Persulfidation and phosphorylation of transcription factor SlWRKY6 differentially regulate tomato fruit ripening.

Cysteine desulfhydrase catalyses the generation of the signaling molecule hydrogen sulfide (H2S) in plants. In this study, we found that H2S can inhibit tomato (Solanum lycopersicum) fruit ripening and SlWRKY6 undergoes differential protein persulfidation in SlLCD1-overexpressing leaves. Then, further study indicated that SlWRKY6 could be persulfidated by H2S at Cys396. By construction of slwrky6 mutants and SlWRKY6-OE lines, we found that SlWRKY6 positively regulates leaf senescence and fruit ripening by activating the transcription of ripening-related genes STAYGREEN 1 (SlSGR1) and Senescence-Associated Gene 12 (SlSAG12). In addition, SlWRKY6 interacted with kinase SlMAPK4 and was phosphorylated at Ser33. Dual-luciferase transient expression assays and electrophoretic mobility shift assays indicated that SlWRKY6 persulfidation attenuated its transcriptional regulation of target genes SlSGR1 and SlSAG12, whereas SlWRKY6 phosphorylation by SlMAPK4 activated the transcription of target genes to promote fruit ripening. Moreover, we provided evidence that SlWRKY6 persulfidation attenuated its SlMAPK4-mediated phosphorylation to inhibit tomato fruit ripening. By transient expression of SlWRKY6, SlWRKY6C396A, SlWRKY6S33A, and SlWRKY6S33D in slwrky6 fruits, we found that SlWRKY6 persulfidation attenuated the expression of SlSGR1 and SlSAG12 thereby delaying tomato fruit ripening, while SlWRKY6 phosphorylation increased the expression of target genes. As tomato fruits ripened, endogenous H2S production decreased, while SlMAPK4 expression increased. Therefore, our findings reveal a model in which SlWRKY6 persulfidation due to higher endogenous H2S levels in un-ripened fruit inhibits its ability to activate SlSGR1 and SlSAG12 expression, while SlWRKY6 phosphorylation by SlMAPK4 activates its transcriptional activity, thereby promoting tomato fruit ripening.

ETHYLENE RESPONSE FACTORS 4.1/4.2 with an EAR motif repress anthocyanin biosynthesis in red-skinned pears.

Red-skinned pears (Pyrus L.) are preferred to consumers for their attractive color and abundant anthocyanins. Pyrus ETHYLENE RESPONSE FACTOR 3 (PyERF3) positively regulates anthocyanin biosynthesis through interacting with Pyrus myeloblastosis family 114 (PyMYB114) and Pyrus basic helix-loop-helix 3 (PybHLH3) in red-skinned pears. However, the role of APETALA2/ethylene response factors (AP2/ERFs), which negatively regulate anthocyanin biosynthesis, remains unclear in red-skinned pears. Here, we validated that 2 AP2/ERFs, PyERF4.1 and PyERF4.2, screened from the transcriptome data of 'Starkrimson' pear (Pyrus communis L.) and its green mutant, inhibit anthocyanin biosynthesis in transgenic pear calli, as well as in overexpression and gene-edited tomato (Solanum lycopersicum) fruits. Meanwhile, the co-transformation of PyERF4.1/PyERF4.2 with PyERF3-PyMYB114-PybHLH3 inhibited anthocyanin biosynthesis in pear fruits and strawberry (Fragaria vesca) receptacles. Further assays showed that PyMYB114 activated the transcription of PyERF4.1/PyERF4.2; PyERF4.1/PyERF4.2 then interacted with PyERF3 to affect the stability of the PyERF3-PyMYB114-PybHLH3 complex, thereby inhibiting the transcription of the anthocyanin biosynthesis gene Pyrus anthocyanidin synthase (PyANS). Furthermore, deletion of the ERF-associated-amphiphilic repression (EAR) motif eliminated the inhibitory effect of PyERF4.1/PyERF4.2 on anthocyanin biosynthesis, and a mutation of the PyERF4.2-EAR motif (LxLxM to LxLxL) strengthened the inhibitory effect, demonstrating that the EAR motif is indispensable for the inhibitory effect of PyERF4.1/PyERF4.2 on anthocyanin biosynthesis in pears. Our study has shed light on a feedback regulatory loop mechanism that balances the excessive accumulation of anthocyanins in red-skinned pears, providing insights into the regulatory mechanism of anthocyanin biosynthesis and the regulatory network of coloration in red-skinned pears.

