Salt-tolerant and -sensitive seedlings exhibit noteworthy differences in lipolytic events in response to salt stress

ABSTRACT

Present findings hypothesize that salt-tolerant and -sensitive oilseed plants are expected to exhibit deviant patterns of growth through lipolytic events in seedling cotyledons. It reports the growth response and different lipolytic mechanisms operating during oil body (OB) mobilization in the seedling cotyledons of salt-tolerant (DRSH 1) and salt-sensitive (PSH 1962) varieties of sunflower (Helianthus annuus L.). Salt tolerance or sensitivity to 120 mM NaCl correlates with high proteolytic degradation of OB membrane proteins, particularly oleosins, whereas salt-sensitive seedling cotyledons exhibit negligible proteolytic activity, thereby retaining OB membrane integrity for a longer time. High lipoxygenase (LOX) activity and its further upregulation by salt stress are the unique features of salt-sensitive sunflower seedlings. Salt-tolerant seedling cotyledons exhibit noteworthy modulation of phospholipase-D (PLD) activity by salt stress. Salt-sensitive seedling cotyledons exhibit higher lipase activity than salt-sensitive ones and enzyme activity is downregulated by salt stress. Salt-sensitive variety exhibits higher lipid accumulation and faster lipid mobilization with seedling development than salt-tolerant variety. Accumulation of oleic and linoleic acid in the seedling cotyledons of salt-tolerant and sensitive varieties exhibits differential sensitivity to salt stress. Novel detection of hexanoic acid (6:0) is a noteworthy feature as a response to salt stress in salt-sensitive variety. These findings, thus, provide new information on long-distance salt stress sensing mechanisms at seedling stage of plant development.

Based on extensive past investigations undertaken in the author’s laboratory at Delhi University on understanding the various biochemical mechanisms operating during seed germination in sunflower (Helianthus annuus L.), present work reports the growth response and different biochemical mechanisms operating during oil body (OB) mobilization in cotyledons of salt-tolerant (DRSH 1) and salt-sensitive (PSH 1962) varieties. As a part of preliminary investigations, growth response of four varieties of sunflower seeds was assessed during early phase of germination and compared with that of the semi salt-tolerant variety already under investigation. A concentration of 120 mM NaCl was adopted to monitor the impact of salt stress in four sunflower varieties to begin with, in view of the fact that at this concentration of NaCl, the semi salt-tolerant variety (KBSH 53) has already been established in the laboratory to demonstrate semi salt-tolerance in sunflower seedlings during early phase of growth.¹ The data provided in Table 1 show germination response and hypocotyl length attained by different sunflower varieties during early phase of seedling growth (up to 6d, in dark). It is evident from these preliminary investigations that the varieties DRSH 1 and PSH 1962 could be referred as ‘salt-tolerant’ and ‘salt-sensitive’ with reference to variety KBSH 53 already in use and designated as ‘semi salt-tolerant’ variety. Salt-tolerant variety exhibited much higher seed germination response and better hypocotyl elongation than the semi-tolerant one. In contrast, salt-sensitive variety showed lesser germination response and hypocotyl extension than semi salt-tolerant one, in response to salt stress. Based on these observations and subsequent selection, all further investigations on the biochemical mechanisms of salt tolerance and sensitivity in sunflower were focused on varieties DRSH 1 and PSH 1962. The observations obtained from these two varieties have been discussed with reference to the response pattern of semi salt-tolerant variety.

Table 1.

Germination response and seedling growth in terms of hypocotyl length (cm) in five sunflower varieties in control or under salt stress* conditions.

