Search

Disruption of either the auxin transporter PIN-FORMED 1 (PIN1) or the protein kinase PINOID (PID) leads to the development of pin-like inflorescences. Previous studies have shown that phosphoregulation of PIN1 by AGC kinases including PID directs auxin flux to drive organ initiation. Here, we report unexpected findings on the genetic interactions between these two genes. We deleted the first 2/3 of the PIN1 coding sequence using CRISPR/Cas9, and the resulting pin1 mutant (pin1-27) was a strong allele. Surprisingly, heterozygous pin1-27 suppressed two independent pid null mutants, whereas homozygous pin1-27 enhanced the phenotypes of the pid mutants during embryogenesis. Furthermore, we show that deletion of either the hydrophilic loop or the second half of PIN1 also abolished PIN1 function, yet those heterozygous pin1 mutants were also capable of rescuing pid nulls. Moreover, we inserted green fluorescent protein (GFP) into the hydrophilic loop of PIN1 through CRISPR-mediated homology-directed repair (HDR). The GFP signal and pattern in the PIN1-GFPHDR line are similar to those in the previously reported PIN1-GFP transgenic lines. Interestingly, the PIN1-GFPHDR line also rescued various pid null mutant alleles in a semidominant fashion. We conclude that decreasing the number of functional PIN1 copies is sufficient to suppress the pid mutant phenotype, suggesting that PIN1 is likely part of a larger protein complex required for organogenesis.

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.

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.

The phytohormone auxin, indole-3-acetic acid (IAA), plays a prominent role in plant development. Auxin homeostasis is coordinately regulated by auxin synthesis, transport, and inactivation; however, the physiological contribution of auxin inactivation to auxin homeostasis has not been determined. The GH3 IAA-amino acid conjugating enzymes play a central role in auxin inactivation. Chemical inhibition of GH3 proteins in planta is challenging because the inhibition of these enzymes leads to IAA overaccumulation that rapidly induces GH3 expression. Here, we report the characterization of a potent GH3 inhibitor, kakeimide, that selectively targets IAA-conjugating GH3 proteins. Chemical knockdown of the auxin inactivation pathway demonstrates that auxin turnover is very rapid (about 10 min) and indicates that both auxin biosynthesis and inactivation dynamically regulate auxin homeostasis.

Genome editing technologies provide a powerful tool for genetic improvement of perennial ryegrass, an important forage and turfgrass species worldwide. The sole publication for gene editing in perennial ryegrass used gene-gun for plant transformation and a dual promoter based CRISPR/Cas9 system for editing. However, their editing efficiency was low (5.9% or only one gene-edited plant produced). To test the suitability of the maize Ubiquitin 1 (ZmUbi1) promoter in gene editing of perennial ryegrass, we produced ZmUbi1 promoter:RUBY transgenic plants. We observed that ZmUbi1 promoter was active in callus tissue prior to shoot regeneration, suggesting that the promoter is suitable for Cas9 and sgRNA expression in perennial ryegrass for high-efficiency production of bi-allelic mutant plants. We then used the ZmUbi1 promoter for controlling Cas9 and sgRNA expression in perennial ryegrass. A ribozyme cleavage target site between the Cas9 and sgRNA sequences allowed production of functional Cas9 mRNA and sgRNA after transcription. Using Agrobacterium for genetic transformation, we observed a 29% efficiency for editing the PHYTOENE DESATURASE gene in perennial ryegrass. DNA sequencing analyses revealed that most pds plants contained bi-allelic mutations. These results demonstrate that the expression of a single Cas9 and sgRNA transcript unit controlled by the ZmUbi1 promoter provides a highly efficient system for production of bi-allelic mutants of perennial ryegrass and should also be applicable in other related grass species.

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.

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 genetic 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.

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.

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.

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.

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.

