Screening a transposon-mutagenized soybean population led to the discovery of a recessively inherited chlorotic phenotype. This "y24" phenotype results in smaller stature, weaker stems, and a smaller root system. Genome sequencing identified 15 candidate genes with mutations likely to result in a loss of function. Amplicon sequencing of a segregating population was then used to narrow the list to a single candidate mutation, a single-base change in that disrupts splicing of the second intron. Single cell transcriptomic profiling indicates that this gene is expressed primarily in mesophyll cells, and RNA sequencing data indicate that it is upregulated in germinating seedlings by cold stress. Previous studies have shown that mutations to , the rice ortholog of , produced a chlorotic phenotype that was more pronounced in cool temperatures. Growing soybean y24 mutants at lower temperatures also resulted in a more severe phenotype. In addition, transgenic expression of wild-type in the knockout mutant of the Arabidopsis ortholog rescues the chlorotic phenotype, further supporting the hypothesis that the mutation in is causal of the y24 phenotype. The variant analysis strategy used to identify the genes underlying this phenotype provides a template for the study of other soybean mutants.

Cannabis plants produce a spectrum of secondary metabolites, encompassing cannabinoids and more than 300 non-cannabinoid compounds. Among these, anthocyanins have important functions in plants and also have well documented health benefits. Anthocyanins are largely responsible for the red/purple color phenotypes in plants. Although some well-known Cannabis varieties display a wide range of red/purple pigmentation, the genetic underpinnings of anthocyanin biosynthesis have not been well characterized in Cannabis. This study unveils the genetic diversity of anthocyanin biosynthesis genes found in Cannabis, and we characterize the diversity of anthocyanins and related phenolics found in four differently pigmented Cannabis varieties. Our investigation revealed that the genes , , , , , , , and exhibited the strongest correlation with anthocyanin accumulation in Cannabis leaves. The results of this study enhance our understanding of the anthocyanin biosynthetic pathway and shed light on the molecular mechanisms governing Cannabis leaf pigmentation.

Thylakoid membranes in chloroplasts and cyanobacteria harbor the multisubunit protein complexes that catalyze the light reactions of photosynthesis. In plant chloroplasts, the thylakoid membrane system comprises a highly organized network with several subcompartments that differ in composition and morphology: grana stacks, unstacked stromal lamellae, and grana margins at the interface between stacked and unstacked regions. The localization of components of the photosynthetic apparatus among these subcompartments has been well characterized. However, less is known about the localization of proteins involved in the biogenesis and repair of the photosynthetic apparatus, the partitioning of proteins between two recently resolved components of the traditional margin fraction (refined margins and curvature), and the effects of light on these features. In this study, we analyzed the partitioning of numerous thylakoid biogenesis and repair factors among grana, curvature, refined margin, and stromal lamellae fractions of thylakoid membranes, comparing the results from illuminated and dark-adapted plants. Several proteins previously shown to localize to a margin fraction partitioned in varying ways among the resolved curvature and refined margin fractions. For example, the ALB3 insertase and FtsH protease involved in photosystem II (PSII) repair were concentrated in the refined margin fraction, whereas TAT translocon subunits and proteins involved in early steps in photosystem assembly were concentrated in the curvature fraction. By contrast, two photosystem assembly factors that facilitate late assembly steps were depleted from the curvature fraction. The enrichment of the PSII subunit OE23/PsbP in the curvature fraction set it apart from other PSII subunits, supporting the previous conjecture that OE23/PsbP assists in PSII biogenesis and/or repair. The PSII assembly factor PAM68 partitioned differently among thylakoid fractions from dark-adapted plants and illuminated plants and was the only analyzed protein to convincingly do so. These results demonstrate an unanticipated spatial heterogeneity of photosystem biogenesis and repair functions in thylakoid membranes and reveal the curvature fraction to be a focal point of early photosystem biogenesis.

