Screening Candidate Genes at the Co Locus Conferring to the Columnar Growth Habit in Apple (Malus × Domestica Borkh.).

The columnar growth trait of apple (Malus × domestica Borkh.) is genetically controlled by the Columnar (Co) locus on 10 chromosomes, including several candidate genes. Except for MdCo31, other candidate genes at the Co locus are less elucidated. In this study, a strategy of step-by-step screening was adopted to select 11 candidate genes by experimental cloning, transient expression, and genetic transformation. There existed several SNPs in four genes by sequence alignment in columnar and non-columnar apples. Two genes were detected in the nucleus and three genes in the cell membrane, other genes were located in multiple cellular structures by subcellular location. Ectopic expression demonstrated that more branching occurred in MdCo38-OE by upregulating NtPIN1 and NtGA2ox and enlarged leaves in MdCo41-OE tobaccos by upregulating NtCCDs. Transcripts of MdCo38 and MdCo41 were associated with the Co genotypes in apples. The results indicate that MdCo38 and MdCo41 are involved in the columnar growth phenotype in apple, probably through altering polar auxin transport, active gibberellin levels, and strigolactone biosynthesis.

MdCo31 interacts with an RNA polymerase II transcription subunit 32 to regulate dwarf growth with short internodes in columnar apple.

The dominant Co locus controls the columnar growth phenotype of apple (Malus × domestica) trees. Candidate gene MdCo31, encoding 2-oxoglutarate-dependent dioxygenase, causes dwarf growth with short internodes in transgenic plants by reducing the abundance of biologically active gibberellin. However, the pathway regulating MdCo31 in the dwarfism of apple trees remains unclear. In this study, expression of MdCo31 was proved to be negatively correlated with internode length in F1 populations created by crossing columnar parents, and with dwarfism in transgenic apple plantlets. Yeast (Saccharomyces cerevisiae) two-hybrid screening identified the RNA polymerase II transcription subunit MdMED32 as putative interactor of MdCo31. Bimolecular fluorescence complementation, co-immunoprecipitation, and dual-luciferase reporter assays confirmed this interaction both in vivo and in vitro. Ectopic expression of MdMED32 in Nicotiana tabacum led to a dwarf phenotype, similar to that of MdCo31 transgenic apple plants. Expression of GA2ox1 and GA20ox1, encoding key enzymes of gibberellin metabolism, was upregulated in transgenic plants. Transient transcriptional activity demonstrated that MdMED32 functioned as an activator, promoting expression of MdGA2ox1 and MdGA20ox1. These findings indicate that the interaction between MdCo31 and MdMED32 functions in the regulation of internode length in columnar apple.

Functional Characterization of MdTAC1a Gene Related to Branch Angle in Apple (Malus x domestica Borkh.).

The Tiller Angle Control 1 (TAC1) gene belongs to the IGT family, which mainly controls plant branch angle, thereby affecting plant form. Two members of MdTAC1 are identified in apple; the regulation of apple branch angle by MdTAC1 is still unclear. In this study, a subcellular localization analysis detected MdTAC1a in the nucleus and cell membrane, but MdTAC1b was detected in the cell membrane. Transgenic tobacco by overexpression of MdTAC1a or MdTAC1b showed enlarged leaf angles, the upregulation of several genes, such as GA 2-oxidase (GA2ox), and a sensitive response to light and gravity. According to a qRT-PCR analysis, MdTAC1a and MdTAC1b were strongly expressed in shoot tips and vegetative buds of weeping cultivars but were weakly expressed in columnar cultivars. In the MdTAC1a promoter, there were losses of 2 bp in spur cultivars and 6 bp in weeping cultivar compared with standard and columnar cultivars. An InDel marker specific to the MdTAC1a promoter was developed to distinguish apple cultivars and F1 progeny. We identified a protein, MdSRC2, that interacts with MdTAC1a, whose encoding gene which was highly expressed in trees with large branch angles. Our results indicate that differences in the MdTAC1a promoter are major contributors to branch-angle variation in apple, and the MdTAC1a interacts with MdSRC2 to affect this trait.

