Transcription factor OsWRKY11 induces rice heading at low concentrations but inhibits rice heading at high concentrations.

The heading date of rice is a crucial agronomic characteristic that influences its adaptability to different regions and its productivity potential. Despite the involvement of WRKY transcription factors in various biological processes related to development, the precise mechanisms through which these transcription factors regulate the heading date in rice have not been well elucidated. The present study identified OsWRKY11 as a WRKY transcription factor which exhibits a pivotal function in the regulation of the heading date in rice through a comprehensive screening of a clustered regularly interspaced palindromic repeats (CRISPR) ‒ CRISPR-associated nuclease 9 mutant library that specifically targets the WRKY genes in rice. The heading date of oswrky11 mutant plants and OsWRKY11-overexpressing plants was delayed compared with that of the wild-type plants under short-day and long-day conditions. Mechanistic investigation revealed that OsWRKY11 exerts dual effects on transcriptional promotion and suppression through direct and indirect DNA binding, respectively. Under normal conditions, OsWRKY11 facilitates flowering by directly inducing the expression of OsMADS14 and OsMADS15. The presence of elevated levels of OsWRKY11 protein promote formation of a ternary protein complex involving OsWRKY11, Heading date 1 (Hd1), and Days to heading date 8 (DTH8), and this complex then suppresses the expression of Ehd1, which leads to a delay in the heading date. Subsequent investigation revealed that a mild drought condition resulted in a modest increase in OsWRKY11 expression, promoting heading. Conversely, under severe drought conditions, a significant upregulation of OsWRKY11 led to the suppression of Ehd1 expression, ultimately causing a delay in heading date. Our findings uncover a previously unacknowledged mechanism through which the transcription factor OsWRKY11 exerts a dual impact on the heading date by directly and indirectly binding to the promoters of target genes.

Transcription factor OsWRKY72 controls rice leaf angle by regulating LAZY1-mediated shoot gravitropism.

Leaf angle is a major trait of ideal architecture, which is considered to influence rice (Oryza sativa) cultivation and grain yield. Although a few mutants with altered rice leaf inclination angles have been reported, the underlying molecular mechanism remains unclear. In this study, we showed that a WRKY transcription factor gene, OsWRKY72, was highly expressed in the leaf sheath and lamina joint. Phenotypic analyses showed that oswrky72 mutants had smaller leaf angles than the wild type, while OsWRKY72 overexpression lines exhibited an increased leaf angle. This observation suggests that OsWRKY72 functions as a positive regulator, promoting the enlargement of the leaf angle. Our bioinformatics analysis identified LAZY1 as the downstream gene of OsWRKY72. Electrophoretic mobility shift assays and dual-luciferase analysis revealed that OsWRKY72 directly inhibited LAZY1 by binding to its promoter. Moreover, knocking out OsWRKY72 enhanced shoot gravitropism, which contrasted with the phenotype of lazy1 plants. These results imply that OsWRKY72 regulates the leaf angle through gravitropism by reducing the expression of LAZY1. In addition, OsWRKY72 could directly regulate the expression of other leaf angle-related genes such as FLOWERING LOCUS T-LIKE 12 (OsFTL12) and WALL-ASSOCIATED KINASE 11 (OsWAK11). Our study indicates that OsWRKY72 contributes positively to the expansion of the leaf angle by interfering with shoot gravitropism in rice.

Rice transcriptional repressor OsTIE1 controls anther dehiscence and male sterility by regulating JA biosynthesis.

