Chromatography affinity resin with photosynthetically-sourced protein A ligand.

Green, photosynthesizing plants can be proficiently used as cost-effective, single-use, fully biodegradable bioreactors for environmentally-friendly production of a variety of valuable recombinant proteins. Being near-infinitely scalable and most energy-efficient in generating biomass, plants represent profoundly valid alternatives to conventionally used stationary fermenters. To validate this, we produced a plastome-engineered tobacco bioreactor line expressing a recombinant variant of the protein A from Staphylococcus aureus, an affinity ligand widely useful in antibody purification processes, reaching accumulation levels up to ~ 250 mg per 1 kg of fresh leaf biomass. Chromatography resin manufactured from photosynthetically-sourced recombinant protein A ligand conjugated to agarose beads demonstrated the innate pH-driven ability to bind and elute IgG-type antibodies and allowed one-step efficient purification of functional monoclonal antibodies from the supernatants of the producing hybridomas. The results of this study emphasize the versatility of plant-based recombinant protein production and illustrate its vast potential in reducing the cost of diverse biotechnological applications, particularly the downstream processing and purification of monoclonal antibodies.

The EIN3 transcription factor GmEIL1 improves soybean resistance to Phytophthora sojae.

Phytophthora root and stem rot of soybean (Glycine max), caused by the oomycete Phytophthora sojae, is an extremely destructive disease worldwide. In this study, we identified GmEIL1, which encodes an ethylene-insensitive3 (EIN3) transcription factor. GmEIL1 was significantly induced following P. sojae infection of soybean plants. Compared to wild-type soybean plants, transgenic soybean plants overexpressing GmEIL1 showed enhanced resistance to P. sojae and GmEIL1-silenced RNA-interference lines showed more severe symptoms when infected with P. sojae. We screened for target genes of GmEIL1 and confirmed that GmEIL1 bound directly to the GmERF113 promoter and regulated GmERF113 expression. Moreover, GmEIL1 positively regulated the expression of the pathogenesis-related gene GmPR1. The GmEIL1-regulated defence response to P. sojae involved both ethylene biosynthesis and the ethylene signalling pathway. These findings suggest that the GmEIL1-GmERF113 module plays an important role in P. sojae resistance via the ethylene signalling pathway.

HD-Zip proteins modify floral structures for self-pollination in tomato.

Cleistogamy is a type of self-pollination that relies on the formation of a stigma-enclosing floral structure. We identify three homeodomain-leucine zipper IV (HD-Zip IV) genes that coordinately promote the formation of interlocking trichomes at the anther margin to unite neighboring anthers, generating a closed anther cone and cleistogamy (flower morphology necessitating strict self-pollination). These HD-Zip IV genes also control style length by regulating the transition from cell division to endoreduplication. The expression of these HD-Zip IV genes and their downstream gene, Style 2.1, was sequentially modified to shape the cleistogamy morphology during tomato evolution and domestication. Our results provide insights into the molecular basis of cleistogamy in modern tomato and suggest targets for improving fruit set and preventing pollen contamination in genetically modified crops.

MicroRNA Regulatory Pattern in Diabetic Mouse Cortex at Different Stages Following Ischemic Stroke.

After ischemic stroke, microRNAs (miRNAs) participate in various processes, including immune responses, inflammation, and angiogenesis. Diabetes is a key factor increasing the risk of ischemic stroke; however, the regulatory pattern of miRNAs at different stages of diabetic stroke remains unclear. This study comprehensively analyzed the miRNA expression profiles in diabetic mice at 1, 3, and 7 days post-reperfusion following the middle cerebral artery occlusion (MCAO). We identified differentially expressed (DE) miRNAs in diabetic stroke and found significant dysregulation of some novel miRNAs (novel_mir310, novel_mir89, and novel_mir396) post-stroke. These DEmiRNAs were involved in apoptosis and the formation of tight junctions. Finally, we identified three groups of time-dependent DE miRNAs (miR-6240, miR-135b-3p, and miR-672-5p). These have the potential to serve as biomarkers of diabetic stroke. These findings provide a new perspective for future research, emphasizing the dynamic changes in miRNA expression after diabetic stroke and offering potential candidates as biomarkers for future clinical applications.

Editing of SlWRKY29 by CRISPR-activation promotes somatic embryogenesis in Solanum lycopersicum cv. Micro-Tom.

