Fine-tuning phosphatidic acid production for optimal plant stress responses

Abstract

Phosphatidic acid (PA) is involved in biotic and abiotic stress responses in plants. Here, we summarize quantitative lipidomics and real-time imaging used in PA studies and highlight recent studies of diacylglycerol kinase 5 (DGK5), an enzyme involved in PA biosynthesis, facilitating fine-tuning PA production for optimal stress responses in plants.

Phosphatidic acid (PA) metabolism

PA, the simplest phospholipid, is a component of biological membranes and acts as a second messenger in multiple signaling pathways [1]. PA production is mediated by multiple biochemical routes: it can be biosynthesized de novo through lysophosphatidic acid acyltransferases (LPAATs) in the glycerol 3-phosphate pathway, and PA also can be produced through the phosphorylation of diacylglycerol (DAG) catalyzed by diacylglycerol kinases (DGKs) and the hydrolysis of phospholipids, such as phosphatidylcholine (PC), catalyzed by phospholipase Ds (PLDs). In contrast, PA can be converted back into DAG by specific phosphatidic acid phosphohydrolases (PAHs) and nonspecific lipid phosphate phosphatases (LPPs), and cytidine diphosphate diacylglycerol (CDP-DAG) by CDP-DAG synthases (CDSs), respectively. CDP-DAG is the precursor of multiple phospholipids, including phosphatidylinositol and its derivatives, phosphoinositides. In Arabidopsis leaf, PA accounts for approximately 2% of all phospholipids, phosphatidylserine (PS) about 1%, and all phosphoinositides less than 1% [2]. Despite the low abundance, PA plays an important role in plant stress signaling and responses, and its content fluctuates under various stress conditions [1].

Enabling technologies for PA studies

The cellular PA level is quite low, which is about 50–100 μM in Arabidopsis plants [3], but it can be quickly produced or removed by its metabolic enzymes in different stress responses. Therefore, cellular PA functions dynamically. In other words, quantifying PA at cellular and subcellular levels is critical to elucidate PA’s action in the plant cell. Single-cell lipidomics can be used to analyze diverse phospholipid species and their relative abundance in individual cells [4]. Single-cell lipidomics coupled with single-cell transcriptomics will be powerful in precisely dissecting genes involved in PA metabolism (Figure 1). Additionally, PA content varies in different cellular organelles. Using subcellular lipidomics, PA content in subcellular compartments, like nuclei [1, 5], can be analyzed.

Figure 1.

Technologies and tools that facilitate studies and fine-tuning of cellular PA production for optimal stress responses. Detecting and quantifying PA in situ enables mechanistic studies of PA dynamics. Sigle-cell and subcellular lipidomics quantify PA content at the single-cell and organellar levels. Advances in lipid imaging enable us to observe cellular PA changes in situ. The PA biosensors, based on the yeast SNARE protein Spo20p, are the first and most widely used PA sensors. The ratiometric biosensor PAleon, coupled with advanced microscopy techniques, facilitates the study of PA dynamics. Various genome-editing technologies are capable of genetic engineering of DGK5 for fine-tuning PA production, facilitated by stress-inducible cis-elements identified by single-cell transcriptomics. CRISPR/Cas9 can be used to optimize the coding region and the promoter of DGK5. PrimeRoot enables precise insertions of large DNA fragments into plant genomes, and with PrimeRoot, an optimized allele of DGK5 can be engineered into plants. The CRISPR interference (CRISPRi) system, containing a catalytically dead Cas9 (dCas9) fused with the Krüppel-associated box (KRAB) repression domain, can be used to target key cis-elements in the promoter of DGK5 to downregulate its expression. To achieve optimal plant stress responses, coordinated genetic engineering of DGK5 is needed.