Proper anther dehiscence is essential for successful pollination and reproduction in angiosperms, and jasmonic acid (JA) is crucial for the process. However, the mechanisms underlying the tight regulation of JA biosynthesis during anther development remain largely unknown. Here, we demonstrate that the rice (Oryza sativa L.) ethylene-response factor-associated amphiphilic repression (EAR) motif-containing protein TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTORS (TCP) INTERACTOR CONTAINING EAR MOTIF PROTEIN1 (OsTIE1) tightly regulates JA biosynthesis by repressing TCP transcription factor OsTCP1/PCF5 during anther development. The loss of OsTIE1 function in Ostie1 mutants causes male sterility. The Ostie1 mutants display inviable pollen, early stamen filament elongation, and precocious anther dehiscence. In addition, JA biosynthesis is activated earlier and JA abundance is precociously increased in Ostie1 anthers. OsTIE1 is expressed during anther development, and OsTIE1 is localized in nuclei and has transcriptional repression activity. OsTIE1 directly interacts with OsTCP1, and overexpression of OsTCP1 caused early anther dehiscence resembling that of Ostie1. JA biosynthesis genes including rice LIPOXYGENASE are regulated by the OsTIE1-OsTCP1 complex. Our findings reveal that the OsTIE1-OsTCP1 module plays a critical role in anther development by finely tuning JA biosynthesis and provide a foundation for the generation of male sterile plants for hybrid seed production.

Multi-omics analysis reveals the evolutionary origin of diterpenoid alkaloid biosynthesis pathways in Aconitum.

Diterpenoid alkaloids (DAs) have been often utilized in clinical practice due to their analgesic and anti-inflammatory properties. Natural DAs are prevalent in the family Ranunculaceae, notably in the Aconitum genus. Nevertheless, the evolutionary origin of the biosynthesis pathway responsible for DA production remains unknown. In this study, we successfully assembled a high-quality, pseudochromosome-level genome of the DA-rich species Aconitum vilmorinianum (A. vilmorinianum) (5.76 Gb). An A. vilmorinianum-specific whole-genome duplication event was discovered using comparative genomic analysis, which may aid in the evolution of the DA biosynthesis pathway. We identified several genes involved in DA biosynthesis via integrated genomic, transcriptomic, and metabolomic analyses. These genes included enzymes encoding target ent-kaurene oxidases and aminotransferases, which facilitated the activation of diterpenes and insertion of nitrogen atoms into diterpene skeletons, thereby mediating the transformation of diterpenes into DAs. The divergence periods of these genes in A. vilmorinianum were further assessed, and it was shown that two major types of genes were involved in the establishment of the DA biosynthesis pathway. Our integrated analysis offers fresh insights into the evolutionary origin of DAs in A. vilmorinianum as well as suggestions for engineering the biosynthetic pathways to obtain desired DAs.

Functions of WRKYs in plant growth and development.

As sessile organisms, plants must overcome various stresses. Accordingly, they have evolved several plant-specific growth and developmental processes. These plant processes may be related to the evolution of plant-specific protein families. The WRKY transcription factors originated in eukaryotes and expanded in plants, but are not present in animals. Over the past two decades, there have been many studies on WRKYs in plants, with much of the research concentrated on their roles in stress responses. Nevertheless, recent findings have revealed that WRKYs are also required for seed dormancy and germination, postembryonic morphogenesis, flowering, gametophyte development, and seed production. Thus, WRKYs may be important for plant adaptations to a sessile lifestyle because they simultaneously regulate stress resistance and plant-specific growth and development.

Molecular characterization reveals that OsSAPK3 improves drought tolerance and grain yield in rice.

BACKGROUND: Many data suggest that the sucrose non-fermenting 1-related kinases 2 (SnRK2s) are very important to abiotic stress for plants. In rice, these kinases are known as osmotic stress/ABA-activated protein kinases (SAPKs). Osmotic stress/ABA-activated protein kinase 3 (OsSAPK3) is a member of SnRK2II in rice, but its function is still unclear. RESULTS: The expression of OsSAPK3 was up regulated by drought, NaCl, PEG and ABA. OsSAPK3 mutated seedings (sapk3-1 and sapk3-2) showed reduced hypersensitivity to exogenous ABA. In addition, under drought conditions, sapk3-1 and sapk3-2 showed more intolerance to drought, including decreased survival rate, increased water loss rate, increased stomatal conductance and significantly decreased expression levels of SLAC1 and SLAC7. Physiological and metabolic analyses showed that OsSAPK3 might play an important role in drought stress signaling pathway by affecting osmotic adjustment and osmolytes, ROS detoxification and expression of ABA dependent and independent dehydration-responsive genes. All gronomic traits analyses demonstrated that OsSAPK3 could improve rice yield by affecting the regulation of tiller numbers and grain size. CONCLUSION: OsSAPK3 plays an important role in both ABA-dependent and ABA-independent drought stress responses. More interestingly, OsSAPK3 could improve rice yield by indirectly regulating tiller number and grain size. These findings provide new insight for the development of drought-resistant rice.

