Zhongchi Liu

A major bottleneck in genome editing of many perennial plants is their recalcitrance to transformation and regeneration. To boost genome editing in such perennial crops, transcriptional reprogramming of morphogenic genes is introduced by CRISPR-Combo, a versatile system for simultaneous genome editing and transcriptional activation in plant cells. In potato, we screen 17 morphogenic genes and identify 4 genes (WOX11/12, ARF5, ABI3-1, and ABI3-2) that promote regeneration of genome-edited hairy roots, and 3 of the 4 genes are also found to boost shoot regeneration by Agrobacterium-mediated stable transformation. Similarly, screening of 10 morphogenic genes in citrus leads to the identification of 5 genes (BBM3, FUS3, IPT1, SERK1, and STM) that enhance plant regeneration upon activation. In wild strawberry, we demonstrate that simultaneous activation of Baby Boom genes (BBM1 and BBM2) or of GRF3 and GIF1 reduces the generation time of genome-edited plants by over one month. Moreover, in poplar, we show that simultaneous activation of WUS and WOX11 synergistically promotes plant regeneration without exogenous plant hormones, which leads to a protocol of generating genome-edited poplar shoots in less than one month. Collectively, this study provides efficient strategies for boosting genome editing in four perennial crops.

Light strongly affects strawberry fruit quality. The ubiquitin E3 ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) negatively regulates light signaling. However, its role in fruit quality remains unclear. This study demonstrates that FvCOP1 plays a negative role in strawberry fruit quality. Here, we generated fvcop1 knockout mutants in diploid strawberry (Fragaria vesca). The mutants showed substantial changes in fruit color and shape. Metabolomic analyses revealed elevated levels of sugars, anthocyanins, vitamins, lignin, flavonoids, and phenolic acids in fvcop1 mutants compared with wild type. Transcriptome analysis identified key genes in sugar, anthocyanin, flavonoid, and lignin biosynthesis pathways. Moreover, FvCOP1 suppresses the accumulation of sugars, anthocyanins, and flavonoids by inhibiting transcriptional activation of downstream genes regulated by FvHY5, FvRIF, and FvMYB10. We demonstrated that FvHY5 and FvRIF directly bind to promoters of sugar/anthocyanin biosynthesis genes to activate their expression. Furthermore, FvCOP1 ubiquitinates FvHY5, FvMYB10, and FvRIF, leading to their destabilization and thereby repressing sugar and anthocyanin production. These findings establish FvCOP1 as a molecular brake on light-mediated fruit quality improvement. Targeting FvCOP1 in breeding programs could enhance strawberry nutritional and commercial value by optimizing light-responsive metabolite accumulation.

Leaf morphology impacts physiological activities and contributes to environment adaptation. Plant hormones auxin and cytokinin both regulate leaf shape, but how they act together to specify leaf complexity is not fully understood. In woodland strawberry (Fragaria vesca), the wild type develops trifoliate leaves, whereas the myb117a mutants produce 1 to 5 leaflets with fewer serrations. Transcriptome analysis revealed that the auxin biosynthesis gene FveYUC4, the cytokinin biosynthesis gene FveIPT2, the cytokinin degradation gene FveCKX1, and the transcription factor gene FveCUC2a are altered in the myb117a mutant. Accordingly, the myb117a leaves contain lower auxin levels and higher cytokinin levels compared to wild type. Moreover, treatment with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) produced simple leaves with smooth margins, whereas exogenous cytokinin application resulted in a higher percentage of 4–5 leaflets in myb117a. Several lines of evidence showed that FveMYB117a can directly bind to the promoters of FveYUC4 and FveCUC2a and influence their expression. Both myb117a yuc4 and myb117a cuc2a double mutants have fewer leaflets with greatly reduced or no serrations compared to myb117a, suggesting that these genes function in the same pathway. Overall, our results indicate that FveMYB117a is a transcription factor that coordinates auxin and cytokinin homeostasis in young leaves, thereby contributing to robust leaf morphogenesis in strawberry.

Hidden hunger, caused by chronic micronutrient deficiencies, affects billions of people worldwide and remains a critical public health issue despite progress in food production. Biofortification offers a promising solution by enhancing nutrient levels within plant tissues through traditional breeding or advanced biotechnologies. Recent advancements in plant synthetic biology have significantly improved biofortification strategies, enabling precise and targeted nutrient enrichment. This mini‐review outlines five core strategies in synthetic biology‐based biofortification: overexpression of endogenous biosynthetic genes, introduction of heterologous biosynthetic pathways, expression of nutrient‐specific transporters, optimization of transcriptional regulation, and protein (directed) evolution. Vitamin B₁ biofortification serves as a primary illustrative example due to its historical importance and ongoing relevance. Recent breakthroughs, particularly from Chinese research teams, are also highlighted. Together, these strategies offer transformative potential for addressing global nutritional challenges through precise, sustainable and innovative plant‐based approaches.

