Disruption of microtubules in plants suppresses macroautophagy and triggers starch excess-associated chloroplast autophagy

Microtubules, the major components of cytoskeleton, are involved in various fundamental biological processes in plants. Recent studies in mammalian cells have revealed the importance of microtubule cytoskeleton in autophagy. However, little is known about the roles of microtubules in plant autophagy. Here, we found that ATG6 interacts with TUB8/β-tubulin 8 and colocalizes with microtubules in Nicotiana benthamiana. Disruption of microtubules by either silencing of tubulin genes or treatment with microtubule-depolymerizing agents in N. benthamiana reduces autophagosome formation during upregulation of nocturnal or oxidation-induced macroautophagy. Furthermore, a blockage of leaf starch degradation occurred in microtubule-disrupted cells and triggered a distinct ATG6-, ATG5- and ATG7-independent autophagic pathway termed starch excess-associated chloroplast autophagy (SEX chlorophagy) for clearance of dysfunctional chloroplasts. Our findings reveal that an intact microtubule network is important for efficient macroautophagy and leaf starch degradation.     

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.     

Photosynthate Partitioning into Starch in Soybean Leaves: I. Effects of Photoperiod versus Photosynthetic Period Duration

Photosynthesis, photosynthate partitioning into foliar starch, and translocation were investigated in soybean plants (Glycine max (L.) Merr. cv. Amsoy 71), grown under different photoperiods and photosynthetic periods to determine the controls of leaf starch accumulation. Starch accumulation rates in soybean leaves were inversely related to the length of the daily photosynthetic period under which the plants were grown. Photosynthetic period and not photoperiod per se appears to be the important factor. Plants grown in a 14-hour photosynthetic period partitioned approximately 60% of the daily foliar accumulation into starch whereas 7-hour plants partitioned about 90% of their daily foliar accumulation into starch. The difference in starch accumulation resulted from a change in photosynthate partitioning between starch and leaf residual dry weight. Residual dry weight is defined as leaf dry weight minus the weight of total nonstructural carbohydrates. Differences in photosynthate partitioning into starch were also associated with changes in photosynthetic and translocation rates, as well as with leaf and whole plant morphology. It is concluded that leaf starch accumulation is a programmed process and not simply the result of a limitation in translocation.     

From Arabidopsis to cereal crops: Conservation of chloroplast protein degradation by autophagy indicates its fundamental role in plant productivity

Autophagy is an evolutionarily conserved process leading to the degradation of intracellular components in eukaryotes, which is important for nutrient recycling especially in response to starvation conditions. Nutrient recycling is an essential process that underpins productivity in crop plants, such that remobilized nitrogen derived from older organs supports the formation of new organs or grain-filling within a plant. We extended our understanding of autophagy in a model plant, Arabidopsis thaliana, to an important cereal, rice (Oryza sativa). Through analysis of transgenic rice plants stably expressing fluorescent marker proteins for autophagy or chloroplast stroma, we revealed that chloroplast proteins are partially degraded in the vacuole via Rubisco-containing bodies (RCBs), a type of autophagosomes containing stroma. We further reported evidence that the RCB pathway functions during natural leaf senescence to facilitate subsequent nitrogen remobilization into newly expanding leaves. Thus, our recent studies establish the importance of autophagy in biomass production of cereals.     

Effect of Day Length and Night Temperature on Starch Accumulation and Degradation in Soybean

The effect of the day length on the accumulation and the degradationof the starch in leaf, stem and root tissues of prefloweringsoybean plants was determined by growing plants under a 7 or14 h light regime. As has been reported previously, the rateof starch accumulation by leaves was inversely related to daylength. High sucrose content was associated with a high rateof starch accumulation. Stem tissue showed diurnal fluctuationsin starch content and the rate of accumulation was also inverselyrelated to day length. This starch resulted from photosynthesiswithin the stem itself. A negligible amount of starch was foundin root tissue of both sets of plants. The rate of starch breakdown in leaves of 7 h plants was significantlyless than that in 14 h plants. Nevertheless, leaf starch inshort day length plants was depleted at least 4 h prior to theend of the dark period. In both sets of plants, degradationof stem starch started simultaneously with that in the leavesand continued throughout the dark period, although at a muchlower rate than that of leaves. Thus, stem starch acted as abuffer once leaf starch was depleted, providing carbohydratesto the plant, although in small quantities. To determine if soybean leaves adjust their rate of starch accumulationduring the light period to different dark period temperatures,plants were grown under temperature regimes of 30/20 ^°Cand 30/30 ^°C. Plants did not differ in rate of starch accumulationor CO₂ exchange rate, but did show large differences in growthcharacteristics. High temperature plants had significantly greaterleaf area and tended to have greater leaf area ratio. Thus,despite similar rates of starch accumulation on a leaf areabasis, high temperature plants accumulated greater amounts ofstarch on a per plant basis. Glycine max(L.)Merr., soybean reserve carbohydrates, remobilization, source-sink realtionships     

