Exploring crop genomes: assembly features, gene prediction accuracy, and implications for proteomics studies.

Plant genomics plays a pivotal role in enhancing global food security and sustainability by offering innovative solutions for improving crop yield, disease resistance, and stress tolerance. As the number of sequenced genomes grows and the accuracy and contiguity of genome assemblies improve, structural annotation of plant genomes continues to be a significant challenge due to their large size, polyploidy, and rich repeat content. In this paper, we present an overview of the current landscape in crop genomics research, highlighting the diversity of genomic characteristics across various crop species. We also assessed the accuracy of popular gene prediction tools in identifying genes within crop genomes and examined the factors that impact their performance. Our findings highlight the strengths and limitations of BRAKER2 and Helixer as leading structural genome annotation tools and underscore the impact of genome complexity, fragmentation, and repeat content on their performance. Furthermore, we evaluated the suitability of the predicted proteins as a reliable search space in proteomics studies using mass spectrometry data. Our results provide valuable insights for future efforts to refine and advance the field of structural genome annotation.

ALTERED MERISTEM PROGRAM1 sustains cellular differentiation by limiting HD-ZIP III transcription factor gene expression.

Plants show remarkable developmental and regenerative plasticity through the sustained activity of stem cells in meristems. Under certain conditions, pluripotency can even be re-established in cells that have already entered differentiation. Mutation of the putative carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) in Arabidopsis (Arabidopsis thaliana) causes a set of hypertrophic phenotypes, indicating a defect in the suppression of pluripotency. A role of AMP1 in the miRNA-mediated inhibition of translation has previously been reported, however, how this activity is related to its developmental functions is unclear. Here we examined the functional interaction between AMP1 and the Class III homeodomain-leucine zipper (HD-ZIP III) transcription factors, which are miRNA-controlled determinants of shoot meristem specification. We found that the HD-ZIP III transcriptional output is enhanced in the amp1 mutant and that plant lines with increased HD-ZIP III activity not only developed amp1 mutant-like phenotypes but also showed a synergistic genetic interaction with the mutant. Conversely, the reduction of HD-ZIP III function suppressed the shoot hypertrophy defects of the amp1 mutant. We further provide evidence that the expression domains of HD-ZIP III family members are expanded in the amp1 mutant and that this misexpression occurs at the transcriptional level and does not involve the function of miRNA165/166. Finally, amp1 mutant-specific phenotypes cannot be mimicked by a general inhibition of miRNA function in the AMP1 expression domain. These findings lead us to a model in which AMP1 restricts cellular pluripotency upstream of HD-ZIP III proteins and this control appears to be not directly mediated by the canonical miRNA pathway.

Generation of high oleic acid sunflower lines using gamma radiation mutagenesis and high-throughput fatty acid profiling.

Sunflower (Helianthus annuus L.) is the second most important oil seed crop in Europe. The seeds are used as confection seeds and, more importantly, to generate an edible vegetable oil, which in normal varieties is rich in the polyunsaturated fatty acid linoleic acid. Linoleic acid is biosynthesized from oleic acid through activity of the oleate desaturase FATTY ACID DESATURASE 2 (FAD2), which in seeds is encoded by FAD2-1, a gene that's present in single copy in sunflowers. Defective FAD2-1 expression enriches oleic acid, yielding the high oleic (HO) acid trait, which is of great interest in oil seed crops, since HO oil bears benefits for both food and non-food applications. Chemical mutagenesis has previously been used to generate sunflower mutants with reduced FAD2-1 expression and here it was aimed to produce further genetic material in which FAD2-1 activity is lost and the HO trait is stably expressed. For this purpose, a sunflower mutant population was created using gamma irradiation and screened for fad2-1 mutants with a newly developed HPLC-based fatty-acid profiling system that's suitable for high-throughput analyses. With this approach fad2-1 knock-out mutants could be isolated, which stably hyper-accumulate oleic acid in concentrations of 85-90% of the total fatty acid pool. The genetic nature of these new sunflower lines was characterized and will facilitate marker development, for the rapid introgression of the trait into elite sunflower breeding material.

