Young Flower Buds Research Articles

ArPAOs from A. roxburghii showed essential roles in polyamine mediating water stress tolerance

Anoectochilus roxburghii is a precious Chinese herb with excellent pharmacological effects. When meet stress, plants produced polyamines (PAs) which are identified in active tissues and play crucial roles in stress responses. Nevertheless, the molecular mechanism of PAs responding to stress in A. roxburghii were largely unknown. Here, for the first time, three PAOs in A. roxburghii were identified from transcriptome data and functionally analyzed. Expression analysis was performed using fluorescence quantitative PCR and eventually, transgenic Arabidopsis thaliana were created to confirm the biological function of ArPAOs. It was found that the highest expression was in young flower buds and the expression levels of three ArPAO genes in various tissues were also different, which indicated their different catalytic preferences and functions. Under the treatment of exogenous spermine and spermidine, the expression of three ArPAOs fluctuated and the overall expression level increased. Overexpression (OE) of ArPAOs could enhance water stress tolerance through mediating polyamine catabolism. It might because the excessive expression of ArPAOs reduce the content of spermine and spermidine, creating a negative feedback regulation upstream and causes plants manufacturing PAs which reinforce water stress tolerance. Taken together, the study firstly reported the overexpression of ArPAOs enhance plant water stress tolerance and regulate the content of polyamines. This work will provide a vital theoretical foundation for uncovering the operation and regulatory mechanism of ArPAOs in promoting plant development and managing stress.

Aphid alarm pheromone mimicry in transgenic Chrysanthemum morifolium: insights into the potential of (E)-β-farnesene for aphid resistance.

(E)-β-Farnesene (EBF) serves as the primary component of the alarm pheromone used by most aphid pest species. Pyrethrum (Tanacetum cinerariifolium) exhibits tissue-specific regulation of EBF accumulation and release, effectively mimicking the aphid alarm signal, deterring aphid attacks while attracting aphid predators. However, cultivated chrysanthemum (Chrysanthemum morifolium), a popular and economically significant flower, is highly vulnerable to aphid infestations. In this study, we investigated the high expression of the pyrethrum EBF synthase (TcEbFS) gene promoter in the flower head and stem, particularly in the parenchyma cells. Subsequently, we introduced the TcEbFS gene, under the control of its native promoter, into cultivated chrysanthemum. This genetic modification led to increased EBF accumulation in the flower stem and young flower bud, which are the most susceptible tissues to aphid attacks. Analysis revealed that aphids feeding on transgenic chrysanthemum exhibited prolonged probing times and extended salivation durations during the phloem phase, indicating that EBF in the cortex cells hindered their host-location behavior. Interestingly, the heightened emission of EBF was only observed in transgenic chrysanthemum flowers after mechanical damage. Furthermore, we explored the potential of this transgenic chrysanthemum for aphid resistance by comparing the spatial distribution and storage of terpene volatiles in different organs and tissues of pyrethrum and chrysanthemum. This study provides valuable insights into future trials aiming for a more accurate replication of alarm pheromone release in plants. It highlights the complexities of utilizing EBF for aphid resistance in cultivated chrysanthemum and calls for further investigations to enhance our understanding of this defense mechanism.

A dated phylogeny of the Neotropical Dipterygeae clade reveals 30 million years of winged papilionate floral conservatism in the otherwise florally labile early-branching papilionoid legumes

