MiR397b regulates both lignin content and seed number in Arabidopsis via modulating a laccase involved in lignin biosynthesis

Plant laccase (LAC) enzymes belong to the blue copper oxidase family and polymerize monolignols into lignin. Recent studies have established the involvement of microRNAs in this process; however, physiological functions and regulation of plant laccases remain poorly understood. Here, we show that a laccase gene, LAC4, regulated by a microRNA, miR397b, controls both lignin biosynthesis and seed yield in Arabidopsis. In transgenic plants, overexpression of miR397b (OXmiR397b) reduced lignin deposition. The secondary wall thickness of vessels and the fibres was reduced in the OXmiR397b line, and both syringyl and guaiacyl subunits are decreased, leading to weakening of vascular tissues. In contrast, overexpression of miR397b‐resistant laccase mRNA results in an opposite phenotype. Plants overexpressing miR397b develop more than two inflorescence shoots and have an increased silique number and silique length, resulting in higher seed numbers. In addition, enlarged seeds and more seeds are formed in these miR397b overexpression plants. The study suggests that miR397‐mediated development via regulating laccase genes might be a common mechanism in flowering plants and that the modulation of laccase by miR397 may be potential for engineering plant biomass production with less lignin.     

棉花miR397 LAC4模块调控棉铃虫抗性研究

miRNA(microRNA)通过调控其靶标基因在植物的生长、发育和抗逆过程中扮演着重要的角色。该研究采用分子生物学和生物化学等方法,探讨棉花miR397 LAC4参与植株木质素生物合成和对棉铃虫抗性响应机制。结果发现：(1)棉花miR397(ghr miR397)在转录后调控漆酶基因(GhLAC4)的表达,GhLAC4属于蓝铜氧化酶家族,通过调控木质素合成,抵御棉铃虫入侵棉花。(2)GUS报告基因融合表达和酶活性测定表明,ghr miR397在转录后切割靶标基因GhLAC4抑制其表达。(3)利用VIGS(virus induced gene silencing)技术在棉花中沉默和过表达ghr miR397,棉铃虫抗性检测分析表明,沉默miR397表达会增加棉花对棉铃虫的抗性,但过表达ghr miR397则会降低棉花的抗性。(4)选择性和非选择性棉铃虫实验分析、组织化学染色和木质素含量测定表明,沉默GhLAC4表达会减少木质素的积累,增加棉花对棉铃虫的敏感性。研究表明,ghr miR397 GhLAC4模块共同微调棉花木质素合成来参与棉花抗虫性调控,同时也为棉花抗虫育种提供了新思路。     

Overexpression of microRNA319 impacts leaf morphogenesis and leads to enhanced cold tolerance in rice (Oryza sativa L.)

MicroRNA319 (miR319) family is one of the conserved microRNA (miRNA) families among diverse plant species. It has been reported that miR319 regulates plant development in dicotyledons, but little is known at present about its functions in monocotyledons. In rice (Oryza sativa L.), the MIR319 gene family comprises two members, Osa‐MIR319a and Osa‐MIR319b. Here, we report an expression pattern analysis and a functional characterization of the two Osa‐MIR319 genes in rice. We found that overexpressing Osa‐MIR319a and Osa‐MIR319b in rice both resulted in wider leaf blades. Leaves of osa‐miR319 overexpression transgenic plants showed an increased number of longitudinal small veins, which probably accounted for the increased leaf blade width. In addition, we observed that overexpressing osa‐miR319 led to enhanced cold tolerance (4 °C) after chilling acclimation (12 °C) in transgenic rice seedlings. Notably, under both 4 and 12 °C low temperatures, Osa‐MIR319a and Osa‐MIR319b were down‐regulated while the expression of miR319‐targeted genes was induced. Furthermore, genetically down‐regulating the expression of either of the two miR319‐targeted genes, OsPCF5 and OsPCF8, in RNA interference (RNAi) plants also resulted in enhanced cold tolerance after chilling acclimation. Our findings in this study demonstrate that miR319 plays important roles in leaf morphogenesis and cold tolerance in rice.     

Simultaneous regulation of F5H in COMT‐RNAi transgenic switchgrass alters effects of COMT suppression on syringyl lignin biosynthesis

Ferulate 5‐hydroxylase (F5H) catalyses the hydroxylation of coniferyl alcohol and coniferaldehyde for the biosynthesis of syringyl (S) lignin in angiosperms. However, the coordinated effects of F5H with caffeic acid O‐methyltransferase (COMT) on the metabolic flux towards S units are largely unknown. We concomitantly regulated F5H expression in COMT‐down‐regulated transgenic switchgrass (Panicum virgatum L.) lines and studied the coordination of F5H and COMT in lignin biosynthesis. Down‐regulation of F5H in COMT‐RNAi transgenic switchgrass plants further impeded S lignin biosynthesis and, consequently, increased guaiacyl (G) units and reduced 5‐OH G units. Conversely, overexpression of F5H in COMT‐RNAi transgenic plants reduced G units and increased 5‐OH units, whereas the deficiency of S lignin biosynthesis was partially compensated or fully restored, depending on the extent of COMT down‐regulation in switchgrass. Moreover, simultaneous regulation of F5H and COMT expression had different effects on cell wall digestibility of switchgrass without biomass loss. Our results indicate that up‐regulation and down‐regulation of F5H expression, respectively, have antagonistic and synergistic effects on the reduction in S lignin resulting from COMT suppression. The coordinated effects between lignin genes should be taken into account in future studies aimed at cell wall bioengineering.     

