A nuclear-localized cysteine desulfhydrase, LCD1, plays a crucial role in mediating endogenous hydrogen sulfide production in tomatoes. However, the mechanism underlying the nuclear localization of SlLCD1 is not yet fully understood. In this study, it was found that SlLCD1 specifically interacted with nuclear import receptor importin α3 (SlIMPA3). Furthermore, it was demonstrated that silencing SlIMPA3 through virus-induced gene silencing or introducing mutations in SlIMPA3 via CRISPR/Cas9 significantly accelerated fruit ripening. Moreover, enhanced chlorophyll degradation, carotenoid accumulation, and premature upregulation of ripening-associated genes in the slimpa3 mutant indicated SlIMPA3 to be a negative regulator of fruit ripening and leaf senescence. Besides, SlIMPA3 deletion resulted in excessive hydrogen peroxide accumulation in fruits and leaves, potentially leading to premature leaf senescence and accelerated fruit ripening in the slimpa3 mutant. SlIMPA3 exhibited pronounced nuclear localization with weak distribution in the cytoplasm. SlLCD1 showed specific nuclear localization; however, after GFP tagging in slimpa3-edited tomato leaves, it migrated to the cytoplasm, suggesting that SlIMPA3 mediated the nuclear localization of SlLCD1. SlLCD1 transient expression in slimpa3 mutant fruits indicated that it did not inhibit tomato ripening following the SlIMPA3 mutation. In summary, our study revealed that SlIMPA3 interacted with SlLCD1 to facilitate its nuclear entry. Mutations in SlIMPA3 led to premature fruit ripening and leaf senescence, likely due to disrupted reactive oxygen species homeostasis resulting from SlLCD1 mislocalization in the slimpa3 mutant.

H2S is a well-known gaseous signaling molecule that plays important roles in plant response to biotic stresses. Pseudomonas syringae pv tomato (Pst) could cause enormous loss, while whether H2S could modulate plant defense against Pst is still unclear. By CRISPR/Cas9, the Sldcd1 gene editing mutant showed reduced endogenous H2S content and attenuated resistance, whereas treatment with exogenous H2S could enhance the resistance. A transcription factor, SlWRKY71, was screened and identified to promote the transcription of SlDCD1 via yeast one-hybrid, dual-luciferase reporter system, electrophoretic mobility shift assays, and transient overexpression. Here, it was found that exogenous H2S relieved the symptoms of bacterial speck disease in tomato leaves, conferring tolerance to Pst. DC3000, and the expression of the H2S-producing enzyme SlDCD1 was significantly induced. The Slwrky71 mutant also showed reduced defense in tomato leaves against Pst. DC3000, whereas SlWRKY71-OE tomato leaves showed increased tolerance. Transient overexpression of SlDCD1 in the context of Slwrky71 with exogenous H2S treatment has stronger resistance, and the overexpression of SlWRKY71 in the context of Sldcd1 showed relatively weak disease resistance, and with the addition of H2S enhanced the effect. Therefore, we concluded that SlWRKY71 could activate SlDCD1 expression and promote endogenous H2S production, thereby improving tomato leaves resistance to Pst. DC3000.

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 maybe 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.

Tomato is one of the most popular horticultural crops, and many commercial tomato cultivars are particularly susceptible to Botrytis cinerea. Hydrogen sulfide (H2S) is an important gaseous molecule in various plant stress responses. In this study, it was found that endogenous H2S increases in tomato leaves in response to B. cinerea infection, along with a 3.8-fold increase in gene expression of DCD1 which encodes a H2S-generating enzyme D-cysteine desulfhydrase 1 in tomato at 3 DPI. Then we investigated the role of DCD1 in resistance of tomato leaves and fruits to B. cinerea. The mutation of DCD1 by CRIPSR/Cas9 greatly reduced the resistance of tomato leaves and breaker and red fruits to B. cinerea accompanied with increased reactive oxygen species (ROS) especially hydrogen peroxide (H2O2) and malondialdehyde (MDA) content increased by 1.2 and 1.4 times respectively at 5 DPI of leaves. Further investigation showed that DCD1 mutation caused decreased activity of antioxidative enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) in both leaves and fruits, in particular, CAT activity in dcd1 mutant was 25.0 % and 41.7 % of that in WT at leaves and red fruits at 5 DPI. DCD1 mutation also caused decreased expression of defense-related genes PAL (encoding phenylalanine ammonia-lyase) and PUB24, and their expression in the dcd1 red fruit is approximately 1.3 and 1.8 times higher than in wild-type red fruit at 5 DPI, respectively. Thus, the work emphasizes the positive role of DCD1 and H2S in plant responses to necrotrophic fungal pathogens. In addition, the work provides strong evidence that fruit at ripened stage is more susceptible to B. cinerea infection compared with green fruit, suggesting that senescence of plant tissues is more favorable to B. cinerea infection.

