Programmed cell death (PCD) induced by endoplasmic reticulum (ER) stress is implicated in variousplant physiological processes, yet its mechanism is still elusive. An activation of caspase-3-like ...enzymatic activity was clearly demonstrated but the role of the two known plant proteases with caspase-3-like activity, cathepsin B and proteasome subunit PBA1, remains to be established.
Both genetic downregulation and chemical inhibition were used to investigate the function of cathepsin B and PBA1 in ER-stress-induced PCD (ERSID). Transcript level and activity labelling of cathepsin B were used to assess activation. To study tonoplast rupture, a plant PCD feature, both confocal and electronic microscopies were used.
Cathepsin B downregulation reduced reactive oxygen species (ROS) accumulation and ERSID without affecting the induction of the unfolded protein response (UPR), but downregulation of PBA1 increased UPR and ERSID. Tonoplast rupture was not altered in the cathepsin B mutant and cathepsin B activation was independent of vacuolar processing enzyme (VPE). VPE activity was independent of cathepsin B.
ERSID is regulated positively by cathepsin B and negatively by PBA1, revealing a complex picture behind caspase-3-like activity in plants. Cathepsin B may execute its function after tonoplast rupture and works in parallel with VPE.
Programmed cell death (PCD) has a key role in defence and development of all multicellular organisms. In plants, there is a large gap in our knowledge of the molecular machinery involved at the ...various stages of PCD, especially the early steps. Here, we identify kiss of death (KOD) encoding a 25‐amino‐acid peptide that activates a PCD pathway in Arabidopsis thaliana. Two mutant alleles of KOD exhibited a reduced PCD of the suspensor, a single file of cells that support embryo development, and a reduced PCD of root hairs after a 55°C heat shock. KOD expression was found to be inducible by biotic and abiotic stresses. Furthermore, KOD expression was sufficient to cause death in leaves or seedlings and to activate caspase‐like activities. In addition, KOD‐induced PCD required light in leaves and was repressed by the PCD‐suppressor genes AtBax inhibitor 1 and p35. KOD expression resulted in depolarization of the mitochondrial membrane, placing KOD above mitochondria dysfunction, an early step in plant PCD. A KOD∷GFP fusion, however, localized in the cytosol of cells and not mitochondria.
This study identifies a novel regulator of cell death in plants and shows that the 25‐amino‐acid peptide kiss of death regulates programmed cell death at an early step in the cell death‐signalling cascade.
Plant cells, like cells from other kingdoms, have the ability to self-destruct in a genetically controlled manner. This process is defined as Programmed cell death (PCD). PCD can be triggered by ...various stimuli in plants including by endoplasmic reticulum (ER) stress. Research in the past two decades discovered that disruption of protein homeostasis in the ER could cause ER stress, which when prolonged/unresolved leads cells into PCD. ER stress-induced PCD is part of several plant processes, for instance, drought and heat stress have been found to elicit ER stress-induced PCD. Despite the importance of ER stress-induced PCD in plants, its regulation remains largely unknown, when compared with its counterpart in animal cells. In mammalian cells, several pro-apoptotic proteases called caspases were found to play a crucial role in ER stress-induced PCD. Over the past decade, several key proteases with caspase-like enzymatic activity have been discovered in plants and implicated in PCD regulation. This review covers what is known about caspase-like enzymatic activities during plant ER stress-induced PCD and discusses possible regulation pathways leading to the activation of relevant proteases in plants.
Phytobricks are standardized DNA parts for plants that can be assembled hierarchically into transcriptional units and, subsequently, into multigene constructs. Phytobricks each contain the sequences ...of one or more functional elements that comprise eukaryotic transcription units, with sequence features that enable them to be used interchangeably in one-step cloning reactions to facilitate combinatorial assembly. The simplicity and efficiency of this one-step reaction has enabled Phytobrick assembly to be miniaturized and automated on liquid handing platforms. In this method, we describe how to design and construct new Phytobricks as well as how to assemble them in both manual and nanoscale automated one-step reactions. Finally, we describe a high-throughput method for sequence verification of assembled plasmids.
Transgenic plants are produced both to investigate gene function and to confer desirable traits into crops. Transgene copy number is known to influence expression levels, and consequently, ...phenotypes. Similarly, knowledge of transgene zygosity is desirable for making quantitative assessments of phenotype and tracking the inheritance of transgenes in progeny generations. Since the first transgenic plants were produced, several methods for determining copy number have been applied, including Southern blotting, quantitative real-time PCR, and more recently, sequencing methods; however, each method has specific disadvantages, compromising throughput, accuracy, or expense. Digital PCR (dPCR) divides reactions into partitions, converting the exponential, analogue nature of PCR into a linear, digital signal that allows the frequency of occurrence of specific sequences to be accurately estimated. Confidence increases with the number of partitions; therefore, the availability of emulsion technologies that enable reactions to be divided into tens of thousands of nanodroplets allows accurate determination of copy number in what has become known as digital droplet PCR (ddPCR). ddPCR offers similar benefits of low costs and scalability as other PCR techniques but with superior accuracy and reliability. Graphic abstract: Digital PCR (dPCR) divides reactions into partitions, converting the exponential, analogue nature of PCR into a linear, digital signal that allows the frequency of transgene copy number to be accurately assessed.
