Regulating bulb dormancy release and flowering in lily through chemical modulation of intercellular communication.

Lily is a bulbous plant with an endogenous dormancy trait. Fine-tuning bulb dormancy release is still a challenge in the development of bulb storage technology. In this study, we identified three regulators of symplastic transport, 2,3-Butanedione oxime (BDM), N-Ethyl maleimide (NEM), and 2-Deoxy-D-glucose (DDG), that also regulate bulb dormancy release. We demonstrated that BDM and DDG inhibited callose synthesis between cells and promoted symplastic transport and soluble sugars in the shoot apical meristem (SAM), eventually accelerating bulb dormancy release and flowering in lilies. Conversely, NEM had the opposite effect. These three regulators can be flexibly applied to either accelerate or delay lily bulb dormancy release.

Epigenetic silencing of callose synthase by VIL1 promotes bud-growth transition in lily bulbs.

In plants, restoring intercellular communication is required for cell activity in buds during the growth transition from slow to fast growth after dormancy release. However, the epigenetic regulation of this phenomenon is far from understood. Here we demonstrate that lily VERNALIZATION INSENSITIVE 3-LIKE 1 (LoVIL1) confers growth transition by mediating plasmodesmata opening via epigenetic repression of CALLOSE SYNTHASE 3 (LoCALS3). Moreover, we found that a novel transcription factor, NUCLEAR FACTOR Y, SUBUNIT A7 (LoNFYA7), is capable of recruiting the LoVIL1-Polycomb Repressive Complex 2 (PRC2) and enhancing H3K27me3 at the LoCALS3 locus by recognizing the CCAAT cis-element (Cce) of its promoter. The LoNFYA7-LoVIL1 module serves as a key player in orchestrating the phase transition from slow to fast growth in lily bulbs. These studies also indicate that LoVIL1 is a suitable marker for the bud-growth-transition trait following dormancy release in lily cultivars.

GhbZIP30-GhCCCH17 module accelerates corm dormancy release by reducing endogenous ABA under cold storage in Gladiolus.

Gladiolus hybridus is one of the most popular flowers worldwide. However, its corm dormancy characteristic largely limits its off-season production. Long-term cold treatment (LT), which increases sugar content and reduces abscisic acid (ABA), is an efficient approach to accelerate corm dormancy release (CDR). Here, we identified a GhbZIP30-GhCCCH17 module that mediates the antagonism between sugars and ABA during CDR. We showed that sugars promoted CDR by reducing ABA levels in Gladiolus. Our data demonstrated that GhbZIP30 transcription factor directly binds the GhCCCH17 zinc finger promoter and activates its transcription, confirmed by yeast one-hybrid, dual-luciferase (Dual-LUC), chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assay (EMSA). GhCCCH17 is a transcriptional activator, and its nuclear localisation is altered by surcose and cytokinin treatments. Both GhbZIP30 and GhCCCH17 positively respond to LT, sugars, and cytokinin treatments. Silencing GhbZIP30 or GhCCCH17 resulted in delayed CDR by regulating ABA metabolic genes, while their overexpression promoted CDR. Taken together, we propose that the GhbZIP30-GhCCCH17 module is involved in cold- and glucose-induced CDR by regulating ABA metabolic genes.

Transcriptomic analysis reveals candidate genes associated with anther development in Lilium Oriental Hybrid 'Siberia'.

