Local conjugation of auxin by the GH3 amido synthetases is required for normal development of roots and flowers in Arabidopsis.

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. 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. Moreover, this work provides a method for elucidating functions of individual members of a gene family, whose members have overlapping functions.

The main oxidative inactivation pathway of the plant hormone auxin.

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.

PIEZO ion channel is required for root mechanotransduction in Arabidopsis thaliana.

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.

An amiRNA screen uncovers redundant CBF and ERF34/35 transcription factors that differentially regulate arsenite and cadmium responses.

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.

Role of Arabidopsis INDOLE-3-ACETIC ACID CARBOXYL METHYLTRANSFERASE 1 in auxin metabolism.

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.

MAP3Kinase-dependent SnRK2-kinase activation is required for abscisic acid signal transduction and rapid osmotic stress response.

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.

An Essential Role for miRNA167 in Maternal Control of Embryonic and Seed Development.

Maternal cells play a critical role in ensuring the normal development of embryos, endosperms, and seeds. Mutations that disrupt the maternal control of embryogenesis and seed development are difficult to identify. Here, we completely deleted four MICRORNA167 (MIR167) genes in Arabidopsis (Arabidopsis thaliana) using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Cas9) genome-editing technology. We found that plants with a deletion of MIR167A phenocopied plants overexpressing miRNA167-resistant versions of Auxin Response Factor6 (ARF6) or ARF8, two miRNA167 targets. Both the mir167a mutant and the ARF overexpression lines were defective in anther dehiscence and ovule development. Serendipitously, we found that the mir167a (♀) × wild type (♂) crosses failed to produce normal embryos and endosperms, despite the findings that embryos with either mir167a+/- or mir167a-/- genotypes developed normally when mir167a+/- plants were self-pollinated, revealing a central role of MIR167A in maternal control of seed development. The mir167a phenotype is 100% penetrant, providing a great genetic tool for studying the roles of miRNAs and auxin in maternal control. Moreover, we found that mir167a mutants flowered significantly later than wild-type plants, a phenotype that was not observed in the ARF overexpression lines. We show that the reproductive defects of mir167a mutants were suppressed by a decrease of activities of ARF6, ARF8, or both. Our results clearly demonstrate that MIR167A is the predominant MIR167 member in regulating Arabidopsis reproduction and that MIR167A acts as a maternal gene that functions largely through ARF6 and ARF8.

PINOID Is Required for Formation of the Stigma and Style in Rice.

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.

ESCRT-dependent vacuolar sorting and degradation of the auxin biosynthetic enzyme YUC1 flavin monooxygenase.

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.