The dynamic arms race during the early invasion of woodland strawberry by Botrytis cinerea revealed by dual dense high-resolution RNA-seq analyses.

Necrotrophic pathogens replicate massively upon colonizing plants, causing large-scale wilting and death of plant tissues. Understanding both mechanisms of pathogen invasion and host response processes prior to symptom appearance and their key regulatory networks is therefore important for defense against pathogen attack. Here, we investigated the mechanisms of interaction between woodland strawberry (Fragaria vesca) leaves and gray mold pathogen (Botrytis cinerea) at 14 infection time points during the first 12 hours of the infection period using a dense, high-resolution time series dual transcriptomic analysis, characterizing the arms race between strawberry F. vesca and B. cinerea before the appearance of localized lesions. Strawberry leaves rapidly initiated strong systemic defenses at the first sign of external stimulation and showed lower levels of transcriptomic change later in the infection process. Unlike the host plants, B. cinerea showed larger-scale transcriptomic changes that persisted throughout the infection process. Weighted gene co-expression network analysis identified highly correlated genes in 32 gene expression modules between B. cinerea and strawberry. Yeast two-hybrid and bimolecular fluorescence complementation assays revealed that the disease response protein FvRLP2 from woodland strawberry interacted with the cell death inducing proteins BcXYG1 and BcPG3 from B. cinerea. Overexpression of FvRLP2 in both strawberry and Arabidopsis inhibited B. cinerea infection, confirming these genes' respective functions. These findings shed light on the arms race process by which B. cinerea invades host plants and strawberry to defend against pathogen infection.

Genome-Wide Identification, Characterization, and Expression Analysis of Long-Chain Acyl-CoA Synthetases in Carya illinoinensis under Different Treatments.

As crucial enzymes in the lipid metabolic network, long-chain acyl-CoA synthases (LACSs) are members of the acyl-activated enzyme superfamily and play a crucial role in epidermal wax synthesis, plant lipid anabolic metabolism, and stress tolerance. In this study, 11 pecan LACS genes were identified and categorized into five groups and located on nine chromosomes. The significant degree of conservation in the AtLACS and CiLACS protein sequences was demonstrated by multiple sequence alignment and conserved motif analysis. Cis-acting element analysis identified numerous stress-responsive and hormone-inducible elements in the promoter regions of CiLACS genes. The expression levels of CiLACS9 and CiLACS9-1 were considerably up-regulated under salt and drought stress, according to the qRT-RCR study. Treatment with ABA also led to increased expression levels of CiLACS1, CiLACS1-1, CiLACS2, and CiLACS9-1. Notably, CiLACS4, CiLACS4-1, CiLACS9, and CiLACS9-1 exhibited peak expression levels at 135 days after anthesis and are likely to have been crucial in the accumulation of seed kernel oil. Moreover, the CiLACS9 gene was shown to be located in the cytoplasm. These findings offer a theoretical framework for clarifying the roles of LACS genes in the processes of pecan kernel oil synthesis and response to abiotic stressors.

Asparagine-rich protein (NRP) mediates stress response by regulating biosynthesis of plant secondary metabolites in Arabidopsis.

The plant-specific stress response protein NRP (asparagine-rich protein) is characterized by an asparagine-rich domain at its N-terminus and a conserved development and cell death (DCD) domain at its C-terminus. Previous transcriptional studies and phenotypic analyses have demonstrated the involvement of NRP in response to severe stress conditions, such as high salt and ER Endoplasmic reticulum-stress. We have recently identified distinct roles for NRP in biotic- and abiotic-stress signaling pathways, in which NRP interacts with different signaling proteins to change their subcellular localizations and stability. Here, to further explore the function of NRP, a transcriptome analysis was carried out on nrp1nrp2 knock-out lines at different life stages or under different growing conditions. The most significant changes in the transcriptome at both stages and conditions turned out to be the induction of the synthesis of secondary metabolites (SMs). Such an observation implicates that NRP is a general stress-responsive protein involved in various challenges faced by plants during their life cycle, which might involve a broad alteration in the distribution of SMs.

