Arabidopsis Leaf Research Articles

Leaf growth in third dimension: a perspective of leaf thickness from genetic regulation to ecophysiology.

Leaf thickness, the leaf growth in the third dimension as quantified by the distance between the adaxial and abaxial surface, is an indispensable aspect of leaf development. The fitness of a plant is strongly influenced by leaf thickness via modulation of major physiological processes, including photosynthesis and water use efficiency. The cellular basis of leaf thickness by alterations in either cell size or the number of cell layers is envisaged using Arabidopsis leaf thickness mutants, such as angustifolia (an) and rotundifolia (rot). Environmental factors coordinate with endogenous signaling mechanisms to exhibit leaf thickness plasticity. Plants growing in different ecological and environmental regimes show different leaf thickness attributes. However, genetic and molecular understandings of leaf thickness regulation remain largely limited. In this review, we highlight how cellular growth is transposed to fine-tune the leaf thickness via the integration of potential cues and molecular players. We further discuss the physiological significance of leaf thickness plasticity to the environmental cues that might serve as ecological adaptation enabling the plants to withstand future climatic conditions. Taken together, we seek to bridge the genetics and molecular biology of leaf thickness to its physiological significance so that leaf thickness can be systemically targeted in crop improvement programs.

Fast chlorophyll fluorescence rise kinetics as a high-throughput diagnostic tool for evaluating the capacity of 2-amino-3-methylhexanoic acid at inducing plant resistance against high temperature

Plant resistant induction is considered as a promising strategy for protecting crops against extreme high temperature (HT). However, a high-throughput method to accurately estimate the capacity of plant resistance inducers (PRIs) for HT resistance has not been developed. Here, we present a simple approach using fast chlorophyll fluorescence kinetics in Arabidopsis leaf discs to assess PRI efficacy in inducing HT resistance. Both 2-amino-3-methylhexanoic acid (AMHA) and salicylic acid (SA) significantly alleviated the temperature-dependent increase in the K-peak of the OJIP curve and variations in amplitude of heat-responsive JIP-test parameters within the elevated-temperature range of 25 to 42℃. The PIABS (performance index on absorption basis) and WK (relative variable fluorescence at the K-step to the amplitude FJ - FO) as two classical heat-responsive characteristic parameters were used to produce a novel hypersensitive parameter HT sensitivity indicator, PIABS/WK (named Hs). Based on the correlation of logHs with elevated temperatures, a model for quantifying the capacity of HT-resistance induction (called Ci) by AMHA or SA was established. A three-grade classification according to the Ci value was proposed as low (0<Ci ≤ 1℃), moderate (1℃ <Ci ≤ 2℃), and high resistance (Ci > 2℃). AMHA at 1µM and SA at 100µM had Ci values of 2.49℃ and 4.09℃ in Arabidopsis plants, respectively, associated with their high level of HT resistance induction. Additionally, the EC50 derived from the relative stimulation ratio (Kc) was also introduced as a quantitative index for measuring the ability of AMHA and SA to induce HT resistance. The EC50 value of AMHA is about 0.1µM in Arabidopsis and 0.35µM in tomato, being much lower than that of SA (approximately 63µM in Arabidopsis). Thus, AMHA is a more potent plant inducer than SA. The model was validated through additional experimental evidence, demonstrating its reliability and applicability. This study provides an expeditious high-throughput method for screening promising PRI candidates.

Insights into physiological roles of flavonoids in plant coldacclimation.

Flavonoids represent a diverse group of plant specialised metabolites which are also discussed in the context of dietary health and inflammatory response. Numerous studies have revealed that flavonoids play a central role in plant acclimation to abiotic factors like low temperature or high light, but their structural and functional diversity frequently prevents a detailed mechanistic understanding. Further complexity in analysing flavonoid metabolism arises from the different subcellular compartments which are involved in biosynthesis and storage. In the present study, non-aqueous fractionation of Arabidopsis leaf tissue was combined with metabolomics and proteomics analysis to reveal the effects of flavonoid deficiencies on subcellular metabolism during cold acclimation. During the first 3 days of a 2-week cold acclimation period, flavonoid deficiency was observed to affect pyruvate, citrate and glutamate metabolism which indicated a role in stabilising C/N metabolism and photosynthesis. Also, tetrahydrofolate metabolism was found to be affected, which had significant effects on the proteome of the photorespiratory pathway. In the late stage of cold acclimation, flavonoid deficiency was found to affect protein stability, folding and proteasomal degradation, which resulted in a significant decrease in total protein amounts in both mutants. In summary, these findings suggest that flavonoid metabolism plays different roles in the early and late stages of plant cold acclimation and significantly contributes to establishing a new protein homeostasis in a changing environment.

