Auxin apical dominance governed by the OsAsp1-OsTIF1 complex determines distinctive rice caryopses development on different branches.

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.

MPK3/MPK6-mediated phosphorylation of ERF72 positively regulates resistance to Botrytis cinerea through directly and indirectly activating the transcription of camalexin biosynthesis enzymes.

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.

Systematic Identification of Methyl Jasmonate-Responsive Long Noncoding RNAs and Their Nearby Coding Genes Unveils Their Potential Defence Roles in Tobacco BY-2 Cells.

Long noncoding RNAs (lncRNAs) are distributed in various species and play critical roles in plant growth, development, and defence against stimuli. However, the lncRNA response to methyl jasmonate (MeJA) treatment has not been well characterized in Nicotiana tabacum Bright Yellow-2 (BY-2) cells, and their roles in plant defence remain elusive. Here, 7848 reliably expressed lncRNAs were identified in BY-2 cells, of which 629 differentially expressed (DE) lncRNAs were characterized as MeJA-responsive lncRNAs. The lncRNAs in BY-2 cells had a strong genus specificity in Nicotiana. The combined analysis of the cis-regulated lncRNAs and their target genes revealed the potential up- and downregulated target genes that are responsible for different biological functions and metabolic patterns. In addition, some lncRNAs for response-associated target genes might be involved in plant defence and stress resistance via their MeJA- and defence-related cis-regulatory elements. Moreover, some MeJA-responsive lncRNA target genes were related to quinolinate phosphoribosyltransferase, lipoxygenases, and endopeptidase inhibitors, which may contribute to nicotine synthesis and disease and insect resistance, indicating that MeJA-responsive lncRNAs regulate nicotine biosynthesis and disease resistance by regulating their potential target genes in BY-2 cells. Therefore, our results provide more targets for genetically engineering the nicotine content and plant defence in tobacco plants.

Functional analysis of rice OSCA genes overexpressed in the arabidopsis osca1 mutant due to drought and salt stresses.

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.

ERF72 interacts with ARF6 and BZR1 to regulate hypocotyl elongation in Arabidopsis.

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.

Subcellular Distribution of NTL Transcription Factors in Arabidopsis thaliana.

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.

Calcium-dependent protein kinase 21 phosphorylates 14-3-3 proteins in response to ABA signaling and salt stress in rice.

The calcium-dependent protein kinases (CDPKs) are a class of plant-specific kinase that directly bind Ca2+ 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.

Genome-wide survey and expression analysis of the OSCA gene family in rice.

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 [Ca(2+)]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.

Identification of inositol 1,4,5-trisphosphate-binding proteins by heparin-agarose affinity purification and LTQ ORBITRAP MS in Oryza sativa.

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).

Genome-Wide Comprehensive Survey of the Subtilisin-Like Proteases Gene Family Associated With Rice Caryopsis Development.

Subtilisin-like proteases (SUBs), which are extensively distributed in three life domains, affect all aspects of the plant life cycle, from embryogenesis and organogenesis to senescence. To explore the role of SUBs in rice caryopsis development, we recharacterized the OsSUB gene family in rice (Oryza sativa ssp. japonica). In addition, investigation of the SUBs was conducted across cultivated and wild rice in seven other Oryza diploid species (O. brachyantha, O. glaberrima, O. meridionalis, O. nivara, O. punctata, O. rufipogon, and O. sativa ssp. indica). Sixty-two OsSUBs were identified in the latest O. sativa ssp. japonica genome, which was higher than that observed in wild species. The SUB gene family was classified into six evolutionary branches, and SUB1 and SUB3 possessed all tandem duplication (TD) genes. All paralogous SUBs in eight Oryza plants underwent significant purifying selection. The expansion of SUBs in cultivated rice was primarily associated with the occurrence of tandem duplication events and purifying selection and may be the result of rice domestication. Combining the expression patterns of OsSUBs in different rice tissues and qRT-PCR verification, four OsSUBs were expressed in rice caryopses. Moreover, OsSUBs expressed in rice caryopses possessed an earlier origin in Oryza, and the gene cluster formed by OsSUBs together with the surrounding gene blocks may be responsible for the specific expression of OsSUBs in caryopses. All the above insights were inseparable from the continuous evolution and domestication of Oryza. Together, our findings not only contribute to the understanding of the evolution of SUBs in cultivated and wild rice but also lay the molecular foundation of caryopsis development and engineering improvement of crop yield.

OsOSCA1.1 Mediates Hyperosmolality and Salt Stress Sensing in Oryza sativa.