Abstract The early-branching clades of Fabaceae subfamily Papilionoideae are characterized by their remarkable lability in floral architecture. In contrast, more derived papilionoid lineages are marked by evolutionary conservatism towards strongly bilateral, papilionate flowers. Here, we show an unexpected example of conservatism of a unique floral architecture during the early diversification history of the papilionoids. We built the most comprehensively sampled molecular phylogenetic tree with a focus on the early-diverging papilionoid Dipterygeae clade to evaluate conservatism of the winged papilionate architecture and associated traits related to flower specialization (e.g. zygomorphy, petal differentiation, stable stamen number and stamen sheath). Dipterygeae comprise c. 22 species of mostly giant trees from across tropical forests in Central America and the Amazon, but they are also ecologically dominant in the savannas of the Brazilian Central Plateau. Phylogenetic analyses of nuclear ribosomal ITS/5.8S and plastid matK and trnL intron sequences strongly supported inter-relationships and the monophyly of each genus (Dipteryx, Monopteryx, Pterodon and Taralea). Bayesian relaxed-clock dating and a Bayesian model of ancestral character estimation revealed c. 30 Myr of conservatism of all winged papilionate-related flower traits in a clade comprising the most recent common ancestor of Dipteryx, Pterodon and Taralea, but lability in fruit morphology during the diversification of the entire Dipterygeae clade. Despite Monopteryx and remaining Dipterygeae being florally discrepant, they are collectively defined by a floral synapomorphy that is unique among all papilionoid Fabaceae: the highly differentiated calyx, where the two upper lobes are enlarged and wing-like, whereas the other three lower lobes are reduced. We suggest that the different dispersal strategies and the ancient winged papilionate floral conservatism in Dipterygeae, which has maintained effective ecological interactions with specialized pollinators and ensured the protection of young flower buds and developing fruits, may explain successful evolutionary and ecological persistence of the clade across the main Neotropical biomes.

Effect of silver nanoparticles on sterilization of different explant sources of Gerbera jamesonii cultured in vitro

In this study, silver nanoparticles effects on the sterilization of different sources of explants (young leaves, young flower stalks and young flower buds) of Gerbera as well as on the in vitro morphogenesis and their growth were investigated. The explants were sterilized and cut transversally (1 mm) with the flower stalk, square (0.5 × 0.5 cm) for the leaf sample, longitudinally (0.5 mm) for the flowers (removed the petals) and cultured on MS medium; then, the explants (contamination-free or no browning/necrosis) were transferred into MS medium supplemented with 0.02 mg/L TDZ plus 0.8 mg/L adenine, 10% coconut water, 30 g/L sucrose and 8 g/L agar in 15 days. The results showed that AgNPs at the appropriate concentration and duration treatment was effective in explant sterilization of flower bud (0.02% AgNPs and 20 min), flower stalks (0.02% AgNPs and 30 min) and young leaves (0.05% AgNPs and 20 min) after 15 days of culture. In addition, 3 types of morphogenesis including callus induction, somatic embryogenesis and direct shoot regeneration of explants derived from sterilization by AgNPs were improved as compared to that of HgCl2. In addition, research on the optimal medium for shoot multiplication, rooting as well as evaluation of acclimatization and the growth at the greenhouse were also studied. Results showed that MS medium supplemented with 2 mg/L NAA combined with 0.5 mg/L BA and 2 mg/L KIN is suitable for shoot multiplication; meanwhile, MS medium supplemented with 2 mg/L NAA improved rooting ability as well as quality of plantlets and to improving survival rate and acclimatization of Gerbera cultured in vitro.

Overexpression of LtKNOX1 from Lilium tsingtauense in Nicotiana benthamiana affects the development of leaf morphology

ABSTRACT Leaves are the main vegetative organs of the aboveground part of plants and play an important role in plant morphogenesis. KNOTTED-LIKE HOMEOBOX (KNOX) plays a crucial role in regulating leaf cell fate and maintaining leaf development. In this study, we analyzed LtKNOX1 from Lilium tsingtauense and illustrated its function in transgenic plants. Tissue-specific expression analysis indicated that LtKNOX1 was highly expressed in stems, young flower buds, and shoot apical meristems (SAMs). Ectopic overexpression of LtKNOX1 in Nicotiana benthamiana suggested that transformants with mild phenotypes were characterized by foliar wrinkles and mildly curled leaves; transformants with intermediate phenotypes showed severely crimped blades and narrow leaf angles, and the most severe phenotypes lacked normal SAMs and leaves. Moreover, the expression levels of genes involved in the regulation of KNOX in transgenic plants were detected, including ASYMMETRIC LEAVES1, PIN-FORMED 1, GA20-oxidase, CUP-SHAPED COTYLEDON 2, CLAVATA 1 and WUSCHEL(WUS), and the expression of other genes were down-regulated except WUS. This study contributes to our understanding of the LtKNOX1 function.

