. Besides providing a model for the induction phase of embryogenesis in vitro, these studies propose previously undescribed functions for plant hemoglobins.

Vitamin C deficiency improves somatic embryo development through distinct gene regulatory networks in Arabidopsis.

Changes in the endogenous ascorbate redox status through genetic manipulation of cellular ascorbate levels were shown to accelerate cell proliferation during the induction phase and improve maturation of somatic embryos in Arabidopsis. Mutants defective in ascorbate biosynthesis such as vtc2-5 contained ~70 % less cellular ascorbate compared with their wild-type (WT; Columbia-0) counterparts. Depletion of cellular ascorbate accelerated cell division processes and cellular reorganization and improved the number and quality of mature somatic embryos grown in culture by 6-fold compared with WT tissues. To gain insight into the molecular mechanisms underlying somatic embryogenesis (SE), we profiled dynamic changes in the transcriptome and analysed dominant patterns of gene activity in the WT and vtc2-5 lines across the somatic embryo culturing process. Our results provide insight into the gene regulatory networks controlling SE in Arabidopsis based on the association of transcription factors with DNA sequence motifs enriched in biological processes of large co-expressed gene sets. These data provide the first detailed account of temporal changes in the somatic embryo transcriptome starting with the zygotic embryo, through tissue dedifferentiation, and ending with the mature somatic embryo, and impart insight into possible mechanisms for the improved culture of somatic embryos in the vtc2-5 mutant line.

Enhancing in vitro regeneration via somatic embryogenesis and Fusarium wilt resistance of Egyptian cucumber (Cucumis sativus L.) cultivars.

BACKGROUND: Somatic embryogenesis offers a reliable method for cucumber (Cucumis sativus L.) regeneration and genetic enhancement against Fusarium wilt. This study aimed to establish a tailored somatic embryogenesis system for Egyptian cultivars, fostering genetic improvements and Fusarium wilt-resistance lines. RESULTS: Employing the Optimal Arbitrary Design (OAD) approach, we optimized the induction medium, initiating prolific embryogenic calli (53.3 %) at 1 mg/L 2,4-D. The cotyledonary leaf (CL) was the preferred explant, showing 60 % embryogenic callus development. Bieth Alpha exhibited higher responsiveness, generating âˆ¼ 18 somatic embryos per explant compared to Prince's âˆ¼ 10. Somatic embryogenesis system validation used quantitative RT-PCR, showing Cucumis sativus splicing factor 3B subunit (CUS1) and an embryogenesis marker gene expression exclusively within embryogenic calli and mainly during embryogenesis initiation. Evaluating fungal toxin filtrate concentrations for selecting embryogenic calli, the S2 selection (25 % filtrate, four subculture cycles) was chosen for somatic embryo development. To gauge the ramifications of selection at the genetic stratum, an in-depth analysis was executed. A cluster analysis grounded in ISSR banding patterns revealed a distinct separation between in vivo-cultivated plants of the two cultivars and regenerated plants devoid of pathogen filtrate treatment or those regenerated post-filtrate treatment. This segregation distinctly underscores the discernible genetic impact of the selection process. CONCLUSIONS: The highest embryogenic capacity (53.3%) was achieved in this study by optimizing the induction stage, which demonstrated the optimal concentrations of BA and 2,4-D for induced proembryonic masses. Moreover, consistent gene expression throughout both stages of embryogenesis suggests that our system unequivocally follows the somatic embryogenesis pathway.

Gene expression analysis in microdissected shoot meristems of Brassica napus microspore-derived embryos with altered SHOOTMERISTEMLESS levels.

Altered expression of Brassica napus (Bn) SHOOTMERISTEMLESS (STM) affects the morphology and behaviour of microspore-derived embryos (MDEs). While down-regulation of BnSTM repressed the formation of the shoot meristem (SAM) and reduced the number of Brassica MDEs able to regenerate viable plants at germination, over-expression of BnSTM enhanced the structure of the SAM and improved regeneration frequency. Within dissected SAMs, the induction of BnSTM up-regulated the expression of many transcription factors (TFs) some of which directly involved in the formation of the meristem, i.e. CUP-SHAPED COTYLEDON1 and WUSCHEL, and regulatory components of the antioxidant response, hormone signalling, and cell wall synthesis and modification. Opposite expression patterns for some of these genes were observed in the SAMs of MDEs down-regulating BnSTM. Altered expression of BnSTM affected transcription of cell wall and lignin biosynthetic genes. The expression of PHENYLALANINE AMMONIA LYASE2, CINNAMATE 4-4HYDROXYLASE, and CINNAMYL ALCOHOL DEHYDROGENASE were repressed in SAMs over-expressing BnSTM. Since lignin formation is a feature of irreversible cell differentiation, these results suggest that one way in which BnSTM promotes indeterminate cell fate may be by preventing the expression of components of biochemical pathways involved in the accumulation of lignin in the meristematic cells. Overall, these studies provide evidence for a novel function of BnSTM in enhancing the quality of in vitro produced meristems, and propose that this gene can be used as a potential target to improve regeneration of cultured embryos.

