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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.

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.

The stigma is the entry point for sexual reproduction in plants, but the mechanisms underlying stigma development are largely unknown. Here, we disrupted putative auxin biosynthetic and signaling genes to evaluate their roles in rice (Oryza sativa) development. Disruption of the rice PINOID (OsPID) gene completely eliminated the development of stigmas, and overexpression of OsPID led to overproliferation of stigmas, suggesting that OsPID is a key determinant for stigma development. Interestingly, ospid mutants did not display defects in flower initiation, nor did they develop any pin-like inflorescences, a characteristic phenotype observed in pid mutants in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). We constructed double mutants of OsPID and its closest homolog, OsPIDb, yet the double mutants still did not develop any pin-like inflorescences, indicating that either ospid is compensated by additional homologous genes or OsPID has different functions in rice compared with PID in other organisms. We then knocked out one of the NAKED PINS IN YUC MUTANTS (NPY) genes, which cause the formation of pin-like inflorescences in Arabidopsis when compromised, in the ospid background. The ospid osnpy2 double mutants developed pin-like inflorescences, which were phenotypically similar to pid mutants in Arabidopsis and maize, demonstrating that the roles of OsPID in inflorescence development are likely masked by redundant partners. This work identified a key determinant for stigma development in rice and revealed a complex picture of the PID gene in rice development. Furthermore, the stigma-less ospid mutants are potentially useful in producing hybrid rice.

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.

Dynamic changes in the transcriptional regulatory circuit can influence the specification of distinct cell types. Numerous transcription factors (TFs) have been shown to function through dynamic rewiring during embryonic development but a comprehensive survey on the global regulatory network is still lacking. Here, we performed an integrated analysis of epigenomic and transcriptomic data to reveal key regulators from 2 cells to postnatal day 0 in mouse embryogenesis. We predicted 3D chromatin interactions including enhancer-promoter interactions in 12 tissues across 8 developmental stages, which facilitates linking TFs to their target genes for constructing genetic networks. To identify driver TFs particularly those not necessarily differentially expressed ones, we developed a new algorithm, dubbed as Taiji, to assess the global importance of TFs in development. Through comparative analysis across tissues and developmental stages, we systematically uncovered TFs that are critical for lineage-specific and stage-dependent tissue specification. Most interestingly, we have identified TF combinations that function in spatiotemporal order to form transcriptional waves regulating developmental progress and differentiation. Not only does our analysis provide the first comprehensive map of transcriptional regulatory circuits during mouse embryonic development, the identified novel regulators and the predicted 3D chromatin interactions also provide a valuable resource to guide further mechanistic studies.

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.

Plants optimize their growth and development in response to changes in light quality, quantity, and direction. In dark conditions, Arabidopsis seedlings elongate their hypocotyls while suppressing the expansion and growth of their cotyledons. Fast stem elongation in the dark can ensure that seedlings quickly emerge from underneath the surface of soil to reach light. Once they perceive light, Arabidopsis seedlings expand their cotyledons and gain the capacity to start photosynthesis while greatly reducing the elongation of their hypocotyls. The differential growth rates between cotyledons and hypocotyls in both dark and light are important for plants to grow properly in different light environments, yet the molecular mechanisms by which light, or a lack thereof, can differentially regulate the growth of stems and leaves are not understood. In PNAS, Sun et al. (1) uncover the key roles of a group of Small Auxin Up RNA ( SAUR ) genes in determining the differential growth between stems and leaves (Fig. 1). Fig. 1. Differential expression of lirSAUR s is a key determinant for the different growth rates of cotyledons and hypocotyls under various light conditions. ( A ) High lirSAUR levels stimulate growth, and low lirSAUR expression represses growth. Dark-grown plants have elevated lirSAUR expression in the elongated hypocotyls (red) and decreased expression of lirSAUR s in the closed cotyledons (light blue). In contrast, light-grown seedlings have expanded cotyledons and short hypocotyls, which correlate with high lirSAUR expression in cotyledons and low lirSAUR expression in hypocotyls. Plants grown under shade have long hypocotyls, extended petioles, and small cotyledons. It is not clear whether lirSAUR s also play a role in the differential growth of plants grown under shade. ( B ) Some SAUR genes are differentially expressed in plants grown under shade. A 1-h shade treatment is sufficient to induce the expression of many SAUR genes. Microarray data were previously … [↵][1]1To whom correspondence should be addressed. Email: yundezhao{at}ucsd.edu. [1]: #xref-corresp-1-1