MIR164B ensures robust Arabidopsis leaf development by compensating for compromised POLYCOMB REPRESSIVE COMPLEX2 function.

Robustness is pervasive throughout biological systems, enabling them to maintain persistent outputs despite perturbations in their components. Here, we reveal a mechanism contributing to leaf morphology robustness in the face of genetic perturbations. In Arabidopsis (Arabidopsis thaliana), leaf shape is established during early development through the quantitative action of the CUP-SHAPED COTYLEDON2 (CUC2) protein, whose encoding gene is negatively regulated by the co-expressed MICRORNA164A (MIR164A) gene. Compromised epigenetic regulation due to defective Polycomb Repressive Complex 2 (PRC2) function results in the transcriptional derepression of CUC2 but has no impact on CUC2 protein dynamics or early morphogenesis. We solve this apparent paradox by showing that compromised PRC2 function simultaneously derepresses the expression of another member of the MIR164 gene family, MIR164B. This mechanism dampens CUC2 protein levels, thereby compensating for compromised PRC2 function and canalizing early leaf morphogenesis. Furthermore, we show that this compensation mechanism is active under different environmental conditions. Our findings shed light on how the interplay between different steps of gene expression regulation can contribute to developmental robustness.

The AtMINPP Gene, Encoding a Multiple Inositol Polyphosphate Phosphatase, Coordinates a Novel Crosstalk between Phytic Acid Metabolism and Ethylene Signal Transduction in Leaf Senescence.

Plant senescence is a highly coordinated process that is intricately regulated by numerous endogenous and environmental signals. The involvement of phytic acid in various cell signaling and plant processes has been recognized, but the specific roles of phytic acid metabolism in Arabidopsis leaf senescence remain unclear. Here, we demonstrate that in Arabidopsis thaliana the multiple inositol phosphate phosphatase (AtMINPP) gene, encoding an enzyme with phytase activity, plays a crucial role in regulating leaf senescence by coordinating the ethylene signal transduction pathway. Through overexpressing AtMINPP (AtMINPP-OE), we observed early leaf senescence and reduced chlorophyll contents. Conversely, a loss-of-function heterozygous mutant (atminpp/+) exhibited the opposite phenotype. Correspondingly, the expression of senescence-associated genes (SAGs) was significantly upregulated in AtMINPP-OE but markedly decreased in atminpp/+. Yeast one-hybrid and chromatin immunoprecipitation assays indicated that the EIN3 transcription factor directly binds to the promoter of AtMINPP. Genetic analysis further revealed that AtMINPP-OE could accelerate the senescence of ein3-1eil1-3 mutants. These findings elucidate the mechanism by which AtMINPP regulates ethylene-induced leaf senescence in Arabidopsis, providing insights into the genetic manipulation of leaf senescence and plant growth.

BHLH transcription factors cooperate with chromatin remodelers to regulate cell fate decisions during Arabidopsis stomatal development.

The development of multicellular organisms requires coordinated changes in gene expression that are often mediated by the interaction between transcription factors (TFs) and their corresponding cis-regulatory elements (CREs). During development and differentiation, the accessibility of CREs is dynamically modulated by the epigenome. How the epigenome, CREs, and TFs together exert control over cell fate commitment remains to be fully understood. In the Arabidopsis leaf epidermis, meristemoids undergo a series of stereotyped cell divisions, then switch fate to commit to stomatal differentiation. Newly created or reanalyzed scRNA-seq and ChIP-seq data confirm that stomatal development involves distinctive phases of transcriptional regulation and that differentially regulated genes are bound by the stomatal basic helix-loop-helix (bHLH) TFs. Targets of the bHLHs often reside in repressive chromatin before activation. MNase-seq evidence further suggests that the repressive state can be overcome and remodeled upon activation by specific stomatal bHLHs. We propose that chromatin remodeling is mediated through the recruitment of a set of physical interactors that we identified through proximity labeling-the ATPase-dependent chromatin remodeling SWI/SNF complex and the histone acetyltransferase HAC1. The bHLHs and chromatin remodelers localize to overlapping genomic regions in a hierarchical order. Furthermore, plants with stage-specific knockdown of the SWI/SNF components or HAC1 fail to activate specific bHLH targets and display stomatal development defects. Together, these data converge on a model for how stomatal TFs and epigenetic machinery cooperatively regulate transcription and chromatin remodeling during progressive fate specification.

