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Abstract The objective of this work was to determine the base temperature of six lisianthus cultivars, grown at seven planting seasons in the municipality of Capão do Leão, in the state of Rio Grande do Sul, Brazil. The following cultivars were evaluated: DBL Echo Blue, Robella 2 Pink, Excalibur White, Excalibur 2 Blue Picotee, DBL Echo Champagne, and Arena III Red. Base temperature was determined from the air temperature data collected daily, in each planting season, fusing the lowest variability and development ratio methods. The base temperature for lisianthus cultivars is between 14.0 and 15.0ºC, by the lowest variability method, and between 13.9 and 15.0ºC by the development ratio method. The highest thermal accumulation was obseerved in the second planting season (from 11/23/2017 to 02/22/2018) for all cultivars, and lower air temperatures during the growing season resulted in longer production cycles. In the seven planting seasons, the highest base temperature is 15.0ºC, obtained for cultivar Arena III Red, and the is 14°C, for all lisianthus cultivars.

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Eustoma grandiflorum (Raf.) Shinn. is an annual herbaceous plant native to the southern United States, Mexico, and the Greater Antilles. It has a large flower with a variety of colors and is an important flower crop. In this study, we established a chromosome-scale de novo assembly of E. grandiflorum genome sequences by integrating four genomic and genetic approaches: (1) Pacific Biosciences (PacBio) Sequel deep sequencing, (2) error correction of the assembly by Illumina short reads, (3) scaffolding by chromatin conformation capture sequencing (Hi-C), and (4) genetic linkage maps derived from an F2 mapping population. Thirty-six pseudomolecules and 64 unplaced scaffolds were created, with a total length of 1,324.8 Mb. A total of 36,619 genes were predicted on the genome as high-confidence genes. A comparison of genome structure between E. grandiflorum and C. canephora or O. pumila suggested whole-genome duplication after the divergence between the families Gentianaceae and Rubiaceae. Phylogenetic analysis with single-copy genes suggested that the divergence time between Gentianaceae and Rubiaceae was 74.94 MYA. Genetic diversity analysis was performed for nine commercial E. grandiflorum varieties bred in Japan, from which 254,205 variants were identified. This first report on the construction of a reference genome sequence in the genus Eustoma is expected to contribute to genetic and genomic studies in this genus and in the family Gentianaceae. [ABSTRACT FROM AUTHOR]

Relatively mild xerophytic or hardening treatments can induce healthy development of plants. In the present study, as one of xerophytophysiological applications, sub-irrigation was applied to a flower plant of Eustoma grandiflorum to confirm whether the sub-irrigation improved flowering quality in addition to plant growth and physiology. As shown by the results, long-term sub-irrigation induced osmotic adjustment, with osmolyte concentration increasing 32.8 osmol m−3 (p ≤ 0.01), improved leaf photosynthetic activities, with more than 10% (p ≤ 0.05) increase in photosynthetic capacity, and promoted plant growth, with a shoot biomass increase by 27.5% (p ≤ 0.01) and a root increase by 50.5% (p ≤ 0.01). These improvements were attributed to turgor maintenance and cell water re-compartmentation into the symplasm, which were both the consequence of osmotic adjustment. The lower osmotic potential and lower relative leaf water potential at incipient plasmolysis suggested that plants in sub-irrigation plots might be more resistant to environmental stresses. Sub-irrigation also improved flower quality shown by increased anthocyanin concentration (16% up, p ≤ 0.01). Flower quality improvement might be attributed to up-regulation of the PAL gene, which could catalyze the synthesis of anthocyanins. PAL gene up-regulation might be associated with a concentration increase in salicylic acid (SA), which was suggested as a plant hormone for signaling. Sub-irrigation also affected the flower opening and closing oscillations with less changed opening size or oscillation amplitude. We adopted mathematical models and thoroughly analyzed dynamic changes in photosynthesis, plant growth, and flower opening oscillations. In conclusion, sub-irrigation was a feasible practice and could be used in E. grandiflorum culture to improve plant growth and flower opening quality. [ABSTRACT FROM AUTHOR]

