Flax is a crucial fiber crop that exhibits excellent textile properties and serves as a model plant for investigating phloem fiber development.  The regulation of multiple genes significantly influences fiber development, notably involving NAC (NAM, ATAF1/2, CUC2) transcription factors in forming the fiber secondary cell wall (SCW).  Overexpression of LuNAC61 in flax resulted in sparse top meristematic zone leaves and significantly reduced stem cellulose content.  Scanning electron microscopy and staining observations revealed a significant reduction in fiber bundles.  β-Glucuronidase (GUS) staining analysis demonstrated high activity of the LuNAC61 promoter in the bast fibers of the flax stem.  Additionally, several members of the LuPLATZ and LuCesA families exhibited significant coexpression with LuNAC61.  Subcellular localization indicated the presence of LuPLATZ24 protein in the nucleus and cytoplasm, LuNAC61 protein exclusively in the nucleus, and LuCesA10 in the nucleus and endoplasmic reticulum.  LuPLATZ24 positively regulates LuNAC61, whereas LuNAC61 negatively affects LuCesA10, suggesting the involvement of a metabolic network in regulating flax fiber development.  In conclusion, this study provides a critical opportunity for a comprehensive and in-depth analysis of the mechanisms governing flax fiber development and the potential use of biotechnology to enhance flax fiber yield.

Cotton is a pivotal economic crop for natural textile fibers that also serves as an important source of edible oil (Long et al. 2023). Cottonseed oil contains approximately 14% oleic acid and 59% linoleic acid. An increase in monounsaturated fatty acids, particularly oleic acid, enhances the oxidative stability and nutritional value of edible oil (Chen et al. 2021). Currently, the demand for edible oil in China is increasing in terms of both production and nutrition. Improving cottonseed oil’s storability and nutritional value is crucial for the comprehensive utilization of cotton. However, cottonseed has long been regarded as a by-product in the cotton industry, so research on improving the content and quality of cottonseed oil has lagged compared to other crop attributes.

Phosphatidylcholine: diacylglycerol cholinephospho-transferase (PDCT) is the gate-keeping enzyme for the conversion between phosphatidylcholine and diacylglycerol (Lu et al. 2009). Studies in multiple plants have revealed increases in monounsaturated fatty acids in seeds with PDCT knock-out. To clone the PDCTs of upland cotton (Gossypium hirsutum), the protein sequences of PDCT from Arabidopsis (Lu et al. 2009), oilseed rape (Brassica napus; Bai et al. 2020), soybean (Glycine max; Li et al. 2023), peanut (Arachis hypogaea), and sesame (Sesamum indicum) were used as references for BLAST searches in CottonMD (https://yanglab.hzau.edu.cn/CottonMD; Yang et al. 2023). Four PDCT homologs in cotton were obtained and named GhPDCT1 (Gh_D06G1990), GhPDCT2 (Gh_A06G1621), GhPDCT3 (Gh_A05G3864), and GhPDCT4 (Gh_D05G1178) (Fig. 1-A). The sequence similarities between the four GhPDCTs and AtPDCT are 58.47, 60.13, 45.18, and 58.61%, respectively. Further, the phylogenetic analysis revealed that the GhPDCTs are clustered with the PDCTs of Brassica napus (Fig. 1-A).

The heatmap of GhPDCTs in cotton tissues was built using released transcriptome data. The results showed that GhPDCT3 and GhPDCT4 had very little expression in all tissues (Fig. 1-B). GhPDCT2 was expressed in roots, stems, leaves and ovules at different developmental stages, but at relatively low levels. GhPDCT1 shared similar basal expression with GhPDCT2, but the transcript level of GhPDCT1 in ovules was significantly higher than that of GhPDCT2. Notably, the expression of GhPDCT1 was sharply up-regulated in ovules at 20 and 25 days post anthesis (DPA). The expression pattern of GhPDCT1 was further verified by RT-qPCR, which indicated that GhPDCT1 was up-regulated in the late stage of ovule development and peaked around 25 DPA. Previous reports highlighted the rapid accumulation of oil content in cotton seeds at 20–30 DPA (Zhao et al. 2018). Therefore, GhPDCT1 is considered the key candidate for regulating the seed oil content of cotton (Fig. 1-B).

