Effects of intracellular Ca2+ on developmental potential and ultrastructure of cryopreserved-warmed oocyte in mouse.

Maintaining appropriate intracellular calcium of oocytes is necessary to prevent ultrastructure and organelle damage caused by freezing and cryoprotectants. The present study aimed to investigate whether cryoprotectant-induced changes in the calcium concentrations of oocytes can be regulated to reduce damage to developmental potential and ultrastructure. A total of 33 mice and 1381 oocytes were used to explore the effects of intracellular calcium on the development and ultrastructures of oocytes subjected to 2-aminoethoxydiphenyl borate (2-APB) inhibition or thapsigargin (TG) stimulation. Results suggested that high levels intracellular calcium interfered with TG compromised oocyte survival (84.4 % vs. 93.4 %, p < 0.01) and blastocyst formation in fresh and cryopreservation oocytes (78.1 % vs. 86.4 %, and 60.5 % vs. 72.5 %, p < 0.05) compared with that of 2-APB pretreated oocytes in which Ca2+ was stabilized even though no differences in fertilization and cleavage was detected (p > 0.05). Examination by transmission electron microscopy indicated that the microvilli decreased and shortened, cortical granules considerably decreased in the cortex area, mitochondrial vesicles and vacuoles increased, and the proportion of vacuole mitochondria increased after oocytes were exposed to cryoprotectants. The cryopreservation-warming process deteriorated the negative effects on organelles of survival oocytes. By contrast, a low level of intracellular calcium mediated with 2-APB was supposed to contribute to the protection of organelles. These findings suggested oocyte injuries induced by cryoprotectants and low temperatures can be alleviated. More studies are necessary to confirm the relationship among Ca2+ concentration of the cytoplasm, ultrastructural injuries, and disrupted developmental potential in oocytes subjected to cryopreservation and warming.

Identification novel mutations and phenotypic spectrum expanding in PATL2 in infertile women with IVF/ICSI failure.

AIM: Abnormalities in oocyte maturation, fertilization, and early embryonic development are major causes of primary infertility in women who are undergoing IVF/ICSI attempts. Although many genetic factors responsible for these abnormal phenotypes have been identified, there are more additional pathogenic genes and variants yet to be discovered. Previous studies confirmed that bi-allelic PATL2 deficiency is an important factor for female infertility. In this study, 935 infertile patients with IVF/ICSI failure were selected for whole-exome sequencing, and 18 probands carrying PATL2 variants with a recessive inheritance pattern were identified. METHODS: We estimated that the prevalence contributed by PATL2 was 1.93% (18/935) in our study cohort. RESULTS: 15 novel variants were found in those families, including c.1093C > T, c.1609dupA, c.1204C > T, c.643dupG, c.877-2A > G, c.1228C > G, c.925G > A, c.958G > A, c.4A > G, c.1258T > C, c.1337G > A, c.1264dupA, c.88G > T, c.1065-2A > G, and c.1271T > C. The amino acids altered by the corresponding variants were highly conserved in mammals, and in silico analysis and 3D molecular modeling suggested that the PATL2 mutants impaired the physiologic function of the resulting proteins. Diverse clinical phenotypes, including oocyte maturation defect, fertilization failure, and early embryonic arrest might result from different variants of PATL2. CONCLUSIONS: These results expand the spectrum of PATL2 variants and provide an important reference for genetic counseling for female infertility, and they increase our understanding of the mechanisms of oocyte maturation arrest caused by PATL2 deficiency.

Homozygous Mutations in BTG4 Cause Zygotic Cleavage Failure and Female Infertility.

