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Ensuring food security for an ever-growing global population while adapting to climate change is the main challenge for agriculture in the 21st century. Though new technologies are being applied to tackle the problem, we are approaching a plateau in crop improvement using conventional breeding. Recent advances in gene engineering via the CRISPR/Cas technology pave the way to accelerate plant breeding and meet this increasing demand. Here, we present a gene discovery pipeline named ‘BREEDIT’ that combines multiplex genome editing of whole gene families with crossing schemes to improve complex traits such as yield and drought resistance. We induced gene knockouts in 48 growth-related genes using CRISPR/Cas9 and generated a collection of over 1000 gene-edited maize plants. Edited populations displayed, on average, significant increases of 5 to 10% for leaf length and up to 20% for leaf width compared with controls. For each gene family, edits in subsets of genes could be associated with increased traits, allowing us to reduce the gene space needed to focus on for trait improvement. We propose BREEDIT as a gene discovery pipeline which can be rapidly applied to generate a diverse collection of mutants to identify subsets of promising candidates that could be later incorporated in breeding programs.

SAMBA has been identified as a plant-specific regulator of the anaphase-promoting complex/cyclosome (APC/C) that controls unidirectional cell cycle progression in Arabidopsis (Arabidopsis thaliana), but so far its role has not been studied in monocots. Here, we show the association of SAMBA with the APC/C is conserved in maize (Zea mays). Two samba genome edited mutants showed growth defects, such as reduced internode length, shortened upper leaves with erect leaf architecture, and reduced leaf size due to an altered cell division rate and cell expansion, which aggravated with plant age. The two mutants differed in the severity and developmental onset of the phenotypes, because samba-1 represented a knockout allele, while translation re-initiation in samba-3 resulted in a truncated protein that was still able to interact with the APC/C and regulate its function, albeit with altered APC/C activity and efficiency. Our data are consistent with a dosage-dependent role for SAMBA to control developmental processes for which a change in growth rate is pivotal.

Modern agriculture is struggling to meet the increasing food, silage and raw material demands due to the rapid growth of population and climate change. In Arabidopsis, DA1 and DAR1 are proteases that negatively regulate cell proliferation and control organ size. DA1 and DAR1 are activated by ubiquitination catalyzed by the E3 ligase BIG BROTHER (BB). Here, we characterized the DA1, DAR 1 and BB gene families in maize and analyzed whether perturbation of these genes regulates organ size similar to what was observed in Arabidopsis. We generated da1_dar1a_dar1b triple CRISPR maize mutants and bb1_bb2 double mutants. Detailed phenotypic analysis showed that the size of leaf, stem, cob, and seed was not consistently enlarged in these mutants. Also overexpression of a dominant-negative DA1R333K allele, resembling the da1-1 allele of Arabidopsis which has larger leaves and seeds, did not alter the maize phenotype. The mild negative effects on plant height of the DA1R333K_bb1_bb2 mutant indicate that the genes in the DA1 pathway may control organ size in maize, albeit less obvious than in Arabidopsis.

The plant shoot apex houses the shoot apical meristem, a highly organized and active stem-cell tissue where molecular signaling in discrete cells determines when and where leaves are initiated. We optimized a spatial transcriptomics approach, in situ sequencing (ISS), to colocalize the transcripts of 90 genes simultaneously on the same section of tissue from the maize (Zea mays) shoot apex. The RNA ISS technology reported expression profiles that were highly comparable with those obtained by in situ hybridizations (ISHs) and allowed the discrimination between tissue domains. Furthermore, the application of spatial transcriptomics to the shoot apex, which inherently comprised phytomers that are in gradual developmental stages, provided a spatiotemporal sequence of transcriptional events. We illustrate the power of the technology through PLASTOCHRON1 (PLA1), which was specifically expressed at the boundary between indeterminate and determinate cells and partially overlapped with ROUGH SHEATH1 and OUTER CELL LAYER4 transcripts. Also, in the inflorescence, PLA1 transcripts localized in cells subtending the lateral primordia or bordering the newly established meristematic region, suggesting a more general role of PLA1 in signaling between indeterminate and determinate cells during the formation of lateral organs. Spatial transcriptomics builds on RNA ISH, which assays relatively few transcripts at a time and provides a powerful complement to single-cell transcriptomics that inherently removes cells from their native spatial context. Further improvements in resolution and sensitivity will greatly advance research in plant developmental biology.

