Trehalose-6-phosphate (Tre6P) is the intermediate in the two-step pathway of trehalose biosynthesis mediated by Tre6P-synthases (TPSs) and Tre6P-phosphatases (TPPs). Plants harbor small families of TPS and TPP genes, however most plant TPSs lack enzymatic activity, suggesting they have regulatory functions. The classical mutant ramosa3 (ra3) increases inflorescence branching in maize, and RA3 encodes a catalytic TPP. We found that RA3 interacts with maize ZmTPS1, a non-catalytic TPS. Mutants in ZmTPS1 and its close paralog ZmTPS12 enhance ra3 phenotypes, suggesting their physical interaction is biologically significant. ZmTPS1 also interacts with the two catalytically active maize TPSs, ZmTPS11 and ZmTPS14, however zmtps11;zmtps14 double mutants fail to complete embryogenesis, suggesting that they are essential, as in arabidopsis. Interestingly, the non-catalytic ZmTPS1 protein stimulated the coupled activity of RA3 and ZmTPS14, suggesting that RA3, ZmTPS1, and ZmTPS14 form a complex, and we confirmed this by expressing and purifying the three proteins and by Alphafold predictions. Our results suggest that non-catalytic TPSs form a complex with catalytic TPSs and TPPs to stimulate catalytic activity and regulate plant development.

Transition metals play essential roles as coenzymes and catalysts in plants and are vital for chloroplast function and photosynthetic efficiency. Nickel (Ni) and cobalt (Co) are two important trace transition metals that are present in chloroplasts. However, the functions of Ni and Co in maize and the mechanisms regulating their homeostasis in chloroplasts remain poorly understood. In this study, we identified a maize zebra lesion (zl1) mutant characterized by leaf chlorosis, severe defects in chloroplast development, and reduced chlorophyll accumulation. Bulked segregant RNA-seq analysis and positional cloning revealed that zl1 carries a non-synonymous mutation in GRMZM2G141636, which encodes a putative nickel/cobalt transporter. Two additional non-synonymous mutations in this gene exhibit similar phenotypes and failed to complement the zl1 mutant. CRISPR-generated knockout mutants were albino and died at an early developmental stage, indicating that ZL1 is crucial for maize development. ZL1-eGFP fusion proteins localized to the chloroplasts and other plastids, and zl1 chloroplasts had significantly reduced Ni and Co levels, while supplementation with Ni2+ or Co2+ partially rescued the chlorosis phenotype, suggesting the leaf chlorosis phenotype is caused by a deficiency of Ni2+ or Co2+ in chloroplasts. RNA profiling revealed widespread misregulation of photosynthesis-related genes in the zl1 mutant. Taken together, these results highlight the crucial role of ZL1 in maize development and photosynthesis, likely through the regulation of cobalt and nickel homeostasis in chloroplasts.

The proper initiation of lateral organs is crucial for plant growth and crop yield determination. The discovery of the boundary-specific CUP-SHAPED COTYLEDON (CUC) transcription factors provides new insights into this intriguing biological process. Although CUC functions have been extensively studied in eudicots, it is not clear how they control development in monocot crops, such as maize (Zea mays). We identified three maize CUC genes (ZmCUC1, ZmCUC2 and ZmCUC3) that are specifically expressed in the diverse boundary domains of maize and redundantly control leaf separation, shoot apical meristem (SAM) maintenance and axillary meristem (AM) initiation. The three genes have clear functional divergence, with ZmCUC1 and ZmCUC2 primarily regulating leaf margin separation and AM initiation. By contrast, ZmCUC3 plays a broader role, governing the formation of all boundary tissues. All three CUC genes redundantly maintain meristem activity and leaf initiation by regulating multiple pathways, including modulation of the redox regulator MALE STERILE CONVERTED ANTHER1. This study provides direct evidence that CUC genes are crucial for the formation of diverse shoot boundary domains and meristem development in maize, highlighting their roles in the balance between organ initiation and differentiation, and meristem maintenance.

