Biology subjects

Niyogi, K. K.

Publications and source records attributed to Niyogi, K. K..

At least 19 recordsLinked to original sources

bioRxiv2026

A Natural qE- and qZ-Deficient Alga Retains Functional Plant-Like and Chlorophycean Violaxanthin De-Epoxidases

Photosynthetic organisms protect themselves from excess light through non-photochemical quenching (NPQ), a process that safely dissipates excess light energy as heat. Current understanding of NPQ is largely drawn from a few model systems, leaving it unclear how universal NPQ mechanisms are across the green lineage. We characterize the slow, sustained NPQ of the green alga, Auxenochlorella protothecoides x symbiontica (UTEX 250-A). The Auxenochlorella genome lacks Light-Harvesting Complex Stress Related (LHCSR), one of the two proteins required for the fast energy-dependent quenching (qE) component of NPQ. Mutants lacking the Photosystem II Subunit S (PSBS)-like gene (psbsl1) in Auxenochlorella had no effect on NPQ, indicating this alga lacks canonical qE. We identified two candidate enzymes associated with zeaxanthin-dependent quenching (qZ), which has slower kinetics: a plant-type Violaxanthin De-Epoxidase (VDE) and an algal-type Chlorophycean Violaxanthin De-Epoxidase (CVDE). High light did not trigger the expected conversion of violaxanthin to zeaxanthin, a pigment change that is a hallmark of qZ. Mutants lacking VDE (vde1), CVDE (cvde1), or both enzymes (vde1 cvde1) still displayed slow, reversible NPQ. However, when the Auxenochlorella VDE1 and CVDE1 were expressed in a VDE-deficient mutant of Nicotiana benthamiana, both enzymes restored zeaxanthin production and NPQ, confirming they are catalytically functional de-epoxidases. Together, these results reveal an alga that retains two functional, evolutionarily distinct xanthophyll-cycle enzymes yet does not rely on typical xanthophyll pigment dynamics for photoprotection. This points to a biological function that is decoupled from de-epoxidase activity, highlighting unexplored diversity in green algal photoprotection.

plant biology↗

bioRxiv2026

Simple Electroporation of Chlamydomonas reinhardtii Strains with an Intact Cell Wall

Chlamydomonas reinhardtii is a model green alga extensively used to study photosynthesis and cilia using molecular biology and genetics. Electroporation is a very common technique to transform DNA into the nuclear genome, which is essential to generate mutant collections and express transgenes. Here, we describe a simple, fast, and efficient protocol to transform strains with an intact cell wall. It achieves a good transformation efficiency without cell wall digestion or use of commercial kits and is compatible with the widely available Gene Pulser electroporation system. Key featuresO_LIHigh transformation efficiency of Chlamydomonas reinhardtii strains with an intact cell wall. C_LIO_LIFaster than currently available electroporation protocols. C_LI

molecular biology↗

bioRxiv2026

Ovothiol A mediates singlet oxygen resistance and acclimation in Chlamydomonas

Light is essential for photosynthetic organisms, but excess light can generate toxic levels of reactive oxygen species (ROS). To neutralize these ROS, plants and algae produce a variety of antioxidants like carotenoids, tocopherols, and glutathione. However, the role of alternative ROS scavengers, such as ovothiols, has not been studied in the context of oxidative stress in photosynthetic organisms. Here, we report that many algal groups have the potential for the biosynthesis of ovothiols, a group of thiohistidines. We discovered that the model green microalga Chlamydomonas reinhardtii produces millimolar concentrations of ovothiol A, whose biosynthesis is mediated by the ovothiol synthase OVOA1. Using CRISPR-generated ovoa1 knockout mutants, we found that ovothiol production is essential for resistance and acclimation to singlet oxygen, a prominent ROS in photosynthetic organisms. Finally, we demonstrated that OVOA1 expression is activated by singlet oxygen and light signaling pathways in which we identified the major regulatory factors. Overall, our results show that ovothiol A is a major, previously overlooked antioxidant in Chlamydomonas. This work broadens our understanding of cellular mechanisms that combat the damaging effects of oxidative stress. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/702910v2_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@cddb9corg.highwire.dtl.DTLVardef@10d0a43org.highwire.dtl.DTLVardef@11cc087org.highwire.dtl.DTLVardef@a40cc5_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2026