Zygotic cleavage failure (ZCF) is a unique early embryonic phenotype resulting in female infertility and recurrent failure of in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI). With this phenotype, morphologically normal oocytes can be retrieved and successfully fertilized, but they fail to undergo cleavage. Until now, whether this phenotype has a Mendelian inheritance pattern and which underlying genetic factors play a role in its development remained to be elucidated. B cell translocation gene 4 (BTG4) is a key adaptor of the CCR4-NOT deadenylase complex, which is involved in maternal mRNA decay in mice, but no human diseases caused by mutations in BTG4 have previously been reported. Here, we identified four homozygous mutations in BTG4 (GenBank: NM_017589.4) that are responsible for the phenotype of ZCF, and we found they followed a recessive inheritance pattern. Three of them-c.73C>T (p.Gln25Ter), c.1A>G (p.?), and c.475_478del (p.Ile159LeufsTer15)-resulted in complete loss of full-length BTG4 protein. For c.166G>A (p.Ala56Thr), although the protein level and distribution of mutant BTG4 was not altered in zygotes from affected individuals or in HeLa cells, the interaction between BTG4 and CNOT7 was abolished. In vivo studies further demonstrated that the process of maternal mRNA decay was disrupted in the zygotes of the affected individuals, which provides a mechanistic explanation for the phenotype of ZCF. Thus, we provide evidence that ZCF is a Mendelian phenotype resulting from mutations in BTG4. These findings contribute to our understanding of the role of BTG4 in human early embryonic development and provide a genetic marker for female infertility.

The extensin protein SAE1 plays a role in leaf senescence and is targeted by the ubiquitin ligase SINA4 in tomato.

Extensins are hydroxyproline-rich glycoproteins and generally play a structural role in cell wall integrity. In this study, we determined a novel role of tomato (Solanum lycopersicum) SENESCENCE-ASSOCIATED EXTENSIN1 (SAE1) in leaf senescence. Both gain- and loss-of-function analyses suggest that SAE1 plays a positive role in leaf senescence in tomato. Transgenic plants overexpressing SAE1 (SAE1-OX) exhibited premature leaf senescence and enhanced dark-induced senescence, whereas SAE1 knockout (SAE1-KO) plants displayed delayed development-dependent and dark-induced leaf senescence. Heterologous overexpression of SlSAE1 in Arabidopsis also led to premature leaf senescence and enhanced dark-induced senescence. In addition, the SAE1 protein was found to interact with the tomato ubiquitin ligase SlSINA4, and SlSINA4 promoted SAE1 degradation in a ligase-dependent manner when co-expressed in Nicotiana benthamiana leaves, suggesting that SlSINA4 controls SAE1 protein levels via the ubiquitin-proteasome pathway. Introduction of an SlSINA4-overexpression construct into the SAE1-OX tomato plants consistently completely eliminated accumulation of the SAE1 protein and suppressed the phenotypes conferred by overexpression of SAE1. Taken together, our results suggest that the tomato extensin SAE1 plays a positive role in leaf senescence and is regulated by the ubiquitin ligase SINA4.

Leptin Receptor Gln223Arg Polymorphism of Human Spermatozoa Associated with Male Infertility in a Chinese Population.

Background: Leptin (LEP) is believed to play a crucial role in male reproduction, while the molecular mechanisms through which LEP affects the male reproductive system are unclear. LEP acts by binding to a leptin receptor (LEPR) which mediates its physiological action, but there are only limited studies on the function of LEPR in human sperm. Purpose: This study aimed to determine the Gln223Arg polymorphisms of the LEPR gene in human spermatozoa and evaluate their possible relationship with semen variables. Methods: The study was performed on Chinese men: 115 healthy subjects and 108 patients with primary and 98 with secondary infertility. Semen samples were obtained from all patients, and semen variables were analyzed. The genotypic and allelic frequencies of Gln223Arg polymorphism in spermatozoa were determined by PCR and restriction fragment length polymorphism (RFLP) analyses. Statistical analyses were performed using the chi-square test, the Kruskal-Wallis test, and the Mann-Whitney test. Results: There were no significant differences in genotypic or allelic frequency distributions of Gln223Arg polymorphism among men with primary infertility, secondary infertility, and controls. Similarly, semen volume and sperm concentration did not differ with the different genotypes in all groups of men. The percentages of motile sperm for AA + AG genotypes in men with primary infertility (31.98%) were significantly lower than those in secondary infertility, and control men with GG genotypes were 34.41% and 59.36%, respectively. At the same time, the percentages of normal morphology sperm for AA + AG genotypes in men with primary infertility (2.93%) were significantly lower than those in secondary infertility and control men with GG genotypes 3.71% and 6.54%, respectively. Conclusion: This study reveals a possible association between the Gln223Arg polymorphism of the LEPR gene in spermatozoa affecting spermatozoal membrane integrity and having a direct role in sperm motility.

