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Mediating induced abscisic acid (ABA) biosynthesis is important for enhancing plant stress tolerance. Here, we found that rice (Oryza sativa L.) osa–miR2105 (miR2105) and the Stress/ABA-activated protein kinase (OsSAPK10) coordinately regulate the rice basic region-leucine zipper transcription factor (bZIP TF; OsbZIP86) at the posttranscriptional and posttranslational levels to control drought-induced ABA biosynthesis via modulation of rice 9-cis-epoxycarotenoid dioxygenase (OsNCED3) expression. OsbZIP86 expression is regulated by miR2105-directed cleavage of the OsbZIP86 mRNA. OsbZIP86 encodes a nuclear TF that binds to the promoter of the ABA biosynthetic gene OsNCED3. OsSAPK10 can phosphorylate and activate OsbZIP86 to enhance the expression of OsNCED3. Under normal growth conditions, altered expression of miR2105 and OsbZIP86 displayed no substantial effect on rice growth. However, under drought conditions, miR2105 knockdown or OsbZIP86 overexpression transgenic rice plants showed higher ABA content, enhanced tolerance to drought, lower rates of water loss, and more stomatal closure of seedlings, compared with wild-type rice Zhonghua 11; in contrast, miR2105 overexpression, OsbZIP86 downregulation, and OsbZIP86 knockout plants displayed opposite phenotypes. Collectively, our results show that the “miR2105-(OsSAPK10)-OsbZIP86-OsNCED3” module regulates the drought-induced ABA biosynthesis without penalty on rice growth under normal conditions, suggesting candidates for improving drought tolerance in rice.

Pitaya (Hylocereus polyrhizus) is cultivated in a broad ecological range, due to its tolerance to drought, heat, and poor soil. The zinc finger proteins regulate gene expression at the transcriptional and post-transcriptional levels, by interacting with DNA, RNA, and proteins, to play roles in plant growth and development, and stress response. Here, a total of 81 CCCH-type zinc finger protein genes were identified from the pitaya genome. Transcriptomic analysis showed that nine of them, including HuTZF3, responded to both salt and heat stress. RT-qPCR results showed that HuTZF3 is expressed in all tested organs of pitaya, with a high level in the roots and stems, and confirmed that expression of HuTZF3 is induced by salt and heat stress. Subcellular localization showed that HuTZF3 is targeted in the processing bodies (PBs) and stress granules (SGs). Heterologous expression of HuTZF3 could improve both salt and heat tolerance in Arabidopsis, reduce oxidative stress, and improve the activity of catalase and peroxidase. Therefore, HuTZF3 may be involved in post-transcriptional regulation via localizing to PBs and SGs, contributing to both salt and heat tolerance in pitaya.

Some members of the CYP51G subfamily has been shown to be obtusifoliol 14α-demethylase, key enzyme of the sterol and brassinosteroid (BR) biosynthesis, which mediate plant development and response to stresses. However, little is known about the functions of CYP51H subfamily in rice. Here, OsCYP51H3, an ortholog of rice OsCYP51G1 was identified. Compared with wild type, the mutants oscyp51H3 and OsCYP51H3-RNAi showed dwarf phenotype, late flowering, erected leaves, lower seed-setting rate, and smaller and shorter seeds. In contrast, the phenotypic changes of OsCYP51H3-OE plants are not obvious. Metabolomic analysis of oscyp51H3 mutant indicated that OsCYP51H3 may also encode an obtusifoliol 14α-demethylase involved in phytosterol and BR biosynthesis, but possibly not that of triterpenes. The RNA-seq results showed that OsCYP51H3 may affect the expression of a lot of genes related to rice development. These findings showed that OsCYP51H3 codes for a putative obtusifoliol 14α-demethylase involved in phytosterol and BR biosynthesis, and mediates rice development.

