First Report of Fusarium verticillioides causing Stem Blight of Schizonepeta tenuifolia in China.

Schizonepeta tenuifolia is an important medicinal plant in China. Over 10000 ha of S. tenuifolia is cultivated in the country annually. However, fungal diseases are a major limiting factor in S. tenuifolia production. In 2022, 50 ha in several S. tenuifolia fields in Hebei province were observed to be severely affected by a disease causing a yield loss of 30%. Results from field surveys suggested an epidemic during seedlings stages that affected S. tenuifolia stems, causing irregularly watery brown lesions. Lesions ranged from 1.5 to 2 × 2.5 to 3 cm. To isolate the causal agent, tissue was removed from the border of lesions and surface sterilized in 75% ethanol for 30 sec and 0.1% HgCl2 for 1 min, then rinsed three times with steriled distilled water(SDW), plated on potato dextrose agar(PDA) at 25℃, and incubated in the dark for 7 days. Five putative isolates of the genus Fusarium were hyphal-tipped on new PDA plates. Isolates were cultured on synthetic low-nutrient agar(SNA) with a ~ 1 × 2-cm strip of sterile filter paper on the agar surface(Nirenberg 1976). Cultures were incubated for 7 to 10 days at 20℃ in dark conditions. When sporulation was observed, agar blocks were mounted on a microscopic slide with a drop of lactophenol cotton blue and examined at 400×. Colonies grew rapidly with abundant pink to violet aerial hyphae. Sporodochia formed on the agar, and the aerial conidiophores branched sparsely, often alternately or oppositely, terminating with up to three verticillate phialides. Microconidia produced on polyphialides and aggregating in heads were unicellular, ovoidal or ellipsoidal, 4.4 to 17 × 1.5 to 4.5 µm. Macroconidia were abundant, falcate to straight, three to five septate, with a distinct foot cell, 27 to 73 × 3.1 to 5.6 µm. Based on morphological characteristics, isolates were tentatively identified as F. verticillioides(A1-Hatmi et al. 2016; Guarro 2013). Pathogenicity tests were performed by injection inoculation of 0.1 mL of conidial suspensions(1×106 conidia/mL) into three S. tenuifolia stems using a disposable needle and syringe. Distilled water was injected into three mock controls. Inoculated plants were placed in a greenhouse at 32 to 34℃ and 95% relative humidity. Typical lesions were observed 7 days after inoculation, except in the control samples. Each treatment was replicated three times. The suspected pathogen was consistently reisolated from diseased tissue according to Koch's postulates, and was found to be morphologically similar to F. verticillioides. Preliminary morphological identification of the pathogen was further confirmed by using genomic DNA extracted from the mycelia of a 7-day-old culture grown on PDA at 25℃. The translation elongation factor 1-α gene(TEF1) was amplified(O'Donnell et al. 1998) and the TEF region(Genbank Accession No. OR105502) was sequenced by Sangon Biotech Co., Ltd.(Shanghai, China) and displayed 100% nucleotide similarity with rDNA-TEF of F. verticillioides(JF740717) separately after a BLASTn search in Genbank. Based on the symptoms, fungal morphology, TEF sequence, and pathogenicity testing, this fungus was identified as F. verticillioides. to our knowledge, this is the first report of F. verticillioides infecting S. tenuifolia in China. This report will promote further research of F. verticillioides on this host and lead to better understanding of disease prevalence, extent of damage, and possible management options.

Design of high-oleic tobacco (Nicotiana tabacum L.) seed oil by CRISPR-Cas9-mediated knockout of NtFAD2-2.

BACKGROUND: Tobacco seed oil could be used as an appropriate feedstock for biodiesel production. However, the high linoleic acid content of tobacco seed oil makes it susceptible to oxidation. Altering the fatty acid profile by increasing the content of oleic acid could improve the properties of biodiesel produced from tobacco seed oil. RESULTS: Four FAD2 genes, NtFAD2-1a, NtFAD2-1b, NtFAD2-2a, and NtFAD2-2b, were identified in allotetraploid tobacco genome. Phylogenetic analysis of protein sequences showed that NtFAD2-1a and NtFAD2-2a originated from N. tomentosiformis, while NtFAD2-1b and NtFAD2-2b from N. sylvestris. Expression analysis revealed that NtFAD2-2a and NtFAD2-2b transcripts were more abundant in developing seeds than in other tissues, while NtFAD2-1a and NtFAD2-1b showed low transcript levels in developing seed. Phylogenic analysis showed that NtFAD2-2a and NtFAD2-2b were seed-type FAD2 genes. Heterologous expression in yeast cells demonstrated that both NtFAD2-2a and NtFAD2-2b protein could introduce a double bond at the Δ12 position of fatty acid chain. The fatty acid profile analysis of tobacco fad2-2 mutant seeds derived from CRISPR-Cas9 edited plants showed dramatic increase of oleic acid content from 11% to over 79%, whereas linoleic acid decreased from 72 to 7%. In addition, the fatty acid composition of leaf was not affected in fad2-2 mutant plants. CONCLUSION: Our data showed that knockout of seed-type FAD2 genes in tobacco could significantly increase the oleic acid content in seed oil. This research suggests that CRISPR-Cas9 system offers a rapid and highly efficient method in the tobacco seed lipid engineering programs.

