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In plants, jasmonate ZIM-domain proteins (JAZs) act as critical regulators, interacting physically with transcription factors (TFs) and other transcriptional regulators to modulate jasmonate (JA)-responsive gene expression and participate in crosstalk with other hormone signalling pathways. Identifying novel JAZ-interacting proteins will provide new insights into JA signalling cascades in plants. Here, we performed yeast two-hybrid screening to identify 70 NtJAZ1-interacting proteins, including an A/T-rich interaction domain containing protein 1 (NtAIDP1) from JA-treated tobacco Bright Yellow-2 (BY-2) cells. NtAIDP1 is localised in the nucleus and interacts with NtJAZ1 via its C-terminal heat shock protein 20 (HSP) domain. Aside from NtJAZ1, NtAIDP1 also interacts with other JA-inducible NtJAZs, including NtJAZ2b, NtJAZ2b.2, NtJAZ5, NtJAZ7, NtJAZ11 and NtJAZ12, but not with NtJAZ3, NtJAZ3b or NtJAZ10, and interacts with NtNINJA, NtDELLA1 and NtDELLA2 in the yeast two-hybrid assay. Furthermore, NtAIDP1 binds to the AT-rich region of the GAG fragment of the putrescine N-methyltransferase 1a (NtPMT1a) promoter and activates the transcriptional activity of the GAG fragment, whereas NtMYC2a interacts with and competitively inhibits the transactivational activity of NtAIDP1 in Arabidopsis mesophyll protoplasts. Overexpression of NtAIDP1 promotes the transcription of NtPDF1.2 and NtJAZ1, but has little effect on the expression of NtPMT1a, quinolinate phosphoribosyltransferase 2 (NtQPT2), and NtMYC2a in tobacco. These results indicate that NtAIDP1 is a new component of the JA signalling pathway and is involved in JA-regulated gene expression.

Drought and salt are two major abiotic stresses that severely impact plant growth and development, as well as crop production. A previous study showed that OsOSCA1.4, one of eleven rice OSCAs (OsOSCAs), complements hyperosmolality-induced [Ca2+]cyt increases (OICIcyt), salt stress-induced [Ca2+]cyt increases (SICIcyt) and the associated growth phenotype in Arabidopsis osca1 (reduced hyperosmolality-induced [Ca2+]cyt increase 1). In this study, Except for OsOSCA2.3 and OsOSCA4.1, we generated independent transgenic lines overexpressing eight other OsOSCAs in the osca1 to explore their functions in osmotic Ca2+ signalling, stomatal movement, leaf water loss, and root growth in response to hyperosmolality and salt stress. Similar to OsOSCA1.4, overexpression of OsOSCA1.1 or OsOSCA2.2 in osca1 complemented OICIcyt and SICIcyt, as well as stomatal closure and root growth in response to hyperosmolality and salt stress treatments, and drought-related leaf water loss. In addition, overexpression of OsOSCA1.2, OsOSCA1.3 or OsOSCA2.1 in osca1 restored OICIcyt and SICIcyt, whereas overexpression of OsOSCA2.5 or OsOSCA3.1 did not. Moreover, osca1 overexpressing these five OsOSCAs exhibited various abiotic stress-associated growth phenotypes. However, overexpression of OsOSCA2.4 did not have any of these effects. These results indicated that multiple members of the OsOSCA family have redundant functions in osmotic sensing and diverse roles in stress adaption.

Ethylene response factor (ERF) Group VII members generally function in regulating plant growth and development, abiotic stress responses, and plant immunity in Arabidopsis; however, the details of the regulatory mechanism by which Group VII ERFs mediate plant immune responses remain elusive. Here, we characterized one such member, ERF72, as a positive regulator that mediates resistance to the necrotrophic pathogen Botrytis cinerea. Compared with the wild-type (WT), the erf72 mutant showed lower camalexin concentration and was more susceptible to B. cinerea, while complementation of ERF72 in erf72 rescued the susceptibility phenotype. Moreover, overexpression of ERF72 in the WT promoted camalexin biosynthesis and increased resistance to B. cinerea. We identified the camalexin-biosynthesis genes PAD3 and CYP71A13 and the transcription factor WRKY33 as target genes of ERF72. We also determined that MPK3 and MPK6 phosphorylated ERF72 at Ser151 and improved its transactivation activity, resulting in increased camalexin concentration and increased resistance to B. cinerea. Thus, ERF72 acts in plant immunity to coordinate camalexin biosynthesis both directly by regulating the expression of biosynthetic genes and indirectly by targeting WRKK33.

