A single amino acid substitution in MdLAZY1A dominantly impairs shoot gravitropism in Malus.

Plant architecture is 1 of the most important factors that determines crop yield potential and productivity. In apple (Malus domestica), genetic improvement of tree architecture has been challenging due to a long juvenile phase and growth as complex trees composed of a distinct scion and a rootstock. To better understand the genetic control of apple tree architecture, the dominant weeping growth phenotype was investigated. We report the identification of MdLAZY1A (MD13G1122400) as the genetic determinant underpinning the Weeping (W) locus that largely controls weeping growth in Malus. MdLAZY1A is 1 of the 4 paralogs in apple that are most closely related to AtLAZY1 involved in gravitropism in Arabidopsis (Arabidopsis thaliana). The weeping allele (MdLAZY1A-W) contains a single nucleotide mutation c.584T>C that leads to a leucine to proline (L195P) substitution within a predicted transmembrane domain that colocalizes with Region III, 1 of the 5 conserved regions in LAZY1-like proteins. Subcellular localization revealed that MdLAZY1A localizes to the plasma membrane and nucleus in plant cells. Overexpressing the weeping allele in apple cultivar Royal Gala (RG) with standard growth habit impaired its gravitropic response and altered the growth to weeping-like. Suppressing the standard allele (MdLAZY1A-S) by RNA interference (RNAi) in RG similarly changed the branch growth direction to downward. Overall, the L195P mutation in MdLAZY1A is genetically causal for weeping growth, underscoring not only the crucial roles of residue L195 and Region III in MdLAZY1A-mediated gravitropic response but also a potential DNA base editing target for tree architecture improvement in Malus and other crops.

SnRK1 kinase-mediated phosphorylation of transcription factor bZIP39 regulates sorbitol metabolism in apple.

Sorbitol is a major photosynthate produced in leaves and transported through the phloem of apple (Malus domestica) and other tree fruits in Rosaceae. Sorbitol stimulates its own metabolism, but the underlying molecular mechanism remains unknown. Here, we show that sucrose nonfermenting 1 (SNF1)-related protein kinase 1 (SnRK1) is involved in regulating the sorbitol-responsive expression of both SORBITOL DEHYDROGENASE 1 (SDH1) and ALDOSE-6-PHOSPHATE REDUCTASE (A6PR), encoding 2 key enzymes in sorbitol metabolism. SnRK1 expression is increased by feeding of exogenous sorbitol but decreased by sucrose. SnRK1 interacts with and phosphorylates the basic leucine zipper (bZIP) transcription factor bZIP39. bZIP39 binds to the promoters of both SDH1 and A6PR and activates their expression. Overexpression of SnRK1 in 'Royal Gala' apple increases its protein level and activity, upregulating transcript levels of both SDH1 and A6PR without altering the expression of bZIP39. Of all the sugars tested, sorbitol is the only 1 that stimulates SDH1 and A6PR expression, and this stimulation is blocked by RNA interference (RNAi)-induced repression of either SnRK1 or bZIP39. These findings reveal that sorbitol acts as a signal regulating its own metabolism via SnRK1-mediated phosphorylation of bZIP39, which integrates sorbitol signaling into the SnRK1-mediated sugar signaling network to modulate plant carbohydrate metabolism.

Anthocyanin Accumulation Provides Protection against High Light Stress While Reducing Photosynthesis in Apple Leaves.

The photoprotective role of anthocyanin remains controversial. In this study, we explored the effects of anthocyanin on photosynthesis and photoprotection using transgenic 'Galaxy Gala' apple plants overexpressing MdMYB10 under high light stress. The overexpression of MdMYB10 dramatically enhanced leaf anthocyanin accumulation, allowing more visible light to be absorbed, particularly in the green region. However, through post-transcriptional regulation, anthocyanin accumulation lowered leaf photosynthesis in both photochemical reaction and CO2 fixation capacities. Anthocyanin accumulation also led to a decreased de-epoxidation state of the xanthophyll cycle and antioxidant capacities, but this is most likely a response to the light-shielding effect of anthocyanin, as indicated by a higher chlorophyll concentration and lower chlorophyll a/b ratio. Under laboratory conditions when detached leaves lost carbon fixation capacity due to the limitation of CO2 supply, the photoinhibition of detached transgenic red leaves was less severe under strong white, green, or blue light, but it became more severe in response to strong red light compared with that of the wild type. In field conditions when photosynthesis was performed normally in both green and transgenic red leaves, the degree of photoinhibition was comparable between transgenic red leaves and wild type leaves, but it was less severe in transgenic young shoot bark compared with the wild type. Taken together, these data show that anthocyanin protects plants from high light stress by absorbing excessive visible light despite reducing photosynthesis.

