Low-Phosphate Induction of Plastidal Stromules Is Dependent on Strigolactones But Not on the Canonical Strigolactone Signaling Component MAX21[OPEN] Gilles Vismans, Tom van der Meer, Olivier Langevoort, Marielle Schreuder, Harro Bouwmeester, Helga Peisker, Peter Dörman, Tijs Ketelaar, and Alexander van der Krol* Laboratory of Plant Physiology (G.V., T.v.d.M., O.L., M.S., A.v.d.K.) and Laboratory of Cell Biology (G.V., T.v.d.M., O.L., T.K), Wageningen University, 6708 PB Wageningen, The Netherlands; and Institute of Molecular Physiology and Biotechnology of Plants, University of Bonn, 53113 Bonn, Germany (H.P., P.D.) ORCID IDs: 0000-0002-7535-0190 (T.v.d.M.); 0000-0002-3231-2071 (O.L.); 0000-0001-9506-7264 (T.K.); 0000-0001-6585-7572 (A.v.d.K.).
Stromules are highly dynamic protrusions of the plastids in plants. Several factors, such as drought and light conditions, influence the stromule frequency (SF) in a positive or negative way. A relatively recently discovered class of plant hormones are the strigolactones; strigolactones inhibit branching of the shoots and promote beneficial interactions between roots and arbuscular mycorrhizal fungi. Here, we investigate the link between the formation of stromules and strigolactones. This research shows a strong link between strigolactones and the formation of stromules: SF correlates with strigolactone levels in the wild type and strigolactone mutants (max2-1 max3-9), and SF is stimulated by strigolactone GR24 and reduced by strigolactone inhibitor D2.
Strigolactones are a class of phytohormones, which only recently have been recognized to function both in plant development and as signal molecules from plants to arbuscular mycorrhizal fungi (AMF; RuyterSpira et al., 2011). The induction of strigolactone biosynthesis and secretion by roots of plants under low-phosphate conditions is thought to stimulate the interaction with AMF, as strigolactones induce branching in AMF (Toh et al., 2012). The highly branched structure of the AMF filaments may aid the plant in the uptake of scarcely available soil phosphate. The biosynthesis and exudation of strigolactones is also strongly increased under low nutrient availability in Arabidopsis (Arabidopsis thaliana), even though Arabidopsis is not host to AMF (Yoneyama et al., 2007), indicating that strigolactones may have other roles in plant responses to low phosphate besides stimulation of AMF. Phosphate is essential for cell function as it is required for the
1 Part of this research was funded by STW project 13149 “Novel sustainable treatments for compact plants in greenhouse spaces.” * Address correspondence to [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Alexander van der Krol ([email protected]). T.K. and A.v.d.K. conceived the original research and supervised experiments; G.V., T.v.d.M., and O.L. performed most of the practical work and data analysis; H.P. and P.D. performed the phospholipid quantification; M.S. provided technical assistance; A.v.d.K. and G.V. wrote the article with contributions of all authors; H.B. supervised the project. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.16.01146
production of phospholipids, energy production, and regulation of protein activity, and much of the plant’s phosphate is stored in the phospholipids of the cell membranes. When phosphate availability is limiting for the plant cell, phosphor may be mobilized from membrane phospholipids, in which case the lipids are replaced by galactolipids derived from plastids (Tjellström et al., 2008; Lambers et al., 2012). This exchange of lipids between the plastid and other membranes occurs via the endoplasmic reticulum (ER) through plastid ER membrane contact sites (Dörmann and Benning, 2002; Tjellström et al., 2008), and indeed this frequency of these contact sites is increased under low Pi conditions (Tjellström et al., 2008). Stromules are highly dynamic tubular extensions that protrude from the plastid, filled with stroma and surrounded by the plastid double membrane. Thus, stromules give a large increase in the potential contact between plastid and other cell compartments. Indeed, stromules seem to be intercalated between ER membranes (Schattat et al., 2011a, 2011b), but stromules have also been observed in infoldings of the nuclear envelope (Kwok and Hanson, 2004). In a high-throughput screen of cDNA-GFP fusions, many proteins were found to be localized to the stroma of chloroplasts, but not all of them were also localized to stromules (Escobar et al., 2003). The formation of stromules is cell type specific and developmentally regulated (Köhler and Hanson, 2000). Although it has been suggested that stromules may function in the exchange of galactolipids from the plastid to other membranes under low-phosphate conditions, this has never been investigated. Because low-phosphate conditions stimulate the production of strigolactones
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and strigolactone biosynthesis starts in plastids, this raises the intriguing question whether strigolactones are causal to stromule formation. Here, we used Arabidopsis plants expressing FNRtp-EGFP, which labels stroma-filled stromules (Marques et al., 2003), to image stromule activity in relation to low phosphate and strigolactones in the wild type, the strigolactone signaling mutant max2-1 (Nelson et al., 2011), and the strigolactone biosynthesis mutant max3-9 (Booker et al., 2004). In addition, stromule activity was analyzed in tobacco (Nicotiana tabacum) seedlings expressing a CT(RecA)S65TmGFP4, which also labels plastids, including stromules (Köhler et al., 1997). The aim of the investigation was to determine (1) if there is a correlation between stromule activity and low-phosphate stress in plants and (2) if there is a relationship between the induction of strigolactones under low-phosphate stress and (putative) stromule formation under lowphosphate stress. Stromule formation was quantified in different cell types, and the stromule frequency (SF) in Arabidopsis seedling hypocotyl cells was used to study the effect of low phosphate and strigolactones. Results show a significant increase of SF in wild-type and max2-1 Arabidopsis seedlings when grown on lowphosphate medium, confirming that stromules are part of the low-phosphate response of plants. Moreover, frequency of stromule formation was not increased in the max3-9 background, indicating that strigolactone biosynthesis is required for the low-phosphate induction of stromules. Results were confirmed by induction of stromules by exogenous applied strigolactone GR24 in Arabidopsis and tobacco and by inhibition of stromule formation by inhibitors of strigolactone biosynthesis in wild-type Arabidopsis and tobacco. The role of strigolactones in stromule formation and phosphate metabolism in plants is discussed. RESULTS Different Stromule Frequency in Different Cell Types
The occurrence of stromules differs for different cell types in plants (Köhler and Hanson, 2000). Therefore, we first established growth and imaging conditions for reliable comparison of quantified SF in order to study the effect of different treatments. We used Arabidopsis plants in which stromules are labeled using a ferredoxin NADP(H) oxidoreductase (FNR) transit peptide fused to enhanced GFP (FNR-EGFP). For the chemical treatment experiments, freshly germinated 3-d-old seedlings were used, FNRtp-EGFP activity in plastids of root, hypocotyl, and leaf guard cells was imaged, and plastid shape and SF were measured. The SFs were studied in three different cell types: root epidermal cells, hypocotyl epidermal cells, and guard cells. In the three studied cell types in wild-type Arabidopsis seedlings, the SF is highest in root epidermal cells, with an SF of SF = 0.29 6 0.04. Guard cells have a lower SF (SF = 0.16 6 0.06), and hypocotyl epidermal cells have 2236
Figure 1. SFs differ per cell type. Col-0 seedlings were studied after 48 h of cold dark treatment followed by 3 d at 24°C with 16 h light. t test, error bars represent the SE. Statistical differences are indicated by A, B, and C. Hypocotyl epidermal cells (SF = 0.05 6 0.01, n = 12). Root epidermal cells (SF = 0.29 6 0.04; n = 12) and guard cells (SF = 0.16 6 0.06; n = 12).
the lowest SF (SF = 0.05 6 0.01; Fig. 1). SFs were similar to those previously reported for Arabidopsis (Holzinger et al., 2008; Newell et al., 2012). Because hypocotyl epidermal cells were used to study stromules in numerous previous publications (Hanson and Sattarzadeh, 2013; Kwok and Hanson, 2004; Holzinger, Kwok et al., 2008; Newell et al., 2012), we selected these cells to evaluate the effect of different treatments involving low phosphate, sugars, or strigolactones. SF Is Increased by Low-Phosphate Treatment
Next, we tested whether low-phosphate conditions result in an increase in SF. To observe the effect of low phosphate on plastid SF, germinated Arabidopsis seedlings were transferred to solid medium containing either 0.53 Murashige and Skoog (MS) with phosphate or 0.53 MS without phosphate. In order to deplete phosphate reserves in the seedlings, seedlings were kept on media for 7 d, after which SF in hypocotyl cells was quantified. In 7-d-old seedlings, the SF in hypocotyl cells was slightly lower compared than in 3-d-old seedlings (from SF = 0.05 6 0.01 at 3 d to SF = 0.027 6 0.01 at 7 d), indicating that in comparing SF not only cell type but also developmental stage should be taken into account. For hypocotyl cells in seedlings on medium without phosphate, the SF was increased to SF = 0.11 6 0.036 (Fig. 2), showing that indeed SF increases in response to low phosphate. Since low-phosphate conditions also result in an increase in strigolactone production, we next investigated whether there is a link between strigolactones and stromules. SF Relates to Endogenous SL Levels in Arabidopsis
To study the effect of strigolactones on SF, we made use of two types of strigolactone mutants: the max2-1 mutant, which is mutated in the F-box protein MAX2 and which lacks conventional D14/MAX2-dependent strigolactone signaling (Nelson et al., 2011), and the max3-9 mutant, which is mutated in the CCD9 gene and Plant Physiol. Vol. 172, 2016
Induction of Stromules Is Dependent on Strigolactones
Figure 2. Increase in SF under phosphate starvation. Col-0 seedlings placed in the cold and dark for 2 d, followed by 7 d at 24°C with 16 h light. Error bars represent the SE. Pi, SF = 0.027 6 0.01; n = 4. No Pi, SF = 0.11 6 0.036; n = 4. Phosphate-starved seedlings have an increased number of stromules (ANOVA, LSD; P = 0.045). Statistical differences are indicated by A and B.