Control of leaf expansion: a developmental switch from metabolics to hydraulics

Leaf expansion is the central process by which plants colonize space, allowing energy capture and carbon acquisition. Water and carbon emerge as main limiting factors of leaf expansion, but the literature remains controversial about their respective contributions. Here, we tested the hypothesis that the importance of hydraulics and metabolics is organized according to both dark/light fluctuations and leaf ontogeny. For this purpose, we established the developmental pattern of individual leaf expansion during days and nights in the model plant Arabidopsis (Arabidopsis thaliana). Under control conditions, decreases in leaf expansion were observed at night immediately after emergence, when starch reserves were lowest. These nocturnal decreases were strongly exaggerated in a set of starch mutants, consistent with an early carbon limitation. However, low-light treatment of wild-type plants had no influence on these early decreases, implying that expansion can be uncoupled from changes in carbon availability. From 4 d after leaf emergence onward, decreases of leaf expansion were observed in the daytime. Using mutants impaired in stomatal control of transpiration as well as plants grown under soil water deficit or high air humidity, we gathered evidence that these diurnal decreases were the signature of a hydraulic limitation that gradually set up as the leaf developed. Changes in leaf turgor were consistent with this pattern. It is concluded that during the course of leaf ontogeny, the predominant control of leaf expansion switches from metabolics to hydraulics. We suggest that the leaf is better armed to buffer variations in the former than in the latter.     

热带次生林不同林层植物叶片非结构性碳水化合物的季节变化及其对氮磷添加的响应

非结构性碳水化合物(Non-structural Carbohydrates, NSCs)是植物生长代谢过程中重要的能量来源。通过在华南热带次生林进行氮磷添加试验,探究不同林层植物叶片NSCs的季节变化及其对氮磷添加的响应,取样时间为2019年1月、4月、7月和10月。结果表明:1)植物叶片NSCs存在显著的种间差异,磷(P)添加对叶片淀粉和NSCs含量具有显著影响,且物种与磷添加的交互作用显著影响叶片淀粉含量。2)黑嘴蒲桃和紫玉盘叶片NSCs含量对氮(N)添加的响应较为敏感,而白车和竹节叶片NSCs含量对P添加的响应较为敏感,氮磷同时添加(+NP)对植物叶片NSCs的增效作用最好。3)植物叶片NSCs存在显著的季节性变化,且季节与林层间的交互作用对叶片可溶性糖和NSCs含量具有显著影响。4)不同林层植物对氮磷添加的响应不同,氮磷添加使林下层植物叶片可溶性糖含量增高,林冠层降低,在干季,N添加会使林下层植物叶片淀粉含量增高,林冠层降低。P添加的影响恰好与之相反。在湿季,氮磷添加使林下层和林冠层植物叶片的淀粉含量增加。5)林冠层植物叶片NSCs含量高于林下层,且林下层植物叶片NSCs含量...     

Sucrose and Starch Synthesis in Spinach Plants Grown under Long and Short Photosynthetic Periods

The flow of carbon into sucrose and starch was investigated in fully expanded primary leaves of spinach using the long to short day transition and partial defoliation as tools to manipulate sucrose/starch synthesis. Transfer from 12 hour to 7 hour photosynthetic periods resulted in a 4-fold increase in the initial rate of starch synthesis, a 50% increase in the initial rate of sucrose synthesis, a 30% increase in leaf sucrose, and a 40% decrease in fructose, 2,6-biphosphate. In addition, sucrose synthesis rates in cells isolated from shortened daylength plants are 80% higher than in cells isolated from control plants. These results show that, in spinach, an increase in the rates of both sucrose and starch synthesis can occur under short day conditions. In contrast, when short day plants are partially defoliated, starch levels remain high, fructose 2,6-biphosphate levels remain low, but the level of leaf sucrose drops by 50%. Thus, when demand exceeds supply, starch synthesis has priority over filling of leaf sucrose pools in the short day plant.     