SAMBA has been identified as a plant-specific regulator of the anaphase-promoting complex (APC/C) which controls unidirectional cell cycle progression in Arabidopsis, but so far its role was not studied in monocots. Here, the association of SAMBA with APC/C was shown to be conserved in maize. Two samba CRISPR alleles showed growth defects that aggravated with plant age such as dwarfed plants due to shortened upper leaf length, erect leaf architecture, and reduced leaf size due to an altered cell division rate and cell expansion. Despite the fact that in both alleles the frameshift occurred at the same position, the two alleles differed in the severity and developmental onset of the phenotypes, because samba-1 represented a knock-out allele, while translation re-initiation in samba-3 resulted in a truncated protein that was still able to interact with the APC/C and regulate its function, albeit with altered APC/C activity or efficiency. Our data are consistent with a dosage-dependent role for SAMBA to control developmental processes for which a change in growth rate is pivotal.

Agrobacterium spp. are important plant pathogens that are the causative agents of crown gall or hairy root disease. Their unique infection strategy depends on the delivery of part of their DNA to plant cells. Thanks to this capacity, these phytopathogens became a powerful and indispensable tool for plant genetic engineering and agricultural biotechnology. Although Agrobacterium spp. are standard tools for plant molecular biologists, current laboratory strains have remained unchanged for decades and functional gene analysis of Agrobacterium has been hampered by time-consuming mutation strategies. Here, we developed clustered regularly interspaced short palindromic repeats (CRISPR)-mediated base editing to enable the efficient introduction of targeted point mutations into the genomes of both Agrobacterium tumefaciens and Agrobacterium rhizogenes. As an example, we generated EHA105 strains with loss-of-function mutations in recA, which were fully functional for maize (Zea mays) transformation and confirmed the importance of RolB and RolC for hairy root development by A. rhizogenes K599. Our method is highly effective in 9 of 10 colonies after transformation, with edits in at least 80% of the cells. The genomes of EHA105 and K599 were resequenced, and genome-wide off-target analysis was applied to investigate the edited strains after curing of the base editor plasmid. The off-targets present were characteristic of Cas9-independent off-targeting and point to TC motifs as activity hotspots of the cytidine deaminase used. We anticipate that CRISPR-mediated base editing is the start of “engineering the engineer,” leading to improved Agrobacterium strains for more efficient plant transformation and gene editing.

The ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) kinases coordinate the DNA damage response. The roles described for Arabidopsis thaliana ATR and ATM are assumed to be conserved over other plant species, but molecular evidence is scarce. Here, we demonstrate that the functions of ATR and ATM are only partially conserved between Arabidopsis and maize (Zea mays). In both species, ATR and ATM play a key role in DNA repair and cell cycle checkpoint activation, but whereas Arabidopsis plants do not suffer from the absence of ATR under control growth conditions, maize mutant plants accumulate replication defects, likely due to their large genome size. Moreover, contrarily to Arabidopsis, maize ATM deficiency does not trigger meiotic defects, whereas the ATR kinase appears to be crucial for the maternal fertility. Strikingly, ATR is required to repress premature endocycle onset and cell death in the maize endosperm. Its absence results in a reduction of kernel size, protein and starch content, and a stochastic death of kernels, a process being counteracted by ATM. Additionally, while Arabidopsis atr atm double mutants are viable, no such mutants could be obtained for maize. Therefore, our data highlight that the mechanisms maintaining genome integrity may be more important for vegetative and reproductive development than previously anticipated.

