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Clubroot disease, which is caused by the obligate biotrophic protist Plasmodiophora brassicae, leads to the formation of galls, commonly known as pathogen-induced tumors, on the roots of infected plants. The identification of crucial regulators of host tumor formation is essential to unravel the mechanisms underlying the proliferation and differentiation of P. brassicae within plant cells. To gain insight into this process, transcriptomic analysis was conducted to identify key genes associated with both primary and secondary infection of P. brassicae in Chinese cabbage. Our results demonstrate that the k-means clustering of subclass 1, which exhibited specific trends, was closely linked to the infection process of P. brassicae. Of the 1610 differentially expressed genes (DEGs) annotated in subclass 1, 782 were identified as transcription factors belonging to 49 transcription factor families, including bHLH, B3, NAC, MYB_related, WRKY, bZIP, C2H2, and ERF. In the primary infection, several genes, including the predicted Brassica rapa probable pectate lyase, RPM1-interacting protein 4-like, L-type lectin-domain-containing receptor kinase, G-type lectin S-receptor-like serine, B. rapa photosystem II 22 kDa protein, and MLP-like protein, showed significant upregulation. In the secondary infection stage, 45 of 50 overlapping DEGs were upregulated. These upregulated DEGs included the predicted B. rapa endoglucanase, long-chain acyl-CoA synthetase, WRKY transcription factor, NAC domain-containing protein, cell division control protein, auxin-induced protein, and protein variation in compound-triggered root growth response-like and xyloglucan glycosyltransferases. In both the primary and secondary infection stages, the DEGs were predicted to be Brassica rapa putative disease resistance proteins, L-type lectin domain-containing receptor kinases, ferredoxin-NADP reductases, 1-aminocyclopropane-1-carboxylate synthases, histone deacetylases, UDP-glycosyltransferases, putative glycerol-3-phosphate transporters, and chlorophyll a-binding proteins, which are closely associated with plant defense responses, biosynthetic processes, carbohydrate transport, and photosynthesis. This study revealed the pivotal role of transcription factors in the initiation of infection and establishment of intracellular parasitic relationships during the primary infection stage, as well as the proliferation and differentiation of the pathogen within the host cell during the secondary infection stage.

IntroductionSaline-alkali stress is one of the main abiotic factors limiting rice production worldwide. With the widespread use of rice direct seeding technology, it has become increasingly important to improve rice saline-alkali tolerance at the germination stage. MethodsTo understand the genetic basis of saline-alkali tolerance and facilitate breeding efforts for developing saline-alkali tolerant rice varieties, the genetic basis of rice saline-alkali tolerance was dissected by phenotyping seven germination-related traits of 736 diverse rice accessions under the saline-alkali stress and control conditions using genome-wide association and epistasis analysis (GWAES).ResultsTotally, 165 main-effect quantitative trait nucleotides (QTNs) and 124 additional epistatic QTNs were identified as significantly associated with saline-alkali tolerance, which explained a significant portion of the total phenotypic variation of the saline-alkali tolerance traits in the 736 rice accessions. Most of these QTNs were located in genomic regions either harboring saline-alkali tolerance QTNs or known genes for saline-alkali tolerance reported previously. Epistasis as an important genetic basis of rice saline-alkali tolerance was validated by genomic best linear unbiased prediction in which inclusion of both main-effect and epistatic QTNs showed a consistently better prediction accuracy than either main-effect or epistatic QTNs alone. Candidate genes for two pairs of important epistatic QTNs were suggested based on combined evidence from the high-resolution mapping plus their reported molecular functions. The first pair included a glycosyltransferase gene LOC_Os02g51900 (UGT85E1) and an E3 ligase gene LOC_Os04g01490 (OsSIRP4), while the second pair comprised an ethylene-responsive transcriptional factor, AP59 (LOC_Os02g43790), and a Bcl-2-associated athanogene gene, OsBAG1 (LOC_Os09g35630) for salt tolerance. Detailed haplotype analyses at both gene promoter and CDS regions of these candidate genes for important QTNs identified favorable haplotype combinations with large effects on saline-alkali tolerance, which can be used to improve rice saline-alkali tolerance by selective introgression. DiscussionOur findings provided saline-alkali tolerant germplasm resources and valuable genetic information to be used in future functional genomic and breeding efforts of rice saline-alkali tolerance at the germination stage.

