GmDFB1, an ARM-repeat superfamily protein, regulates floral organ identity through repressing siRNA- and miRNA-mediated gene silencing in soybean.

The development of flowers in soybean (Glycine max) is essential for determining the yield potential of the plant. Gene silencing pathways are involved in modulating flower development, but their full elucidation is still incomplete. Here, we conducted a forward genetic screen and identified an abnormal flower mutant, deformed floral bud1-1 (Gmdfb1-1), in soybean. We mapped and identified the causal gene, which encodes a member of the armadillo (ARM)-repeat superfamily. Using small RNA sequencing (sRNA-seq), we found an abnormal accumulation of small interfering RNAs (siRNAs) and microRNA (miRNAs) in the Gmdfb1 mutants. We further demonstrated that GmDFB1 interacts with the RNA exosome cofactor SUPER KILLER7 (GmSKI7). Additionally, GmDFB1 interacts with the PIWI domain of ARGONAUTE 1 (GmAGO1) to inhibit the cleavage efficiency on the target genes of sRNAs. The enhanced gene silencing mediated by siRNA and miRNA in the Gmdfb1 mutants leads to the downregulation of their target genes associated with flower development. This study revealed the crucial role of GmDFB1 in regulating floral organ identity in soybean probably by participating in two distinct gene silencing pathways.

miR394 modulates brassinosteroid signaling to regulate hypocotyl elongation in Arabidopsis.

The interplay between microRNAs (miRNAs) and phytohormones allows plants to integrate multiple internal and external signals to optimize their survival of different environmental conditions. Here, we report that miR394 and its target gene LEAF CURLING RESPONSIVENESS (LCR), which are transcriptionally responsive to BR, participate in BR signaling to regulate hypocotyl elongation in Arabidopsis thaliana. Phenotypic analysis of various transgenic and mutant lines revealed that miR394 negatively regulates BR signaling during hypocotyl elongation, whereas LCR positively regulates this process. Genetically, miR394 functions upstream of BRASSINOSTEROID INSENSITIVE2 (BIN2), BRASSINAZOLEs RESISTANT1 (BZR1), and BRI1-EMS-SUPPRESSOR1 (BES1), but interacts with BRASSINOSTEROID INSENSITIVE1 (BRI1) and BRI1 SUPRESSOR PROTEIN (BSU1). RNA-sequencing analysis suggested that miR394 inhibits BR signaling through BIN2, as miR394 regulates a significant number of genes in common with BIN2. Additionally, miR394 increases the accumulation of BIN2 but decreases the accumulation of BZR1 and BES1, which are phosphorylated by BIN2. MiR394 also represses the transcription of PACLOBUTRAZOL RESISTANCE1/5/6 and EXPANSIN8, key genes that regulate hypocotyl elongation and are targets of BZR1/BES1. These findings reveal a new role for a miRNA in BR signaling in Arabidopsis.

Rewiring of a KNOXI regulatory network mediated by UFO underlies the compound leaf development in Medicago truncatula.

Class I KNOTTED-like homeobox (KNOXI) genes are parts of the regulatory network that control the evolutionary diversification of leaf morphology. Their specific spatiotemporal expression patterns in developing leaves correlate with the degrees of leaf complexity between simple-leafed and compound-leafed species. However, KNOXI genes are not involved in compound leaf formation in several legume species. Here, we identify a pathway for dual repression of MtKNOXI function in Medicago truncatula. PINNATE-LIKE PENTAFOLIATA1 (PINNA1) represses the expression of MtKNOXI, while PINNA1 interacts with MtKNOXI and sequesters it to the cytoplasm. Further investigations reveal that UNUSUAL FLORAL ORGANS (MtUFO) is the direct target of MtKNOXI, and mediates the transition from trifoliate to pinnate-like pentafoliate leaves. These data suggest a new layer of regulation for morphological diversity in compound-leafed species, in which the conserved regulators of floral development, MtUFO, and leaf development, MtKNOXI, are involved in variation of pinnate-like compound leaves in M. truncatula.

