Highlights in gibberellin research: A tale of the dwarf and the slender.

It has been almost a century since biologically active gibberellin (GA) was isolated. Here, we give a historical overview of the early efforts in establishing the GA biosynthesis and catabolism pathway, characterizing the enzymes for GA metabolism, and elucidating their corresponding genes. We then highlight more recent studies that have identified the GA receptors and early GA signaling components (DELLA repressors and F-box activators), determined the molecular mechanism of DELLA-mediated transcription reprograming, and revealed how DELLAs integrate multiple signaling pathways to regulate plant vegetative and reproductive development in response to internal and external cues. Finally, we discuss the GA transporters and their roles in GA-mediated plant development.

SPINDLY O-fucosylates nuclear and cytoplasmic proteins involved in diverse cellular processes in plants.

SPINDLY (SPY) is a novel nucleocytoplasmic protein O-fucosyltransferase that regulates target protein activity or stability via O-fucosylation of specific Ser/Thr residues. Previous genetic studies indicate that AtSPY regulates plant development during vegetative and reproductive growth by modulating gibberellin and cytokinin responses. AtSPY also regulates the circadian clock and plant responses to biotic and abiotic stresses. The pleiotropic phenotypes of spy mutants point to the likely role of AtSPY in regulating key proteins functioning in diverse cellular pathways. However, very few AtSPY targets are known. Here, we identified 88 SPY targets from Arabidopsis (Arabidopsis thaliana) and Nicotiana benthamiana via the purification of O-fucosylated peptides using Aleuria aurantia lectin followed by electron transfer dissociation-MS/MS analysis. Most AtSPY targets were nuclear proteins that function in DNA repair, transcription, RNA splicing, and nucleocytoplasmic transport. Cytoplasmic AtSPY targets were involved in microtubule-mediated cell division/growth and protein folding. A comparison with the published O-linked-N-acetylglucosamine (O-GlcNAc) proteome revealed that 30% of AtSPY targets were also O-GlcNAcylated, indicating that these distinct glycosylations could co-regulate many protein functions. This study unveiled the roles of O-fucosylation in modulating many key nuclear and cytoplasmic proteins and provided a valuable resource for elucidating the regulatory mechanisms involved.

O-GlcNAcylation of master growth repressor DELLA by SECRET AGENT modulates multiple signaling pathways in Arabidopsis.

The DELLA family of transcription regulators functions as master growth repressors in plants by inhibiting phytohormone gibberellin (GA) signaling in response to developmental and environmental cues. DELLAs also play a central role in mediating cross-talk between GA and other signaling pathways via antagonistic direct interactions with key transcription factors. However, how these crucial protein-protein interactions can be dynamically regulated during plant development remains unclear. Here, we show that DELLAs are modified by the O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) SECRET AGENT (SEC) in Arabidopsis. O-GlcNAcylation of the DELLA protein REPRESSOR OF ga1-3 (RGA) inhibits RGA binding to four of its interactors-PHYTOCHROME-INTERACTING FACTOR3 (PIF3), PIF4, JASMONATE-ZIM DOMAIN1, and BRASSINAZOLE-RESISTANT1 (BZR1)-that are key regulators in light, jasmonate, and brassinosteroid signaling pathways, respectively. Consistent with this, the sec-null mutant displayed reduced responses to GA and brassinosteroid and showed decreased expression of several common target genes of DELLAs, BZR1, and PIFs. Our results reveal a direct role of OGT in repressing DELLA activity and indicate that O-GlcNAcylation of DELLAs provides a fine-tuning mechanism in coordinating multiple signaling activities during plant development.

The Interaction between DELLA and ARF/IAA Mediates Crosstalk between Gibberellin and Auxin Signaling to Control Fruit Initiation in Tomato.

