The tgd5 Mutation Affects Plastid Structure and Causes Giant Lipid Droplet Formation in Trichomes of Arabidopsis

Abstract

Trichomes, epidermal protrusions in terrestrial plants, play diverse roles in plant defense, metabolism, and development. Arabidopsis thaliana, a model plant with single-celled and non-glandular trichomes, is a valuable system for studying cell differentiation in plants. However, organelle biology in Arabidopsis trichomes is relatively underexplored. Using light and transmission electron microscopy, we investigated the phenotypes of intracellular structures in Arabidopsis trichomes caused by tgd5 mutations, which are known to disrupt lipid transfer from the endoplasmic reticulum to plastids and have a large impact on chloroplast morphology in pavement and guard cells. Significant phenotypic changes in the plastid structure were observed in tgd5 trichome cells, including the absence of plastoglobuli, the emergence of clusters of electron-dense particles in the stroma, and the possibly cup-shaped morphology of plastids. Additionally, the tgd5 mutations triggered the formation of giant, up to 15 µm in diameter, neutral lipid-containing droplets in the trichome cells, as revealed using histochemical staining with lipophilic dyes. These lipid droplets were substantially larger and more frequent in trichome cells than in other types of cells in tgd5. These findings highlight the role of TGD5 in maintaining plastid structure and implicate the unique activity of lipid metabolism in Arabidopsis trichomes.

1. Introduction

Trichomes are epidermal protrusions comprising a single cell or multiple cells found on the shoots of many terrestrial plants [1]. Various trichome functions have been postulated, including protecting plants against abiotic and biotic stresses, the synthesis and emission of specialized metabolites, developmental regulation, and facilitating interaction with insects [1]. Trichomes have attracted attention not only for their functional aspects but also as a model system for cell differentiation in plants. This is partly because the trichome assumes a predictable form on the plant surface; hence, any morphological changes can be easily detected, and because mutants defective in trichome development can be obtained owing to the trichomes’ dispensability for plant growth under ambient laboratory conditions [2]. In particular, the molecular mechanisms underlying trichome development have been systematically investigated in the model plant Arabidopsis thaliana, which has single-celled trichomes with two to three branches, using various developmental mutants [3]. A detailed molecular network model that mainly focuses on the interplay of transcription factors has been proposed to explain trichome formation in Arabidopsis. Compared to this progress in the study of trichome cell differentiation, there has not been much research on the intracellular structures of trichome cells despite their possible importance in understanding the roles of trichomes. While intracellular structures of “glandular” trichome cells, which are the accumulation and excretion sites of large quantities of specialized metabolites, have been well documented in relation to the accumulated products and their secretions (see [4] for a recent example), studies on the intracellular structures of “non-glandular” trichome cells that do not have a secretory mechanism but are involved in protective and defensive roles are limited [5]. This situation is similar in Arabidopsis, which has non-glandular trichomes, despite its widespread use as a research material for trichome differentiation.

Plastids are key to plant life, serving as the hubs for crucial processes like photosynthesis and nutrient storage. Their multifaceted functions in energy production and metabolic synthesis make them essential for plant growth and adaptation to environmental changes. We examined plastid morphology in trichome cells of the Arabidopsis parc6 mutant using a transgene encoded, plastid-targeted cyan fluorescent protein (CFP) used in our previous study [6] because trichome plastids in Arabidopsis are otherwise colorless (non-pigmented). PARC6 was originally identified as a regulator gene of division site placement in mesophyll chloroplasts [7,8,9]. Later, we identified PARC6 as the causal gene for one of our plastid morphology mutants (suba mutants), in which chloroplasts in the pavement cells of the leaf epidermis frequently emanated one or more extremely long extension(s), known as stromules (stroma-filled tubules) [10]. We found grape-like aggregations of plastids in both the trichome and pavement cells of parc6 (suba2) mutants but not in their mesophyll cells [6], implying a similarity between the regulatory mechanisms of plastid division in both trichome and pavement cells. This was expected, considering that both cell types share the same developmental origin. We also examined plastid morphology in pavement and stomatal guard cells of the other suba mutant, suba1, using plastid-targeted CFPs and transmission electron microscopy (TEM), and noticed some unique and distinctive phenotypes, including stromule hyperformation in pavement cells and the dismantling of chloroplasts in guard cells [11]. The causally defective gene in suba1 was identified as TGD5 [11], which encodes a subunit protein of the plastid envelope localized TGD complex [12]. It has been suggested that the TGD complex is involved in the endoplasmic reticulum (ER)-to-plastid lipid transfer, that is required to assemble thylakoid lipids [13]. These results indicate that ER-to-plastid lipid trafficking plays a critical role in maintaining plastid morphology in epidermal cells. In this context, we were interested in the morphology of trichome plastids in tgd5. In the present study, we observed these plastids using fluorescence microscopy and TEM. We also report on the formation and staining properties of giant spherical structures in tgd5 trichomes, which were unexpectedly found in our observations, and discuss their possible implications.