First Report of Bacterial Canker on Blueberry (Vaccinium corymbosum) Caused by Pseudomonas syringae pv. syringae in Serbia.

During May 2021, necrosis of young twigs and flower buds were observed on two-year-old highbush blueberry plants (Vaccinium corymbosum) cv. Draper, in a 1 hectare orchard in the municipality of Šabac, Serbia. Disease symptoms included reddish-brown to black irregularly shaped cankers developing on the shoot tips that extended downwards along the branches. In some plants, cankers surrounded the stem, causing shoot-tip dieback and necrosis of the buds. Beneath the bark, a distinct margin between diseased and healthy tissue was visible. A few weeks before symptoms development, seven freezing events with temperature from -3°C to -1°C, and five near-freezing temperatures were recorded in this area, leading to the hypothesis that symptoms were associated to the presence of ice nucleating bacteria belonging to Pseudomonas syringae. The observed disease incidence was 80%, while 10% of the plants died. Bacteria were isolated from symptomatic tissue on King's medium B (KB). After 2 to 3 days of incubation at 27°C, predominantly grey-whitish, shiny, round, convex bacterial colonies were observed on agar plates. Ten isolates producing a fluorescent pigment on KB were selected for further characterization by biochemical and molecular tests. The isolates were Gram, oxidase and arginine-dihydrolase negative, levan positive, induced hypersensitive response on tobacco leaves and showed no pectinolytic activity on potato slices. Based on the results of API 20E and API 20NE tests (BioMerieux, France), and the fact that isolates did not utilize tartrate nor had tyrosinase activity, they were preliminarly identified as Pseudomonas syrinage pv. syringae (Braun-Kiewnick and Sands 2001). Additionally, all tested isolates had ice-nucleation activity at -5°C. The syrB gene responsible for syringomycin synthesis, was amplified in all isoaltes with the specific primer pair B1/B2 (Sorensen et al. 1998). The 16S rRNA gene sequences of five selected isolates (GenBank MZ410287 to 91) showed 100% identity to P. s. pv. syringae isolated from Prunus avium in United Kingdom (GenBank CP026568) and France (GenBank LT962480). Sequences of gyrB gene (Sarkar and Guttman 2004) of two selected isolates (GenBank MZ420633 and MZ420634) showed 98,44% identity to the P. s. pv. syrinage strain isolated in France (GenBank LT962480). Pathogenicity of the isolates was confirmed on 2-year-old blueberry plants cv. Draper, by inoculating two plants per isolate. One-cm long wounds were made on branches using a scalpel and 20 µl of bacterial suspension (106 CFU/ml) was infiltrated into the tissue. The cuts were then covered with moist sterile cotton pads and wraped in parafilm for 3 days. Inoculation was also performed on two leaves per plant by needleless syringe infiltration (106 CFU/ml). Sterile distilled water was used as a negative control. Plants were maintained in greenhouse at 27°C day and 15°C night temperature. Three weeks after inoculation, the inoculated branches and leaves developed necrosis, leaves spots and cankers respectively, resembling the natural infection. Symptoms were not observed on the control plants. Bacteria were reisolated from symptomatic tissue and their identity was confirmed by amplifying the syrB gene sequence and additional biochemical tests. This is the first report of bacterial canker of highbush blueberry caused by P. s. pv. syringae in Serbia. In Europe, there was only one report on Pseudomonas spp. causing disease on blueberry leaves in Poland (Kaluzna et al. 2013). Due to market demands and export potential, blueberry production in Serbia has been rapidly increasing. In 2015, highbush blueberry was cultivated on 220 ha, while in 2020 the area increased to 1899 ha. However, under favourable environmental conditions, blueberry production might be severely affected by bacterial canker.

