Plants had to develop multiple survival strategies over the ages to cope with the ever changing environment. The production of Late Embryogenesis Abundant Proteins (LEAs) therefore must be one of these evolutionary developments with an important role towards drought tolerance. These LEA proteins are heat stable, hydrophilic and desiccation linked. LEA proteins are also expressed under osmotic and cold stresses. We studied two desiccation linked LEAs (XhLEA1-4 and XhLEA0797) which were discovered in Xerophyta humilis, add full name. In this chapter we present a literature account on LEA proteins with a particular emphasis on the desiccation linked ones. The rest of the chapters deal with the expression and functional analysis of the two LEAs from X. humilis.
As the name implies, LEA proteins were originally discovered to be upregulated in the late stages of orthodox seed development, coincident with the onset of desiccation tolerance in these seeds (Dure et al., 1981; Galau and Dure, 1981; Galau et al., 1986). Subsequent reports indicate that LEAs also occur in vegetative tissues of plants in response to a range of abiotic stresses such as hyperosmotic stress induced by partial loss of water, salt or freezing (Imai et al., 1996; Swire-Clark and Marcotte, 1999; Cheng et al., 2002; Houde et al., 2004; Riera et al., 2004).
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The common feature of LEAs is that they are low complexity, water deficit inducible proteins, unfolded in the hydrated state, extremely hydrophilic and heat stable (with the exception of Group 5); have no catalytic activity or structural domains; and most of them lack cysteine and tryptophan residues (Baker et al., 1988; Lin et al., 1990; Ingram and Bartels, 1997; Curry and Walker-Simmons, 1993; Close 1996; Bray 1997). LEA proteins are boiling-soluble indicating that the proteins are hydrated and non-globular. These are the characteristics that led to the suggestion that LEA proteins are involved in the protection of plant cells from dehydration, and their accumulation also coincides with desiccation and freezing tolerance of plants (Zhang et al., 2000).
The association of LEAs with different stresses might mean that either there are multipurpose LEAs or they should have a subordinate role in stress tolerance, may be stabilizing other important macromolecules or enhancing their protective role (Goyal et al, 2005; Tunnacliffe and Wise, 2007).
Genetic expression studies in plants revealed that LEA proteins are generally associated with water-deficit stress such as desiccation of seeds, dehydration of vegetative tissues, low temperature, increased salt solutions or application of the plant growth regulator ABA (Bartels, 1999). LEA proteins disappear from tissue during the first hours of seed germination or in response to stress relief in plant tissues which is an indication that their expression is either developmentally or environmentally regulated (Bartels, 1999).
LEA proteins also occur outside the plant kingdom in anhydrobiotic animals and microorganisms such as in bacteria, Bacillus subtilis (Stacy and Aalen 1998), Rotifers (Tunnacliffe et al. 2005 ), in Polypedilum vanderplanki (Arthropod) (Kikawada et al.,, 2006) in Collembola species (Insect) (Bahrndorff et al., 2009), in Artemia franciscana (desiccation-tolerant crustacean) (Hand et al., 2007) in Nematodes (Browne et. al., 2002; Goyal et al., 2005). The expression of LEAs in all these organisms was correlated with desiccation tolerance, and it is now well established that these proteins are not restricted to plants but widely distributed across species.
It was also reported that Arabidopsis mutants lacking one or two LEA proteins belonging to group 1 LEA produced desiccation tolerant seeds (Caroles et al., 2002). This is not a surprising result, since it is known that desiccation tolerance is likely to involve upregulation of a number of genes and not just 1 LEA type protein. It is also proposed that different types of LEAs might protect tissues against different types of subcellular insults. It thus becomes imperative that individual LEAs should be tested for different functions to determine their precise role in stress tolerance.
The bulk source of evidence of the role of LEA proteins in desiccation tolerance has mainly been from observation of high levels of expression of corresponding mRNA during drying and from direct detection of such proteins in heat stable protein extracts, from western blots using antibodies to known LEA proteins (eg Close et al., 1993; Close 1996; 1997) as well as from mutants lacking the corresponding genes. Inactivation of group 3 LEA proteins in bacterium, Deinococcus radiodurant, for example, reduced viability of desiccated cultures by 75% (Battista et al., 2001).
