Transcription factors MhDREB2A/MhZAT10 play a role in drought and cold stress response crosstalk in apple.

Drought and cold stresses seriously affect tree growth and fruit yield during apple (Malus domestica) production, with combined stress causing injury such as shoot shriveling. However, the molecular mechanism underlying crosstalk between responses to drought and cold stress remains to be clarified. In this study, we characterized the zinc finger transcription factor ZINC FINGER OF ARABIDOPSIS THALIANA 10 (ZAT10) through comparative analysis of shoot-shriveling tolerance between tolerant and sensitive apple rootstocks. MhZAT10 responded to both drought and cold stresses. Heterologous expression of MhZAT10 in the sensitive rootstock 'G935' from domesticated apple (Malus domestica) promoted shoot-shriveling tolerance, while silencing of MhZAT10 expression in the tolerant rootstock 'SH6' of Malus honanensis reduced stress tolerance. We determined that the apple transcription factor DEHYDRATION RESPONSE ELEMENT-BINDING PROTEIN 2A (DREB2A) is a direct regulator activating the expression of MhZAT10 in response to drought stress. Apple plants overexpressing both MhDREB2A and MhZAT10 genes exhibited enhanced tolerance to drought and cold stress, while plants overexpressing MhDREB2A but with silenced expression of MhZAT10 showed reduced tolerance, suggesting a critical role of MhDREB2A-MhZAT10 in the crosstalk between drought and cold stress responses. We further identified drought-tolerant MhWRKY31 and cold-tolerant MhMYB88 and MhMYB124 as downstream regulatory target genes of MhZAT10. Our findings reveal a MhDREB2A-MhZAT10 module involved in crosstalk between drought and cold stress responses, which may have applications in apple rootstock breeding programs aimed at developing shoot-shriveling tolerance.

Methylome and transcriptome analyses of three different degrees of albinism in apple seedlings.

BACKGROUND: Leaf colour mutations are universally expressed at the seedling stage and are ideal materials for exploring the chlorophyll biosynthesis pathway, carotenoid metabolism and the flavonoid biosynthesis pathway in plants. RESULTS: In this research, we analysed the different degrees of albinism in apple (Malus domestica) seedlings, including white-leaf mutants (WM), piebald leaf mutants (PM), light-green leaf mutants (LM) and normal leaves (NL) using bisulfite sequencing (BS-seq) and RNA sequencing (RNA-seq). There were 61,755, 79,824, and 74,899 differentially methylated regions (DMRs) and 7566, 3660, and 3546 differentially expressed genes (DEGs) identified in the WM/NL, PM/NL and LM/NL comparisons, respectively. CONCLUSION: The analysis of the methylome and transcriptome showed that 9 DMR-associated DEGs were involved in the carotenoid metabolism and flavonoid biosynthesis pathway. The expression of different transcription factors (TFs) may also influence the chlorophyll biosynthesis pathway, carotenoid metabolism and the flavonoid biosynthesis pathway in apple leaf mutants. This study provides a new method for understanding the differences in the formation of apple seedlings with different degrees of albinism.

The effect of auxin and strigolactone on ATP/ADP isopentenyltransferase expression and the regulation of apical dominance in peach.

KEY MESSAGE: We confirmed the roles of auxin, CK, and strigolactones in apical dominance in peach and established a model of plant hormonal control of apical dominance in peach. Auxin, cytokinin, and strigolactone play important roles in apical dominance. In this study, we analyzed the effect of auxin and strigolactone on the expression of ATP/ADP isopentenyltransferase (IPT) genes (key cytokinin biosynthesis genes) and the regulation of apical dominance in peach. After decapitation, the expression levels of PpIPT1, PpIPT3, and PpIPT5a in nodal stems sharply increased. This observation is consistent with the changes in tZ-type and iP-type cytokinin levels in nodal stems and axillary buds observed after treatment; these changes are required to promote the outgrowth of axillary buds in peach. These results suggest that ATP/ADP PpIPT genes in nodal stems are key genes for cytokinin biosynthesis, as they promote the outgrowth of axillary buds. We also found that auxin and strigolactone inhibited the outgrowth of axillary buds. After decapitation, IAA treatment inhibited the expression of ATP/ADP PpIPTs in nodal stems to impede the increase in cytokinin levels. By contrast, after GR24 (GR24 strigolactone) treatment, the expression of ATP/ADP IPT genes and cytokinin levels still increased markedly, but the rate of increase in gene expression was markedly lower than that observed after decapitation in the absence of IAA (indole-3-acetic acid) treatment. In addition, GR24 inhibited basipetal auxin transport at the nodes (by limiting the expression of PpPIN1a in nodal stems), thereby inhibiting ATP/ADP PpIPT expression in nodal stems. Therefore, strigolactone inhibits the outgrowth of axillary buds in peach only when terminal buds are present.

