A LlWRKY33-LlHSFA4-LlCAT2 module confers resistance to Botrytis cinerea in lily.

Gray mold caused by Botrytis cinerea is one of the major threats in lily production. However, limited information is available about the underlying defense mechanism against B. cinerea in lily. Here, we characterized a nuclear-localized class A heat stress transcription factor (HSF)-LlHSFA4 from lily (Lilium longiflorum), which positively regulated the response to B. cinerea infection. LlHSFA4 transcript and its promoter activity were increased by B. cinerea infection in lily, indicating its involvement in the response to B. cinerea. Virus-induced gene silencing (VIGS) of LlHSFA4 impaired the resistance of lily to B. cinerea. Consistent with its role in lily, overexpression of LlHSFA4 in Arabidopsis (Arabidopsis thaliana) enhanced the resistance of transgenic Arabidopsis to B. cinerea infection. Further analysis showed that LlWRKY33 directly activated LlHSFA4 expression. We also found that both LlHSFA4 and LlWRKY33 positively regulated plant response to B. cinerea through reducing cell death and H2O2 accumulation and activating the expression of the reactive oxygen species (ROS) scavenging enzyme gene LlCAT2 (Catalase 2) by binding its prompter, which might contribute to reducing H2O2 accumulation in the infected area. Taken together, our data suggested that there may be a LlWRKY33-LlHSFA4-LlCAT2 regulatory module which confers B. cinerea resistance via reducing cell death and the ROS accumulation.

Lily LlHSFC2 coordinates with HSFAs to balance heat stress response and improve thermotolerance.

Heat stress transcription factors (HSFs) are core regulators of plant heat stress response. Much research has focused on class A and B HSFs, leaving those of class C relatively understudied. Here, we reported a lily (Lilium longiflorum) heat-inducible HSFC2 homology involved in thermotolerance. LlHSFC2 was located in the nucleus and cytoplasm and exhibited a repression ability by binding heat stress element. Overexpression of LlHSFC2 in Arabidopsis, tobacco (Nicotiana benthamiana), and lily, all increased the thermotolerance. Conversely, silencing of LlHSFC2 in lily reduced its thermotolerance. LlHSFC2 could interact with itself, or interact with LlHSFA1, LlHSFA2, LlHSFA3A, and LlHSFA3B of lily, AtHSFA1e and AtHSFA2 of Arabidopsis, and NbHSFA2 of tobacco. LlHSFC2 interacted with HSFAs to accelerate their transactivation ability and act as a transcriptional coactivator. Notably, compared with the separate LlHSFA3A overexpression, co-overexpression of LlHSFC2/LlHSFA3A further enhanced thermotolerance of transgenic plants. In addition, after suffering HS, the homologous interaction of LlHSFC2 was repressed, but its heterologous interaction with the heat-inducible HSFAs was promoted, enabling it to exert its co-activation effect for thermotolerance establishment and maintenance. Taken together, we identified that LlHSFC2 plays an active role in the general balance and maintenance of heat stress response by cooperating with HSFAs, and provided an important candidate for the enhanced thermotolerance breeding of crops and horticulture plants.

HD-Zip I protein LlHOX6 antagonizes homeobox protein LlHB16 to attenuate basal thermotolerance in lily.

Homeodomain-leucine zipper (HD-Zip) I transcription factors are crucial for plant responses to drought, salt, and cold stresses. However, how they are associated with thermotolerance remains mostly unknown. We previously demonstrated that lily (Lilium longiflorum) LlHB16 (HOMEOBOX PROTEIN 16) promotes thermotolerance, whereas the roles of other HD-Zip I members are still unclear. Here, we conducted a transcriptomic analysis and identified a heat-responsive HD-Zip I gene, LlHOX6 (HOMEOBOX 6). We showed that LlHOX6 represses the establishment of basal thermotolerance in lily. LlHOX6 expression was rapidly activated by high temperature, and its protein localized to the nucleus. Heterologous expression of LlHOX6 in Arabidopsis (Arabidopsis thaliana) and overexpression in lily reduced their basal thermotolerance. In contrast, silencing LlHOX6 in lily elevated basal thermotolerance. Cooverexpressing or cosilencing LlHOX6 and LlHB16 in vivo compromised their functions in modulating basal thermotolerance. LlHOX6 interacted with itself and with LlHB16, although heterologous interactions were stronger than homologous ones. Notably, LlHOX6 directly bounds DNA elements to repress the expression of the LlHB16 target genes LlHSFA2 (HEAT STRESS TRANSCRIPTION FACTOR A2) and LlMBF1c (MULTIPROTEIN BRIDGING FACTOR 1C). Moreover, LlHB16 activated itself to form a positive feedback loop, while LlHOX6 repressed LlHB16 expression. The LlHOX6-LlHB16 heterooligomers exhibited stronger DNA binding to compete for LlHB16 homooligomers, thus weakening the transactivation ability of LlHB16 for LlHSFA2 and LlMBF1c and reducing its autoactivation. Altogether, our findings demonstrate that LlHOX6 interacts with LlHB16 to limit its transactivation, thereby impairing heat stress responses in lily.

