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1.
Plant Cell ; 36(5): 1868-1891, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38299382

RESUMO

Carotenoids are essential for photosynthesis and photoprotection. Plants must evolve multifaceted regulatory mechanisms to control carotenoid biosynthesis. However, the regulatory mechanisms and the regulators conserved among plant species remain elusive. Phytoene synthase (PSY) catalyzes the highly regulated step of carotenogenesis and geranylgeranyl diphosphate synthase (GGPPS) acts as a hub to interact with GGPP-utilizing enzymes for the synthesis of specific downstream isoprenoids. Here, we report a function of Nudix hydrolase 23 (NUDX23), a Nudix domain-containing protein, in post-translational regulation of PSY and GGPPS for carotenoid biosynthesis. NUDX23 expresses highly in Arabidopsis (Arabidopsis thaliana) leaves. Overexpression of NUDX23 significantly increases PSY and GGPPS protein levels and carotenoid production, whereas knockout of NUDX23 dramatically reduces their abundances and carotenoid accumulation in Arabidopsis. NUDX23 regulates carotenoid biosynthesis via direct interactions with PSY and GGPPS in chloroplasts, which enhances PSY and GGPPS protein stability in a large PSY-GGPPS enzyme complex. NUDX23 was found to co-migrate with PSY and GGPPS proteins and to be required for the enzyme complex assembly. Our findings uncover a regulatory mechanism underlying carotenoid biosynthesis in plants and offer promising genetic tools for developing carotenoid-enriched food crops.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Carotenoides , Regulação da Expressão Gênica de Plantas , Carotenoides/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Nudix Hidrolases , Cloroplastos/metabolismo , Geranil-Geranildifosfato Geranil-Geraniltransferase/metabolismo , Geranil-Geranildifosfato Geranil-Geraniltransferase/genética , Farnesiltranstransferase/metabolismo , Farnesiltranstransferase/genética , Pirofosfatases/metabolismo , Pirofosfatases/genética , Processamento de Proteína Pós-Traducional , Plantas Geneticamente Modificadas , Folhas de Planta/metabolismo , Folhas de Planta/genética
2.
Plant Cell ; 34(5): 1745-1767, 2022 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-34791448

RESUMO

Primary metabolism provides energy for growth and development as well as secondary metabolites for diverse environmental responses. Here we describe an unexpected consequence of disruption of a glycolytic enzyme enolase named LOW EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 2 (LOS2) in causing constitutive defense responses or autoimmunity in Arabidopsis thaliana. The autoimmunity in the los2 mutant is accompanied by a higher expression of about one-quarter of intracellular immune receptor nucleotide-binding leucine-rich repeat (NLR) genes in the genome and is partially dependent on one of these NLR genes. The LOS2 gene was hypothesized to produce an alternatively translated protein c-Myc Binding Protein (MBP-1) that functions as a transcriptional repressor. Complementation tests show that LOS2 executes its function in growth and immunity regulation through the canonical enolase activity but not the production of MBP-1. In addition, the autoimmunity in the los2 mutants leads to a higher accumulation of sugars and organic acids and a depletion of glycolytic metabolites. These findings indicate that LOS2 does not exert its function in immune responses through an alternatively translated protein MBP-1. Rather, they show that a perturbation of glycolysis from the reduction of the enolase activity results in activation of NLR-involved immune responses which further influences primary metabolism and plant growth, highlighting the complex interaction between primary metabolism and plant immunity.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Glicólise/genética , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Imunidade Vegetal/genética
3.
Plant Physiol ; 192(3): 2123-2142, 2023 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-37067900

RESUMO

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.


