Publications by authors named "Yunde Zhao"

99 Publications

PIEZO ion channel is required for root mechanotransduction in .

Proc Natl Acad Sci U S A 2021 May;118(20)

Department of Neuroscience, Dorris Neuroscience Center, The Scripps Research Institute, La Jolla, CA 92037;

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 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. 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 PIEZO1 plays an important role in root mechanotransduction and establish PIEZOs as physiologically relevant mechanosensitive ion channels across animal and plant kingdoms.
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http://dx.doi.org/10.1073/pnas.2102188118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8158017PMC
May 2021

Natural allelic variation in a modulator of auxin homeostasis improves grain yield and nitrogen use efficiency in rice.

Plant Cell 2021 May;33(3):566-580

State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China.

The external application of nitrogen (N) fertilizers is an important practice for increasing crop production. However, the excessive use of fertilizers significantly increases production costs and causes environmental problems, making the improvement of crop N-use efficiency (NUE) crucial for sustainable agriculture in the future. Here we show that the rice (Oryza sativa) NUE quantitative trait locus DULL NITROGEN RESPONSE1 (qDNR1), which is involved in auxin homeostasis, reflects the differences in nitrate (NO3-) uptake, N assimilation, and yield enhancement between indica and japonica rice varieties. Rice plants carrying the DNR1indica allele exhibit reduced N-responsive transcription and protein abundance of DNR1. This, in turn, promotes auxin biosynthesis, thereby inducing AUXIN RESPONSE FACTOR-mediated activation of NO3- transporter and N-metabolism genes, resulting in improved NUE and grain yield. We also show that a loss-of-function mutation at the DNR1 locus is associated with increased N uptake and assimilation, resulting in improved rice yield under moderate levels of N fertilizer input. Therefore, modulating the DNR1-mediated auxin response represents a promising strategy for achieving environmentally sustainable improvements in rice yield.
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http://dx.doi.org/10.1093/plcell/koaa037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8136903PMC
May 2021

Cell kinetics of auxin transport and activity in Arabidopsis root growth and skewing.

Nat Commun 2021 03 12;12(1):1657. Epub 2021 Mar 12.

School of Plant Sciences and Food Security, Tel Aviv University, Tel Aviv, Israel.

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.
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http://dx.doi.org/10.1038/s41467-021-21802-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954861PMC
March 2021

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

Plant Cell Environ 2021 May 25;44(5):1692-1706. Epub 2021 Feb 25.

Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, California, USA.

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.
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http://dx.doi.org/10.1111/pce.14023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8068611PMC
May 2021

A reporter for noninvasively monitoring gene expression and plant transformation.

Hortic Res 2020 19;7:152. Epub 2020 Sep 19.

Section of Cell and Developmental Biology, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0116 USA.

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 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 can be used to noninvasively monitor gene expression in plants. Furthermore, we show that 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.
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http://dx.doi.org/10.1038/s41438-020-00390-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502077PMC
September 2020

UDP-glucosyltransferase UGT84B1 regulates the levels of indole-3-acetic acid and phenylacetic acid in Arabidopsis.

Biochem Biophys Res Commun 2020 11 28;532(2):244-250. Epub 2020 Aug 28.

Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, 183-8509, Japan; RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, 230-0045, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbrc.2020.08.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641881PMC
November 2020

Homeobox transcription factor OsZHD2 promotes root meristem activity in rice by inducing ethylene biosynthesis.

J Exp Bot 2020 09;71(18):5348-5364

Crop Biotech Institute and Graduate School of Biotechnology, Kyung Hee University, Yongin, Korea.

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.
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http://dx.doi.org/10.1093/jxb/eraa209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501826PMC
September 2020

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

Biochem Biophys Res Commun 2020 07 20;527(4):1033-1038. Epub 2020 May 20.

RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, 230-0045, Japan; Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, Fuchu, 183-8509, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbrc.2020.05.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7331827PMC
July 2020

Two homologous INDOLE-3-ACETAMIDE (IAM) HYDROLASE genes are required for the auxin effects of IAM in Arabidopsis.

J Genet Genomics 2020 03 19;47(3):157-165. Epub 2020 Mar 19.

Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, 92093-0116, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jgg.2020.02.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7231657PMC
March 2020

Update on Receptors and Signaling.