Altered seed oil and glucosinolate levels in transgenic plants overexpressing the Brassica napus SHOOTMERISTEMLESS gene.

SHOOTMERISTEMLESS (STM) is a homeobox gene conserved among plant species which is required for the formation and maintenance of the shoot meristem by suppressing differentiation and maintaining an undetermined cell fate within the apical pole. To assess further the role of this gene during seed storage accumulation, transgenic Brassica napus (Bn) plants overexpressing or down-regulating BnSTM under the control of the 35S promoter were generated. Overexpression of BnSTM increased seed oil content without affecting the protein and sucrose level. These changes were accompanied by the induction of genes encoding several transcription factors promoting fatty acid (FA) synthesis: LEAFY COTYLEDON1 (BnLEC1), BnLEC2, and WRINKLE1 (BnWRI1). In addition, expression of key representative enzymes involved in sucrose metabolism, glycolysis, and FA biosynthesis was up-regulated in developing seeds ectopically expressing BnSTM. These distinctive expression patterns support the view of an increased carbon flux to the FA biosynthetic pathway in developing transformed seeds. The overexpression of BnSTM also resulted in a desirable reduction of seed glucosinolate (GLS) levels ascribed to a transcriptional repression of key enzymes participating in the GLS biosynthetic pathway, and possibly to the differential utilization of common precursors for GLS and indole-3-acetic acid synthesis. No changes in oil and GLS levels were observed in lines down-regulating BnSTM. Taken together, these findings provide evidence for a novel function for BnSTM in promoting desirable changes in seed oil and GLS levels when overexpressed in B. napus plants, and demonstrate that this gene can be used as a target for genetic improvement of oilseed species.

Transduction of Signals during Somatic Embryogenesis.

Somatic embryogenesis (SE) is an in vitro biological process in which bipolar structures (somatic embryos) can be induced to form from somatic cells and regenerate into whole plants. Acquisition of the embryogenic potential in culture is initiated when some competent cells within the explants respond to inductive signals (mostly plant growth regulators, PRGs), and de-differentiate into embryogenic cells. Such cells, "canalized" into the embryogenic developmental pathway, are able to generate embryos comparable in structure and physiology to their in vivo counterparts. Genomic and transcriptomic studies have identified several pathways governing the initial stages of the embryogenic process. In this review, the authors emphasize the importance of the developmental signals required for the progression of embryo development, starting with the de-differentiation of somatic cells and culminating with tissue patterning during the formation of the embryo body. The action and interaction of PGRs are highlighted, along with the participation of master regulators, mostly transcription factors (TFs), and proteins involved in stress responses and the signal transduction required for the initiation of the embryogenic process.

Brassinolide-improved development of Brassica napus microspore-derived embryos is associated with increased activities of purine and pyrimidine salvage pathways.

Cellular brassinolide (BL) levels regulate the development of Brassica napus microspore-derived embryos (MDEs). Synthesis and degradation of nucleotides were measured on developing MDEs treated with BL or brassinazole (BrZ), a biosynthetic inhibitor of BL. Purine metabolism was investigated by following the metabolic fate of (14)C-labelled adenine and adenosine, substrates of the salvage pathway, and inosine, an intermediate of both salvage and degradation pathways. For pyrimidine, orotic acid, uridine and uracil were employed as markers for the de novo (orotic acid), salvage (uridine and uracil), and degradation (uracil) pathways. Our results indicate that utilization of adenine, adenosine, and uridine for nucleotides and nucleic acids increased significantly in BL-treated embryos at day 15 and remained high throughout the culture period. These metabolic changes were ascribed to the activities of the respective salvage enzymes: adenine phosphoribosyltransferase (EC 2.4.2.7), adenosine kinase (EC 2.7.1.20), and uridine kinase (EC 2.7.1.48), which were induced by BL applications. The BL promotion of salvage synthesis was accompanied by a reduction in the activities of the degradation pathways, suggesting the presence of competitive anabolic and catabolic mechanisms utilizing the labelled precursors. In BrZ-treated embryos, with depleted BL levels, the salvage activity of both purine and pyrimidine nucleotides was reduced and this was associated to structural abnormalities and poor embryonic performance. In these embryos, the activities of major salvage enzymes were consistently lower to those measured in their control (untreated) counterparts.

