Improving recombinant Rubisco biogenesis,plant photosynthesis and growth by coexpressing its ancillary RAF1 chaperone

Enabling improvements to crop yield and resource use by enhancing the catalysis of the photosynthetic CO₂-fixing enzyme Rubisco has been a longstanding challenge. Efforts toward realization of this goal have been greatly assisted by advances in understanding the complexities of Rubisco’s biogenesis in plastids and the development of tailored chloroplast transformation tools. Here we generate transplastomic tobacco genotypes expressing Arabidopsis Rubisco large subunits (AtL), both on their own (producing tob^AtL plants) and with a cognate Rubisco accumulation factor 1 (AtRAF1) chaperone (producing tob^AtL-R1 plants) that has undergone parallel functional coevolution with AtL. We show AtRAF1 assembles as a dimer and is produced in tob^AtL-R1 and Arabidopsis leaves at 10–15 nmol AtRAF1 monomers per square meter. Consistent with a postchaperonin large (L)-subunit assembly role, the AtRAF1 facilitated two to threefold improvements in the amount and biogenesis rate of hybrid L₈^AS₈^t Rubisco [comprising AtL and tobacco small (S) subunits] in tob^AtL-R1 leaves compared with tob^AtL, despite >threefold lower steady-state Rubisco mRNA levels in tob^AtL-R1. Accompanying twofold increases in photosynthetic CO₂-assimilation rate and plant growth were measured for tob^AtL-R1 lines. These findings highlight the importance of ancillary protein complementarity during Rubisco biogenesis in plastids, the possible constraints this has imposed on Rubisco adaptive evolution, and the likely need for such interaction specificity to be considered when optimizing recombinant Rubisco bioengineering in plants.The increasing global demands for food supply, bioenergy production, and CO₂-sequestration have placed a high need on improving agriculture yields and resource use (1, 2). It is now widely recognized that yield increases are possible by enhancing the light harvesting and CO₂-fixation processes of photosynthesis (3–5). A major target for improvement is the enzyme Rubisco [ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase] whose deficiencies in CO₂-fixing speed and efficiency pose a key limitation to photosynthetic CO₂ capture (6, 7). In plants, the complex, multistep catalytic mechanism of Rubisco to bind its 5-carbon substrate RuBP, orient its C-2 for carboxylation, and then process the 6-carbon product into two 3-phosphoglycerate (3PGA) products, limits its throughput to one to four catalytic cycles per second (8). The mechanism also makes Rubisco prone to competitive inhibition by O₂ that produces only one 3PGA and 2-phosphoglycolate (2PG). Metabolic recycling of 2PG by photorespiration requires energy and results in most plants losing 30% of their fixed CO₂ (5). To compensate for these catalytic limitations, plants like rice and wheat invest up to 50% of the leaf protein into Rubisco, which accounts for ∼25% of their leaf nitrogen (9).Natural diversity in Rubisco catalysis demonstrates that plant Rubisco is not the pinnacle of evolution (6, 7). Better-performing versions in some red algae have the potential to raise the yield of crops like rice and wheat by as much as 30% (10). Bioengineering Rubisco in leaves therefore faces two key challenges: identifying the structural changes that promote performance and identifying ways to efficiently transplant these changes into Rubisco within a target plant. A significant hurdle to both challenges is the complex biogenesis requirements of Rubisco in plant chloroplasts (7, 11). A number of ancillary proteins are required to correctly process and assemble the chloroplast made Rubisco large (L) subunit (coded by the plastome rbcL gene) and cytosol made small (S) subunits (coded by multiple RbcS genes in the nucleus) into L₈S₈ complexes in the chloroplast stroma. The complicated assembly requirements of Rubisco in chloroplasts prevent their functional testing in Escherichia coli and conversely impedes, sometimes prevents, the biogenesis of Rubisco from other higher plants, cyanobacteria, and algae (12–14). For example, the L-subunits from sunflower and varying Flaveria sp. showed fivefold differences in their capacity to form hybrid L₈S₈ Rubisco (that comprise tobacco S-subunits) in tobacco chloroplasts despite each rbcL transgene sharing the same genetic regulatory sequences and showing >92% amino acid identity (13, 14). Evidently, evolution of Rubisco function may have been constrained to maintain compatibility with the molecular chaperones required for its biogenesis (7, 15).The necessity of chloroplast chaperonin (CPN) complexes for Rubisco biogenesis has been known for some time (16). Upon release from the hetero-oligomeric CPN ring structures in chloroplasts (17) the folded L-subunits are thought to sequentially assemble into dimers (L₂) then octamers (L₂)₄ before S-subunit binding (18). The molecular details of this process remain unclear. The maize Photosynthetic Mutant Library has provided useful insight by identifying three chaperones with roles associated with Rubisco synthesis, assembly, and stability: Rubisco accumulation factors-1 (RAF1) (19) and-2 (RAF2; a Pterin-4a-Carbinolamine Dehydratase-like protein) (20) and Bundle Sheath Defective-2 (BSDII; a DnaJ-like protein) (21). Results of chemical cross-linking experiments in maize leaves suggest all three proteins might associate with the S-subunit during Rubisco biogenesis (20). Other studies, however, suggest RAF1 interacts with post-CPN folded L-subunits to assist in L₂ then (L₂)₄ formation (19, 22). This function mirrors that shown for RbcX, a Rubisco chaperone that acts as a “molecular staple” to assemble folded L-subunits into L₂ units for (L₂)₄ assembly before S-subunit binding to displace the RbcX and trigger catalytic potential (18). Although the function of RbcX in L₈S₈ Rubisco biogenesis has been resolved in exquisite molecular detail in vitro and in E. coli, its functional role in cyanobacteria and in leaf chloroplasts remain unresolved. Comparable molecular details on RAF1, RAF2, and BSDII structure and function remain incomplete, making it difficult to reliably assign their roles and interactions with Rubisco in chloroplasts.Targeted transformation of the chloroplast genome (plastome) provides a reliable but time-consuming tool for engineering Rubisco (23). This technology is best developed in tobacco with the ^cmtrL genotype specifically made for bioengineering Rubisco and testing its effects on leaf photosynthesis and growth (6, 7, 13, 14). Here we use chloroplast transformation in ^cmtrL to examine the function of RAF1 from Arabidopsis (AtRAF1) in Rubisco biogenesis. We show that AtRAF1 forms a stable dimer that, when coexpressed with its cognate Arabidopsis Rubisco L-subunits (AtL), enhances hybrid L₈^AS₈^t Rubisco (containing Arabidopsis L- and tobacco S-subunits) assembly in tobacco chloroplasts and concomitantly improves leaf photosynthesis and plant growth by more than twofold.     

