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Rating pome fruit quality traits using deep learning and image processing. 利用深度学习和图像处理评定梨果的品质特征。
IF 2.3 3区 生物学 Q2 PLANT SCIENCES Pub Date : 2024-10-08 eCollection Date: 2024-10-01 DOI: 10.1002/pld3.70005
Nhan H Nguyen, Joseph Michaud, Rene Mogollon, Huiting Zhang, Heidi Hargarten, Rachel Leisso, Carolina A Torres, Loren Honaas, Stephen Ficklin

Quality assessment of pome fruits (i.e. apples and pears) is used not only for determining the optimal harvest time but also for the progression of fruit-quality attributes during storage. Therefore, it is typical to repeatedly evaluate fruits during the course of a postharvest experiment. This evaluation often includes careful visual assessments of fruit for apparent defects and physiological symptoms. A general best practice for quality assessment is to rate fruit using the same individual rater or group of individual raters to reduce bias. However, such consistency across labs, facilities, and experiments is often not feasible or attainable. Moreover, while these visual assessments are critical empirical data, they are often coarse-grained and lack consistent objective criteria. Granny, is a tool designed for rating fruit using machine-learning and image-processing to address rater bias and improve resolution. Additionally, Granny supports backward compatibility by providing ratings compatible with long-established standards and references, promoting research program continuity. Current Granny ratings include starch content assessment, rating levels of peel defects, and peel color analyses. Integrative analyses enhanced by Granny's improved resolution and reduced bias, such as linking fruit outcomes to global scale -omics data, environmental changes, and other quantitative fruit quality metrics like soluble solids content and flesh firmness, will further enrich our understanding of fruit quality dynamics. Lastly, Granny is open-source and freely available.

梨果(即苹果和梨)的质量评估不仅用于确定最佳采收时间,还用于评估贮藏期间果实质量属性的变化。因此,典型的做法是在采后实验过程中反复评估水果。这种评估通常包括对果实的明显缺陷和生理症状进行仔细的目测评估。质量评估的一般最佳做法是使用相同的单个评定者或单个评定者小组对水果进行评定,以减少偏差。然而,在不同的实验室、设施和实验中,这种一致性往往是不可行或无法实现的。此外,虽然这些目测评估是重要的经验数据,但它们通常粒度较粗,缺乏一致的客观标准。Granny 是一款专为水果评级而设计的工具,它利用机器学习和图像处理技术来解决评级者的偏差并提高分辨率。此外,Granny 还支持向后兼容,提供与长期确立的标准和参考兼容的评级,促进研究计划的连续性。Granny 目前的评级包括淀粉含量评估、果皮缺陷评级和果皮颜色分析。Granny 提高了分辨率,减少了偏差,从而加强了综合分析,例如将水果结果与全球范围的组学数据、环境变化以及可溶性固形物含量和果肉硬度等其他定量水果质量指标联系起来,这将进一步丰富我们对水果质量动态的了解。最后,Granny 是开源的,可以免费获取。
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引用次数: 0
GABA promotes peroxisome proliferation in Triticum monococcum leaves. GABA 可促进单粒小麦叶片中过氧物酶体的增殖。
IF 2.3 3区 生物学 Q2 PLANT SCIENCES Pub Date : 2024-10-04 eCollection Date: 2024-10-01 DOI: 10.1002/pld3.70007
Yunus Şahin, Taras Nazarov, Ercan Selçuk Ünlü, Andrei Smertenko, Nusret Zencrici

