Vegetable biology and breeding in the genomics era

Vegetable crops provide a rich source of essential nutrients for humanity and represent critical economic values to global rural societies. However, genetic studies of vegetable crops have lagged behind major food crops, such as rice, wheat and maize, thereby limiting the application of molecular breeding. In the past decades, genome sequencing technologies have been increasingly applied in genetic studies and breeding of vegetables. In this review, we recapitulate recent progress on reference genome construction, population genomics and the exploitation of multi-omics datasets in vegetable crops. These advances have enabled an in-depth understanding of their domestication and evolution, and facilitated the genetic dissection of numerous agronomic traits, which jointly expedites the exploitation of state-of-the-art biotechnologies in vegetable breeding. We further provide perspectives of further directions for vegetable genomics and indicate how the ever-increasing omics data could accelerate genetic, biological studies and breeding in vegetable crops.

     

Mercuric chloride induced brain toxicity in mice: The protective effects of puerarin-loaded PLGA nanoparticles

Mercury is a toxic, environmentally heavy metal that can cause severe damage to all organs, including the nervous system. The functions of puerarin include antioxidant, anti-inflammatory, nerve cell repair, regulation of autophagy, and so forth. But because of the limited oral absorption of puerarin, it affects the protective effect on brain tissue. The nano-encapsulation of Pue can improve its limitation. Therefore, this study investigated the protective effect of Pue drug-loaded PLGA nanoparticles (Pue-PLGA-nps) on brain injury induced by mercuric chloride (HgCl₂) in mice. The mice were divided into normal saline (NS) group, HgCl₂ (4 mg/kg) group, Pue-PLGA-nps (50 mg/kg) group, HgCl₂ + Pue (4 mg/kg + 30 mg/kg) group, and HgCl₂ + Pue-PLGA-nps (4 mg/kg + 50 mg/kg) group. After 28 days of treatment, the mice were observed for behavioral changes, antioxidant capacity, autophagy and inflammatory response, and mercury levels in the brain, blood, and urine were measured. The results showed that HgCl₂ toxicity caused learning and memory dysfunction in mice, increased mercury content in brain and blood, and increased serum levels of interleukin (IL-6), IL-1β, and tumor necrosis factor-α in the mice. HgCl₂ exposure decreased the activity of T-AOC, superoxide dismutase, and glutathione peroxidase, and increased the expression of malondialdehyde in the brain of mice. Moreover, the expression levels of TRIM32, toll-like receptor 4 (TLR4), and LC3 proteins were upregulated. Both Pue and Pue-PLGA-nps interventions mitigated the changes caused by HgCl₂ exposure, and Pue-PLGA-nps further enhanced this effect. Our results suggest that Pue-PLGA-nps can ameliorate HgCl₂-induced brain injury and reduce Hg accumulation, which is associated with inhibition of oxidative stress, inflammatory response, and TLR4/TRIM32/LC3 signaling pathway.     

TBC1D25 alleviates nonalcoholic steatohepatitis by inhibiting abnormal lipid accumulation and inflammation

Nonalcoholic fatty liver disease (NAFLD) is a strong stimulant of cardiovascular diseases, affecting one-quarter of the world's population. TBC1 domain family member 25 (TBC1D25) regulates the development of myocardial hypertrophy and cerebral ischemia–reperfusion injury; however, its effect on NAFLD/nonalcoholic steatohepatitis (NASH) has not been reported. In this study, we demonstrated that TBC1D25 expression is upregulated in NASH. TBC1D25 deficiency aggravated hepatic steatosis, inflammation, and fibrosis in NASH. In vitro tests revealed that TBC1D25 overexpression restrained NASH responses. Subsequent mechanistic validation experiments demonstrated that TBC1D25 interfered with NASH progression by inhibiting abnormal lipid accumulation and inflammation. TBC1D25 deficiency significantly promoted NASH occurrence and development. Therefore, TBC1D25 may potentially be used as a clinical therapeutic target for NASH treatment.     

湖南发现渡濑氏鼠耳蝠与中管鼻蝠

2022年7月和9月,本研究组在湖南开展翼手目多样性资源调查时,于常德市桃源县、湘西州保靖县、永州市双牌县和邵阳市绥宁县使用蝙蝠竖琴网分别采集到9只鼠耳蝠（7♂,2♀）和5只管鼻蝠（2♂,3♀）,结合传统形态学和分子系统发育学方法对上述标本进行物种鉴定。该批鼠耳蝠体型中等,前臂长43.41～50.89 mm,全身毛发呈红褐色,耳廓端部、鼻孔周围、五趾与爪及尾末端均呈黑褐色,翼膜掌指间有三角形状的红褐色斑直达翼缘;头骨顶部平缓,颧弓发达,脑颅高而凸显,其形态特征和度量数据均与渡濑氏鼠耳蝠（Myotis rufoniger）相符。同时,采集的管鼻蝠标本体型中等,前臂长33.83～37.53 mm,背毛棕褐色,腹毛呈浅黄色;颅全长16.74～17.29 mm,颧弓平直而宽厚,其外形特征和度量数据均与中管鼻蝠（Murinahuttoni）相符。基于Cytb和COI基因构建的系统发育树均支持以上形态学鉴定结果。该报道为渡濑氏鼠耳蝠和中管鼻蝠在湖南省分布的新发现。标本现存于广州大学华南生物多样性保护与利用重点实验室,其中渡濑氏鼠耳蝠标本号为GZHU22429、GZHU22430、GZHU 2245...     