UV-B irradiation-activated E3 ligase GmILPA1 modulates gibberellin catabolism to increase plant height in soybean.

Plant height is a key agronomic trait that affects yield and is controlled by both phytohormone gibberellin (GA) and ultraviolet-B (UV-B) irradiation. However, whether and how plant height is modulated by UV-B-mediated changes in GA metabolism are not well understood. It has not been reported that the E3 ubiquitin ligase Anaphase Promoting Complex/Cyclosome (APC/C) is involved in the regulation of plant growth in response to environmental factors. We perform a forward genetic screen in soybean and find that a mutation in Glycine max Increased Leaf Petiole Angle1 (GmILPA1), encoding a subunit of the APC/C, lead to dwarfism under UV-B irradiation. UV-B promotes the accumulation of GmILPA1, which ubiquitinate the GA catabolic enzyme GA2 OXIDASE-like (GmGA2ox-like), resulting in its degradation in a UV-B-dependent manner. Another E3 ligase, GmUBL1, also ubiquitinate GmGA2ox-like and enhance the GmILPA1-mediated degradation of GmGA2ox-like, which suggest that GmILPA1-GmGA2ox-like module counteract the UV-B-mediated reduction of bioactive GAs. We also determine that GmILPA1 is a target of selection during soybean domestication and breeding. The deletion (Indel-665) in the promoter might facilitate the adaptation of soybean to high UV-B irradiation. This study indicates that an evolutionary GmILPA1 variant has the capability to develop ideal plant architecture with soybean cultivars.

H2O2-dependent oxidation of the transcription factor GmNTL1 promotes salt tolerance in soybean.

Reactive oxygen species (ROS) play an essential role in plant growth and responses to environmental stresses. Plant cells sense and transduce ROS signaling directly via hydrogen peroxide (H2O2)-mediated posttranslational modifications (PTMs) on protein cysteine residues. Here, we show that the H2O2-mediated cysteine oxidation of NAC WITH TRANS-MEMBRANE MOTIF1-LIKE 1 (GmNTL1) in soybean (Glycine max) during salt stress promotes its release from the endoplasmic reticulum (ER) membrane and translocation to the nucleus. We further show that an oxidative posttranslational modification on GmNTL1 residue Cys-247 steers downstream amplification of ROS production by binding to and activating the promoters of RESPIRATORY BURST OXIDASE HOMOLOG B (GmRbohB) genes, thereby creating a feed-forward loop to fine-tune GmNTL1 activity. In addition, oxidation of GmNTL1 Cys-247 directly promotes the expression of CATION H+ EXCHANGER 1 (GmCHX1)/SALT TOLERANCE-ASSOCIATED GENE ON CHROMOSOME 3 (GmSALT3) and Na+/H+ Antiporter 1 (GmNHX1). Accordingly, transgenic overexpression of GmNTL1 in soybean increases the H2O2 levels and K+/Na+ ratio in the cell, promotes salt tolerance, and increases yield under salt stress, while an RNA interference-mediated knockdown of GmNTL1 elicits the opposite effects. Our results reveal that the salt-induced oxidation of GmNTL1 promotes its relocation and transcriptional activity through an H2O2-mediated posttranslational modification on cysteine that improves resilience of soybean against salt stress.

HY5 inhibits in vitro shoot stem cell niches initiation via directly repressing pluripotency and cytokinin pathways.