Dependence of starch storage on nutrient availability and photon flux density in small birch Betula pendula Roth)

Abstract Small birch plants (Betula pendula Roth) were grown in a climate chamber at different levels of nutrient availability and at two photon flux densities. The extent to which starch storage was dependent upon nutrient availability and photon flux density was investigated. Acclimated values of starch concentration in leaves were highest at low nutrient availability and high photon flux density. Starch storage in roots was only found at the lowest nutrient availability. However, the relative rate of starch storage (starch stored per unit plant dry weight and time) was higher in plants with good nutrition. The data suggest that, at sub-optimal nutrient availability, the momentary rate of net shoot photosynthesis is unlikely to limit the structural (as opposed to carbon storage) growth of the plant. Although photosynthetic rate per unit leaf area (as measured at the growth climate) was slightly lower in plants with poor nutrient availability, photosynthetic rate per unit leaf nitrogen was higher. These data suggest a priority of leaf nitrogen usage in photosynthesis, with limiting amounts of leaf nitrogen (and possibly other nutrients) for subsequent growth processes. This argument is consistent with the higher concentrations of starch found in plants with poor nutrient availability.     

Evidence for contribution of autophagy to Rubisco degradation during leaf senescence in Arabidopsis thaliana

During leaf senescence, Rubisco is gradually degraded and its components are recycled within the plant. Although Rubisco can be mobilized to the vacuole by autophagy via specific autophagic bodies, the importance of this process in Rubisco degradation has not been shown directly. Here, we monitored Rubisco autophagy during leaf senescence by fusing synthetic green fluorescent protein (sGFP) or monomeric red fluorescent protein (mRFP) with Rubisco in Arabidopsis (Arabidopsis thaliana). When attached leaves were individually exposed to darkness to promote their senescence, the fluorescence of Rubisco‐sGFP was observed in the vacuolar lumen as well as chloroplasts. In addition, release of free‐sGFP due to the processing of Rubisco‐sGFP was observed in the vacuole of individually darkened leaves. This vacuolar transfer and processing of Rubisco‐sGFP was not observed in autophagy‐deficient atg5 mutants. Unlike sGFP, mRFP was resistant to proteolysis in the leaf vacuole of light‐grown plants. The vacuolar transfer and processing of Rubisco‐mRFP was observed at an early stage of natural leaf senescence and was also obvious in leaves naturally covered by other leaves. These results indicate that autophagy contributes substantially to Rubisco degradation during natural leaf senescence as well as dark‐promoted senescence.     

Molecular identification and comparison of the starch synthase bound to starch granules between endosperm and leaf blades in rice plants

Normal (nonglutinous) rice plants (Oryza sativa andO. glaberrima) contain more than 18% amylose in endosperm starch, whilewaxy (glutinous) plants lack it in this starch. In contrast, leaf starch contained more than 3.6% amylose even inwaxy plants. SDS-PAGE analysis of proteins bound to endosperm starch granules in the normal plants revealed a single band with aMr of 60 kd, whereaswaxy plants did not exhibit a similar band. The activity of starch synthase (NDP-glucose-starch glucosyltransferase) was completely inhibited by antibody against the 60-kd protein. Thus, we conclude that the 60-kd protein is thewaxy protein encoded by theWx allele, which also plays a role in the synthesis of nonglutinous starch in endosperm tissue. In leaf blades, the proteins bound to starch granules separated into five bands withMr's of 53.6 to 64.9 kd on SDS-PAGE. Analysis of these proteins by immunoblotting using antiserum againstWx protein and inhibition of starch synthase activity by the synthase antibody revealed that none of these proteins was homologous toWx protein. We suggest that the synthesis of amylose in leaf blades is brought about by a protein encoded by a gene(s) different from theWx gene expressed in the endosperm.     

Photosynthate Partitioning into Starch in Soybean Leaves: II. IRRADIANCE LEVEL AND DAILY PHOTOSYNTHETIC PERIOD DURATION EFFECTS

Two photosynthetic periods and photosynthetic photon flux densities (PPFD) were used to study the relationship between the rate of photosynthesis and starch accumulation in vegetative soybean leaves (Merr. cv Amsoy 71). Plants grown in short daily photosynthetic periods (7 hours) had higher rates of CO₂ fixation per unit leaf dry weight and of leaf starch accumulation than plants grown in long daily photosynthetic periods (14 hours) irrespective of PPFD. CO₂ fixation rates per unit leaf area were similar in 7-hour and 14-hour plants grown at low PPFD but were highest in 14-hour plants at the high PPFD. When single leaves of 14-hour plants were given 7-hour photosynthetic periods, their rates of starch accumulation remained unchanged. The programming of starch accumulation rate and possibly of photosynthetic rate by the length of the daily photosynthetic period is apparently a whole-plant, not an individual leaf, phenomenon. Programming of chloroplast starch accumulation rate by length of the daily photosynthetic and/or dark periods was independent of PPFD within the ranges used in this experiment.     