First report of Fusarium ussurianum causing leaf spot on Hemerocallis citrina in Sichuan Basin of China.

Hemerocallis citrina is a popular vegetable crop in China, due to abundant nutrients in its edible flower buds. In March 2021, serious symptoms of leaf spot were observed on nearly 90% cultivated H. citrina seedlings in the fields of Dazhou city (31°17'56″ N, 107°31'59″ E), Sichuan, China. Symptomatic leaves were collected from 15 seedlings in five different sampling sites (3 seedlings per site). Small pieces (5 × 3 mm) of lesion margin were excised, surface disinfected in 70% ethanol for 20 s and 1% sodium hypochlorite (NaClO) for 40 s, washed, dried, placed on potato dextrose agar (PDA) amended with streptomycin sulfate (50 mg/L) and incubated in dark at 25 ℃ for two days. Finally, eight purified isolates, HHC-FL22, HHC-FL23, HHC-FL25, HHC-FL26, HHC-FL27, HHC-FL28, HHC-FL29 and HHC-FL30, showing similar morphology were obtained through transferring hyphal tips to fresh PDA plates. On PDA plates, mycelia were initially white but gradually became light yellow, and scarlet diffusible pigments were also produced with time. On carnation leaf agar, our isolates produced slightly curved macroconidia with 4 to 8 septa that measured 3.1 to 5.7 × 36.8 to 69.3 µm (n = 30). Microconidia and chlamydospores were not observed. Our isolates were initially identified as Fusarium species based on morphological features (Leslie and Summerell 2006). To further confirm accurate identity, primers EF1/EF2 (O'Donnell et al. 2010), TRI1015B/TRI1013E (Hao et al. 2017), RPB1-F5/RPB1-G2R (O'Donnell et al. 2010), and fRPB2-5F/fRPB2-11aR and RPB2-5f2/RPB2-7cr (O'Donnell et al. 2012) were used to amplify gene sequences of translation elongation factor-1 alpha (TEF1), 3-O-acetyltransferase (Tri101), and DNA-directed RNA polymerase II largest (RPB1) and second largest subunit (RPB2), respectively. Our sequences were deposited in GenBank under accession numbers OQ860946 to OQ860953 (TEF1), OR393245 to OR393252 (Tri101), OP131893 to OP131900 (RPB1), and OQ860954 to OQ860961 and OP131885 to OP131892 (RPB2), respectively. BLASTN searches of our sequences showed 99 ~ 100% identity with TEF1 (FJ240301.1), Tri101 (FJ240345.1), RPB1 (MW233297.1) and RPB2 (KM361666.1) of F. ussurianum NRRL 45681, and 99.05 ~ 100% identity with TEF1 (FJ240305.1) and Tri101 (FJ240349.1) of F. ussurianum NRRL 45833, respectively. Two independent maximum-likelihood phylogenetic trees based on different combined datasets of TEF1, Tri101, RPB1 and RPB2 of Fusarium species confirmed that our isolates were F. ussurianum. To test pathogenicity, conidial suspension from HHC-FL23 (106 conidia / mL) were sprayed to seedlings of cultivar "chuanhuanghua No.1" (n = 3) and incubated in a greenhouse (25°C under 90% relative humidity, 16/8 h light/dark cycle). Controls were treated with ddH2O. Ten days post-inoculation, natural symptoms appeared on leaves inoculated with HHC-FL23, but control group seedlings remained disease-free. This experiment was repeated three times. All re-isolated pathogens from diseased leaves were molecularly and morphologically identified using methods described above. Consequently, the re-isolated fungi were identical to these inoculated. The leaf spot disease could cause foliar damage and even drastic yield loss of flower buds under severe conditions. To our knowledge, this is the first report of F. ussurianum causing leaf spot in H. citrina worldwide. Our study will assist in monitoring causal agent diversity of leaf spot and breeding new resistant varieties in H. citrina.

dCas9-BE3 and dCas12a-BE3 Systems Mediated Base Editing in Kiwifruit Canker Causal Agent Pseudomonas syringae pv. actinidiae.

