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.

Macadamia is an economically significant crop, with its kernel oil being abundant in monounsaturated fatty acids (MUFA). Analyzing the expression of genes related to MUFA biosynthesis is essential for understanding the complex regulatory networks in Macadamia. However, there are few reports on the identification of suitable reference genes for use as internal controls in this species. Consequently, selecting a reliable reference gene for gene expression studies under various conditions is critical. In this study, we evaluated the expression stability of 11 traditional housekeeping genes: α-tubulin (TUBa), β-tubulin (TUBb), malate dehydrogenase (MDH), 18S ribosomal 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) under abiotic stresses, hormonal treatments and in variety of plant tissues using the online tool RefFinder, which integrates four commonly used software programs: ΔCt, geNorm (version 3.4), NormFinder (version 0953), and BestKeeper (version 1.0). A comprehensive expression stability ranking was established by integrating results from these four methods based on the geometric mean. The findings indicated that ACT was the most stable gene across all samples, including those subjected to cold stress, NaCl stress, PEG stress, ABA treatment, MeJA treatment, and both stem and leaf tissues. EF1b was identified as the most stable gene in GA treatment and heat stress samples, while UBC and CYP were ranked highest in ethrel treatment and root tissue samples, respectively. Finally, the reliability of these findings was further validated using the target gene SAD through qRT-PCR. In summary, this study evaluated and validated appropriate reference genes for qRT-PCR, which will facilitate future investigations into the molecular mechanisms in Macadamia.

Watercore disease, a physiological disorder in pineapple (Ananas comosus), manifests during late fruit development. Affected fruits develop water-soaked flesh and reduced storability. (1) Background: To explore underlying molecular mechanisms, comparative proteomic profiling was conducted in this study. (2) Methods: Data-independent acquisition (DIA) strategy was employed for comparative analysis between the resistant germplasm “35-1” and the susceptible germplasm “29-3”, as well as between the healthy and diseased “Paris”. (3) Results: Resistant (“35-1”) versus susceptible (“29-3”) germplasm analysis revealed differentially expressed proteins (DEPs) and unique proteins (SEPs) enriched in cell walls, secretory vesicles, and apoplast, functioning in cell wall loosening, hormone response, isoflavonoid biosynthesis, and farnesyl diphosphate biosynthesis. Healthy versus diseased “Paris” pulp analysis showed DEPs/SEPs enrichment in ribosomal small subunit biogenesis. These proteins form a central regulatory network potentially orchestrating tRNA synthesis, tubulin biosynthesis, and other carbohydrate metabolism. Partial protein overlap occurred in germplasm- and disease-derived differences. Resistant germplasm (“35-1”) and healthy “Paris” accumulated stress-responsive/resistant proteins and cell wall-modifying enzymes (e.g., phenylalanine ammonia-lyase, raffinose synthase, expansins, and mannan hydrolase). Susceptible germplasm (“29-3”) and diseased “Paris” exhibited prominent stress-responsive protein accumulation, such as alcohol dehydrogenase, 1-aminocyclopropane-1-carboxylate oxidase, and hypoxia-induced protein. (4) Conclusions: This comparative proteomics study identifies pineapple watercore resistance/susceptibility-associated proteins, providing a molecular basis for resistant germplasm development and disorder control.