Arabidopsis PROTODERMAL FACTOR2 binds lysophosphatidylcholines and transcriptionally regulates phospholipid metabolism.

Plant homeodomain leucine zipper IV (HD-Zip IV) transcription factors (TFs) contain an evolutionarily conserved steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. While the START domain is required for TF activity, its presumed role as a lipid sensor is not clear. Here we used tandem affinity purification from Arabidopsis cell cultures to demonstrate that PROTODERMAL FACTOR2 (PDF2), a representative member that controls epidermal differentiation, recruits lysophosphatidylcholines (LysoPCs) in a START-dependent manner. Microscale thermophoresis assays confirmed that a missense mutation in a predicted ligand contact site reduces lysophospholipid binding. We additionally found that PDF2 acts as a transcriptional regulator of phospholipid- and phosphate (Pi) starvation-related genes and binds to a palindromic octamer with consensus to a Pi response element. Phospholipid homeostasis and elongation growth were altered in pdf2 mutants according to Pi availability. Cycloheximide chase experiments revealed a role for START in maintaining protein levels, and Pi starvation resulted in enhanced protein destabilization, suggesting a mechanism by which lipid binding controls TF activity. We propose that the START domain serves as a molecular sensor for membrane phospholipid status in the epidermis. Our data provide insights toward understanding how the lipid metabolome integrates Pi availability with gene expression.

Nuclear localization of Arabidopsis HD-Zip IV transcription factor GLABRA is driven by Importin α.

GLABRA2 (GL2), a class IV homeodomain leucine-zipper (HD-Zip IV) transcription factor (TF) from Arabidopsis, is a developmental regulator of specialized cell types in the epidermis. GL2 contains a monopartite nuclear localization sequence (NLS) that is conserved in most HD-Zip IV members across the plants. We demonstrate that NLS mutations affect nuclear transport and result in a loss-of-function phenotypes. NLS fusions to EYFP show that it is sufficient for nuclear localization in roots and trichomes. Despite partial overlap of the NLS with the homeodomain, genetic dissection indicates that nuclear localization and DNA binding are separable functions. Affinity purification of GL2 from plants followed by MS-based proteomics identified Importin α (IMPα) isoforms as potential GL2 interactors. NLS structural prediction and molecular docking studies with IMPα-3 revealed major interacting residues. Cytosolic yeast two-hybrid assays and co-immunoprecipitation experiments with recombinant proteins verified NLS-dependent interactions between GL2 and several IMPα isoforms. IMPα triple mutants (impα-1,2,3) exhibit abnormal trichome formation and defects in GL2 nuclear localization in trichomes, consistent tissue-specific and redundant functions of IMPα isoforms. Taken together, our findings provide mechanistic evidence for IMPα-dependent nuclear localization of GL2 in Arabidopsis, a process that is critical for cell-type differentiation of the epidermis.

Nuclear localization of HD-Zip IV transcription factor GLABRA2 is driven by Importin α.

GLABRA2 (GL2), a class IV homeodomain leucine-zipper (HD-Zip IV) transcription factor (TF) from Arabidopsis , is a developmental regulator of specialized cell types in the epidermis. GL2 contains a putative monopartite nuclear localization sequence (NLS) partially overlapping with its homeodomain (HD). We demonstrate that NLS deletion or alanine substitution of its basic residues (KRKRKK) affects nuclear localization and results in a loss-of-function phenotype. Fusion of the predicted NLS (GTNKRKRKKYHRH) to the fluorescent protein EYFP is sufficient for its nuclear localization in roots and trichomes. The functional NLS is evolutionarily conserved in a distinct subset of HD-Zip IV members including PROTODERMAL FACTOR2 (PDF2). Despite partial overlap of the NLS with the HD, genetic dissection of the NLS from PDF2 indicates that nuclear localization and DNA binding are separable functions. Affinity purification of GL2 from plant tissues followed by mass spectrometry-based proteomics identified Importin α (IMPα) isoforms as potential GL2 interactors. NLS structural prediction and molecular docking studies with IMPα-3 revealed major interacting residues. Split-ubiquitin cytosolic yeast two-hybrid assays suggest interaction between GL2 and four IMPα isoforms from Arabidopsis. Direct interactions were verified in vitro by co-immunoprecipitation with recombinant proteins. IMPα triple mutants ( impα- 1,2,3 ) exhibit defects in EYFP:GL2 nuclear localization in trichomes but not in roots, consistent with tissue-specific and redundant functions of IMPα isoforms in Arabidopsis . Taken together, our findings provide mechanistic evidence for IMPα-dependent nuclear localization of GL2 and other HD-Zip IV TFs in plants. One sentence summary: GLABRA2, a representative HD-Zip IV transcription factor from Arabidopsis , contains an evolutionarily conserved monopartite nuclear localization sequence that is recognized by Importin α for translocation to the nucleus, a process that is necessary for cell-type differentiation of the epidermis.

Metabolic synergy in Camelina reproductive tissues for seed development.

