The liverwort Marchantia polymorpha is a widely used model organism for studying land-plant biology, and it has also proven to be a promising testbed for bioengineering. CRISPR/Cas9 technology has become a transformative tool for precise genome modifications in M. polymorpha; however, a robust method for the simultaneous expression of multiple gRNAs, which is crucial for enhancing the versatility of CRISPR/Cas9-based genome editing, has yet to be fully developed. In this study, we introduce an adaptation from the OpenPlant kit CRISPR/Cas9 tools that facilitates expression of multiple gRNAs from a single transcript through incorporation of tRNA sequences. The ability to deliver multiple gRNAs simultaneously significantly improves the capacity and scalability of genome editing in M. polymorpha. Additionally, by combining this vector system with a simplified and optimized protocol for thallus transformation, we further streamline the generation of CRISPR/Cas9 mutants in M. polymorpha. The resulting gene-editing system offers a multipurpose, time-saving, and straightforward tool for advancing functional genomics in M. polymorpha, enabling more comprehensive genetic modifications and genome engineering.

Gynandropsis gynandra is a nutrient-rich, climate-resilient, C4 under-utilised crop with potential to enhance food security in low-input farming systems. To support its genetic improvement, we performed linkage mapping using two F2 populations (213 and 187 individuals) derived from Malaysian and Malawian founder lines. Under controlled greenhouse conditions, populations were phenotyped for agronomic traits (plant height, leaf area, flowering time), nutritional content (carotenoids, tocopherols), and anatomical features linked to C4 photosynthesis (vein density, bundle sheath size). High-density SNP genotyping enabled construction of linkage maps and identification of 15 QTL. Shared QTL for plant size and flowering time across both populations suggest stable genetic control suitable for marker-assisted selection. Additional QTL for vein density and vitamin content provide insights into the genetic basis of agronomic traits and C4 physiology. These results offer foundational tools for G. gynandra pre-breeding and reinforce its potential as a model for C4 photosynthesis research and sustainable agriculture.

During photosynthetic development, GOLDEN2-LIKE (GLK) factors drive the expression of photosynthesis-associated nuclear (PhAN) genes. Simultaneous loss of GLK1 and GLK2 in Arabidopsis leads to pale-green plants with small, incompletely developed chloroplasts, revealing involvement of additional components. Following mutagenesis of a glk1 -1 knock-down, glk2 -1 knock-out (KO) mutant we identified suppressor of glks ( sgl ) 2 , sgl2 . The sgl2 glk1 glk2 triple mutant showed increases in chlorophyll, PhAN gene transcripts and thylakoid stacking in mesophyll cell chloroplasts, and enhanced cellular content of chloroplasts in mesophyll and bundle-sheath cells. The suppressor mutation delayed flowering but greening phenotypes preceded flowering. sgl2 was caused by loss of function of AUXIN RESPONSE FACTOR2 , ARF2 . arf2sgl2 boosted chloroplast content. It dramatically suppressed the developmental and chloroplast delay of the phytochrome-deficient long hypocotyl 1 ( hy1 ) mutant. We observed in glk1 -1 glk2 -1 low but detectable expression of GLK1 , which was elevated in arf2sgl2 glk1 -1 glk2 -1, and saw reduced suppression by arf2sgl2 of a glk1 -2 glk2 -2 mutant carrying KO alleles, revealing partial epistasis. Overexpression of Growth Regulating Factor 5 ( GRF5 ), upregulated in arf2sgl2 , rescued chlorophyll levels in glk1-1 glk2- 1, consistently with ARF2 and GRF5 as a transcription module. In developing cereal leaves presumed orthologs of ARF2 and GRF5 are expressed at early stages of plastid proliferation and assembly, preceding GLK expression during greening stage. ARF2 is therefore a negative switch modulating GRF5 early in chloroplast development in photosynthetic cells, upstream of the GLKs. Our results reveal a hierarchy of transcription factor action and feed-forward regulation in chloroplast biogenesis during leaf development. ### Competing Interest Statement The authors have declared no competing interest. Biotechnology and Biological Sciences Research Council, BBP0031171 Indian Council of Agricultural Research, 18(01)/2018-EQR/Edn Royal Holloway University of London, https://ror.org/04g2vpn86, Doctoral fees studentship

Accelerating stomatal kinetics through synthetic optogenetics and mutations that enhance guard cell K+ flux has proven a viable strategy to improve water use efficiency and biomass production. Stomata of the model C4 species Gynandropsis gynandra, a relative of the C3 plant Arabidopsis thaliana, are similarly fast to open and close. We identified and cloned the guard cell rectifying outward K+ channel (GROK) of Gynandropsis and showed that GROK is preferentially expressed in stomatal guard cells. GROK is homologous to the Arabidopsis guard cell K+ channel GORK and, expressed in oocytes, yields a K+ current consistent with that of Gynandropsis guard cells. Complementing the Arabidopsis gork mutant with GROK promoted K+ channel gating and K+ flux, increasing stomatal kinetics and yielding gains in water use efficiency and biomass with varying light, especially under water limitation. Our findings demonstrate the potential for engineering a C4 K+ channel into guard cells of a C3 species, and they speak to the puzzle of how C4 species have evolved mechanisms that enhance water use efficiency and growth under stress.