therefore is blocked in biosynthesis of strigolactones. Compared to wild-type plants, these max mutants have different levels of endogenous of strigolactones: max3-9 has lower levels of strigolactones compared to the wild type and, because of lack of feedback regulation, the max2-1 mutant has higher strigolactone levels than both the wild type and max3-9 (Mashiguchi et al., 2009). The FNR-eGFP stromule marker gene was introduced into the max3-9 and max2-1 mutant background, and SF was quantified in hypocotyl cells of 3-d-old seedlings. As seen in Figure 3, the relative SF was significantly higher in max2-1 (ANOVA, LSD; n = 12, P # 0.001) and lower in max3-9 (ANOVA, LSD; n = 12, P = 0.02), compared to the SF in wild-type Arabidopsis. To determine whether the SF in the wild type, max2-1, and max3-9 is also affected in other cell types of these genotypes, the SF of hypocotyl cells was compared to those of root cells and cotyledon guard cells. Although the SF in guard cells
and roots cells show similar trends (higher in max2-1 and lower in max3-9), the differences with SF in the wild type in these cell types were not significantly different (Fig. 4A). Overall, the SF does seem to correlate with endogenous strigolactone levels, but the strength of the effect differs per cell type. Our results indicate that formation of stromules is possible in the absence of strigolactones but that conditions in a cell that produces strigolactones favor stromule formation. To confirm the relationship between strigolactones and stromule formation, we tested the effect of strigolactone application. For this, 1 h before imaging, seedlings of the strigolactone biosynthesis mutant max3-9 were submerged in solutions containing 0, 10, or 25 mM of GR24. Images of plastids were taken after 1 to 2 h, and SF in hypocotyl epidermal cells was determined. Indeed, exogenous applied strigolactones result in an increase of SF (Fig. 4B). To get evidence that the difference in SF in wild type, max2-1, and max3-9 depends on endogenous strigolactones, we subsequently tested the effect of a specific inhibitor of strigolactone biosynthesis D2 (López-Ráez et al., 2010). SF Is Reduced by CCD7 Inhibitor D2
Endogenous strigolactone biosynthesis is dependent on biosynthesis enzymes carotenoid cleavage dioxygenases CCD7 and CCD8, which can be inhibited by the compound D2 (López-Ráez et al., 2010). To demonstrate that the SF observed is dependent on the endogenous strigolactone biosynthesis, seedlings were treated with 5 mM of D2 or mock treated with 5 mL/mL acetone. For this, approximately 36 h prior to imaging, seedlings were transferred from solid medium with 0.53 MS to medium with 0.53 MS containing either 5 mM D2 or the mock treatment. In response to D2, the SF decreased significantly in Col-0 (ANOVA, LSD; Figure 3. Stromules in wild-type and SL mutants of Arabidopsis. Representative images of plastids and stromules in Arabidopsis hypocotyl epidermal cells in (A) Col-0, (B) max2-1, and (C) max3-9. D, The relative SF in hypocotyl epidermal cells of 3-d-old seedlings (24°C, 16 h light) in the three different genotypes. Col-0 (SF = 0.5 6 0.1; n = 12), max2-1 (SF = 0.12 6 0.02; n = 12), and max3-9 (SF = 0.01 6 0.005; n = 12). max2-1 has a significantly higher SF than both Col-0 (ANOVA, LSD; P , 0.001) and max3 (ANOVA, LSD; 0.02). max3 has also a significantly lower SF than Col-0 (ANOVA, LSD; P = 0.02). Statistical differences are indicated by A, B, and C. Error bars represent the SE.
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Stromule Induction by Low Phosphate Is Absent in Strigolactone Biosynthesis Mutant max3-9
Figure 4. Strigolactone GR24 results in increased stromule frequency. A, The SFs in several cell types and genotypes of 3-d-old seedlings. No significant differences are found in root cells nor guard cells, but the frequencies show similar trends as in hypocotyl epidermal cells. B, max3-9 seedlings grown for 2 d at 4°C in the dark, followed by 3 d at 24°C and 16 h of light. The appropriate concentrations of GR24 are applied 1 h before and during imaging. SF in max3-9 on different concentrations of the synthetic strigolactone GR24. A significant increase in SF is observed between 0 mM (SF = 0.02 6 0.004) and 10 mM (SF = 0.05 6 0.008), n = 10 and P = 0.033, ANOVA and LSD. An increase of 10 mM GR24 to 25 mM GR24 does not lead to a significant increase in the SF. Statistical differences are indicated by different letters. Error bars represent the SE.
n = 12, P = 0.043) and max2-1 (ANOVA, LSD; n = 12, P = 0.029), but not in max3-9, due to the already low endogenous SF in max3-9 (Fig. 5). Because results so far indicate that endogenous strigolactones are involved in stromule formation, we subsequently tested whether addition of exogenous strigolactones (GR24) can also increase SF in cells.
We showed that SF is significantly increased by lowphosphate conditions (Fig. 2). Therefore, we subsequently tested whether this induction requires endogenous strigolactone biosynthesis. Seedlings of the wild type, max2-1, and max3-9 were kept on medium with or without phosphate for 7 d, after which SF in hypocotyl cells was quantified. Results show that SF was increased in the wild type and in max2-1, but not in max3-9 (Fig. 7), indicating that the effect of low phosphate on SF requires active strigolactone biosynthesis. Representative images of plastids with stromules in the hypocotyl cells of the wild type, max2-1, and max3-9 under low phosphate are shown in Supplemental Figure S2. The fact that in the max2-1 background the SF also shows a significant increase suggests that under low phosphate the endogenous strigolactone biosynthesis may be higher in max2-1 than in max2-1 under normal phosphate conditions. This implies that induction of strigolactone biosynthesis under low phosphate occurs independent of MAX2 feedback regulation on strigolactone biosynthesis gene expression. Combined, the results indicate a clear role for strigolactones in the formation of plastidial stromules. Next, we determined whether the effect of strigolactones on stromule formation also occurs in other plant species. Strigolactones Also Involved in Stromule Formation in Tobacco
To study the relationship between strigolactones and stromule formation in another plant species, we used tobacco plants with a CaMV 35S-CT(RecA)S65TmGFP4nos expression construct, which targets GFP to the plastids’ stroma and thus labels stromules (Köhler et al., 1997). Because we do not have strigolactone mutants of tobacco, strigolactones levels were manipulated using GR24 or chemical inhibitors of endogenous strigolactone
Strigolactones Stimulate Stromule Formation Independent of MAX2
To test whether SF can be increased by addition of the synthetic strigolactone GR24, 3-d-old germinated seedlings of wild-type, max2-1, and max3-9 Arabidopsis plants were treated with GR24. For all three genotypes, the SF was significantly increased in response to GR24 (Fig. 6). In the wild type, SF increased from SF = 0.05 6 0.01 to SF = 0.13 6 0.04. Remarkably, SF also increased in max2-1 (from SF = 0.17 6 0.3 to SF = 0.29 6 0.05), indicating that this response to strigolactones does not require the conventional MAX2-dependent strigolactone signaling pathway. 2238
Figure 5. Three-day-old, 36-h, 5-mM D2-treated seedlings. A decrease in SF can be seen in both Col-0 (ANOVA, LSD; n = 12, P = 0.43) and max2-1 (ANOVA, LSD; n = 12, P = 0.029). Due to the low endogenous SF in max3-9 no decrease in SF is observed. Statistical differences are indicated by different letters. Error bars represent the SE. Plant Physiol. Vol. 172, 2016
Induction of Stromules Is Dependent on Strigolactones
Figure 6. Effect of synthetic strigolactone GR24 (10 mM) on stromule formation in hypocotyl epidermal cells of 3-d-old seedlings of wild-type Col-0, max2-1, and max3-9 mutant Arabidopsis. A significant increase in SF is seen for each genotype. n = 15 per treatment. Significance of the SF increase is determined using a t test: wild type, P , 0.001; max2-1, P = 0.038; max3-9, P = 0.002. Statistical differences are indicated by different letters. Error bars represent the SE.
total lipid content was determined and expressed as mol %. Results show that in response to low-phosphate conditions the relative level of some galactolipids is increased (DGDG and SQDG), while the level of different species of phospholipids is decreased (PI, PG, PE, and PC; Fig. 11). This confirms the exchange of phospholipids by galactolipids under low Pi conditions. However, there was no significant difference between the galactolipid-phospholipid exchange in the wild type, max2-1, and max3-9, indicating that the difference in stromule activity in these genotypes does not have a big influence on overall lipid exchange when measured after 10 d on low Pi. DISCUSSION Low-Phosphate Stimulation of Stromules Requires Strigolactones
Stromules Do Not Affect Long-Term GalactolipidPhospholipid Exchange
Induction of strigolactone biosynthesis is part of the response of plants to low-phosphate conditions. Lowphosphate conditions also results in the enhanced exchange of phospholipids by galactolipids, and it has been suggested the stromules may play a role in this exchange. Here, we have shown that low-phosphate stress in plants increases the frequency of stromule formation from plastids (Fig. 7). Moreover, we have shown that strigolactones play an important role in stromule formation by manipulating endogenous strigolactone biosynthesis either in mutants (max3-9, max2-1) by low-phosphate stress or by use of inhibitors of strigolactone biosynthesis (D2) or by adding exogenous GR24. In each case, an increase in strigolactone biosynthesis activity coincided with an increase in SF, while a decrease in strigolactone biosynthesis coincided with a decrease in SF. In wild-type tobacco seedlings 1 mM D2 was already sufficient to inhibit stromule formation (Fig. 10), but in Arabidopsis max2-9 seedlings, 5 mM D2 was needed to inhibit stromule formation (Fig. 5), most
With the low-phosphate treatment of wild-type Arabidopsis and the two strigolactone mutants we can now test if stromules function in the exchange of phospholipids by galactolipids for plants grown under low Pi conditions. Assuming a role of stromules in the lipid exchange under low phosphate and the different stromule activity of the wild type, max2-1, and max3-9 under low Pi conditions (Fig. 7), we predict that under low Pi galactolipid content in max2-1 may be higher and in max3-9 lower compared to that in the wild type. We therefore tested whether under low-phosphate conditions the phospholipid and galactolipid content differs between wild-type Arabidopsis (normal increase in stromule activity under low Pi) and max2-1 mutants (highest stromule activity under low Pi) and max3-9 mutants (no induction stromule activity under low Pi). Seven-day-old seedlings were grown for 10 d under normal or Pi limiting conditions after which lipids were extracted and quantified (see “Materials and Methods”). The relative contribution of individual lipid species to
Figure 7. Effect of low phosphate on the SF in hypocotyl epidermal cells of 7-d-old Arabidopsis seedlings. In the biosynthesis mutant max3, low phosphate does not increase the SF. The SF in Col-0 shows an increase of 0.058 6 0.016; n = 20, P = 0.001. The SF in max2 is increased by 0.16 6 0.018; n = 15, P # 0.001. Statistical differences are indicated by A, B, and C. Error bars represent the SE.
biosynthesis. First, we tested SF in different cell types (Supplemental Table S1), and for evaluation of manipulation of SF we used either the SF of cells in the elongation zone from the seedling root or SF in guard cells (Köhler et al., 1997). Subsequently, SF was quantified in response to minimal medium (Fig. 8), in response to exogenous applied synthetic strigolactone GR24 (Fig. 9) and in response to treatment with D2 (inhibitor of strigolactone biosynthesis; Fig. 10), Results indicate that strigolactones seem to have a similar function in the induction of stromules in tobacco: SF was increased when seedlings were grown on medium without micronutrients (Fig. 8), SF was increased by applying GR24 in dosage-dependent manner (Fig. 9), and SF was decreased by inhibitors of strigolactone biosynthesis (D2; Fig. 10).