Photosynthesis in fruits is well documented, but its contribution to seed development and yield remains largely unquantified. In oilseeds, the pods are green and elevated with direct access to sunlight. With 13C labeling in planta and through an intact pod labeling system, a unique multi-tissue comprehensive flux model mechanistically described how pods assimilate up to one-half (33 to 45%) of seed carbon by proximal photosynthesis in Camelina sativa. By capturing integrated tissue metabolism, the studies reveal the contribution of plant architecture beyond leaves, to enable seed filling and maximize the number of viable seeds. The latent capacity of the pod wall in the absence of leaves contributes approximately 79% of seed biomass, supporting greater seed sink capacity and higher theoretical yields that suggest an opportunity for crop productivity gains.

The START domain mediates Arabidopsis GLABRA2 dimerization and turnover independently of homeodomain DNA binding.

Class IV homeodomain leucine-zipper transcription factors (HD-Zip IV TFs) are key regulators of epidermal differentiation that are characterized by a DNA-binding HD in conjunction with a lipid-binding domain termed steroidogenic acute regulatory-related lipid transfer (START). Previous work established that the START domain of GLABRA2 (GL2), a HD-Zip IV member from Arabidopsis (Arabidopsis thaliana), is required for TF activity. Here, we addressed the functions and possible interactions of START and the HD in DNA binding, dimerization, and protein turnover. Deletion analysis of the HD and missense mutations of a conserved lysine (K146) resulted in phenotypic defects in leaf trichomes, root hairs, and seed mucilage, similar to those observed for START domain mutants, despite nuclear localization of the respective proteins. In vitro and in vivo experiments demonstrated that while HD mutations impair binding to target DNA, the START domain is dispensable for DNA binding. Vice versa, protein interaction assays revealed impaired GL2 dimerization for multiple alleles of START mutants, but not HD mutants. Using in vivo cycloheximide chase experiments, we provided evidence for the role of START, but not HD, in maintaining protein stability. This work advances our mechanistic understanding of HD-Zip TFs as multidomain regulators of epidermal development in plants.

Suppression of SDP1 Improves Soybean Seed Composition by Increasing Oil and Reducing Undigestible Oligosaccharides.

In developing soybean seeds, carbon is partitioned between oil, protein and carbohydrates. Here, we demonstrate that suppression of lipase-mediated turnover of triacylglycerols (TAG) during late seed development increases fatty acid content and decreases the presence of undigestible oligosaccharides. During late stages of embryo development, the fatty acid content of soybean seed decreases while the levels of the oligosaccharides raffinose and stachyose increase. Three soybean genes orthologous to the Arabidopsis lipase gene SUGAR-DEPENDENT1 (SDP1) are upregulated at this time. Suppression of these genes resulted in higher oil levels, with lipid levels in the best lines exceeding 24% of seed weight. In addition, lipase-suppressed lines produced larger seeds compared to wild-type plants, resulting in increases of over 20% in total lipid per seed. Levels of raffinose and stachyose were lower in the transgenic lines, with average reductions of 15% in total raffinose family oligosaccharides observed. Despite the increase in oil, protein content was not negatively impacted and trended higher in the transgenic lines. These results are consistent with a role for SDP1 in turning over TAG to supply carbon for other needs, including the synthesis of oligosaccharides, and offer new strategies to further improve the composition of soybean seeds.

Making glue from seeds and gums: Working with plant-based polymers to introduce students to plant biochemistry.

Plants and plant products are key to the survival of life on earth. Despite this fact, the significance of plant biochemistry is often underrepresented in science curricula. We designed an innovative laboratory activity to engage students in learning about the biochemical properties of natural polymers produced by plants. The focus of the hands-on activity is on mucilages and gums, which contain complex polysaccharides that have applications in industry. The 1.5-h activity is organized into three laboratory exercises. It begins with a demonstration of the water absorption property of seed coat mucilage upon hydration of seeds from psyllium, a plant that is grown commercially for mucilage production. The second exercise involves microscopy of a variety of plant seeds stained with ruthenium red dye to visualize pectin polysaccharides of the seed mucilage. Students learn about phenotypic variation among plant species and how the seed coat mucilage is beneficial to keep seeds hydrated during germination. The third exercise highlights an industrial application of plant gums as adhesives. The students prepare edible glue made with gum arabic, a type of plant polymer from the dried exudate of the Acacia plant. This three-part activity has been implemented in conjunction with a Girls Researching Our World (GROW) summer workshop for sixth to eighth graders over a 4-year period. It may be adapted as a laboratory activity for students of all ages, for example, to enhance biochemistry education for high-school students or undergraduate non-majors. © 2019 International Union of Biochemistry and Molecular Biology, 47(4):468-475, 2019.

Bioorthogonal click chemistry for fluorescence imaging of choline phospholipids in plants.

