HD-Zip I Several specific HD-Zip I family members have been characterized with respect to their roles in regulating drought responses. Of the Arabidopsis γ-clade (Fig. 2), AtHB7 and AtHB12 transcripts are expressed in young expanding tissues and transcript levels are dramatically increased, especially in the vasculature, upon application of exogenous ABA or water deficit (Olsson et al., 2004). When over-expressed in Arabidopsis, AtHB7 and AtHB12 confer a reduced growth phenotype typical of water-limiting conditions (Olsson et al., 2004). Conversely, it has been observed that athb12 mutant plants have slight but reproducible increases in stem length (Son et al., 2010). Hjellstrom et al. (2003) have shown that retardation of stem growth in AtHB7 over-expressing plants is caused by decreased cell elongation. In AtHB12 over-expression lines, stem retardation is associated with down-regulation of GA20 oxidase 1 activity, which is responsible for GA-stimulated cell elongation (Son et al., 2010). It has been proposed that the role of AtHB7 and AtHB12 is to reduce growth under water deficit, and the inhibition of GA biosynthesis is consistent with this role (Hjellstrom et al., 2003; Olsson et al., 2004; Son et al., 2010).
The sunflower (Helianthus annuus) HB4 (HaHB4) gene clusters within the drought/ABA inducible γ-clade (Fig. 2) and is strongly induced by water deficit and ABA (Gago et al., 2002). Work with two forms of the HaHB4 promoter identified an ABA response element (ABRE) that responds to ABA application and also an ABRE that responds to NaCl treatment via an ABA-independent mechanism (Manavella et al., 2008a). When over-expressed in Arabidopsis, HaHB4 confers a similar reduced growth phenotype as seen when AtHB7 and AtHB12 are over-expressed (Olsson et al., 2004; Dezar et al., 2005). When tested under drought stress, Arabidopsis plants expressing HaHB4 constitutively or under a drought-inducible promoter showed increased survival through a mechanism that inhibited drought-related senescence (Dezar et al., 2005; Manavella et al., 2006). Arabidopsis plants over-expressing HaHB4 that were grown under well-watered conditions also showed a delay in the developmental senescence that occurs at the end of the life cycle (Manavella et al., 2006). Microarray and Q-PCR data suggested that the role of HaHB4 in Arabidopsis and H. annuus is to suppress the expression of ethylene-related genes and delay senescence. It was then found that the increasing levels of HaHB4 expression in sunflower correlated with the developmental age of leaves, as older leaves approached senescence (Manavella et al., 2006). It was concluded that HaHB4 was delaying the onset of senescence in Arabidopsis under drought stress and also acting as an antagonist to developmental senescence in both species. A delay in senescence has not been reported in transgenic Arabidopsis over-expressing AtHB7 and AtHB12 genes under well-watered or water-deficit conditions, making it difficult to compare the roles of these genes in the two species.
The Medicago truncatula HD-Zip I member HB1 (MtHB1), belonging to the γ clade, (Fig. 2) is induced by ABA and salinity stress in roots and plays a role in lateral root emergence (Ariel et al., 2010). When MtHB1 is over-expressed in the roots of composite plants, the primary root is longer and lateral root emergence is reduced, a phenotype typically seen in wild-type plants exposed to severe salt stress. Two TILLING-derived mthb1 mutant lines showed a reciprocal phenotype and had shorter roots with an increased number of emerged lateral roots (Ariel et al., 2010). The molecular mechanism responsible for the lateral root emergence phenotypes involves suppression of LOB-binding domain 1 (LBD1) by MtHB1. The lateral organ boundaries (LOB) domain TFs of Arabidopsis and rice play a role in auxin-regulated lateral root initiation and adventitious root formation, respectively (Liu et al., 2005; Okushima et al., 2007). It is proposed that the role of MtHB1 is to inhibit lateral root emergence, when roots are exposed to adverse conditions, which reduces the surface area exposed to soil stress (Ariel et al., 2010).
NaHD20, a Nicotiana attenuataγ-clade homolog, is also induced in roots and leaves by exogenous ABA and soil water deficit (Réet al., 2011). It was observed that under water stress N. attenuata plants with reduced NaHD20 transcripts, through virus-induced gene silencing, had reduced levels of ABA, N. attenuata 9-cis-epoxycarotenoid dioxygenase 1 (NaNCED1; an ABA biosynthesis gene) and N. attenuata osmotin 1 (NaOSM1) but that N. attenuata lipid transfer protein 1 (NaLTP1) induction was not affected (Réet al., 2011).
OsHOX6 (O. sativa Homeobox 6), OsHOX22 and OsHOX24, which are considered the rice homologs of AtHB7 and AtHB12 (Fig. 2), are also up-regulated by water deficit but there are no reports clarifying the functional roles of the corresponding proteins in the rice drought adaptation response (Agalou et al., 2008). These rice genes differ from their Arabidopsis counterparts in their basal expression patterns and there are also differences between the rice paralogs themselves. Under well-watered conditions, OsHOX6 has a relatively high basal level of expression in all tissues, while OsHOX22 transcript is expressed mainly in the blade and panicles. Further, OsHOX24 is strongly expressed in panicles and weakly in other tissues (Agalou et al., 2008). Under extended drought, OsHOX22 and OsHOX24 transcripts are increasingly up-regulated in leaf tissues in both drought-resistant and drought-sensitive cultivars, whereas OsHOX6 is only slightly up-regulated in a drought-sensitive cultivar (Agalou et al., 2008).
