Loblolly pine (Pinus taeda L.) is one of the most important species for oleoresin (a mixture of terpenoids) in South China. The high oleoresin content of loblolly pine is associated with resistance to bark beetles and other economic benefits. In this study, we conducted transcriptome analyses of loblolly pine secondary xylem to gain insight into the genes involved in terpenoid biosynthesis. A total of 372 unigenes were identified as being critical for oleoresin production, including genes for ATP-binding cassette (ABC) transporters, the cytochrome P450 (CYP) protein family, and terpenoid backbone biosynthesis enzymes. Six key genes involved in terpenoid biosynthetic pathways were selected for multiple sequence alignment, conserved motif prediction, and phylogenetic and expression profile analyses. The protein sequences of all six genes exhibited a higher degree of sequence conservation, and upstream genes were relatively more conserved than downstream genes in terpenoid biosynthetic pathways. The N-terminal regions of these sequences were less conserved than the C-terminal ends, as the N-terminals were quite diverse in both length and composition. The phylogenetic analyses revealed that most genes originated from gene duplication after species divergence, and partial genes exhibited incomplete lineage sorting. In addition, the expression profile analyses showed that all six genes exhibited high expression levels during the high-oleoresin-yielding phase.

Abscission in plants is tightly controlled by multiple phytohormones and the expression of various genes. However, whether the plant hormone brassinosteroids (BRs) are involved in this process is largely unknown. Here, we found that exogenous application of BRs reduced the ethylene-induced fruitlet abscission of litchi due to lower ethylene (ET) production and suppressed the expression of the ethylene biosynthetic genes LcACS1/4 and LcACO2/3 in the fruitlet abscission zone (FAZ). Two genes that encode the BR core signaling components brassinazole resistant (BZR) proteins, namely, LcBZR1 and LcBZR2, were characterized. LcBZR1/2 were localized to the nucleus and acted as transcription repressors. Interestingly, the LcBZR1/2 transcript levels were not changed during ET-induced fruitlet abscission, while their expression levels were significantly increased after BR application. Moreover, gel shift and transient expression assays indicated that LcBZR1/2 could suppress the transcription of LcACS1/4 and LcACO2/3 by specifically binding to their promoters. Importantly, ectopic expression of LcBZR1/2 in Arabidopsis significantly delayed floral organ abscission and suppressed ethylene biosynthesis. Collectively, our results suggest that BRs suppress ET-induced fruitlet abscission through LcBZR1/2-controlled expression of genes related to ethylene biosynthesis in litchi. In addition, similar results were observed in the Arabidopsis gain-of-function mutant bzr1-1D, which showed delayed floral organ abscission in parallel with lower expression of ACS/ACO genes and reduced ethylene production, suggesting that the mechanism of BZR-controlled organ abscission via regulation of ethylene biosynthesis might be conserved in Arabidopsis.

Large and premature organ abscission may limit the industrial development of fruit crops by causing serious economic losses. It is well accepted that ethylene (ET) is a strong inducer of organ abscission in plants. However, the mechanisms underlying the control of organ abscission by ET are largely unknown. We previously revealed that LcKNAT1, a KNOTTED-LIKE FROM ARABIDOPSIS THALIANA1 (KNAT1)-like protein, acted as a negative regulator in control of fruitlet abscission through suppressing the expression of ET biosynthetic genes in litchi. In this study, we further reported that LcKNAT1 could also directly repress the transcription of LcEIL2 and LcEIL3, two ETHYLENE INSENSITIVE 3-like (EIL) homologs in litchi, which functioned as positive regulators in ET-activated fruitlet abscission by directly promoting the expression of genes responsible for ET biosynthesis and cell wall degradation. The expression level of LcKNAT1 was downregulated, while LcEIL2/3 was upregulated at the abscission zone (AZ) accompanying the fruitlet abscission in litchi. The results of electrophoretic mobility shift assays (EMSAs) and transient expression showed that LcKNAT1 could directly bind to the promoters of LcEIL2 and LcEIL3 and repress their expression. Furthermore, the genetic cross demonstrated that the β-glucuronidase (GUS) expression driven by the promoters of LcEIL2 or LcEIL3 at the floral AZ was obviously suppressed by LcKNAT1 under stable transformation in Arabidopsis. Taken together, our findings suggest that the LcKNAT1-LcEIL2/3 regulatory module is likely involved in the fruitlet abscission in litchi, and we propose that LcKNAT1 could suppress both ET biosynthesis and signaling to regulate litchi fruit abscission.

SUMMARY Reactive oxygen species (ROS) have been emerging as a key regulator in plant organ abscission. However, the mechanism underlying the regulation of ROS homeostasis in the abscission zone (AZ) is not completely established. Here, we report that a DOF (DNA binding with one finger) transcription factor LcDOF5.6 can suppress the litchi fruitlet abscission through repressing the ROS accumulation in fruitlet AZ (FAZ). The expression of LcRbohD, a homolog of the Arabidopsis RBOHs that are critical for ROS production, was significantly increased during the litchi fruitlet abscission, in parallel with an increased accumulation of ROS in FAZ. In contrast, silencing of LcRbohD reduced the ROS accumulation in FAZ and decreased the fruitlet abscission in litchi. Using in vitro and in vivo assays, we revealed that LcDOF5.6 was shown to inhibit the expression of LcRbohD via direct binding to its promoter. Consistently, silencing of LcDOF5.6 increased the expression of LcRbohD, concurrently with higher ROS accumulation in FAZ and increased fruitlet abscission. Furthermore, the expression of key genes (LcIDL1, LcHSL2, LcACO2, LcACS1, and LcEIL3) in INFLORESCENCE DEFICIENT IN ABSCISSION signaling and ethylene pathways were altered in LcRbohD‐silenced and LcDOF5.6‐silenced FAZ cells. Taken together, our results demonstrate an important role of the LcDOF5.6–LcRbohD module during litchi fruitlet abscission. Our findings provide new insights into the molecular regulatory network of organ abscission.