Summary
Autophagy is a major and conserved pathway for delivering and recycling unwanted proteins or damaged organelles to be degraded in the vacuoles. AuTophaGy‐related (ATG) protein 18a has been ...established as one of the essential components for autophagy occurrence in Arabidopsis thaliana. We previously cloned the ATG18a homolog from Malus domestica (MdATG18a) and monitored its responsiveness to various abiotic stresses at the transcriptional level. However, it is still unclear what its function is under abiotic stress in apple. Here, we found that heterologous expression of MdATG18a in tomato plants markedly enhanced their tolerance to drought. Overexpression (OE) of that gene in apple plants improved their drought tolerance as well. Under drought conditions, the photosynthesis rate and antioxidant capacity were significantly elevated in OE lines when compared with the untransformed wild type (WT). Transcript levels of other important apple ATG genes were more strongly up‐regulated in transgenic MdATG18a OE lines than in the WT. The percentage of insoluble protein in proportion to total protein was lower and less oxidized protein accumulated in the OE lines than in the WT under drought stress. This was probably due to more autophagosomes being formed in the former. These results demonstrate that overexpression of MdATG18a in apple plants enhances their tolerance to drought stress, probably because of greater autophagosome production and a higher frequency of autophagy. Those processes help degrade protein aggregation and limit the oxidation damage, thereby suggesting that autophagy plays important roles in the drought response.
: The objectives of this study were to test the effects of exogenous melatonin on apple (Malus domestica Borkh. cv. Golden Delicious) leaves and investigate its possible physiological role in ...delaying leaf senescence. Detached leaves treated with 10 mm melatonin solutions clearly showed a slowing in their process of dark‐induced senescence, as evidenced by both biochemical and molecular parameters. Melatonin delayed the normal reduction in chlorophyll content and maximum potential photosystem II efficiency (Fv/Fm). It also suppressed the transcript levels of a key chlorophyll degradation gene, pheide a oxygenase (PAO), and the senescence‐associated gene 12 (SAG12). This outcome was thought to be because of the enhanced antioxidant capabilities of melatonin. Indeed, H2O2 accumulation was inhibited by exogenous melatonin, which might have resulted from direct reactive oxygen species scavenging by melatonin and a great enhancement of ascorbate peroxidase (APX; EC 1.11.1.11), which acted on both mRNA and protein activity levels. Melatonin treatment led to the maintenance of higher contents of ascorbic acid (AsA) and glutathione (GSH) but less dehydroascorbate (DHA) and oxidized glutathione (GSSG) compared with the control, possibly through its regulation of the AsA–GSH cycle.
Nitrogen (N) availability is an essential factor for plant growth. Recycling and remobilization of N have strong impacts on crop yield and quality under N deficiency. Autophagy is a critical ...nutrient‐recycling process that facilitates remobilization under starvation. We previously showed that an important AuTophaGy (ATG) protein from apple, MdATG18a, has a positive role in drought tolerance. In this study, we explored its biological role in response to low‐N. Overexpression of MdATG18a in both Arabidopsis and apple improved tolerance to N‐depletion and caused a greater accumulation of anthocyanin. The increased anthocyanin concentration in transgenic apple was possibly due to up‐regulating flavonoid biosynthetic and regulatory genes (MdCHI, MdCHS, MdANS, MdPAL, MdUFGT, and MdMYB1) and higher soluble sugars concentration. MdATG18a overexpression enhanced starch degradation with up‐regulating amylase gene (MdAM1) and up‐regulated sugar metabolism related genes (MdSS1, MdHXKs, MdFK1, and MdNINVs). Furthermore, MdATG18a functioned in nitrate uptake and assimilation by up‐regulating nitrate reductase MdNIA2 and 3 high‐affinity nitrate transporters MdNRT2.1/2.4/2.5. MdATG18a overexpression also elevated other important MdATG genes expression and autophagosomes formation under N‐depletion, which play key contributions to above changes. Together, these results demonstrate that overexpression of MdATG18a enhances tolerance to N‐deficiencies and plays positive roles in anthocyanin biosynthesis through greater autophagic activity.
Autophagy plays a critical role in recycling and remobilization of nitrogen. Here, overexpression of MdATG18a enhanced tolerance to N‐depletion in both Arabidopsis and apple. Overexpression of MdATG18a in apple accumulated more anthocyanin and improved nitrate uptake by up‐regulating anthocyanin‐related genes and some nitrate transporters, respectively. These results were possibly because of increased autophagy activity in transgenic lines under N deficiency.
•Apple MdATG3a and MdATG3b homologues share conserved domains and motifs of ATG3.•Both genes are responsive to environmental stress at transcriptional level.•Overexpression of either in Arabidopsis ...afford tolerance to multiple stresses.•MdATG3b overexpression in apple callus improves growth performance under starvation.
