Comparative functional analyses of PHR1, PHL1, and PHL4 transcription factors in regulating Arabidopsis responses to phosphate starvation.

To cope with phosphate (Pi) starvation, plants trigger an array of adaptive responses to sustain their growth and development. These responses are largely controlled at transcriptional levels. In Arabidopsis (Arabidopsis thaliana), PHOSPHATE RESPONSE 1 (PHR1) is a key regulator of plant physiological and transcriptional responses to Pi starvation. PHR1 belongs to a MYB-CC-type transcription factor family which contains 15 members. In this PHR1 family, PHR1/PHR1-like 1(PHL1) and PHL2/PHL3 form two distinct modules in regulating plant development and transcriptional responses to Pi starvation. PHL4 is the most closely related member to PHR1. Previously, using the phr1phl4 mutant, we showed that PHL4 is also involved in regulating plant Pi responses. However, the precise roles of PHL1 and PHL4 in regulating plant Pi responses and their functional relationships with PHR1 have not been clearly defined. In this work, we further used the phl1phl4 and phr1phl1phl4 mutants to perform comparative phenotypic and transcriptomic analyses with phr1, phr1phl1, and phr1phl4. The results showed that both PHL1 and PHL4 act redundantly and equally with PHR1 to regulate leaf senescence, Pi starvation induced-inhibition of primary root growth, and accumulation of anthocyanins in shoots. Unlike PHR1 and PHL1, however, the role of PHL4 in maintaining Pi homeostasis is negligible. In regulating transcriptional responses to Pi starvation at genomic levels, both PHL1 and PHL4 play minor roles when acts alone, however, they act synergistically with PHR1. In regulating Pi starvation-responsive genes, PHL4 also function less than PHL1 in terms of the number of the genes it regulates and the magnitude of gene transcription it affects. Furthermore, no synergistic interaction was found between PHL1 and PHL4 in regulating plant response to Pi starvation. Therefore, our results clarified the roles of PHL1 and PHL4 in regulating plant responses to Pi starvation. In addition, this work revealed a new function of these three transcription factors in regulating flowering time.

Knockout of miR396 genes increases seed size and yield in soybean.

Yield improvement has long been an important task for soybean breeding in the world in order to meet the increasing demand for food and animal feed. miR396 genes have been shown to negatively regulate grain size in rice, but whether miR396 family members may function in a similar manner in soybean is unknown. Here, we generated eight soybean mutants harboring different combinations of homozygous mutations in the six soybean miR396 genes through genome editing with clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas)12SF01 in the elite soybean cultivar Zhonghuang 302 (ZH302). Four triple mutants (mir396aci, mir396acd, mir396adf, and mir396cdf), two quadruple mutants (mir396abcd and mir396acfi), and two quintuple mutants (mir396abcdf and mir396bcdfi) were characterized. We found that plants of all the mir396 mutants produced larger seeds compared to ZH302 plants. Field tests showed that mir396adf and mir396cdf plants have significantly increased yield in growth zones with relatively high latitude which are suited for ZH302 and moderately increased yield in lower latitude. In contrast, mir396abcdf and mir396bcdfi plants have increased plant height and decreased yield in growth zones with relatively high latitude due to lodging issues, but they are suited for low latitude growth zones with increased yield without lodging problems. Taken together, our study demonstrated that loss-of-function of miR396 genes leads to significantly enlarged seed size and increased yield in soybean, providing valuable germplasms for breeding high-yield soybean.

Efficient and heritable A-to-K base editing in rice and tomato.