Low LdMYB12 expression contributes to petal spot deficiency in Lilium davidii var. unicolor.

Petal spots are widespread in plants, they are important for attracting pollinators and as economic traits in crop breeding. However, the genetic and developmental control of petal spots has seldom been investigated. To further clarify the development of petal spots formation, we performed comparative transcriptome analysis of Lilium davidii var. unicolor and Lilium davidii petals at the full-bloom stage. In comparison with the parental species L. davidii, petals of the lily variety L. davidii var. unicolor do not have the distinct anthocyanin spots. We show that among 7846 differentially expressed genes detected, LdMYB12 was identified as a candidate gene contributing to spot formation in lily petals. The expression level of LdMYB12 in the petals of L. davidii was higher than that in L. davidii var. unicolor petals. Moreover, overexpression of LdMYB12 led to the appearance of spots on the petals of L. davidii var. unicolor, accompanied by increased expression of anthocyanin synthesis-related genes. Taken together, these results indicate that abnormal expression of LdMYB12 contributes to petal spot deficiency in L. davidii var. unicolor.

A LlMYB305-LlC3H18-LlWRKY33 module regulates thermotolerance in lily.

The CCCH proteins play important roles in plant growth and development, hormone response, pathogen defense and abiotic stress tolerance. However, the knowledge of their roles in thermotolerance are scarce. Here, we identified a heat-inducible CCCH gene LlC3H18 from lily. LlC3H18 was localized in the cytoplasm and nucleus under normal conditions, while it translocated in the cytoplasmic foci and co-located with the markers of two messenger ribonucleoprotein (mRNP) granules, processing bodies (PBs) and stress granules (SGs) under heat stress conditions, and it also exhibited RNA-binding ability. In addition, LlC3H18 exhibited transactivation activity in both yeast and plant cells. In lily and Arabidopsis, overexpression of LlC3H18 damaged their thermotolerances, and silencing of LlC3H18 in lily also impaired its thermotolerance. Similarly, Arabidopsis atc3h18 mutant also showed decreased thermotolerance. These results indicated that the appropriate expression of C3H18 was crucial for establishing thermotolerance. Further analysis found that LlC3H18 directly bound to the promoter of LlWRKY33 and activated its expression. Besides, it was found that LlMYB305 acted as an upstream factor of LlC3H18 and activated its expression. In conclusion, we demonstrated that there may be a LlMYB305-LlC3H18-LlWRKY33 regulatory module in lily that is involved in the establishment of thermotolerance and finely regulates heat stress response.

A lily membrane-associated NAC transcription factor LlNAC014 is involved in thermotolerance via activation of the DREB2-HSFA3 module.