At present, the development of plants with improved traits like superior quality, high yield, or stress resistance, are highly desirable in agriculture. Accelerated crop improvement, however, must capitalize on revolutionary new plant breeding technologies, like genetically modified and gene-edited crops, to heighten food crop traits. Genome editing still faces ineffective methods for the transformation and regeneration of different plant species and must surpass the genotype dependency of the transformation process. Tomato is considered an alternative plant model system to rice and Arabidopsis, and a model organism for fleshy-fruited plants. Furthermore, tomato cultivars like Micro-Tom are excellent models for tomato research due to its short life cycle, small size, and capacity to grow at high density. Therefore, we developed an indirect somatic embryo protocol from cotyledonary tomato explants and used this to generate epigenetically edited tomato plants for the SlWRKY29 gene via CRISPR-activation (CRISPRa). We found that epigenetic reprogramming for SlWRKY29 establishes a transcriptionally permissive chromatin state, as determined by an enrichment of the H3K4me3 mark. A whole transcriptome analysis of CRISPRa-edited pro-embryogenic masses and mature somatic embryos allowed us to characterize the mechanism driving somatic embryo induction in the edited tomato cv. Micro-Tom. Furthermore, we show that enhanced embryo induction and maturation are influenced by the transcriptional effector employed during CRISPRa, as well as by the medium composition and in vitro environmental conditions such as osmotic components, plant growth regulators, and light intensity.

Pepper U-Box E3 ubiquitin ligase 24, CaPUB24, negatively regulates drought stress response.

Under stress conditions, plants modulate their internal states and initiate various defence mechanisms to survive. The ubiquitin-proteasome system is one of the critical modules in these mechanisms, and Plant U-Box proteins play an important role in this process as E3 ubiquitin ligases. Here, we isolated the Plant U-box 24 gene CaPUB24 (Capsicum annuum Plant U-Box 24) from pepper and characterized its functions in response to drought stress. We found that, compared to the other CaPUBs in the same group, the expression of CaPUB24 was significantly induced by drought stress. We also found that CaPUB24 was localized to the nucleus and cytoplasm and had E3 ubiquitin ligase activity. To investigate the biological role of CaPUB24 in response to drought stress further, we generated CaPUB24-silenced pepper plants and CaPUB24-overexpressing Arabidopsis transgenic plants. CaPUB24-silenced pepper plants exhibited enhanced drought tolerance compared to the control plants due to reduced transpirational water loss and increased abscisic acid (ABA) sensitivity. In contrast, CaPUB24-overexpressing Arabidopsis transgenic plants exhibited reduced drought tolerance and ABA-insensitive phenotypes. Our findings suggest that CaPUB24 negatively modulates drought stress response in an ABA-dependent manner.

CmDOF18 positively regulates salinity tolerance in Chrysanthemum morifolium by activating the oxidoreductase system.

BACKGROUND: Chrysanthemum, one of the four major cut flowers all over the world, is very sensitive to salinity during cultivation. DNA binding with one finger (DOF) transcription factors play important roles in biological processes in plants. The response mechanism of CmDOF18 from chrysanthemum to salt stress remains unclear. RESULTS: In this study, CmDOF18 was cloned from Chrysanthemum morifolium, and its expression was induced by salinity stress. The gene encodes a 291-amino acid protein with a typical DOF domain. CmDOF18 was localized to the nucleus in onion epidermal cells and showed transcriptional activation in yeast. CmDOF18 transgenic plants were generated to identify the role of this gene in resistance to salinity treatment. Chrysanthemum plants overexpressing CmDOF18 were more resistant to salinity stress than wild-type plants. Under salinity stress, the malondialdehyde content and leaf electrolyte conductivity in CmDOF18-overexpressing transgenic plants were lower than those in wild-type plants, while the proline content, chlorophyll content, superoxide dismutase activity and peroxidase activity were higher than those in wild-type plants. The opposite findings were observed in gene-silenced plants compared with wild-type plants. The gene expression levels of oxidoreductase increased in CmDOF18-overexpressing transgenic plants but decreased in CmDOF18-SRDX gene-silenced transgenic plants. CONCLUSION: In summary, we analyzed the function of CmDOF18 from chrysanthemum, which may regulate salinity stress in plants, possibly due to its role in the regulation of oxidoreductase.