Although peroxisomes are integral for both primary and secondary metabolism, how developmental changes affect activity of peroxisomes remains poorly understood. Here, we used published RNA-seq data to analyze the expression patterns of genes encoding 21 peroxisome metabolic pathways at successive developmental stages of Zea mays and Oryza sativa. Photorespiration was the most represented pathway in adult leaf relative to the juvenile stages. Components of reactive oxygen species (ROS)/reactive nitrogen species (RNS) metabolism, NADPH regeneration, and catabolism of polyamines were also enriched at later stages of leaf differentiation. The most commonly upregulated gene in differentiated leaves across all datasets of both species was BETAINE ALANINE DEHYDROGENASE (BADH). BADH functions in catabolism of polyamines where it converts 4-aminobutyraldehyde (ABAL) to 4-aminobutyrate (GABA). We tested the outcome of RNA-seq analysis by qRT-PCR in developing Triticum monococcum ssp. monococcum (Einkorn) seedlings. Consistent with the outcomes of RNA-seq analysis, transcription of BADH and CATALASE3 (CAT3) were upregulated in older seedlings. CAT3 is an essential peroxisome biogenesis factor and a key enzyme of ROS homeostasis. Furthermore, exogenous application of GABA resulted in higher peroxisome abundance and transcriptional upregulation of BADH and a gene encoding another peroxisome biogenesis factor responsible for peroxisome fission, PEROXIN11C (PEX11C), in leaves. We propose that GABA contributes to regulation of peroxisome fission machinery during leaf differentiation.

虽然过氧物酶体在初级和次级代谢中都不可或缺,但人们对发育变化如何影响过氧物酶体的活性仍然知之甚少。在这里,我们利用已发表的 RNA-seq 数据分析了编码 21 种过氧物酶体代谢途径的基因在玉米和大豆不同发育阶段的表达模式。与幼叶阶段相比,光呼吸是成叶阶段表达最多的途径。活性氧(ROS)/活性氮(RNS)代谢、NADPH 再生和多胺分解代谢的成分在叶片分化后期也有所丰富。在两个物种的所有数据集中,分化叶片中最常上调的基因是乙氨酸丙氨酸脱氢酶(BADH)。BADH 在多胺的分解代谢中发挥作用,它将 4-氨基丁醛(ABAL)转化为 4-氨基丁酸(GABA)。我们在发育中的单粒小麦(Einkorn)幼苗中通过 qRT-PCR 测试了 RNA 序列分析的结果。与 RNA-seq 分析的结果一致,BADH 和 CATALASE3 (CAT3) 的转录在较老的幼苗中上调。CAT3 是一种重要的过氧化物酶体生物发生因子,也是 ROS 平衡的关键酶。此外,外源施用 GABA 会导致叶片中过氧化物酶体丰度增加、BADH 和编码另一种过氧化物酶体生物发生因子 PEROXIN11C(PEX11C)的基因转录上调。我们认为 GABA 在叶片分化过程中有助于过氧物酶体裂变机制的调控。
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引用次数: 0
Arabidopsis apoplast TET8 positively correlates to leaf senescence, and tet3tet8 double mutants are delayed in leaf senescence. 拟南芥细胞质 TET8 与叶片衰老呈正相关,tet3tet8 双突变体叶片衰老延迟。
IF 2.3 3区 生物学 Q2 PLANT SCIENCES Pub Date : 2024-09-24 eCollection Date: 2024-09-01 DOI: 10.1002/pld3.70006
Jayde A Zimmerman, Benjamin Verboonen, Andrew P Harrison Hanson, Luis R Arballo, Judy A Brusslan

Extracellular vesicles (EVs) are membrane-bound exosomes secreted into the apoplast. Two distinct populations of EVs have been described in Arabidopsis: PEN1-associated and TET8-associated. We previously noted early leaf senescence in the pen1 single and pen1pen3 double mutant. Both PEN1 and PEN3 are abundant in EV proteomes suggesting that EVs might regulate leaf senescence in soil-grown plants. We observed that TET8 is more abundant in the apoplast of early senescing pen1 and pen1pen3 mutant rosettes and in older wild-type (WT) rosettes. The increase in apoplast TET8 in the pen1 mutant did not correspond to increased TET8 mRNA levels. In addition, apoplast TET8 was more abundant in the early leaf senescence myb59 mutant, meaning the increase in apoplast TET8 protein during leaf senescence is not dependent on pen1 or pen3. Genetic analysis showed a significant delay in leaf senescence in tet3tet8 double mutants after 6 weeks of growth suggesting that these two tetraspanin paralogs operate additively and are positive regulators of leaf senescence. This is opposite of the effect of pen1 and pen1pen3 mutants that show early senescence and suggest PEN1 to be a negative regulator of leaf senescence. Our work provides initial support that apoplast-localized TET8 in combination with TET3 positively regulates age-related leaf senescence in soil-grown Arabidopsis plants.