Monitoring plant growth by analyzing their morphology using microfocus X‐ray CT

AbstractThe growth and development of embryophytes is deeply influenced by environmental stimuli, such as light, temperature, and soil nutrients. Understanding the mechanisms underlying the growth response of plants to environmental stimuli is crucial for agriculture. In this study, we examined the morphology of a flowering plant, Arabidopsis thaliana, using microfocus X‐ray computed tomography (µCT), which enables non‐destructive analysis of the external and internal structures of plants. Three‐dimensional (3D) images of the plant, which were reconstructed from X‐ray scanned data, clearly showed the shapes of its leaves, stems, and buds from any angle. At a higher magnification, the µCT also revealed the small hair‐like structures called trichomes on the Arabidopsis leaf epidermis. However, motion artifacts found in the 3D‐reconstructed images indicated that plant's growth rate was faster than scanning speed. Thus, scan parameters must be accordingly optimized. Additionally, CT‐based 3D printing can be used to design micro devices that can be further used to monitor plant growth. These results suggest that µCT is a useful technique for analyzing morphology of growing plants.

Plasticity of the Arabidopsis leaf lipidome and proteome in response to pathogen infection and heat stress.

Plants must cope with a variety of stressors during their life cycle, and the adaptive responses to these environmental cues involve all cellular organelles. Among them, comparatively little is known about the contribution of cytosolic lipid droplets (LDs) and their core set of neutral lipids and associated surface proteins to the rewiring of cellular processes in response to stress. Here, we analyzed the changes that occur in the lipidome and proteome of Arabidopsis (Arabidopsis thaliana) leaves after pathogen infection with Botrytis cinerea or Pseudomonas syringae, or after heat stress. Analyses were carried out in wild-type plants and the oil-rich double mutant trigalactosyldiacylglycerol1-1 sugar dependent 1-4 (tgd1-1 sdp1-4) that allowed for an allied study of the LD proteome in stressed leaves. Using liquid chromatography-tandem mass spectrometry-based methods, we showed that a hyperaccumulation of the primary LD core lipid triacylglycerol is a general response to stress and that acyl chain and sterol composition are remodeled during cellular adaptation. Likewise, comparative analysis of the LD protein composition in stress-treated leaves highlighted the plasticity of the LD proteome as part of the general stress response. We further identified at least two additional LD-associated proteins, whose localization to LDs in leaves was confirmed by confocal microscopy of fluorescent protein fusions. Taken together, these results highlight LDs as dynamic contributors to the cellular adaptation processes that underlie how plants respond to environmental stress.

A tale of two pumps: Blue light and abscisic acid alter Arabidopsis leaf hydraulics via bundle sheath cell H+-ATPases.

The bundle sheath cell (BSC) layer tightly enveloping the xylem throughout the leaf is recognized as a major signal-perceiving "valve" in series with stomata, regulating leaf hydraulic conductance (Kleaf) and thereby radial water flow via the transpiring leaf. The BSC blue light (BL) signaling pathway increases Kleaf and the underlying BSC water permeability. Here, we explored the hypothesis that BSCs also harbor a Kleaf-downregulating signaling pathway related to the stress phytohormone abscisic acid (ABA). We employed fluorescence imaging of xylem sap in detached leaves and BSC protoplasts from different genotypes of Arabidopsis (Arabidopsis thaliana) plants, using pH and membrane potential probes to monitor physiological responses to ABA and BL in combination with pharmacological agents. We found that BL-enhanced Kleaf required elevated BSC cytosolic Ca2+. ABA inhibited BL-activated xylem-sap-acidifying BSC H+-ATPase AHA2 (Arabidopsis H+-ATPase 2), resulting in depolarized BSCs and alkalinized xylem sap. ABA also stimulated BSC vacuolar H+-ATPase (VHA), which alkalinized the BSC cytosol. Each pump stimulation, AHA2 by BL and VHA by ABA (under BL), also required Ca2+. ABA stimulated VHA in the dark depending on Ca2+, but only in an alkaline external medium. Taken together with earlier findings on the pH sensitivity of BSC osmotic water permeability (i.e. aquaporin activity), our results suggest a Ca2+-dependent and pH-mediated causative link between the BL- and ABA-regulated activities of two BSC H+-ATPases and Kleaf.