The NTL (NAC with transmembrane motif 1-like) transcription factors with a conserved transmembrane motif are members of the NAC family and are important in plant development and in response to stress. However, knowledge of their regulatory pathways is scarce, especially under heat stress. Here, we cloned and identified a novel lily (Lilium longiflorum) NTL gene, LlNAC014, that increases thermotolerance. High temperature repressed LlNAC014 expression but activated its protein. LlNAC014 contained a typical transmembrane motif at its far C-terminus and was normally located on membranes, but under heat stress it entered the nucleus as a transcription factor. LlNAC014 also has a transactivation domain at its C-terminus, and its active form, LlNAC014ΔC, could function as a trans-activator in both yeast and plant cells. LlNAC014ΔC overexpression in lily and Arabidopsis increased thermotolerance, and also caused growth defects; silencing LlNAC014 in lily decreased thermotolerance. LlNAC014ΔC could constitutively activate the heat stress response by inducing the expression of heat-responsive genes, some of which were dependent on the HSF (heat stress transcription factor) pathway. Further analysis showed that LlNAC014 was a direct regulator of the DREB2-HSFA3 module, and bound to the CTT(N7)AAG element in the promoters of LlHSFA3A, LlHSFA3B, and LlDREB2B to activate their expression. Thus, LlNAC014 increased thermotolerance by sensing high temperature and translocating to the nucleus to activate the DREB2-HSFA3 module.

The nature and genomic landscape of repetitive DNA classes in Chrysanthemum nankingense shows recent genomic changes.

BACKGROUND AND AIMS: Tandemly repeated DNA and transposable elements represent most of the DNA in higher plant genomes. High-throughput sequencing allows a survey of the DNA in a genome, but whole-genome assembly can miss a substantial fraction of highly repeated sequence motifs. Chrysanthemum nankingense (2n = 2x = 18; genome size = 3.07 Gb; Asteraceae), a diploid reference for the many auto- and allopolyploids in the genus, was considered as an ancestral species and serves as an ornamental plant and high-value food. We aimed to characterize the major repetitive DNA motifs, understand their structure and identify key features that are shaped by genome and sequence evolution. METHODS: Graph-based clustering with RepeatExplorer was used to identify and classify repetitive motifs in 2.14 millions of 250-bp paired-end Illumina reads from total genomic DNA of C. nankingense. Independently, the frequency of all canonical motifs k-bases long was counted in the raw read data and abundant k-mers (16, 21, 32, 64 and 128) were extracted and assembled to generate longer contigs for repetitive motif identification. For comparison, long terminal repeat retrotransposons were checked in the published C. nankingense reference genome. Fluorescent in situ hybridization was performed to show the chromosomal distribution of the main types of repetitive motifs. KEY RESULTS: Apart from rDNA (0.86 % of the total genome), a few microsatellites (0.16 %), and telomeric sequences, no highly abundant tandem repeats were identified. There were many transposable elements: 40 % of the genome had sequences with recognizable domains related to transposable elements. Long terminal repeat retrotransposons showed widespread distribution over chromosomes, although different sequence families had characteristic features such as abundance at or exclusion from centromeric or subtelomeric regions. Another group of very abundant repetitive motifs, including those most identified as low-complexity sequences (9.07 %) in the genome, showed no similarity to known sequence motifs or tandemly repeated elements. CONCLUSIONS: The Chrysanthemum genome has an unusual structure with a very low proportion of tandemly repeated sequences (~1.02 %) in the genome, and a high proportion of low-complexity sequences, most likely degenerated remains of transposable elements. Identifying the presence, nature and genomic organization of major genome fractions enables inference of the evolutionary history of sequences, including degeneration and loss, critical to understanding biodiversity and diversification processes in the genomes of diploid and polyploid Chrysanthemum, Asteraceae and plants more widely.

Lily WRKY factor LlWRKY22 promotes thermotolerance through autoactivation and activation of LlDREB2B.

Most of WRKY transcription factors play important roles in plant development, protection against disease, and response to abiotic stress; however, their roles in lily are largely unknown. Transcriptome analysis in lily (Lilium longiflorum) led to the identification and isolation of a WRKY-IIe gene, LlWRKY22, which was found to be activated at high temperature and play a positive role in thermotolerance regulation. LlWRKY22 expression was continuously activated by heat stress. We further found that LlWRKY22 protein localized to the nucleus and exhibited transactivation activity in both yeast and plant cells, and that its C terminus contributed to its transactivation activity. Meanwhile, overexpression of LlWRKY22 in lily improved thermotolerance and activated the expression of heat-related LlDREB2B gene; however, silencing of LlWRKY22 exerted the opposite effects. Further analysis revealed that LlWRKY22 directly activated the expression of LlDREB2B by binding to two tandem W-box elements on its promoter. Simultaneously, we also found that LlWRKY22 can directly bind its own promoter, thereby activating its own expression and forming a positive regulatory loop. Combined, our findings demonstrated that LlWRKY22 may be a new regulator of heat stress response and positively participates in the establishment of thermotolerance by activating itself and LlDREB2B.

