Effects of exogenous calcium and calcium inhibitor on physiological characteristics of winter turnip rape (Brassica rapa) under low temperature stress.

Low temperature is one of the environmental factors that restrict the growth and geographical distribution of Brassica. To investigate the effects of exogenous calcium and calcium inhibitor on the ability of winter turnip rapeseed (Brassica rapa L.) to withstand low temperatures (4℃), we used a strong cold-resistant variety Longyou 7 (L7) and a weak cold-resistant variety Longyou 99 (L99) as the materials. The seedlings were treated with CaCl2 (20 mmol·L-1) and calcium inhibitor LaCl3 (10 mmol·L-1) at 0 h (CK), 6 h, 12 h, 24 h and 48 h after 4℃ treatments. Physiological characteristics, Ca2+ flux and Ca2+ concentration in roots after treatments were analyzed. Results illustrated that under 4℃ treatment, activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) increased by both CK and exogenous CaCl2 treatments. Contents of soluble protein (SP) and proline (Pro) increased, while contents of malondialdehyde (MDA) decreased, resulting in reduced membrane lipid peroxidation. But enzyme activity decreased and MDA content increased following treatment with exogenous LaCl3. The rate of Ca2+ flow showed a higher uptake in L7 roots compared with L99. L99 showed Ca2+ efflux with a rate of 30.21 pmol‧cm-2‧s-1, whereas L7 showed short efflux then returned to influx. Calcium ion content in roots decreased in both cultivars after CaCl2 treatment. Results of RNA-seq revealed that genes were differentially expressed in response to low temperatures, hormones, photosystem II, chloroplasts, DNA replication, ribosomal RNA processing, and translation. This study found significant expression genes related to cellular signal transduction (MAPK signaling pathway) and material metabolism (nitrogen metabolism, glycerol ester metabolism).It was also analyzed by WGCNA that two modules had the strongest correlation with physiological indicators. Eight candidate genes were identified among MAPK signaling pathway and the two modules.

Transcription factor ZmDof22 enhances drought tolerance by regulating stomatal movement and antioxidant enzymes activities in maize (Zea mays L.).

KEY MESSAGE: The Dof22 gene encoding a deoxyribonucleic acid binding with one finger in maize, which is associated with its drought tolerance. The identification of drought stress regulatory genes is essential for the genetic improvement of maize yield. Deoxyribonucleic acid binding with one finger (Dof), a plant-specific transcription factor family, is involved in signal transduction, morphogenesis, and environmental stress responses. In present study, by weighted correlation network analysis (WGCNA) and gene co-expression network analysis, 15 putative Dof genes were identified from maize that respond to drought and rewatering. A real-time fluorescence quantitative PCR showed that these 15 genes were strongly induced by drought and ABA treatment, and among them ZmDof22 was highly induced by drought and ABA treatment. Its expression level increased by nearly 200 times after drought stress and more than 50 times after ABA treatment. After the normal conditions were restored, the expression levels were nearly 100 times and 40 times of those before treatment, respectively. The Gal4-LexA/UAS system and transcriptional activation analysis indicate that ZmDof22 is a transcriptional activator regulating drought tolerance and recovery ability in maize. Further, overexpressed transgenic and mutant plants of ZmDof22 by CRISPR/Cas9, indicates that the ZmDof22, improves maize drought tolerance by promoting stomatal closure, reduces water loss, and enhances antioxidant enzyme activity by participating in the ABA pathways. Taken together, our findings laid a foundation for further functional studies of the ZmDof gene family and provided insights into the role of the ZmDof22 regulatory network in controlling drought tolerance and recovery ability of maize.

Systematic Analysis of the BrHAT Gene Family and Physiological Characteristics of Brassica rapa L. Treated with Histone Acetylase and Deacetylase Inhibitors under Low Temperature.

