Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 5 uppsatser.

LOBO, A. K. M. Photosynthesis regulation by sucrose metabolism under water deficit and source-sink alterations in sugarcane. 2016. 118 f. Tese (Doutorado em Bioquímica)-Universidade Federal do Ceará, Fortaleza, 2016. / Submitted by Anderson Silva Pereira (anderson.pereiraaa@gmail.com) on 2017-01-03T20:20:30Z No. of bitstreams: 1 2016_tese_akmlobo.pdf: 4541762 bytes, checksum: e3450d87e286b1a5213eb866921b6c94 (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2017-01-09T21:32:52Z (GMT) No. of bitstreams: 1 2016_tese_akmlobo.pdf: 4541762 bytes, checksum: e3450d87e286b1a5213eb866921b6c94 (MD5) / Made available in DSpace on 2017-01-09T21:32:52Z (GMT). No. of bitstreams: 1 2016_tese_akmlobo.pdf: 4541762 bytes, checksum: e3450d87e286b1a5213eb866921b6c94 (MD5) Previous issue date: 2016 / Water deficit stress is the major limiting factor for plant growth and development, constraining food production. In order to survive in such dry conditions, many biochemical and physiological changes must be triggered by plants. In general, the responses to drought are loss of water content, reductions of stomatal conductance and photosynthesis and increase of carbohydrates. Soluble sugars play a key role in plant metabolism, acting as substrates and modulators of enzyme activity in carbon-related pathways and controlling the expression of different genes related to carbon, lipid and nitrogen routs. However, the mechanisms involved with photosynthesis down-regulation by drought and sugars in C4 plants are not fully understood. The aim of this study was to investigate how drought and source-sink perturbation regulate photosynthesis in sugarcane plants. Therefore, two studies were conducted with sugarcane plants with four months old cultivated under greenhouse conditions. In the first study sugarcane plants (cv. IACSP94-2094) were subjected to water deficit for 5 days (WD) with concomitant spraying of 50 mM exogenous sucrose (WD + Suc). While in the second study source-sink relationship was perturbed in two sugarcane cultivars (cv. IACSP94-2094 and cv. IACSP95-5000) by imposing partial darkness, spraying 50 mM exogenous sucrose and their combination for 5 days. The negative effects of WD in the gas exchange and photochemical parameters were aggravated by exogenous sucrose. Photosynthesis reductions were related to both stomatal and biochemical limitations, but exogenous sucrose intensified metabolic restrictions mainly through down-regulation of Rubisco initial activity and PSII effective quantum efficiency in drought-stressed plants. In addition, Rubisco activation state was decreased by WD + Suc, indicating perhaps that the activity of this enzyme was reduced by tight-binding inhibitors, such as sugars phosphates. Sucrose metabolism enzymes and sugars amount were also differently altered by WD and WD + Suc in leaves, sheath and stalk in WD and WD +Suc plants. Interestingly, Sucrose/hexose ratio decreased in both leaf and sheath whereas it was increased in stalk, suggesting that sucrose and related sugars were intensely metabolized and transported in drought-stressed plants. In well-watered conditions, photosynthesis was inhibited by sucrose spraying in both genotypes, through decreases in maximum Rubisco carboxylation rate (Vcmax), initial slope of A-Ci curve (k), stomatal conductance (gs) and ATP production driven by electron transport (Jatp). The partial darkness and sucrose spraying combination did not change photosynthesis in both genotypes. Significant increases in Vcmax, gs and Jatp and marginal increases in k were noticed when combining partial darkness and sucrose spraying compared with sucrose spraying alone. Altogether, these results suggest that CO2 assimilation impairment is aggravated by exogenous sucrose in drought-stressed plants. This limitation was mainly related to biochemical restrictions, specially associated with Rubisco initial activity and PSII quantum efficiency. In contrast, in vitro PEPCase activity and amount were increased in sucrose-treated plants, suggesting that C4 cycle efficiency was reduced in vivo by C3 cycle inhibition under drought conditions. Moreover, sucrose amount was increased in the stalk, suggesting the feedback regulation from stalk to source leaves in drought-stressed plants. Our data also revealed that increases in