Expression Analysis of AUX/IAA Family Genes in Apple Under Salt Stress.

Members of the auxin/indoleacetic acid (Aux/IAA) gene family in plants are primary auxin-responsive genes that play important roles in many aspects of plant development and in responses to abiotic stress. Recently, 33 Aux/IAA have been identified in the apple genome. The biological responses of MdIAAs to salt stress are still unknown. In this study, Malus zumi, Malus baccata, and Malus × domestica 'Fuji' plantlets were subjected to salt stress by supplementing hydroponic media with NaCl at various concentrations. M. zumi showed the strongest salt resistance, followed by 'Fuji', and M. baccata was the most sensitive to salt stress. Tissue-specific expression profiles of MdIAAs were determined by quantitative real-time polymerase chain reaction. When apple plantlets were subjected to salt stress, most of salt-responsive MdIAAs were up-regulated by 1 h, 3 h, and 6 h in roots, shoot tips, and leaves, respectively. Highly expressed MdIAAs in roots, especially for M. zumi, consisted with the salt tolerance of apple rootstocks. Transgenic apple calli were tolerant to salt stress when over-expressed salt-responsive genes, MdIAA8, -9, and -25. These results provide clues about salt resistance in these three Malus species, which helps apple breeding of salt tolerance by genetic transformation.

Genome-Wide Identification of the Aux/IAA Family Genes (MdIAA) and Functional Analysis of MdIAA18 for Apple Tree Ideotype.

The Aux/IAA (auxin/indole-3-acetic acid) gene family is one of the early auxin-responsive gene families, which play a central role in auxin response. Few reports are involved in Aux/IAA genes in fruit trees, especially in apple (Malus × domestica Borkh.). A total of 33 MdIAA members were identified, of which 27 members contained four conserved domains, whereas the others lost one or two conserved domains. Several cis-elements in promoters of MdIAAs were predicted responsive to hormones and abiotic stress. Tissue-specific expression patterns of MdIAAs in different apple tree ideotypes were investigated by quantitative real-time PCR. A large number of MdIAAs were highly expressed in leaf buds and reproductive organs, and MdIAAs clustered in same group showed similar expression profiles. Overexpression of MdIAA18 in Arabidopsis resulted in compact phenotype. These results indicated that MdIAA genes may be involved in vegetative and reproductive growth of apple. Taken together, the results provide useful clues to reveal the function of MdIAAs in apple and control apple tree architecture by manipulation of MdIAAs.

Characterization of a SUPERMAN-like Gene, MdSUP11, in apple (Malus × domestica Borkh.).

Arabidopsis SUPERMAN and its family members of its family play important roles in plant growth and floral organ development; yet much less is known about their functions expanding in apple tree development. Previous work has identified 12 SUP-like genes in the apple (Malus × domestica Borkh.) genome, and the MdSUP11 which is expressed in both vegetative and reproductive organs of apple. However, the function of MdSUP11 remains obscure. In this study, the ß-glucuronidase expression driven by the MdSUP11 native promoter was detected in roots, young leaves, and floral organs of transgenic Arabidopsis. In transgenic tobacco, overexpression of MdSUP11 lead to dwarfism, aberrant leaf shapes, and morphological changes of floral organs. Endogenous concentrations of auxin (indole-3-acetic acid), abscisic acid, isopentenyl adenosine and zeatin riboside were significantly higher in young MdSUP11-transformed tobacco plants than in non-transformed plants. Gene expression analysis using real-time quantitative PCR showed up-regulation of NtDFR2 and NtANS1 expression in unopened transgenic flowers, whereas NtCHS expression was not changed significantly. Together, these results suggest that MdSUP11 is associated with apple's vegetative and reproductive development. Its overexpression in tobacco affects leaf and flower organ development and plant height; potentially by changing NtDFR2 and NtANS1 expression and endogenous levels of indole-3-acetic acid, cytokinins and abscisic acid.

