Water deficit and high calcium (Ca²⁺) content and are two typical soil characteristics in the Karst region. However, the problem of whether high Ca²⁺ in Karst calcareous soil could increase drought tolerance in calcicole plants has not been solved. We investigated the ecophysiological responses of Cyclobalanopsis glauca (Thunb.) Oerst. cuttings to short-term drought stress and Ca²⁺ application. Drought stress (10% PEG-6000) markedly reduced relative water content (RWC) and water potential (WP), and enhanced the levels of reactive oxygen species (ROS) production (H₂O₂ and O₂^•−) and malondialdehyde (MDA) content in C. glauca leaves. Under drought treatment, exogenous Ca²⁺ application (20 mM CaCl₂) markedly increased the RWC and WP, and reduced the H₂O₂, O₂^•−, and MDA content. Furthermore, water deficit induced a significant increase in the activities of antioxidant enzymes such as peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), and glutathione peroxidase (GPX), and increased the accumulation of osmoregulation substances. External Ca²⁺ alleviated drought-induced oxidative stress and osmotic stress with further increased activities of antioxidant enzymes, and enhanced the accumulation of osmoregulation substances. In addition, exogenous Ca²⁺ treatment alleviated the reduction of the photosynthesis rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and chlorophyll content (SPAD), and further increased water use efficiency (WUE) under drought stress. This study confirms that exogenouos Ca²⁺ application induces improvements in the water status, osmotic adjustment, antioxidant defense, and photosynthesis efficiency of C. glauca under drought stress.

Phenolics are a group of important plant secondary metabolites that have been proven to possess remarkable antioxidant activity and to be beneficial for human health. Pecan nuts are an excellent source of dietary phenolics. In recent years, many studies have focused on the separation and biochemical analysis of pecan phenolics, but the molecular mechanisms of phenolic metabolism in pecans have not been fully elucidated, which significantly hinders quality breeding research for this plant. Chalcone synthase (CHS) plays crucial roles in phenolic biosynthesis. In this study, three Carya illinoinensis CHSs (CiCHS1, CiCHS2, and CiCHS3), were isolated and analyzed. CiCHS2 and CiCHS3 present high expression levels in different tissues, and they are also highly expressed at the initial developmental stages of kernels in three pecan genotypes. A correlation analysis was performed between the phenolic content and CHSs expression values during kernel development. The results indicated that the expression variations of CiCHS2 and CiCHS3 are significantly related to changes in total phenolic content. Therefore, CiCHSs play crucial roles in phenolic components synthesis in pecan. We believe that the isolation of CiCHSs is helpful for understanding phenolic metabolism in C. illinoinensis, which will improve quality breeding and resistance breeding studies in this plant.

A comprehensive method for evaluating kernel quality was established by estimating in detail the kernel quality of 27 pecan cultivars introduced into China and by exploring the major trait differences among the different cultivars of pecan. The contents of crude fat, crude protein, soluble sugar, and tannin; the fatty acid composition; the amino acid composition; and the mineral element composition of 27 pecan cultivars were analyzed and tested using the national standards for principal component analysis (PCA), and orthogonal partial least-squares discriminant analysis (OPLS-DA) was used to comprehensively evaluate the 34 characteristic indicators. The fatty acids in the kernel were dominated by unsaturated fats, with oleic acid having the highest content relative to linoleic acid; the amino acid composition was dominated by medicinal amino acids; and potassium, magnesium, and calcium were the predominant mineral elements. Systematic cluster analysis revealed that the 27 pecan cultivars could be classified into three categories (relatively optimal, general, and poorest kernel quality) and 21 indexes, including oleic, linoleic, linolenic, crude fat, soluble sugars, aspartic acid, and threonine, served as indexes of the differences among the 27 tested cultivars. Combined with principal component analysis and stepwise regression analysis, a kernel quality evaluation model was established and verified, and reliable evaluation results were obtained. In addition, Nos. 9, 65, 66, 72, 8, and 21, which had the best quality, were further ranked using the probability ranking method within the third category of optimal quality cultivars. There were obvious differences in kernel characteristics among the different cultivars of pecan. Six cultivars with potential to be developed into excellent cultivars were preliminarily screened out. It was found that the organic combination of cluster analysis, principal component analysis, and orthogonal partial least-squares discriminant analysis can provide a reliable method for the quality evaluation of pecans.

