The autoactivation of human single-chain urokinase-type plasminogen activator (uPA).

Most serine proteases are synthesized as inactive zymogens that are activated by cleavage by another protease in a tightly regulated mechanism. The urokinase-type plasminogen activator (uPA) and plasmin cleave and activate each other, constituting a positive feedback loop. How this mutual activation cycle begins has remained a mystery. We used hydrogen deuterium exchange mass spectrometry to characterize the dynamic differences between the inactive single-chain uPA (scuPA) and its active form two-chain uPA (tcuPA). The results show that the C-terminal ß-barrel and the area around the new N terminus have significantly reduced dynamics in tcuPA as compared with scuPA. We also show that the zymogen scuPA is inactive but can, upon storage, become active in the absence of external proteases. In addition to plasmin, the tcuPA can activate scuPA by cleavage at K158, a process called autoactivation. Unexpectedly, tcuPA can cleave at position 158 even when this site is mutated. TcuPA can also cleave scuPA after K135 or K136 in the disordered linker, which generates the soluble protease domain of uPA. Plasmin cleaves scuPA exclusively after K158 and at a faster rate than tcuPA. We propose a mechanism by which the uPA receptor dimerization could promote autoactivation of scuPA on cell surfaces. These results resolve long-standing controversies in the literature surrounding the mechanism of uPA activation.

Effects of airflow superfine pulverization on the structure, functional properties, and flavor quality of wheat bran.

BACKGROUND: Wheat bran (WB) is a byproduct of refined wheat flour production with poor edible taste and low economic value. Herein, the WB was micronized via airflow superfine pulverization (ASP), and the effects of the ASP conditions on its particle size, nutritive compositions, whiteness, hydration characteristics, moisture distribution, microstructure, cation exchange capacity, volatile flavor components, and other characteristics were investigated. RESULTS: Reducing the rotational speed of the ASP screw and increasing the number of pulverizations significantly decreased the median particle size Dx(50) of WB to a minimum of 12.97 ± 0.19 µm (P < 0.05), increased the soluble dietary fiber content from 55.05 ± 2.94 to 106.86 ± 1.60 mg g-1, and improved the whiteness and water solubility index. In addition, the water holding capacity and oil holding capacity were significantly reduced (P < 0.05), while the cation exchange and swelling capacities first increased and then decreased. Up to about 70% of water in WB exists as bound water. As the Dx(50) of WB decreased, the content of bound and immobile water increased, while the free water decreased from 14.37 ± 1.21% to 7.59 ± 1.03%. Furthermore, WB was micronized and the particles became smaller and more evenly distributed. Using gas chromatography-ion mobility spectrometry, a total of 37 volatile compounds in micronized WB (including 10 aldehydes, 9 esters, 7 alcohols, and several acids, furans, ethers, aldehydes, esters, and alcohols) were identified as the main volatile compounds of WB. CONCLUSION: Collectively, ASP improved the physicochemical properties of WB. This study provides theoretical references for the use of ASP to improve the utilization and edibility of WB. © 2024 Society of Chemical Industry.

Deficiency of autism-related Scn2a gene in mice disrupts sleep patterns and circadian rhythms.

Autism spectrum disorder (ASD) affects ~2% of the population in the US, and monogenic forms of ASD often result in the most severe manifestation of the disorder. Recently, SCN2A has emerged as a leading gene associated with ASD, of which abnormal sleep pattern is a common comorbidity. SCN2A encodes the voltage-gated sodium channel NaV1.2. Predominantly expressed in the brain, NaV1.2 mediates the action potential firing of neurons. Clinical studies found that a large portion of children with SCN2A deficiency have sleep disorders, which severely impact the quality of life of affected individuals and their caregivers. The underlying mechanism of sleep disturbances related to NaV1.2 deficiency, however, is not known. Using a gene-trap Scn2a-deficient mouse model (Scn2atrap), we found that Scn2a deficiency results in increased wakefulness and reduced non-rapid-eye-movement (NREM) sleep. Brain region-specific Scn2a deficiency in the suprachiasmatic nucleus (SCN) containing region, which is involved in circadian rhythms, partially recapitulates the sleep disturbance phenotypes. At the cellular level, we found that Scn2a deficiency disrupted the firing pattern of spontaneously firing neurons in the SCN region. At the molecular level, RNA-sequencing analysis revealed differentially expressed genes in the circadian entrainment pathway including core clock genes Per1 and Per2. Performing a transcriptome-based compound discovery, we identified dexanabinol (HU-211), a putative glutamate receptor modulator, that can partially reverse the sleep disturbance in mice. Overall, our study reveals possible molecular and cellular mechanisms underlying Scn2a deficiency-related sleep disturbances, which may inform the development of potential pharmacogenetic interventions for the affected individuals.