The efficiency of plant regeneration from explants is influenced by phytohormones and environmental conditions. Light has a particularly marked effect on in vitro shoot regeneration, and some light signaling factors are involved in shoot regeneration, while the underlying molecular mechanism remains elusive. Here, ELONGATED HYPOCOTYL5 (HY5), as the key transcription factor of light signaling, was found to inhibit shoot regeneration under a range of light conditions. The heightened shoot regeneration capacity of the hy5-215 mutant was less marked in the dark than in the light, showing that HY5-mediated inhibition of shoot regeneration is partly light dependent. The co-localization of WUSCHEL (WUS) and CLAVATA3 (CLV3) expressions was found to coincide with the initiation of stem cell niches in root explants during shoot regeneration. HY5 could directly repress CLV3 and WUS expression by binding to their respective promoters. In parallel, HY5 indirectly repressed CLV3 and WUS by binding to the ARABIDOPSIS RESPONSE REGULATOR12 (ARR12) promoter. The resulting dual regulation exerted by HY5 on WUS and CLV3 impeded the initiation of shoot stem cell niches. A HY5-mediated inhibitory pathway was identified that links cytokinin signaling and the pluripotency pathway during shoot regeneration.

Cytosine methylation of the FWA promoter promotes direct in vitro shoot regeneration in Arabidopsis thaliana.

Epigenetic modifications within promoter sequences can act as regulators of gene expression. Shoot regeneration is influenced by both DNA methylation and histone methylation, but the mechanistic basis of this regulation is obscure. Here, we identified 218 genes related to the regeneration capacity of callus that were differentially transcribed between regenerable calli (RC) and non-regenerable calli (NRC) in Arabidopsis thaliana. An analysis of the promoters of five of the differentially expressed genes (FWA, ACC1, TFL1, MAX3, and GRP3) pointed to an inverse relationship between cytosine methylation and transcription. The FWA promoter was demethylated and highly expressed in NRC, whereas it was methylated and expressed at low levels in RC. Explants of the hypomethylation mutants fwa-1 and fwa-2 showed strong levels of FWA expression and regenerated less readily than the wild type, suggesting that FWA inhibits direct in vitro shoot regeneration. WUSCHEL-RELATED HOMEOBOX 9 (WOX9), which is required for shoot apical meristem formation, was directly repressed by FWA. Overexpressing WOX9 partly rescued the shoot regeneration defect of fwa-2 plants. These findings suggest that cytosine methylation of the FWA promoter forms part of the regulatory system governing callus regenerability and direct in vitro shoot regeneration.

Arabidopsis WRKY71 regulates ethylene-mediated leaf senescence by directly activating EIN2, ORE1 and ACS2 genes.

Leaf senescence is a pivotal step in the last stage of the plant life cycle and is influenced by various external and endogenous cues. A series of reports have indicated the involvement of the WRKY transcription factors in regulating leaf senescence, but the molecular mechanisms and signaling pathways remain largely unclear. Here we provide evidence demonstrating that WRKY71 acts as a positive regulator of leaf senescence in Arabidopsis. WRKY71-1D, an overexpressor of WRKY71, exhibited early leaf senescence, while wrky71-1, the WRKY71 loss-of-function mutant, displayed delayed leaf senescence. Accordingly, a set of senescence-associated genes (SAGs) were substantially elevated in WRKY71-1D but markedly decreased in wrky71-1. Chromatin immunoprecipitation assays indicated that WRKY71 can bind directly to the promoters of SAG13 and SAG201. Transcriptome analysis suggested that WRKY71 might mediate multiple cues to accelerate leaf senescence, such as abiotic stresses, dark and ethylene. WRKY71 was ethylene inducible, and treatment with the ethylene precursor 1-amino-cyclopropane-1-carboxylic acid enhanced leaf senescence in WRKY71-1D but caused only a marginal delay in leaf senescence in wrky71-1. In vitro and in vivo assays demonstrated that WRKY71 can directly regulate ETHYLENE INSENSITIVE2 (EIN2) and ORESARA1 (ORE1), genes of the ethylene signaling pathway. Consistently, leaf senescence of WRKY71-1D was obviously retarded in the ein2-5 and nac2-1 mutants. Moreover, WRKY71 was also proved to interact with ACS2 in vitro and in vivo. Treatment with AgNO3 and aminoethoxyvinylglycine and acs2-1 could greatly arrest the leaf senescence of WRKY71-1D. In conclusion, our data revealed that WRKY71 mediates ethylene signaling and synthesis to hasten leaf senescence in Arabidopsis.

