Plasma membrane aquaporins regulate root hydraulic conductivity in the model plant Setaria viridis.

The high rate of productivity observed in panicoid crops is in part due to their extensive root system. Recently, green foxtail (Setaria viridis) has emerged as a genetic model system for panicoid grasses. Natural accessions of S. viridis originating from different parts of the world, with differential leaf physiological behavior, have been identified. This work focused on understanding the physiological and molecular mechanisms controlling root hydraulic conductivity and root-to-shoot gas exchange signaling in S. viridis. We identified 2 accessions, SHA and ZHA, with contrasting behavior at the leaf, root, and whole-plant levels. Our results indicated a role for root aquaporin (AQP) plasma membrane (PM) intrinsic proteins in the differential behavior of SHA and ZHA. Moreover, a different root hydraulic response to low levels of abscisic acid between SHA and ZHA was observed, which was associated with root AQPs. Using cell imaging, biochemical, and reverse genetic approaches, we identified PM intrinsic protein 1;6 (PIP1;6) as a possible PIP1 candidate that regulates radial root hydraulics and root-to-shoot signaling of gas exchange in S. viridis. In heterologous systems, PIP1;6 localized in the endoplasmic reticulum, and upon interaction with PIP2s, relocalization to the PM was observed. PIP1;6 was predominantly expressed at the root endodermis. Generation of knockout PIP1;6 plants (KO-PIP1;6) in S. viridis showed altered root hydraulic conductivity, altered gas exchange, and alteration of root transcriptional patterns. Our results indicate that PIPs are essential in regulating whole-plant water homeostasis in S. viridis. We conclude that root hydraulic conductivity and gas exchange are positively associated and are regulated by AQPs.

Guard cell activity of PIF4 and HY5 control transpiration.

Whole-plant transpiration, controlled by plant hydraulics and stomatal movement, is regulated by endogenous and environmental signals, with the light playing a dominant role. Stomatal pore size continuously adjusts to changes in light intensity and quality to ensure optimal CO2 intake for photosynthesis on the one hand, together with minimal water loss on the other. The link between light and transpiration is well established, but the genetic knowledge of how guard cells perceive those signals to affect stomatal conductance is still somewhat limited. In the current study, we evaluated the role of two central light-responsive transcription factors; a bZIP-family transcription factor ELONGATED HYPOCOTYL5 (HY5) and the basic helix-loop-helix (BHLH) transcription factor PHYTOCHROME INTERACTING FACTOR4 (PIF4), in the regulation of steady-state transpiration. We show that overexpression of PIF4 exclusively in guard cells (GCPIF4) decreases transpiration, and can restrain the high transpiration of the pif4 mutant. Expression of HY5 specifically in guard cells (GCHY5) had the opposite effect of enhancing transpiration rates of WT- Arabidopsis and tobacco plants and of the hy5 mutant in Arabidopsis. In addition, we show that GCHY5 can reverse the low transpiration caused by guard cell overexpression of the sugar sensor HEXOKINASE1 (HXK1, GCHXK), an established low transpiring genotype. Finally, we suggest that the GCHY5 reversion of low transpiration by GCHXK requires the auto-activation of the endogenous HY5 in other tissues. These findings support the existence of an ongoing diurnal regulation of transpiration by the light-responsive transcription factors HY5 and PIF4 in the stomata, which ultimately determine the whole-plant water use efficiency.

Measuring the Hydraulic Conductivity of Grass Root Systems.

Root-system hydraulic conductivity (RSHC) is an important physiological characteristic that describes the inherent ability of roots to conduct water across a water-potential gradient between the root and the stem xylem. RSHC is commonly used as an indicator of plant functioning and adaptability to a given environment. A simple, fast, and easy-to-use protocol is described for the quantification of RSHC at the seedling stage in two important monocot species grown in hydroponic solution: Setaria viridis, a C4 model plant, and wheat, a C3 crop plant. This protocol can also be easily modified for use with almost any grass species and environmental treatments, such as salinity or hormone treatments. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Setaria hydrostatic root-system hydraulic conductivity Alternate Protocol: Measuring the root conductivity of young plants with soft stems.