Phytoglobins in the nuclei, cytoplasm and chloroplasts modulate nitric oxide signaling and interact with abscisic acid.

Symbiotic hemoglobins provide O2 to N2 -fixing bacteria within legume nodules, but the functions of non-symbiotic hemoglobins or phytoglobins (Glbs) are much less defined. Immunolabeling combined with confocal microscopy of the Glbs tagged at the C-terminus with green fluorescent protein was used to determine their subcellular localizations in Arabidopsis and Lotus japonicus. Recombinant proteins were used to examine nitric oxide (NO) scavenging in vitro and transgenic plants to show S-nitrosylation and other in vivo interactions with NO and abscisic acid (ABA) responses. We found that Glbs occur in the nuclei, chloroplasts and amyloplasts of both model plants, and also in the cytoplasm of Arabidopsis cells. The proteins show similar NO dioxygenase activities in vitro, are nitrosylated in Cys residues in vivo, and scavenge NO in the stomatal cells. The Cys/Ser mutation does not affect NO dioxygenase activity, and S-nitrosylation does not significantly consume NO. We demonstrate an interaction between Glbs and ABA on several grounds: Glb1 and Glb2 scavenge NO produced in stomatal guard cells following ABA supply; plants overexpressing Glb1 show higher constitutive expression of the ABA responsive genes Responsive to ABA (RAB18), Responsive to Dehydration (RD29A) and Highly ABA-Induced 2 (HAI2), and are more tolerant to dehydration; and ABA strongly upregulates class 1 Glbs. We conclude that Glbs modulate NO and interact with ABA in crucial physiological processes such as the plant's response to dessication.

Transcriptional and Metabolic Changes Associated with Phytoglobin Expression during Germination of Barley Seeds.

To understand how the class 1 phytoglobin is involved in germination process via the modulation of the nitric oxide (NO) metabolism, we performed the analysis of physiological and molecular parameters in the embryos of transgenic barley (Hordeum vulgare L. cv Golden Promise) plants differing in expression levels of the phytoglobin (Pgb1) gene during the first 48 h of germination. Overexpression of Pgb1 resulted in a higher rate of germination, higher protein content and higher ATP/ADP ratios. This was accompanied by a lower rate of NO emission after radicle protrusion, as compared to the wild type and downregulating line, and a lower rate of S-nitrosylation of proteins in the first hours postimbibition. The rate of fermentation estimated by the expression and activity of alcohol dehydrogenase was significantly higher in the Pgb1 downregulating line, the same tendency was observed for nitrate reductase expression. The genes encoding succinate dehydrogenase and pyruvate dehydrogenase complex subunits were more actively expressed in embryos of the seeds overexpressing Pgb1. It is concluded that Pgb1 expression in embryo is essential for the maintenance of redox and energy balance before radicle protrusion, when seeds experience low internal oxygen concentration and exerts the effect on metabolism during the initial development of seedlings.

The Hypoxic Proteome and Metabolome of Barley (Hordeum vulgare L.) with and without Phytoglobin Priming.

Overexpression of phytoglobins (formerly plant hemoglobins) increases the survival rate of plant tissues under hypoxia stress by the following two known mechanisms: (1) scavenging of nitric oxide (NO) in the phytoglobin/NO cycle and (2) mimicking ethylene priming to hypoxia when NO scavenging activates transcription factors that are regulated by levels of NO and O2 in the N-end rule pathway. To map the cellular and metabolic effects of hypoxia in barley (Hordeum vulgare L., cv. Golden Promise), with or without priming to hypoxia, we studied the proteome and metabolome of wild type (WT) and hemoglobin overexpressing (HO) plants in normoxia and after 24 h hypoxia (WT24, HO24). The WT plants were more susceptible to hypoxia than HO plants. The chlorophyll a + b content was lowered by 50% and biomass by 30% in WT24 compared to WT, while HO plants were unaffected. We observed an increase in ROS production during hypoxia treatment in WT seedlings that was not observed in HO seedlings. We identified and quantified 9694 proteins out of which 1107 changed significantly in abundance. Many proteins, such as ion transporters, Ca2+-signal transduction, and proteins related to protein degradation were downregulated in HO plants during hypoxia, but not in WT plants. Changes in the levels of histones indicates that chromatin restructuring plays a role in the priming of hypoxia. We also identified and quantified 1470 metabolites, of which the abundance of >500 changed significantly. In summary the data confirm known mechanisms of hypoxia priming by ethylene priming and N-end rule activation; however, the data also indicate the existence of other mechanisms for hypoxia priming in plants.

