aBIOTECH

Benzylisoquinoline alkaloids (BIAs) are a diverse family of plant natural products with extensive pharmacological properties, but the yield of BIAs from plant is limited. The understanding of BIA biosynthetic mechanism in plant and the development of synthetic biology enable the possibility to produce BIAs through microbial fermentation, as an alternative to agriculture-based supply chains. In this review, we discussed the engineering strategies to synthesize BIAs in Saccharomyces cerevisiae (yeast) and improve BIA production level, including heterologous pathway reconstruction, enzyme engineering, expression regulation, host engineering and fermentation engineering. We also highlight recent metabolic engineering advances in the production of BIAs in yeast.

Jianing Han

Yinan Wu

Yilun Zhou

Sijin Li

Engineering Saccharomyces cerevisiae to produce plant benzylisoquinoline alkaloids.

The tyrosine metabolism pathway serves as a starting point for the production of a variety of structurally diverse natural compounds in plants, such as tocopherols, plastoquinone, ubiquinone, betalains, salidroside, benzylisoquinoline alkaloids, and so on. Among these, tyrosine-derived metabolites, tocopherols, plastoquinone, and ubiquinone are essential to plant survival. In addition, this pathway provides us essential micronutrients (e.g., vitamin E and ubiquinone) and medicine (e.g., morphine, salidroside, and salvianolic acid B). However, our knowledge of the plant tyrosine metabolism pathway remains rudimentary, and genes encoding the pathway enzymes have not been fully defined. In this review, we summarize and discuss recent advances in the tyrosine metabolism pathway, key enzymes, and important tyrosine-derived metabolites in plants.

Jing-Jing Xu

Xin Fang

Chen-Yi Li

Lei Yang

Xiao-Ya Chen

General and specialized tyrosine metabolism pathways in plants.

Plants absorb light energy for photosynthesis via photosystem complexes in their chloroplasts. However, excess light can damage the photosystems and decrease photosynthetic output, thereby inhibiting plant growth and development. Plants have developed a series of light acclimation strategies that allow them to withstand high light. In the first line of defense against excess light, leaves and chloroplasts move away from the light and the plant accumulates compounds that filter and reflect the light. In the second line of defense, known as photoprotection, plants dissipate excess light energy through non-photochemical quenching, cyclic electron transport, photorespiration, and scavenging of excess reactive oxygen species. In the third line of defense, which occurs after photodamage, plants initiate a cycle of photosystem (mainly photosystem II) repair. In addition to being the site of photosynthesis, chloroplasts sense stress, especially light stress, and transduce the stress signal to the nucleus, where it modulates the expression of genes involved in the stress response. In this review, we discuss current progress in our understanding of the strategies and mechanisms employed by plants to withstand high light at the whole-plant, cellular, physiological, and molecular levels across the three lines of defense.

Man Zhang

Yu Ming

Hong-Bin Wang

Hong-Lei Jin

Strategies for adaptation to high light in plants

植物适应高光环境的策略

Targeted gene manipulation is highly desirable for fundamental plant research, plant synthetic biology, and molecular breeding. The clustered regularly interspaced short palindromic repeats-associated (Cas) nuclease is a revolutionary tool for genome editing, and has received snowballing popularity for gene knockout applications in diverse organisms including plants. Recently, the nuclease-dead Cas (dCas) proteins have been repurposed as programmable transcriptional regulators through translational fusion with portable transcriptional repression or activation domains, which has paved new ways for flexible and multiplex control over the activities of target genes of interest without the need to generate DNA lesions. Here, we review the most important breakthroughs of dCas transcriptional regulators in non-plant organisms and recent accomplishments of this growing field in plants. We also provide perspectives on future development directions of dCas transcriptional regulators in plant research in hope to stimulate their quick evolution and broad applications.

Zhenxiang Li

Xiangyu Xiong

Jian-Feng Li

The working dead: repurposing inactive CRISPR-associated nucleases as programmable transcriptional regulators in plants.

工作中的死者：将失活的CRISPR相关核酸酶重新用作植物中的可编程转录调控因子。

The CRISPR-Cas genome editing tools are revolutionizing agriculture and basic biology with their simplicity and precision ability to modify target genomic loci. Software-predicted guide RNAs (gRNAs) often fail to induce efficient cleavage at target loci. Many target loci are inaccessible due to complex chromatin structure. Currently, there is no suitable tool available to predict the architecture of genomic target sites and their accessibility. Hence, significant time and resources are spent on performing editing experiments with inefficient guides. Although in vitro-cleavage assay could provide a rough assessment of gRNA efficiency, it largely excludes the interference of native genomic context. Transient in-vivo testing gives a proper assessment of the cleavage ability of editing reagents in a native genomic context. Here, we developed a modified protocol that offers highly efficient protoplast isolation from rice,Arabidopsis,and chickpea, using a sucrose gradient, transfection using PEG (polyethylene glycol), and validation of single guide RNAs (sgRNAs) cleavage efficiency of CRISPR-Cas9. We have optimized various parameters for PEG-mediated protoplast transfection and achieved high transfection efficiency using our protocol in both monocots and dicots. We introduced plasmid vectors containing Cas9 and sgRNAs targeting genes in rice,Arabidopsis,and chickpea protoplasts. Using dual sgRNAs, our CRISPR-deletion strategy offers straightforward detection of genome editing success by simple agarose gel electrophoresis. Sanger sequencing of PCR products confirmed the editing efficiency of specific sgRNAs. Notably, we demonstrated that isolated protoplasts can be stored for up to 24/48 h with little loss of viability, allowing a pause between isolation and transfection. This high-efficiency protocol for protoplast isolation and transfection enables rapid (less than 7 days) validation of sgRNA cleavage efficiency before proceeding with stable transformation. The isolation and transfection method can also be utilized for rapid validation of editing strategies, evaluating diverse editing reagents, regenerating plants from transfected protoplasts, gene expression studies, protein localization and functional analysis, and other applications.

