FUNCTIONAL STUDIES OF HETEROTRIMERIC G PROTEIN BETA SUBUNIT, AGB1, IN SALINITY AND GUARD CELL RESPONSES
Open Access
- Author:
- Yu, Yunqing
- Graduate Program:
- Plant Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 03, 2016
- Committee Members:
- Sarah Mary Assmann, Dissertation Advisor/Co-Advisor
Gabriele Brigitte Monshausen, Committee Chair/Co-Chair
Sarah Mary Assmann, Committee Member
Timothy J Jegla, Committee Member
Kathleen Marie Brown, Outside Member - Keywords:
- Heterotrimeric G proteins
AGB1
Salinity
stomatal movement
Feronia
Arabidopsis - Abstract:
- Heterotrimeric G-proteins are composed of Gα, Gβ and Gγ subunits. Plants have relatively fewer numbers of each of the subunits compared to animals. In Arabidopsis, there are one canonical Gα (GPA1), three extra-large Gα (XLG1, XLG2 and XLG3), one Gβ (AGB1), and three Gγ subunits (AGG1, AGG2 and AGG3). Plant G proteins have been shown to play numerous roles in plant development and in response to biotic and abiotic stresses. My Ph.D. research mainly focuses on the function of the Gβ subunit, AGB1, in salinity and stomatal movement responses. Salinity is one of major agricultural problems that diminish plant growth and crop yield. In plants, Na+ is a toxic ion that limits mineral nutrient acquisition, impairs metabolic activity and causes oxidative stress. Na+ fluxes include Na+ uptake and extrusion across the plasma membrane, Na+ compartmentation into the vacuole, and long distance translocation of Na+ from root to shoot via the transpiration stream. The regulation of Na+ transport has been elucidated in the Sodium Overly Sensitive (SOS) pathway, which involves a plasma membrane Na+/H+ antiporter, SOS1/NHX7, a serine/threonine protein kinase, SOS2/CIPK24, and a Ca2+ binding protein, SOS3/CBL4. Other Na+/H+ antiporters and CBLs such as NHX1 and CBL10 are also involved in Na+ transport regulation. In chapter 1, I review literature of Arabidopsis G proteins, plant salinity, and a receptor-like kinase (FERONIA), which is found in AGB1 associated proteins. In Chapter 2, I showed that the knockout mutant of the agb1 is hypersensitive to salt, exhibiting a leaf bleaching phenotype. I showed that the hypersensitivity to salt tress in agb1 is due to the ionic toxicity component of salinity stress. agb1 mutants accumulate more Na+ and less K+ in both shoots and roots of hydroponically grown plants, as measured by Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES). agb1 plants have higher root to shoot translocation rates of radiolabelled 24Na+ under transpiring conditions, as a result of larger stomatal apertures and increased stomatal conductance. 24Na+ tracer experiments also show that 24Na+ uptake rates by excised roots of agb1 and wild-type are initially equal, but that agb1 has higher net Na+ uptake at 90 min, implicating possible involvement of AGB1 in regulation of Na+ efflux. Calcium alleviates the salt hypersensitivity of agb1 by reducing Na+ accumulation to below the toxicity threshold. My results provide an example of G proteins playing multiple roles in different tissues in stress tolerance. In Chapter 3, to elucidate AGB1 molecular signaling, I performed co-immunoprecipitation (co-IP) and mass spectrometry to identify the interactors of AGB1. Plasma membrane protein-enriched fractions obtained using the two-phase partitioning method were used for the co-IP assays. I performed co-IP using transgenic Arabidopsis expressing 35S::FLAG-AGB1 in the agb1-2 background and using wild-type Col plants as a control. After eliminating proteins present in the control IP, commonly identified contaminants, and organellar proteins, a total of 103 candidate AGB1-associated proteins were confidently identified. As expected, proteins with the highest frequency of occurrence in replicate experiments were G protein subunits. All of the Gγ subunits (AGG1, AGG2 and AGG3), and most of the Gα subunits (GPA1, XLG2 and XLG3) were identified. I also found receptor-like kinases, Ca2+ signaling related proteins including Calmodulin 2, Annexin D4, and IQ-domain 31 protein, and 14-3-3-like proteins, which may couple with G protein signaling. In recent years, there is increasing evidence that receptor-like kinases may