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Although strongly influenced by environmental conditions, lateral root (LR) positioning along the primary root appears to follow obediently an internal spacing mechanism dictated by auxin oscillations that prepattern the primary root, referred to as the root clock. Surprisingly, none of the hitherto characterized PIN- and ABCB-type auxin transporters seem to be involved in this LR prepatterning mechanism. Here, we characterize ABCB15, 16, 17, 18, and 22 (ABCB15-22) as novel auxin-transporting ABCBs. Knock-down and genome editing of this genetically linked group of ABCBs caused strongly reduced LR densities. These phenotypes were correlated with reduced amplitude, but not reduced frequency of the root clock oscillation. High-resolution auxin transport assays and tissue-specific silencing revealed contributions of ABCB15-22 to shootward auxin transport in the lateral root cap (LRC) and epidermis, thereby explaining the reduced auxin oscillation. Jointly, these data support a model in which LRC-derived auxin contributes to the root clock amplitude.

Transgene residuals in edited plants affect genetic analysis, pose off-target risks, and cause regulatory concerns. Several strategies have been developed to efficiently edit target genes without leaving any transgenes in plants. Some approaches directly address this issue by editing plant genomes with DNA-free reagents. On the other hand, DNA-based techniques require another step for ensuring plants are transgene-free. Fluorescent markers, pigments, and chemical treatments have all been employed as tools to distinguish transgenic plants from transgene-free plants quickly and easily. Moreover, suicide genes have been used to trigger self-elimination of transgenic plants, greatly improving the efficiency of isolating the desired transgene-free plants. Transgenes can also be excised from plant genomes using site-specific recombination, transposition or gene editing nucleases, providing a strategy for editing asexually produced plants. Finally, haploid induction coupled with gene editing may make it feasible to edit plants that are recalcitrant to transformation. Here, we evaluate the strengths and weaknesses of recently developed approaches for obtaining edited plants without transgene residuals.

Inactivation of the phytohormone auxin plays important roles in plant development, and several enzymes have been implicated in auxin inactivation. In this study, we show that the predominant natural auxin, indole-3-acetic acid (IAA), is mainly inactivated via the GH3-ILR1-DAO pathway. IAA is first converted to IAA-amino acid conjugates by GH3 IAA-amidosynthetases. The IAA-amino acid conjugates IAA-aspartate (IAA-Asp) and IAA-glutamate (IAA-Glu) are storage forms of IAA and can be converted back to IAA by ILR1/ILL amidohydrolases. We further show that DAO1 dioxygenase irreversibly oxidizes IAA-Asp and IAA-Glu into 2-oxindole-3-acetic acid-aspartate (oxIAA-Asp) and oxIAA-Glu, which are subsequently hydrolyzed by ILR1 to release inactive oxIAA. This work established a complete pathway for the oxidative inactivation of auxin and defines the roles played by auxin homeostasis in plant development.

Auxin inactivation is critical for plant growth and development. To develop plant growth regulators functioning in auxin inactivation pathway, we performed a phenotype-based chemical screen in Arabidopsis and identified a chemical, nalacin, that partially mimicked the effects of auxin. Genetic, pharmacological, and biochemical approaches demonstrated that nalacin exerts its auxin-like activities by inhibiting indole-3-acetic acid (IAA) conjugation that is mediated by Gretchen Hagen 3 (GH3) acyl acid amido synthetases. The crystal structure of Arabidopsis GH3.6 in complex with D4 (a derivative of nalacin) together with docking simulation analysis revealed the molecular basis of the inhibition of group II GH3 by nalacin. Sequence alignment analysis indicated broad bioactivities of nalacin and D4 as inhibitors of GH3s in vascular plants, which were confirmed, at least, in tomato and rice. In summary, our work identifies nalacin as a potent inhibitor of IAA conjugation mediated by group II GH3 that plays versatile roles in hormone-regulated plant development and has potential applications in both basic research and agriculture.