Rice microRNA168a (osa-miR168a) plays important roles in mediating flowering time, grain yield and vigor, seeding growth, and immunity by targeting the RNA-induced silencing complex component Argonaute 1 (AGO1). However, the functions of miR168a exerted by targeting other genes require further clarification before it could be used in rice molecular breeding. In this study, we identified a new target gene of osa-miR168a-5p (miR168a-5p) in rice called OsOFP3 (ovate family protein 3) and investigated the roles of miR168a-5p in response to brassinosteroids (BRs), salt stress, and nitrogen allocation. Up- and downregulated miR168a-5p expression respectively decreased and increased the expression of the BR-negative regulator OsOPF3. The results of RNA ligase-mediated rapid amplification of cDNA ends (5'RLM-RACE) revealed cleavage sites in OsOPF3 and OsNPF2.4 mRNAs. The phenotype of miR168a-5p transgenic rice was BR-associated and included the lamina bending response to BR, short seeds, and low 1000-grain weight. MicroRNA 168a-5p also regulated the expression of the nitrate transporter, OsNPF2.4, which affected nitrogen allocation, and regulated OsAGO1a expression in response to salt stress. Taken together, rice miR168a-5p regulates BR-associated pathways, nitrogen transport, and stress by targeting OsOFP3, OsNPF2.4, and OsAGO1a, respectively, resulting in a series of important agronomic traits for rice breeding.

Background Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a devastating rice disease in Southeast Asia and West Africa. OsSWEET14, encoding a sugar transporter, is known to be a major susceptible gene of bacterial blight targeted by four different transcription activator-like (TAL) effectors from either Asian or African Xoo strains. However, the OsSWEET14 single knockout or promoter mutants in the Kitaake background are moderately resistant or even susceptible to African Xoo strains. Therefore, in this study, we knocked out OsSWEET14 in rice cv. Zhonghua 11 background for disease assessment. Results In this study, CRISPR/Cas9 was utilized to disrupt the function of OsSWEET14 by modifying its corresponding coding region in the genome of rice cv. Zhonghua 11 (CR-S14). In total, we obtained nine different OsSWEET14-mutant alleles. Besides conferring broad-spectrum resistance to Asian Xoo strains, tested mutant alleles also showed strong resistance to African Xoo strain AXO1947. Moreover, the expression of OsSWEET14 was detected in vascular tissues, including the stem, leaf sheath, leaf blade and root. The disruption of OsSWEET14 led to increased plant height without a reduction in yield. Conclusions Disruption of OsSWEET14 in the Zhonghua 11 background is able to confer strong resistance to African Xoo strain AXO1947 and Asian Xoo strain PXO86. CR-S14 has normal reproductive growth and enhanced plant height under normal growth conditions. These results imply that CR-S14 may serve as a better tester line than sweet14 single-knockout mutant in the Kitaake background for the diagnostic kit for rice blight resistance. The genetic background and increased plant height need to be taken into consideration when utilizing OsSWEET14 for resistant rice breeding.

Red pitaya (Hylocereus polyrhizus) is a significant functional food that is largely planted in Southeast Asia. Heat stress (HS) induced by high temperatures is likely to restrict the growth and survival of red pitaya. Although pitaya can tolerate temperatures as high as 40 °C, little is known of how it can withstand HS. In this study, the transcriptomic and metabolomic responses of red pitaya seedlings to HS were analyzed. A total of 198 transcripts (122 upregulated and 76 downregulated) were significantly differentially expressed after 24 h and 72 h of exposure to 42 °C compared with a control grown at 28 °C. We also identified 64 differentially accumulated metabolites in pitaya under HS (37 increased and 27 decreased). These differential metabolites, especially amino acids, organic acids, and sugars, are involved in metabolic pathways and the biosynthesis of amino acids. Interaction network analysis of the heat-responsive genes and metabolites suggested that similar pathways and complex response mechanisms are involved in the response of pitaya to HS. Overexpression of one of the upregulated genes (contig10820) in Arabidopsis, which is a homolog of PR-1 and named HuPR-1, significantly increased tolerance to HS. This is the first study showing that HuPR-1 plays a role in the response of pitaya to abiotic stress. These findings provide valuable insights that will aid future studies examining adaptation to HS in pitaya.

