Excessive Starch Accumulation Research Articles

Comparative transcriptomic analysis on compatible/incompatible grafts in citrus.

Grafting is a useful cultivation technology to resist abiotic and biotic stresses and is an integral part of citrus production. However, some widely utilized rootstocks may still exhibit graft incompatibility in the orchard. ‘Hongmian miyou’ (Citrus maxima (Burm.) Merrill) is mutated from ‘Guanxi miyou’, but these two scions showed different compatibility with available Poncirus trifoliata rootstock. Foliage etiolation is an observed symptom of graft incompatibility, but its mechanism remains poorly understood. This study is the first to investigate the morphological, physiological, and anatomical differences between compatible and incompatible grafts, and perform transcriptome profiling at crucial stages of the foliage etiolation process. Based on comprehensive analyses, hormonal balance was disordered, and two rate-limiting genes, NCED3 (9-cis-epoxycarotenoid dioxygenase 3) and NCED5, being responsible for ABA (abscisic acid) accumulation, were highlighted. Further correlation analysis indicated that IAA (indole-3-acetic acid) and ABA were the most likely inducers of the expression of stress-related genes. In addition, excessive starch accumulation was observed in the lamina and midribs of leaves of incompatible grafts. These results provide a new insight into the role of hormonal balance and ABA biosynthesis genes in regulating and contributing to graft incompatibility, and will further define and deploy candidate genes to explore the mechanisms underlying citrus rootstock–scion interactions.

Plasma membrane-localized SEM1 protein mediates sugar movement to sink rice tissues.

The translocation of photosynthate carbohydrates, such as sucrose, is critical for plant growth and crop yield. Previous studies have revealed that sugar transporters, plasmodesmata and sieve plates act as important controllers in sucrose loading into and unloading from phloem in the vascular system. However, other pivotal steps for the regulation of sucrose movement remain largely elusive. In this study, characterization of two starch excesses in mesophyll (sem) mutants and dye and sucrose export assays were performed to provide insights into the regulatory networks that drive source-sink relations in rice. Map-based cloning identified two allelic mutations in a gene encoding a GLUCAN SYNTHASE-LIKE (GSL) protein, thus indicating a role for SEM1 in callose biosynthesis. Subcellular localization in rice showed that SEM1 localized to the plasma membrane. In situ expression analysis and GUS staining showed that SEM1 was mainly expressed in vascular phloem cells. Reduced sucrose transport was found in the sem1-1/1-2 mutant, which led to excessive starch accumulation in source leaves and inhibited photosynthesis. Paraffin section and transmission electron microscopy experiments revealed that less-developed vascular cells (VCs) in sem1-1/1-2 potentially disturbed sugar movement. Moreover, dye and sugar trafficking experiments revealed that aberrant VC development was the main reason for the pleiotropic phenotype of sem1-1/1-2. In total, efficient sucrose loading into the phloem benefits from an optional number of VCs with a large vacuole that could act as a buffer holding tank for sucrose passing from the vascular bundle sheath.

Metabolomic analysis elucidates how shade conditions ameliorate the deleterious effects of greening (Huanglongbing) disease in citrus.

Under tropical and subtropical environments, citrus leaves are exposed to excess sunlight, inducing photoinhibition. Huanglongbing (HLB, citrus greening), a devastating phloem-limited disease putatively caused by Candidatus Liberibacter asiaticus, exacerbates this challenge with additional photosynthetic loss and excessive starch accumulation. A combined metabolomics and physiological approach was used to elucidate whether shade alleviates the deleterious effects of HLB in field-grown citrus trees, and to understand the underlying metabolic mechanisms related to shade-induced morpho-physiological changes in citrus. Using metabolite profiling and multinomial logistic regression, we identified pivotal metabolites altered in response to shade. A core metabolic network associated with shade conditions was identified through pathway enrichment analysis and metabolite mapping. We measured physio-biochemical responses and growth and yield characteristics. With these, the relationships between metabolic network and the variables measured above were investigated. We found that moderate-shade alleviates sink limitation by preventing excessive starch accumulation and increasing foliar sucrose levels. Increased growth and fruit yield in shaded compared with non-shaded trees were associated with increased photosystem II efficiency and leaf carbon fixation pathway metabolites. Our study also shows that, in HLB-affected trees under shade, the signaling of plant hormones (auxins and cytokinins) and nitrogen supply were downregulated with reducing new shoot production likely due to diminished needs of cell damage repair and tissue regeneration under shade. Overall, our findings provide the first glimpse of the complex dynamics between cellular metabolites and leaf physiological functions in citrus HLB pathosystem under shade, and reveal the mechanistic basis of how shade ameliorates HLB disease.

