Developing barley grains must balance the use of imported sucrose between soluble sugars and storage starch, but how this balance is controlled remains unclear. Here we show that loss of the plastidial α-glucan phosphorylase HvPHO1 causes starch and soluble sugars to accumulate concurrently in barley grains. Developing pho1 grains showed sustained increases in glucose 1-phosphate (G1P) and adenosine diphosphate glucose (ADP-glucose), and exogenous G1P also promoted starch accumulation, supporting a stimulatory effect of elevated G1P on starch synthesis. G1P directly bound sucrose non-fermenting 1-related protein kinase 1 (HvSnRK1), suppressing its kinase activity and its phosphorylation of sucrose synthase 2 (HvSUSY2); the metabolic phenotypes of snrk1 and susy2 mutants supported a role for this phosphorylation module in sucrose utilization and soluble-sugar homeostasis. HvPHO1 also interacted with the starch-granule initiation factor starch synthase 4 (HvSS4). Loss of HvPHO1 increased the abundance of B-type starch granules and shifted their size distribution, indicating a parallel role in storage-starch organization. pho1 lines showed greater grain dry weight and test weight. Thus, loss of HvPHO1 initiates G1P-mediated metabolic feedback that reconfigures the balance between sucrose utilization and starch accumulation and is associated with enhanced grain carbon deposition and grain weight. These findings reveal G1P-dependent feedback linking starch-precursor status to SnRK1-mediated carbon allocation in barley endosperm.
Goji is recognized as a nutritionally rich superfruit with medicinal and industrial importance. However, its inherently indeterminate growth habit causes asynchronous flowering and uneven fruit ripening, resulting in harvesting inefficiency with high labour costs and low fruit yield. To elucidate the molecular basis of shoot determinacy and early flowering, comparative transcriptome analysis of indeterminate and determinate shoot apices in an F1 hybrid population of Lycium barbarum identified 15 candidate genes associated with shoot growth termination and early flowering, including an SP-like gene (LbSP1, Lba0102749) and a goji homologue of SlSP5G (LbSP5G1, Lba0501086) from the phosphatidylethanolamine-binding protein (PEBP) family. CRISPR/Cas9-mediated knockout of LbSP1 and LbSP5G1 in black goji berry produced homozygous single (CRLbSP1, CRLbSP5G1) and double determinate (2gCR LbSP1 + LbSP5G1) lines exhibiting compact shoot architecture, reduced day-length sensitivity, terminal flowering, and improved fruit yield. The double determinate line showed a significantly reduced number of leaves to the first inflorescence (7.25) compared to wild-type (WT) (18.6), with fruit yield per plant of 174.94 g, followed by CRLbSP5G1 (64.55 g), CRLbSP1 (55.17 g), and WT (22.09 g). Transcriptomic and co-expression analysis revealed extensive reprogramming of flowering regulatory networks, with LbSP1 and LbSP5G1 functioning as central regulatory hubs. Protein-protein interaction assays further established functional divergence between the two genes within a PEBP-centred network governing meristem fate. These findings provide the first functional characterization of terminal flowering genes in goji, establishing a molecular framework for breeding compact, early-flowering, and high-yield cultivars optimized for mechanized harvesting.
Understanding how numerous heterotic quantitative trait loci (hQTL) shape heterosis and shifting from hQTL to candidate genes are central challenges. To address these challenges, we performed a systematic, genome-wide mapping in a biparental population and a population of diverse hybrids. Leveraging both a triple testcross and immortalized F2 designs, we examined the genetic basis of the heterosis of the wheat hybrid Piko × Hermann. We detected a major hQTL on chromosome 4B, primarily driven by epistasis. This region contains the Green Revolution gene Rht-B1 as the primary candidate gene. Then, we used a population of ~6000 wheat hybrids, derived from crosses between diverse Central European inbred lines, to perform a one-dimensional scan for hQTL. This scan identified 174 hQTL for grain yield, 70 for heading date, and 166 for plant height. Further dissection of these hQTL revealed that epistatic interactions predominantly contribute to heterosis. We discovered an epistatic hub at the distal end of chromosome 4A coinciding with an alien introgression region from emmer wheat. A data-driven, integrative analysis combining high-resolution SNP data, sequence variant annotation, and hQTL signals from the population uncovered TraesCS7B03G1341000 as the candidate gene underlying grain yield heterosis. Our findings deepen the understanding of the genetic architecture of heterosis in wheat by shifting from hQTL to candidate genes and provide insights into their application in hybrid wheat breeding.