Ammonium-nitrate mixtures dominated by NH4+-N promote the growth of pecan (Carya illinoinensis) through enhanced N uptake and assimilation.

Nitrogen (N) limits plant productivity, and its uptake and assimilation may be regulated by N sources, N assimilating enzymes, and N assimilation genes. Mastering the regulatory mechanisms of N uptake and assimilation is a key way to improve plant nitrogen use efficiency (NUE). However, it is poorly known how these factors interact to influence the growth process of pecans. In this study, the growth, nutrient uptake and N assimilation characteristics of pecan were analyzed by aeroponic cultivation at varying NH4 +/NO3 - ratios (0/0, 0/100,25/75, 50/50, 75/25,100/0 as CK, T1, T2, T3, T4, and T5). The results showed that T4 and T5 treatments optimally promoted the growth, nutrient uptake and N assimilating enzyme activities of pecan, which significantly increased aboveground biomass, average relative growth rate (RGR), root area, root activity, free amino acid (FAA) and total organic carbon (TOC) concentrations, nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (Fd-GOGAT and NADH-GOGAT), and glutamate dehydrogenase (GDH) activities. According to the qRT-PCR results, most of the N assimilation genes were expressed at higher levels in leaves and were mainly significantly up-regulated under T1 and T4 treatments. Correlation analysis showed that a correlation between N assimilating enzymes and N assimilating genes did not necessarily exist. The results of partial least squares path model (PLS-PM) analysis indicated that N assimilation genes could affect the growth of pecan by regulating N assimilation enzymes and nutrients. In summary, we suggested that the NH4 +/NO3 - ratio of 75:25 was more beneficial to improve the growth and NUE of pecan. Meanwhile, we believe that the determination of plant N assimilation capacity should be the result of a comprehensive analysis of N concentration, N assimilation enzymes and related genes.

Identification and Expression Analysis of MPK and MKK Gene Families in Pecan (Carya illinoinensis).

Mitogen-activated protein kinases consist of three kinase modules composed of MPKs, MKKs, and MPKKKs. As members of the protein kinase (PK) superfamily, they are involved in various processes, such as developmental programs, cell division, hormonal progression, and signaling responses to biotic and abiotic stresses. In this study, a total of 18 MPKs and 10 MKKs were annotated on the pecan genome, all of which could be classified into four subgroups, respectively. The gene structures and conserved sequences of family members in the same branch were relatively similar. All MPK proteins had a conserved motif TxY, and D(L/I/V)K and VGTxxYMSPER existed in all MKK proteins. Duplication events contributed largely to the expansion of the pecan MPK and MKK gene families. Phylogenetic analysis of protein sequences from six plants indicated that species evolution occurred in pecan. Organ-specific expression profiles of MPK and MKK showed functional diversity. Ka/Ks values indicated that all genes with duplicated events underwent strong negative selection. Seven CiPawMPK and four CiPawMKK genes with high expression levels were screened by transcriptomic data from different organs, and these candidates were validated by qRT-PCR analysis of hormone-treated and stressed samples.

Evolution and functional analysis of the GRAS family genes in six Rosaceae species.

