RESUMO
The pyrenoid is a chloroplastic microcompartment in which most algae and some terrestrial plants condense the primary carboxylase, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) as part of a CO2-concentrating mechanism that improves the efficiency of CO2 capture. Engineering a pyrenoid-based CO2-concentrating mechanism (pCCM) into C3 crop plants is a promising strategy to enhance yield capacities and resilience to the changing climate. Many pyrenoids are characterized by a sheath of starch plates that is proposed to act as a barrier to limit CO2 diffusion. Recently, we have reconstituted a phase-separated "proto-pyrenoid" Rubisco matrix in the model C3 plant Arabidopsis thaliana using proteins from the alga with the most well-studied pyrenoid, Chlamydomonas reinhardtii [N. Atkinson, Y. Mao, K. X. Chan, A. J. McCormick, Nat. Commun. 11, 6303 (2020)]. Here, we describe the impact of introducing the Chlamydomonas proteins StArch Granules Abnormal 1 (SAGA1) and SAGA2, which are associated with the regulation of pyrenoid starch biogenesis and morphology. We show that SAGA1 localizes to the proto-pyrenoid in engineered Arabidopsis plants, which results in the formation of atypical spherical starch granules enclosed within the proto-pyrenoid condensate and adjacent plate-like granules that partially cover the condensate, but without modifying the total amount of chloroplastic starch accrued. Additional expression of SAGA2 further increases the proportion of starch synthesized as adjacent plate-like granules that fully encircle the proto-pyrenoid. Our findings pave the way to assembling a diffusion barrier as part of a functional pCCM in vascular plants, while also advancing our understanding of the roles of SAGA1 and SAGA2 in starch sheath formation and broadening the avenues for engineering starch morphology.
Assuntos
Arabidopsis , Chlamydomonas reinhardtii , Arabidopsis/genética , Arabidopsis/metabolismo , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Dióxido de Carbono/metabolismo , Cloroplastos/metabolismo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Fotossíntese , Amido/metabolismoRESUMO
The plastidial α-glucan phosphorylase (PHS1) can elongate and degrade maltooligosaccharides (MOSs), but its exact physiological role in plants is poorly understood. Here, we discover a specialized role of PHS1 in establishing the unique bimodal characteristic of starch granules in wheat (Triticum spp.) endosperm. Wheat endosperm contains large A-type granules that initiate at early grain development and small B-type granules that initiate in later grain development. We demonstrate that PHS1 interacts with B-GRANULE CONTENT1 (BGC1), a carbohydrate-binding protein essential for normal B-type granule initiation. Mutants of tetraploid durum wheat (Triticum turgidum) deficient in all homoeologs of PHS1 had normal A-type granules but fewer and larger B-type granules. Grain size and starch content were not affected by the mutations. Further, by assessing granule numbers during grain development in the phs1 mutant and using a double mutant defective in both PHS1 and BGC1, we demonstrate that PHS1 is exclusively involved in B-type granule initiation. The total starch content and number of starch granules per chloroplast in leaves were not affected by loss of PHS1, suggesting that its role in granule initiation in wheat is limited to the endosperm. We therefore propose that the initiation of A- and B-type granules occurs via distinct biochemical mechanisms, where PHS1 plays an exclusive role in B-type granule initiation.
Assuntos
Endosperma , Triticum , Endosperma/genética , Endosperma/metabolismo , Triticum/genética , Triticum/metabolismo , Amido/metabolismo , Plastídeos/metabolismo , Cloroplastos/metabolismo , Grão ComestívelRESUMO
Molecular factors that contribute to the diverse spatial and temporal patterns of starch granule initiation between species and organs are poorly understood. Wheat (Triticum sp.) endosperm contains both large A-type granules initiated during early grain development and small B-type granules that initiate about 10-15 days later. Here we identify that the MYOSIN-RESEMBLING CHLOROPLAST PROTEIN (MRC) is required for the correct timing of B-type granule initiation in wheat endosperm during grain development. MRC is expressed in the endosperm exclusively in early grain development, before B-type granule initiation. We isolated three independent TILLING mutants of tetraploid wheat (Triticum turgidum cv. Kronos) with premature stop or missense mutations in the A-genome homoeolog, which we showed to be the only active homoeolog in tetraploid wheat due to a disruption of the B-genome homoeolog. The mrc mutants had significantly smaller A-type granules and a higher relative volume of B-type granules in the endosperm than the wild type. Whereas B-type granules initiated 15 - 20 days post anthesis (dpa) in the wild type, they appeared as early as 10 dpa in the mrc-1 mutant. These results suggest a temporal role for MRC in repressing B-type granule initiation, providing insight into how the distinct biochemical mechanisms that control A- and B-type granule initiation are regulated. This role of MRC in the wheat endosperm is distinct from the previously described role of Arabidopsis (Arabidopsis thaliana) MRC in promoting granule initiation in leaves, providing an example of functional diversification among granule initiation proteins.
