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Calcium acts as a universal secondary messenger that transfers developmental cues and stress signals for gene expression and adaptive growth. A prior study showed that abiotic stresses induce mutually independent cytosolic Ca2+ ([Ca2+]cyt) and nucleosolic Ca2+ ([Ca2+]nuc) increases in Arabidopsis thaliana root cells. However, gene expression networks deciphering [Ca2+]cyt and [Ca2+]nuc signalling pathways remain elusive. Here, using transgenic A. thaliana to selectively impair abscisic acid (ABA)- or methyl jasmonate (MeJA)-induced [Ca2+]cyt and [Ca2+]nuc increases, we identified [Ca2+]cyt- and [Ca2+]nuc-regulated ABA- or MeJA-responsive genes with a genome oligo-array. Gene co-expression network analysis revealed four Ca2+ signal-decoding genes, CAM1, CIPK8, GAD1, and CPN20, as hub genes co-expressed with Ca2+-regulated hormone-responsive genes and hormone signalling genes. Luciferase complementation imaging assays showed interactions among CAM1, CIPK8, and GAD1; they also showed interactions with several proteins encoded by Ca2+-regulated hormone-responsive genes. Furthermore, CAM1 and CIPK8 were required for MeJA-induced stomatal closure; they were associated with ABA-inhibited seed germination. Quantitative reverse transcription polymerase chain reaction analysis showed the unique expression pattern of [Ca2+]-regulated hormone-responsive genes in cam1, cipk8, and gad1. This comprehensive understanding of distinct Ca2+ and hormonal signalling will allow the application of approaches to uncover novel molecular foundations for responses to developmental and stress signals in plants.

Long noncoding RNAs (lncRNAs) play important regulatory roles in caryopsis development and grain size in rice. However, whether there exist differences in lncRNA expression between caryopses located on primary branches (CPB) and caryopses located on secondary branches (CSB) that contribute to their differential development remains elusive. Here, we performed transcriptome-wide analysis to identify 2,273 lncRNAs expressed in CPB and CSB at 0, 5, 12, and 20 days after flowering (DAF). Although these lncRNAs were widely distributed, the majority were located in intergenic regions of the 12 rice chromosomes. Based on gene expression cluster analysis, lncRNAs expressed in CPB and CSB were clustered into two subtypes in a position-independent manner: one includes 0- and 5-DAF CPB and CSB, and 12-DAF CSB; the second includes 12-DAF CPB and 20-DAF CPB and CSB. Furthermore, according to the expression value of each lncRNA, K-means cluster analysis revealed 135 early-stage, 116 middle-stage, and 114 late-stage expression-delayed lncRNAs in CSB. Then, we analyzed the expression values of the expression-delayed lncRNAs and nearby coding genes (100 kb upstream and downstream of the lncRNAs), and found 631 lncRNA-mRNA pairs, including 258 lncRNAs and 571 nearby coding genes, some of which are related to hormone-regulated grain development. These results suggested that expression-delayed lncRNAs in CSB may regulate the development of CPB and CSB, providing insight into the mechanism underlying the developmental differences between CPB and CSB, and the differences in grain yield.

Rice calcium-dependent protein kinase 21 (OsCPK21) is specifically and highly expressed throughout reproductive development and plays a critical role in rice pollen development by indirectly regulating the MIKC*-type MADS box transcription factor. However, little is known about the function of OsCPK21 in rice caryopsis development. In this study, we performed an in vitro pull-down experiment followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and identified hydroxysteroid dehydrogenase 2 (HSD2) as a candidate OsCPK21-interacting protein in 25 DAF (days after flowering) rice caryopses. Then, we verified the interaction between OsCPK21 and OsHSD2 using yeast two-hybrid and bimolecular fluorescence assays and revealed the in vitro phosphorylation of OsHSD2 by OsCPK21. Furthermore, oscpk21 and oshsd2 mutants were generated by the CRISPR/Cas9 technique, and we found that the lipid profiles were drastically changed in both oscpk21 and oshsd2, implying that OsHSD2 phosphorylated by OsCPK21 regulates lipid abundance in caryopsis development, thereby providing a potential target for the genetic improvement of rice grain quality in future lipid-related breeding and biotechnology applications.

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.

