Strigolactone regulates adventitious root formation via the MdSMXL7-MdWRKY6-MdBRC1 signaling cascade in apple.

Propagation through stem cuttings is a popular method worldwide for species such as fruit tree rootstocks and forest trees. Adventitious root (AR) formation from stem cuttings is crucial for effective and successful clonal propagation of apple rootstocks. Strigolactones (SLs) are newly identified hormones involved in AR formation. However, the regulatory mechanisms underpinning this process remain elusive. In the present study, weighted gene co-expression network analysis, as well as rooting assays using stable transgenic apple materials, revealed that MdBRC1 served as a key gene in the inhibition of AR formation by SLs. We have demonstrated that MdSMXL7 and MdWRKY6 synergistically regulated MdBRC1 expression, depending on the interactions of MdSMXL7 and MdWRKY6 at the protein level downstream of SLs as well as the direct promoter binding on MdBRC1 by MdWRKY6. Furthermore, biochemical studies and genetic analysis revealed that MdBRC1 inhibited AR formation by triggering the expression of MdGH3.1 in a transcriptional activation pathway. Finally, the present study not only proposes a component, MdWRKY6, that enables MdSMXL7 to regulate MdBRC1 during the process of SL-controlled AR formation in apple, but also provides prospective target genes to enhance AR formation capacity using CRISPR (i.e. clustered regularly interspaced short palindromic repeats) technology, particularly in woody plants.

Transcription Factor IAA27 Positively Regulates P Uptake through Promoted Adventitious Root Development in Apple Plants.

Phosphate (P) deficiency severely limits the growth and production of plants. Adventitious root development plays an essential role in responding to low phosphorus stress for apple plants. However, the molecular mechanisms regulating adventitious root growth and development in response to low phosphorus stress have remained elusive. In this study, a mutation (C-T) in the coding region of the apple AUXIN/INDOLE-3-ACETIC ACID 27 (IAA27) gene was identified. MdIAA27T-overexpressing transgenic apple improved the tolerance to phosphorus deficiency, which grew longer and denser adventitious roots and presented higher phosphorous content than the control plants under low phosphorus conditions, while the overexpression of MdIAA27C displayed the opposite trend. Moreover, the heterologous overexpression of MdIAA27 in tobacco yielded the same results, supporting the aforementioned findings. In vitro and in vivo assays showed that MdIAA27 directly interacted with AUXIN RESPONSE FACTOR (ARF8), ARF26 and ARF27, which regulated Small Auxin-Up RNA 76 (MdSAUR76) and lateral organ boundaries domain 16 (MdLBD16) transcription. The mutation in IAA27 resulted in altered interaction modes, which in turn promoted the release of positive ARFs to upregulate SAUR76 and LBD16 expression in low phosphorus conditions. Altogether, our studies provide insights into how the allelic variation of IAA27 affects adventitious root development in response to low phosphorus stress.

Integrated multi-omics analysis uncovers roles of mdm-miR164b-MdORE1 in strigolactone-mediated inhibition of adventitious root formation in apple.

Apple is one of the most important fruit crops in temperate regions and largely relies on cutting propagation. Adventitious root formation is crucial for the success of cutting propagation. Strigolactones have been reported to function in rooting of woody plants. In this study, we determined that strigolactones have inhibitory effects on adventitious root formation in apple. Transcriptome analysis identified 12 051 differentially expressed genes over the course of adventitious root initiation, with functions related to organogenesis, cell wall biogenesis or plant development. Further analysis indicated that strigolactones might inhibit adventitious root formation through repressing two core hub genes, MdLAC3 and MdORE1. Combining small RNA and degradome sequencing, as well as dual-luciferase sensor assays, we identified and validated three negatively correlated miRNA-mRNA pairs, including mdm-miR397-MdLAC3 and mdm-miR164a/b-MdORE1. Overexpression of mdm-miR164b and silencing MdORE1 exhibited enhanced adventitious root formation in tobacco and apple, respectively. Finally, we verified the role of mdm-miR164b-MdORE1 in strigolactone-mediated repression of rooting ability. Overall, the identified comprehensive regulatory network in apple not only provides insight into strigolactone-mediated adventitious root formation in other woody plants, but also points to a potential strategy for genetic improvement of rooting capacity in woody plants.

An HD-ZIP transcription factor, MxHB13, integrates auxin-regulated and juvenility-determined control of adventitious rooting in Malus xiaojinensis.