FAX2 Mediates Fatty Acid Export from Plastids in Developing Arabidopsis Seeds.

Vegetable oils are mainly stored in the form of triacylglycerol (TAG) in oilseeds. Fatty acids (FAs), one of the building blocks for TAG assembly, are synthesized in plastids and then exported to the endoplasmic reticulum for storage oil synthesis. A recent study demonstrated that the export of FAs from plastids was mediated by a FAX (FA export) family protein. However, the significance of FAs export from plastid during seed oil accumulation has not been investigated. In this study, we found that FAX2 was highly expressed in developing Arabidopsis seeds and the expression level was consistent with FAs synthesis activity. FAX2 mutant seeds showed an approximately 18% reduction of lipid levels compared with wild-type seeds. By contrast, overexpression of FAX2 enhanced seed lipid accumulation by up to 30%. The FAs export activity of FAX2 was confirmed by yeast mutant cell complementation analysis. Our results showed that FAX2 could interact with other proteins to facilitate FAs transport. Taken together, these results indicate that FAX2-mediated FA export from plastids is important for seed oil accumulation, and that FAX2 can be used as a target gene for increasing lipid production in oilseeds.

Seed-specific overexpression of AtFAX1 increases seed oil content in Arabidopsis.

Biosynthesis of plant seed oil is accomplished through the coordinate action of multiple enzymes in multiple subcellular compartments. Fatty acid (FA) has to be transported from plastid to endoplasmic reticulum (ER) for TAG synthesis. However, the role of plastid FA transportation during seed oil accumulation has not been evaluated. AtFAX1 (Arabidopsis fatty acid export1) mediated the FA export from plastid. In this study, we overexpressed AtFAX1 under the control of a seed specific promoter in Arabidopsis. The resultant overexpression lines (OEs) produced seeds which contained 21-33% more oil and 24-30% more protein per seed than those of the wild type (WT). The increased oil content was probably because of the enhanced FA and TAG synthetic activity. The seed size and weight were both increased accordingly. In addition, the seed number per silique and silique number per plant had no changes in transgenic plants. Taken together, our results demonstrated that seed specific overexpression of AtFAX1 could promote oil accumulation in Arabidopsis seeds and manipulating FA transportation is a feasible strategy for increasing the seed oil content.

Genome-wide identification and expression analysis of EPF/EPFL gene family in Populus trichocarpa.

The Epidermal Patterning Factor/EPF-like (EPF/EPFL) family encodes a specific type of secreted protein in plants and plays an important role in plant growth and development, especially in the process of morphogenesis. To investigate the characteristics of EPF/EPFL gene family members and their regulatory functions in stomatal development of Populus trichocarpa, a total of 15 EPF/EPFL family genes were identified. Then the gene structure, chromosome location, phylogenetic relationship, protein conserved domain and gene expression profile were analyzed. According to phylogenetic analysis, PtEPF/EPFL can be classified into four groups. The gene structure and protein conservation motifs within the EPF family indicate the high conservation of the PtEPF/EPFL sequence. The promoter region of PtEPF/EPFL was found to contain cis-elements in response to stress and plant hormones. In addition, RT-qPCR results indicated that the PtEPF/EPFL have a differentially expressed in different tissues. Under drought stress treatment, a substantial upregulation was observed in the majority of PtEPF/EPFL members, suggesting their potential involvement in drought response. These results provide a theoretical basis for future exploration of the characteristics and functions of more PtEPF/EPFL genes.

Effects of elevated CO2 concentration and experimental warming on morphological, physiological, and biochemical responses of winter wheat under soil water deficiency.