Brassica rapa L. is an important overwintering oilseed crop in Northwest China. Histone acetyltransferases (HATs) play an important role in epigenetic regulation, as well as the regulation of plant growth, development, and responses to abiotic stresses. To clarify the role of histone acetylation in the low-temperature response of B. rapa L., we identified 29 HAT genes in B. rapa L. using bioinformatics tools. We also conducted a comprehensive analysis of the physicochemical properties, gene structure, chromosomal localization, conserved structural domains and motifs, cis-acting regulatory elements, and evolutionary relationships of these genes. Using transcriptome data, we analyzed the expression patterns of BrHAT family members and predicted interactions between proteins; the results indicated that BrHATs play an important role in the low-temperature response of B. rapa L. HAT inhibitor (curcumin; CUR) and histone deacetylase inhibitor (Trichostatin A; TSA) were applied to four B. rapa L. varieties varying in cold resistance under the same low-temperature conditions, and changes in the physiological indexes of these four varieties were analyzed. The inhibitor treatment attenuated the effect of low temperature on seed germination, and curcumin treatment was most effective, indicating that the germination period was primarily regulated by histone acetylase. Both inhibitor treatments increased the activity of protective enzymes and the content of osmoregulatory substances in plants, suggesting that histone acetylation and deacetylation play a significant role in the response of B. rapa L. to low-temperature stress. The qRT-PCR analyses showed that the expression patterns of BrHATs were altered under different inhibitor treatments and low-temperature stress; meanwhile, we found three significantly differentially expressed genes. In sum, the process of histone acetylation is involved in the cold response and the BrHATs gene plays a role in the cold stress response.

Integrated miRNA and mRNA Transcriptome Analysis Reveals Regulatory Mechanisms in the Response of Winter Brassica rapa to Drought Stress.

Drought is a major abiotic stress factor that reduces agricultural productivity. Understanding the molecular regulatory network of drought response in winter rape is of great significance for molecular Brassica rapa. In order to comprehensively analyze the network expression of DEGs and DEMIs in winter rape under drought stress, in this study we used Longyou 7 as the experimental material to identify DEGs and DEMIs related to drought stress by transcriptome and miRNA sequencing. A total of 14-15 key differential mRNA genes related to drought stress and biological stress were screened out under different treatments in the three groups. and 32 differential miRNAs were identified through targeted regulatory relationships, and the mRNA expression of 20 target genes was negatively regulated by the targeting regulatory relationship. It is mainly enriched in starch and sucrose metabolism, carbon metabolism and other pathways. Among them, gra-MIR8731-p3_2ss13GA18GA regulated the expression of multiple mRNAs in the three treatments. miRNA is mainly involved in the drought resistance of Chinese cabbage winter rape by regulating the expression of target genes, such as starch and sucrose metabolism, amino acid biosynthesis, and carbon metabolism. These miRNAs and their target genes play an indispensable role in winter rapeseed drought stress tolerance regulation.

Evolutionary QTL-allele changes in main stem node number among geographic and seasonal subpopulations of Chinese cultivated soybeans.

The main stem node number (MSN) is a trait related to geographic adaptation, plant architecture and yield potential of soybean. The QTL-allele constitution of the Chinese Cultivated Soybean Population (CCSP) was identified using the RTM-GWAS (restricted two-stage multi-locus genome-wide association study) procedure, from which a QTL-allele matrix was established and then separated into submatrices to explore the genetic structure, evolutionary differentiation, breeding potential and candidate genes of MSN in CCSP. The MSN of 821 accessions varied from 8.8 to 31.1, with an average of 16.3 in Nanjing, China (32.07° N, 118.62° E), where the MSNs of all the materials could be evaluated in a standardized manner. Among the six geo-seasonal subpopulations, the MSN varied from 21.7 in a southern summer-autumn-sowing subpopulation (SA-IV) down to 13.5 in a northeastern spring-sowing subpopulation (SP-I). The materials were genotyped with restriction site-associated DNA-sequencing. Totally 142 main-effect QTLs (73.24% new) with 560 alleles contributing 72.98% to the phenotypic variance were identified. The evolutionary QTL-allele changes in MSN from SA-IV through SP-I showed that inheritance (78.93% of alleles) was the primary factor influencing the evolution of this trait, followed by allele emergence (19.64% alleles), allele exclusion (1.43% alleles), and recombination among retained alleles. In the evolutionary changes, 70 QTLs, including 12 newly emerged QTLs, with 118 alleles were involved. An increase potential of 2-8 nodes was predicted and 112 candidate genes were annotated and preliminarily verified with χ2-tests in the CCSP. The RTM-GWAS showed powerful in detecting QTL-allele system, assessing evolution factors, predicting optimal crosses and identifying candidate genes in a germplasm population.