Sunflower varieties	% Germination response		Stage of seedling growth (days after germination)
			2		4		6
	(-)	(+)	(-)	(+)	(-)	(+)	(-)	(+)
KBSH 53	83.26 ±0.967	58.46 ±0.357**	1.82 ±0.032	0.98 ±0.032***	5.12 ±0.092	2.55 ±0.075***	9.2 ±0.094	4.2 ±0.068***
KBSH 44	36.24 ±1.439	16.4 ±0.588**	1.26 ±0.089	0.427 ±0.061**	2.43 ±0.143	1.35 ±0.099*	4.6 ±0.175	-
DRSH 1	93 ±0.641	75.73 ±0.613***	3.36 ±0.195	2.06 ±0.196**	6.46 ±0.191	4.02 ±0.072***	8.7 ±0.047	5.2 ±0.047**
LFSH 171	10.5 ±0.448	-	2.1 ±0.094	-	-	-	-	-
PSH 1962	82 ±0.593	44.93 ±0.944***	3.16 ±0.166	0.86 ±0.098***	6.03 ±0.195	2.7 ±0.141***	9.6 ±0.222	3.3 ±0.217***

*120 mM NaCl provided in half-strength Hoagland solution used for irrigation germination trays.

(+) indicates salt stress (120 mM NaCl).

± indicates standard error of the sample.

Statistical analysis was carried out using SPSS 22 (*p < 0.05, **p < 0.01 and ***p < 0.001) as compared to control (without NaCl), according to one-way ANOVA.