Real-time detection and quantification of PA changes in situ are key to our mechanistic understanding and subsequent engineering of PA metabolism-related genes. Lipid biosensors containing specific lipid-binding domains are often used to measure the spatiotemporal dynamics of phospholipids in vivo [1, 2]. The PA biosensors based on a PA-binding motif from the yeast SNARE protein Spo20p are the first and most widely used [6], and the newly ratiometric PA biosensor PAleon, contained a PA-binding domain sandwiched between the donor and acceptor of the fluorescence resonance energy transfer (FRET) system, can monitor PA concentration change in living plant cells [3, 7, 8] (Figure 1).

PA and DGK5 in cellular stress responses

With advanced technologies, recent works demonstrate that the PA dynamics mediated by DGKs and PLDs is important for responses to biotic and abiotic stresses. Reactive oxygen species (ROS) play a vital role in plant immunity against pathogen invasion, and PA production mediated by DGKs is associated with ROS signaling [9, 10], but how PA levels are fined-tuned in plant immune responses remains elusive. Kong et al. recently uncovered one mechanism for regulation: PA binds to and stabilizes respiratory burst oxidase homolog D (RBOHD), a NADPH oxidase, decreasing its ubiquitination to promote ROS production in Arabidopsis [8]. The study further showed that PA production from DAG upon immune elicitation is regulated by the differential, dual phosphorylation of DGK5 by Botrytis-induced kinase 1 (BIK1) and mitogen-activated protein kinase 4 (MPK4) (Figure 2). BIK1 phosphorylates DGK5 at Ser⁵⁰⁶ and increases DGK5’s activity, whereas MPK4 phosphorylates DGK5 at Thr⁴⁴⁶ and suppresses its activity. Additionally, they found that DGK5-mediated PA production is involved in both basal and effector-triggered immunity. In another study, Qi et al. observed that one of the transcripts of DGK5, DGK5β, instead of DGK5α that encodes proteins lacking the calmodulin-binding domain, mediates PA production under the treatment of chitin, a fungus-associated molecular pattern [7]. DGK5β-generated PA also enhances RBOHD stability by suppressing its vacuolar degradation (Figure 2). Taken together, DGK5-mediated PA production contributes to resistance to biotic stresses such as bacterial and fungal pathogens.

Figure 2.

DGK5 and PA for abiotic and biotic stress responses. Under biotic stress, bacterial flg22 and fungal chitin are recognized by receptors FLS2 and CERK1, respectively, which activate BIK1 and RIPK. Phosphorylated BIK1 and RIPK activate DGK5 by phosphorylating Ser⁵⁰⁶ to produce PA. But phosphorylated MPK4 induced by bacterial infection suppresses DGK5 activity by phosphorylating Thr⁴⁴⁶. The dual phosphorylation of DGK5 regulates PA homeostasis. PA binds to RBOHD, stabilizes RBOHD by suppressing PIRE/PBL13-mediated vacuolar degradation of RBOHD, and increases its plasma membrane localization, promoting ROS production. The calmodulin-binding domain is key to DGK5 functions. Under abiotic stress, DGK5-mediated PA production inhibits the activity of ABA2, a dehydrogenase in ABA biosynthesis, and decreases ABA production. Additionally, PA promotes the nuclear localization of ABA2, reducing ABA production. ABA, abscisic acid; ABA2, ABA insensitive 2; BIK1, BOTRYTIS-INDUCED KINASE 1; CERK1, chitin elicitor receptor kinase 1; DGK, diacylglycerol (DAG) kinase; ETI, effector-triggered immunity; FLS2, FLAGELLIN SENSING 2; MPK, mitogen-activated protein kinase; MEK, MPK kinase; MEKK, MEK kinase; PA, phosphatidic acid; PBL13, AvrPphB susceptible 1-like 13; PIRE, PBL13 interacting RING domain E3 ubiquitin ligase; PTI, pattern-triggered immunity; ROS, reactive oxygen species; RBOHD, respiratory burst oxidase homolog D, a NADPH oxidase; RIPK, RPM1-induced protein kinase.