• Continuous cropping of lisianthus led to soil salinity and soil function loss. • Fungal community experienced significantly decline in biomass and diversity. • No apparent accumulation of pathogens with continuous cropping. • Loss of prebiotics and accumulation of autotoxicants with continuous cropping. • Soil metabolites alteration is the main driver in the development of soil sickness. Continuous cropping obstacle (CCO) is becoming a serious threat to the quality production of lisianthus (Eustoma grandiflorum). However, investigations of the causal mechanism of CCO in lisianthus are still lacking. To this end, soil biotic dynamics and metabolites alterations were illuminated using metagenomic sequencing and metabolomic profiling along a chronosequence that has cultivated lisianthus from 2 weeks to 6 years. Soil EC and nutrient contents increased significantly as the cultivation duration prolonged. Fungal community has undergone significant decline in their diversity and biomass. Nevertheless, no apparent accumulation of pathogenicity-related microbes was observed along the investigated chronosequence. The alterations of soil metabolites along the chronosequence reflect the decline of microbial activity, the accumulation of phytotoxic compounds and the loss of metabolite involved in beneficial plant-microbe interactions. As a result, lisianthus plants in the longer continuous cropping soil experienced stressful condition as evidenced by the increasing trend of stress responsive metabolites and the overall soil function deterioration as indicated by soil enzymes. Hierarchical partitioning analysis showed that the investigated factors can explain a total of 92.25 % of the variations in the soil function degradation. Of which, soil metabolite alteration was the most important factor in explaining the CCO development, covering 24.55 % of the explainable variations. Spearman's correlation results further implied that soil metabolite alteration is the main driver in the development of CCO in lisianthus, especially by mediating the plant-microbes interactions. Our findings revealed a general pattern of soil biotic and abiotic response to the continuous monocropping of lisianthus and suggest that the overcome of the CCO in lisianthus requires further studies focusing on the crucial metabolites and their interactions with microbes. [ABSTRACT FROM AUTHOR]

Nitrogen (N) and Sulfur (S) are nutrients that have a strong uptake interaction. Therefore, the addition of these nutrients in the right ratio has a positive influence in plant development. This work is aiming to assess the effects of the interaction between NH4+ and SO42- at different concentrations, on the growth, physiology, and macronutrient concentration in Lisianthus (Eustoma grandiflorum (Raf.) Shinn.) cv. Dark Pink ABC 1-2. The trial treatments included three concentrations of NH4+ (0, 2.5 and 5 meq L-1) and two concentrations of SO42- (7 y 10 meq L-1). The different concentrations of SO42- y NH4+ were based on Steiner’s nutrient solution modifications. We used 10-liter black polyethylene containers and perlite substrate with particles size of 0.2-0.5mm in diameter. The results showed that the stem diameter and dry biomass production of roots, stems, flowers, and total biomass were higher with 5 meq L-1 of NH4+ and 7 meq L-1 of SO42-. On the other hand, total leaf dry weight and plant height were higher with 2.5 meq L-1 of NH4+ and 7 meq L-1 of SO42-. The plants showed the same behavior in terms of photosynthesis, transpiration, and stomatal conductance. We obtained the highest concentration of N and P by applying 2.5 meq L-1 of NH4+ with 10 meq L-1 of SO42. Furthermore, Mg, and S concentrations were higher when we supplied 2.5 meq L-1 of NH4+ with the two concentrations of SO42; while K and Ca were higher when we increased the concentration of NH4+ with 7 meq L-1 of SO42-. We obtained the best growth rate, biomass production and nutrient concentration (K and Ca) in Lisianthus plants with 5 meq L-1 of NH4+/ 7 meq L-1 of SO42-; while physiological activity improved with 2.5 meq L-1 of NH4+ / 7 meq L-1 of SO42- ratio. [ABSTRACT FROM AUTHOR]

