Capping invertase activity by its inhibitor: Roles and implications in sugar signaling,carbon allocation,senescence and evolution

Since the initial biochemical study of a putative invertase inhibitor half a century ago, it has remained as a puzzle as whether such an inhibitory protein indeed limits invertase activity in vivo and, if it does, what is the developmental or physiological significance of such an interaction? Recently, we demonstrated that an invertase inhibitor, INVINH1, specifically inhibited cell wall invertase activity in tomato and Arabidopsis. Silencing INVINH1 expression in tomato released a significant amount of extra cell wall invertase activity. This posttranslational elevation of invertase activity resulted in a blockage of ABA-induced leaf senescence and an increase in fruit sugar levels and seed weight. Here, we discuss the implication of the findings and propose a model that the invertse inhibitor may act as a key modulator in controlling leaf longevity and seed development to ensure success during plant evolution. This may be achieved by optimizing carbon and nitrogen allocation and sugar signaling via interaction between invertase and inhibitor. The discoveries open up exciting new areas for exploring fundamental questions in sugar signaling, carbon allocation and plant development as well as avenues for improving crop productivity.Key words: carbon allocation, evolution, invertase, invertase inhibitor, leaf senescence, seed development, sugar signaling     

大花百子莲的结实和结籽格局及种子产量影响因素分析

在2007和2008年对大花百子莲(Agapanthus praecox subsp.orientalis‘Big Blue’)在群体、果序和单果水平上的结实和结籽格局进行了观测,并依据每花序单花数、单花胚珠数和单粒种子质量推算其单株潜在种子产量;此外,采用人工补充授粉方法研究了不同授粉方式对大花百子莲结实和结籽的影响,并对其种子产量的影响因素进行了分析。结果表明:从始花期、盛花期至末花期,随开花时间的延迟,大花百子莲植株的每花序单花数和座果率均逐渐降低,其中始花期开花的植株每花序单花数和座果率均最高。从果序基部、中部至顶端,座果率和结籽率均逐渐减小且差异显著。在果序基部和中部,果实内种子败育率沿果实基部至顶端依次降低,而单粒种子质量则依次增加;且果序基部的果实各部位的种子败育率均低于果序中部的果实,而单粒种子质量则差异不大。大花百子莲的单株潜在种子产量约为43.18 g,而其单株实际种子产量约为2.87 g,远低于其潜在种子产量。自然授粉的大花百子莲每柱头花粉数为27.5粒,而自花授粉、同株异花授粉和异株授粉的每柱头花粉数均超过100粒;与自然授粉、自花授粉和同株异花授粉植株相比,异株授粉植株的座果率和结籽率均显著增加。研究结果显示:大花百子莲的结实和结籽格局呈现非随机分布规律,在引种地存在有性繁殖障碍;养分和授粉状况是制约大花百子莲种子产量的重要因素。     

Posttranslational Elevation of Cell Wall Invertase Activity by Silencing Its Inhibitor in Tomato Delays Leaf Senescence and Increases Seed Weight and Fruit Hexose Level

Invertase plays multiple pivotal roles in plant development. Thus, its activity must be tightly regulated in vivo. Emerging evidence suggests that a group of small proteins that inhibit invertase activity in vitro appears to exist in a wide variety of plants. However, little is known regarding their roles in planta. Here, we examined the function of INVINH1, a putative invertase inhibitor, in tomato (Solanum lycopersicum). Expression of a INVINH1:green fluorescent protein fusion revealed its apoplasmic localization. Ectopic overexpression of INVINH1 in Arabidopsis thaliana specifically reduced cell wall invertase activity. By contrast, silencing its expression in tomato significantly increased the activity of cell wall invertase without altering activities of cytoplasmic and vacuolar invertases. Elevation of cell wall invertase activity in RNA interference transgenic tomato led to (1) a prolonged leaf life span involving in a blockage of abscisic acid–induced senescence and (2) an increase in seed weight and fruit hexose level, which is likely achieved through enhanced sucrose hydrolysis in the apoplasm of the fruit vasculature. This assertion is based on (1) coexpression of INVINH1 and a fruit-specific cell wall invertase Lin5 in phloem parenchyma cells of young fruit, including the placenta regions connecting developing seeds; (2) a physical interaction between INVINH1 and Lin5 in vivo; and (3) a symplasmic discontinuity at the interface between placenta and seeds. Together, the results demonstrate that INVINH1 encodes a protein that specifically inhibits the activity of cell wall invertase and regulates leaf senescence and seed and fruit development in tomato by limiting the invertase activity in planta.     