A chromosome-level genome assembly of the soybean pod borer: insights into larval transcriptional response to transgenic soybean expressing the pesticidal Cry1Ac protein.

BACKGROUND: Genetically modified (GM) crop plants with transgenic expression of Bacillus thuringiensis (Bt) pesticidal proteins are used to manage feeding damage by pest insects. The durability of this technology is threatened by the selection for resistance in pest populations. The molecular mechanism(s) involved in insect physiological response or evolution of resistance to Bt is not fully understood. RESULTS: To investigate the response of a susceptible target insect to Bt, the soybean pod borer, Leguminivora glycinivorella (Lepidoptera: Tortricidae), was exposed to soybean, Glycine max, expressing Cry1Ac pesticidal protein or the non-transgenic parental cultivar. Assessment of larval changes in gene expression was facilitated by a third-generation sequenced and scaffolded chromosome-level assembly of the L. glycinivorella genome (657.4 Mb; 27 autosomes + Z chromosome), and subsequent structural annotation of 18,197 RefSeq gene models encoding 23,735 putative mRNA transcripts. Exposure of L. glycinivorella larvae to transgenic Cry1Ac G. max resulted in prediction of significant differential gene expression for 204 gene models (64 up- and 140 down-regulated) and differential splicing among isoforms for 10 genes compared to unexposed cohorts. Differentially expressed genes (DEGs) included putative peritrophic membrane constituents, orthologs of Bt receptor-encoding genes previously linked or associated with Bt resistance, and those involved in stress responses. Putative functional Gene Ontology (GO) annotations assigned to DEGs were significantly enriched for 36 categories at GO level 2, respectively. Most significantly enriched cellular component (CC), biological process (BP), and molecular function (MF) categories corresponded to vacuolar and microbody, transport and metabolic processes, and binding and reductase activities. The DEGs in enriched GO categories were biased for those that were down-regulated (≥ 0.783), with only MF categories GTPase and iron binding activities were bias for up-regulation genes. CONCLUSIONS: This study provides insights into pathways and processes involved larval response to Bt intoxication, which may inform future unbiased investigations into mechanisms of resistance that show no evidence of alteration in midgut receptors.

Vesicle formation-related protein CaSec16 and its ankyrin protein partner CaANK2B jointly enhance salt tolerance in pepper.

Vesicle transport plays important roles in plant tolerance against abiotic stresses. However, the contribution of a vesicle formation related protein CaSec16 (COPII coat assembly protein Sec16-like) in pepper tolerance to salt stress remains unclear. In this study, we report that the expression of CaSec16 was upregulated by salt stress. Compared to the control, the salt tolerance of pepper with CaSec16-silenced was compromised, which was shown by the corresponding phenotypes and physiological indexes, such as the death of growing point, the aggravated leaf wilting, the higher increment of relative electric leakage (REL), the lower content of total chlorophyll, the higher accumulation of dead cells, H2O2, malonaldehyde (MDA), and proline (Pro), and the inhibited induction of marker genes for salt-tolerance and vesicle transport. In contrast, the salt tolerance of pepper was enhanced by the transient overexpression of CaSec16. In addition, heterogeneously induced CaSec16 protein did not enhance the salt tolerance of Escherichia coli, an organism lacking the vesicle transport system. By yeast two-hybrid method, an ankyrin protein, CaANK2B, was identified as the interacting protein of CaSec16. The expression of CaANK2B showed a downward trend during the process of salt stress. Compared with the control, pepper plants with transient-overexpression of CaANK2B displayed increased salt tolerance, whereas those with CaANK2B-silenced exhibited reduced salt tolerance. Taken together, both the vesicle formation related protein CaSec16 and its interaction partner CaANK2B can improve the pepper tolerance to salt stress.

Activation of the native PHYTOENE SYNTHASE 1 promoter by modifying near-miss cis-acting elements induces carotenoid biosynthesis in embryogenic rice callus.