Carotenoids are vital photosynthetic pigments for plants and provide essential nutrients for humans. However, our knowledge of the regulatory control of carotenoid biosynthesis remains limited. Golden2-like transcription factors (GLKs) are widely recognized as essential and conserved factors for chloroplast development and the major regulators of chlorophyll biosynthesis. Yet the molecular mechanisms by which GLKs transcriptionally activate their target genes are unclear. Here, we report that GLKs directly regulate carotenoid biosynthesis in a G-box Binding Factor (GBF)-dependent manner. Both in vitro and in vivo studies reveal that GLKs physically interact with GBFs. Through the direct binding of GBFs to the G-box motif, the GLK-GBF regulatory module transcriptionally activates phytoene synthase (PSY), the gene encoding the rate-limiting enzyme for carotenoid biosynthesis. The ability of GLKs to promote carotenoid and chlorophyll biosynthesis is greatly diminished in the Arabidopsis gbf1/2/3 triple knockout mutants, showing the requirement of GBFs for GLK function. GLKs and GBFs form liquid-liquid phase separation-mediated nuclear condensates as the compartmented and concentrated transcriptional complexes. Our findings uncover a novel and conserved regulatory module for photosynthetic pigment biosynthesis through formation of GLK-GBF transcriptional complexes and nuclear biomolecular condensates in plants.One-sentence summaryGLKs transcriptionally regulate photosynthetic pigment synthesis in a GBF-dependent manner and are associated with the formation of phase separation-mediated nuclear condensates.

Pollen grains stock up on starch to power germination and pollen tube growth upon pollination. New findings in barley show that localized auxin biosynthesis by a YUC flavin monooxygenase leads to reprogramming energy metabolism during pollen maturation.

The regulatory factor ZHD2 promotes root meristem activity and affects grain yield in rice by inducing ethylene biosynthesis., Root meristem activity is the most critical process influencing root development. Although several factors that regulate meristem activity have been identified in rice, studies on the enhancement of meristem activity in roots are limited. We identified a T-DNA activation tagging line of a zinc-finger homeobox gene, OsZHD2, which has longer seminal and lateral roots due to increased meristem activity. The phenotypes were confirmed in transgenic plants overexpressing OsZHD2. In addition, the overexpressing plants showed enhanced grain yield under low nutrient and paddy field conditions. OsZHD2 was preferentially expressed in the shoot apical meristem and root tips. Transcriptome analyses and quantitative real-time PCR experiments on roots from the activation tagging line and the wild type showed that genes for ethylene biosynthesis were up-regulated in the activation line. Ethylene levels were higher in the activation lines compared with the wild type. ChIP assay results suggested that OsZHD2 induces ethylene biosynthesis by controlling ACS5 directly. Treatment with ACC (1-aminocyclopropane-1-carboxylic acid), an ethylene precursor, induced the expression of the DR5 reporter at the root tip and stele, whereas treatment with an ethylene biosynthesis inhibitor, AVG (aminoethoxyvinylglycine), decreased that expression in both the wild type and the OsZHD2 overexpression line. These observations suggest that OsZHD2 enhances root meristem activity by influencing ethylene biosynthesis and, in turn, auxin.

Indole-3-acetamide (IAM) is the first confirmed auxin biosynthetic intermediate in some plant pathogenic bacteria. Exogenously applied IAM or production of IAM by overexpressing the bacterial iaaM gene in Arabidopsis causes auxin overproduction phenotypes. However, it is still inconclusive whether plants use IAM as a key precursor for auxin biosynthesis. Herein, we reported the isolation IAMHYDROLASE1 (IAMH1) gene in Arabidopsis from a forward genetic screen for IAM-insensitive mutants that display normal auxin sensitivities. IAMH1 has a close homolog named IAMH2 that is located right next to IAMH1 on chromosome IV in Arabidopsis. We generated iamh1 iamh2 double mutants using our CRISPR/Cas9 gene editing technology. We showed that disruption of the IAMH genes rendered Arabidopsis plants resistant to IAM treatments and also suppressed the iaaM overexpression phenotypes, suggesting that IAMH1 and IAMH2 are the main enzymes responsible for converting IAM into indole-3-acetic acid (IAA) in Arabidopsis. The iamh double mutants did not display obvious developmental defects, indicating that IAM does not play a major role in auxin biosynthesis under normal growth conditions. Our findings provide a solid foundation for clarifying the roles of IAM in auxin biosynthesis and plant development.

