miR156-PvSPL2 controls culm development by transcriptional repression of switchgrass CYTOKININ OXIDASE/DEHYDROGENASE4.

Culm development in grasses can be controlled by both miR156 and cytokinin. However, the crosstalk between the miR156-SPL module and the cytokinin metabolic pathway remains largely unknown. Here, we found CYTOKININ OXIDASE/DEHYDROGENASE4 (PvCKX4) plays a negative regulatory role in culm development of the bioenergy grass Panicum virgatum (switchgrass). Overexpression of PvCKX4 in switchgrass reduced the internode diameter and length without affecting tiller number. Interestingly, we also found that PvCKX4 was always upregulated in miR156 overexpressing (miR156OE) transgenic switchgrass lines. Additionally, upregulation of either miR156 or PvCKX4 in switchgrass reduced the content of isopentenyl adenine (iP) without affecting trans-zeatin (tZ) accumulation. It is consistent with the evidence that the recombinant PvCKX4 protein exhibited much higher catalytic activity against iP than tZ in vitro. Furthermore, our results showed that miR156-targeted SPL2 bound directly to the promoter of PvCKX4 to repress its expression. Thus, alleviating the SPL2-mediated transcriptional repression of PvCKX4 through miR156 overexpression resulted in a significant increase in cytokinin degradation and impaired culm development in switchgrass. On the contrary, suppressing PvCKX4 in miR156OE transgenic plants restored iP content, internode diameter, and length to wild-type levels. Most strikingly, the double transgenic lines retained the same increased tiller numbers as the miR156OE transgenic line, which yielded more biomass than the wild type. These findings indicate that the miR156-SPL module can control culm development through transcriptional repression of PvCKX4 in switchgrass, which provides a promising target for precise design of shoot architecture to yield more biomass from grasses.

Genome-wide identification, evolution, and role of SPL gene family in beet (Beta vulgaris L.) under cold stress.

BACKGROUND: SPL transcription factors play vital roles in regulating plant growth, development, and abiotic stress responses. Sugar beet (Beta vulgaris L.), one of the world's main sugar-producing crops, is a major source of edible and industrial sugars for humans. Although the SPL gene family has been extensively identified in other species, no reports on the SPL gene family in sugar beet are available. RESULTS: Eight BvSPL genes were identified at the whole-genome level and were renamed based on their positions on the chromosome. The gene structure, SBP domain sequences, and phylogenetic relationship with Arabidopsis were analyzed for the sugar beet SPL gene family. The eight BvSPL genes were divided into six groups (II, IV, V, VI, VII, and VIII). Of the BvSPL genes, no tandem duplication events were found, but one pair of segmental duplications was present. Multiple cis-regulatory elements related to growth and development were identified in the 2000-bp region upstream of the BvSPL gene start codon (ATG). Using quantitative real-time polymerase chain reaction (qRT-PCR), the expression profiles of the eight BvSPL genes were examined under eight types of abiotic stress and during the maturation stage. BvSPL transcription factors played a vital role in abiotic stress, with BvSPL3 and BvSPL6 being particularly noteworthy. CONCLUSION: Eight sugar beet SPL genes were identified at the whole-genome level. Phylogenetic trees, gene structures, gene duplication events, and expression profiles were investigated. The qRT-PCR analysis indicated that BvSPLs play a substantial role in the growth and development of sugar beet, potentially participating in the regulation of root expansion and sugar accumulation.

Genome-wide identification and expression analysis of the SPL transcription factor family and its response to abiotic stress in Pisum sativum L.

