WRKY6 transcription factor modulates root potassium acquisition through promoting expression of AKT1 in Arabidopsis.

Potassium (K+), being an essential macronutrient in plants, plays a central role in many aspects. Root growth is highly plastic and is affected by many different abiotic stresses including nutrient deficiency. The Shaker-type K+ channel Arabidopsis (Arabidopsis thaliana) K+ Transporter 1 (AKT1) is responsible for K+ uptake under both low and high external K+ conditions. However, the upstream transcription factor of AKT1 is not clear. Here, we demonstrated that the WRKY6 transcription factor modulates root growth to low potassium (LK) stress in Arabidopsis. WRKY6 showed a quick response to LK stress and also to many other abiotic stress treatments. The two wrky6 T-DNA insertion mutants were highly sensitive to LK treatment, whose primary root lengths were much shorter, less biomass and lower K+ content in roots than those of wild-type plants, while WRKY6-overexpression lines showed opposite phenotypes. A further investigation showed that WRKY6 regulated the expression of the AKT1 gene via directly binding to the W-box elements in its promoter through EMSA and ChIP-qPCR assays. A dual luciferase reporter analysis further demonstrated that WRKY6 enhanced the transcription of AKT1. Genetic analysis further revealed that the overexpression of AKT1 greatly rescued the short root phenotype of the wrky6 mutant under LK stress, suggesting AKT1 is epistatic to WRKY6 in the control of LK response. Further transcriptome profiling suggested that WRKY6 modulates LK response through a complex regulatory network. Thus, this study unveils a transcription factor that modulates root growth under potassium deficiency conditions by affecting the potassium channel gene AKT1 expression.

The abscisic acid-responsive element binding factors MAPKKK18 module regulates abscisic acid-induced leaf senescence in Arabidopsis.

The mitogen-activated protein kinase kinase kinase 18 (MAPKKK18) has been reported to play a role in abiotic stress priming in long-term abscisic acid (ABA) response including drought tolerance and leaf senescence. However, the upstream transcriptional regulators of MAPKKK18 remain to be determined. Here, we report ABA-responsive element binding factors (ABFs) as upstream transcription factors of MAPKKK18 expression. Mutants of abf2, abf3, abf4, and abf2abf3abf4 dramatically reduced the transcription of MAPKKK18. Our electrophoresis mobility shift assay and dual-luciferase reporter assay demonstrated that ABF2, ABF3, and ABF4 bound to ABA-responsive element cis-elements within the promoter of MAPKKK18 to transactivate its expression. Furthermore, enrichments of the promoter region of MAPKKK18 by ABF2, ABF3, and ABF4 were confirmed by in vivo chromatin immunoprecipitation coupled with quantitative PCR. In addition, we found that mutants of mapkkk18 exhibited obvious delayed leaf senescence. Moreover, a genetic study showed that overexpression of ABF2, ABF3, and ABF4 in the background of mapkkk18 mostly phenocopied the stay-green phenotype of mapkkk18 and, expression levels of five target genes of ABFs, that is, NYE1, NYE2, NYC1, PAO, and SAG29, were attenuated as a result of MAPKKK18 mutation. These findings demonstrate that ABF2, ABF3, and ABF4 act as transcription regulators of MAPKKK18 and also suggest that, at least in part, ABA acts in priming leaf senescence via ABF-induced expression of MAPKKK18.

Genome-Wide Identification, Characterization, and Expression Analysis of Four Subgroup Members of the GH13 Family in Wheat (Triticum aestivum L.).