Assuntos
Malus , Malus/metabolismo , Fosforilação , Fatores de Transcrição/metabolismo , Metabolismo dos Carboidratos/genética , Sorbitol/farmacologia , Sorbitol/metabolismo , Sacarose/metabolismo , Regulação da Expressão Gênica de Plantas
4.
Plant Physiol ; 193(1): 643-660, 2023 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-37233026

RESUMO

Chromoplasts are plant organelles with a unique ability to sequester and store massive carotenoids. Chromoplasts have been hypothesized to enable high levels of carotenoid accumulation due to enhanced sequestration ability or sequestration substructure formation. However, the regulators that control the substructure component accumulation and substructure formation in chromoplasts remain unknown. In melon (Cucumis melo) fruit, ß-carotene accumulation in chromoplasts is governed by ORANGE (OR), a key regulator for carotenoid accumulation in chromoplasts. By using comparative proteomic analysis of a high ß-carotene melon variety and its isogenic line low-ß mutant that is defective in CmOr with impaired chromoplast formation, we identified carotenoid sequestration protein FIBRILLIN1 (CmFBN1) as differentially expressed. CmFBN1 expresses highly in melon fruit tissue. Overexpression of CmFBN1 in transgenic Arabidopsis (Arabidopsis thaliana) containing ORHis that genetically mimics CmOr significantly enhances carotenoid accumulation, demonstrating its involvement in CmOR-induced carotenoid accumulation. Both in vitro and in vivo evidence showed that CmOR physically interacts with CmFBN1. Such an interaction occurs in plastoglobules and results in promoting CmFBN1 accumulation. CmOR greatly stabilizes CmFBN1, which stimulates plastoglobule proliferation and subsequently carotenoid accumulation in chromoplasts. Our findings show that CmOR directly regulates CmFBN1 protein levels and suggest a fundamental role of CmFBN1 in facilitating plastoglobule proliferation for carotenoid sequestration. This study also reveals an important genetic tool to further enhance OR-induced carotenoid accumulation in chromoplasts in crops.


Assuntos
Arabidopsis , Cucurbitaceae , beta Caroteno/metabolismo , Cucurbitaceae/metabolismo , Fibrilinas/metabolismo , Proteômica , Carotenoides/metabolismo , Plastídeos/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Frutas/genética
5.
New Phytol ; 239(3): 1014-1034, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-36747049

RESUMO

Malic acid accumulation in the vacuole largely determines acidity and perception of sweetness of apple. It has long been observed that reduction in malate level is associated with increase in ethylene production during the ripening process of climacteric fruits, but the molecular mechanism linking ethylene to malate reduction is unclear. Here, we show that ethylene-modulated WRKY transcription factor 31 (WRKY31)-Ethylene Response Factor 72 (ERF72)-ALUMINUM ACTIVATED MALATE TRANSPORTER 9 (Ma1) network regulates malate accumulation in apple fruit. ERF72 binds to the promoter of ALMT9, a key tonoplast transporter for malate accumulation of apple, transcriptionally repressing ALMT9 expression in response to ethylene. WRKY31 interacts with ERF72, suppressing its transcriptional inhibition activity on ALMT9. In addition, WRKY31 directly binds to the promoters of ERF72 and ALMT9, transcriptionally repressing and activating ERF72 and ALMT9, respectively. The expression of WRKY31 decreases in response to ethylene, lowering the transcription of ALMT9 directly and via its interactions with ERF72. These findings reveal that the regulatory complex WRKY31 forms with ERF72 responds to ethylene, linking the ethylene signal to ALMT9 expression in reducing malate transport into the vacuole during fruit ripening.


Assuntos
Malus , Malus/genética , Malus/metabolismo , Malatos/metabolismo , Alumínio/metabolismo , Frutas/genética , Frutas/metabolismo , Etilenos/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
6.
Plant Cell ; 32(2): 449-469, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31826966