Persulfidation of transcription factor MYB10 inhibits anthocyanin synthesis in red-skinned pear.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that delays color change during fruit ripening. Whether H2S affects anthocyanin biosynthesis in red-skinned pears (Pyrus L.) remains unclear. Here, we found that H2S substantially inhibits anthocyanin accumulation in red-skinned pears and the expression of several genes encoding transcription factors is affected in response to H2S signaling. For example, PyMYB10 and PyMYB73 were down-regulated, whereas PyMYB114 and PyMYB6 were up-regulated. Bioinformatics analysis showed that PyMYB73 and PyMYB6, each containing an EAR motif, may negatively regulate anthocyanin accumulation. Transient expression analysis showed that PyMYB73 substantially promotes anthocyanin biosynthesis by co-transforming with PyMYB10/PyMYB114 + PybHLH3; however, PyMYB6 inhibited anthocyanin biosynthesis in strawberry (Fragaria vesca) receptacles and pear fruits, and PyMYB73 interacted with PyMYB10 and PyMYB6 but not PyMYB114 or PybHLH3. Further investigation showed that Cys194 and Cys218 of PyMYB10 were modified by persulfidation and that PyMYB10Cys218Ala substantially increased anthocyanin accumulation by a transient transformation system. Co-transformation of PyMYB10Cys218Ala + PyMYB73/PyMYB6 also promoted anthocyanin accumulation in pear fruits. Yeast two-hybrid assays showed that the mutation of PyMYB10 did not affect the interaction between PyMYB10 and PyMYB73, but it inhibited interaction with PyMYB6. Moreover, H2S weakened the interaction between PyMYB10 and PyMYB73 but enhanced the interaction with PyMYB6. Thus, we provided a model in which PyMYB10 undergoes persulfidation at Cys218, enhancing the interaction with PyMYB6 and reducing the interaction with PyMYB73. These subsequently results in lower expression of the anthocyanin biosynthesis-related genes Pyrus dihydroflavonol 4-reductase (PyDFR), Pyrus anthocyanidin synthase (PyANS), Pyrus UDP-glucose: flavonoid 3-glucosyl transferase (PyUFGT) and Pyrus glutathione S-transferase (PyGST), thereby inhibiting anthocyanin accumulation in red-skinned pears. Our findings provided a molecular mechanism for H2S-mediated anthocyanin biosynthesis in red-skinned pears.

E3 ligase BRG3 persulfidation delays tomato ripening by reducing ubiquitination of the repressor WRKY71.

Hydrogen sulfide (H2S) is a gaseous signaling molecule reported to play multiple roles in fruit ripening. However, the molecular mechanisms underlying H2S-mediated delay in fruit ripening remain to be established. Here, the gene encoding a WRKY transcription factor, WRKY71, was identified as substantially upregulated in H2S-treated tomato (Solanum lycopersicum) via transcriptome profiling. The expression of WRKY71 was negatively associated with that of CYANOALANINE SYNTHASE1 (CAS1). Transient and stable genetic modification experiments disclosed that WRKY71 acts as a repressor of the tomato ripening process. CAS1 appears to play an opposite role, based on the finding that the ripening process was delayed in the cas1 mutant and accelerated in CAS1-OE tomatoes. Dual-luciferase reporter assay, yeast one-hybrid, electrophoretic mobility shift assay, and transient transformation experiments showed that WRKY71 bound to the CAS1 promoter and suppressed its activation. Moreover, the persulfidation of WRKY71 enhanced its binding ability to the CAS1 promoter. Data from luciferase complementation and Y2H assays confirmed that WRKY71 interacts with a BOI-related E3 ubiquitin-protein ligase 3 (BRG3) and is ubiquitinated in vitro. Further experiments showed that modification of BRG3 via persulfidation at Cys206 and Cys212 led to reduced ubiquitination activity. Our findings support a model whereby BRG3 undergoes persulfidation at Cys206 and Cys212, leading to reduced ubiquitination activity and decreased interactions with the WRKY71 transcript, with a subsequent increase in binding activity of the persulfidated WRKY71 to the CAS1 promoter, resulting in its transcriptional inhibition and thereby delayed ripening of tomatoes. Our collective findings provide insights into a mechanism of H2S-mediated regulation of tomato fruit ripening.

SlERF109-like and SlNAC1 Coordinately Regulated Tomato Ripening by Inhibiting ACO1 Transcription.