Lily (Lilium spp. and hybrids) is an important cut flower crop worldwide. Lily flowers have large anthers, which release a large amount of pollen that stains the tepals or clothing and thus can affect the commercial value of cut flowers. In this study, lily Oriental 'Siberia' was used to investigate the regulatory mechanism of lily anther development, which may provide information to prevent pollen pollution in the future. Based on the flower bud length, anther length and color, and anatomical observations, lily anther development was categorized into five stages: green (G), green-to-yellow 1 (GY1), green-to-yellow 2 (GY2), yellow (Y), and purple (P). Total RNA was extracted from the anthers at each stage for transcriptomic analysis. A total of 268.92-Gb clean reads were generated, and 81,287 unigenes were assembled and annotated. The number of differentially expressed genes (DEGs) and unique genes were largest for the pairwise comparison between the G and GY1 stages. The G and P samples were clustered separately, whereas the GY1, GY2, and Y samples were clustered together in scatter plots from a principal component analysis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses of DEGs detected in the GY1, GY2, and Y stages revealed that the pectin catabolic process, hormone levels, and phenylpropanoid biosynthesis were enriched. The DEGs associated with jasmonic acid biosynthesis and signaling were highly expressed at the early stages (G and GY1), whereas the DEGs associated with phenylpropanoid biosynthesis were mainly expressed in the intermediate stages (GY1, GY2, and Y). The DEGs involved in the pectin catabolic process were expressed at advanced stages (Y and P). Cucumber mosaic virus-induced gene silencing of LoMYB21 and LoAMS caused a strongly inhibited anther dehiscence phenotype, but without affecting the development of other floral organs. These results provide novel insights for understanding the regulatory mechanism of anther development in lily and other plants.

BASIC PENTACYSTEINE2 fine-tunes corm dormancy release in Gladiolus.

Bud dormancy is an important trait in geophytes that largely affects their flowering process and vegetative growth after dormancy release. Compared with seed dormancy, the regulation of bud dormancy is still largely unclear. Abscisic acid (ABA) acts as the predominant hormone that regulates the whole dormancy process. In Gladiolus (Gladiolus hybridus), cold storage promotes corm dormancy release (CDR) by repressing ABA biosynthesis and signaling. However, the mechanisms governing ABA-related processes during CDR via epigenetics are poorly understood. Here, we show that class I BASIC PENTACYSTEINE2, (GhBPC2) directly binds to 9-CIS-EPOXYCAROTENOID DIOXYGENASE (GhNCED) and ABA INSENSITIVE5 (GhABI5) loci and down-regulates their expression to accelerate CDR. During CDR, histone modifications change dramatically at the GhBPC2-binding loci of GhABI5 with an increase in H3K27me3 and a decrease in H3K4me3. GhBPC2 is involved in both H3K27me3 and H3K4me3 and fine-tunes GhABI5 expression by recruiting polycomb repressive complex 2 (PRC2) and the chromatin remodeling factor EARLY BOLTING IN SHORT DAYS (GhEBS). These results show GhBPC2 epigenetically regulates CDR in Gladiolus by mediating GhABI5 expression with PRC2 and GhEBS.

Ethylene Response Factor LlERF110 Mediates Heat Stress Response via Regulation of LlHsfA3A Expression and Interaction with LlHsfA2 in Lilies (Lilium longiflorum).

Heat stress seriously affects the quality of cut lily flowers. The ethylene response factors (ERFs) participate in heat stress response in many plants. In this study, heat treatment increased the production of ethylene in lily leaves, and exogenous ethylene treatment enhanced the heat resistance of lilies. LlERF110, an important transcription factor in the ethylene signaling pathway, was found in the high-temperature transcriptome. The coding region of LlERF110 (969 bp) encodes 322 amino acids and LlERF110 contains an AP2/ERF typical domain belonging to the ERF subfamily group X. LlERF110 was induced by ethylene and was expressed constitutively in all tissues. LlERF110 is localized in the nucleus and has transactivation activity. Virus-induced gene silencing of LlERF110 in lilies reduced the basal thermotolerance phenotypes and significantly decreased the expression of genes involved in the HSF-HSP pathway, such as LlHsfA2, LlHsfA3A, and LlHsfA5, which may activate other heat stress response genes; and LlHsp17.6 and LlHsp22, which may protect proteins under heat stress. LlERF110 could directly bind to the promoter of LlHsfA3A and activate its expression according to the yeast one hybrid and dual-luciferase reporter assays. LlERF110 interacts with LlHsfA2 in the nucleus according to BiFC and the yeast two-hybrid assays. In conclusion, these results indicate that LlERF110 plays an important role in the basal thermotolerance of lilies via regulation of the HSF-HSP pathway, which could be the junction of the heat stress response pathway and the ethylene signaling pathway.

