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1. Overexpression of VIRE2-INTERACTING PROTEIN2 in Arabidopsis regulates genes involved in Agrobacterium-mediated plant transformation and abiotic stresses

   https://doi.org/10.1038/s41598-019-49590-3  

   Raman, Vidhyavathi ; Anand, Ajith ; Vasudevan, Balaji ; Morsy, Mustafa R. ; Pant, Bikram D. ; Lee, Hee-Kyung ; Tang, Yuhong ; Mysore, Kirankumar S. ( September 2019 , Scientific Reports)

   ArabidopsisVIRE2-INTERACTINGPROTEIN2 (VIP2) was previously described as a protein with a NOT domain, and Arabidopsisvip2mutants are recalcitrant toAgrobacterium-mediated root transformation. Here we show that VIP2 is a transcription regulator and the C-terminal NOT2 domain of VIP2 interacts with VirE2. Interestingly,AtVIP2overexpressor lines in Arabidopsis did not show an improvement inAgrobacterium-mediated stable root transformation, but the transcriptome analysis identified 1,634 differentially expressed genes compared to wild-type. These differentially expressed genes belonged to various functional categories such as membrane proteins, circadian rhythm, signaling, response to stimulus, regulation of plant hypersensitive response, sequence-specific DNA binding transcription factor activity and transcription regulatory region binding. In addition to regulating genes involved inAgrobacterium-mediated plant transformation,AtVIP2overexpressor line showed differential expression of genes involved in abiotic stresses. The majority of the genes involved in abscisic acid (ABA) response pathway, containing the Abscisic Acid Responsive Element (ABRE) element within their promoters, were down-regulated inAtVIP2overexpressor lines. Consistent with this observation,AtVIP2overexpressor lines were more susceptible to ABA and other abiotic stresses. Based on the above findings, we hypothesize that VIP2 not only plays a role inAgrobacterium-mediated plant transformation but also acts as a general transcriptional regulator in plants.
2. Tailoring a Global Iron Regulon to a Uropathogen

   https://doi.org/10.1128/mBio.00351-20  

   Banerjee, Rajdeep ; Weisenhorn, Erin ; Schwartz, Kevin J. ; Myers, Kevin S. ; Glasner, Jeremy D. ; Perna, Nicole T. ; Coon, Joshua J. ; Welch, Rodney A. ; Kiley, Patricia J. ( April 2020 , mBio)

   Gottesman, Susan (Ed.)

   ABSTRACT Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O 2 -dependent synthesis of themore »siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O 2 to produce aerobactin. IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes.« less
3. Advancing Crop Transformation in the Era of Genome Editing

   https://doi.org/10.1105/tpc.16.00196  

   Altpeter, F. ; Springer, N.M. ; Bartley, L.E. ; Blechl, A.E. ; Brutnell, T.P. ; Citovsky, V. ; Conrad, L.J. ; Gelvin, S.B. ; Jackson, D.P. ; Kausch, A.P. ; et al ( July 2016 , The Plant cell)

   Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than thirty years of technological advances. Genome editing provides new opportunities to enhance crop productivity, but relies on genetic transformation and plant regeneration, which are bottlenecks in the process. Herein we review the state of plant transformation and point to innovations needed to enable genome editing in crops. Plant tissue culture methods need optimization and simplification for efficiency and minimize time in culture. Currently, specialized facilities exist for crop transformation. Single cell and robotic techniques should be developed for high throughput genomic screens. Utilization of plant genes involved in developmental reprogramming, wound response, and/or homologous recombination could boost recovery of transformed plants. Engineering universal Agrobacterium strains and recruitment of other microbes, such as Ensifer or Rhizobium, could facilitate delivery of DNA and proteins into plant cells. Synthetic biology should be employed for de novo design of transformation systems. Genome editing is a potential game-changer in crop genetics when plant transformation systems are optimized.
4. Identification of Genes Encoding Enzymes Catalyzing the Early Steps of Carrot Polyacetylene Biosynthesis

   https://doi.org/https://doi.org/10.1104/pp.18.01195  

   Lucas Busta, Won Cheol ( December 2018 , Plant physiology)