The formation and accumulation of protein-networks by physical interactions in the rapid occlusion of laticifer cells in rubber tree undergoing successive mechanical wounding.

BACKGROUND: Although the wound response of plants has been extensively studied, little is known of the rapid occlusion of wounded cell itself. The laticifer in rubber tree is a specific type of tissue for natural rubber biosynthesis and storage. In natural rubber production, tapping is used to harvest the latex which flows out from the severed laticifer in the bark. Therefore, study of the rapid wound-occlusion of severed laticifer cells is important for understanding the rubber tree being protected from the continuously mechanical wounding. RESULTS: Using cytological and biochemical techniques, we revealed a biochemical mechanism for the rapid occlusion of severed laticifer cells. A protein-network appeared rapidly after tapping and accumulated gradually along with the latex loss at the severed site of laticifer cells. Triple immunofluorescence histochemical localization showed that the primary components of the protein-network were chitinase, ß-1,3-glucanase and hevein together with pro-hevein (ProH) and its carboxyl-terminal part. Molecular sieve chromatography showed that the physical interactions among these proteins occurred under the condition of neutral pH. The interaction of ß-1,3-glucanase respectively with hevein, chitinase and ProH was testified by surface plasmon resonance (SPR). The interaction between actin and ß-1,3-glucanase out of the protein inclusions of lutoids was revealed by pull-down. This interaction was pharmacologically verified by cytochalasin B-caused significant prolongation of the duration of latex flow in the field. CONCLUSIONS: The formation of protein-network by interactions of the proteins with anti-pathogen activity released from lutoids and accumulation of protein-network by binding to the cytoskeleton are crucial for the rapid occlusion of laticifer cells in rubber tree. The protein-network at the wounded site of laticifer cells provides not only a physical barrier but also a biochemical barrier to protect the wounded laticifer cells from pathogen invasion.

Comparative transcriptome analysis of latex from rubber tree clone CATAS8-79 and PR107 reveals new cues for the regulation of latex regeneration and duration of latex flow.

BACKGROUND: Rubber tree (Hevea brasiliensis Muell. Arg.) is the primarily commercial source of natural rubber in the world. Latex regeneration and duration of latex flow after tapping are the two factors that determine rubber yield of rubber tree, and exhibit a huge variation between rubber tree clones CATAS8-79 and PR107. RESULTS: To dissect the molecular mechanism for the regulation of latex regeneration and duration of latex flow, we sequenced and comparatively analyzed latex of rubber tree clone CATAS8-79 and PR107 at transriptome level. More than 26 million clean reads were generated in each pool and 51,829 all-unigenes were totally assembled. A total of 6,726 unigenes with differential expression patterns were detected between CATAS8-79 and PR107. Functional analysis showed that genes related to mass of categories were differentially enriched between the two clones. Expression pattern of genes which were involved in latex regeneration and duration of latex flow upon successive tapping was analyzed by quantitative PCR. Several genes related to rubber biosynthesis, cellulose and lignin biosynthesis and rubber particle aggregation were differentially expressed between CATAS8-79 and PR107. CONCLUSIONS: This is the first report about probing latex regeneration and duration of latex flow by comparative transcriptome analysis. Among all the suggested factors, it is more important that the level of endogenous jasmonates, carbohydrate metabolism, hydroxymethylglutaryl-CoA reductase (HMGR) and Hevea rubber transferase (HRT) in mevalonate (MVA) parthway for latex regeneration while the level of endogenous ethylene (ETH), lignin content of laticifer cell wall, antioxidants and glucanases for the duration of latex flow. These data will provide new cues for understanding the molecular mechanism for the regulation of latex regeneration and duration of latex flow in rubber tree.

A role for circuitry of the cortical amygdala in excessive alcohol drinking, withdrawal, and alcohol use disorder.