Exogenous putrescine regulates leaf starch overaccumulation in cucumber under salt stress

To understand the mechanism by which exogenous putrescine (Put) alleviated starch overaccumulation in the leaves of cucumber subjected to salt stress, a greenhouse experiment was conducted. Our research findings focused on NaCl stress leading to an increase in the number of starch granules in the chloroplast and starch content in the leaves, resulting in the destruction of photosynthetic organs and reduced photosynthesis. However, exogenous Put reduced the number of starch granules and protected the photosynthetic organs, thereby enhancing photosynthesis. On the other hand, Put-treated seedlings reduced AGPase activity by downregulating AGPase and increased β-amylase activity by upregulating BAM1, resulting in relief from excess accumulation of leaf starch. The application of d-Arg under salt stress decreased the expression of ADC, inhibited Put synthesis, reduced the endogenous polyamine (PAs) content, and inhibited the growth of seedlings; exogenous Put partly alleviated the inhibitory effect of d-Arg and significantly increased the endogenous PAs content, indicating that the ADC pathway is important for Put synthesis in plants suffering from salt stress. Taken together, exogenous application of PAs could increase levels of endogenous PAs via important ADC pathways, mainly inducing AGPase and BAM1 expression, leading to changes in AGPase and β-amylase activities involved in starch metabolism to reduce excessive starch accumulation in leaves, relieve photosynthetic feedback repression and protect photosynthetic organs, thus enhancing cucumber seedling salt stress tolerance.

Phloem Regeneration Is a Mechanism for Huanglongbing-Tolerance of "Bearss" Lemon and "LB8-9" Sugar Belle® Mandarin.

Huanglongbing (HLB) is an extremely destructive and lethal disease of citrus worldwide, presumably caused by phloem-limited bacteria, Candidatus Liberibacter asiaticus (CLas). The widespread invasiveness of the HLB pathogen and lack of natural HLB-resistant citrus cultivars have underscored the need for identifying tolerant citrus genotypes to support the current citrus industry’s survival and potentially to lead to future natural HLB resistance. In this study, transverse sections of leaf lamina and midribs were examined with light and epifluorescence microscopy to determine anatomical characteristics that underlie HLB-tolerant mechanisms operating among “Bearss” lemon, “LB8-9” Sugar Belle® mandarin, and its sibling trees compared with HLB-sensitive “Valencia” sweet orange. The common anatomical aberrations observed in all CLas-infected varieties are as follows: phloem necrosis, hypertrophic phloem parenchyma cells, phloem plugging with abundant callose depositions, phloem collapse with cell wall distortion and thickening, excessive starch accumulation, and sometimes even cambium degeneration. Anatomical distribution of starch accumulation even extended to tracheid elements. Although there were physical, morphological, and pathological similarities in the examined foliage, internal structural preservation in “Bearss” lemon and “LB8-9” Sugar Belle® mandarin was superior compared with HLB-sensitive “Valencia” sweet orange and siblings of “LB8-9” Sugar Belle® mandarin. Intriguingly, there was substantial phloem regeneration in the tolerant types that may compensate for the dysfunctional phloem, in comparison with the sensitive selections. The lower levels of phloem disruption, together with greater phloem regeneration, are two key elements that contribute to HLB tolerance in diverse citrus cultivars.