HBI transcription factor-mediated ROS homeostasis regulates nitrate signal transduction.

Nitrate is both an important nutrient and a critical signaling molecule that regulates plant metabolism, growth, and development. Although several components of the nitrate signaling pathway have been identified, the molecular mechanism of nitrate signaling remains unclear. Here, we showed that the growth-related transcription factors HOMOLOG OF BRASSINOSTEROID ENHANCED EXPRESSION2 INTERACTING WITH IBH1 (HBI1) and its three closest homologs (HBIs) positively regulate nitrate signaling in Arabidopsis thaliana. HBI1 is rapidly induced by nitrate through NLP6 and NLP7, which are master regulators of nitrate signaling. Mutations in HBIs result in the reduced effects of nitrate on plant growth and ∼22% nitrate-responsive genes no longer to be regulated by nitrate. HBIs increase the expression levels of a set of antioxidant genes to reduce the accumulation of reactive oxygen species (ROS) in plants. Nitrate treatment induces the nuclear localization of NLP7, whereas such promoting effects of nitrate are significantly impaired in the hbi-q and cat2 cat3 mutants, which accumulate high levels of H2O2. These results demonstrate that HBI-mediated ROS homeostasis regulates nitrate signal transduction through modulating the nucleocytoplasmic shuttling of NLP7. Overall, our findings reveal that nitrate treatment reduces the accumulation of H2O2, and H2O2 inhibits nitrate signaling, thereby forming a feedback regulatory loop to regulate plant growth and development.

MiR160 and its target genes ARF10, ARF16 and ARF17 modulate hypocotyl elongation in a light, BRZ, or PAC-dependent manner in Arabidopsis: miR160 promotes hypocotyl elongation.

Multiple hormonal and environmental signals participate in the regulation of plant hypocotyl elongation, which allow the plants to optimize their survival strategy from seed germination to seedling establishment. Auxin plays key roles in cell elongation via auxin signaling transduction and its interactions with other hormonal and environmental signals. However, the roles of auxin response factor (ARF) family in cross-talk between auxin and other hormonal or environmental signals during hypocotyl elongation are not fully understood. Here we show that miR160 and its target genes ARF10, ARF16 and ARF17 modulate hypocotyl elongation in a light, brassinazole (BRZ, a BR biosynthesis inhibitor), or paclobutrazol (PAC, a GA biosynthesis inhibitor)-dependent manner in Arabidopsis. miR160, ARF10, ARF16 and ARF17 have no effects on hypocotyl elongation in the dark. However, in the presence of either light, BRZ, or PAC, ARF10, ARF16 and ARF17 inhibit hypocotyl elongation, and miR160 promotes hypocotyl elongation via cleavage of their mRNA. miR160 and ARF10 are both expressed in the hypocotyl. ARF10 represses the expression of PACLOBUTRAZOL RESISTANCE1 (PRE1) and 35S::PRE1 could partly rescue the phenotype of mARF10 (a miR160-resistant form of ARF10), suggesting that PRE1 acts downstream of ARF10 in regulating hypocotyl elongation. In conclusion, our results indicate that miR160-ARF10/16/17 might serve as a molecular link in cross-talk of auxin, light, BR, and GA in hypocotyl elongation.

WinRoots: A High-Throughput Cultivation and Phenotyping System for Plant Phenomics Studies Under Soil Stress.

