Selection of Stable Reference Genes for QRT-PCR in Tree Peony 'Doulv' and Functional Analysis of PsCUC3.

(1) Background: Tree peonies display extensive cultivar diversity due to widespread hybridization, resulting in a complex genetic architecture. This complexity complicates the selection of universal reference genes across different cultivars for qRT-PCR analyses. Paeonia suffruticosa 'Doulv', notable for its unique green blooms in China, exhibits chlorosis post-flowering and features petaloid stamens and pistils. (2) Methods: Based on published literature and RNA-seq data from 'Doulv', nine candidate reference genes-ACT (Actin), TUB (ß-Tubulin), UBC (Ubiquitin Conjugating Enzyme), UBQ (Ubiquitin), UPL (Ubiquitin Protein Ligase), PP2A (Protein Phosphatase 2A), PP2C (Protein Phosphatase 2C), MBF1A (Multiprotein Bridging Factor 1A), and GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase)-were selected. Their expression stability was assessed across various tissues and developmental stages of 'Doulv' flowers using qRT-PCR, with evaluations conducted via GeNorm_v3.5, NormFinder_v20, and BestKeeper_v1.0. Gene cloning and expression analyses of PsCUC3, including its subcellular localization, were performed. (3) Results: GAPDH and ACT were identified as the most stable reference genes in petaloid stamens across various developmental stages of 'Doulv', whereas UBC and MBF1A were optimal across different tissues. Notably, specific conserved amino acids in PsCUC3 from 'Doulv' diverged from those in NAM/CUC3 proteins of other species, impacting its protein structure. PsCUC3 expression analysis revealed no correlation with chlorophyll content in petaloid stamens but an association with petaloid organ development. Furthermore, PsCUC3 was predominantly localized in the nucleus. (4) Conclusions: This study comprehensively evaluated suitable reference genes using GeNorm_v3.5, NormFinder_v20, and BestKeeper_v1.0 software, establishing a robust qRT-PCR detection system for 'Doulv' peony. These results provide a solid experimental foundation for further research on 'Doulv' peony. Building on this experimental foundation, the functional analysis of the PsCUC3 gene was conducted. The findings suggest a potential association between the PsCUC3 gene and floral morphology alterations in 'Doulv', identifying PsCUC3 as crucial for understanding the molecular mechanisms influencing floral structure in tree peonies.

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We measured the morphological index, nutritional composition and the expression analysis of key genes during grain development of Paeonia suffruticosa cv. 'Fengdan' grown at altitudes of 100, 650 and 1010 m in Luo-yang. The aim of this study was to examine differences in grain yield traits and the transformation of soluble sugar, starch, soluble protein and fatty acid contents, as well as the related enzyme activity and differential expression of key genes in oil metabolism. The results showed that grain yield traits increased with altitudes and that the growth period of grain at the higher altitudes was longer than that at low and mid altitudes. The soluble sugar and starch in mature grains increased with altitudes, while soluble protein and crude fat did not change. During grain development, the activities of sucrose synthase (SS) and sucrose phosphate synthase (SPS) first decreased and then increased, with the lowest occurred at 90 d after flowering. The activities of pyruvate dehydrogenase (PDH), glutamic-pyruvic transaminase (GPT) and glutamic-oxalacetic transaminease (GOT) increased rapidly during 50-90 d after flowering and peaked at 90 d. The relative expression of acetyl-CoA carboxylase (ACCase) and stearoyl-ACP desaturase (SAD) peaked at 50 d after flowering, and ω-6 fatty acid desaturase 2 (FAD2) peaked at 90 d, in oil tree peony grain at different altitudes. There was a negative correlation of soluble sugar and starch with the accumulation of soluble protein and crude fat. SPS activity was positively correlated with the contents of soluble sugar and starch, and negatively correlated with the contents of soluble protein and crude fat during grain development. Activities of GPT and GOT were negatively associated with the content of soluble sugar and the content of starch, and had a highly significant positive correlation with the contents of soluble protein and crude fat. Activity of PDH was positively correlated with the content of soluble proteins and activities of GPT and GOT, and negatively correlated with the contents of soluble sugar and starch. It suggested that nutrient accumulation in the process of grain development of tree peony was transformed from sugar to crude fat and protein, and that metabolic enzymes, such as SPS, PDH, GPT and GOT, played an important role in this process. Palmitate acid, stearic acid and linoleic acid were negatively correlated with the relative increment of α-linolenic acid, indicating that fatty acid desaturation process in the grain development of tree peony was towards the direction of α-linolenic acid synthesis. The relative expression of ACCase, SAD, and FAD2 was positively correlated with the relative increment of α-linolenic acid accumulation, which played an important role in α-linolenic acid synthesis. The oil quality of tree peony grain was relatively stable at different altitudes, but grain production increased with altitude. Planting oil tree peony at mid to high altitudes could be an important strategy for the efficient use of marginal land in Luoyang.

