Major advances in crop yields are needed in the coming decades. However, plant breeding is currently limited by incremental improvements in quantitative traits that often rely on laborious selection of rare naturally occurring mutations in gene-regulatory regions. Here, we demonstrate that CRISPR/Cas9 genome editing of promoters generates diverse cis-regulatory alleles that provide beneficial quantitative variation for breeding. We devised a simple genetic scheme, which exploits trans-generational heritability of Cas9 activity in heterozygous loss-of-function mutant backgrounds, to rapidly evaluate the phenotypic impact of numerous promoter variants for genes regulating three major productivity traits in tomato: fruit size, inflorescence branching, and plant architecture. Our approach allows immediate selection and fixation of novel alleles in transgene-free plants and fine manipulation of yield components. Beyond a platform to enhance variation for diverse agricultural traits, our findings provide a foundation for dissecting complex relationships between gene-regulatory changes and control of quantitative traits.

Precise control of plant stem cell proliferation is necessary for the continuous and reproducible development of plant organs 1,2 . The peptide ligand CLAVATA3 (CLV3) and its receptor CLV1 maintain stem cell homeostasis within a deeply conserved negative feedback circuit 1,2 . In Arabidopsis, CLV1 paralogs also contribute to homeostasis, by compensating for the loss of CLV1 through transcriptional upregulation 3 . Here we show that compensation 4,5 operates in diverse

Cells are continuously exposed to chemical signals that they must discriminate between and respond to appropriately. In embryophytes, the leucine-rich repeat receptor-like kinases (LRR-RLKs) are signal receptors critical in development and defense. LRR-RLKs have diversified to hundreds of genes in many plant genomes. Although intensively studied, a well-resolved LRR-RLK gene tree has remained elusive.To resolve the LRR-RLK gene tree, we developed an improved gene discovery method based on iterative hidden Markov model searching and phylogenetic inference. We used this method to infer complete gene trees for each of the LRR-RLK subclades and reconstructed the deepest nodes of the full gene family.We discovered that the LRR-RLK gene family is even larger than previously thought, and that protein domain gains and losses are prevalent. These structural modifications, some of which likely predate embryophyte diversification, led to misclassification of some LRR-RLK variants as members of other gene families. Our work corrects this misclassification.Our results reveal ongoing structural evolution generating novel LRR-RLK genes. These new genes are raw material for the diversification of signaling in development and defense. Our methods also enable phylogenetic reconstruction in any large gene family.

Interactions between MADS box transcription factors are critical in the regulation of floral development, and shifting MADS box protein-protein interactions are predicted to have influenced floral evolution. However, precisely how evolutionary variation in protein-protein interactions affects MADS box protein function remains unknown. To assess the impact of changing MADS box protein-protein interactions on transcription factor function, we turned to the grasses, where interactions between B-class MADS box proteins vary. We tested the functional consequences of this evolutionary variability using maize (Zea mays) as an experimental system. We found that differential B-class dimerization was associated with subtle, quantitative differences in stamen shape. In contrast, differential dimerization resulted in large-scale changes to downstream gene expression. Differential dimerization also affected B-class complex composition and abundance, independent of transcript levels. This indicates that differential B-class dimerization affects protein degradation, revealing an important consequence for evolutionary variability in MADS box interactions. Our results highlight complexity in the evolution of developmental gene networks: changing protein-protein interactions could affect not only the composition of transcription factor complexes but also their degradation and persistence in developing flowers. Our results also show how coding change in a pleiotropic master regulator could have small, quantitative effects on development.

There is intense interest in using genome editing technologies to domesticate wild plants, or accelerate the improvement of weakly domesticated crops, in de novo domestication. Here, we discuss promising genetic strategies, with a focus on plant development. Importantly, genome editing releases us from dependence on random mutagenesis or intraspecific diversity, allowing us to draw solutions more broadly from diversity. However, sparse understanding of the complex genetics of diversity limits innovation. Beyond genetics, we urge the ethical use of indigenous knowledge, indigenous plants, and ethnobotany. De novo domestication still requires conventional breeding by phenotypic selection, especially in the development of crops for diverse environments and cultures. Indeed, uniting genome editing with selective breeding could facilitate faster and better outcomes than either technology alone. Domestication is complex and incompletely understood, involving changes to many aspects of plant biology and human culture. Success in de novo domestication requires careful attention to history and collaboration across traditional boundaries. Expected final online publication date for the Annual Review of Plant Biology, Volume 74 is May 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