Plant Physiol 2020 04;182(4):1527-1530

Department of Plant and Microbial Biology, Zürich-Basel Plant Science Center, University of Zürich, Zürich 8008, Switzerland and The Sainsbury Laboratory, University of East Anglia, Norwich Research Park, Norwich NR4 7UH, United Kingdom.

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http://dx.doi.org/10.1104/pp.20.00275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140928PMC
April 2020

Non-intrinsic ATP-binding cassette proteins ABCI19, ABCI20 and ABCI21 modulate cytokinin response at the endoplasmic reticulum in Arabidopsis thaliana.

Plant Cell Rep 2020 Apr 3;39(4):473-487. Epub 2020 Feb 3.

Division of Integrative Bioscience and Biotechnology, POSTECH, Pohang, 37673, Republic of Korea.

Key Message: The 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.
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http://dx.doi.org/10.1007/s00299-019-02503-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7346704PMC
April 2020

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

Nat Commun 2020 01 2;11(1):12. Epub 2020 Jan 2.

Division of Biological Sciences, Cell and Developmental Biology Section, University of California San Diego, La Jolla, CA, 92093, USA.

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.
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http://dx.doi.org/10.1038/s41467-019-13875-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6940395PMC
January 2020

Editorial: Organ Modification for Edible Parts of Horticultural Crops.

Front Plant Sci 2019 23;10:961. Epub 2019 Jul 23.

Section of Cell and Developmental Biology, University of California, San Diego, San Diego, CA, United States.

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http://dx.doi.org/10.3389/fpls.2019.00961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6664054PMC
July 2019

Gibberellins Play a Role in Regulating Tomato Fruit Ripening.

Plant Cell Physiol 2019 Jul;60(7):1619-1629

State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing, China.

Although exogenous applications of gibberellins (GAs) delay tomato ripening, the regulatory mechanisms of GAs in the process have never been well recognized. Here, we report that the concentration of endogenous GAs is declined before the increase of ethylene production in mature-green to breaker stage fruits. We further demonstrate that reductions in GA levels via overexpression of a GA catabolism gene SlGA2ox1 specifically in fruit tissues lead to early ripening. Consistently, we have also observed that application of a GA biosynthetic inhibitor, prohexadione-calcium, at the mature-green stage accelerates fruit ripening, while exogenous GA3 application delays the process. Furthermore, we demonstrate that ethylene biosynthetic gene expressions and ethylene production are activated prematurely in GA-deficient fruits but delayed/reduced in exogenous GA3-treated WT fruits. We also show that the GA deficiency-mediated activation of ethylene biosynthesis is due to the activation of the ripening regulator genes RIN, NOR and CNR. In conclusion, our results demonstrate that GAs play a negative role in tomato fruit ripening.
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http://dx.doi.org/10.1093/pcp/pcz069DOI Listing
July 2019

Plant genome editing using xCas9 with expanded PAM compatibility.

J Genet Genomics 2019 05 5;46(5):277-280. Epub 2019 Apr 5.

Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100081, China. Electronic address:

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http://dx.doi.org/10.1016/j.jgg.2019.03.004DOI Listing
May 2019

The plant ESCRT component FREE1 shuttles to the nucleus to attenuate abscisic acid signalling.

Nat Plants 2019 05 8;5(5):512-524. Epub 2019 Apr 8.

Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University (SCNU), Guangzhou, China.

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.
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http://dx.doi.org/10.1038/s41477-019-0400-5DOI Listing
May 2019

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

Plant Physiol 2019 06 27;180(2):926-936. Epub 2019 Mar 27.

National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China

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 () development. Disruption of the rice () gene completely eliminated the development of stigmas, and overexpression of led to overproliferation of stigmas, suggesting that is a key determinant for stigma development. Interestingly, mutants did not display defects in flower initiation, nor did they develop any pin-like inflorescences, a characteristic phenotype observed in mutants in Arabidopsis () and maize (). We constructed double mutants of and its closest homolog, , yet the double mutants still did not develop any pin-like inflorescences, indicating that either is compensated by additional homologous genes or has different functions in rice compared with in other organisms. We then knocked out one of the () genes, which cause the formation of pin-like inflorescences in Arabidopsis when compromised, in the background. The double mutants developed pin-like inflorescences, which were phenotypically similar to mutants in Arabidopsis and maize, demonstrating that the roles of 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 gene in rice development. Furthermore, the stigma-less mutants are potentially useful in producing hybrid rice.
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http://dx.doi.org/10.1104/pp.18.01389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548269PMC
June 2019

Precise gene replacement in rice by RNA transcript-templated homologous recombination.