RESUMO
BACKGROUND: Durum wheat (Triticum turgidum subsp. durum) is widely grown for pasta production, and more recently, is gaining additional interest due to its resilience to warm, dry climates and its use as an experimental model for wheat research. Like in bread wheat, the starch and protein accumulated in the endosperm during grain development are the primary contributors to the calorific value of durum grains. RESULTS: To enable further research into endosperm development and storage reserve synthesis, we generated a high-quality transcriptomics dataset from developing endosperms of variety Kronos, to complement the extensive mutant resources available for this variety. Endosperms were dissected from grains harvested at eight timepoints during grain development (6 to 30 days post anthesis (dpa)), then RNA sequencing was used to profile the transcriptome at each stage. The largest changes in gene expression profile were observed between the earlier timepoints, prior to 15 dpa. We detected a total of 29,925 genes that were significantly differentially expressed between at least two timepoints, and clustering analysis revealed nine distinct expression patterns. We demonstrate the potential of our dataset to provide new insights into key processes that occur during endosperm development, using starch metabolism as an example. CONCLUSION: We provide a valuable resource for studying endosperm development in this increasingly important crop species.
Assuntos
Endosperma , Triticum , Endosperma/genética , Endosperma/metabolismo , Triticum/metabolismo , Transcriptoma , Grão Comestível , Amido/metabolismoRESUMO
The determination of starch granule morphology in plants is poorly understood. The amyloplasts of wheat endosperm contain large discoid A-type granules and small spherical B-type granules. To study the influence of amyloplast structure on these distinct morphological types, we isolated a mutant in durum wheat (Triticum turgidum) defective in the plastid division protein PARC6, which had giant plastids in both leaves and endosperm. Endosperm amyloplasts of the mutant contained more A- and B-type granules than those of the wild-type. The mutant had increased A- and B-type granule size in mature grains, and its A-type granules had a highly aberrant, lobed surface. This morphological defect was already evident at early stages of grain development and occurred without alterations in polymer structure and composition. Plant growth and grain size, number and starch content were not affected in the mutants despite the large plastid size. Interestingly, mutation of the PARC6 paralog, ARC6, did not increase plastid or starch granule size. We suggest TtPARC6 can complement disrupted TtARC6 function by interacting with PDV2, the outer plastid envelope protein that typically interacts with ARC6 to promote plastid division. We therefore reveal an important role of amyloplast structure in starch granule morphogenesis in wheat.
