Neolamarckia cadamba is an economically-important fast-growing tree species in South China and Southeast Asia. As a prerequisite first step for future gene expression studies, we have identified and characterized a series of stable reference genes that can be used as controls for quantitative real time PCR (qRT-PCR) expression analysis in this study. The expression stability of 15 candidate reference genes in various tissues and mature leaves under different conditions was evaluated using four different algorithms, i.e., geNorm, NormFinder, BestKeeper and RefFinder. Our results showed that SAMDC was the most stable of the selected reference genes across the set of all samples, mature leaves at different photosynthetic cycles and under drought stress, whereas RPL10A had the most stable expression in various tissues. PGK and RPS25 were considered the most suitable reference for mature leaves at different developmental stages and under cold treatment, respectively. Additionally, the gene expression profiles of sucrose transporter 4 (NcSUT4), and 9‐cis‐epoxycarotenoid dioxygenase 3 (NcNCED3) were used to confirm the validity of candidate reference genes. Collectively, our study is the first report to validate the optimal reference genes for normalization under various conditions in N. cadamba and will benefit the future discovery of gene function in this species.

The WRKY transcription factor family plays important regulatory roles in multiple biological processes in higher plants. They have been identified and functionally characterized in a number of plant species, but very little is known in Neolamarckia cadamba, a ‘miracle tree’ for its fast growth and potential medicinal resource in Southeast Asia. In this study, a total of 85 WRKY genes were identified in the genome of N. cadamba. They were divided into three groups according to their phylogenetic features, with the support of the characteristics of gene structures and conserved motifs of protein. The NcWRKY genes were unevenly distributed on 22 chromosomes, and there were two pairs of segmentally duplicated events. In addition, a number of putative cis-elements were identified in the promoter regions, of which hormone- and stress-related elements were shared in many NcWRKYs. The transcript levels of NcWRKY were analyzed using the RNA-seq data, revealing distinct expression patterns in various tissues and at different stages of vascular development. Furthermore, 16 and 12 NcWRKY genes were confirmed to respond to various hormone treatments and two different abiotic stress treatments, respectively. Moreover, the content of cadambine, the active metabolite used for the various pharmacological activities found in N. cadamba, significantly increased after Methyl jasmonate treatment. In addition, expression of NcWRKY64/74 was obviously upregulated, suggesting that they may have a potential function of regulating the biosynthesis of cadambine in response to MeJA. Taken together, this study provides clues into the regulatory roles of the WRKY gene family in N. cadamba.

Sucrose transporters (SUCs/SUTs) play crucial roles in apoplast transport and long-distance distribution of sucrose throughout the whole plant. However, whether and how the Arabidopsis AtSUC4 modulates sucrose import from apoplast to cytosol remains unclear. In the present study, we found that AtSUC4 protein was localized to the plasma membrane in the root. Expression of AtSUC4 in roots was gradually induced with the increasing sucrose concentrations (0%, 2%, 4% and 6%). When feeding high concentrations (4% and 6%) of sucrose, the primary root growth of seedling was inhibited. Interestingly, atsuc4 mutants exhibited longer primary root than the wild type under these conditions, indicating that atsuc4 mutants were less sensitive to excess sucrose. Moreover, the root of atsuc4 mutants accumulated less sucrose and abscisic acid (ABA) and more indole-3-acetic acid (IAA) on 4% and 6% sucrose supplementation. Transcriptomic analysis revealed that numerous genes associated with sugar transport and metabolism, as well as ABA signalling were down-regulated, whereas many IAA signaling-related genes were up-regulated in mutant plants relative to the wild type under 6% sucrose treatment. Collectively, our finding demonstrated that the deficiency of AtSUC4 reduced the inhibition of primary root growth under high sucrose condition, probably through reducing the sucrose transportation and metabolism, and subsequent alteration in IAA and ABA signalling.

Neolamarckia cadamba is an indigenous, timber-producing tree species in Southern China that plays an important role in the sustainable development of the local forestry industry. However, the geographic genetic variation across its natural distribution area in Southern China has yet to be characterized for best utilization. Here, we report the geographic genetic variation in growth and wood properties of N. cadamba from 10 provenances that represent the entire natural distribution of N. cadamba in Southern China. There was significant geographic variation in diameter breast height (DBH), height (H), volume (V), vessel length (VL), vessel diameter (VD), VL/VD, and wood basic density (WBD). The variation in tree volume across provenances was greater than that of other growth traits, indicating that volume has a greater potential for selection in provenance trials. The provenance heritabilities of growth traits and wood properties ranged from 0.59 to 0.67 and from 0.40 to 0.45, respectively. Trend surface analysis revealed that patterns of geographic variation associated with growth traits were weakly negatively correlated with those of wood properties. The pattern of geographic variation in growth traits showed a gradual increase from the periphery to the central region, whereas wood properties showed the opposite pattern, and latitude had the greatest effect on both. Wood property measurements suggested that the YNMS provenance produced superior timber wood, whereas the GXLZ, GXFCG, and GXNN provenances produced the best pulpwood. These provenances could potentially provide more valuable breeding materials for the genetic improvement of N. cadamba.