Nat Biotechnol 2019 04 18;37(4):445-450. Epub 2019 Mar 18.

Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.

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.
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http://dx.doi.org/10.1038/s41587-019-0065-7DOI Listing
April 2019

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

Plant Physiol 2019 05 13;180(1):453-464. Epub 2019 Mar 13.

Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California 92093-0116

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 () genes in Arabidopsis () using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Cas9) genome-editing technology. We found that plants with a deletion of phenocopied plants overexpressing miRNA167-resistant versions of () or , two miRNA167 targets. Both the mutant and the overexpression lines were defective in anther dehiscence and ovule development. Serendipitously, we found that the (♀) × wild type (♂) crosses failed to produce normal embryos and endosperms, despite the findings that embryos with either or genotypes developed normally when plants were self-pollinated, revealing a central role of in maternal control of seed development. The phenotype is 100% penetrant, providing a great genetic tool for studying the roles of miRNAs and auxin in maternal control. Moreover, we found that mutants flowered significantly later than wild-type plants, a phenotype that was not observed in the overexpression lines. We show that the reproductive defects of mutants were suppressed by a decrease of activities of , , or both. Our results clearly demonstrate that is the predominant member in regulating Arabidopsis reproduction and that acts as a maternal gene that functions largely through and .
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http://dx.doi.org/10.1104/pp.19.00127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6501067PMC
May 2019

Fluorescence Marker-Assisted Isolation of Cas9-Free and CRISPR-Edited Arabidopsis Plants.

Methods Mol Biol 2019 ;1917:147-154

Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, USA.

CRISPR/Cas9 gene editing technology has successfully introduced modifications at target DNA sequences in many plant species including Arabidopsis. After the target gene is edited, the CRISPR/Cas9 construct needs to be removed to ensure genetic stability and to gain any regulatory approval for commercial applications. However, removal of the transgenes by genetic segregation, backcross, and genotyping is very laborious and time-consuming. The methods we report here allow fast and effective isolation of transgene-free T2 Arabidopsis plants with the desired modifications at the target genes. We express a fluorescence protein mCherry under the control of a seed-specific promoter At2S3 and placed the cassette into the CRISPR/Cas9 vector. Therefore, we can use mCherry as a proxy for the presence of Cas9, and we are able to visually isolate the Cas9-free Arabidopsis plants with heritable mutations at the T2 generation. We targeted two sites in the ABP1 gene to demonstrate the effectiveness of our approach.
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http://dx.doi.org/10.1007/978-1-4939-8991-1_11DOI Listing
June 2019

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

J Integr Plant Biol 2019 Sep 19;61(9):968-973. Epub 2019 Mar 19.

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA 92093-0116, USA.

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.
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http://dx.doi.org/10.1111/jipb.12760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588437PMC
September 2019

Modulation of Auxin Signaling and Development by Polyadenylation Machinery.

Plant Physiol 2019 02 28;179(2):686-699. Epub 2018 Nov 28.

Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China

Polyadenylation influences gene expression by affecting mRNA stability, transport, and translatability. Here, we report that Cleavage stimulation Factor 77 (AtCstF77), a component of the pre-mRNA 3'-end polyadenylation machinery, affects polyadenylation site (PAS) selection in transcripts of some auxin signaling genes in Arabidopsis (). Disruption of reduced auxin sensitivity and decreased the expression of the auxin reporter - Null mutations of caused severe developmental defects, but were not lethal as previously reported. - genetically interacted with - auxin receptor double mutants, further supporting that polyadenylation affects auxin signaling. was ubiquitously expressed in embryos, seedlings, and adult plants. The AtCstF77 protein was localized in the nucleus, which is consistent with its function in pre-mRNA processing. We observed that PASs in transcripts from approximately 2,400 genes were shifted in the - mutant. Moreover, most of the PAS shifts were from proximal to distal sites. Auxin treatment also caused PAS shifts in transcripts from a small number of genes. Several auxin signaling or homeostasis genes had different PASs in their transcripts in the - mutant. The expression levels of /-- were significantly increased in the - mutant, which can partially account for the auxin resistance phenotype of this mutant. Our results demonstrate that AtCstF77 plays pleiotropic and critical roles in Arabidopsis development. Moreover, disruption of AtCstF64, another component of the polyadenylation machinery, led to developmental defects and reduced auxin response, similar to those of the - mutant. We conclude that AtCstF77 affects auxin responses, likely by controlling PAS selection of transcripts of some auxin signaling components.
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http://dx.doi.org/10.1104/pp.18.00782DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6426405PMC
February 2019

Editorial: Hormonal Control of Important Agronomic Traits.