Melatonin Enhances Heat Tolerance via Increasing Antioxidant Enzyme Activities and Osmotic Regulatory Substances by Upregulating zmeno1 Expression in Maize (Zea mays L.).

Heat stress severely affects the yield and quality of maize. Melatonin (N-acetyl-5-methoxy-tryptamin, MT) plays an important role in various types of stress resistance in plants, including heat tolerance. Enolase (ENO, 2-phospho-D-glycerate hydrolyase) contributes to plant growth, development, and stress response. As of now, the molecular mechanisms by which MT and ENO1 affect heat tolerance are unknown. In our research, we have revealed that heat stress (H) and heat stress + MT (MH) treatment upregulate ZmENO1 expression levels by 15 and 20 times, respectively. ZmENO1 overexpression and mutant maize lines were created by transgenic and genome editing. These results illustrate that heat stress has a significant impact on the growth of maize at the seedling stage. However, ZmENO1-OE lines showed a lower degree of susceptibility to heat stress, whereas the mutant exhibited the most severe effects. Under heat stress, exogenous application of MT improves heat resistance in maize. The ZmENO1-OE lines exhibited the best growth and highest survival rate, while the zmeno1 mutants showed the least desirable results. Following treatment with H and MH, the level of MT in ZmENO1-OE lines exhibited the greatest increase and reached the maximum value, whereas the level of MT in the zmeno1 mutant was the lowest. Heat stress decreased the maize's relative water content and fresh weight, although ZmENO1-OE lines had the highest and zmeno1 mutants had the lowest. Heat stress led to an increase in the levels of MDA, hydrogen peroxide, and superoxide in all plants. Additionally, the ionic permeability and osmotic potential of the plants were significantly increased. However, the levels of MT were decreased in all plants, with the greatest decrease observed in the ZmENO1-OE lines. Interestingly, the zmeno1 mutant plants had the highest expression levels of MT. Heat stress-induced upregulation of ZmSOD, ZmPOD, ZmAPX, ZmCAT, ZmP5CS, and ZmProDH in all plants. However, the ZmENO1-OE lines exhibited the greatest increase in expression levels, while the zmeno1 mutants showed the lowest increase following MT spraying. The patterns of SOD, POD, APX, and CAT enzyme activity, as well as proline and soluble protein content, aligned with the variations in the expression levels of these genes. Our findings indicate that MT can upregulate the expression of the ZmENO1 gene. Upregulating the ZmENO1 gene resulted in elevated expression levels of ZmSOD, ZmPOD, ZmAPX, ZmCAT, ZmP5CS, and ZmProDH. This led to increased activity of antioxidant enzymes and higher levels of osmoregulatory substances. Consequently, it mitigated the cell membrane damage caused by heat stress and ultimately improved the heat resistance of maize. The results of this study provide genetic resources for molecular design breeding and lay a solid foundation for further exploring the molecular mechanism of MT regulation of heat stress tolerance in maize.

Impact of Straw Incorporation on the Physicochemical Profile and Fungal Ecology of Saline-Alkaline Soil.

Improving the soil structure and fertility of saline-alkali land is a major issue in establishing a sustainable agro-ecosystem. To explore the potential of different straw returning in improving saline-alkaline land, we utilized native saline-alkaline soil (SCK), wheat straw-returned saline-alkaline soil (SXM) and rapeseed straw-returned saline-alkaline soil (SYC) as our research objects. Soil physicochemical properties, fungal community structure and diversity of saline-alkaline soils were investigated in different treatments at 0-10 cm, 10-20 cm and 20-30 cm soil depths. The results showed that SXM and SYC reduced soil pH and total salinity but increased soil organic matter, alkali-hydrolyzable nitrogen, available phosphorus, total potassium, etc., and the enhancement effect of SYC was more significant. The total salinity of the 0-10 cm SCK soil layer was much higher than that of the 10-30 cm soil layers. Fungal diversity and abundance were similar in different soil layers in the same treatment. SXM and SYC soil had higher fungal diversity and abundance than SCK. At the genus level, Plectosphaerella, Mortierella and Ascomycota were the dominant groups of fungal communities in SXM and SYC. The fungal diversity and abundance in SXM and SYC soils were higher than in SCK soils. Correlation network analysis of fungal communities with environmental factors showed that organic matter, alkali-hydrolyzable nitrogen and available phosphorus were the main environmental factors for the structural composition of fungal communities of Mortierella, Typhula, Wickerhamomyces, Trichosporon and Candida. In summary, straw returning to the field played an effective role in improving saline-alkaline land, improving soil fertility, affecting the structure and diversity of the fungal community and changing the interactions between microorganisms.