MYB transcription factors play crucial roles in regulating plant growth and development, but the functions of the MYB-related subfamily in woody plants remain poorly understood. In this study, we characterized PagMYBR028, an MYB-related transcription factor from poplar '84 K' (Populus alba × Populus glandulosa), which was predominantly expressed in leaves and xylem. Compared with WT plants, PagMYBR028-overexpressing transgenic poplar exhibited reduced leaf area and retarded secondary xylem development, whereas PagMYBR028-RNAi lines displayed the opposite phenotypes. Further analysis revealed that PagMYBR028 interacts with PagMYBR005, a related regulator with overlapping functions in poplar development. Both proteins directly repress the expression of PagGRF12b, and their interaction enhances this transcriptional repression. In addition, overexpression of PagGRF12b promoted secondary cell wall deposition. Taken together, our results delineate a PagMYBR028-PagMYBR005-PagGRF12b module that coordinately regulates poplar growth and secondary wall development, providing new insights into the transcriptional network underlying wood formation and a theoretical basis for molecular breeding in trees.
Fruit cuticles regulate the diffusion of water, gases and solutes, functioning as essential protective interfaces against environmental and biotic stresses. Engineering cuticle structure and mechanics therefore represents a promising strategy for improving fruit quality and resilience. To enhance biosynthesis of cutin, the major structural component of the cuticle, we generated transgenic tomato lines expressing the Arabidopsis thaliana GLYCEROL-3-PHOSPHATE sn-2-ACYLTRANSFERASE 4 (AtGPAT4) gene specifically in exocarp tissues. AtGPAT4 is a key enzyme providing acylglycerol intermediates for cutin polymer assembly. We performed comprehensive phenotypic, microscopic, spectroscopic, transcriptional and surface nanomechanical analyses to monitor the structural consequences of AtGPAT4 overexpression and evaluated functional traits including water loss and susceptibility to the Botrytis cinerea fungus. Contrary to expectations, exocarp-targeted expression of AtGPAT4 generated fruits with cuticles that had diminished levels of cutin and epicuticular waxes, together with decreased cuticular stiffness and resistance to deformation, and whole fruit firmness. Evidently, these structural and chemical alterations did not affect thermal stability or water retention, but they increased susceptibility to fungal infection by B. cinerea. Although the engineering outcomes were unintended, they offered mechanistic insights into how manipulation of cutin biosynthesis pathways reshapes cuticle architecture, polymer composition and nanomechanical performance. This work underscores the complexity of engineering fruit surface traits and offers new perspectives for future biotechnological strategies aimed at improving cuticle robustness and pathogen resistance.
Rice fragrance is a key determinant of grain quality, yet the aroma profile of cultivated rice is highly uniform and largely dominated by the popcorn-like compound 2-acetyl-1-pyrroline. Here we expanded the aromatic repertoire of rice by engineering a rose-like fragrance through reconstruction of the phenylalanine-derived 2-phenylethanol (2-PE) pathway in the endosperm. To increase phenylalanine precursor supply, we co-expressed an engineered G211R/G212S variant of the rice 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase isoform OsDHS2 (LOC_Os08g37790; mOsDHS2) with heterologous phenylacetaldehyde synthase (PAAS) and phenylacetaldehyde reductase (PAR) in the endosperm. As a result, the engineered grains accumulated up to 2014 μg/kg 2-PE, conferring a distinct rose-like aroma. Unexpectedly, metabolic rewiring also reshaped grain composition, resulting in nearly doubled soluble protein content and increased levels of several vitamin B6-related metabolites, including the active coenzyme forms pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate. These findings reveal extensive associated metabolic coordination between aromatic amino acid pathways and central seed metabolism. Our work establishes a strategy for creating designer fragrances in staple crops while simultaneously improving nutritional traits, providing a versatile platform for producing high-value metabolites in cereal grains.