RESUMO

Rapid pollen tube growth requires uptake of Suc or its hydrolytic products, hexoses, from the apoplast of surrounding tissues in the style. Due to species-specific sugar requirements, reliance of pollen germination and tube growth on cell wall invertase and Suc or hexose transporters varies between species, but it is not known if plants have a sugar transporter that mediates the uptake of both hexose and Suc for pollen tube growth. Here, we show that a sugar transporter protein in apple (Malus domestica), MdSTP13a, takes up both hexose and Suc when expressed in yeast, and is essential for pollen tube growth on Glc and Suc but not on maltose. MdSTP13a-mediated direct uptake of Suc is primarily responsible for apple pollen tube growth on Suc medium. Sorbitol, a major photosynthate and transport carbohydrate in apple, modulates pollen tube growth via the MYB transcription factor MdMYB39L, which binds to the promoter of MdSTP13a to activate its expression. Antisense repression of MdSTP13a blocks sorbitol-modulated pollen tube growth. These findings demonstrate that MdSTP13a takes up both hexose and Suc for sorbitol-modulated pollen tube growth in apple, revealing a situation where acquisition of sugars for pollen tube growth is regulated by a sugar alcohol.


Assuntos
Transporte Biológico/fisiologia , Hexoses/metabolismo , Malus/metabolismo , Proteínas de Plantas/metabolismo , Tubo Polínico/crescimento & desenvolvimento , Sorbitol/metabolismo , Sacarose/metabolismo , Transporte Biológico/genética , Regulação da Expressão Gênica de Plantas , Maltose/metabolismo , Malus/genética , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Tubo Polínico/genética , Polinização/genética , Polinização/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Simportadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , beta-Frutofuranosidase/metabolismo
7.
Int J Mol Sci ; 23(20)2022 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-36293472

RESUMO

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.


Assuntos
Fabaceae , Malus , Antocianinas/metabolismo , Clorofila A , Malus/genética , Malus/metabolismo , Antioxidantes/metabolismo , Dióxido de Carbono , Fotossíntese/fisiologia , Clorofila , Folhas de Planta/metabolismo , Luz , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Fabaceae/metabolismo , Xantofilas/metabolismo
8.
Plant Physiol ; 182(2): 992-1006, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31772076

RESUMO

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.


Assuntos
Frutas/genética , Frutas/metabolismo , Malatos/metabolismo , Malus/genética , Proteínas de Plantas/metabolismo , Vacúolos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico/genética , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Malus/metabolismo , Mutação , Oócitos/metabolismo , Oócitos/fisiologia , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Domínios Proteicos , Interferência de RNA , Nicotiana/metabolismo , Nicotiana/fisiologia , Vacúolos/genética , Vacúolos/fisiologia , Xenopus laevis
9.
Plant Physiol ; 183(2): 750-764, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32241879

RESUMO

Excessive application of nitrate, an essential macronutrient and a signal regulating diverse physiological processes, decreases malate accumulation in apple (Malus domestica) fruit, but the underlying mechanism remains poorly understood. Here, we show that an apple BTB/TAZ protein, MdBT2, is involved in regulating malate accumulation and vacuolar pH in response to nitrate. In vitro and in vivo assays indicate that MdBT2 interacts directly with and ubiquitinates a bHLH transcription factor, MdCIbHLH1, via the ubiquitin/26S proteasome pathway in response to nitrate. This ubiquitination results in the degradation of MdCIbHLH1 protein and reduces the transcription of MdCIbHLH1-targeted genes involved in malate accumulation and vacuolar acidification, including MdVHA-A, which encodes a vacuolar H+-ATPase, and MdVHP1, which encodes a vacuolar H+-pyrophosphatase, as well as MdALMT9, which encodes an aluminum-activated malate transporter. A series of transgenic analyses in apple materials including fruits, plantlets, and calli demonstrate that MdBT2 controls nitrate-mediated malate accumulation and vacuolar pH at least partially, if not completely, via regulating the MdCIbHLH1 protein level. Taken together, these findings reveal that MdBT2 regulates the stability of MdCIbHLH1 via ubiquitination in response to nitrate, which in succession transcriptionally reduces the expression of malate-associated genes, thereby controlling malate accumulation and vacuolar acidification in apples under high nitrate supply.