细胞外囊泡(EVs)是分泌到细胞外质的膜结合外泌体。拟南芥中有两种不同的外泌体:与 PEN1 相关的和与 TET8 相关的。我们以前曾注意到 pen1 单突变体和 pen1pen3 双突变体的叶片提前衰老。PEN1 和 PEN3 在 EV 蛋白组中都很丰富,这表明 EV 可能会调节土壤生长植物的叶片衰老。我们观察到,在早期衰老的 pen1 和 pen1pen3 突变体莲座丛以及较老的野生型(WT)莲座丛中,TET8 的含量更高。在 pen1 突变体中,细胞质 TET8 的增加与 TET8 mRNA 水平的增加并不一致。此外,在早期叶片衰老的 myb59 突变体中,凋落体 TET8 的含量更高,这意味着叶片衰老过程中凋落体 TET8 蛋白的增加并不依赖于 pen1 或 pen3。遗传分析表明,生长 6 周后,tet3tet8 双突变体的叶片衰老明显延迟,这表明这两种四泛素旁系亲和作用是相加的,是叶片衰老的正调控因子。这与表现出早期衰老的 pen1 和 pen1pen3 突变体的效果相反,表明 PEN1 是叶片衰老的负调控因子。我们的研究初步证明,凋亡体定位的 TET8 与 TET3 结合在一起,对土壤生长的拟南芥植物中与年龄相关的叶片衰老具有正向调节作用。
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引用次数: 0
Deciphering chemical diversity among five variants of Abeliophyllum distichum flowers through metabolomics analysis. 通过代谢组学分析解密 Abeliophyllum distichum 花的五个变体之间的化学多样性。
IF 2.3 3区 生物学 Q2 PLANT SCIENCES Pub Date : 2024-09-19 eCollection Date: 2024-09-01 DOI: 10.1002/pld3.616
Yeong-Geun Lee, Jeong Eun Kwon, Won-Sil Choi, Nam-In Baek, Se Chan Kang

Abeliophyllum distichum (Oleaceae), endemic to the Korean Peninsula and the sole member of its genus and species, possesses high scarcity value, escalating its importance under the Nagoya Protocol. Despite its significance, their metabolites and activities of A. distichum flowers remain unexplored. This study employs an integrated metabolomic approach utilizing NMR, LC/MS, GC/MS, and FTIR techniques to comprehensively analyze the metabolite profile of A. distichum flowers. By combining these methods, we identified 35 metabolites, 43 secondary metabolites, and 108 hydrophobic primary metabolites. Notably, distinct concentration patterns of these compounds were observed across five variants, classified based on morphological characteristics. Correlation analyses of primary and secondary metabolites unveiled varietal metabolic flux, providing insights into A. distichum flower metabolism. Additionally, the reconstruction of metabolic pathways based on dissimilarities in morphological traits elucidates variant-specific metabolic signatures. These findings not only enhance our understanding of chemical differences between varieties but also underscore the importance of considering varietal differences in future research and conservation efforts.