Сезонний ритм розвитку рослин актинідії (Actinidia arguta) в умовах Північного Степу Причорномор’я

The relevance of research on actinidia plants, which is a non-traditional uncommon fruit crop for Ukraine, is highlighted. In the conditions of introduction of the northern steppe of the Black Sea coast as a result of experimental researches dates of passing of phenological phases of two forms - female and male French selection are established. Female plants begin flowering 7 days earlier than males and bloom three days longer, regardless of the type of maturity. It was found that the biological maturity of actinidia fruits begins in the second decade of September (early - in the first), their full maturity was recorded in the late second to early third decade of September. The duration of the vegetation period of plants of different varieties and forms in the 2020 season was 229-236 days, in 2021 221-232 days, which indicates a direct dependence of plant growth and development on weather conditions. In terms of varieties, plants of medium type of development formed a yield higher than the early by 4464 g on average for two years of research. Assessment of resistance to pests and diseases in the 2020-2021 seasons showed that these objects are absent on actinidia plants, which is probably due to their absence in the new environment. The decrease in air temperature to -2… -5 oC at night in 2020 led to the freezing of young shoots and flower buds in plants of both forms. As a result, the fruit harvest was much lower than next year - 2021. For the successful cultivation of actinidia in the conditions studied, developed agronomic recommendations for the prevention of damage by low temperatures. The conclusion is made about carrying out selection works on creation of new grades of this culture, with a high level of plasticity and resistance to frosts during the growing season.

Nanovector-mediated exogenous delivery of dsRNA induces silencing of target genes in very young tomato flower buds.

RNA interference (RNAi) is a post-translational regulatory mechanism that controls gene expression in plants. This process can be artificially induced by double-stranded RNA (dsRNA) molecules with sequence homology to target mRNAs. Exogenously applied dsRNA on leaves has been shown to silence virulence genes of fungi and viruses, conferring protection to plants. Coupling dsRNA to nanoparticles has been demonstrated to prolong the silencing effect. The ability of exogenous dsRNA to silence endogenous genes in plants is currently under debate, mainly due to the difficulty in delivering dsRNA into plant tissues and organs. Our study aims to develop a method based on the exogenous application of dsRNA on tomato flowers for silencing endogenous genes controlling ovary growth. Two methods of dsRNA delivery into tomato flower buds (i.e., pedicel soaking and injection) were compared to test their efficacy in silencing the tomato Aux/IAA9 (SlIAA9) gene, which encodes for a known repressor of ovary growth. We examined the silencing effect of dsRNA alone and coupled to layered double hydroxide (LDHs) nanoparticles. We found that injection into the pedicel led to the silencing of SlIAA9 and the efficacy of the method was confirmed by choosing a different ovary growth repressor gene (SlAGAMOUS-like 6; SlAGL6). The coupling of dsRNA to LDHs increased the silencing effect in the case of SlIAA9. Silencing of the two repressors caused an increase in ovary size only when flower buds were treated with dsRNA coupled to LDHs. RNA-Seq of small RNAs showed that induction of RNAi was caused by the processing of injected dsRNA. In this work, we demonstrate for the first time that exogenous dsRNA coupled to LDHs can induce post-transcriptional gene silencing in the young tomato ovary by injection into the flower pedicel. This method represents a silencing tool for the study of the molecular changes occurring during the early stages of ovary/fruit growth as a consequence of downregulation of target genes, without the need to produce transgenic plants stably expressing RNAi constructs.

Sequential Deposition and Remodeling of Cell Wall Polymers During Tomato Pollen Development.