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Figure 8. Mean SF in tobacco root epidermal cells of seedlings grown for 14 d on control (MS agar) or nutrient-poor (water agar) medium. SF is significantly increased from 0.29 6 0.02 in the control compared to 0.41 6 0.03 in the nutrient-poor-grown plants. n = 15, P = 0.008, t test. Statistical differences are indicated by A and B. Error bars represent the SE.
likely due to a higher strigolactone production in the max2-9 background. These results were further confirmed by experiments where exogenous strigolactones were supplied, which resulted in an increase is SF, both in Arabidopsis and in tobacco. For tobacco seedlings, we also tested effects of the inhibitor fluridone, which inhibits both SL and ABA biosynthesis. Results show that fluridone treatment leads to a dramatic decrease in SF in tobacco root cells (Supplemental Fig. S3). We note that 33 mM GR24 actually inhibits SF in tobacco guard cells (Fig. 9), even though this is close to the 25 mM that caused an increase in SF in Arabidopsis (Fig. 4). We suspect that differences in effects of GR24 on stromules between tobacco and Arabidopsis may be related to difference in sensitivity to the synthetic strigolactone GR24 in tobacco versus Arabidopsis, which may also relate to difference in uptake. Most importantly, besides an artificial manipulation of strigolactone levels by exogenous supplied GR24, also physiological manipulation of endogenous strigolactone levels by low-phosphate treatments indicate a relation between strigolactone levels and stromule formation (Figs. 6–9). Therefore, stromules are increasing in response to strigolactones from either endogenous or exogenous source.
protein, which then interacts with the SCFMAX2 complex to target SMAX type proteins for degradation. In future research, we will determine whether the known strigolactone receptor D14 protein is still required for the strigolactone-mediated induction of stromules. If so, this could indicate that upon binding of strigolactones, the D14 protein can also interact with another signaling pathway (e.g. another F-box protein besides MAX2). Multiple F-box proteins functioning in the same hormone-signaling pathway have also been shown for auxin signaling where both the F-box protein TIR1 and related AFB F-box proteins are involved in auxin perception. It is also possible that the effect of strigolactones on stromules represents an ancient strigolactone signaling response through KAI2, which was already present in chlorophytes and which precedes the development of a D14/MAX2 signaling in higher plants (Janssen and Snowden, 2012). Galactolipid-Phospholipid Exchange under Low PI Not Affected in max Mutants
One putative function of stromules may be to increase the surface area of chloroplasts to aid in the transport to and from the cytosol or organelles (Natesan et al., 2005). Although it was suggested that stromules may therefore be of importance in the low-phosphateresponse-related exchange of galactolipids and phospholipids, the results of our lipid measurements did not show an effect of the different SF in the wild type, max2-1, and max3-9 and phospholipid exchange. It is possible that stromules do participate in this process or even are required to mediate this process, but without mutants that are completely devoid of stromule formation, this hypothesis cannot currently not be tested. We note that stromules are always only in a fraction of the plastids, in hypocotyl epidermal cells varying from 0.2% to 25% of the plastids in the field of view. We did
Stimulation of Stromule Formation Does Not Require MAX2
Surprisingly, the correlation between endogenous strigolactone levels in the wild type and the max mutants and the SF was maintained in the max2-1 mutant (high endogenous strigolactone levels and high SF). Moreover, SF was also increased by addition of GR24 and by low-phosphate stress in the max2-1 mutant background, which supposedly increases endogenous strigolactone levels. Combined, these results indicate that the effect of strigolactones on SF does not require the conventional strigolactone signaling pathway component MAX2. Conventional strigolactone signaling is through binding of strigolactones to the D14 2240
Figure 9. Mean SF in tobacco guard cells during treatment with increasing GR24 concentrations. Significant increases in SF can be seen using 0.3 mM GR14, 0.02 6 0.01 to 0.23 6 0.05 (n = 15, P = 0.001; posthoc: LSD) and 3.3 mM GR24 from 0.02 6 0.01 to 0.77 6 0.07 (n = 15, P = 0.000; post-hoc: LSD) Statistical differences are indicated by A, B, and C. Error bars represent the SE. Plant Physiol. Vol. 172, 2016
Induction of Stromules Is Dependent on Strigolactones
Figure 10. Inhibition of stromule formation in tobacco root cells by the Strigolactone biosynthesis inhibitor D2. The SF at 0 mM D2 is 0.34 6 0.02 whereas at 1 and 5 mM D2 the average SF is 0.23 6 0.03 (n = 9, P = 0.008, post hoc: LSD). Statistical differences are indicated by A and B.
not do long-term imaging of the same cells to determine whether the population of plastids that form stromules is constant or that over time all plastids in a cell can form stromules. It is possible that only a subpopulation of plastids can form stromules and that the low-phosphate stress increases the stromule lifetime. Previous studies have shown that the dynamic behavior of stromules strongly coincides with that of cortical ER tubules (Schattat et al., 2011a, 2011b), which is in support of a role in exchange between plastid and other cellular membranes. It could be that the different SF in the wild type, max2-1, and max3-9 is reflected in different rates in lipid exchange, which after prolonged time reaches the same end equilibrium in phospholipids and galactolipids. The strong increase in SF upon low-phosphate stress suggests that this is an important part of the low-phosphate
response in plants. Recently, the low-phosphate growth response of the strigolactone signaling mutant max2-1 and strigolactone biosynthesis mutant max1 have been characterized (Ito et al., 2015). Assuming that the max1 mutant also has reduced stromule activity as seen in max3-9, these two mutants presumably show the same difference in intrinsic stromule activity as the max2-1 and max3-9 mutants. The morphological growth phenotypes that enable plants to maintain Pi homeostasis when grown under low-Pi conditions were similar in max2-1 and max1 (root hair elongation, induction of PHO1). This suggests that these lowphosphate-related phenotypes in max2-1 and max1 are not a function of presumed differences in stromule activity in max2-1 and max1. Moreover, all low-phosphaterelated phenotypes in max2-1 and max1 were shown to be dependent on MAX2 signaling, while no difference in overall phosphate content was detected between the max2-1 and max1 mutants. This would be in agreement with our measurements on phospholipid content in max2-1 and max3-9, which do not show significant differences either under normal growth or under low Pi conditions (Fig. 11). Reinterpreting the Effect of ABA on Stromule Formation
Stromules were shown to be induced by ABA in wheat (Triticum aestivum; Gray et al., 2012). Moreover, inhibition of stromule formation by norflurazon in wheat was interpreted as ABA being the causal agent of stromule formation (Gray et al., 2012). However, these results from Gray et al. (2012) can now also be explained by inhibition of strigolactone biosynthesis by Figure 11. Lipid composition of wild-type, max2-1, and max3-9 seedlings grown on medium with (+P) our without phosphate (2P). Error bars represent the SE. DGDG, Digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; PA, phosphatidic acid; PS, phosphatidyl serine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; SQDG, sulfoquinovosyldiacylglycerol.
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norflurazon and thus are consistent with our results. Gray et al. (2012) also used Abamine, a competitive inhibitor of 9-cis-epoxycarotenoid dioxygenase, which function in the ABA biosynthesis pathway, but not in the strigolactone biosynthesis pathway. Induction of stromules in wheat root hairs was blocked by Abamine; however, also here treatment was for 16 h, and results could thus be also be explained by an indirect effect of ABA biosynthesis on strigolactone biosynthesis. In our hands, ABA was also able to induce stromule activity within 4 h in Arabidopsis wild type and max2-1, but not in max 3-9 (Supplemental Fig. S1). Therefore, the results of Gray et al. (2012) can now be reinterpreted as indirect effects of ABA, which require active strigolactone biosynthesis. Indeed, a complex interaction between ABA and strigolactones has been observed before (López‐ Ráez et al., 2010). What Is the Role of Stromules in Plant Growth?
Although our results have shown that the induction of stromule formation by low-phosphate stress is absent in the max3-9 mutant and that stromule activity is extremely high in max2-1 under low phosphate, this difference is not yet clearly reflected in a difference in growth phenotype in these mutants, either under normal or under low-phosphate conditions. Stromule induction has been observed in relation to biotic stresses like plant viral infection (Krenz et al., 2012) and abiotic stresses like heat (Holzinger et al., 2007). In many instances, stromules were observed in close contact with nuclei (Kwok and Hanson, 2004; Holzinger et al., 2007; Caplan et al., 2015). In this context, stromules could have an important role in chloroplast-to-nucleus or chloroplast-to-cytoplasm retrograde signaling. Recently, it was shown that SF is increased by induction of ROS stress in chloroplasts and by a mutation in the plastidial protein NADPH-dependent thioredoxin reductase, which supposedly induces plastidial ROS stress (Brunkard et al., 2015). NADPH-dependent thioredoxin reductase is a central hub in chloroplast redox signaling pathways, and in future research we will determine whether the induction of stromules by plastidial ROS stress does require biosynthesis of strigolactones. However, at present, the function of stromules and their impact on the plant physiology remains to be determined. MATERIALS AND METHODS Plant Transformation For all experiments involving stromules in Arabidopsis (Arabidopsis thaliana; Col-0, max2-1, and max3-9), transgenic plants transformed with pGreenII0129::35S::FNRtp::EGFP were used, which were kindly provided by Schattat et al. (Marques et al., 2003, 2004). Plant transformation of Arabidopsis wild type (Col-0), max2-1, and max3-9 was with the standard floral-dip method (modified from Bechtold et al. [1993]). Seedlings from T0 germinated seed expressing the FNRtp-EGFP could be selected under UV light and transgenic lines with good expression of FNRtp-EGF were made homozygous. 2242
Plant Material and Growth Conditions For all experiments involving stromule visualization in Arabidopsis, we used transgenic Col-0, max2-1, and max3-9, expressing FNRtp-EGFP. Seeds were germinated on 0.53 MS medium containing 0.8% agar (Duchefa Bio) after stratification at 4°C in the dark for at least 48 h. After stratification, plates were transferred to a climate chamber kept at 24°C with a 16-h-light/9-h-dark photoperiod for 3 d. For the phosphate starvation experiment, the seeds were placed on 0.53 MS with or without phosphate and stratified at 4°C in the dark for 48 h. After stratification, the plates were transferred to a climate chamber kept at 24°C with a 16 h light/9 h dark photoperiod for 7 d rather than 3 d.
Arabidopsis Microscopy Sample Preparations GR24 Experiments For experiments using GR24, seedlings were grown as described. One hour before imaging, the seedlings were submerged in 100 mL perfluordecalin (PFD; Sigma-Aldrich) containing the concentration (0 , 10 , or 25 mM) of GR24 diluted from a stock solution in acetone. Images were taken between 1 and 2 h after submerging in PFD as indicated. The control seedlings were submerged in PFD containing 10 mL/mL acetone. For imaging, the seedlings were mounted in PFD supplemented with the corresponding concentration of GR24 or acetone.
D2 Experiments D2 is an inhibitor of CCDs and inhibits strigolactone biosynthesis but not ABA biosynthesis (López-Ráez et al., 2010). For the D2 experiments, seedlings were grown as described previously, with the exception that the 36-h-old seedlings approximately 36 h prior to imaging were transferred from 0.53 MS medium to 0.53 MS medium containing 0 or 5 mM D2. The control contained 0.5 mL/mL acetone. This resulted in 36 h treated, 3-d-old seedlings. Samples were mounted in PFD water. For imaging, seedlings were mounted in water.
Phosphate Experiment For the phosphate experiment, seedlings were grown on 0.53 MS + 0.8% agar with or without phosphate for 7 d at 24°C, 16 h light, after being stratified for 48 h at 4°C. During imaging, seedlings were mounted in water.
ABA Experiment The wild-type, max2-1, and max3-9 genotypes were infiltrated with 20 mM ABA in demineralized water, using a needleless syringe. Seedlings were grown as described previously. Six hours before imaging, the seedlings were vacuum infiltrated by pulling a vacuum in the syringe five times. After infiltration, seedlings were kept in the dark at 24°C.
Lipid Quantification Seeds of the wild type, max2-1, and max3-9 were germinated on 1% agar plates containing 0.53 MS (12 h light/12 h dark), and after 1 week, seedlings were transferred to 1% agar plate containing either 0.53 MS (including phosphate) or 0.53 MS without phosphate. After 2 weeks of growth on this new medium, lipids were extracted from the shoot tissue and lipid content was quantified. Lipids were extracted from frozen leaves and lipids separated by thin-layer chromatography (Dörmann et al., 1995). Fatty acids from isolated lipids were converted into methyl esters, which were quantified by gas chromatography using pentadecanoic acid (15:0) as the internal standard (Browse et al., 1986).