Squamous promoter binding protein-like (SPL) genes encode plant-specific transcription factors (TFs) that play essential roles in modulating plant growth, development, and stress response. Pea (Pisum sativum L.) is a coarse grain crop of great importance in food production, biodiversity conservation and molecular genetic research, providing genetic information and nutritional resources for improving agricultural production and promoting human health. However, only limited researches on the structure and functions of SPL genes exist in pea (PsSPLs). In this study, we identified 22 PsSPLs and conducted a genome-wide analysis of their physical characteristics, chromosome distribution, gene structure, phylogenetic evolution and gene expression patterns. As a result, the PsSPLs were unevenly distributed on the seven chromosomes of pea and harbored the SBP domain, which is composed of approximately 76 amino acid residues. The phylogenetic analysis revealed that the PsSPLs clustered into eight subfamilies and showed high homology with SPL genes in soybean. Further analysis showed the presence of segmental duplications in the PsSPLs. The expression patterns of 22 PsSPLs at different tissues, developmental stages and under various stimulus conditions were evaluated by qRT-PCR method. It was found that the expression patterns of PsSPLs from the same subfamily were similar in different tissues, the transcripts of most PsSPLs reached the maximum peak value at 14 days after anthesis in the pod. Abiotic stresses can cause significantly up-regulated PsSPL19 expression with spatiotemporal specificity, in addition, four plant hormones can cause the up-regulated expression of most PsSPLs including PsSPL19 in a time-dependent manner. Therefore, PsSPL19 could be a key candidate gene for signal transduction during pea growth and development, pod formation, abiotic stress and plant hormone response. Our findings should provide insights for the elucidating of development regulation mechanism and breeding for resistance to abiotic stress pea.

Genome-wide identification, stress- and hormone-responsive expression characteristics, and regulatory pattern analysis of Scutellaria baicalensis SbSPLs.

SQUAMOSA PROMOTER BINDING PROTEIN-LIKEs (SPLs) encode plant-specific transcription factors that regulate plant growth and development, stress response, and metabolite accumulation. However, there is limited information on Scutellaria baicalensis SPLs. In this study, 14 SbSPLs were identified and divided into 8 groups based on phylogenetic relationships. SbSPLs in the same group had similar structures. Abscisic acid-responsive (ABRE) and MYB binding site (MBS) cis-acting elements were found in the promoters of 8 and 6 SbSPLs. Segmental duplications and transposable duplications were the main causes of SbSPL expansion. Expression analysis based on transcriptional profiling showed that SbSPL1, SbSPL10, and SbSPL13 were highly expressed in roots, stems, and flowers, respectively. Expression analysis based on quantitative real-time polymerase chain reaction (RT‒qPCR) showed that most SbSPLs responded to low temperature, drought, abscisic acid (ABA) and salicylic acid (SA), among which the expression levels of SbSPL7/9/10/12 were significantly upregulated in response to abiotic stress. These results indicate that SbSPLs are involved in the growth, development and stress response of S. baicalensis. In addition, 8 Sba-miR156/157 s were identified, and SbSPL1-5 was a potential target of Sba-miR156/157 s. The results of target gene prediction and coexpression analysis together indicated that SbSPLs may be involved in the regulation of L-phenylalanine (L-Phe), lignin and jasmonic acid (JA) biosynthesis. In summary, the identification and characterization of the SbSPL gene family lays the foundation for functional research and provides a reference for improved breeding of S. baicalensis stress resistance and quality traits.

Inhibiting sphingosine 1-phosphate lyase: From efficacy to mechanism.

Sphingosine-1 phosphate (S1P) is a lipid metabolite regulating diverse biological processes, including proliferation, differentiation, migration, and apoptosis, highlighting its physiological and therapeutic significance. Current S1P-based therapeutic approaches primarily focus on modulating the downstream signalling via targeting S1P receptors, however, this is challenged by incomplete receptor internalisation. Sphingosine-1-phosphate lyase (SPL) is a highly conserved enzyme that "gatekeeps" the final step of S1P degradation. Cognisant of the complex ligand and receptor interaction and dynamic metabolic networks, the selective modulation of SPL activity presents a new opportunity to regulate S1P biosynthesis and reveal its role in various systems. Over the past decade, an evolving effort has been made to identify new molecules that could block SPL activity in vitro or in vivo. This review focuses on summarising the current understanding of the reported SPL inhibitors identified through various screening approaches, discussing their efficacy in diverse model systems and the possible mechanism of action. Whilst effective modulation of S1P levels via inhibiting SPL is feasible, the specificity of those inhibitors remains inconclusive, presenting a clear challenge for future implications. Yet, none of the currently available SPL inhibitors is proven effective in elevating S1P levels within the central nervous system. This review article embraces future research focusing on investigating selective SPL inhibitors with high potency and possibly blood-brain-barrier permeability, which would aid the development of new S1P-based therapeutics for neurological disorders.