The biosynthesis and transport of nicotine has been shown to be coordinately upregulated by jasmonate (JA). MYC2, a member of basic helix-loop-helix (bHLH) transcription factor family, is well-documented as the core player in the JA signalling pathway to regulate diverse plant development processes. Four MYC2 genes were found in the tobacco genome, NtMYC2a/2b and 1a/1b. In this study, we tested whether one of them, NtMYC2a, acts as a 'master switch' in the regulation of nicotine biosynthesis and transport in tobacco. We generated NtMYC2a knockout tobacco plants using the CRISPR-Cas9 technique and analysed the effect of NtMYC2a knockout on expression of the nicotine biosynthesis genes (NtAO, NtQS, NtPMT1a, NtQPT2, NtODC2, NtMPO1, NtA622 and NtBBLa) and transport genes (NtMATE2 and NtJAT1), as well as leaf accumulation of nicotine in the NtMYC2a knockout plants. We found that all the nicotine biosynthesis and transport genes tested in this study were significantly downregulated (>50% reduction compared with wild-type control) in the NtMYC2a knockout plants. Moreover, the leaf nicotine content in knockout plants was dramatically reduced by ca 80% compared with the wild-type control. These results clearly show that NtMYC2a acts as a 'master switch' to coordinate JA-induced nicotine accumulation in tobacco and suggests that NtMYC2a might play an important role in tobacco nicotine-mediated defence against herbivory.

Generally, the caryopses located on proximal secondary branches (CSB) have smaller grain size and slower and poorer filling rate than those on apical primary branches (CPB) in rice, which greatly limits the grain yield potential fulfillment. However, the key regulators determining the developmental differences between CPB and CSB remain elusive. Here, we have performed transcriptomic analysis in CPB and CSB at four developmental stages [0, 5, 12 and 20 days after fertilization (DAF)] using high-throughput RNA-sequencing technique. Based on gene expression cluster analysis, the genes expressed in CPB and CSB were clustered into two subtypes in a positional-independent manner: one includes 0- and 5-DAF CPB and CSB, and 12-DAF CSB; another includes 12-DAF CPB, 20-DAF CPB and CSB. Moreover, according to the expression value of each gene, K-mean cluster analysis showed that the K4 to K6 classifiers contain the genes highly expressed in 5-DAF CPB and 12-DAF CSB, which were enriched in DNA synthesis, protein synthesis and cell proliferation mainly responsible for grain size decision. Then, functional enrichment analysis in Gene Ontology database showed that auxin-related genes were relatively enriched, indicating that auxin might be the key determinant for gene expression in K4 to K6 classifiers. Finally, the application of exogenous IAA in CSB before fertilization promoted gene expression, caryopsis development and grain weight closer to that in CPB, providing a molecular framework to optimize CSB development and potential targets for increasing grain yield.

In plants, both hyperosmolality and salt stress induce cytosolic calcium increases within seconds, referred to as the hyperosmolality-induced [Ca2+]cyt increases, OICIcyt, and salt stress-induced [Ca2+]cyt increases, SICIcyt. Previous studies have shown that Arabidopsis reduced hyperosmolality-induced [Ca2+]i increase 1 (OSCA1.1) encodes a hyperosmolality-gated calcium-permeable channel that mediates OICIcyt in guard cells and root cells. Multiple OSCA members exist in plants; for example, Oryza sativa has 11 OsOSCAs genes, indicating that OSCAs have diverse biological functions. Here, except for OsOSCA4.1, ten full-length OsOSCAs were separately subcloned, in which OsOSCA1.4 was exclusively localised to the plasma membrane and other nine OsOSCAs-eYFP co-localised with an endoplasmic reticulum marker in Arabidopsis mesophyll protoplasts. OsOSCA1.4 was further identified as a calcium-permeable ion channel that activates an inward current after receiving an osmotic signal exerted by hyperosmolality or salt stress, and mediates OICIcyt and SICIcyt in human embryonic kidney 293 (HEK293) cells. Moreover, overexpression of OsOSCA1.4 in Arabidopsis osca1 mutant complemented osmotic Ca2+ signalling, root growth, and stomatal movement in response to hyperosmolality and salt stress. These results will facilitate further study of OsOSCA-mediated calcium signalling and its distinct roles in rice growth and development.