PA is also involved in plant responses to abiotic stress, including drought, salinity and freezing. The plant hormone abscisic acid (ABA) is crucial to plant abiotic stress responses, and ABA DEFICIENT2 (ABA2) is a key enzyme in the major ABA biosynthesis pathway. A new study shows that DGK5 binds to ABA2 and generates PA to suppress ABA2’s activity, as well as promote ABA2’s nuclear sequestration [5] (Figure 2). The results indicate that DGK5-mediated lipid phosphorylation suppresses ABA biosynthesis and negatively regulates Arabidopsis’s response to abiotic stress, including drought and high salinity. It is also reported that the Arabidopsis dgk5 knockout mutant showed improved tolerance and decreased PA production in response to freezing stress [11].

Spatiotemporally fine-tuning PA production by engineering DGK5 may yield optimal plant stress responses

Cellular PA dynamics is regulated by multiple enzymes encoded by many different genes [1]. Here, we focus on DGK5 engineering for PA production to achieve optimal stress responses in plants. With detailed biochemical studies of the role of DGK5 in PA production and cellular stress responses [5, 7, 8], we learn that DGK5 is involved in a tradeoff in the response to abiotic and biotic stress conditions. The tradeoff between two traits is not uncommon in plants. Another example is the immunity-growth tradeoff, in which the energy balance tilting towards one causes the loss in the other.

One of the strategies to break the tradeoff is to use genome editing to fine-tune gene function. For example, from a rice mutant collection, Sha et al. identified a mutant named rbl1, which shows robust immunity but a severe yield penalty, a typical immunity-growth tradeoff [12]. The causal gene RBL1 encodes a PA-metabolizing enzyme, converting PA into CDP-DAG. Through multiplexed genome editing of RBL1, an optimal allele named RBL1^Δ12, harboring a 12-bp deletion in the coding region, was obtained. rbl1^Δ12 shows broad-spectrum disease resistance without affecting the yield, breaking the immunity-growth tradeoff [12]. Similarly, we believe that targeted multiplexed mutagenesis mediated by genome editing can be applied to DGK5 to fine-tune its function to achieve both abiotic and biotic stress resilience, or at least to achieve resilience to one stress without negatively affecting the other (Figure 1). More efficiently, we can use promoter-editing in fine-tuning the expression of DGK5, the consequence of which can be more predictable, as cis-elements in the promoter and their effects on gene expression are readily predictable with advanced bioinformatic tools [13].

Plant stress responses need to be spatiotemporally regulated to achieve optimal outcomes. Stress-induced or -suppressed expression of DGK5 is one such strategy. For example, we can use a pathogen-induced promoter to drive an engineered DGK5 allele that mimics the BIK1-phosphorylated variant (DGK5^S506D) and alters the MPK4-phosphorylated residue (DGK5^T446A) to enhance plant immunity [8] (Figure 1). The above-optimized allele can be integrated into a genomic safe harbor site, enabled by the genome-editing technology named PrimeRoot that can precisely insert large DNA fragments (~11 kb) into genomes [14]. For abiotic stress responses, CRISPR interference (CRISPRi), containing a catalytically inactive dCas9 fused to a transcriptional repression domain, and CRISPR/Cas13a mediated RNA interference [15], are feasible approaches to downregulate DGK5 expression via guide RNAs specifically binding to key cis-elements in the promoter or mRNAs of DGK5, and abiotic stress-induced expression of the systems is preferred (Figure 1). In summary, to optimally modulate the opposite roles of DGK5 in biotic and abiotic stress responses, orchestrated precise genetic engineering is needed.

Concluding remarks

The function of phospholipids is multifaceted, and mutation of phospholipid metabolism-related genes often causes significant consequences, but the biochemical mechanisms of these resultant changes are difficult to dissect, as phospholipids are highly interconnected and multiple phospholipid metabolism pathways are entangled, as well as constant phospholipid replenishment from the membrane trafficking [2]. However, with technical advances, three recent studies, together with others [1], showcase the exact role of PA-producing gene DGK5 in cellular abiotic and biotic stress responses. These advances facilitate the designing of precise metabolic engineering to achieve optimal stress responses and stress-resilient crops.