Assuntos
Malatos/metabolismo , Nitratos/farmacologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Pirofosfatase Inorgânica/genética , Pirofosfatase Inorgânica/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo
10.
Plant Cell ; 30(7): 1562-1581, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29871985

RESUMO

In plant-microbe interactions, plant sugars produced by photosynthesis are not only a carbon source for pathogens, but may also act as signals that modulate plant defense responses. Here, we report that decreasing sorbitol synthesis in apple (Malus domestica) leaves by antisense suppression of ALDOSE-6-PHOSPHATE REDUCTASE (A6PR) leads to downregulation of 56 NUCLEOTIDE BINDING/LEUCINE-RICH REPEAT (NLR) genes and converts the phenotypic response to Alternaria alternata from resistant to susceptible. We identified a resistance protein encoded by the apple MdNLR16 gene and a small protein encoded by the fungal HRIP1 gene that interact in both a yeast two-hybrid assay and a bimolecular fluorescence complementation assay. Deletion of HRIP1 in A. alternata enables gain of virulence on the wild-type control plant. Overexpression of MdNLR16 in two antisense A6PR lines increases resistance, whereas RNAi suppression of MdNLR16 in the wild-type control decreases resistance against A. alternata MdWRKY79 transcriptionally regulates MdNLR16 by binding to the promoter of MdNLR16 in response to sorbitol, and exogenous sorbitol feeding partially restores resistance of the antisense A6PR lines to A. alternata These findings indicate that sorbitol modulates resistance to A. alternata via the MdNLR16 protein that interacts with the fungal effector in a classic gene-for-gene manner in apple.


Assuntos
Alternaria/patogenicidade , Malus/metabolismo , Malus/microbiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Sorbitol/farmacologia , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Malus/genética , Proteínas de Plantas/genética
11.
Plant Biotechnol J ; 18(2): 540-552, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31350935

RESUMO

Sugar transporters are necessary to transfer hexose from cell wall spaces into parenchyma cells to boost hexose accumulation to high concentrations in fruit. Here, we have identified an apple hexose transporter (HTs), MdHT2.2, located in the plasma membrane, which is highly expressed in mature fruit. In a yeast system, the MdHT2.2 protein exhibited high 14 C-fructose and 14 C-glucose transport activity. In transgenic tomato heterologously expressing MdHT2.2, the levels of both fructose and glucose increased significantly in mature fruit, with sugar being unloaded via the apoplastic pathway, but the level of sucrose decreased significantly. Analysis of enzyme activity and the expression of genes related to sugar metabolism and transport revealed greatly up-regulated expression of SlLIN5, a key gene encoding cell wall invertase (CWINV), as well as increased CWINV activity in tomatoes transformed with MdHT2.2. Moreover, the levels of fructose, glucose and sucrose recovered nearly to those of the wild type in the sllin5-edited mutant of the MdHT2.2-expressing lines. However, the overexpression of MdHT2.2 decreased hexose levels and increased sucrose levels in mature leaves and young fruit, suggesting that the response pathway for the apoplastic hexose signal differs among tomato tissues. The present study identifies a new HTs in apple that is able to take up fructose and glucose into cells and confirms that the apoplastic hexose levels regulated by HT controls CWINV activity to alter carbohydrate partitioning and sugar content.


Assuntos
Frutas , Malus , Proteínas de Plantas , Solanum lycopersicum , Parede Celular/enzimologia , Frutas/química , Frutas/genética , Solanum lycopersicum/química , Solanum lycopersicum/genética , Malus/genética , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Açúcares/metabolismo , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismo
12.
New Phytol ; 228(6): 1897-1913, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32712992

RESUMO

Climate-driven phenological change across local spatial gradients leads to leaf shape variation. At higher elevations, leaves of broadleaf species tend to become narrower, but the underlying molecular mechanism is largely unknown. In this study, a series of morphometric analyses and biochemical assays, combined with functional identification in apple, were performed. We show that the decrease in apple leaf width with increasing altitude is controlled by a basic/helix-loop-helix transcription factor (bHLH TF), MdbHLH3. The MdbHLH3-overexpressing lines have a lower transcript abundance of MdPIN1 encoding an auxin efflux carrier but a higher transcript abundance of MdGH3-2 encoding a putative auxin amido conjugate synthase, resulting in a lower free auxin concentration; feeding the transgenic leaves with exogenous auxin partially restores leaf width. MdbHLH3 transcriptionally suppresses and activates MdPIN1 and MdGH3-2, respectively, by specifically binding to their promoters. This alters auxin homeostasis and transport, consequently leading to changes in leaf shape. These findings suggest that the bHLH TF MdbHLH3 directly modulates auxin signaling in controlling leaf shape in response to local spatial gradients in apple.