Jasmonate-regulated seed germination and crosstalk with other phytohormones.

Seed plants have evolved mechanisms that maintain the dormancy of mature seeds until the time is appropriate for germination. Seed germination is a critical step in the plant life cycle, and it is an important trait in relation to agricultural production. The process is precisely regulated by various internal and external factors, and in particular by diverse endogenous hormones. Jasmonates (JAs) are one of the main plant hormones that mediate stress responses, and recent studies have provided evidence of their inhibitory effects on seed germination. In this review, we summarize our current understanding of the molecular mechanisms underlying the regulatory roles of JAs during the seed germination stage. We describe the crosstalk between JA and other phytohormones that influence seed germination, such as abscisic acid and gibberellic acid.

Identification and fine mapping of rtms1-D, a gene responsible for reverse thermosensitive genic male sterility from Diannong S-1X.

Thermosensitive genic male sterility (TGMS) has been widely used in two-line hybrid rice breeding. Due to hybrid seed production being highly affected by changeable environments, its application scope is limited to some extent. Thus, it is of great importance to identify potential TGMS genes in specific rice varieties. Here, Diannong S-1 xuan (DNS-1X), a reverse TGMS (RTGMS) japonica male sterile line, was identified from Diannong S-1. Genetic analysis showed that male sterility was tightly controlled by a single recessive gene, which was supported by the phenotype of the F1 and F2:3 populations derived from the cross between DNS-1X and Yunjing 26 (YJ26). Combining simple sequence repeat (SSR) markers and bulked segregation analysis (BSA), we identified a 215 kb region on chromosome 10 as a candidate reverse TGMS region, which was designated as rtms1-D. It was narrower than the previously reported RTGMS genes rtms1 and tms6(t). The fertility conversion detected in the natural environment showed that DNS-1X was sterile below 28-30 °C; otherwise, it was fertile. Histological analysis further indicated that the pollen abortion was occurred in the young microspore stage. This study will provide new resources for two-line hybrid rice and pave the way for molecular breeding of RTGMS lines.

Metabolomics and microbiome reveal potential root microbiota affecting the alkaloidal metabolome in Aconitum vilmorinianum Kom.

BACKGROUND: The plant microbiome is vital for plant health, fitness, and productivity. Interestingly, plant metabolites and the plant microbiome can influence each other. The combination of metabolomics and microbiome may reveal the critical links between the plant and its microbiome. It is of great significance to agricultural production and human health, especially for Chinese medicine research. Aconitum vilmorinianum Kom. is a herb with alkaloid activities, and its roots are the raw material for some Chinese medicines. Former studies have investigated alkaloidal metabolites and antibacterial activities of endophytes in A. vilmorinianum roots. However, there are limited reports on the root microbiota that can influence the alkaloidal metabolome of A. vilmorinianum. RESULTS: This research used ultra performance liquid chromatography-tandem mass spectrometry technology and high-throughput sequencing to examine the alkaloidal metabolome, bacterial microbiota, and fungal microbiota in A. vilmorinianum roots at two different sites in China. The results revealed that the samples from the two sites were rich in distinct alkaloidal metabolites and recruited significantly different root microbiota. Based on bioinformatics analysis, we found the potential bacterial and fungal microbiota impacting the alkaloidal metabolome in A. vilmorinianum. CONCLUSION: Our findings reveal the composition of the alkaloidal metabolome, bacterial root microbiota, and fungal root microbiota in A. vilmorinianum roots at two different sites. Potential root microbiota that can influence the alkaloidal metabolome of A. vilmorinianum are indicated. This study provides a strategy for the cultivation and research of A. vilmorinianum and other Chinese herbs.