BACKGROUND: Phospholipids are important structural and signaling molecules in plant membranes. Some fluorescent dyes can stain general lipids of membranes, but labeling and visualization of specific lipid classes have yet to be developed for most components of the membrane. New techniques for visualizing membrane lipids are needed to further delineate their dynamic structural and signaling roles in plant cells. In this study we examined whether propargylcholine, a bioortholog of choline, can be used to label the major membrane lipid, phosphatidylcholine, and other choline phospholipids in plants. We established that propargylcholine is readily taken up by roots, and that its incorporation is not detrimental to plant growth. After plant tissue is harvested and fixed, a click-chemistry reaction covalently links the alkyne group of propargylcholine to a fluorescently-tagged azide, resulting in specific labeling of choline phospholipids. RESULTS: Uptake of propargylcholine, followed by click chemistry with fluorescein or Alexa Fluor 594 azide was used to visualize choline phospholipids in cells of root, leaf, stem, silique and seed tissues from Arabidopsis thaliana. Co-localization with various subcellular markers indicated coinciding fluorescent signals in cell membranes, such as the tonoplast and the ER. Among different cell types in the leaf epidermis, guard cells displayed strong labeling. Mass spectrometry-based lipidomic analysis of the various plant tissues revealed that incorporation of propargylcholine was strongest in roots with approximately 50% of total choline phospholipids being labeled, but it was also incorporated in the other tissues including seeds. Phospholipid profiling confirmed that, in each tissue analyzed, incorporation of the bioortholog had little impact on the pool of choline plus choline-like phospholipids or other lipid species. CONCLUSION: We developed and validated a click-chemistry based method for fluorescence imaging of choline phospholipids using a bioortholog of choline, propargylcholine, in various cell-types and tissues from Arabidopsis. This click-chemistry method provides a direct way to metabolically tag and visualize specific lipid molecules in plant cells. This work paves the way for future studies addressing in situ localization of specific lipids in plants.

A four-gene operon in Bacillus cereus produces two rare spore-decorating sugars.

Bacterial glycan structures on cell surfaces are critical for cell-cell recognition and adhesion and in host-pathogen interactions. Accordingly, unraveling the sugar composition of bacterial cell surfaces can shed light on bacterial growth and pathogenesis. Here, we found that two rare sugars with a 3-C-methyl-6-deoxyhexose structure were linked to spore glycans in Bacillus cereus ATCC 14579 and ATCC 10876. Moreover, we identified a four-gene operon in B. cereus ATCC 14579 that encodes proteins with the following sequential enzyme activities as determined by mass spectrometry and one- and two-dimensional NMR methods: CTP:glucose-1-phosphate cytidylyltransferase, CDP-Glc 4,6-dehydratase, NADH-dependent SAM:C-methyltransferase, and NADPH-dependent CDP-3-C-methyl-6-deoxyhexose 4-reductase. The last enzyme predominantly yielded CDP-3-C-methyl-6-deoxygulose (CDP-cereose) and likely generated a 4-epimer CDP-3-C-methyl-6-deoxyallose (CDP-cillose). Some members of the B. cereus sensu lato group produce CDP-3-C-methyl-6-deoxy sugars for the formation of cereose-containing glycans on spores, whereas others such as Bacillus anthracis do not. Gene knockouts of the Bacillus C-methyltransferase and the 4-reductase confirmed their involvement in the formation of cereose-containing glycan on B. cereus spores. We also found that cereose represented 0.2-1% spore dry weight. Moreover, mutants lacking cereose germinated faster than the wild type, yet the mutants exhibited no changes in sporulation or spore resistance to heat. The findings reported here may provide new insights into the roles of the uncommon 3-C-methyl-6-deoxy sugars in cell-surface recognition and host-pathogen interactions of the genus Bacillus.

Constitutively overexpressing a tomato fructokinase gene (LeFRK1) in cotton (Gossypium hirsutum L. cv. Coker 312) positively affects plant vegetative growth, boll number and seed cotton yield.

Increasing fructokinase (FRK) activity in cotton (Gossypium hirsutum L.) plants may reduce fructose inhibition of sucrose synthase (Sus) and lead to improved fibre yield and quality. Cotton was transformed with a tomato (Solanum lycopersicum L.) fructokinase gene (LeFRK1) under the control of the CMV 35S promoter. In a greenhouse, the LeFRK1 plants had increased fibre and leaf FRK activity over nonexpressing nulls, but not improved fibre length and strength. Compared with the nulls, LeFRK1 plants yielded 13-100% more seed-cotton mass per boll and more bolls per plant, and therefore more seed cotton and fibre yield per plant. The enhanced yield was related to a greater seed number per boll for LeFRK1 plants. Photosynthetic rates were not appreciably different among genotypes. However, more area per leaf and leaf number (in some instances) for LeFRK1 plants than for nulls enhanced the capacity for C gain. Larger leaf areas for LeFRK1 plants were associated with larger stem diameters. Lower sucrose levels in developing leaves of LeFRK1 plants suggest that LeFRK1 overexpression leads to improved in vivo Sus activity in developing leaves and possibly in developing seeds. The improvement in yield for LeFRK1 plants may also be the result of improvements in photosynthate supply as a consequence of greater leaf area.