Cytosine and adenosine base editors (CBE and ABE) have been widely used in plants, greatly accelerating gene function research and crop breeding. Current base editors can achieve efficient A-to-G and C-to-T/G/A editing. However, efficient and heritable A-to-Y (A-to-T/C) editing remains to be developed in plants. In this study, a series of A-to-K base editor (AKBE) systems were constructed for monocot and dicot plants. Furthermore, nSpCas9 was replaced with the PAM-less Cas9 variant (nSpRY) to expand the target range of the AKBEs. Analysis of 228 T0 rice plants and 121 T0 tomato plants edited using AKBEs at 18 endogenous loci revealed that, in addition to highly efficient A-to-G substitution (41.0% on average), the plant AKBEs can achieve A-to-T conversion with efficiencies of up to 25.9 and 10.5% in rice and tomato, respectively. Moreover, the rice-optimized AKBE generates A-to-C conversion in rice, with an average efficiency of 1.8%, revealing the significant value of plant-optimized AKBE in creating genetic diversity. Although most of the A-to-T and A-to-C edits were chimeric, desired editing types could be transmitted to the T1 offspring, similar to the edits generated by the traditional ABE8e. Besides, using AKBEs to target tyrosine (Y, TAT) or cysteine (C, TGT) achieved the introduction of an early stop codon (TAG/TAA/TGA) of target genes, demonstrating its potential use in gene disruption.

Overexpression of AHL proteins enhances root hair production by altering the transcription of RHD6-downstream genes.

AT-HOOK MOTIF NUCLEAR LOCALIZED (AHL) proteins occur in all sequenced plant species. They bind to the AT-rich DNA sequences in chromosomes and regulate gene transcription related to diverse biological processes. However, the molecular mechanism underlying how AHL proteins regulate gene transcription is poorly understood. In this research, we used root hair production as a readout to study the function of two Arabidopsis AHL proteins, AHL17, and its closest homolog AHL28. Overexpression of AHL17 or AHL28 greatly enhanced root hair production by increasing the transcription of an array of genes downstream of RHD6. RHD6 is a key transcription factor that regulates root hair development. Mutation of RHD6 completely suppressed the overproduction of root hairs by blocking the transcription of AHL17-activated genes. The overexpression of AHL17 or AHL28, however, neither affected the transcription of RHD6 nor the accumulation of RHD6 protein. These two AHL proteins also did not directly interact with RHD6. Furthermore, we found that three members of the Heat Shock Protein70 family, which have been annotated as the subunits of the plant Mediator complex, could form a complex with both AHL17 and RHD6. Our research might reveal a previously unrecognized mechanism of how AHL proteins regulate gene transcription.

PHOSPHATE RESPONSE 1 family members act distinctly to regulate transcriptional responses to phosphate starvation.

To sustain growth when facing phosphate (Pi) starvation, plants trigger an array of adaptive responses that are largely controlled at transcriptional levels. In Arabidopsis (Arabidopsis thaliana), the four transcription factors of the PHOSPHATE RESPONSE 1 (PHR1) family, PHR1 and its homologs PHR1-like 1 (PHL1), PHL2, and PHL3 form the central regulatory system that controls the expression of Pi starvation-responsive (PSR) genes. However, how each of these four proteins function in regulating the transcription of PSR genes remains largely unknown. In this work, we performed comparative phenotypic and transcriptomic analyses using Arabidopsis mutants with various combinations of mutations in these four genes. The results showed that PHR1/PHL1 and PHL2/PHL3 do not physically interact with each other and function as two distinct modules in regulating plant development and transcriptional responses to Pi starvation. In the PHR1/PHL1 module, PHR1 plays a dominant role, whereas, in the PHL2/PHL3 module, PHL2 and PHL3 contribute similarly to the regulation of PSR gene transcription. By analyzing their common and specific targets, we showed that these PHR proteins could function as both positive and negative regulators of PSR gene expression depending on their targets. Some interactions between PHR1 and PHL2/PHL3 in regulating PSR gene expression were also observed. In addition, we identified a large set of defense-related genes whose expression is not affected in wild-type plants but is altered in the mutant plants under Pi starvation. These results increase our understanding of the molecular mechanism underlying plant transcriptional responses to Pi starvation.

SHORT-ROOT stabilizes PHOSPHATE1 to regulate phosphate allocation in Arabidopsis.