Leymus chinensis (Trin.) Tzvel is a rhizomatous grass species in the Eastern Eurasian steppe zone that is often limited by low soil nitrogen availability. Although a previous study showed that the rhizomes of L. chinensis have the capacity to take up nitrogen, the importance of such uptake for nitrogen nutrition is unclear. Moreover, little is known regarding the inorganic nitrogen uptake kinetics of roots and rhizomes in response to nitrogen status. Here, we first found that ammonium is preferred over nitrate and glycine for L. chinensis growth. Using the 15N-labelling method, we found that the rate of ion influx into roots was approximately five-fold higher than into rhizomes under the same nitrogen content, and the ion influxes into roots and rhizomes under 0.05 mM N were greater than in the presence of 3 mM N, especially in the form of NH4+. Using a non-invasive micro-test technique, we characterised the patterns of NH4+ and NO3– fluxes in the root mature zone, root tip, rhizome mature zone, and rhizome tip following incubation in the solution with different N compounds and different N concentrations. These results suggest a dynamic balance between the uptake, utilisation, and excretion of nitrogen in L. chinensis.

Jasmonates (JAs) are well-known regulators of stress, defence, and secondary metabolism in plants, with JA perception triggering extensive transcriptional reprogramming, including both activation and/or repression of entire metabolic pathways. We performed RNA sequencing based transcriptomic profiling of tobacco BY-2 cells before and after treatment with methyl jasmonate (MeJA) to identify novel transcriptional regulators associated with alkaloid formation. A total of 107,140 unigenes were obtained through de novo assembly, and at least 33,213 transcripts (31%) encode proteins, in which 3419 transcription factors (TFs) were identified, representing 72 gene families, as well as 840 transcriptional regulators (TRs) distributed among 19 gene families. After MeJA treatment BY-2 cells, 7260 differentially expressed transcripts were characterised, which include 4443 MeJA-upregulated and 2817 MeJA-downregulated genes. Of these, 227 TFs/TRs in 36 families were specifically upregulated, and 102 TFs/TRs in 38 families were downregulated in MeJA-treated BY-2 cells. We further showed that the expression of 12 ethylene response factors and four basic helix-loop-helix factors increased at the transcriptional level after MeJA treatment in BY-2 cells and displayed specific expression patterns in nic mutants with or without MeJA treatments. Our data provide a catalogue of transcripts of tobacco BY-2 cells and benefit future study of JA-modulated regulation of secondary metabolism in tobacco.

Hexokinase (HXK) as a moonlighting protein involves in glucose metabolism and signalling to regulate growth and development in plants. Therefore, the clarification for the structural properties of OsHXK7 (Oryza sativa Hexokinase 7) is essential to understand its role mechanism associated with the Glc signalling and metabolism. In this study, the structural characteristics of OsHXK7 (Oryza sativa Hexokinase 7) were identified. In the fluorescence spectrum, the Trp peak representing OsHXK7 binding to D-glucose (D-Glc) and 2-deoxyglucose (2-dG) showed an obvious blue shift. The distinct change in the secondary structure of OsHXK7 after binding to Glc was also detected in circular dichroism spectra. Using superimposed modelling, OsHXK7 showed a Glc-induced structural change, in which the 76th glycine, 148th serine and 256th tryptophan were contained within the pocket region. It was further shown by site-directed mutagenesis that the 76th glycine and the 256th tryptophan, but not the 148th serine, are the pivotal sites of OsHXK7 that maintain its catalytic activity and intrinsic blue shift fluorescence. These results suggest that OsHXK7 binding to Glc leads to a conformational change, that is likely essential for the function of OsHXK7 in Glc signalling and metabolism during plant growth and development.

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.

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.

N-terminal-TM-C2 domain proteins (NTMC2), which share domain architecture and sequence similarity to synaptotagmins (Syts) in mammals and FAM62 (extended Syts) in metazoans, form a small gene family in plants. Previous studies showed that the Arabidopsis thaliana NTMC2 type 1.1 protein (NTMC2T1.1, named AtSyt1) possesses calcium- and membrane-binding activities that allow it to function in a plasma membrane repair pathway induced by stress. However, we lack understanding of the diverse biological roles of plant NTMC2 family genes. In this study, a total of 13 OsNTMC2 genes was identified through a comprehensive bioinformatics analysis of the rice (Oryza sativa L.) genome and classified into six OsNTMC2 groups (OsNTMC2T1 to OsNTMC2T6) based on phylogeny and motif constitution. OsNTMC2T1 to OsNTMC2T3 have two calcium-binding domains (C2A and C2B), but OsNTMC2T4 to OsNTMC2T6 have single C2 domain. The expression profiles of OsNTMC2 genes were analysed at different stages of vegetative and reproductive development. This analysis revealed that at least one OsNTMC2 gene was abundantly expressed at each stage of development. These results should facilitate research on this gene family and provide new insights elucidating their functions in higher plants.