Tobacco (Nicotiana tabacum) GR24 Experiments To determine the optimal GR24 concentration, leaf discs were incubated in 100 mL PFD supplemented with GR24 1.5 h prior to imaging. Three different concentrations of GR24 (prepared using 3.3 mM stock solution in acetone) were used: 0.3, 3.3, and 33 mM. Samples used for control measurements were incubated in PFD supplemented with 10 mL/mL acetone. The leaf discs were mounted on a microscope slide in PFD supplemented with the corresponding concentration of GR24. Plant Physiol. Vol. 172, 2016
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Tobacco Fluridone/D2 Experiments
LITERATURE CITED
Tobacco seedlings were grown on MS agar medium. Thirty-six hours prior to imaging, the seedlings were transferred to fresh MS media either supplemented with the correct concentration of fluridone (1, 5, or 100 mM) or with no additional supplements (control treatment). During imaging, the roots were mounted in PFD. The same setup was used to test the effect of D2, a specific SL biosynthesis inhibitor. The D2 stock (27.6 mg/mL in acetone), kindly provided by Yanxia Zhang, was diluted to a final concentration of 1 mM. Again, seedlings grown on MS media for 2 weeks were transferred to fresh MS media supplemented with D2 (1 and 5 mM) 36 h prior to imaging. Other seedlings were transferred to fresh MS media with 0.5 mL/mL acetone to serve as a control.
Bechtold N, Ellis J, Pelletier G (1993). In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C R Acad Sci III 316: 1194–1199 Booker J, Auldridge M, Wills S, McCarty D, Klee H, Leyser O (2004) MAX3/CCD7 is a carotenoid cleavage dioxygenase required for the synthesis of a novel plant signaling molecule. Curr Biol 14: 1232– 1238 Browse J, McCourt PJ, Somerville CR (1986) Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Anal Biochem 152: 141–145 Brunkard JO, Runkel AM, Zambryski PC (2015) Chloroplasts extend stromules independently and in response to internal redox signals. Proc Natl Acad Sci USA 112: 10044–10049 Caplan JL, Kumar AS, Park E, Padmanabhan MS, Hoban K, Modla S, Czymmek K, Dinesh-Kumar SP (2015) Chloroplast stromules function during innate immunity. Dev Cell 34: 45–57 Dörmann P, Benning C (2002) Galactolipids rule in seed plants. Trends Plant Sci 7: 112–118 Dörmann P, Hoffmann-Benning S, Balbo I, Benning C (1995) Isolation and characterization of an Arabidopsis mutant deficient in the thylakoid lipid digalactosyl diacetylglycerol. Plant Cell 7: 1801–1810 Escobar NM, Haupt S, Thow G, Boevink P, Chapman S, Oparka K (2003) High-throughput viral expression of cDNA-green fluorescent protein fusions reveals novel subcellular addresses and identifies unique proteins that interact with plasmodesmata. Plant Cell 15: 1507–1523 Gray JC, Hansen MR, Shaw DJ, Graham K, Dale R, Smallman P, Natesan SK, Newell CA (2012) Plastid stromules are induced by stress treatments acting through abscisic acid. Plant J 69: 387–398 Hanson MR, Sattarzadeh A (2013) Trafficking of proteins through plastid stromules. Plant Cell 25: 2774–2782 Holzinger A, Buchner O, Lütz C, Hanson MR (2007) Temperature-sensitive formation of chloroplast protrusions and stromules in mesophyll cells of Arabidopsis thaliana. Protoplasma 230: 23–30 Holzinger A, Kwok EY, Hanson MR (2008) Effects of arc3, arc5 and arc6 mutations on plastid morphology and stromule formation in green and nongreen tissues of Arabidopsis thaliana. Photochem Photobiol 84: 1324–1335 Ito S, Nozoye T, Sasaki E, Imai M, Shiwa Y, Shibata-Hatta M, Ishige T, Fukui K, Ito K, Nakanishi H, et al (2015) Strigolactone regulates anthocyanin accumulation, acid phosphatases production and plant growth under low phosphate condition in Arabidopsis. PLoS One 10: e0119724 Janssen BJ, Snowden KC (2012) Strigolactone and karrikin signal perception: receptors, enzymes, or both? Front Plant Sci 3: 296 Köhler RH, Cao J, Zipfel WR, Webb WW, Hanson MR (1997) Exchange of protein molecules through connections between higher plant plastids. Science 276: 2039–2042 Köhler RH, Hanson MR (2000) Plastid tubules of higher plants are tissuespecific and developmentally regulated. J Cell Sci 113: 81–89 Krenz B, Jeske H, Kleinow T (2012) The induction of stromule formation by a plant DNA-virus in epidermal leaf tissues suggests a novel intra- and intercellular macromolecular trafficking route. Front Plant Sci 3: 291 Kwok EY, Hanson MR (2004) Plastids and stromules interact with the nucleus and cell membrane in vascular plants. Plant Cell Rep 23: 188– 195 Lambers H, Cawthray GR, Giavalisco P, Kuo J, Laliberté E, Pearse SJ, Scheible WR, Stitt M, Teste F, Turner BL (2012) Proteaceae from severely phosphorus-impoverished soils extensively replace phospholipids with galactolipids and sulfolipids during leaf development to achieve a high photosynthetic phosphorus-use-efficiency. New Phytol 196: 1098–1108 López-Ráez JA, Kohlen W, Charnikhova T, Mulder P, Undas AK, Sergeant MJ, Verstappen F, Bugg TD, Thompson AJ, Ruyter-Spira C, et al (2010) Does abscisic acid affect strigolactone biosynthesis? New Phytol 187: 343–354 Marques JP, Dudeck I, Klösgen RB (2003) Targeting of EGFP chimeras within chloroplasts. Mol Genet Genomics 269: 381–387 Marques JP, Schattat MH, Hause G, Dudeck I, Klösgen RB (2004) In vivo transport of folded EGFP by the DeltapH/TAT-dependent pathway in chloroplasts of Arabidopsis thaliana. J Exp Bot 55: 1697–1706
Fluorescence Microscopy and Image Processing Three- or seven-day-old Arabidopsis seedlings were imaged on a Nikon Eclipse Ti inverted microscope connected to a Roper Scientific spinning disk system, consisting of a CSU-X1 spinning disk head (Yokogawa), Evolve EM-CCD camera (Roper Scientific), and a 31.2 magnifying lens between the spinning disk head and the camera. GFP was excited using a 491-nm laser line. For imaging, we used a Plan Apo 603 oil immersion objective (NA 1.4). Metamorph imaging software was used to operate the microscope and collect images. Z-stacks of 10 slices with 0.5-mm intervals were collected from the hypocotyl epidermis. All image processing was performed using Fiji (ImageJ). Maximum intensity projections of these Z-stacks were made, and a threshold was applied to these images using the “analyze particle” plug-in. The threshold was applied by using the threshold tool in Fiji. The automatic threshold was manually adjusted to only highlight plastids that were in focus. The thresholding tool automatically inverts the image color to enhance contrast. Following application of the threshold, the analyze particles tool of Fiji was used to count the number of in-focus plastids. A minimum surface size of 40 pixels was used with no upper surface limit. Values that were clear image analysis artifacts were discarded from further measurements. SFs and stromule lengths were determined manually. The line drawing tool in Fiji was used to measure the length of the stromule. Plastid protrusions were classified as a stromule using the guidelines of Schattat et al. (2015). Briefly, only thin protrusions with a length of over 0.5 mm extending from a clearly wider plastid body were counted as a stromule. Thin protrusions between two plastid bodies were excluded, because no differentiation could be made between stromules or plastids in a stage of division. Representative images of stromules (and what is not counted as stromule) can be found in Supplemental Figure S2. For each experiment, at least ten plants per genotype per treatment were used, averaging to approximately 600 to 800 analyzed plastids for each genotype per treatment. To determine the SF, the number of stromules per picture is divided by the total number of plastids in the same picture. All error bars represent SEs, and significance was determined using the Student’s t test or a one-way ANOVA with LSD correction when more than two groups were compared using IBM SPSS statistics version 23.
Supplemental Data The following supplemental materials are available. Supplemental Figure S1. Stromule frequency in the wild type with and without added ABA. Supplemental Figure S2. Induction of stromules by GR24 in tobacco epidermal pavement cells. Supplemental Figure S3. Representative image of stromules and what is not counted as stromule. Supplemental Table S1. Stromule frequency in different cell types of tobacco.
ACKNOWLEDGMENT We thank Martin Schattat (Martin-Luther-University, Halle, Germany) for providing the FNRtp-EGFP construct and tobacco reporter plants. Received August 2, 2016; accepted October 13, 2016; published October 19, 2016. Plant Physiol. Vol. 172, 2016
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Mashiguchi K, Sasaki E, Shimada Y, Nagae M, Ueno K, Nakano T, Yoneyama K, Suzuki Y, Asami T (2009) Feedback-regulation of strigolactone biosynthetic genes and strigolactone-regulated genes in Arabidopsis. Biosci Biotechnol Biochem 73: 2460–2465 Natesan SKA, Sullivan JA, Gray JC (2005) Stromules: a characteristic cellspecific feature of plastid morphology. J Exp Bot 56: 787–797 Nelson DC, Scaffidi A, Dun EA, Waters MT, Flematti GR, Dixon KW, Beveridge CA, Ghisalberti EL, Smith SM (2011) F-box protein MAX2 has dual roles in karrikin and strigolactone signaling in Arabidopsis thaliana. Proc Natl Acad Sci USA 108: 8897–8902 Newell CA, Natesan SK, Sullivan JA, Jouhet J, Kavanagh TA, Gray JC (2012) Exclusion of plastid nucleoids and ribosomes from stromules in tobacco and Arabidopsis. Plant J 69: 399–410 Ruyter-Spira C, Al-Babili S, van der Krol S, Bouwmeester H (2013) The biology of strigolactones. Trends Plant Sci 18: 72–83 Ruyter-Spira C, Kohlen W, Charnikhova T, van Zeijl A, van Bezouwen L, de Ruijter N, Cardoso C, Lopez-Raez JA, Matusova R, Bours R, et al (2011) Physiological effects of the synthetic strigolactone analog GR24 on root system architecture in Arabidopsis: another belowground role for strigolactones? Plant Physiol 155: 721–734
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Schattat M, Barton K, Baudisch B, Klösgen RB, Mathur J (2011a) Plastid stromule branching coincides with contiguous endoplasmic reticulum dynamics. Plant Physiol 155: 1667–1677 Schattat M, Barton K, Mathur J (2011b) Correlated behavior implicates stromules in increasing the interactive surface between plastids and ER tubules. Plant Signal Behav 6: 715–718 Schattat MH, Barton KA, Mathur J (2015) The myth of interconnected plastids and related phenomena. Protoplasma 252: 359–371 Tjellström H, Andersson MX, Larsson KE, Sandelius AS (2008) Membrane phospholipids as a phosphate reserve: the dynamic nature of phospholipid-to-digalactosyl diacylglycerol exchange in higher plants. Plant Cell Environ 31: 1388–1398 Toh S, Kamiya Y, Kawakami N, Nambara E, McCourt P, Tsuchiya Y (2012) Thermoinhibition uncovers a role for strigolactones in Arabidopsis seed germination. Plant Cell Physiol 53: 107–117 Yoneyama K, Xie X, Kusumoto D, Sekimoto H, Sugimoto Y, Takeuchi Y, Yoneyama K (2007) Nitrogen deficiency as well as phosphorous deficiency in sorghum promotes the production and exudation of 5-deoxystrigol, the host recognition signal for arbuscular mycorrhizal fungi and root parasites. Planta 227: 125–132
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Stromules are highly dynamic protrusions of the plastids in plants. Several factors, such as drought and light conditions, influence the stromule frequency (SF) in a positive or negative way. A relatively recently discovered class of plant hormones are the strigolactones; strigolactones inhibit branching of the shoots and promote beneficial interactions between roots and arbuscular mycorrhizal fungi. Here, we investigate the link between the formation of stromules and strigolactones. This research shows a strong link between strigolactones and the formation of stromules: SF correlates with strigolactone levels in the wild type and strigolactone mutants (max2-1 max3-9), and SF is stimulated by strigolactone GR24 and reduced by strigolactone inhibitor D2.