El nitrógeno (N) y azufre (S) son nutrimentos que tienen una fuerte interacción en sus vías de asimilación. Por lo tanto, la adición de estos en una adecuada proporción influye positivamente en el desarrollo de las plantas. El objetivo de este trabajo fue evaluar el efecto de la interacción de diferentes concentraciones de NH4+ y SO42- en variables de crecimiento, fisiológicas y de concentración de macronutrimentos en las plantas de lisianthus (Eustoma grandiflorum (Raf.) Shinn.) cv. ABC 1-2 Rosado Oscuro. Los tratamientos evaluados consistieron en tres concentraciones de NH4+ (0, 2.5 y 5 meq L-1) y dos concentraciones de SO42- (7 y 10 meq L-1). Las diferentes concentraciones de SO42- y NH4+ se diseñaron a partir de modificaciones de la solución nutritiva de Steiner. Se utilizaron contenedores de polietileno negro de 10 L y sustrato perlita con partículas de 0.2-0.5 mm de diámetro. Los resultados mostraron que el diámetro de tallo y la producción de biomasa seca en raíz, tallo, flor y total fueron mayores al suministrar 5 meq L-1 de NH4+ con 7 meq L-1 de SO42-. Por otro lado, el peso seco de hoja y altura de planta fue mayor al emplear 2.5 meq L-1 de NH4+ con 7 meq L-1 de SO42-. Este mismo comportamiento se observó en los parámetros de fotosíntesis, transpiración y conductancia estomática. La mayor concentración de N y P se obtuvo al emplear 2.5 meq L-1 de NH4+ con 10 meq L-1 de SO42-; asimismo, la concentración de Mg y S fue mayor con 2.5 meq L-1 de NH4+ en ambas concentraciones de SO42; mientras que K y Ca fue mayor al incrementar la concentración de NH4+ con 7 meq L-1 de SO42-. El mejor crecimiento, producción de biomasa y concentración de nutrimentos (K y Ca) de las plantas de lisianthus se obtiene con la proporción 5 meq L-1 de NH4+/ 7 meq L-1 de SO42-; mientras que, la actividad fisiológica es favorecida por la proporción 2.5 meq L-1 de NH4+ / 7 meq L-1 de SO42-.

Morphotypes of arbuscular mycorrhizal (AM) symbiosis, Arum , Paris , and Intermediate types, are mainly determined by host plant lineages. It was reported that the phytohormone gibberellin (GA) inhibits the establishment of Arum -type AM symbiosis in legume plants. In contrast, we previously reported that GA promotes the establishment of Paris -type AM symbiosis in Eustoma grandiflorum , while suppressing Arum -type AM symbiosis in a legume model plant, Lotus japonicus. This raises a hitherto unexplored possibility that GA-mediated transcriptional reprogramming during AM symbiosis is different among plant lineages as the AM morphotypes are distinct. Here, our comparative transcriptomics revealed that several symbiosis-related genes were commonly upregulated upon AM fungal colonization in L. japonicus (Arum -type), Daucus carota (Intermediate-type), and E. grandiflorum (Paris -type). Despite of the similarities, the fungal colonization levels and the expression of symbiosis-related genes were suppressed in L. japonicus and D. carota but were promoted in E. grandiflorum in the presence of GA. Moreover, exogenous GA inhibited the expression of genes involved in biosynthetic process of the pre-symbiotic signal component, strigolactone, which resulted in the reduction of its endogenous accumulation in L. japonicus and E. grandiflorum. Additionally, differential regulation of genes involved in sugar metabolism suggested that disaccharides metabolized in AM roots would be different between L. japonicus and D. carota / E. grandiflorum. Therefore, this study uncovered the conserved transcriptional responses during mycorrhization regardless of the distinct AM morphotype. Meanwhile, we also found diverse responses to GA among phylogenetically distant AM host plants. [ABSTRACT FROM AUTHOR]