Architecting cis-regulation to quantitatively tune gene expression in cereals

Precise modulation of gene expression via cis-regulatory editing holds promise for non-transgenic crop improvement, but the sequence-to-function relationships that govern plant promoter activity remain poorly understood. Here, we develop a massively parallel reporter assay (MPRA) in Sorghum bicolor to systematically measure the effects of >30,000 CRISPR-like mutations-deletions, substitutions, and motif insertions-across entire native promoters and 5' untranslated regions (UTRs) of three photosynthesis genes: PsbS, Raf1, and SBPase. We find that gene expression is most tunable within a [~]500 base pair core promoter region, where mutational effects are reproducible across biological replicates and predictive of protein output. Within these regions, we identify compact deletions and motif insertions that strongly increase protein production (>30-fold relative to wild type), exceeding the performance of transgenic enhancer elements. Mutation-effect relationships are gene-specific, highlighting the need for tailored regulatory maps. Our results establish a high-throughput strategy for cis-regulatory fine-mapping that enables crop improvements via minimal, precise, and non-transgenic gene edits.

synthetic biology↗

bioRxiv2025

Biosynthesis of minimal C-phycocyanin chromophore assemblies in E. coli provides a platform to dissect protein-mediated tuning of exciton transfer

Cyanobacteria are arguably among the most evolutionarily successful organisms on Earth, inhabiting a wide range of ocean, fresh water, soil, and even desert environments on every continent. The cyanobacterial phycobilisome consists of stacks of disk-like light-collecting moieties, allophycocyanin (APC) and phycocyanin (CPC), with covalently bound phycocyanobilin (PCB) pigments. The ways in which the energies of the specific chromophores in these complexes are tuned by the protein to achieve its highly efficient and directional energy transfer are not fully understood, as complex combinations of decay pathways are occurring simultaneously and competitively through this elaborate light-harvesting system. This makes it difficult to extract information about isolated protein-pigment interactions. We provide herein a description of a useful new experimental platform in which we have recombinantly expressed a fully functioning CPC complex and selectively created minimal chromophore sets to study their individual contributions to the overall CPC spectra. Structural and computational analysis of this protein system have provided a greater understanding of how the protein environment serves to alter the photophysics of each of these chromophores. Introduction of a quencher into various positions within CPC confirmed the ability of the protein environment to tune the directionality of energy transport in this assembly. Further mutational analysis suggested the roles of key amino acids surrounding the chromophores, showcasing the utility of heterologous expression techniques for understanding the effects of structure on EET mechanisms in the phycobilisome.

bioengineering↗

bioRxiv2025

TOR inhibition drives accumulation of amino acids through transcriptional activation in algae

Cellular homeostasis is maintained by the balance between energy production and breakdown and is fundamental to all forms of life. The conserved, ancient target of rapamycin (TOR) kinase is a central metabolic regulator in eukaryotes that integrates carbon and nitrogen to maintain homeostasis and promote growth and development through protein synthesis. While TOR regulatory mechanisms of amino acid accumulation are well known in yeast and mammals, they remain unknown in photosynthetic organisms. Here, we developed the unicellular green alga Chromochloris zofingiensis as a simpler model system for understanding TOR function. Multiomics experiments showed that TOR inhibition leads to an increase in amino acid levels independent of hexokinase-mediated glucose signaling. We observed upregulation of selective amino acid biosynthesis pathways at the transcript and protein levels as potential mechanisms driving the increase in amino acids. Transcriptomics and proteomics experiments identified a basic helix-loop-helix (bHLH) transcription factor with rapid upregulation during TOR inhibition. DAP-seq analysis demonstrated that bHLH can bind directly to the promoters of amino acid biosynthesis genes, potentially regulating their transcription in response to TOR inhibition. We found high conservation of the bHLH-binding motif in the genomes of other green algae and plants, suggesting a conserved regulatory mechanism for amino acid biosynthesis across Viridiplantae. Phosphoproteomics experiments also revealed novel conserved targets that are not currently recognized as part of the TOR pathway. Altogether, our findings elucidate the transcriptional regulation of amino acid metabolism and explain how TOR regulates nitrogen metabolism to support growth and development in photosynthetic organisms. Significance StatementCarbon and nitrogen metabolism play key roles in enhancing plant yield and reducing fertilizer use. Thus, improving nitrogen utilization can significantly boost crop productivity and algal biotechnology. From yeast to plants to mammals, the protein target of rapamycin (TOR) kinase is an essential metabolic regulator. Here, we developed the unicellular green alga Chromochloris zofingiensis as a simpler system to study conserved mechanisms in TOR signaling. Using a multiomics approach, we showed transcriptional regulation of amino acid accumulation upon TOR inhibition and identified a transcription factor with evolutionarily conserved DNA binding sites in nitrogen metabolism genes. We also discovered novel conserved targets of TOR. Our study demonstrates the role of TOR in regulating nitrogen metabolism to support growth and development in photosynthetic organisms.