Strigolactones are a class of phytohormones, which only recently have been recognized to function both in plant development and as signal molecules from plants to arbuscular mycorrhizal fungi (AMF; RuyterSpira et al., 2011). The induction of strigolactone biosynthesis and secretion by roots of plants under low-phosphate conditions is thought to stimulate the interaction with AMF, as strigolactones induce branching in AMF (Toh et al., 2012). The highly branched structure of the AMF filaments may aid the plant in the uptake of scarcely available soil phosphate. The biosynthesis and exudation of strigolactones is also strongly increased under low nutrient availability in Arabidopsis (Arabidopsis thaliana), even though Arabidopsis is not host to AMF (Yoneyama et al., 2007), indicating that strigolactones may have other roles in plant responses to low phosphate besides stimulation of AMF. Phosphate is essential for cell function as it is required for the
1 Part of this research was funded by STW project 13149 “Novel sustainable treatments for compact plants in greenhouse spaces.” * Address correspondence to [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Alexander van der Krol ([email protected]). T.K. and A.v.d.K. conceived the original research and supervised experiments; G.V., T.v.d.M., and O.L. performed most of the practical work and data analysis; H.P. and P.D. performed the phospholipid quantification; M.S. provided technical assistance; A.v.d.K. and G.V. wrote the article with contributions of all authors; H.B. supervised the project. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.16.01146
production of phospholipids, energy production, and regulation of protein activity, and much of the plant’s phosphate is stored in the phospholipids of the cell membranes. When phosphate availability is limiting for the plant cell, phosphor may be mobilized from membrane phospholipids, in which case the lipids are replaced by galactolipids derived from plastids (Tjellström et al., 2008; Lambers et al., 2012). This exchange of lipids between the plastid and other membranes occurs via the endoplasmic reticulum (ER) through plastid ER membrane contact sites (Dörmann and Benning, 2002; Tjellström et al., 2008), and indeed this frequency of these contact sites is increased under low Pi conditions (Tjellström et al., 2008). Stromules are highly dynamic tubular extensions that protrude from the plastid, filled with stroma and surrounded by the plastid double membrane. Thus, stromules give a large increase in the potential contact between plastid and other cell compartments. Indeed, stromules seem to be intercalated between ER membranes (Schattat et al., 2011a, 2011b), but stromules have also been observed in infoldings of the nuclear envelope (Kwok and Hanson, 2004). In a high-throughput screen of cDNA-GFP fusions, many proteins were found to be localized to the stroma of chloroplasts, but not all of them were also localized to stromules (Escobar et al., 2003). The formation of stromules is cell type specific and developmentally regulated (Köhler and Hanson, 2000). Although it has been suggested that stromules may function in the exchange of galactolipids from the plastid to other membranes under low-phosphate conditions, this has never been investigated. Because low-phosphate conditions stimulate the production of strigolactones
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and strigolactone biosynthesis starts in plastids, this raises the intriguing question whether strigolactones are causal to stromule formation. Here, we used Arabidopsis plants expressing FNRtp-EGFP, which labels stroma-filled stromules (Marques et al., 2003), to image stromule activity in relation to low phosphate and strigolactones in the wild type, the strigolactone signaling mutant max2-1 (Nelson et al., 2011), and the strigolactone biosynthesis mutant max3-9 (Booker et al., 2004). In addition, stromule activity was analyzed in tobacco (Nicotiana tabacum) seedlings expressing a CT(RecA)S65TmGFP4, which also labels plastids, including stromules (Köhler et al., 1997). The aim of the investigation was to determine (1) if there is a correlation between stromule activity and low-phosphate stress in plants and (2) if there is a relationship between the induction of strigolactones under low-phosphate stress and (putative) stromule formation under lowphosphate stress. Stromule formation was quantified in different cell types, and the stromule frequency (SF) in Arabidopsis seedling hypocotyl cells was used to study the effect of low phosphate and strigolactones. Results show a significant increase of SF in wild-type and max2-1 Arabidopsis seedlings when grown on lowphosphate medium, confirming that stromules are part of the low-phosphate response of plants. Moreover, frequency of stromule formation was not increased in the max3-9 background, indicating that strigolactone biosynthesis is required for the low-phosphate induction of stromules. Results were confirmed by induction of stromules by exogenous applied strigolactone GR24 in Arabidopsis and tobacco and by inhibition of stromule formation by inhibitors of strigolactone biosynthesis in wild-type Arabidopsis and tobacco. The role of strigolactones in stromule formation and phosphate metabolism in plants is discussed. RESULTS Different Stromule Frequency in Different Cell Types
The occurrence of stromules differs for different cell types in plants (Köhler and Hanson, 2000). Therefore, we first established growth and imaging conditions for reliable comparison of quantified SF in order to study the effect of different treatments. We used Arabidopsis plants in which stromules are labeled using a ferredoxin NADP(H) oxidoreductase (FNR) transit peptide fused to enhanced GFP (FNR-EGFP). For the chemical treatment experiments, freshly germinated 3-d-old seedlings were used, FNRtp-EGFP activity in plastids of root, hypocotyl, and leaf guard cells was imaged, and plastid shape and SF were measured. The SFs were studied in three different cell types: root epidermal cells, hypocotyl epidermal cells, and guard cells. In the three studied cell types in wild-type Arabidopsis seedlings, the SF is highest in root epidermal cells, with an SF of SF = 0.29 6 0.04. Guard cells have a lower SF (SF = 0.16 6 0.06), and hypocotyl epidermal cells have 2236
Figure 1. SFs differ per cell type. Col-0 seedlings were studied after 48 h of cold dark treatment followed by 3 d at 24°C with 16 h light. t test, error bars represent the SE. Statistical differences are indicated by A, B, and C. Hypocotyl epidermal cells (SF = 0.05 6 0.01, n = 12). Root epidermal cells (SF = 0.29 6 0.04; n = 12) and guard cells (SF = 0.16 6 0.06; n = 12).
the lowest SF (SF = 0.05 6 0.01; Fig. 1). SFs were similar to those previously reported for Arabidopsis (Holzinger et al., 2008; Newell et al., 2012). Because hypocotyl epidermal cells were used to study stromules in numerous previous publications (Hanson and Sattarzadeh, 2013; Kwok and Hanson, 2004; Holzinger, Kwok et al., 2008; Newell et al., 2012), we selected these cells to evaluate the effect of different treatments involving low phosphate, sugars, or strigolactones. SF Is Increased by Low-Phosphate Treatment
Next, we tested whether low-phosphate conditions result in an increase in SF. To observe the effect of low phosphate on plastid SF, germinated Arabidopsis seedlings were transferred to solid medium containing either 0.53 Murashige and Skoog (MS) with phosphate or 0.53 MS without phosphate. In order to deplete phosphate reserves in the seedlings, seedlings were kept on media for 7 d, after which SF in hypocotyl cells was quantified. In 7-d-old seedlings, the SF in hypocotyl cells was slightly lower compared than in 3-d-old seedlings (from SF = 0.05 6 0.01 at 3 d to SF = 0.027 6 0.01 at 7 d), indicating that in comparing SF not only cell type but also developmental stage should be taken into account. For hypocotyl cells in seedlings on medium without phosphate, the SF was increased to SF = 0.11 6 0.036 (Fig. 2), showing that indeed SF increases in response to low phosphate. Since low-phosphate conditions also result in an increase in strigolactone production, we next investigated whether there is a link between strigolactones and stromules. SF Relates to Endogenous SL Levels in Arabidopsis
To study the effect of strigolactones on SF, we made use of two types of strigolactone mutants: the max2-1 mutant, which is mutated in the F-box protein MAX2 and which lacks conventional D14/MAX2-dependent strigolactone signaling (Nelson et al., 2011), and the max3-9 mutant, which is mutated in the CCD9 gene and Plant Physiol. Vol. 172, 2016
Induction of Stromules Is Dependent on Strigolactones
Figure 2. Increase in SF under phosphate starvation. Col-0 seedlings placed in the cold and dark for 2 d, followed by 7 d at 24°C with 16 h light. Error bars represent the SE. Pi, SF = 0.027 6 0.01; n = 4. No Pi, SF = 0.11 6 0.036; n = 4. Phosphate-starved seedlings have an increased number of stromules (ANOVA, LSD; P = 0.045). Statistical differences are indicated by A and B.
therefore is blocked in biosynthesis of strigolactones. Compared to wild-type plants, these max mutants have different levels of endogenous of strigolactones: max3-9 has lower levels of strigolactones compared to the wild type and, because of lack of feedback regulation, the max2-1 mutant has higher strigolactone levels than both the wild type and max3-9 (Mashiguchi et al., 2009). The FNR-eGFP stromule marker gene was introduced into the max3-9 and max2-1 mutant background, and SF was quantified in hypocotyl cells of 3-d-old seedlings. As seen in Figure 3, the relative SF was significantly higher in max2-1 (ANOVA, LSD; n = 12, P # 0.001) and lower in max3-9 (ANOVA, LSD; n = 12, P = 0.02), compared to the SF in wild-type Arabidopsis. To determine whether the SF in the wild type, max2-1, and max3-9 is also affected in other cell types of these genotypes, the SF of hypocotyl cells was compared to those of root cells and cotyledon guard cells. Although the SF in guard cells
and roots cells show similar trends (higher in max2-1 and lower in max3-9), the differences with SF in the wild type in these cell types were not significantly different (Fig. 4A). Overall, the SF does seem to correlate with endogenous strigolactone levels, but the strength of the effect differs per cell type. Our results indicate that formation of stromules is possible in the absence of strigolactones but that conditions in a cell that produces strigolactones favor stromule formation. To confirm the relationship between strigolactones and stromule formation, we tested the effect of strigolactone application. For this, 1 h before imaging, seedlings of the strigolactone biosynthesis mutant max3-9 were submerged in solutions containing 0, 10, or 25 mM of GR24. Images of plastids were taken after 1 to 2 h, and SF in hypocotyl epidermal cells was determined. Indeed, exogenous applied strigolactones result in an increase of SF (Fig. 4B). To get evidence that the difference in SF in wild type, max2-1, and max3-9 depends on endogenous strigolactones, we subsequently tested the effect of a specific inhibitor of strigolactone biosynthesis D2 (López-Ráez et al., 2010). SF Is Reduced by CCD7 Inhibitor D2
Endogenous strigolactone biosynthesis is dependent on biosynthesis enzymes carotenoid cleavage dioxygenases CCD7 and CCD8, which can be inhibited by the compound D2 (López-Ráez et al., 2010). To demonstrate that the SF observed is dependent on the endogenous strigolactone biosynthesis, seedlings were treated with 5 mM of D2 or mock treated with 5 mL/mL acetone. For this, approximately 36 h prior to imaging, seedlings were transferred from solid medium with 0.53 MS to medium with 0.53 MS containing either 5 mM D2 or the mock treatment. In response to D2, the SF decreased significantly in Col-0 (ANOVA, LSD; Figure 3. Stromules in wild-type and SL mutants of Arabidopsis. Representative images of plastids and stromules in Arabidopsis hypocotyl epidermal cells in (A) Col-0, (B) max2-1, and (C) max3-9. D, The relative SF in hypocotyl epidermal cells of 3-d-old seedlings (24°C, 16 h light) in the three different genotypes. Col-0 (SF = 0.5 6 0.1; n = 12), max2-1 (SF = 0.12 6 0.02; n = 12), and max3-9 (SF = 0.01 6 0.005; n = 12). max2-1 has a significantly higher SF than both Col-0 (ANOVA, LSD; P , 0.001) and max3 (ANOVA, LSD; 0.02). max3 has also a significantly lower SF than Col-0 (ANOVA, LSD; P = 0.02). Statistical differences are indicated by A, B, and C. Error bars represent the SE.