Changes in cancer incidence in teenagers and young adults (ages 13 to 24 years) in England 1979-2003

BACKGROUND: Cancer for teenagers and young adults represents a major source of morbidity and mortality. Trends in cancer incidence can provide pointers concerning how changes in the environment and in personal behavior affect cancer risks. METHODS: Data on 39,129 neoplasms in individuals ages 13 to 24 years who were diagnosed in England from 1979 to 2003 were analyzed. Variability in incidence by time period and differences in the time trends by age group, sex, and geographic region were analyzed using generalized linear models. RESULTS: Incidence rates of leukemias, lymphomas, central nervous system, bone, and germ cell tumors; melanoma; and carcinomas of the thyroid, ovary, cervix, and colon/rectum increased over time (all P < .01); whereas the incidence of carcinomas of the stomach and bladder decreased (both P < .01). These changes were consistent by age, sex, and region for most neoplasms. Melanoma incidence stabilized in southern England by 1993 but continued to increase in northern England (P = .001). The increase in non-Hodgkin lymphoma was greater in individuals ages 20 to 24 year than in younger individuals, but the increase in Hodgkin lymphoma was confined to individuals ages 13 to 14 years. CONCLUSIONS: The changes in incidence rates may have been caused in part by environmental changes and in part by behavioral changes in young individuals. Some of these results can be used to inform public health campaigns, which can be constructed to encourage better lifestyle choices by young individuals.     

