Polyamine depletion enhances oil body mobilization through possible regulation of oleosin degradation and aquaporin abundance on its membrane

ABSTRACT

Oil body (OB) mobilization, a crucial event associated with early seedling growth, is delayed in response to salt stress. Previous reports suggest that careful regulation of polyamine (PA) metabolism is essential for salt stress tolerance in plants. Many aspects of PA-mediated regulation of metabolism have been uncovered. However, their role in the process of OB mobilization remains unexplored. Interestingly, the present investigations reveal a possible influence of PA homeostasis on OB mobilization, while implicating complex regulation of oleosin degradation and aquaporin abundance in OB membranes in the process. Application of PA inhibitors resulted in the accumulation of smaller OBs when compared to control (−NaCl) and the salt-stressed counterparts, suggesting a faster rate of mobilization. PA deficit also resulted in reduced retention of some larger oleosins under controlled conditions but enhanced retention of all oleosins under salt stress. Additionally, with respect to aquaporins, a higher abundance of PIP2 under PA deficit both under control and saline conditions, is correlated with a faster mobilization of OBs. Contrarily, TIP1s, and TIP2s remained almost undetectable in response to PA depletion and were differentially regulated by salt stress. The present work, thus, provides novel insights into PA homeostasis-mediated regulation of OB mobilization, oleosin degradation, and aquaporin abundance on OB membranes.

KEYWORDS:

• Aquaporins
• oil body
• oleosin
• polyamine depletion
• salt stress

Introduction

Sunflower (Helianthus annuus L.) is one of the major oilseed crops that yields high-quality edible oil known for its appreciable amounts of unsaturated fatty acids (oleic and linoleic acids) and vitamin E¹. Like other oilseeds, sunflower also accumulates ubiquitous lipid-containing bodies in the seeds, known as the oil bodies (OBs). Bounded by a phospholipid monolayer, OBs, also known as lipid droplets or oleosomes, are specialized spherical inclusions originating from the ER during seed development and storing lipid reserves in their core, primarily composed of tricylglycerides (TAGs)². These highly dynamic subcellular entities have garnered increasing attention owing to their multifarious biological functions in lipid and energy metabolism, seed germination, pollen development, pollen tube growth, stomatal opening, hormone signaling, and stress responses^2–5. In the context of germinating seeds, OBs constitute a vital energy reservoir in the form of stored TAGs. Hydrolysis of these TAGs provides precursors for carbohydrate production and fuels subsequent seedling growth during the post-germinative phase before the seedling switches to photosynthesis for energy requirement^2,6. Mobilization of OBs associated with seed germination, therefore, marks a major metabolic event in oilseed species. OB mobilization begins when the seed starts to germinate and the OBs gradually become depleted during seedling development, and almost all TAGs are consumed⁷. TAG hydrolysis is mediated by lipid hydrolytic enzymes, including lipases and lipoxygenases (LOX)^8,9. Two pathways of OB mobilization have been identified in plants, i.e., lipolysis and more recently described lipophagy^3,4. During lipolysis, lipases catalyze hydrolytic cleavage of the fatty acid ester bonds in TAGs, producing glycerol and free fatty acids as a result. The free fatty acids so produced are subjected to β-oxidation and are ultimately converted to sucrose, which serves as the energy source in the growing seedling^2,6. Two lipases, SDP1 (Sugar-Dependent 1) and SDP1L (SDP1-Like), have been implicated in TAG hydrolysis^10,11. Of these, SDP1 has been demonstrated to be delivered to the OB surface by tubulations extending from peroxisomes¹². The action of lipoxygenase (LOX) may also be significant for the hydrolysis of storage lipids during seed germination, as has been demonstrated in sunflowers⁹. The second pathway, i.e., lipophagy, is a peculiar form of OB degradation that occurs via autophagy wherein the OBs are engulfed by the vacuoles with subsequent decomposition of lipid reserves¹³. Lipophagy has potential roles in seed germination and the succeeding developmental phase as well as pollen grain maturation and pollen tube growth^3–5.

