Plastidic phosphoglucose isomerase is an important determinant of starch accumulation in mesophyll cells, growth, photosynthetic capacity, and biosynthesis of plastidic cytokinins in Arabidopsis

Póster presentado en el XXI Reunión de la Sociedad Española de Fisiología Vegetal y el XIV Congreso Hispano-Luso Fisiología Vegetal, celebrado en Toledo del 14 al 17 de junio de 2015.Peer Reviewe

Carbon Dynamics, Development and Stress Responses in Arabidopsis: Involvement of the APL4 Subunit of ADP-Glucose Pyrophosphorylase (Starch Synthesis)

An Arabidopsis thaliana T-DNA insertional mutant was identified and characterized for enhanced tolerance to the singlet-oxygen-generating herbicide atrazine in comparison to wild-type. This enhanced atrazine tolerance mutant was shown to be affected in the promoter structure and in the regulation of expression of the APL4 isoform of ADP-glucose pyrophosphorylase, a key enzyme of the starch biosynthesis pathway, thus resulting in decrease of APL4 mRNA levels. The impact of this regulatory mutation was confirmed by the analysis of an independent T-DNA insertional mutant also affected in the promoter of the APL4 gene. The resulting tissue-specific modifications of carbon partitioning in plantlets and the effects on plantlet growth and stress tolerance point out to specific and non-redundant roles of APL4 in root carbon dynamics, shoot-root relationships and sink regulations of photosynthesis. Given the effects of exogenous sugar treatments and of endogenous sugar levels on atrazine tolerance in wild-type Arabidopsis plantlets, atrazine tolerance of this apl4 mutant is discussed in terms of perception of carbon status and of investment of sugar allocation in xenobiotic and oxidative stress responses

Carbon allocation in aspen trees

Trees allocate assimilated carbon between growth and storage. In this PhD thesis, Iinvestigated the regulation of carbon allocation during tree growth both attranscriptional as well as whole-tree level, and with a focus on wood formation.I performed a large-scale DNA affinity purification sequencing (DAP-seq)screen on transcription factor proteins that regulate gene expression in developingwood of aspen (Populus tremula). Together with bioinformaticians, I identified bothnovel and previously reported interactions. The results were integrated into apublicly available database, providing a novel resource for wood biology. We alsopresent a practical guide for the analysis of DAP-seq data to facilitate similar studies.Next, I investigated carbon partitioning between growth and storage in aspen,focusing on the role of starch as the major storage compound. We report that aspengrowth is not limited by starch reserves and suggest a passive starch storagemechanism where sink tissues are the growth-limiting factor.In a study on Arabidopsis (Arabidopsis thaliana), I address the debate on whethersucrose synthase (SUS) enzymes are required in the biosynthesis of cellulose, themost abundant component of wood. As mutants lacking all SUS isoforms grewnormally and their cellulose content was comparable to that of wild-type, I concludethat SUS activity is not required for cellulose biosynthesis in Arabidopsis.Taken together, the results of this PhD study fill key knowledge gaps in the fieldand provide new starting points for future research projects on carbon allocation intrees

Aspen growth is not limited by starch reserves

All photosynthetic organisms balance CO2 assimilation with growth and carbon storage. Stored carbon is used for growth at night and when demand exceeds assimilation. Gaining a mechanistic understanding of carbon partitioning between storage and growth in trees is important for biological studies and for estimating the potential of terrestrial photosynthesis to sequester anthropogenic CO2 emissions.(1,2) Starch represents the main carbon storage in plants.(3,4) To examine the carbon storage mechanism and role of starch during tree growth, we generated and characterized low-starch hybrid aspen (Populus tremula x tremuloides) trees using CRISPR-Cas9-mediated gene editing of two PHOSPHOGLUCOMUTASE (PGM) genes coding for plastidial PGM isoforms essential for starch biosynthesis. We demonstrate that starch deficiency does not reduce tree growth even in short days, showing that starch is not a critical carbon reserve during diel growth of aspen. The low-starch trees assimilated up to similar to 30% less CO2 compared to the wild type under a range of irradiance levels, but this did not reduce growth or wood density. This implies that aspen growth is not limited by carbon assimilation under benign growth conditions. Moreover, the timing of bud set and bud flush in the low-starch trees was not altered, implying that starch reserves are not critical for the seasonal growth-dormancy cycle. The findings are consistent with a passive starch storage mechanism that contrasts with the annual Arabidopsis and indicate that the capacity of the aspen to absorb CO2 is limited by the rate of sink tissue growth

Genetic and physiological analysis of juvenility in plants

One of the distinguishable plant developmental events is the transition from the vegetative to reproductive phase (RP) of development. This stage is preceded by the juvenile to adult transition within the vegetative phase. During the juvenile vegetative phase (JVP) plants are incompetent to initiate reproductive development and are effectively insensitive to photoperiod. With the change to the adult vegetative phase (AVP), plants attain competence to respond to floral inducers, which is required for the transition to the RP. This study exploits Antirrhinum and Arabidopsis as model systems to understand the genetic and environmental factors that regulate floral incompetence during the JVP. Determinants such as irradiance and [CO2] were found to be key modifiers of the JVP. A relationship between photosynthetic assimilate levels and vegetative phase transition was revealed by analysis of carbohydrates in plants at defined developmental stages. Experimental data suggest that carbohydrate levels may be required to reach a specific threshold before plants undergo the transition from a juvenile to an adult phase of vegetative growth. This may be necessary in order to sustain a steady supply of sugars for sufficient bulk flow from the leaves to the shoot apical meristem (SAM), via the phloem, to enable delivery of florigen, which thus renders the SAM competent to flower. Determination of the JVP in Arabidopsis mutants impaired in different genetic pathways has shown that multiple inputs influence the timing of the vegetative phase transition. Carbohydrates have been demonstrated to be involved possibly through their function as nutrients or signals or by their interaction with hormones. Physiological analysis of flowering time mutants has shown that a variety of signals act to promote and enable the juvenile to adult phase transition that involves both floral activators and repressors