SlCESTA Is a Brassinosteroid-Regulated bHLH Transcription Factor of Tomato That Promotes Chilling Tolerance and Fruit Growth When Over-Expressed.

Brassinosteroids (BRs) are required for various aspects of plant growth and development, but also participate in stress responses. The hormones convey their activity through transcriptional regulation and posttranslational modification of transcription factors and one class are basic helix-loop-helix (bHLH) proteins of the BR Enhanced Expression (BEE) subfamily, which in Arabidopsis thaliana include BEE1-3 and CESTA (CES). CES and the BEEs promote the expression of different BR-responsive genes, including genes encoding gibberellin (GA) biosynthetic and catabolizing enzymes, as well as cold-responsive genes. Interestingly, in terms of an application, CES could promote both fruit growth and cold stress tolerance when over-expressed in A. thaliana and here it was investigated, if this function is conserved in the fruit crop Solanum lycopersicum (cultivated tomato). Based on amino acid sequence similarity and the presence of regulatory motifs, a CES orthologue of S. lycopersicum, SlCES, was identified and the effects of its over-expression were analysed in tomato. This showed that SlCES, like AtCES, was re-localized to nuclear bodies in response to BR signaling activation and that it effected GA homeostasis, with related phenotypes, when over-expressed. In addition, over-expression lines showed an increased chilling tolerance and had altered fruit characteristics. The possibilities and potential limitations of a gain of SlCES function as a breeding strategy for tomato are discussed.

Species-Specific Variation in Abscisic Acid Homeostasis and Responses Impacts Important Traits in Crassocephalum Orphan Crops.

Crassocephalum rubens and Crassocephalum crepidioides are plant species native to Africa, but grow in most tropical and subtropical regions of the world. They are rich in vitamins, minerals, and essential oils and are traditional leafy vegetables and medicinal plants in Sub-Saharan Africa. The plants are still mainly collected from the wild but shall be taken into cultivation and an important aim in the domestication of these species is to improve traits that are relevant for crop production. Here, seed formation and germination capacities in C. crepidioides and C. rubens were investigated, and it was found that C. crepidioides exhibits a higher level of seed dormancy, which could be broken with light, and was correlated with higher amounts of abscisic acid (ABA), a plant hormone that promotes seed dormancy. ABA is also very well-known for its role in abiotic stress tolerance, and it is shown that tetraploid C. crepidioides exhibits a higher level of resistance against drought and heat stress than diploid C. rubens, traits that will benefit the cultivation of these plants, particularly in rain-fed cropping systems. The potential of Crassocephalum to improve nutrition and increase the resilience of marginal cropping systems in Africa is discussed.

A novel chemical inhibitor of polar auxin transport promotes shoot regeneration by local enhancement of HD-ZIP III transcription.

De novo shoot organogenesis is a prerequisite for numerous applications in plant research and breeding but is often a limiting factor, for example, in genome editing approaches. Class III homeodomain-leucine zipper (HD-ZIP III) transcription factors have been characterized as crucial regulators of shoot specification, however up-stream components controlling their activity during shoot regeneration are only partially identified. In a chemical genetic screen, we isolated ZIC2, a novel activator of HD-ZIP III activity. Using molecular, physiological and hormone transport analyses in Arabidopsis and sunflower (Helianthus annuus), we examined the molecular mechanism by which the drug promotes HD-ZIP III expression. ZIC2-dependent upregulation of HD-ZIP III transcription promotes shoot regeneration in Arabidopsis and is accompanied by the induction of shoot specifying factors WUS and RAP2.6L and a subset of cytokinin biosynthesis enzymes. ZIC2's effect on HD-ZIP III expression and regeneration is based on its ability to limit polar auxin transport. We further provide evidence that chemical modulation of auxin efflux can enhance de novo shoot formation in the regeneration recalcitrant species sunflower. Activation of HD-ZIP III transcription during shoot regeneration depends on the local distribution of auxin and chemical modulation of auxin transport can be used to overcome poor shoot organogenesis in tissue culture.