Oil bodies (OBs) isolated from the cotyledons of sunflower seedlings exhibit longer retention as a response to salt stress in salt-sensitive variety. In salt-tolerant variety, however, the retention of oil bodies was not evident with advancing seedling age. The bicarbonate-washed oil body membrane proteins,² analyzed electrophoretically, showed longer retention of proteins on the membrane of salt-sensitive variety than on salt-tolerant ones, both in response to salt stress and also with increasing age of the seedlings (Figure 1). It is thus evident from that salt tolerance or sensitivity in sunflower seedlings is associated with noteworthy changes in the extent of OB mobilization through modulation of the ability of oil bodies to retain major intrinsic OB membrane proteins. A number of enzymes are expected to transiently express themselves on the outer surface (facing cytosol) of OB membranes in order to facilitate triacylglyceride (TAG) hydrolysis by lipase action.³ Principally, these include lipoxygenase (LOX) for facilitating oxygenation of fatty acids within the oil bodies, expression of thiol protease activity for degradation of oleosins, changes in phospholipase-D (PLD) activity, which is a crucial signaling intermediate and lipase activity, which leads TAG hydrolysis within the OB matrix.⁴ Comparative analysis of LOX activity in the cytosolic supernatant (10,000 g) of salt-sensitive seedling cotyledons shows upregulation of LOX activity by salt stress in salt-sensitive variety than in salt-tolerant ones (Figure 2). The peak of LOX activity is evident in 4 d old, salt-sensitive seedling cotyledons in response to salt stress. In salt-tolerant variety, LOX activity remains relatively low up to 4 d of seedling development irrespective of salt stress. In 6 d old seedlings, however, control (unstressed) seedling cotyledons exhibit relatively higher LOX activity which is brought down by salt tress. This appears to be an unusual observation of LOX activity as a response to salt stress in salt-tolerant variety. Thus, higher LOX activity and its further upregulation by salt stress are the unique features of salt-sensitive sunflower seedlings. Salt-tolerance, thus, appears to correlate with lower LOX activity irrespective of salt tress. A 65 kDa thiol protease present in the cytosol of sunflower seedling cotyledons is known to act on oleosin isoforms on the OB membranes in order to trigger their systematic degradation leading to lipase access from the cytosol to the TAG matrix caused by enhanced porosity of the membrane due to gradual proteolytic action.^5,6 A comparison of thiol protease activity in the salt-tolerant and sensitive varieties reveals very high protease activity in salt-tolerant seedling cotyledons and negligible activity in salt-sensitive ones (Figure 2). The high thiol protease activity evident in seedling cotyledons of salt-tolerant variety is apparently not affected by salt tress. In contrast, the low thiol protease activity evident in salt-sensitive variety appears to be downregulated by salt stress with increasing age of the seedling cotyledons (Figure 2). Thus, salt-tolerance correlates with high proteolytic degradation of oleosins and, salt-sensitive seedling cotyledons exhibit negligible proteolytic activity, thereby retaining OB membrane integrity for a longer time. Phasing of phosphatidic acid (PA) production is regulated by multiple PLD isoforms in response to abiotic stress conditions.⁷ PLD activity shows contrasting effect of salt stress in salt-tolerant and sensitive varieties of sunflower seedlings. In both the varieties, PLD activity shows a gradual decline with age. In salt-sensitive variety, the enzyme activity remains unaffected up to 4 d stage of seedling development and the activity marginally increases in 6 d old seedling cotyledons. In salt-tolerant variety, however, PLD activity significantly declines in 2 and 4 d old seedling cotyledons as a response to salt stress, but in 6 d old seedlings, the activity increases significantly and is at highest level (Figure 2). Thus, salt-tolerant sunflower seedlings exhibit noteworthy sensitivity in terms of PLD activity modulation by salt. This, however, is not evident in salt-sensitive seedlings. Lipase activity gradually increases with seedling age in salt-tolerant variety but not in salt-sensitive ones where activity shows a peak at 4 d stage. The enzyme activity is much higher in salt-tolerant seedlings raised in the absence of salt, as compared to salt-sensitive variety at the respective age (Figure 2). Salt stress, in general, is detrimental to lipase activity to some extent in most stages of seedling development both in salt-tolerant and salt-sensitive varieties. Thus, salt-sensitive sunflower seedlings exhibit higher lipase activity than salt-sensitive ones and the enzyme activity is negatively modulated by salt stress. The total neutral lipid content of salt-tolerant seedlings gradually declines with seedling age. In salt-tolerant variety, this decline is less evident. In both the varieties, salt stress leads to significantly higher accumulation of total lipids in seedling cotyledons at all stages of development with reference to the respective controls. Thus, accumulation of total neutral lipids is much higher in salt-sensitive variety than in salt-tolerant ones (Figure 3). Thin layer chromatographic (TLC) analysis of total lipids shows slower mobilization of TAGs by way of expression of monoacylglycerides (MAGs), diacylglycerides (DAGs) and free fatty acids (FFAs) in salt-tolerant variety than in sensitive ones. Thus, salt-sensitive variety shows the accumulation of higher amount of MAGs, two isoforms of DAGs and free fatty acids at 2 d of seedling development stage itself, and this mobilization pattern seemingly does not appear to be modulated by salt stress. At the later stage of development (6 d), salt-stressed seedling cotyledons of salt-sensitive variety show higher accumulation of DAGs and FFAs than in unstressed condition. It is thus evident from these observations that both in terms of total neutral lipid accumulation and lipid mobilization, the salt-sensitive variety of sunflower seedlings exhibit higher lipid accumulation and faster mobilization with seedling development than cotyledons from salt-tolerant variety at the respective stage of development. Gas chromatographic-mass spectroscopic (GC-MS) analysis of TAG composition shows that oleic (18:1 Δ⁹) and linoleic (18:2 Δ ^9,12) acids constitute the major fractions of TAG in seedling cotyledons both in salt-tolerant and salt-sensitive varieties, followed by palmitic (16:0) and stearic (18:0) acids, in terms of relative abundance. Salt stress brings about a noteworthy decline in oleic acid accumulation during early stage of seed germination (2 d) in salt-tolerant variety which is not evident in salt-sensitive variety at the same stage of development. This decline in oleic acid content in response to salt stress is evident at later stages of development (4 and 6 days) salt-sensitive variety. In contrast to oleic acid, linoleic acid content significantly increases in response to salt stress in 2 d old seedlings of salt-tolerant variety, which subsequently shows a reverse response (decline) at 4 d stage and increase in 6 d stage. In contrast, in salt-sensitive variety, linoleic acid content is upregulated at all stages of development at all stages of salt stress (Figure 3). Thus, accumulation of oleic and linoleic acid in the seedlings of salt-tolerant and sensitive varieties exhibits differential sensitivity to salt stress. Palmitic and stearic acid contents are not significantly affected by seedling age and salt stress in both salt-tolerant and sensitive varieties except for a noteworthy higher accumulation of stearic acid in response to salt stress in salt-tolerant variety. In addition to the above-stated four major fatty acids detected in seedling cotyledons of sunflower, at least six fatty acids have been found to exist as minor fractions (less than 2%). Interestingly, even among these minor fatty acids, regulation by salt stress is evident (data not shown). Novel detection of hexanoic acid (6:0) is a noteworthy feature as a response to salt stress in salt-sensitive sunflower seedlings. It is thus evident that salt stress not only differentially modulates two major fatty acid contents (oleic and linoleic) but it also regulates the levels of some minor fatty acids as well.