Adventitious root (AR) formation is a critical factor in the vegetative propagation of forestry and horticultural plants. Competence for AR formation declines in many species during the miR156/SPL-mediated vegetative phase change. Auxin also plays a regulatory role in AR formation. In apple rootstock, both high miR156 expression and exogenous auxin application are prerequisites for AR formation. However, the mechanism by which the miR156/SPL module interacts with auxin in controlling AR formation is unclear. In this paper, leafy cuttings of juvenile (Mx-J) and adult (Mx-A) phase Malus xiaojinensis were used in an RNA-sequencing experiment. The results revealed that numerous genes involved in phytohormone signaling, carbohydrate metabolism, cell dedifferentiation, and reactivation were downregulated in Mx-A cuttings in response to indole butyric acid treatment. Among the differentially expressed genes, an HD-ZIP transcription factor gene, MxHB13, was found to be under negative regulation of MdSPL26 by directly binding to MxHB13 promoter. MxTIFY9 interacts with MxSPL26 and may play a role in co-repressing the expression of MxHB13. The expression of MxTIFY9 was induced by exogenous indole butyric acid. MxHB13 binds to the promoter of MxABCB19-2 and positively affects the expression. A model is proposed in which MxHB13 links juvenility-limited and auxin-limited AR recalcitrance mechanisms in Mx-A.

Application of genome-wide insertion/deletion markers on genetic structure analysis and identity signature of Malus accessions.

BACKGROUND: Apple (Malus ssp.), one of the most important temperate fruit crops, has a long cultivation history and is economically important. To identify the genetic relationships among the apple germplasm accessions, whole-genome structural variants identified between M. domestica cultivars 'Jonathan' and 'Golden Delicious' were used. RESULTS: A total of 25,924 insertions and deletions (InDels) were obtained, from which 102 InDel markers were developed. Using the InDel markers, we found that 942 (75.3%) of the 1251 Malus accessions from 35 species exhibited a unique identity signature due to their distinct genotype combinations. The 102 InDel markers could distinguish 16.7-71.4% of the 331 bud sports derived from 'Fuji', 'Red Delicious', 'Gala', 'Golden Delicious', and other cultivars. Five distinct genetic patterns were found in 1002 diploid accessions based on 78 bi-allele InDel markers. Genetic structure analysis indicated that M. domestica showed higher genetic diversity than the other species. Malus underwent a relatively high level of wild-to-crop or crop-to-wild gene flow. M. sieversii was closely related to both M. domestica and cultivated Chinese cultivars. CONCLUSIONS: The identity signatures of Malus accessions can be used to determine distinctness, uniformity, and stability. The results of this study may also provide better insight into the genetic relationships among Malus species.

Genomics-assisted prediction of salt and alkali tolerances and functional marker development in apple rootstocks.

BACKGROUND: Saline, alkaline, and saline-alkaline stress severely affect plant growth and development. The tolerance of plants to these stressors has long been important breeding objectives, especially for woody perennials like apple. The aims of this study were to identify quantitative trait loci (QTLs) and to develop genomics-assisted prediction models for salt, alkali, and salt-alkali tolerance in apple rootstock. RESULTS: A total of 3258 hybrids derived from the apple rootstock cultivars 'Baleng Crab' (Malus robusta Rehd., tolerant) × 'M9' (M. pumila Mill., sensitive) were used to identify 17, 13, and two QTLs for injury indices of salt, alkali, and salt-alkali stress via bulked segregant analysis. The genotype effects of single nucleotide polymorphism (SNP) markers designed on candidate genes in each QTL interval were estimated. The genomic predicted value of an individual hybrid was calculated by adding the sum of all marker genotype effects to the mean phenotype value of the population. The prediction accuracy was 0.6569, 0.6695, and 0.5834 for injury indices of salt, alkali, and salt-alkali stress, respectively. SNP182G on MdRGLG3, which changes a leucine to an arginine at the vWFA-domain, conferred tolerance to salt, alkali, and salt-alkali stress. SNP761A on MdKCAB, affecting the Kv_beta domain that cooperated with the linked allelic variation SNP11, contributed to salt, alkali, and salt-alkali tolerance in apple rootstock. CONCLUSIONS: The genomics-assisted prediction models can potentially be used in breeding saline, alkaline, and saline-alkaline tolerant apple rootstocks. The QTLs and the functional markers may provide insight for future studies into the genetic variation of plant abiotic stress tolerance.

The Artificial Promoter rMdAG2I Confers Flower-specific Activity in Malus.