Alcohol use disorder (AUD) poses a significant public health challenge. Individuals with AUD engage in chronic and excessive alcohol consumption, leading to cycles of intoxication, withdrawal, and craving behaviors. This review explores the involvement of the cortical amygdala (CoA), a cortical brain region that has primarily been examined in relation to olfactory behavior, in the expression of alcohol dependence and excessive alcohol drinking. While extensive research has identified the involvement of numerous brain regions in AUD, the CoA has emerged as a relatively understudied yet promising candidate for future study. The CoA plays a vital role in rewarding and aversive signaling and olfactory-related behaviors and has recently been shown to be involved in alcohol-dependent drinking in mice. The CoA projects directly to brain regions that are critically important for AUD, such as the central amygdala, bed nucleus of the stria terminalis, and basolateral amygdala. These projections may convey key modulatory signaling that drives excessive alcohol drinking in alcohol-dependent subjects. This review summarizes existing knowledge on the structure and connectivity of the CoA and its potential involvement in AUD. Understanding the contribution of this region to excessive drinking behavior could offer novel insights into the etiology of AUD and potential therapeutic targets.

Development of a new class of potent and highly selective G protein-coupled receptor kinase 5 inhibitors and structural insight from crystal structures of inhibitor complexes.

G protein-coupled receptor kinase 5 (GRK5) is an important drug development target for heart failure, cardiac hypertrophy, and cancer. We have designed and developed a new class of highly selective, potent, and non-covalent GRK5 inhibitors. One of the inhibitors displayed GRK5 IC50 value of 10 nM and exhibited >100,000-fold selectivity over GRK2. The X-ray structure of a ketoamide-derived inhibitor-bound GRK5 showed the formation of a hemithioketal intermediate with active site Cys474 in the GRK5 active site and provided new insights into the ligand-binding site interactions responsible for high selectivity. The current studies serve as an important guide to therapeutic GRK5 inhibitor drug development.

Sex Differences in Neural Networks Recruited by Frontloaded Binge Alcohol Drinking.

Frontloading is an alcohol drinking pattern where intake is skewed toward the onset of access. The goal of the current study was to identify brain regions involved in frontloading. Whole brain imaging was performed in 63 C57Bl/6J (32 female and 31 male) mice that underwent 8 days of binge drinking using the drinking-in-the-dark (DID) model. On days 1-7, three hours into the dark cycle, mice received 20% (v/v) alcohol or water for two hours. Intake was measured in 1-minute bins using volumetric sippers, which facilitated analyses of drinking patterns. On day 8 mice were perfused 80 minutes into the DID session and brains were extracted. Brains were then processed to stain for Fos protein using iDISCO+. Following light sheet imaging, ClearMap2.1 was used to register brains to the Allen Brain Atlas and detect Fos+ cells. For brain network analyses, day 8 drinking patterns were used to characterize mice as frontloaders or non-frontloaders using a recently developed change-point analysis. Based on this analysis the groups were female frontloaders (n = 20), female non-frontloaders (n = 2), male frontloaders (n = 13) and male non-frontloaders (n = 8). There were no differences in total alcohol intake in animals that frontloaded versus those that did not. Only two female mice were characterized as non-frontloaders, thus preventing brain network analysis of this group. Functional correlation matrices were calculated for each group from log10 Fos values. Euclidean distances were calculated from these R values and hierarchical clustering was used to determine modules (highly connected groups of brain regions). In males, alcohol access decreased modularity (3 modules in both frontloaders and non-frontloaders) as compared to water drinkers (7 modules). In females, an opposite effect was observed. Alcohol access (9 modules for frontloaders) increased modularity as compared to water drinkers (5 modules). These results suggest sex differences in how alcohol consumption reorganizes the functional architecture of neural networks. Next, key brain regions in each network were identified. Connector hubs, which primarily facilitate communication between modules, and provincial hubs, which facilitate communication within modules, were of specific interest for their important and differing roles. In males, 4 connector hubs and 17 provincial hubs were uniquely identified in frontloaders (i.e., were brain regions that did not have this status in male non-frontloaders or water drinkers). These represented a group of hindbrain regions (e.g., locus coeruleus and the pontine gray) functionally connected to striatal/cortical regions (e.g., cortical amygdalar area) by the paraventricular nucleus of the thalamus. In females, 16 connector and 17 provincial hubs were uniquely identified which were distributed across 8 of the 9 modules in the female frontloader alcohol drinker network. Only one brain region (the nucleus raphe pontis) was a connector hub in both sexes, suggesting that frontloading in males and females may be driven by different brain regions. In conclusion, alcohol consumption led to fewer, but more densely connected, groups of brain regions in males but not females, and recruited different hub brain regions between the sexes. These results suggest that alcohol frontloading leads to a reduction in network efficiency in male mice.