As a typical climacteric fruit, tomato (Solanum lycopersicum) is widely used for studying the ripening process. The negative regulation of tomato fruits by transcription factor SlNAC1 has been reported, but its regulatory network was unclear. In the present study, we screened a transcription factor, SlERF109-like, and found it had a stronger relationship with SlNAC1 at the early stage of tomato fruit development through the use of transcriptome data, RT-qPCR, and correlation analysis. We inferred that SlERF109-like could interact with SlNAC1 to become a regulatory complex that co-regulates the tomato fruit ripening process. Results of transient silencing (VIGS) and transient overexpression showed that SlERF109-like and SlNAC1 could regulate chlorophyll degradation-related genes (NYC1, PAO, PPH, SGR1), carotenoids accumulation-related genes (PSY1, PDS, ZDS), ETH-related genes (ACO1, E4, E8), and cell wall metabolism-related genes expression levels (CEL2, EXP, PG, TBG4, XTH5) to inhibit tomato fruit ripening. A dual-luciferase reporter and yeast one-hybrid (Y1H) showed that SlNAC1 could bind to the SlACO1 promoter, but SlERF109-like could not. Furthermore, SlERF109-like could interact with SlNAC1 to increase the transcription for ACO1 by a yeast two-hybrid (Y2H) assay, a luciferase complementation assay, and a dual-luciferase reporter. A correlation analysis showed that SlERF109-like and SlNAC1 were positively correlated with chlorophyll contents, and negatively correlated with carotenoid content and ripening-related genes. Thus, we provide a model in which SlERF109-like could interact with SlNAC1 to become a regulatory complex that negatively regulates the tomato ripening process by inhibiting SlACO1 expression. Our study provided a new regulatory network of tomato fruit ripening and effectively reduced the waste of resources.

Comparative physiological and transcriptomic analysis reveal MdWRKY75 associated with sucrose accumulation in postharvest 'Honeycrisp' apples with bitter pit.

BACKGROUND: Calcium (Ca) deficiency can cause apple bitter pit, reduce the quality and shelf life. WRKY transcription factors play essential role in plant response to multiple disorders. However, the underlying mechanisms causing bitter pit in apple fruit due to Ca deficiency during storage is extremely limited. RESULTS: In the present study, the nutritional metabolites and reactive oxygen species (ROS) were compared in Ca-deficient and healthy apple fruit (CK) during storage. Results showed that Ca-deficient apples sustained significantly higher production of ROS, PPO activity, flavonoids, total phenol, total soluble solids (TSS), and sucrose contents, but the contents of Ca, H2O2, titratable acids (TA), glucose and fructose were significantly lower than those of CK during storage. Principal component analysis (PCA) showed that TSS, •O2-, PPO, malondialdehyde (MDA) and Ca were the main factors, and TSS had a positive correlation with sucrose. Furthermore, transcriptome analysis revealed that WRKYs were co-expressed with sucrose metabolism-related enzymes (SWEETs, SS, SPS). qRT-PCR and correlation analysis indicated that MdWRKY75 was correlated positively with MdSWEET1. Moreover, transient overexpression of MdWRKY75 could significantly increase the sucrose content and promote the expression of MdSWEET1 in apple fruit. CONCLUSIONS: Calcium deficiency could decrease antioxidant capacity, accelerate nutritional metabolism and up-regulate the expression of WRKYs in apple with bitter pit. Overexpression of MdWRKY75 significantly increased sucrose accumulation and the expression of MdSWEET1. These findings further strengthened knowledge of the basic molecular mechanisms in calcium deficiency apple flesh and contributed to improving the nutritional quality of apple fruit.

A Hydrogen-Sulfide-Repressed Methionine Synthase SlMS1 Acts as a Positive Regulator for Fruit Ripening in Tomato.