Auxin and carbohydrate control flower bud development in Anthurium andraeanum during early stage of sexual reproduction.

BACKGROUND: Flower buds of Anthurium andraeanum frequently cease to grow and abort during the early flowering stage, resulting in prolonged planting times and increased commercialization costs. Nevertheless, limited knowledge exists of the mechanism of flower development after initiation in A. andraeanum. RESULTS: In this study, the measurement of carbohydrate flow and intensity between leaves and flowers during different growth stages showed that tender leaves are strong sinks and their concomitant flowers are weak ones. This suggested that the tender leaves compete with their concomitant flower buds for carbohydrates during the early growth stages, potentially causing the abortion of the flower buds. The analysis of transcriptomic differentially expressed genes suggested that genes related to sucrose metabolism and auxin response play an important role during flower bud development. Particularly, co-expression network analysis found that AaSPL12 is a hub gene engaged in flower development by collaborating carbohydrate and auxin signals. Yeast Two Hybrid assays revealed that AaSPL12 can interact with AaARP, a protein that serves as an indicator of dormancy. Additionally, the application of exogenous IAA and sucrose can suppress the expression of AaARP, augment the transcriptional abundance of AaSPL12, and consequently expedite flower development in Anthurium andraeanum. CONCLUSIONS: Collectively, our findings indicated that the combination of auxin and sugar signals could potentially suppress the repression of AaARP protein to AaSPL12, thus advancing the development of flower buds in Anthurium andraeanum.

Sequential activation of strigolactone and salicylate biosynthesis promotes leaf senescence.

Leaf senescence is a complex process strictly regulated by various external and endogenous factors. However, the key signaling pathway mediating leaf senescence remains unknown. Here, we show that Arabidopsis SPX1/2 negatively regulate leaf senescence genetically downstream of the strigolactone (SL) pathway. We demonstrate that the SL receptor AtD14 and MAX2 mediate the age-dependent degradation of SPX1/2. Intriguingly, we uncover an age-dependent accumulation of SLs in leaves via transcriptional activation of SL biosynthetic genes by the transcription factors (TFs) SPL9/15. Furthermore, we reveal that SPX1/2 interact with the WRKY75 subclade TFs to inhibit their DNA-binding ability and thus repress transcriptional activation of salicylic acid (SA) biosynthetic gene SA Induction-Deficient 2, gating the age-dependent SA accumulation in leaves at the leaf senescence onset stage. Collectively, our new findings reveal a signaling pathway mediating sequential activation of SL and salicylate biosynthesis for the onset of leaf senescence in Arabidopsis.

SPL16 and SPL23 mediate photoperiodic control of seasonal growth in Populus trees.

Perennial trees in boreal and temperate regions undergo growth cessation and bud set under short photoperiods, which are regulated by phytochrome B (phyB) photoreceptors and PHYTOCHROME INTERACTING FACTOR 8 (PIF8) proteins. However, the direct signaling components downstream of the phyB-PIF8 module remain unclear. We found that short photoperiods suppressed the expression of miR156, while upregulated the expression of miR156-targeted SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE 16 (SPL16) and SPL23 in leaves and shoot apices of Populus trees. Accordingly, either overexpression of MIR156a/c or mutagenesis of SPL16/23 resulted in the attenuation of growth cessation and bud set under short days (SD), whereas overexpression of SPL16 and SPL23 conferred early growth cessation. We further showed that SPL16 and SPL23 directly suppressed FLOWERING LOCUS T2 (FT2) expression while promoted BRANCHED1 (BRC1.1 and BRC1.2) expression. Moreover, we revealed that PIF8.1/8.2, positive regulators of growth cessation, directly bound to promoters of MIR156a and MIR156c and inhibited their expression to modulate downstream pathways. Our results reveal a connection between the phyB-PIF8 module-mediated photoperiod perception and the miR156-SPL16/23-FT2/BRC1 regulatory cascades in SD-induced growth cessation. Our study provides insights into the rewiring of a conserved miR156-SPL module in the regulation of seasonal growth in Populus trees.