BACKGROUND: GRAS genes formed one of the important transcription factor gene families in plants, had been identified in several plant species. The family genes were involved in plant growth, development, and stress resistance. However, the comparative analysis of GRAS genes in Rosaceae species was insufficient. RESULTS: In this study, a total of 333 GRAS genes were identified in six Rosaceae species, including 51 in strawberry (Fragaria vesca), 78 in apple (Malus domestica), 41 in black raspberry (Rubus occidentalis), 59 in European pear (Pyrus communis), 56 in Chinese rose (Rosa chinensis), and 48 in peach (Prunus persica). Motif analysis showed the VHIID domain, SAW motif, LR I region, and PFYRE motif were considerably conserved in the six Rosaceae species. All GRAS genes were divided into 10 subgroups according to phylogenetic analysis. A total of 15 species-specific duplicated clades and 3 lineage-specific duplicated clades were identified in six Rosaceae species. Chromosomal localization presented the uneven distribution of GRAS genes in six Rosaceae species. Duplication events contributed to the expression of the GRAS genes, and Ka/Ks analysis suggested the purification selection as a major force during the evolution process in six Rosaceae species. Cis-acting elements and GO analysis revealed that most of the GRAS genes were associated with various environmental stress in six Rosaceae species. Coexpression network analysis showed the mutual regulatory relationship between GRAS and bZIP genes, suggesting the ability of the GRAS gene to regulate abiotic stress in woodland strawberry. The expression pattern elucidated the transcriptional levels of FvGRAS genes in various tissues and the drought and salt stress in woodland strawberry, which were verified by RT-qPCR analysis. CONCLUSIONS: The evolution and functional analysis of GRAS genes provided insights into the further understanding of GRAS genes on the abiotic stress of Rosaceae species.

Genome-Wide Identification and Expression Analysis of AMT and NRT Gene Family in Pecan (Carya illinoinensis) Seedlings Revealed a Preference for NH4+-N.

Nitrogen (N) is a major limiting factor for plant growth and crop production. The use of N fertilizer in forestry production is increasing each year, but the loss is substantial. Mastering the regulatory mechanisms of N uptake and transport is a key way to improve plant nitrogen use efficiency (NUE). However, this has rarely been studied in pecans. In this study, 10 AMT and 69 NRT gene family members were identified and systematically analyzed from the whole pecan genome using a bioinformatics approach, and the expression patterns of AMT and NRT genes and the uptake characteristics of NH4+ and NO3- in pecan were analyzed by aeroponic cultivation at varying NH4+/NO3- ratios (0/0, 0/100,25/75, 50/50, 75/25,100/0 as CK, T1, T2, T3, T4, and T5). The results showed that gene duplication was the main reason for the amplification of the AMT and NRT gene families in pecan, both of which experienced purifying selection. Based on qRT-PCR results, CiAMTs were primarily expressed in roots, and CiNRTs were majorly expressed in leaves, which were consistent with the distribution of pecan NH4+ and NO3- concentrations in the organs. The expression levels of CiAMTs and CiNRTs were mainly significantly upregulated under N deficiency and T4 treatment. Meanwhile, T4 treatment significantly increased the NH4+, NO3-, and NO2- concentrations as well as the Vmax and Km values of NH4+ and NO3- in pecans, and Vmax/Km indicated that pecan seedlings preferred to absorb NH4+. In summary, considering the single N source of T5, we suggested that the NH4+/NO3- ratio of 75:25 was more beneficial to improve the NUE of pecan, thus increasing pecan yield, which provides a theoretical basis for promoting the scale development of pecan and provides a basis for further identification of the functions of AMT and NRT genes in the N uptake and transport process of pecan.

Insight into the CBL and CIPK gene families in pecan (Carya illinoinensis): identification, evolution and expression patterns in drought response.

