An integrated multi-omics analysis of dwarf and normal-height red tangerine × trifoliate orange hybrid seedlings revealed a PtARF6/8-PtGH3.1 transcriptional module that regulates auxin homeostasis through IAA conjugation, providing genetic insights into GH3.1-mediated dwarfism.
Abstract
Dwarfing rootstocks are crucial for high-density and labor-efficient citrus cultivation, yet the molecular mechanisms underlying plant height regulation remain poorly understood. Using an integrated multi-omics analysis of dwarf and normal-height red tangerine (Citrus reticulata) × trifoliate orange (Poncirus trifoliata) hybrid seedlings, we identified the Gretchen Hagen 3.1 gene PtGH3.1, an auxin-conjugating enzyme, as an important regulator of dwarfism. Compared with normal-height hybrid seedlings, PtGH3.1 was significantly upregulated in the shoot tips of dwarf hybrid seedlings, correlating with reduced free auxin (indole-3-acetic acid, IAA) levels. Functional studies in a model citrus system (Citrus macrocarpa) demonstrated that heterologous overexpression of PtGH3.1 decreased free IAA and inhibited plant growth, whereas CRISPR/Cas9-mediated knockout of its homolog CmGH3.1 with editing ratio below 40% promoted growth. Mechanistically, the transcription factors auxin response factor 6 (PtARF6) and auxin response factor 8 (PtARF8) physically interact, forming a heterodimer that activates PtGH3.1 transcription. Dwarf hybrid seedlings exhibited elevated PtARF6 but reduced PtARF8 transcript levels. Heterologous overexpression of either PtARF6 or PtARF8 in Citrus macrocarpa induced dwarfism by activating CmGH3.1 expression. This study reveals a PtARF6/8-PtGH3.1 transcriptional module that regulates auxin homeostasis through IAA conjugation, providing genetic insights into GH3.1-mediated dwarfism and identifying precise targets (PtGH3.1, PtARF6, and PtARF8) for breeding dwarfing rootstocks in citrus.
Drought stress limits forest tree growth and adaptation, with xylem vessels critical for hydraulic transport and structural integrity. However, the molecular mechanisms of abscisic acid (ABA)-auxin interaction in regulating vessel morphogenesis under water-deficit conditions remain unclear. Here, we identified PtomiR393a, a drought-responsive microRNA in Populus tomentosa that mediates crosstalk between ABA and auxin signaling pathways under drought stress. Suppressing PtomiR393 enhanced drought tolerance and growth, whereas its overexpression had the opposite effect. Under drought conditions, suppression of PtomiR393 resulted in reduced vessel size (12.18-13.57%) and increased vessel density (27.22-30.14%), while its overexpression exhibited increased vessel size (15.42-16.01%) and reduced vessel density (19.80-20.62%). Functional assays showed that PtomiR393 specifically targets PtoFBL4, an F-box auxin receptor, modulating auxin signaling in response to drought stress. Expression analyses further revealed that PtomiR393 downregulates genes involved in vessel and fiber formation and secondary cell wall biosynthesis by repressing PtoFBL4-mediated auxin signaling. Furthermore, drought-induced ABA signaling activated PtoERF1 expression via PtoAREB13, thereby inhibiting PtomiR393a expression. The study revealed a PtoERF1-PtomiR393a-PtoFBL4 cascade that links ABA-auxin crosstalk and regulates vessel development under drought stress. These findings offer new insights into drought tolerance mechanisms in trees and suggest potential strategies to enhance forest tree resilience to water-deficit conditions.
Yong-Ming Chen, Mingyang Quan, Dan Wang et al.· New Phytologist· 0 citations
Integrated transcriptomic and metabolomic characterization of knockout mutants (Stgame9) revealed extensive reprogramming of gene expression and metabolism, affecting not only SGA and sterol pathways but also a broader range of metabolic processes, with stress-related metabolic responses being attenuated in Stgame9 tubers.
Ying Liu, Irene Merino, Lizel Potgieter et al.· Plant physiology and biochem...· 0 citations
Salt stress severely limits global crop productivity. Poncirus trifoliata, a widely used citrus rootstock valued for its disease resistance, exhibits sensitivity to salt stress, presenting a major constraint for sustainable citriculture. A stepwise screening strategy identified PtWRKY20 as a key negative regulator of salt tolerance in Poncirus trifoliata. Transcript analysis revealed that PtWRKY20 expression is rapidly induced within 1 h of exposure to salt stress, peaks at 3 h, and then gradually declines from 6 to 24 h, before dropping below the control level by 48 h. Subcellular localization confirmed that PtWRKY20 is a nuclear-localized transcription factor. Functional validation via CRISPR/Cas9-mediated knockout in hairy roots demonstrated enhanced salt tolerance in Ptwrky20-KO plants, evidenced by reduced chlorosis, higher chlorophyll content, lower electrolyte leakage, and decreased malondialdehyde accumulation under salt stress. Conversely, PtWRKY20 overexpression increased sensitivity, showing opposite physiological trends. Integrated DNA affinity purification sequencing and RNA-seq analyses, validated by yeast one-hybrid and dual-luciferase assays, identified 10 direct target genes bound and transcriptionally regulated by PtWRKY20, including six transcription factor genes (e.g., PtMYB78), a sugar metabolism gene (PtHXK3), an ion homeostasis mediator (PtAMT2) and 2 unannotated genes. Our findings establish PtWRKY20 as a negative regulator of the salt stress response and suggest that its targeted knockout via rootstock-adapted CRISPR is a promising strategy for developing salt-tolerant citrus rootstocks.