Induced abscisic acid (ABA) biosynthesis plays an important role in plant tolerance to abiotic stresses, including drought, cold and salinity. However, regulation pathway of the ABA biosynthesis in response to stresses is unclear. Here, we identified a rice miRNA, osa-miR2105 (miR2105), which plays a crucial role in ABA biosynthesis under drought stress. Analysis of expression, transgenic rice and cleavage site showed that OsbZIP86 is a target gene of miR2105. Subcellular localization and luciferase activity assays showed that OsbZIP86 is a nuclear transcription factor. In vivo and in vitro analyses showed that OsbZIP86 directly binds to the promoter of OsNCED3, and interacts with OsSAPK10, resulting in enhanced-expression of OsNCED3. Transgenic rice plants with knock-down of miR2105 or overexpression of OsbZIP86 showed higher ABA content, more tolerance to drought, a lower rate of water loss, more stomatal closure than wild type rice ZH11 under drought stress. These rice plants showed no penalty with respect to agronomic traits under normal conditions. By contrast, transgenic rice plants with miR2105 overexpression, OsbZIP86 downregulation, or OsbZIP86 knockout displayed less tolerance to drought stress and other phenotypes. Collectively, our results show that a regulatory network of ‘miR2105-OsSAPK10/OsbZIP86-OsNCED3’ control ABA biosynthesis in response to drought stress.One-sentence summary‘miR2105-OsbZIP86-OsNCED3’ module plays crucial role in mediating ABA biosynthesis to contribute to drought tolerance with no penalty with respect to agronomic traits under normal conditions.

Reverse‐transcription quantitative real‐time PCR (RT‐qPCR) is a primary tool for measuring gene expression levels, and selection of appropriate reference genes is crucial for accurate and reproducible results of gene expression under various experimental conditions. However, no systematic evaluation of reference genes in pitaya (Hylocereus undatus Britt.) has been performed. Here, we examined the expression of five candidate reference genes, namely elongation factor 1‐alpha (HuEF1‐α), 18S ribosomal RNA (Hu18S rRNA), ubiquitin (HuUBQ), actin (HuACT), and ubiquitin‐conjugating enzyme (HuUQT), under different conditions in pitaya. The expression stabilities of these five genes were evaluated using two computation programs: geNorm and NormFinder. The results were further validated by normalizing the expression of the phosphoglycerate kinase (HuPGK) and ethylene‐responsive transcription factor (HuERF) genes. Our results indicate that combined use of HuUBQ and HuUQT is the most stable reference under all of the experimental conditions examined. HuEF1‐α, HuUBQ, and HuUQT are the top three most stable reference genes under salt stress, drought stress, and heat stress, and across different cultivars. HuEF1‐α, HuACT, and HuUQT exhibited the most stable expression patterns across different tissues. Our results will allow researchers to select the most appropriate reference genes for gene expression studies of pitaya under different conditions.

Pitaya (Hylocereus polyrhizus) is a highly valued functional food that is widely planted in Southeast Asia. However, with increased soil salinization in cultivation areas, a better understanding of the molecular mechanisms associated with salt stress responses in red pitaya is necessary. Herein, RNA-Seq was used to de novo assemble and characterize the transcriptomic profiles of red pitaya roots in response to salt stress. A total of 73,589 transcripts were obtained and the average sequence length was 1308 bp. From these transcripts, 26,878 unique transcripts were successfully matched to 13,519 Swiss-prot proteins. Gene ontology (GO) annotations showed that within the categories of molecular function, biological function, and cell component, catalytic activity (GO:0003824), metabolic process (GO:0008152), and cell part (GO:0044464) were the most enriched, respectively. 2624 transcripts were significantly differentially expressed among three time points (3 h, 7 h, and 30 h) following exposure to 450 mM NaCl. Furthermore, 261 genes were up-regulated and 61 down-regulated in all three of the time points. Glycolysis/gluconeogenesis was one of the most significantly modulated pathways. The findings presented herein provide further insight into salt stress responses in pitaya and will provide a valuable resource for future functional studies examining salt adaptations.

Pitaya (Hylocereus undatus) is a high salt-tolerant fruit, and ethylene response factors (ERFs) play important roles in transcription-regulating abiotic tolerance. To clarify the function of HuERF1 in the salt tolerance of pitaya, HuERF1 was heterogeneously expressed in Arabidopsis. HuERF1 had nuclear localization when HuERF1::GFP was expressed in Arabidopsis protoplasts and had transactivation activity when HuERF1 was expressed in yeast. The expression of HuERF1 in pitaya seedlings was significantly induced after exposure to ethylene and high salinity. Overexpression of HuERF1 in Arabidopsis conferred enhanced tolerance to salt stress, reduced the accumulation of superoxide (O2∙) and hydrogen peroxide (H2O2), and improved antioxidant enzyme activities. These results indicate that HuERF1 is involved in ethylene-mediated salt stress tolerance, which may contribute to the salt tolerance of pitaya.