RNA interference (RNAi) is a crucial mechanism in immunity against infectious microbes through the action of DICER-LIKE (DCL) and ARGONAUTE (AGO) proteins. In the case of the taxonomically diverse fungal pathogen and the oomycete , plant DCL and AGO proteins have proven roles as negative regulators of immunity, suggesting functional specialization of these proteins. To address this aspect in a broader taxonomic context, we characterized the colonization pattern of an informative set of and loss-of-function mutants in upon infection with a panel of pathogenic microbes with different lifestyles, and a fungal mutualist. Our results revealed that, depending on the interacting pathogen, AGO1 acts as a positive or negative regulator of immunity, while AGO4 functions as a positive regulator. Additionally, AGO2 and AGO10 positively modulated the colonization by a fungal mutualist. Therefore, analyzing the role of RNAi across a broader range of plant-microbe interactions has identified previously unknown functions for AGO proteins. For some pathogen interactions, however, all tested mutants exhibited wild-type-like infection phenotypes, suggesting that the roles of AGO and DCL proteins in these interactions may be more complex to elucidate.

Many land plants have evolved such that the transition from vegetative to reproductive development is synchronized with environmental cues. Examples of reproduction in response to seasonal cues can be found in both vascular and nonvascular species; however, most of our understanding of the molecular events controlling this timing has been worked out in angiosperm model systems. While the organism-level mechanisms of sexual reproduction vary dramatically between vascular and nonvascular plants, phylogenetic and transcriptomic evidence suggest paralogs in nonvascular plants may have conserved function with their vascular counterparts. Given that undergoes sexual reproductive development in response to photoperiodic and cold temperature cues, it is well-suited for studying evolutionarily conserved mechanisms of seasonal control of reproduction. Thus, we used publicly available microarray data to identify genes differentially expressed in response to temperature cues. We identified two () genes in the genome that are the most like the angiosperm CDFs based on conservation of protein motifs and diurnal expression patterns. In angiosperms, DNA-One Finger Transcription Factors (DOFs) play an important role in regulating photoperiodic flowering, regulating physiological changes in response to seasonal temperature changes, and mediating the cold stress response. We created knockout mutations and tested their impact on sexual reproduction and response to cold stress. Unexpectedly, the timing of sexual reproduction in the -double mutants did not differ significantly from wild type, suggesting that the are not necessary for seasonal regulation of this developmental transition. We also found that there was no change in expression of downstream cold-regulated genes in response to cold stress and no change in freezing tolerance in the knockout mutant plants. Finally, we observed no interaction between PpCDLs and the partial homologs of FKF1, an repressor of CDFs. This is different from what is observed in angiosperms, which suggests that the functions of CDF proteins in angiosperms are not conserved in .

Callus and cell suspension culture techniques are valuable tools in plant biotechnology and are widely used in fundamental and applied research. For studies in callus and cell suspension cultures to be relevant, it is essential to know if the underlying biochemistry is similar to intact plants. This study examined the expression of core circadian genes in Arabidopsis callus from the cell suspension named AT2 and found that the circadian rhythms were impaired. The circadian waveforms were like intact plants in the light/dark cycles, but the circadian expression in the AT2 callus became weaker in the free-running, constant light conditions. Temperature cycles could drive the rhythmic expression in constant conditions, but there were novel peaks at the point of temperature transitions unique to each clock gene. We found that callus freshly induced from seedlings had normal oscillations, like intact plants, suggesting that the loss of the circadian oscillation in the AT2 callus was specific to this callus. We determined that neither the media composition nor the source of the AT2 callus caused this disruption. We observed that expression was not differentially expressed between dawn and dusk in both entrained, light-dark cycles and constant light conditions. Overexpression of in the AT2 callus partially recovers the circadian oscillation in the AT2 callus. This work shows that while callus and cell suspension cultures can be valuable tools for investigating plant responses, careful evaluation of their phenotype is important. Moreover, the altered circadian rhythms under constant light and temperature cycles in the AT2 callus could be useful backgrounds to understand the connections driving circadian oscillators and light and temperature sensing at the cellular level.

Quality assessment of pome fruits (i.e. apples and pears) is used not only for determining the optimal harvest time but also for the progression of fruit-quality attributes during storage. Therefore, it is typical to repeatedly evaluate fruits during the course of a postharvest experiment. This evaluation often includes careful visual assessments of fruit for apparent defects and physiological symptoms. A general best practice for quality assessment is to rate fruit using the same individual rater or group of individual raters to reduce bias. However, such consistency across labs, facilities, and experiments is often not feasible or attainable. Moreover, while these visual assessments are critical empirical data, they are often coarse-grained and lack consistent objective criteria. Granny, is a tool designed for rating fruit using machine-learning and image-processing to address rater bias and improve resolution. Additionally, Granny supports backward compatibility by providing ratings compatible with long-established standards and references, promoting research program continuity. Current Granny ratings include starch content assessment, rating levels of peel defects, and peel color analyses. Integrative analyses enhanced by Granny's improved resolution and reduced bias, such as linking fruit outcomes to global scale -omics data, environmental changes, and other quantitative fruit quality metrics like soluble solids content and flesh firmness, will further enrich our understanding of fruit quality dynamics. Lastly, Granny is open-source and freely available.

