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Auxin is a key regulator of plant growth and development. Local auxin biosynthesis and intercellular transport generates regional gradients in the root that are instructive for processes such as specification of developmental zones that maintain root growth and tropic responses. Here we present a toolbox to study auxin-mediated root development that features: (i) the ability to control auxin synthesis with high spatio-temporal resolution and (ii) single-cell nucleus tracking and morphokinetic analysis infrastructure. Integration of these two features enables cutting-edge analysis of root development at single-cell resolution based on morphokinetic parameters under normal growth conditions and during cell-type-specific induction of auxin biosynthesis. We show directional auxin flow in the root and refine the contributions of key players in this process. In addition, we determine the quantitative kinetics of Arabidopsis root meristem skewing, which depends on local auxin gradients but does not require PIN2 and AUX1 auxin transporter activities. Beyond the mechanistic insights into root development, the tools developed here will enable biologists to study kinetics and morphology of various critical processes at the single cell-level in whole organisms. Auxin gradients regulate plant root growth and development. Here the authors manipulate auxin synthesis in specific root cell types and use single-cell nucleus tracking and morphokinetics to map directional auxin flow in the root and quantify the kinetics of meristem skewing.

Plants have evolved a unique and conserved developmental program that enables the conversion of leaves into floral organs. Elegant genetic and molecular work has identified key regulators of flower meristem identity. However, further understanding of flower meristem specification has been hampered by redundancy and by pleiotropic effects. The KNOXI transcription factor SHOOT MERISTEMLESS (STM) is a well-characterized regulator of shoot apical meristem maintenance. Arabidopsis thaliana stm loss-of-function mutants arrest shortly after germination; therefore, the knowledge on later roles of STM in later processes, including flower development, is limited. Here, we uncover a role for STM in the specification of flower meristem identity. Silencing STM in the APETALA1 (AP1) expression domain in the ap1-4 mutant background resulted in a leafy-flower phenotype, and an intermediate stm-2 allele enhanced the flower meristem identity phenotype of ap1-4. Transcriptional profiling of STM perturbation suggested that STM activity affects multiple floral fate genes, among them the F-box protein-encoding gene UNUSUAL FLORAL ORGANS (UFO). In agreement with this notion, stm-2 enhanced the ufo-2 floral fate phenotype, and ectopic UFO expression rescued the leafy flowers in genetic backgrounds with compromised AP1 and STM activities. This work suggests a genetic mechanism that underlies the activity of STM in the specification of flower meristem identity.

Plant hormones are small-molecule signaling compounds that are collectively involved in all aspects of plant growth and development. Unlike animals, plants actively regulate the spatial distribution of several of their hormones. For example, auxin transport results in the formation of auxin maxima that have a key role in developmental patterning. However, the spatial distribution of the other plant hormones, including gibberellic acid (GA), is largely unknown. To address this, we generated two bioactive fluorescent GA compounds and studied their distribution in Arabidopsis thaliana roots. The labeled GAs specifically accumulated in the endodermal cells of the root elongation zone. Pharmacological studies, along with examination of mutants affected in endodermal specification, indicate that GA accumulation is an active and highly regulated process. Our results strongly suggest the presence of an active GA transport mechanism that would represent an additional level of GA regulation.

Fruit taste is determined by sugars, acids and in some species, bitter chemicals. Attraction of seed-dispersing organisms in nature and breeding for consumer preferences requires reduced fruit bitterness. A key metabolic shift during ripening prevents tomato fruit bitterness by eliminating α -tomatine, a renowned defence-associated Solanum alkaloid. Here, we combined fine mapping with information from 150 resequenced genomes and genotyping a 650-tomato core collection to identify nine bitter-tasting accessions including the ‘high tomatine’ Peruvian landraces reported in the literature. These ‘bitter’ accessions contain a deletion in GORKY, a nitrate/peptide family transporter mediating α -tomatine subcellular localization during fruit ripening. GORKY exports α -tomatine and its derivatives from the vacuole to the cytosol and this facilitates the conversion of the entire α -tomatine pool to non-bitter forms, rendering the fruit palatable. Hence, GORKY activity was a notable innovation in the process of tomato fruit domestication and breeding. Bitterness is one of the fruit traits that are most disliked by consumers. In this study, the authors identified and characterized a tonoplast membrane transporter in tomato fruit, which is responsible for the translocation of bitter α-tomatine and other derivatives from the vacuole to the cytoplasm for non-bitter conversion.

