Rice acyl-CoA-binding proteins OsACBP4 and OsACBP5 are differentially localized in the endoplasmic reticulum of transgenic Arabidopsis

Abstract

Acyl-CoA-binding proteins (ACBPs) are known to bind and transport acyl-CoA esters and phospholipids intracellularly. In our recent paper in the New Phytologist, we reported that the six acyl-CoA-binding proteins (OsACBPs) in rice (Oryza sativa) are distributed across various subcellular compartments in transgenic Arabidopsis (Arabidopsis thaliana) such as the cytosol (OsACBP1, OsACBP2 and OsACBP3), the endoplasmic reticulum (ER) including the tubules (OsACBP4 and OsACBP5) and the cisternae (OsACBP4), and the peroxisomes (OsACBP6). Localization of OsACBP4::GFP to the peripheral ER cisternae and the central cisternal ER-like structures in transgenic Arabidopsis distinguished it from OsACBP5::GFP. We further report that besides the ER, OsACBP4::GFP and OsACBP5::GFP were also targeted to the membrane of ER bodies and ER-derived spherical structures, respectively, in transgenic Arabidopsis. These findings support our previous conclusion that OsACBP4 and OsACBP5 are not redundant proteins in the ER.

Acyl-CoA-binding proteins (ACBPs) contain a conserved acyl-CoA-binding domain.¹ Through their ability to bind acyl-CoA esters and phospholipids they can maintain acyl-CoA pools and facilitate subcellular lipid trafficking.² Plant ACBPs are grouped into 4 classes: class I (small ACBPs) show a molecular mass of about 10 kD, class III (large ACBPs) consists of proteins with a C-terminal acyl-CoA-binding domain, and multi-domain ACBPs which fall into class II (ankyrin-ACBPs) and class IV (kelch-ACBPs), contain C-terminal ankyrin repeats and kelch motifs, respectively.³ Previous phylogenetic analysis has indicated an independent evolution of class I and class IV, while class II and III are closely related.³ The Arabidopsis (Arabidopsis thaliana) ACBP family is composed of 6 members, designated as AtACBP1 to AtACBP6.⁴ They are subcellularly localized to the cytosol (AtACBP4, AtACBP5, and AtACBP6), plasma membrane (AtACBP1 and AtACBP2), endoplasmic reticulum (ER) (AtACBP1 and AtACBP2), and apoplast (AtACBP3).^5-9 Diverse functions have been assigned to AtACBPs in embryo development, leaf senescence, freezing stress, drought tolerance, defense responses, and phytoremediation.^9-18 The rice (Oryza sativa) ACBP family also consists of 6 members (OsACBP1-OsACBP6).³ Conservation in abilities to bind acyl-CoA esters and phospholipids in rice and Arabidopsis ACBPs is attributed to the presence of an acyl-CoA-binding domain. It has been reported that some ACBPs show uniqueness in their affinity for lipid substrates.^2,19 Although the specific functions of OsACBPs are not fully understood, their induced expression by various stresses, such as drought, high salinity, wounding, and pathogen infection, suggests that they are involved in stress responses.³

Localization of OsACBP4::GFP and OsACBP5::GFP to the endoplasmic reticulum (ER) body membrane in transgenic Arabidopsis

Although OsACBP4 and OsACBP5 belong to different classes of the ACBP family, they are both subcellularly localized to the ER.^3,19 OsACBP4 contains C-terminal ankyrin repeats, falling into class II and resembling AtACBP1 and AtACBP2, while OsACBP5 is a large ACBP similar to AtACBP3 and belongs to class III.³ Our recent findings have shown that the localization of OsACBP4 and OsACBP5 overlapped at the peripheral tubular ER (but not cisternal ER) in transgenic Arabidopsis.¹⁹

The ER is a network composed of several domains, including the nuclear envelope, the central cisternal ER (cec ER), and the peripheral tubular and cisternal ER.²⁰ Species-specific ER-derived structures that are known include ER bodies from the order Brassicales and protein bodies from maize and rice.^21-23 In Arabidopsis, it has been reported that ER bodies occur in the cells of young seedlings and mature roots, but not rosette leaves.²¹ Their formation in rosette leaves is induced by wounding and jasmonic acid treatment.²² In 35S::OsACBP4::GFP transgenic Arabidopsis seedlings, the OsACBP4::GFP fluorescent signals colocalized with the ER marker, ER-Tracker Red (10 μM, E34250, Invitrogen) (Fig. 1A). OsACBP4::GFP signals were observed to surround ER bodies (Fig. 1B), indicating that OsACBP4::GFP is localized to the ER body membrane. Furthermore, OsACBP4::GFP was also located to the membrane of ER-derived spherical structures (Fig. 1C). A similar expression was observed for OsACBP5::GFP in 35S::OsACBP5::GFP-transformed Arabidopsis seedlings (Fig. 1D-F).

