The subfamily Agavoideae comprises crassulacean acid metabolism (CAM), C3, and C4 plants with a young age of speciation and slower mutation accumulation, making it a model crop for studying CAM evolution. However, the genetic mechanism underlying CAM evolution remains unclear because of lacking genomic information. This study assembled the genome of Agave hybrid NO.11648, a constitutive CAM plant belonging to subfamily Agavoideae, at the chromosome level using data generated from high-throughput chromosome conformation capture, Nanopore, and Illumina techniques, resulting in 30 pseudo-chromosomes with a size of 4.87 Gb and scaffold N₅₀ of 186.42 Mb. The genome annotation revealed 58 841 protein-coding genes and 76.91% repetitive sequences, with the dominant repetitive sequences being the I-type repeats (Copia and Gypsy accounting for 18.34% and 13.5% of the genome, respectively). Our findings also provide support for a whole genome duplication event in the lineage leading to A. hybrid, which occurred after its divergence from subfamily Asparagoideae. Moreover, we identified a gene duplication event in the phosphoenolpyruvate carboxylase kinase (PEPCK) gene family and revealed that three PEPCK genes (PEPCK3, PEPCK5, and PEPCK12) were involved in the CAM pathway. More importantly, we identified transcription factors enriched in the circadian rhythm, MAPK signaling, and plant hormone signal pathway that regulate the PEPCK3 expression by analysing the transcriptome and using yeast one-hybrid assays. Our results shed light on CAM evolution and offer an essential resource for the molecular breeding program of Agave spp.

Sisal ( Agave sisalana ) productivity is severely threatened by purple leaf roll and zebra stripe diseases. The SQUAMOSA PROMOTER BINDING PROTEIN (SBP)-box gene family is an important regulator of plant defense against biotic stresses, yet its role in sisal immunity remains poorly understood. We performed a genome-wide identification of AhSBP genes in the A. sisalana genome and analyzed their expansion mechanisms through phylogenetic and synteny modeling. Comparative transcriptomics, promoter cis-element analysis, and RT-qPCR validation were further integrated to elucidate the regulatory mechanisms of the miR156-AhSBP module under pathogen stress. A total of 45 AhSBP genes were identified, revealing a lineage-specific expansion mainly driven by segmental duplication (70%). Among these, 18 AhSBP genes were identified as putative miR156 targets. Transcriptomic and qPCR analyses showed that asi-miR156 is significantly induced in susceptible cultivars, leading to the suppression of its direct targets, such as AhSBP9 and AhSBP38 . In contrast, resistant cultivars maintain low miR156 levels, allowing the activation of the SBP-mediated defense program. Our findings suggest that the miR156-AhSBP module serves as a primary regulatory hub coordinating sisal's immune homeostasis, while members like AhSBP17 and AhSBP34 likely function as downstream effectors. This study provides a comprehensive understanding of the AhSBP gene family and offers potential genetic targets for engineering broad-spectrum disease resistance in sisal.

In tropical rubber-growing regions, Corynespora leaf fall disease stands as a predominant and economically significant threat to rubber trees. The toxin protein encoded by the cas5 gene is the main pathogenic factor of Corynespora cassiicola. To identify transcription factors capable of binding with the cas5 gene promoter sequence of C. cassiicola, the promoter of the cas5 gene was predicted by bioinformatics, and the 1000 bp promoter of the cas5 gene was isolated to construct a yeast one-hybrid bait vector for self-activation detection. A yeast one-hybrid cDNA expression library of C. cassiicola was constructed to screen for potential transcriptional regulators interacting with the 1000 bp promoter of the cas5 gene. The transcriptional regulators interacting with the cas5 gene were determined by the yeast one-hybrid (Y1H) point-to-point verification experiment. Y1H results showed that the bait vector did not have self-activation. The cDNA library had a titer of 2.516 × 10⁸ cfu/mL and a total clone count of 5.032 × 10⁸. Screening identified 30 candidate transcriptional regulators. Through point-to-point yeast one-hybrid verification, only one of the 30 candidate transcription factors (named CcbZIP3629) showed interaction with cas5. Molecular docking was performed using the AlphaFold3-predicted structure of CcbZIP3629, which revealed its binding to two ACGT core motifs within the promoter. These findings provide the groundwork for elucidating the regulatory mechanism of the cas5 gene, particularly by which CcbZIP3629 mediates the expression of the Cc-Cas5 toxin.

Background: Macadamia integrifolia, a new economically important crop, the kernel oil is rich in bioactive compound and monounsaturated fatty acid. Gene expression analysis of qRT-PCR is beneficial to understand the complex regulatory networks of macadamia.Results: In this study, the expression stability of 11 traditional housekeeping genes including α-tubulin (TUBa), β–tubulin (TUBb), malate dehydrogenase (MDH), 18S ribosome RNA (18S), glyceraldehyde-3- phosphate dehydrogenase (GAPDH), α-elongation factor 1 (EF1a), β- elongation factor 1 (EF1b), ubiquitin (UBQ), ubiquitin-conjugating enzyme (UBC), cyclophilin (CYP) and actin (ACT) were accessed by qRT-PCR in macadamia seedlings under different experimental conditions and tissues. The expression stability of the 11 reference genes was evaluated by the online tool RefFinder, which include ΔCt, geNorm, NormFinder, BestKeeper four commonly software, and then determinated a comprehensive expression stability ranking by integrating above four ranking results based on the geometric mean. Our results show that ACT was the best stable genes for all samples, cold stress, NaCl sress, PEG stress, ABA treatment, MeJA treatment, stem and leaf tissue samples; EF1b is the most stable gene in GA treatment and heat stress samples; UBC and CYP were respectively ranked top in ethylene treatment and root tissue samples. Finally, the reliability of these results was further validated with a target gene SAD by qRT-PCR. Conclusions: In summary, this study evaluated and validated the suitable reference genes for qRT-PCR under different experiment treatment and tissues, and will be useful for further gene expression studies on the molecular mechanisms in Macadamia.