Soybean ( L.) is an important leguminous plant, in which pests trigger significant damage every year. Important members of this community are insects with piercing-sucking mouthpart, especially the southern green stinkbug, L.. This insect with its extraoral digestion causes visible alterations (morphological and color changes) in the seeds. We aimed to obtain precise information about the extent and nature of damage in soybeans caused by using nondestructive imaging methods. Two infestation conditions were applied: one with controlled numbers of pests (six insects/15 pods) and another with naturally occurring pests (samples collected from the apical part of the plant and samples from whole plants). An intact control group was also included, resulting in four treatment groups. Seed samples were analyzed by computed tomography (CT) and image color analysis under laboratory conditions. According to our CT findings, the damage caused by changed the radiodensity, volume, and shape (Solidity) of the soybean seeds during the pod-filling and maturing period. Radiodensity was significantly reduced in all three damaged categories compared to the intact sample; the mean radiodensity reduction range was 49-412 HU. The seed volume also decreased significantly (25%-80% decrease), with a threefold reduction for samples exposed to regulated damage compared to natural ones. The samples exposed to natural damage showed significant but minor reduction in solidity, while samples exposed to regulated damage showed a prominent decrease (~12%). Image color analysis showed that the damaged samples were well distinguishable, and the differences were statistically verifiable. The achieved data derived from our external and internal imaging approaches contribute to a better understanding of the internal chemical processes, and CT analysis helps to understand the alteration trends of the hidden structure of seeds caused by a pest. Our results can contribute to the development of a practically applicable system based on image analysis, which can identify lots damaged by insects.

Sunflower ( L.) plays an essential role in meeting the demand for edible oil worldwide. The yield of sunflower seeds encompasses several component traits, including the disc diameter. Over three consecutive years, 2019, 2020, and 2022, we assessed phenotypic variation in disc diameter across a diverse set of sunflower accessions (N = 342) in replicated field trials. Upon aggregating the phenotypic data from multiple years, we estimated the broad sense heritability ( ) of the disc diameter trait to be 0.88. A subset of N = 274 accessions was genotyped by using the tunable genotyping-by-sequencing (tGBS) method, resulting in 226,779 high-quality SNPs. Using these SNPs and the disc diameter phenotype, we conducted a genome-wide association study (GWAS) employing two statistical approaches: the mixed linear model (MLM) and the fixed and random model circulating probability unification (farmCPU). The MLM and farmCPU GWAS approaches identified 106 and 8 significant SNPs located close to 53 and 21 genes, respectively. The MLM analysis identified two significant peaks: a prominent signal on chromosome 10 and a relatively weaker signal on chromosome 16, both of which were also detected by farmCPU. The genetic loci associated with disc diameter, as well as the related candidate genes, present promising avenues for further functional validation and serve as a basis for sunflower oil yield improvement.

The Cysteine-rich secretory proteins (CRISPS), Antigen 5 (Ag5), and Pathogenesis-related 1 (PR-1) protein (CAP) superfamily members are found in multiple eukaryotic organisms, including yeasts, animals, and plants. Although one of the plant CAP family genes, is known to respond to pathogen infection in plants, the functions of other CAP family genes in remain largely unknown. In this study, we conducted a comprehensive analysis of the similarities, loci, and expression patterns of 22 Arabidopsis CAP genes/proteins, providing a clue to elucidate their molecular functions. According to the promoter-β-glucuronidase (GUS) analysis, members of the CAP family were expressed in various young tissues or organs, such as root and shoot meristems, reproductive tissues, and particularly at the lateral root initiation site before the formation of the lateral root primordium, with distinct expression specificity. In particular, , , and were specifically expressed in the cortical cells at the lateral root developing regions, suggesting that these genes may function in lateral root development. Thus, the expression patterns of Arabidopsis CAP family genes suggest that CAP family proteins may have certain function in the expressed organs or tissues in plant.