Small HSPs play an important role in crosstalk between HSF-HSP and ROS pathways in heat stress response through transcriptomic analysis in lilies (Lilium longiflorum).

BACKGROUND: High temperature seriously limits the annual production of fresh cut lilies, which is one of the four major cut flowers in the global cut flower market. There were few transcriptomes focused on the gene expression of lilies under heat stress. In order to reveal the potential heat response patterns in bulbous plants and provide important genes for further genetic engineering techniques to improve thermotolerance of lily, RNA sequencing of lilies under heat treatments were conducted. RESULTS: In this study, seedlings of Lilium longiflorum 'White Heaven' were heat-treated at 37 °C for different lengths of time (0 h, 0.5 h, 1 h, 3 h, 6 h, and 12 h with a 12 h-light/12 h-dark cycle). The leaves of these lily seedlings were immediately collected after heat treatments and quickly put into liquid nitrogen for RNA sequencing. 109,364,486-171,487,430 clean reads and 55,044 unigenes including 21,608 differentially expressed genes (DEGs) (fold change ≥2) were obtained after heat treatment. The number of DEGs increased sharply during the heat treatments of 0.5 h-1 h and 1 h-3 h compared to that of other periods. Genes of the heat stress transcription factor (HSF) family and the small heat shock proteins (small HSPs, also known as HSP20) family responded to heat stress early and quickly. Compared to that of the calcium signal and hormone pathways, DEGs of the HSF-HSP pathway and reactive oxygen species (ROS) pathway were significantly and highly induced. Moreover, they had the similar expression pattern in response to heat stress. Small HSPs family genes were the major components in the 50 most highly induced genes at each heat stress treatment and involved in ROS pathway in the rapid response to heat stress. Furthermore, the barley stripe mosaic virus induced gene silencing (BSMV-VIGS) of LlHsfA2 caused a significantly reduced thermotolerance phenotype in Lilium longiflorum 'White Heaven', meanwhile decreasing the expression of small HSPs family genes and increasing the ROS scavenging enzyme ascorbate peroxidase (APX) genes, indicating the potential interplay between these two pathways. CONCLUSIONS: Based on our transcriptomic analysis, we provide a new finding that small HSPs play important roles in crosstalk between HSF-HSP and ROS pathways in heat stress response of lily, which also supply the groundwork for understanding the mechanism of heat stress in bulbous plants.

The Heat Stress Transcription Factor LlHsfA4 Enhanced Basic Thermotolerance through Regulating ROS Metabolism in Lilies (Lilium Longiflorum).

Heat stress severely affects the annual agricultural production. Heat stress transcription factors (HSFs) represent a critical regulatory juncture in the heat stress response (HSR) of plants. The HsfA1-dependent pathway has been explored well, but the regulatory mechanism of the HsfA1-independent pathway is still under-investigated. In the present research, HsfA4, an important gene of the HsfA1-independent pathway, was isolated from lilies (Lilium longiflorum) using the RACE method, which encodes 435 amino acids. LlHsfA4 contains a typical domain of HSFs and belongs to the HSF A4 family, according to homology comparisons and phylogenetic analysis. LlHsfA4 was mainly expressed in leaves and was induced by heat stress and H2O2 using qRT-PCR and GUS staining in transgenic Arabidopsis. LlHsfA4 had transactivation activity and was located in the nucleus and cytoplasm through a yeast one hybrid system and through transient expression in lily protoplasts. Over expressing LlHsfA4 in Arabidopsis enhanced its basic thermotolerance, but acquired thermotolerance was not achieved. Further research found that heat stress could increase H2O2 content in lily leaves and reduced H2O2 accumulation in transgenic plants, which was consistent with the up-regulation of HSR downstream genes such as Heat stress proteins (HSPs), Galactinol synthase1 (GolS1), WRKY DNA binding protein 30 (WRKY30), Zinc finger of Arabidopsis thaliana 6 (ZAT6) and the ROS-scavenging enzyme Ascorbate peroxidase 2 (APX2). In conclusion, these results indicate that LlHsfA4 plays important roles in heat stress response through regulating the ROS metabolism in lilies.