Gene mapping of starch accumulation and premature leaf senescence in the ossac3 mutant of rice

Leaf senescence is the final stage of plant leaf development. Early leaf senescence leads to a reduction in yield and quality of rice (Oryza sativa). Reduction in yield and quality of rice will cause direct economic losses to farmers, and even insufficient food supply worldwide. Elucidation of the molecular mechanism of premature senescence is important for the genetic improvement of rice. The rice mutant ossac3 (sugar accumulating 3) characterized in this study was raised from seeds of the indica maintainer line Xinong 1B treated with ethyl methanesulfonate. The mutant showed premature leaf senescence associated with excessive starch accumulation in the leaf. Under field conditions, the mutant showed the early senescence phenotype at the seedling stage. Apart from newly developing leaves, the leaves began to turn yellow from the leaf tip extending to the middle of the leaf, and the base of the leaves was pale green. The mutant phenotype was maintained to maturity. Compared to the wild type, photosynthetic pigment content and photosynthetic rate in leaves of the mutant were significantly decreased. Concentrations of reactive oxygen species were significantly increased, antioxidant enzyme activities were significantly reduced, and all analyzed agronomic traits decreased in the mutant. Iodine-potassium iodide staining and starch content analysis showed that starch accumulated in ossac3 leaves. Quantitative real-time PCR analysis revealed changes in the expression levels of starch metabolism-related genes. The mutant phenotype was controlled by a pair of recessive nuclear genes and ossac3 was localized on chromosome 3 between the markers RM15243 and HJY8 with a physical distance of 374.2 kb.

LasΔ5315 Effector Induces Extreme Starch Accumulation and Chlorosis as Ca. Liberibacter asiaticus Infection in Nicotiana benthamiana.

Huanglongbing (HLB), a destructive plant bacterial disease, severely impedes worldwide citrus production. HLB is associated with a phloem-limited α-proteobacterium, Candidatus Liberibacter asiaticus (Las). Las infection causes yellow shoots and blotchy mottle on leaves and is associated with excessive starch accumulation. However, the mechanisms underlying the starch accumulation remain unknown. We previously showed that the Las5315mp effector induced callose deposition and cell death in Nicotiana benthamiana. In this study, we demonstrated that Las can experimentally infect N. benthamiana via dodder transmission. Furthermore, we revealed another key function of the Las5315 effector by demonstrating that transient expression of the truncated form of the effector, LasΔ5315, induced excessive starch accumulation by 6 fold after 8 dpi in N. benthamiana after removal of the chloroplast transit peptide from the Las5315mp. The induction mechanisms of LasΔ5315 in N. benthamiana were attributed to the up-regulation of ADP-glucose pyrophosphorylase, granule-bound starch synthase, soluble starch synthase, and starch branching enzyme for increasing starch production, and to the significant down-regulation of the starch degradation enzymes: alpha-glucosidase, alpha-amylase, and glycosyl hydrolase for decreasing starch degradation. This is the first report that Las can infect the model plant N. benthamiana. Using this model plant, we demonstrated that the LasΔ5315 effector caused the most prominent HLB symptoms, starch accumulation and chlorosis as Las infection in N. benthamiana. Altogether the Las 5315 effector is critical for Las pathogenesis, and therefore, an important target for interference.

Sex-specific carbon and nitrogen partitioning under N deposition in Populus cathayana

Females of Populus cathayana allocated increased N to soluble proteins, while males keep N allocation pattern unchanged under N enrichment. In our study, Populus cathayana as a model species is employed to detect the sex-specific responses in growth, photosynthetic nitrogen (N) use efficiency (PNUE), carbon (C) and N partitioning when exposed to 0, 7.5 and 15 g N m−2 year−1 on the basis of local N deposition level. Our results showed that females had higher responses in photosynthesis and growth than males when exposed to N deposition, and also exhibited higher PNUE than males when exposed to high level of N deposition, suggesting that plasticity in the females may enhance the capacity of young seedlings to acquire resources. Moreover, we found that females mainly allocated increased N to soluble proteins and detergent-soluble proteins, but not to cell-wall proteins, while males maintained original partitioning pattern of N and also accumulated excessive N in the form of free amino acids. Females also allocated more fraction of leaf N to carboxylation (P C), bioenergetics (P B), and then more fraction of leaf N to all components of photosynthetic machinery (P T) than males under high level of N deposition. Such sex-specific N allocation strategy may correlate with sex-related PNUE. These results indicated that there is a higher critical N demand in females, and females could use N nutrient more efficiently than males under high N deposition. We also found that higher shift from starch to soluble sugars, such as sucrose, occurred in females than in males under N enrichment. By contrast, excessive accumulation of starch and non-structural carbohydrate in females relative to males was observed under control conditions, which might inhibit female photosynthesis rate. Accordingly, we suggested that the different leaf C and N partitioning patterns could explain the sex-specific responses in growth. Therefore, females may obtain advantageous position in the process of intraspecific competition when exposed to high level of N deposition because they have higher light capture (total leaf area) ability and utilization efficiency (PNUE) than the males that confer the ability for fast growth and thus are likely to be more responsive to N enrichment. Our results suggested that it could be important to look at the stronger growth response of the females over the males under N enrichment at both the leaf and the plant scale.

