Arabidopsis leaf extracellular vesicles in wound-induced jasmonate accumulation

ABSTRACT

The plant extracellular vesicles (EVs) are lipid-enveloped nano-particles containing proteins, nucleic acids and metabolites and function in plant development and response. The Arabidopsis four transmembrane protein TETRASPANIN 8 (TET8) knock-out mutant tet8 secreted less EVs than the wild-type (WT). In this report, we show that the tet8 mutant was attenuated in the plant hormone jasmonate (JA) accumulation in response to mechanical wounding treatment. We also noticed that the EVs contained a high level of phospholipids phosphatidic acids (PAs) which may serve as precursors of JA biosynthesis during wound-triggered-self-healing processes. Thus, we propose an open question about a potential role of EVs or TET8 or both in damage-associated JA response.

Introduction

The plant extracellular vesicles (EVs) were firstly reported during 1960s and later demonstrated to be associated with plant immune response.^1,2–6 The EVs containing small RNAs were proposed to contribute to the plant resistance to the necrotrophic fungus Botrytis cinerea and tet8 mutant showed enhanced susceptibility to the pathogen.¹ In our recent report,⁷ we revealed the lipidomic profiles of Arabidopsis leaf EVs and observed that the tet8 mutant secreted a significant less amount of EVs, which was associated with the reduced production of oxygen species (ROS) upon stress treatments using salicylic acid or bacterial pathogen-associated molecular pattern molecule flg22.

In animal cells, such as injured neurons in peripheral nervous system, EVs containing functional NADPH oxidase 2 complexes could deliver ROS signaling to the surrounding cells to participate in axonal regeneration and functional recovery via stimulating phosphoinositide 3-kinase-phosporylated (p-)Akt signaling and regenerative outgrowth after spinal injury.⁸ Stem cells (like mesenchymal stromal cells) were reported to release EVs to the repairing injured cells or organs.⁹ However, the role of EVs in plant response toward wounding remains enigmatic.

Wound-induced JA production was compromised in tet8 mutant

Plants are sessile and vulnerable to environmental factors. Wounded plants produce damage-associated signals such as the phytohormone JA to initiate self-healing and organ regeneration processes.^10,11 Commonly, mechanical tissue damage trigger a rapid local JA burst,¹² which could act as mobile signals in the systemic wound response.¹³ The Lacking nervous system, plants also deliver signals to neighboring cells as well as long distant tissues via different manners, such as diffusion through symplast via plasmodesmata or through the apoplast or electrical signals (as the systemic JA accumulation) ^14,15. To examine whether the wounding signals transport were also associated with the EVs, we performed mechanical wounding treatment of rosette leaves of the 4-week-old WT and tet8 mutant Arabidopsis plants, and measured the JA content in the wounded (local) as well as the neighboring (distal) leaves (Figure 1a). We found that, compared to the WT, the tet8 local leaves displayed a significantly reduced response in JA accumulation toward wound treatment (Figure 1b). In both the WT and the tet8 leaves, the wound-induction of JA level in distal leaves was clear but weaker, but the systemic response was deferred in tet8 leaves compared with the WT at the beginning and reached the level as the WT until 1 h post-treatment (Figure 1c). The tet8 mutant had a starting JA content similar to that the WT but an attenuated JA burst upon mechanical injury, raising a question of whether the EVs or TET8 or both are relevant to JA accumulation.

Figure 1.

Changes in JA contents after wounding treatment. (a) Graphic show of wound treatment of leaves and sample collection for jasmonic acid (JA) measurements. The 4-week-old Arabidopsis plants were analyzed. Wounded sites were marked with two lines. The wounded (local) and the intact neighboring (distal) leaves were marked with yellow or red dots, respectively. (b) JA content of the wounded leaves post-treatment, representing the local response. (c) JA content of the intact neighboring leaves, representing the systemic response toward wounding treatment. For b and c, six leaves were harvested for each sample and four repeats were conducted for JA measurements (n = 4). The time course post-treatment was indicated above the figure. Values are reported as mean, and error bars represent SDs. Statistical significance was determined using two-tail Student’s t test. *, P < .05; **, P < .01

The high level of phosphatidic acids (PAs) in EVs might be associated with JA biosynthesis