Genetic modifications of floral organs are important in the breeding of Malus species. Flower-specific promoters can be used to improve floral organs specifically, without affecting vegetative organs, and therefore developing such promoters is highly desirable. Here, we characterized two paralogs of the Arabidopsis thaliana gene AGAMOUS (AG) from Malus domestica (apple): MdAG1 and MdAG2. We then isolated the second-intron sequences for both genes, and created four artificial promoters by fusing each intron sequence to a minimal 35S promoter sequence in both the forward and reverse directions. When transferred into tobacco (Nicotiana benthamiana) by Agrobacterium tumefaciens-mediated stable transformation, one promoter, rMdAG2I, exhibited activity specifically in flowers, whereas the other three also showed detectable activity in vegetative organs. A test of the four promoters' activities in the ornamental species Malus micromalus by Agrobacterium-mediated transient transformation showed that, as in tobacco, only rMdAG2I exhibited a flower-specific expression pattern. Through particle bombardment transformation, we demonstrated that rMdAG2I also had flower-specific activity in the apple cultivar 'Golden Delicious'. The flower-specific promoter rMdAG2I, derived from M. domestica, thus has great potential for use in improving the floral characteristics of ornamental plants, especially the Malus species.

Persistent negative temperature response of mesophyll conductance in red raspberry (Rubus idaeus L.) leaves under both high and low vapour pressure deficits: a role for abscisic acid?

The temperature dependence of mesophyll conductance (gm ) was measured in well-watered red raspberry (Rubus idaeus L.) plants acclimated to leaf-to-air vapour pressure deficit (VPDL) daytime differentials of contrasting amplitude, keeping a fixed diurnal leaf temperature (Tleaf ) rise from 20 to 35 °C. Contrary to the great majority of gm temperature responses published to date, we found a pronounced reduction of gm with increasing Tleaf irrespective of leaf chamber O2 level and diurnal VPDL regime. Leaf hydraulic conductance was greatly enhanced during the warmer afternoon periods under both low (0.75 to 1.5 kPa) and high (0.75 to 3.5 kPa) diurnal VPDL regimes, unlike stomatal conductance (gs ), which decreased in the afternoon. Consequently, the leaf water status remained largely isohydric throughout the day, and therefore cannot be evoked to explain the diurnal decrease of gm . However, the concerted diurnal reductions of gm and gs were well correlated with increases in leaf abscisic acid (ABA) content, thus suggesting that ABA can induce a significant depression of gm under favourable leaf water status. Our results challenge the view that the temperature dependence of gm can be explained solely from dynamic leaf anatomical adjustments and/or from the known thermodynamic properties of aqueous solutions and lipid membranes.​.

Dynamics of phytohormone and DNA methylation patterns changes during dormancy induction in strawberry (Fragaria × ananassa Duch.).

Changes in endogenous phytohormone levels, DNA methylation patterns, and expression levels of related genes during induction of dormancy in two strawberry cultivars, Darselect and All Star, were studied under controlled environmental conditions. At 12°C, regardless of day length, potted, runner-derived plants of both cultivars gradually exhibited morphological traits typical of dormancy after treatment for 8 weeks. These morphological changes were accompanied by a synchronous significant decline in indole-3-acetic acid (IAA) level and increases in abscisic acid (ABA) content and global genomic DNA methylation in young leaves. Exposed at 15°C and a short-day photoperiod, the changes in morphology, phytohormone levels and DNA methylation of both cultivars were similar to those observed at 12°C. Slight but non-significant changes in IAA and ABA levels and genomic DNA methylation occurred in young leaves at both 15°C with long days and 18°C with short days. These results indicated that temperature alone was sufficient to induce strawberry to enter the typical dormant phase, and day length had no impact at 12°C. The higher temperature permissible for dormancy induction in strawberry was 15°C, but at this temperature dormancy induction was modified by day length. The expression patterns of FaPIN1, FaNCED1, FaDRM and FaROS1 were coincident with the changes in phytohormone levels and DNA methylation. Although the two tested cultivars have different temporal responses with the different degree of cold tolerance and depth of dormancy, both the endogenous phytohormone and DNA methylation were changed when induced by external environmental factors.

Comparison of cadmium-induced iron-deficiency responses and genuine iron-deficiency responses in Malus xiaojinensis.

The effects of the heavy metal Cd in Malus xiaojinensis were investigated using hydroponic cultures. Chlorophyll and Fe concentrations in young leaves were markedly decreased by Cd treatment, although Fe concentration was significantly enhanced in the roots. A comparative examination of the Fe-deficiency responses due to Fe deficiency and Cd treatment was also performed. Both Fe deficiency and Cd treatment induced responses similar to those of Fe-deficiency in M. xiaojinensis, including acidification of the rhizosphere, enhanced Fe(III) chelate reductase activity, and upregulation of the Fe-deficiency-responsive genes MxIRT1 and MxFRO2-Like. However, the Fe-deficiency responses induced by Cd treatment were different in intensity and timing from those induced by Fe deficiency.