Being the largest the Ca2+ store in mammalian cells, endoplasmic reticulum (ER)-mediated Ca2+ signalling often involves both Ca2+ release via inositol 1, 4, 5-trisphosphate receptors (IP3R) and store operated Ca2+ entries (SOCE) through Ca2+ release activated Ca2+ (CRAC) channels on plasma membrane (PM). IP3Rs are functionally coupled with CRAC channels and other Ca2+ handling proteins. However, it still remains less well defined as to whether IP3Rs could regulate ER-mediated Ca2+ signals independent of their Ca2+ releasing ability. To address this, we generated IP3Rs triple and double knockout human embryonic kidney (HEK) cell lines (IP3Rs-TKO, IP3Rs-DKO), and systemically examined ER Ca2+ dynamics and CRAC channel activity in these cells. The results showed that the rate of ER Ca2+ leakage and refilling, as well as SOCE were all significantly reduced in IP3Rs-TKO cells. And these TKO effects could be rescued by over-expression of IP3R3. Further, results showed that the diminished SOCE was caused by NEDD4L-mediated ubiquitination of Orai1 protein. Together, our findings indicate that IP3R3 is one crucial player in coordinating ER-mediated Ca2+ signalling.

Hypocotyl cell elongation related to photomorphogenesis in Arabidopsis seedlings is regulated by a network involving ethylene, auxin, and brassinosteroid signalling that is mediated by interactions among ERF72, ARF6, and BZR1, forming a revised BZR-ARF-PIF/DELLA-ERF (BAP/DE) module., The phytohormones brassinosteroid (BR), auxin, and gibberellin (GA) regulate photomorphogenesis-related hypocotyl elongation in Arabidopsis via the co-operative interaction of BZR-ARF-PIF/DELLA (BAP/D) transcription factors/regulators. In addition, ethylene activates the PIF3 or ERF1 pathway through EIN3/EIL1 to balance hypocotyl elongation in Arabidopsis seedlings. However, the mechanism by which ethylene is co-ordinated with other phytohormones to produce light-regulated hypocotyl growth remains elusive. In this study, we found that hypocotyl cell elongation is regulated by a network involving ethylene, auxin, and BR signalling, which is mediated by interactions among ERF72, ARF6, and BZR1. ERF72 interacted directly with ARF6 and BZR1 in vitro and in vivo, and it antagonised regulation by ARF6 and BZR1 of the transcription of BEE3 and XTH7. In addition, light modulated the subcellular localisation of ERF72 and transcription of ERF72 through the EIN2-EIN3/EIL1 pathway, facilitating the function of ERF72 in photomorphogenesis. The expression of BEE3 and XTH7 was also regulated by the EIN2-EIN3/EIL1 pathway. Our findings indicate that a revised BZR-ARF-PIF/DELLA-ERF (BAP/DE) module integrates light and hormone signals to regulate hypocotyl elongation in Arabidopsis.

In flowering plants, pollen development is a critical step for reproductive success and necessarily involves complex genetic regulatory networks. Calcium-dependent protein kinases (CPKs) are plant-specific calcium sensors involved in the regulation of plant development and adaption to the environment; however, whether they play a role in regulating male reproduction remains elusive. Here, we found that the knockdown of spikelet-specific OsCPK21 causes pollen abortion in OsCPK21-RNAi transgenic plants. Severe defects in pollen development initiated at stage 10 of anther development and simultaneous cell death occurred in the pollen cells of OsCPK21-RNAi plants. Microarray analysis and qRT-PCR revealed that the transcription of OsCPK21 is coordinated with that of MIKC*-type MADS box transcription factors OsMADS62, OsMADS63, and OsMADS68 during rice anther development. We further showed that OsCPK21 indirectly up-regulates the transcription of OsMADS62, OsMADS63, and OsMADS68 through the potential MYB binding site, DRE/CRT element, and/or new ERF binding motif localised in the promoter region of these three MADS genes. These findings suggest that OsCPK21 plays an essential role in pollengenesis, possibly via indirectly regulating the transcription of MIKC*-type MADS box proteins.