Lily HD-Zip I Transcription Factor LlHB16 Promotes Thermotolerance by Activating LlHSFA2 and LlMBF1c.

HD-Zip I transcription factors play important roles in plant development and response to abiotic stresses; however, their roles in thermotolerance are largely unknown. Through transcriptome analysis in lily (Lilium longiflorum), we isolated and identified a HD-Zip I gene differentially expressed at high temperatures, LlHB16, which belongs to the ß2 subgroup and positively regulates thermotolerance. The expression of LlHB16 was rapidly and continuously activated by heat stress. LlHB16 protein localized to the nucleus and exhibited transactivation activity in both plant and yeast cells, and its C-terminus contributed to its transcriptional activity. Overexpressing LlHB16 in Arabidopsis and lily improved thermotolerance and activated the expression of heat-related genes in both plants, especially that of HSFA2 and MBF1c. In addition, LlHB16 overexpression in Arabidopsis also caused growth defects, delayed flowering and abscisic acid (ABA) insensitivity. Further analysis revealed that LlHB16 directly binds to the promoters of LlHSFA2 and LlMBF1c and activates their expressions. Similarly, the expression of AtHSFA2 and AtMBF1c was also elevated in LlHB16 transgenic Arabidopsis lines. Together, our findings demonstrate that LlHB16 participates in the establishment of thermotolerance involved in activating LlHSFA2 and LlMBF1c, and LlHB16 overexpression resulted in ABA insensitivity in transgenic plants, suggesting that LlHB16 links the basal heat-responsive pathway and ABA signal to collaboratively regulate thermotolerance.

Starch Degradation and Sucrose Accumulation of Lily Bulbs after Cold Storage.

Functional lilies are a group of edible lily cultivars with great potential for landscape application. Low-temperature storage can significantly improve their taste, but the knowledge of this process is largely unknown. In this study, we used the functional lilies 'Fly Shaohua' and 'Fly Tiancheng' as materials. Through physiological observation and transcriptome analysis during the bulbs' cold storage, it was found that the starch degradation and sucrose accumulation in bulbs contributed to taste improvement. After 60 d of cold storage, the sucrose accumulation was highest and the starch content was lower in the bulbs, suggesting this time-point was optimal for consumption. Accompanying the fluctuation of sucrose content during cold storage, the enzyme activities of sucrose phosphate synthase and sucrose synthase for sucrose synthesis were increased. Transcriptome analysis showed that many differentially expressed genes (DEGs) were involved in the starch and sucrose metabolism pathway, which might promote the conversion of starch to sucrose in bulbs. In addition, the DEGs involved in dormancy and stress response were also determined during cold storage, which might explain the decreased sucrose accumulation with extended storage time over 60 d due to the energy consumption for dormancy release. Taken together, our results indicated sucrose accumulation was a main factor in the taste improvement of lily bulbs after cold storage, which is attributable to the different gene expression of starch and sucrose metabolism pathways in this process.

Overexpression of a novel heat-inducible ethylene-responsive factor gene LlERF110 from Lilium longiflorum decreases thermotolerance.

AP2/ERF (APETALA2/ethylene-responsive factor) family transcription factors are involved in various plant-specific processes, especially in plant development and response to abiotic stress. However, their roles in thermotolerance are still largely unknown. In the current study, we identified a heat-inducible ERF member LlERF110 from Lilium longiflorum that was rapidly induced by high temperature. Its protein was localized in the nucleus, and transcriptional activation activity was observed in yeast and plant cells. In addition, LlERF110 was able to bind to GCC- and CGG-elements, but not to DRE-elements. Overexpression of LlERF110 conferred delayed bolting and bushy phenotype, with decreased thermotolerance accompanied by a disrupted ROS (reactive oxygen species) homeostasis in transgenic plants. The accumulation of LlERF110 may activate certain repressors related to heat stress response (HSR) and indirectly damage the normal expression of heat stress (HS)-protective genes such as AtHSFA2, which consequently leads to reduced thermotolerance. Our results implied that LlERF110 might function as a heat-inducible gene but may hinder the establishment of thermotolerance.