OSCA (reduced hyperosmolality-induced [Ca2+]i increase) is a family of mechanosensitive calcium-permeable channels that play a role in osmosensing and stomatal immunity in plants. Oryza sativa has 11 OsOSCA genes; some of these were shown to complement hyperosmolality-induced [Ca2+]cyt increases (OICIcyt), salt stress-induced [Ca2+]cyt increases (SICIcyt), and the associated growth phenotype in the Arabidopsis thaliana mutant osca1. However, their biological functions in rice remain unclear. In this paper, we found that OsOSCA1.1 mediates OICIcyt and SICIcyt in rice roots, which are critical for stomatal closure, plant survival, and gene expression in shoots, in response to hyperosmolality and the salt stress treatment of roots. Compared with wild-type (Zhonghua11, ZH11) plants, OICIcyt and SICIcyt were abolished in the roots of 10-day-old ososca1.1 seedlings, in response to treatment with 250 mM of sorbitol and 100 mM of NaCl, respectively. Moreover, hyperosmolality- and salt stress-induced stomatal closure were also disrupted in a 30-day-old ososca1.1 mutant, resulting in lower stomatal resistance and survival rates than that in ZH11. However, overexpression of OsOSCA1.1 in ososca1.1 complemented stomatal movement and survival, in response to hyperosmolality and salt stress. The transcriptomic analysis further revealed the following three types of OsOSCA1.1-regulated genes in the shoots: 2416 sorbitol-responsive, 2349 NaCl-responsive and 1844 common osmotic stress-responsive genes after treated with 250 mM of sorbitol and 125 mM NaCl of in 30-day-old rice roots for 24 h. The Gene Ontology enrichment analysis showed that these OsOSCA1.1-regulated genes were relatively enriched in transcription regulation, hormone response, and phosphorylation terms of the biological processes category, which is consistent with the Cis-regulatory elements ABRE, ARE, MYB and MYC binding motifs that were overrepresented in 2000-bp promoter regions of these OsOSCA1.1-regulated genes. These results indicate that OsOSCA-mediated calcium signaling specifically regulates gene expression, in response to drought and salt stress in rice.

Isolation and characterization of a mycosubtilin homologue antagonizing Verticillium dahliae produced by Bacillus subtilis strain Z15.

Bacillus subtilis strain Z15 (BS-Z15) was isolated from the cotton field of Xinjiang, China, and characterized as an effective biocontrol agent antagonizing plant pathogen Verticillium dahliae 991 (VD-991). However, the chemical substance produced by BS-Z15 for resistance to VD-991 remains elusive. Here, a serial purification methods including HCl precipitation, organic solvent extraction, and separation by semi-preparative High-Performance Liquid Chromatography were performed to obtain a single compound about 3.5 mg/L from the fermentation broth of BS-Z15, which has an antifungal activity against VD-991. Moreover, Fourier Transform Infrared spectrum, Nuclear Magnetic Resonance Spectroscopy, and Tandem Mass Spectrometry analyses were carried out to finally confirm that the active compound from BS-Z15 is a mycosubtilin homologue with C17 fatty acid chain. Genomic sequence prediction and PCR verification further showed that the BS-Z15 genome contains the whole mycosubtilin operon comprising four ORFs: fenF, mycA, mycB, and mycC, and the expression levels of mycA-N, mycB-Y and mycC-N reached a peak at 32-h fermentation. Although mycosubtilin homologue at 1 µg/mL promoted the germination of cotton seed, that with high concentration at 10 µg/mL had no significant effect on seed germination, plant height and dry weight. Furthermore, mycosubtilin homologue sprayed at 10 µg/mL on two-week-old cotton leaves promotes the expression of pathogen-associated genes and gossypol accumulation, and greatly decreases VD-991 infection in cotton with disease index statistics. This study provides an efficient purification strategy for mycosubtilin homologue from BS-Z15, which can potentially be used as a biocontrol agent for controlling verticillium wilt in cotton.

Transcriptomic identification of long noncoding RNAs and their hormone-associated nearby coding genes involved in the differential development of caryopses localized on different branches in rice.

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.

Cytosolic and Nucleosolic Calcium-Regulated Molecular Networks in Response to Long-Term Treatment with Abscisic Acid and Methyl Jasmonate in Arabidopsis thaliana.

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.

OsHSD2 interaction with and phosphorylation by OsCPK21 is essential for lipid metabolism during rice caryopsis development.

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.

Adenosine diphosphate ribosylation factor-GTPase-activating protein stimulates the transport of AUX1 endosome, which relies on actin cytoskeletal organization in rice root development.

Polar auxin transport, which depends on polarized subcellular distribution of AUXIN RESISTANT 1/LIKE AUX1 (AUX1/LAX) influx carriers and PIN-FORMED (PIN) efflux carriers, mediates various processes of plant growth and development. Endosomal recycling of PIN1 is mediated by an adenosine diphosphate (ADP)ribosylation factor (ARF)-GTPase exchange factor protein, GNOM. However, the mediation of auxin influx carrier recycling is poorly understood. Here, we report that overexpression of OsAGAP, an ARF-GTPase-activating protein in rice, stimulates vesicle transport from the plasma membrane to the Golgi apparatus in protoplasts and transgenic plants and induces the accumulation of early endosomes and AUX1. AUX1 endosomes could partially colocalize with FM4-64 labeled early endosome after actin disruption. Furthermore, OsAGAP is involved in actin cytoskeletal organization, and its overexpression tends to reduce the thickness and bundling of actin filaments. Fluorescence recovery after photobleaching analysis revealed exocytosis of the AUX1 recycling endosome was not affected in the OsAGAP overexpression cells, and was only slightly promoted when the actin filaments were completely disrupted by Lat B. Thus, we propose that AUX1 accumulation in the OsAGAP overexpression and actin disrupted cells may be due to the fact that endocytosis of the auxin influx carrier AUX1 early endosome was greatly promoted by actin cytoskeleton disruption.

Transcriptomic analysis reveals the contribution of auxin on the differentially developed caryopses on primary and secondary branches in rice.

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.

NtAIDP1, a novel NtJAZ interacting protein, binds to an AT-rich region to activate the transcription of jasmonate-inducible genes in tobacco.

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.

Intraorganellar calcium imaging in Arabidopsis seedling roots using the GCaMP variants GCaMP6m and R-CEPIA1er.

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.

Heterogeneous expression of plasma-membrane-localised OsOSCA1.4 complements osmotic sensing based on hyperosmolality and salt stress in Arabidopsis osca1 mutant.

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.