C photosynthesis can be complemented with a C carbon concentrating mechanism (CCM) to minimize photorespiratory losses. C photosynthesis is often more efficient than C under steady-state conditions. However, the C CCM depends on inter-cellular metabolite concentration gradients, which must increase following increases in light intensity and could decrease rates of C photosynthesis under fluctuating light. Additionally, incomplete flux through photorespiration could prove beneficial to C assimilation during light induction of the CCM. Here, we compare metabolic profiles in the closely related C and C during a light transient from low to high light to determine if these non-steady state accumulation patterns provide insight to the induction of the metabolite gradients needed to drive C4 intermediate transport and if there is incomplete cycling of photorespiratory intermediates. In these C and C species, metabolite steady-state pool sizes suggest that C transport acids maintain concentration gradients across the bundle sheath and mesophyll cell types under these light fluctuations. However, there was incomplete flux through photorespiration in the C , which could reduce photorespiratory CO loss via glycine decarboxylation and help maintain higher rates of assimilation during following induction periods.

Sequence-specific endonucleases have been key to the study of the mechanisms and control of DNA double-strand break (DSB) repair and recombination, and the availability of CRISPR-Cas nucleases over the last decade has driven rapid progress in the understanding and application of targeted recombination in many organisms, including plants. We present here an analysis of recombination at targeted chromosomal 5' overhang DSB generated by the FnCas12a endonuclease in the plant, . The much-studied Cas9 nuclease cleaves DNA to generate blunt-ended DSBs, but relatively less is known about the repair of other types of breaks, such as those with 5'-overhanging ends. Sequencing the repaired breaks clearly shows that the majority of repaired DSB carry small deletions and are thus repaired locally by end-joining recombination, confirmed by Nanopore sequencing of larger amplicons. Paired DSBs generate deletions at one or both cut-sites, as well as deletions and reinsertions of the deleted segment between the two cuts, visible as inversions. While differences are seen in the details, overall the deletion patterns are similar between repair at single-cut and double-cut events, notwithstanding the fact that only the former involve cohesive DNA overhangs. A strikingly different repair pattern is however observed at breaks flanked by direct repeats. This change in sequence context results in the presence of a major alternative class of repair events, corresponding to highly efficient repair by single-strand annealing recombination.

Anthropogenic increase in carbon dioxide (CO) affects plant physiology. Plant responses to elevated CO typically include: (1) enhanced photosynthesis and increased primary productivity due to carbon fertilization and (2) suppression of leaf transpiration due to CO-driven decrease in stomatal conductance. The combined effect of these responses on the total plant transpiration and on evapotranspiration (ET) has a wide range of implications on local, regional, and global hydrological cycles, and thus needs to be better understood. Here, we investigated the net effect of CO-driven perennial ryegrass () physiological responses on transpiration and evapotranspiration by integrating physiological and hydrological (water budget) methods, under a controlled environment. Measurements of the net photosynthetic rate, stomatal conductance, transpiration rate, leaf mass per area, aboveground biomass, and water balance components were recorded. Measured variables under elevated CO were compared with those of ambient CO. As expected, our results show that elevated CO significantly decreases whole-plant transpiration rates (38% lower in the final week) which is a result of lower stomatal conductance (57% lower in the final week) despite a slight increase in aboveground biomass. Additionally, there was an overall decline in evapotranspiration (ET) under elevated CO, indicating the impact of CO-mediated suppression of transpiration on the overall water balance. Although studies with larger sample sizes are needed for more robust conclusions, our findings have significant implications for global environmental change. Reductions in ET from ryegrass-dominated grasslands and pastures could increase soil moisture and groundwater recharge, potentially leading to increased surface runoff and flooding.