Chrysanthemum embryo development is negatively affected by a novel ERF transcription factor, CmERF12.

Embryo abortion often occurs during distant hybridization events. Apetala 2/ethylene-responsive factor (AP2/ERF) proteins are key transcription factor (TF) regulators of plant development and stress resistance, but their roles in hybrid embryo development are poorly understood. In this study, we isolated a novel AP2/ERF TF, CmERF12, from chrysanthemum and show that it adversely affects embryo development during distant hybridization. Transcriptome and real-time quantitative PCR demonstrate that CmERF12 is expressed at significantly higher levels in aborted ovaries compared with normal ones. CmERF12 localizes to the cell nucleus and contains a conserved EAR motif that mediates its transcription repressor function in yeast and plant cells. We generated artificial microRNA (amiR) CmERF12 transgenic lines of Chrysanthemum morifolium var. 'Yuhualuoying' and conducted distant hybridization with the wild-type tetraploid, Chrysanthemum nankingense, and found that CmERF12-knock down significantly promoted embryo development and increased the seed-setting rates during hybridization. The expression of various genes related to embryo development was up-regulated in developing ovaries from the cross between female amiR-CmERF12 C. morifolium var. 'Yuhualuoying'× male C. nankingense. Furthermore, CmERF12 directly interacted with CmSUF4, which is known to affect flower development and embryogenesis, and significantly reduced its ability to activate its target gene CmEC1 (EGG CELL1). Our study provides a novel method to overcome barriers to distant hybridization in plants and reveals the mechanism by which CmERF12 negatively affects chrysanthemum embryo development.

Transcriptome and Metabolome Analyses Provide Insights into the Stomium Degeneration Mechanism in Lily.

Lily (Lilium spp.) is a widely cultivated horticultural crop that has high ornamental and commercial value but also the serious problem of pollen pollution. However, mechanisms of anther dehiscence in lily remain largely unknown. In this study, the morphological characteristics of the stomium zone (SZ) from different developmental stages of 'Siberia' lily anthers were investigated. In addition, transcriptomic and metabolomic data were analyzed to identify the differentially expressed genes (DEGs) and secondary metabolites involved in stomium degeneration. According to morphological observations, SZ lysis occurred when flower buds were 6-8 cm in length and was completed in 9 cm. Transcriptomic analysis identified the genes involved in SZ degeneration, including those associated with hormone signal transduction, cell structure, reactive oxygen species (ROS), and transcription factors. A weighted co-expression network showed strong correlations between transcription factors. In addition, TUNEL (TdT-mediated dUTP nick-end labeling) assays showed that programmed cell death was important during anther SZ degeneration. Jasmonates might also have key roles in anther dehiscence by affecting the expression of the genes involved in pectin lysis, water transport, and cysteine protease. Collectively, the results of this study improve our understanding of anther dehiscence in lily and provide a data platform from which the molecular mechanisms of SZ degeneration can be revealed.

A Novel R2R3-MYB Gene LoMYB33 From Lily Is Specifically Expressed in Anthers and Plays a Role in Pollen Development.

Lily (Lilium spp.) is an important commercial flower crop, but its market popularity and applications are adversely affected by severe pollen pollution. Many studies have examined pollen development in model plants, but few studies have been conducted on flower crops such as lily. GAMYBs are a class of R2R3-MYB transcription factors and play important roles in plant development and biotic resistance; their functions vary in different pathways, and many of them are involved in anther development. However, their function and regulatory role in lily remain unclear. Here, the GAMYB homolog LoMYB33 was isolated and identified from lily. The open reading frame of LoMYB33 was 1620 bp and encoded a protein with 539 amino acids localized in the nucleus and cytoplasm. Protein sequence alignment showed that LoMYB33 contained a conserved R2R3 domain and three BOX motifs (BOX1, BOX2, and BOX3), which were unique to the GAMYB family. LoMYB33 had transcriptional activation activity, and its transactivation domain was located within 90 amino acids of the C-terminal. LoMYB33 was highly expressed during the late stages of anther development, especially in pollen. Analysis of the promoter activity of LoMYB33 in transgenic Arabidopsis revealed that the LoMYB33 promoter was highly activated in the pollen of stage 12 to 13 flowers. Overexpression of LoMYB33 in Arabidopsis significantly retarded growth; the excess accumulation of LoMYB33 also negatively affected normal anther development, which generated fewer pollen grains and resulted in partial male sterility in transgenic plants. Silencing of LoMYB33 in lily also greatly decreased the amount of pollen. Overall, our results suggested that LoMYB33 might play an important role in the anther development and pollen formation of lily.