Brassinosteroid-regulated bHLH transcription factor CESTA induces the gibberellin 2-oxidase GA2ox7.

Brassinosteroids (BRs) are plant steroids that have growth-promoting capacities, which are partly enabled by an ability to induce biosynthesis of gibberellins (GAs), a second class of plant hormones. In addition, BRs can also activate GA catabolism; here we show that in Arabidopsis (Arabidopsis thaliana) the basic helix-loop-helix transcription factor CESTA (CES) and its homologues BRASSINOSTEROID-ENHANCED EXPRESSION (BEE) 1 and 3 contribute to this activity. CES and the BEEs are BR-regulated at the transcriptional and posttranslational level and participate in different physiological processes, including vegetative and reproduction development, shade avoidance, and cold stress responses. We show that CES/BEEs can induce the expression of the class III GA 2-oxidase GA2ox7 and that this activity is increased by BRs. In BR signaling - and CES/BEE-deficient mutants, GA2ox7 expression decreased, yielding reduced levels of GA110, a product of GA2ox7 activity. In plants that over-express CES, GA2ox7 expression is hyper-responsive to BR, GA110 levels are elevated and amounts of bioactive GA are reduced. We provide evidence that CES directly binds to the GA2ox7 promoter and is activated by BRs, but can also act by BR-independent means. Based on these results, we propose a model for CES activity in GA catabolism where CES can be recruited for GA2ox7 induction not only by BR, but also by other factors.

Transcription factor BES1 interacts with HSFA1 to promote heat stress resistance of plants.

Heat stress is a major environmental stress type that can limit plant growth and development. To survive sudden temperature increases, plants utilize the heat shock response, an ancient signaling pathway. Initial results had suggested a role for brassinosteroids (BRs) in this response. Brassinosteroids are growth-promoting steroid hormones whose activity is mediated by transcription factors of the BES1/BZR1 subfamily. Here, we provide evidence that BES1 can contribute to heat stress signaling. In response to heat, BES1 is activated even in the absence of BRs and directly binds to heat shock elements (HSEs), known binding sites of heat shock transcription factors (HSFs). HSFs of the HSFA1 type can interact with BES1 and facilitate its activity in HSE binding. These findings lead us to propose an extended model of the heat stress response in plants, in which the recruitment of BES1 is a means of heat stress signaling cross-talk with a central growth regulatory pathway.

The Orphan Crop Crassocephalum crepidioides Accumulates the Pyrrolizidine Alkaloid Jacobine in Response to Nitrogen Starvation.

Crassocephalum crepidioides is an African orphan crop that is used as a leafy vegetable and medicinal plant. Although it is of high regional importance in Sub-Saharan Africa, the plant is still mainly collected from the wild and therefore efforts are made to promote its domestication. However, in addition to beneficial properties, there was first evidence that C. crepidioides can accumulate the highly toxic pyrrolizidine alkaloid (PA) jacobine and here it was investigated, how jacobine production is controlled. Using ecotypes from Africa and Asia that were characterized in terms of their PA profiles, it is shown that the tetraploid C. crepidioides forms jacobine, an ability that its diploid close relative Crassocephalum rubens appears to lack. Evidence is provided that nitrogen (N) deficiency strongly increases jacobine in the leaves of C. crepidioides, that this capacity depends more strongly on the shoot than the root system, and that homospermidine synthase (HSS) activity is not rate-limiting for this reaction. A characterization of HSS gene representation and transcription showed that C. crepidioides and C. rubens possess two functional versions, one of which is conserved, that the HSS transcript is mainly present in roots and that its abundance is not controlled by N deficiency. In summary, this work improves our understanding of how environmental cues impact PA biosynthesis in plants and provides a basis for the development of PA-free C. crepidioides cultivars, which will aid its domestication and safe use.

The acyltransferase PMAT1 malonylates brassinolide glucoside.