Global climate change and freshwater scarcity have become two major environmental issues that constrain the sustainable development of the world economy. Climate warming caused by increasing atmospheric CO2 concentration can change global/regional rainfall patterns, leading to uneven global seasonal precipitation distribution and frequent regional extreme drought events, resulting in a drastic reduction of available water resources during the critical crop reproduction period, thus causing many important food-producing regions to face severe water deficiency problems. Understanding the potential processes and mechanisms of crops in response to elevated CO2 concentration and temperature under soil water deficiency may further shed lights on the potential risks of climate change on the primary productivity and grain yield of agriculture. We examined the effects of elevated CO2 concentration (e[CO2]) and temperature (experimental warming) on plant biomass and leaf area, stomatal morphology and distribution, leaf gas exchange and mesophyll anatomy, rubisco activity and gene expression level of winter wheat grown at soil water deficiency with environmental growth chambers. We found that e[CO2] × water × warming sharply reduced plant biomass by 57% and leaf photosynthesis (P n) 50%, although elevated [CO2] could alleviated the stress from water × warming at the amount of gene expression in RbcL3 (128%) and RbcS2 (215%). At ambient [CO2], the combined stress of warming and water deficiency resulted in a significant decrease in biomass (52%), leaf area (50%), P n (71%), and G s (90%) of winter wheat. Furthermore, the total nonstructural carbohydrates were accumulated 10% and 27% and increased R d by 127% and 99% when subjected to water × warming and e[CO2] × water × warming. These results suggest that water × warming may cause irreversible damage in winter wheat and thus the effect of "CO2 fertilization effect" may be overestimated by the current process-based ecological model.

The CO2 fertilization effect on leaf photosynthesis of maize (Zea mays L.) depends on growth temperatures with changes in leaf anatomy and soluble sugars.

Understanding the potential mechanisms and processes of leaf photosynthesis in response to elevated CO2 concentration ([CO2]) and temperature is critical for estimating the impacts of climatic change on the growth and yield in crops such as maize (Zea mays L.), which is a widely cultivated C4 crop all over the world. We examined the combined effect of elevated [CO2] and temperature on plant growth, leaf photosynthesis, stomatal traits, and biochemical compositions of maize with six environmental growth chambers controlling two CO2 levels (400 and 800 µmol mol-1) and three temperature regimes (25/19°C, 31/25°C, and 37/31°C). We found that leaf photosynthesis was significantly enhanced by increasing growth temperature from 25/19°C to 31/25°C independent of [CO2]. However, leaf photosynthesis drastically declined when the growth temperature was continually increased to 37/31°C at both ambient CO2 concentration (400 µmol mol-1, a[CO2]) and elevated CO2 concentration (800 µmol mol-1, e[CO2]). Meanwhile, we also found strong CO2 fertilization effect on maize plants grown at the highest temperature (37/31°C), as evidenced by the higher leaf photosynthesis at e[CO2] than that at a[CO2], although leaf photosynthesis was similar between a[CO2] and e[CO2] under the other two temperature regimes of 25/19°C and 31/25°C. Furthermore, we also found that e[CO2] resulted in an increase in leaf soluble sugar, which was positively related with leaf photosynthesis under the high temperature regime of 37/31°C (R 2 = 0.77). In addition, our results showed that e[CO2] substantially decreased leaf transpiration rates of maize plants, which might be partially attributed to the reduced stomatal openness as demonstrated by the declined stomatal width and stomatal area. These results suggest that the CO2 fertilization effect on plant growth and leaf photosynthesis of maize depends on growth temperatures through changing stomatal traits, leaf anatomy, and soluble sugar contents.

Overexpressing Jatropha curcas CBF2 in Nicotiana benthamiana improved plant tolerance to drought stress.

Jatropha curcas is an important bioenergy oil plant, and often planted on barren land to save the area of arable land. It is significant to improve the adaptability of J. curcas to various abiotic stresses. In the present study, we transferred a J. curcas gene, encoding a CBF2 transcription factor, into Nicotiana benthamiana. Under drought treatment, the JcCBF2 transgenic lines showed improved survival rate, leaf water retention and active oxygen scavenging capacity, but reduced photosynthesis and transpiration rate, suggesting that JcCBF2 played an important role in improving plant drought tolerance. Overexpressing JcCBF2 decreased leaf area and increased leaf thickness. To explore the possible mechanisms for the change of leaf anatomical structure, the leaves of wild-type and overexpression lines under drought stress were RNA sequenced. Genes involved in the plant hormones signal transduction were found to be enriched. Cytokinin and indole-3-acetic acid were the major plant hormones whose abundance increased. Quantitative RT-PCR analysis showed expression of NbMYB21, NbMYB86 and NbMYB44 and both abscisic acid (ABA) and jasmonic acid (JA) related genes in the overexpression lines were increased under drought stress. These results indicated that JcCBF2 was able to positively regulate plant drought response by changing the leaf anatomical structure and possibly through JA and ABA signalling pathways. Our work may help us to understand the drought tolerant mechanism.

Enhancement of Tobacco (Nicotiana tabacum L.) Seed Lipid Content for Biodiesel Production by CRISPR-Cas9-Mediated Knockout of NtAn1.