A Novel Lateral Organ Boundary-domain Factor CmLBD2 Positively Regulates Pollen Development by Activating CmACOS5 in Chrysanthemum morifolium.

Male sterility, as a common reproductive characteristic in plants, plays an important role in breeding, in which pollen abortion is a key factor leading to male sterility. Here, based on a low expression level gene CmACOS5 in transcriptome of pollen abortive chrysanthemum, a new transcription factor CmLBD2 of the Lateral Organ Boundaries Domain family, which could bind the promoter of CmACOS5 by yeast one-hybrid library was screened. This study revealed the origin and expression pattern of CmLBD2 in chrysanthemum and verified the functions of two genes in pollen development by transgenic means. Inhibiting the expression of CmACOS5 or CmLBD2 can lead to a large reduction in pollen and even abortion in chrysanthemum. Using yeast one-/two-hybrid, electrophoretic mobility shift assays, and luciferase reporter assays, it was verified that CmLBD2 directly binds to the promoter of CmACOS5. These results suggest that LBD2 is a novel, key transcription factor regulating pollen development. This result will provide a new research background for enriching the function of LBD family proteins and also lay a new foundation for the breeding of male sterile lines and the mechanism of pollen development.

The transcription factor CmLEC1 positively regulates the seed-setting rate in hybridization breeding of chrysanthemum.

Distant hybridization is widely used to develop crop cultivars, whereas the hybridization process of embryo abortion often severely reduces the sought-after breeding effect. The LEAFY COTYLEDON1 (LEC1) gene has been extensively investigated as a central regulator of seed development, but it is far less studied in crop hybridization breeding. Here we investigated the function and regulation mechanism of CmLEC1 from Chrysanthemum morifolium during its seed development in chrysanthemum hybridization. CmLEC1 encodes a nucleic protein and is specifically expressed in embryos. CmLEC1's overexpression significantly promoted the seed-setting rate of the cross, while the rate was significantly decreased in the amiR-CmLEC1 transgenic chrysanthemum. The RNA-Seq analysis of the developing hybrid embryos revealed that regulatory genes involved in seed development, namely, CmLEA (late embryogenesis abundant protein), CmOLE (oleosin), CmSSP (seed storage protein), and CmEM (embryonic protein), were upregulated in the OE (overexpressing) lines but downregulated in the amiR lines vs. wild-type lines. Future analysis demonstrated that CmLEC1 directly activated CmLEA expression and interacted with CmC3H, and this CmLEC1-CmC3H interaction could enhance the transactivation ability of CmLEC1 for the expression of CmLEA. Further, CmLEC1 was able to induce several other key genes related to embryo development. Taken together, our results show that CmLEC1 plays a positive role in the hybrid embryo development of chrysanthemum plants, which might involve activating CmLEA's expression and interacting with CmC3H. This may be a new pathway in the LEC1 regulatory network to promote seed development, one perhaps leading to a novel strategy to not only overcome embryo abortion during crop breeding but also increase the seed yield.

Characterization and functional analysis of LoUDT1, a bHLH transcription factor related to anther development in the lily oriental hybrid Siberia (Lilium spp.).