Figure 1. OsACBP4::GFP and OsACBP5::GFP are localized to the endoplasmic reticulum (ER) body membrane and ER-derived spherical structure membrane, respectively. (A) Colocalization of OsACBP4::GFP (green) with ER-Tracker Red (red) (E34250, Invitrogen) in the hypocotyl cells of 2-d-old transgenic Arabidopsis. (B, C) OsACBP4::GFP expression in the root cells of 7-d-old transgenic Arabidopsis. (D) Colocalization of OsACBP5::GFP (green) with ER-Tracker Red (red) in the hypocotyl cells of 2-d-old transgenic Arabidopsis. (E, F) OsACBP5::GFP expression in the cotyledonary and root cells of 7-d-old transgenic Arabidopsis. White arrows indicate ER body membrane; red arrows, ER-derived spherical structure membrane; arrowheads, nuclear envelope. Bar = 10 μm.

Localization of OsACBP4::GFP in the central cisternal ER-like structures in transgenic Arabidopsis

OsACBP4 was hallmarked by its localization to the peripheral cisternae of the ER in comparison to OsACBP5, when it was overexpressed from the 35S promoter in the cotyledonary cells of 7-d-old transgenic Arabidopsis.¹⁹ In 35S::OsACBP4::GFP-transformed Arabidopsis, OsACBP4::GFP fluorescence was located close to the nuclear envelope in many root cells (Fig. 2A), in contrast to the clear edge of the nuclear envelope for OsACBP5::GFP (Fig. 2B). The signals on this nuclear envelope-cisternal ER connection is reminiscent of the cecER in yeast (Saccharomyces cerevisiae),²⁴ but they were not identical. The structure of the cecER, first reported in yeast, consists of a large integral piece of flat cisterna connected to the nuclear envelope.²⁴ Considering that the cecER has a larger volume to surface area ratio, it possibly functions within the lumen.²⁴ Yeast cecER is known to be directed toward the buds and presumably contributes to the ER by shaping the buds.²⁴ In comparison, OsACBP4::GFP fluorescence could be seen in the vicinity surrounding the nuclear envelope (Fig. 2A). These OsACBP4::GFP- signals at the ER coincided with the massive aggregated cisternal ER with fenestrated space (Fig. 2C, D). Taken together, OsACBP4::GFP was demonstrated herein to be localized to both the central and peripheral cisternal ER.

Figure 2. OsACBP4::GFP and OsACBP5::GFP are localized to different endoplasmic reticulum (ER) domains. Confocal images of root cells of 7-d-old transgenic Arabidopsis. (A) OsACBP4::GFP; (B) OsACBP5::GFP; (C) High resolution confocal image of OsACBP4::GFP; (D) Merged image of (C) with transmitted light image. White arrows indicate nuclear envelope; red arrows, central cisternal ER-like structures. Bar = 10 μm.

Conclusions and perspectives

Monocot rice ACBPs do show some differences in subcellular localization in comparison to ACBPs from the eudicot Arabidopsis.¹⁹ With the exception of the cytosolic localization of class I ACBPs, the remaining classes demonstrate differential localization between rice and Arabidopsis.¹⁹ Both Arabidopsis and rice class II and class III members have been predicted by computational analysis to be targeted to the endomembranes; however, experimental verification has proven otherwise.¹⁹ Although OsACBP4 and OsACBP5 are not phylogenetically grouped in the same class, they are confined to the ER.^3,19 We showed that OsACBP4 and OsACBP5 share localization at the membrane of the ER body and ER-derived spherical structure, but OsACBP4 is also present in the central ER cisternae. These findings further support our earlier findings as reported in Meng et al.¹⁹ that OsACBP4 and OsACBP5 are non-redundant ER proteins. Phospholipids biosynthesized in the ER are transported to other subcellular locations dependent on the need of the cell.²⁵ Hence, the specific functions of OsACBP4 and OsACBP5 in the ER in relation to lipid-trafficking await deciphering in future studies.

Previous investigations have revealed that ER bodies are involved in defense responses triggered by the endophytic fungus Piriformospora indica.²⁶ Also, the accumulation of ER bodies was induced by wounding which was used to mimic herbivore bites.²² The relationship between ER bodies and stress was further substansiated by observations that the overexpression of the ER body membrane protein MEB1 or MEB2 in the yeast iron transporter mutant protected it from toxic metals.^27,28 It is noteworthy to mention that the expression of OsACBP4 and OsACBP5 has been previously shown in real-time-polymerase chain reactions to be drought- and wound-inducible, respectively.³ Taken together with results herein on the association of OsACBP4 and OsACBP5 with ER bodies, the potential involvement of these 2 ER proteins in biotic and abiotic stresses seems imminent.