Brassinosteroids (BRs) are steroid hormones of plants that coordinate fundamental growth and development processes. Their homeostasis is controlled by diverse means, including glucosylation of the bioactive BR brassinolide (BL), which is catalyzed by the UDP-glycosyltransferases (UGTs) UGT73C5 and UGT73C6 and occurs mainly at the C-23 position. Additional evidence had suggested that the resultant BL-23-O-glucoside (BL-23-O-Glc) can be malonylated, but the physiological significance of and enzyme required for this reaction had remained unknown. Here, we show that in Arabidopsis thaliana malonylation of BL-23-O-Glc is catalyzed by the acyltransferase phenolic glucoside malonyl-transferase 1 (PMAT1), which is also known to malonylate phenolic glucosides and lipid amides. Loss of PMAT1 abolished BL-23-O-malonylglucoside formation and enriched BL-23-O-Glc, showing that the enzyme acts on the glucoside. An overexpression of PMAT1 in plants where UGT73C6 was also overexpressed, and thus, BL-23-O-Glc formation was promoted, enhanced the symptoms of BR-deficiency of UGT73C6oe plants, providing evidence that PMAT1 contributes to BL inactivation. Based on these results, a model is proposed in which PMAT1 acts in the conversion of both endogenous and xenobiotic glucosides to adjust metabolic homeostasis in spatial and temporal modes.

Modes of Brassinosteroid Activity in Cold Stress Tolerance.

Cold stress is a significant environmental factor that negatively affects plant growth and development in particular when it occurs during the growth phase. Plants have evolved means to protect themselves from damage caused by chilling or freezing temperatures and some plant species, in particular those from temperate geographical zones, can increase their basal level of freezing tolerance in a process termed cold acclimation. Cold acclimation improves plant survival, but also represses growth, since it inhibits activity of the growth-promoting hormones gibberellins (GAs). In addition to GAs, the steroid hormones brassinosteroids (BRs) also take part in growth promotion and cold stress signaling; however, in contrast to Gas, BRs can improve cold stress tolerance with fewer trade-offs in terms of growth and yields. Here we summarize our current understanding of the roles of BRs in cold stress responses with a focus on freezing tolerance and cold acclimation pathways.

AMP1 and CYP78A5/7 act through a common pathway to govern cell fate maintenance in Arabidopsis thaliana.

Higher plants can continuously form new organs by the sustained activity of pluripotent stem cells. These stem cells are embedded in meristems, where they produce descendants, which undergo cell proliferation and differentiation programs in a spatiotemporally-controlled manner. Under certain conditions, pluripotency can be reestablished in descending cells and this reversion in cell fate appears to be actively suppressed by the existing stem cell pool. Mutation of the putative carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) in Arabidopsis causes defects in the suppression of pluripotency in cells normally programmed for differentiation, giving rise to unique hypertrophic phenotypes during embryogenesis as well as in the shoot apical meristem. A role of AMP1 in the miRNA-dependent control of translation has recently been established, however, how this activity is connected to its developmental functions is not resolved. Here we identify members of the cytochrome P450 clade CYP78A to act in parallel with AMP1 to control cell fate in Arabidopsis. Mutation of CYP78A5 and its close homolog CYP78A7 in a cyp78a5,7 double mutant caused suspensor-to-embryo conversion and ectopic stem cell pool formation in the shoot meristem, phenotypes characteristic for amp1. The tissues affected in the mutants showed pronounced expression levels of AMP1 and CYP78A5 in wild type. A comparison of mutant transcriptomic responses revealed an intriguing degree of overlap and highlighted alterations in protein lipidation processes. Moreover, we also found elevated protein levels of selected miRNA targets in cyp78a5,7. Based on comprehensive genetic interaction studies we propose a model in which both enzyme classes act on a common downstream process to sustain cell fate decisions in the early embryo and the shoot apical meristem.

Comparative analysis of epigenetic inhibitors reveals different degrees of interference with transcriptional gene silencing and induction of DNA damage.

