Cloning and characterization of the Rubisco activase gene from <Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam

A full-length cDNA of Rubisco activase (IBrcaI) was cloned from sweet potato (Ipomoea batatas (L.) Lam) using Rapid-Amplification of cDNA Ends (RACE). IBrcaI contains a 1,347 bp open reading frame encoding a protein of 439 amino acids. The sequence alignment of multiple Rubisco activase genes from sweet potato and other plants showed high homology at two previously described ATP-binding sites. Western blot analysis indicated that there are two Rubisco activase proteins in sweet potato. Expression of IBrcaI was only detected in leaves. In the 14 h light and 10 h dark photoperiods, maximal and minimal IBrcaI mRNA expression levels were detected at 8:00 in the morning and at midnight, respectively.     

Alterations in Rubisco activity and in stomatal behavior induce a daily rhythm in photosynthesis of aerial leaves in the amphibious-plant Nuphar lutea

Nuphar lutea is an amphibious plant with submerged and aerial foliage, which raises the question how do both leaf types perform photosynthetically in two different environments. We found that the aerial leaves function like terrestrial sun-leaves in that their photosynthetic capability was high and saturated under high irradiance (ca. 1,500 μmol photons m⁻² s⁻¹). We show that stomatal opening and Rubisco activity in these leaves co-limited photosynthesis at saturating irradiance fluctuating in a daily rhythm. In the morning, sunlight stimulated stomatal opening, Rubisco synthesis, and the neutralization of a night-accumulated Rubisco inhibitor. Consequently, the light-saturated quantum efficiency and rate of photosynthesis increased 10-fold by midday. During the afternoon, gradual closure of the stomata and a decrease in Rubisco content reduced the light-saturated photosynthetic rate. However, at limited irradiance, stomatal behavior and Rubisco content had only a marginal effect on the photosynthetic rate, which did not change during the day. In contrast to the aerial leaves, the photosynthesis rate of the submerged leaves, adapted to a shaded environment, was saturated under lower irradiance. The light-saturated quantum efficiency of these leaves was much lower and did not change during the day. Due to their low photosynthetic affinity for CO₂ (35 μM) and inability to utilize other inorganic carbon species, their photosynthetic rate at air-equilibrated water was CO₂-limited. These results reveal differences in the photosynthetic performance of the two types of Nuphar leaves and unravel how photosynthetic daily rhythm in the aerial leaves is controlled.     

Plasticity in carbon acquisition of the heterophyllous Luronium natans: An endangered freshwater species in Europe

Luronium natans (L.) Raf. (Floating Water-plantain) is an endangered amphibious freshwater species endemic to Europe. We examined the plasticity in carbon acquisition and photosynthesis in L. natans to assess if lack of plasticity could contribute to explain the low competitive ability of the species. The plasticity of photosynthesis in submerged leaves towards inorganic carbon availability was examined and the photosynthesis of submerged, floating and aerial leaves was contrasted. L. natans was shown to be plastic in inorganic carbon uptake, as it was able to effectively acclimate to changed concentrations of free-CO₂. The photosynthetic apparatus was down-regulated in plants grown at high CO₂. Chlorophyll concentration, Rubisco activity and maximum photosynthesis were significantly lower in submerged leaves of plants grown at high CO₂ (200 μM free-CO₂) compared to plants grown at low CO₂ (18 μM free-CO₂). Furthermore, bicarbonate utilization was down-regulated in response to high CO₂. Carbon acquisition of submerged, floating and aerial leaves of L. natans differed significantly. The aerial leaves were superior in photosynthesising in air and, surprisingly, the floating leaves had the highest rates of photosynthesis in water. The study did not support the hypothesis that the low competitive ability of L. natans is caused by inefficient photosynthesis or a lack of plasticity in photosynthesis. However, the somewhat low photosynthetic performance of the submerged leaves may be a contributing factor.     

Characterization and expression of Rubisco activase genes in Ipomoea batatas

Two-dimensional electrophoresis, coupled with MALDI-TOF-MS, was used to identify differentially expressed proteins between young and mature leaves of sweet potato [Ipomoea batatas (L.) Lam]. The results showed that there were 25 differential proteins between young and mature leaves. The Rubisco activase (RCA) that catalyzes the activation of Rubisco in vivo and plays a crucial role in photosynthesis was among these 25 proteins. So far, little was known about the molecular biology of RCA in sweet potato. Here, this research reports the cloning and characterization of two genes encoding the short isoform and the long isoform of sweet potato RCAs. Analysis of DNA sequences of RCA suggested that the corresponding mRNAs were transcribed from two different genes. To study the roles of these two RCA isoforms in photosynthesis, we investigated the expression patterns of these RCA genes at the mRNA and protein levels every 2 h in a photoperiod and under different temperatures conditions. The results indicated that these two RCA isoforms may play different roles in regulating photosynthesis and they may be regulated by light, heat or both. In addition, there were interactions between Rubisco large subunit (RBCl) and short isoform RCA (RCAs) as well as RCAs and long isoform RCA (RCAl), but no interaction between RBCl and RCAl, implying they might form a sandwich-like structure (RBCl–RCAs–RCAl), at least in yeast cells. These results provided new information on the modulation of RCA genes in sweet potato, which could be useful in improving photosynthesis and plant growth in sweet potato.     