During flower development, different floral organs are formed to ensure fertilization and fruit set. Although the genetic networks underlying flower development are increasingly well understood, less is known about the mechanistic basis in different species. Here, we identified a mutant of woodland strawberry (Fragaria vesca), bare receptacle (bre), which produces flowers with greatly reduced carpels and other floral organs. Genetic analysis revealed that BRE encodes an APETALA2 (AP2) transcription factor. BRE was highly expressed in floral meristems and floral organ primordia. BRE could directly bind the GCC-box motif in the YUCCA (YUC) auxin biosynthesis genes FveYUC4 and FveYUC2 and promote their expression. The yuc4 mutant had fewer floral organs, and the bre yuc4 double mutant had similar numbers of petals and carpels to bre. Auxin homeostasis and distribution were severely disrupted in bre. Although auxin application or FveYUC4 overexpression did not rescue the bre phenotypes, bre was hypersensitive to treatment with the polar auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). In addition, BRE was able to directly bind and regulate the expression of five other auxin pathway genes. Overall, these results demonstrate that BRE is required for floral organogenesis, particularly carpel initiation, and acts through the auxin pathway in strawberry.

Fertilization is a fundamental process that triggers seed and fruit development, but the molecular mechanisms underlying fertilization-induced seed development are poorly understood. Previous research has established AGamous-Like62 (AGL62) activation and auxin biosynthesis in the endosperm as key events following fertilization in Arabidopsis (Arabidopsis thaliana) and wild strawberry (Fragaria vesca). To test the hypothesis that epigenetic mechanisms are critical in mediating the effect of fertilization on the activation of AGL62 and auxin biosynthesis in the endosperm, we first identified and analyzed imprinted genes from the endosperm of wild strawberry. We isolated endosperm tissues from F1 seeds of two wild strawberry Fragaria vesca subspecies, generated endosperm-enriched transcriptomes, and identified candidate Maternally-Expressed and Paternally-Expressed Genes (MEGs and PEGs). Through bioinformatic analyses, we identified four imprinted genes that may be involved in regulating the expression of FveAGL62 and auxin biosynthesis genes. We conducted functional analysis of a maternally expressed gene FveMYB98 through CRISPR-knockout and overexpression in transgenic strawberry as well as analysis in heterologous systems. FveMYB98 directly repressed FveAGL62 at stage 3 endosperm, which likely serves to limit auxin synthesis and endosperm proliferation. These results provide an inroad into the regulation of early stage seed development by imprinted genes in strawberry, suggest potential function of imprinted genes in parental conflict, and identify FveMYB98 as a regulator of a key transition point in endosperm development.

Background: The plant hormone auxin plays a crucial role in regulating important functions in strawberry fruit development. Although a few studies have described the complex auxin biosynthetic and signaling pathway in wild diploid strawberry (Fragaria vesca), the molecular mechanisms underlying auxin biosynthesis and crosstalk in octoploid strawberry fruit development are not fully characterized. To address this knowledge gap, comprehensive transcriptomic analyses were conducted at different stages of fruit development and compared between the achene and receptacle to identify developmentally regulated auxin biosynthetic genes and transcription factors during the fruit ripening process. Similar to wild diploid strawberry, octoploid strawberry accumulates high levels of auxin in achene compared to receptacle. Results: Genes involved in auxin biosynthesis and conjugation, such as Tryptophan Aminotransferase of Arabidopsis (TAAs), YUCCA (YUCs), and Gretchen Hagen 3 (GH3s), were found to be primarily expressed in the achene, with low expression in the receptacle. Interestingly, several genes involved in auxin transport and signaling like Pin-Formed (PINs), Auxin/Indole-3-Acetic Acid Proteins (Aux/IAAs), Transport Inhibitor Response 1 / Auxin-Signaling F-Box (TIR/AFBs) and Auxin Response Factor (ARFs) were more abundantly expressed in the receptacle. Moreover, by examining DEGs and their transcriptional profiles across all six developmental stages, we identified key auxin-related genes co-clustered with transcription factors from the NAM-ATAF1,2-CUC2/ WRKYGQK motif (NAC/WYKY), Heat Shock Transcription Factor and Heat Shock Proteins (HSF/HSP), APETALA2/Ethylene Responsive Factor (AP2/ERF) and MYB transcription factor groups. Conclusions: These results elucidate the complex regulatory network of auxin biosynthesis and its intricate crosstalk within the achene and receptacle, enriching our understanding of fruit development in octoploid strawberries.

Summary: Cis‐regulatory elements (CREs) are integral to the spatiotemporal and quantitative expression dynamics of target genes, thus directly influencing phenotypic variation and evolution. However, many of these CREs become highly susceptible to transcriptional silencing when in a transgenic state, particularly when organised as tandem repeats. We investigated the mechanism of this phenomenon and found that three of the six selected flower‐specific CREs were prone to transcriptional silencing when in a transgenic context. We determined that this silencing was caused by the ectopic expression of non‐coding RNAs (ncRNAs), which were processed into 24‐nt small interfering RNAs (siRNAs) that drove RNA‐directed DNA methylation (RdDM). Detailed analyses revealed that aberrant ncRNA transcription within the AGAMOUS enhancer (AGe) in a transgenic context was significantly enhanced by an adjacent CaMV35S enhancer (35Se). This particular enhancer is known to mis‐activate the regulatory activities of various CREs, including the AGe. Furthermore, an insertion of 35Se approximately 3.5 kb upstream of the AGe in its genomic locus also resulted in the ectopic induction of ncRNA/siRNA production and de novo methylation specifically in the AGe, but not other regions, as well as the production of mutant flowers. This confirmed that interactions between the 35Se and AGe can induce RdDM activity in both genomic and transgenic states. These findings highlight a novel epigenetic role for CRE–CRE interactions in plants, shedding light on the underlying forces driving hypermethylation in transgenes, duplicate genes/enhancers, and repetitive transposons, in which interactions between CREs are inevitable.