Soil stress, such as salinity, is a primary cause of global crop yield reduction. Existing crop phenotyping platforms cannot fully meet the specific needs of phenomics studies of plant response to soil stress in terms of throughput, environmental controllability, or root phenotypic acquisition. Here, we report the WinRoots, a low-cost and high-throughput plant soil cultivation and phenotyping system that can provide uniform, controlled soil stress conditions and accurately quantify the whole-plant phenome, including roots. Using soybean seedlings exposed to salt stress as an example, we demonstrate the uniformity and controllability of the soil environment in this system. A high-throughput multiple-phenotypic assay among 178 soybean cultivars reveals that the cotyledon character can serve as a non-destructive indicator of the whole-seedling salt tolerance. Our results demonstrate that WinRoots is an effective tool for high-throughput plant cultivation and soil stress phenomics studies.

HBI1-TCP20 interaction positively regulates the CEPs-mediated systemic nitrate acquisition.

Nitrate is the main source of nitrogen for plants but often distributed heterogeneously in soil. Plants have evolved sophisticated strategies to achieve adequate nitrate by modulating the root system architecture. The nitrate acquisition system is triggered by the short mobile peptides C-TERMINALLY ENCODED PEPTIDES (CEPs) that are synthesized on the nitrate-starved roots, but induce the expression of nitrate transporters on the other nitrate-rich roots through an unclear signal transduction pathway. Here, we demonstrate that the transcription factors HBI1 and TCP20 play important roles in plant growth and development in response to fluctuating nitrate supply. HBI1 physically interacts with TCP20, and this interaction was enhanced by the nitrate starvation. HBI1 and TCP20 directly bind to the promoters of CEPs and cooperatively induce their expression. Mutation in HBIs and/or TCP20 resulted in impaired systemic nitrate acquisition response. Our solid genetic and molecular evidence strongly indicate that the HBI1-TCP20 module positively regulates the CEPs-mediated systemic nitrate acquisition.

The Type-B Cytokinin Response Regulator ARR1 Inhibits Shoot Regeneration in an ARR12-Dependent Manner in Arabidopsis.

Exogenous cytokinin is critical for in vitro shoot regeneration. Proteins involved in the cytokinin signal transduction pathway, including type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs), participate in shoot regeneration in Arabidopsis (Arabidopsis thaliana). Some type-B ARRs (e.g., ARR1 and ARR12) promote shoot regeneration by directly activating WUSCHEL (WUS) expression; however, it is unclear how type-B ARRs inhibit shoot regeneration. Here, we show that ARR12 is a central enhancer of callus formation and shoot regeneration, whereas ARR1 is a strong inhibitor of this process that counteracts the positive effect of ARR12. ARR1 indirectly represses CLAVATA3 (CLV3) expression in an ARR12-dependent manner via competing with ARR12 for binding to the CLV3 promoter, which contributes to its ARR12-dependent inhibitory effect on callus formation and shoot regeneration. In parallel, ARR1 inhibits shoot regeneration through transcriptional activation of INDOLE-3-ACETIC ACID INDUCIBLE17, an auxin response repressor gene, and the consequent indirect repression of WUS expression. Thus, type-B ARRs have diverse effects on callus formation and shoot regeneration. Our study reveals novel molecular pathways linking cytokinin signaling, the CLV3 regulator, and auxin signaling, and sheds light on the mechanism underlying cytokinin-regulated shoot regeneration.

MYB94 and MYB96 additively inhibit callus formation via directly repressing LBD29 expression in Arabidopsis thaliana.

Plant somatic cells can be reprogrammed during in vitro culture. Callus induction is the initial step of a typical plant regeneration system. Recent studies showed that auxin-induced callus formation in multiple organs occurs from the pericycle or pericycle-like cells via a root developmental pathway. However, the molecular control of callus formation is largely unknown. Here, two MYB transcription factors, MYB94 and MYB96, were shown to play negative roles in auxin-induced callus formation in Arabidopsis. MYB94 and MYB96 were expressed in the newly formed callus. myb96, myb94, and myb94 myb96 generated more calli than the WT, with myb94 myb96 producing the most. MYB94 and MYB96 repressed expression of LATERAL ORGAN BOUNDARIES-DOMAIN 29 (LBD29) via directly binding to the gene's promoter. The loss of function of LBD29 partly rescued the callus formation defect of myb94 myb96. Our findings found MYB94 and MYB96 to be important repressors of callus formation and MYB94/96-LBD29 as a new regulatory pathway acting in parallel with ARF7/19-LBDs' pathway to modulate in vitro callus formation.

