A long non-coding RNA functions as a competitive endogenous RNA to modulate TaNAC018 by acting as a decoy for tae-miR6206.

Increasing evidence indicates a strong correlation between the deposition of cuticular waxes and drought tolerance. However, the precise regulatory mechanism remains elusive. Here, we conducted a comprehensive transcriptome analysis of two wheat (Triticum aestivum) near-isogenic lines, the glaucous line G-JM38 rich in cuticular waxes and the non-glaucous line NG-JM31. We identified 85,143 protein-coding mRNAs, 4,485 lncRNAs, and 1,130 miRNAs. Using the lncRNA-miRNA-mRNA network and endogenous target mimic (eTM) prediction, we discovered that lncRNA35557 acted as an eTM for the miRNA tae-miR6206, effectively preventing tae-miR6206 from cleaving the NAC transcription factor gene TaNAC018. This lncRNA-miRNA interaction led to higher transcript abundance for TaNAC018 and enhanced drought-stress tolerance. Additionally, treatment with mannitol and abscisic acid (ABA) each influenced the levels of tae-miR6206, lncRNA35557, and TaNAC018 transcript. The ectopic expression of TaNAC018 in Arabidopsis also improved tolerance toward mannitol and ABA treatment, whereas knocking down TaNAC018 transcript levels via virus-induced gene silencing in wheat rendered seedlings more sensitive to mannitol stress. Our results indicate that lncRNA35557 functions as a competing endogenous RNA to modulate TaNAC018 expression by acting as a decoy target for tae-miR6206 in glaucous wheat, suggesting that non-coding RNA has important roles in the regulatory mechanisms responsible for wheat stress tolerance.

Intronic microRNA-directed regulation of mitochondrial reactive oxygen species enhances plant stress tolerance in Arabidopsis.

MicroRNAs (miRNAs) play crucial roles in regulating plant development and stress responses. However, the functions and mechanism of intronic miRNAs in plants are poorly understood. This study reports a stress-responsive RNA splicing mechanism for intronic miR400 production, whereby miR400 modulates reactive oxygen species (ROS) accumulation and improves plant tolerance by downregulating its target expression. To monitor the intron splicing events, we used an intronic miR400 splicing-dependent luciferase transgenic line. Luciferase activity was observed to decrease after high cadmium concentration treatment due to the retention of the miR400-containing intron, which inhibited the production of mature miR400. Furthermore, we demonstrated that under Cd treatments, Pentatricopeptide Repeat Protein 1 (PPR1), the target of miR400, acts as a positive regulator by inducing ROS accumulation. Ppr1 mutation affected the Complex III activity in the electron transport chain and RNA editing of the mitochondrial gene ccmB. This study illustrates intron splicing as a key step in intronic miR400 production and highlights the function of intronic miRNAs as a 'signal transducer' in enhancing plant stress tolerance.

Salt responsive alternative splicing of a RING finger E3 ligase modulates the salt stress tolerance by fine-tuning the balance of COP9 signalosome subunit 5A.

Increasing evidence points to the tight relationship between alternative splicing (AS) and the salt stress response in plants. However, the mechanisms linking these two phenomena remain unclear. In this study, we have found that Salt-Responsive Alternatively Spliced gene 1 (SRAS1), encoding a RING-Type E3 ligase, generates two splicing variants: SRAS1.1 and SRAS1.2, which exhibit opposing responses to salt stress. The salt stress-responsive AS event resulted in greater accumulation of SRAS1.1 and a lower level of SRAS1.2. Comprehensive phenotype analysis showed that overexpression of SRAS1.1 made the plants more tolerant to salt stress, whereas overexpression of SRAS1.2 made them more sensitive. In addition, we successfully identified the COP9 signalosome 5A (CSN5A) as the target of SRAS1. CSN5A is an essential player in the regulation of plant development and stress. The full-length SRAS1.1 promoted degradation of CSN5A by the 26S proteasome. By contrast, SRAS1.2 protected CSN5A by competing with SRAS1.1 on the same binding site. Thus, the salt stress-triggered AS controls the ratio of SRAS1.1/SRAS1.2 and switches on and off the degradation of CSN5A to balance the plant development and salt tolerance. Together, these results provide insights that salt-responsive AS acts as post-transcriptional regulation in mediating the function of E3 ligase.