The cell wall of a mature pollen grain is a highly specialized, multilayered structure. The outer, sporopollenin-based exine provides protection and support to the pollen grain, while the inner intine, composed primarily of cellulose, is important for pollen germination. The formation of the mature pollen grain wall takes place within the anther with contributions of cell wall material from both the developing pollen grain as well as the surrounding cells of the tapetum. The process of wall development is complex; multiple cell wall polymers are deposited, some transiently, in a controlled sequence of events. Tomato (Solanum lycopersicum) is an important agricultural crop, which requires successful fertilization for fruit production as do many other members of the Solanaceae family. Despite the importance of pollen development for tomato, little is known about the detailed pollen gain wall developmental process. Here, we describe the structure of the tomato pollen wall and establish a developmental timeline of its formation. Mature tomato pollen is released from the anther in a dehydrated state and is tricolpate, with three long apertures without overlaying exine from which the pollen tube may emerge. Using histology and immunostaining, we determined the order in which key cell wall polymers were deposited with respect to overall pollen and anther development. Pollen development began in young flower buds when the premeiotic microspore mother cells (MMCs) began losing their cellulose primary cell wall. Following meiosis, the still conjoined microspores progressed to the tetrad stage characterized by a temporary, thick callose wall. Breakdown of the callose wall released the individual early microspores. Exine deposition began with the secretion of the sporopollenin foot layer. At the late microspore stage, exine deposition was completed and the tapetum degenerated. The pollen underwent mitosis to produce bicellular pollen; at which point, intine formation began, continuing through to pollen maturation. The entire cell wall development process was also punctuated by dynamic changes in pectin composition, particularly changes in methyl-esterified and de-methyl-esterified homogalacturonan.

Evaluation of the Impact of Eustenopus villosus on Centaurea solstitialis Seed Production in California.

Simple SummaryThe exotic thistle, Centaurea solstitialis, has become overly abundant in the western USA. A seed weevil, Eustenopus villosus, was released in the early 1990s to reduce seed production and abundance of the thistle. A first step in evaluating success of the biological control program is to evaluate the amount of seed destroyed by the weevil in its new area of infestation. At two sites for two years, we found that adult weevils killed 60–70% of young flower buds, forcing plants to regrow new buds which delayed flowering by 9 days and extended flowering by 4 weeks at season end. Flower heads varied in size and seed production increased linearly by size of the flower head. The seed weevil attacked larger flower heads more frequently than smaller flower heads but the probability of a flower head being attacked did not vary with plant size. Weevil larvae occurred in 27% to 49% of seed heads and resulted in 34% to 47% of the annual seed crop being destroyed. We recommend that another survey be performed to see if the seed weevil has increased in abundance since its introduction.The impact of the capitulum weevil Eustenopus villosus on Centaurea solstitialis seed production was examined at two field sites in central California. The study occurred in 1993–1995 during the early phases of the biological control program on C. solstitialis and before the current guild of capitulum insects had become widespread. Results showed that adult feeding on early flower buds resulted in 60–70% of buds failing to develop. Regrowth delayed capitulum production by 9 days and extended production by 4 weeks at season end. Between 69% and 92% of capitula were punctured from feeding or oviposition but the occurrence of larvae in capitula ranged from 27% to 49%. Seed production in C. solstitialis capitula increased linearly with size. The occurrence of larvae was proportionally higher in larger capitula (>8 mm) but the probability of attack for individual capitula did not vary with plant size. Total seed loss from larval feeding ranged from 34 to 47%. It is recommended that another survey be performed to determine if the level of infestation of E. villosus has increased since its initial introduction.

L-3,4-dihydroxyphenylalanine Accumulation in Faba Bean (Vicia faba L.) Tissues during Different Growth Stages