The soils in karst regions, with carbonate rocks as the background material, are characterized by two main factors, drought and high calcium levels. Handeliodendron bodinieri (Levl.) Rehd is an endemic plant species in this area of China. However, few studies have been carried out on the adaptation mechanism of H. bodinieri to drought and high calcium soil. To reveal the physiological responses of H. bodinieri to exogenous calcium under drought stress, 10% PEG-6000 was used to simulate drought stress, and the effects of exogenous calcium at different concentrations on the physiology of H. bodinieri seedlings under drought stress were studied. The results showed that drought stress significantly reduced the relative water content and water potential of H. bodinieri seedlings. Malondialdehyde (MDA) content, O₂− and H₂O₂ production rates significantly increased under drought stress. The addition of exogenous calcium significantly reduced MDA content and O₂− and H₂O₂ production rates. Moreover, peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), ascorbic acid peroxidase (APX) and glutathione peroxidase (GPX) activities increased significantly under drought stress. At the same time, the accumulation of osmotic regulators such as soluble sugar, betaine and free proline also increased significantly. The addition of exogenous calcium further increased the activity of antioxidant enzymes and the accumulation of osmotic regulatory substances. Consequently, the oxidative stress and osmotic stress induced by drought decreased. Finally, exogenous calcium enhanced the photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) of H. bodinieri under drought stress and significantly improved water use efficiency (WUE). This study confirmed that the application of exogenous calcium can enhance the water condition, photosynthetic capacity, osmotic regulation and antioxidant capacity of H. bodinieri under drought stress. Of the treatments, the 10 mmol·L⁻¹ CaCl₂ treatment is more likely to improve survival of H. bodinieri under drought tolerance. This study provides an important reference for describing the adaptation mechanism and appropriate conservation of H. bodinieri under drought and high calcium conditions.

Research Highlights: The distribution of simple sequence repeat (SSR) motifs in two draft genomes of pecan was evaluated. Sixty-six SSR loci were validated by PCR amplification in pecan. Twenty-two new development markers can be used for genetic study in genus Carya. Background and Objectives: Pecan has good nutritional and health benefits and is an important crop worldwide. However, the genetic research in this species is insufficient. One of the main reasons for this is the lack of enough accurate, convenient, and economical molecular markers. Among different marker types, SSR loci are enormously useful in genetic studies. However, the number of SSRs in C. illinoinensis (Wangenh.) K. Koch is limited. Materials and Methods: The distribution of SSR motifs in the pecan genome was analyzed. Then, the primers for each SSR were designed. To evaluate their availability, 74 SSR loci were randomly selected and amplified in pecan. Finally, 22 new SSRs and eight former ones were picked to evaluate the genetic diversity in 60 pecan genotypes and to determine their transferability in other Carya species. Results: 145,714 and 143,041 SSR motifs were obtained from two draft genomes of ‘87MX3-2’ and ‘Pawnee’, respectively. In total, 9145 candidate primers were obtained. Sixty-six (89.19%) primers amplified the target products. Among the 30 SSRs, 29 loci showed polymorphism in 60 pecan genotypes. The polymorphic information content (PIC) values ranged from 0.012 to 0.906. In total, 26, 25, and 22 SSRs can be used in C. cathayensis Sarg., C. dabieshanensis W. C. Cheng & R. H. Chang, and C. hunanensis W.C. Liu, respectively. Finally, the dendrogram of all individuals was constructed. The results agree with the geographic origin of the four species and the pedigree relationships between different pecan cultivars. Conclusions: The characterization of SSRs in the pecan genome and the new SSRs will promote the progress of genetic study and breeding in pecan, as well as other species of genus Carya.

Pecan is rich in bioactive components such as fatty acids (FAs) and flavonoids and is an important nut type worldwide. Therefore, the molecular mechanisms of phytochemical biosynthesis in pecan are a focus of research. Recently, a draft genome and several transcriptomes have been published. However, the full-length mRNA transcripts remain unclear, and the regulatory mechanisms behind the quality components biosynthesis and accumulation have not been fully investigated. In this study, single-molecule long-read sequencing technology was used to obtain full-length transcripts of pecan kernels. In total, 37,504 isoforms of 16,702 genes were mapped to the reference genome. The numbers of known isoforms, new isoforms, and novel isoforms were 9013 (24.03%), 26,080 (69.54%), and 2411 (6.51%), respectively. Over 80% of the transcripts (30,751, 81.99%) had functional annotations. A total of 15,465 alternative splicing (AS) events and 65,761 alternative polyadenylation events were detected; wherein, the retained intron was the predominant type (5652, 36.55%) of AS. Furthermore, 1894 long noncoding RNAs and 1643 transcription factors were predicted using bioinformatics methods. Finally, the structural genes associated with FA and flavonoid biosynthesis were characterized. A high frequency of AS accuracy (70.31%) was observed in FA synthesis-associated genes. This study provides a full-length transcriptome data set of pecan kernels, which will significantly enhance the understanding of the regulatory basis of phytochemical biosynthesis during pecan kernel maturation.