plant biology↗

bioRxiv2025

Light on its feet: Acclimation to high and low diurnal light is flexible in Chlamydomonas reinhardtii

Chlamydomonas acclimates to repeated low (LL) or high light (HL) days by changing the abundance of photosynthetic complexes and the ultrastructure of its thylakoid membranes. These phenotypes persist through the night phases, suggesting a readiness for the daylight environment that is routinely experienced despite the intervening dark periods (Dupuis & Ojeda et al. 2025). Here, we investigate how prior acclimation impacts algal fitness upon a change in daylight intensity and how quickly Chlamydomonas can reprogram its photoprotective strategy in a diurnal context. We performed a systems analysis of synchronized populations acclimated to diurnal LL when subjected to HL days and of populations acclimated to diurnal HL when subjected to LL days. In the latter case, diurnal photoacclimation decreased fitness during the first day at a new light intensity: HL-acclimated cells barely increased in size over the first LL period, and they failed to complete a cell cycle. However, although LL-acclimated cells showed severe photodamage after 6 hours of HL, they recovered chloroplast form and function later that afternoon and successfully divided at nightfall. These cells rapidly altered their thylakoid membrane ultrastructure, increased their photoprotective quenching capacity, and decreased their inventory of photosystem and antenna proteins by the end of the first HL day. Transcriptomic and proteomic analyses revealed rapid induction of thousands of genes, including those encoding proteases, chaperones, and other proteins involved in the chloroplast unfolded protein response. These results show that the alga is highly flexible and competent to rapidly acclimate to changes in diurnal light intensity.

plant biology↗

bioRxiv2025

Powdery mildew exploits host plastoglobuli functions via DGAT3 and FBN2 for proliferation

Plastoglobuli (PG) are specialized lipid-containing compartments attached to thylakoid membranes within chloroplasts. PGs participate in various metabolic pathways in response to environmental stresses such as high light, nitrogen deficiency, and heat stress. However, their roles in biotic stress remain largely unexplored. In this study, two distinct lines of investigation converge on the importance of PGs to the powdery mildew-Arabidopsis interaction. First, powdery mildew infection results in the accumulation of PGs and PG-localized Arabidopsis Diacylglycerol Acyltransferase 3 (DGAT3), a triacylglycerol biosynthetic enzyme that supports powdery mildew proliferation. Second, a bioinformatic analysis led to the identification of a powdery mildew effector that is PG-localized and interacts specifically with FIBRILLIN 2 (FBN2), one of the most abundant PG core proteins. While silencing the effector limits powdery mildew proliferation, an FBN2 knockout results in increased spore production, providing a direct link between PG function and pathogen manipulation of the host. These findings underscore the significance of PGs in host-pathogen interactions and offer new insights into the intricate mechanisms underlying the interaction of an obligate biotroph with its host. SIGNIFICANCEPlastoglobuli (PG) functions are largely unexplored in the context of host-pathogen interactions. Using the Arabidopsis-powdery mildew system, we uncover new roles for PGs in host-pathogen dynamics through integrated genetic, biochemical, and microscopic analyses. Mutants in PG-localized proteins impact powdery mildew proliferation. Moreover, a powdery mildew effector that interacts with the core PG protein FBN2 is identified and found to contribute to virulence. These findings suggest this obligate biotrophic pathogen exploits PGs for its own gain.