BACKGROUND: Calcium (Ca2+) serves as a ubiquitous second messenger and plays a pivotal role in signal transduction. Calcineurin B-like proteins (CBLs) are plant-specific Ca2+ sensors that interact with CBL-interacting protein kinases (CIPKs) to transmit Ca2+ signals. CBL-CIPK complexes have been reported to play pivotal roles in plant development and response to drought stress; however, limited information is available about the CBL and CIPK genes in pecan, an important nut crop. RESULTS: In the present study, a total of 9 CBL and 30 CIPK genes were identified from the pecan genome and divided into four and five clades based on phylogeny, respectively. Gene structure and distribution of conserved sequence motif analysis suggested that family members in the same clade commonly exhibited similar exon-intron structures and motif compositions. The segmental duplication events contributed largely to the expansion of pecan CBL and CIPK gene families, and Ka/Ks values revealed that all of them experienced strong negative selection. Phylogenetic analysis of CIPK proteins from 14 plant species revealed that CIPKs in the intron-poor clade originated in seed plants. Tissue-specific expression profiles of CiCBLs and CiCIPKs were analysed, presenting functional diversity. Expression profiles derived from RNA-Seq revealed distinct expression patterns of CiCBLs and CiCIPKs under drought treatment in pecan. Moreover, coexpression network analysis helped to elucidate the relationships between these genes and identify potential candidates for the regulation of drought response, which were verified by qRT-PCR analysis. CONCLUSIONS: The characterization and analysis of CBL and CIPK genes in pecan genome could provide a basis for further functional analysis of CiCBLs and CiCIPKs in the drought stress response of pecan.

Nanopore long-read RNAseq reveals transcriptional variations in citrus species.

The number of studies on plant transcriptomes using ONT RNAseq technology is rapidly increasing in recent. It is a powerful method to decipher transcriptomic complexity, particularly alternative splicing (AS) event detection. Citrus plants are the most important widely grown fruit crops. Exploring different AS events in citrus contributes to transcriptome improvement and functional genome study. Here, we performed ONT RNAseq in 9 species (Atalantia buxifolia, Citrus clementina, C. grandis, C. ichangensis, C. reticulata, C. sinensis, Clausena lansium, Fortunella hindsii, and Poncirus trifoliata), accompanied with Illumina sequencing. Non-redundant full-length isoforms were identified between 41,957 and 76,974 per species. Systematic analysis including different types of isoforms, number of isoforms per gene locus, isoform distribution, ORFs and lncRNA prediction and functional annotation were performed mainly focused on novel isoforms, unraveling the capability of novel isoforms detection and characterization. For AS events prediction, A3, RI, and AF were overwhelming types across 9 species. We analyzed isoform similarity and evolutionary relationships in all species. We identified that multiple isoforms derived from orthologous single copy genes among different species were annotated as enzymes, nuclear-related proteins or receptors. Isoforms with extending sequences on 5', 3', or both compared with reference genome were filtered out to provide information for transcriptome improvement. Our results provide novel insight into comprehending complex transcriptomes in citrus and valuable information for further investigation on the function of genes with diverse isoforms.

Discovery of Dosimertinib, a Highly Potent, Selective, and Orally Efficacious Deuterated EGFR Targeting Clinical Candidate for the Treatment of Non-Small-Cell Lung Cancer.

Osimertinib is a highly potent and selective third-generation epidermal growth factor receptor (EGFR) inhibitor, which provides excellent clinical benefits and is now a standard-of-care therapy for advanced EGFR mutation-positive non-small-cell lung cancer (NSCLC). However, AZ5104, a primary toxic metabolite of osimertinib, has caused unwanted toxicities. To address this unmet medical need, we initiated an iterative program focusing on structural optimizations of osimertinib and preclinical characterization, leading to the discovery of a highly potent, selective, and orally efficacious deuterated EGFR-targeting clinical candidate, dosimertinib. Preclinical studies revealed that dosimertinib demonstrated robust in vivo antitumor efficacy and favorable PK profiles, but with lower toxicity than osimertinib. These preclinical data support further clinical development of dosimertinib for the treatment of NSCLC. Dosimertinib has received official approval in China to initiate the phase I clinical trial (registration numbers: CXHL2000060 and CXHL2000061).

The emergence and evolution of intron-poor and intronless genes in intron-rich plant gene families.