ESR2–HDA6 complex negatively regulates auxin biosynthesis to delay callus initiation in Arabidopsis leaf explants during tissue culture

Plants exhibit an astonishing ability to regulate organ regeneration upon wounding. Excision of leaf explants promotes the biosynthesis of indole-3-acetic acid (IAA), which is polar-transported to excised regions, where cell fate transition leads to root founder cell specification to induce de novo root regeneration. The regeneration capacity of plants has been utilized to develop in vitro tissue culture technologies. Here, we report that IAA accumulation near the wounded site of leaf explants is essential for callus formation on 2,4-dichlorophenoxyacetic acid (2,4-D)-rich callus-inducing medium (CIM). Notably, a high concentration of 2,4-D does not compensate for the action of IAA because of its limited efflux; rather, it lowers IAA biosynthesis via a negative feedback mechanism at an early stage of in vitro tissue culture, delaying callus initiation. The auxin negative feedback loop in CIM-cultured leaf explants is mediated by an auxin-inducible APETALA2 transcription factor, ENHANCER OF SHOOT REGENERATION 2 (ESR2), along with its interacting partner HISTONE DEACETYLASE 6 (HDA6). The ESR2–HDA6 complex binds directly to, and removes the H3ac mark from, the YUCCA1 (YUC1), YUC7, and YUC9 loci, consequently repressing auxin biosynthesis and inhibiting cell fate transition on 2,4-D-rich CIM. These findings indicate that negative feedback regulation of auxin biosynthesis by ESR2 and HDA6 interferes with proper cell fate transition and callus initiation.

AraLeTA: An Arabidopsis leaf expression atlas across diurnal and developmental scales.

Mature plant leaves are a composite of distinct cell types, including epidermal, mesophyll, and vascular cells. Notably, the proportion of these cells and the relative transcript concentrations within different cell types may change over time. While gene expression data at a single-cell level can provide cell-type-specific expression values, it is often too expensive to obtain these data for high-resolution time series. Although bulk RNA-seq can be performed in a high-resolution time series, RNA-seq using whole leaves measures average gene expression values across all cell types in each sample. In this study, we combined single-cell RNA-seq data with time-series data from whole leaves to assemble an atlas of cell-type-specific changes in gene expression over time for Arabidopsis (Arabidopsis thaliana). We inferred how the relative transcript concentrations of different cell types vary across diurnal and developmental timescales. Importantly, this analysis revealed 3 subgroups of mesophyll cells with distinct temporal profiles of expression. Finally, we developed tissue-specific gene networks that form a community resource: an Arabidopsis Leaf Time-dependent Atlas (AraLeTa). This allows users to extract gene networks that are confirmed by transcription factor-binding data and specific to certain cell types at certain times of day and at certain developmental stages. AraLeTa is available at https://regulatorynet.shinyapps.io/araleta/.

A low-cost open-source imaging platform reveals spatiotemporal insight into leaf elongation and movement.

Plant organs move throughout the diurnal cycle, changing leaf and petiole positions to balance light capture, leaf temperature, and water loss under dynamic environmental conditions. Upward movement of the petiole, called hyponasty, is one of several traits of the shade avoidance syndrome (SAS). SAS traits are elicited upon perception of vegetation shade signals such as far-red light (FR) and improve light capture in dense vegetation. Monitoring plant movement at a high temporal resolution allows studying functionality and molecular regulation of hyponasty. However, high temporal resolution imaging solutions are often very expensive, making this unavailable to many researchers. Here, we present a modular and low-cost imaging set-up, based on small Raspberry Pi computers, that can track leaf movements and elongation growth with high temporal resolution. We also developed an open-source, semi-automated image analysis pipeline. Using this setup we followed responses to FR enrichment, light intensity, and their interactions. Tracking both elongation and the angle of the petiole, lamina, and entire leaf in Arabidopsis (Arabidopsis thaliana) revealed insight into R:FR sensitivities of leaf growth and movement dynamics and the interactions of R:FR with background light intensity. The detailed imaging options of this system allowed us to identify spatially separate bending points for petiole and lamina positioning of the leaf.