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 13 (SLP13) together with SPL9 redundantly regulates wax biosynthesis under drought stress.

Wax biosynthesis is closely controlled by many regulators under different environmental conditions. We have previously shown that the module miR156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE9 (SPL9)-DEWAX is involved in the diurnal regulation of wax production; however, it was not determined whether other SPLs are also involved in wax synthesis. Here, we report that SPL13 also regulates drought-induced wax production, by directly and indirectly affecting the expression of the two wax biosynthesis genes ECERIFERUM1 (CER1) and CER4, respectively. In addition, we show that SPL13 together with SPL9 redundantly regulates wax accumulation under both normal and drought stress conditions, and that simultaneous mutation of both genes additively increases cuticle permeability and decreases drought tolerance. However, in contrast to SPL9, SPL13 does not seem to participate in the DEWAX-mediated diurnal regulation of wax production.

The transcription factor OsSPL9 endows rice with copper deficiency resilience.

Copper (Cu) is a crucial micronutrient essential for the growth and development of plants. Rice exhibits remarkable resistance to Cu deficiency, but the underlying molecular mechanisms are not well understood. In this study, we reveal that the plant's ability to withstand Cu deficiency is orchestrated by a transcription factor known as OsSPL9. We have demonstrated that OsSPL9 functions as a central regulator of Cu homeostasis. Disrupting OsSPL9 through knockout significantly reduces the plant's tolerance to Cu deficiency. As a result, the spl9 mutants exhibit reduced Cu accumulation in their shoots when compared to wild-type plants. This reduction is linked to a disruption in the transport of Cu from older leaves to younger ones. Furthermore, we show that OsSPL9 directly binds to GTAC motifs in the promoters of key genes involved in Cu uptake and transport, as well as Cu-miRNAs, and enhances their transcription under Cu-deficient conditions. Overall, our findings shed light on the molecular basis of rice resilience to Cu deficiency stress and place the transcription factor OsSPL9 as a master regulator of this response.

Formation of low-pressure reaction textures during near-isothermal exhumation of hot orogenic crust (Bohemian Massif, Austria).

Two types of aluminous paragneiss from the Loosdorf complex (Bohemian Massif, NE Austria) contain coarse-grained granulite assemblages and retrograde reaction textures that are investigated to constrain the post-peak history of the Gföhl unit in the southern Bohemian Massif. Both types have a peak assemblage garnet-biotite-sillimanite-plagioclase-K-feldspar-quartz-granitic melt ± kyanite ± ilmenite ± rutile, recording peak metamorphic conditions of ∼0.9-1.1 GPa and ∼780-820°C estimated by isochemical phase equilibrium modelling. The first sample type (Ysper paragneiss) developed (i) cordierite coronae around garnet and (ii) cordierite-spinel and cordierite-quartz reaction textures at former garnet-sillimanite interfaces. Calculated chemical potential relationships indicate that the textures formed in the course of a post-peak near-isothermal decompression path reaching ∼0.4 GPa. Texture formation follows a two-step process. Initially, cordierite coronae grow between garnet and sillimanite. As these coronae thicken, they facilitate the development of local compositional domains, leading to the formation of cordierite-spinel and cordierite-quartz symplectites. The second sample type (Pielach paragneiss) exhibits only discontinuous cordierite coronae around garnet porphyroblasts but lacks symplectites. The formation of cordierite there also indicates near-isothermal decompression to 0.4-0.5 GPa and 750-800°C. This relatively hot decompression path is explained by the contemporaneous exhumation of a large HP-UHT granulite body now underlying the Loosdorf complex. The timing of regional metamorphism in the granulites and the southern Bohemian Massif in general is well constrained and has its peak at ∼340 Ma. Monazite from Loosdorf paragneiss samples yield a slightly younger age of ∼335 Ma. Although the ages overlap within error, they are interpreted to reflect near-isothermal decompression and exhumation resulting in the formation of the observed reaction textures.