L-3,4-dihydroxyphenylalanine (L-dopa)-rich faba (Vicia faba L.) tissues are a potentially useful source for its pharmaceutical purpose, although the species contains health curious vicine and convicine in the seeds. We determined the contents of L-dopa, vicine, and convicine in different tissues of faba plants throughout the entire growth period. The three compounds accumulated in germinating sprouts and showed high contents at 8 days of germination, especially in the epicotyl containing 132.33 mg∙g−1 DW of L-dopa, 19.81 mg∙g−1 DW of vicine, and 10.38 mg∙g−1 DW of convicine, respectively. We compared the contents of the three compounds among leaves of different ages in plants at different growth stages. The samples could be ranked, from highest to lowest content, ranging from 11.11 to 81.82 mg∙g−1 DW, as follows: new leaves > young leaves > old leaves; and vegetative > flowering > ripening. Vicine and convicine were not detected in leaves or flowers. The L-dopa content was high in flowers, especially young flower buds, ranging from 36.51 to 100.70 mg∙g−1 DW. In older flowers, the L-dopa content tended to decrease as the calyx and petal parts developed. In addition, the three compounds showed decreasing contents in pods, stems, and roots with increasing plant age. The results of this study provide information on the timing and extent of L-dopa, vicine, and convicine accumulation in different faba tissues, and indicate that various by-products, especially new/young leaves and flowers, are potential sources of natural L-dopa.

Asymmetric epigenome maps of subgenomes reveal imbalanced transcription and distinct evolutionary trends in Brassica napus

The complexity of the epigenome landscape and transcriptional regulation is significantly increased during plant polyploidization, which drives genome evolution and contributes to the increased adaptability to diverse environments. However, a comprehensive epigenome map of Brassica napus is still unavailable. In this study, we performed integrative analysis of five histone modifications, RNA polymerase II occupancy, DNA methylation, and transcriptomes in two B. napus lines (2063A and B409), and established global maps of regulatory elements, chromatin states, and their dynamics for the whole genome (including the An and Cn subgenomes) in four tissue types (young leaf, flower bud, silique, and root) of these two lines. Approximately 65.8% of the genome was annotated with different epigenomic signals. Compared with the Cn subgenome, the An subgenome possesses a higher level of active epigenetic marks and lower level of repressive epigenetic marks. Genes from subgenome-unique regions contribute to the major differences between the An and Cn subgenomes. Asymmetric histone modifications between homeologous gene pairs reflect their biased expression patterns. We identified a novel bivalent chromatin state (with H3K4me1 and H3K27me3) in B. napus that is associated with tissue-specific gene expression. Furthermore, we observed that different types of duplicated genes have discrepant patterns of histone modification and DNA methylation levels. Collectively, our findings provide a valuable epigenetic resource for allopolyploid plants.

Electrophysiological and behavioral responses of gall midge, Jaapiella sp. to volatiles of host plant Lycium barbarum

Plant-derived volatiles are important for guiding herbivorous insects to their host plants. Jaapiella sp. (Diptera: Cecidomyiidae) is the most serious pest of Lycium barbarum. The young flower buds of L. barbarum are attractive to females of Jaapiella sp. for oviposition in the field. In this study, we compared response of Jaapiella sp. to volatiles from flower buds of L. barbarum at different stages, and clarified the crucial semiochemicals attracting Jaapiella sp. to lay eggs. Volatiles from young and mature flower buds were collected using SPME, and then identified and quantified by gas chromatography-mass spectrometry (GC-MS). Both electrophysiological and behavior experiments were conducted to measure the attraction of eight synthetic compounds to females and screen the crucial components based on gas chromatography-electroantennogram detection (GC-EAD) and Y-tube bioassays, respectively. Results showed that qualitative and quantitative diffe-rences between two odor profiles. Consistent electroantennographic response was observed for eight compounds from headspace collections, of which five synthetic compounds (3-carene, camphene, terpinolene, d-limonene, and (+)-pinene) were essential for significant preference or avoidance. Our results indicated that these compounds from L. barbarum flower buds could be effective candidates as oviposition attractant or repellents of Jaapiella sp., which would be helpful in the exploitation of new control agent to Jaapiella sp. in the field.