sink strength due to partial darkness offset the inhibition of sugarcane photosynthesis caused by sucrose spraying, enhancing the knowledge on endogenous regulation of sugarcane photosynthesis through the source-sink relationship. / A deficiência hídrica é o principal fator limitante para o crescimento e desenvolvimento das culturas. Para sobreviver nessas condições adversas, várias modificações bioquímicas e fisiológicas são desencadeadas pelas plantas. Em geral, os efeitos da seca em plantas são diminuição do status hídrico, reduções da condutância estomática, fotossíntese e crescimentos e aumentos nos níveis de carboidratos. Os açúcares solúveis desempenham papéis chave no metabolismo das plantas, atuando como substratos e moduladores da atividade enzimática em vias relacionadas com o carbono. Além disso, os açúcares controlam a expressão de genes associados com as rotas do metabolismo do carbono, lipídios e nitrogênio. Entretanto, os mecanismos envolvidos com a regulação negativa da fotossíntese por deficiência hídrica e açúcares em plantas C4 não estão totalmente entendidos. O objetivo deste estudo foi investigar como a deficiência hídrica e perturbações na relação fonte-dreno regulam a fotossíntese em plantas de cana-de-açúcar. Dois estudos foram conduzidos com plantas de cana-de-açúcar com quatro meses de idade cultivadas sob condições de casa de vegetação. No primeiro estudo, plantas de cana-de-açúcar (cv. IACSP94-2094) foram submetidas a deficiência hídrica por 5 dias (WD) com subsequente aplicação de sacarose exógena 50 mM (WD + Suc). Enquanto que no segundo estudo a relação fonte-dreno foi perturbada em duas cultivares de cana-de-açúcar (cv. IACSP94-2094 and cv. IACSP95-5000) pela imposição parcial de sombreamento, aplicação de sacarose exógena 50 mM e por suas combinações por 5 dias. Os efeitos negativos de WD nos parâmetros de trocas gasosas e fotoquímicos foram agravados por sacarose exógena. As reduções na fotossíntese foram relacionadas com limitações estomáticas e bioquímicas, porém a sacarose exógena intensificou as restrições bioquímicas principalmente por reduções na atividade inicial de Rubisco e eficiência quântica do PSII em plantas sob seca. Além disso, o estado de ativação de Rubisco foi inibido por WD + Suc, sugerindo que a atividade inicial dessa enzima foi possivelmente reduzida por inibidores que se ligam fortemente em seu sitio ativo, tais como açúcares fosfato. As enzimas do metabolismo de sacarose e a concentração de açúcares foram modificados diferentemente por WD e WD + Suc em folhas, bainha e colmo. Interessantemente, a relação sacarose/hexose decresceu em folhas e bainha, enquanto que no colmo essa relação aumentou, sugerindo que sacarose e outros açúcares relacionados foram intensamente metabolizados e transportados. Em condições irrigadas a fotossíntese foi inibida pela aplicação de sacarose nos dois genótipos, através de decréscimos da taxa máxima de carboxilação de Rubisco (Vcmax), inclinação inicial da curva A-Ci (k), condutância estomática (gs) e produção de ATP direcionada pelo transporte de elétrons (Jatp). A combinação de sombreamento parcial e sacarose não alterou a fotossíntese em ambos os genótipos. Significantes aumentos em Vcmax, gs, Jatp e k foram observados quando sombreamento parcial e sacarose foram combinados em comparação com plantas tratadas apenas com sacarose. Em conclusão, esses resultados sugerem que o impedimento da assimilação de CO2 é agravada por adição de sacarose exógena em plantas sob estresse hídrico. Essa limitação foi relacionada principalmente com restrições bioquímicas, especialmente associadas com reduções na atividade inicial de Rubisco e eficiência quântica do FSII. Em contraste, a atividade in vivo e concentração de PEPCase foram aumentadas em plantas tratadas com sacarose e estresse hídrico, sugerindo que a eficiência do ciclo C4 foi reduzida in vivo por inibições do ciclo C3 sob condições de seca. Além disso, o conteúdo de sacarose aumentou no colmo, indicando uma regulação de feedback do colmo para as folhas em plantas sob seca. Nossos dados revelam ainda que aumentos na força do dreno devido ao sombreamento parcial aliviaram os efeitos inibitórios na fotossíntese de cana-de-açúcar causados pela aplicação de sacarose, aumentando o conhecimento na regulação endógena da fotossíntese de cana-de-açúcar através da relação fonte-dreno.