Lily (Lilium spp.), with its beautiful flower, is an important horticultural crop and a popular ornamental plant, but because the abundant pollen pollutes the flowers and surroundings, its use is restricted. To solve this problem, the mechanism of pollen development in lily needs to be analyzed. However, the complex and delicate process of anther development in lily remains largely unknown. In this study, LoUDT1, a bHLH transcription factor (TF), was isolated and identified in lily. LoUDT1 was closely related to OsUDT1 of Oryza sativa and AtDYT1 of Arabidopsis. It was localized in the cytoplasm and nucleus and showed no transcriptional activation in yeast cells. LoUDT1 interacted with another bHLH TF, LoAMS, and the interaction depended on their BIF domains. LoUDT1 and LoAMS were both expressed in the anthers but showed different expression patterns. LoUDT1 was continuously expressed during the entire development of anthers, whereas LoAMS was only highly expressed early in anther development. With overexpression of LoUDT1 in Arabidopsis, normal anther development was affected and defective pollens were produced, which caused partial male sterility of transgenic plants. These defects depended on the level of LoUDT1 accumulation. By contrast, with the appropriate expression of LoUDT1 in a dyt1-3 mutant, normal pollen grains were produced, showing partial fertility. Thus, LoUDT1 might be a key regulator of anther development in lily. By further increasing the understanding of anther development, the results of this study can provide a theoretical basis for the molecular breeding of pollen-free lilies.

Genome-wide DNA mutations in Arabidopsis plants after multigenerational exposure to high temperatures.

BACKGROUND: Elevated temperatures can cause physiological, biochemical, and molecular responses in plants that can greatly affect their growth and development. Mutations are the most fundamental force driving biological evolution. However, how long-term elevations in temperature influence the accumulation of mutations in plants remains unknown. RESULTS: Multigenerational exposure of Arabidopsis MA (mutation accumulation) lines and MA populations to extreme heat and moderate warming results in significantly increased mutation rates in single-nucleotide variants (SNVs) and small indels. We observe distinctive mutational spectra under extreme and moderately elevated temperatures, with significant increases in transition and transversion frequencies. Mutation occurs more frequently in intergenic regions, coding regions, and transposable elements in plants grown under elevated temperatures. At elevated temperatures, more mutations accumulate in genes associated with defense responses, DNA repair, and signaling. Notably, the distribution patterns of mutations among all progeny differ between MA populations and MA lines, suggesting that stronger selection effects occurred in populations. Methylation is observed more frequently at mutation sites, indicating its contribution to the mutation process at elevated temperatures. Mutations occurring within the same genome under elevated temperatures are significantly biased toward low gene density regions, special trinucleotides, tandem repeats, and adjacent simple repeats. Additionally, mutations found in all progeny overlap significantly with genetic variations reported in 1001 Genomes, suggesting non-uniform distribution of de novo mutations through the genome. CONCLUSION: Collectively, our results suggest that elevated temperatures can accelerate the accumulation, and alter the molecular profiles, of DNA mutations in plants, thus providing significant insight into how environmental temperatures fuel plant evolution.

LlWRKY39 is involved in thermotolerance by activating LlMBF1c and interacting with LlCaM3 in lily (Lilium longiflorum).

WRKY transcription factors (TFs) are of great importance in plant responses to different abiotic stresses. However, research on their roles in the regulation of thermotolerance remains limited. Here, we investigated the function of LlWRKY39 in the thermotolerance of lily (Lilium longiflorum 'white heaven'). According to multiple alignment analyses, LlWRKY39 is in the WRKY IId subclass and contains a potential calmodulin (CaM)-binding domain. Further analysis has shown that LlCaM3 interacts with LlWRKY39 by binding to its CaM-binding domain, and this interaction depends on Ca2+. LlWRKY39 was induced by heat stress (HS), and the LlWRKY39-GFP fusion protein was detected in the nucleus. The thermotolerance of lily and Arabidopsis was increased with the ectopic overexpression of LlWRKY39. The expression of heat-related genes AtHSFA1, AtHSFA2, AtMBF1c, AtGolS1, AtDREB2A, AtWRKY39, and AtHSP101 was significantly elevated in transgenic Arabidopsis lines, which might have promoted an increase in thermotolerance. Then, the promoter of LlMBF1c was isolated from lily, and LlWRKY39 was found to bind to the conserved W-box element in its promoter to activate its activity, suggesting that LlWRKY39 is an upstream regulator of LlMBF1c. In addition, a dual-luciferase reporter assay showed that via protein interaction, LlCaM3 negatively affected LlWRKY39 in the transcriptional activation of LlMBF1c, which might be an important feedback regulation pathway to balance the LlWRKY39-mediated heat stress response (HSR). Collectively, these results imply that LlWRKY39 might participate in the HSR as an important regulator through Ca2+-CaM and multiprotein bridging factor pathways.