About

Marcela Rojas-Pierce is an Associate Department Head and Professor in the Department of Plant and Microbial Biology at North Carolina State University. Her research focuses on the molecular mechanisms that regulate vacuole biogenesis and dynamics in plant cells, particularly in the model plant Arabidopsis thaliana. Her work investigates how vacuoles function as major storage compartments and their roles in plant growth, development, and physiological responses such as stomatal regulation and gravitropism. She employs chemical and classical genetic approaches to elucidate the processes involved in vacuole formation and function. Her research also explores vacuole fusion dynamics during stomatal movements, which are critical for gas exchange, water regulation, and overall plant productivity. She has contributed to understanding the molecular control of vacuole membrane dynamics, including the regulation of vacuole fusion through SNARE and HOPS complexes. Additionally, her work extends to developing innovative tools such as inducible protein degradation systems for plant biology and applying bio-electrochemical technologies for sustainable agriculture. Her research has been supported by multiple grants, including a notable NSF-funded project on vacuole remodeling during stomata movements, and she has contributed to advancing plant cell biology through her investigations into membrane contact sites involved in gravity perception.

Selected publications

• Regulation of vacuole fusion in stomata by dephosphorylation of the <scp>HOPS</scp> subunit <scp>VPS39</scp>

  The Plant Journal · 2025-12-01

  articleOpen accessSenior authorCorresponding

  while mitigating water vapor loss. The opening of stomata is regulated in part by homotypic vacuole fusion, which is mediated by conserved homotypic vacuole protein sorting (HOPS) and vacuolar SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) complexes. HOPS tethers apposing vacuole membranes and promotes the formation of trans-SNARE complexes to mediate fusion. In yeast, HOPS dissociates from the assembled SNARE complex to complete vacuole fusion, but little is known about this process in plants. HOPS-specific subunits VACUOLE PROTEIN SORTING39 (VPS39) and VPS41 are required for homotypic plant vacuole fusion, and a computational model predicted that post-translational modifications of HOPS may be needed for plant stomatal vacuole fusion. Here, we characterized a viable T-DNA insertion allele of VPS39 which demonstrated a critical role of VPS39 in stomatal vacuole fusion. We found that VPS39 has increased levels of phosphorylation at S413 when stomata are closed versus open, and that VPS39 function in stomata and embryonic development requires dynamic changes in phosphorylation. Among all HOPS and vacuolar SNARE subunits, only VPS39 showed differential levels of phosphorylation between open and closed stomata. Moreover, regions containing S413 are not conserved between plants and other organisms, suggesting plant-specific mechanisms. Our data are consistent with VPS39 phosphorylation altering vacuole dynamics in response to environmental cues, similar to well-established phosphorylation cascades that regulate ion transport during stomatal opening.

  PublisherOA PDFDOI

• Non-Thermal Plasma Activated Water is an Effective Nitrogen Fertilizer Alternative for <i>Arabidopsis thaliana</i>

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-06-17 · 1 citations

  preprintOpen accessSenior author

  Abstract Nitrogen (N) fixation with non-thermal plasmas has been proposed as a sustainable alternative to meet growing N fertilizer demands for agriculture. This technology generates Plasma Activated Water (PAW) with a range of chemical compositions, including different concentrations of nitrate (NO₃⁻) and hydrogen peroxide (H 2 O 2 ), among other compounds. Potential use of PAW as an effective crop fertilizer necessitates a robust understanding of the underlying biology of the plant, which is not yet available. The lack of a unified standard in PAW production and the varying chemical make-up that results from different devices and protocols hampers comparative studies and adoption of this technology. The objective of this study was to compare the efficacy of two PAW solutions with differing concentrations of H 2 O 2 produced from a Radio Frequency (RF) glow discharge plasma source. The effect of these solutions on plant growth, ROS accumulation, gene expression and heat stress response were compared to N-equivalent controls in the model plant Arabidopsis to assess their potential as an alternative N fertilizer. While PAW solutions lacking detectable H 2 O 2 enhanced seedling growth, those containing approximately 0.3 µM of H 2 O 2 did not. ROS accumulation in root tissues was similar between PAW and chemically equivalent solutions, suggesting H 2 O 2 is the primary ROS present in the PAW at the time of treatment. Gene expression studies showed induction of genes involved in N uptake and assimilation in PAW-treated seedlings. Pre- treatment of seedlings with PAW solutions containing H 2 O 2 improved root growth under heat stress which indicates that this treatment may induce plant stress response pathways. Finally, mature plants showed similar growth when fertilized with PAW lacking H 2 O 2 or NO 3 - control regimes for over 5 weeks indicating equivalency in chemical composition, plant nutrient uptake and utilization. Overall, these results demonstrate that PAW is an effective alternative to NO 3 - fertilizers for plant cultivation but the levels of H 2 O 2 need to be carefully controlled.

