Cyanide is produced along with ethylene biosynthesis and beta-cyanoalanine synthase (CAS) plays a crucial role in cyanide detoxification in plants, and its activity increases with fruit ripening. However, the involvement of CAS in pigment metabolism and ethylene biosynthesis during fruit ripening has not been previously reported. In this study, SlCAS1/2 were identified as highly homologous to StCAS1/2 in potato. Among them, SlCAS1 had higher expression level than SlCAS2 during tomato fruit ripening. The silencing of SlCAS1 expression by virus induced gene silencing (VIGS) increased chlorophyll content, decreased anthocyanins accumulation and affected the expression of SlNYC1, SlPPH, SlPAO and SlACO1/3 by comparing with the control. Conversely, overexpression of SlCAS1 (SlCAS1-OE) altered pigment accumulation, upregulated the expression of key anthocyanin biosynthesis genes such as SlDFR and SlANS, and increased the expression of key ethylene metabolism-related genes including SlACS2, SlACS4, SlRIN, and SlNOR, compared to the wild type. In contrast, Slcas1 mutant fruit (Delta SlCAS1) generated by CRISPR/Cas9 technology showed the opposite trend. Transcriptomic analysis comparing the wild type, SlCAS1-OE and Delta SlCAS1 fruit, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that these DEGs were mainly involved in ethylene biosynthesis, fruit ripening, and flavonoid biosynthesis. Principal component analysis (PCA) identified anthocyanin, SlACS2 and SlACO3 as the main factors. Overall, these results showed that SlCAS1 mediated tomato fruit ripening by enhancing anthocyanin and ethylene biosynthesis. These findings provide a new basis for improving tomato fruit color and ripening, offering potential strategies for improving tomato fruit quality.

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.

Abstract 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.

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.

甘薯采后贮藏期短,在采后贮运过程中极易腐败变质,从而影响甘薯的品质,造成大量损失.因此,研究不同耐贮性甘薯的生理生化指标差异具有重要的理论与实践价值.以耐贮藏甘薯品种南瑞苕、奥墨红、徐55-2、新大紫、徐薯18和不耐贮藏甘薯品种徐薯23、大红花、南瓜薯、满村香、万二、金吊薯为试验材料,考察甘薯叶片中抗氧化酶活性及活性氧(ROS)含量的差异,解析抗氧化代谢与甘薯耐贮性的相关性.结果表明,与不耐贮藏甘薯品种相比,耐贮的甘薯品种抗氧化酶如超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)活性较高,脂氧合酶(LOX)活性、丙二醛(MDA)含量、过氧化氢(H2O2)含量和超氧阴离子(O2-·)产生速率相对较低.热图分析结果显示,耐贮藏甘薯品种的叶片抗氧化酶活性相对较高,活性氧含量相对较低,不耐贮藏甘薯品种的叶片抗氧化酶活性相对较低,活性氧含量相对较高.主成分分析结果表明,不同耐贮性的甘薯品种中抗氧化能力和贮藏性能之间呈正相关.相关性分析结果显示,不同抗氧化酶之间有着较高的正相关系数,而与LOX活性、H2O2含量、O2-·产生速率、MDA含量呈明显负相关.由此可见,耐贮藏甘薯品种较不耐贮藏甘薯品种有着更好的抗氧化能力.研究结果为耐贮甘薯的选育提供了理论参考.

Purple-fleshed sweetpotato (Ipomoea batatas（L.）Lam.) is rich in anthocyanins. R2R3-type MYB transcription factors（TFs）with EAR motifs inhibiting anthocyanin biosynthesis have been reported, and there is still a lack of information on how mutations in the EAR motifs of MYBs affect anthocyanin accumulation. In this study, we obtained three IbMYB44 TFs by bioinformatics. Among these TFs, IbMYB44.1, IbMYB44.3 with a complete EAR motif and IbMYB44.2 with a single amino acid mutant in the EAR motif caused an amino acid substitution from leucine to valine. RT-qPCR analysis showed that IbMYB44s was expressed at lower levels in the purple-fleshed sweetpotato than in nonpurple-fleshed sweetpotato (P < 0.01). Transient expression assays showed that the inhibitory effect of IbMYB44.1/3 was stronger than IbMYB44.2 in tobacco leaves and red-skinned pears. RT-qPCR analysis further proved that IbMYB44.1/3 significantly inhibited the expression of anthocyanin biosynthesis-related genes compared with IbMYB44.2 in tobacco leaves and red-skinned pears. A dual luciferase reporter assay showed that IbMYB44s cannot directly activate the IbANS promoter, and the result was also verified by yeast one-hybrid (Y1H) experiments. Moreover, we identified the interaction of IbMYB340 with IbMYB44.1, IbMYB44.2 and IbMYB44.3 via yeast two-hybrid (Y2H) assays. Thus, IbMYB44.1/3 could interact with IbMYB340 to negatively regulate anthocyanin biosynthesis. This study enriched the regulatory network of anthocyanins and also provided a theoretical basis for a single amino acid mutant from leucine to valine in the EAR motif of IbMYB44.2 affecting anthocyanin biosynthesis in the purple-fleshed sweetpotato.

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.