Phytochrome B regulates jasmonic acid-mediated defense response against Botrytis cinerea in Arabidopsis.

The phytochrome B mediated light signaling integrates with various phytohormone signalings to control plant immune response. However, it is still unclear whether phyB-mediated light signaling has an effect on the biosynthesis of jasmonate during plant defense response against Botrytis cinerea. In this study, we demonstrated that phyB-mediated light signaling has a role in this process. Initially, we confirmed that phyb plants were obviously less resistant to B. cinerea while phyB overexpressing plants showed significantly enhanced resistance. We also found that the expression of numerous JA biosynthesis genes was promoted upon treatment with red or white light when compared to that of darkness, and that this promotion is dependent on phyB. Consistent with the gene expression results, phyb plants accumulated reduced pool of JA-Ile, indicating that phyB-mediated light signaling indeed increased JA biosynthesis. Further genetic analysis showed that light-mediated JAZ9 degradation and phyB-enhanced resistance were dependent on the receptor COI1, and that pif1/3/4/5 (pifq) can largely rescue the severe symptom of phyb. Taken together, our study demonstrates that phyB may participate in plant defense against B. cinerea through the modulation of the biosynthesis of JA.

Natural alleles of a uridine 5'-diphospho-glucosyltransferase gene responsible for differential endosperm development between upland rice and paddy rice.

Traditional upland rice generally exhibits insufficient grains resulting from abnormal endosperm development compared to paddy rice. However, the underlying molecular mechanism of this trait is poorly understood. Here, we cloned the uridine 5'-diphospho (UDP)-glucosyltransferase gene EDR1 (Endosperm Development in Rice) responsible for differential endosperm development between upland rice and paddy rice by performing quantitative trait loci analysis and map-based cloning. EDR1 was highly expressed in developing seeds during grain filling. Natural variations in EDR1 significantly reduced the UDP-glucosyltransferase activity of EDR1YZN compared to EDR1YD1 , resulting in abnormal endosperm development in the near-isogenic line, accompanied by insufficient grains and changes in grain quality. By analyzing the distribution of the two alleles EDR1YD1 and EDR1YZN among diverse paddy rice and upland rice varieties, we discovered that EDR1 was conserved in upland rice, but segregated in paddy rice. Further analyses of grain chalkiness in the alleles of EDR1YD1 and EDR1YZN varieties indicated that rice varieties harboring EDR1YZN and EDR1YD1 preferentially showed high chalkiness, and low chalkiness, respectively. Taken together, these results suggest that the UDP-glucosyltransferase gene EDR1 is an important determinant controlling differential endosperm development between upland rice and paddy rice.

Arabidopsis SIGMA FACTOR BINDING PROTEIN1 (SIB1) and SIB2 inhibit WRKY75 function in abscisic acid-mediated leaf senescence and seed germination.

The plant-specific VQ gene family participates in diverse physiological processes but little information is available on their role in leaf senescence. Here, we show that the VQ motif-containing proteins, Arabidopsis SIGMA FACTOR BINDING PROTEIN1 (SIB1) and SIB2 are negative regulators of abscisic acid (ABA)-mediated leaf senescence. Loss of SIB1 and SIB2 function resulted in increased sensitivity of ABA-induced leaf senescence. In contrast, overexpression of SIB1 significantly delayed this process. Moreover, biochemical studies revealed that SIBs interact with WRKY75 transcription factor. Loss of WRKY75 function decreased sensitivity to ABA-induced leaf senescence, while overexpression of WRKY75 significantly accelerated this process. Chromatin immunoprecipitation assays revealed that WRKY75 directly binds to the promoters of GOLDEN 2-LIKE1(GLK1) and GLK2, to repress their expression. SIBs repress the transcriptional function of WRKY75 and negatively regulate ABA-induced leaf senescence in a WRKY75-dependent manner. In contrast, WRKY75 positively modulates ABA-mediated leaf senescence in a GLK-dependent manner. In addition, SIBs inhibit WRKY75 function in ABA-mediated seed germination. These results demonstrate that SIBs can form a complex with WRKY75 to regulate ABA-mediated leaf senescence and seed germination.