The transcription factor MhZAT10 enhances antioxidant capacity by directly activating the antioxidant genes MhMSD1, MhAPX3a and MhCAT1 in apple rootstock SH6 (Malus honanensis × M. domestica).

Stress tolerance in apple (Malus domestica) can be improved by grafting to a stress-tolerant rootstock, such as 'SH6' (Malus honanensis × M. domestica 'Ralls Genet'). However, the mechanisms of stress tolerance in this rootstock are unclear. In Arabidopsis (Arabidopsis thaliana), the transcription factor ZINC FINGER OF ARABIDOPSIS THALIANA 10 is a key component of plant tolerance to multiple abiotic stresses and positively regulates antioxidant enzymes. However, how reactive oxygen species are eliminated upon activation of ZINC FINGER OF ARABIDOPSIS THALIANA 10 in response to abiotic stress remains elusive. Here, we report that MhZAT10 in the rootstock SH6 directly activates the transcription of three genes encoding the antioxidant enzymes MANGANESE SUPEROXIDE DISMUTASE 1 (MhMSD1), ASCORBATE PEROXIDASE 3A (MhAPX3a) and CATALASE 1 (MhCAT1) by binding to their promoters. Heterologous expression in Arabidopsis protoplasts showed that MhMSD1, MhAPX3a and MhCAT1 localize in multiple subcellular compartments. Overexpressing MhMSD1, MhAPX3a or MhCAT1 in SH6 fruit calli resulted in higher superoxide dismutase, ascorbate peroxidase and catalase enzyme activities in their respective overexpressing calli than in those overexpressing MhZAT10. Notably, the calli overexpressing MhZAT10 exhibited better growth and lower reactive oxygen species levels under simulated osmotic stress. Apple SH6 plants overexpressing MhZAT10 in their roots via Agrobacterium rhizogenes-mediated transformation also showed enhanced tolerance to osmotic stress, with higher leaf photosynthetic capacity, relative water content in roots and antioxidant enzyme activity, as well as less reactive oxygen species accumulation. Overall, our study demonstrates that the transcription factor MhZAT10 synergistically regulates the transcription of multiple antioxidant-related genes and elevates reactive oxygen species detoxification.

Transcriptional and metabolic responses of apple to different potassium environments.

Potassium (K) is one of the most important macronutrients for plant development and growth. The influence mechanism of different potassium stresses on the molecular regulation and metabolites of apple remains largely unknown. In this research, physiological, transcriptome, and metabolite analyses were compared under different K conditions in apple seedlings. The results showed that K deficiency and excess conditions influenced apple phenotypic characteristics, soil plant analytical development (SPAD) values, and photosynthesis. Hydrogen peroxide (H2O2) content, peroxidase (POD) activity, catalase (CAT) activity, abscisic acid (ABA) content, and indoleacetic acid (IAA) content were regulated by different K stresses. Transcriptome analysis indicated that there were 2,409 and 778 differentially expressed genes (DEGs) in apple leaves and roots under K deficiency conditions in addition to 1,393 and 1,205 DEGs in apple leaves and roots under potassium excess conditions, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment showed that the DEGs were involved in flavonoid biosynthesis, photosynthesis, and plant hormone signal transduction metabolite biosynthetic processes in response to different K conditions. There were 527 and 166 differential metabolites (DMAs) in leaves and roots under low-K stress as well as 228 and 150 DMAs in apple leaves and roots under high-K stress, respectively. Apple plants regulate carbon metabolism and the flavonoid pathway to respond to low-K and high-K stresses. This study provides a basis for understanding the metabolic processes underlying different K responses and provides a foundation to improve the utilization efficiency of K in apples.

