Biotin carboxyl carrier protein (BCCP) is a subunit of the heteromeric acetyl-CoA carboxylase (htACCase), and it chemically links the two half-reactions that constitute the formation of malonyl-CoA from acetyl-CoA, a critical reaction in fatty acid biosynthesis. Because plants are a major source of edible fats and oils, it is important to understand the structural organization of the plant htACCase, relative to its potential to regulate fatty acid biosynthesis in plant plastids. Moreover, unique to the plant htACCase, noncatalytic subunits called biotin attachment domain-containing (BADC) proteins are important in the assembly of the holoenzyme, and they specifically interact with the bcCP and the biotin carboxylase (BC) subunits. We report herein NMR structural studies of the Arabidopsis BCCP isozymes (bcCP1 and BCCP2). We calculated the structure of C-terminal domain of BCCP1 (K200-P280) and explored structural changes in the BCCP1 protein upon its interactions with bc and BADC. The chemical shift perturbation experiments identified potential surface residues on the BCCP1 protein that may facilitate physical interactions between BC and BADC proteins. These studies indicate that the BADC protein interacts with a "thumb"-like protrusion, which is a common structural feature of the bacterial and plant bcCPs, and thereby acts as a potential "cap" to facilitate the assembly of a BC-BCCP-BADC complex.

Accurate and nondestructive estimation of plant biomass is crucial for optimizing plant productivity, but existing methods are often expensive and require complex experimental setups. To address this challenge, we developed an automated system for estimating plant root and shoot biomass over the plant's lifecycle in hydroponic systems. This system employs a robotic arm and turntable to capture 40 images at equidistant angles around a hydroponically grown lettuce plant. These images are then processed into silhouettes and used in voxel-based volumetric 3D reconstruction to produce detailed 3D models. We utilize a space carving method along with a raytracing-based optical correction technique to create high-accuracy reconstructions. Analysis of these models demonstrates that our system accurately reconstructs the plant root structure and provides precise measurements of root volume, which can be calibrated to indicate biomass.

The presence of a selection marker in transgenic plants has raised public concerns regarding health safety. We have developed a CRISPR/Cas9-based DNA delivery system termed transgenic selection-associated fragment elimination (T-SAFE). The T-SAFE system comprises four cassettes: the selection marker, CRISPR/Cas9, spacer-plus-protospacer adjacent motif (SP), and the cargo. The first two cassettes, the selection marker and CRISPR/Cas9, are collectively referred to as SCC. The SCC is flanked by two identical SPs derived from the fruit fly Ebony gene, which efficiently facilitate the SCC cleavage and subsequently lead to self-elimination of the selection marker upon integration of exogenous DNA into the plant genome. To inhibit the production of a functional Cas9 protein in bacteria, the IV2 intron of the potato ST-LS1 gene has been incorporated into the Cas9 gene. Additionally, the Cas9 gene is driven by a reproductive cell-specific or inducible promoter to avoid SCC cleavage in nonreproductive plant cells. These innovative features allow the T-SAFE system to achieve an elimination efficiency of the selection marker ranging from 10%-30% in Arabidopsis and 5%-8% in rice, with a DNA delivery capacity of approximately 10 kb. This approach offers a safe means for genetically modifying plants.

The APETALA2/ethylene-responsive factor (AP2/ERF) superfamily represents a class of transcription factors involved in plant growth, development, and stress responses. Carthamus tinctorius L., also known as safflower, is an important plant whose flowers contain carthamin, an expensive aromatic pigment with various medicinal and flavoring properties. This study aimed to elucidate the roles of these transcription factors in plant growth, metabolic regulation, and environmental adaptation in safflower, providing foundational information and theoretical support for genetic improvement and stress resilience research in this crop. In this study, we identified and characterized the AP2/ERF family genes in safflower through a comprehensive genomic analysis. A total of 127 AP2/ERF genes were identified and clustered into seven groups and 14 subgroups based on phylogenetic analysis. Multiple sequence alignment revealed that the basic region and two helical structures were highly conserved in most AP2/ERF proteins. Cis-acting elements in the promoters of the AP2/ERF genes were analyzed, and a degree of safflower specificity was observed among different safflower species. Tissue-specific expression analysis showed that 23, 21, 15, and 9 genes were most abundantly expressed in the roots, leaves, flowers, and buds, respectively, while only eight genes were highly expressed in all tissues examined. These results indicate that the AP2/ERF family genes in safflower are diverse and complex, with distinct expression patterns for different genes in different safflower species. The findings provide important fundamental data for in-depth studies of the growth, development, and stress response mechanisms in safflower.

