Evolutionary role of seed oils in plants : The case of Jatropha curcas L .

Seed oils in higher plants function as an energy source for germination, emergence and establishing as a new plant. Then, the seed oil must be a trait subjected to natural selection. In this work we revise the evolutionary function of seed oils in angiosperms, analyzing the patterns of seed oil accumulation and their fatty acids composition among species differing in habit, habitat and relatedness. We review some relevant hypotheses about the evolutionary role of seed oils and their influence on plant fitness, using as case study the, apparently, inconsistent fatty acid profile of the tropical biofuel plant Jatropha curcas L. (Euphorbiaceae).


INTRODUCTION
The storage of oils in seeds is a generalized characteristic in higher plants, which has the main function of serve as an energy source to the embryo during the heterotrophic stage (Pujar et al., 2006), previous to the activation of the photosynthetic machinery.Such a stage is fundamental in the success or the failure of the embryo to germinate, emerge and establish as a new plant (Bewley and Black, 1994); therefore, seed traits like size, weight, thickness and hardiness of the coat, and the content of the endosperm determine, in part, the plant reproductive success.Then, the seed oil (considering the total content and the quality) must be a trait subjected to natural selection.
Probably not all the angiosperms accumulate large amounts of oil in their seeds, but this depends on the ecology and physiology of each species; however, even in the majority of the members of the most diverse family of plants, the Orchidaceae, which have tiny dust-like seeds lacking of endosperm (Vinogradova and Andronova, 2002) and, consequently, their seed reserves are scarce, the oil content can be as high as 32% of the seed weight (Arditti, 1967).Epiphytic orchids must produce too small seeds in order to colonize the canopy of forests (Prutsch et al., 2000), they need light during the germination, depend of mycorrhizal fungi for the initial growth and there is evidence of the inability of the embryo to use its oil reserves (oil droplets inside embryo cells) in absence of an external source of simple sugars (Manning and Staden, 1987).The cotyledons and endosperm disappearance seems to have led to the loss *Corresponding author.E-mail: maria.adriano@unach.mx,Tel: +52-962-642-7972 (Ext.100).Fax: +52-962-642-7972.Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License of several biochemical capabilities of orchid seeds (Arditti and Ernst, 1984), including the ability to catabolize oils.In cases like this probably the seed oil is not subjected to selection, but other seed characteristics, like the size and architecture.
To elucidate the evolutionary function of seed oils in plants two issues have been explored: the patterns in the total content and in the fatty acids composition of the oil among species differing in habit, habitat and relatedness.Thus, some hypotheses have been proposed to explain the patterns found.
There is a number of oleaginous plants accumulating seed oils; one of them is Jatropha curcas, a tropical plant native of the Mesoamerican region (Salvador-Figueroa et al., 2014).One interesting characteristic of this species is that their seed oil contains more than 70% of unsaturated fatty acids (Ovando-Medina et al., 2011), while most of tropical species have mainly saturated fatty acids, as the coconut oil (approximately 85% of saturated fats; Bezard et al., 1971) and the African oil palm (approximately 50% of saturation; Sambanthamurthi et al., 2000).Other species of Jatropha, such as J. gossypifolia have about 50% of unsaturated fatty acids (Hosamani and Katagi, 2008).Here, we review some relevant hypotheses centered in angiosperms and propose explanations for the apparently inconsistent fatty acid profile of the tropical biofuel plant J. curcas L. (Euphorbiaceae).

EXPLAINING THE PATTERNS IN THE TOTAL CONTENT OF OILS IN SEEDS
Seeds accumulate principally neutral oils (Lersten, 2006), that is, not volatiles, which are composed of triglycerides or three molecules of fatty acids attached to a molecule of glycerol through ester bonds (Coleman and Lee, 2004).Oil content can vary from around 1% in rice (Oryza sativa L.; Muzafarov and Mazhidov, 1997) to more than 55% in Myristaceae.Although determinations of oil contents have been realized since decades ago (Woodworth et al., 1952;Canvin, 1965), perhaps the first study searching for relations between ecological parameters and oil accumulation was that by Donald Levin in the seventies of the twentieth century.Implicitly, the bases of evolutionary function of seed oil were depicted.Levin (1974) studied the relationships between seed oil content and plant habit and habitat (latitudinal origin and illumination of sites) of over one thousand species of angiosperms.He found that plants have increased their oil content concomitantly with the development of woodiness and shade tolerance, but there is no a pattern of variation in oil content as related to latitudinal origin (Figure 1).
Explanations to the patterns were related with the reproductive strategies (r or K) of the habit group.According to Levin, herbs, shrubs and trees accumulate progressively more seed oil, at the same time, they devote a gradually smaller proportion of their resources (counting, of course, reserves of oil) to reproduction, constituting gradually more K-selected forms.The author mentions that in tropics there is a prevalence of Kselected forms, leading to a selection of seeds rich in reserves.However, is its recognized that trees have characteristics of both r and K-selected organisms, because are perennials with high longevity at time produce large amounts of seeds.
Other simple explanation is that trees, living the first part of their life history in the understory, must accumulate more reserve lipids to have a strong initial growth; that's why the selection has acted on seeds with more oil content.To this respect, Ichie et al. (2001) demonstrated that germinating seeds of Dryobalanops lanceolata (Dipterocarpaceae), an enormous emergent tree of Borneo, use almost all their reserves to form a relatively big stem, which improve their establishing.The same is true for shade-tolerant species (Westoby et al., 1992).Studies analyzing the total content of seed oil of an arrangement of species, in a phylogenetic context, are needed in order to elucidate the evolutionary trends in this subject.