The glycoside hydrolase 13 (GH13) family is crucial for catalyzing α-glucoside linkages, and plays a key role in plant growth, development, and stress responses. Despite its significance, its role in plants remains understudied. This study targeted four GH13 subgroups in wheat, identifying 66 GH13 members from the latest wheat database (IWGSC RefSeq v2.1), including 36 α-amylase (AMY) members, 18 1,4-α-glucan-branching enzyme (SBE) members, 9 isoamylase (ISA) members, and 3 pullulanase (PU) members. Chromosomal distribution reveals a concentration of wheat group 7 chromosomes. Phylogenetic analysis underscores significant evolutionary distance variations among the subgroups, with distinct molecular structures. Replication events shaped subgroup evolution, particularly in regard to AMY members. Subcellular localization indicates AMY member predominance in extracellular and chloroplast regions, while others localize solely in chloroplasts, confirmed by the heterologous expression of TaSEB16 and TaAMY1 in tobacco. Moreover, 3D structural analysis shows the consistency of GH13 across species. Promoter cis-acting elements are suggested to be involved in growth, stress tolerance, and starch metabolism signaling. The RNA-seq data revealed TaGH13 expression changes under drought and submergence stress, and significant expression variation was observed between strong and weak gluten varieties during seed germination using quantitative real-time PCR (qRT-PCR), correlating with seed starch content. These findings demonstrate the pivotal role of GH13 family gene expression in wheat germination, concerning variety preference and environmental stress. Overall, this study advances the understanding of wheat GH13 subgroups, laying the groundwork for further functional studies.

Perceptions of Oncology Nurses Regarding Fertility Preservation and Providing Oncofertility Services for Men of Childbearing Age with Cancer.

This study investigated the knowledge, thoughts, and attitudes of oncology nurses in China regarding fertility preservation for male cancer patients of childbearing age, and for offering counseling or oncofertility services for the men in their care. Data was collected from 18 oncology nurses in Southwest China through voluntary self-report and in-depth interviews. The qualitative interview data were analyzed using a descriptive phenomenology method based on the lived experience of the nurses. The interviewees commonly reported 6 main concerns regarding fertility preservation (FP): their insufficient knowledge and inadequate nursing education; the importance of offering such services to cancer patients; legal vulnerability if FP information is withheld from patients; the role of the nurse in counseling; and barriers to discussing FP in practice. Nurses had a positive attitude toward FP, but most had no practical role in routinely informing male patients of their options, and the nurses believed that discussion of FP was outside their scope of practice. This study offers insight into the perceptions of oncology nurses in a developing country regarding the provision of FP services for adult male cancer patients. These results lead us to recommend that local fertility nurses should be given new training regarding FP. Furthermore, nurse-led clinics are desirable. Future research should focus on the effectiveness of nurse participation in FP counseling and referral, and how to improve the professional confidence of oncology nurses for addressing FP issues.

Dynamic Expression, Differential Regulation and Functional Diversity of the CNGC Family Genes in Cotton.

Cyclic nucleotide-gated channels (CNGCs) constitute a family of non-selective cation channels that are primarily permeable to Ca2+ and activated by the direct binding of cyclic nucleotides (i.e., cAMP and cGMP) to mediate cellular signaling, both in animals and plants. Until now, our understanding of CNGCs in cotton (Gossypium spp.) remains poorly addressed. In the present study, we have identified 40, 41, 20, 20, and 20 CNGC genes in G. hirsutum, G. barbadense, G. herbaceum, G. arboreum, and G. raimondii, respectively, and demonstrated characteristics of the phylogenetic relationships, gene structures, chromosomal localization, gene duplication, and synteny. Further investigation of CNGC genes in G. hirsutum, named GhCNGC1-40, indicated that they are not only extensively expressed in various tissues and at different developmental stages, but also display diverse expression patterns in response to hormones (abscisic acid, salicylic acid, methyl jasmonate, ethylene), abiotic (salt stress) and biotic (Verticillium dahlia infection) stimuli, which conform with a variety of cis-acting regulatory elements residing in the promoter regions; moreover, a set of GhCNGCs are responsive to cAMP signaling during cotton fiber development. Protein-protein interactions supported the functional aspects of GhCNGCs in plant growth, development, and stress responses. Accordingly, the silencing of the homoeologous gene pair GhCNGC1&18 and GhCNGC12&31 impaired plant growth and development; however, GhCNGC1&18-silenced plants enhanced Verticillium wilt resistance and salt tolerance, whereas GhCNGC12&31-silenced plants had opposite effects. Together, these results unveiled the dynamic expression, differential regulation, and functional diversity of the CNGC family genes in cotton. The present work has laid the foundation for further studies and the utilization of CNGCs in cotton genetic improvement.