   Polyacetylenic lipids accumulate in various Apiaceae species after pathogen attack, suggesting that these compounds are naturally occurring pesticides and potentially valuable resources for crop improvement. These compounds also promote human health and slow tumor growth. Even though polyacetylenic lipids were discovered decades ago, the biosynthetic pathway underlying their production is largely unknown. To begin filling this gap and ultimately enable polyacetylene engineering, we studied polyacetylenes and their biosynthesis in the major Apiaceae crop carrot (Daucus carota subsp. sativus). Using gas chromatography and mass spectrometry, we identified three known polyacetylenes and assigned provisional structures to two novel polyacetylenes. We also quantified these compounds in carrot leaf, petiole, root xylem, root phloem, and root periderm extracts. Falcarindiol and falcarinol predominated and accumulated primarily in the root periderm. Since the multiple double and triple carbon-carbon bonds that distinguish polyacetylenes from ubiquitous fatty acids are often introduced by Δ12 oleic acid desaturase (FAD2)-type enzymes, we mined the carrot genome for FAD2 genes. We identified a FAD2 family with an unprecedented 24 members and analyzed public, tissue-specific carrot RNA-Seq data to identify coexpressed members with root periderm-enhanced expression. Six candidate genes were heterologously expressed individually and in combination in yeast and Arabidopsis (Arabidopsis thaliana), resultingmore »in the identification of one canonical FAD2 that converts oleic to linoleic acid, three divergent FAD2-like acetylenases that convert linoleic into crepenynic acid, and two bifunctional FAD2s with Δ12 and Δ14 desaturase activity that convert crepenynic into the further desaturated dehydrocrepenynic acid, a polyacetylene pathway intermediate. These genes can now be used as a basis for discovering other steps of falcarin-type polyacetylene biosynthesis, to modulate polyacetylene levels in plants, and to test the in planta function of these molecules. Many organisms implement specialized biochemical pathways to convert ubiquitous metabolites into bioactive chemical compounds. Since plants comprise the majority of the human diet, specialized plant metabolites play crucial roles not only in crop biology but also in human nutrition. Some asterids produce lipid compounds called polyacetylenes (for review, see Negri, 2015) that exhibit antifungal activity (Garrod et al., 1978; Kemp, 1978; Harding and Heale, 1980, 1981; Olsson and Svensson, 1996) and accumulate in response to fungal phytopathogen attack (De Wit and Kodde, 1981; Elgersma and Liem, 1989). These observations have led to the longstanding hypothesis that polyacetylenes are natural pesticides. These same lipid compounds exhibit cytotoxic activity against human cancer cell lines and slow tumor growth (Fujimoto and Satoh, 1988; Matsunaga et al., 1989, 1990; Cunsolo et al., 1993; Bernart et al., 1996; Kobaek-Larsen et al., 2005; Zidorn et al., 2005), making them important nutritional compounds. The major source of polyacetylenes in the human diet is carrot (Daucus carota L.). Carrot is one of the most important crop species in the Apiaceae, with rapidly increasing worldwide cultivation (Rubatzky et al., 1999; Dawid et al., 2015). The most common carrot polyacetylenes are C17 linear aliphatic compounds containing two conjugated carbon-carbon triple bonds, one or two carbon-carbon double bonds, and a diversity of additional in-chain oxygen-containing functional groups. In carrot, the most abundant of these compounds are falcarinol and falcarindiol (Dawid et al., 2015). Based on their structures, it has been hypothesized that these compounds (alias falcarin-type polyacetylenes) are derived from ubiquitous fatty acids. Indeed, biochemical investigations (Haigh et al., 1968; Bohlman, 1988), radio-chemical tracer studies (Barley et al., 1988), and the discovery of pathway intermediates (Jones et al., 1966; Kawazu et al., 1973) implicate a diversion of flux away from linolenate biosynthesis as the entry point into falcarin-type polyacetylene biosynthesis (for review, see Minto and Blacklock, 2008). The final steps of linolenate biosynthesis are the conversion of oleate to linoleate, mediated by fatty acid desaturase 2 (FAD2), and linoleate to linolenate, catalyzed by FAD3. Some plant species contain divergent forms of FAD2 that, instead of or in addition to converting oleate to linoleate, catalyze the installation of unusual in-chain functional groups such as hydroxyl groups, epoxy groups, conjugated double bonds, or carbon-carbon triple bonds into the acyl chain (Badami and Patil, 1980) and thus divert flux from linolenate production into the accumulation of unusual fatty acids. Previous work in parsley (Petroselinum crispum; Apiaceae) identified a divergent form of FAD2 that (1) was up-regulated in response to pathogen treatment and (2) when expressed in soybean embryos resulted in production of the monoyne crepenynate and, by the action of an unassigned enzyme, dehydrocrepenynate (Kirsch et al., 1997; Cahoon et al., 2003). The results of the parsley studies are consistent with a pathogen-responsive, divergent FAD2-mediated pathway that leads to acetylenic fatty acids. However, information regarding the branch point into acetylenic fatty acid production in agriculturally relevant carrot is still largely missing, in particular, the identification and functional characterization of enzymes that can divert carbon flux away from linolenate biosynthesis into the production of dehydrocrepenynate and ultimately falcarin-type polyacetylenes. Such genes, once identified, could be used in the future design of transgenic carrot lines with altered polyacetylene content, enabling direct testing of in planta polyacetylene function and potentially the engineering of pathogen-resistant, more nutritious carrots. These genes could also provide the foundation for further investigations of more basic aspects of plant biology, including the evolution of fatty acid-derived natural product biosynthesis pathways across the Asterid clade, as well as the role of these pathways and compounds in plant ecology and plant defense. Recently, a high-quality carrot genome assembly was released (Iorizzo et al., 2016), providing a foundation for genome-enabled studies of Apiaceous species. This study also provided publicly accessible RNA sequencing (RNA-Seq) data from diverse carrot tissues. Using these resources, this study aimed to provide a detailed gas chromatography-based quantification of polyacetylenes in carrot tissues for which RNA-Seq data are available, then combine this information with bioinformatics analysis and heterologous expression to identify and characterize biosynthetic genes that underlie the major entry point into carrot polyacetylene biosynthesis. To achieve these goals, thin-layer chromatography (TLC) was combined with gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detection to identify and quantify polyacetylenic metabolites in five different carrot tissues. Then the sequences and tissue expression profiles of potential FAD2 and FAD2-like genes annotated in the D. carota genome were compared with the metabolite data to identify candidate pathway genes, followed by biochemical functionality tests using yeast (Saccharomyces cerevisae) and Arabidopsis (Arabidopsis thaliana) as heterologous expression systems.« less
5. Deletion of maize RDM4 suggests a role in endosperm maturation as well as vegetative and stress-responsive growth