Comparative proteomics of primary and secondary lutoids reveals that chitinase and glucanase play a crucial combined role in rubber particle aggregation in Hevea brasiliensis.

Lutoids are specific vacuole-based organelles within the latex-producing laticifers in rubber tree Hevea brasiliensis. Primary and secondary lutoids are found in the primary and secondary laticifers, respectively. Although both lutoid types perform similar roles in rubber particle aggregation (RPA) and latex coagulation, they vary greatly at the morphological and proteomic levels. To compare the differential proteins and determine the shared proteins of the two lutoid types, a proteomic analysis of lutoid membranes and inclusions was performed, revealing 169 proteins that were functionally classified into 14 families. Biological function analysis revealed that most of the proteins are involved in pathogen defense, chitin catabolism, and proton transport. Comparison of the gene and protein changed patterns and determination of the specific roles of several main lutoid proteins, such as glucanase, hevamine, and hevein, demonstrated that Chitinase and glucanase appeared to play crucial synergistic roles in RPA. Integrative analysis revealed a protein-based metabolic network mediating pH and ion homeostasis, defense response, and RPA in lutoids. From these findings, we developed a modified regulation model for lutoid-mediated RPA that will deepen our understanding of potential mechanisms involved in lutoid-mediated RPA and consequent latex coagulation.

Prior experience with flavored alcohol increases preference for flavored alcohol but flavor does not influence binge-like drinking behavior in mice.

BACKGROUND: Binge drinking can lead to various negative consequences and in non-experimental settings, alcohol usually contains flavoring, which may promote increased binge drinking. Preclinical models of binge-like drinking have been well established, however, the influence of flavor on alcohol preference and binge-like drinking has not been fully explored. METHODS: Male and female C57BL/6 J mice were tested via two-bottle choice with alcohol flavored with different concentrations of unsweetened Cherry flavor Kool-Aid and water. Next, mice were tested for preference for flavored alcohol over plain alcohol. Consumption of flavored alcohol versus water was examined over 48 h. Binge-like drinking with flavored alcohol was validated via drinking in the dark (DID). A separate cohort of mice underwent chronic DID for 6 weeks with either flavored or plain alcohol. After chronic DID, mice were then tested for preference for flavored versus plain alcohol and then alcohol consumption despite adverse effects was examined using the quinine adulteration test. RESULTS: The 0.1% Kool-Aid concentration was chosen to use for further testing based on intake. Mice preferred Kool-Aid flavored alcohol over plain alcohol after the concentration test, but mice with no prior exposure to plain or flavored alcohol preferred plain over flavored alcohol. Throughout all initial testing, female mice showed increased alcohol intake compared to male mice. Both male and female mice showed binge-like drinking of flavored alcohol, with females having higher intake and blood alcohol levels. Kool-Aid flavor did not increase alcohol intake during chronic binge-like drinking. Previous exposure to flavored alcohol during DID increased the preference for flavored alcohol over plain alcohol but did not influence alcohol consumption despite adverse effects. CONCLUSION: The present study indicates that prior experience with flavored alcohol increases preference and intake, suggesting an effect of learned safety from neophobia. However, flavor does not impact binge-like alcohol consumption or alcohol drinking despite negative consequences. Additionally, the current study shows that female mice will consume more flavored alcohol than males, similar to findings from other alcohol studies.

Chronic Intermittent Ethanol Vapor Exposure Paired with Two-Bottle Choice to Model Alcohol Use Disorder.

Alcohol use disorder (AUD) is a chronic alcohol-related disorder that typically presents as uncontrolled drinking and preoccupation with alcohol. A key component of AUD research is using translationally relevant preclinical models. Over the past several decades, a variety of animal models have been used to study AUD. One prominent model of AUD is the chronic intermittent ethanol vapor exposure (CIE) model, which is a well-established approach for inducing alcohol dependence in rodents through repeated cycles of ethanol exposure via inhalation. To model AUD in mice, the CIE exposure is paired with a voluntary two-bottle choice (2BC) of alcohol drinking and water to measure the escalation of alcohol drinking. The 2BC/CIE procedure involves alternating weeks of 2BC drinking and CIE, which repeat until the escalation of alcohol drinking is achieved. In the present study, we outline the procedures for performing 2BC/CIE, including the daily use of the CIE vapor chamber, and provide an example of escalated alcohol drinking in C57BL/6J mice using this approach.

