Plant synthetic biology holds great promise for engineering plants to meet future demands. Genetic circuits are being designed, built and tested in plants to demonstrate the proof of concept. However, developing these components in monocots, which the world relies on for grain, lags behind dicot models, such as Arabidopsis thaliana and Nicotiana benthamiana. Here, we show the successful adaptation of a ligand-inducible sensor to activate an endogenous anthocyanin pathway in the C4 monocot model Setaria viridis. We identify two transcription factors that can be expressed as a single transcript that are sufficient to induce endogenous anthocyanin production in S. viridis protoplasts and whole plants in a constitutive or ligand-inducible manner. We also test multiple ligands to overcome physical barriers to ligand uptake, identifying triamcinolone acetonide (TA) as a highly potent inducer of this system. Using hyperspectral imaging and a discriminative target characterisation method in a near-remote configuration, we can non-destructively detect anthocyanin production in leaves in response to ligands. This work demonstrates the use of inducible expression systems in monocots to manipulate endogenous pigmentation production for remote detection. Applying inducible anthocyanin production coupled with sensitive detection algorithms could enable crop plants to report on the status of field contamination or detect undesirable chemicals impacting agriculture, ushering in an era of agriculture-based sensor systems.

Flower colour variation is pivotal for plant evolution, ecological adaptation, and pollinator attraction. In the Liriodendron genus, L. tulipifera features a distinct orange petal stripe which is absent in L. chinense, driven by differential carotenoid accumulation, yet the underlying genetic mechanism remains unclear. To address this, we assembled a high-quality 1.57 Gb genome of L. tulipifera 'MSL' and analysed comparative transcriptome data to identify carotenoid biosynthesis-related regulatory factors. Transcriptomic analysis revealed specific activation of the carotenoid biosynthesis pathway in the orange stripe region of L. tulipifera, with specific expression of the lycopene ε-cyclase gene (LCYE). A subgroup 2 R2R3-MYB transcription factor, designated RCP (Regulated Carotenoid Pigment), was found to bind the LCYE promoter and promote carotenoid biosynthesis. Notably, two indel variants (24 and 15 bp) in the third exon of L. chinense LcRCP altered its function, reducing LCYE activation and carotenoid content, and these variants are widespread in natural L. chinense populations. Transient expression assays in N. benthamiana and Liriodendron leaves showed L. tulipifera LtRCP promoted carotenoid accumulation, while LcRCP inhibited it. Our findings uncover a natural regulatory variation governing interspecific flower colour divergence in Liriodendron, providing new insights into carotenoid pathway regulation and its role in floral trait evolution.

Phosphorus (P) is an essential macronutrient for various biological processes in plant growth. Modern agricultural science has advanced the knowledge of regulatory mechanisms underlying phosphorus starvation responses (PSRs), aiming to develop phosphate-efficient crops with sustainable production under reduced Pi fertilizer application. However, information regarding coordinated shoot and root adaptations in response to combined nutrient stresses is limited. This study investigated the role of Phloem Phosphate Stress Repressed 1 (PPSR1) in modulating PSRs and other nutrient deficiency adaptations. The Arabidopsis functional homologue of Cucumis sativus PPSR1 (CsPPSR1), designated AtPPSR1, was identified. AtPPSR1 encodes a glycine-rich domain-containing protein, and its ectopic expression confers enhanced growth performance to plants. Transcriptomic analyses revealed AtPPSR1 as a regulatory mediator of PSRs, photosynthesis, and root development. AtPPSR1 interacted with PHOSPHATE STARVATION RESPONSE 1 (PHR1) to regulate PHR1-target genes for adaptive root development in response to Pi-starvation stress. Additionally, AtPPSR1 was graft-transmissible, and shoot-borne AtPPSR1 played a role in restoring the root phenotype of the ppsr1 mutant. Physiological analyses revealed that enhanced AtPPSR1 expression enabled resilience to nitrogen (N) and potassium (K)-starvation, as well as to Pi-deficiency. Furthermore, we identified homologues of CsPPSR1 and AtPPSR1 in Brassica napus (canola), which displayed similar expression patterns in response to Pi-starvation stress. Overexpression of PPSR1, identified from Arabidopsis, cucumber, and canola, improved growth performance and seed production in canola under N-, Pi-, or K-deficient conditions, within the controlled environment. These findings provide novel insights into PPSR1-mediated molecular coordination to enhance plant resilience to mineral nutrient deficiency.