Enzymatic modification using glycosyltransferases offers a green route to produce highly branched starch to enhance starch functionality in the food industry, yet this approach faces challenges including the high cost of enzyme production and inefficiency. Here, we developed a viable in planta strategy by heterologously expressing an α-glucan branching enzyme (GBE) from Geobacillus thermoglucosidans STB02 (GtGBE) in rice, aiming to tailor starch branching structures directly in a living plant system. Several independent transgenic lines were generated, expressing the GtGBE gene under the control of either the endosperm-specific rice starch branching enzyme IIb (BEIIb) promoter (pBEIIb) or the constitutive maize Ubiquitin promoter (pUbi). GtGBE generated distinct promoter-dependent branching patterns. The pBEIIb-driven construct partially recapitulated BEI-deficient-like profiles through the combined effect of downregulated endogenous BEI and relatively low GtGBE expression levels. In contrast, the pUbi-driven construct enhanced BEIIb-like activity through sustained high GtGBE accumulation and upregulation of endogenous BEIIb. Both strategies increased short amylopectin chains at the expense of intermediate/long chains and amylose, resulting in decreased relative crystallinity, apparent amylose content, gelatinization temperatures, and peak viscosity, alongside improved retrogradation resistance and rheological properties. Notably, GtGBE expression driven by the BEIIb promoter showed greater stability under heat stress. Moreover, pUbi-GtGBE transgenic lines exhibited a significant increase in 1000-grain weight and filled grain number per panicle without compromising other agronomic traits, highlighting the dual potential of this strategy for agricultural and industrial applications. This work establishes GtGBE as an effective molecular tool for the direct and tailored bioengineering of starch in a living plant system.
The precise enhancement of nutritional quality in silage maize is a core strategy for increasing livestock production efficiency. Through evolutionary analysis of multiple plant species, we identified two functionally synergistic upstream open reading frames (uORF1 and uORF2) within the 5' untranslated region of the gene encoding GDP-L-galactose phosphorylase (GGP). By leveraging a natural translation-enhancing haplotype of uORF1 (Hap2) and performing CRISPR/Cas9-mediated targeted mutagenesis of the highly conserved uORF2, we successfully engineered an elite dual-uORFs variant. This variant significantly increased vitamin C (Vc) content and concurrently improved key silage quality traits, including crude protein and phosphorus levels, without inducing growth penalties. Transcriptomic profiling further elucidated the molecular mechanisms by which the dual-uORFs variation coordinately regulates Vc biosynthesis and the improvement of silage quality. Our findings deepen the understanding of the conventional paradigm of single-uORF regulation and provide a novel strategy and superior germplasm resources for the precision breeding of high-Vc, high-quality silage maize.
Drought stress is a major limitation to global wheat production. Here, we demonstrate that the wheat gene TaCOMT1A, encoding a caffeic acid O-methyltransferase, plays a crucial role in enhancing drought tolerance in wheat. Overexpression of TaCOMT1A significantly improved drought tolerance at the seedling stage, as evidenced by higher survival rates, biomass, and antioxidant capacity, along with reduced oxidative damage in transgenic lines. Field trials demonstrated that these lines maintained superior grain yield under limited irrigation. We identified TaCOMT1A as a multifunctional enzyme capable of synthesising both the flavonoid sakuranetin and melatonin in vitro. Metabolomic and functional analyses confirmed that sakuranetin is a key downstream metabolite mediating the drought tolerance conferred by TaCOMT1A. Exogenous application of sakuranetin enhanced drought tolerance across diverse wheat cultivars and, importantly, rescued the susceptible phenotype of TaCOMT1A EMS mutants (E829 and E830). Mechanistically, sakuranetin treatment bolstered the antioxidant system and attenuated oxidative stress under drought. Furthermore, both TaCOMT1A overexpression and sakuranetin application reduced plant height by suppressing gibberellic acid (GA3) biosynthesis. Crucially, field application of sakuranetin increased grain yield under both well-irrigated and drought conditions. Our results establish a novel pathway where TaCOMT1A enhances drought tolerance and modulates plant architecture primarily through the production of sakuranetin, positioning this metabolite as a promising plant-based priming agent for sustainable wheat cultivation.
