Although ammonia–nitrogen (NH₄⁺–N) and nitrate–nitrogen (NO₃⁻–N) are the two main forms of N absorbed and utilized by plants, the preferences of plants for these forms are still unclear. In this study, we analyzed the growth, photosynthesis, and nutrients of pecan under different NH₄⁺:NO₃⁻ ratios (0/0, 0/100, 25/75, 50/50, 75/25, 100/0) by indoor aerosol incubation. The results showed that additions of different N forms promoted the growth and development of pecan seedlings. When NO₃⁻–N was used as the sole N source, it significantly promoted the ground diameter growth of pecan and increased the leaf pigment content and photosynthetic rate. The NH₄⁺:NO₃⁻ ratio of 75:25 and NH₄⁺–N as the sole N source significantly increased the soluble sugars in stems and roots, starch in leaves, stems and roots, soluble protein in leaves and stems, and soluble phenols in stems and roots. Additionally, the NH₄⁺:NO₃⁻ ratio of 75:25 increased plant height, leaf number, root soluble protein, and leaf soluble phenol contents. In conclusion, regarding the physiological aspects of pecan growth, pecans are more inclined to use NH₄⁺–N. Considering that the NH₄⁺–N as the only N source may lead to nutrient imbalance or even toxicity, the NH₄⁺:NO₃⁻ ratio of 75:25 was most favorable for the growth and development of pecan seedlings.

Research Highlights: This study identifies the effect of nitrogen (N) and phosphorus (P) addition on stoichiometry correlations between understory plants and soil in subtropical Chinese fir plantations. Background and Objectives: Nitrogen and P are two nutrients limiting forest ecosystem production. To obtain more wood production, N and P are usually applied in plantation management. Changes in soil N and P will generally alter the stoichiometric characteristics of understory plants, which control carbon (C) and nutrient cycles between plants and soil. However, different correlations between plant and soil stoichiometry among functional groups of understory plants have not been investigated, which also impacted element cycling between plants and soil. Materials and Methods: Subtropical Chinese fir plantations were selected for N (100 kg ha⁻¹ year⁻¹) and P (50 kg ha⁻¹ year⁻¹) addition study. We collected fresh litter and the corresponding soil of four understory plants (Lophatherum gracile Brongn., Woodwardia japonica (L.f.) Sm., Dryopteris atrata (Kunze) Ching and Dicranopteris dichotoma (Thunb.) Berhn.) for study of C, N, and P stoichiometric ratios. Results: Nitrogen and P addition affected C, N, and P concentrations and stoichiometric ratios in litter and soil as well as correlations between litter and soil stoichiometric ratios. Understory plant species with different functional types impacted the correlations between plants and soil in C, N, and P stoichiometric ratios, especially correlations between litter C and soil C and N. Conclusions: Changes in soil N and P affect the stoichiometric ratios of understory plants. Functional groups impacted the correlation in C, N, and P stoichiometric ratios between plants and soil, indicating functional groups varied in their impacts on element cycling between plants and soil in plantations with exogenous nutrient addition, which should be considered in future management of plantations with intensive fertilization practice.

As crucial enzymes in the lipid metabolic network, long-chain acyl-CoA synthases (LACSs) are members of the acyl-activated enzyme superfamily and play a crucial role in epidermal wax synthesis, plant lipid anabolic metabolism, and stress tolerance. In this study, 11 pecan LACS genes were identified and categorized into five groups and located on nine chromosomes. The significant degree of conservation in the AtLACS and CiLACS protein sequences was demonstrated by multiple sequence alignment and conserved motif analysis. Cis-acting element analysis identified numerous stress-responsive and hormone-inducible elements in the promoter regions of CiLACS genes. The expression levels of CiLACS9 and CiLACS9-1 were considerably up-regulated under salt and drought stress, according to the qRT-RCR study. Treatment with ABA also led to increased expression levels of CiLACS1, CiLACS1-1, CiLACS2, and CiLACS9-1. Notably, CiLACS4, CiLACS4-1, CiLACS9, and CiLACS9-1 exhibited peak expression levels at 135 days after anthesis and are likely to have been crucial in the accumulation of seed kernel oil. Moreover, the CiLACS9 gene was shown to be located in the cytoplasm. These findings offer a theoretical framework for clarifying the roles of LACS genes in the processes of pecan kernel oil synthesis and response to abiotic stressors.