Abeliophyllum distichum(油茶科)是朝鲜半岛特有的植物,也是其属和种的唯一成员,具有极高的稀缺价值,使其在名古屋议定书中的重要性不断提升。尽管其重要性不言而喻,但 A. distichum 花的代谢物和活性仍未得到研究。本研究采用综合代谢组学方法,利用 NMR、LC/MS、GC/MS 和 FTIR 技术全面分析了 A. distichum 花的代谢物谱。结合这些方法,我们确定了 35 种代谢物、43 种次级代谢物和 108 种疏水初级代谢物。值得注意的是,在根据形态特征分类的五个变体中观察到了这些化合物不同的浓度模式。初级和次级代谢物的相关性分析揭示了变种的代谢通量,为深入了解 A. distichum 花的代谢提供了线索。此外,根据形态特征的差异重建代谢途径也阐明了变种特有的代谢特征。这些发现不仅加深了我们对不同品种间化学差异的理解,还强调了在未来的研究和保护工作中考虑品种差异的重要性。
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引用次数: 0
Immunolocalization of hordein synthesis and transport in developing barley endosperm. 发育中的大麦胚乳中角蛋白合成和运输的免疫定位。
IF 2.3 3区 生物学 Q2 PLANT SCIENCES Pub Date : 2024-09-05 eCollection Date: 2024-09-01 DOI: 10.1002/pld3.591
Gregory Tanner, Allison van de Meene, Anthony Bacic

The spatial accumulation of hordeins in the developing endosperm of barley grains was examined by immunofluorescence microscopy (immunolight microscopy [iLM]) and immunoelectron microscopy (iEM) to establish the timing and subcellular pattern of hordein synthesis and deposition. The pattern seen for hordeins was compared to other abundant grain proteins, such as serpin Z4 and lipid transfer protein 1 (LTP1). Hordein accumulates throughout grain development, from 6 to 37 days post-anthesis (DPA). In contrast, serpin Z4 was present at 6 DPA, but the greatest synthesis and accumulation occurred during the middle of seed development, from 15 to 30 DPA. LTP1 accumulated later in seed development, from 15 to 30 DPA. Hordeins accumulated within the lumen of the endoplasmic reticulum (ER), were exocytosed from the ER membrane, and accumulated in protein bodies, which then fused either with the protein storage vacuoles or with other protein bodies, which also later fused with the protein storage vacuoles. iEM showed hordein, and LTP1 appeared not to traverse the Golgi apparatus (GA). Hordein, LTP1, and serpin Z4 colocalized to the same protein bodies and were co-transported to the protein storage vacuole in the same protein bodies. It is likely that this represents a general transport mechanism common to storage proteins in developing grains.