Distinct fluctuations in nucleotide metabolism accompany the enhanced in vitro embryogenic capacity of Brassica cells over-expressing SHOOTMERISTEMLESS.

Besides regulating meristem formation and maintenance in vivo, SHOOTMERISTEMLESS (STM) has been shown to affect embryogenesis. While the over-expression of Brassica napus (Bn)STM enhances the number of microspore-derived embryos produced in culture and their ability to regenerate viable plants, a down-regulation of this gene represses the embryogenic process (Elhiti et al., J Exp Bot, 61:4069-4085, 2010). Synthesis and degradation of pyrimidine and purine nucleotides were measured in developing microspore-derived embryos (MDEs) generated from B. napus lines ectopically expressing or down-regulating BnSTM. Pyrimidine metabolism was investigated by following the metabolic fate of exogenously supplied (14)C-uridine, uracil and orotic acid, whereas purine metabolism was estimated by using (14)C-adenine, adenosine and inosine. The improvement in embryo number and quality affected by the ectopic expression of BnSTM was linked to the increased pyrimidine and purine salvage activity during the early phases of embryogenesis and the enlargement of the adenylate pool (ATP + ADP) required for the active growth of the embryos. This was due to an increase in transcriptional and enzymatic activity of several salvage enzymes, including adenine phosphoribosyltransferase (APRT) and adenosine kinase (ADK). The highly operative salvage pathway induced by the ectopic expression of BnSTM was associated with a slow catabolism of nucleotides, suggesting the presence of an antagonist mechanism controlling the rate of salvage and degradation pathways. During the second half of embryogenesis utilization of uridine for UTP + UDPglucose (UDPG) synthesis increased in the embryos over-expressing BnSTM, and this coincided with a better post-germination performance. All these events were precluded by the down-regulation of BnSTM which repressed the formation of the embryos and their post-embryonic performance. Overall, this work provides evidence that precise metabolic changes are associated with proper embryo development in culture.

Synthetic Strigolactone GR24 Improves Arabidopsis Somatic Embryogenesis through Changes in Auxin Responses.

Somatic embryogenesis in Arabidopsis encompasses an induction phase requiring auxin as the inductive signal to promote cellular dedifferentiation and formation of the embryogenic tissue, and a developmental phase favoring the maturation of the embryos. Strigolactones (SLs) have been categorized as a novel group of plant hormones based on their ability to affect physiological phenomena in plants. The study analyzed the effects of synthetic strigolactone GR24, applied during the induction phase, on auxin response and formation of somatic embryos. The expression level of two SL biosynthetic genes, MOREAXILLARY GROWTH 3 and 4 (MAX3 and MAX4), which are responsible for the conversion of carotene to carotenal, increased during the induction phase of embryogenesis. Arabidopsis mutant studies indicated that the somatic embryo number was inhibited in max3 and max4 mutants, and this effect was reversed by applications of GR24, a synthetic strigolactone, and exacerbated by TIS108, a SL biosynthetic inhibitor. The transcriptional studies revealed that the regulation of GR24 and TIS108 on somatic embryogenesis correlated with changes in expression of AUXIN RESPONSIVE FACTORs 5, 8, 10, and 16, known to be required for the production of the embryogenic tissue, as well as the expression of WUSCHEL (WUS) and Somatic Embryogenesis Receptor-like Kinase 1 (SERK1), which are markers of cell dedifferentiation and embryogenic tissue formation. Collectively, this work demonstrated the novel role of SL in enhancing the embryogenic process in Arabidopsis and its requirement for inducing the expression of genes related to auxin signaling and production of embryogenic tissue.

Medical Cannabis and Industrial Hemp Tissue Culture: Present Status and Future Potential.