Assuntos
Endosperma , Triticum , Endosperma/genética , Endosperma/metabolismo , Triticum/genética , Triticum/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Amido/metabolismo , Plastídeos/genética , Plastídeos/metabolismo , Mutação/genéticaRESUMO
What determines the number of starch granules in plastids is an enigmatic aspect of starch metabolism. Several structurally and functionally diverse proteins have been implicated in the granule initiation process in Arabidopsis (Arabidopsis thaliana), with each protein exerting a varying degree of influence. Here, we show that a conserved starch synthase-like protein, STARCH SYNTHASE5 (SS5), regulates the number of starch granules that form in Arabidopsis chloroplasts. Among the starch synthases, SS5 is most closely related to SS4, a major determinant of granule initiation and morphology. However, unlike SS4 and the other starch synthases, SS5 is a noncanonical isoform that lacks catalytic glycosyltransferase activity. Nevertheless, loss of SS5 reduces starch granule numbers that form per chloroplast in Arabidopsis, and ss5 mutant starch granules are larger than wild-type granules. Like SS4, SS5 has a conserved putative surface binding site for glucans and also interacts with MYOSIN-RESEMBLING CHLOROPLAST PROTEIN, a proposed structural protein influential in starch granule initiation. Phenotypic analysis of a suite of double mutants lacking both SS5 and other proteins implicated in starch granule initiation allows us to propose how SS5 may act in this process.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas de Cloroplastos/metabolismo , Glicosiltransferases/metabolismo , Sintase do Amido/metabolismo , Amido/metabolismo , Proteínas de Arabidopsis/química , Sítios de Ligação , Proteínas de Cloroplastos/química , Cloroplastos/metabolismo , Sequência Conservada , Glucanos/metabolismo , Glicosiltransferases/química , Modelos Moleculares , Mutação/genética , Fenótipo , Folhas de Planta/enzimologia , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Sintase do Amido/químicaRESUMO
Recent work has identified several proteins involved in starch granule initiation, the first step of starch synthesis. However, the degree of conservation in the granule initiation process remains poorly understood, especially among grass species differing in patterns of carbohydrate turnover in leaves, and granule morphology in the endosperm. We therefore compared mutant phenotypes of Hordeum vulgare (barley), Triticum turgidum (durum wheat), and Brachypodium distachyon defective in PROTEIN TARGETING TO STARCH 2 (PTST2), a key granule initiation protein. We report striking differences across species and organs. Loss of PTST2 from leaves resulted in fewer, larger starch granules per chloroplast and normal starch content in wheat, fewer granules per chloroplast and lower starch content in barley, and almost complete loss of starch in Brachypodium. The loss of starch in Brachypodium leaves was accompanied by high levels of ADP-glucose and detrimental effects on growth and physiology. Additionally, we found that loss of PTST2 increased granule initiation in Brachypodium amyloplasts, resulting in abnormal compound granule formation throughout the seed. These findings suggest that the importance of PTST2 varies greatly with the genetic and developmental background and inform the extent to which the gene can be targeted to improve starch in crops.
Assuntos
Brachypodium , Hordeum , Sintase do Amido , Amido/metabolismo , Sintase do Amido/genética , Endosperma/metabolismo , Hordeum/genética , Hordeum/metabolismo , Triticum/genética , Triticum/metabolismo , Glucose/metabolismo , Difosfato de Adenosina/metabolismoRESUMO
In Arabidopsis (Arabidopsis thaliana) leaves, starch is synthesized during the day and degraded at night to fuel growth and metabolism. Starch is degraded primarily by ß-amylases, liberating maltose, but this activity is preceded by glucan phosphorylation and is accompanied by dephosphorylation. A glucan phosphatase family member, LIKE SEX4 1 (LSF1), binds starch and is required for normal starch degradation, but its exact role is unclear. Here, we show that LSF1 does not dephosphorylate glucans. The recombinant dual specificity phosphatase (DSP) domain of LSF1 had no detectable phosphatase activity. Furthermore, a variant of LSF1 mutated in the catalytic cysteine of the DSP domain complemented the starch-excess phenotype of the lsf1 mutant. By contrast, a variant of LSF1 with mutations in the carbohydrate binding module did not complement lsf1 Thus, glucan binding, but not phosphatase activity, is required for the function of LSF1 in starch degradation. LSF1 interacts with the ß-amylases BAM1 and BAM3, and the BAM1-LSF1 complex shows amylolytic but not glucan phosphatase activity. Nighttime maltose levels are reduced in lsf1, and genetic analysis indicated that the starch-excess phenotype of lsf1 is dependent on bam1 and bam3 We propose that LSF1 binds ß-amylases at the starch granule surface, thereby promoting starch degradation.