通过免疫荧光显微镜(immunolight microscopy [iLM])和免疫电子显微镜(immunoelectron microscopy [iEM])对大麦粒胚乳发育过程中胚乳蛋白的空间积累进行了研究,以确定胚乳蛋白合成和沉积的时间和亚细胞模式。将谷粒角蛋白的模式与其他丰富的谷粒蛋白(如丝胶蛋白 Z4 和脂质转移蛋白 1 (LTP1))进行了比较。角蛋白在谷粒的整个发育过程中都会积累,从开花后 6 天到 37 天(DPA)不等。与此相反,丝胶蛋白 Z4 在播种后 6 天就出现了,但在种子发育中期(播种后 15 天至 30 天)合成和积累最多。LTP1 在种子发育后期积累,从 15 到 30 DPA。角蛋白在内质网(ER)腔内积累,从ER膜外排,并在蛋白体中积累,然后与蛋白储存液泡或其他蛋白体融合,这些蛋白体后来也与蛋白储存液泡融合。Hordein、LTP1 和 serpin Z4 定位于相同的蛋白体,并在相同的蛋白体中被共同转运到蛋白储存液泡。这很可能代表了发育中谷粒中贮藏蛋白共有的一般运输机制。
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引用次数: 0
Effects of light regimes on circadian gene co‐expression networks in Arabidopsis thaliana 光照制度对拟南芥昼夜节律基因共表达网络的影响
IF 3 3区 生物学 Q2 PLANT SCIENCES Pub Date : 2024-08-27 DOI: 10.1002/pld3.70001
Quentin Rivière, Virginie Raskin, Romário de Melo, Stéphanie Boutet, Massimiliano Corso, Matthieu Defrance, Alex A. R. Webb, Nathalie Verbruggen, Armand D. Anoman
Light/dark (LD) cycles are responsible for oscillations in gene expression, which modulate several aspects of plant physiology. Those oscillations can persist under constant conditions due to regulation by the circadian oscillator. The response of the transcriptome to light regimes is dynamic and allows plants to adapt rapidly to changing environmental conditions. We compared the transcriptome of Arabidopsis under LD and constant light (LL) for 3 days and identified different gene co‐expression networks in the two light regimes. Our studies yielded unforeseen insights into circadian regulation. Intuitively, we anticipated that gene clusters regulated by the circadian oscillator would display oscillations under LD cycles. However, we found transcripts encoding components of the flavonoid metabolism pathway that were rhythmic in LL but not in LD. We also discovered that the expressions of many stress‐related genes were significantly increased during the dark period in LD relative to the subjective night in LL, whereas the expression of these genes in the light period was similar. The nocturnal pattern of these stress‐related gene expressions suggested a form of “skotoprotection.” The transcriptomics data were made available in a web application named Cyclath, which we believe will be a useful tool to contribute to a better understanding of the impact of light regimes on plants.
光/暗(LD)周期是基因表达振荡的原因,它调节着植物生理的多个方面。由于昼夜节律振荡器的调节,这些振荡在恒定条件下可以持续。转录组对光照制度的反应是动态的,可使植物迅速适应不断变化的环境条件。我们比较了拟南芥在 LD 和恒定光照(LL)条件下 3 天的转录组,发现了两种光照条件下不同的基因共表达网络。我们的研究为昼夜节律调控提供了意想不到的启示。凭直觉,我们预计受昼夜节律振荡器调控的基因簇在 LD 周期下会出现振荡。然而,我们发现编码类黄酮代谢途径成分的转录本在 LL 条件下具有节律性,而在 LD 条件下则没有。我们还发现,许多应激相关基因的表达量在 LD 的黑暗期相对于 LL 的主观夜间显著增加,而这些基因在光照期的表达量则相似。这些应激相关基因表达的夜间模式表明了一种 "光照保护 "形式。转录组学数据可在一个名为 Cyclath 的网络应用程序中获得,我们相信它将是一个有用的工具,有助于更好地理解光照制度对植物的影响。
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引用次数: 0
Expression of a mammalian RNA demethylase increases flower number and floral stem branching in Arabidopsis thaliana 表达哺乳动物 RNA 去甲基化酶可增加拟南芥的花朵数量和花茎分枝
IF 3 3区 生物学 Q2 PLANT SCIENCES Pub Date : 2024-08-22 DOI: 10.1002/pld3.70000
Kasey Markel, Lucas Waldburger, Patrick M. Shih
RNA methylation plays a central regulatory role in plant biology and is a relatively new target for plant improvement efforts. In nearly all cases, perturbation of the RNA methylation machinery results in deleterious phenotypes. However, a recent landmark paper reported that transcriptome‐wide use of the human RNA demethylase FTO substantially increased the yield of rice and potatoes. Here, we have performed the first independent replication of those results and demonstrated broader transferability of the trait, finding increased flower and fruit count in the model species Arabidopsis thaliana. We also performed RNA‐seq of our FTO‐transgenic plants, which we analyzed in conjunction with previously published datasets to detect several previously unrecognized patterns in the functional and structural classification of the upregulated and downregulated genes. From these, we present mechanistic hypotheses to explain these surprising results with the goal of spurring more widespread interest in this promising new approach to plant engineering.
RNA 甲基化在植物生物学中起着核心调控作用,是植物改良工作中一个相对较新的目标。几乎在所有情况下,干扰 RNA 甲基化机制都会导致有害表型。然而,最近一篇具有里程碑意义的论文报道,在转录组范围内使用人类 RNA 去甲基化酶 FTO 可大幅提高水稻和马铃薯的产量。在这里,我们对这些结果进行了首次独立复制,并证明了该性状更广泛的可转移性,在模式物种拟南芥中发现花和果的数量增加了。我们还对FTO转基因植物进行了RNA-seq分析,并结合以前发表的数据集进行了分析,在上调和下调基因的功能和结构分类中发现了几种以前未曾认识到的模式。在此基础上,我们提出了一些机理假设来解释这些令人惊讶的结果,目的是激发人们对这一前景广阔的植物工程新方法的兴趣。
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引用次数: 0
Association mapping and candidate gene identification for yield traits in European hazelnut (Corylus avellana L.). 欧洲榛子(Corylus avellana L.)产量性状的关联图谱和候选基因鉴定。
IF 2.3 3区 生物学 Q2 PLANT SCIENCES Pub Date : 2024-08-20 eCollection Date: 2024-09-01 DOI: 10.1002/pld3.625
Asena Akköse Baytar, Ertuğrul Gazi Yanar, Anne Frary, Sami Doğanlar