The emerging role of jasmonate in the control of flowering time.

Plants dynamically synchronize their flowering time with changes in the internal and external environments through a variety of signaling pathways to maximize fitness. In the last two decades, the major pathways associated with flowering, including the photoperiod, vernalization, age, autonomous, gibberellin, and ambient temperature pathways, have been extensively analyzed. In recent years, an increasing number of signals, such as sugar, thermosensory, stress, and certain hormones, have been shown to be involved in fine-tuning flowering time. Among these signals, the jasmonate signaling pathway has a function in the determination of flowering time that has not been systematically summarized. In this review, we present an overview of current knowledge of jasmonate control of flowering and discuss jasmonate crosstalk with other signals (such as gibberellin, defense, and touch) during floral transition.

Phytomelatonin inhibits seed germination by regulating germination-related hormone signaling in Arabidopsis.

Seed germination is a vital initial stage in the life cycle of a plant, which determines subsequent vegetative growth and reproduction. Melatonin acts as a plant's master regulator and is also involved in the process of seed germination. In a recent study, we show that the high concentration melatonin inhibited seed germination in Arabidopsis. Transcriptome and phenotype analysis implied that melatonin-mediated seed germination interacted with phytohormones abscisic acid (ABA), gibberellin (GA), and auxin. In this short communication, we discuss the mechanism of phytomelatonin that inhibits seed germination through ABA, GA, and IAA in Arabidopsis.

AtWRKY75 positively regulates age-triggered leaf senescence through gibberellin pathway.

WRKY transcription factors play essential roles during leaf senescence. However, the mechanisms by which they regulate this process remains largely unknown. Here, we identified the transcription factor WRKY75 as a positive regulator during leaf senescence. Mutations of WRKY75 caused a delay in age-triggered leaf senescence, whereas overexpression of WRKY75 markedly accelerated this process. Expression of senescence-associated genes (SAGs) was suppressed in WRKY75 mutants but increased in WRKY75-overexpressing plants. Further analysis demonstrated that WRKY75 directly associates with the promoters of SAG12 and SAG29, to activate their expression. Conversely, GAI and RGL1, two DELLA proteins, can suppress the WRKY75-mediated activation, thereby attenuating SAG expression during leaf senescence. Genetic analyses showed that GAI gain-of-function or RGL1 overexpression can partially rescue the accelerated senescence phenotype caused by WRKY75 overexpression. Furthermore, WRKY75 can positively regulate WRKY45 expression during leaf senescence. Our data thus imply that WRKY75 may positively modulate age-triggered leaf senescence through the gibberellin-mediated signaling pathway.

ERF1 delays flowering through direct inhibition of FLOWERING LOCUS T expression in Arabidopsis.

ETHYLENE RESPONSE FACTOR1 (ERF1) is a key component in ethylene signaling, playing crucial roles in both biotic and abiotic stress responses. Here, we demonstrate that ERF1 also has an important role during floral initiation in Arabidopsis thaliana. Knockdown or knockout of ERF1 accelerated floral initiation, whereas overexpression of ERF1 dramatically delayed floral transition. These contrasting phenotypes were correlated with opposite transcript levels of FLOWERING LOCUS T (FT). Chromatin immunoprecipitation (ChIP) assays revealed that ERF1 associates with genomic regions of the FT gene to repress its transcription. ft-10/ERF1RNAi plants showed a similar flowering phenotype to the ft-10 mutant, whereas the flowering of FTox/ERF1ox mimicked that of FTox plants, suggesting that ERF1 acts upstream of FT during floral initiation. Similarly, altered floral transition in ethylene-related mutants was also correlated with FT expression. Further analysis suggested that ERF1 also participates in delay in flowering-time control mediated by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. Thus, ERF1 may act as a negative modulator of flowering-time control by repressing FT transcription in Arabidopsis.

Iron deficiency-induced transcription factors bHLH38/100/101 negatively modulate flowering time in Arabidopsis thaliana.