Apple ALMT9 Requires a Conserved C-Terminal Domain for Malate Transport Underlying Fruit Acidity.

Malate accumulation in the vacuole largely determines apple (Malus domestica) fruit acidity, and low fruit acidity is strongly associated with truncation of Ma1, an ortholog of ALUMINUM-ACTIVATED MALATE TRANSPORTER9 (ALMT9) in Arabidopsis (Arabidopsis thaliana). A mutation at base 1,455 in the open reading frame of Ma1 leads to a premature stop codon that truncates the protein by 84 amino acids at its C-terminal end. Here, we report that both the full-length protein, Ma1, and its naturally occurring truncated protein, ma1, localize to the tonoplast; when expressed in Xenopus laevis oocytes and Nicotiana benthamiana cells, Ma1 mediates a malate-dependent inward-rectifying current, whereas the ma1-mediated transmembrane current is much weaker, indicating that ma1 has significantly lower malate transport activity than Ma1. RNA interference suppression of Ma1 expression in 'McIntosh' apple leaves, 'Empire' apple fruit, and 'Orin' apple calli results in a significant decrease in malate level. Genotyping and phenotyping of 186 apple accessions from a diverse genetic background of 17 Malus species combined with the functional analyses described above indicate that Ma1 plays a key role in determining fruit acidity and that the truncation of Ma1 to ma1 is genetically responsible for low fruit acidity in apple. Furthermore, we identified a C-terminal domain conserved in all tonoplast-localized ALMTs essential for Ma1 function; protein truncations into this conserved domain significantly lower Ma1 transport activity. We conclude that the truncation of Ma1 to ma1 reduces its malate transport function by removing a conserved C-terminal domain, leading to low fruit acidity in apple.

HIPM Is a Susceptibility Gene of Malus spp.: Reduced Expression Reduces Susceptibility to Erwinia amylovora.

Fire blight, a devastating disease caused by the bacterium Erwinia amylovora, is a major threat to apple crop production. To improve our understanding of the fire blight disease and to identify potential strategies to control the pathogen, we studied the apple protein HIPM (for HrpN-interacting protein from Malus spp.), which has previously been identified as interacting with the E. amylovora effector protein HrpN. Transgenic apple plants were generated with reduced HIPM expression, using an RNA interference construct, and were subsequently analyzed for susceptibility to E. amylovora infection. Lines exhibiting a greater than 50% silencing of HIPM expression showed a significant decrease in susceptibility to E. amylovora infection. Indeed, a correlation between HIPM expression and E. amylovora infection was identified, demonstrating the crucial role of HIPM during fire blight disease progression. Furthermore, an apple oxygen-evolving enhancer-like protein (MdOEE) was identified via a yeast two-hybrid screen to interact with HIPM. This result was confirmed with bimolecular fluorescence complementation assays and leads to new hypotheses concerning the response mechanism of the plant to E. amylovora as well as the mechanism of infection of the bacterium. These results suggest that MdOEE and, particularly, HIPM are promising targets for further investigations toward the genetic improvement of apple.

Transgenic Suppression of AGAMOUS Genes in Apple Reduces Fertility and Increases Floral Attractiveness.