IntroductionRice (Oryza sativa L.) production is being challenged by global warming. Identifying new loci and favorable alleles associated with heat tolerance is crucial to developing rice heat-tolerant varieties.MethodsWe evaluated the heat tolerance at the seedling stage using 620 diverse rice accessions. A total of six loci associated with heat tolerance were identified by a genome-wide association study (GWAS) with ~2.8 million single nucleotide polymorphisms (SNPs). ResultsAmong the six detected loci, qHT7 harbored the strongest association signal and the most associated SNPs. By comparing the transcriptomes of two representative accessions with contrasting heat tolerance, LOC_Os07g48710 (OsVQ30) was selected as a promising candidate gene in qHT7 due to the significant difference in its expression level between the two accessions. Haplotype 4 (Hap4) of LOC_Os07g48710 was determined as the favorable haplotype for heat tolerance via the gene-based haplotype analysis. The heat-tolerant haplotype LOC_Os07g48710Hap4 is highly enriched in the tropical Geng/Japonica accessions, and its frequency has decreased significantly during the improvement process of rice varieties. DiscussionBased on the GWAS and transcriptomics integrated results, a hypothetical model modulated by qHT7 in response to heat stress was proposed. Our results provide valuable candidate genes for improving rice heat tolerance through molecular breeding.

Intercropping systems have been studied as a sustainable agricultural planting pattern to increase soil quality and crop yields. However, the relationships between metabolites and soil physicochemical properties remain poorly understood under sugarcane/peanut intercropping system. Thus, we determined the rhizosphere soil physicochemical properties, and analyzed rhizosphere soil metabolites and root metabolites by metabolomics method under monoculture and intercropping patterns of sugarcane and peanut. The results showed that pH, the contents of total phosphorus (P), total potassium (K), available nitrogen (N), available phosphorus (P), and available potassium (K) were higher in rhizosphere soil of intercropping peanut than monoculture peanut, and the content of total P was higher in rhizosphere soil of intercropping sugarcane than monoculture sugarcane. Sugarcane/peanut intercropping also significantly increased the activities of acid phosphatase and urease in rhizosphere soil. The metabolomics results showed that 32 metabolites, mainly organic acids and their derivatives (25.00%), nucleotides and their metabolites (18.75%), were detected in root and rhizosphere soil samples. In the MP-S (rhizosphere soil of monoculture peanut) vs. IP-S (rhizosphere soil of intercropping peanut) comparison, 47 differential metabolites (42 upregulated) were screened, including glycerolipids (19.15%), organic acids and their derivatives (17.89%), and amino acids and their metabolites (12.77%). In the MS-S (rhizosphere soil of monoculture sugarcane) vs. IS-S (rhizosphere soil of intercropping sugarcane) comparison, 51 differential metabolites (26 upregulated) were screened, including heterocyclic compounds (15.69%), glycerolipids (11.76%), and organic acids and their derivatives (9.80%). The metabolite species from MP-S, MS-S, IP-S, and IS-S were similar, but some metabolite contents were significantly different, such as adenine, adenosine, maltotriose, thermozeaxanthin-13 and PE-NMe (20:0/24:0). Adenine and adenosine were detected in root and rhizosphere soils, and their levels were increased in the intercropping treatment, which were mainly related to enhanced purine metabolism in root and rhizosphere soils under the sugarcane/peanut intercropping system. Importantly, adenine and adenosine were significantly positively correlated with total P and total K contents, acid phosphatase and urease activities, and pH. This study clarified that the sugarcane/peanut intercropping system could improve soil nutrients and enzymes and was related to purine metabolism.