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The effect climate change has on forest trees is a large scaled topic. It is to believe that one of the largest threat to the environment today is global warming (IPPC, 2007). The use of fossil fuels seems to be the big threat with its greenhouse gas (GHG) emission and therefore forest is of interest. Forest contributes in several ways. Forest trees work as a renewable source of numerus materials and as it takes up CO 2 from the greenhouse gasses it gives us oxygen (O2). The important process of photosynthesis, to able production of more trees and creation of more forests, tells us the vitality of understanding the tree physiology to the fullest. The response of photosynthesis to temperature is a central facet of trees’ response to climate change. With its photosynthesis plants play a large role in the carbon cycle as they store the hazardous carbon dioxide helping us humans to deal with problems directly linked to climatic change and in the same time they build up biomass that can be used as a renewable source. To understand, and to find the key, how plants can achieve optimum potential of photosynthesis several observations were made using plant material from fertilized and unfertilized Picea abies. Well acknowledged leaf gas exchange measurements were used to see the limitations of photosynthesis, observing the net CO2 uptake rate (Anet), the maximum Rubisco carboxylation (Vcmax), maximum rate of electron transport for regeneration of RuBP (Jmax) and their unique response to temperatures. For three days observations were conducted at the Slu Asa field research station in Lammhult, Sweden. In addition to gas exchange measurements, nitrogen (N), phosphorus (P) and chlorophyll content was measured in needles of the fertilized and unfertilized P. abies to see if the content somehow made an impact on photosynthetic parameters and the influence nutrients might have on the specific leaf area (SLA). Results from the observations showed that optimum temperature for photosynthesis varies to be by fertilized 22°C and unfertilized 19°C. The net photosynthetic rate responded to the influence by added fertilizers to almost a double, 9.10 μmol m−2 sec−1 than of the unfertilized, 5.36 μmol m−2 sec−1. These results indicate that a fertilized P. abies has a greater potency to capture carbon than of an unfertilized P. abies. The result also reveals the future prospect of adding fertilizer to a P. abies as a potential of growth in biomass as well as a carbon sink when atmospheric CO2 levels rise. There were no great differences in the behaviour between Vcmax and Jmax to the added or non-added fertilized P. abies. Perhaps the fact that given fertilizer contained both nitrogen and phosphorous could have in their combinations influenced the sensitivity of the relationship between them two and therefore also the result. The presence of N and P in the fertilized P. abies affected the concentration of chlorophyll positively, paving the way for photosynthesis, accumulated biomass and possibly for trees defence against abiotic stress factors.

abstract: Rubisco activase (Rca) from higher plants is a stromal ATPase essential for reactivating Rubiscos rendered catalytically inactive by endogenous inhibitors. Rca’s functional state is thought to consist of ring-like hexameric assemblies, similar to other members of the AAA+ protein superfamily. However, unlike other members, it does not form obligate hexamers and is quite polydisperse in solution, making elucidation of its self-association pathway challenging. This polydispersity also makes interpretation of traditional biochemical approaches difficult, prompting use of a fluorescence-based technique (Fluorescence Correlation Spectroscopy) to investigate the relationship between quaternary structure and function. Like cotton β Rca, tobacco β Rca appears to assemble in a step-wise and nucleotide-dependent manner. Incubation in varying nucleotides appears to alter the equilibrium between varying oligomers, either promoting or minimizing the formation of larger oligomers. High concentrations of ADP seem to favor continuous assembly towards larger oligomers, while assembly in the presence of ATP-yS (an ATP analog) appears to halt continuous assembly in favor of hexameric species. In contrast, assembly in the “Active ATP Turnover” condition (a mixture of ATP and ADP) appears to favor an almost equal distribution of tetramer and hexamer, which when compared with ATPase activity, shows great alignment with maximum activity in the low µM range. Despite this alignment, the decrease in ATPase activity does not follow any particular oligomer, but rather decreases with increasing aggregation, suggesting that assembly dynamics may regulate ATPase activity, rather than the formation/disappearance of one specific