The effectiveness of reed-biochar in mitigating phosphorus losses and enhancing microbially-driven phosphorus dynamics in paddy soil.

Biochar is a promising novel material for mitigating phosphorus (P) loss and enhancing P retention in chemical-amended agricultural soils. However, the optimal application rate for aforesaid effectiveness and potential drivers of the process are not well understood. Herein, a column-based pot experiment was carried out to investigate how and to what extent reed-biochar is effective in positively triggering P loss and availability in paddy soils treated by chemical fertilizer. Compared with chemical-only treatment, the accumulated leakage of total P, dissoluble P, and particulate P in chemical fertilizer coupled with 1-4% reed-biochar treatment decreased by 5.3-13.3%, 8.3-10.4%, and 3.0-15.4%, respectively. The accumulated leakage of total P and dissoluble P in 6-8% rate treatments was increased by 5.6-7.5% and 18.3-32.9%, respectively. Increasing reed-biochar rate from 1% to 8% caused an enhancement in soil total P and available P content and P activation coefficient, and the 4% rate achieved a similar effectiveness to the higher rate. Reed-biochar application increased the abundance and diversty of soil phoD-harboring microbes (P < 0.05), while the increment had little to do with the application rate. Soil phoD-harboring community composition and total C content were the main predictors of the P leaching losses, and meanwhile, the total C content was the dominated predictor of soil P retention and availability. These results suggest that adding 1-4% reed-biochar was more beneficial to mitigate paddy P loss and to enhance soil P availability. This study highlights the importance of understanding how microbial populations mediate P transformation to decipher the biochar-driven improvement of soil P utilization.

The Educational Program for Healthcare Providers Regarding Fertility Preservation for Cancer Patients: a Systematic Review.

The emerging discipline of oncofertility advocates for the timely provision of fertility preservation (FP) to all cancer patients of childbearing age by healthcare providers. A lack of practice due to limited FP-related knowledge was found among healthcare providers. A systematic review was undertaken on the educational programs on FP for healthcare providers. An initial search was performed in MEDLINE, PsycINFO, CINAHL, Web of Science, PubMed, and Scopus databases in October 2019. This review was guided by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement. Among the 160 articles that were identified, five relevant articles published between 2009 and 2019 were reviewed. Of the five studies, three were quantitative nonrandomized studies, one was a randomized controlled trial, and one was a qualitative study. Three programs were relevant to oncology nurses, one was relevant to social workers as well as nurses, and one was relevant to oncology fellows and residents. The four programs significantly increased healthcare providers' knowledge about FP, but clinical practice was only improved in the Educating Nurses about Reproductive Issues in Cancer Healthcare program (P < 0.01). Nevertheless, most of the studies used a self-made questionnaire or tool to assess the effects of the training programs. The educational programs improved the FP-related knowledge of healthcare providers but lacked the high-quality randomized controlled trials needed to provide robust evidence on the effectiveness of training programs using standard tools. More training projects should be developed based on learning theories or models to improve oncofertility care in clinical practice.

Arabidopsis CPK6 positively regulates ABA signaling and drought tolerance through phosphorylating ABA-responsive element-binding factors.

Abscisic acid (ABA) regulates numerous developmental processes and drought tolerance in plants. Calcium-dependent protein kinases (CPKs) are important Ca2+ sensors playing crucial roles in plant growth and development as well as responses to stresses. However, the molecular mechanisms of many CPKs in ABA signaling and drought tolerance remain largely unknown. Here we combined protein interaction studies, and biochemical and genetic approaches to identify and characterize substrates that were phosphorylated by CPK6 and elucidated the mechanism that underlines the role of CPK6 in ABA signaling and drought tolerance. The expression of CPK6 is induced by ABA and dehydration. Two cpk6 T-DNA insertion mutants are insensitive to ABA during seed germination and root elongation of seedlings; in contrast, overexpression of CPK6 showed the opposite phenotype. Moreover, CPK6-overexpressing lines showed enhanced drought tolerance. CPK6 interacts with and phosphorylates a subset of core ABA signaling-related transcription factors, ABA-responsive element-binding factors (ABFs/AREBs), and enhances their transcriptional activities. The phosphorylation sites in ABF3 and ABI5 were also identified through MS and mutational analyses. Taken together, we present evidence that CPK6 mediates ABA signaling and drought tolerance through phosphorylating ABFs/AREBs. This work thus uncovers a rather conserved mechanism of calcium-dependent Ser/Thr kinases in ABA signaling.