  PublisherDOI

• Regulation of Vacuole Fusion in Stomata by Dephosphorylation of the HOPS subunit VPS39

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-10-02 · 1 citations

  preprintOpen accessSenior authorCorresponding

  ABSTRACT Understanding how plants regulate water loss is important for improving crop productivity. Tight control of stomatal opening and closing is essential for the uptake of CO 2 while mitigating water vapor loss. The opening of stomata is regulated in part by homotypic vacuole fusion, which is mediated by conserved ho motypic vacuole p rotein s orting (HOPS) and vacuolar SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) complexes. HOPS tethers apposing vacuole membranes and promotes the formation of trans -SNARE complexes to mediate fusion. In yeast, HOPS dissociates from the assembled SNARE complex to complete vacuole fusion, but little is known about this process in plants. HOPS-specific subunits VACUOLE PROTEIN SORTING39 (VPS39) and VPS41 are required for homotypic plant vacuole fusion, and a computational model predicted that post-translational modifications of HOPS may be needed for plant stomatal vacuole fusion. Here, we characterized a viable T-DNA insertion allele of VPS39 which demonstrated a critical role of VPS39 in stomatal vacuole fusion. We found that VPS39 has increased levels of phosphorylation when stomata are closed versus open, and that VPS39 function in stomata and embryonic development requires dynamic changes in phosphorylation. Our data are consistent with VPS39 phosphorylation altering vacuole dynamics in response to environmental cues, similar to well-established phosphorylation cascades that regulate ion transport during stomatal opening. SIGNIFICANCE STATEMENT Vacuole fusion is important for stomata opening but how it is regulated in response of stomata opening signals is not characterized. This research demonstrated the role of the HOPS complex in vacuole fusion in stomata, and it identified phosphorylation sites in the HOPS subunit VPS39 that are critical for vacuole fusion. One Ser residue was enriched in closed stomata and represents a putative site for control of vacuole fusion downstream of stomata opening signals.

  PublisherOA PDFDOI

• Manipulation of targeted protein degradation in plant biology

  Biochemical Society Transactions · 2025-04-01 · 3 citations

  reviewOpen access1st authorCorresponding

  Inducible protein degradation systems are an important but untapped resource for the study of protein function in plant cells. Unlike mutagenesis or transcriptional control, regulated degradation of proteins of interest allows the study of the biological mechanisms of highly dynamic cellular processes involving essential proteins. While systems for targeted protein degradation are available for research and therapeutics in animals, there are currently limited options in plant biology. Targeted protein degradation systems rely on target ubiquitination by E3 ubiquitin ligases. Systems that are available or being developed in plants can be distinguished primarily by the type of E3 ubiquitin ligase involved, including those that utilize Cullin-RING ligases, bacterial novel E3 ligases, and N-end rule pathway E3 ligases, or they can be controlled by proteolysis targeting chimeras. Target protein ubiquitination leads to degradation by the proteasome or targeting to the vacuole, with both pathways being ubiquitous and important for the endogenous control of protein abundance in plants. Targeted proteolysis approaches for plants will likely be an important tool for basic research and to yield novel traits for crop biotechnology.

  PublisherOA PDFDOI

• Non-thermal plasma activated water is an effective nitrogen fertilizer alternative for Arabidopsis thaliana