DNA repair in plant mitochondria - a complete base excision repair pathway in potato tuber mitochondria.

Mitochondria are one of the major sites of reactive oxygen species (ROS) production in the plant cell. ROS can damage DNA, and this damage is in many organisms mainly repaired by the base excision repair (BER) pathway. We know very little about DNA repair in plants especially in the mitochondria. Combining proteomics, bioinformatics, western blot and enzyme assays, we here demonstrate that the complete BER pathway is found in mitochondria isolated from potato (Solanum tuberosum) tubers. The enzyme activities of three DNA glycosylases and an apurinic/apyrimidinic (AP) endonuclease (APE) were characterized with respect to Mg2+ dependence and, in the case of the APE, temperature sensitivity. Evidence for the presence of the DNA polymerase and the DNA ligase, which complete the repair pathway by replacing the excised base and closing the gap, was also obtained. We tested the effect of oxidative stress on the mitochondrial BER pathway by incubating potato tubers under hypoxia. Protein carbonylation increased significantly in hypoxic tuber mitochondria indicative of increased oxidative stress. The activity of two BER enzymes increased significantly in response to this oxidative stress consistent with the role of the BER pathway in the repair of oxidative damage to mitochondrial DNA.

Waxy and non-waxy barley cultivars exhibit differences in the targeting and catalytic activity of GBSS1a.

Amylose synthesis is strictly associated with activity of granule-bound starch synthase (GBSS) enzymes. Among several crops there are cultivars containing starch types with either little or no amylose known as near-waxy or waxy. This (near) amylose-free phenotype is associated with a single locus (waxy) which has been mapped to GBSS-type genes in different crops. Most waxy varieties are a result of either low or no expression of a GBSS gene. However, there are some waxy cultivars where the GBSS enzymes are expressed normally. For these types, single nucleotide polymorphisms have been hypothesized to represent amino-acid substitutions leading to loss of catalytic activity. We here confirm that the HvGBSSIa enzyme from one such waxy barley variety, CDC_Alamo, has a 90% reduction in catalytic activity. We also engineered plants with expression of transgenic C-terminal green fluorescent protein-tagged HvGBSSIa of both the non-waxy type and of the CDC_Alamo type to monitor their subcellular localization patterns in grain endosperm. HvGBSSIa from non-waxy cultivars was found to localize in discrete concentric spheres strictly within starch granules. In contrast, HvGBSSIa from waxy CDC_Alamo showed deficient starch targeting mostly into unknown subcellular bodies of 0.5-3 µm in size, indicating that the waxy phenotype of CDC_Alamo is associated with deficient targeting of HvGBSSIa into starch granules.

Cross-Linked Amylose Bio-Plastic: A Transgenic-Based Compostable Plastic Alternative.

Bio-plastics and bio-materials are composed of natural or biomass derived polymers, offering solutions to solve immediate environmental issues. Polysaccharide-based bio-plastics represent important alternatives to conventional plastic because of their intrinsic biodegradable nature. Amylose-only (AO), an engineered barley starch with 99% amylose, was tested to produce cross-linked all-natural bioplastic using normal barley starch as a control. Glycerol was used as plasticizer and citrate cross-linking was used to improve the mechanical properties of cross-linked AO starch extrudates. Extrusion converted the control starch from A-type to Vh- and B-type crystals, showing a complete melting of the starch crystals in the raw starch granules. The cross-linked AO and control starch specimens displayed an additional wide-angle diffraction reflection. Phospholipids complexed with Vh-type single helices constituted an integrated part of the AO starch specimens. Gas permeability tests of selected starch-based prototypes demonstrated properties comparable to that of commercial Mater-Bi© plastic. The cross-linked AO prototypes had composting characteristics not different from the control, indicating that the modified starch behaves the same as normal starch. The data shows the feasibility of producing all-natural bioplastic using designer starch as raw material.

Function of type-2 Arabidopsis hemoglobin in the auxin-mediated formation of embryogenic cells during morphogenesis.