In recent years high-THC (psychoactive) and low-THC (industrial hemp) type cannabis (Cannabis sativa L.) have gained immense attention in medical, food, and a plethora of other consumer product markets. Among the planting materials used for cultivation, tissue culture clones provide various advantages such as economies of scale, production of disease-free and true-to-type plants for reducing the risk of GMP-EuGMP level medical cannabis production, as well as the development and application of various technologies for genetic improvement. Various tissue culture methods have the potential application with cannabis for research, breeding, and novel trait development, as well as commercial mass propagation. Although tissue culture techniques for plant regeneration and micropropagation have been reported for different cannabis genotypes and explant sources, there are significant variations in the response of cultures and the morphogenic pathway. Methods for many high-yielding elite strains are still rudimentary, and protocols are not established. With a recent focus on sequencing and genomics in cannabis, genetic transformation systems are applied to medical cannabis and hemp for functional gene annotation via traditional and transient transformation methods to create novel phenotypes by gene expression modulation and to validate gene function. This review presents the current status of research focusing on different aspects of tissue culture, including micropropagation, transformation, and the regeneration of medicinal cannabis and industrial hemp transformants. Potential future tissue culture research strategies helping elite cannabis breeding and propagation are also presented.

Depletion of cellular brassinolide decreases embryo production and disrupts the architecture of the apical meristems in Brassica napus microspore-derived embryos.

Exogenous applications of brassinolide (BL) increased the number and quality of microspore-derived embryos (MDEs) whereas treatments with brassinazole (BrZ), a BL biosynthetic inhibitor, had the opposite effect. At the optimal concentration (4x10(-6) M) BrZ decreased both embryo yield and conversion to less than half the value of control embryos. Metabolic studies revealed that BL levels had profound effects on glutathione and ascorbate metabolism by altering the amounts of their reduced forms (ASC and GSH) and oxidized forms [dehydroascorbate (DHA), ascorbate free radicals (AFRs), and GSSG]. Applications of BL switched the glutathione and ascorbate pools towards the oxidized forms, thereby lowering the ASC/ASC+DHA+AFR and GSH/GSH+GSSG ratios. These changes were ascribed to the ability of BL to increase the activity of ascorbate peroxidase (APX) and decrease that of glutathione reductase (GR). This trend was reversed in a BL-depleted environment, effected by BrZ applications. These metabolic alterations were associated with changes in embryo structure and performance. BL-treated MDEs developed zygotic-like shoot apical meristems (SAMs) whereas embryos treated with BrZ developed abnormal meristems. In the presence of BrZ, embryos either lacked a visible SAM, or formed SAMs in which the meristematic cells showed signs of differentiation, such as vacuolation and storage product accumulation. These abnormalities were accompanied by the lack or misexpression of three meristem marker genes isolated from Brassica napus (denoted as BnSTM, BnCLV1, and BnZLL-1) homologous to the Arabidopsis SHOOTMERISTEMLESS (STM), CLAVATA 1 (CLV1), and ZWILLE (ZLL). The expression of BnSTM and BnCLV1 increased after a few days in cultures in embryos treated with BL whereas an opposite tendency was observed with applications of BrZ. Compared with control embryos where these two genes exhibited abnormal localization patterns, BnSTM and BnCLV1 always localized throughout the subapical domains of BL-treated embryos in a zygotic-like fashion. Expression of both genes was often lost in the SAM of BrZ-treated embryos. The results suggest that maintenance of cellular BL levels is required to modulate the ascorbate and glutathione redox status during embryogenesis to ensure proper development of the embryos and formation of functional apical meristems.

Modulation of embryo-forming capacity in culture through the expression of Brassica genes involved in the regulation of the shoot apical meristem.