Auxin is a key plant growth regulator for diverse developmental processes in plants. Indole-3-acetic acid (IAA) is a primary plant auxin that regulates the formation of various organs. Plants also produce phenylacetic acid (PAA), another natural auxin, which occurs more abundantly than IAA in various plant species. Although it has been demonstrated that the two auxins have distinct transport characteristics, the metabolic pathways and physiological roles of PAA in plants remain unsolved. In this study, we investigated the role of Arabidopsis UDP-glucosyltransferase UGT84B1 in IAA and PAA metabolism. We demonstrated that UGT84B1, which converts IAA to IAA-glucoside (IAA-Glc), can also catalyze the conversion of PAA to PAA-glucoside (PAA-Glc), with a higher catalytic activity in vitro. Furthermore, we showed a significant increase in both the IAA and PAA levels in the ugt84b1 null mutants. However, no obvious developmental phenotypes were observed in the ugt84b1 mutants under laboratory growth conditions. Moreover, the overexpression of UGT84B1 resulted in auxin-deficient root phenotypes and changes in the IAA and PAA levels. Our results indicate that UGT84B1 plays an important role in IAA and PAA homeostasis in Arabidopsis.

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.

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.

Key messageThe non-intrinsic ABC proteins ABCI20 and ABCI21 are induced by light under HY5 regulation, localize to the ER, and ameliorate cytokinin-driven growth inhibition in young Arabidopsis thaliana seedlings. The plant ATP-binding cassette (ABC) I subfamily (ABCIs) comprises heterogeneous proteins containing any of the domains found in other ABC proteins. Some ABCIs are known to function in basic metabolism and stress responses, but many remain functionally uncharacterized. ABCI19, ABCI20, and ABCI21 of Arabidopsis thaliana cluster together in a phylogenetic tree, and are suggested to be targets of the transcription factor ELONGATED HYPOCOTYL 5 (HY5). Here, we reveal that these three ABCIs are involved in modulating cytokinin responses during early seedling development. The ABCI19, ABCI20 and ABCI21 promoters harbor HY5-binding motifs, and ABCI20 and ABCI21 expression was induced by light in a HY5-dependent manner. abci19 abci20 abci21 triple and abci20 abci21 double knockout mutants were hypersensitive to cytokinin in seedling growth retardation assays, but did not show phenotypic differences from the wild type in either control medium or auxin-, ABA-, GA-, ACC- or BR-containing media. ABCI19, ABCI20, and ABCI21 were expressed in young seedlings and the three proteins interacted with each other, forming a large protein complex at the endoplasmic reticulum (ER) membrane. These results suggest that ABCI19, ABCI20, and ABCI21 fine-tune the cytokinin response at the ER under the control of HY5 at the young seedling stage.

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.

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.

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 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.

CRISPR/Cas9 gene editing technology has been very effective in editing genes in many plant species including rice. Here we further improve the current CRISPR/Cas9 gene editing technology in both efficiency and time needed for isolation of transgene-free and target gene-edited plants. We coupled the CRISPR/Cas9 cassette with a unit that activates anthocyanin biosynthesis, providing a visible marker for detecting the presence of transgenes. The anthocyanin-marker assisted CRISPR (AAC) technology enables us to identify transgenic events even at calli stage, to select transformants with elevated Cas9 expression, and to identify transgene-free plants in the field. We used the AAC technology to edit LAZY1 and G1 and successfully generated many transgene-free and target gene-edited plants at T1 generation. The AAC technology greatly reduced the labor, time, and costs needed for editing target genes in rice.

YUC flavin monooxygenases catalyze the rate-limiting step of auxin biosynthesis. Here we report the vacuolar targeting and degradation of GFP-YUC1. GFP-YUC1 fusion expressed in Arabidopsis protoplasts or transgenic plants was primarily localized in vacuoles. Surprisingly, we established that GFP-YUC1, a soluble protein, was sorted to vacuoles through the ESCRT pathway, which has long been recognized for sorting and targeting integral membrane proteins. We further show that GFP-YUC1 was ubiquitinated and in this form GFP-YUC1 was targeted for degradation, a process that was also stimulated by elevated auxin levels. Our findings revealed a molecular mechanism of GFP-YUC1 degradation and demonstrate that the ESCRT pathway can recognize both soluble and integral membrane proteins as cargoes.