plant biology↗

bioRxiv2025

Natural Variation in Photoprotection: Rapid NPQ Kinetics in Ferns

Land plants perform oxygenic photosynthesis but are unable to use all of the solar radiation that they absorb on a daily basis. To minimize the production of reactive oxygen species in excess light, photosynthetic organisms use non-photochemical quenching (NPQ) mechanisms to dissipate excess excitation energy. However, the on-off transition of these mechanisms is slower than the light fluctuations themselves. In high-to-low light transitions, this can be costly to the overall productivity and carbon gain of the organism across its lifetime, because useful light energy is wasted. Here, we characterize the rapid kinetics of NPQ found in species across the fern lineage. Most of the 23 examined fern species showed faster NPQ induction and faster and more complete NPQ relaxation. Curve fitting suggested that energy-dependent quenching was the dominant contributor to the kinetics. The xerophytic fern Astrolepis windhamii exhibited rapid, dithiothreitol-resistant accumulation of zeaxanthin during NPQ induction, and it maintained low residual NPQ after NPQ relaxation, which however was not associated with rapid re-epoxidation of zeaxanthin. Rapid NPQ kinetics might have been an adaptive trait as ferns radiated in sunflecked forest understories during angiosperm diversification and expansion during the Cretaceous.

plant biology↗

bioRxiv2025

Modification of Non-photochemical Quenching Pathways in the C4 Model Plant Setaria viridis Revealed Shared and Unique Photoprotection Mechanisms as Compared to C3 Plants

Light is essential for photosynthesis; however, excess light can increase the accumulation of photoinhibitory reactive oxygen species that reduce photosynthetic efficiency. Plants have evolved photoprotective non-photochemical quenching (NPQ) pathways to dissipate excess light energy. In tobacco and soybean (C3 plants), overexpression of three NPQ genes, violaxanthin de-epoxidase (VDE), Photosystem II Subunit S (PsbS), and zeaxanthin epoxidase (ZEP), hereafter VPZ, resulted in faster NPQ induction and relaxation kinetics, and increased crop yields in field conditions. NPQ is well-studied in C3 plants; however, NPQ and the translatability of the VPZ approach in C4 plants is poorly understood. The green foxtail Setaria viridis is an excellent model to study photosynthesis and photoprotection in C4 plants. To understand the regulation of NPQ and photosynthesis in C4 plants, we performed transient overexpression in Setaria protoplasts and generated (and employed) stable transgenic Setaria plants overexpressing one of the three Arabidopsis NPQ genes or all three NPQ genes (AtVPZ lines). Overexpressing (OE) AtVDE and AtZEP in Setaria produced similar results as in C3 plants, with increased or reduced zeaxanthin (thus NPQ), respectively. However, overexpressing AtPsbS appeared to be challenging in Setaria, with largely reduced NPQ in protoplasts and under-represented homozygous AtPsbS-OE lines, potentially due to competitive and tight heterodimerization of AtPsbS and SvPsbS proteins. Furthermore, Setaria AtVPZ lines had increased zeaxanthin, faster NPQ induction, higher NPQ level, but slower NPQ relaxation. Despite this, AtVPZ lines had improved growth as compared to wildtype under several conditions, especially high temperatures, which is not related to the faster relaxation of NPQ but may be attributable to increased zeaxanthin and NPQ in C4 plants. Our results identified shared and unique characteristics of the NPQ pathway in C4 model Setaria as compared to C3 plants and provide insights to improve C4 crop yields under fluctuating environmental conditions.

plant biology↗

bioRxiv2024

The cell biology and genome of Stentor pyriformis, a giant cell that embeds symbiotic algae in a microtubule meshwork