OBs also harbor specialized integral proteins that are anchored within their membrane, namely oleosins, steroleosins, and caleosins. These OB membrane proteins are known to play crucial structural and/or regulatory roles¹⁴. Among these, oleosins are the most abundant and primarily play structural roles, such as maintaining OB integrity by preventing fusion and regulating their turnover¹⁵. Oleosin degradation precedes lipid hydrolysis or lipolysis and is believed to allow lipases and other hydrolytic enzymes to access the TAG core during seedling growth post-germination^7,14,16. A few plausible mechanisms underlying oleosin degradation have been proposed, including the proteolytic action of a thiol protease and ubiquitin-mediated proteasomal degradation^7,14,16–18. The latter pathway relies on the post-translational modification of oleosins, especially the K48-diubiquitination (K48Ub₂), which marks them for selective extraction from the OB coat and degradation by the proteasome¹⁶.

In addition to their developmental roles, OBs have also been linked to plant responses to abiotic and biotic stresses^3,4. Exposure to abiotic stresses induces accumulation of OBs as that seen in the leaves of heat- or cold-stressed plants, possibly as a mechanism of removal of excess toxic-free fatty acids and also as a reservoir for energy molecules under nutrient deficiency to support a restart with the onset of favorable conditions^3,4. OBs are also implicated in the production of oxylipins and anti-pathogenic compounds as defense mechanisms for biotic agents as well as in serving as a platform for the detoxification of xenobiotics^19–21. OB-associated proteins, particularly caleosins, have been reported to be crucial players in mediating responses to abiotic stresses, such as salt, drought, and cold²⁰.

With advancing technologies, newer functions of OB metabolism are uncovered. A prominent line of investigation in OB metabolism has been focused on analyzing OB proteomes to identify OB-associated proteins and their role in various OB-mediated processes. It has been revealed that OB proteomes are subject to considerable alterations with different stages, like seed development, germination, and seedling growth, which further reinforces the dynamic nature of these organelles or may, by contrast, be indicative of their transformation into different forms concomitant with the progressive stages of the process⁴. Interestingly, a few proteomic studies have also reported the occurrence of aquaporins, the water channels, on OB. For instance, Jolivet et al.^22,23 reported the presence of a putative aquaporin belonging to the family of TIPs (TIPα now TIP3) among the proteins recovered from OBs of Arabidopsis thaliana and Arachis hypogaea. TIP1 and TIP2 correspond to the marker TIP (Tonoplast Intrinsic Protein) isoforms associated with the lytic vacuoles (LVs) and protein storage vacuoles (PSVs), respectively²⁴. Likewise, the occurrence of aquaporins in the membranes of purified OBs has also been recorded in other plant species^25,26. During their lifetime, OBs physically interact with several subcellular organelles, including the progenitor ER, mitochondria, vacuoles, peroxisomes (glyoxysomes in seedlings), and even plasma membrane and plasmodesmata^{2,12,15,27–30}. These interactions are essential for the biogenesis of OBs, degradation of storage lipids, and delivery of cargo to the plasmodesmata and adjacent areas on plasma membrane^8,28,29. It has been conjectured that the presence of aquaporins on the OB membrane may also signify this interaction with other subcellular organelles. In concert with this, co-localization of the TIP marker of the tonoplast and OB membrane-associated proteins (oleosins and caleosins) has also been demonstrated³¹.

The present set of investigations was designed to determine the influence of polyamines (PAs) on OB mobilization and oleosin degradation under normal as well as salt stress conditions. Additionally, taking inkling from previous reports suggesting the presence of aquaporins on OB membranes, immunodetection of aquaporin isoforms was undertaken. Salt stress is a major abiotic stress that adversely affects all aspects of plant growth and development by imposing dual stressful conditions, ionic and osmotic stresses, which negatively impact various physiological and metabolic processes³². In concurrence with salt stress-induced alteration in numerous other metabolic events, the process of OB mobilization concomitant with seedling growth has also been demonstrated to be influenced by prevailing saline conditions^33–35. A delayed degradation of oil bodies (OBs) and their associated membrane proteins have been shown previously in seedlings of sunflower and Jatropha curcas exposed to salt stress^33–35. Polyamines (PAs) represent a class of protective amine compounds that are increasingly accumulated in plants exposed to stresses, including salt stress^36,37. Enhanced accumulation of PAs is known to exert many positive effects on plant growth and survival under salt stress^37,38. Perturbation of PA homeostasis is often associated with increased sensitivity to salt stress³⁹. So far, no reports exist concerning the influence of PA homeostasis on lipid metabolism in plants along with other aspects investigated in the present work.