oligomer. Work presented here also indicates that all oligomers larger than hexamers are catalytically inactive, thus providing support for the idea that they may serve as a storage mechanism to minimize wasteful hydrolysis. These findings are also supported by assembly work carried out on an Assembly Mutant (R294V), known for favoring formation of closed-ring hexamers. Similar assembly studies were carried out on spinach Rca, however, due to its aggregation propensity, FCS results were more difficult to interpret. Based on these findings, one could argue that assembly dynamics are essential for Rca function, both in ATPase and in regulation of Rubisco carboxylation activity, thus providing a rational for Rca’s high degree of polydispersity. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2018

abstract: Ribulose-1, 5-bisphosphate carboxylase oxygenase, commonly known as RuBisCO, is an enzyme involved in carbon fixation in photosynthetic organisms. The enzyme is subject to a mechanism-based deactivation during its catalytic cycle. RuBisCO activase (Rca), an ancillary enzyme belonging to the AAA+ family of the ATP-ases, rescues RuBisCO by facilitating the removal of the tightly bound sugar phosphates from the active sites of RuBisCO. In this work, we investigated the ATP/ADP dependent oligomerization equilibrium of fluorescently tagged Rca for a wide range of concentrations using fluorescence correlation spectroscopy. Results show that in the presence of ADP-Mg2+, the oligomerization state of Rca gradually changes in steps of two subunits. The most probable association model supports the dissociation constants (K_d) of ∼4, 1, 1 μM for the monomer-dimer, dimer-tetramer, and tetramer-hexamer equlibria, respectively. Rca continues to assemble at higher concentrations which are indicative of the formation of aggregates. In the presence of ATP-Mg2+, a similar stepwise assembly is observed. However, at higher concentrations (30-75 µM), the average oligomeric size remains relatively unchanged around six subunits per oligomer. This is in sharp contrast with observations in ADP-Mg2+, where a marked decrease in the diffusion coefficient of Rca was observed, consistent with the formation of aggregates. The estimated K_d values obtained from the analysis of the FCS decays were similar for the first steps of the assembly process in both ADP-Mg2+ and ATP-Mg2+. However, the formation of the hexamer from the tetramer is much more favored in ATP-Mg2+, as evidenced from 20 fold lower K_d associated with this assembly step. This suggests that the formation of a hexameric ring in the presence of ATP-Mg2+. In addition to that, Rca aggregation is largely suppressed in the presence of ATP-Mg2+, as evidenced from the 1000 fold larger K_d value for the hexamer-24 mer association step. In essence, a fluorescence-based method was developed to monitor in vitro protein oligomerization and was successfully applied with Rca. The results provide a strong hint at the active oligomeric structure of Rca, and this information will hopefully help the ongoing research on the mechanistic enzymology of Rca. / Dissertation/Thesis / Ph.D. Chemistry 2014

Proteases play a crucial role in plant defence mechanisms as well as acclimation to changing metabolic demands and environmental cues. Proteases regulate the development of a plant from germination through to senescence and plant death. In this thesis the role of proteases and their inhibitors in plant response to cold stress and CO2 enrichment were investigated. The activity and inhibition of cysteine proteases (CP), as well as degradation of their potential target proteins was investigated in transgenic tobacco plants expressing the rice cystatin, OC-I. Expression of OC-I caused a longer life span; delayed senescence; significant decrease in in vitro CP activity; a concurrent increase in protein content; and protection from chilling-induced decreases in photosynthesis. An initial proteomics study identified altered abundance of a cyclophilin, a histone, a peptidyl-prolyl cis-trans isomerase and two RuBisCO activase isoforms in OC-I expressing leaves. Immunogold labelling studies revealed that RuBisCO and OC-I is present in RuBisCO vesicular bodies (RVB) that appear to be important in RuBisCO degradation in leaves under optimal and stress conditions. Plants need to respond quickly to changes in the environment that cause changes in the demand for photosynthesis. In this study the effect of CO2 enrichment on photosynthesis-related genes and novel proteases and protease inhibitors regulated by CO2 enrichment and/or development, was investigated. Maize plants grown to maturity with CO2 enrichment showed significant changes in leaf chlorophyll and protein content, increased epidermal cell size, and decreased epidermal cell density. An increased stomatal index in leaves grown at high-CO2 indicates that leaves adjust their stomatal densities through changes in epidermal cell numbers rather than stomatal numbers. Photosynthesis and carbohydrate metabolism were not significantly affected. Developmental stage affected over 3000 transcripts between leaf ranks 3 and 12, while 142 and 90 transcripts were modified by high CO2 in the same leaf ranks respectively. Only 18 transcripts were affected by CO2 enrichment exclusively. Particularly, two novel CO2 -modulated serine protease inhibitors modulated by both sugars and pro-oxidants, were identified. Growth with high CO2 decreased oxidative damage to leaf proteins. / Thesis (PhD)--University of Pretoria, 2009. / Plant Science / unrestricted