Arabidopsis encodes ten TREHALOSE-6-PHOSPHATE PHOSPHATASE ( TPP ) genes, homologous to maize RAMOSA3 (I), which controls shoot branching. To explore the roles of the arabidopsis TPPs , we analyzed their expression in shoot apices and found distinct spatial patterns, including TPPI and TPPJ expressed in shoot meristem boundaries, reminiscent of RA3 expression. Single and double TPP mutants lacked dramatic phenotypes, however a CRISPR-Cas9 knockout of all ten TPP genes resulted in increased branching, mirroring ra3 mutants in maize, as well as reduced size and earlier flowering. Expression of GFP-tagged TPPI under its native promoter partially complemented these defects, with protein localization in meristems, vascular tissues and in nuclei. Metabolite profiling revealed higher trehalose 6-phosphate (Tre6P), lower trehalose, and altered sugar and iron-associated metabolites. The mutants also developed chlorosis and grew poorly on low-nutrient media, linked to low iron levels, and reversible with iron supplementation. Consistent with these findings, developmental and iron-responsive genes were up-regulated in the mutants, while photosynthesis-related genes were repressed. Our findings suggest that TPP genes redundantly regulate shoot architecture, sugar metabolism, iron homeostasis and photosynthesis in arabidopsis, and support a role for TPP-mediated Tre6P signaling in coordinating developmental and physiological pathways. ### Competing Interest Statement The authors have declared no competing interest. National Science Foundation, IOS 2131631 Max Planck Society, https://ror.org/01hhn8329

The CLAVATA signaling pathway regulates plant development and plant-environment interactions. CLAVATA signaling consists of mobile, cell-type or environment-specific CLAVATA3/ESR-related (CLE) peptides, which are perceived by a receptor complex consisting of leucine-rich repeat receptor-like kinases such as CLAVATA1 and receptor-like proteins such as CLAVATA2, which often functions with the pseudokinase CORYNE (CRN). CLAVATA signaling has been extensively studied in various plant species for its developmental role in meristem maintenance. In addition, CLAVATA signaling was implicated in plant-microbe interactions, including root nodule symbiosis and plant interactions with mutualistic arbuscular mycorrhizal (AM) fungi. However, knowledge on AM symbiosis regulation by CLAVATA signaling is limited. Here, we report a dual role for Medicago truncatula CRN in development and plant-microbe interactions. In shoots, MtCRN modulates inflorescence meristem branching. In roots, the MtCRN promoter is active in vascular tissues and meristematic regions. In addition, MtCRN expression is activated in cortex cells colonized by AM fungi and negatively regulates root interactions with these microbes in a nitrogen-dependent manner; negative AM symbiosis regulation by CRN was also observed in the monocot Zea mays, suggesting this function is conserved across plant clades. We further report that MtCRN functions partially independently of the AM autoregulation signal MtCLE53. Transcriptomic data revealed that M. truncatula crn roots display signs of perturbed nutrient, symbiosis, and stress signaling, suggesting that MtCRN plays various roles in plant development and interactions with the environment.

Plant shoot stem cells generate organs essential for food, feed, and biofuels. However, plant single-cell analyses struggled to capture these rare cells or to detect stem cell regulators like CLAVATA3 and WUSCHEL. Here, we dissected stem cell-enriched shoot tissues from maize and Arabidopsis for single-cell RNA sequencing (scRNA-seq), and we optimized protocols to recover thousands of CLAVATA3- and WUSCHEL-expressing cells. A cross-species comparison identified deeply conserved stem cell regulators. We also profiled maize stem cell mutants, validated candidate regulators with spatial transcriptomics, and confirmed their roles in shoot development. These include a family of RNA-binding protein genes and two families of sugar kinase genes. Finally, we show how coupling large-scale single-cell profiling with allelic variation analysis in diverse maize germplasm serves as a valuable resource for identifying stem cell regulators significantly associated with grain yield components. Our discoveries advance the study of shoot stem cells and open avenues for rational crop engineering.

