Transcriptomic Data Analysis Using the Galaxy Platform: Coffee (Coffea arabica L.) Flowers as Example.

Coffee, an important agricultural product for tropical producing countries, is facing challenges due to climate change, including periods of drought, irregular rain distribution, and high temperatures. These changes result in plant water stress, leading to significant losses in coffee productivity and quality. Understanding the processes that affect coffee flowering is crucial for improving productivity and quality. In this chapter, we describe a protocol for transcriptome analysis using available Internet software, mainly in the Galaxy Platform, using RNA-Seq data from flowers collected from different parts of the coffee tree. The methods presented in this chapter provide a comprehensive protocol for transcriptome analysis of differentially expressed genes from flowers of coffee plant. This knowledge can be utilized in coffee genetic improvement programs, particularly in the selection of cultivars that are tolerant to water deficit.

Unveiling the aesthetic secrets: exploring connections between genetic makeup, chemical, and environmental factors for enhancing/improving the color and fragrance/aroma of Chimonanthus praecox.

Floral color and scent profiles vary across species, geographical locations, and developmental stages. The exclusive floral color and fragrance of Chimonanthus praecox is contributed by a range of endogenous chemicals that distinguish it from other flowers and present amazing ornamental value. This comprehensive review explores the intricate interplay of environmental factors, chemicals and genes shaping the flower color and fragrance of Chimonanthus praecox. Genetic and physiological factors control morpho-anatomical attributes as well as pigment synthesis, while environmental factors such as temperature, light intensity, and soil composition influence flower characteristics. Specific genes control pigment synthesis, and environmental factors such as temperature, light intensity, and soil composition influence flower characteristics. Physiological processes including plant hormone contribute to flower color and fragrance. Hormones, notably ethylene, exert a profound influence on varioustraits. Pigment investigations have spotlighted specific flavonoids, including kaempferol 3-O-rutinoside, quercetin, and rutin. Red tepals exhibit unique composition with cyanidin-3-O-rutinoside and cyanidin-3-O-glucoside being distinctive components. Elucidating the molecular basis of tepal color variation, particularly in red and yellow varieties, involves the identification of crucial regulatory genes. In conclusion, this review unravels the mysteries of Chimonanthus praecox, providing a holistic understanding of its flower color and fragrance for landscape applications. This comprehensive review uniquely explores the genetic intricacies, chemical and environmental influences that govern the mesmerizing flower color and fragrance of Chimonanthus praecox, providing valuable insights for its landscape applications. This review article is designed for a diverse audience, including plant geneticists, horticulturists, environmental scientists, urban planners, and students, offering understandings into the genetic intricacies, ecological significance, and practical applications of Chimonanthus praecox across various disciplines. Its appeal extends to professionals and enthusiasts interested in plant biology, conservation, and industries dependent on unique floral characteristics.

Identification of microRNAs and their target genes associated with chasmogamous and cleistogamous flower development in Viola prionantha.

MAIN CONCLUSION: Differentially expressed microRNAs were found associated with the development of chasmogamous and cleistogamous flowers in Viola prionantha, revealing potential roles of microRNAs in the developmental evolution of dimorphic flowers. In Viola prionantha, chasmogamous (CH) flowers are induced by short daylight, while cleistogamous (CL) flowers are triggered by long daylight. How environmental factors and microRNAs (miRNAs) affect dimorphic flower formation remains unknown. In this study, small RNA sequencing was performed on CH and CL floral buds at different developmental stages in V. prionantha, differentially expressed miRNAs (DEmiRNAs) were identified, and their target genes were predicted. In CL flowers, Viola prionantha miR393 (vpr-miR393a/b) and vpr-miRN3366 were highly expressed, while in CH flowers, vpr-miRN2005, vpr-miR172e-2, vpr-miR166m-3, vpr-miR396f-2, and vpr-miR482d-2 were highly expressed. In the auxin-activated signaling pathway, vpr-miR393a/b and vpr-miRN2005 could target Vpr-TIR1/AFB and Vpr-ARF2, respectively, and other DEmiRNAs could target genes involved in the regulation of transcription, e.g., Vpr-AP2-7. Moreover, Vpr-UFO and Vpr-YAB5, the main regulators in petal and stamen development, were co-expressed with Vpr-TIR1/AFB and Vpr-ARF2 and showed lower expression in CL flowers than in CH flowers. Some V. prionantha genes relating to the stress/defense responses were co-expressed with Vpr-TIR1/AFB, Vpr-ARF2, and Vpr-AP2-7 and highly expressed in CL flowers. Therefore, in V. prionantha, CH-CL flower development may be regulated by the identified DEmiRNAs and their target genes, thus providing the first insight into the formation of dimorphic flowers in Viola.