Arabidopsis synaptotagmin 2 (SYT2) has been reported to participate in an unconventional secretory pathway in somatic cells. Our results showed that SYT2 was expressed mainly in the pollen of Arabidopsis thaliana. The pollen of syt2 T-DNA and RNA interference mutant lines exhibited reduced total germination and impeded pollen tube growth. Analysis of the expression of SYT2-GFP fusion protein in the pollen tube indicates that SYT2 was localized to distinct, patchy compartments but could co-localize with the Golgi markers, BODIPY TR C5 ceramide and GmMan1-mCherry. However, SYT2-DsRed-E5 was localized to the plasma membrane in Arabidopsis suspension cells, in addition to the Golgi apparatus. The localization of SYT2 at the plasma membrane was further supported by immunofluorescence staining in pollen tubes. Moreover, brefeldin A treatment inhibited the transport of SYT2 to the plasma membrane and caused SYT2 to aggregate and form enlarged compartments. Truncation of the SYT2-C2AB domains also resulted in retention of SYT2 in the Golgi apparatus. An in vitro phospholipid-binding assay showed that SYT2-C2AB domains bind to the phospholipid membrane in a calcium-dependent manner. Take together, our results indicated that SYT2 was required for pollen germination and pollen tube growth, and was involved in conventional exocytosis.

In rice (Oryza sativa), caryopses located on proximal secondary branches (CSBs) have smaller grain size and poorer grain filling than those located on apical primary branches (CPBs), greatly limiting grain yield. However, the molecular mechanism responsible for developmental differences between CPBs and CSBs remains elusive. In this transcriptome-wide expression study, we identified the gene Aspartic Protease 1 (OsAsp1), which reaches an earlier and higher transcriptional peak in CPBs than in CSBs after pollination. Disruption of OsAsp1 expression in the heterozygous T-DNA line asp1-1+/–eliminated developmental differences between CPBs and CSBs. OsAsp1 negatively regulated the transcriptional inhibitor of auxin biosynthesis, OsTAA1 transcriptional inhibition factor 1 (OsTIF1), to preserve indole-3-acetic acid (IAA) apical dominance in CPBs and CSBs. IAA also facilitated OsTIF1 translocation from the endoplasmic reticulum (ER) to the nucleus by releasing the interaction of OsTIF1 with OsAsp1 to regulate caryopses IAA levels via a feedback loop. IAA promoted transcription of OsAsp1 through MADS29 to maintain an OsAsp1 differential between CPBs and CSBs during pollination. Together, these findings provide a mechanistic explanation for the distributed auxin differential between CPBs and CSBs to regulate distinct caryopses development in different rice branches and potential targets for engineering yield improvement in crops., Author summary Rice is a major food crop and an important model plant. Compared with caryopses on apical primary branches (CPBs) of rice, those located on proximal secondary branches (CSBs) display smaller grains and poor grain filling, which greatly limit rice yield potential fulfilment, especially among ‘super’ rice cultivars. In this study, we demonstrated that high indole-3-acetic (IAA) levels upregulated Aspartic Protease 1 (OsAsp1) transcription via MADS29 post-pollination to produce higher OsAsp1 levels in CPBs than in CSBs. OsAsp1 then interacted with OsTAA1 transcriptional inhibition factor 1 (OsTIF1) in the endoplasmic reticulum (ER) to dispel OsTIF1 transcriptional inhibition of OsTAA1, causing IAA content to peak in CPBs at 5 days after fertilisation (DAF). IAA facilitated OsTIF1 translocation from the ER to the nucleus by reducing its interaction with OsAsp1 as feedback regulation of IAA levels in caryopses. Thus, differential auxin levels between CPBs and CSBs are determined by the OsAsp1-OsTIF1 complex, and are essential for the distinct development of CPBs and CSBs, providing potential targets for engineering yield improvement in crops.