Maize develops separate ear and tassel inflorescences with initially similar morphology but ultimately different architecture and sexuality. The detailed regulatory mechanisms underlying these changes still remain largely unclear. In this study, through analyzing the time-course meristem transcriptomes and floret single-cell transcriptomes of ear and tassel, we revealed the regulatory dynamics and pathways underlying inflorescence development and sex differentiation. We identified 16 diverse gene clusters with differential spatiotemporal expression patterns and revealed biased regulation of redox, programmed cell death, and hormone signals during meristem differentiation between ear and tassel. Notably, based on their dynamic expression patterns, we revealed the roles of two RNA-binding proteins in regulating inflorescence meristem activity and axillary meristem formation. Moreover, using the transcriptional profiles of 53 910 single cells, we uncovered the cellular heterogeneity between ear and tassel florets. We found that multiple signals associated with either enhanced cell death or reduced growth are responsible for tassel pistil suppression, while part of the gibberellic acid signal may act non-cell-autonomously to regulate ear stamen arrest during sex differentiation. We further showed that the pistil-protection gene SILKLESS 1 (SK1) functions antagonistically to the known pistil-suppression genes through regulating common molecular pathways, and constructed a regulatory network for pistil-fate determination. Collectively, our study provides a deep understanding of the regulatory mechanisms underlying inflorescence development and sex differentiation in maize, laying the foundation for identifying new regulators and pathways for maize hybrid breeding and improvement.

Meristems are stem cell-containing structures that produce all plant organs and are therefore important targets for crop improvement. Developmental regulators control the balance and rate of cell divisions within the meristem. Altering these regulators impacts meristem architecture and, as a consequence, plant form. In this review, we discuss genes involved in regulating the shoot apical meristem, inflorescence meristem, axillary meristem, root apical meristem, and vascular cambium in plants. We highlight several examples showing how crop breeders have manipulated developmental regulators to modify meristem growth and alter crop traits such as inflorescence size and branching patterns. Plant transformation techniques are another innovation related to plant meristem research because they make crop genome engineering possible. We discuss recent advances on plant transformation made possible by studying genes controlling meristem development. Finally, we conclude with discussions about how meristem research can contribute to crop improvement in the coming decades.

The CLAVATA signaling pathway regulates plant development and plant-environment interactions. CLAVATA signaling consists of mobile, cell-type or environment-specific CLAVATA3/ESR-related (CLE) peptides, which are perceived by a receptor complex consisting of leucine-rich repeat receptor-like kinases such as CLAVATA1 and receptor-like proteins such as CLAVATA2, which often functions with the pseudokinase CORYNE (CRN). CLAVATA signaling has been extensively studied in various plant species for its role in meristem maintenance and in legumes for modulating root interactions with nitrogen-fixing rhizobia. Some signaling proteins involved in development and nodulation, including CLAVATA1, also regulate plant interactions with mutualistic arbuscular mycorrhizal (AM) fungi. However, our knowledge on AM symbiosis regulation by CLAVATA signaling remains limited and only a handful of genetic regulators have been identified. Here we report that Medicago truncatula CRN controls inflorescence meristem branching and negatively regulates root interactions with AM fungi. MtCRN functions partially independently of the AM autoregulation signal MtCLE53. Transcriptomic data revealed that crn roots display signs of perturbed signaling related to a range of responses to biotic and abiotic factors, suggesting that MtCRN plays various roles in plant development and interactions with the environment. ### Competing Interest Statement The authors have declared no competing interest.

Messenger RNAs (mRNAs) in multicellular organisms can act as signals transported cell-to-cell and over long distances. In plants, mRNAs traffic cellto-cell via plasmodesmata (PDs) and over long distances via the phloem vascular system to control diverse biological processes - such as cell fate and tissue patterning - in destination organs. Research on long-distance transport of mRNAs plants has made remarkable progress, including the cataloguing of many mobile mRNAs, characterization of mRNA features important for transport, identification of mRNA-binding proteins involved in their transport, and understanding of the physiological roles of mRNA transport. However, information on short-range mRNA cell-to-cell transport is still limited. This review discusses the regulatory mechanisms and physiological functions of mRNA transport at the cellular and whole plant levels.

