Postharvest Biology and Technology of Fruits, Vegetables, and Flowers

BIOTECHNOLOGICAL APPROACHES 377
process known as cold acclimation (Thomashow, 1999). Plants use a wide array of proteins
to protect themselves against low temperature and freezing conditions (Shinozaki et al.,
2003). There have been many approaches aimed at inducing tolerance to low temperatures,
based on traditional breeding as well as on horticultural practices and genetic manipulation.
Two examples of the latter (the use of desaturases and the alternate oxidase) are discussed
elsewhere in this chapter. Many cold-responsive genes have been identified, and some of
them are also induced in response to other types of stress, which suggest that they belong
to a family of genes responding to a stress signals in general. However, there also genes
specifically involved in the response to low temperatures such as the small CBF gene family
encoding transcription factors (Zarka et al., 2003). In Arabidopsis, the transcriptional factors
CBF1, CBF2, and CBF3 (also referred to as DREB1b, DREB1c, and DREB1a, respectively)
are rapidly induced by low temperature followed by expression of CBF-targeted genes, the
CBF regulon, which acts to bring about an increase in freezing tolerance (Sharma et al.,
2005). The genes induced by the CBF family contain the CCGAC core sequence also named
C-repeat (Baker et al., 1994), which has been found to be essential for the low-temperature
responsiveness of additional cold-induced plant genes, including the Arabidopsis gene
COR15A (Baker et al., 1994), the Brassica napus gene BN115 (Jiang et al., 1996), and
the wheat gene WCS120 (Ouellet et al., 1998).
Overexpression of CBF3 in Arabidopsis mimics the response of the plant during cold
acclimation (Gilmour et al., 2000). The CBF1, 2, and 3 proteins, though highly similar
in amino acid sequence, are not identical but they share redundant functional activities
(Gilmour et al., 2004). Most of the work on CBF genes has been performed in Arabidopsis,
and even though tomato contains three CBF homolog genes, tomato cannot cold acclimatize
raising the question whether it has a functional CBF cold response pathway. Only the
tomato LeCBF1 gene, however, was found to be cold inducible, and constitutive overexpression
of LeCBF1 in transgenic Arabidopsis plants induced the expression of CBF-targeted
genes and increased freezing tolerance indicating that LeCBF1 encodes a functional homolog
of the Arabidopsis CBF1-3 proteins (Zhang et al., 2004). However, constitutive
overexpression of either LeCBF1 or AtCBF3 in transgenic tomato plants did not increase
freezing tolerance (Zhang et al., 2004). It is concluded that tomato has a complete CBF
cold response pathway, but that the tomato CBF regulon differs from that of Arabidopsis
and appears to be considerably smaller and less diverse in function. It remains to be
seen whether the other fruit crops contain the CBF regulon and whether it works as in
Arabidopsis.
Antifreeze proteins are found in a wide range of overwintering plants where they inhibit
the growth and recrystallization of ice that forms in intercellular spaces (Griffith and Yaish,
2004). Unlike antifreeze proteins found in fish and insects, plant antifreeze proteins have
multiple, hydrophilic ice-binding domains. Surprisingly, antifreeze proteins from plants
are homologous to pathogenesis-related proteins and also provide protection against psychrophilic
pathogens (Sharma et al., 2005). Transferring single genes encoding antifreeze
proteins to freezing-sensitive plants lowered their freezing temperatures by approximately
1 ◦ C (Breton et al., 2000). The identification of these freezing tolerance-associated proteins
and the elucidation of their cryoprotective functions will have important applications in
several fields (Atici and Nalbantoglu, 2003). Designing new strategies to improve cold tolerance
in crop varieties could increase the plant productivity and also expand the area under
cultivation.