Co-overproducing Rubisco and Rubisco activase enhances photosynthesis in the optimal temperature range in rice

Rubisco limits C₃ photosynthesis under some conditions and is therefore a potential target for improving photosynthetic efficiency. The overproduction of Rubisco is often accompanied by a decline in Rubisco activation, and the protein ratio of Rubisco activase (RCA) to Rubisco (RCA/Rubisco) greatly decreases in Rubisco-overproducing plants (RBCS-ox). Here, we produced transgenic rice (Oryza sativa) plants co-overproducing both Rubisco and RCA (RBCS-RCA-ox). Rubisco content in RBCS-RCA-ox plants increased by 23%–44%, and RCA/Rubisco levels were similar or higher than those of wild-type plants. However, although the activation state of Rubisco in RBCS-RCA-ox plants was enhanced, the rates of CO₂ assimilation at 25°C in RBCS-RCA-ox plants did not differ from that of wild-type plants. Alternatively, at a moderately high temperature (optimal range of 32°C–36°C), the rates of CO₂ assimilation in RBCS-ox and RBCS-RCA-ox plants were higher than in wild-type plants under conditions equal to or lower than current atmospheric CO₂ levels. The activation state of Rubisco in RBCS-RCA-ox remained higher than that of RBCS-ox plants, and activated Rubisco content in RCA overproducing, RBCS-ox, RBCS-RCA-ox, and wild-type plants was highly correlated with the initial slope of CO₂ assimilation against intercellular CO₂ pressures (A:Ci) at 36°C. Thus, a simultaneous increase in Rubisco and RCA contents leads to enhanced photosynthesis within the optimal temperature range.

A simultaneous increase in Rubisco and RCA contents in transgenic rice leads to an enhancement of photosynthesis at moderately high temperatures within the optimal temperature range.     

Performance of two isolates of Isaria fumosorosea from hot climate zones in solid and submerged cultures and thermotolerance of their propagules

Isaria fumosorosea frequently causes mycosis of agricultural pests in the hot semiarid and dry tropical regions of Mexico. Because temperature tolerance restricts the use of fungal biopesticides, we investigated two isolates from these areas for possible development into mycoinsecticides for use in hot weather agricultural zones. We studied the effects of culture system (solid or submerged cultures) and temperature on the fungal growth, extracellular enzyme production, pathogenicity, and thermotolerance of the produced propagules. Between 20 and 28 °C, the specific growth rates of the isolate PCC were higher on solid media, but in the submerged culture, the isolate P43A grew faster even at temperatures of up to 34 °C. On solid media, P43A produced 1.5-fold more proteases than PCC, but in the submerged culture, both strains had similar activities. Under the same culture conditions, PCC produced a blastospore:conidia ratio of 1:2, and P43A produced a ratio of 1:5. PCC aerial conidia had the shortest Lethal Time 50 (LT₅₀, the time to reach 50 % mortality) against Galleria mellonella larvae, but LT₅₀ was equal for the aerial conidia and the submerged propagules of P43A and PCC. The submerged and aerial propagules of P43A were more thermotolerant than those of PCC. Each isolate performed differently in each culture system, and we concluded that the intended production method should be included as a criterion for screening of entomopathogenic fungus. We found that thermotolerance is a specific characteristic of an isolate from a given species. Because of its specific characteristics, P43A shows more promise for the development of a submerged conidia-based mycoinsecticide for foliar application in aqueous form in hot climate regions.     