Messenger RNAs (mRNAs) are the templates for protein translation but can also act as non-cell-autonomous signaling molecules. Plants input endogenous and exogenous cues to mobile mRNAs and output them to local or systemic target cells and organs to support specific plant responses. Mobile mRNAs form ribonucleoprotein (RNP) complexes with proteins during transport. Components of these RNP complexes could interact with plasmodesmata (PDs), a major mediator of mRNA transport, to ensure mRNA mobility and transport selectivity. Based on advances in the last two to three years, this review summarizes mRNA transport mechanisms in local and systemic signaling from the perspective of RNP complex formation and PD transport. We also discuss the physiological roles of endogenous mRNA transport and the recently revealed roles of non-cell-autonomous mRNAs in inter-organism communication.

A comprehensive understanding of inflorescence development is crucial for crop genetic improvement, as inflorescence meristems give rise to reproductive organs and determine grain yield. However, dissecting inflorescence development at the cellular level has been challenging owing to a lack of specific marker genes to distinguish among cell types, particularly in different types of meristems that are vital for organ formation. In this study, we used spatial enhanced resolution omics-sequencing (Stereo-seq) to construct a precise spatial transcriptome map of the developing maize ear primordium, identifying 12 cell types, including 4 newly defined cell types found mainly in the inflorescence meristem. By extracting the meristem components for detailed clustering, we identified three subtypes of meristem and validated two MADS-box genes that were specifically expressed at the apex of determinate meristems and involved in stem cell determinacy. Furthermore, by integrating single-cell RNA transcriptomes, we identified a series of spatially specific networks and hub genes that may provide new insights into the formation of different tissues. In summary, this study provides a valuable resource for research on cereal inflorescence development, offering new clues for yield improvement. The authors integrate spatial (Stereo-seq) and single-cell transcriptomes of the developing maize ear to produce an atlas of maize ear cells and their developmental trajectories. They also identify a pair of transcription factors involved in inflorescence development.

Heterozygous mutations in two genes encoding key regulators of development improve kernel row number in inbred and hybrid maize, revealing their potential for yield improvement.

We have developed a flexible undergraduate curriculum that leverages the place-based research of environmental microbiomes to increase the number of Indigenous researchers in microbiology, data science and scientific computing. Monitoring Environmental Microbiomes (MEM) provides a curriculum and research framework designed to integrate an Indigenous approach when conducting authentic scientific research and to build interest and confidence at the undergraduate level. MEM has been successfully implemented as a short summer workshop to introduce computing practices in microbiome analysis. Based on self-assessed student knowledge of topics and skills, increased scientific confidence and interest in genomics careers were observed. We propose MEM be incorporated in a scalable course-based research experience for undergraduate institutions, including tribal colleges and universities, community colleges and other minority serving institutions. This coupled curricular and research framework explicitly considers cultural perspectives, access and equity to train a diverse future workforce that is more informed to engage in microbiome research and to translate microbiome science to benefit community and environmental health.

Different plant species within the grasses were parallel targets of domestication, giving rise to crops with distinct evolutionary histories and traits 1 . Key traits that distinguish these species are mediated by specialized cell types 2 . Here we compare the transcriptomes of root cells in three grass species— Zea mays , Sorghum bicolor and Setaria viridis . We show that single-cell and single-nucleus RNA sequencing provide complementary readouts of cell identity in dicots and monocots, warranting a combined analysis. Cell types were mapped across species to identify robust, orthologous marker genes. The comparative cellular analysis shows that the transcriptomes of some cell types diverged more rapidly than those of others—driven, in part, by recruitment of gene modules from other cell types. The data also show that a recent whole-genome duplication provides a rich source of new, highly localized gene expression domains that favour fast-evolving cell types. Together, the cell-by-cell comparative analysis shows how fine-scale cellular profiling can extract conserved modules from a pan transcriptome and provide insight on the evolution of cells that mediate key functions in crops.