Figure 1.

Pattern of oil body (OB) (a) and OB membrane protein mobilization (b) in salt-tolerant and salt-sensitive sunflower seedlings.

Figure 2.

Development associated pre-lipolysis enzymatic changes in sunflower seedling cotyledons (10,000 g supernatant) known to facilitate oil body (OB) mobilization in salt-tolerant and salt-sensitive varieties. Statistical analysis was carried out using SPSS 22 (*p < .05, **p < .01 and ***p < .001) as compared to control (without NaCl), according to one-way ANOVA. All the experiments have been carried out at least thrice.

Figure 3.

Lipid mobilization patterns in sunflower seedling cotyledons (7,000 g supernatant) of salt-tolerant and salt-sensitive varieties. Gas-chromatographic-mass spectroscopic (GC-MS) analysis of fatty acid composition changes under salt stress (120 mM NaCl). Statistical analysis was carried out using SPSS 22 (*p < .05, **p < .01 and ***p < .001) as compared to control (without NaCl), according to one-way ANOVA. All the experimental set-ups were repeated at least thrice.

Discussion

Salt-tolerance or sensitivity in sunflower seedlings has been observed to be associated with noteworthy changes in the extent of time-dependent OB mobilization through modulation of the ability of OBs to retain major intrinsic OB membrane proteins. It is believed that differential proteolytic sensitivity of different oleosins on OB membrane could be a determinant of oil body longevity during seed germination under salt stress. The present work, thus, provides new information on the differences in the extent of retention of OB membrane proteins in salt-tolerant and sensitive seedlings. Thus, retention of OBs was evident for longer period in response to salt stress in salt-tolerant variety. In salt-sensitive variety, salt stress leads to better retention of OBs during early stage of seedling growth than in unstressed conditions. Present work further demonstrates strong differences in the expression of 65 kDa, thiol protease activity. Protease activity is very high in salt-tolerant variety and is not apparently significantly affected by salt stress, thereby, indicating stronger proteolysis of OB membrane proteins. In contrast, the protease activity is very low in salt-sensitive seedling cotyledons and it is further scaled down in seedlings raised in presence of 120 mM NaCl. Seed germination in oilseeds also accompanies the expression and activity of certain enzymes as pre-requisite for lipolytic degradation of TAGs. These include lipoxygenase (LOX), phospholipase A₂ (PLA₂), thiol protease, and lipase.³ Of these, lipase is the principal enzyme leading to TAG hydrolysis, releasing FFA and glycerol. LOX activity results in the oxygenation of TAGs to their corresponding hydroxyl-peroxy derivatives in some oilseeds. These derivatives are then acted upon by lipases.⁸ LOX may also play a role in the reconstitution of lipid composition of cell membranes when the cells expand during seed germination.^9,10 Earlier work from Delhi University has demonstrated that LOX activity is not detectable in ungerminated sunflower seeds but it subsequently becomes detectable in seedling cotyledons, hypocotyls, and roots.¹¹ Changes in LOX activity during seedling growth are further modulated by light and calcium in seedling cotyledons. In sunflower seedling cotyledons, LOX activity is evident both on OB membranes and in the cytosol although its major fraction resides in cytosol.¹¹ Present investigations have demonstrated enhanced LOX activity and its further upregulation under salt stress in salt-sensitive sunflower seedlings. Salt-tolerance, on the contrary, appears to correlate with lower LOX activity, irrespective of salt stress. The observed enhanced LOX activity in salt-sensitive sunflower seedlings in response to salt stress could possibly have a correlation with varying composition of fatty acid which could serve as the potential substrates for LOX. In this context, it may be noted that LOX in Arabidopsis exhibits almost 2.5 times higher activity with linolenic acid (18:3 Δ ^{9, 12, 15}) as compared with linoleic acid.¹² On the other hand, low LOX activity in salt-tolerant sunflower seedlings could possibly be explained as a response to noteworthy changes in the calcium ion content of tissue since LOX is known to be regulated by calcium in a negative way not only in sunflower seedlings¹¹ but also in Vigna unguiculata and in cucumber.^13,14 PA is one of the primary signaling molecules involved in the complex network of lipid signaling under stress conditions, such as drought and salt stress.