Wound treatment induced rapid increase in PAs and decrease in other phospholipids (phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols and phosphatidylinositols) in Arabidopsis¹⁶ and castor bean,¹⁷ and PAs were considered an important source of JA biosynthesis via a lipolytic pathway in which the acyl-hydrolyzing enzymes produce the JA precursor linolenic acid.¹⁸ Tomato plants were shown to exhibit a wound-inducible phospholipase A (PLA) activity and largely release linolenic acid as well as JA from membranes.^19,20 Besides, it was suggested that phospholipase D (PLD), which hydrolyzes phospholipids at the terminal phosphoesteric bond to produce PA, may also play a role in mediating wound-induced lipid hydrolysis.²¹ The published Arabidopsis EV proteomics showed several phospholipases in the EVs, including phosphoinositide phospholipase C2, PLC2, phospholipase Dδ, PLDδ, PLDα1 and PLDγ1.²² And our lipidomic data⁷ showed that the EVs contained a significantly higher level of PAs (more than 31% of total phospholipids, especially PA-c34:3, PA-c34:2, PA-c36:6, PA-c36:5, PA-c36:4 and PA-c36:3) compared with the total leaf tissue (no more than 5% of total phospholipids) (Figure 2) and other membrane fractions [such as plasma membrane, ²³] which also underwent massive isolation procedures. The above data together indicates a functional role of PAs in the EV. Although we could not deny that excess PAs may partly owe to the tedious procedure of sample preparation,²² we prefer the explanation that PAs in EVs are the convenient and mobile PA-enriched cargos. The tet8 mutant showed decreased EV secretion but unchanged contents of total PAs⁷ and the attenuated JA response in the local and distant tet8 leaves prompted us to question that whether the EVs might provide an extracellular source of PAs which could be rapidly delivered to adjacent cells for JA biosynthesis.

Figure 2.

Distribution of PAs in WT Arabidopsis leaf EVs and leaf tissues. Data are expressed in mol% of total phospholipids. The x-axle represents the fatty acid composition of PAs. Phs is short for phospholipids. Four independent repeats were conducted. Black dots represent each repeats. Values are means ± SDs. Statistical significance was determined using two-tail Student’s t test. **, P < .01; ***, P < .001

Our findings provide a new insight into plant EVs in wounding response or even in callus formation and tissue regeneration. Beyond that, JAs are the mast regulator of plant defense against insect herbivores and necrotrophic fungi, thus the EV lipids play important roles in plant–insect and plant–microbe interactions.

Experimental procedures

Plant materials and growth conditions

Seeds of Arabidopsis thaliana ecotype Columbia-0 (Col-0) and the T-DNA insertion mutant tet8 (AT2G23810, SALK_136039 C, obtained from the European Arabidopsis Stock Center) were germinated on 1/2 Murashige Skoog (MS) agar medium and the seedlings were transferred to soil and grown in a controlled chamber at 16-h light/8-h dark cycle, 22°C and 70% relative humidity.

JA isolation and measurement

For measurement of jasmonic acid (JA), about six fresh leaves for each sample were weighted and grounded into powder in the liquid nitrogen. Ethyl acetate with internal standard (2HJA) was added into the powder and vortex roughly to fully isolate JA. Suspensions were collected after 10,000 x g centrifuge into new tubes. Residues were isolated with additional ethyl acetate again. Suspensions were combined and dried with rotary evaporator. The final pellets were dissolved in 70% methanol and filtered before LC/MS (UPLC-QTRAP 6500 Plus, Sciex) analysis. The measurements were conducted with Mobile phase A: 0.05% formic acid in water and Mobile phase B: methanol with the 1.8 µm C18 column (internal diameter 2.1 × 50 mm, The ACQUITY UPLC).

Funding Statement

The research was supported by grants from the National Natural Science Foundation of China (31788103), and the Chinese Academy of Sciences (QYZDY-SSW-SMC026)

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Author contributions

N.-J.L and X.-Y.C. conceived the research. N.-J.L. performed most of the experiments. J.-J.B. and L.-J.W assisted with plant cultivation. N.-J.L. and X.-Y.C. wrote the manuscript.