Assuntos
Arabidopsis/metabolismo , Metabolismo dos Carboidratos/fisiologia , Fosfatases de Especificidade Dupla/metabolismo , Amido/metabolismo , beta-Amilase/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Metabolismo dos Carboidratos/genética , Proteínas de Transporte , Clonagem Molecular , Fosfatases de Especificidade Dupla/genética , Regulação da Expressão Gênica de Plantas , Glucanos/metabolismo , Fosforilação , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes , Alinhamento de Sequência , Nicotiana/genética , Nicotiana/metabolismo , beta-Amilase/genéticaRESUMO
Starch granule initiation is poorly understood at the molecular level. The glucosyltransferase, STARCH SYNTHASE 4 (SS4), plays a central role in granule initiation in Arabidopsis leaves, but its function in cereal endosperms is unknown. We investigated the role of SS4 in wheat, which has a distinct spatiotemporal pattern of granule initiation during grain development. We generated TILLING mutants in tetraploid wheat (Triticum turgidum) that are defective in both SS4 homoeologs. The morphology of endosperm starch was examined in developing and mature grains. SS4 deficiency led to severe alterations in endosperm starch granule morphology. During early grain development, while the wild-type initiated single 'A-type' granules per amyloplast, most amyloplasts in the mutant formed compound granules due to multiple initiations. This phenotype was similar to mutants deficient in B-GRANULE CONTENT 1 (BGC1). SS4 deficiency also reduced starch content in leaves and pollen grains. We propose that SS4 and BGC1 are required for the proper control of granule initiation during early grain development that leads to a single A-type granule per amyloplast. The absence of either protein results in a variable number of initiations per amyloplast and compound granule formation.
Assuntos
Sintase do Amido , Endosperma/genética , Proteínas de Plantas/genética , Plastídeos/genética , Amido , Sintase do Amido/genética , Triticum/genéticaRESUMO
Starch granules contain two Glc polymers, amylopectin and amylose. Amylose makes up approximately 10% to 30% (w/w) of all natural starches thus far examined, but mutants of crop and model plants that produce amylose-free starch are generally indistinguishable from their wild-type counterparts with respect to growth, starch content, and granule morphology. Since the function and adaptive significance of amylose are unknown, we asked whether there is natural genetic variation in amylose synthesis within a wild, uncultivated species. We examined polymorphisms among the 1,135 sequenced accessions of Arabidopsis (Arabidopsis thaliana) in GRANULE-BOUND STARCH SYNTHASE (GBSS), encoding the enzyme responsible for amylose synthesis. We identified 18 accessions that are predicted to have polymorphisms in GBSS that affect protein function, and five of these accessions produced starch with no or extremely low amylose (< 0.5% [w/w]). Eight further accessions had amylose contents that were significantly lower or higher than that of Col-0 (9% [w/w]), ranging from 5% to 12% (w/w). We examined the effect of the polymorphisms on GBSS function and uncovered three mechanisms by which GBSS sequence variation led to different amylose contents: (1) altered GBSS abundance, (2) altered GBSS activity, and (3) altered affinity of GBSS for binding PROTEIN TARGETING TO STARCH1-a protein that targets GBSS to starch granules. These findings demonstrate that amylose in leaves is not essential for the viability of some naturally occurring Arabidopsis genotypes, at least over short timescales and under some environmental conditions and open an opportunity to explore the adaptive significance of amylose.
Assuntos
Amilose/biossíntese , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Sintase do Amido/genética , Sintase do Amido/metabolismo , Amido/análise , Amilopectina/análise , Amilopectina/genética , Amilopectina/metabolismo , Amilose/análise , Amilose/genética , Amilose/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Grânulos Citoplasmáticos/enzimologia , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Variação Genética , Genótipo , Fenótipo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Polimorfismo de Nucleotídeo Único , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Amido/metabolismoRESUMO