Pseudomonas syringae pv. actinidiae (Psa) causes bacterial canker of kiwifruit with heavy economic losses. However, little is known about the pathogenic genes of Psa. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas-mediated genome editing technology has dramatically facilitated the characterization of gene function in various organisms. However, CRISPR genome editing could not be efficiently employed in Psa due to lacking homologous recombination repair. The base editor (BE) system, which depends on CRISPR/Cas, directly induces single nucleoside C to T without homology recombination repair. Here, we used dCas9-BE3 and dCas12a-BE3 systems to create substitutions of C to T and to convert CAG/CAA/CGA codons to stop codons (TAG/TAA/TGA) in Psa. The dCas9-BE3 system-induced single C-to-T conversion frequency of 3 to 10 base positions ranged from 0% to 100%, with a mean of 77%. The dCas12a-BE3 system-induced single C-to-T conversion frequency of 8 to 14 base positions in the spacer region ranged from 0% to 100%, with a mean of 76%. In addition, a relatively saturated Psa gene knockout system covering more than 95% of genes was developed based on dCas9-BE3 and dCas12a-BE3, which could knock out two or three genes at the same time in the Psa genome. We also found that hopF2 and hopAO2 were involved in the Psa virulence of kiwifruit. The HopF2 effector can potentially interact with proteins such as RIN, MKK5, and BAK1, while the HopAO2 effector can potentially interact with the EFR protein to reduce the host's immune response. In conclusion, for the first time, we established a PSA.AH.01 gene knockout library that may promote research on elucidating the gene function and pathogenesis of Psa.

Manipulation of the transcription factor SlNAC1 for improved tolerance to abiotic stress in tomato.

The tomato transcription factor SlNAC1 plays an important role in abiotic stress response and is fine-tuned at both transcriptional and posttranslational levels. The SlNAC1 gene is strongly induced by multiple abiotic stresses and the SlNAC1 protein is subjected to ubiquitin proteasome-mediated degradation. We found here that SlNAC1 possesses two distinct transactivation domains (TADs), TAD1 and TAD2. Significantly, the instability of SlNAC1 was attributed to the acidic amino acid-rich TAD1, in which the instability and transcriptional potential of TAD1 functionally overlapped; whereas the glutamine-rich TAD2 was stable and accounted for the abiotic stress signalling mediated by SlNAC1. Towards the goal of enhanced tolerance to abiotic stress in tomatoes, we manipulated SlNAC1 at both gene and protein levels: we generated a stable and functional SlNAC1 mutant SlNAC1∆191-270 by removing TAD1 and further engineered it to be stress-controllable by fusing the corresponding cDNA with the abiotic stress-inducible promoter ProStNAC1 . Transgenic tomato plants expressing the ProStNAC1 ::SlNAC1∆191-270 transgene did not display any undesired traits and exhibited enhanced tolerance to cold, drought and salt stresses. Taken together, our manipulation of the stress-related transcription factor via conditional expression of its derived stable and functional mutant provides a successful example for developing crops dynamically adapted to abiotic stress.

Genomic Variation and Host Interaction among Pseudomonas syringae pv. actinidiae Strains in Actinidia chinensis 'Hongyang'.

Kiwifruit bacterial canker is a recent epidemic disease caused by Pseudomonas syringae pv. actinidiae (Psa), which has undergone worldwide expansion in a short time and resulted in significant economic losses. 'Hongyang' (Actinidia chinensis), a widely grown cultivar because of its health-beneficial nutrients and appreciated red-centered inner pericarp, is highly sensitive to Psa. In this work, ten Psa strains were isolated from 'Hongyang' and sequenced for genome analysis. The results indicated divergences in pathogenicity and pathogenic-related genes among the Psa strains. Significantly, the interruption at the 596 bp of HrpR in two low-pathogenicity strains reemphasized this gene, expressing a transcriptional regulator for the effector secretion system, as an important pathogenicity-associated locus of Psa. The transcriptome analysis of 'Hongyang' infected with different Psa strains was performed by RNA-seq of stem tissues locally (at the inoculation site) and systemically. Psa infection re-programmed the host genes expression, and the susceptibility to Psa might be attributed to the down-regulation of several genes involved in plant-pathogen interactions, especially calcium signaling transduction, as well as fatty acid elongation. This suppression was found in both low- and high-pathogenicity Psa inoculated tissues, but the effect was stronger with more virulent strains. Taken together, the divergences of P. syringae pv. actinidiae in pathogenicity, genome, and resulting transcriptomic response of A. chinensis provide insights into unraveling the molecular mechanism of Psa-kiwifruit interactions and resistance improvement in the kiwifruit crop.