A better understanding of the extent of convergent selection among crops could greatly improve breeding programs. We found that the quantitative trait locus KRN2 in maize and its rice ortholog, OsKRN2, experienced convergent selection. These orthologs encode WD40 proteins and interact with a gene of unknown function, DUF1644, to negatively regulate grain number in both crops. Knockout of KRN2 in maize or OsKRN2 in rice increased grain yield by ~10% and ~8%, respectively, with no apparent trade-offs in other agronomic traits. Furthermore, genome-wide scans identified 490 pairs of orthologous genes that underwent convergent selection during maize and rice evolution, and these were enriched for two shared molecular pathways. KRN2, together with other convergently selected genes, provides an excellent target for future crop improvement.

Carpels in maize undergo programmed cell death in half of the flowers initiated in ears and in all flowers in tassels. The HD-ZIP I transcription factor gene GRASSY TILLERS1 (GT1) is one of only a few genes known to regulate this process. To identify additional regulators of carpel suppression, we performed a gt1 enhancer screen and found a genetic interaction between gt1 and ramosa3 (ra3). RA3 is a classic inflorescence meristem determinacy gene that encodes a trehalose-6-phosphate (T6P) phosphatase (TPP). Dissection of floral development revealed that ra3 single mutants have partially derepressed carpels, whereas gt1;ra3 double mutants have completely derepressed carpels. Surprisingly, gt1 suppresses ra3 inflorescence branching, revealing a role for gt1 in meristem determinacy. Supporting these genetic interactions, GT1 and RA3 proteins colocalize to carpel nuclei in developing flowers. Global expression profiling revealed common genes misregulated in single and double mutant flowers, as well as in derepressed gt1 axillary meristems. Indeed, we found that ra3 enhances gt1 vegetative branching, similar to the roles for the trehalose pathway and GT1 homologs in the eudicots. This functional conservation over ∼160 million years of evolution reveals ancient roles for GT1-like genes and the trehalose pathway in regulating axillary meristem suppression, later recruited to mediate carpel suppression. Our findings expose hidden pleiotropy of classic maize genes and show how an ancient developmental program was redeployed to sculpt floral form.

Plasmodesmata (PD) are channels in the walls of plant cells which enable cell-to-cell information transfer. This includes the selective transport of specific transcription factors that control cell fate during plant development. KNOTTED1 (KN1) homeobox (KNOX) family transcription factors that are essential for the maintenance and function of stem cells in shoot meristems use this trafficking pathway, but its mechanism is largely unknown. Here we describe a forward genetic approach to the identification of regulators of selective KN1 trafficking through PD, using a trichome rescue system that permits simple visual analysis in Arabidopsis leaves. A KN1 trafficking regulator identified in this approach had the capacity to regulate the transport not only of KN1 but also of another mobile regulatory protein, TRANSPARENT TESTA GLABRA1 (TTG1). Our system could be easily adapted to reveal the mechanism underlying the selective transport of additional mobile signals through PD.

Native Americans domesticated maize (Zea mays ssp. mays) from lowland teosinte parviglumis (Zea mays ssp. parviglumis) in the warm Mexican southwest and brought it to the highlands of Mexico and South America where it was exposed to lower temperatures that imposed strong selection on flowering time. Phospholipids are important metabolites in plant responses to low-temperature and phosphorus availability and have been suggested to influence flowering time. Here, we combined linkage mapping with genome scans to identify High PhosphatidylCholine 1 (HPC1), a gene that encodes a phospholipase A1 enzyme, as a major driver of phospholipid variation in highland maize. Common garden experiments demonstrated strong genotype-by-environment interactions associated with variation at HPC1, with the highland HPC1 allele leading to higher fitness in highlands, possibly by hastening flowering. The highland maize HPC1 variant resulted in impaired function of the encoded protein due to a polymorphism in a highly conserved sequence. A meta-analysis across HPC1 orthologs indicated a strong association between the identity of the amino acid at this position and optimal growth in prokaryotes. Mutagenesis of HPC1 via genome editing validated its role in regulating phospholipid metabolism. Finally, we showed that the highland HPC1 allele entered cultivated maize by introgression from the wild highland teosinte Zea mays ssp. mexicana and has been maintained in maize breeding lines from the Northern United States, Canada, and Europe. Thus, HPC1 introgressed from teosinte mexicana underlies a large metabolic QTL that modulates phosphatidylcholine levels and has an adaptive effect at least in part via induction of early flowering time.