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Stromule Induction by Low Phosphate Is Absent in Strigolactone Biosynthesis Mutant max3-9
Figure 4. Strigolactone GR24 results in increased stromule frequency. A, The SFs in several cell types and genotypes of 3-d-old seedlings. No significant differences are found in root cells nor guard cells, but the frequencies show similar trends as in hypocotyl epidermal cells. B, max3-9 seedlings grown for 2 d at 4°C in the dark, followed by 3 d at 24°C and 16 h of light. The appropriate concentrations of GR24 are applied 1 h before and during imaging. SF in max3-9 on different concentrations of the synthetic strigolactone GR24. A significant increase in SF is observed between 0 mM (SF = 0.02 6 0.004) and 10 mM (SF = 0.05 6 0.008), n = 10 and P = 0.033, ANOVA and LSD. An increase of 10 mM GR24 to 25 mM GR24 does not lead to a significant increase in the SF. Statistical differences are indicated by different letters. Error bars represent the SE.
n = 12, P = 0.043) and max2-1 (ANOVA, LSD; n = 12, P = 0.029), but not in max3-9, due to the already low endogenous SF in max3-9 (Fig. 5). Because results so far indicate that endogenous strigolactones are involved in stromule formation, we subsequently tested whether addition of exogenous strigolactones (GR24) can also increase SF in cells.
We showed that SF is significantly increased by lowphosphate conditions (Fig. 2). Therefore, we subsequently tested whether this induction requires endogenous strigolactone biosynthesis. Seedlings of the wild type, max2-1, and max3-9 were kept on medium with or without phosphate for 7 d, after which SF in hypocotyl cells was quantified. Results show that SF was increased in the wild type and in max2-1, but not in max3-9 (Fig. 7), indicating that the effect of low phosphate on SF requires active strigolactone biosynthesis. Representative images of plastids with stromules in the hypocotyl cells of the wild type, max2-1, and max3-9 under low phosphate are shown in Supplemental Figure S2. The fact that in the max2-1 background the SF also shows a significant increase suggests that under low phosphate the endogenous strigolactone biosynthesis may be higher in max2-1 than in max2-1 under normal phosphate conditions. This implies that induction of strigolactone biosynthesis under low phosphate occurs independent of MAX2 feedback regulation on strigolactone biosynthesis gene expression. Combined, the results indicate a clear role for strigolactones in the formation of plastidial stromules. Next, we determined whether the effect of strigolactones on stromule formation also occurs in other plant species. Strigolactones Also Involved in Stromule Formation in Tobacco
To study the relationship between strigolactones and stromule formation in another plant species, we used tobacco plants with a CaMV 35S-CT(RecA)S65TmGFP4nos expression construct, which targets GFP to the plastids’ stroma and thus labels stromules (Köhler et al., 1997). Because we do not have strigolactone mutants of tobacco, strigolactones levels were manipulated using GR24 or chemical inhibitors of endogenous strigolactone
Strigolactones Stimulate Stromule Formation Independent of MAX2
To test whether SF can be increased by addition of the synthetic strigolactone GR24, 3-d-old germinated seedlings of wild-type, max2-1, and max3-9 Arabidopsis plants were treated with GR24. For all three genotypes, the SF was significantly increased in response to GR24 (Fig. 6). In the wild type, SF increased from SF = 0.05 6 0.01 to SF = 0.13 6 0.04. Remarkably, SF also increased in max2-1 (from SF = 0.17 6 0.3 to SF = 0.29 6 0.05), indicating that this response to strigolactones does not require the conventional MAX2-dependent strigolactone signaling pathway. 2238
Figure 5. Three-day-old, 36-h, 5-mM D2-treated seedlings. A decrease in SF can be seen in both Col-0 (ANOVA, LSD; n = 12, P = 0.43) and max2-1 (ANOVA, LSD; n = 12, P = 0.029). Due to the low endogenous SF in max3-9 no decrease in SF is observed. Statistical differences are indicated by different letters. Error bars represent the SE. Plant Physiol. Vol. 172, 2016
Induction of Stromules Is Dependent on Strigolactones
Figure 6. Effect of synthetic strigolactone GR24 (10 mM) on stromule formation in hypocotyl epidermal cells of 3-d-old seedlings of wild-type Col-0, max2-1, and max3-9 mutant Arabidopsis. A significant increase in SF is seen for each genotype. n = 15 per treatment. Significance of the SF increase is determined using a t test: wild type, P , 0.001; max2-1, P = 0.038; max3-9, P = 0.002. Statistical differences are indicated by different letters. Error bars represent the SE.
total lipid content was determined and expressed as mol %. Results show that in response to low-phosphate conditions the relative level of some galactolipids is increased (DGDG and SQDG), while the level of different species of phospholipids is decreased (PI, PG, PE, and PC; Fig. 11). This confirms the exchange of phospholipids by galactolipids under low Pi conditions. However, there was no significant difference between the galactolipid-phospholipid exchange in the wild type, max2-1, and max3-9, indicating that the difference in stromule activity in these genotypes does not have a big influence on overall lipid exchange when measured after 10 d on low Pi. DISCUSSION Low-Phosphate Stimulation of Stromules Requires Strigolactones
Stromules Do Not Affect Long-Term GalactolipidPhospholipid Exchange
Induction of strigolactone biosynthesis is part of the response of plants to low-phosphate conditions. Lowphosphate conditions also results in the enhanced exchange of phospholipids by galactolipids, and it has been suggested the stromules may play a role in this exchange. Here, we have shown that low-phosphate stress in plants increases the frequency of stromule formation from plastids (Fig. 7). Moreover, we have shown that strigolactones play an important role in stromule formation by manipulating endogenous strigolactone biosynthesis either in mutants (max3-9, max2-1) by low-phosphate stress or by use of inhibitors of strigolactone biosynthesis (D2) or by adding exogenous GR24. In each case, an increase in strigolactone biosynthesis activity coincided with an increase in SF, while a decrease in strigolactone biosynthesis coincided with a decrease in SF. In wild-type tobacco seedlings 1 mM D2 was already sufficient to inhibit stromule formation (Fig. 10), but in Arabidopsis max2-9 seedlings, 5 mM D2 was needed to inhibit stromule formation (Fig. 5), most
With the low-phosphate treatment of wild-type Arabidopsis and the two strigolactone mutants we can now test if stromules function in the exchange of phospholipids by galactolipids for plants grown under low Pi conditions. Assuming a role of stromules in the lipid exchange under low phosphate and the different stromule activity of the wild type, max2-1, and max3-9 under low Pi conditions (Fig. 7), we predict that under low Pi galactolipid content in max2-1 may be higher and in max3-9 lower compared to that in the wild type. We therefore tested whether under low-phosphate conditions the phospholipid and galactolipid content differs between wild-type Arabidopsis (normal increase in stromule activity under low Pi) and max2-1 mutants (highest stromule activity under low Pi) and max3-9 mutants (no induction stromule activity under low Pi). Seven-day-old seedlings were grown for 10 d under normal or Pi limiting conditions after which lipids were extracted and quantified (see “Materials and Methods”). The relative contribution of individual lipid species to
Figure 7. Effect of low phosphate on the SF in hypocotyl epidermal cells of 7-d-old Arabidopsis seedlings. In the biosynthesis mutant max3, low phosphate does not increase the SF. The SF in Col-0 shows an increase of 0.058 6 0.016; n = 20, P = 0.001. The SF in max2 is increased by 0.16 6 0.018; n = 15, P # 0.001. Statistical differences are indicated by A, B, and C. Error bars represent the SE.
biosynthesis. First, we tested SF in different cell types (Supplemental Table S1), and for evaluation of manipulation of SF we used either the SF of cells in the elongation zone from the seedling root or SF in guard cells (Köhler et al., 1997). Subsequently, SF was quantified in response to minimal medium (Fig. 8), in response to exogenous applied synthetic strigolactone GR24 (Fig. 9) and in response to treatment with D2 (inhibitor of strigolactone biosynthesis; Fig. 10), Results indicate that strigolactones seem to have a similar function in the induction of stromules in tobacco: SF was increased when seedlings were grown on medium without micronutrients (Fig. 8), SF was increased by applying GR24 in dosage-dependent manner (Fig. 9), and SF was decreased by inhibitors of strigolactone biosynthesis (D2; Fig. 10).
