Screening acacia mearnsii (black wattle) seedlings for frost tolerance using an artificial frost technique, combined with proteomic analysis.

Abstract

Acacia mearnsii de Wild. (commonly known as black wattle), was first introduced to South Africa in the 1800’s. Today, it is one of the leading commercially grown forestry crops in South Africa due to its usefulness and versatility. Black wattle is a source of high-quality tannin and raw material for wood pulp. It is also excellent for building and as a source of firewood. These uses have contributed to its popularity as a crop for both commercial farmers and small growers. Since its introduction to South Africa, abiotic stresses such as frost damage have affected the silviculture of black wattle, and this has resulted in major financial losses for the forestry industry. Over several decades, breeding research has been conducted with the aim of developing genetically improved seed for frost prone areas. However, this has yet to be achieved. Screening for frost tolerance in black wattle has mainly been conducted using field trials. While this provides the most realistic means of screening, it has several challenges. Field trials are time consuming, expensive and require much effort. Furthermore, frost events are unpredictable in timing and in magnitude, thus affecting frost damage screening trials, with levels of zero to extreme frost occurring in any one year at a selected site. This does not allow for a productive plant breeding program to consistently screen for frost tolerance. The current study focussed on the development of an artificial frost screening method for black wattle. A protocol comprising of eight days of moderately cold temperatures (adaptation) and one day of extremely cold temperature (frost tolerance) was established using a temperature-controlled chamber. The frost damage that resulted from the implementation of this protocol produced similar levels of damage as those experienced in previous field trials. This protocol was thereafter tested in two separate trials involving 100 families of wattle accessions that had previously been ranked in field trials that had been run in frost prone areas. A weak correlation was observed between the results of the artificial frost screening trials and the corresponding field results (r=0.24 to 0.28, rs=0.20 to 0.28). Kruskal-Wallis tests showed a statistical similarity between the medians of one of the artificial frost screening trials and the field frost damage evaluations, and a significant difference between the medians of the two artificial frost screening trials that were conducted. The understanding of the molecular aspects that contribute towards frost tolerance in black wattle is extremely limited. Several studies involving a molecular approach to understanding this trait in other woody species have shown promising results. Frost tolerance is a multigenic trait and therefore a proteomic approach was chosen as the best option to identify biomarkers associated with it. Protein extraction from black wattle has not been previously conducted and therefore a protocol that dealt with the interference of phenolics and that was compatible with downstream proteomic techniques was developed. During the protein extraction protocol development, three different protein extraction methods were compared. These methods differed in terms of their precipitation agent combinations. These combinations included acetone and methanol, phenol and ammonium acetate and ammonium acetate and methanol. The combination of phenol and ammonium acetate produced the highest protein yield, as well as the most distinct protein spots after separation by two-dimensional gel electrophoresis. This method was used to extract proteins from 40 black wattle families with varying levels of frost tolerance, before and after cold stress treatment. These extracted proteins were thereafter separated using two-dimensional gel electrophoresis so that changes in protein expression as a result of cold exposure could be analysed. Multivariate analyses of the proteomic data revealed that six proteins were upregulated in frost-tolerant black wattle families. The identified proteins were: two isoforms of oxygen-evolving enhancer protein 1, probable 1-acyl-sn-glycerol-3-phosphate acyltransferase 4, ribulose bisphosphate carboxylase/oxygenase activate, chaperonin 60 subunit alpha 1 and stromal 70 kDa heat shock-related protein. After identification by mass spectrometry, it was established that these proteins have previously been shown to contribute to the protection of cellular membranes, maintenance of photosynthetic processes and the prevention of protein misfolding and aggregation. These proteomic functions have been observed in previous studies to be associated with the process of cold acclimation in plants and thus seem to play a role in frost tolerance in black wattle. The establishment of an artificial frost screening protocol and the proteomic profiling of black wattle for frost tolerance are important tools for preliminary screening of families prior to field trials. By using these protocols fewer black wattle families will require field testing. This will be beneficial both in terms of cost, effort and time associated with field trials. The development of artificial methods for the induction of stresses and the proteomic changes that result from these are valuable tools for understanding the mechanisms that plants use to cope with abiotic and biotic threats.