Characterization of HbEREBP1, a wound-responsive transcription factor gene in laticifers of Hevea brasiliensis Muell. Arg.

AP2/ERF transcription factors play an important role in regulation of the cross-talk between ethylene and jasmonate signaling pathways mediating defense responses of plants to biotic and abiotic stresses. In this study, an AP2/ERF transcription factor gene was isolated and characterized from laticifers of rubber tree by using RACE and real time PCR. The full length cDNA, referred to as HbEREBP1, was 1,095 bp in length and contained a 732 bp open reading frame encoding a putative protein of 243 amino acid residues. The molecular mass of the putative protein is 26.4 kDa with a pI of 9.46. The deduced amino acid sequence had a specific domain of AP2 superfamily and an ethylene-responsive element binding factor-associated amphiphilic repression motif, sharing 42.4, 39.1, and 38.0% identity with that of AtERF11, AtERF4, and AtERF8 in Arabidopsis, respectively. HbEREBP1 expression was down-regulated by tapping and mechanical wounding in the laticifers of adult trees. It was also down-regulated at early stage while up-regulated at late stage upon treatment with exogenous ethephon or methyl jasmonate, which was reverse to the case of defense genes in laticifers of epicormic shoots of rubber tree. Our results suggest that HbEREBP1 may be a negative regulator of defense genes in laticifers.

Zebrafish Embryos Display Characteristic Bioelectric Signals during Early Development.

Bioelectricity is defined as endogenous electrical signaling mediated by the dynamic distribution of charged molecules. Recently, increasing evidence has revealed that cellular bioelectric signaling is critical for regulating embryonic development, regeneration, and congenital diseases. However, systematic real-time in vivo dynamic electrical activity monitoring of whole organisms has been limited, mainly due to the lack of a suitable model system and voltage measurement tools for in vivo biology. Here, we addressed this gap by utilizing a genetically stable zebrafish line, Tg (ubiquitin: ASAP1), and ASAP1 (Accelerated sensor of action potentials 1), a genetically encoded voltage indicator (GEVI). With light-sheet microscopy, we systematically investigated cell membrane potential (Vm) signals during different embryonic stages. We found cells of zebrafish embryos showed local membrane hyperpolarization at the cleavage furrows during the cleavage period of embryogenesis. This signal appeared before cytokinesis and fluctuated as it progressed. In contrast, whole-cell transient hyperpolarization was observed during the blastula and gastrula stages. These signals were generally limited to the superficial blastomere, but they could be detected within the deeper cells during the gastrulation period. Moreover, the zebrafish embryos exhibit tissue-level cell Vm signals during the segmentation period. Middle-aged somites had strong and dynamic Vm fluctuations starting at about the 12-somite stage. These embryonic stage-specific characteristic cellular bioelectric signals suggest that they might play a diverse role in zebrafish embryogenesis that could underlie human congenital diseases.

Hierarchical porous biochars with controlled pore structures derived from co-pyrolysis of potassium/calcium carbonate with cotton straw for efficient sorption of diethyl phthalate from aqueous solution.

A one-pot co-pyrolysis of potassium/calcium carbonate with biowaste-derived hydrochar strategy was proposed to prepare hierarchical porous biochars (HPBs) for the first time. The pore structure, especially the pore size distribution, could be designed by adjusting the mass ratios of different carbonates. HPBs were hydrophobic, nitrogen doped, graphitized, and contained surface functional groups. HPBs showed unexpected sorption quantity for diethyl phthalate (DEP) that reached 657 mg g-1, which much higher than that of the reported sorbents. The sorption was multilayered and had multiple action modes, and was limited by the chemical sorption and the sorption quantity was dominated by the physical sorption. Lewis acid-base interaction, π-π stacking interaction, hydrogen bonding interaction, partitioning and pore filling were the potential sorption mechanisms. This work proposed a simple, environmentally friendly and low-cost method to convert biowaste into advanced HPBs and confirmed that produced HPBs represent ideal sorbents for the removal of organic pollutants.

The Hidden Brain: Uncovering Previously Overlooked Brain Regions by Employing Novel Preclinical Unbiased Network Approaches.

