In our study, the effects of water stress on photosynthesis and photosynthetic electron transport chain (PETC) were studied in several ways, including monitoring the change of gas exchange parameters, modulated chlorophyll fluorescence, rapid fluorescence induction kinetics, reactive oxygen species (ROS), antioxidant enzyme activities and D1 protein levels in apple leaves. Our results show that when leaf water potential (ψ_w) is above –1.5 MPa, the stomatal limitation should be the main reason for a drop of photosynthesis. In this period, photosynthetic rate (P_N), stomatal conductance (G_s), transpiration rate (E) and intercellular CO₂ concentration (C_i) all showed a strong positive correlation with ψ_w. Modulated chlorophyll fluorescence parameters related to photosynthetic biochemistry activity including maximum photochemical efficiency (F_v/F_m), actual photochemical efficiency of PSII (Φ_PSII), photochemical quenching coefficient (q_P) and coefficient of photochemical fluorescence quenching assuming interconnected PSII antennae (q_L) also showed a strong positive correlation as ψ_w gradually decreased. On the other hand, in this period, Stern-Volmer type non-photochemical quenching coefficient (NPQ) and quantum yield of light-induced non-photochemical fluorescence quenching [Y_(NPQ)] kept going up, which shows an attempt to dissipate excess energy to avoid damage to plants. When ψ_w was below –1.5 MPa, P_N continued to decrease linearly, while C_i increased and a ‘V’ model presents the correlation between C_i and ψ_w by polynomial regression. This implies that, in this period, the drop in photosynthesis activity might be caused by non-stomatal limitation. F_v/F_m, Φ_PSII, q_P and q_L in apple leaves treated with water stress were much lower than in control, while NPQ and Y_(NPQ) started to go down. This demonstrates that excess energy might exceed the tolerance ability of apple leaves. Consistent with changes of these parameters, excess energy led to an increase in the production of ROS including H₂O₂ and O₂•⁻. Although the activities of antioxidant enzymes like catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) increased dramatically and ascorbate peroxidase (APX) decreased in apple leaves with drought stress, it was still not sufficient to scavenge ROS. Consequently, the accumulation of ROS triggered a reduction of net D1 protein content, a core protein in the PSII reaction center. As D1 is responsible for the photosynthetic electron transport from plastoquinone A (Q_A) to plastoquinone B (Q_B), the capacity of PETC between Q_A and Q_B was considerably downregulated. The decline of photosynthesis and activity of PETC may result in the shortage of adenosine triphosphate (ATP) and limitation the regeneration of RuBP (J_max), a key enzyme in CO₂ assimilation. These are all non-stomatal factors and together contributed to decreased CO₂ assimilation under severe water stress.

Transcription factors (TFs) play essential roles in the regulatory networks controlling many developmental processes in plants. Members of the basic leucine (Leu) zipper (bZIP) TF family, which is unique to eukaryotes, are involved in regulating diverse processes, including flower and vascular development, seed maturation, stress signaling and defense responses to pathogens. The bZIP proteins have a characteristic bZIP domain composed of a DNA-binding basic region and a Leu zipper dimerization region. In this study, we identified 112 apple (Malus domestica Borkh) bZIP TF-encoding genes, termed MdbZIP genes. Synteny analysis indicated that segmental and tandem duplication events, as well as whole genome duplication, have contributed to the expansion of the apple bZIP family. The family could be divided into 11 groups based on structural features of the encoded proteins, as well as on the phylogenetic relationship of the apple bZIP proteins to those of the model plant Arabidopsis thaliana (AtbZIP genes). Synteny analysis revealed that several paired MdbZIP genes and AtbZIP gene homologs were located in syntenic genomic regions. Furthermore, expression analyses of group A MdbZIP genes showed distinct expression levels in ten different organs. Moreover, changes in these expression profiles in response to abiotic stress conditions and various hormone treatments identified MdbZIP genes that were responsive to high salinity and drought, as well as to different phytohormones.

Basic helix-loop-helix (bHLH) proteins, which are characterized by a conserved bHLH domain, comprise one of the largest families of transcription factors in both plants and animals, and have been shown to have a wide range of biological functions. However, there have been very few studies of bHLH proteins from perennial tree species. We describe here the identification and characterization of 175 bHLH transcription factors from apple (Malus × domestica). Phylogenetic analysis of apple bHLH (MdbHLH) genes and their Arabidopsis thaliana (Arabidopsis) orthologs indicated that they can be classified into 23 subgroups. Moreover, integrated synteny analysis suggested that the large-scale expansion of the bHLH transcription factor family occurred before the divergence of apple and Arabidopsis. An analysis of the exon/intron structure and protein domains was conducted to suggest their functional roles. Finally, we observed that MdbHLH subgroup III and IV genes displayed diverse expression profiles in various organs, as well as in response to abiotic stresses and various hormone treatments. Taken together, these data provide new information regarding the composition and diversity of the apple bHLH transcription factor family that will provide a platform for future targeted functional characterization.