KEY MESSAGE: Modification of silent latent endosperm-enabled promoters (SLEEPERs) allows the ectopic activation of non-expressed metabolic genes in rice callus Metabolic engineering in plants typically involves transgene expression or the mutation of endogenous genes. An alternative is promoter modification, where small changes in the promoter sequence allow genes to be switched on or off in particular tissues. To activate silent genes in rice endosperm, we screened native promoters for near-miss cis-acting elements that can be converted to endosperm-active regulatory motifs. We chose rice PHYTOENE SYNTHASE 1 (PSY1), encoding the enzyme responsible for the first committed step in the carotenoid biosynthesis pathway, because it is not expressed in rice endosperm. We identified six motifs within a 120-bp region, upstream of the transcriptional start site, which differed from endosperm-active elements by up to four nucleotides. We mutated four motifs to match functional elements in the endosperm-active BCH2 promoter, and this promoter was able to drive GFP expression in callus and in seeds of regenerated plants. The 4 M promoter was not sufficient to drive PSY1 expression, so we mutated the remaining two elements and used the resulting 6 M promoter to drive PSY1 expression in combination with a PDS transgene. This resulted in deep orange callus tissue indicating the accumulation of carotenoids, which was subsequently confirmed by targeted metabolomics analysis. PSY1 expression driven by the uncorrected or 4 M variants of the promoter plus a PDS transgene produced callus that lacked carotenoids. These results confirm that the adjustment of promoter elements can facilitate the ectopic activation of endogenous plant promoters in rice callus and endosperm and most likely in other tissues and plant species.

Understanding the role of the fructose-1,6-bisphosphatase gene for enhancing the photosynthetic rate in Arabidopsis thaliana.

Transgenic Arabidopsis thaliana (ecotype Columbia) was successfully transformed with the gene fructose-1,6-bisphosphatase (FBPas e) and named as AtFBPase plants. Transgenic plants exhibited stable transformation, integration and significantly higher expressions for the transformed gene. Morphological evaluation of transgenic plants showed increased plant height (35cm), number of leaves (25), chlorophyll contents (28%), water use efficiency (increased from 1.5 to 2.6µmol CO2 µmol-1 H2 O) and stomatal conductance (20%), which all resulted in an enhanced photosynthetic rate (2.7µmolm-2 s-1 ) compared to wild type plants. This study suggests the vital role of FBPase gene in the modification of regulatory pathways to enhance the photosynthetic rate, which can also be utilised for economic crops in future.

Unraveling the Contribution of MulSOS2 in Conferring Salinity Tolerance in Mulberry (Morus atropurpurea Roxb).

Salinity is one of the most serious threats to sustainable agriculture. The Salt Overly Sensitive (SOS) signaling pathway plays an important role in salinity tolerance in plants, and the SOS2 gene plays a critical role in this pathway. Mulberry not only has important economic value but also is an important ecological tree species; however, the roles of the SOS2 gene associated with salt stress have not been reported in mulberry. To gain insight into the response of mulberry to salt stress, SOS2 (designated MulSOS2) was cloned from mulberry (Morus atropurpurea Roxb), and sequence analysis of the amino acids of MulSOS2 showed that it shares some conserved domains with its homologs from other plant species. Our data showed that the MulSOS2 gene was expressed at different levels in different tissues of mulberry, and its expression was induced substantially not only by NaCl but also by ABA. In addition, MulSOS2 was exogenously expressed in Arabidopsis, and the results showed that under salt stress, transgenic MulSOS2 plants accumulated more proline and less malondialdehyde than the wild-type plants and exhibited increased tolerance to salt stress. Moreover, the MulSOS2 gene was transiently overexpressed in mulberry leaves and stably overexpressed in the hairy roots, and similar results were obtained for resistance to salt stress in transgenic mulberry plants. Taken together, the results of this study are helpful to further explore the function of the MulSOS2 gene, which provides a valuable gene for the genetic breeding of salt tolerance in mulberry.

Setting up Agrobacterium tumefaciens-mediated transformation of the tropical legume Aeschynomene evenia, a powerful tool for studying gene function in Nod Factor-independent symbiosis.