Maize is the highest yielding cereal crop grown worldwide for grain or silage. Here, we show that modulating the expression of the maize PLASTOCHRON1 (ZmPLA1) gene, encoding a cytochrome P450 (CYP78A1), results in increased organ growth, seedling vigour, stover biomass and seed yield. The engineered trait is robust as it improves yield in an inbred as well as in a panel of hybrids, at several locations and over multiple seasons in the field. Transcriptome studies, hormone measurements and the expression of the auxin responsive DR5rev:mRFPer marker suggest that PLA1 may function through an increase in auxin. Detailed analysis of growth over time demonstrates that PLA1 stimulates the duration of leaf elongation by maintaining dividing cells in a proliferative, undifferentiated state for a longer period of time. The prolonged duration of growth also compensates for growth rate reduction caused by abiotic stresses., Maize is the highest yielding cereal crop grown worldwide. Here Sun et al. show that maize growth can be further enhanced by prolonging the duration of leaf elongation by targeted ectopic expression of the PLASTOCHRON1 gene and show that this leads to increased yield in field trials.

HISTONE MONOUBIQUITINATION1 (HUB1) and its paralog HUB2 act in a conserved heterotetrameric complex in the chromatin-mediated transcriptional modulation of developmental programs, such as flowering time, dormancy, and the circadian clock. The KHD1 and SPEN3 proteins were identified as interactors of the HUB1 and HUB2 proteins with in vitro RNA-binding activity. Mutants in SPEN3 and KHD1 had reduced rosette and leaf areas. Strikingly, in spen3 mutants, the flowering time was slightly, but significantly, delayed, as opposed to the early flowering time in the hub1-4 mutant. The mutant phenotypes in biomass and flowering time suggested a deregulation of their respective regulatory genes CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) and FLOWERING LOCUS C (FLC) that are known targets of the HUB1-mediated histone H2B monoubiquitination (H2Bub). Indeed, in the spen3-1 and hub1-4 mutants, the circadian clock period was shortened as observed by luciferase reporter assays, the levels of the CCA1α and CCA1β splice forms were altered, and the CCA1 expression and H2Bub levels were reduced. In the spen3-1 mutant, the delay in flowering time was correlated with an enhanced FLC expression, possibly due to an increased distal versus proximal ratio of its antisense COOLAIR transcript. Together with transcriptomic and double-mutant analyses, our data revealed that the HUB1 interaction with SPEN3 links H2Bub during transcript elongation with pre-mRNA processing at CCA1. Furthermore, the presence of an intact HUB1 at the FLC is required for SPEN3 function in the formation of the FLC-derived antisense COOLAIR transcripts., The work was supported by the European Commission Marie Curie Initial Research Training network (FP7-PEOPLE-2013-ITN-607880) (to P.M., K.D.G., and M.V.L.); the Spanish Ministry of Economy and Competitiveness, by the Generalitat de Catalunya and by the Spanish Ministry of Economy and Competitiveness through the “Severo Ochoa Program for Centers of Excellence in R&D” 2016–2019 (to P.M.); and Deutsche Forschungsgemeinschaft Grant SFB960 (to K.D.G.). M.W. was the recipient of a Marie Curie Intra-European fellowship (FP7-PEOPLE-2010-IEF-273068; acronym, LightEr) and S.D. was a postdoctoral fellow of the Research Foundation-Flanders.

The conserved poly(ADP-ribosyl)ation (PAR) pathway consists of three genetic components that are potential targets to modulate the plant’s energy homeostasis upon stress with the aim to improve yield stability in crops and help secure food supply. We studied the role of the PAR pathway component ADP-ribose/NADH pyrophosphohydrolase (AtNUDX7) in yield and mild drought stress by using a transgenic approach in Arabidopsis thaliana and maize (Zea mays). Arabidopsis AtNUDX7 cDNA was overexpressed in Arabidopsis and maize by means of the constitutive Cauliflower Mosaic Virus 35S promoter and the strong constitutive Brachypodium distachyon pBdEF1α promoter, respectively. Overexpression of AtNUDX7 in Arabidopsis improved seed parameters that were measured by a novel, automated method, accelerated flowering and reduced inflorescence height. This combination of beneficial traits suggested that AtNUDX7 overexpression in Arabidopsis might enhance the ADP-ribose recycling step and maintain energy levels by supplying an ATP source in the poly(ADP-ribosyl)ation energy homeostasis pathway. Arabidopsis and maize lines with high, medium and low overexpression levels of the AtNUDX7 gene were analysed in automated platforms and the inhibition of several growth parameters was determined under mild drought stress conditions. The data showed that the constitutive overexpression of the Arabidopsis AtNUDX7 gene in Arabidopsis and maize at varying levels did not improve tolerance to mild drought stress, but knocking down AtNUDX7 expression did, however at the expense of general growth under normal conditions.