Autophagy Contributes to Leaf Starch Degradation

Transitory starch, a major photosynthetic product in the leaves of land plants, accumulates in chloroplasts during the day and is hydrolyzed to maltose and Glc at night to support respiration and metabolism. Previous studies in Arabidopsis thaliana indicated that the degradation of transitory starch only occurs in the chloroplasts. Here, we report that autophagy, a nonplastidial process, participates in leaf starch degradation. Excessive starch accumulation was observed in Nicotiana benthamiana seedlings treated with an autophagy inhibitor and in autophagy-related (ATG) gene-silenced N. benthamiana and in Arabidopsis atg mutants. Autophagic activity in the leaves responded to the dynamic starch contents during the night. Microscopy showed that a type of small starch granule-like structure (SSGL) was localized outside the chloroplast and was sequestered by autophagic bodies. Moreover, an increased number of SSGLs was observed during starch depletion, and disruption of autophagy reduced the number of vacuole-localized SSGLs. These data suggest that autophagy contributes to transitory starch degradation by sequestering SSGLs to the vacuole for their subsequent breakdown.

Callose deposition in the phloem plasmodesmata and inhibition of phloem transport in citrus leaves infected with “Candidatus Liberibacter asiaticus”

Huanglongbing (HLB) is a destructive disease of citrus trees caused by phloem-limited bacteria, Candidatus Liberibacter spp. One of the early microscopic manifestations of HLB is excessive starch accumulation in leaf chloroplasts. We hypothesize that the causative bacteria in the phloem may intervene photoassimilate export, causing the starch to over-accumulate. We examined citrus leaf phloem cells by microscopy methods to characterize plant responses to Liberibacter infection and the contribution of these responses to the pathogenicity of HLB. Plasmodesmata pore units (PPUs) connecting companion cells and sieve elements were stained with a callose-specific dye in the Liberibacter-infected leaf phloem cells; callose accumulated around PPUs before starch began to accumulate in the chloroplasts. When examined by transmission electron microscopy, PPUs with abnormally large callose deposits were more abundant in the Liberibacter-infected samples than in the uninfected samples. We demonstrated an impairment of symplastic dye movement into the vascular tissue and delayed photoassimilate export in the Liberibacter-infected leaves. Liberibacter infection was also linked to callose deposition in the sieve plates, which effectively reduced the sizes of sieve pores. Our results indicate that Liberibacter infection is accompanied by callose deposition in PPUs and sieve pores of the sieve tubes and suggest that the phloem plugging by callose inhibits phloem transport, contributing to the development of HLB symptoms.

CO 2 assimilation, photosystem II photochemistry, carbohydrate metabolism and antioxidant system of citrus leaves in response to boron stress

