Cytokinin-responsive MdTCP17 interacts with MdWOX11 to repress adventitious root primordium formation in apple rootstocks.

Adventitious root (AR) formation plays an important role in vegetatively propagated plants. Cytokinin (CK) inhibits AR formation, but the molecular mechanisms driving this process remain unknown. In this study, we confirmed that CK content is related to AR formation and further revealed that a high auxin/CK ratio was beneficial to AR formation in apple (Malus domestica). A correlation between expression of CK-responsive TEOSINTE BRANCHED1, CYCLOIDEA, and PCF17 (MdTCP17) and AR formation in response to CK was identified, and overexpression of MdTCP17 in transgenic apple inhibited AR formation. Yeast two-hybrid, bimolecular fluorescence complementation, and co-immunoprecipitation assays revealed an interaction between MdTCP17 and WUSCHEL-RELATED HOMEOBOX11 (MdWOX11), and a significant correlation between the expression of MdWOX11 and AR ability. Overexpression of MdWOX11 promoted AR primordium formation in apple, while interference of MdWOX11 inhibited AR primordium production. Moreover, a positive correlation was found between MdWOX11 and LATERAL ORGAN BOUNDARIES DOMAIN29 (MdLBD29) expression, and yeast one-hybrid, dual luciferase reporter, and ChIP-qPCR assays verified the binding of MdWOX11 to the MdLBD29 promoter with a WOX-box element in the binding sequence. Furthermore, MdTCP17 reduced the binding of MdWOX11 and MdLBD29 promoters, and coexpression of MdTCP17 and MdWOX11 reduced MdLBD29 expression. Together, these results explain the function and molecular mechanism of MdTCP17-mediated CK inhibition of AR primordium formation, which could be used to improve apple rootstocks genetically.

Insights into the complicated networks contribute to adventitious rooting in transgenic MdWOX11 apple microshoots under nitrate treatments.

Adventitious root formation is a bottleneck for the mass propagation of microshoots, and nitrate is an essential nutrient regulating adventitious roots. WOX11 is involved in adventitious rooting. But the crosstalk between nitrate and WOX11 is completely unknown. In this study, MdWOX11 transgenic apple microshoots were grown on different nitrate treatments. Low nitrate promotes adventitious rooting in overexpressed microshoots more than wild type and RNA interference microshoots. In contrast, medium nitrate significantly inhibits it in overexpressed and RNA interference microshoots compared with wild type microshoots. Stem anatomy indicated that medium nitrate delays root primordia formation compared with low nitrate. Methyl jasmonate and zeatin riboside played positive and negative roles in adventitious rooting, respectively. Transcriptomic analysis was conducted to understand the molecular mechanisms behind the phenotypes better. Hormone signalling, sugar metabolism, nitrogen metabolism, cell cycle and root development pathway-related genes were selected for their potential involvement in adventitious rooting. Results suggest that nitrogen signaling and MdWOX11 expression affect cytokinin accumulation and response to cytokinin through regulating the expression of genes related to cytokinin synthesis and transduction pathways, which ultimately affect adventitious rooting. This study provided important insights into the complicated networks involved in adventitious rooting in transgenic microshoots under nitrate treatments.

Transcriptome analysis reveals that cytokinins inhibit adventitious root formation through the MdRR12-MdCRF8 module in apple rootstock.

