Plant expression of NifD protein variants resistant to mitochondrial degradation.

To engineer Mo-dependent nitrogenase function in plants, expression of the structural proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here, we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in Escherichia coli identified a subset that retained function, including a single amino acid variant (Y100Q). We found that other naturally occurring NifD proteins containing alternate amino acids at the Y100 position were also less susceptible to degradation. The Y100Q variant also enabled expression of a NifD(Y100Q)-linker-NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)-linker-NifK retained function in bacterial nitrogenase assays, demonstrating that this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.

Seed-specific RNAi in safflower generates a superhigh oleic oil with extended oxidative stability.

Vegetable oils extracted from oilseeds are an important component of foods, but are also used in a range of high value oleochemical applications. Despite being biodegradable, nontoxic and renewable current plant oils suffer from the presence of residual polyunsaturated fatty acids that are prone to free radical formation that limit their oxidative stability, and consequently shelf life and functionality. Many decades of plant breeding have been successful in raising the oleic content to ~90%, but have come at the expense of overall field performance, including poor yields. Here, we engineer superhigh oleic (SHO) safflower producing a seed oil with 93% oleic generated from seed produced in multisite field trials spanning five generations. SHO safflower oil is the result of seed-specific hairpin-based RNA interference of two safflower lipid biosynthetic genes, FAD2.2 and FATB, producing seed oil containing less than 1.5% polyunsaturates and only 4% saturates but with no impact on lipid profiles of leaves and roots. Transgenic SHO events were compared to non-GM safflower in multisite trial plots with a wide range of growing season conditions, which showed no evidence of impact on seed yield. The oxidative stability of the field-grown SHO oil produced from various sites was 50 h at 110°C compared to 13 h for conventional ~80% oleic safflower oils. SHO safflower produces a uniquely stable vegetable oil across different field conditions that can provide the scale of production that is required for meeting the global demands for high stability oils in food and the oleochemical industry.

The microRNA159-regulated GAMYB-like genes inhibit growth and promote programmed cell death in Arabidopsis.

The microRNA159 (miR159) family represses the conserved GAMYB-like genes that encode R2R3 MYB domain transcription factors that have been implicated in gibberellin (GA) signaling in anthers and germinating seeds. In Arabidopsis (Arabidopsis thaliana), the two major miR159 family members, miR159a and miR159b, are functionally specific for two GAMYB-like genes, MYB33 and MYB65. These transcription factors have been shown to be involved in anther development, but there are differing reports about their role in the promotion of flowering and little is known about their function in seed germination. To understand the function of this pathway, we identified the genes and processes controlled by these GAMYB-like genes. First, we demonstrate that miR159 completely represses MYB33 and MYB65 in vegetative tissues. We show that GA does not release this repression and that these transcription factors are not required for flowering or growth. By contrast, in the absence of miR159, the deregulation of MYB33 and MYB65 in vegetative tissues up-regulates genes that are highly expressed in the aleurone and GA induced during seed germination. Confirming that these genes are GAMYB-like regulated, their expression was reduced in myb33.myb65.myb101 seeds. Aleurone vacuolation, a GA-mediated programmed cell death process required for germination, was impaired in these seeds. Finally, the deregulation of MYB33 and MYB65 in vegetative tissues inhibits growth by reducing cell proliferation. Therefore, we conclude that miR159 acts as a molecular switch, only permitting the expression of GAMYB-like genes in anthers and seeds. In seeds, these transcription factors participate in GA-induced pathways required for aleurone development and death.

Insights into Nitrogenase Bioelectrocatalysis for Green Ammonia Production.

There is a growing interest in using ammonia as a liquid carrier of hydrogen for energy applications. Currently, ammonia is produced industrially by the Haber-Bosch process, which requires high temperature and high pressure. In contrast, bacteria have naturally evolved an enzyme known as nitrogenase, that is capable of producing ammonia and hydrogen at ambient temperature and pressure. Therefore, nitrogenases are attractive as a potentially more efficient means to produce ammonia via harnessing the unique properties of this enzyme. In recent years, exciting progress has been made in bioelectrocatalysis using nitrogenases to produce ammonia. Here, the prospects for developing biological ammonia production are outlined, key advances in bioelectrocatalysis by nitrogenases are highlighted, and possible solutions to the obstacles faced in realising this goal are discussed.