Catalytic mechanism of rhomboid protease GlpG probed by 3,4-dichloroisocoumarin and diisopropyl fluorophosphonate

Rhomboid proteases have many important biological functions. Unlike soluble serine proteases such as chymotrypsin, the active site of rhomboid protease, which contains a Ser-His catalytic dyad, is submerged in the membrane and surrounded by membrane-spanning helices. Previous crystallographic analyses of GlpG, a bacterial rhomboid protease, and its complex with isocoumarin have provided insights into the mechanism of the membrane protease. Here, we studied the interaction of GlpG with 3,4-dichloroisocoumarin and diisopropyl fluorophosphonate, both mechanism-based inhibitors for the serine protease, and describe the crystal structure of the covalent adduct between GlpG and diisopropyl fluorophosphonate, which mimics the oxyanion-containing tetrahedral intermediate of the hydrolytic reaction. The crystal structure confirms that the oxyanion is stabilized by the main chain amide of Ser-201 and by the side chains of His-150 and Asn-154. The phosphorylation of the catalytic Ser-201 weakens its interaction with His-254, causing the catalytic histidine to rotate away from the serine. The rotation of His-254 is accompanied by further rearrangement of the side chains of Tyr-205 and Trp-236 within the substrate-binding groove. The formation of the tetrahedral adduct is also accompanied by opening of the L5 cap and movement of transmembrane helix S5 toward S6 in a direction different from that predicted by the lateral gating model. Combining the new structural data with those on the isocoumarin complex sheds further light on the plasticity of the active site of rhomboid membrane protease.     

A new genotyping method for detecting low abundance single nucleotide mutations based on gap ligase chain reaction and quantitative PCR assay

We tested applicability of a new genotyping technique to detect a low abundance CD17 (A → T) mutation of β-globin gene. The technique utilized a combined gap ligase chain reaction (Gap-LCR) and quantitative PCR (qPCR) methods. One pair of Gap-LCR primers was modified by adding specific sequences to the 5′ end of the upstream and the 3′ end of the downstream primer which served as a combining sequence for qPCR. First, specific mutation is detected using Gap-LCR; then, ligation products are detected by qPCR. Our results show that the amount of LCR products is directly proportional to the amount of template DNA. We further demonstrate that this technique detects a low abundance mutant DNA with a mutant/normal allele ratio as low as 1:10000. This technique was applied to detect a paternally inherited CD17 mutation from 53 maternal plasma samples. The results were consistent with those obtained by PCR/reverse dot blot of amniotic fluid cell DNA. In conclusion, by combining Gap-LCR and qPCR technology we successfully established a highly sensitive technique to detect low abundance point mutations. This technique can be applied to detect fetal DNA point mutation in maternal plasma.     

PPARα activation inhibits endothelin-1-induced cardiomyocyte hypertrophy by prevention of NFATc4 binding to GATA-4

Peroxisome proliferator-activated receptor alpha (PPARα) has been implicated in the pathogenesis of cardiac hypertrophy, although its mechanism of action remains largely unknown. To determine the effect of PPARα activation on endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy and explore its molecular mechanisms, we evaluated the interaction of PPARα with nuclear factor of activated T-cells c4 (NFATc4) in nuclei of cardiomyocytes from neonatal rats in primary culture. In ET-1-stimulated cardiomyocytes, data from electrophoretic mobility-shift assays (EMSA) and co-immunoprecipitation (co-IP) revealed that fenofibrate (Fen), a PPARα activator, in a concentration-dependent manner, enhanced the association of NFATc4 with PPARα and decreased its interaction with GATA-4, in promoter complexes involved in activation of the rat brain natriuretic peptide (rBNP) gene. Effects of PPARα overexpression were similar to those of its activation by Fen. PPARα depletion by small interfering RNA abolished inhibitory effects of Fen on NFATc4 binding to GATA-4 and the rBNP DNA. Quantitative RT-PCR and confocal microscopy confirmed inhibitory effects of PPARα activation on elevation of rBNP mRNA levels and ET-1-induced cardiomyocyte hypertrophy. Our results suggest that activated PPARα can compete with GATA-4 binding to NFATc4, thereby decreasing transactivation of NFATc4, and interfering with ET-1 induced cardiomyocyte hypertrophy.     