Most legumes are able to develop a root nodule symbiosis in association with proteobacteria collectively called rhizobia. Among them, the tropical species Aeschynomene evenia has the remarkable property of being nodulated by photosynthetic Rhizobia without the intervention of Nod Factors (NodF). Thereby, A. evenia has emerged as a working model for investigating the NodF-independent symbiosis. Despite the availability of numerous resources and tools to study the molecular basis of this atypical symbiosis, the lack of a transformation system based on Agrobacterium tumefaciens significantly limits the range of functional approaches. In this report, we present the development of a stable genetic transformation procedure for A. evenia. We first assessed its regeneration capability and found that a combination of two growth regulators, NAA (= Naphthalene Acetic Acid) and BAP (= 6-BenzylAminoPurine) allows the induction of budding calli from epicotyls, hypocotyls and cotyledons with a high efficiency in media containing 0,5 µM NAA (up to 100% of calli with continuous stem proliferation). To optimize the generation of transgenic lines, we employed A. tumefaciens strain EHA105 harboring a binary vector carrying the hygromycin resistance gene and the mCherry fluorescent marker. Epicotyls and hypocotyls were used as the starting material for this process. We have found that one growth medium containing a combination of NAA (0,5 µM) and BAP (2,2 µM) was sufficient to induce callogenesis and A. tumefaciens strain EHA105 was sufficiently virulent to yield a high number of transformed calli. This simple and efficient method constitutes a valuable tool that will greatly facilitate the functional studies in NodF-independent symbiosis.

Overexpression of soybean GmDHN9 gene enhances drought resistance of transgenic Arabidopsis.

Soybean is one of the important oil crops and a major source of protein and lipids. Drought can cause severe soybean yields. Dehydrin protein (DHN) is a subfamily of LEA proteins that play an important role in plant responses to abiotic stresses. In this study, the soybean GmDHN9 gene was cloned and induced under a variety of abiotic stresses. Results showed that the GmDHN9 gene response was more pronounced under drought induction. Subcellular localization results indicated that the protein was localized in the cytoplasm. The role of transgenic Arabidopsis plants in drought stress response was further studied. Under drought stress, the germination rate, root length, chlorophyll, proline, relative water content, and antioxidant enzyme content of transgenic Arabidopsis thaliana transgenic genes were higher than those of wild-type plants, and transgenic plants contained less O2-, H2O2 and MDA contents. In short, the GmDHN9 gene can regulate the homeostasis of ROS and enhance the drought resistance of plants.

Feedback regulation of the isoprenoid pathway by SsdTPS overexpression has the potential to enhance plant tolerance to drought stress.

In order to maintain the dynamic physiological balance, plants are compelled to adjust their energy metabolism and signal transduction to cope with the abiotic stresses caused by complex and changeable environments. The diterpenoid natural compound and secondary metabolites, sclareol, derived from Salvia sclarea, has gained significant attention owing to its economic value as a spice material and diverse physiological activities. Here, we focused on the roles and regulatory mechanisms of the sclareol diterpene synthase gene SsdTPS in the resistance of S. sclarea to abiotic stresses. Our results suggested that abiotic stresses could induce the response and upregulation of SsdTPS expression and isoprenoid pathway in S. sclarea. Ectopic expression of SsdTPS conferred drought tolerance in transgenic Arabidopsis, compared with wild-type. Overexpression of SsdTPS enhanced the transcription of ABA signal transduction synthetic regulators and induced the positive feedback upregulating key regulatory genes in the MEP pathway, thereby promoting the increase of ABA content and improving drought tolerance in transgenic plants. In addition, SsdTPS-overexpressed transgenic Arabidopsis improved the responses of stomatal regulatory genes and ROS scavenging enzyme activities and gene expression to drought stress. This promoted the stomatal closure and ROS reduction, thus enhancing water retention capacity and reducing oxidative stress damage. These findings unveil the potentially positive role of SsdTPS in orchestrating multiple regulatory mechanisms and maintaining homeostasis for improved abiotic stress resistance in S. sclarea, providing a novel insight into strategies for promoting drought resistance and cultivating highly tolerant plants.

AnDREB5.1, a A5 group DREB gene from desert shrub Ammopiptanthus nanus, confers osmotic and cold stress tolerances in transgenic tobacco.

The Dehydration-Responsive Element Binding (DREB) subfamily of transcription factors plays crucial roles in plant abiotic stress response. Ammopiptanthus nanus (A. nanus) is an eremophyte exhibiting remarkable tolerance to environmental stress and DREB proteins may contribute to its tolerance to water deficit and low-temperature stress. In the present study, an A. nanus DREB A5 group transcription factor gene, AnDREB5.1, was isolated and characterized in terms of structure and function in abiotic stress tolerance. AnDREB5.1 protein is distributed in the nucleus, possesses transactivation capacity, and is capable of binding to DRE core cis-acting element. The transcription of AnDREB5.1 was induced under osmotic and cold stress. Tobacco seedlings overexpressing AnDREB5.1 displayed higher tolerance to cold stress, osmotic stress, and oxidative stress compared to wild-type tobacco (WT). Under osmotic and cold stress, overexpression of AnDREB5.1 increased antioxidant enzyme activity in tobacco leaves, inhibiting excessive elevation of ROS levels. Transcriptome sequencing analysis showed that overexpression of AnDREB5.1 raised the tolerance of transgenic tobacco seedlings to abiotic stress by regulating multiple genes, including antioxidant enzymes, transcription factors, and stress-tolerant related functional genes like NtCOR413 and NtLEA14. This study provides new evidence for understanding the potential roles of the DREB A5 subgroup members in plants.