Acclimation of Photosynthate Partitioning and Photosynthetic Rates to Changes in Length of the Daily Photosynthetic Period

Pangola, soya bean and spinach plants were grown in long andshort day photosynthetic periods. Reciprocal shifts betweenlong and short day grown plants were made to study acclimationin the rate of leaf starch synthesis with change in daylength.The rate of leaf starch accumulation is a function of the lengthof the daily photosynthetic period. Acclimation, that is a changein partitioning with a change in length of the photosyntheticperiod, occurs in a variety of species. Acclimation in the rateof starch accumulation occurs rapidly in pangola and is apparentlycomplete the day after a change in length of the daily photosyntheticperiod. Soya bean and spinach leaves require a few days in thenew environment for an acclimation to occur. Digitaria decumbens Stent., Glycine max (L.) Merr., Spinacia oleracea L., pangola, soya bean, spinach, specific leaf weight, starch, photosynthesis     

Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene

Autophagy is an intracellular process for vacuolar bulk degradation of cytoplasmic components. The molecular machinery responsible for yeast and mammalian autophagy has recently begun to be elucidated at the cellular level, but the role that autophagy plays at the organismal level has yet to be determined. In this study, a genome-wide search revealed significant conservation between yeast and plant autophagy genes. Twenty-five plant genes that are homologous to 12 yeast genes essential for autophagy were discovered. We identified an Arabidopsis mutant carrying a T-DNA insertion within AtAPG9, which is the only ortholog of yeast Apg9 in Arabidopsis (atapg9-1). AtAPG9 is transcribed in every wild-type organ tested but not in the atapg9-1 mutant. Under nitrogen or carbon-starvation conditions, chlorosis was observed earlier in atapg9-1 cotyledons and rosette leaves compared with wild-type plants. Furthermore, atapg9-1 exhibited a reduction in seed set when nitrogen starved. Even under nutrient growth conditions, bolting and natural leaf senescence were accelerated in atapg9-1 plants. Senescence-associated genes SEN1 and YSL4 were up-regulated in atapg9-1 before induction of senescence, unlike in wild type. All of these phenotypes were complemented by the expression of wild-type AtAPG9 in atapg9-1 plants. These results imply that autophagy is required for maintenance of the cellular viability under nutrient-limited conditions and for efficient nutrient use as a whole plant.     

Determining the role of Tie-dyed1 in starch metabolism: epistasis analysis with a maize ADP-glucose pyrophosphorylase mutant lacking leaf starch

In regions of their leaves, tdy1-R mutants hyperaccumulate starch. We propose 2 alternative hypotheses to account for the data, that Tdy1 functions in starch catabolism or that Tdy1 promotes sucrose export from leaves. To determine whether Tdy1 might function in starch breakdown, we exposed plants to extended darkness. We found that the tdy1-R mutant leaves retain large amounts of starch on prolonged dark treatment, consistent with a defect in starch catabolism. To further test this hypothesis, we identified a mutant allele of the leaf expressed small subunit of ADP-glucose pyrophosphorylase (agps-m1), an enzyme required for starch synthesis. We determined that the agps-m1 mutant allele is a molecular null and that plants homozygous for the mutation lack transitory leaf starch. Epistasis analysis of tdy1-R; agps-m1 double mutants demonstrates that Tdy1 function is independent of starch metabolism. These data suggest that Tdy1 may function in sucrose export from leaves.     

Exploiting leaf starch synthesis as a transient sink to elevate photosynthesis, plant productivity and yields

Improvements in plant productivity (biomass) and yield have centered on increasing the efficiency of leaf CO₂ fixation and utilization of products by non-photosynthetic sink organs. We had previously demonstrated a correlation between photosynthetic capacity, plant growth, and the extent of leaf starch synthesis utilizing starch-deficient mutants. This finding suggested that leaf starch is used as a transient photosynthetic sink to recycle inorganic phosphate and, in turn, maximize photosynthesis. To test this hypothesis, Arabidopsis thaliana and rice (Oryza sativa L.) lines were generated with enhanced capacity to make leaf starch with minimal impact on carbon partitioning to sucrose. The Arabidopsis engineered plants exhibited enhanced photosynthetic capacity; this translated into increased growth and biomass. These enhanced phenotypes were displayed by similarly engineered rice lines. Manipulation of leaf starch is a viable alternative strategy to increase photosynthesis and, in turn, the growth and yields of crop and bioenergy plants.     