The Mutation of Glu at Amino Acid 3838 of AtMDN1 Provokes Pleiotropic Developmental Phenotypes in Arabidopsis.

MDN1/Rea1, as an AAA-type ATPase, is predicted to be the largest protein involved in pre-ribosome maturation in most organisms. However, its function in plant growth and development is poorly understood. Here, we characterized a novel Arabidopsis mutant, dwarf &short root (dsr) 1, which shows pleiotropic developmental phenotypes, such as slow germination, short root, dwarf shoot, and reduced seed set under normal growth conditions. Using positional cloning, we revealed that the AtMDN1 function is impaired by a 'glutamic acid' to 'lysine' change at position 3838 of the amino acid sequence in dsr1. Multiple sequence alignment analysis revealed that the mutated Glu residue, which located in the linker domain of AtMDN1, is extremely conserved among organisms. AtMDN1 is expressed in various tissues, particularly in the shoot apex and root tip. Moreover, the results of transcript profile analyses showed that the dysfunction of AtMDN1 in dsr1 impairs the expression of genes related to plant growth and development, which is tightly associated with the pleiotropic phenotypes of dsr1. Thus, we concluded that the Glu residue plays a vital role in maintaining AtMDN1 functions, which are essential for plant growth and development.

Arabidopsis YL1/BPG2 Is Involved in Seedling Shoot Response to Salt Stress through ABI4.

The chloroplast-localized proteins play roles in plant salt stress response, but their mechanisms remain largely unknown. In this study, we screened a yellow leaf mutant, yl1-1, whose shoots exhibited hypersensitivity to salt stress. We mapped YL1 to AT3G57180, which encodes a YqeH-type GTPase. YL1, as a chloroplast stroma-localized protein, could be markedly reduced by high salinity. Upon exposure to high salinity, seedling shoots of yl1-1 and yl1-2 accumulated significantly higher levels of Na(+) than wild type. Expression analysis of factors involved in plant salt stress response showed that the expression of ABI4 was increased and HKT1 was evidently suppressed in mutant shoots compared with the wild type under normal growth conditions. Moreover, salinity effects on ABI4 and HKT1 were clearly weakened in the mutant shoots, suggesting that the loss of YL1 function impairs ABI4 and HKT1 expression. Notably, the shoots of yl1-2 abi4 double mutant exhibited stronger resistance to salt stress and accumulated less Na(+) levels after salt treatment compared with the yl1-2 single mutant, suggesting the salt-sensitive phenotype of yl1-2 seedlings could be rescued via loss of ABI4 function. These results reveal that YL1 is involved in the salt stress response of seedling shoots through ABI4.

A spindle pole antigen gene MoSPA2 is important for polar cell growth of vegetative hyphae and conidia, but is dispensable for pathogenicity in Magnaporthe oryzae.

Spa2 is an important component of the multiprotein complex polarisome, which is involved in the establishment, maintenance, termination of polarized cell growth and is important for defining tip growth of filamentous fungi. In this study, we isolated an insertional mutant of the rice blast fungus Magnaporthe oryzae that formed smaller colony and conidia compared with the wild type. In the mutant, a spindle pole antigen gene MoSPA2 was disrupted by the integration of an exogenous plasmid. Targeted gene deletion and complementation assays demonstrated the gene disruption was responsible for the defects of the insertional mutant. Interestingly, the MoSpa2-GFP fusion protein was found to accumulate as a spot at hyphal tips, septa of hyphae and conidial tip cells where germ tubes are usually produced, but not in appressoria, infection hyphae or at the septa of conidia. Furthermore, the deletion mutants of MoSPA2 exhibited slower hyphal tip growth, more hyphal branches, and smaller size of conidial tip cells. However, MoSPA2 is not required for plant infection. These results indicate that MoSPA2 is required for vegetative hyphal growth and maintaining conidium morphology and that spotted accumulation of MoSpa2 is important for its functions during cell polar growth.