Adventitious root (AR) formation is great significance for apple rootstock breeding. Transcriptome analyses were performed with cytokinins (CTKs) signal treatments to analyze the mechanism of AR formation. The results showed that 6-benzyadenine (6-BA) treatment inhibited AR formation. Histological analysis also observed that AR primordium cell formation was significantly suppressed by 6-BA treatment; the ratio of auxin/cytokinins exhibited the lowest values at 1 and 3 day (d) in the 6-BA treatment group. Furthermore, the differentially expressed genes were divided into five categories, including auxin, cytokinins, other hormones, cell cycle, and carbohydrate metabolism pathways. Due to the study of cytokinins signal treatment, it is important to understand the particular module mediated by the cytokinins pathway. The expression level of MdRR12 (a family member of B-type cytokinins-responsive factors) was significantly upregulated at 3 d by 6-BA treatment. Compared to the wild type, the 35S::MdRR12 transgenic tobaccos suppressed AR formation. The promoter sequence of MdCRF8 contains AGATT motif elements that respond to MdRR12. RNA-seq and RT-qPCR assays predicted cytokinins response factor (MdCRF8) to be a downstream gene regulated by MdRR12. The activity of the pro-MdCRF8-GUS promoter was obviously induced by 6-BA treatment and inhibited by lovastatin (Lov) treatment. Yeast one-hybrid, dual-luciferase reporter, and GUS coexpression assays revealed that MdRR12 could directly bind to the MdCRF8 promoter. Additionally, 35S::MdCRF8 transgenic tobaccos also blocked AR growth. Compared to the wild type, 35S::MdRR12 and 35S::MdCRF8 transgenic tobaccos enhanced sensitivity to cytokinins. Thus, we describe that MdRR12 and MdCRF8 function as integrators of cytokinins signals that affect cell cycle- and carbohydrate metabolism-related genes to regulate cell fate transition during AR formation. On the basis of these results, we concluded that the MdRR12-MdCRF8 module is involved in the negative regulation of AR formation in apple rootstock and can potentially be applied in agriculture using genetic approaches.

Transcriptome Analysis Reveals Multiple Genes and Complex Hormonal-Mediated Interactions with PEG during Adventitious Root Formation in Apple.

Adventitious root (AR) formation is a bottleneck for the mass propagation of apple rootstocks, and water stress severely restricts it. Different hormones and sugar signaling pathways in apple clones determine AR formation under water stress, but these are not entirely understood. To identify them, GL-3 stem cuttings were cultured on polyethylene glycol (PEG) treatment. The AR formation was dramatically decreased compared with the PEG-free control (CK) cuttings by increasing the endogenous contents of abscisic acid (ABA), zeatin riboside (ZR), and methyl jasmonate (JA-me) and reducing the indole-3-acetic acid (IAA) and gibberellic acid 3 (GA3) contents. We performed a transcriptomic analysis to identify the responses behind the phenotype. A total of 3204 differentially expressed genes (DEGs) were identified between CK and PEG, with 1702 upregulated and 1502 downregulated genes. Investigation revealed that approximately 312 DEGs were strongly enriched in hormone signaling, sugar metabolism, root development, and cell cycle-related pathways. Thus, they were selected for their possible involvement in adventitious rooting. However, the higher accumulation of ABA, ZR, and JA-me contents and the upregulation of their related genes, as well as the downregulation of sugar metabolism-related genes, lead to the inhibition of ARs. These results indicate that AR formation is a complicated biological process chiefly influenced by multiple hormonal signaling pathways and sugar metabolism. This is the first study to demonstrate how PEG inhibits AR formation in apple plants.

Transcriptome analysis reveals the inhibitory nature of high nitrate during adventitious roots formation in the apple rootstock.

In the process of vegetative propagation of apple rootstocks, the development of adventitious roots (ARs) has crucial importance. Nitrate is an essential nutrient necessary for plant growth; however, the inhibitory effect of high nitrate on ARs formation has not been explored. The physiological and molecular mechanisms underlying ARs inhibition were examined in this study. Stem cuttings of B9 apple rootstock were cultured on two nitrate treatments (T1 = 18.7 mM L-1 and T2 = 37.5 mM L-1 ), where T2 was identified as ARs inhibiting treatment. Morphological and anatomical observations advocating that high availability of nitrate inhibited AR formation by delaying the ARs initiation and emergence stages, where the root number was 287%, and the length was 604.6% lower than the T1 cuttings. Moreover, the contents of endogenous hormones were also elevated in response to T2 at most of the time points, which may cause a hormonal imbalance within the plant body and drive toward ARs inhibition. Furthermore, 3686 genes were differentially expressed by high-throughput sequencing. Out of these, 1797 genes were upregulated, and 1889 genes were downregulated. Approximately 238 genes related to nitrate, hormones, root development, and cell-cycle induction pathways were selected according to their potential to be involved in ARs regulation. This is the first study providing information regarding the inhibitory effect of high nitrate on ARs formation in apple rootstock.

Mdm-MIR393b-mediated adventitious root formation by targeted regulation of MdTIR1A expression and weakened sensitivity to auxin in apple rootstock.