Leaves are one of the vegetative organs of plants that are essential for plant growth and development. PIN-FORMED (PINs) gene is an indoleacetic acid (IAA) transporter that plays a critical role in leaf development. To determine the function of BpPIN3 in leaf polarity formation in Betula pendula, the transgenic lines with BpPIN3 overexpression (OE) and BpPIN3-reduced expression (RE) were analyzed using the Agrobacterium-mediated method. The RE lines displayed the characteristics of leaf margin adaxial upward curling, with lower expression of BpPIN3 resulting in greater rolling. Tissue localization of IAA in the auxin GUS reporter system proved that auxin in the RE was mainly distributed in the secondary veins, palisade tissues, and epidermal cells in the leaf margin area. The auxin content in the leaf margin area was significantly greater than that in the main vein tissue. The cell density of the palisade tissue and the ratio of palisade tissue to spongy tissue in the curled leaf margin of the RE lines were found to be significantly decreased. RNA-seq analysis revealed that the RE hormone-signaling pathway genes were significantly enriched compared with those of the OE and WT lines; in particular, the auxin response-related genes SAURs (i.e., SAUR23, SAUR24, SAUR28, and SAUR50) and GH3.10 were found to be significantly upregulated. qRT-PCR analysis indicated that BpPIN3 expression at the leaf margin was significantly lower than that near the main vein in the RE lines. In contrast, the expression levels of SAURs and GH3.10 were significantly higher than those near the midrib. In conclusion, BpPIN3 regulates the expression of auxin response-related genes and the polar transport of auxin to change the polar form of the proximal and distal axes of birch leaves.

Salinity–alkalinity stress is one of the main abiotic factors limiting rice production worldwide. With the widespread use of rice direct seeding technology, it has become increasingly important to improve the tolerance to salinity–alkalinity of rice varieties at the germination stage. Although we have a more comprehensive understanding of salt tolerance in rice, the genetic basis of alkali tolerance in rice is still poorly understood. In this study, we measured seven germination-related traits under alkali stress and control conditions using 428 diverse rice accessions. The alkali tolerance levels of rice germplasms varied considerably during germination. Xian/indica accessions had generally higher tolerance to alkali stress than Geng/japonica accessions at the germination stage. Using genome-wide association analysis, 90 loci were identified as significantly associated with alkali tolerance. Eight genes (LOC_Os01g12000, LOC_Os03g60240, LOC_Os03g08960, LOC_Os04g41410, LOC_Os09g25060, LOC_Os11g35350, LOC_Os12g09350, and LOC_Os12g13300) were selected as important candidate genes for alkali tolerance based on the gene functional annotation and gene-CDS-haplotype analysis. According to the expression levels of LOC_Os09g25060 (OsWRKY76), it is likely to play a negative regulatory role in alkali tolerance during rice germination. An effective strategy for improving rice alkali tolerance may be to pyramid alkali-tolerant haplotypes of multiple candidate genes to obtain the optimal haplotype combination. Our findings may provide valuable genetic information and expand the use of alkali tolerance germplasm resources in rice molecular breeding to improve the alkali tolerance at the germination stage.