The role of transitory starch in C(3), CAM, and C(4) metabolism and opportunities for engineering leaf starch accumulation

Essentially all plants store starch in their leaves during the day and break it down the following night. This transitory starch accumulation acts as an overflow mechanism when the sucrose synthesis capacity is limiting, and transitory starch also acts as a carbon store to provide sugar at night. Transitory starch breakdown can occur by either of two pathways; significant progress has been made in understanding these pathways in C(3) plants. The hydrolytic (amylolytic) pathway generating maltose appears to be the primary source of sugar for export from C(3) chloroplasts at night, whereas the phosphorolytic pathway supplies carbon for chloroplast reactions, in particular in the light. In crassulacean acid metabolism (CAM) plants, the hydrolytic pathway predominates when plants operate in C(3) mode, but the phosphorolytic pathway predominates when they operate in CAM mode. Information on transitory starch metabolism in C(4) plants has now become available as a result of combined microscopy and proteome studies. Starch accumulates in all cell types in immature maize leaf tissue, but in mature leaf tissues starch accumulation ceases in mesophyll cells except when sugar export from leaves is blocked. Proper regulation of the amount of carbon that goes into starch, the pathway of starch breakdown, and the location of starch accumulation could help ensure that engineering of C(4) metabolism is coordinated with the downstream reactions required for efficient photosynthesis.     

EFFECT OF PETIOLE PHLOEM DISRUPTION ON STARCH AND MINERAL DISTRIBUTION IN SENESCING SOYBEAN LEAVES

Normally, starch (sugars) and minerals are redistributed from the leaves to the pods during monocarpic senescence in maturing soybean plants. Petiole phloem destruction (steam girdling), which blocked this redistribution by interrupting export through the petiole, altered the foliar senescence pattern producing a distinctive interveinal yellowing with green areas along the veins on pod-bearing plants. This suggests that blockage of the petiole phloem may cause nutrients to accumulate in the green zones along the leaf veins instead of being redistributed to the pods. In the leaves of untreated plants, starch showed the same distribution pattern as chlorophyll; however, starch was preserved in yellow areas as well as the green zones of the steam-girdled leaves. Mineral analyses of the veinal and interveinal zones of treated leaves and controls showed that the veinal green zones and interveinal yellowing in treated plants were not respectively enriched and depleted in minerals corresponding to a redistribution of minerals within the leaves. Depodding also blocked leaf yellowing, net mineral redistribution and starch breakdown. Thus, the pods are able to induce chlorophyll breakdown without net mineral redistribution or starch loss in leaves with petiole phloem destruction. This shows that chlorophyll breakdown is not obligatorily coupled with mineral redistribution or starch breakdown.     

Biochemical Basis for Partitioning of Photosynthetically Fixed Carbon between Starch and Sucrose in Soybean (Glycine max Merr.) Leaves

The control of photosynthetic starch/sucrose formation in leaves of soybean (Glycine max L. Merr.) cultivars was studied in relation to stage of plant development, photosynthetic photoperiod, and nitrogen source. At each sampling, leaf tissue was analyzed for starch content, activities of sucrose-metabolizing enzymes, and labeling of starch and sucrose (by ¹⁴CO₂ assimilation) in isolated cells. In three of the four varieties tested, nodulated plants had lower leaf starch levels and higher activities of sucrose phosphate synthetase (SPS), and isolated mesophyll cells incorporated more carbon (percentage of total ¹⁴CO₂ fixed) into sucrose and less into starch as compared to nonnodulated (nitrate-dependent) plants. The variation among cultivars and nitrogen treatments observed in the activity of SPS in leaf extracts was positively correlated with labeling of sucrose in isolated cells (r = 0.81) and negatively correlated with whole leaf starch content (r = −0.66). The results suggested that increased demand for assimilates by nodulated roots may be accommodated by greater partitioning of carbon into sucrose in the mesophyll cells. We have also confirmed the earlier report (Chatterton, Silvius 1979 Plant Physiol 64: 749-753) that photoperiod affects partitioning of fixed carbon into starch. Within two days of transfer of nodulated soybean Ransom plants from a 14-hour to a 7-hour photoperiod, leaf starch accumulation rates doubled, and this effect was associated with increased labeling of starch and decreased labeling of sucrose in isolated cells. Concurrently, activities of SPS, sucrose synthase, and uridine diphosphatase in leaves were decreased.