Adventitious root (AR) formation is of great significance for apple rootstock breeding. It is widely accepted that miR393 influences AR formation in many plant species; however, the molecular mechanism by which factors regulate AR formation remains insufficient. In this study, the evolutionary relationship of mdm-miR393 and candidate target genes MdTIR1/AFB was systematically identified, and the expression patterns were analysed. Multisequence alignment analysis of miR393 family members suggests that miR393 conservatively evolved between different species. The evolutionary relationship of the TIR1/AFBs can be divided into G1, G2 and G3 subgroups. During AR formation, the expression level of mdm-miR393a/b/c was significantly upregulated at 1 d and 7 d by exogenous auxin treatment. Furthermore, the expression levels of MdTIR1A, MdTIR1D, MdAFB1, MdAFB2, MdAFB3, MdAFB4 and MdAFB8 also appeared to be significantly changed by exogenous auxin induction. Subsequently, tissue-specific expression analysis showed that the expression levels of mdm-miR393 and MdTIR1/AFBs in different tissues exhibited significant differences. The promoter of mdm-miR393 contains multiple elements that respond to ABA, adversity and light signals; auxin treatment can activate the mdm-MIR393b promoter but is obviously inhibited by NPA treatment. The targeting relationship between mdm-MIR393b and MdTIR1A was verified by expression patterns, degradation group data, transient tobacco conversion results, and genes functions experiments. Heterologous overexpression of mdm-MIR393b (35S::mdm-MIR393b) decreased the number of ARs in the phenotype and reduced the expression level of the target gene NtTIR1 in tobacco. Compared to the wild type, the 35S::mdm-MIR393b transgenic plants demonstrated insensitivity to auxin. Furthermore, tir1 mutant exhibited reduced root system structure relative to the control. The above results illustrated that mdm-MIR393b is involved in mediating AR formation by targeted regulation of MdTIR1A expression in apple rootstock.

Transcriptome analysis reveals the promotive effect of potassium by hormones and sugar signaling pathways during adventitious roots formation in the apple rootstock.

Apples are economically valuable and widely consumed fruits. The adventitious roots (ARs) formation is gridlock for apple trees mass propagation. The possible function of multiple hormones and sugar signaling pathways regulating ARs formation has not been completely understood in apple. In this study, B9 stem cuttings were treated with KCl treatment, where the highest root numbers (220) and maximum root length of 731.2 cm were noticed in KCl-treated cuttings, which were 98.2% and 215% higher than control cuttings. The content of endogenous hormones: IAA, ZR, JA, GA, and ABA were detected higher in response to KCl at most time-points. To figure out the molecular mechanisms underlying this effect, we investigated transcriptome analysis. In total, 4631 DEGs were determined, from which about 202 DEGs were considerably enriched in pathways associated with hormone signaling, sugar metabolism, root development, and cell cycle-related and were thereupon picked out on their potential involvements in ARs formation. Though, IAA accumulation and up-regulation of various genes contribute to induce AR formation. These results suggest that AR formation is a complex biological process in apple rootstocks, influenced mainly by the auxin signaling pathway and sugar metabolism.

A direct hippocampo-cerebellar projection in chicken.

Our previous studies suggested that a direct hippocampo-cerebellar projection might exist in the chicken. To confirm such a presumption of hippocampo-cerebellar interactions, horseradish peroxidase (HRP) was used as a retrograde tracer to be injected into the white matter of the folia VI-VIII of the cerebellum in young and adult chickens. In another set of experiments, young chickens were subjected to electrolytic lesions of the hippocampal formation (HF), especially the ventromedial portion, and the cerebellum was observed with the electron microscope to find neuronal degeneration in the HF. Following injections of HRP into the cerebellum, a large number of labeled neurons were found in the area APHm-APHim of the HF in the young and adult chickens. As a result of the electrolytic lesions of the APHm-APHim in the HF, many large degenerated nerve fibers were found in the white matter in the vicinity of the lateral nucleus of the cerebellum, and some small degenerated fibers were found in the white matter of the folia VI-VIII. In the cerebellar cortex of folia VI-VIII, degenerated axonal terminals occurred in both the molecular and Purkinje layers, but not in the granular layer. In the lateral nucleus, some dark degenerating axonal terminals were recognized to connect with the perikarya of neurons of this nucleus. The present experiments demonstrate that the APHm-APHim of the HF directly projected to the cortex of folia VI-VIII and the lateral nucleus of the cerebellum in young chickens.