Assuntos
Malus , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos , Malus/genética , Malus/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
13.
Plant Physiol ; 180(4): 1988-2003, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31221734

RESUMO

Carotenoids exert multifaceted roles to plants and are critically important to humans. Phytoene synthase (PSY) is a major rate-limiting enzyme in the carotenoid biosynthetic pathway. PSY in plants is normally found as a small enzyme family with up to three members. However, knowledge of PSY isoforms in relation to their respective enzyme activities and amino acid residues that are important for PSY activity is limited. In this study, we focused on two tomato (Solanum lycopersicum) PSY isoforms, PSY1 and PSY2, and investigated their abilities to catalyze carotenogenesis via heterologous expression in transgenic Arabidopsis (Arabidopsis thaliana) and bacterial systems. We found that the fruit-specific PSY1 was less effective in promoting carotenoid biosynthesis than the green tissue-specific PSY2. Examination of the PSY proteins by site-directed mutagenesis analysis and three-dimensional structure modeling revealed two key amino acid residues responsible for this activity difference and identified a neighboring aromatic-aromatic combination in one of the PSY core structures as being crucial for high PSY activity. Remarkably, this neighboring aromatic-aromatic combination is evolutionarily conserved among land plant PSYs except PSY1 of tomato and potato (Solanum tuberosum). Strong transcription of tomato PSY1 likely evolved as compensation for its weak enzyme activity to allow for the massive carotenoid biosynthesis in ripe fruit. This study provides insights into the functional divergence of PSY isoforms and highlights the potential to rationally design PSY for the effective development of carotenoid-enriched crops.


Assuntos
Frutas/metabolismo , Geranil-Geranildifosfato Geranil-Geraniltransferase/metabolismo , Proteínas de Plantas/metabolismo , Solanum lycopersicum/metabolismo , Solanum tuberosum/metabolismo , Frutas/enzimologia , Frutas/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Geranil-Geranildifosfato Geranil-Geraniltransferase/genética , Solanum lycopersicum/enzimologia , Proteínas de Plantas/genética , Solanum tuberosum/enzimologia , Solanum tuberosum/genética
14.
Plant Physiol ; 192(3): 1659-1665, 2023 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-37148289
16.
New Phytol ; 217(2): 641-656, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29027668

RESUMO

Sugars produced by photosynthesis not only fuel plant growth and development, but may also act as signals to regulate plant growth and development. This work focuses on the role of sorbitol, a sugar alcohol, in flower development and pollen tube growth of apple (Malus domestica). Transgenic 'Greensleeves' apple trees with decreased sorbitol synthesis had abnormal stamen development, a decreased pollen germination rate and reduced pollen tube growth, which were all closely related to lower sorbitol concentrations in stamens. RNA sequencing and quantitative RT-PCR analyses identified reduced transcript levels during stamen development and pollen tube growth in the transgenic trees of a stamen-specific MYB39-like transcription factor, MdMYB39L, and of its putative target genes involved in hexose uptake, cell wall formation and microsporogenesis. Suppressing MdMYB39L expression in pollen via antisense oligonucleotide transfection significantly reduced the expression of its putative target genes and pollen tube growth. Exogenous sorbitol application during flower development partially restored MdMYB39L expression, stamen development, and pollen germination and tube growth of the transgenic trees. Addition of sorbitol to the germination medium also partially restored pollen germination and tube growth of the transgenic trees. We conclude that sorbitol plays an essential role in stamen development and pollen tube growth via MdMYB39L in apple.