Suppression of Arabidopsis GLB2, a type-2 nonsymbiotic hemoglobin, enhances somatic embryogenesis by increasing auxin production. In the glb2 knock-out line (GLB2-/-), polarization of PIN1 proteins and auxin maxima occurred at the base of the cotyledons of the zygotic explants, which are the sites of embryogenic tissue formation. These changes were also accompanied by a transcriptional upregulation of WUSCHEL (WUS) and SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK1), which are markers of embryogenic competence. The increased auxin levels in the GLB2-/- line were ascribed to the induction of several key enzymes of the tryptophan and IAA biosynthetic pathways, including ANTHRANILATE SYNTHASE (α subunit; ASA1), CYTOCHROME P79B2 (CYP79B2) and AMIDASE1 (AMI1). The effects of GLB2 suppression on somatic embryogenesis and IAA synthesis are mediated by increasing levels of nitric oxide (NO) within the embryogenic cells, which repress the expression of the transcription factor MYC2, a well-characterized repressor of the auxin biosynthetic pathway. A model is proposed in which the suppression of GLB2 reduces the degree of NO scavenging by oxyhemoglobin, thereby increasing the cellular NO concentration. The increased levels of NO repress the expression of MYC2, relieving the inhibition of IAA synthesis and increasing cellular IAA, which is the inductive signal promoting embryogenic competence. Besides providing a model for the induction phase of embryogenesis in vitro, these studies propose previously undescribed functions for plant hemoglobins.

Starch bioengineering affects cereal grain germination and seedling establishment.

Cereal grain germination is central for plant early development, and efficient germination has a major role in crop propagation and malting. Endosperm starch is the prime energy reserve in germination and seedling establishment. In this study, it was hypothesized that optimized starch granule structure, and not only the endosperm starch content per se, is important for germination and seedling establishment. For that purpose, wild-type (WT), and specifically engineered degradable hyperphosphorylated (HP) starch and more resistant amylose-only (AO) starch barley lines were used. The transgenics showed no severe phenotypes and the WT and HP lines degraded the starch similarly, having 30% residual starch after 12 d of germination. However, the AO line showed significant resistance to degradation, having 57% residual starch. Interestingly, protein and ß-glucan (BG) degradation was stimulated for both HP and AO lines as compared with the WT. At late seedling establishment stages, specific sugars were rapidly consumed in the AO line. α-Amylase activity was distinctly suppressed in both the HP and the AO lines. Pre-germination ß-amylase deposition was low in the AO grains and ß-amylase was generally suppressed in both HP and AO lines throughout germination. As further supported by scanning electron microscopy and histochemical analyses on grain and seedlings, it was concluded that inadequate starch granule deposition in combination with the suppressed hydrolase activity leads to temporal and compensating re-direction of starch, sugar, and protein catabolism important to maintain metabolic dynamics during grain germination and seedling establishment.

An assessment of the biotechnological use of hemoglobin modulation in cereals.

Non-symbiotic hemoglobin (nsHb) genes are ubiquitous in plants, but their biological functions have mostly been studied in model plant species rather than in crops. nsHb influences cell signaling and metabolism by modulating the levels of nitric oxide (NO). Class 1 nsHb is upregulated under hypoxia and is involved in various biotic and abiotic stress responses. Ectopic overexpression of nsHb in Arabidopsis thaliana accelerates development, whilst targeted overexpression in seeds can increase seed yield. Such observations suggest that manipulating nsHb could be a valid biotechnological target. We studied the effects of overexpression of class 1 nsHb in the monocotyledonous crop plant barley (Hordeum vulgare cv. Golden Promise). nsHb was shown to be involved in NO metabolism in barley, as ectopic overexpression reduced the amount of NO released during hypoxia. Further, as in Arabidopsis, nsHb overexpression compromised basal resistance toward pathogens in barley. However, unlike Arabidopsis, nsHb ectopic overexpression delayed growth and development in barley, and seed specific overexpression reduced seed yield. Thus, nsHb overexpression in barley does not seem to be an efficient strategy for increasing yield in cereal crops. These findings highlight the necessity for using actual crop plants rather than laboratory model plants when assessing the effects of biotechnological approaches to crop improvement.

A Walk on the Wild Side: Genome Editing of Tuber-Bearing Solanum bulbocastanum.

Solanum bulbocastanum is a wild diploid tuber-bearing plant. We here demonstrate transgene-free genome editing of S. bulbocastanum protoplasts and regeneration of gene-edited plants. We use ribonucleoproteins, consisting of Cas9 and sgRNA, assembled in vitro, to target a gene belonging to the nitrate and peptide transporter family. Four different sgRNAs were designed and we observed efficiency in gene-editing in the protoplast pool between 8.5% and 12.4%. Twenty-one plants were re-generated from microcalli developed from individual protoplasts. In three of the plants we found that the target gene had been edited. Two of the edited plants had deletion mutations introduced into both alleles, whereas one only had a mutation in one of the alleles. Our work demonstrates that protocols for the transformation of Solanum tuberosum can be optimized to be applied to a wild Solanum species.