Ca2+ acts as a universal second messenger in eukaryotes. In animals, a wide variety of environmental and developmental stimuli trigger Ca2+ dynamics in organelles, such as the cytoplasm, nucleus, and endoplasmic reticulum (ER). However, ER Ca2+ ([Ca2+]er) homeostasis and its contributions in cytosolic and/or nucleosolic Ca2+ dynamics in plants remain elusive. GCaMPs are comprised of a circularly permutated form of enhanced green fluorescent protein fused to calmodulin and myosin light-chain kinase M13 and used for monitoring Ca2+ dynamics in mammalian cells. Here, we targeted a high-affinity variant of GCaMP with nuclear export signal in the cytoplasm (NES-GCaMP6m), with a nuclear-localised signal in the nucleus (NLS-GCaMP6m), and a low-affinity variant of GCaMP, also known as calcium-measuring organelle-entrapped protein indicators (CEPIA), with a signal peptide sequence of the ER-localised protein Calreticulin 1a in the ER lumen (CRT1a-R-CEPIA1er) for intraorganellar Ca2+ imaging in Arabidopsis. We found that cytosolic Ca2+ ([Ca2+]cyt) increases induced by 250 mM sorbitol as an osmotic stress stimulus, 50 μM abscisic acid (ABA), or 1 mM carbachol (CCh) were mainly due to extracellular Ca2+ influx, whereas nucleosolic Ca2+ ([Ca2+]nuc) increases triggered by osmotic stress, ABA, or CCh were contributed by [Ca2+]er release. In addition, [Ca2+]er dynamics presented specific patterns in response to different stimuli such as osmotic stress, ABA, or CCh, indicating that Ca2+ signalling occurs in the ER in plants. These results provide valuable insights into subcellular Ca2+ dynamics in response to different stresses in Arabidopsis root cells and prove that GCaMP imaging is a useful tool for furthering our understanding of plant organelle functions.

NAC with a transmembrane (TM) motif1-like (NTL) transcription factors, containing three regions: the N-terminal NAC domain (ND), the middle regulation region (RR), and the C-terminal TM domain, belong to the tail-anchored proteins. Although these NTLs play numerous essential roles in plants, their subcellular distribution and the mechanism of translocation into the nucleus (NU) remain unclear. In this study, we found that most of the full-length NTLs were localized in the endoplasmic reticulum (ER), with the exception of NTL11 and NTL5, which were restricted to the NU. Furthermore, we found that NTL11 contains a TM domain, whereas NTL5 does not. The ND of all of the NTLs was responsible for nuclear localization in plants. After truncation of the TM domain, NTL8_NR, NTL10_NR and NTL13_NR localized in the cytoplasm (CT) and NU, and other NTL_NRs were only localized in the NU, suggesting that the RR of NTL8, NTL10 and NTL13 contains some inhibitory region to mask the nuclear localization signal sequence in the ND domain and permit their diffusion between CT and NU. Furthermore, the N-terminus of NTL11 was translocated to the NU, but the C-terminus was degraded in Arabidopsis mesophyll protoplasts. The chimeric construct of NTL11_ND with NTL10_RR and TM domain (11ND-10RT) was localized exclusively in the ER, and not in the NU. However, 10ND-11RT was found mainly in the NU. Our results indicated that the TM domain is essential for NTL targeting the ER and the N-terminal fragment, including ND and RR, is translocated into the NU after activation through proteolytic cleavage events upon stimulation by internal and external environmental signals.

The ERA (E. coliRAS-like protein)-related GTPase (ERG) is a nuclear-encoded GTPase with two conserved domains: a GTPase domain and a K Homology domain. ERG plays a vital role in early seed development inAntirrhinum majus. However, the mechanism that regulates seed development remains unclear. Blasting the genome sequence revealed two homologies of ERG, AtERG1, and AtERG2 inArabidopsis. In this study, we found that AtERG2 is localised in the mitochondria and binds mitochondrial 18S RNA. Promoter and transcript analyses indicated thatAtERG2was mainly expressed in the leaf vein, trichome, mature pollen, and ovule. The mutants ofAtERG2showed recessive lethal, gametophytic maternal effects, silique shortage, and early seed abortion, in which some seeds arrested in the zygotic stage at 1.5 days after pollination (DAP) and aborted at 2.0 DAP inaterg2-1+/-. Reactive oxygen species (ROS) accumulated at 1.5 DAP in the arrested seeds, and the transcription of several ROS-responsible genes,WRKY40,ANAC017, andAOXla, was up-regulated in theaterg2-1+/- seeds which were arrested 1.5 and 2.0 DAP but not in wild-type (WT) andaterg2-1+/- seeds. The cell death-related gene BAG6 was also transcriptionally activated inaterg2-1+/- seeds arrested at 2.0 DAP. Chloramphenicol treatment during pollination induced a similar phenotype and gene expression pattern but showed no transcriptional changes of ANAC017 in WT. These results suggested thatAtERG2promotes early seed development by affecting the maturation of the mitochondria ribosome small subunit and mitochondrial protein translation inArabidopsis.