The Knowledge, Attitude, and Self-Reported Behaviors of Oncology Physicians Regarding Fertility Preservation in Adult Cancer Patients.

There is a growing concern about the fertility preservation for adult cancer patients of reproductive age. Very little literature exists about fertility preservation of cancer survivors in Chinese text. This study is first to describe the knowledge level, attitude, and practice behaviors among physicians concerning fertility preservation in adult cancer patients in China. A cross-sectional survey with 30-item was conducted to assess Chinese oncology physicians' knowledge, attitude, and behaviors regarding fertility issues. Of 360 oncology physicians, 206 (57.2%) submitted valid questionnaires. With possible overall scores for knowledge and attitude of 9 and 15, respectively, physicians' responses to the questionnaires were 3.91 ± 1.67 and 12.29 ± 1.23. Only 49.5% of physicians routinely informed their cancer patients of childbearing age about the risk of infertility with cancer treatment. The knowledge score of the men physicians was 2-fold that of the women. Physicians aged 20-29 years were significantly more likely than other age groups to prioritize cancer treatment over fertility concerns. Men physicians were significantly more comfortable than the women discussing fertility preservation issues and cooperating with fertility specialists. The oncology physicians in China had limited knowledge of fertility preservation and rarely discussed these issues with their patients, although their attitude was positive. Results suggest that oncology physicians would welcome an in-house fertility-related training program. KEY MESSAGES: This is the first study to address the topic of fertility preservation as it relates to the care that oncologists provide to cancer patients in China. These results revealed the importance of providing fertility-related training program to oncology physicians. Moreover, this study should provide useful information for other Asian countries, and highlight both the similarities and differences between China and Western countries concerning the reproductive rights of patients. This study should encourage international cooperation with institutions of scientific research and education.

Level of Knowledge and Needs on Fertility Preservation in Reproductive-Aged Male Patients with Cancer.

There is a growing concern about the fertility preservation (FP) for cancer patients of childbearing age. This study is the first in China to survey men with cancer, of reproductive age, regarding their knowledge of FP and their related needs. A 12-item cross-sectional survey was conducted of 332 male patients. The score for knowledge of FP was 3.5 ± 0.67, of a possible score of 8. Only 10.6% of the subjects had chosen to preserve fertility before treatments, but during therapy 68.7% wanted more information about FP. Younger patients were more likely have more knowledge concerning FP than older patients (odds ratio [OR] 0.86). The decision to make arrangements for FP before treatments was heavily influenced by being young and without children (OR, 0.78; OR, 0.11). Male cancer patients of reproductive age had limited knowledge of FP, and the majority was disinclined to make FP arrangements before therapy in China. Therefore, male cancer survivors should be well informed about FP soon after diagnosis and programs should be considered to improve the FP-related knowledge of male cancer survivors. We suggest that an assessment of patients' understanding of FP issues, before treatment, should be standard in clinical work.

Rapeseed calcium-dependent protein kinase CPK6L modulates reactive oxygen species and cell death through interacting and phosphorylating RBOHD.