HD-Zip proteins modify floral structures for self-pollination in tomato.

Cleistogamy is a type of self-pollination that relies on the formation of a stigma-enclosing floral structure. We identify three homeodomain-leucine zipper IV (HD-Zip IV) genes that coordinately promote the formation of interlocking trichomes at the anther margin to unite neighboring anthers, generating a closed anther cone and cleistogamy (flower morphology necessitating strict self-pollination). These HD-Zip IV genes also control style length by regulating the transition from cell division to endoreduplication. The expression of these HD-Zip IV genes and their downstream gene, Style 2.1, was sequentially modified to shape the cleistogamy morphology during tomato evolution and domestication. Our results provide insights into the molecular basis of cleistogamy in modern tomato and suggest targets for improving fruit set and preventing pollen contamination in genetically modified crops.

[Brassica juncea BjuWRKY71-1 accelerates flowering by regulating the expression of SOC1].

Brassica juncea (mustard) is a vegetable crop of Brassica, which is widely planted in China. The yield and quality of stem mustard are greatly influenced by the transition from vegetative growth to reproductive growth, i.e., flowering. The WRKY transcription factor family is ubiquitous in higher plants, and its members are involved in the regulation of many growth and development processes, including biological/abiotic stress responses and flowering regulation. WRKY71 is an important member of the WRKY family. However, its function and mechanism in mustard have not been reported. In this study, the BjuWRKY71-1 gene was cloned from B. juncea. Bioinformatics analysis and phylogenetic tree analysis showed that the protein encoded by BjuWRKY71-1 has a conserved WRKY domain, belonging to class Ⅱ WRKY protein, which is closely related to BraWRKY71-1 in Brassica rapa. The expression abundance of BjuWRKY71-1 in leaves and flowers was significantly higher than that in roots and stems, and the expression level increased gradually along with plant development. The result of subcellular localization showed that BjuWRKY71-1 protein was located in nucleus. The flowering time of overexpressing BjuWRKY71-1 Arabidopsis plants was significantly earlier than that of the wild type. Yeast two-hybrid assay and dual-luciferase reporter assay showed that BjuWRKY71-1 interacted with the promoter of the flowering integrator BjuSOC1 and promoted the expression of its downstream genes. In conclusion, BjuWRKY71-1 protein can directly target BjuSOC1 to promote plant flowering. This discovery may facilitate further clarifying the molecular mechanism of BjuWRKY71-1 in flowering time control, and creating new germplasm with bolting and flowering tolerance in mustard.

Analysis of pea mutants reveals the conserved role of FRUITFULL controlling the end of flowering and its potential to boost yield.

Monocarpic plants have a single reproductive phase in their life. Therefore, flower and fruit production are restricted to the length of this period. This reproductive strategy involves the regulation of flowering cessation by a coordinated arrest of the growth of the inflorescence meristems, optimizing resource allocation to ensure seed filling. Flowering cessation appears to be a regulated phenomenon in all monocarpic plants. Early studies in several species identified seed production as a major factor triggering inflorescence proliferative arrest. Recently, genetic factors controlling inflorescence arrest, in parallel to the putative signals elicited by seed production, have started to be uncovered in Arabidopsis, with the MADS-box gene FRUITFULL (FUL) playing a central role in the process. However, whether the genetic network regulating arrest is also at play in other species is completely unknown. Here, we show that this role of FUL is not restricted to Arabidopsis but is conserved in another monocarpic species with a different inflorescence structure, field pea, strongly suggesting that the network controlling the end of flowering is common to other plants. Moreover, field trials with lines carrying mutations in pea FUL genes show that they could be used to boost crop yield.