Tobacco (Nicotiana tabacum L.) seed lipid is a promising non-edible feedstock for biodiesel production. In order to meet the increasing demand, achieving high seed lipid content is one of the major goals in tobacco seed production. The TT8 gene and its homologs negatively regulate seed lipid accumulation in Arabidopsis and Brassica species. We speculated that manipulating the homolog genes of TT8 in tobacco could enhance the accumulation of seed lipid. In this present study, we found that the TT8 homolog genes in tobacco, NtAn1a and NtAn1b, were highly expressed in developing seed. Targeted mutagenesis of NtAn1 genes was created by the CRISPR-Cas9-based gene editing technology. Due to the defect of proanthocyanidin (PA) biosynthesis, mutant seeds showed the phenotype of a yellow seed coat. Seed lipid accumulation was enhanced by about 18 and 15% in two targeted mutant lines. Protein content was also significantly increased in mutant seeds. In addition, the seed yield-related traits were not affected by the targeted mutagenesis of NtAn1 genes. Thus, the overall lipid productivity of the NtAn1 knockout mutants was dramatically enhanced. The results in this present paper indicated that tobacco NtAn1 genes regulate both PAs and lipid accumulation in the process of seed development and that targeted mutagenesis of NtAn1 genes could generate a yellow-seeded tobacco variety with high lipid and protein content. Furthermore, the present results revealed that the CRISPR-Cas9 system could be employed in tobacco seed de novo domestication for biodiesel feedstock production.

Complete mitochondrial genome of striped gourami, Trichogaster fasciata (Perciformes: Osphronemidae).

The complete mitochondrial genome of Trichogaster fasciata is determined in this study. It is 16,635 bp in size and consists of 2 rRNA genes, 13 protein-coding genes, 22 tRNA genes and 1 non-coding control region (D-loop). The overall base composition of the heavy strand of the T. fasciata mitochondrial genome is A: 29.18%, T: 30.22%, C: 25.14%, and G: 15.46%. A 78 bp AT tandem repeats was identified in the control region. This present study will be helpful to bring out the fact of genetic divergence among the genus Trichogaster.

Genetic variation and phylogenetic relationship analysis of Jatropha curcas L. inferred from nrDNA ITS sequences.

Genetic variation and phylogenetic relationships among 102 Jatropha curcas accessions from Asia, Africa, and the Americas were assessed using the internal transcribed spacer region of nuclear ribosomal DNA (nrDNA ITS). The average G+C content (65.04%) was considerably higher than the A+T (34.96%) content. The estimated genetic diversity revealed moderate genetic variation. The pairwise genetic divergences (GD) between haplotypes were evaluated and ranged from 0.000 to 0.017, suggesting a higher level of genetic differentiation in Mexican accessions than those of other regions. Phylogenetic relationships and intraspecific divergence were inferred by Bayesian inference (BI), maximum parsimony (MP), and median joining (MJ) network analysis and were generally resolved. The J. curcas accessions were consistently divided into three lineages, groups A, B, and C, which demonstrated distant geographical isolation and genetic divergence between American accessions and those from other regions. The MJ network analysis confirmed that Central America was the possible center of origin. The putative migration route suggested that J. curcas was distributed from Mexico or Brazil, via Cape Verde and then split into two routes. One route was dispersed to Spain, then migrated to China, eventually spreading to southeastern Asia, while the other route was dispersed to Africa, via Madagascar and migrated to China, later spreading to southeastern Asia.

Over-expression of CYP78A98, a cytochrome P450 gene from Jatropha curcas L, increases seed size of transgenic tobacco

Background: Jatropha curcas L. (further referred to as Jatropha), as a rapidly emerging biofuel crop, has attracted worldwide interest. However, Jatropha is still an undomesticated plant, the true potential of this shrub has not yet been fully realized. To explore the potential of Jatropha, breeding and domestication are needed. Seed size is one of the most important traits of seed yield and has been selected since the beginning of agriculture. Increasing the seed size is a main goal of Jatropha domestication for increasing the seed yield, but the genetic regulation of seed size in Jatropha has not been fully understood. Results: We cloned CYP78A98 gene from Jatropha,a homologue of CYP78A5 in Arabidopsis.Wefound that CYP78A98 was highly expressed in male flower, female flower, stem apex, leaf and developing seed. However, its transcripts were hardly detected in root and stem. CYP78A98 protein localized in endoplasmic reticulum (ER) and the hydrophobic domain at the N-terminus was essential for the correct protein localization. Furthermore, INNER NO OUTER promoter (pINO) drove specific overexpression of CYP78A98 in transgenic tobacco seeds resulted in increased seed size and weight, as well as improved seed protein and fatty acid content. Conclusions: The results indicated that CYP78A98 played a role in Jatropha seed size control. This may help us to better understand the genetic regulation of Jatropha seed development, and accelerate the breeding progress of Jatropha.