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.

文章以多个具有不同贮藏特性的甘薯品种徐55-2、徐32、Z15-1、美99573、商薯9号、Sinjami、徐薯23、徐22-5、Z11-1、烟25为研究对象,考察了其块根贮藏特性的差异,并测定了甘薯叶片中抗氧化相关酶、脂氧合酶(LOX)、活性氧(ROS)及丙二醛(MDA)等指标,解析了它们之间的相关性.结果 表明:徐55-2、徐32、Z15-1为耐贮藏品种;美99573、商薯9号、Sinjami为较耐贮藏品种;徐22-5、Z11-1、烟25为较不耐贮藏品种;徐薯23为极不耐贮藏品种.与不耐贮藏品种相比,耐贮藏品种和较耐贮藏品种叶片中的ROS及M DA的量较低,抗坏血酸过氧化物酶(APX)、过氧化氢酶(CAT)、过氧化物酶(POD)、超氧化物歧化酶(SOD)等抗氧化酶的活性较高,LOX的活性较低;不耐贮藏品种和极不耐贮藏品种的ROS及MDA的量较高,抗氧化酶活性较低,且维持较高的LOX活性.经热图分析、主成分分析及相关性分析,发现10个甘薯品种的贮藏特性与抗氧化酶活性呈显著正相关,而与LOX活性、ROS及MDA的量呈显著负相关.文中结果为甘薯耐贮藏品种的鉴定与筛选提供了理论参考和技术支持.

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.

丝氨酸羟甲基转移酶(serine hydroxymethyltransferase,SHMT)普遍存在于植物中,在高等植物的一碳代谢和光呼吸过程中起着重要作用,然而该家族在甘薯中的功能尚不清楚.文章通过同源比对获取甘薯、番茄、拟南芥中21个SHMT基因家族的信息,通过系统进化树分析,发现21个SHMT蛋白形成4个分支;利用蛋白motif分析、蛋白结构域分析、基因外显子内含子结构分析、蛋白的二级结构和三级结构预测、蛋白质理化性质分析、核定位序列(nuclear localization sequence,NLS)、亚细胞定位分析预测,发现蛋白高级结构和基因结构在同一分支内更为保守,即同源性越高的蛋白,其结构、性质更为接近.通过对番茄中SHMT基因进行表达量分析,发现SHMT基因可能在番茄种子发育、开花、果实成熟方面发挥作用.另外,通过对拟南芥SHMT(AtSHMT)家族在胁迫条件下的表达水平进行分析,推断多个AtSHMT基因对胁迫尤其是盐胁迫、冷胁迫、热胁迫产生应答,表明SHMT基因家族在胁迫应答中起到调控作用.综上,文章将甘薯、番茄、拟南芥中SHMT基因家族的生物信息学分析及基因表达数据进行了挖掘,对深入研究SHMT基因在甘薯、番茄、拟南芥中的作用具有重要意义.

对甘薯、番茄、拟南芥中63个SPL基因家族进行了系统进化树分析、保守蛋白基序(Motif)分析,筛选归纳出同源性较高的2个分支的12个SPL基因进行理化性质分析、核定位预测等,氨基酸序列比对结果表明这些基因的功能可能较为保守.通过对番茄中的SPL基因Solyc05g015510.2、Solyc10g078700.1进行表达量分析,发现这2个基因可能参与调控果实成熟衰老进程.另外,通过对非生物胁迫下的转录水平进行分析得知,拟南芥中的AT5G43270可能参与对盐胁迫、热胁迫条件下的响应,AT1G27360、AT1G27370可能参与热胁迫条件下的响应,AT2G42200可能参与冷胁迫条件下的响应,而AT3 G57920在非生物胁迫条件下表达量没有特别明显的变化,表明AT3 G57920可能不参与非生物胁迫下的响应.

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.