Abstract
Overwintering plants produce ice active agents with low thermal hysteresis but a higher ability to inhibit the recrystallization of ice crystals already formed. Many plants also minimize the damage from freezing by triggering ice formation at high sub-zero temperatures. These processes in plants differ from those in fishes and insects, which prevent intracellular ice formation and maintain a supercooled liquid state by secreting low molecular weight cryoprotectants and thermal hysteresis proteins (THP) to depress the freezing point of body fluid.
Chionochloa tussocks are mainly endemic to New Zealand and dominate the alpine grasslands. Alpine areas in the South Island can experience snow and freezing at any time of the year due to the incursion of polar air masses. Alpine plants here have developed various mechanisms to overcome the cold weather. A previous study showed significantly high ice nucleation activity (INA) in two alpine Chionochloa species but provided no more information about other ice activities in this genus. Moreover, there are only a few studies on the phylogeny of the Chionochloa group based on ribosomal DNA and/or mitochondrial markers and the lack of genetic information has hampered further research in this genus.
Ice activities were investigated in Chionochloa species collected at the Dunedin Botanic Garden and significant seasonal variations were reported with winter collections showing the highest and summer collections showing the lowest ice activities. Non-bacterial intrinsic proteins were essential in maintaining all three types of ice activities, which showed different responses to physical and chemical treatments including heat, pH, high salt, proteinase K, lysosome, reducing and oxidizing agents.
Ice-shell enriched ice active proteins (IAP) were isolated from C. macra and C. spiralis respectively. This technique showed better purification efficiency than the traditional ice-finger method. Recrystallization inhibition proteins (RIP), class II endochitinase antifreeze proteins (EAF) and other potential ice-binding proteins were identified in the ice fraction (IF). Interestingly, ice nucleation activity (INA) was not present in the ice fraction (IF) suggesting weak ice-binding ability of ice nucleation protein (INP) or the requirement of other compounds such as metal ions to maintain the INA. INA was separated by a protocol of 100 kDa MWCO centrifugal device, size exclusion chromatography and ion exchange chromatography. MALDI-TOF mass spectrometry failed to identify any potential INP as fewer peptide fragments were present.
The transcriptome of winter C. macra was annotated for the better understanding of essential proteins and metabolic pathways involved in the species-specific features including high polysaccharide contents, triterpenoid expression, water conservation and cold tolerance in this genus. In addition, alternative splicing events, SSRs markers, SNPs, miRNAs were reported and gene expression profiles were provided for further genetic research in C. macra and other species in this genus. The C. macra transcriptome also served as a personalized MASCOT database in assist with the search for ice-binding proteins in Chionochloa species assessed by mass spectrometry.
Finally, potential ice active protein (IAP) genes in the C. macra transcriptome were investigated by Blast search against current known IAPs. Two putative IAP genes, IRI2 and EAF2, which showed high similarity to the mentioned RIP and EAF respectively, were expressed in the prokaryotic E. coli system and corresponding proteins were purified with ice activities confirmed. Phylogenetic analysis of these IAPs and Pooideae specific cold stress-related genes (C-repeat binding factors and fructosyltransferase) in C. macra indicated these genes from C. macra showed different phylogenetic relationships with core Pooideae and Brachypodium distachyon.
Taken together, these findings confirmed that utilizing transcriptome combined with putative gene expression, and protein isolation combined with ice activity assays, were useful to study IAPs in New Zealand Chionochloa species. The application of a set of improved methods including DSC, ice-shell and transcriptome based MASCOT database not only saved time and labor but also gave accurate results in studying ice activities. Moreover, the first annotated transcriptome of winter C. macra provided a clear genetic map for the further genetic research in Chionochloa specific features and cold tolerance in this genus.