Nano-Sized Antioxidative Trimetallic Complex Based on Maillard Reaction Improves the Mineral Nutrients of Apple (Malus domestica Borkh.).

The metallic complex is widely used in agricultural applications. Due to the oxidation of the metal and environmental unfriendliness of ligand, maintaining an efficient mineral supply for plants without causing environmental damage is difficult. Herein, an antioxidative trimetallic complex with high stability was synthesized by interacting Ca2+, Fe2+, and Zn2+ with the biocompatible ligands from the Maillard reaction. The composite structure elucidation was carried out by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared (FTIR). Thermal stability was measured by thermogravimetric (TG). Antioxidative activities were evaluated by ferric reducing antioxidant power and radical scavenging activity assays. The three metals were successfully fabricated on the Maillard reaction products (MRPs) with contents of Ca (9.01%), Fe (8.25%), and Zn (9.67%). Microscopy images revealed that the three metals were uniformly distributed on the MRPs with partial aggregation of <30 nm. FTIR and XPS results revealed that the metals were interacted with MRPs by metal-O and metal-N bonds. TG and antioxidative activity assays showed that the trimetallic complex meets the requirements of thermodynamics and oxidation resistance of horticultural applications. Additionally, the results of the exogenous spraying experiment showed that the trimetallic complex significantly increased the mineral contents of the "Fuji" apple. By treatment with the complex, the concentrations of Ca, Fe, and Zn were increased by 85.4, 532.5, and 931.1% in the leaf; 16.0, 225.2, and 468.6% in the peel; and 117.6, 217.9, and 19.5% in the flesh, respectively. The MRP-based complexes offered a higher growth rate of the mineral content in apples than ones based on sugars or amino acids. The results of the spraying experiment carried out in 2 years show that the method has high reproducibility. This study thus promotes the development of green metallic complexes and expands the scope of agrochemical strategy.

Exogenous application of acetic acid enhances drought tolerance by influencing the MAPK signaling pathway induced by ABA and JA in apple plants.

The external application of acetic acid (AA) has been shown to improve drought survival in plants, such as Arabidopsis, rice, maize, wheat, rapeseed and cassava, and the application of AA also increased drought tolerance in perennial woody apple (Malus domestica) plants. An understanding of AA-induced drought tolerance in apple plants at the molecular level will contribute to the development of technology that can be used to enhance drought tolerance. In this study, the morphological, physiological and transcriptomic responses to drought stress were analyzed in apple plants after watering without AA (CK), watering with AA (AA), drought treatment (D) and drought treatment with AA (DA). The results suggested that the AA-treated apple plants had a higher tolerance to drought than water-treated plants. Higher levels of chlorophyll and carotenoids were found under the DA conditions than under D stress. The levels of abscisic acid (ABA), jasmonic acid (JA) and methyl jasmonate were increased in AA-treated apple plants. Transcriptomic profiling indicated the key biological pathways involved in metabolic processes, mitogen-activated protein kinase (MAPK) signaling, plant hormone signal transduction and the biosynthesis of secondary metabolites in response to different drought conditions. The 9-cis-epoxycarotenoid dioxygenase, (9S,13S)-cis-oxophytodienoic acid reductase, allene oxide synthase, allene oxide cyclase and lipoxygenase genes participate in the synthase of ABA and JA under drought and AA treatments. Collectively, the results showed that external application of AA enhanced drought tolerance in apple plants by influencing the ABA- and JA-induced MAPK signaling pathways. These data indicated that the application of AA in plants is beneficial for enhancing drought tolerance and decreasing growth inhibition in agricultural fields.

