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The titan arum ( Amorphophallus titanum ), commonly known as the corpse flower, produces the largest unbranched inflorescence in the world. Its rare blooms last only a few days and are notable both for their burst of thermogenic activity and for the odor of rotting flesh by which they attract pollinators. Studies on the titan arum can therefor lend insight into the mechanisms underlying thermogenesis as well as the production of sulfur-based volatiles, about which little is known in plants. Here, we made use of transcriptome and metabolite analyses to uncover underlying mechanisms that enable thermogenesis and volatile production in the titan arum. The ability to perform thermogenesis correlated with the expression of genes involved in bypass steps for the mitochondrial electron transport chain, in particular alternative oxidase expression, and through our analysis is placed within the context of sugar transport and metabolism. The major odorants produced by the titan arum are dimethyl disulfide and dimethyl trisulfide, and we identified pathways for sulfur transport and metabolism that culminate in the production of methionine, which our analysis identifies as the amino acid substrate for production of these odorants. Putrescine, derived from arginine, was identified as an additional and previously unrecognized component of the titan arum's odor. Levels of free methionine and putrescine were rapidly depleted during thermogenesis, consistent with roles in production of the titan arum's odor. Models for how tissues of the titan arum contribute to thermogenesis and volatile production are proposed., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)

The demand for agricultural production is becoming more challenging as climate change increases global temperature and the frequency of extreme weather events. This study examines the phenotypic variation of 149 accessions of Brachypodium distachyon under drought, heat, and the combination of stresses. Heat alone causes the largest amounts of tissue damage while the combination of stresses causes the largest decrease in biomass compared to other treatments. Notably, Bd21-0, the reference line for B. distachyon, did not have robust growth under stress conditions, especially the heat and combined drought and heat treatments. The climate of origin was significantly associated with B. distachyon responses to the assessed stress conditions. Additionally, a GWAS found loci associated with changes in plant height and the amount of damaged tissue under stress. Some of these SNPs were closely located to genes known to be involved in responses to abiotic stresses and point to potential causative loci in plant stress response. However, SNPs found to be significantly associated with a response to heat or drought individually are not also significantly associated with the combination of stresses. This, with the phenotypic data, suggests that the effects of these abiotic stresses are not simply additive, and the responses to the combined stresses differ from drought and heat alone., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Background: Non-invasive reporter systems are powerful tools to query physiological and transcriptional responses in organisms. For example, fluorescent and bioluminescent reporters have revolutionized cellular and organismal assays and have been used to study plant responses to abiotic and biotic stressors. Integrated, cooled charge-coupled device (CCD) camera systems have been developed to image bioluminescent and fluorescent signals in a variety of organisms; however, these integrated long-term imaging systems are expensive., Results: We have developed self-assembled systems for both growing and monitoring plant fluorescence and bioluminescence for long-term experiments under controlled environmental conditions. This system combines environmental growth chambers with high-sensitivity CCD cameras, multi-wavelength LEDs, open-source software, and several options for coordinating lights with imaging. This easy-to-assemble system can be used for short and long-term imaging of bioluminescent reporters, acute light-response, circadian rhythms, delayed fluorescence, and fluorescent-protein-based assays in vivo., Conclusions: We have developed two self-assembled imaging systems that will be useful to researchers interested in continuously monitoring in vivo reporter systems in various plant species., (© 2023. The Author(s).)

Increasing crop yields is complicated by the polyploid nature of our major crops. A recent PLOS Biology study provides a transcriptomic view of the influence of the circadian clock on regulating agriculturally relevant traits in the polyploid bread wheat., Competing Interests: The author has declared that no competing interests exist.