Sucrose transport and starch accumulation are crucial carbohydrate metabolic processes in rice, playing essential roles during late pollen development to ensure pollen maturation, fertility and high grain production. However, the molecular mechanisms regulating sucrose transport and starch accumulation during rice pollen development remain elusive. In this study, we bred and characterized a semi-fertile rice pollen variant, sfp10, which is derived from highland terraced red rice from Yunnan, China. This variant showed insufficient starch accumulation in pollen, reduced pollen fertility and decreased seed setting compared to the wild type. Using map-based cloning, CRISPR/Cas9 mutagenesis and gene complementation, the OsSUT3 gene, which causes the sfp10 variant, was shown to encode a sucrose transporter (SUT) that transports sucrose into pollen for starch synthesis, ensuring pollen viability and fertility. Additionally, transcriptome analysis identified OsWRKY15, a WRKY transcription factor that exhibits a highly similar spatiotemporal expression pattern to OsSUT3 in late-developing panicles. Yeast one-hybrid, EMSA, dual-luciferase reporter and ChIP-seq assays demonstrated that OsWRKY15 directly binds to the promoter region of OsSUT3 and upregulates its expression. Mutation of OsWRKY15 significantly reduced OsSUT3 expression and impaired pollen fertility and seed setting and these defects were further exacerbated in the wrky15/OsSUT3-KO double-knockout lines, while OsSUT3 overexpression in the wrky15 background significantly rescued pollen fertility and seed setting. These findings demonstrate that the OsWRKY15-OsSUT3 regulatory module is essential for sucrose transport and starch accumulation during late pollen development in rice, providing new insights into the transcriptional regulation of carbon allocation to anthers for rice fertility and grain production.
Plants maintain remarkable regenerative potential, yet this capacity declines with developmental age, limiting clonal propagation and biotechnological applications. Here, we show that the miR172-AP2 module serves as a central determinant of regeneration competence in Populus. Regenerative capacity declines progressively along the shoot developmental gradient, correlating with elevated miR172 and reduced AP2 transcript levels. Functional analyses demonstrate that AP2 promotes both shoot and root regeneration by directly activating downstream transcriptional programmes, including AINTEGUMENTA-LIKE1, BIG LEAF and LIKE APETALA1, which regulate organogenic growth and developmental competence. Elevated miR172 or cytokinin signalling represses AP2, reducing regenerative outcomes, indicating that hormonal and developmental signals converge on this module. Genome-wide analyses reveal that AP2 coordinates a broad network of genes controlling wound response, hormone signalling and cell fate specification. Notably, enhanced regeneration via AP2 occurs without detectable developmental abnormalities, highlighting the practical potential of this module. Collectively, these findings uncover a mechanistic link between developmental phase identity and regeneration, revealing a conserved regulatory module that may be leveraged to enhance regeneration across plant species, with broad implications for propagation, tissue culture and biotechnology.
Advancing the utility of plant synthetic biology requires the continued development of protein engineering tools. Self-assembling protein compartments, such as virus-like particles (VLPs), provide versatile scaffolds for synthetic biology. However, few plant-expressed VLPs have demonstrated broad amenability to protein engineering, restricting their applications to specific contexts. Here, the Enterobacteria phage P22 VLP is explored as a novel protein scaffold for plant synthetic biology, demonstrating its production in a eukaryote for the first time. Through transient expression in the biofactory plant Nicotiana benthamiana, the capacity for P22 VLPs to correctly assemble and direct encapsulation of recombinant protein cargo is demonstrated. The durability of this protein scaffold is explored through co-encapsulation of multiple cargo protein species and by encapsulation through direct fusion to the P22 coat protein. Finally, the ability to simultaneously program cargo encapsulation and external protein display on P22 VLPs in vivo is demonstrated through SpyTag/SpyCatcher-mediated protein conjugation. This work demonstrates the broad utility of P22 VLPs as nanoscale protein scaffolds for plant synthetic biology.
