Establishment of a triplex TaqMan quantitative real-time PCR assay for simultaneous detection of Cymbidium mosaic virus, Odontoglossum ringspot virus and Cymbidium ringspot virus.

Orchids are significant ornamental plants whose viral infection results in substantial economic damage. Cymbidium mosaic virus (CymMV), Odontoglossum ringspot virus (ORSV), and Cymbidium ringspot virus (CymRSV) represent three important and prevalent orchid viruses. The detection system proposed in this study uses a triplex TaqMan quantitative real-time PCR assay to identify CymMV, ORSV, and CymRSV in a simultaneous manner. We designed specific primers and probes for CymMV, ORSV, and CymRSV, with amplified sequences of 156 bp, 148 bp, and 145 bp, respectively. The minimum detection limit of the triplex qRT-PCR assay for CymMV and CymRSV was 1 copy/assay, and the minimum detection limit was 10 copies/assay for ORSV. The minimum stable detection limits for CymMV, ORSV, and CymRSV were 10, 102, and 102 copies/assay, respectively. Therefore, this system exhibited higher sensitivity (approximately 10 to 104-fold) than RT-PCR. The intra-and interassay CVs of Cq values are less than 0.55 and 0.95%, respectively, indicating that the triplex assay is highly reliable and accurate. In addition, 66 samples from five different orchid genera were analyzed using the established assay and gene chip. The detection results demonstrated that the triplex probe qRT-PCR demonstrated higher sensitivity than the gene chip, indicating that the triplex real-time PCR assay could be used for the detection of field samples. Our findings suggest that the triplex real-time RT-PCR detection system represents a rapid, simple, and accurate tool for detecting CymMV, ORSV, and CymRSV on orchids.

Arabidopsis lamin-like proteins CRWN1 and CRWN2 interact with SUPPRESSOR OF NPR1-1 INDUCIBLE 1 and RAD51D to prevent DNA damage.

Plants cope with various recurring stress conditions that often induce DNA damage, ultimately affecting plant genome integrity, growth, and productivity. The CROWDED NUCLEI (CRWN) family comprises lamin-like proteins with multiple functions, such as regulating gene expression, genome organization, and DNA damage repair in Arabidopsis (Arabidopsis thaliana). However, the mechanisms and consequences of CRWNs in DNA damage repair are largely unknown. Here, we reveal that CRWNs maintain genome stability by forming repairing nuclear bodies at DNA double-strand breaks. We demonstrate that CRWN1 and CRWN2 physically associate with the DNA damage repair proteins RAD51D and SUPPRESSOR OF NPR1-1 Inducible 1 (SNI1) and act in the same genetic pathway to mediate this process. Moreover, CRWN1 and CRWN2 partially localize at γ-H2AX foci upon DNA damage. Notably, CRWN1 and CRWN2 undergo liquid-liquid phase separation to form highly dynamic droplet-like structures with RAD51D and SNI1 to promote the DNA damage response (DDR). Collectively, our data shed light on the function of plant lamin-like proteins in the DDR and maintenance of genome stability.

The dynamics of H2A.Z on SMALL AUXIN UP RNAs regulate abscisic acid-auxin signaling crosstalk in Arabidopsis.

Extreme environmental changes threaten plant survival and worldwide food production. In response to osmotic stress, the plant hormone abscisic acid (ABA) activates stress responses and restricts plant growth. However, the epigenetic regulation of ABA signaling and crosstalk between ABA and auxin are not well known. Here, we report that the histone variant H2A.Z-knockdown mutant in Arabidopsis Col-0, h2a.z-kd, has altered ABA signaling and stress responses. RNA-sequencing data showed that a majority of stress-related genes are activated in h2a.z-kd. In addition, we found that ABA directly promotes the deposition of H2A.Z on SMALL AUXIN UP RNAs (SAURs), and that this is involved in ABA-repression of SAUR expression. Moreover, we found that ABA represses the transcription of H2A.Z genes through suppressing the ARF7/19-HB22/25 module. Our results shed light on a dynamic and reciprocal regulation hub through H2A.Z deposition on SAURs and ARF7/19-HB22/25-mediated H2A.Z transcription to integrate ABA/auxin signaling and regulate stress responses in Arabidopsis.

Histone H2A monoubiquitination marks are targeted to specific sites by cohesin subunits in Arabidopsis.

