Dual phosphorylation of DGK5-mediated PA burst regulates ROS in plant immunity.

Phosphatidic acid (PA) and reactive oxygen species (ROS) are crucial cellular messengers mediating diverse signaling processes in metazoans and plants. How PA homeostasis is tightly regulated and intertwined with ROS signaling upon immune elicitation remains elusive. We report here that Arabidopsis diacylglycerol kinase 5 (DGK5) regulates plant pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). The pattern recognition receptor (PRR)-associated kinase BIK1 phosphorylates DGK5 at Ser-506, leading to a rapid PA burst and activation of plant immunity, whereas PRR-activated intracellular MPK4 phosphorylates DGK5 at Thr-446, which subsequently suppresses DGK5 activity and PA production, resulting in attenuated plant immunity. PA binds and stabilizes the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), regulating ROS production in plant PTI and ETI, and their potentiation. Our data indicate that distinct phosphorylation of DGK5 by PRR-activated BIK1 and MPK4 balances the homeostasis of cellular PA burst that regulates ROS generation in coordinating two branches of plant immunity.

An unexpected role for tomato threonine deaminase 2 in host defense against bacterial infection.

The hormones salicylic acid (SA) and jasmonic acid (JA) often act antagonistically in controlling plant defense pathways in response to hemibiotrophs/biotrophs (hemi/biotroph) and herbivores/necrotrophs, respectively. Threonine deaminase (TD) converts threonine to α-ketobutyrate and ammonia as the committed step in isoleucine (Ile) biosynthesis and contributes to JA responses by producing the Ile needed to make the bioactive JA-Ile conjugate. Tomato (Solanum lycopersicum) plants have two TD genes: TD1 and TD2. A defensive role for TD2 against herbivores has been characterized in relation to JA-Ile production. However, it remains unknown whether TD2 is also involved in host defense against bacterial hemi/biotrophic and necrotrophic pathogens. Here, we show that in response to the bacterial pathogen-associated molecular pattern (PAMP) flagellin flg22 peptide, an activator of SA-based defense responses, TD2 activity is compromised, possibly through carboxy-terminal cleavage. TD2 knockdown (KD) plants showed increased resistance to the hemibiotrophic bacterial pathogen Pseudomonas syringae but were more susceptible to the necrotrophic fungal pathogen Botrytis cinerea, suggesting TD2 plays opposite roles in response to hemibiotrophic and necrotrophic pathogens. This TD2 KD plant differential response to different pathogens is consistent with SA- and JA-regulated defense gene expression. flg22-treated TD2 KD plants showed high expression levels of SA-responsive genes, whereas TD2 KD plants treated with the fungal PAMP chitin showed low expression levels of JA-responsive genes. This study indicates TD2 acts negatively in defense against hemibiotrophs and positively against necrotrophs and provides insight into a new TD2 function in the elaborate crosstalk between SA and JA signaling induced by pathogen infection.

In Silico and Cellular Differences Related to the Cell Division Process between the A and B Races of the Colonial Microalga Botryococcus braunii.

Botryococcus braunii produce liquid hydrocarbons able to be processed into combustion engine fuels. Depending on the growing conditions, the cell doubling time can be up to 6 days or more, which is a slow growth rate in comparison with other microalgae. Few studies have analyzed the cell cycle of B. braunii. We did a bioinformatic comparison between the protein sequences for retinoblastoma and cyclin-dependent kinases from the A (Yamanaka) and B (Showa) races, with those sequences from other algae and Arabidopsis thaliana. Differences in the number of cyclin-dependent kinases and potential retinoblastoma phosphorylation sites between the A and B races were found. Some cyclin-dependent kinases from both races seemed to be phylogenetically more similar to A. thaliana than to other microalgae. Microscopic observations were done using several staining procedures. Race A colonies, but not race B, showed some multinucleated cells without chlorophyll. An active mitochondrial net was detected in those multinucleated cells, as well as being defined in polyphosphate bodies. These observations suggest differences in the cell division processes between the A and B races of B. braunii.