Down-regulation of an anionic peroxidase in transgenic aspen and its effect on lignin characteristics

It is generally accepted that peroxidases catalyze the final step in the biosynthesis of lignin. In this study, to examine how expression of prxA3a, a gene for an anionic peroxidase, might be related to lignification in plant tissues, we produced transgenic tobacco plants that harbored a gene for β-glucuronidase (GUS) fused to the prxA3a promoter. Histochemical staining for GUS activity indicated that the prxA3a promoter was active mainly in the lignifying cells of stem tissues. Further, to examine the effects of suppressing the expression of prxA3a, we transferred an antisense prxA3a gene construct into the original host, hybrid aspen (Populus sieboldii ×P. gradidentata), under the control of the original promoter of the prxA3a gene. Eleven transformed aspens were obtained and characterized, and the stable integration of the antisense construct was confirmed by PCR and Southern blotting analysis in all these lines. Assays of enzymatic activity showed that both total peroxidase activity and acidic peroxidase activity were lower in most transgenic lines than in the control plants. In addition, the reduction of peroxidase activity was associated with lower lignin content and modified lignin composition. Transgenic lines with the highest reduction of peroxidase activity displayed a higher syringyl/vanillin (S/V) ratio and a lower S+V yield, mainly because of a decreased amount of V units. Thus, our results indicate that prxA3a is involved in the lignification of xylem tissue and that the down-regulation of anionic peroxidase alters both lignin content and composition in hybrid aspen.     

Functional characterization of CCR in birch (Betula platyphylla × Betula pendula) through overexpression and suppression analysis

We cloned a Cinnamoyl‐CoA Reductase gene (BpCCR1) from an apical meristem and first internode of Betula platyphylla and characterized its functions in lignin biosynthesis, wood formation and tree growth through transgenic approaches. We generated overexpression and suppression transgenic lines and analyzed them in comparison with the wild‐type in terms of lignin content, anatomical characteristics, height and biomass. We found that BpCCR1 overexpression could increase lignin content up to 14.6%, and its underexpression decreased lignin content by 6.3%. Surprisingly, modification of BpCCR1 expression led to conspicuous changes in wood characteristics, including xylem vessel number and arrangement, and secondary wall thickness. The growth of transgenic trees in terms of height was also significantly influenced by the modification of BpCCR1 genes. We discuss the functions of BpCCR1 in the context of a phylogenetic tree built with CCR genes from multiple species.     

Time Course Field Analysis of COMT-Downregulated Switchgrass: Lignification,Recalcitrance, and Rust Susceptibility

Modifying plant cell walls by manipulating lignin biosynthesis can improve biofuel yields from lignocellulosic crops. For example, transgenic switchgrass lines with downregulated expression of caffeic acid O-methyltransferase, a lignin biosynthetic enzyme, produce up to 38 % more ethanol than controls. The aim of the present study was to understand cell wall lignification over the second and third growing seasons of COMT-downregulated field-grown switchgrass. COMT gene expression, lignification, and cell wall recalcitrance were assayed for two independent transgenic lines at monthly intervals. Switchgrass rust (Puccinia emaculata) incidence was also tracked across the seasons. Trends in lignification over time differed between the 2 years. In 2012, sampling was initiated in mid-growing season on reproductive-stage plants and there was little variation in the lignin content of all lines (COMT-downregulated and control) over time. COMT-downregulated lines maintained 11–16 % less lignin, 33–40 % lower S/G (syringyl-to-guaiacyl) ratios, and 15–42 % higher sugar release relative to controls for all time points. In 2013, sampling was initiated earlier in the season on elongation-stage plants and the lignin content of all lines steadily increased over time, while sugar release expectedly decreased. S/G ratios increased in non-transgenic control plants as biomass accumulated over the season, while remaining relatively stable across the season in the COMT-downregulated lines. Differences in cell wall chemistry between transgenic and non-transgenic lines were not apparent until plants transitioned to reproductive growth in mid-season, after which the cell walls of COMT-downregulated plants exhibited phenotypes consistent with what was observed in 2012. There were no differences in rust damage between transgenics and controls at any time point. These results provide relevant fundamental insights into the process of lignification in a maturing field-grown biofuel feedstock with downregulated lignin biosynthesis.