Expression of a plant cell wall invertase in roots of Arabidopsis leads to early flowering and an increase in whole plant biomass

In order to enhance sink strength, we expressed a heterologous plant cell wall invertase (CrCIN1) under the control of a root-specific promoter (ppyk10) in Arabidopsis thaliana. Slightly elevated apoplastic invertase activity resulted in apparent phenotypic changes. Transgenic plants developed more secondary roots and subsequently, possibly because of a higher capacity to acquire nutrients, a higher shoot and whole plant biomass. Furthermore, an early flowering phenotype was detected. The data presented here demonstrate that it is possible to modulate carbohydrate metabolism by ectopic expression of cell wall invertases and thereby influence sink organ size and whole plant development.     

土壤养分对菊科一年生入侵种和本地种繁殖性状的影响

入侵植物繁殖性状的研究可为揭示植物入侵机制提供重要的科学依据。为研究土壤养分对入侵植物和本地植物繁殖性状的影响, 并进一步研究养分添加是否更能促进入侵植物的繁殖能力, 我们设置了低、高两个养分水平, 通过同质园实验比较了不同土壤养分对假臭草(Praxelis clematidea)、胜红蓟(Ageratum conyzoides)、三叶鬼针草(Bidens pilosa) 3种菊科一年生入侵种和夜香牛(Vernonia cinerea)、一点红(Emilia sonchifolia)、墨旱莲(Eclipta prostrata) 3种本地种繁殖性状的影响。研究结果显示, 养分添加提高了6种菊科植物的开花株高、株高、地上生物量、单粒种子重量、总花序数、每花序种子数、总种子数量、总种子重量, 并使开花时间提前、花期延长。养分添加对入侵种的开花株高和单粒种子重量的提高幅度要比本地种更显著。相对于部分本地植物(夜香牛、墨旱莲), 养分添加更能促进部分入侵植物(假臭草、胜红蓟)的繁殖能力。三叶鬼针草和一点红的总种子数量和总种子重量在两种土壤养分水平下均较小。本地种墨旱莲的总种子数量和总种子重量在低养分条件下高于3个入侵种。这些结果表明, 高土壤养分仅能促进部分入侵植物相对于部分本地植物的繁殖能力。     

<Emphasis Type="Italic">Arabidopsis HOG1</Emphasis> gene and its petunia homolog <Emphasis Type="Italic">PETCBP</Emphasis> act as key regulators of yield parameters

Plant hormones influence the key parameters that contribute to crop yield, including biomass, branching and seed number. We tested manipulation of cytokinin signaling as an avenue for influencing these growth parameters. Here we report a full-length cDNA coding for a cytokinin binding protein, Petunia cytokinin binding protein (PETCBP) from Petunia hybrida cv. Mitchell. PETCBP encodes for a protein that exhibits high sequence similarity to S-adenosyl-L: -homocysteine hydrolase (SAHH). Transgenic petunia plants expressing this gene in antisense orientation displayed profuse branching, delayed flowering and delayed shoot bud induction from leaf explants in vitro. Homologs were also isolated from Arabidopsis thaliana homology-dependent gene silencing 1 (HOG1) and Orzya sativa (OsCBP). Arabidopsis HOG1 showed high affinity cytokinin binding activity and modified plant architecture similar to PETCBP. Transgenic Arabidopsis plants overexpressing HOG1 showed early flowering with a significantly reduced plant biomass and number of leaves. In contrast, profuse branching, delayed flowering, increased leaf size and higher seed yield were the major phenotypes observed in the antisense suppression lines. These results suggest that genetic manipulation of this cytokinin binding protein or its orthologs could be used for improving crop biomass and seed yield.     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants overexpressing <Emphasis Type="Italic">Ramosa1</Emphasis> maize gene show an increase in organ size due to cell expansion