NFYA1 is involved in regulation of postgermination growth arrest under salt stress in Arabidopsis.

The nuclear factor Y (NF-Y), which is a ubiquitous transcription factor found in eukaryotes, is composed of three distinct subunits, namely, NF-YA, NF-YB, and NF-YC. Here, we firstly characterized the detailed function of the Arabidopsis NFYA1 factor. It is found that the 35S::AtNFYA1-overexpressed lines were hypersensitive to salt stress and Abscisic acid (ABA) during the early-postgermination growth stages. The transgenic lines exhibited a severe postgermination growth arrest compared with the wild-type (WT) under salt stress and ABA treatment. Interestingly, sodium tungstate, which is an ABA synthesis inhibitor, restored the salt-sensitive phenotype of the 35S::AtNFYA1 lines. Results of the qRT-PCR analysis showed that the mRNA levels of ABI3 and ABI5, as well as their downstream genes AtEM1 and AtEM6, were more greatly upregulated under salt stress during seed germination in the transgenic lines compared with those in WT. On the other hand, the NFYA1-RNAi lines were found to be insensitive to salt stress and exhibited decreased levels of ABI3, ABI5, EM1, and EM6 transcripts. Our results provide clear evidence supporting a role of AtNFYA1 in regulating postgermination growth arrest under salt stress.

Transcript profiling of microRNAs during the early development of the maize brace root via Solexa sequencing.

To characterize the microRNAs that contribute to the development of brace root, Solexa high-throughput sequencing of three libraries derived from tissues of node (N), nodes with just-emerged brace roots (NR), and nodes with just-emerged brace roots after IAA treatment (NRI) was performed. Total 650,793, 957,303 and 1,082,948 genome-matched unique reads were obtained in N, NR and NRI libraries, respectively. Further analysis confirmed the authenticity of 137 known miRNAs and the discovery of 159 novel miRNAs in maize. 14 conserved and 16 novel miRNAs differentially expressed in brace root, as well as 15 target genes, were identified and validated by qRT-PCR during maize brace root development. Moreover, we identified 9 miRNA precursor-matched novel sRNAs that may form miRNA clusters, as well as 24 nt siRNAs in the three libraries. In addition, we suggest that auxin represent a regulator in brace root development and can be regulated at the posttranscriptional level by miRNAs.

Stress-induced alternative splicing provides a mechanism for the regulation of microRNA processing in Arabidopsis thaliana.

MicroRNAs (miRNAs) have emerged as a class of regulators of gene expression through posttranscriptional degradation or translational repression in living cells. Increasing evidence points to the important relationship between miRNAs and environmental stress responses, but the regulatory mechanisms in plants are poorly understood. Here, we found that Arabidopsis thaliana intronic miR400 was cotranscribed with its host gene (At1g32583) and downregulated by heat treatment. Intriguingly, an alternative splicing (AS) event that occurred in the intron (306 bp) where MIR400 was located was specifically induced by heat stress. A 100 bp fragment was excised, and the remaining 206 bp intron containing MIR400 transcripts was retained in the host gene. The stress-induced AS event thus resulted in greater accumulation of miR400 primary transcripts and a low level of mature miR400. Together, these results provide the direct evidence that AS acts as a regulatory mechanism linking miRNAs and environmental stress in plants.

Transcript profiling during the early development of the maize brace root via Solexa sequencing.