C4 photosynthesis has arisen from the ancestral C3 state in over sixty lineages of angiosperms. It is widely accepted that an early step in C4 evolution is associated with the appearance of so-called C2 photosynthesis caused by loss of glycine decarboxylase activity from mesophyll cells followed by activation in the bundle sheath. Although changes in cis to a distal enhancer upstream of the P-subunit of GLYCINE DECARBOXYLASE (GLDP) from C2 Moricandia enable loss of expression from mesophyll cells, the mechanism then allowing GLDP expression in the bundle sheath is not known. Here we identify a MYC-MYB transcription factor module previously associated with the control of glucosinolate biosynthesis as the basis of this foundational event in the evolution of C2 photosynthesis. Specifically, we find that in the C3 state this MYC-MYB module already patterns GLDP expression to bundle sheath cells. As a consequence, when GLDP expression is lost from the mesophyll, the MYC-MYB dependent expression in the bundle sheath is revealed. Evolution of C2 photosynthesis is thus associated with a MYC-MYB based transcriptional network already present in the C3 state. This work identifies a molecular genetic mechanism underlying the bundle sheath accumulation of glycine decarboxylase required for C2 photosynthesis and thus a fundamental step in the evolution of C4 photosynthesis.

Research in Arabidopsis thaliana has a powerful influence on our understanding of gene functions and pathways. However, not everything translates from Arabidopsis to crops and other plants. Here, a group of experts consider instances where translation has been lost and why such translation is not possible or is challenging. First, despite great efforts, floral dip transformation has not succeeded in other species outside Brassicaceae. Second, due to gene duplications and losses throughout evolution, it can be complex to establish which genes are orthologs of Arabidopsis genes. Third, during evolution Arabidopsis has lost arbuscular mycorrhizal symbiosis. Fourth, other plants have evolved specialized cell types that are not present in Arabidopsis. Fifth, similarly, C4 photosynthesis cannot be studied in Arabidopsis, which is a C3 plant. Sixth, many other plant species have larger genomes, which has given rise to innovations in transcriptional regulation that are not present in Arabidopsis. Seventh, phenotypes such as acclimation to water stress can be challenging to translate due to different measurement strategies. And eighth, while the circadian oscillator is conserved, there are important nuances in the roles of circadian regulators in crop plants. A key theme emerging across these vignettes is that even when translation is lost, insights can still be gained through comparison with Arabidopsis.

C4 photosynthesis has evolved in over sixty plant lineages and improves photosynthetic efficiency by ~50%. One unifying character of C4 plants is photosynthetic activation of a compartment such as the bundle sheath, but gene regulatory networks controlling this cell type are poorly understood. In Arabidopsis, a bipartite MYC-MYB transcription factor module restricts gene expression to these cells, but in grasses the regulatory logic allowing bundle sheath gene expression has not been defined. Using the global staple and C3 crop rice, we find that the SULFITE REDUCTASE promoter is sufficient for strong bundle sheath expression. This promoter encodes an intricate cis-regulatory logic with multiple activators and repressors acting combinatorially. Within this landscape we identify a distal cis-regulatory module (CRM) activated by an ensemble of transcription factors from the WRKY, G2-like, MYB-related, DOF, IDD and bZIP families. This module is necessary and sufficient to pattern gene expression to the rice bundle sheath. Oligomerisation of the CRM and fusion to core promoters containing Y-patches allow activity to be increased 220-fold. This CRM generates bundle sheath-specific expression in Arabidopsis indicating deep conservation in function between monocotyledons and dicotyledons. In summary, we identify an ancient, short, and tuneable CRM patterning expression to the bundle sheath that we anticipate will be useful for engineering this cell type in various crop species.