Trichomes are crucial for plant resistance to biotic and abiotic stresses. In cucumber, the density and morphology of fruit spines directly influence fruit appearance and market value, making them a key agronomic trait. Nevertheless, the genetic regulators governing trichome initiation and development remain poorly understood. In this study, the AP2/ERF transcription factor ENHANCER OF SHOOT REGENERATION 1 (CsESR1) was identified and shown to be predominantly expressed in the epidermis of stems, leaves, flower buds, and ovaries, as confirmed by quantitative reverse transcriptase-polymerase chain reaction and GUS staining analyses. CRISPR/Cas9-mediated knockout of CsESR1 resulted in a marked decrease in spine density, altered spine morphology, and a glabrous phenotype in vegetative tissues including stems, leaves, and tendrils. Furthermore, the defective development of bloom trichomes in the Csesr1 mutant resulted in increased glossiness of the fruit peel. Protein interaction assays revealed that CsESR1 physically associates with key trichome regulators, CsTOE3, CsGL1, and CsTRY. Moreover, Csesr1 plants exhibited reduced aphid resistance, concomitant with altered expression of defense-related genes, phytohormone levels, and antioxidant enzyme activities. Collectively, this study establishes CsESR1 as a key regulator of trichome formation in cucumber and uncovers its role in plant defense, providing novel insights into the molecular networks coordinating epidermal differentiation and stress adaptation.
Piaoyun Sun, Jinqiang Yan, Wenrui Liu et al.· The Plant Journal· 0 citations
The R1R2R3-MYB (3R-MYB) transcription factor subfamily is associated with stress tolerance; however, the underlying mechanisms in crops remain poorly understood. This study investigates the function of maize MYB3R in regulating seedling drought tolerance. We characterised MYB3R overexpression lines and CRISPR-Cas9 loss-of-function mutants in maize and rice using physiological assays and transcriptome profiling. DNA affinity purification sequencing (DAP-seq) and molecular interaction assays were employed to identify direct downstream targets. MYB3R overexpression enhanced drought tolerance by promoting root development, stomatal closure and antioxidant defence, whereas mutants displayed hypersensitivity. MYB3R binds the mitosis-specific activator (MSA) motif to directly transactivate the B-type cyclin gene CYCB1;2, and cycb1;2 mutants phenocopied the myb3r drought defects. These findings establish that the MYB3R-CYCB1;2 module positively regulates maize drought tolerance by coordinating developmental and physiological adaptations. This pathway provides a valuable molecular target for breeding drought-resilient crops.
Kang Guo, Yingli Jiang, Yuxin Guo et al.· The Plant Journal· 0 citations
Abstract Anthocyanins are the primary determinants of floral pigmentation in Chrysanthemum ×morifolium, and ethylene acts as a key regulator of their biosynthesis. Although the ethylene-mediated regulatory circuitry is functionally important, its underlying mechanism in chrysanthemum has remained unclear. In this study, we identified CmMYB4 as a transcriptional repressor that directly suppresses the expression of key anthocyanin biosynthetic genes, including CmDFR (dihydroflavonol 4-reductase), CmUFGT (flavonoid 3-O-glucosyltransferase), and Cm3MaT (anthocyanin 3-O-glucoside-6″-O-malonyltransferase). Time-ordered gene co-expression network analysis comparing the transcriptomes of CmMYB4-overexpressing and control plants, together with molecular biology experimental results, further revealed CmERF3 (ethylene response factor 3) as a hierarchical upstream regulator of anthocyanin biosynthesis. Exogenous ethylene treatment induced CmERF3 expression while reducing anthocyanin accumulation. Subsequent functional characterization showed that the overexpression of CmERF3 suppresses anthocyanin biosynthesis in both tobacco and chrysanthemum by directly activating CmMYB4 and repressing CmDFR, CmUFGT, and Cm3MaT. Collectively, these findings revealed that ethylene inhibits anthocyanin biosynthesis through a CmERF3−CmMYB4−LBGs (late biosynthetic genes) regulatory module. This study not only elucidates the molecular mechanism governing ethylene-mediated anthocyanin inhibition but also provides new perspectives for the molecular engineering of ornamental traits in chrysanthemum.
Mengling Li, Wenjing Zhao, Shuangda Li et al.· Horticulture Research· 0 citations