Abstract
Many goals in synthetic biology, including the elucidation and refactoring of biosynthetic pathways and the engineering of regulatory circuits and networks, require knowledge of protein ...function. In plants, the prevalence of large gene families means it can be particularly challenging to link specific functions to individual proteins. However, protein characterization has remained a technical bottleneck, often requiring significant effort to optimize expression and purification protocols. To leverage the ability of biofoundries to accelerate design–built–test–learn cycles, we present a workflow for automated DNA assembly and cell-free expression of plant proteins that accelerates optimization and enables rapid screening of enzyme activity. First, we developed a phytobrick-compatible Golden Gate DNA assembly toolbox containing plasmid acceptors for cell-free expression using Escherichiacoli or wheat germ lysates as well as a set of N- and C-terminal tag parts for detection, purification and improved expression/folding. We next optimized automated assembly of miniaturized cell-free reactions using an acoustic liquid handling platform and then compared tag configurations to identify those that increase expression. We additionally developed a luciferase-based system for rapid quantification that requires a minimal 11–amino acid tag and demonstrate facile removal of tags following synthesis. Finally, we show that several functional assays can be performed with cell-free protein synthesis reactions without the need for protein purification. Together, the combination of automated assembly of DNA parts and cell-free expression reactions should significantly increase the throughput of experiments to test and understand plant protein function and enable the direct reuse of DNA parts in downstream plant engineering workflows.
Promoters serve a critical role in establishing baseline transcriptional capacity through the recruitment of proteins, including transcription factors. Previously, a paucity of data for ...cis-regulatory elements in plants meant that it was challenging to determine which sequence elements in plant promoter sequences contributed to transcriptional function. In this study, we have identified functional elements in the promoters of plant genes and plant pathogens that utilize plant transcriptional machinery for gene expression. We have established a quantitative experimental system to investigate transcriptional function, investigating how identity, density and position contribute to regulatory function. We then identified permissive architectures for minimal synthetic plant promoters enabling the computational design of a suite of synthetic promoters of different strengths. These have been used to regulate the relative expression of output genes in simple genetic devices.
AIM: To investigate member 3a of Wingless-type MMTV integration site family(Wnt3a) expression in cancerous and surrounding tissues and the relationship between clinicopathologic features of ...hepatocellular carcinoma(HCC) and Wnt3 a expression.METHODS: Wnt3 a expression and cellular distribution and clinicopathologic characteristics in cancerous tissue and matched surrounding tissues were analyzed in 80 HCC patients from January 2006 to August 2008 by tissue microarrays and immunohistochemistry. The overall and disease-free survival rates were estimated using the Kaplan-Meier method and compared with the log-rank test. The prognostic analysis was carried out with univariate and multivariate Cox regressions models.RESULTS : The incidence of oncogenic Wnt3a expression in the cancerous group was up to 96.25%(77 of 80), which was significantly higher(χ2 = 48.818, P < 0.001) than that in the surrounding group(46.25%, 37 of 80). Brown Wnt3 a staining gradually increased with clinical staging that showed very strong staining in advanced HCC. The clinicopathologic features of high Wnt3 a expression in HCC were related to poorlydifferentiated grade(χ2 = 20.211, P < 0.001), liver cirrhosis(χ2 = 8.467, P < 0.004), hepatitis B virus(HBV) infection(χ2 = 12.957, P < 0.001), higher tumor-nodemetastasis stage(χ2 = 22.960, P < 0.001), and 5-year survival rate(χ2 = 15.469, P < 0.001).CONCLUSION: Oncogenic Wnt3 a expression associated with HBV infection and cirrhotic liver might be an independent prognostic factor for HCC.
Background： Whether cholinergic innervations and/or autophagy have a role in the etiopathology of benign prostatic hyperplasia （BPH） is still unknown. This study aimed to investigate the role of ...cholinergic innervation and autophagy in the etiopathology of BPH. Methods： Male, 13-week-old spontaneous hypertension rats （spontaneous BPH animal model） were divided into three groups： an experimental group （EG, n = 24）, a control group （CG, n = 24）, and a normal control group （NC, n = 10）. The EG animals were intragastrically injected with tolterodine （3.5 mg/kg, twice a day）, CG animals were intragastrically injected with physiological saline, and the NC animals did not receive any treatment. Rats were sacrificed every 4 weeks, and the prostatic gross morphological changes, wet weight/body weight （ww/bw）, dry weight/wet weight （dw/ww）, histological changes, ultrastructural changes, and LC3 immunohistochemistry were continuously observed and compared. Results： The gross morphological and ww/bw changes in the three groups were similar at every stage. The dw/ww （mg/mg） values of the EG at week 17, 21, 25, and 29 were 0.1478 ±0.0034, 0.1653 ± 0.0036, 0.1668 ± 0.0045, and 0.1755±0.0034, respectively, and the CG values were 0.1511 ±0.0029, 0.1734± 0.0020, 0.1837 ±0.0052, and 0.1968 ± 0.0045, respectively. The difference between EG and CG for dw/ww showed statistical significance after 21 weeks of age （week 21： P = 0.016, week 25： P = 0.008, and week 29： P = 0.001）. Both EG and CG, prostatic glandular epithelial cell proliferation, and secretory function improved with age, but in EG, these improvements were slower than those in CG, and all the differences were statistically significant after 21 weeks. An increasing number of autophagosomes in the prostatic glandular cell cytoplasm, attenuation of LC3-I immunohistochemical staining, enhancement of LC3-II staining, and the ratio of LC3-1I/LC3-1 staining were all progressive in both groups, but the rate of change in EG was faster than that in CG, and these differences gained statistical significance after 25 weeks. Comparisons with regard to the above indexes between CG and NC showed no statistical significance at any stage. Conclusions： Cholinergic innervations and activation of autophagy appear to have important functions in the etiopathology of BPH. Drug-mediated blockade of cholinergic innervations could delay the physiopathology processes. Moreover, overactivation of autophagy may also play an important role in this delay.
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