ABA and Bud Dormancy in Perennials: Current Knowledge and Future Perspective.

Bud dormancy is an evolved trait that confers adaptation to harsh environments, and affects flower differentiation, crop yield and vegetative growth in perennials. ABA is a stress hormone and a major regulator of dormancy. Although the physiology of bud dormancy is complex, several advancements have been achieved in this field recently by using genetics, omics and bioinformatics methods. Here, we review the current knowledge on the role of ABA and environmental signals, as well as the interplay of other hormones and sucrose, in the regulation of this process. We also discuss emerging potential mechanisms in this physiological process, including epigenetic regulation.

Antagonism between abscisic acid and gibberellin regulates starch synthesis and corm development in Gladiolus hybridus.

Understanding corm development in flower bulbs is of importance for securing the quality of cut flowers and propagation of commercial stocks. Gladiolus is one of the most popular bulb plants worldwide. Its corm development is characterized by starch accumulation. Previous research has shown that phytohormones (especially gibberellin (GA)) are involved in tuber development. However, the relationship between abscisic acid (ABA)/GA and starch during corm development remains unclear. To gain deeper insights into the biological process of corm development, we performed a detailed anatomical characterization of different stages of corm development and analyzed phytohormone levels. Our study showed that corm development is linked to hormones (ABA and GA) and carbohydrates (sucrose and starch). Exogenous hormone treatment and silencing of endogenous hormone biosynthesis genes indicated that ABA positively regulates corm development, while GA acts as an antagonist of ABA function. A sucrose synthase gene (GhSUS2) was shown to be involved in the antagonism between ABA and GA. GhSUS2 was upregulated by ABA and downregulated by GA. The increase in the transcript level of GhSUS2 coincided with the development of corm/cormels. Silencing of GhSUS2 repressed corm development and starch accumulation. In conclusion, we propose that GhSUS2, an essential enzyme in sucrose degradation, is differentially regulated by ABA and GA and controls corm development in Gladiolus.

Compact dual-gas sensor for simultaneous measurement of atmospheric methane, and water vapor using a 3.38 µm antimonide-distributed feedback laser diode.

A simple, compact sensor involving a continuous-wave 3.38 µm distributed feedback laser in combination with a novel compact dense-pattern multipass cell was demonstrated for simultaneous measurement of atmospheric methane and water vapor. The calibration-free direct absorption spectroscopy approach was adopted for data generation and processing. Allan deviation analysis indicates that minimum detection limits (1σ) of 11.0 ppb for CH4 and 100 ppm for H2O were achieved with a 1-s integration time at an optimum pressure of 50 Torr. Atmospheric environmental mixing ratios of these two gases were recorded and analyzed. This newly developed mid-infrared dual-gas sensor is very suitable for trace gas sensing in weight-limited unmanned aerial vehicle- or balloon-embedded field observations.

Alternative Splicing Provides a Mechanism to Regulate LlHSFA3 Function in Response to Heat Stress in Lily.