Endosymbiotic events in which an endosymbiont is retained within a cell that remains capable of phagocytosis, a situation known as mixotrophy, provide potentially important clues about the evolutionary origins of eukaryotes, particularly regarding the relative evolutionary sequence of phagocytosis and endosymbiosis. Mixotrophy in ciliates is commonplace, but has been investigated at a cellular and molecular level almost entirely in one organism, Paramecium bursaria. Reliance on just one model system makes it difficult to know which cell biological aspects of this system represent general features of ciliate mixotrophy versus accidental features of the specific organism. Here we describe the cell biology and genome of the giant heterotrichous ciliate Stentor pyriformis. We show that this giant unicellular organism contains Chlorella variabilis as its endosymbiont, that the Chlorella can live freely outside the host, that within the host the Chlorella cells are docked near the cell surface, surrounded by microtubule "baskets", and that the photosynthetic efficiency of the Chlorella is reduced inside the Stentor cell compared to when it is free-living outside the host, with photon energy instead being shunted to non-photochemical quenching. Compared to the non-mixotrophic Stentor coeruleus, S. pyriformis has several distinct cellular features that may be related to endosymbiosis: the presence of microtubule baskets, which are absent in S. coeruleus; positive rather than negative phototaxis, which is likely an adaptation to allow the photosynthetic symbionts access to sufficient light; and a lack of pigment in the host cell, which may be an adaptation to tolerate high light levels. Compared to P. bursaria, S. pyriformis has several similar cellular features: in both organisms, the symbiont is a strain of Chlorella variabilis; the Chlorella endosymbiont retains the ability to live freely when separated from the host; and the algal symbionts contained in perialgal vesicles are docked at the cell surface. One potentially informative difference between P. bursaria and S. pyriformis is that S. pyriformis employs a standard genetic code, similar to other Stentor species but different from most other ciliates, including P. bursaria, which use a non-standard code in which one or more stop-codons are respecified to encode amino acids. This difference in genetic code could serve as a barrier to impede gene transfer from symbiont to host in other ciliates, but this would not be a factor in S. pyriformis. A second cell biological difference is that whereas P. bursaria performs phototaxis by a kinetic accumulation mechanism, in which swimming is non-directional but cells slow down in regions of higher light intensity, S. pyriformis performs directed swimming towards the direction of high light intensity. However, as in P. bursaria, phototaxis in S. pyriformis requires the presence of the Chlorella, implying a potential flow of information from the symbiont to direct the orientation and swimming of the host cell. We propose that S. pyriformis will serve as a useful model system for studying the evolution of mixotrophy and endosymbiosis, with unique advantages in terms of size and regenerative ability as well as distinct cellular and genomic features compared with other mixotrophic ciliate models.

cell biology↗

bioRxiv2024

Fe starvation induces a second LHCI tetramer to photosystem I in green algae

Iron (Fe) availability limits photosynthesis at a global scale where Fe-rich photosystem (PS) I abundance is drastically reduced in Fe-poor environments. We used single-particle cryo-electron microscopy to reveal a unique Fe starvation-dependent arrangement of light-harvesting chlorophyll (LHC) proteins where Fe starvation-induced TIDI1 is found in an additional tetramer of LHC proteins associated with PSI in Dunaliella tertiolecta and Dunaliella salina. These cosmopolitan green algae are resilient to poor Fe nutrition. TIDI1 is a distinct LHC protein that co- occurs in diverse algae with flavodoxin (an Fe-independent replacement for the Fe-containing ferredoxin). The antenna expansion in eukaryotic algae we describe here is reminiscent of the iron-starvation induced (isiA-encoding) antenna ring in cyanobacteria, which typically co-occurs with isiB, encoding flavodoxin. Our work showcases the convergent strategies that evolved after the Great Oxidation Event to maintain PSI capacity.

plant biology↗

bioRxiv2024

An algal nutrient-replete, optimized medium for fast growth and high triacylglycerol accumulation