Sequence analysis showed that GhPDCT1/2 and GmPDCT1/2 contain similar conserved motifs, as well as a C-terminal PAP2_3 domain (Fig. 1-C). The GmPDCT1 and GmPDCT2 in soybean were both found to be located in the cytosol (Li et al. 2023). To study the subcellular localization of GhPDCT, a GFP-PDCT1 fusion protein was expressed in the protoplasts of cotton cotyledons (Hu et al. 2022), and the RFP-labeled transcription factor GoPGF (Zhang et al. 2024) was co-expressed to mark the nucleus. Observations with a laser scanning confocal microscope showed the green fluorescence of GFP-PDCT1 expressed in the cytoplasm (Fig. 1-D).

Knock-out of GhPDCT was achieved with the optimized CRISPR/Cas9 system of cotton (Wang et al. 2018). Due to the high similarity (94.2%) of the coding sequences of GhPDCT1 and GhPDCT2, two sgRNAs respectively targeting two different sites of the 1st exon were designed for the simultaneous mutagenesis of GhPDCT1 and GhPDCT2 (Fig. 1-E). The G. hirsutum L. line ‘Jin668’ was used to produce the GhPDCT1/2 mutant of cotton (ghpdct) with Agrobacterium-mediated transformation (Zhu et al. 2023). The DNA of the ghpdct mutant was extracted for Hi-TOM sequencing, and the offspring of ghpdct-5 with the full mutation were planted for further studies. As shown in Fig. 1-F, ghpdct-5 has a 1 nt deletion at target 2 of GhPDCT2 (A subgenome). In addition, two types of mutations were found in GhPDCT1 (D subgenome), one with a 1 nt insertion at target 1, and the other with a 1 nt insertion and a 2 nt deletion at target 1. The wild type (WT) and ghpdct were planted in the field and a phenotypic study was conducted during the whole growing period. No obvious differences in plant growth were observed between WT and ghpdct. For example, the plant height, fiber length, seed weight of WT and ghpdct showed no statistically significant differences (Fig. 1-G–I).

The fatty acids in seeds of WT and ghpdct were measured by gas chromatography-mass spectrometry (GC-MS) (Fig. 1-J). Oleic acid (OA, C18:1) accounted for an average of 14.46% of the total fatty acids in seeds of WT, and 16.49% in seeds of ghpdct, which indicates the up-regulation of oleic acid in the ghpdct mutant. Conversely, linoleic acid (LA, C18:2) was reduced in seeds of ghpdct (52.83%) compared to seeds of WT (59.98%). In addition, knockout of GhPDCT increased the seed content of palmitic acid (PA, C16:0) from 21.24% in WT to 25.85% in ghpdct, and the content of stearic acid (SA, C18:0) increased from 1.70% in WT seeds to 2.39% in ghpdct seeds. These results indicated that the GhPDCT mutation alters the balance of monounsaturated and polyunsaturated fatty acids in cotton seeds, with minimal impacts on growth and development beyond seed oil metabolism.

In conclusion, we have produced the ghpdct mutant of cotton using the CRISPR/Cas9 system. Knock-out of GhPDCT1/2 affects the conversion between phosphatidylcholine and diacylglycerol in cottonseeds, and changes the contents of oleic acid, linoleic acid, palmitic acid, and stearic acid. We obtained a new germplasm with a higher oleic acid content in cottonseed oil, which can be applied to enhance the economic and nutritional value of cotton as an oil crop, thereby contributing to the industrial upgrading of cotton.

In situ mRNA hybridization (ISH) is a powerful tool for examining the spatiotemporal expression of genes in shoot apical meristems and flower buds of cucumber.  The most common ISH protocol uses paraffin wax; however, embedding tissue in paraffin wax can take a long time and might result in RNA degradation and decreased signals.  Here, we developed an optimized protocol to simplify the process and improve RNA sensitivity.  We combined embedding tissue in low melting-point Steedman’s wax with processing tissue sections in solution, as in the whole-mount ISH method in the optimized protocol. Using the optimized protocol, we examined the expression patterns of the CLAVATA3 (CLV3) and WUSCHEL (WUS) genes in shoot apical meristems and floral meristems of Cucumis sativus (cucumber) and Arabidopsis thaliana (Arabidopsis).  The optimized protocol saved 4–5 days of experimental period compared with the standard ISH protocol using paraffin wax.  Moreover, the optimized protocol achieved high signal sensitivity.  The optimized protocol was successful for both cucumber and Arabidopsis, which indicates it might have general applicability to most plants