Metabolic engineering of morphinan alkaloids by over-expression and RNAi suppression of salutaridinol 7-O-acetyltransferase in opium poppy.

We demonstrate that both over-expression and suppression of the gene encoding the morphinan pathway enzyme salutaridinol 7-O-acetyltransferase (SalAT) in opium poppy affects the alkaloid products that accumulate. Over-expression of the gene in most of the transgenic events resulted in an increase in capsule morphine, codeine and thebaine on a dry-weight basis. The transgenic line with the highest alkaloid content had 41%, 37% and 42% greater total alkaloids than the control in three independent trials over 3 years. DNA-encoded hairpin RNA-mediated suppression of SalAT resulted in the novel accumulation of the alkaloid salutaridine at up to 23% of total alkaloid; this alkaloid is not detectable in the parental genotype. Salutaridine is not the substrate of SalAT but the substrate of the previous enzyme in the pathway, salutaridine reductase. RNA transcript analysis of 16 primary T0 transformants and their segregating T1 progeny revealed an average reduction in SalAT transcript to about 12% of the control. Reduction in SalAT transcript was evident in both leaves and latex. Reverse transcriptase PCR and high-performance liquid chromatography analyses confirmed cosegregation of the expressed transgene with the salutaridine accumulating phenotype.

Increasing morphinan alkaloid production by over-expressing codeinone reductase in transgenic Papaver somniferum.

Only plants of the Papaver genus (poppies) are able to synthesize morphinan alkaloids, and cultivation of P. somniferum, opium poppy, remains critical for the production and supply of morphine, codeine and various semi-synthetic analgesics. Opium poppy was transformed with constitutively expressed cDNA of codeinone reductase (PsCor1.1), the penultimate step in morphine synthesis. Most transgenic lines showed significant increases in capsule alkaloid content in replicated glasshouse and field trials over 4 years. The morphinan alkaloid contents on a dry weight basis were between 15% and 30% greater than those in control high-yielding genotypes and control non-transgenic segregants. Transgenic leaves had approximately 10-fold greater levels of Cor transcript compared with non-transgenic controls. Two cycles of crossing of the best transgenic line into an elite high-morphine genotype resulted in significant increases in morphine and total alkaloids relative to the elite recurrent parent. No significant changes in alkaloid profiles or quantities were observed in leaf, roots, pollen and seed.

RNAi-mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy.

We report on the silencing of codeinone reductase (COR) in the opium poppy, Papaver somniferum, using a chimeric hairpin RNA construct designed to silence all members of the multigene COR family through RNA interference (RNAi). After gene silencing, the precursor alkaloid (S)-reticuline-seven enzymatic steps upstream of codeinone-accumulated in transgenic plants at the expense of morphine, codeine, oripavine and thebaine. Methylated derivatives of reticuline also accumulated. Analysis verified loss of Cor gene transcript, appearance of 22-mer degradation products and reduction of enzyme activity. The surprising accumulation of (S)-reticuline suggests a feedback mechanism preventing intermediates from general benzylisoquinoline synthesis entering the morphine-specific branch. However transcript levels for seven other enzymes in the pathway, both before and after (S)-reticuline, were unaffected. This is the first report of gene silencing in transgenic opium poppy and of metabolic engineering to cause the high-yield accumulation of the nonnarcotic alkaloid reticuline.

Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix.

The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world's increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia.

Opium Poppy (Papaver somniferum).

The genetic transformation of opium poppy, Papaver somniferum, offers the opportunity to study the mechanisms involved in the regulation of benzylisoquinoline and morphinan alkaloid biosynthesis. The development of an efficient transformation protocol for opium poppy has allowed us to transform a range of genotypes from all around the world, including previously recalcitrant high-yielding commercial Australian cultivars. The method involves Agrobacterium tumefaciens infection of hypocotyl explants, followed by the production of antibiotic or herbicide resistant embryogenic callus, the subsequent induction of somatic embryos and development into normal plants. The use of different selective agents, binary vectors, and poppy genotypes has demonstrated the robustness and reliability of this protocol in the production of many hundreds of confirmed transgenic poppies.