The plant circadian clock coordinates developmental, physiological, and metabolic processes with diel changes in light and temperature throughout the year. The balance between the persistence and plasticity of the clock in response to predictable and unpredictable environmental changes may be key to the clock's adaptive nature across temporal and spatial scales. Studies under controlled conditions have uncovered critical signaling pathways involved in light and temperature perception by the clock; however, they don't account for the natural lag of temperature behind photoperiod. Studies in natural environments provide key insights into the clock's adaptive advantage under more complex natural settings. Here, we discuss the role of the circadian clock in light and temperature perception and signaling, how the clock integrates these signals for a coordinated and adaptive response, and the adaptive advantage conferred by the clock across time and space in natural environments., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Motivation: Clustering spatial-resolved gene expression is an essential analysis to reveal gene activities in the underlying morphological context by their functional roles. However, conventional clustering analysis does not consider gene expression co-localizations in tissue for detecting spatial expression patterns or functional relationships among the genes for biological interpretation in the spatial context. In this article, we present a convolutional neural network (CNN) regularized by the graph of protein-protein interaction (PPI) network to cluster spatially resolved gene expression. This method improves the coherence of spatial patterns and provides biological interpretation of the gene clusters in the spatial context by exploiting the spatial localization by convolution and gene functional relationships by graph-Laplacian regularization., Results: In this study, we tested clustering the spatially variable genes or all expressed genes in the transcriptome in 22 Visium spatial transcriptomics datasets of different tissue sections publicly available from 10× Genomics and spatialLIBD. The results demonstrate that the PPI-regularized CNN constantly detects gene clusters with coherent spatial patterns and significantly enriched by gene functions with the state-of-the-art performance. Additional case studies on mouse kidney tissue and human breast cancer tissue suggest that the PPI-regularized CNN also detects spatially co-expressed genes to define the corresponding morphological context in the tissue with valuable insights., Availability and Implementation: Source code is available at https://github.com/kuanglab/CNN-PReg., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

As sessile organisms, plants are finely tuned to respond dynamically to developmental, circadian and environmental cues. Genome-wide studies investigating these types of cues have uncovered the intrinsically different ways they can impact gene expression over time. Recent advances in single-cell sequencing and time-based bioinformatic algorithms are now beginning to reveal the dynamics of these time-based responses within individual cells and plant tissues. Here, we review what these techniques have revealed about the spatiotemporal nature of gene regulation, paying particular attention to the three distinct ways in which plant tissues are time sensitive. (i) First, we discuss how studying plant cell identity can reveal developmental trajectories hidden in pseudotime. (ii) Next, we present evidence that indicates that plant cell types keep their own local time through tissue-specific regulation of the circadian clock. (iii) Finally, we review what determines the speed of environmental signaling responses, and how they can be contingent on developmental and circadian time. By these means, this review sheds light on how these different scales of time-based responses can act with tissue and cell-type specificity to elicit changes in whole plant systems., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Changes in gene expression are important for responses to abiotic stress. Transcriptome profiling of heat- or cold-stressed maize genotypes identifies many changes in transcript abundance. We used comparisons of expression responses in multiple genotypes to identify alleles with variable responses to heat or cold stress and to distinguish examples of cis- or trans-regulatory variation for stress-responsive expression changes. We used motifs enriched near the transcription start sites (TSSs) for thermal stress-responsive genes to develop predictive models of gene expression responses. Prediction accuracies can be improved by focusing only on motifs within unmethylated regions near the TSS and vary for genes with different dynamic responses to stress. Models trained on expression responses in a single genotype and promoter sequences provided lower performance when applied to other genotypes but this could be improved by using models trained on data from all three genotypes tested. The analysis of genes with cis-regulatory variation provides evidence for structural variants that result in presence/absence of transcription factor binding sites in creating variable responses. This study provides insights into cis-regulatory motifs for heat- and cold-responsive gene expression and defines a framework for developing models to predict expression responses across multiple genotypes., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

The first descriptions of circadian rhythms were of the rhythmic leaf movements of plants. Rhythmic leaf movements offer a sensitive, noninvasive, nondestructive, and non-transgenic assay of plant circadian rhythms that can be readily automated, greatly facilitating genetic studies. Rhythmic leaf movement is particularly useful for the assessment of standing variation in clock function and can be readily applied to a diverse array of dicotyledonous plants, including both wild species and domesticated crops., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Throughout plant evolution the circadian clock has expanded into a complex signaling network, coordinating physiological and metabolic processes with the environment. Early land plants faced new environmental pressures that required energy-demanding stress responses. Integrating abiotic stress response into the circadian system provides control over daily energy expenditure. Here, we describe the evolution of the circadian clock in plants and the limited, yet compelling, evidence for conserved regulation of abiotic stress. The need to introduce abiotic stress tolerance into current crops has expanded research into wild accessions and revealed extensive variation in circadian clock parameters across monocot and eudicot species. We argue that research into the ancestral links between the clock and abiotic stress will benefit crop improvement efforts., (© 2021 The Authors New Phytologist © 2021 New Phytologist Foundation.)