As structural components of biological membranes, phytosterols are essential not only for a variety of cellular functions but are also precursors for brassinosteroid (BR) biosynthesis. Plant CYP51 is the oldest and most conserved obtusifoliol 14α-demethylase in eukaryotes and is an essential component of the sterol biosynthesis pathway. However, little is known about rice (Oryza sativa L.) CYP51G1. In this study, we showed that rice OsCYP51G1 shared high homology with obtusifoliol 14α-demethylase and OsCYP51G1 was strongly expressed in most of rice organs. Subcellular localization analysis indicated that OsCYP51G1 was localized to the endoplasmic reticulum. Knockdown and knockout of OsCYP51G1 resulted in delayed flowering, impaired membrane integrity, abnormal pollen, and reduced grain yield, whereas OsCYP51G1 overexpression led to increased grain yield. Knockdown of OsCYP51G1 also reduced the levels of end-products (sitosterol and stigmasterol) and increased those of upstream intermediates (24-methylene-cycloartenol and cycloeucalenol) of the OsCYP51G1-mediated sterol biosynthesis step. In contrast, overexpression of OsCYP51G1 increased the sitosterol and stigmasterol content and reduced that of cycloeucalenol. However, knockdown of OsCYP51G1 by RNAi did not elicit these BR deficiency-related phenotypes, such as dwarfism, erect leaves and small seeds, nor was the leaf lamina angle sensitive to brassinolide treatment. These results revealed that rice OsCYP15G1 encodes an obtusifoliol 14α-demethylase for the phytosterols biosynthesis and possible without affecting the biosynthesis of downstream BRs, which was different from its homolog, OsCYP51G3.

Additional file 3. Predicted amino acid sequence of CR-S14.

Additional file 5. CR-S14 confers strong resistance to PXO86.

Additional file 1. Detection of mutations in CR-S14 transcripts.

Additional file 6. Agronomic traits of CR-S14.

Additional file 8. Primers used in this study.

Additional file 4. OsSWEET13ZH11 EBE sequence.

Enhancing nitrogen (N) use efficiency is a potential way to reduce excessive nitrogen application and increase yield. Autophagy is a conserved degradation system in the evolution of eukaryotic cells and plays an important role in plant development and stress response. Autophagic cores have two conjugation pathways that attach the product of autophagy-related gene 8 (ATG8) to phosphatidylethanolamine (PE) and ATG5 to ATG12, respectively, which then help with vesicle elongation and enclosure. Rice has six ATG8 genes, which have not been functionally confirmed so far. We identified the rice gene OsATG8b and characterized its role in N remobilization to affect grain quality by generating transgenic plants with its over-expression and knockdown. Our study confirmed the autophagy activity of OsATG8b through the complementation of the yeast autophagy-defective mutant scatg8 and by observation of autophagosome formation in rice. The autophagy activity is higher in OsATG8b-OE lines and lower in OsATG8b-RNAi than that in wild type (ZH11). 15N pulse-chase analysis revealed that OsATG8b-OE plants conferred higher N recycling efficiency to grains, while OsATG8b-RNAi transgenic plants exhibited lower N recycling efficiency and poorer grain quality. The autophagic role of OsATG8b was experimentally confirmed, and it was concluded that OsATG8b-mediated autophagy is involved in N recycling to grains and contributes to the grain quality, indicating that OsATG8b may be a potential gene for molecular breeding and cultivation of rice.

Background: Enhancing nitrogen (N) use efficiency is a potential way of reducing excessive nitrogen application and increasing yield. Autophagy is a conservde degradation system in the evolution of eukaryotic cells and plays an important role in plant development and stress response. Autophagic cores are two conjugation pathways that attach ATG8 to PE and ATG5 to ATG12, which then help with vesicle elongation and enclosure. Rice has six ATG8 genes, which have not been functionally confirmed so far. Results: Autophagy activity of OsATG8b was confirmed through the complementation of the yeast autophagy defective mutant scatg8 and by observation of autophagosome formation in rice. The autophagy activity is higher in OsATG8b-OE lines and lower in OsATG8b-RNAi than that in ZH11. 15N pulse-chase analysis revealed that OsATG8b-OE plants conferred higher N recycling efficiency to grains, while OsATG8b-RNAi transgenic plants exhibited lower N recycling efficiency and poorer grain quality. Conclusion: Autophagic role of OsATG8b was experimentally confirmed and OsATG8b-mediated autophagy is involved in N recycling to grains and contributes to the grain quality, indicating OsATG8b may be a potential gene for molecular breeding and cultivation of rice.