EXPLAINING THE PATTERNS IN THE FATTY ACID COMPOSITION
Seed oils are formed by an extension of the membranelipid biosynthetic pathway common to all plant tissues (Voelker and Kinney, 2001), but, in contrast to membrane lipids, there is great fatty acid diversity.The acyl chains of fatty acid range from 8 to 24 carbons, varying in degree of saturation (number of double bonds), spatial arrangement (cis, trans) and in functional groups.Many plant species accumulate seed oils with unusual fatty acids (Smith, 1970;Aitzetmüller et al., 1999;Dyer et al., 2002).
A huge amount of information is available on fatty acids diversity in plants, but the main interests are centered in the search for fingerprints useful in plant taxonomy (Sharma, 1993) and in the improvement for high accumulation of seed oils and for the production of novel (unusual) fatty acids or those industrially important (Hosamani and Katagi, 2008).Molecular biology tools are enabling to scientists to elucidate biochemical mechanisms implicated in the fatty acid diversity; for example, Dyer et al. (2002) demonstrated that a single divergent enzyme from Aleurites fordii, named FADX, can use the most common unsaturated fatty acids in plants (oleic, linoleic, and linolenic acids) to produce three different unusual fatty acids (as the 18:29cis, 12trans-eleostearic acid).This suggests a mechanism of accelerated evolution of plant fatty acids.Several other studies report the enzyme diversity involved in the fatty acid synthesis, using Arabidopsis and other plant models.Nowadays, we have a good level of knowledge of the biosynthetic pathways of fatty acids in plants (White et al., 2000;Barker et al., 2007).
Surprisingly, little attention has been paid to the selection factors driving the evolution of the composition of fatty acids in seeds.To this respect, Linder (2000) proposed and tested a hypothesis to predict (and to explain) latitudinal and altitudinal variations in the ratio of saturated/unsaturated fatty acids in seed oils.Table 1 shows the premises and assumptions He used to construct his theory.Linder (2000) suggested that germination temperature is an important selective agent causing seed oils of higher-latitude (or altitude) plants to have proportionately more unsaturated fatty acids than lower-latitude (or altitude) plants.This author designed elegant experiments to test the theory in both phylogenetic and non-phylogenetic contexts.Results showed the validity of the hypothesis at micro and macro-evolutionary scales.
Explanations to the above-mentioned patterns consist in that, in cooler environments (like those of higher latitude), the catabolism of unsaturated fatty acids is more feasible than that of saturated.As a consequence, seeds with more proportion of unsaturated fatty acids are able to germinate earlier and grow more rapidly at low temperatures (increasing their fitness) even though they store less total energy than seeds with more proportion of saturated fatty acids.It is important to take into account that in temperate zones optimal germination conditions are presented in well-defined periods of the year, then, seeds must germinate rapidly in such periods.On the other hand, in warm environments (like those of the tropics) seeds with higher proportions of saturated fatty acids are selectively favored because their oils will provide more energy, without a penalty in the rate of energy acquisition; germination conditions in tropics are more or less stable during the year.

THE CASE OF THE BIOFUEL PLANT J. CURCAS L.
J. curcas, also known in Southern Mexico and Guatemala as "Piñón", is a euphorbiaceous plant with many uses across the world and a great potential in several fields.This plant probably is native to Mesoamerica; however, it currently exists as a crop in both the old and the new world, with excellent adaptation to tropical and subtropical regions.It is a multi-use species, but nowadays is being used to extract oil from its seeds to produce fatty acid methyl esters or biodiesel (Pramanik, 2003;Fairless, 2007).
Since the seed oil is the main product of that plant, many studies have explored the content and composition of the oil of seeds J. curcas from different parts of the tropics.Apparently, the total content (ranging between 20 and 50%; Heller, 1996) is in agreement with the Levin's hypothesis because J. curcas can be considered a shrubby tree (the plant grows a maximum of 5 m).There are 175 species of Jatropha (Dehgan and Webster, 1979),