Ethylene is a key phytohormone that regulates the ripening of climacteric fruits, and methionine is an indirect precursor of ethylene. However, whether methionine synthase plays a role in fruit ripening in Solanum lycopersicum (tomato) is still unknown. In this study, we find that a tomato methionine synthase (named SlMS1), which could be repressed at the transcriptional level by hydrogen sulfide (H2S), acts as a positive regulator for tomato fruit ripening. By a bioinformatics analysis, it is found that SlMS1 and SlMS2 in tomato are highly homologous to methionine synthases in Arabidopsis thaliana. The expression pattern of SlMS1 and SlMS2 is analyzed in tomato, and SlMS1 expression is up-regulated during fruit ripening, suggesting its potential role in regulating fruit ripening. A potential bipartite nuclear localization signal is found in the amino acid sequence of SlMS1; thus, SlMS1 is tagged with GFP and observed in the leaves of Nicotiana benthamiana. Consistently, SlMS1-GFP shows strong nuclear localization and also cytoplasmic localization. The role of SlMS1 in regulating fruit ripening is investigated in tomato fruit by transient silencing (virus-induced gene silencing, VIGS) and transient overexpression. The results show that SlMS1 silencing causes delayed fruit ripening, evidenced by more chlorophyll and less carotenoid accumulation, while SlMS1 overexpression accelerates fruit ripening significantly compared with control. Further investigation shows that SlMS1 overexpression could up-regulate the expression of carotenoid-synthesis-related genes (PSY1, PDS, ZDS), chlorophyll-degradation-related genes (NYC1, PAO, PPH, SGR1), cell-wall-metabolism-related genes (CEL2, EXP, PG, TBG4, XTH5) and ethylene-synthesis-pathway-related genes (ACO1, ACO3, ACS2), while SlMS1 silencing causes the opposite results. The correlation analysis indicates that SlMS1 expression is negatively correlated with chlorophyll content and positively correlated with carotenoid and ripening-related gene expressions. Taken together, our data suggest that SlMS1 is a positive regulator of tomato fruit ripening and a possible target gene for the ripening-delaying effect of H2S.

IbERF71, with IbMYB340 and IbbHLH2, coregulates anthocyanin accumulation by binding to the IbANS1 promoter in purple-fleshed sweet potato (Ipomoea batatas L.).

KEY MESSAGE: The transcription factor (TF) IbERF71 forms a novel complex, IbERF71-IbMYB340-IbbHLH2, to coregulate anthocyanin biosynthesis by binding to the IbANS1 promoter in purple-fleshed sweet potatoes. Purple-fleshed sweet potato (Ipomoea batatas L.) is very popular because of its abundant anthocyanins, which are natural pigments with multiple physiological functions. TFs involved in regulating anthocyanin biosynthesis have been identified in many plants. However, the molecular mechanism of anthocyanin biosynthesis in purple-fleshed sweet potatoes has rarely been examined. In this study, TF IbERF71 and its partners were screened by bioinformatics and RT-qPCR analysis. The results showed that the expression levels of IbERF71 and partners IbMYB340 and IbbHLH2 were higher in purple-fleshed sweet potatoes than in other colors and that the expression levels positively correlated with anthocyanin contents. Moreover, transient expression assays showed that cotransformation of IbMYB340+IbbHLH2 resulted in anthocyanin accumulation in tobacco leaves and strawberry receptacles, and additional IbERF71 significantly increased visual aspects. Furthermore, the combination of the three TFs significantly increased the expression levels of FvANS and FvGST, which are involved in anthocyanin biosynthesis and transport of strawberry receptacles. The dual-luciferase reporter system verified that cotransformation of the three TFs enhanced the transcription activity of IbANS1. In addition, yeast two-hybrid and firefly luciferase complementation assays revealed that IbMYB340 interacted with IbbHLH2 and IbERF71 but IbERF71 could not interact with IbbHLH2 in vitro. In summary, our findings provide novel evidence that IbERF71 and IbMYB340-IbbHLH2 form the regulatory complex IbERF71-IbMYB340-IbbHLH2 that coregulates anthocyanin accumulation by binding to the IbANS1 promoter in purple-fleshed sweet potatoes. Thus, the present study provides a new regulatory network of anthocyanin biosynthesis and strong insight into the color development of purple-fleshed sweet potatoes.

Roles of a Cysteine Desulfhydrase LCD1 in Regulating Leaf Senescence in Tomato.

Hydrogen sulfide (H2S), a novel gasotransmitter in both mammals and plants, plays important roles in plant development and stress responses. Leaf senescence represents the final stage of leaf development. The role of H2S-producing enzyme L-cysteine desulfhydrase in regulating tomato leaf senescence is still unknown. In the present study, the effect of an L-cysteine desulfhydrase LCD1 on leaf senescence in tomato was explored by physiological analysis. LCD1 mutation caused earlier leaf senescence, whereas LCD1 overexpression significantly delayed leaf senescence compared with the wild type in 10-week tomato seedlings. Moreover, LCD1 overexpression was found to delay dark-induced senescence in detached tomato leaves, and the lcd1 mutant showed accelerated senescence. An increasing trend of H2S production was observed in leaves during storage in darkness, while LCD1 deletion reduced H2S production and LCD1 overexpression produced more H2S compared with the wild-type control. Further investigations showed that LCD1 overexpression delayed dark-triggered chlorophyll degradation and reactive oxygen species (ROS) accumulation in detached tomato leaves, and the increase in the expression of chlorophyll degradation genes NYC1, PAO, PPH, SGR1, and senescence-associated genes (SAGs) during senescence was attenuated by LCD1 overexpression, whereas lcd1 mutants showed enhanced senescence-related parameters. Moreover, a correlation analysis indicated that chlorophyll content was negatively correlated with H2O2 and malondialdehyde (MDA) content, and also negatively correlated with the expression of chlorophyll degradation-related genes and SAGs. Therefore, these findings increase our understanding of the physiological functions of the H2S-generating enzyme LCD1 in regulating leaf senescence in tomato.