HDA6 modulates Arabidopsis pavement cell morphogenesis through epigenetic suppression of ROP6 GTPase expression and signaling.

The transcriptional regulation of Rho-related GTPase from plants (ROPs), which determine cell polarity formation and maintenance during plant development, still remains enigmatic. In this study, we elucidated the epigenetic mechanism of histone deacetylase HDA6 in transcriptional repression of ROP6 and its impact on cell polarity and morphogenesis in Arabidopsis leaf epidermal pavement cells (PCs). We found that the hda6 mutant axe1-4 exhibited impaired jigsaw-shaped PCs and convoluted leaves. This correlated with disruptions in the spatial organizations of cortical microtubules and filamentous actin, which is integral to PC indentation and lobe formation. Further transcriptional analyses and chromatin immunoprecipitation assay revealed that HDA6 specifically represses ROP6 expression through histone H3K9K14 deacetylation. Importantly, overexpression of dominant negative-rop6 in axe1-4 restored interdigitated cell morphology. Our study unveils HDA6 as a key regulator in Arabidopsis PC morphogenesis through epigenetic suppression of ROP6. It reveals the pivotal role of HDA6 in the transcriptional regulation of ROP6 and provides compelling evidence for the functional interplay between histone deacetylation and ROP6-mediated cytoskeletal arrangement in the development of interdigitated PCs.

The Arabidopsis leaf quantitative atlas: a cellular and subcellular mapping through unified data integration.

Quantitative analyses and models are required to connect a plant's cellular organisation with its metabolism. However, quantitative data are often scattered over multiple studies, and finding such data and converting them into useful information is time-consuming. Consequently, there is a need to centralise the available data and to highlight the remaining knowledge gaps. Here, we present a step-by-step approach to manually extract quantitative data from various information sources, and to unify the data format. First, data from Arabidopsis leaf were collated, checked for consistency and correctness and curated by cross-checking sources. Second, quantitative data were combined by applying calculation rules. They were then integrated into a unique comprehensive, referenced, modifiable and reusable data compendium representing an Arabidopsis reference leaf. This atlas contains the metrics of the 15 cell types found in leaves at the cellular and subcellular levels.

A computational model of Pseudomonas syringae metabolism unveils a role for branched-chain amino acids in Arabidopsis leaf colonization.

Bacterial pathogens adapt their metabolism to the plant environment to successfully colonize their hosts. In our efforts to uncover the metabolic pathways that contribute to the colonization of Arabidopsis thaliana leaves by Pseudomonas syringae pv tomato DC3000 (Pst DC3000), we created iPst19, an ensemble of 100 genome-scale network reconstructions of Pst DC3000 metabolism. We developed a novel approach for gene essentiality screens, leveraging the predictive power of iPst19 to identify core and ancillary condition-specific essential genes. Constraining the metabolic flux of iPst19 with Pst DC3000 gene expression data obtained from naïve-infected or pre-immunized-infected plants, revealed changes in bacterial metabolism imposed by plant immunity. Machine learning analysis revealed that among other amino acids, branched-chain amino acids (BCAAs) metabolism significantly contributed to the overall metabolic status of each gene-expression-contextualized iPst19 simulation. These predictions were tested and confirmed experimentally. Pst DC3000 growth and gene expression analysis showed that BCAAs suppress virulence gene expression in vitro without affecting bacterial growth. In planta, however, an excess of BCAAs suppress the expression of virulence genes at the early stages of infection and significantly impair the colonization of Arabidopsis leaves. Our findings suggesting that BCAAs catabolism is necessary to express virulence and colonize the host. Overall, this study provides valuable insights into how plant immunity impacts Pst DC3000 metabolism, and how bacterial metabolism impacts the expression of virulence.