Transcriptomic and targeted metabolomic analyses provide insights into the flavonoids biosynthesis in the flowers of Lonicera macranthoides.

BACKGROUND: Flavonoids are one of the bioactive ingredients of Lonicera macranthoides (L. macranthoides), however, their biosynthesis in the flower is still unclear. In this study, combined transcriptomic and targeted metabolomic analyses were performed to clarify the flavonoids biosynthesis during flowering of L. macranthoides. RESULTS: In the three sample groups, GB_vs_WB, GB_vs_WF and GB_vs_GF, there were 25, 22 and 18 differentially expressed genes (DEGs) in flavonoids biosynthetic pathway respectively. A total of 339 flavonoids were detected and quantified at four developmental stages of flower in L. macranthoides. In the three sample groups, 113, 155 and 163 differentially accumulated flavonoids (DAFs) were detected respectively. Among the DAFs, most apigenin derivatives in flavones and most kaempferol derivatives in flavonols were up-regulated. Correlation analysis between DEGs and DAFs showed that the down-regulated expressions of the CHS, DFR, C4H, F3'H, CCoAOMT_32 and the up-regulated expressions of the two HCTs resulted in down-regulated levels of dihydroquercetin, epigallocatechin and up-regulated level of kaempferol-3-O-(6''-O-acetyl)-glucoside, cosmosiin and apigenin-4'-O-glucoside. The down-regulated expressions of F3H and FLS decreased the contents of 7 metabolites, including naringenin chalcone, proanthocyanidin B2, B3, B4, C1, limocitrin-3,7-di-O-glucoside and limocitrin-3-O-sophoroside. CONCLUSION: The findings are helpful for genetic improvement of varieties in L.macranthoides.

Comparative proteomic analysis of papaya bud flowers reveals metabolic signatures and pathways driving hermaphrodite development.

Papaya (Carica papaya) is a trioecious species with female, male, and hermaphrodite plants. Given the sex segregation, selecting hermaphroditic plants is vital for orchard establishment due to their greater commercial value. However, selecting hermaphrodite plants through sexing is laborious and costly. Moreover, environmental stressors can exacerbate the issue by potentially inducing abnormal flower development, thus affecting fruit quality. Despite these challenges, the molecular mechanisms governing sex development in papaya remain poorly understood. Thus, this study aimed to identify proteins associated with sex development in female and hermaphrodite flowers of papaya through comparative proteomic analysis. Proteins from flower buds at the early and late developmental stages of three papaya genotypes (UENF-CALIMAN 01, JS12, and Sunrise Solo 72/12) were studied via proteomic analysis via the combination of the shotgun method and nanoESI-HDMSE technology. In buds at an early stage of development, 496 (35.9%) proteins exhibited significantly different abundances between sexes for the SS72/12 genotype, 139 (10%) for the JS12 genotype, and 165 (11.9%) for the UC-01 genotype. At the final stage of development, there were 181 (13.5%) for SS72/12, 113 (8.4%) for JS12, and 125 (9.1%) for UC-01. The large group of differentially accumulated proteins (DAPs) between the sexes was related to metabolism, as shown by the observation of only the proteins that exhibited the same pattern of accumulation in the three genotypes. Specifically, carbohydrate metabolism proteins were up-regulated in hermaphrodite flower buds early in development, while those linked to monosaccharide and amino acid metabolism increased during late development. Enrichment of sporopollenin and phenylpropanoid biosynthesis pathways characterizes hermaphrodite samples across developmental stages, with predicted protein interactions highlighting the crucial role of phenylpropanoids in sporopollenin biosynthesis for pollen wall formation. Most of the DAPs played key roles in pectin, cellulose, and lignin synthesis and were essential for cell wall formation and male flower structure development, notably in the pollen coat. These findings suggest that hermaphrodite flowers require more energy for development, likely due to complex pollen wall formation. Overall, these insights illuminate the molecular mechanisms of papaya floral development, revealing complex regulatory networks and energetic demands in the formation of male reproductive structures.