The salt-induced transcription factor GmMYB84 confers salinity tolerance in soybean.

Transcription factor activation and DNA methylation are important plant responses to abiotic stress. Here, we established that the salinity stress-induced expression of the soybean (Glycine max) transcription factor-encoding gene GmMYB84 relies on DNA methylation. The level of DNA methylation at sequences 690 nt to 950 nt upstream of the GmMYB84 transcription initiation codon was markedly reduced in plants exposed to salinity stress, resulting in a higher abundance of transcripts. When challenged with salinity stress, plants constitutively expressing GmMYB84 outperformed untransformed plants with respect to their germination rate, primary root elongation, proline accumulation, antioxidant enzyme activity, membrane integrity, and K+ levels. Arabidopsis thaliana plants heterologously expressing GmMYB84 were more tolerant to salt stress and exhibited higher germination rates than the wild type. Electrophoretic mobility shift assays revealed that GmMYB84 binds to the cis-regulatory sequences of GmAKT1, the homolog of ARABIDOPSIS K+ TRANSPORTER 1 (AKT1). Thus, DNA methylation modulates the salinity stress-induced expression of the soybean transcription factor-encoding gene GmMYB84 and thereby confers salinity stress tolerance.

A GmSIN1/GmNCED3s/GmRbohBs Feed-Forward Loop Acts as a Signal Amplifier That Regulates Root Growth in Soybean Exposed to Salt Stress.

Abscisic acid (ABA) and reactive oxygen species (ROS) act as key signaling molecules in the plant response to salt stress; however, how these signals are transduced and amplified remains unclear. Here, a soybean (Glycine max) salinity-induced NAM/ATAF1/2/CUC2 (NAC) transcription factor encoded by SALT INDUCED NAC1 (GmSIN1) was shown to be a key component of this process. Overexpression of GmSIN1 in soybean promoted root growth and salt tolerance and increased yield under salt stress; RNA interference-mediated knockdown of GmSIN1 had the opposite effect. The rapid induction of GmSIN1 in response to salinity required ABA and ROS, and the effect of GmSIN1 on root elongation and salt tolerance was achieved by boosting cellular ABA and ROS contents. GmSIN1 upregulated 9-cis-epoxycarotenoid dioxygenase coding genes in soybean (GmNCED3s, associated with ABA synthesis) and Respiratory burst oxidase homolog B genes in soybean (GmRbohBs, associated with ROS generation) by binding to their promoters at a site that has not been described to date. Together, GmSIN1, GmNCED3s, and GmRbohBs constitute a positive feed-forward system that enables the rapid accumulation of ABA and ROS, effectively amplifying the initial salt stress signal. These findings suggest that the combined modulation of ABA and ROS contents enhances soybean salt tolerance.

AGLF provides C-function in floral organ identity through transcriptional regulation of AGAMOUS in Medicago truncatula.

Floral development is one of the model systems for investigating the mechanisms underlying organogenesis in plants. Floral organ identity is controlled by the well-known ABC model, which has been generalized to many flowering plants. Here, we report a previously uncharacterized MYB-like gene, AGAMOUS-LIKE FLOWER (AGLF), involved in flower development in the model legume Medicago truncatula Loss-of-function of AGLF results in flowers with stamens and carpel transformed into extra whorls of petals and sepals. Compared with the loss-of-function mutant of the class C gene AGAMOUS (MtAG) in M. truncatula, the defects in floral organ identity are similar between aglf and mtag, but the floral indeterminacy is enhanced in the aglf mutant. Knockout of AGLF in the mutants of the class A gene MtAP1 or the class B gene MtPI leads to an addition of a loss-of-C-function phenotype, reflecting a conventional relationship of AGLF with the canonical A and B genes. Furthermore, we demonstrate that AGLF activates MtAG in transcriptional levels in control of floral organ identity. These data shed light on the conserved and diverged molecular mechanisms that control flower development and morphology among plant species.