Histone H2A monoubiquitination (H2Aub1) functions as a conserved posttranslational modification in eukaryotes to maintain gene expression and guarantee cellular identity. Arabidopsis H2Aub1 is catalyzed by the core components AtRING1s and AtBMI1s of polycomb repressive complex 1 (PRC1). Because PRC1 components lack known DNA binding domains, it is unclear how H2Aub1 is established at specific genomic locations. Here, we show that the Arabidopsis cohesin subunits AtSYN4 and AtSCC3 interact with each other, and AtSCC3 binds to AtBMI1s. H2Aub1 levels are reduced in atsyn4 mutant or AtSCC3 artificial microRNA knockdown plants. ChIP-seq assays indicate that most binding events of AtSYN4 and AtSCC3 are associated with H2Aub1 along the genome where transcription is activated independently of H3K27me3. Finally, we show that AtSYN4 binds directly to the G-box motif and directs H2Aub1 to these sites. Our study thus reveals a mechanism for cohesin-mediated recruitment of AtBMI1s to specific genomic loci to mediate H2Aub1.

Feedback regulation of auxin signaling through the transcription of H2A.Z and deposition of H2A.Z to SMALL AUXIN UP RNAs in Arabidopsis.

Auxin is a critical phytohormone that is involved in the regulation of most plant growth and developmental responses. In particular, epigenetic mechanisms, like histone modifications and DNA methylation, were reported to affect auxin biosynthesis and transport. However, the involvement of other epigenetic factors, such as histone variant H2A.Z, in the auxin-related developmental regulation remains unclear. We report that the histone variant H2A.Z knockdown mutant in Arabidopsis Col-0 ecotype, h2a.z-kd, has more lateral roots and weak gravitational responses related to auxin-regulated growth performances. Further study revealed that auxin promotes the eviction of H2A.Z from the auxin-responsive genes SMALL AUXIN-UP RNAs (SAURs) to activate their transcriptions. We found that IAA promotes the transcription of H2A.Z genes through HOMEOBOX PROTEIN 22/25 (AtHB22/25) transcription factors which work as downstream targets of ARF7/19 in auxin signaling. Double mutant of hb22 hb25 showed similar lateral root and gravitropism phenotypes to h2a.z-kd. Our results shed light on a reciprocal regulation hub through INOSITOL AUXOTROPHY 80-mediated H2A.Z eviction and ARF7/19-HB22/25-mediated H2A.Z transcription to modulate the activation of SAURs and plant growth in Arabidopsis.

MYC2-Activated TRICHOME BIREFRINGENCE-LIKE37 Acetylates Cell Walls and Enhances Herbivore Resistance.

O-Acetylation of polysaccharides predominantly modifies plant cell walls by changing the physicochemical properties and, consequently, the structure and function of the cell wall. Expression regulation and specific function of cell wall-acetylating enzymes remain to be fully understood. In this report, we cloned a previously identified stunted growth mutant named sucrose uncoupled1 (sun1) in Arabidopsis (Arabidopsis thaliana). SUN1 encodes a member of the TRICHOME BIREFRINGEN-LIKE family, AtTBL37 AtTBL37 is highly expressed in fast-growing plant tissues and encodes a Golgi apparatus-localized protein that regulates secondary cell wall thickening and acetylation. In sun1, jasmonate signaling and expression of downstream chemical defense genes, including VEGETATIVE STORAGE PROTEIN1 and BRANCHED-CHAIN AMINOTRANSFERASE4, are increased but, unexpectedly, sun1 is more susceptible to insect feeding. The central transcription factor in jasmonate signaling, MYC2, binds to and induces AtTBL37 expression. MYC2 also promotes the expression of many other TBLs Moreover, MYC activity enhances cell wall acetylation. Overexpression of AtTBL37 in the myc2-2 background reduces herbivore feeding. Our study highlights the role of O-acetylation in controlling plant cell wall properties, plant development, and herbivore defense.

Pollen flow of wheat under natural conditions in the Huanghuai River Wheat Region, China.

The transgenic pollen spread is the main pathway of transgenic plant gene flow. The maximum distance of pollen dispersal (horizontal), the spatial dynamics of pollen movement (vertical), and the patterns of pollen dispersal are important considerations in biosafety assessments of genetically modified crops. To evaluate wheat (Triticum aestivum) pollen dispersal, we measured the pollen suspension velocity and analyzed pollen dispersal patterns under natural conditions in the Huanghuai River wheat-growing region in 2009. The pollen suspension velocity was 0.3-0.4 m/s. The highest pollen densities were detected in the north, northwest, and south of the pollen source. Pollen was dispersed over distances greater than 245 m in the northwest and northeast directions. At the pollen source center, pollen density decreased with increasing vertical height. In the north of the pollen source, the pollen density from 65 m to 225 m showed a wave-mode decrease with increasing height. The horizontal transport of pollen over longer distances fitted polynomial equations. In the north, the pollen density was highest at 45 m from the pollen source, and decreased with increasing distance. In the northwest, the pollen density showed a double-peak trend. In the northeast, pollen density was highest from 45 m to 125 m from the source. Wind speeds greater than the pollen suspension velocity and the duration of continuous gusts were the main factors affecting pollen dispersal. This information will be useful for determining the spatial isolation distances for hybrid seed production and for the commercial production of transgenic wheat.