Keywords: Eustoma grandiflorum; genome assembly; whole-genome triplication event; flower coloration; floral development model EN Eustoma grandiflorum genome assembly whole-genome triplication event flower coloration floral development model 1856 1858 3 09/23/22 20221001 NES 221001 Introduction I Eustoma grandiflorum i (lisianthus) is a Gentianaceae-family ornamental plant. In lisianthus, a total of 120 MADS-box genes were identified, including 27 floral organ identity genes (three A-class genes, nine B-class genes, four C-class genes, one D-class genes and 10 E-class genes; Figures 1d, S26-S28). The genome of Eustoma grandiflorum reveals the whole-genome triplication event contributing to ornamental traits in cultivated lisianthus. [Extracted from the article]

Eustoma grandiflorum, commonly known as prairie gentian or Texas bluebells, is among the most popular agriculturally propagated species of cut flowers. Due to its widespread appeal, there is increasing interest in understanding the molecular genetic factors underlying floral development and resistance to abiotic stresses. We analyzed 18 potential reference genes in different organs, at different floral developmental stages and under drought- and salt-stress treatments, for use in RT-qPCR analysis. A total of four analytical tool packages, including geNorm, NormFinder, BestKeeper, and RefFinder were employed to determine the most appropriate reference genes under each treatment condition. The results demonstrate that different reference genes should be used for normalization under different experimental treatments. EgPP and EgPP2A2 were the most stable internal control genes across different organ types, EgPP and Eg18S were the most stable under salt-stress, EgPP and EgACT1 were the most stable across different floral development stages, and EgEF1A and EgTUA were the most stable reference genes under drought-stress. Additional gene expression analyses of EgMIXTA1, EgTOE1, and EgP5CS1 further confirmed the applicability of these reference genes. The results represent a significant contribution to future studies of reference gene selection for the normalization of gene expression in Eustoma grandiflorum.

• The two E. grandiflorum (lisianthus) cultivars exhibiting distinct petal patterns were used to conduct a comparative morphological, anatomical, and transcriptional analysis. • Our study is the first to probe into the genetic mechanisms of petal morphogenesis and construct the petal morphogenesis model in lisianthus. • We identified two potential regulatory genes of petal morphogenesis, including YABBY3 and LFY. • Our findings will contribute to the molecular breeding of lisianthus in the future. Eustoma grandiflorum , also known as lisianthus, is a popular ornamental plant belonging to the Gentianaceae family. Due to its diverse petal shapes, it is an excellent model for studying the genetic mechanisms of petal morphogenesis. In this study, we conducted a comparative morphological, anatomical, and transcriptional analysis of two cultivated varieties of lisianthus, 'Wavy White' with wavy petals and 'Rosita White' with flat petals. Cytological observations showed that the cells of both the epidermis (Ep) and parenchymatous tissue (Pt) at the bud stage, as well as the cell numbers of Ep at the mature stage existed difference between petals in 'Rosita White' and 'Wavy White'. We found that genes involved in cell division, cell expansion, and microtubule-based processes, including genes in the CYCLIN D3 (CYCD3) family, EXPANSIN (EXP) family, and Tubulin / FtsZ family, were highly over-represented during the bud stage in 'Wavy White'. At the bud stage, several specific genes, including EXP1, YABBY3 (YAB3), GROWTH-REGULATING FACTOR 1 (GRF1), LEAFY (LFY), LOB DOMAIN-CONTAINING PROTEIN 40 (LBD40), and PROTODERMAL FACTOR 2 (PDF2) were found to be highly expressed in the petals of 'Wavy White'. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis further confirmed significantly higher expression levels of LFY, PDF2, GRF1 , and YAB3 in 'Wavy White' than those in 'Rosita White' at the bud stage. And we found that YAB3 was more highly expressed in petals in lisianthus cultivars with wavy petals than those in 'Rosita White'; LFY was more highly expressed in petals in lisianthus cultivars with wavy petals except 'Wavy Purple' than those in 'Rosita White'. Overall, our findings shed light on the genetic mechanisms of petal morphogenesis in lisianthus. [ABSTRACT FROM AUTHOR]