<Emphasis Type="Bold">Role of microRNAs and their target genes in salinity response in plant</Emphasis>s

We examined the effects of 2,4-epibrassinolide (EBR) application on photosynthesis, antioxidant enzyme activity, and Rubisco activase (RCA) gene expression in wheat (Triticum aestivum L.) seedlings under a combination of drought and heat stress. The net photosynthetic rates (P_n) of wheat seedlings decreased significantly, the photosynthetic capability was inhibited, and the activities of superoxide (SOD), peroxidase (POD), catalase (CAT), and RCA as well as the initial and total activity of Rubisco declined under the combined stress. These decreases and inhibitory effects were significantly ameliorated by exogenous EBR application. Three subunits (45–46, 41–42, and 38–39 kDa) of RCA were observed in wheat seedlings. The abundances of the 38–39 kDa and 41–42 kDa subunits were significantly lower in plants subjected to stressful conditions than in unstressed plants. Interestingly, a marked increase in 45–46 kDa RCA was observed under heat or heat combined with drought stress. The abundance of 38–39 kDa RCA in seedlings exposed to heat, drought, or their combination was significantly enhanced by EBR pretreatment, which paralleled the changes in initial Rubisco activity and P_n, but was not consistent with observed mRNA abundance. These results indicated that the larger subunit of RCA (45–46 kDa), which is more thermostable and increased in response to moderate heat stress, and the smaller isoform (38–39 kDa) of RCA may play important roles in maintaining the photosynthetic capability by EBR under stress conditions.     

彩叶草Rubisco活化酶基因SsRCA的分子特征及其表达模式

为研究观赏植物彩叶草的光合特性,根据Rubisco活化酶(RCA)的保守区域简并扩增获得的保守片段,采用RACE方法,克隆了RCA全长cDNA,命名为SsRCA(GenBank登录号FJ787730).SsRCA cDNA全长1 548bp,包含1个1 311bp的ORF框,编码436个氨基酸的前体蛋白.其5'-UTR区含有1个终止子TAA,3'-UTR区具有2个mRNA非稳定性相关的DST-like元件和推测的加尾信号AATAAA.SsRCA蛋白具有定位于叶绿体的N端转运肽,具有2个保守的ATP-binding结构域、1个sensor 2基序和多个磷酸化位点.多序列比对和系统进化分析表明,SsRCA与其他植物的RCA蛋白具有较高的一致性,属于RCA的β亚基.表达分析表明,SsRCA基因在含有绿色组织的茎、叶和萼片表达.在9 h黑暗和15 h光照的光周期处理中,正午时表达量最高,午夜时表达量最低,具有明显的光诱导表达特性.     

丽江白雪茶化学成分的研究

核酮糖1,5￣二磷酸羧化酶/加氧酶是光合碳同化作用的关键酶,它对调节铁皮石斛Rubisco活性和光合速率具有直接的作用,对其进行基因等方面的研究,会对改变植物光合速率打下良好基础。该研究以一年生铁皮石斛叶片为材料,采用 RT￣PCR和 RACE技术成功克隆了Rubisco活化酶( RCA)基因,并对其进行生物信息学分析。结果表明：RCA基因全长1730 bp,命名为RCA2( GenBank登录号KT205842),其中5′￣UTR 81 bp、3′￣UTR 326 bp,开放阅读框1323 bp,编码440个氨基酸,分子质量为48.53 kDa,等电点为6.19,包含P￣loop NTPase超家族基因结构。氨基酸多重序列比对发现RCA2核苷酸序列与蝴蝶兰的相似性高达87％。 RCA2编码蛋白为亲水性蛋白;亚细胞定位于叶绿体基质;蛋白质二级结构分析,α螺旋占30.68％,延伸链占25.45％,不规则折叠占43.86％。 RCA2编码蛋白质的功能预测发现,RCA2在中间代谢起到了一个非常重要的角色。该研究发现对铁皮石斛光合作用关键酶Rubisco 的分析可为其光合作用特性的发掘提供理论基础,并为提高温室大棚栽培效率提供理论依据。     

Coordinate Expression of Rubisco Activase and Rubisco during Barley Leaf Cell Development

We have utilized the cellular differentiation gradient and photomorphogenic responses of the first leaf of 7-day-old barley (Hordeum vulgare L.) to examine the accumulation of mRNA and protein encoded by the ribulose-1,5-biphosphate carboxylase holoenzyme (rubisco) activase gene (rca). Previous studies have revealed a pattern of coordinate expression of rubisco subunit polypeptides during development. We compared the expression of rubisco polypeptides and mRNAs with those encoded by rca. The mRNAs encoding both rubisco activase and rubisco are expressed exclusively in leaf tissue of 7-day-old barley seedlings; mRNAs and polypeptides of rca accumulate progressively from the leaf base in a pattern that is qualitatively similar to that of rubisco subunit mRNAs and polypeptides. The parallel pattern of rca protein and mRNA accumulation indicate that a primary control of rca gene expression in this system lies at the level of mRNA production. Light-induced expression of rca in etiolated barley follows a different pattern from that of the acropetal barley leaf gradient, however. Etiolated, 7-day-old barley seedlings contain levels of rca mRNA near the limit of detection in Northern blot hybridization assays. White light induces a 50- to 100-fold accumulation of rca mRNA, which is detectable within 30 min after the onset of illumination. In contrast, steady state levels of mRNAs encoding the small rubisco subunit are affected little by light, and mRNAs encoding the large subunit accumulate about 5-fold in response to illumination. While rca mRNA levels are low in etiolated barley leaves, levels of the protein are approximately 50 to 75% of those found in fully green leaves.     