Transcriptomics Reveals the ERF2-bHLH2-CML5 Module Responses to H2S and ROS in Postharvest Calcium Deficiency Apples.

Calcium deficiency usually causes accelerated quality deterioration in postharvest fruit, whereas the underlining mechanism is still unclear. Here, we report that calcium deficiency induced the development of bitter pit on the surface of apple peels compared with the healthy appearance in control apples during postharvest storage. Physiological analysis indicates that calcium-deficient peels contained higher levels of superoxide anion (O2•-), malondialdehyde (MDA), total phenol, flavonoid contents and polyphenol oxidase (PPO) activity, and reduced calcium, H2S production, anthocyanin, soluble protein content, and peroxidase (POD) activity compared with those in calcium-sufficient peels. The principal component analysis (PCA) results show that calcium content, ROS, and H2S production were the main factors between calcium-deficient and calcium-sufficient apple peels. Transcriptome data indicated that four calmodulin-like proteins (CMLs), seven AP2/ERFs, and three bHLHs transcripts were significantly differentially expressed in calcium-deficient apple peels. RT-qPCR and correlation analyses further revealed that CML5 expression was significantly positively correlated with the expression of ERF2/17, bHLH2, and H2S production related genes. In addition, transcriptional co-activation of CML5 by ERF2 and bHLH2 was demonstrated by apple transient expression assays and dual-luciferase reporter system experiments. Therefore, these findings provide a basis for studying the molecular mechanism of postharvest quality decline in calcium-deficient apples and the potential interaction between Ca2+ and endogenous H2S.

MYB44 competitively inhibits the formation of the MYB340-bHLH2-NAC56 complex to regulate anthocyanin biosynthesis in purple-fleshed sweet potato.

BACKGROUND: Anthocyanins, which have important biological functions and have a beneficial effect on human health, notably account for pigmentation in purple-fleshed sweet potato tuberous roots. Individual regulatory factors of anthocyanin biosynthesis have been identified; however, the regulatory network of anthocyanin biosynthesis in purple-fleshed sweet potato is unclear. RESULTS: We functionally determined that IbMYB340 cotransformed with IbbHLH2 in tobacco and strawberry receptacles induced anthocyanin accumulation, and the addition of IbNAC56a or IbNAC56b caused increased pigmentation. Furthermore, we confirmed the interaction of IbMYB340 with IbbHLH2 and IbNAC56a or IbNAC56b via yeast two-hybrid and firefly luciferase complementation assays; these proteins could form a MYB340-bHLH2-NAC56a or MYB340-bHLH2-NAC56b transcriptional complex to regulate anthocyanin biosynthesis by binding to the IbANS promoter rather than the IbUFGT promoter. Furthermore, it was found by a transient expression system in tobacco leaves that IbMYB44 could decrease anthocyanin accumulation. Moreover, the interaction of IbMYB44 with IbMYB340 and IbNAC56a or IbNAC56b was verified. This result suggested that IbMYB44 acts as a repressor of anthocyanin in sweet potato. CONCLUSIONS: The repressor IbMYB44 affected anthocyanin biosynthesis by competitively inhibiting the IbMYB340-IbbHLH2-IbNAC56a or IbMYB340-IbbHLH2-IbNAC56b regulatory complex formation. Overall, the present study proposed a novel regulatory network whereby several vital TFs play key roles in regulating anthocyanin biosynthesis, and it provides strong insight into the potential mechanism underlying anthocyanin biosynthesis in sweet potato tuberous roots with purple color.

Antioxidative system in sweet potato root is activated by low-temperature storage.