Transcriptome and metabolome analyses revealed the response mechanism of apple to different phosphorus stresses.

Phosphorus (P) is an important element in numerous metabolic reactions and signalling pathways, but the molecular details of these pathways remain largely unknown. In this study, physiological, transcriptome and metabolite analyses of apple leaves and roots were compared under different P conditions. The results showed that different P stresses influenced phenotypic characteristics, soil plant analytical development (SPAD) values and the contents of flavonoids and anthocyanins in apple seedlings. The contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA) and the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), acid phosphatase (ACP) and purple acid phosphatase (PAP) were also affected by different P stresses. In addition, RNA sequencing (RNA-seq) was used to characterize the influence of different P stresses on apple seedlings. Compared with control apple plants, there were 1246 and 1183 differentially expressed genes (DEGs) in leaves and roots under the low-P treatment and 60 and 1030 DEGs in leaves and roots under the high-P treatment, respectively. Gene Ontology (GO) analysis indicated that apple trees might change their responses to metabolic processes, cell proliferation, regulation of biological processes, reactive oxygen species metabolic processes and flavonoid metabolic processes under P stress. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis further indicated that DEGs act on the mitogen-activated protein kinase (MAPK) signalling pathway, flavonoid biosynthesis, phenylpropanoid biosynthesis, and ATP-binding cassette (ABC) transporters. The metabolome analysis revealed that the levels of most amino acids and their derivatives, organic acids and flavonoids in roots treated with low-P stress were higher than those in roots of apple seedlings under control growth conditions. Apple seedlings regulate the flavonoid pathway to respond to different phosphorus environments. The results provide a framework for understanding the metabolic processes underlying different P responses and provide a foundation for improving the utilization efficiency of P in apple trees.

Transcriptome analysis provides insights into the stress response crosstalk in apple (Malus × domestica) subjected to drought, cold and high salinity.

Drought, cold, and high salinity are three major abiotic stresses effecting apple tree growth and fruit production. Understanding the genetic mechanisms of crosstalk between stress responses signalling networks and identifying the genes involved in apple has potential importance for crop improvement and breeding strategies. Here, the transcriptome profiling analysis of in vitro-grown apple plants subjected to drought, cold and high salinity stress, showed a total of 377 upregulated and 211 downregulated common differentially expressed genes (DEGs) to all 3 stress treatments compared with the control. Gene Ontology (GO) analysis indicated that these common DEGs were enriched in 'metabolic process' under the 'biological process' category, as well as in 'binding' and 'catalytic activity' under the 'molecular function' category. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that common DEGs were mainly belong to the 'biological functions' category and 17 DEGs were identified in 'environmental information processing' sub-category which may act as signal transduction components in response crosstalk regulation. Overexpression of 5 upregulated genes individually, out of these 17 common DEGs in apple calli promoted the consistent upregulation of DREB6, CBF1 and ZAT10 and increased the mass weight and antioxidase ability, implying these five common DEGs involved in multiple pathways and improved comprehensive resistance to stress.

Illumina amplicon sequencing of 16S rRNA tag reveals bacterial community development in the rhizosphere of apple nurseries at a replant disease site and a new planting site.

We used a next-generation, Illumina-based sequencing approach to characterize the bacterial community development of apple rhizosphere soil in a replant site (RePlant) and a new planting site (NewPlant) in Beijing. Dwarfing apple nurseries of 'Fuji'/SH6/Pingyitiancha trees were planted in the spring of 2013. Before planting, soil from the apple rhizosphere of the replant site (ReSoil) and from the new planting site (NewSoil) was sampled for analysis on the Illumina MiSeq platform. In late September, the rhizosphere soil from both sites was resampled (RePlant and NewPlant). More than 16,000 valid reads were obtained for each replicate, and the community was composed of five dominant groups (Proteobacteria, Acidobacteria, Bacteroidetes, Gemmatimonadetes and Actinobacteria). The bacterial diversity decreased after apple planting. Principal component analyses revealed that the rhizosphere samples were significantly different among treatments. Apple nursery planting showed a large impact on the soil bacterial community, and the community development was significantly different between the replanted and newly planted soils. Verrucomicrobia were less abundant in RePlant soil, while Pseudomonas and Lysobacter were increased in RePlant compared with ReSoil and NewPlant. Both RePlant and ReSoil showed relatively higher invertase and cellulase activities than NewPlant and NewSoil, but only NewPlant soil showed higher urease activity, and this soil also had the higher plant growth. Our experimental results suggest that planting apple nurseries has a significant impact on soil bacterial community development at both replant and new planting sites, and planting on new site resulted in significantly higher soil urease activity and a different bacterial community composition.