• 7 anthocyanins related to the bicolor flower formation of E. grandiflorum were identified. • The key structural genes and TFs related to flavonoid biosynthesis were screened. • EgCHS1 showed significantly higher expression in the upper tepals than in the tepal bases. The bicolor flowers of Eustoma grandiflorum have a special phenotype and high ornamental value. However, the coloration mechanism of these flowers has not been reported. We used metabolomics and transcriptomics to analyze the accumulation of secondary metabolites and the expression of differential genes in bicolor petals at different developmental stages, to elucidate the molecular mechanism underlying anthocyanin accumulation in these bicolor flowers of E. grandiflorum. The metabolomics analysis revealed that five kinds of anthocyanins in the upper pigmented petals at different developmental stages, and three kinds of anthocyanins were identified in the comparison between the pink upper tepals and the white bases at stage 3, with pelargonidin 3-glucoside 5-(6-coumaroyl) glucoside showing the most significant difference. The differentially expressed genes (DEGs) related to flavonoid biosynthesis in bicolor flowers were further analyzed by transcriptomics, and 25 structural genes and 5 MYB transcription factors (TFs) were screened, among which EgCHS1 showed significantly higher expression (approximately 21.5 times) in the upper tepals than in the tepal bases. Transcriptomic and metabolomic association analysis showed that the EgFLS2 and EgFLS3 genes were related to the largest quantity differentially accumulated metabolites (DAMs), and ferulate was associated with the largest quantity of DEGs. This study provides a basis for anthocyanin accumulation and coloration mechanism during the development of E. grandiflorum bicolor flowers. The results will be conducive for promoting artificial regulation of flower color and breeding new cultivars of E. grandiflorum. [ABSTRACT FROM AUTHOR]

Eustoma grandiflorum (Raf.) Shinn. is an annual herbaceous plant native to the southern United States, Mexico, and the Greater Antilles. It has a large flower with a variety of colors and is an important flower crop. In this study, we established a chromosome-scale de novo assembly of E. grandiflorum genome sequences by integrating four genomic and genetic approaches: (1) Pacific Biosciences (PacBio) Sequel deep sequencing, (2) error correction of the assembly by Illumina short reads, (3) scaffolding by chromatin conformation capture sequencing (Hi-C), and (4) genetic linkage maps derived from an F2 mapping population. Thirty-six pseudomolecules and 64 unplaced scaffolds were created, with a total length of 1,324.8 Mb. A total of 36,619 genes were predicted on the genome as high-confidence genes. A comparison of genome structure between E. grandiflorum and C. canephora or O. pumila suggested whole-genome duplication after the divergence between the families Gentianaceae and Rubiaceae. Phylogenetic analysis with single-copy genes suggested that the divergence time between Gentianaceae and Rubiaceae was 74.94 MYA. Genetic diversity analysis was performed for nine commercial E. grandiflorum varieties bred in Japan, from which 254,205 variants were identified. This first report on the construction of a reference genome sequence in the genus Eustoma is expected to contribute to genetic and genomic studies in this genus and in the family Gentianaceae., Competing Interests: Conflicts of interest The authors declare no conflicts of interest associated with this manuscript., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Genetics Society of America.)