2. Materials and Methods

2.1. Plant Materials and Cultivation

Arabidopsis thaliana (L.) Heynh. accession Columbia (Col) was used in this study. The transgenic Col line FL4-4, which expresses stroma-targeted CFPs, was used to visualize plastids using fluorescence microscopy, as previously described [11]. Hereafter, the non-mutant lines are referred to as wild-type (WT) plants. The TGD5 knockout mutant suba1 was obtained from an ethyl methanesulfonate-mutagenized population of FL4-4 seeds as previously described [10,11]. The T-DNA insertional tgd5 mutant tgd5-3 (SAIL_254_A11; Col background) [12] was obtained from the Arabidopsis Biological Resource Center (ABRC; Ohio State University, Columbus, OH, USA). For the electron microscopy examination of suba1, it was crossed with WT Col plants, and non-fluorescent tgd5^−/− offspring plants (denoted as suba1*) which were obtained at the F₃ generation to exclude the possibility of artifacts due to the CFP expression. Seeds were germinated and grown on soil (Jiffy-7 peat pellets; Jiffy Products International BV, Zwijndrecht, The Netherlands) or Murashige–Skoog agar medium as described previously [11].

2.2. Microscopy and Lipid Staining

CFP fluorescence from plastids was observed using an inverted epifluorescence microscope IX71 (Olympus, Tokyo, Japan), equipped with a CMOS camera ORCA-flash2.8 (Hamamatsu Photonics, Hamamatsu, Japan) with previously described settings [6]. Differential interference contrast images were obtained using an IX71. Aqueous solutions of Sudan Black B (FUJIFILM Wako Pure Chemical, Osaka, Japan) and Sudan III (FUJIFILM Wako) dyes were prepared to a final concentration of 0.1% [w/v] for the lipid staining of trichome cells. Excised leaves were submerged in one of the dye solutions in a 1.5-mL microtube and incubated at room temperature (22–24 °C) for 10 min. Microscopic examinations of non-stained and stained lipid bodies were performed under bright-field optics using a BX51 microscope (Olympus, Tokyo, Japan) equipped with a CCD digital camera DP71 (Olympus) and a 40× UPlanApo objective lens (NA 0.85; Olympus). The ultrastructural analysis of the trichome plastids was performed using TEM (JEM-1400Plus; JEOL, Tokyo, Japan) at 100 kV. The leaf samples used for TEM were prepared as previously described [10].

3. Results

3.1. Morphology and Ultrastructure of Trichome Plastids in the Arabidopsis tgd5 Mutants

First, we examined the morphology of colorless plastids (or leucoplasts) in trichome cells under epifluorescence microscopy using stroma-targeted CFPs (Figure 1). In the trichome cells of Arabidopsis WT plants, plastids generally assumed round or ovoid shapes. However, some plastids exhibited one or more constrictions (Figure 1b), as demonstrated in our previous report [6].

Using TEM, we found that plastoglobuli had developed and were abundant in the trichome plastids of the WT, and that the endomembrane system had also developed to some extent, although no grana-like stacks were observed (Figure 2a,b). In the trichome cells of suba1, a TGD5-knockout mutant [11], the plastid morphology appeared relatively heterogeneous compared to that of the WT under a fluorescence microscope (Figure 1d). Some of these plastids were elongated and tube- or worm-like in shape (double arrowheads in Figure 1d). Unlike the trichome cells of parc6/suba2 [6], no grape-like clusters of plastids were found in those cells of suba1. With TEM, we also found that the trichome plastids in suba1 (suba1*) were slightly enlarged and contained a less-developed endomembrane system compared to those in the WT (Figure 2c,d). In addition, clusters of electron-dense particles were locally present in the stroma of suba1 trichome plastids instead of the mature plastoglobuli seen in WT trichome plastids. Interestingly, we frequently observed doughnut-shaped plastids in the TEM analysis of suba1 trichome cells (Figure 2d), which were presumed to be cross-sections of cup-shaped plastids.