Effects of light quality on leaf morphogenesis of a heterophyllous amphibious plant, Rotala hippuris

Background and Aims

For heterophyllous amphibious plants that experience fluctuating water levels, it is critical to control leaf development precisely in response to environmental cues that can serve as a quantitative index of water depth. Light quality can serve as such a cue because the ratio of red light relative to far-red light (R/FR) increases and blue-light intensity decreases with increasing water depth. Growth experiments were conducted to examine how R/FR and blue-light intensity alter leaf morphology of a heterophyllous amphibious plant, Rotala hippuris.

Methods

Using combinations of far red (730 nm), red (660 nm) and blue (470 nm) light-emitting diodes (LEDs), growth experiments were used to quantitatively evaluate the effects of the R/FR ratio and blue-light intensity on leaf morphology.

Key Results

Under the natural light regime in an outside growth garden, R. hippuris produced distinct leaves under submerged and aerial conditions. R/FR and blue-light intensity were found to markedly affect heterophyllous leaf formation. Higher and lower R/FR caused leaf characters more typical of submerged and aerial leaves, respectively, in both aerial and submerged conditions, in accordance with natural distribution of leaf types and light under water. High blue light caused a shift of trait values toward those of typical aerial leaves, and the response was most prominent under conditions of R/FR that were expected near the water surface.

Conclusions

R/FR and blue-light intensity provides quantitative cues for R. hippuris to detect water depth and determine the developmental fates of leaves, especially near the water surface. The utilization of these quantitative cues is expected to be important in habitats where plants experience water-level fluctuation.     

SDS-dependent proteases induced by ABA and its relation to Rubisco and Rubisco activase contents in rice leaves

Protease activities and its relation to the contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase were investigated in detached leaves of rice (Oryza sativa L.) floated on the solutions containing abscisic acid (ABA) or benzyladenine (BA). Rubisco and Rubisco activase contents were decreased during the time course and the decreases were enhanced by ABA and suppressed by BA. The decrease in Rubisco activase was faster than that in Rubisco. SDS-dependent protease activities at 50–70 kDa (rice SDS-dependent protease: RSP) analyzed by the gelatin containing PAGE were significantly enhanced by ABA. RSPs were also increased in attached leaves during senescence. RSPs had the pH optimum of 5.5, suggesting that RSPs are vacuolar protease. Both decrease in Rubisco and Rubisco activase contents and increase in RSPs activities were suppressed by cycloheximide. These findings indicate that the activities of RSPs are well correlated with the decrease in these protein contents. Immunoblotting analysis showed that Rubisco in the leaf extracts was completely degraded by 5 h at pH 5.5 with SDS where it was optimal condition for RSPs. However, the degradation of Rubisco did not proceed at pH 7.5 without SDS where it is near physiological condition for stromal proteins. Rubisco activase was degraded at similar rate under both conditions. These results suggest that RSPs can functions in a senescence related degradation system of chloroplast protein in rice leaves. Rubisco activase would be more susceptible to proteolysis than Rubisco under physiological condition and this could affect the contents of these proteins in leaves.     

Changes in protein quantities of phosphoenolpyruvate carboxylase and Rubisco activase in various wheat genotypes

In early seedlings of wheat genotypes two isoforms of Rubisco activase with molecular weights of 42 and 46 kDa are expressed. Amounts of both isoforms significantly increase in early seedlings of the durum wheat genotype Barakatli-95 exposed to salt stress. But at the beginning of the tillering stage, the changes in quantities of both RCA isoforms are different in durum and bread wheat genotypes subjected to a 3-day drought stress. In the leaves of the early seedlings of the studied wheat genotypes exposed to drought stress quantities of PEPC subunits increase compared to the control but they remain relatively stable in early roots and germinating seeds. However, quantities of its subunits decrease sharply in roots and germinating seeds of early seedlings under the influence of 100 mM NaCl. In flag leaves and ear elements of the Barakatli-95 genotype grown under normal water supply conditions protein quantities of PEPC subunits change differently depending on time. Changes in protein quantities of RCA, PEPC and Rubisco enzymes have been studied comparatively in ear elements and flag leaves after the fourth day of anthesis.