The structure of the plant inflorescence and flower is an important agronomic and ornamental trait studied for its potential economic applications. In particular, the capacity to modify flower size has always been a breeder’s goal. Genetic and molecular studies have shown that the Zea mays gene Ramosa1 (Ra1) is involved in inflorescence branching regulation. In fact the ra1 loss of function mutation causes extra branching of the inflorescence. In this work we suggest a possible utilization of the Ramosa1 maize gene as a tool to modify inflorescence architecture and flower size in transgenic plants. In fact overexpression of this gene in Arabidopsis plants promotes an increase in reproductive organ size. Pollen, seeds, cotyledons, leaves and roots are also larger than those of the wild type. Analysis of organs from transformants showed that cell expansion was increased without apparently affecting cell division. These results suggest that the RA1 protein is able to up-regulate cell expansion in all organs of Arabidopsis plants.     

Plasticity of reproductive components at different stages of development in the annual plant Thlaspi arvense L.

Summary This study examines the effect of different densities and the removal of all neighbours at different stages of development on all components of reproduction in the inbreeding annual Thlaspi arvense L. A 64-fold increase in density significantly reduced all repooductive components. The number of flower buds per plant was decreased most strongly; the order of decreasing plasticity among the other components was number of capsules per flower, individual seed weight, ovule number per capsule, flowers per flower bud and seeds per ovule. Removing neighbours at all stages of development increased seed yield of plants in comparison to the control without density reduction, but patterns of plasticity depended strongly on time of treatment. The main effect of the removal of neighbours at the vegetative stage was to increase the number of flowers per plant, but the number of ovules per capsule and seed weight increased also, and abortion of capsules decreased. Removing neighbours at the onset of flowering initially failed to affect flower number per plant, instead it resulted in a strong reduction of capsule abortion and an increase in seed weight. However, several weeks after flowering had initially ceased, fresh lateral inflorescences were produced, resulting in a second flush of reproduction. Removing neighbours at the stage of fruit ripening resulted at first only in an increase in seed witht, but later a second reproductive phase occurred. Fresh lateral branches were produced, but the apical meristem was also reactivated. The overall pattern of plasticity among all reproductive components in response to a removal of neighbours was the same as in response to density. The position of a capsule along the inflorescence influenced its number of ovules, the rate of seed abortion and the mean weight of seeds, with the type of effect depending on the developmental stage at which neighbours were removed. Significant negative correlations were found between the mean weight of seeds and the number of seeds in a capsule under all treatments.     

Differential effect of the nitrogen form on the leaf gas exchange,amino acid composition,and antioxidant response of sweet pepper at elevated CO<Subscript>2</Subscript>

Cell wall invertases play an important role in plant growth and development, especially in the grain filling of crop plants. However, their potential in high yield crop breeding has not been investigated. In this study, the main hybrid maize cultivar Zheng Dan 958 (ZD958) was used as a basic variety to assess whether ZmGIF1, a cell wall invertase from maize, can be used to breed new cultivars with higher grain yields. ZmGIF1 expression, cell wall invertase activity, and sugar content in different parental inbred cultivars were compared with those in Zheng 58 and Chang 7-2, the parental inbred cultivars of ZD958. Parental cultivars which showed higher ZmGIF1 expression and invertase activity were selected and intercrossed to improve the expression of ZmGIF1. Compared with the basic cultivar ZD958, higher ZmGIF1 expression and cell wall invertase activity were observed in most hybrid F1 lines, leading to increased grain yield in them. All these results suggest that the expression of ZmGIF1 can be manipulated using different parental cultivars to increase the grain yield of their hybrid F1 progenies.     