European hazelnut (Corylus avellana L.) is an important nut crop due to its nutritional benefits, culinary uses, and economic value. Türkiye is the leading producer of hazelnut, followed by Italy and the United States. Quantitative trait locus studies offer promising opportunities for breeders and geneticists to identify genomic regions controlling desirable traits in hazelnut. A genome-wide association analysis was conducted with 5,567 single nucleotide polymorphisms on a Turkish core set of 86 hazelnut accessions, revealing 189 quantitative trait nucleotides (QTNs) associated with 22 of 31 traits (p < 2.9E-07). These QTNs were associated with plant and leaf, phenological, reproductive, nut, and kernel traits. Based on the close physical distance of QTNs associated with the same trait, we identified 23 quantitative trait loci. Furthermore, we identified 23 loci of multiple QTs comprising chromosome locations associated with more than one trait at the same position or in close proximity. A total of 159 candidate genes were identified for 189 QTNs, with 122 of them containing significant conserved protein domains. Some candidate matches to known proteins/domains were highly significant, suggesting that they have similar functions as their matches. This comprehensive study provides valuable insights for the development of breeding strategies and the improvement of hazelnut and enhances the understanding of the genetic architecture of complex traits by proposing candidate genes and potential functions.

欧洲榛子(Corylus avellana L.)因其营养价值、烹饪用途和经济价值而成为一种重要的坚果作物。土耳其是榛子的主要生产国,其次是意大利和美国。定量性状位点研究为育种者和遗传学家提供了确定控制榛子理想性状的基因组区域的大好机会。一项全基因组关联分析对土耳其核心的 86 个榛子品种进行了 5,567 个单核苷酸多态性分析,结果发现 189 个数量性状核苷酸(QTNs)与 31 个性状中的 22 个性状相关(p<0.05)。
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引用次数: 0
Shade signals activate distinct molecular mechanisms that induce dormancy and inhibit flowering in vegetative axillary buds of sorghum. 遮荫信号会激活不同的分子机制,诱导高粱无性腋芽休眠并抑制其开花。
IF 2.3 3区 生物学 Q2 PLANT SCIENCES Pub Date : 2024-08-19 eCollection Date: 2024-09-01 DOI: 10.1002/pld3.626
Tesfamichael H Kebrom