  PLoS ONE · 2025-09-08 · 6 citations

  articleOpen accessSenior authorCorresponding

  Nitrogen (N) fixation with non-thermal plasmas has been proposed as a sustainable alternative to meet growing N fertilizer demands for agriculture. This technology generates Plasma Activated Water (PAW) with a range of chemical compositions, including different concentrations of nitrate (NO₃⁻) and hydrogen peroxide (H2O2), among other compounds. Potential use of PAW as an effective crop fertilizer necessitates a robust understanding of the underlying biology of the plant, which is not yet available. The lack of a unified standard in PAW production and the varying chemical make-up that results from different devices and protocols hampers comparative studies and adoption of this technology. The objective of this study was to compare the efficacy of two PAW solutions with differing concentrations of H2O2 produced from a Radio Frequency (RF) glow discharge plasma source. The effect of these solutions on plant growth, ROS accumulation, gene expression and heat stress response were compared to N-equivalent controls in the model plant Arabidopsis to assess their potential as an alternative N fertilizer. While PAW solutions lacking detectable H2O2 enhanced seedling growth, those containing approximately 0.3 µM of H2O2 did not. ROS accumulation in root tissues was similar between PAW and chemically equivalent solutions, suggesting H2O2 is the primary ROS present in the PAW at the time of treatment. Gene expression studies showed induction of genes involved in N uptake and assimilation in PAW-treated seedlings. Pre-treatment of seedlings with PAW solutions containing H2O2 improved root growth under heat stress which indicates that this treatment may induce plant stress response pathways. Finally, mature plants showed similar growth when fertilized with PAW lacking H2O2 or NO3- control regimes for over 5 weeks indicating equivalency in chemical composition, plant nutrient uptake and utilization. Overall, these results demonstrate that PAW is an effective alternative to NO3- fertilizers for plant cultivation but the levels of H2O2 need to be carefully controlled.

  PublisherOA PDFDOI

• The Effect of Plasma Activated Water on the Rhizosphere Composition of Arabidopsis and <i>Solanum lycopersicum</i>

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-10-15

  preprintOpen accessSenior authorCorresponding

  ABSTRACT The emerging field of plasma agriculture investigates the potential benefit of non-thermal plasma (NTP) for agricultural practices. NTP-treated water, referred to as plasma activated water (PAW), has been proposed as a sustainable alternative to conventional nitrogen (N) fertilizers. Growing demand for N fertilizer is concomitant with increased global food demands. PAW contains nitrate (NO 3 - ) and reactive oxygen species, such as hydrogen peroxide (H 2 O 2 ), which are fixed from atmospheric molecules via NTP. While early studies report positive effects of PAW on plant growth, its influence on plant-associated microbial communities remains poorly understood. Here, we compared the impacts of PAW or NO 3 - solutions on the rhizosphere microbial community of Arabidopsis thaliana and Solanum lycopersicum . PAW was generated by a radio frequency (RF) glow discharge plasma source and contained no measurable ROS, while the control solution contained an equivalent concentration of NO 3 - . No significant differences in alpha diversity were detected in either plant species microbiome after 5 weeks of treatment when grown in non-commercial potting substrate. Significant dissimilarity was found in terms of beta diversity, but the relative abundance of the sequenced genera suggested no functional differences in rhizosphere communities. Overall, PAW treatment did not adversely impact the rhizosphere microbiome in either Arabidopsis or tomato. These results support the use of PAW as an alternative N-fertilizer, though outcomes may differ for PAW solutions containing ROS.

  PublisherDOI

• Guard Cell-Enriched Phosphoproteome Reveals Phosphorylation of Endomembrane Proteins in Closed Stomata

  Journal of Proteomics · 2025-10-15

  preprintOpen accessSenior authorCorresponding

  ABSTRACT Control of the stomatal aperture is multifaceted, involving a complex interplay of environmental cues and intracellular signaling pathways. It is well established that changes in ion gradients drive water movement into and out of the guard cell, thereby altering cell volume and modulating the opening or closing of the stomatal pore. These rapid responses are often regulated by phosphorylation cascades to efficiently transmit environmental status and either reduce water loss or enhance carbon assimilation. The role of endomembrane trafficking networks in stomatal dynamics is not well characterized. Here, we investigated the regulation of stomatal opening and closing by generating a proteome and phosphoproteome of guard cell-enriched tissue. This deep proteome captured a protein profile that was similar to previously characterized guard cell proteomes. The guard cell-enriched tissue with closed stomata showed greater levels of phosphorylation of proteins related to endomembrane trafficking and vacuoles when compared to both whole leaf tissue with closed stomata and guard cell-enriched tissue with open stomata. These results support the hypothesis that phosphorylation of endomembrane proteins may contribute to the regulation of stomatal movements.