Screening for potential nuclear substrates for the plant cell death suppressor kinase Adi3 using peptide microarrays.

The tomato AGC protein kinase Adi3 is a Ser/Thr kinase that functions as a negative regulator of programmed cell death through cell death suppression (CDS) activity in the nucleus. In this study, to understand the mechanism of Adi3 CDS, peptide microarrays containing random Ser- and Thr-peptide phosphorylation substrates were used to screen for downstream phosphorylation substrates. In the microarray phosphorylation assay, Adi3 showed promiscuous kinase activity more toward Ser-peptides compared to Thr-peptides, and a preference for aromatic and cyclic amino acids on both Ser- and Thr-peptides was seen. The 63 highest phosphorylated peptide sequences from the Ser-peptide microarray were selected as queries for a BLAST search against the tomato proteome. As a result, 294 candidate nuclear Adi3 substrates were selected and categorized based on their functions. Many of these proteins were classified as DNA/RNA polymerases or regulators involved in transcription and translation events. The list of potential Adi3 substrates was narrowed to eleven and four candidates were tested for phosphorylation by Adi3. Two of these candidates, RNA polymerase II 2nd largest subunit (RPB2) and the pathogen defense related transcription factor Pti5, were confirmed as Adi3 phosphorylation substrates by in vitro kinase assays. Using a mutational approach two residues, Thr675 and Thr676, were identified as Adi3 phosphorylation sites on RPB2. This study provides the foundation for understanding Adi3 CDS mechanisms in the nucleus as well as other cellular functions.

In vitro activity characterization of the tomato SnRK1 complex proteins.

Plant Sucrose non-Fermenting 1-Related Protein Kinase1 (SnRK1) complexes are members of the Snf1/AMPK/SnRK protein kinase family and play important roles in many aspects of metabolism. In tomato (Solanum lycopersicum, Sl), only one α-subunit of the SnRK1 complex, SlSnRK1.1, has been characterized to date. In this study, the phylogenetic placement and in vitro kinase activity of a second tomato SnRK1 α-subunit, SlSnRK1.2, were characterized. Interestingly, in the phylogenetic analysis of SnRK1 sequences from monocots and dicots SlSnRK1.2 clusters only with other Solanaceae SnRK1.2 sequences, suggesting possible functional divergence of these kinases from other SnRK1 kinases. For analysis of kinase activity, SlSnRK1.2 was able to autophosphorylate, phosphorylate the complex ß-subunits, and phosphorylate the SnRK1 AMARA peptide substrate, all with drastically lower overall kinase activity compared to SlSnRK1.1. Activation by the upstream kinase SlSnAK was able to increase the kinase activity of both SlSnRK1.1 and SlSnRK1.2, although the increase is less dramatic for SlSnRK1.2. The highest kinase activity on the AMARA peptide for SlSnRK1.2 was seen when reconstituting the complex in vitro with SlSip1 as the ß-subunit. In comparison, SlSnRK1.1 showed the lowest kinase activity on the AMARA peptide when SlSip1 was used. These studies suggest the SlSnRK1.2 phylogenetic divergence and lower SlSnRK1.2 kinase activity compared to SlSnRK1.1 may be indicative of different in vivo roles for each kinase.

Tetraterpene Synthase Substrate and Product Specificity in the Green Microalga Botryococcus braunii Race L.