couple G protein signaling. I found a receptor-like kinase, FERONIA (FER), in the AGB1 associated proteins, and confirmed the direct interaction between FER and AGB1 in BiFC assays. I also showed that FER does not interact with the Gα subunits (GPA1, XLG1, XLG2 and XLG3) in BiFC. Interestingly, FER has been shown to be involved in various responses, just as are G proteins. FER play roles in pollen tube reception, immunity response, mechanical sensing, and hormone regulation including ABA, auxin, ethylene and brassinosteroid. In order to study the genetic relationship between AGB1 and FER, wild-type, agb1, fer and the double mutant of agb1 fer were subjected to different physiological treatments. I found that both agb1 and fer have shorter hypocotyls and are hypersensitive to ABA inhibition of root growth, and that AGB1 and FER act synergistically in hypocotyl elongation and ABA caused leaf chlorosis and root growth inhibition at the seedling stage. However, in guard cell responses, FER is epistatic to AGB1 in ABA and RALF1 (Rapid Alkalinization Factor 1) regulation of stomatal movement. Specifically, agb1 is hyposensitive to ABA inhibition of stomatal opening and displays wild-type response to ABA promotion of stomatal closure. fer is hypersensitive to both ABA effects, and the agb1 fer double mutant resembles fer single mutants in both ABA responses. RALF1 acts as a ligand of FER that induces phosphorylation of FER and other proteins, alkalinizes the apoplast and inhibits cell expansion. I showed that RALF1 inhibits stomatal opening and promotes stomatal closure in wild-type plants. agb1 is hyposensitive to RALF1, and fer and agb1 fer are insensitive to RALF1 in both opening and closure responses. Furthermore, a null mutant of a central protein kinase in ABA signaling, SnRK2.6/OST1, is hyposensitive to RALF1 inhibition of stomatal opening, but shows wild-type response to RALF1 promotion of stomatal closure, suggesting cross-talk between RALF and ABA signaling. Unpublished results from the lab showed direct interaction between AGB1 and ABI1, which is a type A protein phosphatase 2C (PP2C) that interacts with and deactivates OST1. In summary, the research demonstrates a complex genetic relationship between G proteins and FER, and shows that AGB1 is involved in RALF1 effect in guard cells, possibly through the interaction with FER and ABA signaling effectors. In Chapter 4, I investigated the genetic relationship between AGB1 and FER in salinity tolerance, and the RALF1 effect on salt toxicity. Both agb1 and fer are hypersensitive to salt stress, and the kinase activity of FER is not required for salt tolerance. The agb1 fer double mutant shows salt hypersensitivity which is greater than the additive effect of agb1 and fer single mutants, suggesting synergism between AGB1 and FER. fer accumulated comparable amounts of Na+ as Col when grown hydroponically, although fer still showed hypersensitivity to NaCl treatment, suggesting that other mechanisms also contribute to the salt hypersensitivity of fer. I then found that both agb1 and fer overaccumulate ROS in the shoot after 24 h of salt treatment, and both are impaired in salt-induced ROS production in the root after 15 min of salt treatment. And again, the double mutant show synergistic ROS imbalance in both shoot and root. These results suggest that the synergism between AGB1 and FER may occur through salt-induced ROS production. I further showed that RALF1 enhances salt toxicity via increasing Na+ accumulation, and that the kinase activity of FER is required for RALF1 inhibition of root growth, but not for RALF1 enhancement of salt toxicity. agb1 was slightly hyposensitive to RALF1 inhibition of root growth, but showed wild-type response to RALF1 effect on salt toxicity, suggesting that AGB1 may be partially involved in RALF1 effect in seedlings. In summary, this study shows that apart from the epistasis between AGB1 and RALF1 in guard cells, AGB1 does not directly function in FER signaling in salinity response. Finally, in chapter 5, I discussed the roles of G proteins in salinity response and the genetic interactions between G proteins and FER. I then provide potential future directions for this study.