  PublisherDOI

• Rapid depletion of target proteins in plants by an inducible protein degradation system

  The Plant Cell · 2024-03-06 · 22 citations

  articleOpen accessSenior author

  Inducible protein knockdowns are excellent tools to test the function of essential proteins in short time scales and to capture the role of proteins in dynamic events. Current approaches destroy or sequester proteins by exploiting plant biological mechanisms such as the activity of photoreceptors for optogenetics or auxin-mediated ubiquitination in auxin degrons. It follows that these are not applicable for plants as light and auxin are strong signals for plant cells. We describe here an inducible protein degradation system in plants named E3-DART for E3-targeted Degradation of Plant Proteins. The E3-DART system is based on the specific and well-characterized interaction between the Salmonella-secreted protein H1 (SspH1) and its human target protein kinase N1 (PKN1). This system harnesses the E3 catalytic activity of SspH1 and the SspH1-binding activity of the homology region 1b (HR1b) domain from PKN1. Using Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana), we show that a chimeric protein containing the leucine-rich repeat and novel E3 ligase domains of SspH1 efficiently targets protein fusions of varying sizes containing HR1b for degradation. Target protein degradation was induced by transcriptional control of the chimeric E3 ligase using a glucocorticoid transactivation system, and target protein depletion was detected as early as 3 h after induction. This system could be used to study the loss of any plant protein with high-temporal resolution and may become an important tool in plant cell biology.

  PublisherOA PDFDOI

• White LED intensities during co-cultivation affect the Agrobacterium-mediated soybean (Glycine max) transformation using mature half seeds as explants

  PLoS ONE · 2024-11-26

  articleOpen access

  The transition of light fixture from fluorescent light to light-emitting diodes (LEDs) in growth chambers prompts a reevaluation of current practices in plant biotechnology. Agrobacterium-mediated transformation is crucial for genetic engineering and genome editing in soybean (Glycine max). The critical co-cultivation step of soybean transformation occurs under light condition. Current protocols for co-cultivation in soybean transformation lack a standard for light intensity. In the present study, the objective is to investigate the effect of light intensity during co-cultivation on soybean transformation efficiency. Five light intensities were implemented during five days of co-cultivation: 50, 100, 150, 190 μmol∙m-2∙s-1 of white LEDs in addition to 100 μmol∙m-2∙s-1 of fluorescent light. After co-cultivation, all the explants underwent shoot induction and elongation with selection pressure, rooting and acclimation under uniform condition. The experiment was conducted with two selectable markers, hppdPf-4Pa and bar, separately, investigating whether the potential light effects vary due to the marker-associated pathways. The positive PCR analysis of rooted in vitro plants suggested successful transformation events achieved under both selectable markers across all light treatments ranging from 2.4% to 6.9%. Increasing LED light intensity during co-cultivation resulted in different transformation efficiencies between the two selectable markers. Results indicated that increasing the light intensity during co-cultivation led to a linear increase in transformation efficiency when shoot regeneration was under 4-Hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor selection. No difference in transformation efficiency was detected among the treatments under glufosinate selection. Furthermore, when selection occurred with HPPD inhibitor, variation of transformation efficiency was also observed between fluorescent light and white LED at 100 μmol∙m-2∙s-1. The results highlight the significance and potential applications of investigating the impact of light on transformation efficiency.

  PublisherDOI

• Model-based inference of a dual role for HOPS in regulating guard cell vacuole fusion

  in silico Plants · 2024-01-01 · 5 citations

  articleOpen access

  Guard cell movements depend, in part, on the remodelling of vacuoles from a highly fragmented state to a fused morphology during stomata opening. Indeed, full opening of plant stomata requires vacuole fusion to occur. Fusion of vacuole membranes is a highly conserved process in eukaryotes, with key roles played by two multi-subunit complexes: HOPS (homotypic fusion and vacuolar protein sorting) and SNARE (soluble NSF attachment protein receptor). HOPS is a vacuole tethering factor that is thought to chaperone SNAREs from apposing vacuole membranes into a fusion-competent complex capable of rearranging membranes. In plants, recruitment of HOPS subunits to the tonoplast has been shown to require the presence of the phosphoinositide phosphatidylinositol 3-phosphate. However, chemically depleting this lipid induces vacuole fusion. To resolve this counter-intuitive observation regarding the role of HOPS in regulating plant vacuole morphology, we defined a quantitative model of vacuole fusion dynamics and used it to generate testable predictions about HOPS-SNARE interactions. We derived our model by using simulation-based inference to integrate prior knowledge about molecular interactions with limited, qualitative observations of emergent vacuole phenotypes. By constraining the model parameters to yield the emergent outcomes observed for stoma opening-as induced by two distinct chemical treatments-we predicted a dual role for HOPS and identified a stalled form of the SNARE complex that differs from phenomena reported in yeast. We predict that HOPS has contradictory actions at different points in the fusion signalling pathway, promoting the formation of SNARE complexes, but limiting their activity.

  PublisherDOI

Awards & honors

• Vacuole Remodeling in Guard Cells During Stomata Movements (…