A large focus of modern neuroscience has revolved around preselected brain regions of interest based on prior studies. While there are reasons to focus on brain regions implicated in prior work, the result has been a biased assessment of brain function. Thus, many brain regions that may prove crucial in a wide range of neurobiological problems, including neurodegenerative diseases and neuropsychiatric disorders, have been neglected. Advances in neuroimaging and computational neuroscience have made it possible to make unbiased assessments of whole-brain function and identify previously overlooked regions of the brain. This review will discuss the tools that have been developed to advance neuroscience and network-based computational approaches used to further analyze the interconnectivity of the brain. Furthermore, it will survey examples of neural network approaches that assess connectivity in clinical (i.e., human) and preclinical (i.e., animal model) studies and discuss how preclinical studies of neurodegenerative diseases and neuropsychiatric disorders can greatly benefit from the unbiased nature of whole-brain imaging and network neuroscience.

Vegetative storage protein with trypsin inhibitor activity occurs in Sapindus mukorassi, a sapindaceae deciduous tree.

A vegetative storage protein (VSP) with trypsin inhibitor activity in a deciduous tree, Sapindus mukorassi, was characterized by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western-blot, immuno-histochemical localization, light- and electro-microscopy, together with analysis of proteinase inhibitor activity of the purified VSP in vitro. There were two proteins with molecular masses of about 23 and 27 kDa in a relatively high content in the bark tissues of terminal branches of S. mukorassi in leafless periods. The proteins decreased markedly during young shoot development, indicating their role in seasonal nitrogen storage. Immuno-histochemical localization with the polyclonal antibodies raised against the 23 kDa protein demonstrated that the 23 kDa protein was the major component of protein inclusions in protein-storing cells. The protein inclusions were identified by protein-specific staining and should correspond to the electron-dense materials in different forms in the vacuoles of phloem parenchyma cells and phloem ray parenchyma cells under an electron microscope. So, the 23 kDa protein was a typical VSP in S. mukorassi. The 23 and 27 kDa proteins shared no immuno-relatedness, whereas the 23 kDa protein was immuno-related with the 22 kDa VSP in lychee and possessed trypsin inhibitor activity. The 23 kDa protein may confer dual functions: nitrogen storage and defense.

Genome-Wide Identification and Characterization of the JAZ Gene Family in Rubber Tree (Hevea brasiliensis).

Jasmonate signaling plays a vital role in the regulation of secondary laticifer differentiation and natural rubber biosynthesis in Hevea brasiliensis. Jasmonate ZIM-domain (JAZ) proteins are the master regulators of jasmonate signaling. Although several JAZs have been reported in the laticifer cells of H. brasiliensis, the genome-wide screening of HbJAZ members has not yet been explored. In the present study, 18 HbJAZs were identified based on the recent H. brasiliensis genome. Phylogenetic construction revealed that the HbJAZs were clustered into five subgroups and that members within the same subgroup shared highly conserved gene structures and protein motifs. Cis-element analysis of HbJAZ promoters suggested the presence of hormone, stress and development-related cis-elements. HbJAZ1.0, HbJAZ2.0, and HbJAZ5.0 interacted with CORONATINE INSENSITIVE1 (COI1) in the presence of coronatine (COR, a JA mimic). HbJAZ1.0, HbJAZ2.0, HbJAZ5.0, and HbJAZ12.0 could also interact with each other. Of the 18 HbJAZs, transcripts of 15 HbJAZs were present in the vascular cambium region except for that of HbJAZ7.0, HbJAZ8.0d, and HbJAZ13.0. Fourteen of the 15 HbJAZs were significantly up-regulated upon COR treatment. The transcripts of three genes that were absent from vascular cambium region were also absent from the latex. Among the 15 HbJAZs in the latex, the expression patterns of 13 HbJAZs were different between the tapping and ethrel treatments. Eight of the 14 COR-up-regulated HbJAZs in the vascular cambium region were also activated by tapping in latex. Of the eight tapping-activated HbJAZs, 5 HbJAZs were repressed by ethrel application. Based on the computational analyses and gene expression patterns described in this study, the HbJAZ5.0 and HbJAZ10.0b may be associated with laticifer differentiation while the HbJAZ8.0b is a negative regulator for natural rubber biosynthesis in H. brasiliensis.