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.

Inositol 1,4,5-trisphosohate (IP3 ) and its receptors play a pivotal role in calcium signal transduction in mammals. However, no homologs of mammalian IP3 receptors have been found in plants. In this study, we isolated the microsomal fractions from rice cells in suspension culture and further obtained putative IP3 -binding proteins by heparin-agarose affinity purification. The IP3 -binding activities of these protein fractions were determined by [(3) H] IP3 -binding assay. SDS-PAGE and MS analysis were then performed to characterize these proteins. We have identified 297 proteins from the eluates of heparin-agarose column chromatography, which will provide insight into the IP3 signaling pathways in plants. All MS data have been deposited in the ProteomeXchange with identifier PXD000763 (http://proteomecentral.proteomexchange.org/dataset/PXD000763).

Jasmonate (JA) zinc-finger expressed in inflorescence meristem (ZIM)-domain (JAZ) proteins are key regulators of the JA response in plants. Transcriptome profiling of tobacco BY-2 cells was used to identify 17 members of the NtJAZ family, which were divided into 12 distinct groups based on their predicted amino acid sequences and conserved domains. Transcript levels of eight of the NtJAZ groups increased rapidly upon JA treatment, whereas the remaining members did not show a significant response. The majority of JA-induced NtJAZs formed homo- and heteromers and interacted with NtMYC2a (but not NtERF189) in yeast two-hybrid assays. NtJAZ1, NtJAZ3b, NtJAZ7 and NtJAZ10 were localised in the nucleus and degraded rapidly via the 26S proteasome pathway following the treatment of BY-2 with MeJA. RNAi-induced silencing of NtJAZ1, NtJAZ3, NtJAZ7a and NtJAZ10 greatly reduced the levels of NtPMT transcripts and specifically decreased the nicotine content in the four RNAi transgenic BY-2 lines. The levels of transcripts encoding other nicotine biosynthesis enzymes, NtERF189 and NtMYC2a, and other NtJAZs exhibited different expression patterns in RNAi lines with or without MeJA treatment. Our results indicate that cross-talk occurs among different NtJAZs and forms a complex transcription regulatory scheme for JA-induced nicotine biosynthesis in tobacco.

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.

Plant microRNAs modulate diverse developmental processes by regulating expression of their target genes. To explore potential miRNA-guided gene regulation in developing rice (Oryza sativa L.) caryopses, a miRNA microarray was used to identify miRNAs present at the different developmental stages. We found that 27 miRNAs, of which 16 were conserved miRNAs, were present in developing caryopses. High expression levels were detected for miR159, miR167, and miR530 at the morphogenesis stage and for miR169, miR435, and miR528 at the stage of accumulation of metabolites. Next, 26 target genes were predicted for seven of the detected miRNAs and the expression profiles of these miRNAs and their corresponding target genes were examined in developing caryopses. Our results suggest that the miRNAs and their target genes examined at the two distinct stages could contribute to the developmental progress of rice caryopses in concert with phytohormone signalling.

The growth and development of plants under drought stress depends mainly on the expression levels of various genes and modification of proteins. To clarify the molecular mechanism of drought-tolerance of plants, suppression subtractive hybridisation cDNA libraries were screened to identify drought-stress-responsive unigenes in Grimmia pilifera, and a novel E3 ubiquitin ligase gene, GpDSR7, was identified among the 240 responsive unigenes. GpDSR7 expression was induced by various abiotic stresses, particularly by drought. GpDSR7 displayed E3 ubiquitin ligase activity in vitro and was exclusively localised on the ER membrane in Arabidopsis mesophyll protoplasts. GpDSR7-overexpressing transgenic Arabidopsis plants showed a high water content and survival ratio under drought stress. Moreover, the expression levels of some marker genes involved in drought stress were higher in the transgenic plants than in wild-type plants. These results suggest that GpDSR7, an E3 ubiquitin ligase, is involved in tolerance to drought stress at the protein modification level.

