Cold-inducible promoter-driven knockdown of Brachypodium antifreeze proteins confers freeze sensitivity

Abstract

The model forage crop, Brachypodium distachyon, has a family of ice recrystallization inhibition (BdIRI) genes, which encode antifreeze proteins that function by adsorbing to ice crystals and inhibiting their growth. The genes were previously targeted for knockdown using a constitutive CaMV 35S promoter and the resulting transgenic Brachypodium showed reduced antifreeze activity and a greater susceptibility to freezing. However, the transgenic plants also showed developmental defects with shortened stem lengths and were almost completely sterile, raising the possibility that their reduced freeze tolerance could be attributed to developmental deficits. A cold-induced promoter from rice (prOsMYB1R35) has now been substituted for the constitutive promoter to generate temporal miRNA-mediated Brachypodium antifreeze protein knockdowns. Although transgenic lines showed no apparent pleiotropic developmental defects, they demonstrated reduced antifreeze activity as assessed by assays for ice-recrystallization inhibition, thermal hysteresis, electrolyte leakage, leaf infrared thermography, and leaf damage after infection with an ice nucleating phytopathogen. Strikingly, the number of cold-acclimated transgenic plants that survived freezing at -8 °C was reduced by half or killed entirely, depending on the line, compared to cold-acclimated wild type plants. Although these proteins have been studied for almost 60 years, this is the first unequivocal demonstration in any organism of the utility of antifreeze protein function and their contribution to freeze protection, independent of obvious developmental defects. These proteins are thus of potential interest in a wide range of biotechnological applications from accessible cryopreservation, to frozen product additives, to the engineering of transgenic crops with enhanced freezing tolerance.

Brachypodium antifreeze protein gene products inhibit ice recrystallization, attenuate ice nucleation, and reduce immune response

“…No changes in ice nucleation temperature were seen with non-acclimated (NA) lysates, which have no measurable AFP activity. Likewise, nucleation temperatures of lysates from miRNA BdIRI knockdown lines (prOmiRBdIRI-1e or -3c 16 ) with little to no AFP activity were not depressed, irrespective of their CA or NA treatment history, demonstrating that the attenuation of INP activity was dependent on AFP activity, as also shown by IRI assays (Figure 1). Attenuation of INP activity suggests a physical interaction between the two proteins.…”

“…Brachypodium seeds of an inbred line, ecotype Bd21 (RIKEN, Japan) and two transgenic AFP temporal knockdown lines 16 were sown in potting soil and maintained at 4 °C in darkness for four days to synchronise germination. Seeds were moved to a climate-controlled growth chamber (Conviron GEN2000, Controlled Environments Ltd., Winnipeg, MB) at standard Brachypodium growth conditions of 70% relative humidity and 20 h days at ~150 μmol m −2 s −1 at 24 °C followed by 4 h periods with no light at 18 °C.…”

Brachypodium Antifreeze Protein Gene Products Inhibit Ice Recrystallisation, Attenuate Ice Nucleation, and Reduce Immune Response

“…No changes in ice nucleation temperature were seen with non-acclimated (NA) lysates, which have no measurable AFP activity. Likewise, the nucleation temperatures of lysates from miRNA BdIRI knockdown lines (prOmiRBdIRI-1e or -3c [ 8 ]) with little to no AFP activity were not depressed, irrespective of their CA or NA treatment history, demonstrating that the attenuation of INP activity was dependent on AFP activity, as also shown by IRI assays ( Figure 1 d).…”

“…Knockdown of the BdIRI translation products in transgenic Brachypodium as well as the heterologous expression of grass AFP in Arabidopsis have demonstrated that AFP activity confers protection against membrane electrolyte leakage subsequent to sub-zero temperature exposure [ 6 , 7 ]. Unlike AFPs from freeze-susceptible organisms, Bd AFPs do not lower the freezing point of solutions by more than ~0.1 °C, but they are very effective at IRI, or the prevention of ice crystal coalescence at high sub-zero temperatures or under freeze–thaw conditions, a property crucially important for freeze-tolerant grasses [ 6 , 8 ]. Bd AFP has been modelled based on its 67% identity to the AFP of the grass Lolium perenne ( Lp AFP), and it is predicted to have a beta-solenoid structure with two flat ice-binding motifs [ 6 , 9 ].…”

“…These bacteria can kill plants by inducing ice formation at high sub-zero temperatures in order to access intracellular nutrient stores, and they are of commercial concern since they are responsible for up to $7 billion in annual crop losses [ 12 , 13 , 14 , 15 ]. Notably, in addition to their IRI abilities, Bd AFPs appear to disrupt INP activity [ 8 ], but structural uncertainty of the large INPs have hitherto thwarted speculation about the interactions of these two proteins.…”