The globally important crop Brassica rapa, a close relative of Arabidopsis, is an excellent system for modeling our current knowledge of plant growth on a morphologically diverse crop. The long history of B. rapa domestication across Asia and Europe provides a unique collection of locally adapted varieties that span large climatic regions with various abiotic and biotic stress-tolerance traits. This diverse gene pool provides a rich source of targets with the potential for manipulation toward the enhancement of productivity of crops both within and outside the Brassicaceae. To expand the genetic resources available to study natural variation in B. rapa, we constructed an Advanced Intercross Recombinant Inbred Line (AI-RIL) population using B. rapa subsp. trilocularis (Yellow Sarson) R500 and the B. rapa subsp. parachinensis (Cai Xin) variety L58. Our current understanding of genomic structure variation across crops suggests that a single reference genome is insufficient for capturing the genetic diversity within a species. To complement this AI-RIL population and current and future B. rapa genomic resources, we generated a de novo genome assembly of the B. rapa subsp. trilocularis (Yellow Sarson) variety R500, the maternal parent of the AI-RIL population. The genetic map for the R500 x L58 population generated using this de novo genome was used to map Quantitative Trait Loci (QTL) for seed coat color and revealed the improved mapping resolution afforded by this new assembly., Competing Interests: The authors declare no conflict of interest associated with the work described in this manuscript., (© 2020 The Authors. Plant Direct published by American Society of Plant Biologists, Society for Experimental Biology and John Wiley & Sons Ltd.)

Environmental variation along an elevational gradient can yield phenotypic differentiation resulting from varying selection pressures on plant traits related to seasonal responses. Thus, genetic clines can evolve in a suite of traits, including the circadian clock, that drives daily cycling in varied traits and that shares its genetic background with adaptation to seasonality. We used populations of annual Mimulus laciniatus from different elevations in the Sierra Nevada in California to explore among-population differentiation in the circadian clock, flowering responses to photoperiod, and phenological traits (days to cotyledon emergence, days to flowering, and days to seed ripening) in controlled common-garden conditions. Further, we examined correlations of these traits with environmental variables related to temperature and precipitation. We observed that the circadian period in leaf movement was differentiated among populations sampled within about 100 km, with population means varying by 1.6 h. Significant local genetic variation occurred within 2 populations in which circadian period among families varied by up to 1.8 h. Replicated treatments with variable ecologically relevant photoperiods revealed marked population differentiation in critical day length for flowering that ranged from 11.0 to 14.1 h, corresponding to the time period between late February and mid-May in the wild. Flowering time varied among populations in a 14-h photoperiod. Regardless of this substantial population-level diversity, obvious linear clinality in trait variability across elevations could not be determined based on our genotypic sample; it is possible that more complex spatial patterns of variation arise in complex terrains such as those in the Sierra Nevada. Moreover, we did not find statistically significant bivariate correlations between population means of different traits. Our research contributes to the understanding of genetic variation in the circadian clock and in seasonal responses in natural populations, highlighting the need for more comprehensive investigations on the association between the clock and other adaptive traits in plants.

An important challenge of crop improvement strategies is assigning function to paralogs in polyploid crops. Here we describe the circadian transcriptome in the polyploid crop Brassica rapa . Strikingly, almost three-quarters of the expressed genes exhibited circadian rhythmicity. Genetic redundancy resulting from whole genome duplication is thought to facilitate evolutionary change through sub- and neo-functionalization among paralogous gene pairs. We observed genome-wide expansion of the circadian expression phase among retained paralogous pairs. Using gene regulatory network models, we compared transcription factor targets between B. rapa and Arabidopsis circadian networks to reveal evidence for divergence between B. rapa paralogs that may be driven in part by variation in conserved non-coding sequences (CNS). Additionally, differential drought response among retained paralogous pairs suggests further functional diversification. These findings support the rapid expansion and divergence of the transcriptional network in a polyploid crop and offer a new approach for assessing paralog activity at the transcript level., Competing Interests: KG, RS, SZ, PL, TM, CM No competing interests declared, (© 2020, Greenham et al.)