The role of nitric oxide and hemoglobin in plant development and morphogenesis.

Plant morphogenesis is regulated endogenously through phytohormones and other chemical signals, which may act either locally or distant from their place of synthesis. Nitric oxide (NO) is formed by a number of controlled processes in plant cells. It is a central signaling molecule with several effects on control of plant growth and development, such as shoot and root architecture. All plants are able to express non-symbiotic hemoglobins at low concentration. Their function is generally not related to oxygen transport or storage; instead they effectively oxidize NO to NO(3)(-) and thereby control the local cellular NO concentration. In this review, we analyze available data on the role of NO and plant hemoglobins in morphogenetic processes in plants. The comparison of the data suggests that hemoglobin gene expression in plants modulates development and morphogenesis of organs, such as roots and shoots, through the localized control of NO, and that hemoglobin gene expression should always be considered a modulating factor in processes controlled directly or indirectly by NO in plants.

Colorimetric pH indicators based on well-defined amylose and amylopectin matrices enriched with anthocyanins from red cabbage.

This study aimed to prepare a novel colorimetric indicator film from virtually pure (99 %) amylose (AM) and anthocyanins extracted from red cabbage (RCA). The AM used was a unique engineered bulk material extracted from transgenic barley grains. Films produced by solution casting were compared to normal barely starch (NB) and pure barley amylopectin (AP), with amylose contents of 30 % and 0 %, respectively. The pH-indicator films were produced by incorporation of RCA into the different starch support matrices with different amylose contents. Barrier, thermal, and mechanical properties, photo degradation stability, and release behavior data revealed that RCA interact differently through the glucan matrices. Microstructural observations showed that RCA were evenly dispersed in the glucan matrix, and AM+RCA indicator films showed high UV-barrier and mechanical performance over normal starch. FTIR revealed that RCA was properly affected by the AM matrix. Moreover, the AM+RCA films showed sensitive color changes in the pH range (2-11) and a predominant Fickian diffusion release mechanism for RCA. This study provides for the first time data regarding AM films with RCA and their promising potential for application as support matrices in responsive food and other industrial biodegradable packaging materials.

Over-expression of the barley Phytoglobin 1 (HvPgb1) evokes leaf-specific transcriptional responses during root waterlogging.

Oxygen deprivation (hypoxia) in the root due to waterlogging causes profound metabolic changes in the aerial organs depressing growth and limiting plant productivity in barley (Hordeum vulgare L.). Genome-wide analyses in waterlogged wild type (WT) barley (cv. Golden Promise) plants and plants over-expressing the phytoglobin 1 HvPgb1 [HvPgb1(OE)] were performed to determine leaf specific transcriptional responses during waterlogging. Normoxic WT plants outperformed their HvPgb1(OE) counterparts for dry weight biomass, chlorophyll content, photosynthetic rate, stomatal conductance, and transpiration. Root waterlogging severely depressed all these parameters in WT plants but not in HvPgb1(OE) plants, which exhibited an increase in photosynthetic rate. In leaftissue, root waterlogging repressed genes encoding photosynthetic components and chlorophyll biosynthetic enzymes, while induced those of reactive oxygen species (ROS)-generating enzymes. This repression was alleviated in HvPgb1(OE) leaves which also exhibited an induction of enzymes participating in antioxidant responses. In the same leaves, the transcript levels of several genes participating in nitrogen metabolism were also higher relative to WT leaves. Ethylene levels were diminished by root waterlogging in leaves of WT plants, but not in HvPgb1(OE), which were enriched in transcripts of ethylene biosynthetic enzymes and ethylene response factors. Pharmacological treatments increasing the level or action of ethylene further suggested the requirement of ethylene in plant response to root waterlogging. In natural germplasm an elevation in foliar HvPgb1 between 16h and 24h of waterlogging occurred in tolerant genotypes but not in susceptible ones. By integrating morpho-physiological parameters with transcriptome data, this study provides a framework defining leaf responses to root waterlogging and indicates that the induction of HvPgb1 may be used as a selection tool to enhance resilience to excess moisture.

Concerted suppression of all starch branching enzyme genes in barley produces amylose-only starch granules.