Heat stress transcription factors (HSFs) are central regulators of plant responses to heat stress. Their heat-induced transcriptional regulation has been extensively studied; however, their posttranscriptional and posttranslational regulation is poorly understood. In a previous study, we established that there were at least two HSFA3 homologs, LlHSFA3A and LlHSFA3B, in lily (Lilium spp.) and that these genes played distinct roles in thermotolerance. Here, we demonstrate that LlHSFA3B is alternatively spliced under heat stress to produce the heat-inducible splice variant LlHSFA3B-III We further show that LlHSFA3B-III protein localizes in the cytoplasm and nucleus, has no transcriptional activity, and specifically disturbs the protein interactions of intact HSFA3 orthologs LlHSFA3A-I and LlHSFA3B-I. Heterologous expression of LlHSFA3B-III in Arabidopsis (Arabidopsis thaliana) and Nicotiana benthamiana increased plant tolerance of salt and prolonged heat at 40°C, yet reduced plant tolerance of acute heat shock at 45°C. Conversely, heterologous expression of LlHSFA3A-I caused opposing phenotypes, which were substantially ameliorated by coexpression of LlHSFA3B-III LlHSFA3B-III interacted with LlHSFA3A-I to limit its transactivation function and temper the function of LlHSFA3A-I, thus reducing the adverse effects of excessive LlHSFA3A-I accumulation. Based on these observations, we propose a regulatory mechanism of HSFs involving heat-inducible alternative splicing and protein interaction, which might be used in strategies to promote thermotolerance and attenuate the heat stress response in crop plants.

GhTCP19 Transcription Factor Regulates Corm Dormancy Release by Repressing GhNCED Expression in Gladiolus.

Dormancy is one of the least understood phenomena in plant biology; however, bud/corm dormancy is an important economic trait in agricultural/horticultural breeding. In this study, we isolated an ABA biosynthesis gene, GhNCED, from the transcriptome database of corm dormancy release (CDR), and characterized its negative role in regulating CDR. To understand transcriptional regulation of GhNCED, yeast one-hybrid screening was conducted and GhTCP19 was identified and shown to regulate GhNCED expression directly. An in planta assay showed that GhTCP19 negatively regulates GhNCED expression. GhTCP19 is dramatically induced by exogenous cytokinins (CKs) and is induced during CDR. Silencing of GhTCP19 in dormant cormels delayed CDR, resulting in higher expression of GhNCED and ABA levels. Meanwhile, endogenous CK biosynthesis and signaling were inhibited in GhTCP19-silenced cormels. Taken together, our results reveal that GhTCP19 is a positive regulator of the CDR process by repressing expression of an ABA biosynthesis gene (GhNCED), promoting CK biosynthesis (GhIPT) and signal transduction (GhARR) as well as inducing cyclin genes. This study expands our knowledge on CDR which is mediated by TCP family members.

GhNAC83 inhibits corm dormancy release by regulating ABA signaling and cytokinin biosynthesis in Gladiolus hybridus.

Corm dormancy is an important trait for breeding in many bulb flowers, including the most cultivated Gladiolus hybridus. Gladiolus corms are modified underground stems that function as storage organs and remain dormant to survive adverse environmental conditions. Unlike seed dormancy, not much is known about corm dormancy. Here, we characterize the mechanism of corm dormancy release (CDR) in Gladiolus. We identified an important ABA (abscisic acid) signaling regulator, GhPP2C1 (protein phosphatase 2C1), by transcriptome analysis of CDR. GhPP2C1 expression increased during CDR, and silencing of GhPP2C1 expression in dormant cormels delayed CDR. Furthermore, we show that GhPP2C1 expression is directly regulated by GhNAC83, which was identified by yeast one-hybrid library screening. In planta assays show that GhNAC83 is a negative regulator of GhPP2C1, and silencing of GhNAC83 promoted CDR. As expected, silencing of GhNAC83 decreased the ABA level, but also dramatically increased cytokinin (CK; zeatin) content in cormels. Binding assays demonstrate that GhNAC83 associates with the GhIPT (ISOPENTENYLTRANSFERASE) promoter and negatively regulates zeatin biosynthesis. Taken together, our results reveal that GhNAC83 promotes ABA signaling and synthesis, and inhibits CK biosynthesis pathways, thereby inhibiting CDR. These findings demonstrate that GhNAC83 regulates the ABA and CK pathways, and therefore controls corm dormancy.

A Canonical DREB2-Type Transcription Factor in Lily Is Post-translationally Regulated and Mediates Heat Stress Response.