Repetitive DNA sequences and some genes are epigenetically repressed by transcriptional gene silencing (TGS). When genetic mutants are not available or problematic to use, TGS can be suppressed by chemical inhibitors. However, informed use of epigenetic inhibitors is partially hampered by the absence of any systematic comparison. In addition, there is emerging evidence that epigenetic inhibitors cause genomic instability, but the nature of this damage and its repair remain unclear. To bridge these gaps, we compared the effects of 5-azacytidine (AC), 2'-deoxy-5-azacytidine (DAC), zebularine and 3-deazaneplanocin A (DZNep) on TGS and DNA damage repair. The most effective inhibitor of TGS was DAC, followed by DZNep, zebularine and AC. We confirmed that all inhibitors induce DNA damage and suggest that this damage is repaired by multiple pathways with a critical role of homologous recombination and of the SMC5/6 complex. A strong positive link between the degree of cytidine analog-induced DNA demethylation and the amount of DNA damage suggests that DNA damage is an integral part of cytidine analog-induced DNA demethylation. This helps us to understand the function of DNA methylation in plants and opens the possibility of using epigenetic inhibitors in biotechnology.

Inhibitors of Brassinosteroid Biosynthesis and Signal Transduction.

Chemical inhibitors are invaluable tools for investigating protein function in reverse genetic approaches. Their application bears many advantages over mutant generation and characterization. Inhibitors can overcome functional redundancy, their application is not limited to species for which tools of molecular genetics are available and they can be applied to specific tissues or developmental stages, making them highly convenient for addressing biological questions. The use of inhibitors has helped to elucidate hormone biosynthesis and signaling pathways and here we review compounds that were developed for the plant hormones brassinosteroids (BRs). BRs are steroids that have strong growth-promoting capacities, are crucial for all stages of plant development and participate in adaptive growth processes and stress response reactions. In the last two decades, impressive progress has been made in BR inhibitor development and application, which has been instrumental for studying BR modes of activity and identifying and characterizing key players. Both, inhibitors that target biosynthesis, such as brassinazole, and inhibitors that target signaling, such as bikinin, exist and in a comprehensive overview we summarize knowledge and methodology that enabled their design and key findings of their use. In addition, the potential of BR inhibitors for commercial application in plant production is discussed.

Determination of the [15N]-Nitrate/[14N]-Nitrate Ratio in Plant Feeding Studies by GC⁻MS.

Feeding experiments with stable isotopes are helpful tools for investigation of metabolic fluxes and biochemical pathways. For assessing nitrogen metabolism, the heavier nitrogen isotope, [15N], has been frequently used. In plants, it is usually applied in form of [15N]-nitrate, which is assimilated mainly in leaves. Thus, methods for quantification of the [15N]-nitrate/[14N]-nitrate ratio in leaves are useful for the planning and evaluation of feeding and pulse-chase experiments. Here we describe a simple and sensitive method for determining the [15N]-nitrate to [14N]-nitrate ratio in leaves. Leaf discs (8 mm diameter, approximately 10 mg fresh weight) were sufficient for analysis, allowing a single leaf to be sampled multiple times. Nitrate was extracted with hot water and derivatized with mesitylene in the presence of sulfuric acid to nitromesitylene. The derivatization product was analyzed by gas chromatography-mass spectrometry with electron ionization. Separation of the derivatized samples required only 6 min. The method shows excellent repeatability with intraday and interday standard deviations of less than 0.9 mol%. Using the method, we show that [15N]-nitrate declines in leaves of hydroponically grown Crassocephalum crepidioides, an African orphan crop, with a biological half-life of 4.5 days after transfer to medium containing [14N]-nitrate as the sole nitrogen source.

Carbon isotope composition, water use efficiency, and drought sensitivity are controlled by a common genomic segment in maize.