Transient expression of recombinant proteins in leaves of Nicotiana benthamiana is routinely employed for both basic research and manufacturing of biopharmaceutical products in plants. Relying on disarmed strains of the bacterial plant pathogen Agrobacterium tumefaciens as a transgene vector, this safe, cost-effective and easily scalable 'plant molecular farming' approach offers a reliable alternative to classical protein expression platforms. Commonly referred to as agroinfiltration, scaled-up versions of this manufacturing process have now become helpful in the fight against global health issues, such as those rapidly evolving virus strains causing influenza or coronavirus disease 2019. In the past decades, considerable efforts have been deployed to improve the efficacy of Agrobacterium-mediated expression, including through the development of new binary vectors, the design of strong promoters, and the deployment of approaches to increase levels and stability of transgene mRNAs. By comparison, much less attention has been given to understanding the effects that agroinfiltration unavoidably has on host plants, including the infiltration process itself, the perception of Agrobacterium and the subsequent accumulation of recombinant products throughout the expression phase. Using the upregulation profiles of plant receptor genes during the heterologous expression of virus-like particles in N. benthamiana leaves, I here describe how some of these host responses interact with each other to form an intricate signalling interplay at the molecular level. I also review host plant's responses to agroinfiltration and highlight strategies that have emerged to improve the efficacy of plant cell biofactories based on the better understanding of this transient expression system.

The Orobanchaceae family, the largest group of parasitic plants, spans a complete spectrum from autotrophic to holoparasitic species. As a typical endangered holoparasitic species within this family, Cistanche deserticola is a parasitic plant that is widely harvested for traditional medicine in desertic regions, and of growing importance as a cash crop. However, the evolution of C. deserticola at the molecular and cellular level is poorly understood. Here, we constructed the first chromosome-level genome map of C. deserticola. Comparative genomic analyses demonstrated that the C. deserticola genome exhibited a substantial loss of genes related to photosynthesis and immunity (21.58% of the total genes) and contained 115 horizontally transferred genes. This suggested that the genomic evolution of holoparasitic plants was driven by the interplay between the acquisition of functional genes and the loss of genes specific to plant tissues or functions. Additionally, parasitism-related cells were identified using a high-resolution single-cell transcriptomic atlas, revealing stage-specific differentiation during the parasitic process. Early cells (cluster 11) highly expressed dopamine/tyrosine metabolism pathways genes (e.g., polyphenol oxidase), driving phenylethanoid glycoside biosynthesis. By contrast, mature cells (cluster 10) show high levels of gene expression relating to carbohydrate metabolism in association with nutrient acquisition. Connecting these insights, we developed a comprehensive C. deserticola database to integrate multi-omics and ecological data (http://60.30.67.246:7006/Home). This builds a robust molecular foundation for exploring pathways to parasitism in plants more broadly.

Glomerella leaf spot (GLS), a fungal disease caused by Colletotrichum fructicola, is a major destructive disease of apples but research on control measures is limited. Melatonin (MT) is a phytohormone-like compound that affects plant growth and stress response but is prone to light-induced degradation, resulting in low stability and efficacy. Therefore, we developed a melatonin silicon-based nanomaterial (MT@SiO) to enhance the stability of melatonin and increase its potential use on plants. Our results indicated that MT@SiO significantly enhanced apple leaf resistance to GLS. We demonstrated that MT@SiO at an optimal concentration of 50 μM significantly mitigated GLS infection in 'Gala' apples by elevating the level of salicylic acid. The core transcription factor gene MdNAC32 was identified in our transcriptome analysis and found to respond to both GLS infection and MT@SiO treatment. MdNAC32 directly activates the transcription of MdPBS1/2 which promotes the synthesis of SA. Transient overexpression and silencing experiments demonstrated that MdPBS1/2 positively regulates GLS resistance. In addition, we found that the MEK5-MAPK6 module can phosphorylate MdNAC32, which regulates MdPBS1/2 expression. Overall, our results indicate that MT@SiO enhances the activity of the MEK5-MAPK6-NAC32-MdPBS1/2 module by inducing SA accumulation, resulting in enhanced resistance in apples to GLS. The use of the melatonin-based nanomaterial improved the efficacy of MT and highlights the potential use of conjugated nanomaterials to modulate disease resistance in apples. Our study also provides new insights into the involvement of NAC and MAPK pathways in plant defense response to microbial pathogens.

Citrus Huanglongbing (HLB), a devastating disease caused by the unculturable bacterium 'Candidatus Liberibacter asiaticus' (CLas), poses a severe threat to global citrus production. CLas secretes effectors to suppress host immune responses and facilitate its colonisation. Previously, the CLas effector SECP8 (CLIBASIA_05330) has been identified as an immune inhibitor. However, its molecular mechanisms on host immune suppression remain unclear. This study identifies the citrus transcription factor CsTCP15 as a target of SECP8. Transgenic citrus plants overexpressing CsTCP15 enhanced resistance to CLas, whereas CsTCP15-RNAi interference plants became more susceptible, confirming its role as a positive immune regulator. Meanwhile, CsTCP15 was demonstrated to directly bind to cis-elements of salicylic acid (SA)-responsive genes CsPR5 and CsWRKY22, and overexpression of either gene strengthened citrus hairy roots' resistance against CLas. However, SECP8 directly interacts with CsTCP15 and inhibits its homodimerization. Concurrently, mSECP8 facilitates CsBRG3-mediated degradation and further prevents the dimerization of CsTCP15. This two-pronged interference eventually impairs the transcriptional activation of CsPR5 and CsWRKY22, thereby compromising salicylic acid-mediated immunity and promoting CLas infection. Our findings reveal a virulence strategy whereby a CLas effector manipulates a key host immune regulator to establish pathogenesis.