3.2. Formation of Giant Lipid Droplets in Trichomes of the Arabidopsis tgd5 Mutants

During the above observation of plastids, we discovered giant spherical structures in the suba1 trichome cells (arrows in Figure 1c). These giant spheres were approximately 5–15 µm in diameter and often accompanied by tiny spheres (arrowheads in Figure 1c). Given the emphasis in this study on qualitative observations and exploratory findings, a statistical analysis of sphere sizes was not conducted, which can be considered a limitation. They are usually located at or near the branch junction and occasionally in the middle of the branches. While the exact number of spheres per cell could not be obtained, due to the three-dimensional extended structure of the trichome, one to several spheres were typically observed within one trichome cell. These spheres usually aggregated when cells contained more than one. They did not exhibit the rapid cytoplasmic streaming, or the second-by-second morphological changes as observed for trichome plastids, as reported in our previous paper [6]. We examined and compared the frequency of sphere formation in the trichome cells of the WT, suba1, and another tgd5 mutant, tgd5-3 [12] (Figure 3), and confirmed that both tgd5 mutants had a statistically significant higher frequency of sphere formation than the WT (54% in suba1 and 57% in tgd5-3 versus 0% in WT; p < 0.001 using Fisher’s exact test;

Table 1. Occurrence of spherical structures in non-stained trichome cells of Arabidopsis tgd5 mutants. Trichome cells in the adaxial side of foliage leaves from three-week-old plants were examined. Frequencies of sphere occurrence were compared between the wild type (Columbia) and each mutant using Fisher’s exact test with Benjamini–Hochberg correction for multiple testing.

Plant Line	Number of Examined Trichome Cells (Number of Plants)	Number of Trichome Cells with at Least One Sphere	Frequency of Sphere Occurrence (%)	Significant Difference from Wild Type (p < 0.001)
Wild type	191 (5)	0	0	–
suba1	171 (6)	93	54	yes
tgd5-3	168 (6)	96	57	yes

).

We conducted histochemical assays to identify the main components of tgd5-specific spherical structures. We focused on lipid-staining dyes for three reasons. First, the appearance of the spherical structures resembles that of lipid droplets (LDs, also referred to as oil bodies) in various organisms. Second, TGD5 is involved in lipid synthesis and transfer [12]. Last, LD-like structures were also observed in the pavement and stomatal guard cells of suba1 using TEM in our previous study [11], although those LD-like structures were approximately 1–2 µm in diameter and much smaller than the spheres seen in the trichome cells of suba1 and tgd5-3. As a result, the spheres were stained with Sudan Black B (Figure 4) and Sudan III (Figure 5), and the staining was statistically specific to tgd5 mutants (p < 0.001, Fisher’s exact test;

Table 2. Occurrence of lipid-stained spherical structures in trichome cells of Arabidopsis tgd5 mutants. Trichome cells in the adaxial side of foliage leaves from four-week-old plants were stained with the indicated lipophilic dye and examined. Frequencies of occurrence of the positively stained sphere(s) were compared between the wild type (Columbia) and each mutant using Fisher’s exact test with Benjamini–Hochberg correction for multiple testing.

Staining Dye	Plant Line	Number of Examined Trichome Cells (Number of Plants)	Number of Trichome Cells Possessing at Least One Dye-Positive Sphere	Frequency of Dye-Positive Sphere Occurrence (%)	Significant Difference from Wild Type (p < 0.001)
Sudan Black B	Wild type	209 (6)	0	0	–
	suba1	206 (7)	95	46	yes
	tgd5-3	200 (6)	88	44	yes
Sudan III	Wild type	212 (6)	0	0	–
	suba1	192 (6)	70	36	yes
	tgd5-3	198 (6)	72	36	yes

).

As both dyes have the common property of staining neutral lipids, the spheres were identified as LDs containing neutral lipids. We did not find giant LDs larger than 5 µm in diameter, corresponding to those observed with light microscopy, and in TEM images of the suba1* trichome cells. This might be because these giant LDs were usually located at or near the branch junction of trichomes, and it is difficult to include the junction site, which is usually displaced from the axis of the main stalk, in the longitudinal ultrathin sections (80 nm thickness) of trichomes. Nonetheless, we found enhanced development of middle-sized LDs (approximately 2–3 µm in diameter) in epidermal cells around the base of trichomes [14] in suba1* (Figure 2e).