Late embryogenesis abundant (LEA) proteins belong to a large family that exists widely in plants and is mainly involved in desiccation processes during plant development or in the response to abiotic stresses. Here, we reported on an atypical LEA gene (IpLEA) related to salt tolerance from Ipomoea pes-caprae L. (Convolvulaceae). Sequence analysis revealed that IpLEA belongs to the LEA_2 (PF03168) group. IpLEA was shown to have a cytoplasmic localization pattern. Quantitative reverse transcription PCR analysis showed that IpLEA was widely expressed in different organs of the I. pes-caprae plants, and the expression levels increased following salt, osmotic, oxidative, freezing, and abscisic acid treatments. Analysis of the 1,495 bp promoter of IpLEA identified distinct cis-acting regulatory elements involved in abiotic stress. Induction of IpLEA improved Escherichia coli growth performance compared with the control under abiotic stresses. To further assess the function of IpLEA in plants, transgenic Arabidopsis plants overexpressing IpLEA were generated. The IpLEA-overexpressing Arabidopsis seedlings and adult plants showed higher tolerance to salt and drought stress than the wild-type. The transgenic plants also showed higher oxidative stress tolerance than the wild-type Arabidopsis. Furthermore, the expression patterns of a series of stress-responsive genes were affected. The results indicate that IpLEA is involved in the plant response to salt and drought, probably by mediating water homeostasis or by acting as a reactive oxygen species scavenger, thereby influencing physiological processes under various abiotic stresses in microorganisms and plants.

Ipomoea pes-caprae is a seashore halophytic plant and is therefore a good model for studying the molecular mechanisms underlying salt and stress tolerance in plant research. Here, we performed Full-length cDNA Over-eXpressor (FOX) gene hunting with a functional screening of a cDNA library using a salt-sensitive yeast mutant strain to isolate the salt-stress-related genes of I. pes-caprae (IpSR genes). The library was screened for genes that complemented the salt defect of yeast mutant AXT3 and could grow in the presence of 75 mM NaCl. We obtained 38 candidate salt-stress-related full-length cDNA clones from the I. pes-caprae cDNA library. The genes are predicted to encode proteins involved in water deficit, reactive oxygen species (ROS) scavenging, cellular vesicle trafficking, metabolic enzymes, and signal transduction factors. When combined with the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses, several potential functional salt-tolerance-related genes were emphasized. This approach provides a rapid assay system for the large-scale screening of I. pes-caprae genes involved in the salt stress response and supports the identification of genes responsible for the molecular mechanisms of salt tolerance.

Cuticular wax forms a hydrophobic layer covering aerial plant organs and acting as a protective barrier against biotic and abiotic stresses. Compared with well-known wax biosynthetic pathway, molecular regulation of wax biosynthesis is less known. Here, we show that rice OsWS1, a member of the membrane-bound O-acyl transferase gene family, involved in wax biosynthesis and was regulated by an osa-miR1848. OsWS1-tagged green fluorescent protein localized to the endoplasmic reticulum (ER). Compared with wild-type rice, OsWS1 overexpression plants displayed a 3% increase in total wax, especially a 35% increase in very long-chain fatty acids, denser wax papillae around the stoma, more cuticular wax crystals formed on leaf and stem surfaces, pollen coats were thicker and more seedlings survived after water-deficit treatment. In contrast, OsWS1-RNAi and osa-miR1848 overexpression plants exhibited opposing changes. Gene expression analysis showed that overexpression of osa-miR1848 down-regulated OsWS1 transcripts; furthermore, expression profiles of OsWS1 and osa-miR1848 were inversely correlated in the leaf, panicle and stem, and upon water-deficit treatment. These results suggest that OsWS1 is regulated by osa-miR1848 and participates in cuticular wax formation.