Parallel auxin transport via PINs and plasmodesmata during the Arabidopsis leaf hyponasty response

Key messageThe leaf hyponasty response depends on tip-to-petiole auxin transport. This transport can happen through two parallel pathways: active trans-membrane transport mediated by PIN proteins and passive diffusion through plasmodesmata.A plant’s ability to counteract potential shading by neighboring plants depends on transport of the hormone auxin. Neighbor sensing at the leaf tip triggers auxin production. Once this auxin reaches the abaxial petiole epidermis, it causes cell elongation, which leads to leaf hyponasty. Two pathways are known to contribute to this intercellular tip-to-petiole auxin movement: (i) transport facilitated by plasma membrane-localized PIN auxin transporters and (ii) diffusion enabled by plasmodesmata. We tested if these two modes of transport are arranged sequentially or in parallel. Moreover, we investigated if they are functionally linked. Mutants in which one of the two pathways is disrupted indicated that both pathways are necessary for a full hyponasty response. Visualization of PIN3-GFP and PIN7-GFP localization indicated PIN-mediated transport in parallel to plasmodesmata-mediated transport along abaxial midrib epidermis cells. We found plasmodesmata-mediated cell coupling in the pin3pin4pin7 mutant to match wild-type levels, indicating no redundancy between pathways. Similarly, PIN3, PIN4 and PIN7 mRNA levels were unaffected in a mutant with disrupted plasmodesmata pathway. Our results provide mechanistic insight on leaf hyponasty, which might facilitate the manipulation of the shade avoidance response in crops.

Probing the in situ volumes of Arabidopsis leaf plastids using three-dimensional confocal and scanning electron microscopy.

Leaf plastids harbor a plethora of biochemical reactions including photosynthesis, one of the most important metabolic pathways on Earth. Scientists are eager to unveil the physiological processes within the organelle but also their interconnection with the rest of the plant cell. An increasingly important feature of this venture is to use experimental data in the design of metabolic models. A remaining obstacle has been the limited in situ volume information of plastids and other cell organelles. To fill this gap for chloroplasts, we established three microscopy protocols delivering in situ volumes based on: (i) chlorophyll fluorescence emerging from the thylakoid membrane, (ii) a CFP marker embedded in the envelope, and (iii) calculations from serial block-face scanning electron microscopy (SBFSEM). The obtained data were corroborated by comparing wild-type data with two mutant lines affected in the plastid division machinery known to produce small and large mesophyll chloroplasts, respectively. Furthermore, we also determined the volume of the much smaller guard cell plastids. Interestingly, their volume is not governed by the same components of the division machinery which defines mesophyll plastid size. Based on our three approaches, the average volume of a mature Col-0 wild-type mesophyll chloroplasts is 93 μm3 . Wild-type guard cell plastids are approximately 18 μm3 . Lastly, our comparative analysis shows that the chlorophyll fluorescence analysis can accurately determine chloroplast volumes, providing an important tool to research groups without access to transgenic marker lines expressing genetically encoded fluorescence proteins or costly SBFSEM equipment.

Epicuticular wax accumulation and regulation of wax pathway gene expression during bioenergy Sorghum stem development.

Bioenergy sorghum is a drought-tolerant high-biomass C4 grass targeted for production on annual cropland marginal for food crops due primarily to abiotic constraints. To better understand the overall contribution of stem wax to bioenergy sorghum's resilience, the current study characterized sorghum stem cuticular wax loads, composition, morphometrics, wax pathway gene expression and regulation using vegetative phase Wray, R07020, and TX08001 genotypes. Wax loads on sorghum stems (~103-215 µg/cm2) were much higher than Arabidopsis stem and leaf wax loads. Wax on developing sorghum stem internodes was enriched in C28/30 primary alcohols (~65%) while stem wax on fully developed stems was enriched in C28/30 aldehydes (~80%). Scanning Electron Microscopy showed minimal wax on internodes prior to the onset of elongation and that wax tubules first appear associated with cork-silica cell complexes when internode cell elongation is complete. Sorghum homologs of genes involved in wax biosynthesis/transport were differentially expressed in the stem epidermis. Expression of many wax pathway genes (i.e., SbKCS6, SbCER3-1, SbWSD1, SbABCG12, SbABCG11) is low in immature apical internodes then increases at the onset of stem wax accumulation. SbCER4 is expressed relatively early in stem development consistent with accumulation of C28/30 primary alcohols on developing apical internodes. High expression of two SbCER3 homologs in fully elongated internodes is consistent with a role in production of C28/30 aldehydes. Gene regulatory network analysis aided the identification of sorghum homologs of transcription factors that regulate wax biosynthesis (i.e., SbSHN1, SbWRI1/3, SbMYB94/96/30/60, MYS1) and other transcription factors that could regulate and specify expression of the wax pathway in epidermal cells during cuticle development.