The circadian clock plays critical roles in orchestrating temporal regulation of diverse physiological processes. However, its role in mediating root-associated immunity against soil-borne pathogens in crops remains poorly understood. Here, we show that cotton circadian clock gates nighttime resistance to vascular fungal pathogen Verticillium dahliae by shaping rhizosphere microbiome composition and activating jasmonic acid (JA)-dependent immune signalling. Pathogen infection, in turn, accelerates clock pace and perturbs nighttime microbial community structure. We identify GhLUX, an evening complex component, as a key regulator of this defence program. GhLUX enhances immunity by repressing GhJAZ expression, thereby amplifying JA signalling at night. Overexpression of GhLUX in field trials confers enhanced resistance to V. dahliae while simultaneously improving cotton fibre yield and quality. These findings uncover a mechanistic link between the circadian clock, rhizosphere microbiota and root immunity in cotton and suggest that circadian regulators can be harnessed to optimize both disease resistance and agronomic performance.
Terpenes constitute the largest and most structurally diverse class of plant secondary metabolites, with critical roles in plant-environment interactions and broad industrial applications. Although nuclear genome engineering of terpene pathways has been extensively explored, chloroplast genome engineering remains largely undeveloped, with most reported studies restricted to the model plant Nicotiana. Here we report successful chloroplast genome engineering for diterpene production in the crop plant potato (Solanum tuberosum) guided by evolutionary principles. First, we identified the trnT/trnL plastomic locus as a new transgene integration site with minimal integration-associated growth penalties. Insertion of a bifunctional diterpene synthase gene from a fern that is absent in flowering plants into this plastomic site yielded transplastomic potato plants with successful production of new diterpenes, but with reduced growth. The co-expression of an algal geranylgeranyl diphosphate synthase gene of chloroplast genome origin to enhance precursor supply restored normal growth while elevating diterpene production. Transplastomic plants were otherwise agronomically comparable to wild-type. This work expands chloroplast engineering as a viable strategy for evolution-inspired terpene pathway engineering in crop improvement and high-value terpene production.
Our goal is to develop RNA-guided engineering of the chloroplast genome using the CRISPR/Cas9 system. We designed chloroplast minigenes to obtain properly sized single guide RNAs (sgRNAs) in tobacco chloroplasts. The sgRNA 5' end is defined by transcription from an rRNA operon promoter, and its 3' end by processing a downstream tRNA (trnG) or a hepatitis delta virus (HDV) ribozyme. Cas9 is expressed from a nuclear gene and is targeted to chloroplasts by fusion to a transit peptide. Cas9 incorporated the sgRNA and introduced double-strand breaks in the plastid DNA (ptDNA). We report here that the double-strand DNA break in the ndhA and rpoC1 genes was repaired by microhomology-mediated end joining (MMEJ), resulting in deletions in the ptDNA. We further showed that nuclear-expressed sgRNA can be delivered into chloroplasts by fusion with a viroid RNA, as one possible approach for RNA-guided engineering of the ptDNA without direct chloroplast genome transformation. These results are the first step of RNA-guided editing of the chloroplast genome in any crop.
