Identification in Pea Seed Mitochondria of a Late-Embryogenesis Abundant Protein Able to Protect Enzymes from Drying

Abstract

Late-embryogenesis abundant (LEA) proteins are hydrophilic proteins that accumulate to a high level in desiccation-tolerant tissues and are thus prominent in seeds. They are expected to play a protective role during dehydration; however, functional evidence is scarce. We identified a LEA protein of group 3 (PsLEAm) that was localized within the matrix space of pea (Pisum sativum) seed mitochondria. PsLEAm revealed typical LEA features such as high hydrophilicity and repeated motifs, except for the N-terminal transit peptide. Most of the highly charged protein was predicted to fold into amphiphilic a-helixes. PsLEAm was expressed during late seed development and remained in the dry seed and throughout germination. Application of the stress hormone abscisic acid was found to reinduce the expression of PsLEAm transcripts during germination. PsLEAm could not be detected in vegetative tissues; however, its expression could be reinduced in leaves by severe water stress. The recombinant PsLEAm was shown to protect two mitochondrial matrix enzymes, fumarase and rhodanese, during drying in an in vitro assay. The overall results constitute, to our knowledge, the first characterization of a LEA protein in mitochondria and experimental evidence for a beneficial role of a LEA protein with respect to proteins during desiccation.Late-embryogenesis abundant (LEA) proteins are overwhelmingly hydrophilic proteins that accumulate to high levels in the latter stages of seed maturation and disappear following germination (Galau et al., 1986). While almost ubiquitous in the plant kingdom, data mining has revealed the widespread occurrence of LEA proteins in prokaryotes and eucaryotes (GarayArroyo et al., 2000). Historically clustered in five main groups based on primary structure analysis (Dure et al., 1989;Cuming, 1999), the LEA protein classification was recently reexamined using statistically based bioinformatic tools (Wise, 2003).LEA protein expression, which often appears abscisic acid (ABA) dependent, can also occur in vegetative tissues subjected to water deficit associated with drought, salt, or cold stress (for review, see Ingram and Bartels, 1996;Thomashow, 1998;Cuming, 1999). Both the pattern of expression and the structural features of LEA proteins suggest a general protective role in desiccation tolerance (Ingram and Bartels, 1996;Cuming, 1999). This hypothesis was recently supported by the discovery of a LEA protein in an anhydrobiotic nematode (Browne et al., 2002) as well as by the sensitization to desiccation induced by mutational inactivation of LEA genes in the prokaryote Deinococcus radiodurans (Battista et al., 2001). In view of the apparent lack of well-ordered tertiary structure of LEA proteins preventing their use as catalysts, several mechanisms have been proposed to relate their structural features to the protection of cellular structures required by a dehydrated state: water replacement, ion sequestering, macromolecules, and membrane stabilization (Close, 1996(Close, , 1997Cuming, 1999). Experimentally, sev...

Improved tolerance to salt and water stress in Drosophila melanogaster cells conferred by late embryogenesis abundant protein

“…Several LEA proteins belonging to groups 3 and 4 are predicted to reside in mitochondria of the plant Arabidopsis thaliana (Hundertmark and Hincha, 2008). Furthermore, a group 3 LEA protein from pea seeds (PsLEAm) is imported into the mitochondrial matrix and protects mitochondrial matrix enzymes from activity loss and artificial liposomes from vesicle fusion during in vitro desiccation experiments (Grelet et al, 2005;Tolleter et al, 2010;Tolleter et al, 2007). Anhydrobiotic animals apparently have developed similar strategies to survive water loss; multiple LEA proteins belonging to groups 1 and 3 are present in mitochondria of A. franciscana embryos (Menze et al, 2009;Warner et al, 2010).…”

“…Additionally, the repeating motifs of group 1 proteins consist of 20 amino acids, while group 3 proteins are characterized by 11-mer motifs (Battaglia et al, 2008). Mitochondrial targeted LEA proteins may help to maintain the integrity and functionality of the organelle when water is scarce through interactions with proteins and membranes but the mechanisms by which protection is conferred are still unclear (Grelet et al, 2005;Menze et al, 2009;Stupnikova et al, 2006;Tolleter et al, 2010;Tolleter et al, 2007).…”

A Nuclear Gene Encoding the Iron-Sulfur Subunit of Mitochondrial Complex II Is Regulated by B3 Domain Transcription Factors during Seed Development in Arabidopsis

“…A similar pattern has been described only once for a mitochondrial protein, a pea (Pisum sativum) LEA protein. This protein may be involved in protecting the inner mitochondrial membrane during seed desiccation (Grellet et al, 2005;Tolleter et al, 2007); however, no data concerning the regulation of its expression are available. Here, we have performed a detailed characterization of the SDH2-3 promoter and identified key regulatory elements and transcription factors involved in its regula- tion.…”

The Proteome of Seed Development in the Model Legume Lotus japonicus

“…The three LEA proteins identified in Lotus seem to be most similar to LEA proteins belonging to subgroup 3. In pea, a subgroup 3 LEA protein is found in mitochondria (Grelet et al, 2005;Tolleter et al, 2007). One of the wellcharacterized molecular processes of LEA proteins is to protect proteins and other molecules from dehydration (Gilles et al, 2007).…”