Somatic embryogenesis in Arabidopsis is achieved by culturing bending-cotyledon embryos on a 2,4-D-containing induction medium for 14 d followed by a transfer on to a hormone-free development medium. Several genes orthologous to Arabidopsis SHOOTMERISTEMLESS (STM), CLAVATA 1 (CLV1), and ZWILLE (ZLL) were isolated from Brassica oleracea (Bo), B. rapa (Br), and B. napus (Bn), and ectopically expressed in Arabidopsis to assess their effects on somatic embryogenesis. Ectopic expression of BoSTM, BrSTM, and BnSTM increased the number of somatic embryos, whereas a different effect was observed in lines overexpressing BnCLV1 in which somatic embryo formation was severely repressed. The introduction of BnZLL did not have any effects on Arabidopsis somatic embryogenesis. The increased embryo-forming capacity observed in lines overexpressing Brassica STM was associated with a lower requirement for the inductive signal 2,4-D, and a higher expression of WUSCHEL (WUS) which demarcates the formation of embryogenic cells. This was in contrast to the 35S::BnCLV1 lines which showed the highest requirement for exogenous 2,4-D and a reduced WUS expression. Microarray studies were conducted to monitor global changes in transcript levels during Arabidopsis somatic embryogenesis between the wild-type (WT) line and a BoSTM-overexpressing line, which showed the most pronounced enhancement of somatic embryo yield. The introduction of BoSTM affected the expression of many genes involved in hormone perception and signalling, as well as genes encoding DNA methyltransferases and enzymes of glutathione metabolism. Pharmacological experiments performed to confirm some of the microarray results showed that Arabidopsis somatic embryogenesis is encouraged by a global hypomethylation of the DNA during the induction phase and by a switch of the glutathione pool towards an oxidized state during the subsequent development phase. Both events occurred in the 35S::BoSTM line, but not in the WT line. Altered expression of Brassica STM also had profound effects on B. napus microspore-derived embryogenesis. The yield of microspore-derived embryos increased in lines overexpressing BnSTM and significantly decreased in antisense lines down-regulating BnSTM.

A new approach for COVID-19 treatment by micro-RNA.

MicroRNAs (miRNAs) naturally occur in plants and all living organisms. They play an important role in gene regulation through binding toa specific region in open reading frames (ORFs) and/or untranslated regions (UTRs) to block the translation processes through either degrading or blocking mRNA resulting in knocking down or suppression of targeted genes. Plants and many organisms protect themselves from viruses through the production of miRNAs, which are complementary to 3UTR of viruses resulting in degrading the viral mRNA or block the translation on ribosomes. As pandemic, COVID-19, and its consequences on the global economy, we hypothesized a new approach for the treatment of COVID-19 paints. This approach includes designing a mix of miRNAs targeting several regions on COVID-19 open reading frame (ORF) and 3 UTR and suitable delivery system targeting respiratory system tissues. These synthesized miRNAs may be delivered to humansinnon-viral delivery systems such as liposomes like exosome (extracellular vesicle), polymer-based carriers, or inorganic nanoparticles, which are considered to be more suitable for human use.

Identification and characterization of PgHZ1, a novel homeodomain leucine-zipper gene isolated from white spruce (Picea glauca) tissue.

A member of the homeodomain-leucine zipper (HD-Zip) family was isolated from white spruce (Picea glauca) and designated as PgHZ1 (Gene Bank Accession No. DQ201170). The gene has an open reading frame of 1268bp and encodes a protein of 309 amino acid residues. PgHZ1 has all the features of a HD-ZIP protein: a homeodomain composed by three alpha-helices involved in DNA binding and an adjacent leucine zipper motif for protein-protein interaction. Phylogenetic analyses and sequence allignments with several Arabidopsis HD-ZIP members reveal that PgHZ1 belongs to the same monophyletic group of ATHB3, 13, 20, and 23 with which it shares a respective amino acid similarities of 74%, 71%, 68%, and 61%. Expression studies during spruce somatic embryogenesis reveal that the transcript levels of PgHZ1 increase during the late phases of proliferation and remain high during the subsequent embryo growth on the ABA-containing maturation medium. Such an increase does not occur in a non-embryogenic line characterized by a developmental block. Arabidopsis plants with ectopic PgHZ1 expression show an increase sensitivity to ABA, as estimated in seed germination and root growth tests. Compared to wild type plants, plants over-expressing PgHZ1 driven by the CAMV 35S promoter show a variety of phenotypic deviations, including a reduced inflorescence growth, increased branching, small rosette leaves and a delay in flowering. Somatic embryos produced from 35S:PgHZ1 Arabidopsis plants display a heavy accumulation of storage products and remain in a developmental program even if subjected to prolonged culture. This is in contrast to wild type somatic embryos in which storage products are quickly mobilized and the germination program is initiated after only 15 days in maturation. Overall these data support the notion that PgHZ1 confers hypersensitivity to ABA and that proper expression of this gene may be required for proper embryonic growth.

Maize root culture as a model system for studying azoxystrobin biotransformation in plants.