The plant pathogen Agrobacterium tumefaciens infects plants and introduces the transferred-DNA (T-DNA) region of the Ti-plasmid into nuclear DNA of host plants to induce the formation of tumors (crown galls). The T-DNA region carries iaaM and iaaH genes for synthesis of the plant hormone auxin, indole-3-acetic acid (IAA). It has been demonstrated that the iaaM gene encodes a tryptophan 2-monooxygenase which catalyzes the conversion of tryptophan to indole-3-acetamide (IAM), and the iaaH gene encodes an amidase for subsequent conversion of IAM to IAA. In this article, we demonstrate that A. tumefaciens enhances the production of both IAA and phenylacetic acid (PAA), another auxin which does not show polar transport characteristics, in the formation of crown galls. Using liquid chromatography-tandem mass spectroscopy, we found that the endogenous levels of phenylacetamide (PAM) and PAA metabolites, as well as IAM and IAA metabolites, are remarkably increased in crown galls formed on the stem of tomato plants, implying that two distinct auxins are simultaneously synthesized via the IaaM-IaaH pathway. Moreover, we found that the induction of the iaaM gene dramatically elevated the levels of PAM, PAA and its metabolites, along with IAM, IAA and its metabolites, in Arabidopsis and barley. From these results, we conclude that A. tumefaciens enhances biosynthesis of two distinct auxins in the formation of crown galls.

Developing CRISPR/Cas9-mediated non-transgenic mutants in asexually propagated perennial crop plants is challenging but highly desirable. Here, we report a highly useful method using an Agrobacterium-mediated transient CRISPR/Cas9 gene expression system to create non-transgenic mutant plants without the need for sexual segregation. We have also developed a rapid, cost-effective, and high-throughput mutant screening protocol based on Illumina sequencing followed by high-resolution melting (HRM) analysis. Using tetraploid tobacco as a model species and the phytoene desaturase (PDS) gene as a target, we successfully created and expediently identified mutant plants, which were verified as tetra-allelic mutants. We produced pds mutant shoots at a rate of 47.5% from tobacco leaf explants, without the use of antibiotic selection. Among these pds plants, 17.2% were confirmed to be non-transgenic, for an overall non-transgenic mutation rate of 8.2%. Our method is reliable and effective in creating non-transgenic mutant plants without the need to segregate out transgenes through sexual reproduction. This method should be applicable to many economically important, heterozygous, perennial crop species that are more difficult to regenerate.

The recently developed CRISPR (clustered regularly interspaced short palindromic repeats)/Cpf1 system expands the range of genome editing and is emerging as an alternative powerful tool for both plant functional genomics and crop improvement. Cpf1-CRISPR RNA (crRNA) produces double strand DNA breaks (DSBs) with long 5'-protruding ends, which may facilitate the pairing and insertion of repair templates through homology-directed repair (HDR) for targeted gene replacement and introduction of the desired DNA elements at specific gene loci for crop improvement. However, the potential mechanism underlying HDR of DSBs generated by Cpf1-crRNA remains to be investigated, and the inherent low efficiency of HDR and poor availability of exogenous donor DNA as repair templates strongly impede the use of HDR for precise genome editing in crop plants. Here, we provide evidence of synthesis-dependent repair of Cpf1-induced DSBs, which enables us precisely to replace the wild-type ALS gene with the intended mutant version that carries two discrete point mutations conferring herbicide resistance to rice plants. Our observation that the donor repair template (DRT) with only the left homologous arm is sufficient for precise targeted allele replacement offers a better understanding of the mechanism underlying HDR in plants, and greatly simplifies the design of DRTs for precision genome editing in crop improvement.