Assuntos
Malus/crescimento & desenvolvimento , Malus/metabolismo , Proteínas de Plantas/metabolismo , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/metabolismo , Sorbitol/metabolismo , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Germinação , Malus/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Tubo Polínico/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
17.
Plant Physiol ; 170(3): 1315-30, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26637549

RESUMO

Tonoplast transporters, including proton pumps and secondary transporters, are essential for plant cell function and for quality formation of fleshy fruits and ornamentals. Vacuolar transport of anthocyanins, malate, and other metabolites is directly or indirectly dependent on the H(+)-pumping activities of vacuolar H(+)-ATPase (VHA) and/or vacuolar H(+)-pyrophosphatase, but how these proton pumps are regulated in modulating vacuolar transport is largely unknown. Here, we report a transcription factor, MdMYB1, in apples that binds to the promoters of two genes encoding the B subunits of VHA, MdVHA-B1 and MdVHA-B2, to transcriptionally activate its expression, thereby enhancing VHA activity. A series of transgenic analyses in apples demonstrates that MdMYB1/10 controls cell pH and anthocyanin accumulation partially by regulating MdVHA-B1 and MdVHA-B2. Furthermore, several other direct target genes of MdMYB10 are identified, including MdVHA-E2, MdVHP1, MdMATE-LIKE1, and MdtDT, which are involved in H(+)-pumping or in the transport of anthocyanins and malates into vacuoles. Finally, we show that the mechanism by which MYB controls malate and anthocyanin accumulation in apples also operates in Arabidopsis (Arabidopsis thaliana). These findings provide novel insights into how MYB transcription factors directly modulate the vacuolar transport system in addition to anthocyanin biosynthesis, consequently controlling organ coloration and cell pH in plants.


Assuntos
Antocianinas/metabolismo , Malatos/metabolismo , Malus/metabolismo , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Sítios de Ligação/genética , Transporte Biológico Ativo , Genes de Plantas , Concentração de Íons de Hidrogênio , Malus/genética , Modelos Biológicos , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Subunidades Proteicas , Fatores de Transcrição/genética , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Vacúolos/metabolismo
18.
J Exp Bot ; 67(17): 5145-57, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27535992

RESUMO

Understanding the fruit developmental process is critical for fruit quality improvement. Here, we report a comprehensive proteomic analysis of apple fruit development over five growth stages, from young fruit to maturity, coupled with metabolomic profiling. A tandem mass tag (TMT)-based comparative proteomics approach led to the identification and quantification of 7098 and 6247 proteins, respectively. This large-scale proteomic dataset presents a global view of the critical pathways involved in fruit development and metabolism. When linked with metabolomics data, these results provide new insights into the modulation of fruit development, the metabolism and storage of sugars and organic acids (mainly malate), and events within the energy-related pathways for respiration and glycolysis. We suggest that the key steps identified here (e.g. those involving the FK2, TST, EDR6, SPS, mtME and mtMDH switches), can be further targeted to confirm their roles in accumulation and balance of fructose, sucrose and malate. Moreover, our findings imply that the primary reason for decreases in amino acid concentrations during fruit development is related to a reduction in substrate flux via glycolysis, which is mainly regulated by fructose-bisphosphate aldolase and bisphosphoglycerate mutase.


Assuntos
Metabolismo dos Carboidratos , Frutas/crescimento & desenvolvimento , Malus/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Aminoácidos/metabolismo , Frutas/metabolismo , Malatos/metabolismo , Malus/metabolismo , Redes e Vias Metabólicas/fisiologia , Metabolômica/métodos , Proteínas de Plantas/fisiologia , Proteômica/métodos
19.
BMC Genomics ; 16: 612, 2015 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-26276125