The TIR1/AFB auxin co-receptors mediate diverse responses to the plant hormone auxin. The Arabidopsis genome encodes six TIR1/AFB proteins representing three of the four clades that were established prior to angiosperm radiation. To determine the role of these proteins in plant development we performed an extensive genetic analysis involving the generation and characterization of all possible multiply-mutant lines. We find that loss of all six TIR1/AFB proteins results in early embryo defects and eventually seed abortion, and yet a single wild-type allele of TIR1 or AFB2 is sufficient to support growth throughout development. Our analysis reveals extensive functional overlap between even the most distantly related TIR1/AFB genes except for AFB1 . Surprisingly, AFB1 has a specialized function in rapid auxin-dependent inhibition of root growth and early phase of root gravitropism. This activity may be related to a difference in subcellular localization compared to the other members of the family., Competing Interests: MP, MP, NK, YZ, KG, WS, BP, RB, MB, WB, ME No competing interests declared, (© 2020, Prigge et al.)

Competing Interests: The authors declare no conflict of interest.

The dynamics of local climates make development of agricultural strategies challenging. Yield improvement has progressed slowly, especially in drought-prone regions where annual crop production suffers from episodic aridity. Underlying drought responses are circadian and diel control of gene expression that regulate daily variations in metabolic and physiological pathways. To identify transcriptomic changes that occur in the crop Brassica rapa during initial perception of drought, we applied a co-expression network approach to associate rhythmic gene expression changes with physiological responses. Coupled analysis of transcriptome and physiological parameters over a two-day time course in control and drought-stressed plants provided temporal resolution necessary for correlation of network modules with dynamic changes in stomatal conductance, photosynthetic rate, and photosystem II efficiency. This approach enabled the identification of drought-responsive genes based on their differential rhythmic expression profiles in well-watered versus droughted networks and provided new insights into the dynamic physiological changes that occur during drought.

The increasing demand for improved agricultural production will require more efficient breeding for traits that maintain yield under heterogeneous environments. The internal circadian oscillator is essential for perceiving and coordinating environmental cues such as day length, temperature, and abiotic stress responses within physiological processes. To investigate the contribution of the circadian clock to local adaptability, we have analyzed circadian period by leaf movement in natural populations of Mimulus guttatus and domesticated cultivars of Glycine max. We detected consistent variation in circadian period along a latitudinal gradient in annual populations of the wild plant and the selectively bred crop, and this provides novel evidence of natural and artificial selection for circadian performance. These findings provide new support that the circadian clock acts as a central regulator of plant adaptability and further highlight the potential of applying circadian clock gene variation to marker-assisted breeding programs in crops.

Circadian clocks have evolved independently in all three domains of life, and fitness benefits of a functional clock have been demonstrated in experimental genotypes in controlled conditions. Still, little is known about genetic variation in the clock and its fitness consequences in natural populations from heterogeneous environments. Using Wyoming populations of the Arabidopsis relative Boechera stricta as our study system, we demonstrate that genetic variation in the clock can occur at multiple levels: means of circadian period among populations sampled at different elevations differed by less than 1 h, but means among families sampled within populations varied by as much as 3.5 h. Growth traits also varied among and within populations. Within the population with the most circadian variation, we observed evidence for a positive correlation between period and growth and a negative correlation between period and root-to-shoot ratio. We then tested whether performance tradeoffs existed among families of this population across simulated seasonal settings. Growth rankings of families were similar across seasonal environments, but for root-to-shoot ratio, genotype × environment interactions contributed significantly to total variation. Therefore, further experiments are needed to identify evolutionary mechanisms that preserve substantial quantitative genetic diversity in the clock in this and other species., (© 2016 John Wiley & Sons Ltd.)

The plant hormone auxin is perceived by a family of F-box proteins called the TIR1/AFBs. Phylogenetic studies reveal that these proteins fall into four clades in flowering plants called TIR1, AFB2, AFB4, and AFB6. Genetic studies indicate that members of the TIR1 and AFB2 groups act as positive regulators of auxin signaling by promoting the degradation of the Aux/IAA transcriptional repressors. In this report, we demonstrate that both AFB4 and AFB5 also function as auxin receptors based on in vitro assays. We also provide genetic evidence that AFB4 and AFB5 are targets of the picloram family of auxinic herbicides in addition to indole-3-acetic acid. In contrast to previous studies we find that null afb4 alleles do not exhibit obvious defects in seedling morphology or auxin hypersensitivity. We conclude that AFB4 and AFB5 act in a similar fashion to other members of the family but exhibit a distinct auxin specificity., (Copyright © 2016 Prigge et al.)