Apple (Malus × domestica Borkh.) is one of the most important cultivated tree fruit crops worldwide. However, sustainable apple production is threatened by powdery mildew (PM) disease, which is caused by the obligate biotrophic fungus Podosphaera leucotricha. To gain insight into the molecular basis of the PM infection and disease progression, RNA-based transcriptional profiling (RNA-seq) was used to identify differentially expressed genes (DEGs) in apples following inoculation with P. leucotricha. Four RNA-seq libraries were constructed comprising a total of 214 Gb of high-quality sequence. 1177 DEGs (661 upregulated and 629 downregulated) have been identified according to the criteria of a ratio of infection/control fold change > 2, and a false discovery rate (FDR) < 0.001. The majority of DEGs (815) were detected 12 h after inoculation, suggesting that this is an important time point in the response of the PM infection. Gene annotation analysis revealed that DEGs were predominately associated with biological processes, phenylpropanoid biosynthesis, hormone signal transduction and plant-pathogen interactions. Genes activated by infection corresponded to transcription factors (e.g., AP2/ERF, MYB, WRKY and NAC) and synthesis of defense-related metabolites, including pathogenesis-related genes, glucosidase and dehydrin. Overall, the information obtained in this study enriches the resources available for research into the molecular-genetic mechanisms of the apple/powdery mildew interactions, and provides a theoretical basis for the development of new apple varieties with resistance to PM.

Polyploidization results in genome duplication and is an important step in evolution and speciation. The Malus genome confirmed that this genus was derived through auto-polyploidization, yet the genetic and meiotic mechanisms for polyploidization, particularly for aneuploidization, are unclear in this genus or other woody perennials. In fact the contribution of aneuploidization remains poorly understood throughout Plantae. We add to this knowledge by characterization of eupolyploidization and aneuploidization in 27,542 F₁ seedlings from seven diploid Malus populations using cytology and microsatellite markers. We provide the first evidence that aneuploidy exceeds eupolyploidy in the diploid crosses, suggesting aneuploidization is a leading cause of genome duplication. Gametes from diploid Malus had a unique combinational pattern; ova preserved euploidy exclusively, while spermatozoa presented both euploidy and aneuploidy. All non-reduced gametes were genetically heterozygous, indicating first-division restitution was the exclusive mode for Malus eupolyploidization and aneuploidization. Chromosome segregation pattern among aneuploids was non-uniform, however, certain chromosomes were associated for aneuploidization. This study is the first to provide molecular evidence for the contribution of heterozygous non-reduced gametes to fitness in polyploids and aneuploids. Aneuploidization can increase, while eupolyploidization may decrease genetic diversity in their newly established populations. Auto-triploidization is important for speciation in the extant Malus. The features of Malus polyploidization confer genetic stability and diversity, and present heterozygosity, heterosis and adaptability for evolutionary selection. A protocol using co-dominant markers was proposed for accelerating apple triploid breeding program. A path was postulated for evolution of numerically odd basic chromosomes. The model for Malus derivation was considerably revised. Impacts of aneuploidization on speciation and evolution, and potential applications of aneuploids and polyploids in breeding and genetics for other species were evaluated in depth. This study greatly improves our understanding of evolution, speciation, and adaptation of the Malus genus, and provides strategies to exploit polyploidization in other species.

Background: B-box (BBX) zinc-finger transcription factors play important roles in plant growth, development, and stress response. Although these proteins have been studied in model plants such as Arabidopsis thaliana or Oryza sativa, little is known about the evolutionary history or expression patterns of BBX proteins in grapevine (Vitis vinifera L.). Results: We identified a total of 25 VviBBX genes in the grapevine genome and named them according to the homology with Arabidopsis. These proteins were classified into five groups on the basis of their phylogenetic relationships, number of B-box domains, and presence or absence of a CCT domain or VP motif. BBX proteins within the same group showed similar exon-intron structures and were unevenly distributed in grapevine chromosomes. Synteny analyses suggested that only segmental duplication events contributed to the expansion of the VviBBX gene family in grapevine. The observed syntenic relationships between some BBX genes from grapevine and Arabidopsis suggest that they evolved from a common ancestor. Transcriptional analyses showed that the grapevine BBX genes were regulated distinctly in response to powdery mildew infection and various phytohormones. Moreover, the expression levels of a subset of BBX genes in ovules were much higher in seedless grapevine cultivars compared with seeded cultivars during ovule development, implying a potential role in seed abortion. Additionally, VviBBX8, VquBBX15a and VquBBX29b were all located in the nucleus and had transcriptional activity except for VquBBX29b. Conclusions: The results of this study establish the genome-wide analysis of the grapevine BBX family and provide a framework for understanding the biological roles of BBX genes in grapevine.