Seedlings of Citrus grandis were fertilized every other days for 15 weeks with nutrient solution containing 0 (deficiency), 10 μM (control) or 500 μM (excess) H 3BO 3. CO 2 assimilation and chlorophyll (Chl) content decreased to a greater degree in B-deficient than in B-excess leaves, but photosynthetic enzyme activities were similarly decreased. Starch accumulated in B-deficient leaves, but not in B-excess ones. Chlorophyll a fluorescence transient showed that the positive L- and K-steps were more pronounced in B-excess than in B-deficient leaves. Maximum quantum yield of primary photochemistry ( F v/ F m), maximum variable fluorescence ( F v), oxygen-evolving complex (OEC) were less decreased in B-deficient than in B-excess leaves, whereas minimum fluorescence ( F 0) was less increased in B-deficient leaves. Boron-deficient leaves displayed higher or similar antioxidant enzyme activities and higher ascorbate (AsA) and reduced glutathione (GSH) contents compared to B-excess leaves. Content of thiobarbituric acid (TBA) reactive compounds was less increased by B-deficiency than by B-excess. We conclude that B-deficient leaves are less damaged by oxidative stress than B-excess leaves due to their higher ability to scavenge reactive oxygen species. Both the donor (i.e. the OEC) and the acceptor sides of photosystem II were less photoinhibited by B-deficiency than by B-excess. The greater decrease in CO 2 assimilation and Chl content in B-deficient leaves may be caused by the excessive accumulation of starch. The reduction of CO 2 assimilation by B-excess is probably caused by a combination of factors such as oxidative damage, reduced photosynthetic enzyme activities and impaired electron transport capacity.

Boron deficiency decreases growth and photosynthesis, and increases starch and hexoses in leaves of citrus seedlings

Seedlings of sweet orange (Citrus sinensis) were fertilized for 14 weeks with boron (B)-free or B-sufficient (2.5 or 10 microM H(3)BO(3)) nutrient solution every other day. Boron deficiency resulted in an overall inhibition of plant growth, with a reduction in root, stem and leaf dry weight (DW). Boron-starved leaves showed decreased CO(2) assimilation and stomatal conductance, but increased intercellular CO(2) concentrations. Activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH) and stromal fructose-1,6-bisphosphatase (FBPase) were lower in B-deficient leaves than in controls. Contents of glucose, fructose and starch were increased in B-deficient leaves while sucrose was decreased. Boron-deficient leaves displayed higher or similar superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR) and glutathione reductase (GR) activities, while dehydroascorbate reductase (DHAR) and catalase (CAT) activities were lower. Expressed on a leaf area or protein basis, B-deficient leaves showed a higher ascorbate (AsA) concentration, but a similar AsA concentration on a DW basis. For reduced glutathione (GSH), we found a similar GSH concentration on a leaf area or protein basis and an even lower content on a DW basis. Superoxide anion (O(2)(-)) generation, malondialdehyde (MDA) concentration and electrolyte leakage were higher in B-deficient than in control leaves. In conclusion, CO(2) assimilation may be feedback-regulated by the excessive accumulation of starch and hexoses in B-deficient leaves via direct interference with chloroplast function and/or indirect repression of photosynthetic enzymes. Although B-deficient leaves remain high in activity of antioxidant enzymes, their antioxidant system as a whole does not provide sufficient protection from oxidative damage.

Using L‐systems for modeling source–sink interactions, architecture and physiology of growing trees: the L‐PEACH model

Functional-structural plant models simulate the development of plant structure, taking into account plant physiology and environmental factors. The L-PEACH model is based on the development of peach trees. It demonstrates the usefulness of L-systems in constructing functional-structural models. L-PEACH uses L-systems both to simulate the development of tree structure and to solve differential equations for carbohydrate flow and allocation. New L-system-based algorithms are devised for simulating the behavior of dynamically changing structures made of hundreds of interacting, time-varying, nonlinear components. L-PEACH incorporates a carbon-allocation model driven by source-sink interactions between tree components. Storage and mobilization of carbohydrates during the annual life cycle of a tree are taken into account. Carbohydrate production in the leaves is simulated based on the availability of water and light. Apices, internodes, leaves and fruit grow according to the resulting local carbohydrate supply. L-PEACH outputs an animated three-dimensional visual representation of the growing tree and user-specified statistics that characterize selected stages of plant development. The model is applied to simulate a tree's response to fruit thinning and changes in water stress. L-PEACH may be used to assist in horticultural decision-making processes after being calibrated to specific trees.

Growth-Retarding Effect of 2-Aminoindan-2-phosphonic Acid on Spirodela punctata

2-Aminoindan-2-phosphonic acid (AIP) retarded the growth of duckweed, Spirodela punctata, and increased its dry mass. The accumulation of starch was observed at all concentrations of AIP at 8 days after treatment. The increase in starch was inversely proportional to the growth. The retarded growth of Spirodela by AIP was not limited only by excessive starch accumulation.