The coordinated distribution of inorganic phosphate (Pi) between roots and shoots is an important process that plants use to maintain Pi homeostasis. SHORT-ROOT (SHR) is well characterized for its function in root radial patterning. Here we demonstrate a role of SHR in controlling Pi allocation from root to shoot by regulating PHOSPHATE1 in the root differentiation zone. We recovered a weak mutant allele of SHR in Arabidopsis that accumulates much less Pi in the shoot and shows a constitutive Pi starvation response under Pi-sufficient conditions. In addition, Pi starvation suppresses SHR protein accumulation and releases its inhibition on the HD-ZIP III transcription factor PHB. PHB accumulates and directly binds the promoter of PHOSPHATE2 to upregulate its transcription, resulting in PHOSPHATE1 degradation in the xylem-pole pericycle cells. Our findings reveal a previously unrecognized mechanism of how plants regulate Pi translocation from roots to shoots.

Prevalence of mental disorders in the elderly population 5 years after the Lushan earthquake in Ya'an, China.

PURPOSE: This study aimed to explore the prevalence and distribution of mental disorders in the elderly population 5 years after the Lushan earthquake in Ya'an, China. METHODS: A multi-stage, group-matching random sampling method was adopted with 2579 elderly participants (≥ 60 years old) who were interviewed from January to May 2019. Preliminary screening was conducted using the scale by trained psychiatric nurses, followed by a diagnostic interview during the second stage using Chinese Version of the 5th edition of the Diagnostic and Statistical Manual of Mental Disorder by trained psychiatrists. RESULTS: A total of 2561 participants were included in this study with complete data. The weighted lifetime prevalence of all mental disorders in the elderly was 16.2% (95% CI 15.3-17.1), and the weighted 12-month prevalence was 15.2% (95% CI 13.4-17.0). Depressive disorders, anxiety disorders, substance-related and addictive disorders were the most common mental disorders. The 12-month prevalence of all mental disorders were significantly higher in the elderly living alone, with chronic somatic disease, and being poor (P < 0.05). The 12-month prevalence of posttraumatic stress disorder (PTSD) was significantly higher in the elderly in extremely severely earthquake-affected areas (P < 0.001). CONCLUSION: The results of this study show that mental health status of the elderly in Ya'an area differ by socio-economic development, geographical location, and natural disasters. The social and economic development characteristics, the impact of major natural disasters (e.g., earthquakes), and population characteristics should be combined to formulate strategies and interventions to promote the mental health of the elderly.

SIZ1 regulates phosphate deficiency-induced inhibition of primary root growth of Arabidopsis by modulating Fe accumulation and ROS production in its roots.

Inhibition of primary root (PR) growth is a typical developmental response of Arabidopsis to phosphate (Pi) deficiency. Functional disruption of SIZ1, a SUMO E3 ligase, is known to enhance the Pi deficiency-induced inhibition of PR growth. The molecular mechanism of how SIZ1 regulates PR growth under Pi deficiency, however, remains unknown. SIZ1 was recently reported to partially SUMOylate STOP1, a transcription factor that functions in plant tolerance to aluminum toxicity and in plant responses to Pi deficiency by regulating the expression of ALMT1. ALMT1 encodes an aluminum-activated malate transporter, and its expression is induced by Pi deficiency. In siz1, the expression of ALMT1 is enhanced and the removal of Fe from Pi-deficient medium suppressed the siz1 mutant phenotype. In this report, we show that siz1 overaccumulates Fe in its root apoplasts, and consequently, produces more hydroxyl radicals, which are detrimental to root growth. Such physiological changes in siz1 can be completely suppressed by the mutation of STOP1 or ALMT1. Based on previously published work and the results of the current study, we propose that SIZ1 regulates Pi deficiency-mediated PR growth through modulating the accumulation of Fe and the production of hydroxyl radicals by controlling ALMT1 expression.

Mechanism of Laccase Induction via Emodin in Trametes versicolor.