Resource availability regulates reproductive meristem activity, development of reproductive structures and seed set in buckwheat (Fagopyrum esculentum)

Grain yields in buckwheat ( Fagopyrum esculentum ) are consistently low despite a profuse and long-lasting flowering. The mechanisms underlying this reproductive failure are still largely unknown. In this study, performed in strictly controlled conditions, the possible implication of assimilate availability in the regulation of the reproductive development was investigated in the 'La Harpe' cultivar, by manipulating sink/source relationships through excisions of selected leaves and/or selected inflorescences and hand pollinations of selected and precisely identified flowers. Increasing or decreasing source/sink organ ratio in buckwheat, respectively, enhanced or reduced the production of racemes by the shoot apical meristem and of cymes by the racemes, ultimately modulating the number of flowers per plant. The effect on seed production was essentially indirect and related to flower production. Seeds developing on a plant also affected reproductive morphogenesis, limiting flower production. Three stages at which flower development to mature seed may fail have been identified. First, an abnormal morphogenesis resulted in flowers with an undersized and sterile gynoecium. Secondly, after anthesis, numerous flowers with a normal sized gynoecium, were unable to develop a seed after hand pollination. Finally, a small proportion of flowers exhibited a later abortion of the seed. Flower fate is dependent on position in the raceme and time of opening: chance to develop normally and produce a ripe seed is higher for flowers at the base than at the top of the inflorescence. A mechanism, internal to the raceme, regulates flower fate, independently of assimilate availability. This mechanism may be disturbed by drastic reductions in nutrient supply.     

Local plant density,pollination and trait–fitness relationships in a perennial herb

Both differences in local plant density and phenotypic traits may affect pollination and plant reproduction, but little is known about how density affects trait–fitness relationships via changes in pollinator activity. In this study we examined how plant density and traits interact to determine pollinator behaviour and female reproductive success in the self‐incompatible, perennial herb Phyteuma spicatum. Specifically, we hypothesised that limited pollination service in more isolated plants would lead to increased selection for traits that attract pollinators. We conducted pollinator observations and assessed trait–fitness relationships in a natural population, whose individuals were surrounded by a variable number of inflorescences. Both local plant density and plant phenotypic traits affected pollinator foraging behaviour. At low densities, pollinator visitation rates were low, but increased with increasing inflorescence size, while this relationship disappeared at high densities, where visitation rates were higher. Plant fitness, in terms of seed production per plant and per capsule, was related to both floral display size and flowering time. Seed production increased with increasing inflorescence size and was highest at peak flowering. However, trait–fitness relationships were not density‐dependent, and differences in seed production did not appear to be related to differences in pollination. The reasons for this remain unclear, and additional studies are needed to fully understand and explain the observed patterns.     