Reactive oxygen species (ROS) play important roles in plant growth, development, responses to abiotic and biotic stresses. Hypersensitive response (HR)-like cell death is often associated with excess ROS. However, how a calcium-dependent protein kinase (CPK) modulates this process remains elusive in rapeseed (Brassica napus L.). In the present study, we identified and characterized CPK6L from rapeseed as a novel regulator of ROS and cell death. The subcellular localization of BnaCPK6L was investigated through GFP and was found to be located at the endoplasmic reticulum membrane. Overexpression of the constitutively active BnaCPK6LCA resulted in significant accumulation of ROS and HR-like cell death than the full-length. A quantitative RT-PCR survey identified that the expression levels of a few ROS, cell death and defense-related marker genes were up-regulated upon BnaCPK6LCA expression. Mating-based split ubiquitin system (mbSUS) screening revealed that BnaCPK6L interacted with BnaRBOHD (Respiratory Burst Oxidase Homolog D), which was validated by bimolecular fluorescence complementation (BiFC). An in vitro phosphorylation assay indicated that BnaCPK6L phosphorylated BnaRBOHD. Lastly, we also found that three 2C type protein phosphatases (PP2Cs) interacted with BnaCPK6L. Taken together, this study indicates that BnaCPK6L plays an important role in ROS and HR-like cell death through interacting with and phosphorylating RBOHD.

Identification and characterization of plant-specific NAC gene family in canola (Brassica napus L.) reveal novel members involved in cell death.

NAC transcription factors are plant-specific and play important roles in plant development processes, response to biotic and abiotic cues and hormone signaling. However, to date, little is known about the NAC genes in canola (or oilseed rape, Brassica napus L.). In this study, a total of 60 NAC genes were identified from canola through a systematical analysis and mining of expressed sequence tags. Among these, the cDNA sequences of 41 NAC genes were successfully cloned. The translated protein sequences of canola NAC genes with the NAC genes from representative species were phylogenetically clustered into three major groups and multiple subgroups. The transcriptional activities of these BnaNAC proteins were assayed in yeast. In addition, by quantitative real-time RT-PCR, we further observed that some of these BnaNACs were regulated by different hormone stimuli or abiotic stresses. Interestingly, we successfully identified two novel BnaNACs, BnaNAC19 and BnaNAC82, which could elicit hypersensitive response-like cell death when expressed in Nicotiana benthamiana leaves, which was mediated by accumulation of reactive oxygen species. Overall, our work has laid a solid foundation for further characterization of this important NAC gene family in canola.

Identification, expression and interaction analyses of calcium-dependent protein kinase (CPK) genes in canola (Brassica napus L.).

BACKGROUND: Canola (Brassica napus L.) is one of the most important oil-producing crops in China and worldwide. The yield and quality of canola is frequently threatened by environmental stresses including drought, cold and high salinity. Calcium is a well-known ubiquitous intracellular secondary messenger in plants. Calcium-dependent protein kinases (CPKs) are Ser/Thr protein kinases found only in plants and some protozoans. CPKs are Ca2+ sensors that have both Ca2+ sensing function and kinase activity within a single protein and play crucial roles in plant development and responses to various environmental stresses. RESULTS: In this study, we mined the available expressed sequence tags (ESTs) of B. napus and identified a total of 25 CPK genes, among which cDNA sequences of 23 genes were successfully cloned from a double haploid cultivar of canola. Phylogenetic analysis demonstrated that they could be clustered into four subgroups. The subcellular localization of five selected BnaCPKs was determined using green fluorescence protein (GFP) as the reporter. Furthermore, the expression levels of 21 BnaCPK genes in response to salt, drought, cold, heat, abscisic acid (ABA), low potassium (LK) and oxidative stress were studied by quantitative RT-PCR and were found to respond to multiple stimuli, suggesting that canola CPKs may be convergence points of different signaling pathways. We also identified and cloned five and eight Clade A basic leucine zipper (bZIP) and protein phosphatase type 2C (PP2C) genes from canola and, using yeast two-hybrid and bimolecular fluorescence complementation (BiFC), determined the interaction between individual BnaCPKs and BnabZIPs or BnaPP2Cs (Clade A). We identified novel, interesting interaction partners for some of the BnaCPK proteins. CONCLUSION: We present the sequences and characterization of CPK gene family members in canola for the first time. This work provides a foundation for further crop improvement and improved understanding of signal transduction in plants.

Arabidopsis CIPK14 positively regulates glucose response.