Fruit initiation following fertilization in angiosperms is strictly regulated by phytohormones. In tomato (Solanum lycopersicum), auxin and gibberellin (GA) play central roles in promoting fruit initiation. Without fertilization, elevated GA or auxin signaling can induce parthenocarpy (seedless fruit production). The GA-signaling repressor SlDELLA and auxin-signaling components SlIAA9 and SlARF7 repress parthenocarpy, but the underlying mechanism is unknown. Here, we show that SlDELLA and the SlARF7/SlIAA9 complex mediate crosstalk between GA and auxin pathways to regulate fruit initiation. Yeast-two-hybrid and coimmunoprecipitation assays showed that SlARF7 and additional activator SlARFs interact with SlDELLA and SlIAA9 through distinct domains. SlARF7/SlIAA9 and SlDELLA antagonistically modulate the expression of feedback-regulated genes involved in GA and auxin metabolism, whereas SlARF7/SlIAA9 and SlDELLA coregulate the expression of fruit growth-related genes. Analysis of procera (della), SlARF7 RNAi (with downregulated expression of multiple activator SlARFs), and entire (iaa9) single and double mutants indicated that these genes additively affect parthenocarpy, supporting the notion that the SlARFs/SlIAA9 and SlDELLA interaction plays an important role in regulating fruit initiation. Analysis of the GA-deficient mutant gib1 showed that active GA biosynthesis and signaling are required for auxin-induced fruit initiation. Our study reveals how direct crosstalk between auxin- and GA-signaling components is critical for tomato fruit initiation.

Inverse Molecular Sentinel-Integrated Fiberoptic Sensor for Direct and in Situ Detection of miRNA Targets.

Molecular advances have been made in analysis systems for a wide variety of applications ranging from biodiagnostics, biosafety, bioengineering, and biofuel research applications. There are, however, limited practical tools necessary for in situ and accurate detection of nucleic acid targets during field work. New technology is needed to translate these molecular advances from laboratory settings into the real-life practical monitoring realm. The exquisite characteristics (e.g., sensitivity and adaptability) of plasmonic nanosensors have made them attractive candidates for field-ready sensing applications. Herein, we have developed a fiber-based plasmonic sensor capable of direct detection (i.e., no washing steps required) of nucleic acid targets, which can be detected simply by immerging the sensor in the sample solution. This sensor is composed of an optical fiber that is decorated with plasmonic nanoprobes based on silver-coated gold nanostars (AuNS@Ag) to detect target nucleic acids using the surface-enhanced Raman scattering (SERS) sensing mechanism of nanoprobes referred to as inverse molecular sentinels (iMS). These fiber-optrodes can be reused for several detection-regeneration cycles (>6). The usefulness and applicability of the iMS fiber-sensors was tested by detecting target miRNA in extracts from leaves of plants that were induced to have different expression levels of miRNA targets. These fiber-optrodes enable direct detection of miRNA in plant tissue extract without the need for complex assays by simply immersing the fiber in the sample solution. The results indicate the fiber-based sensors developed herein have the potential to be a powerful tool for field and in situ analysis of nucleic acid samples.

The Arabidopsis O-fucosyltransferase SPINDLY activates nuclear growth repressor DELLA.

Plant development requires coordination among complex signaling networks to enhance the plant's adaptation to changing environments. DELLAs, transcription regulators originally identified as repressors of phytohormone gibberellin signaling, play a central role in integrating multiple signaling activities via direct protein interactions with key transcription factors. Here, we found that DELLA is mono-O-fucosylated by the novel O-fucosyltransferase SPINDLY (SPY) in Arabidopsis thaliana. O-fucosylation activates DELLA by promoting its interaction with key regulators in brassinosteroid- and light-signaling pathways, including BRASSINAZOLE-RESISTANT1 (BZR1), PHYTOCHROME-INTERACTING-FACTOR3 (PIF3) and PIF4. Moreover, spy mutants displayed elevated responses to gibberellin and brassinosteroid, and increased expression of common target genes of DELLAs, BZR1 and PIFs. Our study revealed that SPY-dependent protein O-fucosylation plays a key role in regulating plant development. This finding may have broader importance because SPY orthologs are conserved in prokaryotes and eukaryotes, thus suggesting that intracellular O-fucosylation may regulate a wide range of biological processes in diverse organisms.