新疆郁金香营养生长、个体大小和开花次序对繁殖分配的影响

植物有性繁殖与资源分配的关系研究对于揭示植物生活史特征及繁育系统进化具有重要意义。新疆郁金香(Tulipa sinkiangensis)是新疆天山北坡荒漠带特有的一种多年生早春短命植物。在自然生境中,该物种仅以有性繁殖产生后代,每株能产生1-8朵花,且不同植株上的花数及果实数以及花序不同位置上的花与果实大小明显不同。本文通过对新疆郁金香有性繁殖与营养生长及植株大小的关系以及花序中不同位置花及果实间的资源分配研究,旨在揭示营养生长、个体大小及开花次序对其繁殖分配的影响。结果表明:在开花和果实成熟阶段,新疆郁金香植株分配给营养器官(鳞茎和地上营养器官)与繁殖器官的资源间均存在极显著的负相关关系(P<0.01),说明其植株的营养生长与生殖生长间存在权衡关系。多花是新疆郁金香的一个稳定性状,其植株上花数目、花生物量、果实生物量和种子数量与植株生物量间均呈极显著的正相关关系(P<0.01),说明新疆郁金香植株的繁殖分配存在大小依赖性。在具2-5朵花的新疆郁金香植株中,花序内各花的生物量、花粉数和胚珠数、结实率、果实生物量、结籽数、结籽率及种子百粒重按其开花顺序依次递减,说明花序内各花和果实的资源分配符合资源竞争假说。植株通过减少晚发育的花或果实获得的资源来保障早发育的花或果实获得较多的资源,从而达到繁殖成功。     

A test of the reserve meristem hypothesis using Verbascum thapsus (Scrophulariaceae)

The reserve meristem hypothesis predicts that latent meristems may act as a bet-hedging strategy given high-cost, predictable herbivory. Under this hypothesis, damage to a plant should elicit greater branching. This prediction was tested in Verbascum thapsus with three experiments manipulating the intensity and type of damage to reproductive tissue. In the first experiment, seed set was prevented in the treatment group by stigma excision and lanolin application to 80% of the flowers of each plant. In the second experiment, a minimum of two mating pairs of weevils were added to treated plants prior to the onset of flowering. In the third experiment, all fruits were sliced lengthwise twice. All three treatments significantly reduced seed set. In the first two experiments, treated plants significantly increased degree of branching (branch number and total branch length). This supports the reserve meristem hypothesis as an explanation for greater branching in larger plants of V. thapsus. Interestingly, the fruit destruction experiment failed to elicit a branching response, which suggests that the timing of damage is important.     

COMPONENTS OF REPRODUCTIVE EFFORT AND YIELD IN GOLDENRODS

Four components of reproductive yield (the weight of reproductive tissue) were examined in relation to their effect on reproductive effort and their relative contributions to reproductive yield in five species of goldenrods (Solidago, Compositae). The yield components were number of flowing stems per plant, number of flowering branches per stem, number of flowering heads per branch, and number of seeds per seed head. Individuals within populations increase their reproductive effort by increasing their reproductive weight, not by decreasing their vegetative weight. Each species shows a different pattern of positive correlations of yield components with reproductive yield and reproductive effort, indicating that each species has its own mechanisms for regulating reproduction. The yield components were not significantly intercorrelated.     

Pleiotropic effects of flowering time genes in the annual crucifer Arabidopsis thaliana (Brassicaceae)

Variation in flowering time of Arabidopsis thaliana was studied in an experiment with mutant lines. The pleiotropic effects of flowering time genes on morphology and reproductive yield were assessed under three levels of nutrient supply. At all nutrient levels flowering time and number of rosette leaves at flowering varied among mutant lines. The relationship between these two traits depended strongly on nutrient supply. A lower nutrient supply first led to an extension of the vegetative phase, while the mean number of leaves at flowering was hardly affected. A further reduction resulted in no further extension of the vegetative phase and, on average, plants started flowering with a lower leaf number. At low nutrients, early flowering affected the timing of production of siliques rather than the total output, whereas late flowering was favorable at high nutrients. This may explain the fact that many plant species flower at a relatively small size under poor conditions. Flowering time genes had pleiotropic effects on the leaf length, number of rosette and cauline leaves, and number of axillary flowering shoots of the main inflorescence. Silique production was positively correlated with the number of axillary shoots of the main inflorescence; the number of axillary primordia appeared to have a large impact on reproductive yield.     

Changes in the activities of carbon metabolizing enzymes with pod development in lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> L.)