Gametophytic development in Arabidopsis depends on nutrients and cell wall materials from sporophytic cells. However, it is not clear whether hormones and signaling molecules from sporophytic tissues are also required for gametophytic development. Herein, we show that auxin produced by the flavin monooxygenases YUC2 and YUC6 in the sporophytic microsporocytes is essential for early stages of pollen development. The first asymmetric mitotic division (PMI) of haploid microspores is the earliest event in male gametophyte development. Microspore development in yuc2yuc6 double mutants arrests before PMI and consequently yuc2yuc6 fail to produce viable pollens. Our genetic analyses reveal that YUC2 and YUC6 act as sporophytic genes for pollen formation. We further show that ectopic production of auxin in tapetum, which provides nutrients for pollen development, fails to rescue the sterile phenotypes of yuc2yuc6. In contrast, production of auxin in either microsporocytes or microspores rescued the defects of pollen development in yuc2yuc6 double mutants. Our results demonstrate that local auxin biosynthesis in sporophytic microsporocytic cells and microspore controls male gametophyte development during the generation transition from sporophyte to male gametophyte.

A well-developed root system in rice and other crops can ensure plants to efficiently absorb nutrients and water. Auxin is a key regulator for various aspect of root development, but the detailed molecular mechanisms by which auxin controls crown root development in rice are not understood. We show that overexpression of a YUC gene, which encodes the rate-limiting enzyme in auxin biosynthesis, causes massive proliferation of crown roots. On the other hand, we find that disruption of TAA1, which functions upstream of YUC genes, greatly reduces crown root development. We find that YUC overexpression-induced crown root proliferation requires the presence of the transcription factor WOX11. Moreover, the crown rootless phenotype of taa1 mutants was partially rescued by overexpression of WOX11. Furthermore, we show that WOX11 expression is induced in OsYUC1 overexpression lines, but is repressed in the taa1 mutants. Our results indicate that auxin synthesized by the TAA/YUC pathway is necessary and sufficient for crown root development in rice. Auxin activates WOX11 transcription, which subsequently drives crown root initiation and development, establishing the YUC-Auxin-WOX11 module for crown root development in rice.

Cereals high in amylose content (AC) and resistant starch (RS) offer potential health benefits. Previous studies using chemical mutagenesis or RNA interference have demonstrated that starch branching enzyme (SBE) plays a major role in determining the fine structure and physical properties of starch. However, it remains a challenge to control starch branching in commercial lines. Here, we use CRISPR/Cas9 technology to generate targeted mutagenesis in SBEI and SBEIIb in rice. The frequencies of obtained homozygous or bi-allelic mutant lines with indels in SBEI and SBEIIb in T0 generation were from 26.7 to 40%. Mutations in the homozygous T0 lines stably transmitted to the T1 generation and those in the bi-allelic lines segregated in a Mendelian fashion. Transgene-free plants carrying only the frame-shifted mutagenesis were recovered in T1 generation following segregation. Whereas no obvious differences were observed between the sbeI mutants and wild type, sbeII mutants showed higher proportion of long chains presented in debranched amylopectin, significantly increased AC and RS content to as higher as 25.0 and 9.8%, respectively, and thus altered fine structure and nutritional properties of starch. Taken together, our results demonstrated for the first time the feasibility to create high-amylose rice through CRISPR/Cas9-mediated editing of SBEIIb.

CRISPR/Cas9-mediated genome editing relies on a guide RNA (gRNA) molecule to generate sequence-specific DNA cleavage, which is a prerequisite for gene editing. Here we establish a method that enables production of gRNAs from any promoters, in any organisms, and in vitro (Gao and Zhao, 2014). This method also makes it feasible to conduct tissue/cell specific gene editing.

Auxin responses have been arbitrarily divided into two categories: genomic and non-genomic effects. Genomic effects are largely mediated by SCFTIR1/AFB-Aux/IAA auxin receptor complexes whereas it has been postulated that AUXIN BINDING PROTEIN 1 (ABP1) controls the non-genomic effects. However, the roles of ABP1 in auxin signaling and plant development were recently called into question. In this paper, we present recent progress in understanding the SCFTIR1/AFB-Aux/IAA pathway. In more detail, we discuss the current understanding of ABP1 research and provide an updated view of ABP1-related genetic materials. Further, we propose a model in which auxin efflux carriers may play a role in auxin perception and we briefly describe recent insight on processes downstream of auxin perception.