Identification and characterization of miRNAs in two closely related C4 and C3 species of Cleome by high-throughput sequencing.

Cleome gynandra and Cleome hassleriana, which are C4 and C3 plants, respectively, are two species of Cleome. The close genetic relationship between C. gynandra and C. hassleriana provides advantages for discovering the differences in leaf development and physiological processes between C3 and C4 plants. MicroRNAs (miRNAs) are a class of important regulators of various biological processes. In this study, we investigate the differences in the characteristics of miRNAs between C. gynandra and C. hassleriana using high-throughput sequencing technology. In total, 94 and 102 known miRNAs were identified in C. gynandra and C. hassleriana, respectively, of which 3 were specific for C. gynandra and 10 were specific for C. hassleriana. Ninety-one common miRNAs were identified in both species. In addition, 4 novel miRNAs were detected, including three in C. gynandra and three in C. hassleriana. Of these miRNAs, 67 were significantly differentially expressed between these two species and were involved in extensive biological processes, such as glycol-metabolism and photosynthesis. Our study not only provided resources for C. gynandra and C. hassleriana research but also provided useful clues for the understanding of the roles of miRNAs in the alterations of biological processes in leaf tissues during the evolution of the C4 pathway.

Long-term effects of liming on health and growth of a Masson pine stand damaged by soil acidification in Chongqing, China.

In the last decades, the Masson pine (Pinus massoniana) forests in Chongqing, southwest China, have increasingly declined. Soil acidification was believed to be an important cause. Liming is widely used as a measure to alleviate soil acidification and its damage to trees, but little is known about long-term effects of liming on the health and growth of declining Masson pine forests. Soil chemical properties, health condition (defoliation and discoloration), and growth were evaluated following application of limestone powder (0 (unlimed control), 1, 2, 3, and 4 t ha(-1)) in an acidified and declining Masson pine stand at Tieshanping (TSP) of Chongqing. Eight years after liming, in the 0-20 cm and 20-40 cm mineral soil layers, soil pH values, exchangeable calcium (Ca) contents, and Ca/Al molar ratios increased, but exchangeable aluminum (Al) levels decreased, and as a result, length densities of living fine roots of Masson pine increased, with increasing dose. Mean crown defoliation of Masson pines (dominant, codominant and subdominant pines, according to Kraft classes 1-3) decreased with increasing dose, and it linearly decreased with length densities of living fine roots. However, Masson pines (Kraft classes 1-3) in all treatments showed no symptoms of discoloration. Mean current-year twig length, twig dry weight, needle number per twig, needle length per twig, and needle dry weight per twig increased with increasing dose. Over 8 years, mean height increment of Masson pines (Kraft classes 1-3) increased from 5.5 m in the control to 5.8, 6.9, 8.3, and 9.5 m in the 1, 2, 3, and 4 t ha(-1) lime treatments, and their mean DBH (diameter at breast height) increment increased from 3.1 to 3.2, 3.8, 4.9, and 6.2 cm, respectively. The values of all aboveground growth parameters linearly increased with length densities of living fine roots. Our results show that liming improved tree health and growth, and these effects increased with increasing dose.