Characterization of transgenic cotton (Gossypium hirsutum L.) over-expressing Arabidopsis thaliana Related to ABA-insensitive3(ABI3)/Vivparous1 (AtRAV1) and AtABI5 transcription factors: improved water use efficiency through altered guard cell physiology

We investigated select transgenic lines of Gossypium hirsutum that over-express AtRAV1 or AtABI5 transcription factors. The hypothesis is that these lines have enhanced responses to abscisic acid (ABA) resulting in greater water use efficiencies (WUE). We measured leaf surface temperatures (LST), stomatal density, absolute and relative sizes of guard cell apertures, and ABA concentrations in cotyledons. We characterized transgene protein expression and activities in transient assays in Nicotiana benthamiana, in transgenic cotton seeds by immunoblot and by endogenous expression in leaves of an effector of ABA responses (GhDREB). AtRAV1^1-1-5 and AtABI5^1-1-1 over-expression lines had trends of lower levels of ABA in well-watered and drought-stressed cotyledons, and all events tested had higher leaf stomatal conductance and photosynthesis rates under drought in greenhouse, and lower LSTs than control Coker 312 under drought stress conditions. Notably, AtRAV1^1-1-5 cotyledons had significantly higher stomatal densities and 26% smaller guard cell apertures than Coker 312 under drought stress, providing a plausible explanation why LSTs across lines were lower concordant with smaller stomatal apertures. These traits may contribute to intrinsic WUE and assimilate traits of larger leaf areas and longer boll fibers previously shown in these and several independent AtRAV1 and AtABI5 events in the greenhouse and field. These results are consistent with the hypothesis that over-expression of AtRAV1 results in an ABA-hypersensitive phenotype manifest as reduced expression of endogenous GhDREB effector, and lower levels of endogenous ABA in cotyledons associated with greater reductions in pore apertures during stress and increased stomatal density.