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Considerable knowledge has been gained on LEAs that are expressed during seed embryonic development (Shewry and Casey, 1999). However, there has been little comparative knowledge on the characteristics and expression patterns of LEAs produced in seeds during maturation and in tissues of leaves and roots of desiccation tolerant plants when they are exposed to desiccation stress, and whether these LEAs are the same across these systems. To date, most of the existing literature on LEA proteins seems making generalizations and predictions of possible role from sequence homology studies of few LEA cDNA transcripts from stressed tissues of few plant species.
Efforts have been made to sequence genes that are expressed during a cycle of dehydration-rehydration in the resurrection plant Xerophyta humilis. Collett et. al., (2004) have constructed an 11k normalized cDNA desiccation library of roots and leaves and various expression analyses were performed. Walford et al., (unpublished) also analysed dehydration inducible expression of micro-array data of 3400 cDNA's in roots, leaves and seeds of X. humilis. A significant number of upregulated transcripts were annotated as LEAs.
Significantly, most of the LEAs from Walford et al.'s work were clustered in desiccated tissues of roots, leaves and seeds but were not expressed in hydrated tissues. This suggests that: 1) these proteins are highly desiccation induced and/or 2) the same genes are apparently switched on in vegetative tissues of this species as are in the development of desiccation tolerance in its seeds.
1.2 Evolution of LEAs
At this stage research on LEA proteins is mainly focused on the role of these proteins in stress tolerance, and there has not been sufficient explanation as to when and how these proteins might have originated. What is certain is their association with loss of water from tissues and their distribution across variety of species. For instance transcripts of Group 3 LEA proteins have been detected in algae (Joh et al., 1995), in nonvascular plants (Hellwege et al., 1996), in seedless vascular plants (Salmi et al., 2005), and in all seed plants in which they have been looked for. Hence, they are widely distributed in the plant kingdom. But these proteins are also found in organisms other than plants.
Homologues of group 3 LEA protein are found in organisms including the nematodes Caenorhabditis elegans, Steinernema feltiae and Aphelenchus avenae, and the prokaryotes Deinococcus radiodurans, Bacillus subtilis and Haemophilus influenzae (Dure, III, 2001; Solomon et. al. 2000; and Browne et. al., 2002). Other ……LEAs were also reported from soya beans (Lan et al., 2005), from Larvae of an African chironomid, from Collembola species (Insecta) (Bahrndorff et al., 2009), from Xerophyta humilis (Colette et al., 2004). were indeed translated into proteins when was exposed
The discovery of LEA proteins in different organisms might indicate a common origin. Illing et. al., (2005) proposed that desiccation tolerance in vegetative tissues and seeds have the same origin and that the genes involved in seed desiccation tolerance might be developmentally induced whereas the expression of those involved in desiccation tolerance is environmentally induced.
Oliver et al., (2000) also postulated that desiccation tolerance in seeds might have evolved secondarily from primitive forms of vegetative desiccation tolerance that remained in some and become available for induction when vegetative tissues are under desiccation stress. For orthodox seeds, desiccation tolerance is a requirement and is part of natural maturation process. However, the evolution of seeds itself is recent in evolutionary terms, that is vegetative life precedes gametes and embryos. Therefore this hypothesis might not be farfetched.
Tunnaclife and Wise (2007) made yet unsurpassed attempt to draw a phylogenetic tree (Fig. ______) in respect to LEA protein evolution. Based on this draft phylogenetic tree constructed from plant and non plant LEA proteins, these authors suggested that these proteins might have been evolved from bacterial ancestral proteins. The phylogenetic tree shows four multidomain groups. The authors indicated that some groups merg when POPP clustering system was used. POPP clustering identifies closely related proteins and hence results in reduced number of groups. This system identifies relatedness that could provide evolutionary significance, but more work has to be done to draw more inclusive LEA phylogenetic tree.
We are very much convinced that classification of LEAs based on function will prove to be complicated as most of these LEAs are associated with the development of desiccation tolerance. ……..longest propose that LEAs better be classified on the basis of function than similarity in some amino acids and domains. The discovery of LEAs in many organisms is an indication that there are more LEAs than the ones that we know today. Ones LEAs are classified based on function then their diversity could be investigated with a set of parameters.
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