Sunflower seedlings (Helianthus annuus L. cv. KBSH 53) were raised up to 2-d-old stage in dark over germination sheets and provided with a Hoagland nutrient medium. For salt stress, a Hoagland medium supplemented with 120 mM NaCl was used to irrigate the seedlings. To simulate the conditions of PA deprivation, two competitive inhibitors of the PA biosynthetic enzymes ADC (Arginine Decarboxylase) and ODC (Ornithine Decarboxylase) were included in the Hoagland medium, namely DFMA (DL-α-difluoromethylarginine), or DFMO (DL-α-difluoromethylornithine), respectively. Instead of exogenous PA application, present investigations employed PA biosynthetic inhibitors to establish a condition of PA deficit to discern their regulatory role in the events associated with OB degradation. For visualization of OBs, OB pads were separated from tissue homogenate of 2-d-old seedling cotyledons (500 mg) derived from homogenization in 1 ml of extraction buffer [0.1 M Tris-HCl (pH 7.4) containing 1 mM PMSF]. OB preparations were then stained with Nile Red (100 ng.ml⁻¹) and visualized using an epifluorescence microscope at an excitation of 485 nm (em: 525 nm). OB membrane proteins were purified using the bicarbonate (NaHCO₃) washing method, according to Sadeghipour and Bhatla (2002). The isolated proteins were subjected to SDS-PAGE to determine changes in their abundance with the various treatments as well as for detection of aquaporin isoforms by western blotting using polyclonal antibodies against PIP2s, TIP1s, and TIP2s following the protocol of Tailor and Bhatla (2021)⁴⁰.

Limited PA availability results in the accumulation of smaller OBs and may involve oleosin degradation

Oil bodies (OBs) isolated from 2-d-old, dark-grown sunflower seedling cotyledons exhibited variations in size in response to various treatments (Figure 1). Salt stress leads to greater retention of relatively larger OBs. Application of PA inhibitors, by contrast, resulted in the accumulation of smaller OBs when compared to the control (−NaCl). Under the condition of PA depletion combined with salt stress, although numerous larger OBs could be detected, their size was relatively smaller in comparison with those isolated from seedling cotyledons exposed to only NaCl. Enhanced OB degradation in the presence of PA inhibitors reported in the present work may in part support the increased root growth previously reported from the author’s laboratory in 2-d-old sunflower seedlings treated with DFMA or DFMO, irrespective of salt stress⁴⁰. Similar observations have been recorded in Arabidopsis and Pringlea antiscorbutica, wherein the use of D-Arginine, a specific ADC1/2 competitive inhibitor, and DFMO, respectively, was shown to result in a longer primary root length^41,42. It would be interesting to address whether the root growth in these species also entails changes in OB mobilization in response to PA inhibitors.

Figure 1. Nile red staining of oil bodies (OBs). Fluorescence imaging of OBs isolated from cotyledons of 2-d-old, dark-grown seedlings, raised in the absence or presence of 120 mM NaCl, or nutrient medium supplemented with 500 µM of PA biosynthesis inhibitors, DFMA or DFMO, alone or in combination with 120 mM NaCl. OBs were incubated with Nile red stain and fluorescence from them (due to neutral lipids) was visualized (ex: 485 nm; em: 525 nm). Scale bar: 20 μm).