Secondary metabolites of traditional Chinese herbs can prominently stimulate the production of laccase from white rot fungi during submerged fermentation. However, the molecular mechanism through which these natural products induce the production of laccase remains unknown. In this study, the Chinese herbal medicine Polygonum cuspidatum was used to induce laccase production in Trametes versicolor, and the best inducer was identified in emodin, even under conditions of 1000-L, large-scale fermentation. Proteomics analysis identified a selection of proteins that were differentially expressed in the presence of emodin, indicating that emodin may affect the expression of laccase genes through three mechanisms: reducing bioenergy productivity, the aryl hydrocarbon receptor (AHR)/xenobiotic response element (XRE) pathway, and the nuclear erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway. Combined with protoplast flow cytometry and fluorescence, it is revealed that emodin might reduce the synthesis of ATP by lowering the mitochondrial membrane potential, leading to the subsequent responses.

Onopordopicrin from the new genus Shangwua as a novel thioredoxin reductase inhibitor to induce oxidative stress-mediated tumor cell apoptosis.

Isolation and identification of natural products from plants is an essential approach for discovering drug candidates. Herein we report the characterization of three sesquiterpene lactones from a new genus Shangwua, e.g. onopordopicrin (ONP), C2, and C3, and evaluation of their pharmacological functions in interfering cellular redox signaling. Compared to C2 and C3, ONP shows the most potency in killing cancer cells. Further experiments demonstrate that ONP robustly inhibits thioredoxin reductase (TrxR), which leads to perturbation of cellular redox homeostasis with the favor of oxidative stress. Knockdown of the TrxR sensitizes cells to the ONP treatment while overexpression of the enzyme reduces the potency of ONP, underpinning the correlation of TrxR inhibition to the cytotoxicity of ONP. The discovery of ONP expands the library of the natural TrxR inhibitors, and the disclosure of the action mechanism of ONP provides a foundation for the further development of ONP as an anticancer agent.

An epidemiological survey of mental disorders 5 years after the Lushan earthquake in Ya'an, China.

PURPOSE: This study aimed to (1) explore the prevalence and relevant influencing factors of different mental disorders 5 years after the Lushan earthquake in Ya'an, China. METHODS: An epidemiological mental health survey was conducted to identify the prevalence of mental disorders in general population in Ya'an. A multi-stage, group-matching random sampling method was adopted. Face-to-face interviews were done with a two-stage design by trained interviewers and psychiatrists. The 5th edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-V) was used for the diagnosis. RESULTS: There were 8876 participants who were interviewed in this study. The total 12-month and lifetime prevalence of all mental disorders were 12.5% and 14.7%, respectively. There was a significant difference between males and females in the prevalence patterns of several mental disorders. Han ethnic group had higher prevalence of anxiety disorders (2.7%), and the Tibetan group had higher prevalence of alcohol-related disorders (5.0%). Logistic regression analysis showed that the areas severely affected by the earthquake had significantly higher prevalence of depressive disorders, and the extremely severe affected areas had significantly higher prevalence of trauma- and stressor-related disorders. CONCLUSION: Our findings show that the prevalence of a range of mental disorders 5 years after the earthquake in Ya'an are high, and the prevalence of depressive and trauma- and stressor-related disorders may be influenced differently by the various severity of earthquake impact. This study may be crucial for the health policy-making, cultural-specific mental health services and long-term mental recovery after the earthquake.

Intramolecular Annulation of Gossypol by Laccase to Produce Safe Cottonseed Protein.

The presence of the phenol gossypol has severely limited the utilization of cottonseed meal and oil in the food and animal feed industries. Highly efficient means of biodegradation of gossypol and an understanding of the cytotoxicity of its degradation products remain outside current knowledge and are of universal interest. In this work, we showed for the first time that laccase can catalyze the intramolecular annulation of the aldehyde and hydroxyl groups of gossypol for the o-semiquinone radical and originate the released ·OH radical. It was further found that the oxidation of aldehyde groups significantly decreases reproductive toxicity and hepatotoxicity. These results indicate a novel detoxification pathway for gossypol and reveal the crucial role played by radical species in cyclization. This discovery could facilitate the development of safe, convenient, and low-cost industrial methods for the detoxification of cotton protein and oil resources.