Manure refinement affects apple rhizosphere bacterial community structure: a study in sandy soil.

We used DNA-based pyrosequencing to characterize the bacterial community structure of the sandy soil of an apple orchard with different manure ratios. Five manure percentages (5%, 10%, 15%, 20% and 25%) were examined. More than 10,000 valid reads were obtained for each replicate. The communities were composed of five dominant groups (Proteobacteria, Actinobacteria, Chloroflexi, Acidobacteria and Bacteroidetes), of which Proteobacteria content gradually decreased from 41.38% to 37.29% as manure ratio increased from 0% to 25%, respectively. Redundancy analysis showed that 37 classes were highly correlated with manure ratio, 18 of which were positively correlated. Clustering revealed that the rhizosphere samples were grouped into three components: low manure (control, 5%) treatment, medium manure (10%, 15%) treatment and high manure (20%, 25%) treatment. Venn analysis of species types of these three groups revealed that the bacteria community difference was primarily reflected by quantity ratio rather than species variety. Although greater manure content led to higher soil organic matter content, the medium manure improved soil showed the highest urease activity and saccharase activity, while 5% to 20% manure ratio improvement also resulted in higher bacteria diversity than control and 25% manure ratio treatment. Our experimental results suggest that the use of a proper manure ratio results in significantly higher soil enzyme activity and different bacteria community patterns, whereas the use of excessive manure amounts has negative effect on soil quality.

[Effects of debagging time and picking off leaf on fruit quality of "Red Fuji" apple].

This paper studied the effects of debagging time and picking off leaves around fruits on the fruit quality and 1-year old shoots stored nutrition of "Red Fuji" apple. Properly advancing debagging time significantly increased the mean mass, anthocyanin content, and coloration area of the fruits (P < 0.05). Comparing with those of the fruits debagged on September 30, the mean mass, anthocyanin content, and coloration area of the fruits debagged on September 24 increased by 10.98%, 28.15%, and 13.44%, respectively, and the fruit firmness, soluble solid content, and titratable acidity had less differences. When the leaves within 15, 30, and 45 cm away from the fruits were wholly picked off, the light environment around the fruits improved significantly (P < 0.01), with the light intensity increased by 70%, 95%, and 115%, respectively, compared with no leaf picked off treatment. Picking off the leaves within 30-45 cm away from the fruits benefited the fruit coloration and anthocyanin accumulation. No significant effects were observed on the 1-year old shoots stored nutrition and their budding in next year among the treatments of picking off leaves. Advancing debagging time and picking off the leaves within 30-45 cm away from the fruits could improve fruit appearance and quality.

[Effects of different pruning modes on the light distribution characters and fruit yield and quality in densely planted 'Red Fuji' apple orchard].

This paper studied the effects of different pruning modes on the light distribution at different positions of canopy and the fruit yield and quality in different layers in a densely planted 'Red Fuji' apple orchard. Comparing with no pruning, both light and heavy pruning improved the light distribution in the canopy. Under light pruning, the canopy volume with a relative light intensity less than 30% occupied 14. 6% of the total, and that with a relative light intensity more than 80% accounted for 11.2%. Under heavy pruning, the two values were 12.8% and 28.8%, respectively. The fruit yield under light pruning increased in the first year, but that under heavy pruning decreased, with the fruit quality being both improved. The mean fruit mass, firmness, and soluble solid matter content decreased in the order of light pruning > heavy pruning > no pruning, the titratable acid content was in adverse, and the anthocyanin content was in the order of heavy pruning > light pruning > no pruning. From the viewpoint of fruit yield and quality, light pruning was the best modification mode for densely planted orchards.