The phytohormone auxin, specifically indole-3-acetic acid (IAA) plays a prominent role in plant development. Cellular auxin concentration is coordinately regulated by auxin synthesis, transport, and inactivation to maintain auxin homeostasis; however, the physiological contribution of auxin inactivation to auxin homeostasis has remained elusive. The GH3 genes encode auxin amino acid conjugating enzymes that perform a central role in auxin inactivation. The chemical inhibition of GH3s in planta is challenging because the inhibition of GH3 enzymes leads to IAA overaccumulation that rapidly induces GH3 expression. Here, we developed a potent GH3 inhibitor, designated as kakeimide (KKI), that selectively targets auxin-conjugating GH3s. Chemical knockdown of the auxin inactivation pathway demonstrates that auxin turnover is very rapid (about 10 min), indicating auxin biosynthesis and inactivation dynamically regulate auxin homeostasis.

Gretchen Hagen 3 (GH3) amido synthetases conjugate amino acids to a carboxyl group of small molecules including hormones auxin, jasmonate, and salicylic acid. The Arabidopsis genome harbors 19 GH3 genes, whose exact roles in plant development have been difficult to define because of genetic redundancy among the GH3 genes. Here we use CRISPR/Cas9 gene editing technology to delete the Arabidopsis group II GH3 genes, which are able to conjugate indole-3-acetic acid (IAA) to amino acids. We show that plants lacking the eight group II GH3 genes (gh3 octuple mutants) accumulate free IAA and fail to produce IAA-Asp and IAA-Glu conjugates. Consequently, gh3 octuple mutants have extremely short roots, long and dense root hairs, and long hypocotyls and petioles. Our characterization of gh3 septuple mutants, which provide sensitized backgrounds, reveals that GH3.17 and GH3.9 play prominent roles in root elongation and seed production, respectively. We show that GH3 functions correlate with their expression patterns, suggesting that local deactivation of auxin also contributes to maintaining auxin homeostasis and is important for plant development. Moreover, this work provides a method for elucidating functions of individual members of a gene family, whose members have overlapping functions.

CRISPR/Cas9 gene-editing technologies have been very effective in editing target genes in all major crop plants and offer unprecedented potentials in crop improvement. A major challenge in using CRISPR gene-editing technology for agricultural applications is that the target gene-edited crop plants need to be transgene free to maintain trait stability and to gain regulatory approval for commercial production. In this article, we present various strategies for generating transgene-free and target gene-edited crop plants. The CRISPR transgenes can be removed by genetic segregation if the crop plants are reproduced sexually. Marker-assisted tracking and eliminating transgenes greatly decrease the time and labor needed for identifying the ideal transgene-free plants. Transgenes can be programed to undergo self-elimination when CRISPR genes and suicide genes are sequentially activated, greatly accelerating the isolation of transgene-free and target gene-edited plants. Transgene-free plants can also be generated using approaches that are considered non-transgenic such as ribonucleoprotein transfection, transient expression of transgenes without DNA integration, and nano-biotechnology. Here, we discuss the advantages and disadvantages of the various strategies in generating transgene-free plants and provide guidance for adopting the best strategies in editing a crop plant.