4. Discussion

In this study, we examined the plastid phenotypes of trichome cells in Arabidopsis tgd5 mutants. Previously, a lack of functional TGD5 was shown to lead to the occurrence of achlorophyllous or faintly chlorophyllous stomata [15], shrinkage and fragmentation of plastids in the stomatal guard cells, and excessive formation of stromules in leaf pavement cells [11]. In contrast, tgd5 mutations do not appear to affect mesophyll chloroplasts in terms of size, shape, division, or ultrastructure [11]. Thus, we postulated that TGD5 plays an important role in maintaining plastid morphology in epidermal (and other non-mesophyll) tissue, but not in mesophyll tissues in Arabidopsis. The cell type dependent differential effects of tgd5 mutations in Arabidopsis may reflect the differential activity of the ER pathway (or eukaryotic pathway) of plastid lipid assembly, which requires the TGD complex; while mesophyll cells depend on both the ER and plastidial (or prokaryotic) pathways. In contrast, guard cells (and maybe other non-mesophyll cells) exclusively depend on the ER pathway [15]. Our results demonstrate that the loss of functional TGD5 affects the morphology and differentiation (as estimated from the development of the subplastidial membrane system) of plastids in trichome cells (Figure 1 and Figure 2), implying that plastid lipid assembly in Arabidopsis trichomes relies heavily on the ER pathway. Nevertheless, the influence of tgd5 mutations on plastid morphology in trichome cells differed from that in pavement and guard cells, which share the same developmental origins as trichome cells. One of the plastid phenotypes observed in the trichome cells of tgd5 was the disappearance of plastoglobuli and the emergence of clusters of electron-dense particles in the stroma (Figure 2b,c). Because plastoglobuli are known to contain various types of lipids, including galactolipids, diacylglycerol, and triacylglycerol [16], this phenotype may represent a lack of certain lipids and the concomitant accumulation of their reactive substrates in the plastid stroma. This is caused by the inability of ER-to-plastid lipid transfer due to the absence of the functional TGD complex. Another plastid phenotype in tgd5 trichome cells was the appearance of probably cup-shaped plastids (Figure 2c), which has never been observed in other cell types in tgd5 [11] and is thus considered to be a trichome-specific phenotype. While the meaning of this unique shape is not yet understood, this phenotype indicates the large impact of the TGD5 defect on trichome plastid morphology and, therefore, the crucial role of TGD5 in maintaining plastid morphology.

Unexpectedly, we also found that the loss of functional TGD5 led to the formation of neutral lipid-containing giant LDs in trichome cells (Figure 3, Figure 4 and Figure 5), as a new phenotype of Arabidopsis tgd5 mutants. These LDs were large enough to be readily visible without staining under a light microscope (Figure 3). Nile Red-positive LDs have also been reported to exist in the cytosol of leaf cells in tgd5-3 in a former study [12]. Those LDs in leaf cells were much smaller in size (<0.5 µm in diameter) and less frequent (detected in 6 of 42 cells under TEM) than LDs in trichome cells of suba1 and tgd5-3 (approximately 5–15 µm in diameter and >50% frequency of occurrence). Pavement and guard cells in suba1 were also found to contain LD-like structures in our previous study [11], with a size of approximately 1–2 µm in diameter. Thus, we conclude that trichome cells are a particularly prominent site of LD formation due to tgd5 mutations. The observation that giant LDs were found only in the trichome cells of tgd5 plants is intriguing, and the activity of neutral lipid synthesis might be high in Arabidopsis trichomes. To further elucidate the properties of those giant LDs, additional research including time-lapse observation and lipidomic profiling will be necessary in the future. In particular, potential dynamic behaviors of lipid droplets in trichome cells, including growth, merging, movement, and morphological changes, are worth further investigation by employing long-term time-lapse microscopy. Although the functions of trichomes in Arabidopsis are not well understood, a recent study demonstrated that they act as mechanosensory cells that activate a rapid immune response in plants [17]. The trichome-specific phenotypes observed in the present study provide clues for exploring the functions of Arabidopsis trichomes.