The adjustment of stomatal density and clustered ratio on the epidermis is the important strategy for plants to respond to drought, because the stoma-based water loss is directly related to plant growth and survival under drought conditions. But the relevant adjustment mechanism still needs to be explored. 1-Aminocyclopropane-1-carboxylate (ACC) is disclosed to promote stomatal development, while in vivo ACC levels depend on activation of ACC synthase (ACS) family members. Based on the findings of ACS expression involving in drought response and several ACS activity inhibitors reducing stomatal density and cluster in drought response, here we examined how ACS activation is involved in the establishment of stomatal density and cluster on the epidermis under drought conditions. Preliminary data indicated that activation of ACS2 and/or ACS6 (ACS2/6) increased stomatal density and clustered ratio on the Arabidopsis leaf epidermis by accumulating ACC under moderate drought, and raised the survival risk of seedlings under escalated drought. Further exploration indicated that, in Arabidopsis seedlings stressed by drought, the transcription factor SPEECHLESS (SPCH), the initiator of stomatal development, activates ACS2/6 expression and ACC production; and that ACC accumulation induces Ca2+ deficiency in stomatal lineage; this deficiency inactivates a subtilisin-like protease STOMATAL DENSITY AND DISTRIBUTION 1 (SDD1) by stabilizing the inhibition of the transcription factor GT-2 Like 1 (GTL1) on SDD1 expression, resulting in an increases of stomatal density and cluster ratio on the leaf epidermis. This work provides a novel evidence that ACS2/6 activation plays a key role in the establishment of stomatal density and cluster on the leaf epidermis of Arabidopsis in response to drought.

There is a growing consensus on soil conservation by mechanics of plant root system. In order to further study how root system exerts its mechanical properties during soil reinforcing process and which morphological indicator is suitable for reflecting pullout resistance, in-situ vertical pullout test (VPT) and 45° oblique pullout test (OPT) were performed on alfalfa (Medicago sativa L.) roots in the loess area. The results showed that the failure mode of alfalfa roots was pulling out in this study. The peak pullout resistance of the roots increased with root diameter, root length and root surface area, and power law relationships were observed between the pullout resistance and the morphological indices: root diameter, root length and root surface area. The maximum gray relational degree of the morphological indices was 0.841 (VPT) and 0.849 (OPT) for root surface area, suggesting that root surface area was a more significant root morphological index affecting root pullout resistance than root diameter and root length, and was more suitable for characterizing the difference in peak pullout resistance of roots with different size. The index could be used to validate the methods for predicting root pullout capacity. The value of peak pullout resistance was 17.2 ± 2.3 N in VPT test and 28.2 ± 3.8 N (mean ± SE) in OPT test, and a significant difference was observed between the two tests, which showed that the pulling direction significantly affected the peak pullout resistance of alfalfa roots. Vertical pullout test, giving the safety margin, was suggested to determine root pullout resistance for estimate of root reinforcement.

PP2C protein phosphatase family is one of the largest gene families in the plant genome. Many PP2C family members are involved in the regulation of abiotic stress. We found that BpPP2C1 gene has highly up-regulated in root under salt stress in Betula platyphylla. Thus, transgenic plants of Betula platyphylla with overexpression and knockout of BpPP2C1 gene were generated using a zygote transformation system. Under NaCl stress treatment, we measured the phenotypic traits of transgenic plants, chlorophyll-fluorescence parameters, peroxidase (POD) activity, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content. We found that BpPP2C1 overexpressed lines showed obvious salt tolerance, while BpPP2C1 knocked out plants were sensitive to salt stress. Transcriptome analysis identified significantly amount of differentially expressed genes associated with salt stress in BpPP2C1 transgenic lines, especially genes in abscisic acid signaling pathway, flavonoid biosynthetic pathway, oxidative stress and anion transport. Functional study of BpPP2C1 in Betula platyphylla revealed its role in salt stress.

Rice (Oryza sativa L.) is one of the most important staple foods in the world. It is possible to identify candidate genes associated with rice yield using the model of random walk with restart on a functional similarity network. We demonstrated the high performance of this approach by a five-fold cross-validation experiment, as well as the robustness of the parameter r. We also assessed the strength of associations between known seeds and candidate genes in the light of the results scores. The candidates ranking at the top of the results list were considered to be the most relevant rice yield-related genes. This study provides a valuable alternative for rice breeding and biology research. The relevant dataset and script can be downloaded at the website: http://lab.malab.cn/jj/rice.htm.