The endosomal sorting complex required for transport (ESCRT) machinery has been well documented for its function in endosomal sorting in eukaryotes. Here, we demonstrate an up-to-now unknown and non-endosomal function of the ESCRT component in plants. We show that FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1), a recently identified plant-specific ESCRT component essential for multivesicular body biogenesis, plays additional functions in the nucleus in transcriptional inhibition of abscisic acid (ABA) signalling. Following ABA treatment, SNF1-related protein kinase 2 (SnRK2) kinases phosphorylate FREE1, a step requisite for ABA-induced FREE1 nuclear import. In the nucleus, FREE1 interacts with the basic leucine zipper transcription factors ABA-RESPONSIVE ELEMENTS BINDING FACTOR4 and ABA-INSENSITIVE5 to reduce their binding to the cis-regulatory sequences of downstream genes. Collectively, our study demonstrates the crosstalk between endomembrane trafficking and ABA signalling at the transcriptional level and highlights the moonlighting properties of the plant ESCRT subunit FREE1, which has evolved unique non-endosomal functions in the nucleus besides its roles in membrane trafficking in the cytoplasm.

One of the main obstacles to gene replacement in plants is efficient delivery of a donor repair template (DRT) into the nucleus for homology-directed DNA repair (HDR) of double-stranded DNA breaks. Production of RNA templates in vivo for transcript-templated HDR (TT-HDR) could overcome this problem, but primary transcripts are often processed and transported to the cytosol, rendering them unavailable for HDR. We show that coupling CRISPR-Cpf1 (CRISPR from Prevotella and Francisella 1) to a CRISPR RNA (crRNA) array flanked with ribozymes, along with a DRT flanked with either ribozymes or crRNA targets, produces primary transcripts that self-process to release the crRNAs and DRT inside the nucleus. We replaced the rice acetolactate synthase gene (ALS) with a mutated version using a DNA-free ribonucleoprotein complex that contains the recombinant Cpf1, crRNAs, and DRT transcripts. We also produced stable lines with two desired mutations in the ALS gene using TT-HDR.

Sexual reproduction constrains progeny to inherit allelic genes from both parents. Selective acquisition of target genes from only one parent in the F1 generation of plants has many potential applications including the elimination of undesired alleles and acceleration of trait stacking. CRISPR/Cas9-based gene drives can generate biased transmission of a preferred allele and convert heterozygotes to homozygotes in insects and mice, but similar strategies have not been implementable in plants because of a lack of efficient homology-directed repair (HDR). Here, we place a gene drive, which consists of cassettes that produce Cas9, guide RNAs (gRNA), and fluorescent markers, into the CRYPTOCHROME 1 (CRY1) gene through CRISPR/Cas9-mediated HDR, resulting in cry1drive lines. After crossing the cry1drive/cry1drive lines to wild type, we observe F1 plants which have DNA at the CRY1 locus from only the cry1drive/cry1drive parent. Moreover, a non-autonomous trans-acting gene drive, in which the gene drive unit and the target gene are located on different chromosomes, converts a heterozygous mutation in the target gene to homozygous. Our results demonstrate that homozygous F1 plants can be obtained through zygotic conversion using a CRISPR/Cas9-based gene drive.

Plant roots adapt to the mechanical constraints of the soil to grow and absorb water and nutrients. As in animal species, mechanosensitive ion channels in plants are proposed to transduce external mechanical forces into biological signals. However, the identity of these plant root ion channels remains unknown. Here, we show that Arabidopsis thaliana PIEZO1 (PZO1) has preserved the function of its animal relatives and acts as an ion channel. We present evidence that plant PIEZO1 is expressed in the columella and lateral root cap cells of the root tip, which are known to experience robust mechanical strain during root growth. Deleting PZO1 from the whole plant significantly reduced the ability of its roots to penetrate denser barriers compared to wild-type plants. pzo1 mutant root tips exhibited diminished calcium transients in response to mechanical stimulation, supporting a role of PZO1 in root mechanotransduction. Finally, a chimeric PZO1 channel that includes the C-terminal half of PZO1 containing the putative pore region was functional and mechanosensitive when expressed in naive mammalian cells. Collectively, our data suggest that Arabidopsis PIEZO1 plays an important role in root mechanotransduction and establish PIEZOs as physiologically relevant mechanosensitive ion channels across animal and plant kingdoms.