Debasmita Panda

Subhasis Karmakar

Manaswini Dash

Swagat Kumar Tripathy

Priya Das

Sagar Banerjee

Yiping Qi

Sanghamitra Samantaray

Pradipta Kumar Mohapatra

Mirza J. Baig

Kutubuddin A. Molla

Optimized protoplast isolation and transfection with a breakpoint: accelerating Cas9/sgRNA cleavage efficiency validation in monocot and dicot

优化原生质体分离与转染：在单子叶和双子叶植物中加速Cas9/sgRNA切割效率验证的关键点

Abstract The transition from vegetative to reproductive growth is a vital step for the reproductive success of plants. In Arabidopsis thaliana , LEAFY (LFY) plays crucial roles in inflorescence primordium and floral organ development, but little is known about the roles of its homologs in crop plants such as soybean ( Glycine max ). Here, we investigated the expression patterns and functions of the two LFY genes ( LFY1 and LFY2 ) in soybean. Both genes were predominantly expressed in unopened flowers and the shoot apical meristem, with LFY2 having the higher transcript abundance. In an in situ hybridization assay, LFY genes produced strong signals in the floral meristem. We next generated lfy1 and lfy2 knockout lines. The lfy2 mutants showed obvious changes in floral organ morphology, but the lfy1 mutants showed no obvious changes in floral organ morphology or pod development. The lfy1 lfy2 double mutants displayed more serious defects in floral organ development than lfy2 , resulting in complete sterility. Gene expression analysis revealed differences in expression of the A-class APETALA ( AP ) genes AP1a and AP1b in the double mutant lines. These results suggest that LFY2 plays an important role in floral organ formation in soybean by regulating the expression of homeotic genes. Our findings increase the understanding of floral development, which could be useful for flower designs during hybrid soybean breeding.

Lingshuang Wang

Huan Liu

Lei Chen

Tong Su

Shichen Li

Chao Fang

Sijia Lu

Baohui Liu

Hui Yang

Fanjiang Kong

LEAFY1 and 2 are required for floral organ development in soybean

LEAFY1 和 2 是大豆花器官发育所必需的

Honggang Dai

Yaxin Fan

Yichao Mei

Ling-Ling Chen

Junxiang Gao

Correction: Inference and prioritization of tissue-specific regulons in Arabidopsis and Oryza

更正：拟南芥和水稻组织特异性调控因子的推断与优先排序

Abstract The advent of genome editing technologies, particularly CRISPR/Cas9, has significantly advanced the generation of legume mutants for reverse genetic studies and understanding the mechanics of the rhizobial symbiosis. The legume–rhizobia symbiosis is crucial for sustainable agriculture, enhancing nitrogen fixation and improving soil fertility. Numerous genes with a symbiosis-specific expression have been identified, sometimes exclusively expressed in cells forming infection threads or in nitrogen-fixing nodule cells. Typically, mutations in these genes do not affect plant growth. However, in some instances, germline homozygous mutations can be lethal or result in complex pleiotropic phenotypes that are challenging to interpret. To address this issue, a rhizobia-inducible and cell-type-specific CRISPR/Cas9 strategy was developed to knock-out genes in specific legume transgenic root tissues. In this review, we discuss recent advancements in legume genome editing, highlighting the cell-type-specific CRISPR system and its crucial applications in symbiotic nitrogen fixation and beyond.

Jin-Peng Gao

Yangyang Su

Suyu Jiang

Wenjie Liang

Zhijun Lou

Florian Frugier

Ping Xu

Jeremy D. Murray

Applying conventional and cell-type-specific CRISPR/Cas9 genome editing in legume plants

在豆科植物中应用常规和细胞类型特异性CRISPR/Cas9基因组编辑

Arif Ali Khattak

Yingshan Huang

Muhammad Afzal

Xiaolin Wang

MHZ3: a key regulator of ethylene signaling in rice

MHZ3：水稻乙烯信号传导的关键调控因子

Abstract Plastid transformation offers valuable benefits in plant biotechnology, such as high-level transgene expression and the absence of gene silencing. Here we describe the first protocol of a plastid transformation system for a woody vine (liana) kiwifruit ( Actinidia chinensis ). The transgenic DNA carries a spectinomycin-resistance gene ( aadA ) cassette and a green fluorescent protein ( GFP ) reporter gene cassette, flanked by two adjacent kiwifruit plastid genome sequences, thereby allowing targeted insertion between the trnfM and trnG genes. Six spectinomycin-resistant shoots were obtained out of 12 plates subjected to bombardment, and two were positive events, confirmed through PCR and Southern blot analyses. The GFP was localized to plastids as monitored by confocal laser scanning microscopy and reached 2.5% of leaf total soluble protein. Success in kiwifruit extends transplastomic technology of woody species beyond poplar, and will provide an attractive biosynthetic chassis for molecular farming.

Abbreviation	Abiotech.
Journal Impact	4.70
Quartiles(Global)	PLANT SCIENCES(Q1)

ISSN	2096-6326, 2662-1738
h-index	16

Publication Information	Publisher: Springer，Publishing cycle: ，Journal Type: journal，Open Access Journals: No
Basic data	Year of publication: 2020，Proportion of original research papers: 47.83%，Self Citation Rate:， Gold OA Rate: 33.33%

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