Identification of MdDof genes in apple and analysis of their response to biotic or abiotic stress.

As a classic plant-specific transcription factor family - the Dof domain proteins - are involved in a variety of biological processes in organisms ranging from unicellular Chlamydomonas to higher plants. However, there are limited reports of MdDof (Malus domestica Borkh. DNA-binding One Zinc Finger) domain proteins in fruit trees, especially in apple. In this study we identified 54 putative Dof transcription factors in the apple genome. We analysed the gene structures, protein motifs, and chromosome locations of each of the MdDof genes. Next, we characterised all 54 MdDofs their expression patterns under different abiotic and biotic stress conditions. It was found that MdDof6,26 not only played an important role in the biotic/abiotic stress but may also be involved in many molecular functions. Further, both in flower development and pollen tube growth it was found that the relative expression of MdDof24 increased rapidly, also with gene ontology analysis it was indicated that MdDof24 was involved in the chemical reaction and flower development pathways. Taken together, our results provide useful clues as to the function of MdDof genes in apple and serve as a reference for studies of Dof zinc finger genes in other plants.

Assessing the allelotypic effect of two aminocyclopropane carboxylic acid synthase-encoding genes MdACS1 and MdACS3a on fruit ethylene production and softening in Malus.

Phytohormone ethylene largely determines apple fruit shelf life and storability. Previous studies demonstrated that MdACS1 and MdACS3a, which encode 1-aminocyclopropane-1-carboxylic acid synthases (ACS), are crucial in apple fruit ethylene production. MdACS1 is well-known to be intimately involved in the climacteric ethylene burst in fruit ripening, while MdACS3a has been regarded a main regulator for ethylene production transition from system 1 (during fruit development) to system 2 (during fruit ripening). However, MdACS3a was also shown to have limited roles in initiating the ripening process lately. To better assess their roles, fruit ethylene production and softening were evaluated at five time points during a 20-day post-harvest period in 97 Malus accessions and in 34 progeny from 2 controlled crosses. Allelotyping was accomplished using an existing marker (ACS1) for MdACS1 and two markers (CAPS866 and CAPS870) developed here to specifically detect the two null alleles (ACS3a-G289V and Mdacs3a) of MdACS3a. In total, 952 Malus accessions were allelotyped with the three markers. The major findings included: The effect of MdACS1 was significant on fruit ethylene production and softening while that of MdACS3a was less detectable; allele MdACS1-2 was significantly associated with low ethylene and slow softening; under the same background of the MdACS1 allelotypes, null allele Mdacs3a (not ACS3a-G289V) could confer a significant delay of ethylene peak; alleles MdACS1-2 and Mdacs3a (excluding ACS3a-G289V) were highly enriched in M. domestica and M. hybrid when compared with those in M. sieversii. These findings are of practical implications in developing apples of low and delayed ethylene profiles by utilizing the beneficial alleles MdACS1-2 and Mdacs3a.

A novel gene, MdSSK1, as a component of the SCF complex rather than MdSBP1 can mediate the ubiquitination of S-RNase in apple.

As a core factor in S-RNase-based gametophytic self-incompatibility (GSI), the SCF (SKP1-Cullin1-F-box-Rbx1) complex (including pollen determinant SLF, S-locus-F-box) functions as an E3 ubiquitin ligase on non-self S-RNase. The SCF complex is formed by SKP1 bridging between SLF, CUL1, and Rbx1; however, it is not known whether an SCF complex lacking SKP1 can mediate the ubiquitination of S-RNase. Three SKP1-like genes from pollen were cloned based on the structural features of the SLF-interacting-SKP1-like (SSK) gene and the 'Golden Delicious' apple genome. These genes have a motif of five amino acids following the standard 'WAFE' at the C terminal and, in addition, contain eight sheets and two helices. All three genes were expressed exclusively in pollen. In the yeast two-hybrid and pull-down assays only one was found to interact with MdSFBB and MdCUL1, suggesting it is the SLF-interacting SKP1-like gene in apple which was named MdSSK1. In vitro experiments using MdSSK1, S2-MdSFBB1 (S2-Malus domestica S-locus-F-box brother) and MdCUL1 proteins incubated with S 2-RNase and ubiquitin revealed that the SCF complex ubiquitinylates S-RNase in vitro, while MdSBP1 (Malus domestica S-RNase binding protein 1) could not functionally replace MdSSK1 in the SCF complex in ubiquitinylating S-RNase. According to the above experiments, MdSBP1 is probably the only factor responsible for recognition with S-RNase, while not a component of the SCF complex, and an SCF complex containing MdSSK1 is required for mediating the ubiquitination of S-RNase.