Gibberellin Signaling Requires Chromatin Remodeler PICKLE to Promote Vegetative Growth and Phase Transitions.

PICKLE (PKL) is an ATP-dependent chromodomain-helicase-DNA-binding domain (CHD3) chromatin remodeling enzyme in Arabidopsis (Arabidopsis thaliana). Previous studies showed that PKL promotes embryonic-to-vegetative transition by inhibiting expression of seed-specific genes during seed germination. The pkl mutants display a low penetrance of the "pickle root" phenotype, with a thick and green primary root that retains embryonic characteristics. The penetrance of this pickle root phenotype in pkl is dramatically increased in gibberellin (GA)-deficient conditions. At adult stages, the pkl mutants are semidwarfs with delayed flowering time, which resemble reduced GA-signaling mutants. These findings suggest that PKL may play a positive role in regulating GA signaling. A recent biochemical analysis further showed that PKL and GA signaling repressors DELLAs antagonistically regulate hypocotyl cell elongation genes by direct protein-protein interaction. To elucidate further the role of PKL in GA signaling and plant development, we studied the genetic interaction between PKL and DELLAs using the hextuple mutant containing pkl and della pentuple (dP) mutations. Here, we show that PKL is required for most of GA-promoted developmental processes, including vegetative growth such as hypocotyl, leaf, and inflorescence stem elongation, and phase transitions such as juvenile-to-adult leaf and vegetative-to-reproductive phase. The removal of all DELLA functions (in the dP background) cannot rescue these phenotypes in pkl RNA-sequencing analysis using the ga1 (a GA-deficient mutant), pkl, and the ga1 pkl double mutant further shows that expression of 80% of GA-responsive genes in seedlings is PKL dependent, including genes that function in cell elongation, cell division, and phase transitions. These results indicate that the CHD3 chromatin remodeler PKL is required for regulating gene expression during most of GA-regulated developmental processes.

The ERF11 Transcription Factor Promotes Internode Elongation by Activating Gibberellin Biosynthesis and Signaling.

The phytohormone gibberellin (GA) plays a key role in promoting stem elongation in plants. Previous studies show that GA activates its signaling pathway by inducing rapid degradation of DELLA proteins, GA signaling repressors. Using an activation-tagging screen in a reduced-GA mutant ga1-6 background, we identified AtERF11 to be a novel positive regulator of both GA biosynthesis and GA signaling for internode elongation. Overexpression of AtERF11 partially rescued the dwarf phenotype of ga1-6 AtERF11 is a member of the ERF (ETHYLENE RESPONSE FACTOR) subfamily VIII-B-1a of ERF/AP2 transcription factors in Arabidopsis (Arabidopsis thaliana). Overexpression of AtERF11 resulted in elevated bioactive GA levels by up-regulating expression of GA3ox1 and GA20ox genes. Hypocotyl elongation assays further showed that overexpression of AtERF11 conferred elevated GA response, whereas loss-of-function erf11 and erf11 erf4 mutants displayed reduced GA response. In addition, yeast two-hybrid, coimmunoprecipitation, and transient expression assays showed that AtERF11 enhances GA signaling by antagonizing the function of DELLA proteins via direct protein-protein interaction. Interestingly, AtERF11 overexpression also caused a reduction in the levels of another phytohormone ethylene in the growing stem, consistent with recent finding showing that AtERF11 represses transcription of ethylene biosynthesis ACS genes. The effect of AtERF11 on promoting GA biosynthesis gene expression is likely via its repressive function on ethylene biosynthesis. These results suggest that AtERF11 plays a dual role in promoting internode elongation by inhibiting ethylene biosynthesis and activating GA biosynthesis and signaling pathways.

Role of the gibberellin receptors GID1 during fruit-set in Arabidopsis.

