Intracellular localization and gene expression analysis provide new insights on LEA proteins’ diversity in anhydrobiotic midge

Abstract

20Anhydrobiosis, an adaptive ability to withstand complete desiccation, is in insects 21 limited to a single species: the nonbiting midge Polypedilum vanderplanki (the sleeping 22 chironomid). Evolution of anhydrobiosis in a single representative of a large genus is 23 associated with drastic changes in genome structure, including the emergence of new 24 multimember gene families directly involved in desiccation tolerance. Among them, Late 25 Embryogenesis Abundant (LEA) proteins, which protect other proteins from aggregation 26 caused by desiccation, are believed to originate via horizontal gene transfer from a bacterial 27 donor. To obtain new insights on the biological background of the expanded 27-member LEA 28 protein group in P. vanderplanki, we investigated the expression of corresponding genes in a 29 P. vanderplanki-derived cell line, capable of anhydrobiosis, in a normal state and during 30 induction of desiccation tolerance. We found that all LEA proteins genes identified in P.31 vanderplanki's genome except PvLea16 and PvLea17 are also expressed in Pv11 cells. Their 32 expression was elevated in response to anhydrobiosis-inducing trehalose treatment. 33 Expression patterns of PvLea genes were well preserved in Pv11 cells in comparison to P. 34 vanderplanki's larvae both in the control group and during the anhydrobiosis cycle. We also 35 investigated localization of LEA proteins in Pv11 cells and Sf9 cells and found a different 36 level of conservation in intracellular localization of the protein expressed in mammalian and 37 insect cells. 38 39 Introduction 40 One of the most intriguing adaptations to extreme environments is the phenomenon of 41 anhydrobiosis-the ability to withstand complete desiccation in a nonmetabolic state (see 42 review in (1)-which allows living organisms to revive after complete desiccation. 3 43 Organisms capable of entering anhydrobiosis are found across different taxa, and the larvae 44 of the chironomid Polypedilum vanderplanki (Insecta, Diptera) are one of the most complex 45 of these from an evolutionary point of view, as well as being a unique case of emergence of 46 anhydrobiosis in a single species of eukaryotes (2). 47 For the larvae, entering the anhydrobiotic state takes 48 hours and is mediated by 48 several key events including replacement of water with trehalose and vitrification, as well as 49 accumulation of protective biomolecules, including heat shock proteins, antioxidants and 50 enzymes, aquaporins, and Late Embryogenesis Abundant (LEA) proteins (2,3). The latter are 51 highly hydrophilic proteins, well known for their protective effect against water deficits, and 52 they are found in anhydrobiotic organisms in both the plant and animal kingdoms (2). 53 Previously, we identified 27 genes of LEA proteins (PvLea) in the P. vanderplanki 54 genome, all of which are encoding members of LEA group 3. Many of these genes become 55 induced during desiccation in larvae (4). LEA proteins are believed to be one of the main 56 participants in anhydrobiosis mechanisms, ...

New group of transmembrane proteins associated with desiccation tolerance in the anhydrobiotic midge Polypedilum vanderplanki

“…We found a similar pattern of PvLil expression in our recently produced data set that covers anhydrobiosis induction in P. vanderplanki- derived Pv11 cell culture (Fig. 7 c) 21 . All PvLil genes were induced during anhydrobiosis onset in Pv11 cells, which are the only one known animal cell culture model of anhydrobiosis.…”

“…Previous data on PvLEA protein localization in the Chinese hamster ovary cell line or Pv11 cells revealed that most of them are located in the cytosol 19 , 21 . PvLEA1 was detected in the cell membrane and PvLEA3 in the endoplasmic reticulum or Golgi apparatus.…”

“…Here and below in this paragraph p-values were computed in the two-sided Wilcoxon test with continuity correction; n = 13 and 27 for PvLIL and PvLEA, respectively. These characteristics are more variable among PvLEA proteins and the three longest PvLEA www.nature.com/scientificreports/ www.nature.com/scientificreports/ cell line or Pv11 cells revealed that most of them are located in the cytosol 19,21 . PvLEA1 was detected in the cell membrane and PvLEA3 in the endoplasmic reticulum or Golgi apparatus.…”

“…This combination ensures that the localization of PvLEA1 and PvLEA3 proteins in the cell membrane may be related to protection of the membrane against the fusions that occur during desiccation 33 . Since PvLEA3 protein is located in the endoplasmic reticulum or Golgi apparatus, its effect is also related to the membrane of the corresponding subcellular compartment rather than the cell membrane 19,21 .…”

“…P. vanderplanki culture was reared and larvae were slowly dried in accordance with the previously published protocol 24 . The produced RNA-seq data sets were mapped onto P. vanderplanki genome (version 0.9) and AUGUSTUS-produced gene annotations (version 0.91) merged with sequences and annotations of Anhydrobiosis-related gene Island (ARId) to obtain better accuracy of PvLil gene quantification 21 . RNA-seq data were processed via mapping by HISAT2 version 2-2.1.0 46 , sorting of resulting SAM files by SAMtools 1.9-52 47 , obtaining corresponding counts using HTSeq 0.5.4p3 48 and calculation of RPKM values using edgeR 3.26.8 49 .…”