The mechanism of starch granule initiation in chloroplasts is not fully understood. Here, we aimed to build on our recent discovery that PROTEIN TARGETING TO STARCH (PTST) family members, PTST2 and PTST3, are key players in starch granule initiation, by identifying and characterizing additional proteins involved in the process in Arabidopsis thaliana chloroplasts. Using immunoprecipitation and mass spectrometry, we demonstrate that PTST2 interacts with two plastidial coiled-coil proteins. Surprisingly, one of the proteins is the thylakoid-associated MAR BINDING FILAMENT-LIKE PROTEIN1 (MFP1), which was proposed to bind plastid nucleoids. The other protein, MYOSIN-RESEMBLING CHLOROPLAST PROTEIN (MRC), contains long coiled coils and no known domains. Whereas wild-type chloroplasts contained multiple starch granules, only one large granule was observed in most chloroplasts of the mfp1 and mrc mutants. The mfp1 mrc double mutant had a higher proportion of chloroplasts containing no visible granule than either single mutant and accumulated ADP-glucose, the substrate for starch synthesis. PTST2 was partially associated with the thylakoid membranes in wild-type plants, and fluorescently tagged PTST2 was located in numerous discrete patches within the chloroplast in which MFP1 was also located. In the mfp1 mutant, PTST2 was not associated with the thylakoids and formed discrete puncta, suggesting that MFP1 is necessary for normal PTST2 localization. Overall, we reveal that proper granule initiation requires the presence of MFP1 and MRC, and the correct location of PTST2.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Mutação/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Amido/metabolismo , Sintase do Amido/genética , Sintase do Amido/metabolismo , Tilacoides/genética , Tilacoides/metabolismoRESUMO
Starch granules are composed of two distinct glucose polymers - amylose and amylopectin. Amylose constitutes 5-35% of most natural starches and has a major influence over starch properties in foods. Its synthesis and storage occurs within the semicrystalline amylopectin matrix of starch granules, this poses a great challenge for biochemical and structural analyses. However, the last two decades have seen vast progress in understanding amylose synthesis, including new insights into the action of GRANULE BOUND STARCH SYNTHASE (GBSS), the major glucosyltransferase that synthesises amylose, and the discovery of PROTEIN TARGETING TO STARCH1 (PTST1) that targets GBSS to starch granules. Advances in analytical techniques have resolved the fine structure of amylose, raising new questions on how structure is determined during biosynthesis. Furthermore, the discovery of wild plants that do not produce amylose revives a long-standing question of why starch granules contain amylose, rather than amylopectin alone. Overall, these findings contribute towards a full understanding of amylose biosynthesis, structure and function that will be essential for future approaches to improve starch quality in crops.
Assuntos
Amilose , Sintase do Amido , Amilopectina , Glucanos , Amido , Sintase do Amido/genéticaRESUMO
In Triticeae endosperm (e.g. wheat and barley), starch granules have a bimodal size distribution (with A- and B-type granules) whereas in other grasses the endosperm contains starch granules with a unimodal size distribution. Here, we identify the gene, BGC1 (B-GRANULE CONTENT 1), responsible for B-type starch granule content in Aegilops and wheat. Orthologues of this gene are known to influence starch synthesis in diploids such as rice, Arabidopsis, and barley. However, using polyploid Triticeae species, we uncovered a more complex biological role for BGC1 in starch granule initiation: BGC1 represses the initiation of A-granules in early grain development but promotes the initiation of B-granules in mid grain development. We provide evidence that the influence of BGC1 on starch synthesis is dose dependent and show that three very different starch phenotypes are conditioned by the gene dose of BGC1 in polyploid wheat: normal bimodal starch granule morphology; A-granules with few or no B-granules; or polymorphous starch with few normal A- or B-granules. We conclude from this work that BGC1 participates in controlling B-type starch granule initiation in Triticeae endosperm and that its precise effect on granule size and number varies with gene dose and stage of development.