   https://doi.org/10.1093/jxb/eraa325  

   Jia, Shangang ; Yobi, Abou ; Naldrett, Michael J ; Alvarez, Sophie ; Angelovici, Ruthie ; Zhang, Chi ; Holding, David R ( July 2020 , Journal of Experimental Botany)

   Braeutigam, Andrea (Ed.)

   Abstract Opaque kernels in maize may result from mutations in many genes, such as OPAQUE-2. In this study, a maize null mutant of RNA-DIRECTED DNA METHYLATION 4 (RDM4) showed an opaque kernel phenotype, as well as plant developmental delay, male sterility, and altered response to cold stress. We found that in opaque kernels, all zein proteins were reduced and amino acid content was changed, including increased lysine. Transcriptomic and proteomic analysis confirmed the zein reduction and proteomic rebalancing of non-zein proteins, which was quantitatively and qualitatively different from opaque-2. Global transcriptional changes were found in endosperm and leaf, including many transcription factors and tissue-specific expressed genes. Furthermore, of the more than 8000 significantly differentially expressed genes in wild type in response to cold, a significant proportion (25.9% in moderate cold stress and 40.8% in near freezing stress) were not differentially expressed in response to cold in rdm4, suggesting RDM4 may participate in regulation of abiotic stress tolerance. This initial characterization of maize RDM4 provides a basis for further investigating its function in endosperm and leaf, and as a regulator of normal and stress-responsive development.