Time‐Traveling Larvae Ensure Survival in Small, Synchronously Fluctuating Populations

The tiny gall midge Contarinia vincetoxici is a long-lived insect, although its active life, as adult–egg–growing larva, is brief. Eggs are laid in young flower buds of a swallow-wort, which then develop into conspicuous galls harboring the developing larvae. Mature larvae fall to the ground where they enter a long dormancy, which may last over a decade. Gall populations were monitored in a large host plant patch system over 12 years. Despite poor colonization rates, low local gall densities, and large synchronous density fluctuations, almost all patches seemed to be continually inhabited due to the large hidden underground pool of time-traveling larvae. This photograph illustrates the article “Time-traveling larvae ensure survival in small, synchronously fluctuating populations” by Christer Solbreck and Olof Widenfalk published in Ecosphere. https://doi.org/10.1002/ecs2.3086.

Trichomes at the Base of the Petal Are Regulated by the Same Transcription Factors as Cotton Seed Fibers.

Many polypetalous plants have a constriction at the base of the petal that leaves a small gap that can provide entry into the young flower bud before the reproductive organs are fully developed. In cotton (Gossypium hirsutum L.), this gap is occluded by tufts of short unicellular trichomes superficially resembling the fibers found on cotton seeds. We are just beginning to understand the developmental regulation of the seed fibers and have previously characterized several MIXTA-like MYB transcription factors (TFs) that are critical for correct seed fiber development but know little about the molecular regulation of other types of cotton trichomes. Here, using RNAi or dominant suppression transgenic cotton lines and natural fiber mutants, we investigated the development and regulation of the petal base trichomes. Petal base trichomes and seed trichomes were also examined across several different species within and outside of the Malvoideae. We found that the petal base trichomes are regulated by the same MYB TFs as cotton seed fibers and, since they are more widely distributed across different taxa than the seed fibers, could have preceded them in the evolution of these important textile fibers produced by some cotton species.

Haploid Bio-Induction in Plant through Mock Sexual Reproduction.

SummaryHaploidization is invaluable for basic genetic research and crop breeding. The haploid bio-induction principle is an important topic that remains largely unexplored. In this study, both CenH3 RNAi and in vitro inhibition were used to simulate and induce haploids in allopolyploid crop. Notably, in vitro CenH3 inhibition showed that the results were much the same to that of RNAi in phenotype, chromosome behavior, microspore production, and haploid induction. Cytological analyses of RNAi and inhibitor-treated progenies revealed elimination of chromosomes, defective microspores with empty nuclei, thereby giving rise to pseudo male gametes, and haploid parthenogenesis induction. We found distinct defective empty microspores that were positively correlated with the decrease of CenH3 during RNAi manipulation. Investigation through both in vivo and in vitro studies revealed that haploidization was induced through the pseudo male gamete-mediated mock sexual reproduction. The present results provide insights for the haploid parthenogenesis induction process.

Genome divergence in Brassica rapa subspecies revealed by whole genome analysis on a doubled-haploid line of turnip

Subspecies of Brassica rapa are morphologically and genetically diverse, and include a variety of fresh vegetables grown worldwide. Among them, turnip (B. rapa subsp. rapa) produces a large bulbous taproot, and thus is primarily consumed as a root vegetable in Europe and Asia. In comparison to Chinese cabbage (B. rapa subsp. pekinensis), however, genetic analysis and breeding of turnip is hampered in practice due in part to scarcity of useful genetic resources. In this study, we produced a doubled haploid (DH) line of Ganghwa turnip, an heirloom specialty crop in Korea that is usually propagated by open pollination. Microspores were isolated from young flower buds of Ganghwa turnip, and shoots and roots were sequentially regenerated in vitro. Chromosome doubling was induced with the colchicine treatment, and verified by flow cytometry analysis. The G14 DH line displayed uniformity in overall morphology compared to heterogeneous commercial Ganghwa turnips. The whole genome of G14 was sequenced on an Illumina HiSeq 4000 platform, and the reads mapped onto the B. rapa reference genome identified 1,163,399 SNPs and 779,700 indels. Despite high similarity in overall genome sequence, turnips and Chinese cabbage have different compositions of transposable elements (TEs). In particular, long terminal repeat (LTR) retrotransposons are more enriched in turnips than in Chinese cabbage genomes, in which the gypsy elements are classified as major LTR sequences in the turnip genome. These findings suggest that subspecies-specific TE divergence is in part responsible for huge phenotypic variations observed within the same species.