The coding sequences of developmental genes are expected to be conserved over deep time, with cis-regulatory change driving the modulation of gene function. In contrast, proteins with roles in defense are expected to evolve rapidly, in molecular arms-races with pathogens. However, some gene families include both developmental and defense genes. In these families, does the tempo and mode of evolution differ between developmental and defense genes, despite shared ancestry and structure? The leucine-rich repeat receptor-like kinase (LRR-RLKs) protein family includes many members with roles in plant development and defense, thus providing an ideal system for answering this question. LRR-RLKs are receptors that traverse plasma membranes. LRR domains bind extracellular ligands, RLK domains initiate intracellular signaling cascades in response to ligand binding. In LRR-RLKs with roles in defense, LRR domains evolve faster than RLK domains. To determine whether this asymmetry extends to developmental LRR-RLKs, we assessed evolutionary rates and tested for selection acting on eleven clades of LRR-RLK proteins, using deeply sampled protein trees. To assess functional evolution, we performed heterologous complementation assays using Arabidopsis thaliana (arabidopsis) LRR-RLK mutants. We found that the LRR domains of developmental LRR-RLK proteins evolved faster than their cognate RLK domains. LRR-RLKs with roles in development and defense had strikingly similar patterns of molecular evolution. Heterologous transformation experiments revealed that the evolution of developmental LRR-RLKs likely involves multiple mechanisms, including changes to cis-regulation, coding sequence evolution, and escape from adaptive conflict. Our results indicate similar evolutionary pressures acting on developmental and defense signaling proteins, despite divergent organismal functions. In addition, deep understanding of the molecular evolution of developmental receptors can help guide targeted genome engineering in agriculture.

The B-class MADS-box transcription factors STERILE TASSEL SILKY EAR1 (STS1) and SILKY1 (SI1) specify floral organ identity in the grass Zea mays (maize). STS1 and SI1 bind DNA as obligate heterodimers. Obligate heterodimerization between STS1 and SI1 homologs, although common in flowering plants, arose very recently in the grass family. This recent emergence of obligate heterodimerization from STS1 homodimerization provided an opportunity to test the consequences of evolutionary shifts in MADS-box protein-protein interactions. We tested the ability of evolutionary variation in STS1 dimerization to impact floral development, downstream gene regulation, and protein complex formation in maize. We found that STS1 hetero-vs. homodimerization had subtle effects on protein localization and stamen development. In contrast, differential STS1 dimerization resulted in largescale changes to gene expression and protein complex composition. We identified kinases and proteins involved in ubiquitylation as candidate interactors with MADS-box proteins, and found that STS1 was phosphorylated, and in a complex with ubiquitylated proteins. In addition, we found that STS1 homodimers were more abundant than STS1-SI1 heterodimers, independent of RNA levels. Thus, differential dimerization can affect both protein degradation dynamics and combinatorial assembly of MADS-box protein complexes. Our results highlight the robustness of floral development to some molecular change, which may contribute to the evolvability of floral form. Significance StatementInteractions between transcription factors can alter downstream gene expression patterns and, in turn, organismal development. In plants, MADS-box transcription factors specify floral organ identity as part of large complexes. Shifting interactions between MADS-box proteins have been proposed as drivers in the evolution of the flower, and in the diversification of floral form. However, the functional consequences of changes to individual MADS-box protein-protein interactions are unknown. Here, we show that floral development is subtly affected by altered transcription factor protein-protein interactions. We also show that shifting transcription factor interactions can contribute to differential protein degradation. This reveals an important consequence for evolutionary variation in transcription factor interactions, and adds a new layer of complexity to the evolution of developmental gene networks.

CRISPR/Cas9-based genome editing in maize is an effective tool, and researchers commonly wish to target multiple genes simultaneously. Transformation of maize is currently expensive and tedious, so researchers are incentivized to build vectors that target multiple genomic loci from a single transformation event. One way to accomplish this is to arrange Cas9 guides into a multiplex array that is transformed as a single locus to the plant (Char et al., 2017). These arrays can be long and repetitive, are challenging to build with traditional assembly methods such as restriction cloning, and are also difficult and expensive to synthesize. Golden Gate gene assembly (Vad-Nielsen et al., 2016) is a good answer to this challenge, as it is insensitive to the tandem repeats in these arrays. The MoClo system is an elaboration of Golden Gate cloning (Werner et al., 2012) and is particularly well suited for assembling larger multiplexed Cas9 guide arrays. In this protocol, we describe steps for designing and building a custom guide array targeting any number of maize loci using the MoClo standard components and syntax. We provide instructions for using variations of the maize and rice U6 promoters to drive guide RNA expression, but this system is generalizable for constructing guide arrays for other species as well.