Sex determination in plants gives rise to unisexual flowers.  A better understanding of the regulatory mechanism underlying the production of unisexual flowers will help to clarify the process of sex determination in plants and allow researchers and farmers to harness heterosis.  Androecious cucumber (Cucumis sativus L.) plants can be used as the male parent when planted alongside a gynoecious line to produce heterozygous seeds, thus reducing the cost of seed production.  The isolation and characterization of additional androecious genotypes in varied backgrounds will increase the pool of available germplasm for breeding.  Here, we discovered an androecious mutant in a previously generated ethyl methanesulfonate (EMS)-mutagenized library of the cucumber inbred line ‘406’.  Genetic analysis, whole-genome resequencing, and molecular marker-assisted verification demonstrated that a nonsynonymous mutation in the ethylene biosynthetic gene 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE 11 (ACS11) conferred androecy.  The mutation caused an amino acid change from serine (Ser) to phenylalanine (Phe) at position 301 (S301F).  In vitro enzyme activity assays revealed that this S301F mutation leads to a complete loss of enzymatic activity.  This study provides a new germplasm for use in cucumber breeding as the androecious male parent, and it offers new insights into the catalytic mechanism of ACS enzymes.

Planting grass and legume mixtures on improved grasslands has the potential advantage of realizing both higher yields and lower environmental pollution by optimizing the balance between applied N fertilizer and the natural process of legume biological nitrogen fixation. However, the optimal level of N fertilization for grass-legume mixtures, to obtain the highest yield, quality, and contribution of N2 fixation, varies with species. A greenhouse pot experiment was conducted to study the temporal dynamics of N2 fixation of alfalfa (Medicago sativa L.) grown alone and in mixture with smooth bromegrass (Bromus inermis Leyss.) in response to the addition of fertilizer N. Three levels of N (0, 75, and 150 kg ha–1) were examined using 15N-labeled urea to evaluate N2 fixation via the 15N isotope dilution method. Treatments were designated N0 (0.001 g per pot), N75 (1.07 g per pot) and N150 (2.14 g per pot). Alfalfa grown alone did not benefit from the addition of fertilizer N; dry matter was not significantly increased. In contrast, dry weight and N content of smooth bromegrass grown alone was increased significantly by N application. When grown as a mixture, smooth bromegrass biomass was increased significantly by N application, resulted in a decrease in alfalfa biomass. In addition, individual alfalfa plant dry weight (shoots+roots) was significantly lower in the mixture than when grown alone at all N levels. Smooth bromegrass shoot and root dry weight were significantly higher when grown with alfalfa than when grown alone, regardless of N application level. When grown alone, alfalfa’s N2 fixation was reduced with N fertilization (R2=0.9376, P=0.0057). When grown in a mixture with smooth bromegrass, with 75 kg ha–1 of N fertilizer, the percentage of atmospheric N2 fixation contribution to total N in alfalfa (%Ndfa) had a maximum of 84.07 and 83.05% in the 2nd and 3rd harvests, respectively. Total 3-harvest %Ndfa was higher when alfalfa was grown in a mixture than when grown alone (shoots: |t|=3.39, P=0.0096; root: |t|=3.57, P=0.0073). We believe this was due to smooth bromegrass being better able to absorb available soil N (due to its fibrous root system), resulting in lower soil N availability and allowing alfalfa to develop an effective N2 fixing symbiosis prior to the 1st harvest. Once soil N levels were depleted, alfalfa was able to fix N2, resulting in the majority of its tissue N being derived from biological nitrogen fixation (BNF) in the 2nd and 3rd harvests. When grown in a mixture, with added N, alfalfa established an effective symbiosis earlier than when grown alone; in monoculture BNF did not contribute a significant portion of plant N in the N75 and N150 treatments, whereas in the mixture, BNF contributed 17.90 and 16.28% for these treatments respectively. Alfalfa has a higher BNF efficiency when grown in a mixture, initiating BNF earlier, and having higher N2 fixation due to less inhibition by soil-available N. For the greatest N-use-efficiency and sustainable production, grass-legume mixtures are recommended for improving grasslands, using a moderate amount of N fertilizer (75 kg N ha–1) to provide optimum benefits.