The shaping of organs in plants depends on the intercellular flow of the phytohormone auxin, of which the directional signaling is determined by the polar subcellular localization of PIN-FORMED (PIN) auxin transport proteins. Phosphorylation dynamics of PIN proteins are affected by the protein phosphatase 2A (PP2A) and the PINOID kinase, which act antagonistically to mediate their apical-basal polar delivery. Here, we identified the ROTUNDA3 (RON3) protein as a regulator of the PP2A phosphatase activity in Arabidopsis thaliana. The RON3 gene was map-based cloned starting from the ron3-1 leaf mutant and found to be a unique, plant-specific gene coding for a protein with high and dispersed proline content. The ron3-1 and ron3-2 mutant phenotypes [i.e., reduced apical dominance, primary root length, lateral root emergence, and growth; increased ectopic stages II, IV, and V lateral root primordia; decreased auxin maxima in indole-3-acetic acid (IAA)-treated root apical meristems; hypergravitropic root growth and response; increased IAA levels in shoot apices; and reduced auxin accumulation in root meristems] support a role for RON3 in auxin biology. The affinity-purified PP2A complex with RON3 as bait suggested that RON3 might act in PIN transporter trafficking. Indeed, pharmacological interference with vesicle trafficking processes revealed that single ron3-2 and double ron3-2 rcn1 mutants have altered PIN polarity and endocytosis in specific cells. Our data indicate that RON3 contributes to auxin-mediated development by playing a role in PIN recycling and polarity establishment through regulation of the PP2A complex activity.

Abiotic stresses cause crop losses worldwide that reduce the average yield by more than 50%. Due to the high energy consumed to enhance the respiration rates, the excessive reactive oxygen species release provokes cell death and, ultimately, whole plant decay. A metabolic engineering approach in maize ( Zea mays ) altered the expression of two poly(ADP-ribosyl)ation metabolic pathway proteins, poly(ADP-ribose) polymerase ( PARP ) and ADP-ribose-specific Nudix hydrolase ( NUDX ) genes that play a role in the maintenance of the energy homeostasis during stresses. By means of RNAi hairpin silencing and CRISPR/Cas9 gene editing strategies, the PARP expression in maize was downregulated or knocked down. The Arabidopsis NUDX7 gene and its two maize homologs, ZmNUDX2 and ZmNUDX8 , were overexpressed in maize and Arabidopsis . Novel phenotypes were observed, such as significant tolerance to oxidative stress and improved yield in Arabidopsis and a trend of tolerance to mild drought stress in maize and in Arabidopsis . Key words: poly(ADP-ribose) polymerase, Nudix hydrolase, CRISPR/Cas9, maize, oxidative stress, drought stress

In higher plants, genetic transformation, which is part of the toolbox for the study of living organisms, had been reported only 30 years ago, boosting basic plant biology research, generating superior crops, and leading to the new discipline of plant biotechnology. Here, we review its principles and the corresponding molecular tools. In vitro regeneration, through somatic embryogenesis or organogenesis, is discussed because they are prerequisites for the subsequent Agrobacterium tumefaciens-mediated transferred (T)-DNA or direct DNA transfer methods to produce transgenic plants. Important molecular components of the T-DNA are examined, such as selectable marker genes that allow the selection of transformed cells in tissue cultures and are used to follow the gene of interest in the next generations, and reporter genes that have been developed to visualize promoter activities, protein localizations, and protein-protein interactions. Genes of interest are assembled with promoters and termination signals in Escherichia coli by means of GATEWAY-derived binary vectors that represent the current versatile cloning tools. Finally, future promising developments in transgene technology are considered.