The plant circadian clock coordinates the responses to multiple and often simultaneous environmental challenges that the sessile plant cannot avoid. These responses must be integrated efficiently into dynamic metabolic and physiological networks essential for growth and reproduction. Many of the output pathways regulated by the circadian clock feed back to modulate clock function, leading to the appreciation of the clock as a central hub in a sophisticated regulatory network. In this Review, we discuss the circadian regulation of growth, flowering time, abiotic and biotic stress responses, and metabolism, as well as why temporal 'gating' of these processes is important to plant fitness.

Background: A well characterized output of the circadian clock in plants is the daily rhythmic movement of leaves. This process has been used extensively in Arabidopsis to estimate circadian period in natural accessions as well as mutants with known defects in circadian clock function. Current methods for estimating circadian period by leaf movement involve manual steps throughout the analysis and are often limited to analyzing one leaf or cotyledon at a time., Results: In this study, we describe the development of TRiP (Tracking Rhythms in Plants), a new method for estimating circadian period using a motion estimation algorithm that can be applied to whole plant images. To validate this new method, we apply TRiP to a Recombinant Inbred Line (RIL) population in Arabidopsis using our high-throughput imaging platform. We begin imaging at the cotyledon stage and image through the emergence of true leaves. TRiP successfully tracks the movement of cotyledons and leaves without the need to select individual leaves to be analyzed., Conclusions: TRiP is a program for analyzing leaf movement by motion estimation that enables high-throughput analysis of large populations of plants. TRiP is also able to analyze plant species with diverse leaf morphologies. We have used TRiP to estimate period for 150 Arabidopsis RILs as well as 5 diverse plant species, highlighting the broad applicability of this new method.

Several factors affect the yield potential and geographical range of crops including the circadian clock, water availability, and seasonal temperature changes. In order to sustain and increase plant productivity on marginal land in the face of both biotic and abiotic stresses, we need to more efficiently generate stress-resistant crops through marker-assisted breeding, genetic modification, and new genome-editing technologies. To leverage these strategies for producing the next generation of crops, future transcriptomic data acquisition should be pursued with an appropriate temporal design and analyzed with a network-centric approach. The following review focuses on recent developments in abiotic stress transcriptional networks in economically important crops and will highlight the utility of correlation-based network analysis and applications., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The native auxin, indole-3-acetic acid (IAA), is a major regulator of plant growth and development. Its nonuniform distribution between cells and tissues underlies the spatiotemporal coordination of many developmental events and responses to environmental stimuli. The regulation of auxin gradients and the formation of auxin maxima/minima most likely involve the regulation of both metabolic and transport processes. In this article, we have demonstrated that 2-oxindole-3-acetic acid (oxIAA) is a major primary IAA catabolite formed in Arabidopsis thaliana root tissues. OxIAA had little biological activity and was formed rapidly and irreversibly in response to increases in auxin levels. We further showed that there is cell type-specific regulation of oxIAA levels in the Arabidopsis root apex. We propose that oxIAA is an important element in the regulation of output from auxin gradients and, therefore, in the regulation of auxin homeostasis and response mechanisms.

Many processes critical to plant growth and development are regulated by the hormone auxin. Auxin responses are initiated through activation of a transcriptional response mediated by the TIR1/AFB family of F-box protein auxin receptors as well as the AUX/IAA and ARF families of transcriptional regulators. However, there is little information on how auxin regulates a specific cellular response. To begin to address this question, we have focused on auxin regulation of cell expansion in the Arabidopsis hypocotyl. We show that auxin-mediated hypocotyl elongation is dependent upon the TIR1/AFB family of auxin receptors and degradation of AUX/IAA repressors. We also use microarray studies of elongating hypocotyls to show that a number of growth-associated processes are activated by auxin including gibberellin biosynthesis, cell wall reorganization and biogenesis, and others. Our studies indicate that GA biosynthesis is required for normal response to auxin in the hypocotyl but that the overall transcriptional auxin output consists of PIF-dependent and -independent genes. We propose that auxin acts independently from and interdependently with PIF and GA pathways to regulate expression of growth-associated genes in cell expansion.