Flowering onset time is regulated by microRNA-mediated trehalose-6-phosphate signaling in Cajanus cajan L. under elevated CO2.

CO2 levels are known to have an impact on plant development and physiology. In the current study, we have investigated the effect of elevated CO2 on flowering and its regulation through miRNA mediated sugar signaling. We also unraveled small RNA transcriptome of pigeonpea under ambient and elevated CO2 conditions and predicted the targets for crucial miRNAs through computational methods. The results have shown that the delayed flowering in pigeonpea under elevated CO2 was due to an imbalance in C:N stoichiometry and differential expression pattern of aging pathway genes, including SQUAMOSA PROMOTER BINDING PROTEIN-LIKE. Furthermore, qRT PCR analysis has revealed the role of miR156 and miR172 in mediating trehalose-6-phosphate dependent flowering regulation. The current study is crucial in understanding the responses of flowering patterns in a legume crop to elevated CO2 which showed a significant impact on its final yields. Also, these findings are crucial in devising effective crop improvement strategies for developing climate resilient crops, including pigeonpea. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01434-9.

ALTERED MERISTEM PROGRAM1 regulates leaf identity independently of miR156-mediated translational repression.

In Arabidopsis, loss of the carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) produces an increase in the rate of leaf initiation, an enlarged shoot apical meristem and an increase in the number of juvenile leaves. This phenotype is also observed in plants with reduced levels of miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors, suggesting that AMP1 might promote SPL activity. However, we found that the amp1 mutant phenotype is only partially corrected by elevated SPL gene expression, and that amp1 has no significant effect on SPL transcript levels, or on the level or the activity of miR156. Although AMP1 has been reported to promote miRNA-mediated translational repression, amp1 did not prevent the translational repression of the miR156 target SPL9 or the miR159 target MYB33. These results suggest that AMP1 regulates vegetative phase change downstream of, or in parallel to, the miR156/SPL pathway, and that it is not universally required for miRNA-mediated translational repression.

Molecular characterization of SPL gene family during flower morphogenesis and regulation in blueberry.

The SPL gene is a plant-specific transcription factor involved in the regulation of plant growth and development, which have been identified in woody plants. The process of floral bud differentiation affects the timing of flowering and fruit set and regulates plant growth, however, the mechanism of regulation of flower development by SPL genes is less studied. In this study, 56 VcSPL genes were identified in the tetraploid blueberry. The VcSPL gene family was classified into six subfamilies, and analysis of cis-elements showed that VcSPL genes were regulated by light, phytohormones (abscisic acid, MeJA), and low temperature. In the evolutionary analysis, segmental replication may play an important role in VcSPL gene amplification. Interestingly, we also studied diploid blueberry (Bilberry), in which 24 SPL genes were identified, and 36 homologous pairs were found, suggesting a high degree of convergence in the syntenic relationship between blueberry (Vaccinium corymbosum L) and bilberry (Vaccinium darrowii). Based on the expression profile, VcSPL genes were expressed at high levels in flowers, shoots, and roots, indicating a diversity of gene functions. Then we selected 20 differentially-expressed SPL genes to further investigate the role of VcSPL in floral induction and initiation. It showed that the genes VcSPL40, VcSPL35, VcSPL45, and VcSPL53 may play a crucial role in the blueberry floral transition phase (from vegetative growth to flower initiation). These results provided important information for understanding and exploring the role of VcSPLs in flower morphogenesis and plant growth.

CRISPR-induced miRNA156-recognition element mutations in TaSPL13 improve multiple agronomic traits in wheat.