Facile mutant identification via a single parental backcross method and application of whole genome sequencing based mapping pipelines.

Forward genetic screens have identified numerous genes involved in development and metabolism, and remain a cornerstone of biological research. However, to locate a causal mutation, the practice of crossing to a polymorphic background to generate a mapping population can be problematic if the mutant phenotype is difficult to recognize in the hybrid F2 progeny, or dependent on parental specific traits. Here in a screen for leaf hyponasty mutants, we have performed a single backcross of an Ethane Methyl Sulphonate (EMS) generated hyponastic mutant to its parent. Whole genome deep sequencing of a bulked homozygous F2 population and analysis via the Next Generation EMS mutation mapping pipeline (NGM) unambiguously determined the causal mutation to be a single nucleotide polymorphisim (SNP) residing in HASTY, a previously characterized gene involved in microRNA biogenesis. We have evaluated the feasibility of this backcross approach using three additional SNP mapping pipelines; SHOREmap, the GATK pipeline, and the samtools pipeline. Although there was variance in the identification of EMS SNPs, all returned the same outcome in clearly identifying the causal mutation in HASTY. The simplicity of performing a single parental backcross and genome sequencing a small pool of segregating mutants has great promise for identifying mutations that may be difficult to map using conventional approaches.

MicroR159 regulation of most conserved targets in Arabidopsis has negligible phenotypic effects.

BACKGROUND: A current challenge of microRNA (miRNA) research is the identification of biologically relevant miRNA:target gene relationships. In plants, high miRNA:target gene complementarity has enabled accurate target predictions, and slicing of target mRNAs has facilitated target validation through rapid amplification of 5' cDNA ends (5'-RACE) analysis. Together, these approaches have identified more than 20 targets potentially regulated by the deeply conserved miR159 family in Arabidopsis, including eight MYB genes with highly conserved miR159 target sites. However, genetic analysis has revealed the functional specificity of the major family members, miR159a and miR159b is limited to only two targets, MYB33 and MYB65. Here, we examine the functional role of miR159 regulation for the other potential MYB target genes. RESULTS: For these target genes, functional analysis failed to identify miR159 regulation that resulted in any major phenotypic impact, either at the morphological or molecular level. This appears to be mainly due to the quiescent nature of the remaining family member, MIR159c. Although its expression overlaps in a temporal and spatial cell-specific manner with a subset of these targets in anthers, the abundance of miR159c is extremely low and concomitantly a mir159c mutant displays no anther defects. Examination of potential miR159c targets with conserved miR159 binding sites found neither their spatial or temporal expression domains appeared miR159 regulated, despite the detection of miR159-guided cleavage products by 5'-RACE. Moreover, expression of a miR159-resistant target (mMYB101) resulted predominantly in plants that are indistinguishable from wild type. Plants that displayed altered morphological phenotypes were found to be ectopically expressing the mMYB101 transgene, and hence were misrepresentative of the in vivo functional role of miR159. CONCLUSIONS: This study presents a novel explanation for a paradox common to plant and animal miRNA systems, where among many potential miRNA-target relationships usually only a few appear physiologically relevant. The identification of a quiescent miR159c:target gene regulatory module in anthers provides a likely rationale for the presence of conserved miR159 binding sites in many targets for which miR159 regulation has no obvious functional role. Remnants from the demise of such modules may lead to an overestimation of miRNA regulatory complexity when investigated using bioinformatic, 5'-RACE or transgenic approaches.

Genetic analysis reveals functional redundancy and the major target genes of the Arabidopsis miR159 family.