With respect to OB membrane-associated proteins, it has been observed that oleosins, ranging from 10 to 20 kDa, were retained in the OBs isolated from salt-stressed seedling cotyledons. Depletion of PA, on the other hand, resulted in reduced retention of some oleosins, particularly the 17 kDa oleosin, while increasing the retention of the other oleosins (<15 kDa), under non-stressed conditions (Figure 2(a)). However, greater retention of all the oleosin isoforms was evident in salt-stressed seedlings treated with PA biosynthetic inhibitors. This reduction in oleosins in DFMA/DFMO-treated (−NaCl) seedlings does explain the enhanced rate of OB mobilization which, in turn, may support seedling growth. However, their increased retention in salt-stressed and PA-depleted seedlings is slightly contradictory, as it fails to explicate the enhanced root growth in terms of greater OB mobilization when compared to salt stress alone, given that oleosin degradation represents a prerequisite step for lipid hydrolysis. Further experiments may prove beneficial in this regard.

Figure 2. Expression aquaporins on OB membranes. Expression of proteins (A) and aquaporins [PIP2 (B); TIP1 and 2 (C, D)] in the OB membranes (bicarbonate washed) from 2-d-old, dark-grown sunflower seedling cotyledons, raised in the absence or presence of 120 mM NaCl, or PA biosynthetic inhibitors, DFMA and DFMO (500 µM of each), alone or in combination with 120 mM NaCl.

To date, the roles that PAs may have in lipid metabolism and TAG hydrolysis in plant systems remain largely unexplored. Investigations undertaken in animal systems have reported the probable PA-mediated regulation of TAG formation in adipose tissue⁴³. PAs, Spd and Spm in particular, are associated with the activation of several enzymes involved in TAG metabolism and thus, have a stimulatory effect on TAG formation⁴⁴. Some reports have also demonstrated that Spd and Spm inhibit TAG hydrolysis in a dose-dependent manner by inhibiting lipase activity⁴⁵. PAs have also been shown to inhibit lipolysis by negatively regulating cAMP levels and stimulating the conversion of glucose into TAGs⁴⁴. So far, a lack of information from plant systems regarding the role of PAs in lipid metabolism deferred speculations regarding their role, if any, in OB mobilization. The present findings, thus, provide a novel connection between PA and lipid metabolism. It is suggested that the complex regulation of the intracellular PA homeostasis may have an important regulatory role in oleosin degradation and OB mobilization in sunflower seedlings in an early-signaling response both under controlled and salt stress conditions.

Salt stress and PA depletion modulate the expression of aquaporin isoforms on the OB membrane

Western blot analyses undertaken to examine the probable expression of aquaporin isoforms on OB membranes not only signified the presence of PIP2, TIP1, and TIP2 but also revealed interesting changes in their abundance under various treatments. Taking into account the polyclonal nature of the antibodies and the maxima and minima of molecular weights (kDa) inclusive of all the aquaporin isoforms under investigation, polypeptide bands were examined within the region of 25–50 kDa. Abundance changes were thus assessed for polypeptides corresponding to the molecular weights of PIP2, TIP1, and TIP2 isoforms (~25 kDa and ~50 kDa (dimeric) for PIP2s and ~35 kDa for TIP1s and TIP2s) (Figure 2(b-d)). Salt stress did not appear to significantly affect the accumulation of PIP2s. PA depletion was also observed to only slightly affect their abundance in non-stressed seedlings if only the monomeric form was considered; however, a marked increase in bands corresponding to the dimeric form of PIP2 was evident, thereby indicating a greater abundance of the aquaporin on OB membrane in response to PA deficit. The abundance of both monomeric and dimeric forms of PIP2 was further enhanced in OBs extracted from seedling cotyledons subjected to PA biosynthesis inhibitors in combination with 120 mM NaCl stress. This increased PIP2 abundance correlated with a faster mobilization of OBs observed in PA-depleted conditions and may therefore be important for the process of lipolysis. Concerning putative TIP isoforms, salt stress lowered the accumulation of TIP2. TIP1, on the other hand, was barely detectable under both control and NaCl stress conditions. In seedlings treated with PA inhibitors (for both DFMA and DFMO), both TIP1 and TIP2 were almost undetectable in OB proteins in the absence of salt stress. However, in contrast, seedlings subjected to PA inhibitors in combination with 120 mM salt stress revealed an enhanced abundance of TIP1 and a lowered accumulation of TIP2, when compared with non-stressed seedlings.