The Arabidopsis HD-Zip I β-clade gene AtHB6 (Fig. 2) is ubiquitously expressed in all tissues of mature plants, but in leaves it is detected predominantly in the vasculature (Söderman et al., 1999). AtHB6 is thought to play a role in the regulation of cell division or differentiation as its expression in developing leaves declines basipetally, withdrawing with the wave of epidermal cell differentiation, but AtHB6 still remains in the guard cells (Söderman et al., 1999). Expression of AtHB6 in Arabidopsis is increased by water deficit and ABA, but remains within the same cell types as under untreated conditions (Söderman et al., 1999). When AtHB6 is over-expressed in transgenic Arabidopsis, plants show reduced stomatal closure and diminished inhibition of germination by ABA (Himmelbach et al., 2002); these are also characteristics of the ABA-insensitive mutants abi1 and abi2 (Leung et al., 1997). These data imply that AtHB6 plays a role as a negative regulator in the ABA response under water deficit (Deng et al., 2006).
AtHB5 of the β clade (Fig. 2) shows a similar expression pattern to AtHB6 and is found in all major tissues (Söderman et al., 1994; Henriksson et al., 2005). The AtHB5 promoter is active in the hypocotyl of germinating seedlings, but is suppressed by ABA application. After ABA treatment its expression is restricted to a discrete band in the transition zone between the hypocotyl and root (Johannesson et al., 2003). Transgenic Arabidopsis with ectopically increased levels of AtHB5 are more sensitive to root growth inhibition by ABA at the seedling stage and to germination inhibition. The two observations, that AtHB5 is down-regulated by ABA and that increased levels of ABA enhance ABA-specific responses, have led the authors to suggest a role in seedling development under short-term water-limiting conditions that is not sustained through long-term water deficit (Johannesson et al., 2003).
The HD-Zip I TFs of C. plantagineum that group with this β clade under phylogenetic analysis (Fig. 2), CpHB4 and CpHB5, are down-regulated in C. plantagineum leaves and roots upon exposure to water deficit, whereas CpHB6 and CpHB7 are up-regulated (Deng et al., 2002). Under normal growth conditions, CpHB7 promoter activity in Arabidopsis is considered comparable to that of AtHB6. The AtHB6 over-expression phenotype is also paralleled by ectopic expression of CpHB7 in Arabidopsis (Deng et al., 2006).
OsHOX4 of the rice ζ clade (Fig. 2) is also drought responsive but the level of mRNA transcripts decreases upon exposure to water deficit (Agalou et al., 2008). There are contrasting reports regarding the characteristics of transgenic rice over-expressing OsHOX4. While transgenic plants are shorter than wild-type plants, this phenotype is reported to be attributable to a decrease in the number of cells per internode (Agalou et al., 2008) or to a reduction in cell elongation in the stem (Dai et al., 2008). The mechanism behind stem height reduction is thought to involve OsHOX4 up-regulation of YAB1 (YABBY1, encodes a protein with a YABBY domain) expression, a negative regulator of the GA response (Dai et al., 2008). These results, when interpreted in isolation, imply that under water deficit OsHOX4 is down-regulated, leading to a decrease in YAB1 expression, which enables a stronger plant response to GA. However, the implication of a drought response mechanism involving OsHOX4 in the adaptation of rice development is unclear.
The transcript levels of the Arabidopsis δ-clade genes (Fig. 2) AtHB21, AtHB40 and AtHB53 are up-regulated upon exposure to ABA and salinity stress. However, no role in environmental adaptation has been established for these HD-Zip I TFs, although a role in ovule development has been proposed for all three genes (Skinner & Gasser, 2009).
HD-Zip II There is little functional evidence to suggest a role for HD-Zip II TFs in plant growth adaptation responses to water deficit. However, expression studies using microarrays have shown that HAT2 (Homeobox from Arabidopsis thaliana 2) and HAT22 expression is up-regulated under drought in Arabidopsis (Huang et al., 2008).
In rice, OsHOX11 transcripts are dramatically decreased upon drought exposure in a drought-resistant cultivar and OsHOX27 is up-regulated under mild drought, but transcript levels decrease as the severity of the water deficit increases in both drought-resistant and drought-sensitive cultivars (Agalou et al., 2008). OsHOX19 expression is increased by the imposition of drought stress in both drought-sensitive and drought-resistant cultivars.
Transcripts of HD-Zip II TFs from C. plantagineum are also regulated by water availability and ABA. CpHB1 and CpHB2 show tissue-specific differences in expression under water deficit and ABA treatment. CpHB1 expression is induced in leaves by water deficit but not by ABA, and the level of CpHB2 transcript is up-regulated in the roots by water deficit and ABA (Deng et al., 2002).