Gibberellins (GAs) play a critical role in fruit-set and fruit growth. Gibberellin is perceived by its nuclear receptors GA INSENSITIVE DWARF1s (GID1s), which then trigger degradation of downstream repressors DELLAs. To understand the role of the three GA receptor genes (GID1A, GID1B and GID1C) in Arabidopsis during fruit initiation, we have examined their temporal and spatial localization, in combination with analysis of mutant phenotypes. Distinct expression patterns are revealed for each GID1: GID1A is expressed throughout the whole pistil, while GID1B is expressed in ovules, and GID1C is expressed in valves. Functional study of gid1 mutant combinations confirms that GID1A plays a major role during fruit-set and growth, whereas GID1B and GID1C have specific roles in seed development and pod elongation, respectively. Therefore, in ovules, GA perception is mediated by GID1A and GID1B, while GID1A and GID1C are involved in GA perception in valves. To identify tissue-specific interactions between GID1s and DELLAs, we analyzed spatial expression patterns of four DELLA genes that have a role in fruit initiation (GAI, RGA, RGL1 and RGL2). Our data suggest that GID1A can interact with RGA and GAI in all tissues, whereas GID1C-RGL1 and GID1B-RGL2 interactions only occur in valves and ovules, respectively. These results uncover specific functions of each GID1-DELLA in the different GA-dependent processes that occur upon fruit-set. In addition, the distribution of GA receptors in valves along with lack of expression of GA biosynthesis genes in this tissue, strongly suggests transport of GAs from the developing seeds to promote fruit growth.

Functional characterization and developmental expression profiling of gibberellin signalling components in Vitis vinifera.

Gibberellins (GAs) regulate numerous developmental processes in grapevine (Vitis vinifera) such as rachis elongation, fruit set, and fruitlet abscission. The ability of GA to promote berry enlargement has led to its indispensable use in the sternospermocarpic ('seedless') table grape industry worldwide. However, apart from VvGAI1 (VvDELLA1), which regulates internode elongation and fruitfulness, but not berry size of seeded cultivars, little was known about GA signalling in grapevine. We have identified and characterized two additional DELLAs (VvDELLA2 and VvDELLA3), two GA receptors (VvGID1a and VvGID1b), and two GA-specific F-box proteins (VvSLY1a and VvSLY1b), in cv. Thompson seedless. With the exception of VvDELLA3-VvGID1b, all VvDELLAs interacted with the VvGID1s in a GA-dependent manner in yeast two-hybrid assays. Additionally, expression of these grape genes in corresponding Arabidopsis mutants confirmed their functions in planta. Spatiotemporal analysis of VvDELLAs showed that both VvDELLA1 and VvDELLA2 are abundant in most tissues, except in developing fruit where VvDELLA2 is uniquely expressed at high levels, suggesting a key role in fruit development. Our results further suggest that differential organ responses to exogenous GA depend on the levels of VvDELLA proteins and endogenous bioactive GAs. Understanding this interaction will allow better manipulation of GA signalling in grapevine.

Leaf-induced gibberellin signaling is essential for internode elongation, cambial activity, and fiber differentiation in tobacco stems.

The gibberellins (GAs) are a group of endogenous compounds that promote the growth of most plant organs, including stem internodes. We show that in tobacco (Nicotiana tabacum) the presence of leaves is essential for the accumulation of bioactive GAs and their immediate precursors in the stem and consequently for normal stem elongation, cambial proliferation, and xylem fiber differentiation. These processes do not occur in the absence of maturing leaves but can be restored by application of C(19)-GAs, identifying the presence of leaves as a requirement for GA signaling in stems and revealing the fundamental role of GAs in secondary growth regulation. The use of reporter genes for GA activity and GA-directed DELLA protein degradation in Arabidopsis thaliana confirms the presence of a mobile signal from leaves to the stem that induces GA signaling.

Plant hormone jasmonate prioritizes defense over growth by interfering with gibberellin signaling cascade.