Preparation and in Vitro Antitumor Study of Two-Dimensional Muscovite Nanosheets.

Inorganic nanosheets are endowed with many two-dimensional (2D) morphological features including ultra-high specific surface area, ultra-thin thickness, easy functionalization, and so on. They push forward an immense influence on effective cancer diagnosis and therapy, overcoming the inherent limitations of traditional treatment methods. However, long-term toxicity and poor biocompatibility are the critical issues for most inorganic nanosheets, which hinder their further oncological applications and clinical translations. Muscovite, also named white mica (WM), an aluminosilicate, is a major component of traditional Chinese medicine, which can be exfoliated into 2D nanosheets and expected to be a potential drug carrier. In this study, WM powder was exfoliated to prepare WM nanosheets (WMNs) through a polyamine intercalation method. In addition, doxorubicin hydrochloride (Dox) was loaded to WMNs via physical adsorption and electrostatic interaction to prepare Dox-loaded WMNs (Dox@WMNs). Then, we studied that Dox@WMNs released Dox in phosphate buffer saline. We also studied the cellular uptake and cytotoxicity of Dox@WMNs in vitro. The results illustrated that Dox@WMNs cumulatively released Dox much faster and more at acidic pH (6.0 and 4.6) compared with that at physiological pH. In addition, WMNs showed selective cytotoxicity. Within a certain concentration range, WMNs were cytotoxic to Hela cells but non-cytotoxic to RAW 264.7 cells. Compared with cytotoxicity at pH 7.4, the cytotoxicity of Dox@WMNs was significantly enhanced at pH 6.4 and 4.6. WMNs mainly promoted the immunostimulatory polarization of RAW 264.7 cells into M1 macrophages.

[Investigation on the Influencing Factors of Adult Dyslipidemia in Shunqing District of Nanchong City].

OBJECTIVE: To analyze the risk factors of dyslipidemia of adult residents in Shunqing District of Nanchong City. METHODS: A five-stage stratified cluster sampling method was used to select adult residents from 9 communities in the urban area of Shunqing District of Nanchong City from January 2013 to April 2018 for questionnaires survey,physical measurement and laboratory test. Univariate analysis and multivariate logistic regression analysis were used to study the influencing factors of dyslipidemia. RESULTS: A total of 105 956 people was investigated,and the prevalence rate of dyslipidemia was 34.2% (36 272 cases). Among them, the prevalence rate of male was 38.11%, and 31.91% for female ( P<0.01). The proportion of dyslipidemia with hypertension, diabetes, and coronary heart disease was 13.46%, 5.74%, and 0.39%, respectively. The proportion of hypertension with diabetes was 2.79%. Multivariate logistic regression analysis showed that gender (odds ratio ( OR)=1.276, P<0.001), body mass index ( OR=1.052, P<0.001), education level (set ≤elementary school as reference, high school OR=1.094, P<0.001, ≥graduated OR=1.185, P<0.001), smoking history ( OR=1.124, P<0.001), coronary heart disease ( OR=1.189, P<0.001), hypertension ( OR=1.148, P<0.001),sdiabetes ( OR=1.967, P<0.001), and family history of dyslipidemia ( OR=1.760, P<0.001) were the influencing factors of dyslipidemia in residents of this region. Conclusions The dyslipidemia of urban residents in Nanchong area is highly concerned with hypertension, diabetes, and coronary heart disease. Male, obesity, high education level, smoking, coronary heart disease, hypertension, diabetes, and family history of dyslipidemia are risk factors for dyslipidemia in urban residents of Nanchong area. It is necessary to actively target the above risk factors and high-risk groups.