Grain size is a critical agronomic trait, yet the molecular mechanisms governing its determination in crops remain incompletely understood. While recent studies revealed that OsRING80 facilitates DENSE AND ERECT PANICLE 2 (DEP2) degradation to mediate immunity without impacting growth and development, our work identifies a distinct regulatory pathway controlling grain development. We demonstrate that two RING finger E3 ubiquitin ligases, SEVEN IN ABSENTIA 3 (SINA3) and SINA5, post-translationally regulate the stability of DEP2 (also known as SRS1/EP2/OsRELA/SUG1) to modulate grain size. SINA3 and SINA5 physically interact with DEP2, specifically mediated through the C1 structural region of DEP2 containing a coiled-coil domain. These E3 ligases promote K48-linked polyubiquitination of DEP2, targeting it for proteasomal degradation. Liquid chromatography-mass spectrometry (LC-MS) analysis identified six critical lysine residues (K399, K722, K746, K958, K962 and K1344) within DEP2 that are essential for its ubiquitylation and subsequent destabilization. Our genetic evidence further supports this regulatory module: knockout of SINA3 and/or SINA5 leads to DEP2 accumulation, concomitantly increasing grain size and 1000-grain weight significantly, without altering other agronomic traits; conversely, overexpression of SINA3 or SINA5 reduces DEP2 protein levels and diminishes grain size and weight. Therefore, our study uncovers a novel post-translational regulatory module where SINA3 and SINA5 control DEP2 stability to fine-tune grain development. These findings present a promising strategy for optimizing grain yield by manipulating this post-translational regulatory node.
Sesame (Sesamum indicum L., 2n = 26) is one of the oldest oilseed crops and is often called the 'queen of oilseeds' due to its high content of unsaturated fatty acids and natural antioxidants. Despite its long history, the origin and global spread of cultivated sesame remain unresolved. We assembled a telomere-to-telomere (T2T), high-quality reference genome of sesame (cv. Yuzhi11) to investigate sequence differences between genomes and its origin and the local adaptation evolution of flowering time (DF). We generated a 305 Mb T2T sesame reference genome (cv. Yuzhi11) with > 99.99% base-level accuracy, identifying 31 063 protein-coding genes. Repetitive elements accounted for 52.03% of the genome. Population genomic analysis of 927 accessions from 14 regions identified four major groups. Integrative analyses of linkage disequilibrium decay (LD), nucleotide diversity (π), and fixation index (FST) support East Africa as the center of origin, with subsequent migration through the Middle East, to South Asia, South-East Asia, East Asia and ultimately to other parts of the world. Genome-wide association studies (GWAS) and selection scans identified 30 genes associated with flowering time. SiUBP16 is a candidate associated with 7.6% of DF variation. Early-flowering accessions carried up to 225 favourable alleles. A flowering time prediction model for high-latitude regions achieved 96% accuracy. We present a high-quality T2T reference genome for cultivated sesame, shedding light on its origin, evolutionary history, and regional flowering time adaptation. This genome insights valuable tools for breeding programs aimed at improving yield and environmental adaptation in sesame and related crops.
Haploid induction coupled with genome editing (HI-Edit) enables direct modification of commercial crop varieties, bypassing the need for trait introgression or direct transformation of elite lines with CRISPR machinery. However, its widespread application has been constrained by low haploid editing rates (HER), the proportion of haploids carrying edits within the short window between double fertilization and uniparental chromosome elimination. Here, we report substantial improvements in maize HI-Edit efficiency through three complementary strategies: (1) driving an optimized LbCas12a variant (LbCas12aV) using promoters that are highly active in sperm cells and early zygotes; (2) applying a post-pollination heat treatment; and (3) fusing LbCas12aV with the UBA2 domain (ubiquitin-associated domain-2 of Arabidopsis thaliana RAD23) to enhance protein stability during haploid induction. Post-pollination heat treatment alone increased HER to 19.1% (up to 12-fold improvement depending on the target site), providing a simple and effective method to boost the yield of edited doubled haploid (DH) plants. UBA2 fusion improved HER by 6-fold at the Waxy1 (Wx1) locus and 4.5-fold at the Glossy2 (Gl2) locus under normal conditions. Strikingly, combining UBA2 fusion with heat treatment raised the average HER to 25% across multiple events targeting Wx1, with the highest HER reaching 33%. Collectively, these findings demonstrate that increasing CRISPR-Cas protein abundance and modulating environmental conditions can overcome key bottlenecks in HI-Edit. We establish a robust, scalable framework that is readily transferable to other crops for elite-line genome editing.