Auxin and carbohydrate control flower bud development in Anthurium andraeanum during early stage of sexual reproduction.

BACKGROUND: Flower buds of Anthurium andraeanum frequently cease to grow and abort during the early flowering stage, resulting in prolonged planting times and increased commercialization costs. Nevertheless, limited knowledge exists of the mechanism of flower development after initiation in A. andraeanum. RESULTS: In this study, the measurement of carbohydrate flow and intensity between leaves and flowers during different growth stages showed that tender leaves are strong sinks and their concomitant flowers are weak ones. This suggested that the tender leaves compete with their concomitant flower buds for carbohydrates during the early growth stages, potentially causing the abortion of the flower buds. The analysis of transcriptomic differentially expressed genes suggested that genes related to sucrose metabolism and auxin response play an important role during flower bud development. Particularly, co-expression network analysis found that AaSPL12 is a hub gene engaged in flower development by collaborating carbohydrate and auxin signals. Yeast Two Hybrid assays revealed that AaSPL12 can interact with AaARP, a protein that serves as an indicator of dormancy. Additionally, the application of exogenous IAA and sucrose can suppress the expression of AaARP, augment the transcriptional abundance of AaSPL12, and consequently expedite flower development in Anthurium andraeanum. CONCLUSIONS: Collectively, our findings indicated that the combination of auxin and sugar signals could potentially suppress the repression of AaARP protein to AaSPL12, thus advancing the development of flower buds in Anthurium andraeanum.

DIA-Based Quantitative Proteomics in the Flower Buds of Two Malus sieversii (Ledeb.) M. Roem Subtypes at Different Overwintering Stages.

Malus sieversii is considered the ancestor of the modern cultivated apple, with a high value for apple tolerance breeding. Despite studies on the temperature adaptability of M. sieversii carried out at a physiological response and the genome level, information on the proteome changes of M. sieversii during dormancy is limited, especially about the M. sieversii subtypes. In this study, a DIA-based approach was employed to screen and identify differential proteins involved in three overwintering periods of flower buds in two M. sieversii subtypes (Malus sieversii f. luteolus, GL; Malus sieversii f. aromaticus, HC) with different overwintering adaptabilities. The proteomic analysis revealed that the number of the down-regulated differential expression proteins (DEPs) was obviously higher than that of the up-regulated DEPs in the HC vs. GL groups, especially at the dormancy stage and dormancy-release stage. Through functional classification of those DEPs, the majority of the DEPs in the HC vs. GL groups were associated with protein processing in the endoplasmic reticulum, oxidative phosphorylation, starch and sucrose metabolism and ribosomes. Through WGCNA analysis, tricarboxylic acid cycle and pyruvate metabolism were highly correlated with the overwintering stages; oxidative phosphorylation and starch and sucrose metabolism were highly correlated with the Malus sieversii subtypes. This result suggests that the down-regulation of DEPs, which are predominantly enriched in these pathways, could potentially contribute to the lower cold tolerance observed in HC during overwintering stage.

Selection and Validation of qRT-PCR Internal Reference Genes to Study Flower Color Formation in Camellia impressinervis.

Real-time quantitative PCR (qRT-PCR) is a pivotal technique for gene expression analysis. To ensure reliable and accurate results, the internal reference genes must exhibit stable expression across varied experimental conditions. Currently, no internal reference genes for Camellia impressinervis have been established. This study aimed to identify stable internal reference genes from eight candidates derived from different developmental stages of C. impressinervis flowers. We employed geNorm, NormFinder, and BestKeeper to evaluate the expression stability of these candidates, which was followed by a comprehensive stability analysis. The results indicated that CiTUB, a tubulin gene, exhibited the most stable expression among the eight reference gene candidates in the petals. Subsequently, CiTUB was utilized as an internal reference for the qRT-PCR analysis of six genes implicated in the petal pigment synthesis pathway of C. impressinervis. The qRT-PCR results were corroborated by transcriptome sequencing data, affirming the stability and suitability of CiTUB as a reference gene. This study marks the first identification of stable internal reference genes within the entire genome of C. impressinervis, establishing a foundation for future gene expression and functional studies. Identifying such stable reference genes is crucial for advancing molecular research on C. impressinervis.