The efficient degradation system of autophagy in plant cells has important roles in removing and recycling intracellular components during normal development or under environmental stresses. Formation of autophagosomes requires the conjugation of ubiquitin-like protein ATG8 to phosphatidylethanolamine (PE). We isolated two ubiquitin-conjugating enzyme E2-like ATG3 homologues from Malus domestica − MdATG3a and MdATG3b − that are crucial for ATG8-PE conjugation. Both share a conserved N-terminal, as well as the catalytic and C-terminal domains of ATG3 with HPC and FLKF motifs. Each promoter was isolated from genomic DNA and contained several cis-acting elements that are involved in responses to environmental stresses or hormones. In addition to having the same cellular localization in the nucleus and cytoplasm, MdATG3a and MdATG3b showed similar expression patterns toward leaf senescence, nitrogen starvation, drought, salinity, and oxidative stress at the transcriptional level. Ectopic expression of either in Arabidopsis conferred tolerance to osmotic or salinity stress and also improved growth performance under nitrogen- or carbon-starvation. Callus lines of ‘Orin’ apple that over-expressed MdATG3b also displayed better growth performance when nutrient supplies were limited. These overall results demonstrate that, as important autophagy genes, overexpression of MdATG3s can afford tolerance to multiple abiotic stresses at the cellular and whole-plant level.
Since melatonin was identified in plants decades ago, much attention has been devoted to discovering its role in plant science. There is still a great deal to learn about the functional importance of ...melatonin, as well as its functional mode. In this paper, we examine the role of melatonin treatment in the response of
Rehd. to alkaline conditions. Stressed seedlings showed chlorosis and suppressed growth. However, this phenotype was ameliorated when 5 µM melatonin was added to the irrigation solution. This supplementation was also associated with a reduction in cell membrane damage and maintenance of a normal root system architecture. Fewer reactive oxygen species (ROS) were accumulated due to the enhanced scavenging activity of antioxidant enzymes superoxide dismutase, peroxidase, and catalase. In addition, alkaline-stressed seedlings that received the melatonin supplement accumulated more polyamines compared with untreated seedlings. Transcript levels of six genes involved in polyamine synthesis, including
, -
, and -
, and
, -
, and -
, -
, were upregulated in response to melatonin application. All of these results demonstrate that melatonin has a positive function in plant tolerance to alkaline stress because it regulates enzyme activity and the biosynthesis of polyamines.
Aims
Salinity plays a crucial role in numerous environmental processes, with extreme concentrations resulting in the serious decline of global agricultural productivity. Plant-specific HD-Zip I ...transcription factors (TFs), particularly γ-clade HD-Zip I members, are key for the adaption of plants to various abiotic stresses. Our main purpose is to characterize the function of γ-clade HD-Zip I members in apple under salt stress.
Methods
GL-3 and transgenic plants were grown in a hydroponic system or in soil and exposed to the presence or absence of NaCl. Photosynthetic capacity, root damage, Na
+
and K
+
accumulation, reactive oxygen species (ROS) accumulation, and RNA-seq were investigated.
Results
MdHB-7
(γ-clade HD-Zip I TF) expression was induced by salt stress, particularly in the roots. We employed
MdHB-7
overexpression and
MdHB-7
RNAi transgenic apple plants for the treatments to explore the functions of
MdHB-7
under salt stress. Results from the salt treatment and phenotypic evaluation revealed the enhancement of the salt tolerance of apple plants by the
MdHB-7
expression. The overexpression of
MdHB-7
reduced the salt stress-induced damage to root growth and vitality and maintained ion homeostasis. Moreover, the overexpression of
MdHB-7
was observed to promote the detoxification of ROS. Transcriptome and qRT-PCR analysis demonstrate that
MdHB-7
overexpression up-regulated the expression of antioxidant enzyme-related genes.
MdHB-7
-mediated root vitality, antioxidant activity and ion homeostasis were impaired, resulting in
MdHB-7
RNAi lines that were more sensitive to salt stress.
Conclusions
Our results demonstrate the key role of
MdHB-7
as a salinity stress tolerance regulator in apple (
Malus domestica
).
: As an indoleamine molecule, melatonin mediates many physiological processes in plants. We investigated its role in regulating growth, ion homeostasis, and the response to oxidative stress in Malus ...hupehensis Rehd. under high‐salinity conditions. Stressed plants had reduced growth and a marked decline in their net photosynthetic rates and chlorophyll contents. However, pretreatment with 0.1 μm melatonin significantly alleviated this growth inhibition and enabled plants to maintain an improved photosynthetic capacity. The addition of melatonin also lessened the amount of oxidative damage brought on by salinity, perhaps by directly scavenging H2O2 or enhancing the activities of antioxidative enzymes such as ascorbate peroxidase, catalase, and peroxidase. We also investigated whether melatonin might control the expression of ion‐channel genes under salinity. Here, MdNHX1 and MdAKT1 were greatly up‐regulated in the leaves, which possibly contributed to the maintenance of ion homeostasis and, thus, improved salinity resistance in plants exposed to exogenous melatonin.