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.

Arsenic stress causes rapid transcriptional responses in plants. However, transcriptional regulators of arsenic-induced gene expression in plants remain less well known. To date, forward genetic screens have proven limited for dissecting arsenic response mechanisms. We hypothesized that this may be due to the extensive genetic redundancy present in plant genomes. To overcome this limitation, we pursued a forward genetics screen for arsenite tolerance using a randomized library of plants expressing >2,000 artificial microRNAs (amiRNAs). This library was designed to knock-down diverse combinations of homologous gene family members within sub-clades of transcription factor and transporter gene families. We identified six transformant lines showing an altered response to arsenite in root growth assays. Further characterization of an amiRNA line targeting closely homologous CBF and ERF transcription factors show that the CBF1,2 and 3 transcription factors negatively regulate arsenite sensitivity. Furthermore, the ERF34 and ERF35 transcription factors are required for cadmium resistance. Generation of CRISPR lines, higher-order T-DNA mutants, and gene expression analyses, further support our findings. These ERF transcription factors differentially regulate arsenite sensitivity and cadmium tolerance.

Arsenic stress causes rapid transcriptional responses in plants. However, transcriptional regulators of arsenic-induced gene expression in plants remain less well known. To date, forward genetic screens have proven limited for dissecting arsenic response mechanisms. We hypothesized that this may be due to the extensive genetic redundancy present in plant genomes. To overcome this limitation, we pursued a forward genetics screen for arsenite tolerance using a randomized library of plants expressing >2,000 artificial microRNAs (amiRNAs). This library was designed to knock-down diverse combinations of homologous gene family members within sub-clades of transcription factor and transporter gene families. We identified six transformant lines showing an altered response to arsenite in root growth assays. Further characterization of an amiRNA line targeting closely homologous CBF and ERF transcription factors show that the CBF1,2 and 3 transcription factors negatively regulate arsenite sensitivity. Furthermore, the ERF34 and ERF35 transcription factors are required for cadmium resistance. Generation of CRISPR lines, higher-order T-DNA mutants, and gene expression analyses, further support our findings. These ERF transcription factors differentially regulate arsenite sensitivity and cadmium tolerance.

Reporters have been widely used to visualize gene expression, protein localization, and other cellular activities, but the commonly used reporters require special equipment, expensive chemicals, or invasive treatments. Here, we construct a new reporter RUBY that converts tyrosine to vividly red betalain, which is clearly visible to naked eyes without the need of using special equipment or chemical treatments. We show that RUBY can be used to noninvasively monitor gene expression in plants. Furthermore, we show that RUBY is an effective selection marker for transformation events in both rice and Arabidopsis. The new reporter will be especially useful for monitoring cellular activities in large crop plants such as a fruit tree under field conditions and for observing transformation and gene expression in tissue culture under sterile conditions.

SummaryPlant roots adapt to the mechanical constraints of the soil to grow and absorb water and nutrients. As in animal species, mechanosensitive ion channels in plants are proposed to transduce external mechanical forces into biological signals. However, the identity of these plant root ion channels remains unknown. Here, we show that Arabidopsis thaliana PIEZO (AtPIEZO) has preserved the function of its animal relatives and acts as an ion channel. We present evidence that plant PIEZO is highly expressed in the columella and lateral root cap cells of the root tip which experience robust mechanical strain during root growth. Deleting PIEZO from the whole plant significantly reduced the ability of its roots to penetrate denser barriers compared to wild type plants. piezo mutant root tips exhibited diminished calcium transients in response to mechanical stimulation, supporting a role of AtPIEZO in root mechanotransduction. Finally, a chimeric PIEZO channel that includes the C-terminal half of AtPIEZO containing the putative pore region was functional and mechanosensitive when expressed in naive mammalian cells. Collectively, our data suggest that Arabidopsis PIEZO plays an important role in root mechanotransduction and establishes PIEZOs as physiologically relevant mechanosensitive ion channels across animal and plant kingdoms.