Polymorphisms in two key anthocyanic genes of clivia (Clivia miniata L.) reveal evidence of selection and possible association with flower pigmentation.

Members of the genus Clivia show considerable variation in flower pigmentation and morphology. Such variation is affected by mutations that emerge in candidate flower development genes over time. Besides population history, mutations can further illuminate the effects of demographic events in populations in addition to population genetic parameters including selection, recombination, and linkage disequilibrium (LD). The current study aimed to find sequence variants in 2 anthocyanin biosynthetic genes (DFR and bHLH) of Clivia miniata and use the data to assess population genetic factors from a random collection of orange/red- and yellow-flowered specimens. Overall, average nucleotide diversity in the 2 anthocyanin genes was moderate (π = 0.00646), whereas haplotypes differed significantly (Hd ≥ 0.9). Gene evolution was seemingly driven by mutations (CmiDFR) or recombinations (CmibHLH001). LD decayed swiftly within the analyzed gene regions and supported the feasibility of assessing trait-variant associations via the association/linkage mapping approach. In the end, most associations were found to be spurious, but 1 haplotype in CmibHLH001 showed a promising correlation to the orange/red flower phenotype in Clivia specimens. In all, the present study is the first to measure gene-level diversity in C. miniata-data that had never been reported so far. Furthermore, the study also identified allelic and haplotypic variants that may be beneficial in future association genetic studies of Clivia. Such studies, however, consider large diverse populations to control for statistical bias intrinsic to the analysis of small datasets.

Bee-pollination promotes rapid divergent evolution in plants growing in different soils.

Divergent evolution leads to variation among populations and thus promotes diversification. In plants, adaptation to different soils, pollinator guilds, and herbivores is thought to be a key ecological driver of adaptive divergence, but few studies have investigated this process experimentally. Here we use experimental evolution with fast cycling Brassica rapa plants to study the impact of soil, pollination, herbivory, and their interactions on divergent evolution in various traits during eight generations of selection. We found significant evolutionary changes in plant phenotypes caused by all three factors and their interactions. In the richer soil type, plants showed higher evolutionary rates, especially with bumblebee-pollination, which led to the evolution of increased attractiveness of plants to bumblebees. Plants that had experienced aphid-herbivory showed lower attractiveness. We found the strongest evolutionary divergence when plants evolved in different soils with bee-pollination rather than hand-pollination, irrespective of herbivory. This "soil-pollinator effect" impacted divergence in diverse suites of traits, for example leaf size, flowering time, flower petal length, some floral volatiles and leaf glucosinolates. We conclude that the interaction between soil and biotic pollination may be an important cause for divergent evolution of plants growing on different soil types, even without a shift in pollinator guilds.

Mutations overlying the miR172 target site of TOE-type genes are prime candidate variants for the double-flower trait in mei.

Mutations affecting flower shape in many plants have been favored by human selection, and various fruit trees are also grown for ornamental purposes. Mei (Prunus mume) is a dual purpose tree originated in China well known in the Western world for its generous early blooms, often bearing double flowers. Building on the knowledge of its genomic location, a candidate gene approach was used to identify a 49 bp deletion encompassing the miR172 target site of the euAP2 gene pmTOE (PmuVar_Ch1_3490) as a prime variant linked to flower doubleness. Searching within a large dataset of genome sequencing data from Eastern germplasm collections demonstrated a tight variant-trait association, further confirmed in a panel of commercial and non-commercial varieties available in Italy. Moreover, two SNP mutations in the miR172 target site of pmPET (PmuVar_Ch1_1333) were identified in some double flower accessions. The mei orthologue of PETALOSA genes already found responsible for the phenotype in other plants suggests that independent variants may have been selected throughout mei domestication history.

Gh4CL20/20A involved in flavonoid biosynthesis is essential for male fertility in cotton (Gossypium hirsutum L.).