The behaviour of green tea catechins in a full-fat milk system under conditions mimicking the cheesemaking process

Due to their well-known health benefits, green tea catechins have received recent attention as natural additives in foods such as dairy products. However, they may present some irreversible associations with milk components (e.g. protein and milk fat globules). To investigate the behaviour of two important green tea catechins, (+)-catechin (C) and (?)-epigallocatechin gallate (EGCG), in a standard whole milk system under the conditions of cheesemaking, 250 and 500?ppm of each catechin were added to whole milk (3.3% fat). Although both C and EGCG at either concentration increased both total phenolic content and total antioxidant capacity of the subnatants obtained from the milk system, there was a less linear increase when the concentration of the catechins was doubled, whereas C or EGCG were recovered (measured by HPLC) differently. Overall, these results suggest a degree of associations between green tea catechins with milk proteins and milk fat.     

Variable expressivity in patients with autosomal recessive retinitis pigmentosa associated with the gene CNGB1

ABSTRACT

Purpose

In a cohort of eight families (11 patients) with autosomal recessive retinitis pigmentosa (arRP), we clinically characterized disease associated with mutations in CNGB1.     

Incidence and survival of childhood Langerhans cell histiocytosis in Northwest England from 1954 to 1998

BACKGROUND: Langerhans cell histiocytosis (LCH) is a rare proliferative disorder of pathological Langerhans cells, for which the aetiology and pathogenesis remain largely unknown. PROCEDURE: Information on the 101 children with LCH registered with the population-based Manchester Children's Tumour Registry (MCTR) between 1954 and 1998 was extracted from the records of the MCTR. This included age, sex, date of diagnosis, systems affected at diagnosis and follow-up. RESULTS: The overall incidence rate for LCH was 2.6 cases per million child years. In those under 1 year of age the incidence rate was 9.0 cases per million child years, compared to 0.7 cases per million in those aged 10-14 years (P < 0.0001 for age trend). There was no evidence of seasonal variation in presentation by month of birth or first symptom. Bone was the most common site of disease involvement (67% of cases), followed by skin (37%) and soft tissue (22%). The overall survival rate has improved over time, from 57% in 1954-1968 to 74% in 1985-1998. Ninety percent of deaths were due to disease progression, the remainder were due to complications of intensive therapy. The site of LCH lesions and extent of disease present at diagnosis strongly predicted survival outcome. Patients with initial liver involvement had a 5-year survival rate of 25% compared with 93% for those with bone lesions alone at diagnosis. CONCLUSIONS: Incidence rates varied significantly by age at diagnosis, and have been stable over time. Survival has improved considerably over time, but varies strongly by age and systems affected at diagnosis.     

Urban parents' perceptions of healthy infant growth

To examine how urban parents' perceptions of healthy infant size and growth relate to objective weight-for-length percentiles of their children, parents of 222 (69% minority) 6- to 27-month-old infants were surveyed. In all, 41% of parents said growth charts had never been explained to them, and 31% were not confident they understood the meaning of chart percentiles. A total of 20% of parents preferred their child weigh ≥75th percentile, and these children had higher mean weight-for-length percentiles than their peers (P = .05). Similarly, 37% of parents agreed that "a chubby baby is a healthy baby," and these children had higher mean weight-for-length percentiles than others in the cohort ( P = .02). Additionally, 58% of parents ranked a growth curve with consistent growth at the 10th percentile as the "least healthy" of 6 infant growth curves. Growth charts are not consistently explained to or understood by urban parents, and many prefer early life growth patterns associated with later obesity.