Calcium is a ubiquitous intracellular secondary messenger in plants. Calcineurin B-like proteins (CBLs), which contain four Ca(2+)-binding EF hand motifs, are Ca(2+) sensors and regulate a group of Ser/Thr protein kinases called CBL-interacting protein kinases (CIPKs). Although the CBL-CIPK network has been demonstrated to play crucial roles in plant development and responses to various environmental stresses in Arabidopsis, little is known about their function in glucose signaling. In the present study, we identified CIPK14 gene from Arabidopsis that play a role in glucose signaling. The subcellular localization of CIPK14 was determined using green fluorescence protein (GFP) as the reporter. Furthermore, the expression levels of CIPK14 in response to salt, drought, cold, heat, ABA, methyl viologen (MV) and glucose treatments were examined by quantitative RT-PCR and it was found to respond to multiple stimuli, suggesting that CIPK14 may be a point of convergence for several different signaling pathways. Moreover, knock-out mutation of CIPK14 rendered it more sensitive to glucose treatment. Yeast two-hybrid assay demonstrated that CIPK14 interacted with three CBLs and also with two key kinases, sucrose non-fermenting 1-related kinase (SnRK) 1.1 and SnRK1.2 implicated in glucose signaling. This is the first report to demonstrate that CIPK also plays a role in glucose signaling.

Identification and characterization of CBL and CIPK gene families in canola (Brassica napus L.).

BACKGROUND: Canola (Brassica napus L.) is one of the most important oil-producing crops in China and worldwide. The yield and quality of canola is frequently threatened by environmental stresses including drought, cold and high salinity. Calcium is a ubiquitous intracellular secondary messenger in plants. Calcineurin B-like proteins (CBLs) are Ca2+ sensors and regulate a group of Ser/Thr protein kinases called CBL-interacting protein kinases (CIPKs). Although the CBL-CIPK network has been demonstrated to play crucial roles in plant development and responses to various environmental stresses in Arabidopsis, little is known about their function in canola. RESULTS: In the present study, we identified seven CBL and 23 CIPK genes from canola by database mining and cloning of cDNA sequences of six CBLs and 17 CIPKs. Phylogenetic analysis of CBL and CIPK gene families across a variety of species suggested genome duplication and diversification. The subcellular localization of three BnaCBLs and two BnaCIPKs were determined using green fluorescence protein (GFP) as the reporter. We also demonstrated interactions between six BnaCBLs and 17 BnaCIPKs using yeast two-hybrid assay, and a subset of interactions were further confirmed by bimolecular fluorescence complementation (BiFC). Furthermore, the expression levels of six selected BnaCBL and 12 BnaCIPK genes in response to salt, drought, cold, heat, ABA, methyl viologen (MV) and low potassium were examined by quantitative RT-PCR and these CBL or CIPK genes were found to respond to multiple stimuli, suggesting that the canola CBL-CIPK network may be a point of convergence for several different signaling pathways. We also performed a comparison of interaction patterns and expression profiles of CBL and CIPK in Arabidospsis, canola and rice, to examine the differences between orthologs, highlighting the importance of studying CBL-CIPK in canola as a prerequisite for improvement of this crop. CONCLUSIONS: Our findings indicate that CBL and CIPK family members may form a dynamic complex to respond to different abiotic or hormone signaling. Our comparative analyses of the CBL-CIPK network between canola, Arabidopsis and rice highlight functional differences and the necessity to study CBL-CIPK gene functions in canola. Our data constitute a valuable resource for CBL and CPK genomics.

Identification and functional analysis of mitogen-activated protein kinase kinase kinase (MAPKKK) genes in canola (Brassica napus L.).