Papaver somniferum L., a medicinal plant renowned for its pharmaceutical alkaloids, has captivated scientific interest due to its rich secondary metabolite profile. This study explores a novel approach to manipulating alkaloid biosynthesis pathways by integrating virus-induced gene silencing (VIGS) with macerozyme enzyme pretreatment. Targeting key genes in the benzylisoquinoline alkaloid (BIA) pathway (CODM, T6ODM, COR, DIOX2), the research aimed to elucidate the transformative potential of enzymatic preconditioning in somatic embryo cultures. To address the cell wall barrier, a known limitation in genetic manipulation, macerozyme pretreatment was employed, significantly enhancing gene silencing efficacy. Quantitative reverse transcription PCR analyses revealed significant alterations in gene expression profiles with macerozyme pretreatment, whereas no changes were observed in its absence. The T6ODM + DIOX combination was the most effective, reducing CODM, T6ODM, and DIOX2 expression by 72%, 65%, and 60%, respectively. Conversely, T6ODM expression increased by up to 107% in the CODM treatment. Notably, COR expression displayed dual regulatory dynamics, with suppression (47% decrease in T6ODM + DIOX) and enhancement (49% increase in CODM+DIOX) observed under different conditions. These findings underscore the complex interplay of gene regulation in the morphine biosynthesis pathway. This study highlights the critical role of macerozyme enzymatic pretreatment in overcoming cell wall barriers, enabling effective VIGS applications in somatic suspension cultures. The combination of VIGS and enzymatic pretreatment provides a robust platform for targeted metabolic engineering, offering insights into the regulation of morphine biosynthesis and paving the way for advancements in pharmaceutical alkaloid production and functional genomics in medicinal plants.

Basic helix-loop-helix (bHLH) proteins comprise a large family of transcription factors that are involved in plant growth and development, as well as responses to various types of environmental stress. Betula platyphylla (birch) is a pioneer tree species in secondary forest that plays a key role in maintaining ecosystem stability and forest regeneration, but few bHLHs involved in abiotic stress responses have been unveiled in birch. In this study, nine BpbHLH TFs related to stress responses in the birch genome were identified. Quantitative real-time polymerase chain reaction (RT-PCR) analysis indicated that the expression of these TFs can be induced by salt stress, and the expression of BpbHLH1 was higher than that of other BpbHLH genes. Particle bombardment analysis revealed that BpbHLH1 was localized to the nucleus. Yeast transformation found that BpbHLH1 has transcriptional activation activity. We generated BpbHLH1-overexpressing and silencing transgenic birch plants and subjected them to salt stress analysis. BpbHLH1 can enhance the salt tolerance of birch plants by increasing the reactive oxygen species scavenging ability and inhibiting cell death. Yeast one-hybrid, ß-glucuronidase, and chromatin immunoprecipitation assays revealed that BpbHLH1 can regulate the expression of target genes involved in stress resistance by binding to the E-box-1, E-box-2 and G-box elements in their promoters. The results of this study enhanced our understanding of the salt tolerance conferred by BpbHLH TFs in B. platyphylla and identified useful genes for the breeding of novel birch germplasm.

In plants, the nonproteinogenic amino acid β-alanine plays a role in response to hypoxia, flooding, drought, heat, and heavy metal stress conditions. It is also a key intermediate in the synthesis of essential molecules including vitamin B5 and coenzyme A (CoA) through the condensation reaction with pantoate. While the syntheses of pantoate, vitamin B5, and CoA appear to be conserved across plants and bacteria, the synthesis of β-alanine is not. Bacteria and fungi use aspartate, whereas plants can use uracil, spermidine, or propionate to synthesize β-alanine. Given that these three precursors can be formed from the metabolism of glutamine, arginine, isoleucine, and valine, the synthesis of β-alanine could be linked to numerous pathways. Studies of valine catabolism in Arabidopsis suggested that some branched-chain amino acids could in fact serve as precursors for the synthesis of β-alanine. Using GC-MS and isotopically labeled isoleucine and propionate, we linked their metabolism to the synthesis of β-alanine via a proposed transamination of malonate semialdehyde. We then identified three aminotransferases that each catalyzed this final reversible transamination reaction. These results affirm our hypothesis that isoleucine metabolism is also linked to the synthesis of β-alanine via the transamination of metabolic intermediates.

Cannabis plants produce a spectrum of secondary metabolites, encompassing cannabinoids and more than 300 non‐cannabinoid compounds. Among these, anthocyanins have important functions in plants and also have well documented health benefits. Anthocyanins are largely responsible for the red/purple color phenotypes in plants. Although some well‐known Cannabis varieties display a wide range of red/purple pigmentation, the genetic underpinnings of anthocyanin biosynthesis have not been well characterized in Cannabis. This study unveils the genetic diversity of anthocyanin biosynthesis genes found in Cannabis, and we characterize the diversity of anthocyanins and related phenolics found in four differently pigmented Cannabis varieties. Our investigation revealed that the genes 4CL, CHS, F3H, F3′H, FLS, DFR, ANS, and OMT exhibited the strongest correlation with anthocyanin accumulation in Cannabis leaves. The results of this study enhance our understanding of the anthocyanin biosynthetic pathway and shed light on the molecular mechanisms governing Cannabis leaf pigmentation.