The zebrafish van gogh mutation disrupts tbx1, which is involved in the DiGeorge deletion syndrome in humans

The van gogh (vgo) mutant in zebrafish is characterized by defects in the ear, pharyngeal arches and associated structures such as the thymus. We show that vgo is caused by a mutation in tbx1, a member of the large family of T-box genes. tbx1 has been recently suggested to be a major contributor to the cardiovascular defects in DiGeorge deletion syndrome (DGS) in humans, a syndrome in which several neural crest derivatives are affected in the pharyngeal arches. Using cell transplantation studies, we demonstrate that vgo/tbx1 acts cell autonomously in the pharyngeal mesendoderm and influences the development of neural crest-derived cartilages secondarily. Furthermore, we provide evidence for regulatory interactions between vgo/tbx1 and edn1 and hand2, genes that are implicated in the control of pharyngeal arch development and in the etiology of DGS.     

A lead-dependent DNAzyme with a two-step mechanism

A detailed biochemical and mechanistic study of in vitro selected variants of 8-17 DNAzymes is presented. Even though the 8-17 DNAzyme motif has been obtained through in vitro selection under three different conditions involving 10 mM Mg(2+) (called 8-17), 0.5 mM Mg(2+)/50 mM histidine (called Mg5), or 100 microM Zn(2+) (called 17E), all variants are shown to be the most active with Pb(2+) (8-17: k(obs) approximately 0.5 min(-1); Mg5: k(obs) approximately 2 min(-1); 17E: k(obs) approximately 1 min(-1) with 200 microM Pb(2+) at pH 5.0). For the 17E variant of the 8-17 DNAzyme, the single-turnover rate constants followed the order of Pb(2+) > Zn(2+) > Mn(2+) approximately Co(2+) > Ni(2+) > Mg(2+) approximately Ca(2+) > Sr(2+) approximately Ba(2+). The catalytic rate is half-maximal at 13.5 microM Pb(2+), 0.97 mM Zn(2+), or 10.5 mM Mg(2+), suggesting that the metal-binding affinity of the DNAzymes is in the order of Pb(2+) > Zn(2+) > Mg(2+). The Pb(2+)-dependent activity increases linearly with pH and the slope of the plot of log k(obs) versus pH is approximately 1, suggesting a single deprotonation in the rate-limiting step of the reaction. Sequence variations of the DNAzyme confirm the importance of the G*T wobble pair, the two loops and the intervening stem in maintaining the active conformation of the system. While Mg(2+) and Zn(2+) catalyze only a transesterification reaction with formation of a product containing a 2',3'-cyclic phosphate, Pb(2+) catalyzes a transesterification reaction followed by hydrolysis of the 2',3'-cyclic phosphate. Although this two-step mechanism has shown to be operative in protein ribonucleases and in the leadzyme RNAzyme, it is now demonstrated for the first time that this DNAzyme may also use the same mechanism. Therefore, the two-step mechanism is observed in metalloenzymes of all classes, and this 8-17 DNAzyme provides a simple, stable, and cost-effective model system for understanding the structure of Pb(2+)-binding sites and their roles in the two-step mechanism.     

Increase of intracellular Ca(2+) during ischemia/reperfusion injury of heart is mediated by cyclic ADP-ribose

While the molecular mechanisms by which oxidants cause cytotoxicity are still poorly understood, disruption of Ca(2+) homeostasis appears to be one of the critical alterations during the oxidant-induced cytotoxic process. Here, we examined the possibility that oxidative stress may alter the metabolism of cyclic ADP-ribose (cADPR), a potent Ca(2+)-mobilizing second messenger in the heart. Isolated heart perfused by Langendorff technique was subjected to ischemia/reperfusion injury and endogenous cADPR level was determined using a specific radioimmunoassay. Following ischemia/reperfusion injury, a significant increase in intracellular cADPR level was observed. The elevation of cADPR content was closely correlated with the increase in ADP-ribosyl cyclase activity. Inclusion of oxygen free radical scavengers, 2,2,6,6-tetramethyl-1-piperidinyloxy and mannitol, in the reperfusate prevented the ischemia/reperfusion-induced increases in cADPR level and the ADP-ribosyl cyclase activity. Exposure of isolated cardiomyocytes to t-butyl hydroperoxide increased the ADP-ribosyl cyclase activity, cADPR level, and intracellular Ca(2+) concentration ([Ca(2+)](i)) and consequently resulting in cell lethal damage. The oxidant-induced elevation of [Ca(2+)](i) as well as cell lethal damage was blocked by a cADPR antagonist, 8-bromo-cADPR. These results provide evidence for involvement of cADPR and its producing enzyme in alteration of Ca(2+) homeostasis during the ischemia/reperfusion injury of the heart.