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Figure 8. Mean SF in tobacco root epidermal cells of seedlings grown for 14 d on control (MS agar) or nutrient-poor (water agar) medium. SF is significantly increased from 0.29 6 0.02 in the control compared to 0.41 6 0.03 in the nutrient-poor-grown plants. n = 15, P = 0.008, t test. Statistical differences are indicated by A and B. Error bars represent the SE.
likely due to a higher strigolactone production in the max2-9 background. These results were further confirmed by experiments where exogenous strigolactones were supplied, which resulted in an increase is SF, both in Arabidopsis and in tobacco. For tobacco seedlings, we also tested effects of the inhibitor fluridone, which inhibits both SL and ABA biosynthesis. Results show that fluridone treatment leads to a dramatic decrease in SF in tobacco root cells (Supplemental Fig. S3). We note that 33 mM GR24 actually inhibits SF in tobacco guard cells (Fig. 9), even though this is close to the 25 mM that caused an increase in SF in Arabidopsis (Fig. 4). We suspect that differences in effects of GR24 on stromules between tobacco and Arabidopsis may be related to difference in sensitivity to the synthetic strigolactone GR24 in tobacco versus Arabidopsis, which may also relate to difference in uptake. Most importantly, besides an artificial manipulation of strigolactone levels by exogenous supplied GR24, also physiological manipulation of endogenous strigolactone levels by low-phosphate treatments indicate a relation between strigolactone levels and stromule formation (Figs. 6–9). Therefore, stromules are increasing in response to strigolactones from either endogenous or exogenous source.
protein, which then interacts with the SCFMAX2 complex to target SMAX type proteins for degradation. In future research, we will determine whether the known strigolactone receptor D14 protein is still required for the strigolactone-mediated induction of stromules. If so, this could indicate that upon binding of strigolactones, the D14 protein can also interact with another signaling pathway (e.g. another F-box protein besides MAX2). Multiple F-box proteins functioning in the same hormone-signaling pathway have also been shown for auxin signaling where both the F-box protein TIR1 and related AFB F-box proteins are involved in auxin perception. It is also possible that the effect of strigolactones on stromules represents an ancient strigolactone signaling response through KAI2, which was already present in chlorophytes and which precedes the development of a D14/MAX2 signaling in higher plants (Janssen and Snowden, 2012). Galactolipid-Phospholipid Exchange under Low PI Not Affected in max Mutants
One putative function of stromules may be to increase the surface area of chloroplasts to aid in the transport to and from the cytosol or organelles (Natesan et al., 2005). Although it was suggested that stromules may therefore be of importance in the low-phosphateresponse-related exchange of galactolipids and phospholipids, the results of our lipid measurements did not show an effect of the different SF in the wild type, max2-1, and max3-9 and phospholipid exchange. It is possible that stromules do participate in this process or even are required to mediate this process, but without mutants that are completely devoid of stromule formation, this hypothesis cannot currently not be tested. We note that stromules are always only in a fraction of the plastids, in hypocotyl epidermal cells varying from 0.2% to 25% of the plastids in the field of view. We did
Stimulation of Stromule Formation Does Not Require MAX2
Surprisingly, the correlation between endogenous strigolactone levels in the wild type and the max mutants and the SF was maintained in the max2-1 mutant (high endogenous strigolactone levels and high SF). Moreover, SF was also increased by addition of GR24 and by low-phosphate stress in the max2-1 mutant background, which supposedly increases endogenous strigolactone levels. Combined, these results indicate that the effect of strigolactones on SF does not require the conventional strigolactone signaling pathway component MAX2. Conventional strigolactone signaling is through binding of strigolactones to the D14 2240
Figure 9. Mean SF in tobacco guard cells during treatment with increasing GR24 concentrations. Significant increases in SF can be seen using 0.3 mM GR14, 0.02 6 0.01 to 0.23 6 0.05 (n = 15, P = 0.001; posthoc: LSD) and 3.3 mM GR24 from 0.02 6 0.01 to 0.77 6 0.07 (n = 15, P = 0.000; post-hoc: LSD) Statistical differences are indicated by A, B, and C. Error bars represent the SE. Plant Physiol. Vol. 172, 2016
Induction of Stromules Is Dependent on Strigolactones
Figure 10. Inhibition of stromule formation in tobacco root cells by the Strigolactone biosynthesis inhibitor D2. The SF at 0 mM D2 is 0.34 6 0.02 whereas at 1 and 5 mM D2 the average SF is 0.23 6 0.03 (n = 9, P = 0.008, post hoc: LSD). Statistical differences are indicated by A and B.
not do long-term imaging of the same cells to determine whether the population of plastids that form stromules is constant or that over time all plastids in a cell can form stromules. It is possible that only a subpopulation of plastids can form stromules and that the low-phosphate stress increases the stromule lifetime. Previous studies have shown that the dynamic behavior of stromules strongly coincides with that of cortical ER tubules (Schattat et al., 2011a, 2011b), which is in support of a role in exchange between plastid and other cellular membranes. It could be that the different SF in the wild type, max2-1, and max3-9 is reflected in different rates in lipid exchange, which after prolonged time reaches the same end equilibrium in phospholipids and galactolipids. The strong increase in SF upon low-phosphate stress suggests that this is an important part of the low-phosphate
response in plants. Recently, the low-phosphate growth response of the strigolactone signaling mutant max2-1 and strigolactone biosynthesis mutant max1 have been characterized (Ito et al., 2015). Assuming that the max1 mutant also has reduced stromule activity as seen in max3-9, these two mutants presumably show the same difference in intrinsic stromule activity as the max2-1 and max3-9 mutants. The morphological growth phenotypes that enable plants to maintain Pi homeostasis when grown under low-Pi conditions were similar in max2-1 and max1 (root hair elongation, induction of PHO1). This suggests that these lowphosphate-related phenotypes in max2-1 and max1 are not a function of presumed differences in stromule activity in max2-1 and max1. Moreover, all low-phosphaterelated phenotypes in max2-1 and max1 were shown to be dependent on MAX2 signaling, while no difference in overall phosphate content was detected between the max2-1 and max1 mutants. This would be in agreement with our measurements on phospholipid content in max2-1 and max3-9, which do not show significant differences either under normal growth or under low Pi conditions (Fig. 11). Reinterpreting the Effect of ABA on Stromule Formation
Stromules were shown to be induced by ABA in wheat (Triticum aestivum; Gray et al., 2012). Moreover, inhibition of stromule formation by norflurazon in wheat was interpreted as ABA being the causal agent of stromule formation (Gray et al., 2012). However, these results from Gray et al. (2012) can now also be explained by inhibition of strigolactone biosynthesis by Figure 11. Lipid composition of wild-type, max2-1, and max3-9 seedlings grown on medium with (+P) our without phosphate (2P). Error bars represent the SE. DGDG, Digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; PA, phosphatidic acid; PS, phosphatidyl serine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; SQDG, sulfoquinovosyldiacylglycerol.
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norflurazon and thus are consistent with our results. Gray et al. (2012) also used Abamine, a competitive inhibitor of 9-cis-epoxycarotenoid dioxygenase, which function in the ABA biosynthesis pathway, but not in the strigolactone biosynthesis pathway. Induction of stromules in wheat root hairs was blocked by Abamine; however, also here treatment was for 16 h, and results could thus be also be explained by an indirect effect of ABA biosynthesis on strigolactone biosynthesis. In our hands, ABA was also able to induce stromule activity within 4 h in Arabidopsis wild type and max2-1, but not in max 3-9 (Supplemental Fig. S1). Therefore, the results of Gray et al. (2012) can now be reinterpreted as indirect effects of ABA, which require active strigolactone biosynthesis. Indeed, a complex interaction between ABA and strigolactones has been observed before (López‐ Ráez et al., 2010). What Is the Role of Stromules in Plant Growth?
Although our results have shown that the induction of stromule formation by low-phosphate stress is absent in the max3-9 mutant and that stromule activity is extremely high in max2-1 under low phosphate, this difference is not yet clearly reflected in a difference in growth phenotype in these mutants, either under normal or under low-phosphate conditions. Stromule induction has been observed in relation to biotic stresses like plant viral infection (Krenz et al., 2012) and abiotic stresses like heat (Holzinger et al., 2007). In many instances, stromules were observed in close contact with nuclei (Kwok and Hanson, 2004; Holzinger et al., 2007; Caplan et al., 2015). In this context, stromules could have an important role in chloroplast-to-nucleus or chloroplast-to-cytoplasm retrograde signaling. Recently, it was shown that SF is increased by induction of ROS stress in chloroplasts and by a mutation in the plastidial protein NADPH-dependent thioredoxin reductase, which supposedly induces plastidial ROS stress (Brunkard et al., 2015). NADPH-dependent thioredoxin reductase is a central hub in chloroplast redox signaling pathways, and in future research we will determine whether the induction of stromules by plastidial ROS stress does require biosynthesis of strigolactones. However, at present, the function of stromules and their impact on the plant physiology remains to be determined. MATERIALS AND METHODS Plant Transformation For all experiments involving stromules in Arabidopsis (Arabidopsis thaliana; Col-0, max2-1, and max3-9), transgenic plants transformed with pGreenII0129::35S::FNRtp::EGFP were used, which were kindly provided by Schattat et al. (Marques et al., 2003, 2004). Plant transformation of Arabidopsis wild type (Col-0), max2-1, and max3-9 was with the standard floral-dip method (modified from Bechtold et al. [1993]). Seedlings from T0 germinated seed expressing the FNRtp-EGFP could be selected under UV light and transgenic lines with good expression of FNRtp-EGF were made homozygous. 2242
Plant Material and Growth Conditions For all experiments involving stromule visualization in Arabidopsis, we used transgenic Col-0, max2-1, and max3-9, expressing FNRtp-EGFP. Seeds were germinated on 0.53 MS medium containing 0.8% agar (Duchefa Bio) after stratification at 4°C in the dark for at least 48 h. After stratification, plates were transferred to a climate chamber kept at 24°C with a 16-h-light/9-h-dark photoperiod for 3 d. For the phosphate starvation experiment, the seeds were placed on 0.53 MS with or without phosphate and stratified at 4°C in the dark for 48 h. After stratification, the plates were transferred to a climate chamber kept at 24°C with a 16 h light/9 h dark photoperiod for 7 d rather than 3 d.
Arabidopsis Microscopy Sample Preparations GR24 Experiments For experiments using GR24, seedlings were grown as described. One hour before imaging, the seedlings were submerged in 100 mL perfluordecalin (PFD; Sigma-Aldrich) containing the concentration (0 , 10 , or 25 mM) of GR24 diluted from a stock solution in acetone. Images were taken between 1 and 2 h after submerging in PFD as indicated. The control seedlings were submerged in PFD containing 10 mL/mL acetone. For imaging, the seedlings were mounted in PFD supplemented with the corresponding concentration of GR24 or acetone.
D2 Experiments D2 is an inhibitor of CCDs and inhibits strigolactone biosynthesis but not ABA biosynthesis (López-Ráez et al., 2010). For the D2 experiments, seedlings were grown as described previously, with the exception that the 36-h-old seedlings approximately 36 h prior to imaging were transferred from 0.53 MS medium to 0.53 MS medium containing 0 or 5 mM D2. The control contained 0.5 mL/mL acetone. This resulted in 36 h treated, 3-d-old seedlings. Samples were mounted in PFD water. For imaging, seedlings were mounted in water.
Phosphate Experiment For the phosphate experiment, seedlings were grown on 0.53 MS + 0.8% agar with or without phosphate for 7 d at 24°C, 16 h light, after being stratified for 48 h at 4°C. During imaging, seedlings were mounted in water.
ABA Experiment The wild-type, max2-1, and max3-9 genotypes were infiltrated with 20 mM ABA in demineralized water, using a needleless syringe. Seedlings were grown as described previously. Six hours before imaging, the seedlings were vacuum infiltrated by pulling a vacuum in the syringe five times. After infiltration, seedlings were kept in the dark at 24°C.
Lipid Quantification Seeds of the wild type, max2-1, and max3-9 were germinated on 1% agar plates containing 0.53 MS (12 h light/12 h dark), and after 1 week, seedlings were transferred to 1% agar plate containing either 0.53 MS (including phosphate) or 0.53 MS without phosphate. After 2 weeks of growth on this new medium, lipids were extracted from the shoot tissue and lipid content was quantified. Lipids were extracted from frozen leaves and lipids separated by thin-layer chromatography (Dörmann et al., 1995). Fatty acids from isolated lipids were converted into methyl esters, which were quantified by gas chromatography using pentadecanoic acid (15:0) as the internal standard (Browse et al., 1986).