The present work thus presents an intriguing piece of information on the PA metabolism-mediated regulation of aquaporins on the OB membrane and its connection with the process of OB mobilization. It has been surmised that the occurrence of aquaporins on OB membranes may facilitate the maintenance of a hydrophobic lipidic core through the extrusion of water. In contrast, aquaporin may be recruited to OBs whereby they may enable water intake required for lipolysis as well as transport of glycerol from OBs during the seed germination process²². Present observations suggest that the latter assumption might be more plausible, given that higher PIP2 abundance coincides with a faster mobilization of OBs observed under PA depletion. However, further investigations are required in this regard to draw more conclusive evidence.

Conclusion

The present work is the first-ever report underlining a yet undiscovered association between PA metabolism and OB mobilization in plants under normal and stressful conditions, possibly through the regulation of oleosin degradation. The maintenance of intracellular PA homeostasis appears to be involved in maintaining the integrity of OBs in sunflower seedlings. Disruption of this homeostasis with the use of PA inhibitors likely accelerates the process of OB degradation observed in the present work. The promotion of faster OB mobilization under PA deficiency also suggests regulatory roles of PAs on the activities of enzymes involved in TAG metabolism in ways yet to be elucidated. Furthermore, PA deficiency-induced alteration of the abundance of aquaporins on OB membranes as noted under both control and salt stress conditions also suggests that aquaporins may be involved in the process of OB mobilization. Based on insights gained from the present investigations, a model is proposed wherein PA deficit causes enhanced oleosin degradation and faster OB mobilization to facilitate TAG hydrolysis. This enhancement in the rate of OB mobilization may also be accompanied by an increased abundance of PIP2 and differential regulation of TIPs. The presence of aquaporins on the OB membrane also suggests a dynamic interaction of OBs with vacuoles and other organelles to facilitate the transport of water required for OB hydrolysis, or otherwise transport glycerol, the product of lipolysis (Figure 3). The present findings thus open a new avenue of research with a focus on gathering more evidence to directly connect PA metabolism with OB metabolism and also unveil the role of this interaction in regulating plant defense responses. Further investigations into these aspects might provide crucial insights into how the identity of OBs is maintained and how their mobilization is regulated at different stages of seed development and germination and in response to stress. It would also be interesting to further explore and elucidate the underlying mechanism of action of PAs on TAG hydrolysis in plants.

Figure 3. PA depletion causes a faster mobilization of OBs as observed from the retention of OBs of smaller diameter. A model is proposed summarizing present work wherein PA deficit results in 1) an enhanced oleosin degradation, 2) faster OB mobilization to facilitate TAG hydrolysis, 3) increased abundance of PIP2, and 4) differential regulation of TIPs suggesting a dynamic interaction of OBs with vacuoles and other organelles to facilitate the transport of water and glycerol, or maybe other molecules, required for OB hydrolysis. All of this together might support the seedling growth (5) in terms of root length previously observed by the authors.

Abbreviations

ADC	=	Arginine Decarboxylase
DFMA	=	DL-α-difluoromethylarginine
DFMO	=	DL-α-difluoromethylornithine
ER	=	Endoplasmic reticulum
LOX	=	Lipoxygenase
LV	=	Lytic vacuole
OB	=	Oil body
ODC	=	Ornithine Decarboxylase
PA	=	Polyamine
PIP	=	Plasma membrane Intrinsic Protein
PSV	=	Protein storage vacuole
SDP1	=	Sugar-Dependent 1
SDP1L	=	SDP1-Like
TAG	=	Triacylglycerides
TIP	=	Tonoplast Intrinsic Protein

Acknowledgments

The authors gratefully acknowledge the support from the UGC-ISF Joint research project [(F.No-6-9/2017 (IC)]. AT is also grateful to UGC for the Research Fellowship awarded to her vide sanction no. 2061530546; Ref. No. 21/06/2015(i)EU-V dated July 27, 2016.

Disclosure statement

The authors declare no potential conflict of interest.

Additional information

Funding

This work was supported by the UGC-ISF Joint Research Project [F.No. 6-9/2017 (IC)].