Thylakoid Rhodanese-like Protein-Ferredoxin:NADP+ Oxidoreductase Interaction Is Integrated into Plant Redox Homeostasis System.

In vascular plants, the final photosynthetic electron transfer from ferredoxin (Fd) to NADP+ is catalyzed by the flavoenzyme ferredoxin:NADP+ oxidoreductase (FNR). FNR is recruited to thylakoid membranes via an integral membrane protein TROL (thylakoid rhodanese-like protein) and the membrane associated protein Tic62. We have previously demonstrated that the absence of TROL triggers a very efficient superoxide (O2•-) removal mechanism. The dynamic TROL-FNR interaction has been shown to be an apparently overlooked mechanism that maintains linear electron flow before alternative pathway(s) is(are) activated. In this work, we aimed to further test our hypothesis that the FNR-TROL pair could be the source element that triggers various downstream networks of chloroplast ROS scavenging. Tandem affinity purification followed by the MS analysis confirmed the TROL-FNR interaction and revealed possible interaction of TROL with the thylakoid form of the enzyme ascorbate peroxidase (tAPX), which catalyzes the H2O2-dependent oxidation of ascorbate and is, therefore, the crucial component of the redox homeostasis system in plants. Further, EPR analyses using superoxide spin trap DMPO showed that, in comparison with the wild type, plants overexpressing TROL (TROL OX) propagate more O2•- when exposed to high light stress. This indicates an increased sensitivity to oxidative stress in conditions when there is an excess of membrane-bound FNR and less free FNR is found in the stroma. Finally, immunohistochemical analyses of glutathione in different Arabidopsis leaf cell compartments showed highly elevated glutathione levels in TROL OX, indicating an increased demand for this ROS scavenger in these plants, likely needed to prevent the damage of important cellular components caused by reactive oxygen species.

Association of the Arabidopsis oleoyl Δ12-desaturase FAD2 with pre-cis-Golgi stacks at endoplasmic reticulum-Golgi-exit sites.

The unsaturation of phospholipids influences the function of membranes. In Arabidopsis thaliana, the oleoyl Δ12-desaturase FAD2 converts oleic (18:1Δ9 ) to linoleic acid (18:2Δ9,12 ) and influences phospholipid unsaturation in different cellular membranes. Despite its importance, the precise localization of Arabidopsis FAD2 has not been unambiguously described. As FAD2 is thought to modify phospholipid-associated fatty acids at the endoplasmic reticulum (ER), from where unsaturates are distributed to other cellular sites, we hypothesized that FAD2 locates to ER subdomains enabling trafficking of lipid intermediates through the secretory pathway. Fluorescent FAD2 fusions used to test this hypothesis were first assessed for functionality by heterologous expression in yeast (Saccharomyces cerevisiae), and in planta by Arabidopsis fad2 mutant rescue upon ectopic expression from an intrinsic FAD2 promoter fragment. Light sheet fluorescence, laser scanning confocal or spinning disc microscopy of roots, leaves, or mesophyll protoplasts showed the functional fluorescence-tagged FAD2 variants in flattened donut-shaped structures of ~0.5-1 μm diameter, in a pattern not resembling mere ER association. High-resolution imaging of coexpressed organellar markers showed fluorescence-tagged FAD2 in a ring-shaped pattern surrounding ER-proximal Golgi particles, colocalizing with pre-cis-Golgi markers. This localization required the unusual C-terminal retention signal of FAD2, and deletion or substitutions in this protein region resulted in relaxed distribution and diffuse association with the ER. The distinct association of FAD2 with pre-cis-Golgi stacks in Arabidopsis root and leaf tissue is consistent with a contribution of FAD2 to membrane lipid homeostasis through the secretory pathway, as verified by an increased plasma membrane liquid phase order in the fad2 mutant.