Activities of some key enzymes of carbon metabolism sucrose synthase, acid and alkaline invertase, phosphoenol pyruvate carboxylase, malic enzyme and isocitrate dehydrogenase were investigated in relation to the carbohydrate status in lentil pods. Sucrose remained the dominant soluble sugar in the pod wall and seed, with hexoses (glucose and fructose) present at significantly lower levels. Sucrose synthase is the predominant sucrolytic enzyme in the developing seeds of lentil (Lens culinaris L.). Acid invertase was associated with pod elongation and showed little activity in seeds. Sucrose breakdown was dominated by alkaline invertase during the development of podwall, while both the sucrose synthase and alkaline invertase were active in the branch of inflorescence. A substantial increase of sucrolytic enzymes was observed at the time of maximum seed filling stage (10–20 DAF) in lentil seed. The pattern of activity of sucrose synthase highly paralleled the phase of rapid seed filling and therefore, can be correlated with seed sink strength. It seems likely that the fruiting structures of lentil utilize phosphoenol pyruvate carboxylase for recapturing respired carbon dioxide. Higher activities of isocitrate dehydrogenase and malic enzyme in the seed at the time of rapid seed filling could be effectively linked to the deposition of protein reserves.     

Properties of the Invertases of Cultured Sycamore Cells and Changes in Their Activity during Culture Growth

When cultured sycamore cells are homogenised in a phosphate-citrate buffer at pH 7.0 and the homogenate centrifuged two fractions are obtained both of which show the presence of an acid (opt. pH 4.0–4.5) and a neutral (opt. pH 7.0–7.4) invertase. The activity of the insoluble pellet appears to be located in its cell wall fragments. The acid and neutral invertases of the soluble fraction can be separated by fractional precipitation with (NH₄SO₄. The activities of these enzymes are low in stationary phase cells but they increase following subculture to reach peaks of activity towards the end of the period of most active cell growth and division and then decline again as the cells begin to enter stationary phase. The activities of both enzymes are higher in the cell wall than in the soluble fraction and the acid invertase reaches higher levels of activity than the neutral enzyme in both fractions. When cells are subcultured there occurs within a few hours an increase in the acid invertase and a decline in the neutral invertase activity in the cell wall fraction and a decline in the acid invertase of the soluble fraction prior to the large net increases in the activities of both enzymes in both locations which occurs as the cells embark upon cell division. The pattern of changes in the invertase activities through the growth cycle of batch propagated cultures is similar whether the cells are grown in sucrose, or glucose, or sucrose plus glucose; the highest levels of activities were recorded in the glucose-grown cells. The total yield of invertase activities and the distribution of activities between the soluble and cell wall fractions of the homogenates are affected by the pH of the extraction medium (within the range pH 4.0–8.0). It has not proved possible to completely remove the invertases from the cell wall fraction; upwards of 50 % of the acid invertase was recovered from this fraction by treatment with Triton-X followed by urea, but these treatments inactivated a high proportion of the neutral enzyme. These findings are compared with other studies on the activity and intra-cellular distribution of plant invertases and the possible roles of these enzymes discussed.     

Flowering phenology,floral display and reproductive success in dioecious,Aralia nudicaulis L. (Araliaceae)

Summary Aralia nudicaulis L. is a dioecious, perennial, herbaceous plant that is commonly found in the understory vegetation throughout the boreal forest of North America. Female remets have fewer flowers per inflorescence, initiate flowering earlier, and reach peak flowering before male ramets. The consequences of the asynchrony in flowering between the sexes on pollination and seed set were examined during a two-year study. In both years there was significant variation in seed set associated with the flowering times of individual female ramets. In 1983, seed production was highest in the middle of the flowering season. In 1984, seed production was greatest in the later stages of flowering. Variation in seed set was not attributed to lack of pollination in 1983. In 1984, pollination limited seed set per flower during peak flowering. However, seed production never reached the potential five seeds per flower, suggesting that resource limitation was the most important factor affecting fecundity in both years. The asynchronous pattern of flowering is suggested to be the result of the different inflorescence sizes between the sexes.