EFFECTS OF CONTINUOUS LIGHTING ON ENZYME ACTIVITIES OF LEAF CARBON METABOLISM OF TOMATO AND PEPPER PLANTS

Exposure of tomato and pepper plants to long photoperiods (20 hours or more for tomato; 24 hours for pepper) results in leaf chlorosis (tomato), leaf deformities (pepper), and decreased growth and productivity (both species). Some researchers have suggested that excessive starch accumulation in the leaves could be the cause of the negative effects. We observed that tomato and pepper plants do accumulate more starch in their leaves when grown under a long photoperiod (24 hours) compared to a shorter one (16 hours). However, our results indicated that these accumulations were not caused by a limited sink strength but by an alteration of the carbon metabolism at the leaf level. In our last experiment, we studied the activity of enzymes [sucrose phosphate synthase (SPS), sucrose synthase (SS), invertase] of leaf carbon metabolism in tomato and pepper plants grown under different photoperiods (natural, natural + supplemental light of 100 μmol·m-2·s-1 during 16 and 24 hours). We observed a 10% to 15% decrease in leaf SPS activity in tomato (not in pepper) plants grown under a 24-hour photoperiod. In both species, invertase and SS activities were not affected by photoperiod treatments. In tomato plants grown under a 24-hour photoperiod, the decrease in SPS activity corresponded to the appearance of leaf chlorosis (6 to 7 weeks after the beginning of treatments). Therefore, it appears that leaf carbon metabolism could be involved in the development of negative effects of long photoperiod in tomato plants, but not in pepper plants. The fact that photoperiod had no apparent effect on leaf carbon metabolism of pepper may explain why this species can tolerate longer photoperiods than tomato plants.

Reversibility of Photosynthetic Inhibition in Cotton after Long-Term Exposure to Elevated CO2 Concentrations

Cotton (Gossypium hirsutum L. cv Stoneville 213) was grown at 350 and 1000 microliters per liter CO(2). The plants grown at elevated CO(2) concentrations contained large starch pools and showed initial symptoms of visible physical damage. Photosynthetic rates were lower than expected based on instantaneous exposure to high CO(2).A group of plants grown at 1000 microliters per liter CO(2) was switched to 350 microliters per liter CO(2). Starch pools and photosynthetic rates were monitored in the switched plants and in the two unswitched control groups. Photosynthetic rates per unit leaf area recovered to the level of the 350 microliters per liter CO(2) grown control group within four to five days. To assess only nonstomatal limitations to photosynthesis, a measure of photosynthetic efficiencies was calculated (moles CO(2) fixed per square meter per second per mole intercellular CO(2)). Photosynthetic efficiency also recovered to the levels of the 350 microliters per liter CO(2) grown controls within three to four days.Recovery was correlated to a rapid depletion of the starch pool, indicating that the inhibition of photosynthesis is primarily a result of feedback inhibition. However, complete recovery may involve the repair of damage to the chloroplasts caused by excessive starch accumulation. The rapid and complete reversal of photosynthetic inhibition suggests that the appearance of large, strong sinks at certain developmental stages could result in reduction of the large starch accumulations and that photosynthetic rates could recover to near the theoretical capacity during periods of high photosynthate demand.

Investigations on Cytokinins. I. Effect of 6‐Benzylaminopurine on Growth and Starch Content of Lemna minor

AbstractAn excessive starch accumulation in the fronds of Lemna minor L. was observed microscopically and confirmed by direct starch determinations after application of growth inhibiting 6‐benzylaminopurine concentrations. As a high growth rate was coupled with a low starch content and vice versa, the influence of the cytokinin on growth and starch content was compared. It appeared that apart from the expected influence of the growth rate on the starch content, 6‐benzylaminopurine favoured starch accumulation also by itself.

Changes in the starch, protein and RNA content of Lemna minor L. under the influence of kinetin (6-furfurylaminopurine)

Kinetin (6-furfurylaminopurine) is rapidly absorbed byLemna minor L. 3 · 10−8M/ml medium cause an immediate but temporary stimulation of RNA and protein synthesis during the first hour of the treatment. The following excessive accumulation of starch is considered to be more or less a direct consequence of a disturbed RNA and protein metabolism.