Plants rely on a diverse set of small-molecule hormones to regulate every aspect of their biological processes including development, growth, and adaptation. Since the discovery of the first plant hormone, auxin, hormones have always been at the frontier of plant biology. Although the physiological functions of most plant hormones have been studied for decades, the last 15–20 years have seen dramatic progress in our understanding of the molecular mechanisms of hormone actions. The publication of the whole-genome sequences of the model systems of Arabidopsis and rice, together with the advent of multidisciplinary approaches, has opened the door to successful experimentation on plant hormone actions.

The dek18 mutant of maize was previously classified as a collapsed kernel mutant named cp*-931A, which has a decreased auxin content in kernels. Molecular and functional characterization of this mutant line offers the possibility to better understand auxin biology during maize seed development. Seeds of the dek18 mutants are smaller compared to wild-type seeds and the vegetative development of dek18 is delayed. Here we analyzed the expression of several auxin-related genes in dek18 homozygous seeds and normal-sized seeds (Dek18/-) segregating on the same ear. Three genes related to auxin biosynthesis ZmAlliinase/Tar3, ZmTar1, and ZmYuc1 were highly downregulated in the mutant compared to the wild type. Sequence analysis of these genes revealed that no nucleotide difference is present in dek18 homozygous seeds compared to Dek18/-, except for ZmYuc1. Two different ZmYuc1 cDNAs sequences are produced: a normal-sized sequence of 1197 bp and a shorter coding sequence lacking the third exon. Ectopic expression of ZmYuc1 cDNAs in Arabidopsis indicates that (i) the ZmYuc1 gene is functional in Arabidopsis and (ii) the third exon is required for the enzymatic activity of the YUCCA1 protein. Because ZmYuc1, ZmTar1, and ZmAlliinase are barely expressed in dek18 homozygous seeds, it is proposed that the mutation responsible for the dek18 phenotype alters the upstream regulation of the auxin biosynthetic pathway.

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.

We identify an Arabidopsis pyridoxal-phosphate (PLP)-dependent aminotransferase, VAS1, whose loss-of-function simultaneously increases levels of the phytohormone auxin and the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC). VAS1 uses the ethylene biosynthetic intermediate Met as an amino donor and the auxin biosynthetic intermediate, indole-3-pyruvic acid (3-IPA) as an amino acceptor to produce L-Trp and 2-oxo-4-methylthiobutyric acid (KMBA). Our data indicate that VAS1 serves key roles in coordinating the levels of these two vital hormones.

The development and growth of higher plants is highly dependent on the conduction of water and minerals throughout the plant by xylem vessels. In Arabidopsis roots the xylem is organized as an axis of cell files with two distinct cell fates: the central metaxylem and the peripheral protoxylem. During vascular development, high and low expression levels of the class III HD-ZIP transcription factors promote metaxylem and protoxylem identities, respectively. Protoxylem specification is determined by both mobile, ground tissue-emanating miRNA165/6 species, which downregulate, and auxin concentrated by polar transport, which promotes HD-ZIP III expression. However, the factors promoting high HD-ZIP III expression for metaxylem identity have remained elusive. We show here that auxin biosynthesis promotes HD-ZIP III expression and metaxylem specification. Several auxin biosynthesis genes are expressed in the outer layers surrounding the vascular tissue in Arabidopsis root and downregulation of HD-ZIP III expression accompanied by specific defects in metaxylem development is seen in auxin biosynthesis mutants, such as trp2-12, wei8 tar2 or a quintuple yucca mutant, and in plants treated with L-kynurenine, a pharmacological inhibitor of auxin biosynthesis. Some of the patterning defects can be suppressed by synthetically elevated HD-ZIP III expression. Taken together, our results indicate that polar auxin transport, which was earlier shown to be required for protoxylem formation, is not sufficient to establish a proper xylem axis but that root-based auxin biosynthesis is additionally required.