Stomata are microscopic pores that regulate the exchange of CO and water vapour, making them a major target for engineering plants with improved intrinsic water use efficiency (iWUE). Proof-of-concept studies have demonstrated the potential to increase iWUE by reducing stomatal density (SD) and stomatal conductance (g) by ubiquitously expressing EPIDERMAL PATTERNING FACTOR (EPF) family genes. However, unwanted effects on leaf, stem and reproductive traits are often observed when EPFs are misexpressed in this fashion. We sought to test if these effects result from pleiotropy and to identify a targeted promoter that can circumvent the side effects while retaining the desired reduction in SD. A previously reported synthetic EPF (EPF) was expressed in sugarcane (Saccharum spp.) using two putatively tissue-specific promoters from Brachypodium distachyon (BdCESA7p and BdSPCH2p) and a ubiquitous control from Zea mays (ZmUBI4p). BdSPCH2p control reduced SD to statistically equivalent levels as ZmUBI4p on the abaxial (23%) and adaxial (23%) leaf surfaces. ZmUB4p and BdCESA7p induce expression in four tissue types often associated with pleiotropic effects in EPF-expressing low SD plants. Transgenic plants carrying either the BdCESA7p or ZmUBI4p EPF cassettes displayed leaf chlorosis, reduced leaf nitrogen and chlorophyll content, and altered stem architecture. However, transgenic events harboring the BdSPCH2p EPF cassette restricted EPF expression to the stomatal development zone and leaf nodal tissues and produced transgenic plants without the associated pleiotropic effects. These results represent an important step toward engineering low-SD crops since they show that targeted gene expression can engineer stomatal patterning without impairing agronomically important traits.

Yield heterosis has been extensively exploited in hybrid breeding, with intersubspecific hybrids often exhibiting the most pronounced effects. However, developing elite hybrids remains a laborious and time-consuming process. The genetic basis of heterosis has been debated for over a century, hindered largely by the lack of high-quality genomes. Here, we assembled genomes for 12 representative indica, intermediate type and japonica rice accessions. Using sequence variants of the Phr1 gene, we functionally validated two deletions responsible for phenol reaction variation between the subspecies. Comparative genomic analyses revealed extensive sequence variation among these inbred lines and highlighted the pivotal role of structural variants (SVs) in rice subspeciation. Importantly, the number of SVs between parental inbred lines significantly correlated with heterosis across 17 agronomic traits, with distinct correlation patterns for intra- and intersubspecific F hybrids. We identified SVs associated with S5-ORF5 and OsBZR1 and validated their function to heterosis for seed setting rate and yield heterosis, respectively, underscoring the importance of SVs in breeding intersubspecific hybrids. The genomic SVs altered gene expression and these transcriptional changes effectively explained the variance in heterosis. Furthermore, translocations outperformed other SVs and their heterozygous haplotypes exhibited heterosis over homozygous ones. Our findings establish SVs as pivotal drivers in subspeciation and highlight the overdominance model for harnessing rice heterosis.

Selenium (Se) biofortification improves crops' nutritional value, while nano-selenium (nano-Se) offers enhanced bioavailability over traditional Se fertilisers. The quality of tea (Camellia sinensis) depends critically on nitrogen (N) metabolism and amino acid balance, particularly of theanine. This study assessing growth and quality, elucidating molecular mechanisms and evaluating long-term persistence explored the effects of nano-Se on tea through three different experiments. The results showed that nano-Se significantly increased the chlorophyll content, photosynthetic parameters and non-structural carbohydrates, indicating an enhanced photosynthetic capacity. Nitrogen uptake and metabolism were promoted, along with an increased leaf nitrogen content, NO accumulation and upregulation of ammonium transporter genes, thus providing further evidence. Theanine significantly increased in roots and leaves and upregulated theanine transporter genes, while polyphenol and catechin contents decreased, lowering the polyphenol-to-amino acid ratio of tea. The benefits of nano-Se persisted across three harvest times, with a sustained selenium content and an increased root theanine level, although leaf theanine and quality improvements varied. Overall, nano-Se promoted theanine accumulation via a coordinated regulation of N transporters and biosynthetic genes, while concurrently enhancing photosynthesis and Se biofortification. Its long-lasting efficacy positions nano-Se as a sustainable strategy for producing high-quality, Se-enriched tea, particularly in Se-deficient regions.