Shoot branches grow from axillary buds and play a crucial role in shaping shoot architecture and determining crop yield. Shade signals inactivate phytochrome B (phyB) and induce bud dormancy, thereby inhibiting shoot branching. Prior transcriptome profiling of axillary bud dormancy in a phyB-deficient mutant (58M, phyB-1) and bud outgrowth in wild-type (100M, PHYB) sorghum genotypes identified differential expression of genes associated with flowering, plant hormones, and sugars, including SbCN2, SbNCED3, SbCKX1, SbACO1, SbGA2ox1, and SbCwINVs. This study examined the expression of these genes during bud dormancy induced by shade and defoliation in 100M sorghum. The aim was to elucidate the molecular mechanisms activated by shade in axillary buds by comparing them with those activated by defoliation. The expression of marker genes for sugar levels suggests shade and defoliation reduce the sugar supply to the buds and induce bud dormancy. Intriguingly, both shade signals and defoliation downregulated SbNCED3, suggesting that ABA might not play a role in promoting axillary bud dormancy in sorghum. Whereas the cytokinin (CK) degrading gene SbCKX1 was upregulated solely by shade signals in the buds, the CK inducible genes SbCGA1 and SbCwINVs were downregulated during both shade- and defoliation-induced bud dormancy. This indicates a decrease in CK levels in the dormant buds. Shade signals dramatically upregulated SbCN2, an ortholog of the Arabidopsis TFL1 known for inhibiting flowering, whereas defoliation did not increase SbCN2 expression in the buds. Removing shade temporarily downregulated SbCN2 in dormant buds, further indicating its expression is not always correlated with bud dormancy. Because shade signals also trigger a systemic early flowering signal, SbCN2 might be activated to protect the buds from transitioning to flowering before growing into branches. In conclusion, this study demonstrates that shade signals activate two distinct molecular mechanisms in sorghum buds: one induces dormancy by reducing CK and sugars, whereas the other inhibits flowering by activating SbCN2. Given the agricultural significance of TFL1-like genes, the rapid regulation of SbCN2 by light signals in axillary buds revealed in this study warrants further investigation to explore its potential in crop improvement strategies.

嫩枝从腋芽长出,在形成嫩枝结构和决定作物产量方面起着至关重要的作用。遮荫信号会使植物色素 B(phyB)失活,诱导芽休眠,从而抑制芽的分枝。之前对phyB缺陷突变体(58M,phyB-1)的腋芽休眠和野生型(100M,PHYB)高粱基因型的芽萌发进行的转录组分析发现,与开花、植物激素和糖类相关的基因表达存在差异,包括SbCN2、SbNCED3、SbCKX1、SbACO1、SbGA2ox1和SbCwINVs。本研究考察了这些基因在 100M 高粱受遮荫和落叶诱导的芽休眠期的表达情况。目的是通过比较遮荫和落叶对腋芽激活的分子机制,阐明这些机制。糖分水平标记基因的表达表明,遮荫和落叶会减少对芽的糖分供应,诱导芽休眠。耐人寻味的是,遮荫信号和落叶都会下调 SbNCED3,这表明 ABA 在促进高粱腋芽休眠中可能不起作用。细胞分裂素(CK)降解基因 SbCKX1 仅受遮荫信号的影响而上调,而 CK 诱导基因 SbCGA1 和 SbCwINVs 则在遮荫和落叶诱导的芽休眠过程中均出现下调。这表明休眠芽中的 CK 水平下降。遮荫信号显著上调了 SbCN2(拟南芥 TFL1 的直向同源物,已知抑制开花),而落叶并没有增加芽中 SbCN2 的表达。遮荫暂时降低了休眠芽中 SbCN2 的表达,这进一步表明它的表达并不总是与芽的休眠相关。由于遮荫信号也会触发系统性的早花信号,SbCN2可能会被激活,以保护芽在长成分枝之前过渡到开花。总之,本研究表明,遮荫信号激活了高粱花蕾中两种不同的分子机制:一种是通过减少 CK 和糖分诱导休眠,另一种是通过激活 SbCN2 抑制开花。鉴于 TFL1 类基因的农业意义,本研究揭示的腋芽中光信号对 SbCN2 的快速调控值得进一步研究,以探索其在作物改良策略中的潜力。
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引用次数: 0
Impact of predictive selection of LbCas12a CRISPR RNAs upon on- and off-target editing rates in soybean. LbCas12a CRISPR RNA 的预测性选择对大豆靶上和脱靶编辑率的影响。
IF 2.3 3区 生物学 Q2 PLANT SCIENCES Pub Date : 2024-08-16 eCollection Date: 2024-09-01 DOI: 10.1002/pld3.627
Linda Rymarquis, Chenxi Wu, Diane Hohorst, Miguel Vega-Sanchez, Thomas E Mullen, Vijetha Vemulapalli, Douglas R Smith