[Leaf photosynthetic potential in canopy layers of un-thinned and thinned apple orchards].

Through the comparison of leaf photosynthetic potential and of photosynthetically active radiation (PAR), leaf nitrogen concentration (Nl), and mass per unit leaf area (Ml) in different canopy layers of un-thinned orchard (UOD) and thinned orchard (TOD), this paper studied the effects of UOD rebuilding on the use efficiencies of PAR and nitrogen, and their relationships to the fruit yield and quality. Thinning obviously improved the radiation environment in canopy. The radiation distribution in TOD canopy was more uniformly than that in UOD canopy, and the invalid space with relative PAR (PARr) less than 30% in TOD approached to zero, while the minimum mean PARr in UOD was 17%, and the space under 0. 3 of relative canopy height was invalid. The leaf photosynthetic efficiency in TOD was notably improved. Comparing with that in UOD, the photsynthetic rate (Pn) at the middle and bottom of the canopy in TOD was increased by 7.8% and 10.2%, respectively. Meanwhile, the photosynthetic potential parameters such as maximum carboxylation rate (Vmax) and maximum electron transfer rate (Jmax) also increased remarkably in TOD. The leaf photosynthetic potential had significant correlation with Nl, and the Nl was strongly correlated with PARr. As a result, leaf photosynthetic potential and PARr could be estimated according to the spatial distribution of relative leaf nitrogen concentration (Nlr).

[Effects of alternative partial rootzone irrigation on peach growth, productivity, and water use efficiency].

Taking 12-year-old peach (Prunus persica L. Batsch cv. Ruiguang 5) as test material, this paper studied the effects of alternative partial rootzone irrigation (APRI) on its growth, productivity, and water use efficiency under semi-arid climate condition. The results showed that in APRI treatments, the soil water content in the wet side of peach tree decreased with increasing soil depth, while that in the dry side was in adverse, with the maximal difference appeared in 0-25 cm soil layer. In the treatments of APRI with an interval of 2 and 4 weeks, the leaf water potential at pre-dawn was lower than that in the control (sufficient irrigation), but with the time prolonged, this potential in all treatments tended to decrease, and had no significant difference in the afternoon. The peach yield in APRI treatments was 10% lower than that in the control, but the irrigation amount was reduced by 50%, and the water use efficiency was increased by 75%. APRI significantly limited the shoot growth of peach tree, but had no obvious effect on the fruit diameter.

[Optimum meteorological factors and climate divisions of apple for good quality].

The investigation and laboratory analysis were carried out for apple quality of Fuji, Jonagold and Starkrimson grown in various ecological and climate regions of China. Multiple statistical analysis, linear program and computer technology were used to obtain the optimum values and scheme of major meteorological factors affecting the quality components of different apple varieties. The cluster analysis results showed that it was suitable for apple of Fuji, Jonagold and Starkrimson growing in the Loess Plateau and Longdong, Gansu Province. Bohaiwan bay regions were suitable for apple of Fuji and Jonagold and Southwestern mountainous region were suitable for apple Starkrimson. Advantage qualifications and limiting factors in each climate divisions were also pointed out.

[Digital modeling of light distribution within the crop canopy in jujube-wheat interplanting system].

On the basis of field observation and with the principles and methods of agricultural meteorology, the daily variations of the shadow width of jujube tree belts and the relative light intensity were analyzed in the jujube-wheat interplanting system, and the characteristics of vertical distribution of the light within the wheat canopy inside and outside the shadow were studied. The sunshine vertical distribution curves in the wheat canopy inside and outside the shadow were simulated by mathematical methods. The results showed that the shadow area of the south-north planting jujube tree belts was fairly wider, but the relative light illuminance was larger in early morning and late afternoon than at about noon. Although the sunshine vertical distribution in the wheat canopy inside and outside the shadow was obviously different, they both observed the decreasing law for Gauss Curve.