Phytohormone auxin is a master regulator in plant growth and development. Regulation of cellular auxin level plays a central role in plant development. Auxin polar transport system modulates an auxin gradient that determines plant developmental process in response to environmental conditions and developmental programs. Photolabile caged auxins allow optical control of artificial auxin gradients at cellular resolution. Especially, two-photon uncaging system achieves high spatiotemporal control of photolysis reaction at two-photon cross-section. However, the development of caged versions of auxin has been limited by the instability of the caged auxins to higher plant metabolic activities. Here, we describe the synthesis and application of highly stable caged auxins, 4-methoxy-7-nitroindolinyl (MNI)-caged auxins. Natural auxin, indole 3-acetic acid, and two synthetic auxins, 1-NAA and 2,4-D were caged by MNI caging group. MNI-caged auxins showed a high stability in planta and a rapid release the original auxin when photolyzed. We demonstrated that optical control of auxin-responsive gene expression and auxin-related physiological responses by using MNI-caged auxins. We anticipate that MNI-caged auxins will be an effective tool for high-resolution control of endogenous auxin level.

The phytohormone auxin plays a central role in many aspects of plant growth and development. IAA is the most studied natural auxin that possesses the property of polar transport in plants. Phenylacetic acid (PAA) has also been recognized as a natural auxin for >40 years, but its role in plant growth and development remains unclear. In this study, we show that IAA and PAA have overlapping regulatory roles but distinct transport characteristics as auxins in plants. PAA is widely distributed in vascular and non-vascular plants. Although the biological activities of PAA are lower than those of IAA, the endogenous levels of PAA are much higher than those of IAA in various plant tissues in Arabidopsis. PAA and IAA can regulate the same set of auxin-responsive genes through the TIR1/AFB pathway in Arabidopsis. IAA actively forms concentration gradients in maize coleoptiles in response to gravitropic stimulation, whereas PAA does not, indicating that PAA is not actively transported in a polar manner. The induction of the YUCCA (YUC) genes increases PAA metabolite levels in Arabidopsis, indicating that YUC flavin-containing monooxygenases may play a role in PAA biosynthesis. Our results provide new insights into the regulation of plant growth and development by different types of auxins.

Membrane trafficking is essential for plant growth and responses to external signals. The plant unique FYVE domain-containing protein FREE1 is a component of the ESCRT complex (endosomal sorting complex required for transport). FREE1 plays multiple roles in regulating protein trafficking and organelle biogenesis including the formation of intraluminal vesicles of multivesicular body (MVB), vacuolar protein transport and vacuole biogenesis, and autophagic degradation. FREE1 knockout plants show defective MVB formation, abnormal vacuolar transport, fragmented vacuoles, accumulated autophagosomes, and seedling lethality. To further uncover the underlying mechanisms of FREE1 function in plants, we performed a forward genetic screen for mutants that suppressed the seedling lethal phenotype of FREE1-RNAi transgenic plants. The obtained mutants are termed as suppressors of free1 (sof). To date, 229 putative sof mutants have been identified. Barely detecting of FREE1 protein with M3 plants further identified 84 FREE1-related suppressors. Also 145 mutants showing no reduction of FREE1 protein were termed as RNAi-related mutants. Through next-generation sequencing (NGS) of bulked DNA from F2 mapping population of two RNAi-related sof mutants, FREE1-RNAi T-DNA inserted on chromosome 1 was identified and the causal mutation of putative sof mutant is being identified similarly. These FREE1- and RNAi-related sof mutants will be useful tools and resources for illustrating the underlying mechanisms of FREE1 function in intracellular trafficking and organelle biogenesis, as well as for uncovering the new components involved in the regulation of silencing pathways in plants.