Nitrogen (N) is a major limiting factor for plant growth and crop production. The use of N fertilizer in forestry production is increasing each year, but the loss is substantial. Mastering the regulatory mechanisms of N uptake and transport is a key way to improve plant nitrogen use efficiency (NUE). However, this has rarely been studied in pecans. In this study, 10 AMT and 69 NRT gene family members were identified and systematically analyzed from the whole pecan genome using a bioinformatics approach, and the expression patterns of AMT and NRT genes and the uptake characteristics of NH₄⁺ and NO₃⁻ in pecan were analyzed by aeroponic cultivation at varying NH₄⁺/NO₃⁻ ratios (0/0, 0/100,25/75, 50/50, 75/25,100/0 as CK, T1, T2, T3, T4, and T5). The results showed that gene duplication was the main reason for the amplification of the AMT and NRT gene families in pecan, both of which experienced purifying selection. Based on qRT-PCR results, CiAMTs were primarily expressed in roots, and CiNRTs were majorly expressed in leaves, which were consistent with the distribution of pecan NH₄⁺ and NO₃⁻ concentrations in the organs. The expression levels of CiAMTs and CiNRTs were mainly significantly upregulated under N deficiency and T4 treatment. Meanwhile, T4 treatment significantly increased the NH₄⁺, NO₃⁻, and NO₂⁻ concentrations as well as the Vmax and Km values of NH₄⁺ and NO₃⁻ in pecans, and Vmax/Km indicated that pecan seedlings preferred to absorb NH₄⁺. In summary, considering the single N source of T5, we suggested that the NH₄⁺/NO₃⁻ ratio of 75:25 was more beneficial to improve the NUE of pecan, thus increasing pecan yield, which provides a theoretical basis for promoting the scale development of pecan and provides a basis for further identification of the functions of AMT and NRT genes in the N uptake and transport process of pecan.

Pecan seed oil is a valuable source of essential fatty acids and various bioactive compounds; however, the functions of microRNAs and their targets in oil biosynthesis during seed development are still unknown. Here, we found that the oil content increased rapidly in the three early stages in three cultivars, and that oleic acid was the predominant fatty acid component in the mature pecan embryos. We identified, analyzed, and validated the expression levels of miRNAs related to seed development and oil biosynthesis, as well as their potential target genes, using small RNA sequencing data from three stages (120, 135, and 150 days after flowering). During the seed development process, 365 known and 321 novel miRNAs were discovered. In total, 91 known and 181 novel miRNAs were found to be differentially expressed, and 633 target genes were further investigated. The expression trend analysis revealed that the 91 known miRNAs were classified into eight groups, approximately two-thirds of which were up-regulated, whereas most novel miRNAs were down-regulated. The qRT–PCR and degradome sequencing data were used to identify five miRNA- target pairs. Overall, our study provides valuable insights into the molecular regulation of oil biosynthesis in pecan seeds.

Mitogen-activated protein kinases consist of three kinase modules composed of MPKs, MKKs, and MPKKKs. As members of the protein kinase (PK) superfamily, they are involved in various processes, such as developmental programs, cell division, hormonal progression, and signaling responses to biotic and abiotic stresses. In this study, a total of 18 MPKs and 10 MKKs were annotated on the pecan genome, all of which could be classified into four subgroups, respectively. The gene structures and conserved sequences of family members in the same branch were relatively similar. All MPK proteins had a conserved motif TxY, and D(L/I/V)K and VGTxxYMSPER existed in all MKK proteins. Duplication events contributed largely to the expansion of the pecan MPK and MKK gene families. Phylogenetic analysis of protein sequences from six plants indicated that species evolution occurred in pecan. Organ-specific expression profiles of MPK and MKK showed functional diversity. Ka/Ks values indicated that all genes with duplicated events underwent strong negative selection. Seven CiPawMPK and four CiPawMKK genes with high expression levels were screened by transcriptomic data from different organs, and these candidates were validated by qRT-PCR analysis of hormone-treated and stressed samples.