Drought stress is the most severe limitation to plant productivity worldwide and is of increasing concern because of climate change.  Many physiological traits important for growth and maintenance of yield during drought remain surprisingly poorly understood at the cellular and biochemical level.  The Verslues laboratory works to uncover the cellular signaling and biochemical mechanisms that underly plant drought resistance at the physiological level.  More generally, our understanding of plant biology lags behind that of other organisms.  For example, even in Arabidopsis thaliana, the most intensively studied model plant, one third of proteins are of unknown cellular function and up to 70% of those are plant-specific proteins (Reiser et al., 2024, Genetics, 227: iyae027).  This lack of knowledge of how plant cells work is both of academic and practical interest as it restricts the use of tools such as gene editing to improve plant stress resistance and agronomic traits. Our genetic and biochemical analysis of drought (low water potential) resistance often leads us to previously unstudied or poorly annotated proteins.  Thus, another focus of our research is to establish the cellular and biochemical roles of unknown function plant proteins.  Examples include our recent discovery that NPH3-domain proteins are a new, plant-specific type of GTPase (Upadhyay-Tiwari et al. 2024) as well as the unexpected microtubule binding and stabilizing ability of MASP1 (Bhaskara et al., 2017).  By combining drought physiology with cellular and biochemical methods, our research contributes to fundamental plant biology while also revealing cellular mechanisms potentially useful for plant improvement.

Ongoing research in the Verslues laboratory can be summarized in three main foci.

1. NPH3-domain proteins and NRL5: Understanding a new class of GTPase and using it to discover mechanisms of trafficking, cellular polarity, and drought sensing/signaling.

The Verslues laboratory established a forward genetic screen for drought/low y_w-related mutants using a Proline Dehydrogenase1 promoter:luciferase reporter (ProDH1_pro:LUC; Shinde et al., 2016).  ProDH1 was selected because it responds to a combination of metabolic- and stress-related signals and because regulation of ProDH1 is key for the dual roles of proline metabolism as the source of protective proline accumulation or a promoter of ROS accumulation and cell death (Verslues et al., 2023).  Thus, a forward genetic screen based on the ProDH1 promoter offers a new window into stress signaling and metabolic regulation not seen in previous screens.

One of the mutants isolated from our ProDH1_pro:LUC screen has a single amino acid change (P335L) in the Non-Phototrophic Hypocotyl3 (NPH3) domain of NPH3-RPT2-Like5 (NRL5). nrl5 mutants have extreme low y_w hypersensitivity due to mis-regulated proline metabolism (Fig. 1).  We further discovered that NRL5, and other NPH3-domain proteins, are a plant-specific type of GTPase involved in intracellular trafficking (Upadhyay-Tiwari et al. 2024; Verslues laboratory, unpublished data).  A functional NPH3 domain and interaction with trafficking proteins are required for the distinct polar localization of NRL5.  Ongoing work in our laboratory indicates that NRL5 is involved in multiple aspects of protein trafficking and secretion. The extreme low water potential hypersensitivity of nrl5-1 is also an important resource to understand drought-related sensing and signaling mechanisms through approaches such as suppressor screening.  These efforts will uncover new mechanisms of trafficking regulation and polarity of broad interest for cell biology as well as new aspects of drought sensing, signaling and metabolic regulation.

Fig 1: NRL5 trafficking function and the proline cycle. NRL5 has newly discovered GTPase activity and roles in intracellular trafficking. NRL5 mutants are dramatically hypersensitive to water limitation because altered trafficking to the cell wall and plasma membrane leads to mis-perception of the stress and mis-regulation of the proline cycle. Proline Dehydrogenase (ProDH) and P5C Synthetase (P5CS) are key stress-regulated components of the proline metabolism cycle that are mis-regulated in nrl5 and are components of our on-going research.

2. The EGR-MASP1 regulatory network controlling plant growth during drought stress.

Fig. 2: EGR2 and MASP1 control plant growth by regulating meristem cell division and cell expansion.  The EGR protein phosphatases are negative regulators that restrict growth (egr mutants grow more than wild type) while MASP1 is a negative regulator (ectopic MASP1 expression drives increased growth).  MASP1 growth promotion function depends on its phosphorylation at a single site (S670) that is regulated by EGRs.  Phosphomimic MASP1-S670D is hyperactive in promoting growth and preventing the reduction in root meristem size typically seen in drought stressed plants.  Opposing gradients of MASP1 and EGR2 control meristem size and cell division activity during drought stress.

Three previously uncharacterized type 2C protein phosphatases (PP2Cs), which we named as the Clade E Growth-Regulating (EGR) PP2Cs, act as negative regulators to restrict plant growth during moderate severity drought/low y_w stress.  Phosphoproteomics of egr1-1egr2-1 identified Microtubule-Associated Stress Protein 1 (MASP1), that acts to promote growth during drought stress in a manner dependent upon EGR-regulated phosphorylation at a single critical site (S670).  Phosphorylated MASP1 promotes growth by counteracting drought-induced reduction of meristem size (Fig. 2; Bhaskara et al., 2017; Longkumer et al., 2022).  

EGR2 proximity labeling combined with phospho-proteomic analysis of egr1-1egr2-1 identified an interesting set of putative EGR-regulated proteins (Verslues laboratory, unpublished data).  These include multiple classes of proteins involved in plasma membrane organization, transport or signaling (Fig. 3).  Further research is investigating these putative EGR-regulated proteins and their roles in regulating plant growth during drought stress.

Similar experiments found putative MASP1 interaction with microtubule-associated proteins, consistent with MASP1 effect on microtubule stability, as well as potential cell cycle regulators that may preferentially interact with phosphorylated (or dephosphorylated) MASP1.  Future work will identify proteins responsible for the repression of meristem cell division during drought, thus revealing new ways to improve plant growth maintenance during moderate severity water limitation. 

Fig. 3: The EGR2 and MASP1-associated proteins regulate plant growth, plasma membrane signaling, and microtubule dynamics.

3. The mysteries of proline metabolism: its regulation, connection to redox status, and contribution to drought resistance.

Proline metabolism is of interest in its own right as a contributor to plant stress resistance and also offers a window into stress signaling mechanisms.  A focus on proline metabolism led us to identify EGRs and NRL5, thus we plan to continue investigating proline metabolism itself as well as using it to identify mechanisms of drought signaling and metabolic regulation.  In addition to proline acting as a protective solute, the cycle of proline synthesis and catabolism (Fig. 1) is important for maintaining cellular redox status (Sharma et al., 2011; Verslues et al., 2023).  In addition to ProDH1, the other key stress-regulated enzyme that controls flux through the proline cycle and is required for drought-induced proline accumulation is P5CS1.  Little is known about P5CS1 regulation, in part because it has been difficult to express tagged P5CS1 and study its localization or interactions in transgenic plants.  To circumvent this obstacle, the Verslues laboratory generated a P5CS1-YFP knock-in line (in-frame insertion of YFP at the endogenous P5CS1 locus) and demonstrated that P5CS1 is preferentially localized around chloroplasts (Longkumer et al., 2024).  This resolved long standing questions about P5CS1 localization while also re-emphasizing old questions about the connection of proline metabolism to other metabolic pathways and to redox status of the chloroplast.  As P5CS1 has been difficult to study in traditional transgenic plants, the P5CS1 knock-in also provides a new tool to investigate the factors that control P5CS1 expression and subcellular localization and identify P5CS1 interacting proteins.  The Verslues laboratory also continues to investigate other regulators, particularly uncharacterized protein kinases and transporters, that influence drought-induced proline accumulation and drought resistance.