Hairy roots induced by Agrobacterium rhizogenes are well established models to study the metabolism of xenobiotics in plants for phytoremediation purposes. However, the model requires special skills and resources for growing and is a time-consuming process. The roots induction process alters the genetic construct of a plant and is known to express genes that are normally absent from the non-transgenic plants. In this study, we propose and establish a non-transgenic maize root model to study xenobiotic metabolism in plants for phytoremediation purpose using azoxystrobin as a xenobiotic compound. Maize roots were grown aseptically in Murashige and Skoog medium for two weeks and were incubated in 100 µM azoxystrobin solution. Azoxystrobin was taken up by the roots to the highest concentration within 15 min of treatment and its phase I metabolites were also detected at the same time. Conjugated metabolites of azoxystrobin were detected and their identities were confirmed by enzymatic and mass spectrometric methods. Further, azoxystrobin metabolites identified in maize root culture were compared against azoxystrobin metabolites in azoxystrobin sprayed lettuce grown in green house. A very close similarity between metabolites identified in maize root culture and lettuce plant was obtained. The results from this study establish that non-transgenic maize roots can be used for xenobiotic metabolism studies instead of genetically transformed hairy roots due to the ease of growing and handling.

In vitro shoot organogenesis and hormone response are affected by the altered levels of Brassica napus meristem genes.

Arabidopsis shoot meristem activity is regulated by a molecular network involving the participation of several components, including SHOOTMERISTEMLESS (STM), CLAVATA1 (CLV1), and ZWILLE (ZLL). In an effort to identify the role of these genes during in vitro shoot formation Brassica and Arabidopsis plants were transformed with the Brassica napus (Bn) STM, CLV1, ZLL1 and ZLL2 identified in previous work [1]. In both systems shoot organogenesis was promoted by the over-expression of BnSTM, BnZLL1, and BnZLL2, and repressed by the over-expression of BnCLV1. This distinct regulation, analogous to that occurring during in vivo meristem formation where STM and ZLL encourage stem cell formation while CLV1 accelerates transition to differentiation, suggests similar regulatory mechanisms governing shoot formation in vivo and in vitro. While the BnZLL1 and BnZLL2 induction of shoot organogenesis correlated only to changes in auxin signaling, BnSTM and BnCLV1 evoked major transcriptional alterations in cytokinin response. Besides increasing the transcript levels of two cytokinin receptors, ARABIDOPSIS HISTIDINE KINASE4 (AHK4) and CYTOKININ INDEPENDENT KINASE (CKI1), ectopic expression of BnSTM induced Type-B ARABIDOPSIS RESPONSE REGULATORS (ARRs) and repressed Type-A ARRs. Opposite transcriptional patterns occurred in explants over-expressing BnCLV1, characterized by a decreased ability to produce shoots. The role played by Type-A and Type-B ARRs during shoot organogenesis was further examined using a genetic approach which revealed the requirement of ARR12 for the BnSTM positive regulation of shoot organogenesis. Collectively these results expand our knowledge on the function of meristem genes, and provide new tools for enhancing in vitro propagation systems.

Transcriptional changes of antioxidant responses, hormone signalling and developmental processes evoked by the Brassica napus SHOOTMERISTEMLESS during in vitro embryogenesis.

Previous work showed that alterations in Brassica napus (Bn) SHOOTMERISTEMLESS (BnSTM) expression levels influence microspore-derived embryogenesis in B. napus. While over-expression of BnSTM increased microspore-derived embryo (MDE) yield and quality, down-regulation of BnSTM repressed embryo formation [16]. Transcriptional analyses were conducted to investigate the molecular mechanisms underpinning these responses. The induction of BnSTM resulted in a heavy transcriptional activation of genes involved in antioxidant responses, hormone signalling and developmental processes. Several antioxidant enzymes, including catalases, superoxide dismutases, and components of the Halliwell-Asada cycle were induced in embryos ectopically expressing BnSTM and contributed to the removal of reactive oxygen species (ROS). These changes were accompanied by elevated levels of ascorbate and glutathione, which have been shown to promote embryonic growth and development. Induction or repression of BnSTM altered the early cytokinin response, whereas late responses, modulated by Type-A Arabidopsis response regulators (ARRs), were induced in MDEs over-expressing BnSTM. Major differences between transgenic MDEs were also observed in the expression pattern of several auxin transporters and key developmental factors required for normal embryogenesis. While some of these factors, BABYBOOM1 (BBM1) and SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK), play a key role during early embryogeny, others, CYP78A5, LEAFY COTYLEDON1 and 2 (LEC1 and LEC2), as well as WOX2 and 9, are required for proper embryo development. Collectively these results demonstrate the involvement of BnSTM in novel developmental processes which can be utilized to enhance in vitro embryogenesis.