BACKGROUND: Starch is stored in higher plants as granules composed of semi-crystalline amylopectin and amorphous amylose. Starch granules provide energy for the plant during dark periods and for germination of seeds and tubers. Dietary starch is also a highly glycemic carbohydrate being degraded to glucose and rapidly absorbed in the small intestine. But a portion of dietary starch, termed "resistant starch" (RS) escapes digestion and reaches the large intestine, where it is fermented by colonic bacteria producing short chain fatty acids (SCFA) which are linked to several health benefits. The RS is preferentially derived from amylose, which can be increased by suppressing amylopectin synthesis by silencing of starch branching enzymes (SBEs). However all the previous works attempting the production of high RS crops resulted in only partly increased amylose-content and/or significant yield loss. RESULTS: In this study we invented a new method for silencing of multiple genes. Using a chimeric RNAi hairpin we simultaneously suppressed all genes coding for starch branching enzymes (SBE I, SBE IIa, SBE IIb) in barley (Hordeum vulgare L.), resulting in production of amylose-only starch granules in the endosperm. This trait was segregating 3:1. Amylose-only starch granules were irregularly shaped and showed peculiar thermal properties and crystallinity. Transgenic lines retained high-yield possibly due to a pleiotropic upregualtion of other starch biosynthetic genes compensating the SBEs loss. For gelatinized starch, a very high content of RS (65 %) was observed, which is 2.2-fold higher than control (29%). The amylose-only grains germinated with same frequency as control grains. However, initial growth was delayed in young plants. CONCLUSIONS: This is the first time that pure amylose has been generated with high yield in a living organism. This was achieved by a new method of simultaneous suppression of the entire complement of genes encoding starch branching enzymes. We demonstrate that amylopectin is not essential for starch granule crystallinity and integrity. However the slower initial growth of shoots from amylose-only grains may be due to an important physiological role played by amylopectin ordered crystallinity for rapid starch remobilization explaining the broad conservation in the plant kingdom of the amylopectin structure.

Haemoglobin modulates salicylate and jasmonate/ethylene-mediated resistance mechanisms against pathogens.

Nitric oxide (NO) plays a role in defence against hemibiotrophic pathogens mediated by salicylate (SA) and also necrotrophic pathogens influenced by jasmonate/ethylene (JA/Et). This study examined how NO-oxidizing haemoglobins (Hb) encoded by GLB1, GLB2, and GLB3 in Arabidopsis could influence both defence pathways. The impact of Hb on responses to the hemibiotrophic Pseudomonas syringae pathovar tomato (Pst) AvrRpm1 and the necrotrophic Botrytis cinerea were investigated using glb1, glb2, and glb3 mutant lines and also CaMV 35S GLB1 and GLB2 overexpression lines. In glb1, but not glb2 and glb3, increased resistance was observed to both pathogens but was compromised in the 35S-GLB1. A quantum cascade laser-based sensor measured elevated NO production in glb1 infected with Pst AvrRpm1 and B. cinerea, which was reduced in 35S-GLB1 compared to Col-0. SA accumulation was increased in glb1 and reduced in 35S-GLB1 compared to controls following attack by Pst AvrRpm1. Similarly, JA and Et levels were increased in glb1 but decreased in 35S-GLB1 in response to attack by B. cinerea. Quantitative PCR assays indicated reduced GLB1 expression during challenge with either pathogen, thus this may elevate NO concentration and promote a wide-ranging defence against pathogens.

Haemoglobin modulates NO emission and hyponasty under hypoxia-related stress in Arabidopsis thaliana.

Nitric oxide (NO) and ethylene are signalling molecules that are synthesized in response to oxygen depletion. Non-symbiotic plant haemoglobins (Hbs) have been demonstrated to act in roots under oxygen depletion to scavenge NO. Using Arabidopsis thaliana plants, the online emission of NO or ethylene was directly quantified under normoxia, hypoxia (0.1-1.0% O(2)), or full anoxia. The production of both gases was increased with reduced expression of either of the Hb genes GLB1 or GLB2, whereas NO emission decreased in plants overexpressing these genes. NO emission in plants with reduced Hb gene expression represented a major loss of nitrogen equivalent to 0.2mM nitrate per 24h under hypoxic conditions. Hb gene expression was greatly enhanced in flooded roots, suggesting induction by reduced oxygen diffusion. The function could be to limit loss of nitrogen under NO emission. NO reacts with thiols to form S-nitrosylated compounds, and it is demonstrated that hypoxia substantially increased the content of S-nitrosylated compounds. A parallel up-regulation of Hb gene expression in the normoxic shoots of the flooded plants may reflect signal transmission from root to shoot via ethylene and a role for Hb in the shoots. Hb gene expression was correlated with ethylene-induced upward leaf movement (hyponastic growth) but not with hypocotyl growth, which was Hb independent. Taken together the data suggest that Hb can influence flood-induced hyponasty via ethylene-dependent and, possibly, ethylene-independent pathways.