Mitogen-activated protein kinase (MAPK) signalling cascades, consisting of three types of reversibly phosphorylated kinases (MAPKKK, MAPKK, and MAPK), are involved in important processes including plant immunity and hormone responses. The MAPKKKs comprise the largest family in the MAPK cascades, yet only a few of these genes have been associated with physiological functions, even in the model plant Arabidopsis thaliana. Canola (Brassica napus L.) is one of the most important oilseed crops in China and worldwide. To explore MAPKKK functions in biotic and abiotic stress responses in canola, 66 MAPKKK genes were identified and 28 of them were cloned. Phylogenetic analysis of these canola MAPKKKs with homologous genes from representative species classified them into three groups (A-C), comprising four MAPKKKs, seven ZIKs, and 17 Raf genes. A further 15 interaction pairs between these MAPKKKs and the downstream BnaMKKs were identified through a yeast two-hybrid assay. The interactions were further validated through bimolecular fluorescence complementation (BiFC) analysis. In addition, by quantitative real-time reverse transcription-PCR, it was further observed that some of these BnaMAPKKK genes were regulated by different hormone stimuli, abiotic stresses, or fungal pathogen treatments. Interestingly, two novel BnaMAPKKK genes, BnaMAPKKK18 and BnaMAPKKK19, which could elicit hypersensitive response (HR)-like cell death when transiently expressed in Nicotiana benthamiana leaves, were successfully identified. Moreover, it was found that BnaMAPKKK19 probably mediated cell death through BnaMKK9. Overall, the present work has laid the foundation for further characterization of this important MAPKKK gene family in canola.

Endogenous cAMP elevation in Brassica napus causes changes in phytohormone levels.

In higher plants, the regulatory roles of cAMP (cyclic adenosine 3',5'-monophosphate) signaling remain elusive until now. Cellular cAMP levels are generally much lower in higher plants than in animals and transiently elevated for triggering downstream signaling events. Moreover, plant adenylate cyclase (AC) activities are found in different moonlighting multifunctional proteins, which may pose additional complications in distinguishing a specific signaling role for cAMP. Here, we have developed rapeseed (Brassica napus L.) transgenic plants that overexpress an inducible plant-origin AC activity for generating high AC levels much like that in animal cells, which served the genetic model disturbing native cAMP signaling as a whole in plants. We found that overexpression of the soluble AC activity had significant impacts on the contents of indole-3-acetic acid (IAA) and stress phytohormones, i.e. jasmonic acid (JA), abscisic acid (ABA), and salicylic acid (SA) in the transgenic plants. Acute induction of the AC activity caused IAA overaccumulation, and upregulation of TAA1 and CYP83B1 in the IAA biosynthesis pathways, but also simultaneously the hyper-induction of PR4 and KIN2 expression indicating activation of JA and ABA signaling pathways. We observed typical overgrowth phenotypes related to IAA excess in the transgenic plants, including significant increases in plant height, internode length, width of leaf blade, petiole length, root length, and fresh shoot biomass, as well as the precocious seed development, as compared to wild-type plants. In addition, we identified a set of 1465 cAMP-responsive genes (CRGs), which are most significantly enriched in plant hormone signal transduction pathway, and function mainly in relevance to hormonal, abiotic and biotic stress responses, as well as growth and development. Collectively, our results support that cAMP elevation impacts phytohormone homeostasis and signaling, and modulates plant growth and development. We proposed that cAMP signaling may be critical in configuring the coordinated regulation of growth and development in higher plants.

Identification and analysis of MKK and MPK gene families in canola (Brassica napus L.).