### Competing Interest Statement The authors have declared no competing interest.

In the leaves of C3 species such as rice (Oryza sativa), mesophyll cells contain the largest compartment of photosynthetically active chloroplasts. In contrast, plants that use the derived and more efficient C4 photosynthetic pathway have a considerable chloroplast compartment in both bundle sheath and mesophyll cells. Accordingly, the evolution of C4 photosynthesis from the ancestral C3 state required an increased chloroplast compartment in the bundle sheath. Here, we investigated the potential to increase chloroplast compartment size in rice bundle sheath cells by manipulating brassinosteroid signaling. Treatment with brassinazole, a brassinosteroid biosynthesis inhibitor, raised leaf chlorophyll content and increased the number but decreased the area of chloroplasts in bundle sheath cells. Ubiquitous overexpression of the transcription factor-encoding BRASSINAZOLE RESISTANT 1 (OsBZR1) increased bundle sheath chloroplast area by up to 45%, but these plants became chlorotic. However, when OsBZR1 expression was driven by a bundle sheath-specific promoter, the negative effects on growth and viability were alleviated while chloroplast area still increased. In summary, we report a role for brassinosteroids in controlling chloroplast area and number in rice and conclude that cell-specific manipulation of brassinosteroid signaling can be used to manipulate the chloroplast compartment in rice bundle sheath cells.

C4 photosynthesis enhances carbon fixation efficiency by reducing photorespiration through the use of an oxygen-insensitive carboxylase and spatial separation of photosynthesis between mesophyll and bundle sheath cells. The C4 pathway has evolved independently in > 60 plant lineages, but molecular mechanisms underpinning this convergence remain unclear. To explore this, we generated high-resolution transcriptome atlases for two independently evolved C4 dicotyledonous species - Gynandropsis gynandra (NAD-malic enzyme subtype) and Flaveria bidentis (NADP-malic enzyme subtype). We used both single-cell and single-nucleus RNA sequencing to capture gene expression profiles from individual leaf cells, enabling a detailed comparison between cell types and transcriptional signatures. While both approaches produced biologically comparable data for major leaf cell types, transcriptomes from single-nucleus sequencing showed lower stress signatures and were more representative of tissue proportions in the leaf. The single-nucleus data revealed that bundle sheath cells from both C4 species share a gene expression pattern associated with mesophyll cells of C3 plants. A conserved set of transcription factors, including members of the C2H2 and DOF families, was identified in the bundle sheath cells of both species. This study presents the first single-cell-resolution transcriptomes for two independent C4 dicot lineages and provides a valuable resource, including a web-based portal for data visualisation.

The C4 pathway has evolved in over sixty lineages of plants and improves photosynthetic efficiency by ∼50%. One unifying character of C4 plants is photosynthetic activation of a compartment such as the bundle sheath, but gene regulatory networks controlling this cell type are poorly understood. For example, in Arabidopsis thaliana a bipartite MYC-MYB transcription factor module restricts gene expression to this cell type but in grasses the regulatory logic allowing bundle sheath gene expression has not been defined. Using the global staple and C3 crop rice we identified the SULFITE REDUCTASE promoter as sufficient for strong expression in this cell type. This promoter encodes an intricate cis -regulatory logic with multiple activators and repressors acting in a combinatorial manner. However, within this landscape we identified a distal enhancer comprising a module activated by quartet of transcription factors from the G2-like, MYB-related, IDD and bZIP families. This module is necessary and sufficient to pattern gene expression to the rice bundle sheath, and oligomerisation allowed activity to be increased 23-fold. In summary, we identify a short tunable enhancer patterning expression to the rice bundle sheath that we anticipate will be useful for engineering this cell type. The transcription factor module responsible for this patterning is distinct from that previously reported in A. thaliana . ### Competing Interest Statement The authors have declared no competing interest.

Model species continue to underpin groundbreaking plant science research. At the same time, the phylogenetic resolution of the land plant tree of life continues to improve. The intersection of these 2 research paths creates a unique opportunity to further extend the usefulness of model species across larger taxonomic groups. Here we promote the utility of the Arabidopsis thaliana model species, especially the ability to connect its genetic and functional resources, to species across the entire Brassicales order. We focus on the utility of using genomics and phylogenomics to bridge the evolution and diversification of several traits across the Brassicales to the resources in Arabidopsis, thereby extending scope from a model species by establishing a “model clade.” These Brassicales-wide traits are discussed in the context of both the model species Arabidopsis and the family Brassicaceae. We promote the utility of such a “model clade” and make suggestions for building global networks to support future studies in the model order Brassicales.