Increase in grain yield is always a major objective of wheat genetic improvement. The SQUAMOSA promoter-binding protein-like (SPL) genes, coding for a small family of diverse plant-specific transcription factors, represent important targets for improving grain yield and other major agronomic traits in rice. The function of the SPL genes in wheat remains to be investigated in this respect. In this study, we identified 56 wheat orthologues of rice SPL genes belonging to 19 homoeologous groups. Like in rice, nine orthologous TaSPL genes harbour the microRNA156 recognition elements (MRE) in their last exons except for TaSPL13, which harbour the MRE in its 3'-untranslated region (3'UTR). We modified the MRE of TaSPL13 using CRISPR-Cas9 and generated 12 mutations in the three homoeologous genes. As expected, the MRE mutations led to an approximately two-fold increase in the TaSPL13 mutant transcripts. The phenotypic evaluation showed that the MRE mutations in TaSPL13 resulted in a decrease in flowering time, tiller number, and plant height, and a concomitantly increase in grain size and number. The results show that the TaSPL13 mutants exhibit a combination of different phenotypes observed in Arabidopsis AtSPL3/4/5 mutants and rice OsSPL13/14/16 mutants and hold great potential in improving wheat yield by simultaneously increasing grain size and number and by refining plant architecture. The novel TaSPL13 mutations generated can be utilized in wheat breeding programmes to improve these agronomic traits.

A ubiquitin-specific protease functions in regulating cell death and immune responses in rice.

Ubiquitin-specific proteases (UBPs) process deubiquitination in eukaryotic organisms and are widely involved in plant development and responses to environmental stress. However, their role in cell death and plant immunity remains largely unknown. Here, we identified a rice lesion mimic mutant (LMM) and cloned its causative gene, LMM22. Both dysfunction and overexpression of LMM22 gave rise to the hypersensitive response-like cell death, reactive oxygen species bursts, and activated defence responses. LMM22 encodes an active UBP that is localised to the endoplasmic reticulum (ER) and displays a constitutive expression pattern in rice. LMM22 interacts with SPOTTED LEAF 35 (SPL35), a coupling of ubiquitin conjugation to ER degradation domain-containing protein that is known to participate in ubiquitination and the regulation of cell death and disease response in rice. Additional analyses suggest that LMM22 can positively regulate and stabilise the abundance of SPL35 protein likely through its deubiquitination activity. These data therefore improve our understanding of the function of UBP in rice innate immune responses by demonstrating that LMM22 functions as a critical regulator of SPL35 in cell death and disease resistance.

Hydrogen peroxide regulates the Osa-miR156-OsSPL2/OsTIFY11b module in rice.

Field-grown rice (Oryza sativa L.), when exposed to various environmental stresses, produces high amounts of reactive oxygen species, such as H2 O2 . MicroRNAs (miRNAs) play crucial roles in plant stress responses. This study characterized the functions of H2 O2 -regulated miRNAs in rice. Small RNA deep sequencing revealed that miR156 levels decreased following H2 O2 treatment. Searches of the rice transcriptome and degradome databases indicated that OsSPL2 and OsTIFY11b are miR156-target genes. Interactions between miR156 and OsSPL2 and OsTIFY11b were confirmed using transient expression assays through agroinfiltration. In addition, the levels of OsSPL2 and OsTIFY11b transcripts were lower in transgenic rice plants overexpressing miR156 than in wild-type plants. The OsSPL2-GFP and OsTIFY11b-GFP proteins were localized to the nucleus. Yeast two-hybrid and bimolecular fluorescence complementation assays indicated interactions between OsSPL2 and OsTIFY11b. Furthermore, OsTIFY11b interacted with OsMYC2 to regulate the expression of OsRBBI3-3, which encodes a proteinase inhibitor. The results suggested that H2 O2 accumulation in rice suppresses the expression of miR156, and induces the expression of its target genes, OsSPL2 and OsTIFY11b, whose proteins interact in the nucleus to regulate the expression of OsRBBI3-3, which is involved in plant defense.

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 and 13 repress BLADE-ON-PETIOLE 1 and 2 directly to promote adult leaf morphology in Arabidopsis.