Efficient isolation of protoplasts from freesia callus and its application in transient expression assays

Freesia hybrida is one of the most famous cut flower variety domesticated from wild species in Freesia genus, distinguished for its colorful flower colors and sweet scents. It has potential to be a model plant to unravel the molecular basis of plant specialized metabolites biosynthesis in family Iridaceae, which are crucial to genetically modify ornamental traits such as flower color and flower scents. However, the lengthy life cycle propagated from tissue culture and extremely low transformation rate severely decelerate functional characterization of genes through transgenic method in this iridaceous plant. Using calluses induced from young flower buds from one popular cultivar of Freesia hybrida, ‘Red River®’, an efficient protoplast isolation method was established and optimized, which was further employed in the gene transient expression system. Results demonstrated that the newly explored transient system was versatile and could be applied in numerous aspects, such as protein immunoblotting, enzyme activity, protein subcellular localization, protein–protein interaction, protein–DNA interaction and regulatory gene transregulation property assays. Moreover, calluses induced from another cultivar of Freesia hybrida, ‘Ambiance’ could also be used to isolate protoplasts for gene transient transfection analysis, indicating the wide applicability of this system. Results in this study provided new methods for the elucidation of gene function in Freesia in vivo, which might also be inspirational in other species with long life cycles or hardly to be transformed, such as plants in family Iridaceae. Freesia protoplast isolation and transfection methods were established and illustrated the utilization in protein immunoblotting, enzyme activity, subcellular localization, protein-protein or protein-DNA interaction, and investigation of transactivation capacities of regulators.

The Gene ANTHER DEHISCENCE REPRESSOR (ADR) Controls Male Fertility by Suppressing the ROS Accumulation and Anther Cell Wall Thickening in Arabidopsis

Male sterility in plants is caused by various stimuli such as hormone changes, stress, cytoplasmic alterations and nuclear gene mutations. The gene ANTHER DEHISCENCE REPRESSOR (ADR), which is involved in regulating male sterility in Arabidopsis, was functionally analyzed in this study. In ADR::GUS flowers, strong GUS activity was detected in the anthers of young flower buds but was low in mature flowers. ADR + GFP fusion proteins, which can be modified by N-myristoylation, were targeted to peroxisomes. Ectopic expression of ADR in transgenic Arabidopsis plants resulted in male sterility due to anther indehiscence. The defect in anther dehiscence in 35S::ADR flowers is due to the reduction of ROS accumulation, alteration of the secondary thickening in the anther endothecium and suppression of the expression of NST1 and NST2, which are required for anther dehiscence through regulation of secondary wall thickening in anther endothecial cells. This defect could be rescued by external application of hydrogen peroxide (H2O2). These results demonstrated that ADR must be N-myristoylated and targeted to the peroxisome during the early stages of flower development to negatively regulate anther dehiscence by suppressing ROS accumulation and NST1/NST2 expression.

Heat tolerance in garden roses

Roses generally experience a decrease in flower productivity and size in response to summer temperatures greater than 30°C that is common throughout the southern states of the USA. In College Station, Texas, the August mean and mean maximum summer temperatures can be 32 and 40°C, respectively. Under these extreme heat stress conditions, a large rose germplasm collection was evaluated for flowering intensity that takes into account both flower number and flower size. Of the 234 accessions evaluated, most showed moderate flowering during August and about 6% consistently had over 50% flower intensity in August. Experiments done in the growth chamber with potted roses containing young flower buds indicated that a 3-h heat shock (44°C) treatment resulted in decreased flower size and increased flower abscission. Consequently, this approach was used to assess the heat tolerance of 18 rose cultivars in this collection. The results indicated that field ratings of heat tolerance based on flower productivity were negatively correlated to flower abscission and leaf necrosis (r=-0.55* and r=-0.64**) and not correlated with leaf MTS (cell membrane thermostability) of the potted rose plants subjected to the heat shock treatment.