Gibberellins (GAs) are plant hormones that promote a wide range of developmental processes. While GA signalling is well understood, little is known about how GA is transported or how GA distribution is regulated. Here we utilize fluorescently labelled GAs (GA-Fl) to screen for Arabidopsis mutants deficient in GA transport. We show that the NPF3 transporter efficiently transports GA across cell membranes in vitro and GA-Fl in vivo . NPF3 is expressed in root endodermis and repressed by GA. NPF3 is targeted to the plasma membrane and subject to rapid BFA-dependent recycling. We show that abscisic acid (ABA), an antagonist of GA, is also transported by NPF3 in vitro. ABA promotes NPF3 expression and GA-Fl uptake in plants. On the basis of these results, we propose that GA distribution and activity in Arabidopsis is partly regulated by NPF3 acting as an influx carrier and that GA–ABA interaction may occur at the level of transport. Transport of the plant hormone gibberellin is required for normal plant growth and development. Here, Tal et al . show that NPF3 is able to transport gibberellin in vitro , and provide evidence that it is required for normal gibberellin distribution and activity in plants.

Genetic variance is vital for breeding programs and mutant screening, yet traditional mutagenesis methods wrestle with genetic redundancy and a lack of specificity in gene targeting. CRISPR-Cas9 offers precise, site-specific gene editing, but its application in crop improvement has been limited by scalability challenges. In this study, we develop genome-wide multi-targeted CRISPR libraries in tomato, enhancing the scalability of CRISPR gene editing in crops and addressing the challenges of redundancy while maintaining its precision. We design 15,804 unique single guide RNAs (sgRNAs), each targeting multiple genes within the same gene families. These sgRNAs are classified into 10 sub-libraries based on gene function. We generate approximately 1300 independent CRISPR lines and successfully identify mutants with distinct phenotypes related to fruit development, fruit flavor, nutrient uptake, and pathogen response. Additionally, we develop CRISPR-GuideMap, a double-barcode tagging system to enable large-scale sgRNA tracking in generated plants. Our results demonstrate that multi-targeted CRISPR libraries are scalable and effective for large-scale gene editing and offer an approach to overcome gene functional redundancy in basic plant research and crop breeding. Genetic variance is vital for breeding programs and mutant screening, yet traditional mutagenesis methods wrestle with genetic redundancy and a lack of specificity in gene targeting. Here the authors develop a scalable CRISPR system in tomato to edit multiple genes at once, overcoming genetic redundancy and revealing new traits related to fruit quality, nutrient use, and disease resistance.

The hormones gibberellin (GA) and cytokinin (CK) exhibit antagonistic effects on various processes in many species. Previous studies in Arabidopsis have shown that GA inhibits CK signaling. Here, we have investigated the cross-talk between GA and CK in tomato (Solanum lycopersicum). We altered the balance between GA and CK activities by exogenous applications and genetic manipulations, and tested an array of physiological and developmental responses. GA and CK showed antagonistic effects on various developmental and molecular processes during tomato plant growth. GA inhibited all tested CK responses, including the induction of the CK primary response genes, type A Tomato Response Regulators (TRRs). CK also inhibited a subset of GA responses. In contrast with exogenous application of GA, the endogenous GA-independent GA signal generated by the loss of the DELLA gene PROCERA (PRO) did not repress CK-regulated processes, such as anthocyanin accumulation, TRR expression and leaf complexity. Our results suggest a mutual antagonistic interaction between GA and CK in tomato. Although GA may inhibit early steps in the CK response pathway via a DELLA-independent pathway, CK appears to affect downstream branch(es) of the GA signaling pathway. The ratio between the two hormones, rather than their absolute levels, determines the final response.

