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CRISPR/Cas9 is a valuable tool and has been extensively employed to perform gene editing in plants. However, CRISPR/Cas9 has not been successfully used in spinach, an important leafy vegetable crop. Here, we established a CRISPR/Cas9-based gene-editing system for spinach hairy roots and edited two cellulose synthase-like D (CSLD) genes (SoCSLD2 and SoCSLD3) that were involved in root-hair formation of spinach hairy roots. Four mutation types (i.e., replacement, insertion, deletion, and combined mutations) were observed, among which the replacement accounted for the vast majority (about 64.1%). Mutation rate differed largely among different targets. Seven homozygous/bi-allelic and eight heterozygous/chimeric mutants of SoCSLD2 were obtained from 15 independent transgenic hairy root lines. All of the seven homozygous/bi-allelic mutant lines displayed bulking and short root hairs, which resembled the characteristics of Arabidopsis atcsld2 mutants. The transcriptomic analysis further revealed that multiple gene expressions for cell-wall modulation and membrane trafficking were disturbed, which might result in the inhibition of root hair growth in socsld2 mutants. This indicates that SoCSLD2 was successfully knocked out in spinach root hairs using the CRISPR/Cas9-based gene editing system. [ABSTRACT FROM AUTHOR]

Plant laccase genes belong to a multigene family, play key roles in lignin polymerization, and participate in the resistance of plants to biotic and abiotic stresses. Switchgrass is an important resource for forage and bioenergy production, yet information about the switchgrass laccase gene family is scarce. Using bioinformatic approaches, a genome-wide analysis of the laccase multigene family in switchgrass was carried out in this study. In total, 49 laccase genes (PvLac1 to PvLac49) were identified; these can be divided into five subclades, and 20 of them were identified as targets of miR397. The tandem and segmental duplication of laccase genes on Chr05 and Chr08 contributed to the expansion of the laccase family. The laccase proteins shared conserved signature sequences but displayed relatively low sequence similarity, indicating the potential functional diversity of switchgrass laccases. Switchgrass laccases exhibited distinct tissue/organ expression patterns, revealing that some laccases might be involved in the lignification process during stem development. All five of the laccase isoforms selected from different subclades responded to heavy metal. The immediate response of lignin-related laccases, as well as the delayed response of low-abundance laccases, to heavy-metal treatment shed light on the multiple roles of laccase isoforms in response to heavy-metal stress. [ABSTRACT FROM AUTHOR]

Cafferic acid-O-methyltransferases (COMT) down-regulated transgenic and wild-type switchgrass were separated into lignocresols (LCs) and sugars by a phase separation method involving 72% sulfuric acid and cresol. The isolated LCs were characterized by FTIR, GPC, 1H NMR and 2D-HSQC to understand potential structural modification caused by transgenic engineering lignin or phase separation treatment. No significant changes were found in terms of molecular weights and the amount of incorporated p-cresols between transgenic and wild-type switchgrass LCs. However, the compositions, ratios of syringyl (S) units to guaiacyl (G) units, were changed significantly leading to decrease in S units and increase in G units for transgenic switchgrass LC. The benzodioxane structures and 5-hydroxyguaiacyl units were observed in the 2D-HSQC implied that 5-hydroxyconiferyl alcohol was incorporated into lignin as a result of COMT-down-regulation in the transgenic process. [ABSTRACT FROM AUTHOR]