[Effects of different planting patterns on farmland soil quality in Yellow River alluvial plain of Shandong Province].

Taking Chiping County in the Yellow River alluvial plain of Shandong Province as study area, a systematical survey was conducted on the 20 parameters of farmland soil physical and chemical properties under wheat/corn rotation, open air vegetable planting, sunlight greenhouse vegetable planting, and plastic shed vegetable planting, aimed to evaluate the effects of different planting patterns on the farmland soil quality in the plain. Significant differences (P < 0.05 or P < 0.01) were observed in the soil pH, soil moisture content, and the contents of soil organic matter, N, P, K, available S and Zn, and total salt under different planting patterns. The soil available P under all tested planting patterns and the soil available S under sunlight greenhouse vegetable planting presented a comparatively higher variability. Different planting patterns had significant effects on the soil quality, with the trend of protected vegetable planting > open air vegetable planting > wheat/ corn rotation. The effects were higher on soil chemical properties than on soil physical properties, and higher on soil organic matter and macronutrients than on soil secondary nutrients. Soil micronutrients were less affected. The main causes for these were the straw-returning of wheat and corn, the application of sulfur-containing and zinc fertilizers, and the long-term high rate fertilization of protected vegetable planting.

Genome-wide comparative analysis of the metalloprotease ftsH gene families between Arabidopsis thaliana and rice.

Filamentation temperature-sensitive H (FtsH) is an ATP-dependent metalloprotease in prokaryotes and eukaryotes. Homology-based analysis was applied to determine 12 ftsH genes in Arabidopsis genome and 9 members in rice genome. Distribution of these ftsH genes on each chromosome displayed a clear preference for some chromosomes such as chromosome 1, 2, 5 of Arabidopsis and chromosome 1,5 of rice. All 21 FtsH proteins were subcellularly targeted to chloroplast or mitochondria. These members could be phylogenetically assorted as eight groups, of which no ortholog of AtFtsH12 in rice was detected. Paralogs in each group shared similarity higher than 80% and orthologs higher than 70%. This strongly indicated that the members from single group were descended from a common ancestral gene. Four pairs of paralogs, AtftsH1/5, AtftsH2/8, AtftsH7/9 and AftsH3/10 were found in Arabidopsis genome. However, only two pairs of ftsH paralogs, OsftsH3/8 and OsftsH4/5, resided in rice genome. The highly homologous members in each group performed striking conservation of exon-intron boundaries and preference for the variable residues in function domains. By contrast, there was significant difference in base composition and sequence length of introns. The comparative analysis of the ftsH gene families of Arabidopsis and rice provided the basis for characteristic and function research of ftsH genes in other plants.

Constitutive expression of an endoplasmic reticulum small heat shock protein alleviates endoplasmic reticulum stress in transgenic tomato.

To explore the function of endoplasmic reticulum-located small heat-shock proteins (ER-sHSPs) in ER stress, a putative ER-sHSP cDNA (the driven protein was named as LeHSP21.5 with GenBank accession No. AB026983) was isolated from tomato (Lycopersicon esculentum). Fractionation of the crude microsomes by isopycnic sucrose-gradient centrifugation revealed that LeHSP21.5 was distributed on a density corresponding to the fractions with a higher activity of ER marker enzyme, suggesting the localization of LeHSP21.5 in the ER. Overexpressing LeHSP21.5 in transgenic tomato plants (L. esculentum var. Zhongshu 4) greatly attenuated the lethal effect of tunicamycin on tomato seedlings. Moreover, under the tunicamycin treatment, transcripts of BiP, PDI and calnexin in transgenic tomato plants accumulated to a less level than those in non-transgenic tomato plants. These results suggest that LeHSP21.5 can function to alleviate the tunicamycin-induced ER stress.

The involvement of chloroplast HSP100/ClpB in the acquired thermotolerance in tomato.