Microalgae are promising sources to sustainably meet the global needs for energy and products. Algae grow under different trophic conditions, where nutritional status regulates biosynthetic pathways, energy production, and growth. The green alga Chromochloris zofingiensis has strong economic potential because it co-produces biofuel precursors and the high-value antioxidant astaxanthin while accumulating biomass when grown mixotrophically. As an emerging reference alga for photosynthesis, metabolism, and bioproduction, C. zofingiensis needs a defined, optimized medium to standardize experiments during fast growth. Because the interplay of glucose consumption (+Glc) and mineral deficiency influences photosynthesis, growth, and the production of lipids and astaxanthin, we designed a replete nutrient medium tailored to the C. zofingiensis cellular ionome. We combined inductively coupled plasma mass spectrometry (ICP-MS) and +Glc growth curves to determine a medium that is nutrient replete for at least 5 days of +Glc logarithmic growth. We found that there are high nutritional needs for phosphorus and sulfur during mixotrophy. Iron was the only element measured for which the cellular concentration correlated with exogenous concentration and was iteratively adjusted until the internal ionome was consistent through the logarithmic growth phase. This Chromochloris-Optimized Ratio of Elements (CORE) medium supports fast growth and high biomass without causing excess nutrient toxicity. This defined, nutrient-replete standard is important for future C. zofingiensis investigations and can be adapted for other species to support high biomass. The method used to develop CORE medium shows how ionomics informs replicable media design and may be applied in industrial settings to inform cost-effective biofuel production. Significance StatementStudying how carbon sources and mineral nutrients interplay to regulate algal metabolism can be exploited to discover and control pathways in photosynthesis and biofuel production. Here we design a medium from the cellular ionome of Chromochloris zofingiensis, a powerful algal model for photosynthesis, metabolism, and bioproducts, to provide a defined, replete standard for mixotrophic and heterotrophic growth of green algae. These media design principles show how accounting for increased nutritional demands based on carbon substrate can ensure experimental replicability when probing diverse algal metabolisms.

plant biology↗

bioRxiv2024

SOQ1 functions as a methionine sulfoxide reductase in the chloroplast lumen for regulation of photoprotective qH in Arabidopsis

Photosynthetic organisms must balance light absorption and energy dissipation to prevent photo-oxidative damage. Non-photochemical quenching (NPQ) dissipates excess light energy as heat, with the quenching component qH providing sustained photoprotection. However, the molecular mechanism underlying qH induction remains unclear. Our study focuses on the thylakoid membrane protein SUPPRESSOR OF QUENCHING 1 (SOQ1) and its inhibition of qH through interaction with LIPOCALIN IN THE PLASTID (LCNP) in Arabidopsis thaliana. Structural homology of SOQ1 lumenal domains with bacterial disulfide bond protein D suggested potential thiol-disulfide exchange activity. In vitro assays determined that both SOQ1 thioredoxin-like (Trx-like) and C-terminal (CTD) domains contain a redox-active cysteine pair and evidenced electron transfer from Trx-like to CTD. Importantly, we found that SOQ1 lumenal domains exhibit methionine sulfoxide reductase (Msr) activity converting oxidized methionine residues in LCNP back to methionine, which thereby inactivates LCNP and prevents qH formation. Mutational analyses identified cysteine residues in SOQ1-CTD and methionine residues in LCNP as critical for qH suppression, supporting their role in redox regulation. Additionally, we found that the redox state of SOQ1 in vivo is light-dependent, shifting from reduced to oxidized under stress conditions, indicating a dynamic regulation of its activity. We conclude that the Trx-like domain of SOQ1 provides reducing power to its CTD displaying Msr activity. SOQ1 is therefore an unusual example of a protein possessing both a disulfide reductase and Msr domain in tandem. Our findings elucidate the redox-regulation mechanism of qH involving SOQ1-mediated methionine reduction of LCNP, providing insights into the intricate control of photoprotective processes in chloroplasts and enhancing our understanding of plant resilience under environmental stress.

plant biology↗

bioRxiv2024

Too dim, too bright, and just right: Systems analysis of the Chlamydomonas diurnal program upon acclimation to light stress and limitation

Photosynthetic organisms coordinate their metabolism and growth with diurnal light, which can range in intensity from limiting to inhibitory. To gain a comprehensive understanding of how diurnal regulatory circuits interface with sensing and response to various light intensities, we performed a systems analysis of synchronized Chlamydomonas populations acclimated to low, moderate, and high diurnal light. Transcriptomic and proteomic data revealed that the Chlamydomonas rhythmic gene expression program is resilient to limiting and excess light. Although gene expression and photodamage are dynamic over the diurnal cycle, Chlamydomonas populations acclimated to low and high diurnal light exhibit constitutive phenotypes with respect to photosystem abundance, thylakoid architecture, and non-photochemical quenching that persist through the night. This suggests that cells "remember" or anticipate the daylight environment. The integrated data constitute an excellent resource for understanding gene regulatory mechanisms and photoprotection in eukaryotes under environmentally relevant conditions.