Flavonoids have been shown to play an essential role in plant growth and fertility. 4-Coumarate CoA ligase (4CL) is one of the indispensable enzymes involved in the biosynthesis of flavonoids. However, the role of 4CL and flavonoids in impact on cotton fertility is still unknown. In this study, on the basis of identification of an additional Gh4CL gene, Gh4CL20A, by using an updated G. hirsutum genome, we found that Gh4CL20A and its homologous Gh4CL20 were preferentially expressed in petals and stamens. The petals of the loss-of-function Gh4CL20/Gh4CL20A mutant generated by CRISPR/Cas9 gene editing remained white until wilting. Notably, the mutant showed indehiscent anthers, reduced number of pollen grains and pollen viability, leading to male sterility. Histological analysis revealed that abnormal degradation of anther tapetum at the tetrad stage and abnormal pollen grain development at the mature stage caused male sterility of the gene editing mutant. Analysis of the anther transcriptome identified a total of 10574 and 11962 genes up- and down-regulated in the mutant, respectively, compared to the wild-type. GO, KEGG, and WGCNA analyses linked the abnormality of the mutant anthers to the defective flavonoid biosynthetic pathway, leading to decreased activity of 4CL and chalcone isomerase (CHI) and reduced accumulation of flavonoids in the mutant. These results imply a role of Gh4CL20/Gh4CL20A in assuring proper development of cotton anthers by regulating flavonoid metabolism. This study elucidates a molecular mechanism underlying cotton anther development and provides candidate genes for creating cotton male sterile germplasm that has the potential to be used in production of hybrid seeds.

Hybrid Mimulus flowers attract a new pollinator.

Hybridization is common in flowering plants and is believed to be an important force driving adaptation and speciation. The flowers of hybrids often exhibit new trait combinations, which, theoretically, could attract new species of pollinators. In this study, we found that the hybrids between a hummingbird-pollinated species Mimulus cardinalis and a self-pollinated species Mimulus parishii attract bumblebees (Bombus impatiens), a pollinator not attracted to either of the progenitor species. This novel attraction is explained by new combinations of floral traits in hybrids, including, most importantly, petal color, in addition to nectar concentration and corolla size. To understand how petal color variation is perceived by bumblebees, we performed reflectance spectroscopy and multispectral imaging to model the flower appearance in bee vision. This analysis showed that color variation would impact the ease of detection. We also found that YUP, the genetic locus responsible for a large portion of floral color variation and previously shown to be important in bee interactions with other Mimulus species, also played an important role in this novel attraction. These results together suggest that the attraction of new pollinators to hybrid plants could be an underexplored avenue for pollinator shift and speciation.

Comprehensive Embryological Analyses of Common Buckwheat (Fagopyrym esculentum Moench).

Knowledge of detailed reproductive biology of cultivated species is important as requirements for fruit and seed production allow the development of effective management strategies and a sustainable use. Embryological processes of common buckwheat (Fagopyrum esculentum Moench) are difficult to interpret due to the influence of genetic determinants, i.e., dimorphic heterostyly resulting in the production of long- and short-styled flowers, and environmental predisposition, i.e., sensitivity of ovules to thermal stress. Furthermore, the situation is complicated by overproduction of flowers and depletion of resources as the plant ages. Herein we provide protocols that allow to visualize both basic and more specific embryological features and also disturbances in sexual reproduction of common buckwheat resulting from external and internal factors. All stages of plant material fixation, preparation, staining, and observation are described and explained in detail. Technical tips and pictures of properly prepared microscopic sections are also provided.

Canopeo app as image-based phenotyping tool in controlled environment utilizing Arabidopsis mutants.

Canopeo app was developed as a simple, accurate, rapid, and free tool to analyze ground cover fraction (GCF) from red-green-blue (RGB) images and videos captured in the field. With increasing interest in tools for plant phenotyping in controlled environments, the usefulness of Canopeo to identify differences in growth among Arabidopsis thaliana mutants in a controlled environment were explored. A simple imaging system was used to compare Arabidopsis mutants based on the FLAVIN-BINDING, KELCH REPEAT, F-BOX-1 (FKF1) mutation, which has been identified with increased biomass accumulation. Two FKF1 lines such as null expression (fkf1-t) and overexpression (FKF1-OE) lines were used along with wild type (Col-0). Canopeo was used to phenotype plants, based on biomass estimations. Under long-day photoperiod, fkf1-t had increased cellulose biosynthesis, and therefore biomass. Resource partitioning favored seedling vigor and delayed onset of senescence. In contrast, FKF1-OE illustrated a determinative growth habit where plant resources are primarily allocated for seed production. This study demonstrates the use of Canopeo for model plants and highlights its potential for phenotyping broadleaved crops in controlled environments. The value of adapting Canopeo for lab use is those with limited experience and resources have access to phenotyping methodology that is simple, accessible, accurate, and cost-efficient in a controlled environment setting.