Hexokinase (HXK, EC 2.7.1.1) plays an important role in the metabolism and glucose signalling. To examine the characteristics of HXK gene family in rice, the subcellular localizations of ten hexokinases (OsHXK1 — OsHXK10) were determined using OsHXK::GFP fusion proteins in tobacco mesophyll protoplasts. As was previously demonstrated, OsHXK4 was detected in the chloroplast stroma, OsHXK5 and OsHXK6 in the mitochondria, and OsHXK7 and OsHXK10 in the cytoplasm. In the present study, OsHXKs were clearly divided into three types (A, B, C) based on their N-terminal sequences. The new type-C HXKs in plants, OsHXK1, OsHXK7 and OsHXK8, which lack the plastidic transit peptide and the membrane anchor domain, were detected not only in the cytoplasm but also in the nucleus. The type-B HXKs, OsHXK2, OsHXK3, OsHXK9 and OsHXK10, which contained a membrane anchor domain, were distinctly localized in the mitochondria. These results suggest that OsHXKs localized in different cell compartments may be involved in the glucose signalling-related gene expression during growth and development of rice.

ADP-ribosylation factors (ARFs) have been widely identified as being involved in regulation of cell differentiation, intracellular vesicle transport, and cytoskeleton organization in animals and plants. To systematically clarify the ARF gene family in rice, eight putative rice ARF genes (OsARFs) were detected by searching the rice genome database. The OsARFs can be classified into class I (OsARFA1a-OsARFA1e) and class II (OsARFB1a-OsARFB1c) by comparing their deduced amino acid sequences with other known ARFs. Most OsARFs are composed of six exons and five introns, though OsARFB1b has three exons and two introns. The OsARFs are usually expressed in various developing organs, whereas OsARFA1a is predominantly expressed in young roots and in caryopses at early development stages. The histochemical localization of OsARFs in various developing organs was confirmed in young leaves, the pericycle of young roots, and the aleurone layer of developing caryopses. Furthermore, the expression of OsARFA1a was confirmed specifically in the aleurone layer and immature embryos of developing caryopses. The present work provides a foundation for further clarifying the physiological functions of OsARFs in rice growth and development.

Background Reception of and response to exogenous and endogenous osmotic changes is important to sustain plant growth and development, as well as reproductive formation. Hyperosmolality-gated calcium-permeable channels (OSCA) were first characterised as an osmosensor in Arabidopsis and are involved in the perception of extracellular changes to trigger hyperosmolality-induced [Ca2+]i increases (OICI). To explore the potential biological functions of OSCAs in rice, we performed a bioinformatics and expression analysis of the OsOSCA gene family. Results A total of 11 OsOSCA genes were identified from the genome database of Oryza sativa L. Japonica. Based on their sequence composition and phylogenetic relationship, the OsOSCA family was classified into four clades. Gene and protein structure analysis indicated that the 11 OsOSCAs shared similar structures with their homologs in Oryza sativa L. ssp. Indica, Oryza glaberrima, and Oryza brachyantha. Multiple sequence alignment analysis revealed a conserved DUF221 domain in these members, in which the first three TMs were conserved, while the others were not. The expression profiles of OsOSCA genes were analysed at different stages of vegetative growth, reproductive development, and under osmotic-associated abiotic stresses. We found that four and six OsOSCA genes showed a clear correlation between the expression profile and osmotic changes during caryopsis development and seed imbibition, respectively. Orchestrated transcription of three OsOSCAs was strongly associated with the circadian clock. Moreover, osmotic-related abiotic stress differentially induced the expression of 10 genes. Conclusion The entire OSCA family is characterised by the presence of a conserved DUF221 domain, which functions as an osmotic-sensing calcium channel. The phylogenetic tree of OSCA genes showed that two subspecies of cultivated rice, Oryza sativa L. ssp. Japonica and Oryza sativa L. ssp. Indica, are more closely related than wild rice Oryza glaberrima, while Oryza brachyantha was less closely related. OsOSCA expression is organ- and tissue-specific and regulated by different osmotic-related abiotic stresses in rice. These findings will facilitate further research in this gene family and provide potential target genes for generation of genetically modified osmotic-stress-resistant plants. Electronic supplementary material The online version of this article (doi:10.1186/s12870-015-0653-8) contains supplementary material, which is available to authorized users.

Sequences of oligonucleotide primers for qRT-PCR. F: forward; R: reverse. (DOC 52 kb)

Circadian rhythmic expression of the marker gene OsLHY in four-leaf-stage ZH11 seedlings. (DOC 56 kb)

The relative water content of ZH11 seeds during imbibition in solutions containing various PEG concentrations. (DOC 53 kb)

General information and sequence characterisation of 45 OSCA genes from Oryza sativa L. ssp. Japonica, Oryza sativa L. ssp. Indica, Oryza glaberrima, and Oryza brachyantha. (DOC 89 kb)