Eukaryotic genes can be classified into intronless (no introns), intron-poor (three or fewer introns per gene) or intron-rich. Early eukaryotic genes were mostly intron-rich, and their alternative splicing into multiple transcripts, giving rise to different proteins, might have played pivotal roles in adaptation and evolution. Interestingly, extant plant genomes contain many gene families with one or sometimes few sub-families with genes that are intron-poor or intronless, and it remains unknown when and how these intron-poor or intronless genes have originated and evolved, and what their possible functions are. In this study, we identified 33 such gene families that contained intronless and intron-poor sub-families. Intronless genes seemed to have first emerged in early land plant evolution, while intron-poor sub-families seemed first to have appeared in green algae. In contrast to intron-rich genes, intronless genes in intron-poor sub-families occurred later, and were subject to stronger functional constraints. Based on RNA-seq analyses in Arabidopsis and rice, intronless or intron-poor genes in AP2, EF-hand_7, bZIP, FAD_binding_4, STE_STE11, CAMK_CAMKL-CHK1 and C2 gene families were more likely to play a role in response to drought and salt stress, compared with intron-rich genes in the same gene families, whereas intronless genes in the B_lectin and S_locus_glycop gene family were more likely to participate in epigenetic processes and plant development. Understanding the origin and evolutionary trajectory, as well as the potential functions, of intronless and intron-poor sub-families provides further insight into plant genome evolution and the functional divergence of genes.

The wild strawberry kinome: identification, classification and transcript profiling of protein kinases during development and in response to gray mold infection.

BACKGROUND: Protein kinases (PKs) play an important role in signaling cascades and are one of the largest and most conserved protein super families in plants. Despite their importance, the woodland strawberry (Fragaria vesca) kinome and expression patterns of PK genes remain to be characterized. RESULTS: Here, we report on the identification and classification of 954 Fragaria vesca PK genes, which were classified into nine groups and 124 gene families. These genes were distributed unevenly among the seven chromosomes, and the number of introns per gene varied from 0 to 47. Almost half of the putative PKs were predicted to localize to the nucleus and 24.6% were predicted to localize to the cell membrane. The expansion of the woodland strawberry PK gene family occurred via different duplication mechanisms and tandem duplicates occurred relatively late as compared to other duplication types. Moreover, we found that tandem and transposed duplicated PK gene pairs had undergone stronger diversifying selection and evolved relatively faster than WGD genes. The GO enrichment and transcriptome analysis implicates the involvement of strawberry PK genes in multiple biological processes and molecular functions in differential tissues, especially in pollens. Finally, 109 PKs, mostly the receptor-like kinases (RLKs), were found transcriptionally responsive to Botrytis cinerea infection. CONCLUSIONS: The findings of this research expand the understanding of the evolutionary dynamics of PK genes in plant species and provide a potential link between cell signaling pathways and pathogen attack.

Preoperative albumin-to-globulin ratio predicts survival in patients with non-small-cell lung cancer after surgery.

The prognostic value of the preoperative albumin-to-globulin ratio (AGR) has not been investigated in non-small-cell lung cancer (NSCLC). Therefore, we aimed to assess the clinical applicability of the preoperative AGR to predict the prognosis in patients with NSCLC. We retrospectively enrolled 545 patients with stage I/II/III NSCLC who underwent surgery at our institution. The cutoff value for preoperative AGR was calculated by using a receiver operating characteristic curve analysis. A low AGR was associated with several clinicopathological variables related to tumor progression. In the multivariate analyses, the preoperative AGR was identified as an independent prognostic factor for disease-free survival (DFS; P = 0.003) and overall survival (OS; P = 0.005). For patients with stage II and III with a preoperative AGR ≤ 1.43, the surgery plus chemotherapy group had a significantly longer DFS and OS than the surgery alone group (P = 0.002 and P = 0.001, respectively); however, a significant difference in DFS and OS between these two groups was not observed in patients with stage II and III with an AGR > 1.43 (P = 0.808 and P = 0.842, respectively). The preoperative AGR is an independent, significant predictor of DFS and OS in patients with NSCLC. Our results also demonstrate that the preoperative AGR might be a predictive marker of the therapeutic effect of postoperative chemotherapy in patients with stage II and III NSCLC.