Currently, the molecular regulation mechanisms involved in the early development of maize brace root are poorly known. To gain insight into the transcriptome dynamics that are associated with its development, genome-wide gene expression profiling was conducted by Solexa sequencing (Illumina Inc., San Diego, CA, USA). More than five million tags were generated from the stem node tissues without and with just-emerged brace roots, including 149,524 and 178,131 clean tags in the two libraries, respectively. Of these, 82,864 (55.4%) and 91,678 (51.5%) tags were matched to the reference genes. The most differentially expressed tags with a log(2) ratio > 2 or < -2 (P < 0.001) were analyzed further, representing 143 up-regulated and 152 down-regulated genes, except for unknown transcripts, which were classified into 11 functional categories. The most enriched categories were those of metabolism, signal transduction and cellular transport. Many genes or biological pathways were found to be commonly shared between brace root and lateral or adventitious root development, such as genes participating in cell wall degradation and synthesis, auxin transport and signaling, ethylene signaling, etc. Next, the expression patterns of 20 genes were assessed by quantitative real-time PCR, and the results obtained showed general agreement with the Solexa analysis. Furthermore, a comparison of the brace root transcriptome with that of maize primary root revealed substantial differences in the categories and abundances of expressed transcripts. In conclusion, we first reveal the complex changes in the transcriptome during the early development of maize brace root and provide a comprehensive set of data that are essential for understanding its molecular regulation.

Genome-wide profiling of developmental, hormonal or environmental responsiveness of the nucleocytoplasmic transport receptors in Arabidopsis.

Being poikilothermic and sessile organisms, plants have to respond quickly to changing environmental cues, and a higher order of gene regulation is required. The significance of nucleocytoplasmic transport via importinalpha and importinbeta (alpha/beta) has been exhibited in a wide spectrum of biological processes. However, most of these receptors have not been characterized as to which cellular or development processes are required and how their expression is regulated by environmental stimuli. Here we pursued a phylogenetic analysis and investigated the expression patterns of all 8 IMPalphas and 18 IMPbetas in Arabidopsis. The IMPalpha isoforms could be tracked back to a common ancestor, while the IMPbetas derived from different ones. The majority of transport receptor genes were constitutively expressed. Intriguingly, AtIMPalpha5, 7, 8 and AtIMPbeta5 were specifically expressed in different tissues. AtIMPbeta3 was the sole receptor that was obviously modulated by exogenous phytohormones, whereas three IMPalphas and five IMPbetas exhibited responses to environmental stimuli. Furthermore, our RT-PCR data provided direct evidence that AtIMPalpha5, 8 and AtIMPbeta5 are not pseudogenes and we also corrected the open reading frame annotation of AtIMPalpha8. These genome-wide profiling results not only widen our understanding of these transport receptors, but also provide strong evidence supporting the role of transport receptors in multiple signaling pathways and give us an insight into the further analysis of nucleocytoplasmic trafficking in Arabidopsis.

GhZFP1, a novel CCCH-type zinc finger protein from cotton, enhances salt stress tolerance and fungal disease resistance in transgenic tobacco by interacting with GZIRD21A and GZIPR5.

* Zinc finger proteins are a superfamily involved in many aspects of plant growth and development. However, CCCH-type zinc finger proteins involved in plant stress tolerance are poorly understood. * A cDNA clone designated Gossypium hirsutum zinc finger protein 1 (GhZFP1), which encodes a novel CCCH-type zinc finger protein, was isolated from a salt-induced cotton (G. hirsutum) cDNA library using differential hybridization screening and further studied in transgenic tobacco Nicotiana tabacum cv. NC89. Using yeast two-hybrid screening (Y2H), proteins GZIRD21A (GhZFP1 interacting and responsive to dehydration protein 21A) and GZIPR5 (GhZFP1 interacting and pathogenesis-related protein 5), which interacted with GhZFP1, were isolated. * GhZFP1 contains two typical zinc finger motifs (Cx8Cx5Cx3H and Cx5Cx4Cx3H), a putative nuclear export sequence (NES) and a potential nuclear localization signal (NLS). Transient expression analysis using a GhZFP1::GFP fusion gene in onion epidermal cells indicated a nuclear localization for GhZFP1. RNA blot analysis showed that the GhZFP1 transcript was induced by salt (NaCl), drought and salicylic acid (SA). The regions in GhZFP1 that interact with GZIRD21A and GZIPR5 were identified using truncation mutations. * Overexpression of GhZFP1 in transgenic tobacco enhanced tolerance to salt stress and resistance to Rhizoctonia solani. Therefore, it appears that GhZFP1 might be involved as an important regulator in plant responses to abiotic and biotic stresses.