RESUMO

BACKGROUND: Acidity is a major contributor to fruit quality. Several organic acids are present in apple fruit, but malic acid is predominant and determines fruit acidity. The trait is largely controlled by the Malic acid (Ma) locus, underpinning which Ma1 that putatively encodes a vacuolar aluminum-activated malate transporter1 (ALMT1)-like protein is a strong candidate gene. We hypothesize that fruit acidity is governed by a gene network in which Ma1 is key member. The goal of this study is to identify the gene network and the potential mechanisms through which the network operates. RESULTS: Guided by Ma1, we analyzed the transcriptomes of mature fruit of contrasting acidity from six apple accessions of genotype Ma_ (MaMa or Mama) and four of mama using RNA-seq and identified 1301 fruit acidity associated genes, among which 18 were most significant acidity genes (MSAGs). Network inferring using weighted gene co-expression network analysis (WGCNA) revealed five co-expression gene network modules of significant (P < 0.001) correlation with malate. Of these, the Ma1 containing module (Turquoise) of 336 genes showed the highest correlation (0.79). We also identified 12 intramodular hub genes from each of the five modules and 18 enriched gene ontology (GO) terms and MapMan sub-bines, including two GO terms (GO:0015979 and GO:0009765) and two MapMap sub-bins (1.3.4 and 1.1.1.1) related to photosynthesis in module Turquoise. Using Lemon-Tree algorithms, we identified 12 regulator genes of probabilistic scores 35.5-81.0, including MDP0000525602 (a LLR receptor kinase), MDP0000319170 (an IQD2-like CaM binding protein) and MDP0000190273 (an EIN3-like transcription factor) of greater interest for being one of the 18 MSAGs or one of the 12 intramodular hub genes in Turquoise, and/or a regulator to the cluster containing Ma1. CONCLUSIONS: The most relevant finding of this study is the identification of the MSAGs, intramodular hub genes, enriched photosynthesis related processes, and regulator genes in a WGCNA module Turquoise that not only encompasses Ma1 but also shows the highest modular correlation with acidity. Overall, this study provides important insight into the Ma1-mediated gene network controlling acidity in mature apple fruit of diverse genetic background.


Assuntos
Frutas/genética , Redes Reguladoras de Genes , Malatos/metabolismo , Malus/genética , Algoritmos , Frutas/metabolismo , Perfilação da Expressão Gênica , Ontologia Genética , Genes de Plantas , Malus/metabolismo , Análise de Sequência de RNA
20.
Plant Cell Physiol ; 56(9): 1748-61, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26076968

RESUMO

Sorbitol is a major product of photosynthesis in apple (Malus domestica) that is involved in carbohydrate metabolism and stress tolerance. However, little is known about how the global transcript levels in apple leaves respond to decreased sorbitol synthesis. In this study we used RNA sequencing (RNA-seq) profiling to characterize the transcriptome of leaves from transgenic lines of the apple cultivar 'Greensleeves' exhibiting suppressed expression of aldose-6-phosphate reductase (A6PR) to gain insights into sorbitol function and the consequences of decreased sorbitol synthesis on gene expression. We observed that, although the leaves of the low sorbitol transgenic lines accumulate higher levels of various primary metabolites, only very limited changes were found in the levels of transcripts associated with primary metabolism. We suggest that this is indicative of post-transcriptional and/or post-translational regulation of primary metabolite accumulation and central carbon metabolism. However, we identified significantly enriched gene ontology terms belonging to the 'stress related process' category in the antisense lines (P-value < 0.05). These include genes involved in the synthesis/degradation of abscisic acid, salicylic acid and jasmonic acid, nucleotide-binding site leucine-rich repeat (NBS-LRR) disease resistance genes and ATP-binding cassette (ABC) transporter genes. This suggests that sorbitol plays a role in the responses of apple trees to abiotic and biotic stresses.


Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Malus/genética , Malus/fisiologia , Sorbitol/metabolismo , Estresse Fisiológico/genética , Ácido Abscísico/biossíntese , Análise por Conglomerados , Ontologia Genética , Redes e Vias Metabólicas/genética , Fotossíntese/genética , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Análise de Componente Principal , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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