Based on studies of monocot crops and eudicot model plants, the DREB2 class of AP2-type transcription factor has been shown to play crucial roles in various abiotic stresses, especially in the upstream of the heat stress response; however, research on DREB2s has not been reported in non-gramineous monocot plants. Here, we identified a novel DREB2 (LlDREB2B) from lily (Lilium longiflorum), which was homologous to AtDREB2A of Arabidopsis, OsDREB2B of rice, and ZmDREB2A of maize. LlDREB2B was induced by heat, cold, salt, and mannitol stress, and its protein had transcriptional activity, was located in the nucleus, was able to bind to the dehydration-responsive element (DRE), and participated in the heat-responsive pathway of HsfA3. Overexpression of LlDREB2B in Arabidopsis activated expression of downstream genes and improved thermotolerance. LlDREB2B was not regulated by alternative splicing; functional transcripts accumulated under either normal or heat-stress conditions. A potential PEST sequence was predicted in LlDREB2B, but the stability of the LlDREB2B protein was not positively affected when the predicated PEST sequence was deleted. Further analysis revealed that the predicated PEST sequence lacked a SBC or SBC-like motif allowing interaction with BPMs and required for negative regulation. Nevertheless, LlDREB2B was still regulated at the post-translational level by interaction with AtDRIP1 and AtDRIP2 of Arabidopsis. In addition, LlDREB2B also interacted with AtRCD1 and LlRCD1 via a potential RIM motif located at amino acids 215-245. Taken together, our results show that LlDREB2B participated in the establishment of thermotolerance, and its regulation was different from that of the orthologs of gramineous and eudicot plants.

Two-dimensional analysis provides molecular insight into flower scent of Lilium 'Siberia'.

Lily is a popular flower around the world not only because of its elegant appearance, but also due to its appealing scent. Little is known about the regulation of the volatile compound biosynthesis in lily flower scent. Here, we conducted an approach combining two-dimensional analysis and weighted gene co-expression network analysis (WGCNA) to explore candidate genes regulating flower scent production. In the approach, changes of flower volatile emissions and corresponding gene expression profiles at four flower developmental stages and four circadian times were both captured by GC-MS and RNA-seq methods. By overlapping differentially-expressed genes (DEGs) that responded to flower scent changes in flower development and circadian rhythm, 3,426 DEGs were initially identified to be candidates for flower scent production, of which 1,270 were predicted as transcriptional factors (TFs). The DEGs were further correlated to individual flower volatiles by WGCNA. Finally, 37, 41 and 90 genes were identified as candidate TFs likely regulating terpenoids, phenylpropanoids and fatty acid derivatives productions, respectively. Moreover, by WGCNA several genes related to auxin, gibberellins and ABC transporter were revealed to be responsible for flower scent production. Thus, this strategy provides an important foundation for future studies on the molecular mechanisms involved in floral scent production.

Overexpression of lily HsfA3s in Arabidopsis confers increased thermotolerance and salt sensitivity via alterations in proline catabolism.

Although HsfA3 (heat-stress transcription factor A3) is well characterized in heat stress, its roles in other abiotic stresses are less clear. In this study, we isolated two homologous HsfA3 genes, LlHsfA3A and LlHsfA3B, from lily (Lilium longiflorum). Both genes were induced by heat stress, but not by salt stress. Overexpressing LlHsfA3A in Arabidopsis enhanced its basal and acquired thermotolerance, while overexpressing LlHsfA3B just enhanced its acquired thermotolerance. In both cases, overexpressing plants showed hypersensitivity to salt stress, and a lack of sucrose exacerbated this salt sensitivity. Using a transient assay, the opposite effects were observed in lily. Further analysis revealed that either LlHsfA3A or LlHsfA3B overexpression altered normal proline accumulation. During heat treatments, proline increased in wild-type Arabidopsis plants, but no such increase was detected in transgenic plants that showed better basal or acquired thermotolerance. Under salt stress, proline accumulation was decreased in Arabidopsis and lily with the overexpression of LlHsfA3A or LlHsfA3B. Proline catabolism was activated by overexpression, and both LlHsfA3A and LlHsfA3B affected proline oxidation via regulation of AtbZIP11, AtbZIP44, and AtbZIP53 to activate AtproDH1 and AtproDH2 in transgenic Arabidopsis. Taken together, our results suggested that overexpression of LlHsfA3A or LlHsfA3B caused opposite effects on heat and salt tolerance, which may implicate proline catabolism.