• The production of Eustoma to flower in 130 days without exhibiting high temperature induced (HTI) rosette is reported. • HTI rosette is defined as "abnormal growth exhibiting a compressed internodes after forming 3 – 4 pairs of leaves and fail to flower". • HTI rosette growth occurs when seedlings are exposed to 29/27 °C during 34 – 74 days (D) after sowing. • Different forms of HTI rosette growth (A-D) and normal flowering (E) are illustrated. • Optimum growing conditions to flower in 130 days after sowing without showing HTI rosette are sowing seeds at 21/16 °C/0 – 30D and growing seedlings at 25/23 °C/31 – 50D, 21/19 °C/51 – 70D/LD, 23/21 °C/71 – 90D/LD, and finally at 23/21 °C/91 – 130D. The objective of this research is to produce dwarf Eustoma grandiflorum (Raf.) Shinn. 'Little Belle Blue' potted plants to flower in 130 days after sowing without exhibiting high temperature induced (HTI) rosette. High temperature induced rosette is defined as "the abnormal growth and development of plants exhibiting a compressed stem near the ground after forming 3 – 4 pairs of true leaves resulting from exposure of seedlings to high temperatures, and fail to form elongated internodes and to flower". The effect of temperature and photoperiod treatments during various growth and development stages was tested on the accelerated flowering without exhibiting the HTI rosette (Expt. 1, 2, 3), and to induce flowering in 130 days preventing the HTI rosette (Expt. 4, 5, 6). The treatments at 32/27 °C (day/night) during 0 – 30 days after sowing (0 – 30D) (32/27 °C/0 – 30D and 27/22 °C/31 – 60D should be avoided because 39% of plants exhibited HTI rosette (Expt. 1). Lower temperatures and shorter periods (24/22 °C/61 – 90D, 24/22 °C/31 – 60D) prevented rosette growth (Expt. 2). The treatment 23/21 °C/34 – 74D is considered acceptable when coupled with 21/19 °C/74 – 115D to promote flowering and to prevent the HTI rosette (Expt. 3). The earliest flowering (157 days) plants without the HTI rosette suggested that temperatures during various 20-day stages are 25/23 °C/0 – 20D, 21/19 °C/21 – 40D, 12/10 °C/61 – 80D, and 25/23 °C/81 – 100D. Early flowering occurred at 12/10 °C/61 – 80D and 25/23 °C/81 – 100D. During these periods, it was considered that flower bud initiation and development might occur, and this effect was especially pronounced in the treatment 25/23 °C/61 – 80D (Expt. 4). Flowering was the earliest (148 days) when 45-day old seedlings after sowing seeds in January were exposed for 30 days (46 – 75D) to a long-day (LD) photoperiod at 21/18 °C (Expt. 5). The total cropping time was reduced to 133 days without inducing HTI rosette and reducing the number of flowers by the following treatments: Sowing seeds as late as February and growing at 23/21 and 25/23 °C/30 – 50D, either under a short-day (SD) or LD/51 – 70D, and growing at 21/19 and 23/21 °C under LD/71 – 90D (Expt. 6). A clear distinction should be made between seedlings exhibiting physiologically normal growth during the vegetative juvenile stages that last about 60 days forming up to 3 to 4 pairs of small leaves and HTI rosette that occurs predominantly when seedlings are exposed to 29/27 °C/34 – 74D. Eustoma grandiflorum is an annual that does not exhibit seed dormancy since germination occurs in 12 – 14 days at 23/21 °C, and 'Little Belle Blue' does not require vernalization to induce flowering in 130 days after sowing seeds without exhibiting HTI rosette. Seeds are sown at 21/16 °C/0 – 30D in February and growing at 25/23 °C/31 – 50D, 21/19 °C/51 – 70D/LD, 23/21 °C/71 – 90D/LD, and finally at 23/21 °C/91 – 130D is recommended. [ABSTRACT FROM AUTHOR]

Eustoma grandiflorum, commonly known as prairie gentian or Texas bluebells, is among the most popular agriculturally propagated species of cut flowers. Due to its widespread appeal, there is increasing interest in understanding the molecular genetic factors underlying floral development and resistance to abiotic stresses. We analyzed 18 potential reference genes in different organs, at different floral developmental stages and under drought- and salt-stress treatments, for use in RT-qPCR analysis. A total of four analytical tool packages, including geNorm, NormFinder, BestKeeper, and RefFinder were employed to determine the most appropriate reference genes under each treatment condition. The results demonstrate that different reference genes should be used for normalization under different experimental treatments. EgPP and EgPP2A2 were the most stable internal control genes across different organ types, EgPP and Eg18S were the most stable under salt-stress, EgPP and EgACT1 were the most stable across different floral development stages, and EgEF1A and EgTUA were the most stable reference genes under drought-stress. Additional gene expression analyses of EgMIXTA1, EgTOE1, and EgP5CS1 further confirmed the applicability of these reference genes. The results represent a significant contribution to future studies of reference gene selection for the normalization of gene expression in Eustoma grandiflorum. [ABSTRACT FROM AUTHOR]