Recently, the biosynthetic pathway for lycopadiene, a C40 tetraterpenoid hydrocarbon, was deciphered from the L race of Botryococcus braunii, an alga that produces hydrocarbon oils capable of being converted into combustible fuels. The lycopadiene pathway is initiated by the squalene synthase (SS)-like enzyme lycopaoctaene synthase (LOS), which catalyzes the head-to-head condensation of two C20 geranylgeranyl diphosphate (GGPP) molecules to produce C40 lycopaoctaene. LOS shows unusual substrate promiscuity for SS or SS-like enzymes by utilizing C15 farnesyl diphosphate (FPP) and C20 phytyl diphosphate in addition to GGPP as substrates. These three substrates can be combined by LOS individually or in combinations to produce six different hydrocarbons of C30, C35, and C40 chain lengths. To understand LOS substrate and product specificity, rational mutagenesis experiments were conducted based on sequence alignment with several SS proteins as well as a structural comparison with the human SS (HSS) crystal structure. Characterization of the LOS mutants in vitro identified Ser276 and Ala288 in the LOS active site as key amino acids responsible for controlling substrate binding, and thus the promiscuity of this enzyme. Mutating these residues to those found in HSS largely converted LOS from lycopaoctaene production to C30 squalene production. Furthermore, these studies were confirmed in vivo by expressing LOS in E. coli cells metabolically engineered to produce high FPP and GGPP levels. These studies also offer insights into tetraterpene hydrocarbon metabolism in B. braunii and provide a foundation for engineering LOS for robust production of specific hydrocarbons of a desired chain length.

Draft Nuclear Genome Sequence of the Liquid Hydrocarbon-Accumulating Green Microalga Botryococcus braunii Race B (Showa).

Botryococcus braunii has long been known as a prodigious producer of liquid hydrocarbon oils that can be converted into combustion engine fuels. This draft genome for the B race of B. braunii will allow researchers to unravel important hydrocarbon biosynthetic pathways and identify possible regulatory networks controlling this unusual metabolism.

Isolation and Characterization of Cyclic C33 Botryococcenes and a Trimethylsqualene Isomer from Botryococcus braunii Race B.

Three cyclic C33 botryococcenes and one new trimethylsqualene isomer were isolated from the B race, Showa (Berkeley) strain of Botryococcus braunii, which is known to produce large amounts of isoprenoid hydrocarbons ranging in carbon number from 30 to 34. Their purity was determined by GC-MS, and structures were characterized by 1D and 2D NMR. One of these molecules, cyclic C33-1 botryococcene (5), has an unusual connection of a methylenecyclohexane ring to the molecule backbone not seen before in botryococcenes. This report further adds to our knowledge of the wide range of isoprenoid hydrocarbon structures produced by B. braunii.

Raman spectroscopy compatible PDMS droplet microfluidic culture and analysis platform towards on-chip lipidomics.

Lipids produced by microalgae are viewed as a potential renewable alternative to fossil fuels, however, significant improvements in productivity are required for microalgal biofuels to become economically feasible. Here we present a method that allows for the use of Raman spectroscopy with poly(dimethylsiloxane) (PDMS) droplet microfluidic devices, which not only overcomes the high Raman background of PDMS, but also achieves pairing of the high-throughput single-cell resolution advantages of droplet microfluidics with the direct, chemically specific, label-free, and non-destructive nature of Raman spectroscopy. The platform was successfully utilized for in situ characterization of microalgal lipid production over time within droplets, paving the way towards high-throughput microalgal lipidomics assays.

High-throughput droplet microfluidics screening platform for selecting fast-growing and high lipid-producing microalgae from a mutant library.

Biofuels derived from microalgal lipids have demonstrated a promising potential as future renewable bioenergy. However, the production costs for microalgae-based biofuels are not economically competitive, and one strategy to overcome this limitation is to develop better-performing microalgal strains that have faster growth and higher lipid content through genetic screening and metabolic engineering. In this work, we present a high-throughput droplet microfluidics-based screening platform capable of analyzing growth and lipid content in populations derived from single cells of a randomly mutated microalgal library to identify and sort variants that exhibit the desired traits such as higher growth rate and increased lipid content. By encapsulating single cells into water-in-oil emulsion droplets, each variant was separately cultured inside an individual droplet that functioned as an independent bioreactor. In conjunction with an on-chip fluorescent lipid staining process within droplets, microalgal growth and lipid content were characterized by measuring chlorophyll and BODIPY fluorescence intensities through an integrated optical detection system in a flow-through manner. Droplets containing cells with higher growth and lipid content were selectively retrieved and further analyzed off-chip. The growth and lipid content screening capabilities of the developed platform were successfully demonstrated by first carrying out proof-of-concept screening using known Chlamydomonas reinhardtii mutants. The platform was then utilized to screen an ethyl methanesulfonate (EMS)-mutated C. reinhardtii population, where eight potential mutants showing faster growth and higher lipid content were selected from 200,000 examined samples, demonstrating the capability of the platform as a high-throughput screening tool for microalgal biofuel development.