The kinome of pineapple: catalog and insights into functions in crassulacean acid metabolism plants.

BACKGROUND: Crassulacean acid metabolism (CAM) plants use water 20-80% more efficiently by shifting stomata opening and primary CO2 uptake and fixation to the nighttime. Protein kinases (PKs) play pivotal roles in this biological process. However, few PKs have been functionally analyzed precisely due to their abundance and potential functional redundancy (caused by numerous gene duplications). RESULTS: In this study, we systematically identified a total of 758 predicted PK genes in the genome of a CAM plant, pineapple (Ananas comosus). The pineapple kinome was classified into 20 groups and 116 families based on the kinase domain sequences. The RLK was the largest group, containing 480 members, and over half of them were predicted to locate at the plasma membrane. Both segmental and tandem duplications make important contributions to the expansion of pineapple kinome based on the synteny analysis. Ka/Ks ratios showed all of the duplication events were under purifying selection. The global expression analysis revealed that pineapple PKs exhibit different tissue-specific and diurnal expression patterns. Forty PK genes in a cluster performed higher expression levels in green leaf tip than in white leaf base, and fourteen of them had strong differential expression patterns between the photosynthetic green leaf tip and the non-photosynthetic white leaf base tissues. CONCLUSIONS: Our findings provide insights into the evolution and biological function of pineapple PKs and a foundation for further functional analysis of PKs in CAM plants. The gene duplication, expression, and coexpression analysis helped us to rapidly identify the key candidates in pineapple kinome, which may play roles in the carbon fixation process in pineapple and help engineering CAM pathway into C3 crops for improved drought tolerance.

The grapevine kinome: annotation, classification and expression patterns in developmental processes and stress responses.

Protein kinases (PKs) have evolved as the largest family of molecular switches that regulate protein activities associated with almost all essential cellular functions. Only a fraction of plant PKs, however, have been functionally characterized even in model plant species. In the present study, the entire grapevine kinome was identified and annotated using the most recent version of the grapevine genome. A total of 1168 PK-encoding genes were identified and classified into 20 groups and 121 families, with the RLK-Pelle group being the largest, with 872 members. The 1168 kinase genes were unevenly distributed over all 19 chromosomes, and both tandem and segmental duplications contributed to the expansion of the grapevine kinome, especially of the RLK-Pelle group. Ka/Ks values indicated that most of the tandem and segmental duplication events were under purifying selection. The grapevine kinome families exhibited different expression patterns during plant development and in response to various stress treatments, with many being coexpressed. The comprehensive annotation of grapevine kinase genes, their patterns of expression and coexpression, and the related information facilitate a more complete understanding of the roles of various grapevine kinases in growth and development, responses to abiotic stress, and evolutionary history.

Clinical significance of cripto-1 expression in lung adenocarcinoma.

Cripto-1 can promote tumourigenesis and may be a potential prognostic biomarker in several malignancies, yet little is known about this protein in lung adenocarcinoma (LAC). The aim of this study was to evaluate the prognostic value of cripto-1 expression in a cohort of patients with LAC. Tumours from 290 patients with pathologically confirmed LAC were used for an immunohistochemical analysis of cripto-1 expression. The correlation between cripto-1 expression and the clinicopathological parameters of patients, EGFR-TKI sensitivity was analysed. Significant associations between cripto-1 expression and pT status, pN status, pTNM status, E-cadherin expression and EGFR-TKI sensitivity were identified. Compared with patients with low cripto-1 expression, patients with high cripto-1 expression exhibited significantly poorer progression-free survival (PFS) and overall survival (OS). Moreover, multivariate analyses showed that high cripto-1 expression was an independent predictor of worse survival of patients with LAC. The combination of cripto-1 expression and serum CEA level was correlated with both PFS and OS. In conclusion, cripto-1 may be a potential prognostic biomarker of survival in patients with LAC.