In this study, an innovative approach is presented in the field of engineered plant living materials (EPLMs), leveraging a sophisticated interplay between synthetic biology and engineering. We detail a 3D bioprinting technique for the precise spatial patterning and genetic transformation of the tobacco BY-2 cell line within custom-engineered granular hydrogel scaffolds. Our methodology involves the integration of biocompatible hydrogel microparticles (HMPs) primed for 3D bioprinting with Agrobacterium tumefaciens capable of plant cell transfection, serving as the backbone for the simultaneous growth and transformation of tobacco BY-2 cells. This system facilitates the concurrent growth and genetic modification of tobacco BY-2 cells within our specially designed scaffolds. These scaffolds enable the cells to develop into predefined patterns while remaining conducive to the uptake of exogenous DNA. We showcase the versatility of this technology by fabricating EPLMs with unique structural and functional properties, exemplified by EPLMs exhibiting distinct pigmentation patterns. These patterns are achieved through the integration of the betalain biosynthetic pathway into tobacco BY-2 cells. Overall, our study represents a groundbreaking shift in the convergence of materials science and plant synthetic biology, offering promising avenues for the evolution of sustainable, adaptive, and responsive living material systems.

Chloroplasts develop from undifferentiated plastids in response to light. In angiosperms, after the perception of light, the Elongated Hypocotyl 5 (HY5) transcription factor initiates photomorphogenesis, and two families of transcription factors known as GOLDEN2-LIKE (GLK) and GATA are considered master regulators of chloroplast development. In addition, the MIR171-targeted SCARECROW-LIKE GRAS transcription factors also impact chlorophyll biosynthesis. The extent to which these proteins carry out conserved roles in non-seed plants is not known. Using the model liverwort Marchantia polymorpha, we show that GLK controls chloroplast biogenesis, and HY5 shows a small conditional effect on chlorophyll content. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed that MpGLK has a broader set of targets than has been reported in angiosperms. We also identified a functional GLK homolog in green algae. In summary, our data support the hypothesis that GLK carries out a conserved role relating to chloroplast biogenesis in land plants and green algae.

C4 photosynthesis is used by the most productive plants on the planet, and compared with the ancestral C3 pathway, it confers a 50% increase in efficiency1. In more than 60 C4 lineages, CO2 fixation is compartmentalized between tissues, and bundle-sheath cells become photosynthetically activated2. How the bundle sheath acquires this alternate identity that allows efficient photosynthesis is unclear. Here we show that changes to bundle-sheath gene expression in C4 leaves are associated with the gain of a pre-existing cis-code found in the C3 leaf. From single-nucleus gene-expression and chromatin-accessibility atlases, we uncover DNA binding with one finger (DOF) motifs that define bundle-sheath identity in the major crops C3 rice and C4 sorghum. Photosynthesis genes that are rewired to be strongly expressed in the bundle-sheath cells of C4 sorghum acquire cis-elements that are recognized by DOFs. Our findings are consistent with a simple model in which C4 photosynthesis is based on the recruitment of an ancestral cis-code associated with bundle-sheath identity. Gain of such elements harnessed a stable patterning of transcription factors between cell types that are found in both C3 and C4 leaves to activate photosynthesis in the bundle sheath. Our findings provide molecular insights into the evolution of the complex C4 pathway, and might also guide the rational engineering of C4 photosynthesis in C3 crops to improve crop productivity and resilience3,4. Single-nucleus RNA-sequencing and chromatin-accessibility analyses in rice (a C3 plant) and sorghum (a C4 plant) provide insight into how C4 photosynthesis evolved in bundle-sheath cells, revealing that the acquisition of ancestral cis-elements was key.

A sustainable food supply is vital for addressing the challenges of a growing global population and climate change. Gynandropsis gynandra , a nutritious C4 orphan crop native to Africa and Asia, thrives in low-input agricultural systems, making it a valuable candidate for future food security. This crop also serves as a model for studying C4 photosynthesis. However, research on its genetic improvement is limited. In this study, two mapping populations from diverse parental lines were used to identify Quantitative Trait Loci (QTL) linked to agronomically relevant traits like plant height, leaf area, flowering time, nutritional content, and photosynthesis. Fifteen QTL were identified, with two affecting both leaf size and flowering time across populations, which can be applied for marker assisted selection for crop improvement. Additionally, QTL linked to C4 photosynthesis provide insights into the genetic mechanisms behind this pathway. Overall, this research enhances the potential of G. gynandra as a climate-resilient crop. ### Competing Interest Statement The authors have declared no competing interest.