systems biology↗

bioRxiv2024

Resolving an unconventional non-photochemical quenching signature at the light-to-dark transition

Non-photochemical quenching (NPQ) protects photosynthetic organisms via diverse molecular players contributing at varying timescales. However, in the absence of one of the largest contributors to NPQ, energy-dependent quenching (qE), we observe an unusual but universal phenomenon: a transient increase in quenching in the dark following high light exposure. To mechanistically interrogate this light-to-dark (LtD) NPQ phenotype, we performed chlorophyll fluorescence lifetime snapshot measurements across a diverse array of Arabidopsis mutant backgrounds and chemical treatments. We found that the electrochemical gradient across the thylakoid membrane is essential for this phenomenon. Through analysis of higher-order Arabidopsis mutants, we also found that LtD NPQ is independent of the known forms of photoprotective NPQ, as well as the major and minor light-harvesting complexes (LHCII). Our results point to LtD NPQ as a photoinhibition (qI)-related, reaction center quenching with implications for photoprotection in fluctuating light.

plant biology↗

bioRxiv2024

In vivo analysis of the relationship between CP26 and qE-type NPQ via higher-order Arabidopsis cp26 mutants

CP26 is a monomeric minor light-harvesting complex of PSII (LHCII) protein that connects major LHCII trimers to the PSII core in photosynthetic thylakoid membranes. Previous studies have proposed that CP26 is not only involved in light harvesting but could also be involved in non-photochemical quenching (NPQ). Here, we analyzed higher-order Arabidopsis cp26 mutants using biophysical and pharmacological approaches to investigate the nature of NPQ and its relationship to known NPQ regulators (PSII subunit S (PsbS), the xanthophyll-converting enzyme VDE and the pH gradient across the thylakoid membrane). Maximum PSII quantum efficiencies (Fv/Fm) and chlorophyll fluorescence lifetimes in the dark were significantly lower in cp26 mutants, confirming that CP26 deficiency leads to a sustained quenched state even in the absence of light. Destabilized PSII-LHCII supercomplexes as observed with native PAGE analysis are the likely cause for this pre-quenched state, without other antenna proteins being able to rescue this phenotype. Further analyses revealed that cp26 mutants exhibit modest (single mutant) to highly significant (double mutants) reductions in overall NPQ capacity, which do not directly rely on PsbS and VDE (although the effect is more pronounced when these qE components are altered) but depend on thylakoid lumen acidification and protonation of protein residues. Together, these results show that the NPQ component lacking in cp26 mutants acts independently of qE and qZ and is induced in a slower phase of NPQ induction that most likely relies on pH-dependent conformational changes.

plant biology↗

bioRxiv2023

Editing cis-regulatory elements towards generating rice stomatal morphological variation for adaptation to broad and dynamic environments

Cis-regulatory element editing can generate quantitative trait variation while mitigating against extreme phenotypes and harmful pleiotropy associated with coding sequence mutations. Here, we applied a multiplexed guide RNA design approach, informed by bioinformatic datasets, to generate genotypic variation in the promoter of OsSTOMAGEN, a positive regulator of stomatal density in rice. Engineered genotypic variation corresponded to broad and continuous variation in stomatal density, ranging from 70% to 120% of wild-type stomatal density. This near-isogenic panel of stomatal variants was leveraged in physiological assays to establish discrete relationships between stomatal morphological variation and stomatal conductance, carbon assimilation, and intrinsic water use efficiency in steady-state and fluctuating light conditions. Additionally, promoter alleles were subjected to vegetative drought regimes to assay the effects of the edited alleles on developmental response to drought. Notably, the capacity for drought-responsive stomatal density reprogramming in stomagen and two cis-regulatory edited alleles was reduced. Collectively our data demonstrate that cis-regulatory element editing can generate near-isogenic trait variation that can be leveraged for establishing relationships between anatomy, physiology, and crop improvement along diverse environmental clines.

plant biology↗