^15–18 The timing, location, and biochemical pathways of PA production accompanying stress response in plants are regulated by multiple isoforms of phospholipase-D.⁷ Salt stress given via NaCl treatment rapidly triggers the activation of PLD and accumulation of PA in Chlamydomonas moewusii, Medicago sativa, and Solanum lycopersicum. Out of the six main isoforms of PLD reported in Arabidopsis thaliana,¹⁹ isoform α exhibits constitutive expression during NaCl and dehydration treatments whereas PLD isoforms: β and γ, exhibits no noteworthy role in induction.²⁰ PLD is also expected to be a significant signaling molecule in the cytoplasm of seedling cotyledons since its activity exhibits noteworthy variations in the two varieties of sunflower under investigation. Present observations provide strong evidence for a correlation between salt-tolerance/sensitivity and PLD activity induction as a long-distance signaling response in seedling cotyledons. Lipases (triacylglycerol hydrolases; EC 3.1.1.3) are responsible for cleavage of TAGs stored in OB matrix. Subsequent to their biosynthesis in the cell cytoplasm, lipase molecules are expected to cross OB membrane in order to exert their effect. In the germinating kernels of maize, lipase activity appears soon after germination due to de-novo biosynthesis.²¹ Following synthesis, maize lipase gets targeted toward OBs while in castor bean, the enzyme integrates itself into the glyoxysomal membranes.²² In some oilseeds, such as cotton, corn, and rapeseed, the in vitro rate of lipase activity is much more than in-vivo rate of lipid breakdown. In some other oilseeds, lipase activity remains undetectable due to the presence of proteinaceous lipase inhibitors. Such inhibitors have been reported in cucumber, peanut, soybean, and sunflower.^23,24 Salt stress causes lowering of lipase action both in salt-tolerant and salt-sensitive varieties. Salt-sensitivity has been observed to correlate with lower lipase activity, in general, as compared to salt-tolerant variety, thereby indicating enhanced TAG hydrolysis in salt-tolerant variety than in salt-sensitive one. The pattern of gradual decline in total neutral lipids from seedling cotyledons raised in control conditions upto 6 days of growth is similar to past observations from Delhi University on a semi-tolerant sunflower variety (KBSH 53).¹¹ However, in the present case, raising seedlings of salt-tolerant and salt-sensitive varieties in the presence of 120 mM NaCl revealed greater retention of total lipids in salt-sensitive variety than in salt-tolerant one, in response to salt stress. Present observations highlight significance of salt-sensitivity for retention of total neutral lipids. Additionally, salt-sensitive variety showed greater release of free fatty acids, diacyl glycerides, and monoacyl glycerides with age of seedlings than salt-tolerant variety, irrespective of salt stress.

Present observations indicate a noteworthy impact of salt stress on the accumulation of oleic and linoleic acid in seedling cotyledons. However, the impact of salt stress is stronger on the production of linoleic acid, both in salt-tolerant and sensitive varieties of sunflower. These observations are in congruence with recent findings that resistance and sensitivity to drought stress are related to linolenic acid accumulation in oat (Avena sativa) cultivars. Linolenic acid, the precursor of plant hormone, jasmonic acid, is involved in signaling in response to stress.²⁵ α-Linolenic acid is released from plastid lipids by lipase action. Subsequent sequential action of three enzymes, including 1,3-lipoxygenase (LOX), leads to the formation of oxo-phytodienoic acid (OPDA) and its transport to peroxisomes. OPDA is converted to (+)-7-isojasmonic acid. Thus, a probable interaction among lipase, LOX, and linolenic acid generation seems significant in salt stress-induced metabolic events investigated in the present work.²⁵ Interestingly, no change in jasmonic acid concentration was noted in the oat cultivar sensitive to drought stress indicating that linolenic acid and jasmonates have a role in imparting tolerance to plants.²⁶ Levels of linolenic acid (18:3) and palmitoleic acid (16:1) significantly dipped under salt stress. On the other hand, levels of linoleic acid (18:2) and palmitic acid (16:0) were found to be upregulated under stress.^27,28 Also, levels of linolenic acid (18:3) were downregulated in maize cultivars sensitive to salt, suggesting that these fatty acids are essential in maintaining salt tolerance, and could possibly be involved in salt-induced membrane damage.²⁹ Interestingly, in grapevine drought cultivars, the levels of unsaturated fatty acids have been found to be higher under stress conditions.³⁰