Burley tobacco, a chlorophyll-deficient mutant with impaired nitrogen use efficiency (NUE), generally requires three to five times more nitrogen fertilization than flue-cured tobacco to achieve a comparable yield, which generates serious environmental pollution and negatively affects human health. Therefore, exploring the mechanisms underlying NUE is an effective measure to reduce environmental pollution and an essential direction for burley tobacco plant improvement. Physiological and genetic factors affecting tobacco NUE were identified using two tobacco genotypes with contrasting NUE in hydroponic experiments. Nitrogen use inefficient genotype (TN90) had lower nitrogen uptake and transport efficiencies, reduced leaf and root biomass, lower nitrogen assimilation and photosynthesis capacity, and lower nitrogen remobilization ability than the nitrogen use efficient genotype (K326). Transcriptomic analysis revealed that genes associated with photosynthesis, carbon fixation, and nitrogen metabolism are implicated in NUE. Three nitrate transporter genes in the leaves (, , and ) and three nitrate transporter genes (, , and ) in roots were down-regulated by nitrogen starvation, all of which showed lower expression in TN90 than in K326. In addition, the protein-protein interaction (PPI) network diagram identified eight key genes (, , , , , , , and ) that may affect NUE. Less advantageous changes in nitrogen uptake, nitrogen assimilation in combination with nitrogen remobilization, and maintenance of photosynthesis in response to nitrogen deficiency led to a lower NUE in TN90. The key genes (, , , , , , and ) were associated with improving photosynthesis and nitrogen metabolism in tobacco plants grown under N-deficient conditions.

Interdisciplinarity is used to integrate and synthesize new research directions between scientific domains, but it is not the only means by which to generate novelty by bringing diverse perspectives together. Internationality draws upon cultural and linguistic diversity that can potentially impact interdisciplinarity as well. We created an interdisciplinary class originally intended to bridge computational and plant science that eventually became international in scope, including students from the United States and Mexico. We administered a survey over 4 years designed to evaluate student expertise. The first year of the survey included only US students and demonstrated that biology and computational student groups have distinct expertise but can learn the skills of the other group over the course of a semester. Modeling of survey responses shows that biological and computational science expertise is equally distributed between US and Mexico student groups, but that nonetheless, these groups can be predicted based on survey responses due to subspecialization within each domain. Unlike interdisciplinarity, differences arising from internationality are mostly static and do not change with educational intervention and include unique skills such as working across languages. We end by discussing a distinct form of interdisciplinarity that arises through internationality and the implications of globalizing research and education efforts.

Although peroxisomes are integral for both primary and secondary metabolism, how developmental changes affect activity of peroxisomes remains poorly understood. Here, we used published RNA-seq data to analyze the expression patterns of genes encoding 21 peroxisome metabolic pathways at successive developmental stages of and . Photorespiration was the most represented pathway in adult leaf relative to the juvenile stages. Components of reactive oxygen species (ROS)/reactive nitrogen species (RNS) metabolism, NADPH regeneration, and catabolism of polyamines were also enriched at later stages of leaf differentiation. The most commonly upregulated gene in differentiated leaves across all datasets of both species was (). functions in catabolism of polyamines where it converts 4-aminobutyraldehyde (ABAL) to 4-aminobutyrate (GABA). We tested the outcome of RNA-seq analysis by qRT-PCR in developing ssp. (Einkorn) seedlings. Consistent with the outcomes of RNA-seq analysis, transcription of and () were upregulated in older seedlings. CAT3 is an essential peroxisome biogenesis factor and a key enzyme of ROS homeostasis. Furthermore, exogenous application of GABA resulted in higher peroxisome abundance and transcriptional upregulation of and a gene encoding another peroxisome biogenesis factor responsible for peroxisome fission, (), in leaves. We propose that GABA contributes to regulation of peroxisome fission machinery during leaf differentiation.

The flowering time and plant architecture of were significantly associated with yield. In this study, we found that the /() gene regulated the flowering time and plant architecture of . However, the precise regulatory mechanism remains unclear. We cloned two homologous genes, and , from Xiaoyun. The protein sequence analysis showed two proteins containing conserved domains KNOX I, KNOX II, ELK, and HOX of the KONX protein family. The CRISPR/Cas9 knockout lines exhibited early budding and flowering time, coupled with floral organ abscission earlier and a larger leaf angle. On the contrary, overexpression plants displayed a phenotype that was the inverse of these characteristics. Furthermore, we observed upregulation of gibberellin and ethylene biosynthesis genes, as well as floral integrator genes in knocked-out plants. The results revealed that play a role in flowering time, floral organ abscission, and leaf angle as well as germination processes mediated. Additionally, exerted an impact on the biosynthesis pathways of ethylene and GA.