Assuntos
Grão Comestível/crescimento & desenvolvimento , Dosagem de Genes , Proteínas de Plantas/genética , Receptores de Superfície Celular/genética , Amido/metabolismo , Triticum/genética , Grão Comestível/genética , Proteínas de Plantas/metabolismo , Poliploidia , Receptores de Superfície Celular/metabolismo , Triticum/crescimento & desenvolvimentoRESUMO
The molecular mechanism that initiates the synthesis of starch granules is poorly understood. Here, we discovered two plastidial proteins involved in granule initiation in Arabidopsis thaliana leaves. Both contain coiled coils and a family-48 carbohydrate binding module (CBM48) and are homologs of the PROTEIN TARGETING TO STARCH (PTST) protein; thus, we named them PTST2 and PTST3. Chloroplasts in mesophyll cells typically contain five to seven granules, but remarkably, most chloroplasts in ptst2 mutants contained zero or one large granule. Chloroplasts in ptst3 had a slight reduction in granule number compared with the wild type, while those of the ptst2 ptst3 double mutant contained even fewer granules than ptst2 The ptst2 granules were larger but similar in morphology to wild-type granules, but those of the double mutant had an aberrant morphology. Immunoprecipitation showed that PTST2 interacts with STARCH SYNTHASE4 (SS4), which influences granule initiation and morphology. Overexpression of PTST2 resulted in chloroplasts containing many small granules, an effect that was dependent on the presence of SS4. Furthermore, isothermal titration calorimetry revealed that the CBM48 domain of PTST2, which is essential for its function, interacts with long maltooligosaccharides. We propose that PTST2 and PTST3 are critical during granule initiation, as they bind and deliver suitable maltooligosaccharide primers to SS4.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Folhas de Planta/metabolismo , Amido/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Glucanos/metabolismo , Isoamilase/metabolismo , Mutação , Filogenia , Plantas Geneticamente Modificadas , Amido/genética , Sintase do Amido/genética , Sintase do Amido/metabolismoRESUMO
Starch, the major storage carbohydrate in plants, is synthesized in plastids as semi-crystalline, insoluble granules. Many organs and cell types accumulate starch at some point during their development and maturation. The biosynthesis of the starch polymers, amylopectin and amylose, is relatively well understood and mostly conserved between organs and species. However, we are only beginning to understand the mechanism by which starch granules are initiated, and the factors that control the number of granules per plastid and the size/shape of granules. Here, we review recent progress in understanding starch granule initiation and morphogenesis. In Arabidopsis, granule initiation requires several newly discovered proteins with specific locations within the chloroplast, and also on the availability of maltooligosaccharides which act as primers for initiation. We also describe progress in understanding granule biogenesis in the endosperm of cereal grains-within which there is large interspecies variation in granule initiation patterns and morphology. Investigating whether this diversity results from differences between species in the functions of known proteins, and/or from the presence of novel, unidentified proteins, is a promising area of future research. Expanding our knowledge in these areas will lead to new strategies for improving the quality of cereal crops by modifying starch granule size and shape in vivo.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Produtos Agrícolas/crescimento & desenvolvimento , Desenvolvimento Vegetal , Poaceae/genética , Amido/metabolismo , Arabidopsis/metabolismo , Produtos Agrícolas/metabolismo , Grânulos Citoplasmáticos/metabolismo , Grão Comestível/crescimento & desenvolvimento , Grão Comestível/metabolismo , Poaceae/metabolismoRESUMO
To uncover components of the mechanism that adjusts the rate of leaf starch degradation to the length of the night, we devised a screen for mutant Arabidopsis thaliana plants in which starch reserves are prematurely exhausted. The mutation in one such mutant, named early starvation1 (esv1), eliminates a previously uncharacterized protein. Starch in mutant leaves is degraded rapidly and in a nonlinear fashion, so that reserves are exhausted 2 h prior to dawn. The ESV1 protein and a similar uncharacterized Arabidopsis protein (named Like ESV1 [LESV]) are located in the chloroplast stroma and are also bound into starch granules. The region of highest similarity between the two proteins contains a series of near-repeated motifs rich in tryptophan. Both proteins are conserved throughout starch-synthesizing organisms, from angiosperms and monocots to green algae. Analysis of transgenic plants lacking or overexpressing ESV1 or LESV, and of double mutants lacking ESV1 and another protein necessary for starch degradation, leads us to propose that these proteins function in the organization of the starch granule matrix. We argue that their misexpression affects starch degradation indirectly, by altering matrix organization and, thus, accessibility of starch polymers to starch-degrading enzymes.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Folhas de Planta/metabolismo , Amido/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Mutação , Folhas de Planta/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismoRESUMO
Starch serves functions that range over a timescale of minutes to years, according to the cell type from which it is derived. In guard cells, starch is rapidly mobilized by the synergistic action of ß-AMYLASE1 (BAM1) and α-AMYLASE3 (AMY3) to promote stomatal opening. In the leaves, starch typically accumulates gradually during the day and is degraded at night by BAM3 to support heterotrophic metabolism. During osmotic stress, starch is degraded in the light by stress-activated BAM1 to release sugar and sugar-derived osmolytes. Here, we report that AMY3 is also involved in stress-induced starch degradation. Recently isolated Arabidopsis thaliana amy3 bam1 double mutants are hypersensitive to osmotic stress, showing impaired root growth. amy3 bam1 plants close their stomata under osmotic stress at similar rates as the wild type but fail to mobilize starch in the leaves. (14)C labeling showed that amy3 bam1 plants have reduced carbon export to the root, affecting osmolyte accumulation and root growth during stress. Using genetic approaches, we further demonstrate that abscisic acid controls the activity of BAM1 and AMY3 in leaves under osmotic stress through the AREB/ABF-SnRK2 kinase-signaling pathway. We propose that differential regulation and isoform subfunctionalization define starch-adaptive plasticity, ensuring an optimal carbon supply for continued growth under an ever-changing environment.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Folhas de Planta/metabolismo , Amido/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Pressão Osmótica/fisiologia , Folhas de Planta/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaAssuntos
Evolução Biológica , Plastídeos , Plastídeos/metabolismo , Biologia , Fotossíntese , FilogeniaRESUMO
The domestication of starch crops underpinned the development of human civilisation, yet we still do not fully understand how plants make starch. Starch is composed of glucose polymers that are branched (amylopectin) or linear (amylose). The amount of amylose strongly influences the physico-chemical behaviour of starchy foods during cooking and of starch mixtures in non-food manufacturing processes. The GRANULE-BOUND STARCH SYNTHASE (GBSS) is the glucosyltransferase specifically responsible for elongating amylose polymers and was the only protein known to be required for its biosynthesis. Here, we demonstrate that PROTEIN TARGETING TO STARCH (PTST) is also specifically required for amylose synthesis in Arabidopsis. PTST is a plastidial protein possessing an N-terminal coiled coil domain and a C-terminal carbohydrate binding module (CBM). We discovered that Arabidopsis ptst mutants synthesise amylose-free starch and are phenotypically similar to mutants lacking GBSS. Analysis of granule-bound proteins showed a dramatic reduction of GBSS protein in ptst mutant starch granules. Pull-down assays with recombinant proteins in vitro, as well as immunoprecipitation assays in planta, revealed that GBSS physically interacts with PTST via a coiled coil. Furthermore, we show that the CBM domain of PTST, which mediates its interaction with starch granules, is also required for correct GBSS localisation. Fluorescently tagged Arabidopsis GBSS, expressed either in tobacco or Arabidopsis leaves, required the presence of Arabidopsis PTST to localise to starch granules. Mutation of the CBM of PTST caused GBSS to remain in the plastid stroma. PTST fulfils a previously unknown function in targeting GBSS to starch. This sheds new light on the importance of targeting biosynthetic enzymes to sub-cellular sites where their action is required. Importantly, PTST represents a promising new gene target for the biotechnological modification of starch composition, as it is exclusively involved in amylose synthesis.
Assuntos
Amilose/biossíntese , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Sintase do Amido/genética , Amilopectina/metabolismo , Arabidopsis/classificação , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cruzamento , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Estrutura Terciária de Proteína , Sintase do Amido/metabolismoRESUMO
BACKGROUND: Perineural invasion (PNI) is a high-risk feature of cutaneous squamous cell carcinoma (CSCC). Depths at which PNI occurs are unknown. OBJECTIVE: To determine the most superficial depth at which PNI occurs in CSCC and stratify by tumor clinical diameter and body location. METHODS AND MATERIALS: Single-institution retrospective review of CSCC specimens reporting PNI on pathology reports between January 2004 and August 2014. Depth was defined as distance from top of granular layer to middle of nerve invaded by CSCC or distance from erosion to middle of nerve affected by CSCC. RESULTS: Of 66 specimens identified with PNI, 45 specimens were included. Mean histopathologic depth to PNI was 2.7 mm (SD = 1.8 mm, median depth = 2.2 mm, range 0.5-12 mm). Perineural invasion depth varied by anatomic location, with the head associated with most superficial average PNI depth (2.2 mm) and trunk with greatest average PNI depth (4.3 mm). Perineural invasion depth correlated with clinical tumor diameter. The largest percentage of specimens with PNI were of clinical diameter of at least 2 cm (20/45 = 44%). CONCLUSION: Clinicians encountering lesions suspicious for CSCC have the greatest chance of detecting PNI using biopsy techniques that reach at least 3 to 4 mm deep.