Methane protects against polyethylene glycol-induced osmotic stress in maize by improving sugar and ascorbic acid metabolism.

Although aerobic methane (CH4) release from plants leads to an intense scientific and public controversy in the recent years, the potential functions of endogenous CH4 production in plants are still largely unknown. Here, we reported that polyethylene glycol (PEG)-induced osmotic stress significantly increased CH4 production and soluble sugar contents in maize (Zea mays L.) root tissues. These enhancements were more pronounced in the drought stress-tolerant cultivar Zhengdan 958 (ZD958) than in the drought stress-sensitive cultivar Zhongjiangyu No.1 (ZJY1). Exogenously applied 0.65 mM CH4 not only increased endogenous CH4 production, but also decreased the contents of thiobarbituric acid reactive substances. PEG-induced water deficit symptoms, such as decreased biomass and relative water contents in both root and shoot tissues, were also alleviated. These beneficial responses paralleled the increases in the contents of soluble sugar and the reduced ascorbic acid (AsA), and the ratio of AsA/dehydroascorbate (DHA). Further comparison of transcript profiles of some key enzymes in sugar and AsA metabolism suggested that CH4 might participate in sugar signaling, which in turn increased AsA production and recycling. Together, these results suggested that CH4 might function as a gaseous molecule that enhances osmotic stress tolerance in maize by modulating sugar and AsA metabolism.

Methane alleviates copper-induced seed germination inhibition and oxidative stress in Medicago sativa.

Recent results discovered the protective roles of methane (CH4) against oxidative stress in animals. However, the possible physiological roles of CH4 in plants are still unknown. By using physiological, histochemical and molecular approaches, the beneficial role of CH4 in germinating alfalfa seeds upon copper (Cu) stress was evaluated. Endogenous production of CH4 was significantly increased in Cu-stressed alfalfa seeds, which was mimicked by 0.39 mM CH4. The pretreatment with CH4 significantly alleviated the inhibition of seed germination and seedling growth induced by Cu stress. Cu accumulation was obviously blocked as well. Meanwhile, α/ß amylase activities and sugar contents were increased, all of which were consistent with the alleviation of seed germination inhibition triggered by CH4. The Cu-triggered oxidative stress was also mitigated, which was confirmed by the decrease of lipid peroxidation and reduction of Cu-induced loss of plasma membrane integrity in CH4-pretreated alfalfa seedlings. The results of antioxidant enzymes, including ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (POD) total or isozymatic activities, and corresponding transcripts (APX1/2, Cu/Zn SOD and Mn-SOD), indicated that CH4 reestablished cellular redox homeostasis. Further, Cu-induced proline accumulation was partly impaired by CH4, which was supported by the alternation of proline metabolism. Together, these results indicated that CH4 performs an advantageous effect on the alleviation of seed germination inhibition caused by Cu stress, and reestablishment of redox homeostasis mainly via increasing antioxidant defence.

Evolution of an intron-poor cluster of the CIPK gene family and expression in response to drought stress in soybean.

Calcium ion is an intracellular messenger that plays a central role in signal transduction pathways. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) signal network have shown different functions in the Ca(2+) signaling process. In this work, we identified the entire soybean (Glycine max) CIPK gene family, which comprised 52 genes and divided into four subgroups (I to IV) based on phylogeny. The gene structural analysis separated these 52 genes into an intron-rich clade and an intron-poor clade. Chromosomal location analysis resulted in the identification of 22 duplicated blocks and six tandem duplication events. Phylogenetic classification of 193 CIPK proteins from representative plant species suggested that the intron-poor clade of CIPKs originated in seed plants. Analysis of global gene expression patterns of soybean CIPK family revealed that most intron-poor soybean CIPK genes are drought-inducible; a finding that was further confirmed using qRT-PCR. Our study provides a foundation for further functional analysis to reveal the roles that CIPKs and more specifically the intron-poor clade play in drought tolerance in soybean.