Phytosterols are membrane components or precursors for brassinosteroid (BR) biosynthesis. As they cannot be transported long distances, their homeostasis is tightly controlled through their biosynthesis and metabolism. However, it is unknown whether microRNAs are involved in their homeostatic regulation. Rice (Oryza sativa) plants transformed with microRNA osa-miR1848 and its target, the obtusifoliol 14α-demethylase gene, OsCYP51G3, were used to investigate the role of osa-miR1848 in the regulation of phytosterol biosynthesis. osa-miR1848 directs OsCYP51G3 mRNA cleavage to regulate phytosterol and BR biosynthesis in rice. The role of OsCYP51G3 as one of the osa-miR1848 targets is supported by the opposite expression patterns of osa-miR1848 and OsCYP51G3 in transgenic rice plants, and by the identification of OsCYP51G3 mRNA cleavage sites. Increased osa-miR1848 and decreased OsCYP51G3 expression reduced phytosterol and BR concentrations, and caused typical phenotypic changes related to phytosterol and BR deficiency, including dwarf plants, erect leaves, semi-sterile pollen grains, and shorter cells. Circadian expression of osa-miR1848 regulated the diurnal abundance of OsCYP51G3 transcript in developing organs, and the response of OsCYP51G3 to salt stress. We propose that osa-miR1848 regulates OsCYP51G3 expression posttranscriptionally, and mediates phytosterol and BR biosynthesis. osa-miR1848 and OsCYP51G3 might have potential applications in rice breeding to modulate leaf angle, and the size and quality of seeds.

Correct folding of proteins in the endoplasmic reticulum is important for their stability and function under stress. The protein disulfide isomerase (PDI) OsPDIL1;1 is a key protein-folding catalyst in rice (Oryza sativa L.). Here, microRNA5144 (osa-miR5144-3p) is reported to mediate the formation of protein disulfide bonds via targeting OsPDIL1;1 mRNA in rice seeds and seedlings during development and under conditions of abiotic stress, respectively. Expression analysis of transgenic rice and identification of cleavage sites showed that OsPDIL1;1 mRNA is a target of osa-miR5144-3p. Expression of osa-miR5144-3p and OsPDIL1;1 was shown to be inversely regulated in developing organs and under abiotic stress. The down-regulation of osa-miR5144-3p or overexpression of OsPDIL1;1 in transgenic rice showed increased total protein-disulfide bond content, compared with the wild type. This indicates that protein-disulfide bond formation is enhanced by down-regulation of osa-miR5144-3p or overexpression of OsPDIL1;1. These transgenic rice plants also displayed strong resistance to salinity and mercury stress, in comparison with the wild type. In contrast, the transgenic rice plants overexpressing osa-miR5144-3p or down-regulating OsPDIL1;1 had a lower protein-disulfide bond content; they were susceptible to abiotic stress and produced abnormal grains with small and loosely packed starch granules. These results indicate that protein-disulfide bond formation catalyzed by OsPDIL1;1 is modulated by osa-miR5144-3p in rice during development and is involved in resistance to abiotic stress.

Cytokinins play important roles in legume-rhizobia symbiosis. Here we report isolation of six genes encoding isopentenyl transferase (IPT) from Lotus japonicus, which catalyze the rate-limiting step of cytokinin biosynthesis. The LjIPT3 gene was found to be up-regulated in infected roots and mature nodules. Histochemical analysis demonstrated expression of Pro(LjIPT3):GUS (β-glucuronidase) in vegetative and reproductive organs, and was especially high in the vascular bundles of roots. When inoculated with Mesorhizobium loti MAFF303099, LjIPT3 was undetectable in the nodule primordia and developing nodules, and later it was expressed only in the vascular bundles of mature nodules. In addition, knockdown of LjIPT3 (LjIPT3i) by RNA interference reduced levels of endogenous cytokinins, affected plant development and accelerated Chl degradation during dark-induced leaf senescence. Compared with the wild type, LjIPT3i plants produced fewer infection threads and nodules. In addition, expression of downstream nodulation-related transcription factor genes LjNSP1, LjNSP2 and LjNIN decreased dramatically in LjIPT3i plants. These results suggest that LjIPT3 regulates the CRE1-dependent cytokinin pathway, affecting nodule initiation and thereby influencing the number of infection threads and nodules. Detection of nitrogenase activity and observation of nodule structure showed that endogenous cytokinins are required for full development of the infected cells in mature nodules by preventing early senescence. Therefore, our results indicate that the LjIPT3 gene product is required for nodule initiation and development, and does not appear to be involved in early infection events.