The microtubule cytoskeleton and pollen tube Golgi vesicle system are required for in vitro S-RNase internalization and gametic self-incompatibility in apple.

S-RNase is the female determinant of gametophytic self-incompatibility in apple and is usually considered to be the reason for rejection of pollen. In this study, we investigated the role of microtubules (MTs) in internalization of S-RNases by pollen tubes cultured in vitro. The results showed that S-RNase was imported into the pollen tube where it inhibits pollen tube growth, and that S-RNase is co-localized with the Golgi vesicle during the internalization process. Moreover, MT depolymerization is observed following accumulation of S-RNases in the pollen cytosol. On the other hand, S-RNase was prevented from entering the pollen tube when the pollen was treated with the actin filament (AF) inhibitor latrunculin A (LatA), the MT inhibitor oryzalin, or the MT stabilizer taxol at subtoxic concentrations. These hindered the construction of the MT, with pollen tubes capable of growth under these conditions. Pollen tubes showed improved growth in self-pollinated styles that were pre-treated with taxol. This suggests that cytoskeleton antagonists can prevent S-RNase-mediated inhibition of pollen tubes in vivo by blocking S-RNase internalization. These results suggest that an intact and dynamic cytoskeleton is required for the in vitro internalization of S-RNase, as shown by the effects of various cytoskeleton inhibitors. S-RNase internalization takes place via a membrane/cytoskeleton-based Golgi vesicle system, which can also affect self-incompatibility in apple.

Fine genetic mapping of the Co locus controlling columnar growth habit in apple.

Tree architecture is an important, complex and dynamic trait affected by diverse genetic, ontogenetic and environmental factors. 'Wijcik McIntosh', a columnar (reduced branching) sport of 'McIntosh' and a valuable genetic resource, has been used intensively in apple-breeding programs for genetic improvement of tree architecture. The columnar growth habit is primarily controlled by the dominant allele of gene Co (columnar) on linkage group-10. But the Co locus is not well mapped and the Co gene remains unknown. To precisely map the Co locus and to identify candidate genes of Co, a sequence-based approach using both peach and apple genomes was used to develop new markers linked more tightly to Co. Five new simple sequence repeats markers were developed (C1753-3520, C18470-25831, C6536-31519, C7223-38004 and C7629-22009). The first four markers were obtained from apple genomic sequences on chromosome-10, whereas the last (C7629-22009) was from an unanchored apple contig that contains an apple expressed sequence tag CV082943, which was identified through synteny analysis between the peach and apple genomes. Genetic mapping of these five markers in four F(1) populations of 528 genotypes and 290 diverse columnar selections/cultivars (818 genotypes in total) delimited the Co locus in a genetic interval with 0.37 % recombination between markers C1753-3520 and C7629-22009. Marker C18470-25831 co-segregates with Co in the 818 genotypes studied. The Co region is estimated to be 193 kb and contains 26 predicted gene in the 'Golden Delicious' genome. Among the 26 genes, three are putative LATERAL ORGAN BOUNDARIES (LOB) DOMAIN (LBD) containing transcription factor genes known of essential roles in plant lateral organ development, and are therefore considered as strong candidates of Co, designated MdLBD1, MdLBD2, and MdLBD3. Although more comprehensive studies are required to confirm the function of MdLBD1-3, the present work represents an important step forward to better understand the genetic and molecular control of tree architecture in apple.