JXB Volume 73, Issue 5 – Editor’s choice

This article highlights the following publication:

Lysophosphatidic acid acyltransferases: a link with intracellular protein trafficking in Arabidopsis root cells?

Valérie Wattelet-Boyer, Marina Le Guédard, Franziska Dittrich-Domergue, Lilly Maneta-Peyret, Verena Kriechbaumer, Yohann Boutté, Jean-Jacques Bessoule, Patrick Moreau

Journal of Experimental Botany, Volume 73, Issue 5, 2022, Pages 1327–1343, https://doi.org/10.1093/jxb/erab504

The secrets of secretion

The famous Irish author Oscar Wilde once said: “Life is not complex. We are complex. Life is simple, and the simple thing is the right thing.” Clearly, Mr. Wilde must have never peeked through the eyepieces of a microscope to catch sight of the flabbergasting sophistication of the inner workings of a single plant cell, or else his often-quoted worldly wisdom would have sounded very different. Life is complex and it has been for a long time, a very long time. In fact, life became complex about 1.5 – 2 billion years ago when the first eukaryotic cells created a very early fork in the tree of life by establishing morphological complexity, thereby founding a whole new domain of life. The first eukaryotic organisms adopted a phagocytotic lifestyle, they engulfed bacterial endosymbionts that would later evolve into mitochondria and chloroplasts, genetic material was neatly enveloped and stored in the nucleus, and further compartmentalization created an internal cellular machinery that is in stark contrast to unicellular prokaryotes, whose inner makeup is rather simple by comparison.

However, the internal cellular structure in eukaryotes should not be thought of as having a static architecture. Especially the plasma membrane and the endomembrane system that comprises the endoplasmic reticulum, the Golgi apparatus, and the nuclear envelope are in dynamic movement. Constant lava lamp-like budding and fusing of membrane vesicles allows for continuous exchange of content between the different compartments. High cellular complexity demands high levels of organisation, and these trafficking processes are thus coordinated by different sets of proteins that facilitate formation of vesicles at the donor membrane, subsequent directed transport, as well as docking and fusion at the target membrane.

The membranes themselves serve not only as barriers to define substructures, but membrane lipids are also involved in signalling. The membrane component phosphatidic acid (PA), for example, has even been described as a lipid second messenger owing to its regulatory role in diverse processes. Phosphatidic acid is a biosynthetic intermediate of other phospholipids and its amount in membranes is kept low under non-stress conditions but increases in response to internal and external stimuli. The distinct physicochemical properties of PA may shed light on its functionality. Phosphatidic acid has a conical shape, which leads to negative membrane curvature and induces vesicle formation and budding. Furthermore, with its negatively charged monophosphoester headgroup PA acts as a docking site for proteins and can change the activity of target enzymes.

Numerous PA-binding proteins have been identified that are involved in basic metabolic reactions, hormone signalling, as well as abiotic and biotic stress responses. Many of these interactions lack further mechanistic insight to date, and especially how specificity in cellular responses is exerted when PA levels rise remains largely unknown. It has been hypothesised that this is conferred via distinct PA pools that are generated by different enzymes. One such group of enzymes is lysophosphatidic acid acyltransferase (LPAAT) that catalyses the formation of PA from lysophosphatidic acid (LPA). Five membrane-bound LPAAT homologues have been identified in Arabidopsis with AtLPAAT1 residing in plastids and AtLPAAT2, 4, 5 sitting in the ER, where AtLPAAT2 presumably is the main enzyme for de novo PA synthesis.

In this issue of the Journal of Experimental Botany Wattelet-Boyer et al. (2022) set out to investigate the role of LPAAT3-5 in the secretory pathway, the journey that newly synthesised proteins take from the ER to the plasma membrane. They confirmed in E. coli that LPAATs are strict LPA acyltransferases and show no activity with other lysophospholipids. Through fluorescence labelling the authors found that LPAAT4 and LPAAT5 localize exclusively to the ER whereas LPAAT3 was found in ER-associated globular structures that may constitute the ER exit sites. To test the importance of these enzymes in the secretory pathway, different Arabidopsis LPAAT mutants were generated and their root phenotype characterized, since impaired primary root growth is symptomatic for dysfunctional membrane morphodynamics and ER trafficking. The results posed unexpected challenges for the further analysis: LPAAT3-5 single and double mutants showed no or only very week root phenotype, presumably due to functional complementation by the other LPAAT homologues.

The triple mutant deficient in LPAAT3, LPAAT4, and LPAAT5, however, surprised with significantly longer roots compared to the wildtype. The authors showed that transcription of LPAAT2 is up-regulated in these plants, which could have led to increased phospholipid synthesis and stimulated root growth.

Unfortunately, complete elimination of LPAAT2 or reduction via inducible knock-down in the triple mutant was either lethal or showed no significant phenotypic results. Without an appropriate mutant at hand the authors needed to reach into the methodological bag of tricks and combine genetic with pharmacological approaches to investigate a putative role for LPAATs in the secretory pathway. They used the lpaat4;lpaat5 double-mutant that showed wildtype-like transcription levels of LPAAT2 and impaired LPAAT3 activity through treatment with the biochemical inhibitor CI976.

These plants did indeed have a pronounced reduction in primary root growth. Furthermore, PA, the product of the LPAAT-catalysed enzymatic reaction, was significantly reduced in the CI976-treated double mutants, whereas the levels of other major membrane phospholipids remained unchanged. Having established this experimental system in which de novo synthesis of PA is disrupted allowed the authors to address the question of a potential involvement of LPAATs and PA in the functioning of the plant root secretory pathway.
For this, they monitored the trafficking of several plasma membrane proteins by means of immunolocalization or fluorescence labelling. They found increased amounts of the auxin transporter PIN2 and the aquaporin PIP2;7 in intracellular membranes in the CI976-treated lpaat4;lpaat5 double-mutant compared to the wildtype, whereas trafficking of the plasma membrane proton pump was not affected.

These results indicate that PA synthesis by LPAAT3-5 and a critical PA threshold are indeed essential for the correct transport of certain proteins from their place of synthesis to their target membrane, and the authors provide experimental evidence for the existence of specific PA pools with signalling function. However, differences seem to exist among proteins regarding their necessity for LPAAT activity, an intriguing finding that deserves further exploration in future studies.

The work presented by Wattelet-Boyer et al. nicely illustrates the complexity that lies within a single cell, and it shows that trying to grasp this complexity can demand complex experimental approaches. The results presented in this paper contribute to our understanding of the intricate regulatory processes that keep the cellular machinery running, and prove once again that life is not simple, life simply is.