BACKGROUND: Eukaryotic mitogen-activated protein kinase (MAPK/MPK) signaling cascades transduce and amplify environmental signals via three types of reversibly phosphorylated kinases to activate defense gene expression. Canola (oilseed rape, Brassica napus) is a major crop in temperate regions. Identification and characterization of MAPK and MAPK kinases (MAPKK/MKK) of canola will help to elucidate their role in responses to abiotic and biotic stresses. RESULTS: We describe the identification and analysis of seven MKK (BnaMKK) and 12 MPK (BnaMPK) members from canola. Sequence alignments and phylogenetic analyses of the predicted amino acid sequences of BnaMKKs and BnaMPKs classified them into four different groups. We also examined the subcellular localization of four and two members of BnaMKK and BnaMPK gene families, respectively, using green fluorescent protein (GFP) and, found GFP signals in both nuclei and cytoplasm. Furthermore, we identified several interesting interaction pairs through yeast two-hybrid (Y2H) analysis of interactions between BnaMKKs and BnaMPKs, as well as BnaMPK and BnaWRKYs. We defined contiguous signaling modules including BnaMKK9-BnaMPK1/2-BnaWRKY53, BnaMKK2/4/5-BnaMPK3/6-BnaWRKY20/26 and BnaMKK9-BnaMPK5/9/19/20. Of these, several interactions had not been previously described in any species. Selected interactions were validated in vivo by a bimolecular fluorescence complementation (BiFC) assay. Transcriptional responses of a subset of canola MKK and MPK genes to stimuli including fungal pathogens, hormones and abiotic stress treatments were analyzed through real-time RT-PCR and we identified a few of BnaMKKs and BnaMPKs responding to salicylic acid (SA), oxalic acid (OA), Sclerotinia sclerotiorum or other stress conditions. Comparisons of expression patterns of putative orthologs in canola and Arabidopsis showed that transcript expression patterns were generally conserved, with some differences suggestive of sub-functionalization. CONCLUSIONS: We identified seven MKK and 12 MPK genes from canola and examined their phylogenetic relationships, transcript expression patterns, subcellular localization, and protein-protein interactions. Not all expression patterns and interactions were conserved between canola and Arabidopsis, highlighting the limitations of drawing inferences about crops from model species. The data presented here provide the first systematic description of MKK-MPK-WRKY signaling modules in canola and will further improve our understanding of defense responses in general and provide a basis for future crop improvement.

Reactive oxygen species may be involved in the distinctive biological effects of different doses of 12C6+ ion beams on Arabidopsis.

Introduction: Heavy ion beam is a novel approach for crop mutagenesis with the advantage of high energy transfer line density and low repair effect after injury, however, little investigation on the biological effect on plant was performed. 50 Gy irradiation significantly stimulated the growth of Arabidopsis seedlings, as indicated by an increase in root and biomass, while 200 Gy irradiation significantly inhibited the growth of seedlings, causing a visible decrease in plant growth. Methods: The Arabidopsis seeds were irradiated by 12C6+. Monte Carlo simulations were used to calculate the damage to seeds and particle trajectories by ion implantation. The seed epidermis received SEM detection and changes in its organic composition were detected using FTIR. Evidence of ROS and antioxidant systems were analyzed. RNA-seq and qPCR were used to detect changes in seedling transcript levels. Results and discussion: Monte Carlo simulations revealed that high-dose irradiation causes various damage. Evidence of ROS and antioxidant systems implies that the emergence of phenotypes in plant cells may be associated with oxidative stress. Transcriptomic analysis of the seedlings demonstrated that 170 DEGs were present in the 50 Gy and 200 Gy groups and GO enrichment indicated that they were mainly associated with stress resistance and cell wall homeostasis. Further GO enrichment of DEGs unique to 50 Gy and 200 Gy revealed 58 50Gy-exclusive DEGs were enriched in response to oxidative stress and jasmonic acid entries, while 435 200 Gy-exclusive DEGs were enriched in relation to oxidative stress, organic cyclic compounds, and salicylic acid. This investigation advances our insight into the biological effects of heavy ion irradiation and the underlying mechanisms.

Exploring High Thermal Conductivity Amorphous Polymers Using Reinforcement Learning.

Developing amorphous polymers with desirable thermal conductivity has significant implications, as they are ubiquitous in applications where thermal transport is critical. Conventional Edisonian approaches are slow and without guarantee of success in material development. In this work, using a reinforcement learning scheme, we design polymers with thermal conductivity above 0.400 W/m·K. We leverage a machine learning model trained against 469 thermal conductivity data calculated from high-throughput molecular dynamics (MD) simulations as the surrogate for thermal conductivity prediction, and we use a recurrent neural network trained with around one million virtual polymer structures as a polymer generator. For all generated polymers with thermal conductivity ≥0.400 W/m·K, we have evaluated their synthesizability by calculating the synthetic accessibility score and validated the thermal conductivity of selected polymers using MD simulations. The best thermally conductive polymer designed has an MD-calculated thermal conductivity of 0.693 W/m·K, which is also estimated to be easily synthesizable. Our demonstrated inverse design scheme based on reinforcement learning may advance polymer development with target properties, and the scheme can also be generalized to other material development tasks for different applications.