BACKGROUND: Sweet potato is susceptible to chilling injury during low-temperature storage. To explore the correlation between chilling injury and reactive oxygen species (ROS) metabolism, the content of ROS and the activities and gene expression of antioxidant enzymes were analyzed in the typical storage-tolerant cultivar Xushu 32 and storage-sensitive cultivar Yanshu 25. RESULTS: The activities of antioxidant enzymes including ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) were enhanced rapidly in the early period of storage in response to chilling stress. Thereafter, the content of ROS metabolites increased consistently due to gradual decrease in ROS scavenging enzymes. Storage-tolerant cultivar Xushu 32 had higher antioxidant enzyme activities and gene expressions as well as higher content of antioxidant metabolites and lower content of ROS metabolites compared with storage-sensitive cultivar Yanshu 25, suggesting that the capacity of ROS scavenging by antioxidant enzymes and antioxidants is highly associated with the tolerance of sweet potato to chilling stress. CONCLUSION: These results indicated that the antioxidative system is activated in the storage root of sweet potato and the antioxidative capacity is positively associated with better storage performance in the storage-tolerant cultivar. © 2019 Society of Chemical Industry.

Rfa2 is specifically dephosphorylated by Pph3 in Candida albicans.

Rfa2 is a ssDNA (single-stranded DNA)-binding protein that plays an important role in DNA replication, recombination and repair. Rfa2 is regulated by phosphorylation, which alters its protein-protein interaction and protein-DNA interaction. In the present study, we found that the Pph3-Psy2 phosphatase complex is responsible for Rfa2 dephosphorylation both during normal G1-phase and under DNA replication stress in Candida albicans. Phosphorylated Rfa2 extracted from pph3Δ or psy2Δ G1 cells exhibited diminished binding affinity to dsDNA (double-stranded DNA) but not to ssDNA. We also discovered that Cdc28 (cell division cycle 28) and Mec1 are responsible for Rfa2 phosphorylation in G1-phase and under DNA replication stress respectively. Moreover, MS revealed that the domain of Rfa2 that was phosphorylated in G1-phase differed from that phosphorylated under the stress conditions. The results of the present study imply that differential phosphorylation plays a crucial role in RPA (replication protein A) regulation.

Hydrogen Peroxide Promotes Tomato Leaf Senescence by Regulating Antioxidant System and Hydrogen Sulfide Metabolism.

Hydrogen peroxide (H2O2) is relatively stable among ROS (reactive oxygen species) and could act as a signal in plant cells. In the present work, detached tomato leaves were treated with exogenous H2O2 at 10 mmol/L for 8 h to study the mechanism of how H2O2 regulates leaf senescence. The data indicated that H2O2 treatment significantly accelerated the degradation of chlorophyll and led to the upregulation of the expression of leaf senescence-related genes (NYC1, PAO, PPH, SGR1, SAG12 and SAG15) during leaf senescence. H2O2 treatment also induced the accumulation of H2O2 and malondialdehyde (MDA), decreased POD and SOD enzyme activities and inhibited H2S production by reducing the expression of LCD1/2 and DCD1/2. A correlation analysis indicated that H2O2 was significantly and negatively correlated with chlorophyll, the expression of leaf senescence-related genes, and LCD1/2 and DCD1/2. The principal component analysis (PCA) results show that H2S showed the highest load value followed by O2•-, H2O2, DCD1, SAG15, etc. Therefore, these findings provide a basis for studying the role of H2O2 in regulating detached tomato leaf senescence and demonstrated that H2O2 plays a positive role in the senescence of detached leaves by repressing antioxidant enzymes and H2S production.

Application of extracellular matrix cross-linked by microbial transglutaminase to promote wound healing.

One primary focus of skin tissue engineering has been the creation of innovative biomaterials to facilitate rapid wound healing. Extracellular matrix (ECM), an essential biofunctional substance, has recently been discovered to play a crucial role in wound healing. Consequently, we endeavored to decellularize ECM from pig achilles tendon and refine its mechanical and biological properties through modification by utilizing cross-linking agents. Glutaraldehyde (GA), 1-ethyl-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS), double aldol starch (DAS), and microbial transglutaminase (MTG) were utilized to produce crosslinked ECM variants (GA-ECM, EDC/NHS-ECM, DAS-ECM, and MTG-ECM). Comprehensive assessments were conducted to evaluate the physical properties, biocompatibility, and wound healing efficacy of each material. The results indicated that MTG-ECM exhibited superior tensile strength, excellent hydrophilicity, minimal cytotoxicity, and the best pro-healing impact among the four modified scaffolds. Staining analysis of tissue sections further revealed that MTG-ECM impeded the transition from type III collagen to type I collagen in the wound area, potentially reducing the development of wound scar. Therefore, MTG-ECM is expected to be a potential pro-skin repair scaffold material to prevent scar formation.