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has revolutionized creating targeted genetic variation in crops. Although CRISPR enzymes have been reported to have high sequence-specificity, careful design of the editing reagents can also reduce unintended edits at highly homologous sites. This work details the first large-scale study of the heritability of on-target edits and the rate of edits at off-target sites in soybean (Glycine max), assaying ~700 T1 plants each resulting from transformation with LbCas12a constructs containing CRISPR RNAs (crRNAs) predicted to be either "unique" with no off-target sites or "promiscuous" with >10 potential off-targets in the soybean genome. Around 80% of the on-target edits observed in T0 plants were inherited in the T1 generation, and ~49% of the total observed on-target edits in T1 were not observed at T0, indicating continued activity of LbCas12a throughout the life cycle of the plant. In planta editing at off-target sites was observed for the Promiscuous but not the Unique crRNA. Examination of the edited off-target sites revealed that LbCas12a was highly tolerant to mismatches between the crRNA and target site in bases 21-23 relative to the start of the protospacer, but even a single mismatch in the first 20 nt drastically reduced the editing rate. In addition, edits at off-target sites have lower inheritance rates than on-target edits, suggesting that they occur later in the plant's lifecycle. Plants with a desired on-target edit and no off-target edits could be identified in the T1 generation for 100% of the T0 plants edited with the Unique crRNA compared with the 65% of T0 plants edited with the Promiscuous crRNA. This confirms that proper crRNA selection can reduce or eliminate off-target editing. Even when potential off-target sites are predicted, plants containing only the intended edits can still be identified and propagated.

成簇的规则间隔短回文重复序列(CRISPR)技术彻底改变了农作物的定向遗传变异。据报道,尽管CRISPR酶具有高度的序列特异性,但精心设计编辑试剂也能减少高度同源位点的意外编辑。这项工作详细介绍了对大豆(Glycine max)靶上编辑遗传率和非靶点编辑率的首次大规模研究,该研究检测了约 700 株 T1 植株,每株植株都是用 LbCas12a 构建体转化而来,该构建体含有 CRISPR RNA(crRNA),预测这些 CRISPR RNA 在大豆基因组中要么是 "独特的",没有非靶点,要么是 "杂乱的",有 >10 个潜在的非靶点。在 T0 植株中观察到的约 80% 的靶上编辑在 T1 代中得到了遗传,而在 T1 中观察到的总的靶上编辑中约有 49% 在 T0 时没有观察到,这表明 LbCas12a 在植物的整个生命周期中具有持续的活性。在植物体内,Promiscuous 而非 Unique crRNA 在非靶标位点上的编辑被观察到。对已编辑的非靶标位点的研究表明,LbCas12a 对 crRNA 与靶标位点之间相对于原间隔起始位点的 21-23 碱基的错配具有很高的耐受性,但即使是前 20 nt 的单个错配也会大大降低编辑率。此外,非目标位点的编辑比目标编辑的遗传率低,这表明它们发生在植物生命周期的后期。与使用杂合性 crRNA 编辑的 65% 的 T0 植物相比,使用 Unique crRNA 编辑的 T0 植物在 T1 代中 100% 都能鉴定出具有所需的靶上编辑且没有脱靶编辑的植物。这证实了正确的 crRNA 选择可以减少或消除脱靶编辑。即使预测到了潜在的脱靶位点,仍然可以识别和繁殖只含有预期编辑的植株。
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