Rice tillering has an important influence on grain yield, and is promoted by nitrogen (N) fertilizer. Several genes controlling rice tillering, which are regulated by poor N supply, have been identified. However, the molecular mechanism associated with the regulation of tillering based on N supply is poorly understood. Here, we report that rice microRNA393 (OsmiR393) is involved in N-mediated tillering by decreasing auxin signal sensitivity in axillary buds. Expression analysis showed that N fertilizer causes up-regulation of OsmiR393, but down-regulation of two target genes (OsAFB2 and OsTB1). In situ expression analysis showed that OsmiR393 is highly expressed in the lateral axillary meristem. OsmiR393 overexpression mimicked N-mediated tillering in wild type Zhonghua 11 (ZH11). Mutation of OsMIR393 in ZH11 repressed N-promoted tillering, which simulated the effects of limited N, and this could not be restored by supplying N fertilizer. Western blot analysis showed that OsIAA6 was accumulated in both OsmiR393-overexpressing lines and N-treated wild type rice, but was reduced in the OsMIR393 mutant. Therefore, we deduced that N-induced OsmiR393 accumulation reduces the expression of OsTIR1 and OsAFB2, which alleviates sensitivity to auxin in the axillary buds and stabilizes OsIAA6, thereby promoting rice tillering.

The plant PTR/NRT1 (peptide transporter/nitrate transporter 1) gene family comprises di/tripeptide and low-affinity nitrate transporters; some members also recognize other substrates such as carboxylates, phytohormones (auxin and abscisic acid), or defence compounds (glucosinolates). Little is known about the members of this gene family in rice (Oryza sativa L.). Here, we report the influence of altered OsPTR9 expression on nitrogen utilization efficiency, growth, and grain yield. OsPTR9 expression is regulated by exogenous nitrogen and by the day-night cycle. Elevated expression of OsPTR9 in transgenic rice plants resulted in enhanced ammonium uptake, promotion of lateral root formation and increased grain yield. On the other hand, down-regulation of OsPTR9 in a T-DNA insertion line (osptr9) and in OsPTR9-RNAi rice plants had the opposite effect. These results suggest that OsPTR9 might hold potential for improving nitrogen utilization efficiency and grain yield in rice breeding.

SummaryGibberellins (GAs) modulate jasmonate (JA) signaling, which is essential for stress response and development in plants. However, the molecular details of such phytohormone interaction remain largely unknown. Here, we show that the JA ZIM-domain 1 (JAZ1) protein, a key repressor of JA signaling, interacts in vivo with DELLA proteins, repressors of the GA pathway. DELLAs prevent inhibitory JAZ1 interaction with a key transcriptional activator of JA responses, MYC2, and, thus, enhance the ability of MYC2 to regulate its target genes. Conversely, GA triggers degradation of DELLAs, which allows JAZ1 to bind MYC2 and suppress MYC2-dependent JA-signaling outputs. Therefore, our results reveal one means by which GAs suppress cellular competence to respond to JA. Because DELLAs serve as central regulators that mediate the crosstalk of various phytohormones, our model also suggests a candidate mechanism by which JA signaling may be fine-tuned by other signaling pathways through DELLAs.

Peptide transporters (PTRs) have diverse substrates and underlie many biological processes in plants. To better understand their function and regulation, we have analyzed the transport capabilities of eight rice PTRs (OsPTRs) in a yeast ptr2 mutant strain and their expression patterns in plants. A yeast ptr2 complementation assay demonstrated that only OsPTR6 transports Gly–His and Gly–His–Gly, and OsPTR6 shows substrate selectivity for di-/tripeptides; however, the other seven proteins could not transport the five tested di-/tripeptides. The expression patterns for the eight OsPTRs were distinct in tissues under normal growth conditions. In germinating seeds, seven of the eight OsPTRs were expressed at various levels, but OsPTR5 was not expressed. In grain-filling seeds, expression of OsPTR4, OsPTR7 and OsPTR8 increased from early to late stages of seed development and that of OsPTR2, OsPTR3 and OsPTR6 decreased. Expression of OsPTR1, OsPTR2, OsPTR5, OsPTR7 and OsPTR8 were upregulated by drought and salt, and OsPTR4 was downregulated. However, only OsPTR1 displayed a slight upregulation by cold. This study showed that rice OsPTRs have distinctive transport functions for substrates and differing expression patterns during both development and response to stress.