The calcium-dependent protein kinases (CDPKs) are a class of plant-specific kinase that directly bind Ca 2+ and mediate the calcium-signaling pathways to play important physiological roles in growth and development. The rice genome contains 31 CDPK genes, one of which, OsCPK21, is known to modulate the abscisic acid (ABA) and salt stress responses in this crop; however, the molecular mechanisms underlying this regulation are largely unknown. In the present study, we performed yeast two-hybrid screening, glutathione S-transferase pull-down, co-immunoprecipitation, and bimolecular fluorescence complementation assays to confirm the interaction between OsCPK21 and one of its putative targets, Os14-3-3 (OsGF14e). We used an in vitro kinase assay and site-directed mutagenesis to verify that OsCPK21 phosphorylates OsGF14e at Tyr-138. We used real-time PCR to reveal that several ABA and salt inducible genes were more highly expressed in the OsCPK21-OE and OsGF14e WT-OE plants than in the mutant OsGF14e Y138A-OE and wild-type plants. These results suggest that OsCPK21 phosphorylates OsGF14e to facilitate the response to ABA and salt stress.

Calcium acts as a universal second messenger in both developmental processes and responses to environmental stresses. Previous research has shown that a number of stimuli can induce [Ca2+] increases in both the cytoplasm and nucleus in plants. However, the relationship between cytosolic and nucleosolic calcium signaling remains obscure. Here, we generated transgenic plants containing a fusion protein, comprising rat parvalbumin (PV) with either a nuclear export sequence (PV-NES) or a nuclear localization sequence (NLS-PV), to selectively buffer the cytosolic or nucleosolic calcium. Firstly, we found that the osmotic stress-induced cytosolic [Ca2+] increase (OICIcyt) and the salt stress-induced cytosolic [Ca2+] increase (SICIcyt) were impaired in the PV-NES lines compared with the Arabidopsis wildtype (WT). Similarly, the osmotic stress-induced nucleosolic [Ca2+] increase (OICInuc) and salt stress-induced nucleosolic [Ca2+] increase (SICInuc) were also disrupted in the NLS-PV lines. These results indicate that PV can effectively buffer the increase of [Ca2+] in response to various stimuli in Arabidopsis. However, the OICIcyt and SICIcyt in the NLS-PV plants were similar to those in the WT, and the OICInuc and SICInuc in the PV-NES plants were also same as those in the WT, suggesting that the cytosolic and nucleosolic calcium dynamics are mutually independent. Furthermore, we found that osmotic stress- and salt stress-inhibited root growth was reduced dramatically in the PV-NES and NLS-PV lines, while the osmotic stress-induced increase of the lateral root primordia was higher in the PV-NES plants than either the WT or NLS-PV plants. In addition, several stress-responsive genes, namely CML37, DREB2A, MYB2, RD29A, and RD29B, displayed diverse expression patterns in response to osmotic and salt stress in the PV-NES and NLS-PV lines when compared with the WT. Together, these results imply that the cytosolic and nucleosolic calcium signaling coexist to play the pivotol roles in the growth and development of plants and their responses to environment stresses.

Glucose is the primary energy provider and the most important sugar-signalling molecule, regulating metabolites and modulating gene expression from unicellular yeast to multicellular plants and animals. Therefore, monitoring intracellular glucose levels temporally and spatially in living cells is an essential step for decoding the glucose signalling in response to biotic and abiotic stresses. In this study, the genetically encoded FRET (Forster resonance energy transfer) nanosensors, FLIPglu-2μ∆13 and FLIPglu-600μΔ13, were used to measure cytosolic glucose dynamics in rice plants. First, we found that the FRET signal decreased in response to external glucose in a concentration-dependent manner. The glucose concentration at which the cytosolic level corresponded to the K0.5 value for FLIPglu-2μΔ13 was approximately 10.05μM, and that for FLIPglu-600μΔ13 was 0.9mM, respectively. The substrate selectivity of nanosensors for glucose and its analogues is D-Glucose>2-deoxyglucose>3-O-methylglucose>L-Glucose. We further showed that the biotic elicitors (flg22 and chitin) and the abiotic elicitors (osmotic stress, salinity and extreme temperature) induce the intracellular glucose increases in the detached root segments of transgenic rice containing FLIPglu-2μΔ13 in a stimulus-specific manner, but not in FLIPglu-600μΔ13 transgenic lines. These results demonstrated that FRET nanosensors can be used to detect increases in intracellular glucose within the physiological range of 0.2-20μM in response to various stimuli in transgenic rice root cells, which indicated that intracellular glucose may act as a potential secondary messenger to connect extracellular stimuli with cellular physiological responses in plants.