Genome-wide identification of NF-Y gene family in maize (Zea mays L.) and the positive role of ZmNF-YC12 in drought resistance and recovery ability.

Nuclear factor Y (NF-Y) genes play important roles in many biological processes, such as leaf growth, nitrogen nutrition, and drought resistance. However, the biological functions of these transcription factor family members have not been systematically analyzed in maize. In the present study, a total of 52 ZmNF-Y genes were identified and classified into three groups in the maize genome. An analysis of the evolutionary relationship, gene structure, and conserved motifs of these genes supports the evolutionary conservation of NF-Y family genes in maize. The tissue expression profiles based on RNA-seq data showed that all genes apart from ZmNF-Y16, ZmNF-YC15, and ZmNF-YC17 were expressed in different maize tissues. A weighted correlation network analysis was conducted and a gene co expression network method was used to analyze the transcriptome sequencing results; six core genes responding to drought and rewatering were identified. A real time fluorescence quantitative analysis showed that these six genes responded to high temperature, drought, high salt, and abscisic acid (ABA) treatments, and subsequent restoration to normal levels. ZmNF-YC12 was highly induced by drought and rewatering treatments. The ZmNF-YC12 protein was localized in the nucleus, and the Gal4-LexA/UAS system and a transactivation analysis demonstrated that ZmNF-YC12 in maize (Zea mays L.) is a transcriptional activator that regulates drought resistance and recovery ability. Silencing ZmNF-YC12 reduced net photosynthesis, chlorophyll content, antioxidant (superoxide dismutase, catalase, peroxidase and ascorbate peroxidase) system activation, and soluble protein and proline contents; it increased the malondialdehyde content, the relative water content, and the water loss rate, which weakened drought resistance and the recoverability of maize. These results provide insights into understanding the evolution of ZmNF-Y family genes in maize and their potential roles in genetic improvement. Our work provides a foundation for subsequent functional studies of the NF-Y gene family and provides deep insights into the role of the ZmNF-YC12 regulatory network in controlling drought resistance and the recoverability of maize.

Diversity of fungal pathogens associated with loquat and development of novel virulence scales.

Loquat [Eriobotrya japonica (Thunb.) Lindl.] is an important fruit crop in Pakistan; however, a constant decline in its production is noted due biotic and abiotic stresses, particularly disease infestation. Fungal pathogens are the major disease-causing agents; therefore, their identification is necessary for devising management options. This study explored Taxila, Wah-Cantt, Tret, Chatar, Murree, Kalar-Kahar, Choa-Saidan-Shah and Khan-Pur districts in the Punjab and Khyber Paktoon Khawa (KPK) provinces of Pakistan to explore the diversity of fungal pathogens associated with loquat. The samples were collected from these districts and their microscopic characterizations were accomplished for reliable identification. Alternaria alternata, Curvularia lunata, Lasiodiplodia theobromae, Aspergilus flavis, Botrytis cinerea, Chaetomium globosum, Pestalotiopsis mangiferae and Phomopsis sp. were the fungal pathogens infesting loquat in the study area. The isolates of A. alternata and C. lunata were isolated from leaf spots and fruit rot, while the isolates of L. theobromae were associated with twig dieback. The remaining pathogens were allied with fruit rot. The nucleotide evidence of internal transcribed spacer (ITS) regions (ITS1, 5.8S, and ITS2) were computed from all the pathogens and submitted in the database of National Center for Biotechnology Information (NCBI). For multigene analysis, beta-tubulin (BT) gene and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) regions were explored for A. alternata and C. lunata isolates, respectively. The virulence scales of leaf spots, fruit rot, and twig dieback diseases of loquat were developed for the first time through this study. It is the first comprehensive study with morpho-molecular identification, and newly developed virulence scales of the fungal pathogens associated with loquat, which improves the understanding of these destructive diseases.