PuERF008-PuFAD2 module regulates aroma formation via the fatty acid pathway in response to calcium signaling in 'Nanguo' pear.

Calcium acts as a secondary messenger in plants and is essential for plant growth and development. However, studies on the pathway of aroma synthesis in 'Nanguo' pear (Pyrus ussriensis Maxim.) are scarce. In this study, a bioinformatics analysis of transcriptomic data from calcium-treated 'Nanguo' pear was performed, which identified two fatty acid desaturases, PuFAD2 and PuFAD3, and eight AP2/ERF transcription factors, all exhibiting the same expression patterns. Transient expression experiments showed overexpression of PuFAD2 and PuFAD3 significantly increased the levels of aromatic substrates linoleic acid, hexanal, linolenic acid, and (E)-2-hexenal, but RNAi (RNA interference) had the opposite expression. Promoter sequences analysis revealed that PuFAD2 and PuFAD3 have ERE (estrogen response element) motifs on their promoters. The strongest activation of PuFAD2 by PuERF008 was verified using a dual-luciferase reporting system. Additionally, yeast one-hybrid and electrophoretic mobility shift assays revealed PuERF008 could active PuFAD2. Transient overexpression and RNAi analyses of PuERF008 showed a strong correlation with the expression of PuFAD2. This study provides insights into the process of aroma biosynthesis in 'Nanguo' pear and offers a theoretical basis for elucidating the role of calcium signaling in aroma synthesis.

A D-cysteine desulfhydrase, SlDCD2, participates in tomato fruit ripening by modulating ROS homoeostasis and ethylene biosynthesis.

Hydrogen sulfide (H2S) is involved in multiple processes during plant growth and development. D-cysteine desulfhydrase (DCD) can produce H2S with D-cysteine as the substrate; however, the potential developmental roles of DCD have not been explored during the tomato lifecycle. In the present study, SlDCD2 showed increasing expression during fruit ripening. Compared with the control fruits, the silencing of SlDCD2 by pTRV2-SlDCD2 accelerated fruit ripening. A SlDCD2 gene-edited mutant was constructed by CRISPR/Cas9 transformation, and the mutant exhibited accelerated fruit ripening, decreased H2S release, higher total cysteine and ethylene contents, enhanced chlorophyll degradation and increased carotenoid accumulation. Additionally, the expression of multiple ripening-related genes, including NYC1, PAO, SGR1, PDS, PSY1, ACO1, ACS2, E4, CEL2, and EXP was enhanced during the dcd2 mutant tomato fruit ripening. Compared with the wild-type fruits, SlDCD2 mutation induced H2O2 and malondialdehyde (MDA) accumulation in fruits, which led to an imbalance in reactive oxygen species (ROS) metabolism. A correlation analysis indicated that H2O2 content was strongly positively correlated with carotenoids content, ethylene content and ripening-related gene expression and negatively correlated with the chlorophyll content. Additionally, the dcd2 mutant showed earlier leaf senescence, which may be due to disturbed ROS homeostasis. In short, our findings show that SlDCD2 is involved in H2S generation and that the reduction in endogenous H2S production in the dcd2 mutant causes accelerated fruit ripening and premature leaf senescence. Additionally, decreased H2S in the dcd2 mutant causes excessive H2O2 accumulation and increased ethylene release, suggesting a role of H2S and SlDCD2 in modulating ROS homeostasis and ethylene biosynthesis.

Shp1, a regulator of protein phosphatase 1 Glc7, has important roles in cell morphogenesis, cell cycle progression and DNA damage response in Candida albicans.

In yeast, the type 1 protein phosphatase (PP1) catalytic subunit Glc7 is involved in the regulation of multiple cellular processes and thought to achieve specificity through association with different regulatory subunits. Here, we report that the Glc7 regulator Shp1 plays important roles in cell morphogenesis, cell cycle progression and DNA damage response in Candida albicans. SHP1 deletion caused the formation of rod-shaped yeast cells with slow growth. Flow cytometry analysis revealed that shp1Δ cells showed a prolonged G(2)/M phase, which was rescued by deleting the spindle-checkpoint gene MAD2. Furthermore, shp1Δ cells were hypersensitive to heat and genotoxic stresses. Interestingly, depletion of Glc7 caused defects similar to the shp1Δ mutant such as arrest at G(2)/M transition; and the GLC7/glc7Δ heterozygous mutant exhibited increased sensitivity to genotoxic stresses, consistent with the recent finding that Saccharomyces cerevisiae Glc7 has a role in DNA damage response. We also show that Shp1 is required for the nuclear accumulation of Glc7, suggesting that Shp1 executes its cellular function partly by regulating Glc7 localization.