Enhancing both starch and protein accumulation is a key strategy for improving maize yield and quality. Achieving this goal requires an in-depth understanding of the regulatory mechanisms that integrate these pathways. Here, we demonstrate that functionally redundant subclass III SNF1-related protein kinase 2s (SnRK2s) act as central regulators that orchestrate starch synthesis and storage protein accumulation in maize kernels, with ZmSnRK2.10 playing a predominant role. Higher-order SnRK2s mutants lacking ZmSnRK2.10 exhibit defective kernel development, with drastically reduced starch and storage protein content. Mechanistically, ZmSnRK2.10 directly phosphorylates starch synthesis-related enzymes, such as Bt1, enhancing their activities and thereby boosting endosperm starch synthesis. Moreover, it indirectly promotes storage protein synthesis in both endosperm and embryo through modulating the phosphorylation status of downstream transcription factors, specifically Opaque-2 and ZmbZIP75, respectively. Interestingly, our study reveals that ZmSnRK2.10 undergoes sequential activation: initially by sucrose in the endosperm during early kernel filling and subsequently by abscisic acid (ABA) in the embryo during later developmental stages. This spatiotemporal regulation suggests a mechanism facilitating coordinated control of these temporally linked processes. Notably, overexpression of ZmSnRK2.10 leads to significant increases in both starch and protein content, as well as a higher vitreous endosperm ratio, thereby simultaneously enhancing maize yield and quality. Our study thus uncovers a previously unknown regulatory mechanism involving subclass III SnRK2s that govern storage functions in maize kernels and provides potential genetic resources for yield and quality improvement.

Plant biofortification with phytonutrients typically relies on metabolic engineering strategies known as 'push' (enhancing biosynthetic flux), 'block' (inhibiting competing pathways) and 'pull' (promoting metabolite storage). Here, we describe a novel synthetic compound, X57, that simultaneously targets biosynthesis, competition and storage to enhance leaf tocopherol content. Tocopherols protect plants against oxidative stress, have a dietary value as vitamin E and are highly appreciated antioxidants in food and cosmetic formulations. X57 exerts a primary 'push' effect by inducing tocopherol biosynthesis, in part by reactivating a direct pathway that reduces geranylgeranyl diphosphate (GGPP) to phytyl diphosphate, bypassing the need for chlorophyll-derived phytol. Accordingly, X57 promotes tocopherol accumulation in etiolated seedlings and restores tocopherol synthesis in mutants deficient in phytol phosphorylation. The 'block' effect is mediated by down-regulation of GGPP consumption for carotenoid synthesis. X57 also induces a 'pull' effect via proliferation of plastoglobules (PG), plastidial lipoprotein bodies that synthesise and store tocopherols. X57-induced PG proliferation is driven by increased tocopherol levels and up-regulation of genes for PG structural proteins such as fibrillins. The unveiled genetic networks simultaneously coordinating plastidial isoprenoid metabolism and plastid differentiation might only be present in higher plants, because X57 does not promote but reduces tocopherol accumulation in Marchantia polymorpha.

Colletotrichum spp., hemibiotrophic fungal pathogens, threaten global strawberry production. Jasmonate (JA) regulates plant-Colletotrichum interactions, but its mechanisms remain unclear. Here we demonstrate that both exogenous methyl jasmonate (MeJA) treatment and elevated endogenous MeJA levels increase strawberry susceptibility to anthracnose. Two key JA biosynthesis genes, FveAOS2 and FveAOC3, were identified as contributors to Colletotrichum-induced susceptibility. Further analysis revealed that the FveSnRK2.1-FveWRKY50 phosphorylation module functions as an important molecular switch in regulating disease susceptibility. Specifically, Colletotrichum infection or MeJA application activates FveSnRK2.1, which phosphorylates FveWRKY50 at serine residue 88 (S88). This phosphorylation enhances the stability and transcriptional activity of FveWRKY50, leading to increased expression of FveAOS2 and FveAOC3, higher MeJA accumulation and enhanced susceptibility. Notably, the strawberry JASMONATE-ZIM DOMAIN (JAZ) protein FveJAZ5 suppresses susceptibility by directly interacting with FveWRKY50, thereby preventing its interaction with FveSnRK2.1 and inhibiting the activation of FveAOS2 and FveAOC3. Upon pathogen attack or MeJA signalling, FveJAZ5 is degraded, thereby releasing FveWRKY50 from suppression. The study elucidates a Colletotrichum-induced 'JA signaling - JA biosynthesis' positive feedback loop that drives strawberry susceptibility. Knocking out FveWRKY50 and overexpressing FveJAZ5 generated anthracnose-resistant germplasms. These findings deepen understanding of plant-Colletotrichum interactions and provide genes for resistant strawberry breeding.