Blue Light-Triggered Chemical Reactions Underlie Phosphate Deficiency-Induced Inhibition of Root Elongation of Arabidopsis Seedlings Grown in Petri Dishes.

To tolerate phosphate (Pi) deficiency in the environment, plants alter their developmental and metabolic programs. In the past two decades, researchers have extensively used Petri dish-grown seedlings of the model plant Arabidopsis thaliana to study the molecular mechanisms underlying root developmental responses to Pi deficiency. A typical developmental response of the Petri dish-grown Arabidopsis seedlings to Pi deficiency is the inhibited growth of primary root (PR). This response is generally thought to enhance the production of lateral roots and root hairs, which increases the plant's ability to obtain Pi and is therefore regarded as an active cellular response. Here, we report that direct illumination of root surface with blue light is critical and sufficient for Pi deficiency-induced inhibition of PR growth in Arabidopsis seedlings. We further show that a blue light-triggered malate-mediated photo-Fenton reaction and a canonical Fenton reaction form an Fe redox cycle in the root apoplast. This Fe redox cycle results in the production of hydroxyl radicals that inhibit PR growth. In addition to revealing the molecular mechanism underlying Pi deficiency-induced inhibition of PR growth, our work demonstrated that this developmental change is not an active cellular response; instead, it is a phenotype resulting from root growth in transparent Petri dishes. This finding is significant because illuminated, transparent Petri dishes have been routinely used to study Arabidopsis root responses to environmental changes.

Heliaquanoids A-E, Five Sesquiterpenoid Dimers from Inula helianthus-aquatica.

Heliaquanoid A (1), the first exo-2,4-linked Diels-Alder adduct between a pseudoguaianolide dienophile and a guaianolide diene, and heliaquanoids B-E (2-5), four new 2,4-linked Diels-Alder adducts between a xanthanolide dienophile and a guaianolide diene, were isolated from stems and leaves of Inula helianthus-aquatica. Their structures were determined by NMR spectroscopy, a modified Mosher's method, electronic circular dichroism, and X-ray diffraction analysis. Compounds 2 and 3 exhibited moderate cytotoxic activities against HL-60 cells with IC50 values of 7.5 and 4.9 µM, respectively.

Genetic Dissection of Fe-Dependent Signaling in Root Developmental Responses to Phosphate Deficiency.

The inhibition of primary root (PR) growth is a major developmental response of Arabidopsis (Arabidopsis thaliana) to phosphate (Pi) deficiency. Previous studies have independently uncovered key roles of the LOW PHOSPHATE RESPONSE1 (LPR1) ferroxidase, the tonoplast-localized ALUMINUM SENSITIVE3 (ALS3)/SENSITIVE TO ALUMINUM RHIZOTOXICITY1 (STAR1) transporter complex, and the SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1; a transcription factor)-ALUMINUM-ACTIVATED MALATE TRANSPORTER1 (ALMT1; a malate transporter) regulatory module in mediating this response by controlling iron (Fe) homeostasis in roots, but how these three components interact to regulate PR growth under Pi deficiency remains unknown. Here, we dissected genetic relationships among these three key components and found that (1) STOP1, ALMT1, and LPR1 act downstream of ALS3/STAR1 in controlling PR growth under Pi deficiency; (2) ALS3/STAR1 inhibits the STOP1-ALMT1 pathway by repressing STOP1 protein accumulation in the nucleus; and (3) STOP1-ALMT1 and LPR1 control PR growth under Pi deficiency in an interdependent manner involving the promotion of malate-dependent Fe accumulation in roots. Furthermore, this malate-mediated Fe accumulation depends on external Pi availability. We also performed a detailed analysis of the dynamic changes in the tissue-specific Fe accumulation patterns in the root tips of plants exposed to Pi deficiency. The results indicate that the degree of inhibition of PR growth induced by Pi deficiency is not linked to the level of Fe accumulated in the root apical meristem or the elongation zone. Our work provides insights into the molecular mechanism that regulates the root developmental response to Pi deficiency.