We investigated the ability of RNA interference (RNAi) directed against two co-orthologs of AGAMOUS (AG) from Malus domestica (domestic apple, MdAG) to reduce the risks of invasiveness and provide genetic containment of transgenes, while also promoting the attractiveness of flowers for ornamental usage. Suppression of two MdAG-like genes, MdMADS15 and MdMADS22, led to the production of trees with highly showy, polypetalous flowers. These "double-flowers" had strongly reduced expression of both MdAG-like genes. Members of the two other clades within in the MdAG subfamily showed mild to moderate differences in gene expression, or were unchanged, with the level of suppression approximately proportional to the level of sequence identity between the gene analyzed and the RNAi fragment. The double-flowers also exhibited reduced male and female fertility, had few viable pollen grains, a decreased number of stigmas, and produced few viable seeds after cross-pollination. Despite these floral alterations, RNAi-AG trees with double-flowers set full-sized fruit. Suppression or mutation of apple AG-like genes appears to be a promising method for combining genetic containment with improved floral attractiveness.

Stable expression and phenotypic impact of attacin E transgene in orchard grown apple trees over a 12 year period.

BACKGROUND: Transgenic trees currently are being produced by Agrobacterium-mediated transformation and biolistics. The future use of transformed trees on a commercial basis depends upon thorough evaluation of the potential environmental and public health risk of the modified plants, transgene stability over a prolonged period of time and the effect of the gene on tree and fruit characteristics. We studied the stability of expression and the effect on resistance to the fire blight disease of the lytic protein gene, attacin E, in the apple cultivar 'Galaxy' grown in the field for 12 years. RESULTS: Using Southern and western blot analysis, we compared transgene copy number and observed stability of expression of this gene in the leaves and fruit in several transformed lines during a 12 year period. No silenced transgenic plant was detected. Also the expression of this gene resulted in an increase in resistance to fire blight throughout 12 years of orchard trial and did not affect fruit shape, size, acidity, firmness, weight or sugar level, tree morphology, leaf shape or flower morphology or color compared to the control. CONCLUSION: Overall, these results suggest that transgene expression in perennial species, such as fruit trees, remains stable in time and space, over extended periods and in different organs. This report shows that it is possible to improve a desirable trait in apple, such as the resistance to a pathogen, through genetic engineering, without adverse alteration of fruit characteristics and tree shape.

Two receptor-like genes, Vfa1 and Vfa2, confer resistance to the fungal pathogen Venturia inaequalis inciting apple scab disease.

The Vf locus, originating from the crabapple species Malus floribunda 821, confers resistance to five races of the fungal pathogen Venturia inaequalis, the causal agent of apple scab disease. Previously, a cluster of four receptor-like genes, Vfa1, Vfa2, Vfa3, and Vfa4, was identified within the Vf locus. Because the amino-acid sequence of Vfa3 is truncated, it was deemed nonfunctional. In this study, each of the three full-length Vfa genes was introduced into a plant cloning vector, pCAMBIA2301, and used for Agrobacterium-mediated transformation of two apple cultivars, Galaxy and McIntosh, to assess functionality of these genes and to characterize their roles in resistance to V. inaequalis. Transformed apple lines carrying each of Vfa1, Vfa2, or Vfa4 were developed, analyzed for the presence of the transgene using polymerase chain reaction and Southern blotting, and assayed for resistance to apple scab following inoculation with V. inaequalis. Transformed lines expressing Vfa4 were found to be susceptible to apple scab, whereas those expressing either Vfa1 or Vfa2 exhibited partial resistance to apple scab. Based on Western blot analysis as well as microscopic analysis of plant resistance reactions, the roles of Vfa1 and Vfa2 in apple scab disease resistance response are discussed.

Pisatin metabolism in pea (Pisum sativum L.) cell suspension cultures.

Cell suspension cultures were established from germinating pea (Pisum sativum L.) seeds. This cell culture, which accumulated pisatin, consisted mostly of single cells containing a few cell aggregates. The cells responded to treatment with a yeast glucan preparation with transient accumulation of pisatin in both cells and culture media. Addition of pisatin to cell cultures resulted in increased synthesis of pisatin. Phenylalanine ammonia-lyase, chalcone synthase and isoflavone reductase activities were present in untreated cells. Upon treatment with an elicitor preparation the activities of the first two enzymes showed a rapid, transient increase up to 20 hours after treatment. Isoflavone reductase showed a major and minor peak at 16 and 36 h, respectively, after elicitor treatment. The time course of the enzyme activity and pisatin accumulation is consistent with an elicitor-mediated response.