Synthetic Strigolactone GR24 Improves Arabidopsis Somatic Embryogenesis through Changes in Auxin Responses.

Somatic embryogenesis in Arabidopsis encompasses an induction phase requiring auxin as the inductive signal to promote cellular dedifferentiation and formation of the embryogenic tissue, and a developmental phase favoring the maturation of the embryos. Strigolactones (SLs) have been categorized as a novel group of plant hormones based on their ability to affect physiological phenomena in plants. The study analyzed the effects of synthetic strigolactone GR24, applied during the induction phase, on auxin response and formation of somatic embryos. The expression level of two SL biosynthetic genes, MOREAXILLARY GROWTH 3 and 4 (MAX3 and MAX4), which are responsible for the conversion of carotene to carotenal, increased during the induction phase of embryogenesis. Arabidopsis mutant studies indicated that the somatic embryo number was inhibited in max3 and max4 mutants, and this effect was reversed by applications of GR24, a synthetic strigolactone, and exacerbated by TIS108, a SL biosynthetic inhibitor. The transcriptional studies revealed that the regulation of GR24 and TIS108 on somatic embryogenesis correlated with changes in expression of AUXIN RESPONSIVE FACTORs 5, 8, 10, and 16, known to be required for the production of the embryogenic tissue, as well as the expression of WUSCHEL (WUS) and Somatic Embryogenesis Receptor-like Kinase 1 (SERK1), which are markers of cell dedifferentiation and embryogenic tissue formation. Collectively, this work demonstrated the novel role of SL in enhancing the embryogenic process in Arabidopsis and its requirement for inducing the expression of genes related to auxin signaling and production of embryogenic tissue.

Amylose/cellulose nanofiber composites for all-natural, fully biodegradable and flexible bioplastics.

Thermoplastic, polysaccharide-based plastics are environmentally friendly. However, typical shortcomings include lack of water resistance and poor mechanical properties. Nanocomposite manufacturing using pure, highly linear, polysaccharides can overcome such limitations. Cast nanocomposites were fabricated with plant engineered pure amylose (AM), produced in bulk quantity in transgenic barley grain, and cellulose nanofibers (CNF), extracted from agrowaste sugar beet pulp. Morphology, crystallinity, chemical heterogeneity, mechanics, dynamic mechanical, gas and water permeability, and contact angle of the films were investigated. Blending CNF into the AM matrix significantly enhanced the crystallinity, mechanical properties and permeability, whereas glycerol increased elongation at break, mainly by plasticizing the AM. There was significant phase separation between AM and CNF. Dynamic plasticizing and anti-plasticizing effects of both CNF and glycerol were demonstrated by NMR demonstrating high molecular order, but also non-crystalline, and evenly distributed 20 nm-sized glycerol domains. This study demonstrates a new lead in functional polysaccharide-based bioplastic systems.

The Mitogenome of Norway Spruce and a Reappraisal of Mitochondrial Recombination in Plants.

Plant mitogenomes can be difficult to assemble because they are structurally dynamic and prone to intergenomic DNA transfers, leading to the unusual situation where an organelle genome is far outnumbered by its nuclear counterparts. As a result, comparative mitogenome studies are in their infancy and some key aspects of genome evolution are still known mainly from pregenomic, qualitative methods. To help address these limitations, we combined machine learning and in silico enrichment of mitochondrial-like long reads to assemble the bacterial-sized mitogenome of Norway spruce (Pinaceae: Picea abies). We conducted comparative analyses of repeat abundance, intergenomic transfers, substitution and rearrangement rates, and estimated repeat-by-repeat homologous recombination rates. Prompted by our discovery of highly recombinogenic small repeats in P. abies, we assessed the genomic support for the prevailing hypothesis that intramolecular recombination is predominantly driven by repeat length, with larger repeats facilitating DNA exchange more readily. Overall, we found mixed support for this view: Recombination dynamics were heterogeneous across vascular plants and highly active small repeats (ca. 200 bp) were present in about one-third of studied mitogenomes. As in previous studies, we did not observe any robust relationships among commonly studied genome attributes, but we identify variation in recombination rates as a underinvestigated source of plant mitogenome diversity.