The chloroplast HSP100/ClpB is a newly documented member of the ClpB family, but little was known about its role in imparting thermotolerance to cells. A cDNA coding for a HSP100/ClpB homolog has been cloned from Lycopersicon esculentum and termed as Lehsp100/ClpB (the cDNA sequence of Lehsp100/ClpB has been submitted to the GenBank database under accession number: AB219939). The protein encoded by the cDNA was most similar to the putative chloroplast HSP100/ClpBs in higher plants and the ClpB from Cyanobacterium Synechococcus sp. A 97 kDa protein, which matched the predicted size of mature LeHSP100/ClpB, was immunologically detected in chloroplast isolated from heat-treated tomato plants. In addition, the fusion protein, combining the transit sequence of LeHSP100/ClpB and GFP, was found to be located in chloroplast based on the observations of fluorescent microscope images. These results indicated the chloroplast-localization of LeHSP100/ClpB. Both the transcript and the protein of Lehsp100/ClpB were not detected under normal growth conditions, but they were induced by increasingly higher temperatures. An antisense Lehsp100/ClpB cDNA fragment was introduced into the tomato by Agrobacterium-mediated transformation. Antisense lines exhibited an extreme repression of heat-induced expression of Lehsp100/ClpB. The levels of chloroplast HSP60 and small HSP in antisense lines were identical to those of the control plants. After plants preconditioned at 38 degrees C for 2 h were exposed to a lethal heat shock at 46 degrees C for 2 h, the antisense lines were greatly impaired and withered in 21 days of the recovery phase, whereas the untransformed control plants and the vector-transformed plants survived. Furthermore, chlorophyll fluorescence measurements showed that PS II in antisense lines were more susceptible to the thermal irreversible inactivation than the untransformed and vector-transformed control plants. This work provides the first example that induction of chloroplast LeHSP100/ClpB contributes to the acquisition of thermotolerance in higher plants.

[Molecular cloning of tomato LeHsp110/ClpB gene and its effect on the thermotolerance in plant].

The heat shock protein ClpB is a member of the Clp family and functions as molecular chaperones. ClpB is related to the acquired thermotolerance in organisms. A cDNA of 3144 bp was screened out of a tomato cDNA library. The polypeptide deduced from the longest ORF contains 980 amino acid residues, and was classified into HSP100/ClpB family based on the result of molecular phylogenesis analysis. Thus it was named as LeHSP110/ClpB according to its calculated molecular weight. LeHSP110/ClpB was characteristic of heat-inducibility but no constitutive expression, and was demonstrated to locate in chloroplastic stroma. An antisense cDNA fragment of LeHsp110/ClpB under the control of CaMV 35S promoter was introduced into tomato by Agrobacterium tumefactions-mediated method. At high temperature, the mRNA levels of LeHsp110/ClpB in antisense transgenic plants were lower than those in control plants. The PS II of transgenic plants is more sensitive to high temperature than that of control plants according to data of Fv/Fm. These results clearly showed that HSP110/ClpB plays an important role in thermotolerance of high plants.

Cloning and molecular characteristic of the metalloprotease gene LeftsH6 from tomato.

The full-length 2213-bp ftsH (filamentation temperature-sensitive H) cDNA was cloned from the cDNA library of heat-shocked tomato leaves. According to an open reading frame of 2019-bp, the deduced protein precursor was predicted to target chloroplast. The putative AAA (ATPases associated with diverse cellular activities) domain and the Zn(2+)-binding domain, characteristic of FtsH metalloproteases family, were found in the FtsH-like protein. Most similar to the FtsH6 of Arabidopsis thaliana, the tomato ftsH-like gene was named as Lycopersicon esculentum filamentation temperature-sensitive H6 (LeftsH6). Purified FtsH degraded casein but not BSA in vitro, whereas a FtsH mutant with the Glu(472) in the zinc-binding motif replaced by Gln had lost the protease activity. A single copy of LeftsH6 was detected in tomato genome by Southern blot analysis. Northern and Western blot analyses revealed consistently the heat-inducible character of the LeftsH6 gene. No LeftsH6 expression was detected after cold, salt, drought or light stress. The results provided the first experimental evidence of the existence of heat-inducible ftsH gene in higher plants.

Differential regulation of Lehsp23.8 in tomato plants: Analysis of a multiple stress-inducible promoter.

Small heat shock proteins (sHSPs) are the major family of HSP induced by heat stress in plants. In this report, an approximately 1.9kb of Lehsp23.8 5'-flanking sequence was isolated from tomato genome. By using the ß-glucuronidase (GUS) reporter gene system, the developmental and tissue specific expression of the gus gene controlled by the Lehsp23.8 promoter was characterized in transgenic tomato plants. Strong GUS staining was detected in the roots, leaves, flowers, fruits and germinated seeds after heat shock. The heat-induced GUS activity was different in the floral tissues at various developmental stages. Fluorometric GUS assay showed that the heat-induced GUS activity was higher in the pericarp than in the placenta, and it was the lowest in the locular gel. The heat-shock induction of the Lehsp23.8 promoter depended on the different stages of fruit development. The optimal heat-shock temperatures leading to the maximal GUS activity in the pericarp of green, breaker, pink and red fruits were 42, 36, 39 and 39°C, respectively. The heat-induced GUS activity in tomato fruits increased gradually within 48h of treatment and weakened during tomato fruit ripening. Obvious GUS activities under cold, exogenous ABA and heavy metal (Cd(2+), Cu(2+), Pb(2+) or Zn(2+)) stress conditions were also detected. These results show that the Lehsp23.8 promoter is characterized as strongly heat-inducible and multiple-stress responsive.