describes an investigation of the expression of an alfalfa (Medicago
sativa L.) class A4 heat shock transcription factor (HSF), elements
of the heat shock response and the potential utility of engineering
HSFs to protect against low temperature stress. To study HSFs in alfalfa,
a new, cold-inducible HSF homologue, designated MsHSFA4, was isolated
from a field-acclimated cDNA library. Using northern and western macroarrays,
transcript levels for HSF, HSP 18, HSP 86 and protein levels for low
molecular weight HSPs (lmwHSPs) were monitored under controlled chamber
and field conditions. To test the potential to improve low temperature
tolerance through the overexpression of a HSF, an Arabidopsis HSF (AtHSFA4a)
was engineered into alfalfa, and the plants were evaluated for winter
survival and biomass accumulation.
Under both heat and cold stress, HSF, HSP 18, HSP 86 mRNAs and lmwHSP
protein accumulations exhibited a variety of expression patterns. Some
of these patterns exhibited distinct responses that were directly related
to the dormancy classification among 10 alfalfa cultivars. Under both
heat and cold stress, MsHSFA4 expression levels were greater in magnitude
and induction rate in dormant cultivars as compared to non-dormant cultivars.
Under field stress, HSF, HSP 18, HSP 86 mRNAs and lmwHSP protein accumulations
also exhibited a variety of expression patterns. Dormancy cultivar effects
were observed under HSF, HSP 86 mRNAs and lmwHSP protein accumulations.
The most prominent of these effects occurred among HSP 86 mRNA expression
in bud tissue, which exhibited notably higher levels of expression in
non-dormant cultivars as compared to dormant cultivars. The results
herein confirm that MsHSFA4 is a functional transcriptional activator,
active under heat and cold stress, and is most likely involved in activation
of elements of the heat shock response under low temperature stress.
Engineering of AtHSFA4a in alfalfa exhibited effects on winter survival
and fall biomass. Plants containing sense constructs exhibited increased
winter survival and greater fall biomass. Plants containing antisense
constructs exhibited decreased winter survival and a reduction in fall
biomass. This trait of increased and decreased biomass was further observed
in the F1 progeny, indicating that the trait is heritable and a result
of engineered HSF activity.
This research furthered the understanding of the role of the heat shock
response under low temperature by identifying the first cold responsive
HSF. It offers a possible gene selective control mechanism for activating
the expression HSPs under low temperature stress. This work has also
revealed the potential of creating new alfalfa cultivars, expressing
engineered HSFs to enhance stress protection and improve yield performance.