A MYB/bHLH complex regulates tissue-specific anthocyanin biosynthesis in the inner pericarp of red-centered kiwifruit Actinidia chinensis cv. Hongyang.

Many Actinidia cultivars are characterized by anthocyanin accumulation, specifically in the inner pericarp, but the underlying regulatory mechanism remains elusive. Here we report two interacting transcription factors, AcMYB123 and AcbHLH42, that regulate tissue-specific anthocyanin biosynthesis in the inner pericarp of Actinidia chinensis cv. Hongyang. Through transcriptome profiling analysis we identified five MYB and three bHLH transcription factors that were upregulated in the inner pericarp. We show that the combinatorial action of two of them, AcMYB123 and AcbHLH42, is required for activating promoters of AcANS and AcF3GT1 that encode the dedicated enzymes for anthocyanin biosynthesis. The presence of anthocyanin in the inner pericarp appears to be tightly associated with elevated expression of AcMYB123 and AcbHLH42. RNA interference repression of AcMYB123, AcbHLH42, AcF3GT1 and AcANS in 'Hongyang' fruits resulted in significantly reduced anthocyanin biosynthesis. Using both transient assays in Nicotiana tabacum leaves or Actinidia arguta fruits and stable transformation in Arabidopsis, we demonstrate that co-expression of AcMYB123 and AcbHLH42 is a prerequisite for anthocyanin production by activating transcription of AcF3GT1 and AcANS or the homologous genes. Phylogenetic analysis suggests that AcMYB123 or AcbHLH42 are closely related to TT2 or TT8, respectively, which determines proanthocyanidin biosynthesis in Arabidopsis, and to anthocyanin regulators in monocots rather than regulators in dicots. All these experimental results suggest that AcMYB123 and AcbHLH42 are the components involved in spatiotemporal regulation of anthocyanin biosynthesis specifically in the inner pericarp of kiwifruit.

A novel BSD domain-containing transcription factor controls vegetative growth, leaf senescence, and fruit quality in tomato.

BSD (mammalian BTF2-like transcription factors, synapse-associated proteins, and DOS2-like proteins) is a conserved domain that exists in a variety of organisms, but its function has not been well studied. Here, we identified a novel BSD domain-containing protein (SlBSD1) in tomato (Solanum lycopersicum). Biochemical and microscopy assays indicated that SlBSD1 is a functional transcription factor that is predominantly localized in the nucleus. Loss-of-function and overexpression analyses suggested that SlBSD1 is a novel regulator of vegetative growth and leaf senescence in tomato. SlBSD1-knockdown (-KD) plants exhibited retarded vegetative growth and precocious leaf senescence, whereas SlBSD1-overexpression (-OX) plants displayed the opposite phenotypes. The negative role of SlBSD1 in leaf senescence was also supported by RNA-seq analysis comparing leaf tissues from SlBSD1-KD and wild-type plants. In addition, contents of soluble solids were altered in fruits in the SlBSD1-KD and SlBSD1-OX plants. Taken together, our data suggest that the novel transcription factor SlBSD1 plays important roles in controlling fruit quality and other physiological processes in tomato, including vegetative growth and leaf senescence.

Is day 7 culture necessary for in vitro fertilization of cryopreserved/warmed human oocytes?