Plants must effectively defend against biotic and abiotic stresses to survive in nature. However, this defense is costly and is often accompanied by significant growth inhibition. How plants coordinate the fluctuating growth-defense dynamics is not well understood and remains a fundamental question. Jasmonate (JA) and gibberellic acid (GA) are important plant hormones that mediate defense and growth, respectively. Binding of bioactive JA or GA ligands to cognate receptors leads to proteasome-dependent degradation of specific transcriptional repressors (the JAZ or DELLA family of proteins), which, at the resting state, represses cognate transcription factors involved in defense (e.g., MYCs) or growth [e.g. phytochrome interacting factors (PIFs)]. In this study, we found that the coi1 JA receptor mutants of rice (a domesticated monocot crop) and Arabidopsis (a model dicot plant) both exhibit hallmark phenotypes of GA-hypersensitive mutants. JA delays GA-mediated DELLA protein degradation, and the della mutant is less sensitive to JA for growth inhibition. Overexpression of a selected group of JAZ repressors in Arabidopsis plants partially phenocopies GA-associated phenotypes of the coi1 mutant, and JAZ9 inhibits RGA (a DELLA protein) interaction with transcription factor PIF3. Importantly, the pif quadruple (pifq) mutant no longer responds to JA-induced growth inhibition, and overexpression of PIF3 could partially overcome JA-induced growth inhibition. Thus, a molecular cascade involving the COI1-JAZ-DELLA-PIF signaling module, by which angiosperm plants prioritize JA-mediated defense over growth, has been elucidated.

Liver Cancer Detection by a Simple, Inexpensive and Effective Immunosensor with Zinc Oxide Nanoparticles.

Regular monitoring of blood α-fetoprotein (AFP) and/or carcino-embryonic antigen (CEA) levels is important for the routine screening of liver cancer. However, AFP and CEA have a much lower specificity than des-γ-carboxyprothrombin (DCP) to detect liver cancer. Therefore, the study reported here was designed, to develop a screen-printed DCP immunosensor incorporating zinc oxide nanoparticles, for accurate determination of DCP. The designed immunosensor shows low detection limits for the detection of DCP: 0.440 ng/mL (based on impedance measurement), 0.081 ng/mL (based on real part of impedance measurement) and 0.078 ng/mL (based on imaginary part of impedance measurement), within the range of 3.125 ng/mL to 2000 ng/mL. In addition, there was little interference to DCP determination by molecules such as Na⁺, K⁺, Ca(2+), Cl(-), glucose, urea, and uric acid. It is therefore concluded that the DCP immunosensor developed and reported here is simple, inexpensive and effective, and shows promise in the rapid screening of early-stage liver cancer at home with a point-of-care approach.

Rapid and orthogonal logic gating with a gibberellin-induced dimerization system.

Using a newly synthesized gibberellin analog containing an acetoxymethyl group (GA(3)-AM) and its binding proteins, we developed an efficient chemically inducible dimerization (CID) system that is completely orthogonal to existing rapamycin-mediated protein dimerization. Combining the two systems should allow applications that have been difficult or impossible with only one CID system. By using both chemical inputs (rapamycin and GA(3)-AM), we designed and synthesized Boolean logic gates in living mammalian cells. These gates produced output signals such as fluorescence and membrane ruffling on a timescale of seconds, substantially faster than earlier intracellular logic gates. The use of two orthogonal dimerization systems in the same cell also allows for finer modulation of protein perturbations than is possible with a single dimerizer.

Gibberellin-induced DELLA recognition by the gibberellin receptor GID1.

Gibberellins control a range of growth and developmental processes in higher plants and have been widely used in the agricultural industry. By binding to a nuclear receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1), gibberellins regulate gene expression by promoting degradation of the transcriptional regulator DELLA proteins, including GIBBERELLIN INSENSITIVE (GAI). The precise manner in which GID1 discriminates and becomes activated by bioactive gibberellins for specific binding to DELLA proteins remains unclear. Here we present the crystal structure of a ternary complex of Arabidopsis thaliana GID1A, a bioactive gibberellin and the amino-terminal DELLA domain of GAI. In this complex, GID1A occludes gibberellin in a deep binding pocket covered by its N-terminal helical switch region, which in turn interacts with the DELLA domain containing DELLA, VHYNP and LExLE motifs. Our results establish a structural model of a plant hormone receptor that is distinct from the mechanism of the hormone perception and effector recognition of the known auxin receptors.