Currently, there are very few loss-of-function mutations in micro-RNA genes. Here, we characterize two members of the Arabidopsis MIR159 family, miR159a and miR159b, that are predicted to regulate the expression of a family of seven transcription factors that includes the two redundant GAMYB-like genes, MYB33 and MYB65. Using transfer DNA (T-DNA) insertional mutants, we show that a mir159ab double mutant has pleiotropic morphological defects, including altered growth habit, curled leaves, small siliques, and small seeds. Neither mir159a nor mir159b single mutants displayed any of these traits, indicating functional redundancy. By using reporter-gene constructs, it appears that MIR159a and MIR159b are transcribed almost exclusively in the cells in which MYB33 is repressed, as had been previously determined by comparison of MYB33 and mMYB33 (an miR159-resistant allele of MYB33) expression patterns. Consistent with these overlapping transcriptional domains, MYB33 and MYB65 expression levels were elevated throughout mir159ab plants. By contrast, the other five GAMYB-like family members are transcribed predominantly in tissues where miR159a and miR159b are absent, and consequently their expression levels are not markedly elevated in mir159ab. Additionally, mMYB33 transgenic plants can phenocopy the mir159ab phenotype, suggesting that its phenotype is explained by deregulated expression of the redundant gene pair MYB33 and MYB65. This prediction was confirmed; the pleiotropic developmental defects of mir159ab are suppressed through the combined mutations of MYB33 and MYB65, demonstrating the narrow and specific target range of miR159a and miR159b.

Genetic transformation in commercial Tasmanian cultivars of opium poppy, Papaver somniferum, and movement of transgenicpollen in the field.

We report a new transformation protocol for the pharmaceutically important opium poppy, Papaver somniferum L.; the protocol allows transformation for the first time of high yielding commercial cultivars. The method involves Agrobacterium tumefaciens infection of hypocotyl explants, followed by the production of antibiotic- or herbicide-resistant embryogenic callus and the subsequent induction of somatic embryos and plants. Key elements of the improvement are the use of buffering agents to stabilise medium pH and bottom-cooling of the cultures. Transformation was verified by PCR and Southern blot hybridisation. Transcription of transgenes was confirmed by RT-PCR and product sequencing. Expression of transgenes was detected by histochemical GUS staining, phosphinothricin acetyltransferase (PAT) enzyme assays for bar and pat genes, and western analysis of transgenic sunflower seed albumin protein. Expression of various transgenes was detected in stem, leaf, seed, capsule and latex. The pat gene was demonstrated to be stably inherited to the T2 generation and to confer phosphinothricin (PPT) herbicide resistance. Most T0 plants showed normal morphology, were self-fertile and the transgenes displayed the expected Mendelian segregation. The percentage of explants producing somatic embryos that developed into plantlets able to be transplanted to soil, ranged from 6-11% in two Tasmanian cultivars.A field trial using pat transgenic plants was designed to estimate the frequency of hybridisation at various distances into buffer rows of non-transgenic poppies and to related weed species, P. somniferum spp. setigerum and P. dubium. The frequency of hybridisation to completely compatible poppy fell sharply with distance, being 2.6% at 20 cm, 2.13% at 0.5 m and falling to 0% at 2.5 and 5 m. No hybridisation could be detected to two weed species under open pollination conditions, including the compatible P. somniferum spp. setigerum, when grown as close as 20 cm, despite flowering at the same time as the transgenic plants in the presence of foraging bees.

Transformation of opium poppy (Papaver somniferum L.) with antisense berberine bridge enzyme gene (anti-bbe) via somatic embryogenesis results in an altered ratio of alkaloids in latex but not in roots.

The berberine bridge enzyme cDNA bbe from Papaver somniferum L. was transformed in antisense orientation into seedling explants of the industrial elite line C048-6-14-64. In this way, 84 phenotypically normal To plants derived from embryogenic callus cultures were produced. The selfed progeny of these 84 plants yielded several T1 plants with an altered alkaloid profile. One of these plants T1-47, and its siblings T2-1.2 and T2-1.5 are the subject of the present work. The transformation of these plants was evaluated by PCR, and northern and Southern hybridisation. The transgenic plants contained one additional copy of the transgene. The alkaloid content in latex and roots was determined with HPLC and LC-MS. We observed an increased concentration of several pathway intermediates from all biosynthetic branches, e.g., reticuline, laudanine, laudanosine, dehydroreticuline, salutaridine and (S)-scoulerine. The transformation altered the ratio of morphinan and tetrahydrobenzylisoquinoline alkaloids in latex but not the benzophenanthridine alkaloids in roots. The altered alkaloid profile is heritable at least to the T2 generation. These results are the first example of metabolic engineering of the alkaloid pathways in opium poppy and, to our knowledge, the first time that an alkaloid biosynthetic gene has been transformed into the native species, followed by regeneration into a mature plant to enable analyses of the effect of the transgene on metabolism over several generations.