Leaf shape diversity relies on transient morphogenetic activity in leaf margins. However, how this morphogenetic capacity is maintained is still poorly understood. Here, we uncover a role for the hormone cytokinin (CK) in the regulation of morphogenetic activity of compound leaves in tomato (Solanum lycopersicum). Manipulation of CK levels led to alterations in leaf complexity and revealed a unique potential for prolonged growth and morphogenesis in tomato leaves. We further demonstrate that the effect of CK on leaf complexity depends on proper localization of auxin signaling. Genetic analysis showed that reduction of CK levels suppresses the effect of Knotted1 like homeobox (KNOXI) proteins on leaf shape and that CK can substitute for KNOXI activity at the leaf margin, suggesting that CK mediates the activity of KNOXI proteins in the regulation of leaf shape. These results imply that CK regulates flexible leaf patterning by dynamic interaction with additional hormones and transcription factors.

Transport of signaling molecules is of major importance for regulating plant growth, development, and responses to the environment. A prime example is the spatial-distribution of auxin, which is regulated via transporters to govern developmental patterning. A critical limitation in our ability to identify transporters by forward genetic screens is their potential functional redundancy. Here, we overcome part of this functional redundancy via a transportome, multi-targeted forward-genetic screen using artificial-microRNAs (amiRNAs). We generate a library of 3000 plant lines expressing 1777 amiRNAs, designed to target closely homologous genes within subclades of transporter families and identify, genotype and quantitatively phenotype, 80 lines showing reproducible shoot growth phenotypes. Within this population, we discover and characterize a strong redundant role for the unstudied ABCB6 and ABCB20 genes in auxin transport and response. The unique multi-targeted lines generated in this study could serve as a genetic resource that is expected to reveal additional transporters. Characterizing plant membrane transporters via genetic methods is complicated by functional redundancy among multi-gene transporter families. Here Zhang et al. use an artificial microRNA-based screen to overcome this issue and show that ABCB6 and ABCB20 act redundantly to regulate auxin transport.

In plants, changes in local auxin concentrations can trigger a range of developmental processes as distinct tissues respond differently to the same auxin stimulus. However, little is known about how auxin is interpreted by individual cell types. We performed a transcriptomic analysis of responses to auxin within four distinct tissues of the Arabidopsis thaliana root and demonstrate that different cell types show competence for discrete responses. The majority of auxin‐responsive genes displayed a spatial bias in their induction or repression. The novel data set was used to examine how auxin influences tissue‐specific transcriptional regulation of cell‐identity markers. Additionally, the data were used in combination with spatial expression maps of the root to plot a transcriptomic auxin‐response gradient across the apical and basal meristem. The readout revealed a strong correlation for thousands of genes between the relative response to auxin and expression along the longitudinal axis of the root. This data set and comparative analysis provide a transcriptome‐level spatial breakdown of the response to auxin within an organ where this hormone mediates many aspects of development. The transcriptional response to auxin was analyzed in four root cell types. The newly obtained data were cross‐referenced with spatial expression maps to examine auxin's role in regulating gene expression in the root meristem. Synopsis The transcriptional response to auxin was analyzed in four root cell types. The newly obtained data were cross‐referenced with spatial expression maps to examine auxin's role in regulating gene expression in the root meristem. The majority of the thousands of auxin‐responsive genes in the Arabidopsis thaliana root show a spatial bias in their induction or repression by auxin treatment. Auxin promotes the expression of cell‐identity markers for the developing xylem and quiescent center, whereas it inhibits markers for the maturing xylem, cortex and trichoblasts. Relative induction or repression by auxin predicts expression along the longitudinal axis of the root.