WRKY71 Acts Antagonistically Against Salt-Delayed Flowering in Arabidopsis thaliana.

Soil salinity affects various aspects of plant growth and development including flowering. Usually, plants show a delayed flowering phenotype under high salinity conditions, whereas some plants will risk their life to continue to grow, thereby escaping serious salt stress to achieve reproductive success. However, the molecular mechanisms of the escape strategies are not clear yet. In this work, we report that the transcription factor WRKY71 helps escape salt stress in Arabidopsis. The expression of the WRKY71 wild-type (WT) allele was salinity inducible. Compared with Col-0, high salt stress caused only a marginal delay in the flowering time of the activation-tagged mutant WRKY71-1D. However, flowering in the RNA interference (RNAi)-based multiple WRKY knock-out mutant (w71w8 + 28RNAi) was dramatically later than in the WT under high salinity conditions. Meanwhile, expression of FLOWERING LOCUS T (FT) and LEAFY (LFY) was greater in WRKY71-1D than in the WT, and lower in w71w8 + 28RNAi under salinity-stressed conditions. The suggestion is that WRKY71 activity hastens flowering, thereby providing a means for the plant to complete its life cycle in the presence of salt stress.

miR393 inhibits in vitro shoot regeneration in Arabidopsis thaliana via repressing TIR1.

A large number of genes are involved in the control of shoot regeneration from in vitro cultured plant material. The abundance of the miR393 was different between regenerable and non-regenerable calli induced from Arabidopsis thaliana explants. The regenerability of root explants derived from p35S:miR393a (the miR393a over-expressing line) was shown here to be poorer than that of the wild type (WT). Also, explants derived from plants engineered to constitutively express MIM393 (a mutated form of miR393) had an enhanced level of shoot regeneration. The number of newly formed shoot apical meristems (SAMs) was smaller in p35S:miR393a, while it was larger in p35S:MIM393, compared to the WT, indicating that miR393a inhibited shoot regeneration via repressing the de novo formation of SAMs. The capacity to regenerate shown by plants harboring mTIR1 (a form of TIR1 not cleavable by miR393) was similar to that shown by lines constitutively expressing MIM393, while regenerability of tir1-1 (a loss-of-function mutant) was similar to p35S:miR393a. miR393a and TIR1 were both transcribed at high levels in the initiation sites of nascent shoot apical meristems. Thus, the miR393-TIR1 molecular regulation pathway appears to be a component of the regulatory control over shoot regeneration from in vitro culture.

ARGONAUTE10 Inhibits In Vitro Shoot Regeneration Via Repression of miR165/166 in Arabidopsis thaliana.

Many plant cells retain their totipotency when cultured in vitro. The regulation of shoot regeneration from in vitro culture involves a number of gene products, but the nature of the associated post-transcriptional events remains largely unknown. Here, the post-transcriptional regulator ARGONAUTE10 (AGO10), a protein which is specifically expressed in the explant during the period when pro-shoot apical meristems (SAMs) are forming, has been known to inhibit shoot regeneration. In in vitro cultured explants of the loss-of-function mutant ago10, a much larger than normal number of SAMs was formed and, in these, the stem cell marker genes WUSCHEL, CLAVATA3 and SHOOT MERISTEMLESS were all strongly expressed. AGO10 repressed the accumulation of the microRNAs miR165/166, thereby up-regulating a suite of HD-ZIP III genes. The overproduction of miR166 was shown to promote shoot regeneration, while the absence of miR165/166 message resulted in a blockage to shoot regeneration and only a partial rescue of the phenotype of the ago10 mutant. The major conclusion was that the shoot regeneration inhibition determined by AGO10 functions via the repression of miR165/166.