Salt-sensitive sunflower seedling cotyledons also exhibit induction of hexanoic acid (6:0) production in response to salt stress. This is not observed in salt-tolerant seedling cotyledons. Possibly, hexanoic acid, although present in minute quantities, could be involved in the synthesis of some significant signaling molecules required to function in stress conditions in sunflower seedlings. Interestingly, it may be noted that under biotic stress conditons, production of hexanoic acid correlates with linolenic acid production to combat stress. Application of hexanoic acid as a priming agent helps induce defense response in plants under attack from pathogen.³¹ Pharmacologically applied hexanoic acid shows absence or lesser number of necrotic lesions on the leaves of tomato inoculated with Alternaria alternate.³² Exogenous application of hexanoic acid not only activates jasmonic acid pathway³³ but also enhances salicylic acid pathway³⁴ in tomato plant under attack of necrotrophic pathogens. Exogenous application of hexanoic acid 2-(diethylamino) ethyl ester (DA-6) to strawberry (Fragaria ananassa Duch) seedlings in varying concentrations combats chilling stress by lowering the levels of free radicals production (O₂⁻ and H₂O₂) and lipid peroxidation.³⁵ The ability of DA-6 to impart resistance against chilling stress has also been observed in rice³⁶ and tomato.³⁷ Studies using groundnut seedlings subjected to drought stress have also revealed hexanoic acid’s role in overcoming stress.³⁸ Strawberry seedlings subjected to chilling stress demonstrate upregulation in levels of glutathione and ascorbic acid on application of hexanoic acid.^39,40 Thus, a new role of hexanoic acid is evident in sunflower seedling cotyledons in combating salt stress.

To sum up, the present work provides new information on the differences in the extent of retention of OB membrane proteins in salt-tolerant and sensitive sunflower seedlings. Enhanced LOX activity in salt-sensitive sunflower seedlings in response to salt stress could possibly have a correlation with varying composition of fatty acids which could serve as the potential substrates for LOX. A correlation between salt-tolerance/sensitivity and PLD activity induction is also evident as a long-distance signaling response in seedling cotyledons. Salt-sensitivity correlates with lower lipase activity, thereby indicating enhanced TAG hydrolysis in salt-tolerant variety than in salt-sensitive one. Linolenic acid, the precursor of plant hormone, jasmonic acid, is also apparently involved in signaling in response to salt stress. Hexanoic acid, although present in minute quantities, could be involved in the synthesis of some significant signaling molecules required to function in stress conditions in sunflower seedlings. A new role of hexanoic acid is thus, evident in sunflower seedling cotyledons in combating salt stress.

Funding Statement

This work was supported by University of Delhi in the form of Non-NET fellowship to MG (Sanction letter: Sch/139/Non-NET/Botany/Ph.D./2019-20/836)Non-NET fellowship, University of Delhi [Sch/139/Non-NET/Botany/Ph.D./2019-20/836].

Abbreviations

DAG

diacylglyceride

FFA

free fatty acids

LOX

lipoxygenase

MAG

monoacylglyceride

OB

oil body

OPDA

oxo-phytodienoic acid

PA

phosphatidic acid

PLD

phospholipase-D

TAG

triacylglyceride

TLC

thin layer chromatography

Acknowledgments

This work was supported by University of Delhi in the form of Non-NET fellowship to MG (Sanction letter: Sch/139/Non-NET/Botany/Ph.D./2019-20/836)

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.