Maize ZmLAZ1-3 gene negatively regulates drought tolerance in transgenic Arabidopsis.

BACKGROUND: Molecular mechanisms in response to drought stress are important for the genetic improvement of maize. In our previous study, nine ZmLAZ1 members were identified in the maize genome, but the function of ZmLAZ1 was largely unknown. RESULTS: The ZmLAZ1-3 gene was cloned from B73, and its drought-tolerant function was elucidated by expression analysis in transgenic Arabidopsis. The expression of ZmLAZ1-3 was upregulated by drought stress in different maize inbred lines. The driving activity of the ZmLAZ1-3 promoter was induced by drought stress and related to the abiotic stress-responsive elements such as MYB, MBS, and MYC. The results of subcellular localization indicated that the ZmLAZ1-3 protein localized on the plasma membrane and chloroplast. The ectopic expression of the ZmLAZ1-3 gene in Arabidopsis significantly reduced germination ratio and root length, decreased biomass, and relative water content, but increased relative electrical conductivity and malondialdehyde content under drought stress. Moreover, transcriptomics analysis showed that the differentially expressed genes between the transgenic lines and wild-type were mainly associated with response to abiotic stress and biotic stimulus, and related to pathways of hormone signal transduction, phenylpropanoid biosynthesis, mitogen-activated protein kinase signaling, and plant-pathogen interaction. CONCLUSION: The study suggests that the ZmLAZ1-3 gene is a negative regulator in regulating drought tolerance and can be used to improve maize drought tolerance via its silencing or knockout.

Trade-offs constrain the success of glyphosate-free farming.

Glyphosate, the most widely used herbicide, is linked with environmental harm and there is a drive to replace it in agricultural systems. We model the impacts of discontinuing glyphosate use and replacing it with cultural control methods. We simulate winter wheat arable systems reliant on glyphosate and typical in northwest Europe. Removing glyphosate was projected to increase weed abundance, herbicide risk to the environment, and arable plant diversity and decrease food production. Weed communities with evolved resistance to non-glyphosate herbicides were not projected to be disproportionately affected by removing glyphosate, despite the lack of alternative herbicidal control options. Crop rotations with more spring cereals or grass leys for weed control increased arable plant diversity. Stale seedbed techniques such as delayed drilling and choosing ploughing instead of minimum tillage had varying effects on weed abundance, food production, and profitability. Ploughing was the most effective alternative to glyphosate for long-term weed control while maintaining production and profit. Our findings emphasize the need for careful consideration of trade-offs arising in scenarios where glyphosate is removed. Integrated Weed Management (IWM) with more use of cultural control methods offers the potential to reduce chemical use but is sensitive to seasonal variability and can incur negative environmental and economic impacts.

Spatiotemporal formation of glands in plants is modulated by MYB-like transcription factors.

About one third of vascular plants develop glandular trichomes, which produce defensive compounds that repel herbivores and act as a natural biofactory for important pharmaceuticals such as artemisinin and cannabinoids. However, only a few regulators of glandular structures have been characterized so far. Here we have identified two closely-related MYB-like genes that redundantly inhibit the formation of glandular cells in tomatoes, and they are named as GLAND CELL REPRESSOR (GCR) 1 and 2. The GCR genes highly express in the apical cells of tomato trichomes, with expression gradually diminishing as the cells transition into glands. The spatiotemporal expression of GCR genes is coordinated by a two-step inhibition process mediated by SlTOE1B and GCRs. Furthermore, we demonstrate that the GCR genes act by suppressing Leafless (LFS), a gene that promotes gland formation. Intriguingly, homologous GCR genes from tobacco and petunia also inhibit gland formation, suggesting that the GCR-mediated repression mechanism likely represents a conserved regulatory pathway for glands across different plant species.