Scarecrow-like 3 promotes gibberellin signaling by antagonizing master growth repressor DELLA in Arabidopsis.

The diterpenoid phytohormone gibberellin (GA) controls diverse developmental processes throughout the plant life cycle. DELLA proteins are master growth repressors that function immediately downstream of the GA receptor to inhibit GA signaling. By doing so, DELLAs also play pivotal roles as integrators of internal developmental signals from multiple hormone pathways and external cues. DELLAs are likely nuclear transcriptional regulators, which interact with other transcription factors to modulate expression of GA-responsive genes. DELLAs are also involved in maintaining GA homeostasis through feedback up-regulating expression of GA biosynthesis and receptor genes. However, the molecular mechanisms by which DELLAs restrict growth and development are largely unknown. This study reveals an important step of the mechanism. Previous microarray studies identified scarecrow-like 3 (SCL3) as a direct target gene of DELLA in Arabidopsis seedlings. SCL3 expression is induced by DELLA and repressed by GA. Unexpectedly, a scl3 null mutant displays reduced GA responses and elevated expression of GA biosynthesis genes during seed germination and seedling growth, indicating that SCL3 functions as a positive regulator of GA signaling. SCL3 seems to act as an attenuator of DELLA proteins. Transient expression, ChIP, and co-IP studies show that SCL3 autoregulates its own transcription by directly interacting with DELLA. Our data further show that SCL3 and DELLA antagonize each other in controlling both downstream GA responses and upstream GA biosynthetic genes. This work is beginning to shed light on how this complex regulatory network achieves GA homeostasis and controls GA-mediated growth and development in the plant.

Phosphorylation activates master growth regulator DELLA by promoting histone H2A binding at chromatin in Arabidopsis.

DELLA proteins are conserved master growth regulators that play a central role in controlling plant development in response to internal and environmental cues. DELLAs function as transcription regulators, which are recruited to target promoters by binding to transcription factors (TFs) and histone H2A via their GRAS domain. Recent studies showed that DELLA stability is regulated post-translationally via two mechanisms, phytohormone gibberellin-induced polyubiquitination for its rapid degradation, and Small Ubiquitin-like Modifier (SUMO)-conjugation to increase its accumulation. Moreover, DELLA activity is dynamically modulated by two distinct glycosylations: DELLA-TF interactions are enhanced by O-fucosylation, but inhibited by O-linked N-acetylglucosamine (O-GlcNAc) modification. However, the role of DELLA phosphorylation remains unclear as previous studies showing conflicting results ranging from findings that suggest phosphorylation promotes or reduces DELLA degradation to others indicating it has no effect on its stability. Here, we identify phosphorylation sites in REPRESSOR OF ga1-3 (RGA, an AtDELLA) purified from Arabidopsis by mass spectrometry analysis, and show that phosphorylation of two RGA peptides in the PolyS and PolyS/T regions enhances RGA activity by promoting H2A binding and RGA association with target promoters. Notably, phosphorylation does not affect RGA-TF interactions or RGA stability. Our study has uncovered a molecular mechanism of phosphorylation-induced DELLA activity.

Plasmonics nanorod biosensor for in situ intracellular detection of gene expression biomarkers in intact plant systems.