Premise
Consequences for the theory Production and effects of triacylglycerols (TAGs) are exclusive of the seed stage (i) TAGs are independent of the evolutionary forces acting in other plant's life history (ii) TAGs rich in saturated fatty acids should be selected (iii) Something other than selection for maximum energy storage affect the rate saturated/unsaturated (iv) Under cooler temperature conditions unsaturated fatty acids are easily catabolized, then, seeds germinate rapidly (v) Unsaturated fatty acids should increase from low to high latitudes and altitudes (vi) Within a clade, species that germinate at cooler temperatures should have a low ratio saturated/unsaturated Saturated fatty acids yields more energy than unsaturated Some seed TAGs are low in saturated fatty acids

Unsaturated fatty acids have low melting points
The germination temperature is the selective force influencing the rate saturated/unsaturated Constructed with information from Linder (2000).which vary in habit; it would be interesting to test the hypothesis of the correlation between the hardiness and the oil accumulation, but unfortunately information about oil determinations, if exists, is not available.
On the other hand, the composition of fatty acids of the seed oil of J. curcas appears to be in disagreement with the Linder's hypothesis because, being a tropical species, the selection has favored a major proportion of unsaturated fatty acids (Table 2).Linder (2000) mentions alternative hypotheses to explain the latitudinal variation in accumulation of unsaturated fatty acids, but He discarded the herbivory and the photoperiod as the selection forces implicated.Probably, these factors do not explain the trend observed in J. curcas.He also mentions "when the association between the proportion of saturated fatty acids and germination temperature does not hold, there should be either lack of genetic variation for oil composition within one or more species in the clade or other selection processes for alternate oil compositions".We do not have enough data to analyze phylogenetically the saturated/unsaturated ratio in the genus Jatropha or even non-phylogenetically within J. curcas but, based in my chromatographic determinations of the fatty acids profiles of J. curcas from the Mesoamerican region (Ovando-Medina et al., 2011).We think there are other selection processes acting in this particular case.
We hypothesize that the soil humidity is pushing, al least in part, to select unsaturated fatty acids of the seed oil in J. curcas.It is well documented the tolerance of the species to drought; like other Jatropha species, the "piñón" has succulent stems and sheds its leaves during the dry season (Heller, 1996: Gubitz et al., 1999), although it grows well in a range of environments (from humid to semi-arid/arid tropical and subtropical) with optimal annual rainfalls between 300 and 1000 mm.However, it can grow and develop in areas with much more precipitations, if the soil drainage is good (Heller, 1996).
Nevertheless, the plant (and of course the seedling) is susceptible to the inundation; our personal observations of seedlings survival in the State of Chiapas (in the most Southern part of Mexico) showed that a few days of constant flood cause the death of seedlings and fifteen or more days could cause the death of an adult plant.In tropical zones, the flowering and fruiting of J. curcas is achieved during the non-rainy season and at the start of the rainy season; seeds do not present dormancy and germinate fast (typically in five days) and in high percentage.According to Dehgan and Schutzman (1994) J. curcas is present in South America in seasonally dry tropical areas, but are completely absent in the moist Amazon region.
If these observations are generalized facts, taking into account the energetic and catabolic properties of the fatty acids, a seed of drought tolerant plant with more unsaturated fatty acids can germinate and establish rapidly and with more probabilities to survive in a high precipitation zone than one with high proportion of saturated fatty acids.The excess of humidity could be other selection force in favor of a high proportion of unsaturated fatty acids.

CONCLUSION
In order to clarify the evolutionary function of seed oils in plants and to identify more assertively the selection factors driving the patterns in accumulation of oil and in the proportions of saturated/unsaturated fatty acids, it would be interesting to test the Linder's hypothesis in clades distributed along an altitudinal gradient.Besides, it is indispensable the analysis of total content of oils in a phylogenetic context.
To test the hypothesis of the excess of soil humidity as force of selection favoring unsaturated fatty acids accumulation, studies with other tropical drought tolerant plant species are needed.

Figure 1 .
Figure 1.Non-phylogenetic comparison of seed oil contents of angiosperms of five habits and three ecological zones.Constructed with data from Levin (1984).

Table 1 .
Elements of the Linder's theory on the selection of saturated/unsaturated fatty acids of seed oils.

Table 2 .
Proportions of unsaturated fatty acids of J. curcas seed oils determinated by several authors in different parts of the tropical world.

of the seeds Unsaturated fatty acids (Percentage of the complete dry seed) References
*Data of the Standard Deviation is not provided.