Auxin binding protein 1 (ABP1) has been studied for decades. It has been suggested that ABP1 functions as an auxin receptor and has an essential role in many developmental processes. Here we present our unexpected findings that ABP1 is neither required for auxin signaling nor necessary for plant development under normal growth conditions. We used our ribozyme-based CRISPR technology to generate an Arabidopsis abp1 mutant that contains a 5-bp deletion in the first exon of ABP1, which resulted in a frameshift and introduction of early stop codons. We also identified a T-DNA insertion abp1 allele that harbors a T-DNA insertion located 27 bp downstream of the ATG start codon in the first exon. We show that the two new abp1 mutants are null alleles. Surprisingly, our new abp1 mutant plants do not display any obvious developmental defects. In fact, the mutant plants are indistinguishable from wild-type plants at every developmental stage analyzed. Furthermore, the abp1 plants are not resistant to exogenous auxin. At the molecular level, we find that the induction of known auxin-regulated genes is similar in both wild-type and abp1 plants in response to auxin treatments. We conclude that ABP1 is not a key component in auxin signaling or Arabidopsis development.

Decades of research have suggested that AUXIN BINDING PROTEIN 1 (ABP1) is an essential membrane-associated auxin receptor, but recent findings directly contradict this view. Here we show that embryonic lethality observed in abp1-1, which has been a cornerstone of ABP1 studies, is caused by the deletion of the neighbouring BELAYA SMERT (BSM) gene, not by disruption of ABP1. On the basis of our results, we conclude that ABP1 is not essential for Arabidopsis development.

Auxin plays an essential role in root development. It has been a long-held dogma that auxin required for root development is mainly transported from shoots into roots by polarly localized auxin transporters. However, it is known that auxin is also synthesized in roots. Here we demonstrate that a group of YUCCA (YUC) genes, which encode the rate-limiting enzymes for auxin biosynthesis, plays an essential role in Arabidopsis root development. Five YUC genes (YUC3, YUC5, YUC7, YUC8 and YUC9) display distinct expression patterns during root development. Simultaneous inactivation of the five YUC genes (yucQ mutants) leads to the development of very short and agravitropic primary roots. The yucQ phenotypes are rescued by either adding 5 nM of the natural auxin, IAA, in the growth media or by expressing a YUC gene in the roots of yucQ. Interestingly, overexpression of a YUC gene in shoots in yucQ causes the characteristic auxin overproduction phenotypes in shoots; however, the root defects of yucQ are not rescued. Our data demonstrate that localized auxin biosynthesis in roots is required for normal root development and that auxin transported from shoots is not sufficient for supporting root elongation and root gravitropic responses.

lndole-3-acetic acid (IAA), the most important natural auxin in plants, is mainly synthesized from the amino acid tryptophan (Trp). Recent genetic and biochemical studies in Arabidopsis have unambiguously established the first complete Trp-dependent auxin biosynthesis pathway. The first chemical step of auxin biosynthesis is the removal of the amino group from Trp by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) family of transaminases to generate indole-3-pyruvate (IPA). IPA then undergoes oxidative decarboxylation catalyzed by the YUCCA (YUC) family of flavin monooxygenases to produce IAA. This two-step auxin biosynthesis pathway is highly conserved throughout the plant kingdom and is essential for almost all of the major developmental processes. The successful elucidation of a complete auxin biosynthesis pathway provides the necessary tools for effectively modulating auxin concentrations in plants with temporal and spatial precision. The progress in auxin biosynthesis also lays a foundation for understanding polar auxin transport and for dissecting auxin signaling mechanisms during plant development.

Elimination of the CRISPR/Cas9 constructs in edited plants is a prerequisite for assessing genetic stability, conducting phenotypic characterization, and applying for commercialization of the plants. However, removal of the CRISPR/Cas9 transgenes by genetic segregation and by backcross is laborious and time consuming. We previously reported the development of the transgene killer CRISPR (TKC) technology that uses a pair of suicide genes to trigger self-elimination of the transgenes without compromising gene editing efficiency. The TKC technology enables isolation of transgene-free CRISPR-edited plants within a single generation, greatly accelerating crop improvements. Here, we presented two new TKC vectors that show great efficiency in both editing the target gene and in undergoing self-elimination of the transgenes. The new vectors replaced the CaMV35S promoter used in our previous TKC vector with two rice promoters to drive one of the suicide genes, providing advantages over our previous TKC vector under certain conditions. The vectors reported here offered more options and flexibility to conduct gene editing experiments in rice. Keywords: genome editing, suicide gene, transgene killer CRISPR, Cas9, transgene-free