Dynamic transcriptome analysis provides molecular insights into underground floral differentiation in Adonis Amurensis Regel & Radde.

Understanding flower developmental processes is a prerequisite for improving flowering 'plants' production. Adonis amurensis is a fascinating spring ephemeral plant that develops its flower organs underground. Nevertheless, knowledge of the molecular mechanisms driving this particular process is scarce. Herein, we examined transcriptional changes during underground flower differentiation in A. amurensis and unveiled key differently regulated genes and pathways. High-throughput RNA sequencing of meristems at different flower developmental stages, including flower primordium (FP), sepal stage (SE), perianth primordium (PE), stamen stage (ST), and pistil stage (PI), identified 303,234 unigenes that showed 44.79% similarity with sequences in Aquilegia coerulea. Correlations, principal component, and differentially expressed genes (DEGs) analyses revealed that few molecular changes occurred during the transition from PE to ST. Many DEGs exhibited stage-specific regulations. Transcription factor (TF) and phytohormone family genes are critical regulators of the floral differentiation process in A. amurensis. The most differentially regulated TFs were MADS, FAR1, MYBs, AP2/ERF, B3, C2H2, and LOBs. We filtered out 186 candidate genes for future functional studies, including 18 flowering/circadian-related, 32 phytohormone-related, and TF family genes. Our findings deepen our understanding of the underground flower differentiation process and offer critical resources to dissect its regulatory network in A. amurensis. These findings establish a foundational platform for researchers dedicated to exploring the unique phenotypic characteristics of this specific flowering modality and delving into the intricate molecular mechanisms underpinning its regulation and expression.

Laser-induced changes in the gene expression, growth and development of Gladiolus grandiflorus cv. "White Prosperity".

Corms of Gladiolus grandiflorus cv. "White Prosperity" was irradiated via red laser at wavelength 635 nm. Various morphological, flowering, elemental and chemical characterizations were studied. Irradiation with different power (5, 20, and 50 mW) and various irradiation time (0.0, 0.5, 1, 3, 5 and 10 min) was studied. Several characters), totaletermined include vegetative growth parameter (spouting days, plant height (cm), leaves number, leaves fresh and dry weights (g/plant), diameter of plant middle part (mm) and leaf area (cm2), floral parameters (flowering days, vase life (day), fresh and dry weights of inflorescence (g/plant), number of flowers per inflorescence, inflorescence length(cm), flowers diameter(cm), number of corms per plant, corms fresh weight(g/plant), circumference/ corms), pigments [total chlorophylls in leaves (SPAD), anthocyanin content (mg/100 g F.W.) in petals], NPK (%) in new corms and chemical composition in corms; total carbohydrates (%),total phenol (µg CE/g (%),total flavonoid (µg CE/g) (%), antioxidant (DPPH IC50 (µg /ml (%), and proline content (µ moles/g). The results showed that the medium level (20 mW) of He-Ne laser at 5 min caused favorable changes in the leaf anatomical structures and other studied characters followed by the low level (5 mW) of He-Ne laser at 5min. 112 bands emerged from 22 SSR primers, ranging between 130 and 540 bp, with 32 bands having polymorphism ranging from 17-100%. Out of the 22 SSR primers, 3 primers exhibited a high polymorphism percentage, i.e., SSR6, SSR16 and SSR22 which exhibited 7 positive markers. These findings revealed the efficiency of SSR primers for differentiating gladiolus plants and revealed that some alleles were affected by laser in their corms and the expression resulted in color or abnormalities in leaves and/or flowers. Mutation in some alleles could result in abnormalities like mutation in the allele with 410 bp revealed by SSR16.