In this study we produced transgenic tobacco plants by overexpressing a serine proteinase inhibitor gene, SaPIN2a, from the American black nightshade Solanum americanum under the control of the CaMV 35S promoter using Agrobacterium tumefaciens-mediated transformation. SaPIN2a was properly transcribed and translated as indicated by Northern blot and Western blot analyses. Functional integrity of SaPIN2a in transgenic plants was confirmed by proteinase inhibitory activity assay. Bioassays for insect resistance showed that SaPIN2a-overexpressing transgenic tobacco plants were more resistant to cotton bollworm (Helicoverpa armigera) and tobacco cutworm (Spodoptera litura) larvae, two devastating pests of important crop plants, than the control plants. Interestingly, overexpression of SaPIN2a in transgenic tobacco plants resulted in a significant increase in glandular trichome density and a promotion of trichome branching, which could also provide an additional resistance mechanism in transgenic plants against insect pests. Therefore, SaPIN2a could be used as an alternative proteinase inhibitor for the production of insect-resistant transgenic plants.

The complete mitochondrial genome of the yellowfin seabream Acanthopagrus latus was determined in the present study. The genome was 16,609 bp in length and contained 37 genes (2 ribosomal RNA, 22 transfer RNA and 13 protein-coding genes) and the control region (CR), with the content and order of genes being similar to those in typical teleosts. Comparisons of the 37 genes and CR among species indicate the CR was the highest divergent (0.3341), but tRNAGly possesses the lowest genetic variation (0.0542). Much greater p-genetic distances [mean = 0.1559, standard deviation (SD) = 0.0235; n = 1653] for the interspecies level with high frequency (99.4%) than those of the intraspecies level (mean = 0.0098, SD = 0.0090; n = 20) were inferred from 212 Cyt b sequence data, suggesting the Cyt b gene is conserved within Sparidae species and supporting the barcoding validity of Cyt b sequence data for Sparidae species identification. Phylogenetic analysis using amino acid sequences of 13 protein-coding genes supporte...

SaPIN2a, a plant proteinase inhibitor from nightshade (Solanum americanum), was located to the enucleate sieve elements (SEs) of phloem. The expressed SaPIN2a in transgenic lettuce showed inhibition of plant endogenous trypsin- and chymotrypsin-like activities, suggesting that SaPIN2a can regulate proteolysis in plant cells. To further investigate the physiological role of SaPIN2a, we produced transgenic nightshade and lettuce plants overexpressing SaPIN2a from the cauliflower mosaic virus (CaMV) 35S promoter using Agrobacterium-mediated transformation. Overexpression of SaPIN2a in transgenic plants was demonstrated by northern blot and western blot analysis. SaPIN2a-overexpressing transgenic nightshade plants showed significantly lower height than wild-type plants. Transmission electron microscopy analysis showed that chloroplast-like organelles with thylakoids, which are not present in enucleate SEs of wild-type plants, were present in the enucleate SEs of SaPIN2a-overexpressing transgenic plants. This finding is discussed in terms of the possible role played by SaPIN2a in the regulation of proteolysis in SEs.

The complete necleotide sequence of the mitochondrial genome of Parargyrops edita was determined by gene amplification using long polymerase chain reaction and direct sequencing techniques. The genome with 16 640 bp contained 37 genes (two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes). The content and order of genes was identical to those in typical teleosts. The major non-coding region of 964 bp in length with several conserved sequence features were identified as the control region (CR). Both COI and ND4 began with a GTG start codon, which is unusual in other fishes. The genetic variation of the CR from 27 wild samples was evaluated. A total of 536 bp consensus sequence of CR was aligned and 26 unique haplotypes were identified. The haplotype diversity (h) was estimated to be 0.997 (standard deviation [SD] 0.011) and nucleotide diversity (π) was 0.014 (SD 0.008) for the sample collected from coastal waters of Guangdong Province. It showed a very high level of genetic variation in the sample and also suggested that mtDNA CR sequence analysis can be use to evaluate the genetic deversity and genetic structure of P. edita.

Objective: Chilling tolerance of salicylic acid (SA) in banana seedlings (Musa acuminata cv., Williams 8818) was investigated by changes in ultrastructure in this study. Methods: Light and electron microscope observation. Results: Pretreatment with 0.5 mmol/L SA under normal growth conditions (30/22 °C) by foliar spray and root irrigation resulted in many changes in ultrastructure of banana cells, such as cells separation from palisade parenchymas, the appearance of crevices in cell walls, the swelling of grana and stromal thylakoids, and a reduction in the number of starch granules. These results implied that SA treatment at 30/22 °C could be a type of stress. During 3 d of exposure to 7 °C chilling stress under low light, however, cell ultrastructure of SA-pretreated banana seedlings showed less deterioration than those of control seedlings (distilled water-pretreated). Conclusion: SA could provide some protection for cell structure of chilling-stressed banana seedling.