ROS Detection in Botryococcus braunii Colonies with CellROX Green Reagent.

We analyzed the reactive oxygen species (ROS) accumulation in the colony-forming green microalga Botryococcus braunii in response to several stress inducers such as NaCl, NaHCO3, salicylic acid (SA), methyl jasmonate, and acetic acid. A staining assay using the fluorescent dye CellROX Green was used. CellROX Green is a fluorogenic probe used for measuring oxidative stress in live cells. The dye is weakly fluorescent inside cells in a reduced state but exhibits bright green photostable fluorescence upon oxidation by ROS and subsequent binding to DNA. The large amount of liquid hydrocarbons produced and excreted by B. braunii, creates a highly hydrophobic extracellular environment that makes difficult to study short times defense responses on this microalga. The procedure developed here allowed us to detect ROS in this microalga even within a short period of time (in minutes) after treatment of cells with different stress inducers.

Stress responses of the oil-producing green microalga Botryococcus braunii Race B.

Plants react to biotic and abiotic stresses with a variety of responses including the production of reactive oxygen species (ROS), which may result in programmed cell death (PCD). The mechanisms underlying ROS production and PCD have not been well studied in microalgae. Here, we analyzed ROS accumulation, biomass accumulation, and hydrocarbon production in the colony-forming green microalga Botryococcus braunii in response to several stress inducers such as NaCl, NaHCO3, salicylic acid (SA), methyl jasmonate, and acetic acid. We also identified and cloned a single cDNA for the B. braunii ortholog of the Arabidopsis gene defender against cell death 1 (DAD1), a gene that is directly involved in PCD regulation. The function of B. braunii DAD1 was assessed by a complementation assay of the yeast knockout line of the DAD1 ortholog, oligosaccharyl transferase 2. Additionally, we found that DAD1 transcription was induced in response to SA at short times. These results suggest that B. braunii responds to stresses by mechanisms similar to those in land plants and other  organisms.

A squalene synthase-like enzyme initiates production of tetraterpenoid hydrocarbons in Botryococcus braunii Race L.

The green microalga Botryococcus braunii is considered a promising biofuel feedstock producer due to its prodigious accumulation of hydrocarbon oils that can be converted into fuels. B. braunii Race L produces the C40 tetraterpenoid hydrocarbon lycopadiene via an uncharacterized biosynthetic pathway. Structural similarities suggest this pathway follows a biosynthetic mechanism analogous to that of C30 squalene. Confirming this hypothesis, the current study identifies C20 geranylgeranyl diphosphate (GGPP) as a precursor for lycopaoctaene biosynthesis, the first committed intermediate in the production of lycopadiene. Two squalene synthase (SS)-like complementary DNAs are identified in race L with one encoding a true SS and the other encoding an enzyme with lycopaoctaene synthase (LOS) activity. Interestingly, LOS uses alternative C15 and C20 prenyl diphosphate substrates to produce combinatorial hybrid hydrocarbons, but almost exclusively uses GGPP in vivo. This discovery highlights how SS enzyme diversification results in the production of specialized tetraterpenoid oils in race L of B. braunii.

The ubiquitin ligase SEVEN IN ABSENTIA (SINA) ubiquitinates a defense-related NAC transcription factor and is involved in defense signaling.