Chloroplast biogenesis is dependent on master regulators from the GOLDEN2-LIKE (GLK) family of transcription factors. However, glk mutants contain residual chlorophyll, indicating that other proteins must be involved. Here, we identify MYB-related transcription factors as regulators of chloroplast biogenesis in the liverwort Marchantia polymorpha and angiosperm Arabidopsis thaliana. In both species, double-mutant alleles in MYB-related genes show very limited chloroplast development, and photosynthesis gene expression is perturbed to a greater extent than in GLK mutants. Genes encoding enzymes of chlorophyll biosynthesis are controlled by MYB-related and GLK proteins, whereas those allowing CO2 fixation, photorespiration, and photosystem assembly and repair require MYB-related proteins. Regulation between the MYB-related and GLK transcription factors appears more extensive in A. thaliana than in M. polymorpha. Thus, MYB-related and GLK genes have overlapping as well as distinct targets. We conclude that MYB-related and GLK transcription factors orchestrate chloroplast development in land plants.

Plant leaves contain multiple cell types which achieve distinct characteristics whilst still coordinating development within the leaf. The bundle sheath possesses larger individual cells and lower chloroplast content than the adjacent mesophyll, but how this morphology is achieved remains unknown. To identify regulatory mechanisms determining bundle sheath cell morphology we tested the effects of perturbing environmental (light) and endogenous signals (hormones) during leaf development of Oryza sativa (rice). Total chloroplast area in bundle sheath cells was found to increase with cell size as in the mesophyll but did not maintain a 'set-point' relationship, with the longest bundle sheath cells demonstrating the lowest chloroplast content. Application of exogenous cytokinin and gibberellin significantly altered the relationship between cell size and chloroplast biosynthesis in the bundle sheath, increasing chloroplast content of the longest cells. Delayed exposure to light reduced the mean length of bundle sheath cells but increased corresponding leaf length, whereas premature light reduced final leaf length but did not affect bundle sheath cells. This suggests that the plant hormones cytokinin and gibberellin are regulators of the bundle sheath cell-chloroplast relationship and that final bundle sheath length may potentially be affected by light-mediated control of exit from the cell cycle.

Background It has been proposed that engineering the C 4 photosynthetic pathway into C 3 crops could significantly increase yield. This goal requires an increase in the chloroplast compartment of bundle sheath cells in C 3 species. To facilitate large-scale testing of candidate regulators of chloroplast development in the rice bundle sheath, a simple and robust method to phenotype this tissue in C 3 species is required. Results We established a leaf ablation method to accelerate phenotyping of rice bundle sheath cells. The approach allowed bundle sheath cell dimensions, chloroplast area and chloroplast number per cell to be measured. Using this method, bundle sheath cell dimensions of maize were also measured and compared with rice. Our data show that bundle sheath width but not length significantly differed between C 3 rice and C 4 maize. Comparison of paradermal versus transverse bundle sheath cell width indicated that bundle sheath cells were intact after leaf ablation. Moreover, comparisons of planar chloroplast areas and chloroplast numbers per bundle sheath cell between wild-type and transgenic rice lines expressing the maize GOLDEN-2 ( ZmG2 ) showed that the leaf ablation method allowed differences in chloroplast parameters to be detected. Conclusions Leaf ablation is a simple approach to accessing bundle sheath cell files in C 3 species. We show that this method is suitable for obtaining parameters associated with bundle sheath cell size, chloroplast area and chloroplast number per cell.

Common purslane (Portulaca oleracea) integrates both C4 and crassulacean acid metabolism (CAM) photosynthesis pathways and is a promising model plant to explore C4-CAM plasticity. Here, we report a high-quality chromosome-level genome of nicotinamide adenine dinucleotide (NAD)-malic enzyme (ME) subtype common purslane that provides evidence for 2 rounds of whole-genome duplication (WGD) with an ancient WGD (P-β) in the common ancestor to Portulacaceae and Cactaceae around 66.30 million years ago (Mya) and another (Po-α) specific to common purslane lineage around 7.74 Mya. A larger number of gene copies encoding key enzymes/transporters involved in C4 and CAM pathways were detected in common purslane than in related species. Phylogeny, conserved functional site, and collinearity analyses revealed that the Po-α WGD produced the phosphoenolpyruvate carboxylase-encoded gene copies used for photosynthesis in common purslane, while the P-β WGD event produced 2 ancestral genes of functionally differentiated (C4- and CAM-specific) beta carbonic anhydrases involved in the C4 + CAM pathways. Additionally, cis-element enrichment analysis in the promoters showed that CAM-specific genes have recruited both evening and midnight circadian elements as well as the Abscisic acid (ABA)-independent regulatory module mediated by ethylene-response factor cis-elements. Overall, this study provides insights into the origin and evolutionary process of C4 and CAM pathways in common purslane, as well as potential targets for engineering crops by integrating C4 or CAM metabolism.