BACKGROUND: Human embryos are usually cultured to blastocyst stage by Day 5 or 6 after insemination. However, some embryos grow slowly and reach blastocyst stage at Day 7. Acceptable live birth rates have been reported after transfer of Day 7 blastocysts resulted from fresh oocyte in vitro fertilization (IVF). It is unknown whether an extended embryo culture to Day 7 is necessary for cryopreserved oocyte IVF to obtain more transferrable blastocysts. METHODS: In this study, 455 oocytes from 57 cycles were warmed, inseminated, and the resulting embryos were cultured by Day 7 to examine blastocyst development after extended culture. Fifty one blastocysts from 16 cycles were biopsied to examine embryo aneuploidies. RESULTS: It was found that 35.1% of the cycles had Day 7 blastocysts, and 3.5% of the cycles had only Day 7 blastocysts. Day 7 blastocysts accounted for 15.6% of total blastocysts. The proportion of top quality of blastocysts was lower at Day 7 than at Day 5 or 6. However, no differences were observed on aneuploid blastocyst rates among Days 5, 6 and 7. Similar clinical pregnancy, ongoing pregnancy and embryo implantation rates were obtained after Day 7 blastocyst transfer as compared with Day 5 or 6 blastocyst transfer. CONCLUSION: These results indicate that embryos from oocyte warming cycles should be cultured to Day 7 if they do not reach to blastocyst stage by Day 6 so that number of usable blastocysts can be increased.

Functional analysis of the seven in absentia ubiquitin ligase family in tomato.

Seven in absentia (SINA) protein is one subgroup of ubiquitin ligases possessing an N-terminal cysteine-rich really interesting new gene (RING) domain, two zinc-finger motifs, and a C-terminal domain responsible for substrate-binding and dimerization. In tomato (Solanum lycopersicum), the SINA gene family has six members, and we characterize in this study all tomato SINA (SlSINA) genes and the gene products. Our results show that SlSINA genes are differentially regulated in leaf, bud, stem, flower, and root. All SlSINA proteins possess RING-dependent E3 ubiquitin ligase activity, exhibiting similar specificity towards the E2 ubiquitin-conjugating enzyme. SlSINA1/3/4/5/6 are localized in both cytoplasm and nucleus, whereas SlSINA2 is exclusively localized in the nucleus. Moreover, all SlSINAs can interact with each other for homo- or hetero-dimerization. The functionality of SlSINA proteins has been investigated. SlSINA4 plays a positive role in defense signalling, as manifested by elicitation of E3-dependent hypersensitive response-like cell death; the other SlSINAs are negative regulator and capable to suppress hypersensitive response cell death. Transgenic tomato plants overexpressing SlSINA2 exhibit pale-green leaf phenotype, suggesting SlSINA2 regulates chlorophyll level in plant cells, whereas transgenic tomato plants overexpressing SlSINA5 have altered floral structure with exserted stigma, implicating SlSINA5 plays a role in flower development.

Potential use of multispectral imaging technology to identify moisture content and water-holding capacity in cooked pork sausages.

BACKGROUND: The traditional detection methods for moisture content (MC) and water-holding capacity (WHC) in cooked pork sausages (CPS) are destructive, time consuming, require skilled personnel and are not suitable for online industry applications. The goal of this work was to explore the potential of multispectral imaging (MSI) in combination with multivariate analysis for the identification of MC and WHC in CPS. RESULTS: Spectra and textures of 156 CPS treated by six salt concentrations (0-2.5%) were analyzed using different calibration models to find the most optimal results of predicting MC and WHC in CPS. By using the fused data of spectra and textures, partial least squares regression models performed well for determining the MC and WHC, with a correlation coefficient (r) of 0.949 and 0.832, respectively. Additionally, their spatial distribution in CPS could be visualized via applying prediction equations to transfer each pixel in the image. CONCLUSION: Results of satisfactory detection and visualization of the MC and WHC showed that MSI has the potential to serve as a rapid and non-destructive method for use in sausage industry. © 2017 Society of Chemical Industry.

The ubiquitin ligase SEVEN IN ABSENTIA (SINA) ubiquitinates a defense-related NAC transcription factor and is involved in defense signaling.