ADP-glucose pyrophosphorylase gene plays a key role in the quality of corm and yield of cormels in gladiolus.

Starch is the main storage compound in underground organs like corms. ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in storage organs and is likely one of the most important determinant of sink strength. Here, we identify an AGPase gene (GhAGPS1) from gladiolus. The highest transcriptional levels of GhAGPS1 were observed in cormels and corms. Transformation of GhAGPS1 into Arabidopsis rescued the phenotype of aps1 mutant. Silencing GhAGPS1 in gladiolus corms by virus-induced gene silencing (VIGS) decreased the transcriptional levels of two genes and starch content. Transmission electron microscopy analyses of leaf and corm sections confirmed that starch biosynthesis was inhibited. Corm weight and cormel number reduced significantly in the silenced plants. Taken together, these results indicate that inhibiting the expression of AGPase gene could impair starch synthesis, which results in the lowered corm quality and cormel yield in gladiolus.

Cloning and characterization of a novel Gladiolus hybridus AFP family gene (GhAFP-like) related to corm dormancy.

Abscisic acid (ABA) is an important phytohormone controlling seed dormancy. AFPs (ABA INSENSITIVE FIVE BINDING PROTEINS) are reported to be negative regulators of the ABA signaling pathway. The involvement of AFPs in dormant vegetative organs remains poorly understood. Here, we isolated and characterized a novel AFP family member from Gladiolus dormant cormels, GhAFP-like, containing three conserved domains of the AFP family. Quantitative PCR analysis revealed that GhAFP-like was expressed in dormant organs and its expression was down-regulated along with corm storage. GhAFP-like was verified to be a nuclear-localized protein. Overexpressing GhAFP-like in Arabidopsis thaliana not only showed weaker seed dormancy with insensitivity to ABA, but also changed the expression of some ABA related genes. In addition, a primary root elongation assay showed GhAFP-like may involve in auxin signaling response. The results in this study indicate that GhAFP-like acts as a negative regulator in ABA signaling and is related to dormancy.

Gladiolus hybridus ABSCISIC ACID INSENSITIVE 5 (GhABI5) is an important transcription factor in ABA signaling that can enhance Gladiolus corm dormancy and Arabidopsis seed dormancy.

The phytohormone abscisic acid (ABA) regulates plant development and is crucial for abiotic stress response. In this study, cold storage contributes to reducing endogenous ABA content, resulting in dormancy breaking of Gladiolus. The ABA inhibitor fluridone also promotes germination, suggesting that ABA is an important hormone that regulates corm dormancy. Here, we report the identification and functional characterization of the Gladiolus ABI5 homolog (GhABI5), which is a basic leucine zipper motif transcriptional factor (TF). GhABI5 is expressed in dormant vegetative organs (corm, cormel, and stolon) as well as in reproductive organs (stamen), and it is up-regulated by ABA or drought. Complementation analysis reveals that GhABI5 rescues the ABA insensitivity of abi5-3 during seed germination and induces the expression of downstream ABA response genes in Arabidopsis thaliana (EM1, EM6, and RD29B). Down-regulation of GhABI5 in dormant cormels via virus induced gene silence promotes sprouting and reduces the expression of downstream genes (GhLEA and GhRD29B). The results of this study reveal that GhABI5 regulates bud dormancy (vegetative organ) in Gladiolus in addition to its well-studied function in Arabidopsis seeds (reproductive organ).