The intracellular developmental processes in plants, particularly concerning lignin polymer formation and biomass production are regulated by microRNAs (miRNAs). MiRNAs including miR397b are important for developing efficient and cost-effective biofuels. However, traditional methods of monitoring miRNA expression, like PCR, are time-consuming, require sample extraction, and lack spatial and temporal resolution, especially in real-world conditions. We present a novel approach using plasmonics nanosensing to monitor miRNA activity within living plant cells without sample extraction. Plasmonic biosensors using surface-enhanced Raman scattering (SERS) detection offer high sensitivity and precise molecular information. We used the Inverse Molecular Sentinel (iMS) biosensor on unique silver-coated gold nanorods (AuNR@Ag) with a high-aspect ratio to penetrate plant cell walls for detecting miR397b within intact living plant cells. MiR397b overexpression has shown promise in reducing lignin content. Thus, monitoring miR397b is essential for cost-effective biofuel generation. This study demonstrates the infiltration of nanorod iMS biosensors and detection of non-native miRNA 397b within plant cells for the first time. The investigation successfully demonstrates the localization of nanorod iMS biosensors through TEM and XRF-based elemental mapping for miRNA detection within plant cells of Nicotiana benthamiana. The study integrates shifted-excitation Raman difference spectroscopy (SERDS) to decrease background interference and enhance target signal extraction. In vivo SERDS testing confirms the dynamic detection of miR397b in Arabidopsis thaliana leaves after infiltration with iMS nanorods and miR397b target. This proof-of-concept study is an important stepping stone towards spatially resolved, intracellular miRNA mapping to monitor biomarkers and biological pathways for developing efficient renewable biofuel sources.

Development of anodic titania nanotubes for application in high sensitivity amperometric glucose and uric acid biosensors.

The purpose of this study was to develop novel nanoscale biosensors using titania nanotubes (TNTs) made by anodization. Titania nanotubes were produced on pure titanium sheets by anodization at room temperature. In this research, the electrolyte composition ethylene glycol 250 mL/NH4F 1.5 g/DI water 20 mL was found to produce the best titania nanotubes array films for application in amperometric biosensors. The amperometric results exhibit an excellent linearity for uric acid (UA) concentrations in the range between 2 and 14 mg/dL, with 23.3 (µA·cm-2)·(mg/dL)-1 UA sensitivity, and a correlation coefficient of 0.993. The glucose biosensor presented a good linear relationship in the lower glucose concentration range between 50 and 125 mg/dL, and the corresponding sensitivity was approximately 249.6 (µA·cm-2)·(100 mg/dL)-1 glucose, with a correlation coefficient of 0.973.

Four class A AUXIN RESPONSE FACTORs promote tomato fruit growth despite suppressing fruit set.

In flowering plants, auxin produced in seeds after fertilization promotes fruit initiation. The application of auxin to unpollinated ovaries can also induce parthenocarpy (seedless fruit production). Previous studies have shown that auxin signalling components SlIAA9 and SlARF7 (a class A AUXIN RESPONSE FACTOR (ARF)) are key repressors of fruit initiation in tomato (Solanum lycopersicum). A similar repressive role of class A ARFs in fruit set has also been observed in other plant species. However, evidence is lacking for a role of any class A ARF in promoting fruit development as predicted in the current auxin signalling model. Here we generated higher-order tomato mutants of four class A SlARFs (SlARF5, SlARF7, SlARF8A and SlARF8B) and uncovered their precise combinatorial roles that lead to suppressing and promoting fruit development. All four class A SlARFs together with SlIAA9 inhibited fruit initiation but promoted subsequent fruit growth. Transgenic tomato lines expressing truncated SlARF8A/8B lacking the IAA9-interacting PB1 domain displayed strong parthenocarpy, further confirming the promoting role of SlARF8A/8B in fruit growth. Altering the doses of these four SlARFs led to biphasic fruit growth responses, showing their versatile dual roles as both negative and positive regulators. RNA-seq and chromatin immunoprecipitation-quantitative PCR analyses further identified SlARF8A/8B target genes, including those encoding MADS-BOX transcription factors (AG1, MADS2 and AGL6) that are key repressors of fruit set. These results support the idea that SlIAA9/SlARFs directly regulate the transcription of these MADS-BOX genes to inhibit fruit set. Our study reveals the previously unknown dual function of four class A SlARFs in tomato fruit development and illuminates the complex combinatorial effects of multiple ARFs in controlling auxin-mediated fruit set and fruit growth.