European hazelnut ( L.) is an important nut crop due to its nutritional benefits, culinary uses, and economic value. Türkiye is the leading producer of hazelnut, followed by Italy and the United States. Quantitative trait locus studies offer promising opportunities for breeders and geneticists to identify genomic regions controlling desirable traits in hazelnut. A genome-wide association analysis was conducted with 5,567 single nucleotide polymorphisms on a Turkish core set of 86 hazelnut accessions, revealing 189 quantitative trait nucleotides (QTNs) associated with 22 of 31 traits ( < 2.9E-07). These QTNs were associated with plant and leaf, phenological, reproductive, nut, and kernel traits. Based on the close physical distance of QTNs associated with the same trait, we identified 23 quantitative trait loci. Furthermore, we identified 23 loci of multiple QTs comprising chromosome locations associated with more than one trait at the same position or in close proximity. A total of 159 candidate genes were identified for 189 QTNs, with 122 of them containing significant conserved protein domains. Some candidate matches to known proteins/domains were highly significant, suggesting that they have similar functions as their matches. This comprehensive study provides valuable insights for the development of breeding strategies and the improvement of hazelnut and enhances the understanding of the genetic architecture of complex traits by proposing candidate genes and potential functions.

(Oleaceae), endemic to the Korean Peninsula and the sole member of its genus and species, possesses high scarcity value, escalating its importance under the Nagoya Protocol. Despite its significance, their metabolites and activities of flowers remain unexplored. This study employs an integrated metabolomic approach utilizing NMR, LC/MS, GC/MS, and FTIR techniques to comprehensively analyze the metabolite profile of flowers. By combining these methods, we identified 35 metabolites, 43 secondary metabolites, and 108 hydrophobic primary metabolites. Notably, distinct concentration patterns of these compounds were observed across five variants, classified based on morphological characteristics. Correlation analyses of primary and secondary metabolites unveiled varietal metabolic flux, providing insights into flower metabolism. Additionally, the reconstruction of metabolic pathways based on dissimilarities in morphological traits elucidates variant-specific metabolic signatures. These findings not only enhance our understanding of chemical differences between varieties but also underscore the importance of considering varietal differences in future research and conservation efforts.

The spatial accumulation of hordeins in the developing endosperm of barley grains was examined by immunofluorescence microscopy (immunolight microscopy [iLM]) and immunoelectron microscopy (iEM) to establish the timing and subcellular pattern of hordein synthesis and deposition. The pattern seen for hordeins was compared to other abundant grain proteins, such as serpin Z4 and lipid transfer protein 1 (LTP1). Hordein accumulates throughout grain development, from 6 to 37 days post-anthesis (DPA). In contrast, serpin Z4 was present at 6 DPA, but the greatest synthesis and accumulation occurred during the middle of seed development, from 15 to 30 DPA. LTP1 accumulated later in seed development, from 15 to 30 DPA. Hordeins accumulated within the lumen of the endoplasmic reticulum (ER), were exocytosed from the ER membrane, and accumulated in protein bodies, which then fused either with the protein storage vacuoles or with other protein bodies, which also later fused with the protein storage vacuoles. iEM showed hordein, and LTP1 appeared not to traverse the Golgi apparatus (GA). Hordein, LTP1, and serpin Z4 colocalized to the same protein bodies and were co-transported to the protein storage vacuole in the same protein bodies. It is likely that this represents a general transport mechanism common to storage proteins in developing grains.

Extracellular vesicles (EVs) are membrane-bound exosomes secreted into the apoplast. Two distinct populations of EVs have been described in Arabidopsis: PEN1-associated and TET8-associated. We previously noted early leaf senescence in the single and double mutant. Both PEN1 and PEN3 are abundant in EV proteomes suggesting that EVs might regulate leaf senescence in soil-grown plants. We observed that TET8 is more abundant in the apoplast of early senescing and mutant rosettes and in older wild-type (WT) rosettes. The increase in apoplast TET8 in the mutant did not correspond to increased mRNA levels. In addition, apoplast TET8 was more abundant in the early leaf senescence mutant, meaning the increase in apoplast TET8 protein during leaf senescence is not dependent on or . Genetic analysis showed a significant delay in leaf senescence in double mutants after 6 weeks of growth suggesting that these two tetraspanin paralogs operate additively and are positive regulators of leaf senescence. This is opposite of the effect of and mutants that show early senescence and suggest PEN1 to be a negative regulator of leaf senescence. Our work provides initial support that apoplast-localized TET8 in combination with TET3 positively regulates age-related leaf senescence in soil-grown Arabidopsis plants.