Tobacco (Nicotiana tabacum) GR24 Experiments To determine the optimal GR24 concentration, leaf discs were incubated in 100 mL PFD supplemented with GR24 1.5 h prior to imaging. Three different concentrations of GR24 (prepared using 3.3 mM stock solution in acetone) were used: 0.3, 3.3, and 33 mM. Samples used for control measurements were incubated in PFD supplemented with 10 mL/mL acetone. The leaf discs were mounted on a microscope slide in PFD supplemented with the corresponding concentration of GR24. Plant Physiol. Vol. 172, 2016
Induction of Stromules Is Dependent on Strigolactones
Tobacco Fluridone/D2 Experiments
LITERATURE CITED
Tobacco seedlings were grown on MS agar medium. Thirty-six hours prior to imaging, the seedlings were transferred to fresh MS media either supplemented with the correct concentration of fluridone (1, 5, or 100 mM) or with no additional supplements (control treatment). During imaging, the roots were mounted in PFD. The same setup was used to test the effect of D2, a specific SL biosynthesis inhibitor. The D2 stock (27.6 mg/mL in acetone), kindly provided by Yanxia Zhang, was diluted to a final concentration of 1 mM. Again, seedlings grown on MS media for 2 weeks were transferred to fresh MS media supplemented with D2 (1 and 5 mM) 36 h prior to imaging. Other seedlings were transferred to fresh MS media with 0.5 mL/mL acetone to serve as a control.
Bechtold N, Ellis J, Pelletier G (1993). In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C R Acad Sci III 316: 1194–1199 Booker J, Auldridge M, Wills S, McCarty D, Klee H, Leyser O (2004) MAX3/CCD7 is a carotenoid cleavage dioxygenase required for the synthesis of a novel plant signaling molecule. Curr Biol 14: 1232– 1238 Browse J, McCourt PJ, Somerville CR (1986) Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Anal Biochem 152: 141–145 Brunkard JO, Runkel AM, Zambryski PC (2015) Chloroplasts extend stromules independently and in response to internal redox signals. Proc Natl Acad Sci USA 112: 10044–10049 Caplan JL, Kumar AS, Park E, Padmanabhan MS, Hoban K, Modla S, Czymmek K, Dinesh-Kumar SP (2015) Chloroplast stromules function during innate immunity. Dev Cell 34: 45–57 Dörmann P, Benning C (2002) Galactolipids rule in seed plants. Trends Plant Sci 7: 112–118 Dörmann P, Hoffmann-Benning S, Balbo I, Benning C (1995) Isolation and characterization of an Arabidopsis mutant deficient in the thylakoid lipid digalactosyl diacetylglycerol. Plant Cell 7: 1801–1810 Escobar NM, Haupt S, Thow G, Boevink P, Chapman S, Oparka K (2003) High-throughput viral expression of cDNA-green fluorescent protein fusions reveals novel subcellular addresses and identifies unique proteins that interact with plasmodesmata. Plant Cell 15: 1507–1523 Gray JC, Hansen MR, Shaw DJ, Graham K, Dale R, Smallman P, Natesan SK, Newell CA (2012) Plastid stromules are induced by stress treatments acting through abscisic acid. Plant J 69: 387–398 Hanson MR, Sattarzadeh A (2013) Trafficking of proteins through plastid stromules. Plant Cell 25: 2774–2782 Holzinger A, Buchner O, Lütz C, Hanson MR (2007) Temperature-sensitive formation of chloroplast protrusions and stromules in mesophyll cells of Arabidopsis thaliana. Protoplasma 230: 23–30 Holzinger A, Kwok EY, Hanson MR (2008) Effects of arc3, arc5 and arc6 mutations on plastid morphology and stromule formation in green and nongreen tissues of Arabidopsis thaliana. Photochem Photobiol 84: 1324–1335 Ito S, Nozoye T, Sasaki E, Imai M, Shiwa Y, Shibata-Hatta M, Ishige T, Fukui K, Ito K, Nakanishi H, et al (2015) Strigolactone regulates anthocyanin accumulation, acid phosphatases production and plant growth under low phosphate condition in Arabidopsis. PLoS One 10: e0119724 Janssen BJ, Snowden KC (2012) Strigolactone and karrikin signal perception: receptors, enzymes, or both? Front Plant Sci 3: 296 Köhler RH, Cao J, Zipfel WR, Webb WW, Hanson MR (1997) Exchange of protein molecules through connections between higher plant plastids. Science 276: 2039–2042 Köhler RH, Hanson MR (2000) Plastid tubules of higher plants are tissuespecific and developmentally regulated. J Cell Sci 113: 81–89 Krenz B, Jeske H, Kleinow T (2012) The induction of stromule formation by a plant DNA-virus in epidermal leaf tissues suggests a novel intra- and intercellular macromolecular trafficking route. Front Plant Sci 3: 291 Kwok EY, Hanson MR (2004) Plastids and stromules interact with the nucleus and cell membrane in vascular plants. Plant Cell Rep 23: 188– 195 Lambers H, Cawthray GR, Giavalisco P, Kuo J, Laliberté E, Pearse SJ, Scheible WR, Stitt M, Teste F, Turner BL (2012) Proteaceae from severely phosphorus-impoverished soils extensively replace phospholipids with galactolipids and sulfolipids during leaf development to achieve a high photosynthetic phosphorus-use-efficiency. New Phytol 196: 1098–1108 López-Ráez JA, Kohlen W, Charnikhova T, Mulder P, Undas AK, Sergeant MJ, Verstappen F, Bugg TD, Thompson AJ, Ruyter-Spira C, et al (2010) Does abscisic acid affect strigolactone biosynthesis? New Phytol 187: 343–354 Marques JP, Dudeck I, Klösgen RB (2003) Targeting of EGFP chimeras within chloroplasts. Mol Genet Genomics 269: 381–387 Marques JP, Schattat MH, Hause G, Dudeck I, Klösgen RB (2004) In vivo transport of folded EGFP by the DeltapH/TAT-dependent pathway in chloroplasts of Arabidopsis thaliana. J Exp Bot 55: 1697–1706
Fluorescence Microscopy and Image Processing Three- or seven-day-old Arabidopsis seedlings were imaged on a Nikon Eclipse Ti inverted microscope connected to a Roper Scientific spinning disk system, consisting of a CSU-X1 spinning disk head (Yokogawa), Evolve EM-CCD camera (Roper Scientific), and a 31.2 magnifying lens between the spinning disk head and the camera. GFP was excited using a 491-nm laser line. For imaging, we used a Plan Apo 603 oil immersion objective (NA 1.4). Metamorph imaging software was used to operate the microscope and collect images. Z-stacks of 10 slices with 0.5-mm intervals were collected from the hypocotyl epidermis. All image processing was performed using Fiji (ImageJ). Maximum intensity projections of these Z-stacks were made, and a threshold was applied to these images using the “analyze particle” plug-in. The threshold was applied by using the threshold tool in Fiji. The automatic threshold was manually adjusted to only highlight plastids that were in focus. The thresholding tool automatically inverts the image color to enhance contrast. Following application of the threshold, the analyze particles tool of Fiji was used to count the number of in-focus plastids. A minimum surface size of 40 pixels was used with no upper surface limit. Values that were clear image analysis artifacts were discarded from further measurements. SFs and stromule lengths were determined manually. The line drawing tool in Fiji was used to measure the length of the stromule. Plastid protrusions were classified as a stromule using the guidelines of Schattat et al. (2015). Briefly, only thin protrusions with a length of over 0.5 mm extending from a clearly wider plastid body were counted as a stromule. Thin protrusions between two plastid bodies were excluded, because no differentiation could be made between stromules or plastids in a stage of division. Representative images of stromules (and what is not counted as stromule) can be found in Supplemental Figure S2. For each experiment, at least ten plants per genotype per treatment were used, averaging to approximately 600 to 800 analyzed plastids for each genotype per treatment. To determine the SF, the number of stromules per picture is divided by the total number of plastids in the same picture. All error bars represent SEs, and significance was determined using the Student’s t test or a one-way ANOVA with LSD correction when more than two groups were compared using IBM SPSS statistics version 23.
Supplemental Data The following supplemental materials are available. Supplemental Figure S1. Stromule frequency in the wild type with and without added ABA. Supplemental Figure S2. Induction of stromules by GR24 in tobacco epidermal pavement cells. Supplemental Figure S3. Representative image of stromules and what is not counted as stromule. Supplemental Table S1. Stromule frequency in different cell types of tobacco.
ACKNOWLEDGMENT We thank Martin Schattat (Martin-Luther-University, Halle, Germany) for providing the FNRtp-EGFP construct and tobacco reporter plants. Received August 2, 2016; accepted October 13, 2016; published October 19, 2016. Plant Physiol. Vol. 172, 2016
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Mashiguchi K, Sasaki E, Shimada Y, Nagae M, Ueno K, Nakano T, Yoneyama K, Suzuki Y, Asami T (2009) Feedback-regulation of strigolactone biosynthetic genes and strigolactone-regulated genes in Arabidopsis. Biosci Biotechnol Biochem 73: 2460–2465 Natesan SKA, Sullivan JA, Gray JC (2005) Stromules: a characteristic cellspecific feature of plastid morphology. J Exp Bot 56: 787–797 Nelson DC, Scaffidi A, Dun EA, Waters MT, Flematti GR, Dixon KW, Beveridge CA, Ghisalberti EL, Smith SM (2011) F-box protein MAX2 has dual roles in karrikin and strigolactone signaling in Arabidopsis thaliana. Proc Natl Acad Sci USA 108: 8897–8902 Newell CA, Natesan SK, Sullivan JA, Jouhet J, Kavanagh TA, Gray JC (2012) Exclusion of plastid nucleoids and ribosomes from stromules in tobacco and Arabidopsis. Plant J 69: 399–410 Ruyter-Spira C, Al-Babili S, van der Krol S, Bouwmeester H (2013) The biology of strigolactones. Trends Plant Sci 18: 72–83 Ruyter-Spira C, Kohlen W, Charnikhova T, van Zeijl A, van Bezouwen L, de Ruijter N, Cardoso C, Lopez-Raez JA, Matusova R, Bours R, et al (2011) Physiological effects of the synthetic strigolactone analog GR24 on root system architecture in Arabidopsis: another belowground role for strigolactones? Plant Physiol 155: 721–734
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Schattat M, Barton K, Baudisch B, Klösgen RB, Mathur J (2011a) Plastid stromule branching coincides with contiguous endoplasmic reticulum dynamics. Plant Physiol 155: 1667–1677 Schattat M, Barton K, Mathur J (2011b) Correlated behavior implicates stromules in increasing the interactive surface between plastids and ER tubules. Plant Signal Behav 6: 715–718 Schattat MH, Barton KA, Mathur J (2015) The myth of interconnected plastids and related phenomena. Protoplasma 252: 359–371 Tjellström H, Andersson MX, Larsson KE, Sandelius AS (2008) Membrane phospholipids as a phosphate reserve: the dynamic nature of phospholipid-to-digalactosyl diacylglycerol exchange in higher plants. Plant Cell Environ 31: 1388–1398 Toh S, Kamiya Y, Kawakami N, Nambara E, McCourt P, Tsuchiya Y (2012) Thermoinhibition uncovers a role for strigolactones in Arabidopsis seed germination. Plant Cell Physiol 53: 107–117 Yoneyama K, Xie X, Kusumoto D, Sekimoto H, Sugimoto Y, Takeuchi Y, Yoneyama K (2007) Nitrogen deficiency as well as phosphorous deficiency in sorghum promotes the production and exudation of 5-deoxystrigol, the host recognition signal for arbuscular mycorrhizal fungi and root parasites. Planta 227: 125–132
Plant Physiol. Vol. 172, 2016