Comparative transcriptome analysis reveals phytohormone signalings, heat shock module and ROS scavenger mediate the cold-tolerance of rubber tree.

Two contrasting cold response rubber tree clones, the cold-resistant '93-114' and cold-sensitive 'Reken501', were subject to a global transcriptome response assessing via high-throughput RNA-seq technique and comprehensive bioinformatics analysis using the referenced rubber tree genome with the purpose of exploring the potential molecular cues underlying the tolerance of rubber trees to cold stress. As a result, a total of 1919 genes had significantly higher expression, while 2929 genes had significantly lower expression in '93-114' than in 'Reken501' without cold stress. Upon cold stress, the numbers of genes with significantly higher expression decreased to 1501 at 1 h treatment and to 1285 at 24 h treatment in '93-114' than that of 'Reken501', conversely, the numbers of genes with significantly lower expression increased to 7567 at 1 h treatment and to 5482 at 24 h treatment. Functional annotation of the differentially expressed genes between '93-114' and 'Reken501' suggests that down-regulation of auxin and ethylene signaling and activation of heat shock module and ROS scavengers is a primary strategy for H. brasiliensis to cope with cold stress. Our identified vital differentially expressed genes may be beneficial for elucidation of the molecular mechanisms underlying cold tolerance and for genetic improvement of H. brasiliensis clones.

Vegetative storage protein in Litchi chinensis, a subtropical evergreen fruit tree, possesses trypsin inhibitor activity.

BACKGROUND AND AIMS: Vegetative storage proteins (VSPs) are commonly bioactive in herbaceous plants but few VSPs with bioactivity have been identified in trees. In addition, information on the characterization of VSPs in evergreen trees is limited. The objective of this study was to characterize the VSPs with bioactivity in evergreen trees. Methods The VSP in lychee (Litchi chinensis), an evergreen fruit tree, was characterized by a combination of cytological, biochemical and molecular biological techniques. KEY RESULTS: The VSP in lychee was a 22-kDa protein. It accumulated in the large central vacuoles of protein-storing cells (PSCs) in two distinguishable forms, granular and floccular. The PSCs were of a novel type. The 22-kDa protein is distributed in mature leaves, bark tissues of branches, trunk and large roots, paralleling the distribution of PSCs. Its homologues were present in mature seed. During young shoot development and fruiting, the 22-kDa protein decreased apparently, suggesting a nitrogen-storage function. The 22-kDa protein had several isoforms encoded by a small multigene family. One gene member, LcVSP1, was cloned. The LcVSP1 had no intron and contained a 675 bp open reading frame encoding a putative protein of 225 amino acids. LcVSP1 was homologous to Kunitz trypsin inhibitors. The 22-kDa protein inhibited trypsin and chymotrypsin, but had no inhibitory effect on subtilisin. CONCLUSIONS: Lychee is rich in a 22-kDa VSP with trypsin inhibitor activity. The VSP plays an important role in nitrogen storage while its possible defensive function remains to be elucidated.

Transcript Profiling of Hevea brasiliensis during Latex Flow.

Latex exploitation enhances latex regeneration in rubber trees. The latex exploitation-caused latex flow lasts from 10 min to a few hours, which is convenient for exploring the transcript profiling of latex metabolism-related genes at the different stages of latex flow. In the present study, the expression pattern of 62 latex metabolism-related genes involved in water transportation, carbohydrate metabolism, natural rubber biosynthesis, hormone signaling, ROS generation and scavenging, and latex coagulum across three stages of latex flow between rubber tree clones CATAS7-33-97 and CATAS8-79 were comparatively analyzed by quantitative real-time PCR. The two clones show differences in latex regeneration and have a different duration of latex flow. The results showed that the expression levels of 38 genes were significantly higher in CATAS8-79 latex than in CATAS7-33-97 during latex regeneration, while 45 genes had a notably higher expression level in CATAS8-79 latex during latex flow. Together with the activation of the MEP pathway and jasmonate pathway in CATAS8-79 latex, HbPIP1;3, HbPIP1;4, HbSUT3, HbSus3, HbHMGS1-2, HbMK should contribute to the high latex regeneration ability. The up-regulation of ethylene signaling and Hb44KD and the down-regulation of latex coagulation-related genes in CATAS8-79 latex might contribute to its longer latex flow duration. This study provides some cues for revealing the regulation of latex metabolism in rubber trees.