Aquatic photosynthesis accounts for 50% of the global annual net primary production (NPP), despite frequent low availability and limited diffusion of CO2 in the aquatic milieu, and low affinity for CO2 by the primary carboxylating enzyme, Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Many eukaryotic algae, and a single group of land plants, the hornworts, have an inducible carbon concentrating mechanism (CCM), to overcome these limitations. The efficiency of the CCM is improved when RuBisCO is localised to a subcellular compartment, the pyrenoid, which is hypothesised to act as a diffusion barrier for CO2 . Although the pyrenoid is a major player in global carbon balance (we estimate 10-15% of NPP), it is one of the few remaining prominent cellular features without a precise molecular or physiological definition. Under ambient CO2 , at least 90% of the cellular RuBisCO is packed into a dense matrix, together with the chaperone RuBisCO activase. Thylakoid membranes usually traverse the pyrenoid matrix, and the carboxylating substrate is thought to be delivered to the active sites of the enzyme via a carbonic anhydrase located in the lumen of these thylakoids. The mechanism of aggregation of constituents within the pyrenoid, however, still remains largely unknown. Comprehensive mutant screens have yet to reveal mutants incapable of forming pyrenoids other than those mutants with a defective RuBisCO holoenzyme, whereas DNA microarray studies uncovered little with reference to pyrenoid ultrastructure or aggregation. Taken together, this evidence raises the possibility that the basis of pyrenoid ultrastructure and aggregation lies entirely in sequence variations of RuBisCO itself. This work explored, firstly, the advantages conferred by an active CCM in hornworts and in unicellular algae, compared with the passive CO2 acquisition in most terrestrial plants. A physiological framework to CCM and pyrenoid-based photosynthesis, and isotopic discrimination, was provided by comparing the photosynthetic characteristics of selected bryophytes and algae, differing in chloroplast morphology and degrees of internalisation of gas exchanges. The results showed that on-line, carbon isotope discrimination values were a good indicator of CCM occurrence, as well as liquid-phase diffusion limitation, and biochemical limitations resulting from declining RuBisCO activity and electron transport. The methodology was used to diagnose the presence of an active CCM, and the extent of CO2 leakage. Secondly, the effect of RuBisCO sequence variations on the pyrenoid, and associated CCM, was studied using the model alga Chlamydomonas reinhardtii. The starting premise was the report by Nozaki et al. (2002) that, in some species of the family Chlamydomonaceae, a few amino acid residues within the RuBisCO large subunit (LSU) correlated strongly with pyrenoid formation. The specific roles of seven LSU residues were studied by site-directed mutagenesis. Whilst the mutations reduced the affinity of RuBisCO for CO2 and increased CO2 leakage, compared to wild-type Chlamydomonas, there was no effect on the pyrenoid phenotype. Informed by observations that Chlamydomonas mutants with a hybrid RuBisCO, composed of a native LSU, and higher plant small subunit (SSU), lacked a pyrenoid (Genkov et al., 2010), and that defined SSU alterations were neutral with respect to the pyrenoid (Genkov and Spreitzer, 2006), hitherto unexplored SSU domains were modified. A pyrenoid was successfully restored by replacing jointly the two solvent-exposed α-helices, whereas single α-helix replacements had no effect. However, leakage values indicated that the associated CCM was not fully operative, suggesting important correlates between the RuBisCO SSU and the CCM, besides the conditioning of pyrenoid formation. If the pyrenoid is partly defined by simple sequence variations in the RuBisCO SSU, as suggested by the evidence outlined in this thesis, there is the tantalising possibility that transformation of a biophysical CCM into crop plants could be a tractable approach for the future.

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