KEY MESSAGE: A genomic segment on maize chromosome 7 influences carbon isotope composition, water use efficiency, and leaf growth sensitivity to drought, possibly by affecting stomatal properties. Climate change is expected to decrease water availability in many agricultural production areas around the globe. Therefore, plants with improved ability to grow under water deficit are urgently needed. We combined genetic, phenomic, and physiological approaches to understand the relationship between growth, stomatal conductance, water use efficiency, and carbon isotope composition in maize (Zea mays L.). Using near-isogenic lines derived from a maize introgression library, we analysed the effects of a genomic region previously identified as affecting carbon isotope composition. We show stability of trait expression over several years of field trials and demonstrate in the phenotyping platform Phenodyn that the same genomic region also influences the sensitivity of leaf growth to evaporative demand and soil water potential. Our results suggest that the studied genomic region affecting carbon isotope discrimination also harbours quantitative trait loci playing a role in maize drought sensitivity possibly via stomatal behaviour and development. We propose that the observed phenotypes collectively originate from altered stomatal conductance, presumably via abscisic acid.

Cold stress signalling in female reproductive tissues.

Cold stress is a significant threat for plant productivity and impacts on plant distribution and crop production, particularly so when it occurs during the growth phase. A developmental stage at risk is that of flowering, since a single stress event during sensitive stages, such as the full-bloom stage of fruit trees can be fatal for reproductive success. Although pollen development and fertilization are widely viewed as the most critical reproductive phases, the development and function of female reproductive tissues, which in Angiosperms are embedded in the gynoecium, are also affected by cold stress. Today however, we have essentially no understanding of the cold stress response pathways that act during floral organogenesis. In this review, we briefly summarize our current knowledge of cold stress signalling modules active in vegetative tissues that may provide a framework of general principles also transferable to female reproductive tissues. We then align these signalling cascades with those that govern gynoecium development to identify factors that may act in both processes and could thereby contribute to cold stress responses in female reproductive tissues.

Quantification of sugars and organic acids in tomato fruits.

Sugar and organic acid contents are major factors for tomato fruit flavour and are important breeding traits. Here we provide an improved protocol for accurate quantification of the main sugars, glucose and fructose, and the organic acids, citric acid and malic acid, present in tomato. The tomato extract is spiked with lactose and tricarballylic acid as internal standards and loaded onto a NH2 solid phase extraction (SPE) column. The sugars appear in the flow-through and are subsequently analysed by HPLC using a Nucleodur NH2 column and a refractive index detector. The organic acids bind to the SPE column and are eluted with 400 mM phosphoric acid. For analysis, the organic acids are separated by HPLC using a Nucleodur C18ec column and detected by UV absorption at 210 nm. The method shows excellent inter-day and intra-day reproducibility for glucose, fructose and citric acid with standard deviations of 1-5%. Quantification of citric acid by HPLC and GC-MS showed perfect agreement with a deviation of less than 3%. •Simple method for quantification of glucose, fructose, citric acid and malic acid in tomato.•Efficient removal of interfering compounds by solid phase extraction.•High intra and inter-day reproducibility.

Quantification of Glutamate and Aspartate by Ultra-High Performance Liquid Chromatography.

Glutamic and aspartic acid fulfil numerous functions in organisms. They are proteinogenic amino acids, they function as neurotransmitters, and glutamic acid links the citrate cycle with amino acid metabolism. In addition, glutamic acid is a precursor for many bioactive molecules like γ-aminobutyric acid (GABA). In tomatoes, glutamic acid accumulates in ripening fruits. Here we present a simple and rapid method for quantification of glutamate and aspartate in tomatoes. A cleared extract is prepared and 2-aminoadipic acid added as internal standard. Subsequently, the amino acids are derivatised with 2,4-dinitro-1-fluorobenzene under alkaline conditions. The derivatives are separated by ultra-high performance liquid chromatography using a phenyl-hexyl column and 50 mM N-methylmorpholine/acetate buffer pH 7.4 containing 12% acetonitrile as eluent and detected by UV absorption at 363 nm. The whole analysis time including separation and column equilibration takes less than 2.8 min with a flow rate of 1 mL/min and less than 1.6 min with a flow rate of 2 mL/min, making this method suitable for high-throughput applications. The method shows excellent reproducibility with intra- and inter-day SDs of approximately 4% for both aspartic and glutamic acid. Using this method we show that the glutamate/aspartate ratio changes significantly during fruit ripening.