We recently identified a defense-related tomato (Solanum lycopersicum) NAC (NAM, ATAF1,2, CUC2) transcription factor, NAC1, that is subjected to ubiquitin-proteasome system-dependent degradation in plant cells. In this study, we identified a tomato ubiquitin ligase (termed SEVEN IN ABSENTIA3; SINA3) that ubiquitinates NAC1, promoting its degradation. We conducted coimmunoprecipitation and bimolecular fluorescence complementation to determine that SINA3 specifically interacts with the NAC1 transcription factor in the nucleus. Moreover, we found that SINA3 ubiquitinates NAC1 in vitro and promotes NAC1 degradation via polyubiquitination in vivo, indicating that SINA3 is a ubiquitin ligase that ubiquitinates NAC1, promoting its degradation. Our real-time PCR analysis indicated that, in contrast to our previous finding that NAC1 mRNA abundance increases upon Pseudomonas infection, the SINA3 mRNA abundance decreases in response to Pseudomonas infection. Moreover, using Agrobacterium-mediated transient expression, we found that overexpression of SINA3 interferes with the hypersensitive response cell death triggered by multiple plant resistance proteins. These results suggest that SINA3 ubiquitinates a defense-related NAC transcription factor for degradation and plays a negative role in defense signaling.

Ubiquitin-conjugated degradation of golden 2-like transcription factor is mediated by CUL4-DDB1-based E3 ligase complex in tomato.

CULLIN4-RING ubiquitin ligases (CRL4s) as well as their targets are fundamental regulators functioning in many key developmental and stress responses in eukaryotes. In tomato (Solanum lycopersicum), molecular cloning has revealed that the underlying genes of natural spontaneous mutations high pigment 1 (hp1), high pigment 2 (hp2) and uniform ripening (u) encode UV-DAMAGED DNA BINDING PROTEIN 1 (DDB1), DE-ETIOLATED 1 (DET1) and GOLDEN 2-LIKE (GLK2), respectively. However, the molecular basis of the opposite actions of tomato GLK2 vs CUL4-DDB1-DET1 complex on regulating plastid level and fruit quality remains unknown. Here, we provide molecular evidence showing that the tomato GLK2 protein is a substrate of the CUL4-DDB1-DET1 ubiquitin ligase complex for the proteasome degradation. SlGLK2 is degraded by the ubiquitin-proteasome system, which is mainly determined by two lysine residues (K11 and K253). SlGLK2 associates with the CUL4-DDB1-DET1 E3 complex in plant cells. Genetically impairing CUL4, DDB1 or DET1 results in a retardation of SlGLK2 degradation by the 26S proteasome. These findings are relevant to the potential of nutrient accumulation in tomato fruit by mediating the plastid level and contribute to a deeper understanding of an important regulatory loop, linking protein turnover to gene regulation.

Down-regulation of a LBD-like gene, OsIG1, leads to occurrence of unusual double ovules and developmental abnormalities of various floral organs and megagametophyte in rice.

The indeterminate gametophyte1 (ig1) mutation was first characterized to modulate female gametophyte development in maize (Zea mays). However, the function of its rice orthologue, OsIG1, remains unknown. For this, we first analysed OsIG1 localization from differential tissues in rice. Real-time quantitative PCR (qRT-PCR) and histochemical staining results demonstrated that the expression signal of OsIG1 was strongly detected in young inflorescence, moderately in mature flower and weakly in leaf. Furthermore, RNA in situ hybridization analyses exhibited that OsIG1 was strongly expressed in inflorescence meristems, floral meristems, empty-glume- and floret- primordia, especially in the primordia of stamens and immature ovules, and the micropylar side of the mature ovary. In OsIG1-RNAi lines, wrinkled blade formation was accompanied by increased leaf inclination angle. Cross-section further showed that the number of bulliform cells located between the vasculatures was significantly increased, indicating that OsIG1 is involved in division and differentiation of bulliform cell and lateral growth during leaf development. OsIG1-RNAi suppression lines showed pleiotropic phenotypes, including degenerated palea, glume-like features and open hull. In addition, a single OsIG1-RNAi floret is characterized by frequently developing double ovules with abnormal embryo sac development. Additionally, down-regulation of OsIG1 differentially affected the expression of genes associated with the floral organ development including EG1, OsMADS6 and OsMADS1. Taken together, these results demonstrate that OsIG1 plays an essential role in the regulation of empty-glume identity, floral organ number control and female gametophyte development in rice.

Functional characterization of two alternatively spliced transcripts of tomato ABSCISIC ACID INSENSITIVE3 (ABI3) gene.