Plasmodiophora brassicae is a soil borne pathogen and the causal agent of clubroot, a devastating disease of Brassica crops. The pathogen lives inside roots, and hijacks nutrients from the host plants. It is suggested that clubroot galls created an additional nutrient sink in infected roots. However, the molecular mechanism underlying P. brassicae infection and sugar transport is unclear. Here, we analyzed sugar contents in leaves and roots before and after P. brassicae infection using a pair of Chinese cabbage near-isogenic lines (NILs), carrying either a clubroot resistant (CR) or susceptible (CS) allele at the CRb locus. P. brassicae infection caused significant increase of glucose and fructose contents in the root of CS-NIL compared to CR-NIL, suggesting that sugar translocation and P. brassicae growth are closely related. Among 32 B. rapa SWEET homologs, several BrSWEETs belonging to Clade I and III were significantly up-regulated, especially in CS-NIL upon P. brassicae infection. Moreover, Arabidopsis sweet11 mutant exhibited slower gall formation compared to the wild-type plants. Our studies suggest that P. brassicae infection probably triggers active sugar translocation between the sugar producing tissues and the clubbed tissues, and the SWEET family genes are involved in this process.

Superoxide dismutase (SOD) is a very important reactive oxygen species (ROS)-scavenging enzyme. In this study, the functions of a Cu/Zn SOD gene (SaCu/Zn SOD), from Sedum alfredii, a cadmium (Cd)/zinc/lead co-hyperaccumulator of the Crassulaceae, was characterized. The expression of SaCu/Zn SOD was induced by Cd stress. Compared with wild-type (WT) plants, overexpression of SaCu/Zn SOD gene in transgenic Arabidopsis plants enhanced the antioxidative defense capacity, including SOD and peroxidase activities. Additionally, it reduced the damage associated with the overproduction of hydrogen peroxide (H2O2) and superoxide radicals (O2•-). The influence of Cd stress on ion flux across the root surface showed that overexpressing SaCu/Zn SOD in transgenic Arabidopsis plants has greater Cd uptake capacity existed in roots. A co-expression network based on microarray data showed possible oxidative regulation in Arabidopsis after Cd-induced oxidative stress, suggesting that SaCu/Zn SOD may participate in this network and enhance ROS-scavenging capability under Cd stress. Taken together, these results suggest that overexpressing SaCu/Zn SOD increased oxidative stress resistance in transgenic Arabidopsis and provide useful information for understanding the role of SaCu/Zn SOD in response to abiotic stress.

Cadmium is a major toxic heavy-metal pollutant considering their bioaccumulation potential and persistence in the environment. The hyperaccumulating ecotype of Sedum alfredii Hance is a Zn/Cd co-hyperaccumulator inhabiting in a region of China with soils rich in Pb/Zn. Investigations into the underlying molecular regulatory mechanisms of Cd tolerance are of substantial interest. Here, library screening for genes related to cadmium tolerance identified a gene resembling the rubber elongation factor gene designated as SaREFl. The heterologous expression of SaREFl rescued the growth of a transformed Cd-sensitive strain (ycf1). Furthermore, SaREFl-expressing Arabidopsis plants were more tolerant to cadmium stress compared with wild type by measuring parameters of root length, fresh weight and physiological indexes. When under four different heavy metal treatments, we found that SaREFl responded most strongly to Cd and the root was the plant organ most sensitive to this heavy metal. Yeast two-hybrid screening of SaREFl as a bait led to the identification of five possible interacting targets in Sedum alfredii Hance. Among them, a gene annotated as prenylated Rab acceptor 1 (PRA1) domain protein was detected with a high frequency. Moreover, subcellular localization of SaREF1-GFP fusion protein revealed some patchy spots in cytosol suggesting potential association with organelles for its cellular functions. Our findings would further enrich the connotation of REF-like genes and provide theoretical assistance for the application in breeding heavy metal-tolerant plants. [ABSTRACT FROM AUTHOR]