Front Plant Sci 2018 17;9:1504. Epub 2018 Oct 17.

School of Environmental & Life Sciences, University of Newcastle, Callaghan, NSW, Australia.

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http://dx.doi.org/10.3389/fpls.2018.01504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199900PMC
October 2018

Agrobacterium tumefaciens Enhances Biosynthesis of Two Distinct Auxins in the Formation of Crown Galls.

Plant Cell Physiol 2019 Jan;60(1):29-37

RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, Japan.

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.
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http://dx.doi.org/10.1093/pcp/pcy182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343636PMC
January 2019

Synthesis-dependent repair of Cpf1-induced double strand DNA breaks enables targeted gene replacement in rice.

J Exp Bot 2018 09;69(20):4715-4721

Institute of Crop Sciences (ICS), Chinese Academy of Agricultural Sciences (CAAS), Beijing, China.

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.
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http://dx.doi.org/10.1093/jxb/ery245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6137971PMC
September 2018

Programmed Self-Elimination of the CRISPR/Cas9 Construct Greatly Accelerates the Isolation of Edited and Transgene-Free Rice Plants.

Mol Plant 2018 09 29;11(9):1210-1213. Epub 2018 May 29.

National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China; College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA 92093-0116, USA. Electronic address:

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http://dx.doi.org/10.1016/j.molp.2018.05.005DOI Listing
September 2018

Auxin production in diploid microsporocytes is necessary and sufficient for early stages of pollen development.

PLoS Genet 2018 05 29;14(5):e1007397. Epub 2018 May 29.

College of Life and Environment Sciences, Shanghai Normal University, Shanghai, China.

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.
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http://dx.doi.org/10.1371/journal.pgen.1007397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5993292PMC
May 2018

The YUCCA-Auxin-WOX11 Module Controls Crown Root Development in Rice.

Front Plant Sci 2018 23;9:523. Epub 2018 Apr 23.

National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, China.

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 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 , which functions upstream of genes, greatly reduces crown root development. We find that overexpression-induced crown root proliferation requires the presence of the transcription factor WOX11. Moreover, the crown rootless phenotype of mutants was partially rescued by overexpression of . Furthermore, we show that expression is induced in overexpression lines, but is repressed in the mutants. Our results indicate that auxin synthesized by the TAA/YUC pathway is necessary and sufficient for crown root development in rice. Auxin activates transcription, which subsequently drives crown root initiation and development, establishing the YUC-Auxin-WOX11 module for crown root development in rice.
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http://dx.doi.org/10.3389/fpls.2018.00523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5925970PMC
April 2018

Efficient allelic replacement in rice by gene editing: A case study of the NRT1.1B gene.

J Integr Plant Biol 2018 Jul 21;60(7):536-540. Epub 2018 May 21.

Institute of Crop Sciences (ICS), Chinese Academy of Agricultural Sciences (CAAS), Beijing 100081, China.

Precise replacement of an existing allele in commercial cultivars with an elite allele is a major goal in crop breeding. A single nucleotide polymorphism in the NRT1.1B gene between japonica and indica rice is responsible for the improved nitrogen use efficiency in indica rice. Herein, we precisely replaced the japonica NRT1.1B allele with the indica allele, in just one generation, using CRISPR/Cas9 gene-editing technology. No additional selective pressure was needed to enrich the precise replacement events. This work demonstrates the feasibility of replacing any genes with elite alleles within one generation, greatly expanding our ability to improve agriculturally important traits.
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http://dx.doi.org/10.1111/jipb.12650DOI Listing
July 2018

Expanding the Scope of CRISPR/Cpf1-Mediated Genome Editing in Rice.

Mol Plant 2018 07 20;11(7):995-998. Epub 2018 Mar 20.

Institute of Crop Sciences (ICS), Chinese Academy of Agricultural Sciences (CAAS), Beijing 100081, China. Electronic address:

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http://dx.doi.org/10.1016/j.molp.2018.03.009DOI Listing
July 2018