We recently identified a defense-related tomato (Solanum lycopersicum) NAC (NAM, ATAF1,2, CUC2) transcription factor, NAC1, that is subjected to ubiquitin-proteasome system-dependent degradation in plant cells. In this study, we identified a tomato ubiquitin ligase (termed SEVEN IN ABSENTIA3; SINA3) that ubiquitinates NAC1, promoting its degradation. We conducted coimmunoprecipitation and bimolecular fluorescence complementation to determine that SINA3 specifically interacts with the NAC1 transcription factor in the nucleus. Moreover, we found that SINA3 ubiquitinates NAC1 in vitro and promotes NAC1 degradation via polyubiquitination in vivo, indicating that SINA3 is a ubiquitin ligase that ubiquitinates NAC1, promoting its degradation. Our real-time PCR analysis indicated that, in contrast to our previous finding that NAC1 mRNA abundance increases upon Pseudomonas infection, the SINA3 mRNA abundance decreases in response to Pseudomonas infection. Moreover, using Agrobacterium-mediated transient expression, we found that overexpression of SINA3 interferes with the hypersensitive response cell death triggered by multiple plant resistance proteins. These results suggest that SINA3 ubiquitinates a defense-related NAC transcription factor for degradation and plays a negative role in defense signaling.

A droplet microfluidics platform for rapid microalgal growth and oil production analysis.

Microalgae have emerged as a promising source for producing future renewable biofuels. Developing better microalgal strains with faster growth and higher oil production rates is one of the major routes towards economically viable microalgal biofuel production. In this work, we present a droplet microfluidics-based microalgae analysis platform capable of measuring growth and oil content of various microalgal strains with single-cell resolution in a high-throughput manner. The platform allows for encapsulating a single microalgal cell into a water-in-oil emulsion droplet and tracking the growth and division of the encapsulated cell over time, followed by on-chip oil quantification. The key feature of the developed platform is its capability to fluorescently stain microalgae within microdroplets for oil content quantification. The performance of the developed platform was characterized using the unicellular microalga Chlamydomonas reinhardtii and the colonial microalga Botryococcus braunii. The application of the platform in quantifying growth and oil accumulation was successfully confirmed using C. reinhardtii under different culture conditions, namely nitrogen-replete and nitrogen-limited conditions. These results demonstrate the capability of this platform as a rapid screening tool that can be applied to a wide range of microalgal strains for analyzing growth and oil accumulation characteristics relevant to biofuel strain selection and development. Biotechnol. Bioeng. 2016;113: 1691-1701. © 2016 Wiley Periodicals, Inc.

A high-throughput microfluidic single-cell screening platform capable of selective cell extraction.

Microfluidic devices and lab-on-a-chip technologies have been extensively used in high-throughput single-cell analysis applications using their capability to precisely manipulate cells as well as their microenvironment. Although significant technological advances have been made in single-cell capture, culture, and analysis techniques, most microfluidic systems cannot selectively retrieve samples off-chip for additional examinations. Being able to retrieve target cells of interest from large arrays of single-cell culture compartments is especially critical in achieving high-throughput single-cell screening applications, such as a mutant library screening. We present a high-throughput microfluidic single-cell screening platform capable of investigating cell properties, such as growth and biomolecule production, followed by selective extraction of particular cells showing desired traits to off-chip reservoirs for sampling or further analysis. The developed platform consists of 1024 single-cell trapping/culturing sites, where opening and closing of each trap can be individually controlled with a microfluidic OR logic gate. By opening only a specific site out of the 1024 trapping sites and applying backflow, particular cells of interest could be selectively released and collected off-chip. Using a unicellular microalga Chlamydomonas reinhardtii, single-cell capture and selective cell extraction capabilities of the developed platform were successfully demonstrated. The growth profile and intracellular lipid accumulation of the cells were also analyzed inside the platform, where 6-8 hours of doubling time and on-chip stained lipid bodies were successfully identified, demonstrating the compatibility of the system for cell culture and fluorescent tagging assays.

The tomato cell death suppressor Adi3 is restricted to the endosomal system in response to the Pseudomonas syringae effector protein AvrPto.