The phytohormone auxin regulates a wide range of developmental processes in plants. Indole-3-acetic acid (IAA) is the main auxin that moves in a polar manner and forms concentration gradients, whereas phenylacetic acid (PAA), another natural auxin, does not exhibit polar movement. Although these auxins occur widely in plants, the differences between IAA and PAA metabolism remain largely unknown. In this study, we investigated the role of Arabidopsis IAA CARBOXYL METHYLTRANSFERASE 1 (IAMT1) in IAA and PAA metabolism. IAMT1 proteins expressed in Escherichia coli could convert both IAA and PAA to their respective methyl esters. Overexpression of IAMT1 caused severe auxin-deficient phenotypes and reduced the levels of IAA, but not PAA, in the root tips of Arabidopsis, suggesting that IAMT1 exclusively metabolizes IAA in vivo. We generated iamt1 null mutants via CRISPR/Cas9-mediated genome editing and found that the single knockout mutants had normal auxin levels and did not exhibit visibly altered phenotypes. These results suggest that other proteins, namely the IAMT1 homologs in the SABATH family of carboxyl methyltransferases, may also regulate IAA levels in Arabidopsis.

Abiotic stresses, including drought and salinity, trigger a complex osmotic-stress and abscisic acid (ABA) signal transduction network. The core ABA signalling components are snf1-related protein kinase2s (SnRK2s), which are activated by ABA-triggered inhibition of type-2C protein-phosphatases (PP2Cs). SnRK2 kinases are also activated by a rapid, largely unknown, ABA-independent osmotic-stress signalling pathway. Here, through a combination of a redundancy-circumventing genetic screen and biochemical analyses, we have identified functionally-redundant MAPKK-kinases (M3Ks) that are necessary for activation of SnRK2 kinases. These M3Ks phosphorylate a specific SnRK2/OST1 site, which is indispensable for ABA-induced reactivation of PP2C-dephosphorylated SnRK2 kinases. ABA-triggered SnRK2 activation, transcription factor phosphorylation and SLAC1 activation require these M3Ks in vitro and in plants. M3K triple knock-out plants show reduced ABA sensitivity and strongly impaired rapid osmotic-stress-induced SnRK2 activation. These findings demonstrate that this M3K clade is required for ABA- and osmotic-stress-activation of SnRK2 kinases, enabling robust ABA and osmotic stress signal transduction., SnRK2 kinases activate abiotic stress responses in plants following ABA-dependent phosphatase inhibition or ABA-independent osmotic stress signalling. Here Takahashi et al. show that MAPKK-kinases phosphorylate and activate SnRK2s thus enabling robust ABA and osmotic stress signal transduction.

The nuclease-dead Cas9 (dCas9) has been reprogrammed for transcriptional activation by fusing dCas9 to a transcriptional activation domain. In the presence of a guide RNA (gRNA), the dCas9 fusions specifically bind to regions of a promoter to activate transcription. Significant amount of effort has been directed toward the identification and optimization of the fusions of dCas9-activation domain, but very little is known about the impact of gRNA target positions within a promoter in plants on transcriptional activation efficiency. The dCas9–6TAL–VP128 system (dCas9-TV) has been optimized to activate transcription in plants. Here we use the dCas9-TV to activate transcription of OsWOX11 and OsYUC1, two genes that cause dramatic developmental phenotypes when overexpressed. We designed a series of gRNAs targeting the promoters of the two genes. We show that gRNAs that target regions within 350 bp upstream of the transcription start site were most effective in transcriptional activation. Moreover, we show that using two gRNAs that simultaneously target two discrete sites in a promoter can further enhance transcription. This work provides guidelines for designed transcriptional activation through CRISPR/dCas9 systems. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42994-019-00007-9) contains supplementary material, which is available to authorized users.