Apple (Malus × domestica) trees are vulnerable to freezing temperatures. However, there has been only limited success in developing cold-hardy cultivars. This lack of progress is due at least partly ...to lack of understanding of the molecular mechanisms of freezing tolerance in apple.
In this study, we evaluated the potential roles for two R2R3 MYB transcription factors (TFs), MYB88 and the paralogous FLP (MYB124), in cold stress in apple and Arabidopsis. We found that MYB88 and MYB124 positively regulate freezing tolerance and cold-responsive gene expression in both apple and Arabidopsis.
Chromatin-Immunoprecipitation-qPCR and electrophoretic mobility shift assays showed that MdMYB88/MdMYB124 act as direct regulators of the COLD SHOCK DOMAIN PROTEIN 3 (MdCSP3) and CIRCADIAN CLOCK ASSOCIATED 1 (MdCCA1) genes. Dual luciferase reporter assay indicated that MdCCA1 but not MdCSP3 activated the expression of MdCBF3 under cold stress. Moreover, MdMYB88 and MdMYB124 promoted anthocyanin accumulation and H2O2 detoxification in response to cold.
Taken together, our results suggest that MdMYB88 and MdMYB124 positively regulate cold hardiness and cold-responsive gene expression under cold stress by C-REPEAT BINDING FACTOR (CBF)-dependent and CBF-independent pathways.
Ascorbate (Asc) is an important antioxidant in plants and humans that plays key roles in various physiological processes. Understanding the regulation of Asc content in fruit plants is important for ...improving plant resiliency and optimizing Asc in food. Here, we found that both the transcript level and protein abundance of Asc Mannose pathway Regulator 1 Like 1 (MdAMR1L1) was negatively associated with Asc levels during the development of apple (Malus × domestica) fruit. The overexpression or silencing of MdAMR1L1 in apple indicated that MdAMR1L1 negatively regulated Asc levels. However, in the leaves of MdAMR1L1-overexpressing apple lines, the transcript levels of the Asc synthesis gene Guanosine diphosphate-mannose pyrophosphorylase MdGMP1 were increased, while its protein levels and enzyme activity were reduced. This occurred because the MdAMR1L1 protein interacted with MdGMP1 and promoted its degradation via the ubiquitination pathway to inhibit Asc synthesis at the post-translational level. MdERF98, an apple ethylene response factor, whose transcription was modulated by Asc level, is directly bound to the promoter of MdGMP1 to promote the transcription of MdGMP1. These findings provide insights into the regulatory mechanism of Asc biosynthesis in apples and revealed potential opportunities to improve fruit Asc levels.
The shaded peel of ‘Fortune’ (a red cultivar) and ‘Mutsu’ (a yellow/green cultivar) apple (Malus domestica Borkh.) was exposed to full sun by turning fruit 180° at about one week before harvest to ...determine the expression of key genes involved in anthocyanin synthesis in response to sunlight exposure and their relationships with the levels of anthocyanins and other phenolics. For the unturned (control) fruit, the shaded peel had lower expression levels of MdMYB10 (a transcriptional factor in the regulation of anthocyanin biosynthesis) and seven structural genes in anthocyanin synthesis (MdPAL, MdCHS, MdCHI, MdF3H, MdDFR1, MdLDOX, and MdUFGT), and lower levels of anthocyanins and flavonols than the sun-exposed peel in both cultivars. Exposure of the shaded peel to full sun caused marked up-regulation of the expression of MdMYB10 and all seven structural genes, which peaked between 6 h and 30 h after fruit turning, consequently leading to higher levels of anthocyanins, flavonols, and total phenolics than in the shaded peel and even in the sun-exposed peel of control fruit. Interestingly, the levels of flavonols were higher in the shaded peel of turned fruit (the original sun-exposed peel) than in the sun-exposed peel of both control and turned fruit in both cultivars, suggesting that competition for substrates exists in different branches of the phenylpropanoid pathway. These results indicate that sunlight exposure stimulates the expression of MdMYB10 and structural genes in anthocyanin synthesis, thereby elevating the levels of anthocyanins and other phenolic compounds in both red and yellow/green cultivars.
•Sun exposure of shaded peel up-regulated the expression of MdMYB10 & structural genes.•This up-regulation led to higher anthocyanins & flavonols in the original shaded peel.•Fruit turning also elevated the levels of flavonols in the original sun-exposed peel.•Turned fruit had better color and higher total phenolics in the peel than the control.
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