GhDREB1 enhances abiotic stress tolerance, delays GA-mediated development and represses cytokinin signalling in transgenic Arabidopsis.

Plants vary significantly in their ability to tolerate low temperatures. The CBF/DREB1 cold response pathway has been identified in many plant species and plays a pivotal role in low temperature tolerance. Here, we show that GhDREB1 is a functional homologue and elevates the freezing, salt and osmotic stress tolerance of transgenic Arabidopsis. The constitutive expression of GhDREB1 in Arabidopsis caused dwarfism and late flowering phenotypes, which could be rescued by exogenous application of GA(3). Endogenous bioactive GA contents were significantly lower in GhDREB1 overexpressing Arabidopsis than in wild-type plants. RT-PCR analyses revealed that the transcript levels of the GA synthase genes were higher in transgenics than in wild-type plants, whereas the GA deactivating genes were lower. Flowering related genes in different regulatory pathways were also affected by GhDREB1, which may account for the flowering delay phenotype. Moreover, the GhDREB1 overexpressing Arabidopsis exhibited decreased sensitivity to cytokinin (CK) which is associated with repression of expression of type-B and type-A ARRs, two key components in the CK-signalling pathway.

Cotton GhDREB1 increases plant tolerance to low temperature and is negatively regulated by gibberellic acid.

The transcription factors C-repeat binding factors/dehydration-responsive element binding proteins (CBFs/DREBs) control the expression of many stress-inducible genes in Arabidopsis. A cDNA clone, designated GhDREB1, was isolated from cotton (Gossypium hirsutum) by cDNA library screening. Northern blot analysis indicated that mRNA accumulation of GhDREB1 was induced by low temperatures and salt stress, but was not induced by abscisic acid (ABA) or drought stress in cotton seedlings. Transgenic tobacco (Nicotiana tabacum) plants overexpressing GhDREB1 displayed stronger chilling tolerance than wild-type plants. Their leaf chlorophyll fluorescence, net photosynthetic rate and proline concentrations were higher than those of control plants during low-temperature treatment. However, under normal growth conditions, the transgenic tobacco plants exhibited retarded growth and delayed flowering. Interestingly, GhDREB1 transcripts in cotton seedlings were negatively regulated by gibberellic acid (GA(3)) treatment. Analysis of the promoter of the GhDREB1 gene revealed the presence of one low-temperature and four gibberellin-responsive elements. Green fluorescent protein (GFP) signal intensity or beta-glucuronidase (GUS) activity driven by the GhDREB1 promoter was clearly enhanced by low temperature but repressed by GA(3). These results suggest that GhDREB1 functions as a transcription factor and plays an important role in improving cold tolerance, and also affects plant growth and development via GA(3).

[Cloning, expression of the CP gene and antiserum preparation of zucchini yellow mosaic virus infecting Benincasa hispida Cogn. var. chieh-qua How].

A Guangzhou isolate of ZYMV infecting Benincasa hispida Cogn. var. chieh-qua How was identified by indicator tests and partial sequence amplification. The coat protein (CP) gene of this virus was amplified by RT-PCR, and ligated to the expression vector pET-22b(+). The recombinant plasmid pET-ZCP was transformed into E. coli BL21 (DE3) and then induced to express by IPTG. It was shown that the CP gene was highly expressed by SDS-PAGE and Western blot analysis. The molecular weight of the recombinant protein was about 33.0 kD. Antiserum with high specificity was produced after the rabbit was immunized with purified recombinant protein, and the titer was determined to be 1/4096 by antigen coating plate-ELISA (ACP-ELISA).