The mechanisms regulating flowering have been extensively studied and the roles of many environmental signals in this process have been reported. However, little is known on the relationship between iron deficiency and flowering regulation, although the response mechanism to iron deficiency has been studied for decades. In this study, we observed that the flowering time of wild-type Arabidopsis thaliana was significantly repressed by iron deficiency under long days. Phenotype analysis showed that iron deficiency delayed flowering of Arabidopsis through the iron deficiency-induced transcription factors bHLH38, bHLH100, and bHLH101 (bHLH38/100/101), which redundantly regulated flowering time and expression of FLOWERING LOCUS T (FT) specifically under long days. Genetic analysis indicated that disruption of FT expression suppressed the early-flowering phenotype of bhlh38/100/101 triple-mutant plants, indicating that bHLH38/100/101 are dependent on functional FT. Furthermore, bHLH38/100/101 interacted with CONSTANS (CO), thereby interfering with the transcriptional activation of CO to regulate FT expression. Therefore, the results indicated that iron deficiency affects flowering of Arabidopsis under long days through bHLH38/100/101-CO-FT signaling.

Melatonin inhibits seed germination by crosstalk with abscisic acid, gibberellin, and auxin in Arabidopsis.

Seed germination, an important developmental stage in the life cycle of seed plants, is regulated by complex signals. Melatonin is a signaling molecule associated with seed germination under stressful conditions, although the underlying regulatory mechanisms are largely unknown. In this study, we showed that a low concentration (10 µM or 100 µM) of melatonin had no effect on seed germination, but when the concentration of melatonin increased to 500 µM or 1000 µM, seed germination was significantly inhibited in Arabidopsis. RNA sequencing analysis showed that melatonin regulated seed germination correlated to phytohormones abscisic acid (ABA), gibberellin (GA), and auxin. Further investigation revealed that ABA and melatonin synergistically inhibited seed germination, while GA and auxin antagonized the inhibitory effect of seed germination by melatonin. Disruption of the melatonin biosynthesis enzyme gene serotonin N-acetyltransferase (SNAT) or N-acetylserotonin methyltransferase (ASMT) promoted seed germination, while overexpression of ASMT inhibited seed germination. Taken together, our study sheds new light on the function and mechanism of melatonin in modulating seed germination in Arabidopsis.

Functional characterization of the Arabidopsis SERRATE under salt stress.

SERRATE (SE) plays critical roles in RNA metabolism and plant growth regulation. However, its function in stress-response processes remains largely unknown. Here, we examined the regulatory role of SE using the se-1 mutant and its complementation line under saline conditions. The expression of SE was repressed by salt treatment at both mRNA and protein levels. After treatment with different NaCl concentrations, the se-1 mutants showed increased sensitivity to salinity. This heightened sensitivity was evidenced by decreased germination, reduced root growth, more serious chlorosis, and increased conductivity of the mutants compared with the wild type. Further analysis revealed that SE regulates the pre-mRNA splicing of several well-characterized marker genes associated with salt stress tolerance. Our data thus imply that SE may function as a key component in plant response to salt stress by modulating the splicing of salt stress-associated genes.

Phytomelatonin: An Emerging Regulator of Plant Biotic Stress Resistance.

Melatonin has diverse functions in plant development and stress tolerance, with recent evidence showing a beneficial role in plant biotic stress tolerance. It has been hypothesized that pathogenic invasion causes the immediate generation of melatonin, reactive oxygen species (ROS), and reactive nitrogen species (RNS), with these being mutually dependent, forming the integrative melatonin-ROS-RNS feedforward loop. Here we discuss how the loop, possibly located in the mitochondria and chloroplasts, maximizes disease resistance in the early pathogen ingress stage, providing on-site protection. We also review how melatonin interacts with phytohormone signaling pathways to mediate defense responses and discuss the evolutionary context from the beginnings of the melatonin receptor-mitogen-activated protein kinase (MAPK) cascade in unicellular green algae, followed by the occurrence of phytohormone pathways in land plants.