Oak-inspired anti-biofouling shape-memory unidirectional scaffolds with stable solar water evaporation performance.

Biomimetic porous materials have contributed to the enhancement of solar-driven evaporation rate in interfacial desalination and clean water production. However, due to the presence of numerous microbes in water environment, biofouling should occur inside porous materials to clog the channels for water transfer, resulting in obvious inhibition of the solar-driven evaporation efficacy in long-term use. To prevent and control biofouling in porous materials for solar-driven evaporation, a facile and environment-friendly design is required in real application. Oak wood possesses vertically aligned channels for transpiration and polyphenol compounds with antimicrobial activity. In this work, inspired by the oak wood, we developed an anti-biofouling shape-memory chitosan scaffold with unidirectional channels and tannic acid coating (oak-inspired scaffold). The shape-memory property facilitated rapid decoration with oak-inspired photothermal and anti-biofouling coating inside the scaffold, respectively, which also promotes the material durability by avoiding the external force-induced permanent structure failure. More importantly, the oak-inspired tannic acid coating not only prevented bacterial adhesion and colonization, but also inhibited fungal interference. They were subjected to a microbe-rich environment, and after 3 days, the evaporation rates of the untreated chitosan scaffolds were obviously decreased to 1.24, 1.16 and 1.19 kg m-2 h-1 for C. albicans, S. aureus and E. coli, respectively, which were only 65.6, 61.4 and 63.0% of original performance (1.89 kg m-2 h-1). In comparison, the oak-inspired scaffold exhibited a high solar-driven water evaporation rate after incubation in microbial suspensions (1.80, 1.70 and 1.75 kg m-2 h-1 for C. albicans, S. aureus and E. coli after 3 days) and lake water (1.74 kg m-2 h-1 after one month). The bioinspired anti-biofouling scaffolds maintain as high as 86.7-91.8% of the solar-driven water evaporation ability after exposure to a microbe-rich environment, which is conducive to develop a biomimetic long-term durable structure in water treatment.

Storage Property Is Positively Correlated With Antioxidant Capacity in Different Sweet Potato Cultivars.

Sweet potato decays easily due to its high respiration rate and reactive oxygen species (ROS) accumulation during postharvest storage. In this study, we explored the relationship between antioxidant capacity in leaves and storage properties in different sweet potato cultivars, the tuberous roots of 10 sweet potato cultivars were used as the experimental materials to analyze the storage property during storage at 11-15°C. According to the decay percentage after 290 days of storage, Xu 32 was defined as a storage-tolerant cultivar (rot percentage less than 25%); Xu 55-2, Z 15-1, Shangshu 19, Yushu, and Zhezi 3 as above-moderate storage-tolerant cultivars (rot percentage ranging from 25 to 50%); Sushu 16, Yanshu 5, and Hanzi as medium-storable cultivars (rot percentage 50-75%); and Yan 25 as a storage-sensitive cultivar (rot percentage greater than 75%). Meanwhile, analysis of the α-amylase activity in root tubers of the 10 sweet potato cultivars during storage indicated that α-amylase activity was lowest in the storage-tolerant cultivar Xu 32 and highest in the storage-sensitive cultivar Yan 25. Evaluation of antioxidant enzyme activities and ROS content in the leaves of these 10 cultivars demonstrated that cultivar Xu 32, which showed the best storage property, had higher antioxidant enzyme activity [superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxidase (POD)] but lower lipoxygenase (LOX) activity, hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents, and superoxide anion radical (O2⋅-) production rates compared with those of the storage-sensitive cultivar Yan 25 and the medium-storability cultivars Hanzi, Yanshu 5, and Sushu 16. Additionally, principal component analysis (PCA) suggested that sweet potato cultivars with different storage properties were clustered separately. Correlation and heat map analysis further indicated that CAT, APX, POD, and SOD activities were negatively correlated with α-amylase activity, while LOX activity and MDA and H2O2 contents were negatively correlated with the storage property of sweet potato. Combined, our findings revealed that storage property is highly correlated with antioxidant capacity in sweet potato leaves and negatively correlated with α-amylase activity in tuberous roots, which provides a convenient means for the screening of storage-tolerant sweet potato cultivars.