The juvenile-to-adult phase transition during vegetative development is a critical decision point in a plant's life cycle. This transition is mediated by a decline in levels of miR156/157 and an increase in the activities of its direct targets, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) proteins. In Arabidopsis, the juvenile-to-adult transition is characterized by an increase in the length to width ratio of the leaf blade (a change in the distal region of a leaf), but what mediates this change in lamina shape is not known. Here, we show that ectopic expression of SPL9 and SPL13 produces enlarged and elongated leaves, resembling leaves from the blade-on-petiole1 (bop1) bop2 double mutant. The expression of BOP1/BOP2 is down-regulated in successive leaves, correlating with the amount of miR156 and antagonistic to the expression of SPL9 and SPL13 in leaves. SPL9 and SPL13 bind to the promoters of BOP1/BOP2 directly to repress their expression, resulting in delayed establishment of proliferative regions in leaves, which promotes more blade outgrowth (the distal region of a leaf) and suppresses petiole development (the proximal region of a leaf). Our results reveal a mechanism for leaf development along the proximal-distal axis, a heteroblastic character between juvenile leaves and adult leaves.

The transcription factor SPL13 mediates strigolactone suppression of shoot branching by inhibiting cytokinin synthesis in Solanum lycopersicum.

Plant architecture imposes a large impact on crop yield. IDEAL PLANT ARCHITECTURE 1 (IPA1), which encodes a SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor, is a target of molecular design for improving grain yield. However, the roles of SPL transcription factors in regulating tomato (Solanum lycopersicum) plant architecture are unclear. Here, we show that the expression of SPL13 is down-regulated in the lateral buds of strigolactone (SL)-deficient ccd mutants and is induced by GR24 (a synthetic analog of SL). Knockout of SPL13 by CRISPR/Cas9 resulted in higher levels of cytokinins (CKs) and transcripts of the CK synthesis gene ISOPENTENYL TRANSFERASES 1 (IPT1) in the stem nodes, and more growth of lateral buds. GR24 suppresses CK synthesis and lateral bud growth in ccd mutants, but is not effective in spl13 mutants. On the other hand, silencing of the IPT1 gene inhibited bud growth of spl13 mutants. Interestingly, SL levels in root extracts and exudates are significantly increased in spl13 mutants. Molecular studies indicated that SPL13 directly represses the transcription of IPT1 and the SL synthesis genes CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7) and MORE AXILLARY GROWTH 1 (MAX1). The results demonstrate that SPL13 acts downstream of SL to suppress lateral bud growth by inhibiting CK synthesis in tomato. Tuning the expression of SPL13 is a potential approach for decreasing the number of lateral shoots in tomato.

Protein kinase CK2 phosphorylates a conserved motif in the Notch effector E(spl)-Mγ.

Across metazoan animals, the effects of Notch signaling are mediated via the Enhancer of Split (E(spl)/HES) basic Helix-Loop-Helix-Orange (bHLH-O) repressors. Although these repressors are generally conserved, their sequence diversity is, in large part, restricted to the C-terminal domain (CtD), which separates the Orange (O) domain from the penultimate WRPW tetrapeptide motif that binds the obligate co-repressor Groucho. While the kinases CK2 and MAPK target the CtD and regulate Drosophila E(spl)-M8 and mammalian HES6, the generality of this regulation to other E(spl)/HES repressors has remained unknown. To determine the broader impact of phosphorylation on this large family of repressors, we conducted bioinformatics, evolutionary, and biochemical analyses. Our studies identify E(spl)-Mγ as a new target of native CK2 purified from Drosophila embryos, reveal that phosphorylation is specific to CK2 and independent of the regulatory CK2-ß subunit, and identify that the site of phosphorylation is juxtaposed to the WRPW motif, a feature unique to and conserved in the Mγ homologues over 50 × 106 years of Drosophila evolution. Thus, a preponderance of E(spl) homologues (four out of seven total) in Drosophila are targets for CK2, and the distinct positioning of the CK2 and MAPK sites raises the prospect that phosphorylation underlies functional diversity of bHLH-O proteins.