The tomato (Solanum lycopersicum) AGC protein kinase Adi3 functions as a suppressor of cell death and was first identified as an interactor with the tomato resistance protein Pto and the Pseudomonas syringae effector protein AvrPto. Models predict that loss of Adi3 cell death suppression (CDS) activity during Pto/AvrPto interaction leads to the cell death associated with the resistance response initiated from this interaction. Nuclear localization is required for Adi3 CDS. Prevention of nuclear accumulation eliminates Adi3 CDS and induces cell death by localizing Adi3 to intracellular punctate membrane structures. Here we use several markers of the endomembrane system to show that the punctate membrane structures to which non-nuclear Adi3 is localized are endosomal in nature. Wild-type Adi3 also localizes in these punctate endosomal structures. This was confirmed by the use of endosomal trafficking inhibitors, which were capable of trapping wild-type Adi3 in endosomal-like structures similar to the non-nuclear Adi3. This suggests Adi3 may traffic through the cell using the endomembrane system. Additionally, Adi3 was no longer found in the nucleus but was visualized in these punctate endosomal-like membranes during the cell death induced by the Pto/AvrPto interaction. Therefore we propose that inhibiting nuclear import and constraining Adi3 to the endosomal system in response to AvrPto is a mechanism to initiate the cell death associated with resistance.

A microfluidic photobioreactor array demonstrating high-throughput screening for microalgal oil production.

Microalgae are envisioned as a future source of renewable oil. The feasibility of producing high-value biomolecules from microalgae is strongly dependent on developing strains with increased productivity and environmental tolerance, understanding algal gene regulation, and optimizing growth conditions for higher production of target molecules. We present a high-throughput microfluidic microalgal photobioreactor array capable of applying 64 different light conditions to arrays of microscale algal photobioreactors and apply this device to investigate how light conditions influence algal growth and oil production. Using the green colony-forming microalga Botryococcus braunii, the light intensity and light-dark cycle conditions were identified that induced 1.8-fold higher oil accumulation over the typically used culture conditions. Additionally, the studies revealed that the condition under which maximum oil production occurs is significantly different from that of maximum growth. This screening test was accomplished using the developed photobioreactor array at 250 times higher throughput compared to conventional flask-scale photobioreactors.

Two Pdk1 phosphorylation sites on the plant cell death suppressor Adi3 contribute to substrate phosphorylation.

The tomato AGC kinase Adi3 is phosphorylated by Pdk1 for activation of its cell death suppression activity. The Pdk1 phosphorylation site for activation of Adi3 is at Ser539. However, there is at least one additional Pdk1 phosphorylation site on Adi3 that has an unknown function. Here we identify an Arabidopsis thaliana sequence homologue of Adi3 termed AGC1-3. Two Pdk1 phosphorylation sites were identified on AGC1-3, activation site Ser596 and Ser269, and by homology Ser212 on Adi3 was identified as a second Pdk1 phosphorylation site. While Ser212 is not required for Adi3 autophosphorylation, Ser212 was shown to be required for full phosphorylation of the Adi3 substrate Gal83.

Ubiquitination of the tomato cell death suppressor Adi3 by the RING E3 ubiquitin ligase AdBiL.

Programmed cell death (PCD) is an organized process by which organisms selectively remove cells according to developmental needs or in response to biotic or abiotic stress. Despite recent efforts to understand mechanisms by which cell death takes place in plants, several gaps remain in our understanding of the molecular elements involved. The tomato PCD suppressor Adi3 is an AGC kinase that shares functional homology with the mammalian inhibitor of apoptosis PKB. Regulation of PKB stability, cell localization, and activation state is achieved through post-translational modifications such as ubiquitination. In an effort to understand the regulation of Adi3 function, we studied its interaction with the E3 ubiquitin ligase AdBiL. Using in vitro ubiquitination assays we show that AdBiL is an active E3 ubiquitin ligase using the E2 ubiquitin ligase UBC8 to ubiquitinate Adi3. Adi3 is also degraded in a proteasome-dependent manner. Our data draws additional parallels between Adi3 and PKB to support the functional relationship between these two PCD regulators.