Summary Calcium signals act as universal second messengers that trigger many cellular processes in animals and plants, but how specific calcium signals are generated is not well understood. In this study, we determined that AtANN4, a putative calcium-permeable transporter, and its interacting proteins, SCaBP8 and SOS2, generate a calcium signal under salt stress, which initially activates the SOS pathway, a conserved mechanism that modulates ion homeostasis in plants under salt stress. After activation, SCaBP8 promotes the interaction of protein kinase SOS2 with AtANN4, which enhances its phosphorylation by SOS2. This phosphorylation of AtANN4 further increases its interaction with SCaBP8. Both the interaction with and phosphorylation of AtANN4 repress its activity and alter calcium transients and signatures in HEK cells and plants. Our results reveal how downstream targets are required to create a specific calcium signal via a negative feedback regulatory loop, thereby enhancing our understanding of the regulation of calcium signaling.

Spatial control of cytokinesis in plant cells depends on guidance of the cytokinetic apparatus, the phragmoplast, to a cortical “division site” established before mitosis. Previously, we showed that the Tangled1 (Tan1) gene of maize is required for this process during maize leaf development (Cleary, A.L., and L.G. Smith. 1998. Plant Cell. 10:1875–1888.). Here, we show that the Tan1 gene is expressed in dividing cells and encodes a highly basic protein that can directly bind to microtubules (MTs). Moreover, proteins recognized by anti-TAN1 antibodies are preferentially associated with the MT-containing cytoskeletal structures that are misoriented in dividing cells of tan1 mutants. These results suggest that TAN1 protein participates in the orientation of cytoskeletal structures in dividing cells through an association with MTs.

MicroRNA167 (miR167), as a conserved miRNA, has been implicated in auxin signalling by regulating the expression of certain auxin response factor (ARF) genes to determine the plant developmental process. Among the 10 MIR167 genes of rice, the precursor structures derived from MIR167a, MIR167b and MIR167c produce miR167 with high efficiency. To explore the biological function of miR167 in rice, four of its predicted target genes, OsARF6, OsARF12, OsARF17 and OsARF25, were identified in vivo. Although the expression levels of miR167 and its target OsARFs did not show an obvious negative correlation, the enhanced miR167 level in transgenic rice overexpressing miR167 resulted in a substantial decrease in mRNA levels of the four OsARF genes. Moreover, the transgenic rice plants were small in stature with remarkably reduced tiller number. These results suggest that miR167 is important for the appropriate expression of at least four OsARFs, which mediate the auxin response, to contribute to the normal growth and development of rice.

SummaryIn plant cells, the plane of division is anticipated at the onset of mitosis by the presence of a preprophase band (PPB) of microtubules and F-actin at a cortical site that circumscribes the nucleus. During cytokinesis, the microtubule- and F-actin-based phragmoplast facilitates construction of a new cell wall and is guided to the forecast division site. Proper execution of this process is essential for establishing the cellular framework of plant tissues. The microtubule binding protein TANGLED1 (TAN1) of maize is a key player in the determination of division planes [1–3]. Lack of TAN1 leads to misguided phragmoplasts and mispositioned cell walls in maize. In a yeast two-hybrid screen for TAN1-interacting proteins, a pair of related kinesins was identified that shares significant sequence homology with two kinesin-12 genes in Arabidopsis thaliana (A. thaliana): PHRAGMOPLAST ORIENTING KINESIN 1 and 2 (POK1, POK2). POK1 and POK2 are expressed in tissues enriched for dividing cells. The phenotype of pok1;pok2 double mutants strongly resembles that of maize tan1 mutants, characterized by misoriented mitotic cytoskeletal arrays and misplaced cell walls. We propose that POK1 and POK2 participate in the spatial control of cytokinesis, perhaps via an interaction with the A. thaliana TAN1 homolog, ATN.