The plant hormone auxin is perceived by a family of F box proteins called the TIR1/auxin-signaling F box proteins (AFBs). Phylogenetic studies reveal that these proteins fall into four clades in flowering plants called TIR1, AFB2, AFB4, and AFB6. Genetic studies indicate that members of the TIR1 and AFB2 groups act as positive regulators of auxin signaling. In this report, we demonstrate a unique role for the AFB4 clade. Both AFB4 and AFB5 function as auxin receptors based on in vitro assays. However, unlike other members of the family, loss of AFB4 results in a range of growth defects that are consistent with auxin hypersensitivity, including increased hypocotyl and petiole elongation and increased numbers of lateral roots. Indeed, qRT-PCR experiments show that afb4-2 is hypersensitive to indole-3-acetic acid (IAA) in the hypocotyl, indicating that AFB4 is a negative regulator of auxin response. Furthermore, we show that AFB4 has a particularly important role in the response of seedlings to elevated temperature. Finally, we provide evidence that the AFB4 clade is the major target of the picloram family of auxinic herbicides. These results reveal a previously unknown aspect of auxin receptor function., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The plant hormone auxin plays an essential role in plant development. However, only a few auxin biosynthetic genes have been isolated and characterized. Here, we show that the TRANSPORT INHIBITOR RESPONSE2 (TIR2) gene is required for many growth processes. Our studies indicate that the tir2 mutant is hypersensitive to 5-methyl-tryptophan, an inhibitor of tryptophan synthesis. Further, treatment with the proposed auxin biosynthetic intermediate indole-3-pyruvic acid (IPA) and indole-3-acetic acid rescues the tir2 short hypocotyl phenotype, suggesting that tir2 may be affected in the IPA auxin biosynthetic pathway. Molecular characterization revealed that TIR2 is identical to the TAA1 gene encoding a tryptophan aminotransferase. We show that TIR2 is regulated by temperature and is required for temperature-dependent hypocotyl elongation. Further, we find that expression of TIR2 is induced on the lower side of a gravitropically responding root. We propose that TIR2 contributes to a positive regulatory loop required for root gravitropism.

PhycoBiologics is developing an oral vaccine delivery system using vaccines expressed in the chloroplast of microalgae. Despite many advances in plastid transformation technology, levels of expression remain inconsistent. We have concluded that the main factors affecting the level of recombinant protein expression in the chloroplast of Chlamydomonas are: codon optimization, protease activity, protein toxicity and transformation-associated genotypic modification.

Two classes of phosphoenolpyruvate carboxylase (PEPC) sharing the same 107-kDa catalytic subunit (p107) were previously purified from developing castor oil seed (COS) endosperm. The association of p107 with an immunologically unrelated 64-kDa polypeptide (p64) causes pronounced physical and kinetic differences between the Class-1 PEPC p107 homotetramer and Class-2 PEPC p107/p64 hetero-octamer. Tryptic peptide sequencing matched p64 to the deduced C-terminal half of several bacterial-type PEPCs (BTPCs) of vascular plants. Immunoblots probed with anti-(COS p64 peptide or p107)-IgG established that: (i) BTPC exists in vivo as an approximately 118-kDa polypeptide (p118) that is rapidly truncated to p64 by an endogenous cysteine endopeptidase during incubation of COS extracts on ice, and (ii) mature and germinated COS contain Class-1 PEPC and p107, but no detectable Class-2 PEPC nor p118. Non-denaturing PAGE, in-gel PEPC activity staining and immunoblotting of developing COS extracts demonstrated that p118 and p107 are subunits of the non-proteolysed approximately 910-kDa Class-2 PEPC complex. As total PEPC activity of clarified COS extracts was unaffected following p118 truncation to p64, the BTPC p118 may function as a regulatory rather than catalytic subunit of the Class-2 PEPC. Moreover, recombinant AtPPC3 and AtPPC4 (Arabidopsis orthologs of COS p107 and p118) expressed as active and inactive PEPCs, respectively. Cloning of cDNAs encoding p118 (RcPpc4) and p107 (RcPpc3) confirmed their respective designation as bacterial- and plant-type PEPCs. Levels of RcPpc3 and RcPpc4 transcripts generally mirrored the respective amounts of p107 and p118. The collective findings provide insights into the molecular features and functional significance of vascular plant BTPCs.