Alternative splicing can produce transcripts that encode proteins with altered functions. The transcripts of the ABSCISIC ACID INSENSITIVE3 (ABI3)/VIVIPAROUS1 (VP1) gene, which is an important component in abscisic acid (ABA) signaling, are subjected to alternative splicing in both monocotyledons and dicotyledons. We identified two alternatively spliced tomato (Solanum lycopersicum) SlABI3 transcripts, SlABI3-F and SlABI3-T, which encode the nucleus-localized full-length and truncated proteins, respectively. The tissue-specific accumulation of SlABI3-F and SlABI3-T was determined, particularly in seeds at different developmental stages and in response to phytohormonal and abiotic stress. Ectopic over-expression of SlABI3-F and SlABI3-T resulted in the induction of seed-specific genes SlSOM, SlEM1 and SlEM6 in vegetative tissues. However, over-expression of SlABI3-F, but not SlABI3-T, activated expression of the downstream gene SlABI5 and conferred hypersensitivity to exogenous ABA during seed germination and primary root growth. In addition, the SlABI3-F protein interacted with SlABI5 much stronger than SlABI3-T did in the yeast two-hybrid assay. These results suggest that SlABI3-F and SlABI3-T have similar and distinct functionality in the ABA signaling, dependent on which tissue/organ they accumulate in.

Whole transcriptome sequencing reveals genes involved in plastid/chloroplast division and development are regulated by the HP1/DDB1 at an early stage of tomato fruit development.

The phenotype of tomato high pigment-1 (hp1) mutant is characterized by overproduction of pigments including chlorophyll and carotenoids during fruit development and ripening. Although the increased plastid compartment size has been thought to largely attribute to the enhanced pigmentation, the molecular aspects of how the HP1/DDB1 gene manipulates plastid biogenesis and development are largely unknown. In the present study, we compared transcriptome profiles of immature fruit pericarp tissue between tomato cv. Ailsa Craig (WT) and its isogenic hp1 mutant. Over 20 million sequence reads, representing > 1.6 Gb sequence data per sample, were generated and assembled into 21,972 and 22,167 gene models in WT and hp1, respectively, accounting for over 60 % official gene models in both samples. Subsequent analyses revealed that 8,322 and 7,989 alternative splicing events, 8833 or 8510 extended 5'-UTRs, 8,263 or 8,939 extended 3'-UTRs, and 1,136 and 1,133 novel transcripts, exist in WT and hp1, respectively. Significant differences in expression level of 880 genes were detected between the WT and hp1, many of which are involved in signaling transduction, transcription regulation and biotic and abiotic stresses response. Distinctly, RNA-seq datasets, quantitative RT-PCR analyses demonstrate that, in hp1 mutant pericarp tissue at early developmental stage, an apparent expression alteration was found in several regulators directly involved in plastid division and development. These results provide a useful reference for a more accurate and more detailed characterization of the molecular process in the development and pigmentation of tomato fruits.

SlNAC1, a stress-related transcription factor, is fine-tuned on both the transcriptional and the post-translational level.

The plant-specific NAC (NAM, ATAF1,2, CUC2) transcription factors play significant roles in diverse physiological processes. In this study, we determined the regulation of a stress-related tomato (Solanum lycopersicum) NAC1 (SlNAC1) transcription factor at both the transcriptional and the post-translational level. The SlNAC1 protein was found to be stable in the presence of proteasome-specific inhibitor MG132 or MG115 and ubiquitinated in plant cells, suggesting that the SlNAC1 is subject to the ubiquitin-proteasome system-mediated degradation. Deletion analysis identified a short segment of 10 amino acids (aa261-270) that was required for ubiquitin-proteasome system-mediated degradation, among which two leucine residues (L268 and L269) were critical for the protein instability of SlNAC1. Fusion of the degron (SlNAC1(191-270) ) containing these 10 amino acids to green fluorescent protein was found to be sufficient to trigger the degradation of the fusion protein. In addition, the SlNAC1 gene is strongly upregulated during Pseudomonas infection, while repression of the NAC1 ortholog in Nicotiana benthamiana resulted in enhanced susceptibility to Pseudomonas bacteria. These results suggest that rapid upregulation of the NAC1 gene resulting in more protein production is likely one of the strategies plants use to defend themselves against pathogen infection.