In Latin America and sub-Saharan Africa, tropical maize (Zea mays L.) is a major crop for human consumption. To cope with the increasing population and changing environment, there is a need for improving tropical maize germplasm. As part of a biotechnological approach, efficient in vitro regeneration of two tropical maize inbred lines (CML216 and CML244) was established. A number of parameters were optimized, such as age of the immature embryos, plant media and growth regulator concentration. After 6 weeks of culture, somatic embryos that had already reached the coleoptilar stage produced shoots after light induction and developed into fertile plants after acclimation in the soil. The callus induction frequencies and somatic embryo-derived plantlet formation were higher when cultured with the Linsmaier and Skoog medium than those with the Chu’s N6 basal medium. Regeneration of tropical maize shoots depended on the 2,4-dichlorophenoxyacetic acid (2,4-D) concentration at the callus initiation stage from immature embryos. The recalcitrance of the tropical maize inbred line TL26 to in vitro regeneration was overcome in a single-cross hybrid with the CML216 and CML244 genotypes. Remarkably, tropical maize somatic embryos were formed at the abaxial side of the scutellum facing the medium, probably from the axis of the immature embryos, as shown by histological sections. Upon co-cultivation, agrobacteria transiently expressed their intronless β-glucuronidase-encoding gene at the embryogenic tissue, but not with an intron-containing gene, suggesting that virulence genes are induced in Agrobacterium, but that subsequent steps in the T-DNA transfer are inhibited.

The role of translation in the regulation of higher plant growth and development is not well understood. Mutational analysis is a powerful tool to identify and study the function of genes related to a biological process, such as growth. Here we analyzed functionally the angusta3 (ang3) narrow leaf mutant. The AG3 gene was cloned by fine mapping combined with candidate gene sequencing and it corresponded to the ribosomal protein gene RPL5B. Based on amino acid sequence homology, promoter DNA sequence homology and in silico gene expression analysis, RPL5B was found to be putatively functionally redundant with RPL5A. The morphological analysis of ang3 mutants showed that the leaf lamina area was significantly reduced from the third rosette leaf on, mainly because of decreased width. Cellular analysis of the abaxial epidermal cell layer of the third leaf indicated that the cell number in the mutant was similar to that of the wild type, but the cell size was significantly reduced. We postulate that the reduced cell expansion in the epidermis contributes to the narrow shape of ang3 leaves. Growth was also significantly impaired in hypocotyls and primary roots, hinting at a general role for RPL5B in organ growth, unrelated to dorsiventral axis formation. Comparison of the transcriptome of the shoot apices of the mutant and the wild type revealed a limited number of differentially expressed genes, such as MYB23 and MYB5, of which the lower expression in the ang3 mutant correlated with reduced trichome density. Our data suggest that translation is an important level of control of growth and development in plants.

Ensuring food security for an ever-growing global population while adapting to climate change is the main challenge for agriculture in the 21st century. Although new technologies are being applied to tackle this problem, we are approaching a plateau in crop improvement using conventional breeding. Recent advances in CRISPR/Cas9-mediated gene engineering have paved the way to accelerate plant breeding to meet this increasing demand. However, many traits are governed by multiple small-effect genes operating in complex interactive networks. Here, we present the gene discovery pipeline BREEDIT, which combines multiplex genome editing of whole gene families with crossing schemes to improve complex traits such as yield and drought tolerance. We induced gene knockouts in 48 growth-related genes into maize (Zea mays) using CRISPR/Cas9 and generated a collection of over 1,000 gene-edited plants. The edited populations displayed (on average) 5%-10% increases in leaf length and up to 20% increases in leaf width compared with the controls. For each gene family, edits in subsets of genes could be associated with enhanced traits, allowing us to reduce the gene space to be considered for trait improvement. BREEDIT could be rapidly applied to generate a diverse collection of mutants to identify promising gene modifications for later use in breeding programs. BREEDIT is a gene discovery pipeline that combines multiplex CRISPR/Cas9 genome editing of whole gene families with crossing schemes to improve complex quantitative traits.