Diurnal changes of polysome loading track sucrose content in the rosette of wildtype Arabidopsis and the starchless pgm mutant

Growth is driven by newly fixed carbon in the light, but depends at night on reserves, like starch, that are laid down in the light. Unless plants coordinate their growth with diurnal changes in the carbon supply, they will experience acute carbon starvation during the night. Protein synthesis represents a major component of cellular growth. Polysome loading was investigated during the diurnal cycle, an extended night and low CO2 in Arabidopsis Col-0 and in the starchless pgm mutant. In Col-0, polysome loading was 60-70% in the light, 40-45% for much of the night and <20% in an extended night, whilst in pgm it fell to <25% early in the night. Quantification of rRNA species using qRT-PCR revealed that polysome loading remained high for much of the night in the cytosol, was strongly light-dependent in the plastid, and was always high in mitochondria. The rosette sucrose content correlated with overall and with cytosolic polysome loading. Ribosome abundance did not show significant diurnal changes. However, compared to Col-0, pgm had decreased and increased abundance of plastidic and mitochondrial ribosomes, respectively. Incorporation of label from 13CO2 into protein confirmed that protein synthesis continues at a diminished rate in the dark. Modelling revealed that a decrease in polysome loading at night is required to balance protein synthesis with the availability of carbon from starch breakdown. Costs are also reduced by using amino acids that accumulated in previous light period. These results uncover a tight coordination of protein synthesis with the momentary supply of carbon

Blue Light Induces a Distinct Starch Degradation Pathway in Guard Cells for Stomatal Opening

Stomatal pores form a crucial interface between the leaf mesophyll and the atmosphere, controlling water and carbon balance in plants [1]. Major advances have been made in understanding the regulatory networks and ion fluxes in the guard cells surrounding the stomatal pore [2]. However, our knowledge on the role of carbon metabolism in these cells is still fragmentary [3-5]. In particular, the contribution of starch in stomatal opening remains elusive [6]. Here, we used Arabidopsis thaliana as a model plant to provide the first quantitative analysis of starch turnover in guard cells of intact leaves during the diurnal cycle. Starch is present in guard cells at the end of night, unlike in the rest of the leaf, but is rapidly degraded within 30 min of light. This process is critical for the rapidity of stomatal opening and biomass production. We exploited Arabidopsis molecular genetics to define the mechanism and regulation of guard cell starch metabolism, showing it to be mediated by a previously uncharacterized pathway. This involves the synergistic action of β-amylase 1 (BAM1) and α-amylase 3 (AMY3) - enzymes that are normally not required for nighttime starch degradation in other leaf tissues. This pathway is under the control of the phototropin-dependent blue-light signaling cascade and correlated with the activity of the plasma membrane H+-ATPase. Our results show that guard cell starch degradation has an important role in plant growth by driving stomatal responses to light

Global transcript levels respond to small changes of the carbon status during progressive exhaustion of carbohydrates in Arabidopsis rosettes

The balance between the supply and utilization of carbon (C) changes continually. It has been proposed that plants respond in an acclimatory manner, modifying C utilization to minimize harmful periods of C depletion. This hypothesis predicts that signaling events are initiated by small changes in C status. We analyzed the global transcriptional response to a gradual depletion of C during the night and an extension of the night, where C becomes severely limiting from 4 h onward. The response was interpreted using published datasets for sugar, light, and circadian responses. Hundreds of C-responsive genes respond during the night and others very early in the extended night. Pathway analysis reveals that biosynthesis and cellular growth genes are repressed during the night and genes involved in catabolism are induced during the first hours of the extended night. The C response is amplified by an antagonistic interaction with the clock. Light signaling is attenuated during the 24-h light/dark cycle. A model was developed that uses the response of 22K genes during a circadian cycle and their responses to C and light to predict global transcriptional responses during diurnal cycles of wild-type and starchless pgm mutant plants and an extended night in wild-type plants. By identifying sets of genes that respond at different speeds and times during C depletion, our extended dataset and model aid the analysis of candidates for C signaling. This is illustrated for AKIN10 and four bZIP transcription factors, and sets of genes involved in trehalose signaling, protein turnover, and starch breakdown

Enhanced Symbiotic Performance by Rhizobium tropici Glycogen Synthase Mutants

We isolated a Tn5-induced Rhizobium tropici mutant that has enhanced capacity to oxidize N,N-dimethyl-p-phenylendiamine (DMPD) and therefore has enhanced respiration via cytochrome oxidase. The mutant had increased levels of the cytochromes c1 and CycM and a small increase in the amount of cytochrome aa3. In plant tests, the mutant increased the dry weight of Phaseolus vulgaris plants by 20 to 38% compared with the control strain, thus showing significantly enhanced symbiotic performance. The predicted product of the mutated gene is homologous to glycogen synthases from several bacteria, and the mutant lacked glycogen. The DNA sequence of the adjacent gene region revealed six genes predicted to encode products homologous to the following gene products from Escherichia coli: glycogen phosphorylase (glgP), glycogen branching enzyme (glgB), ADP glucose pyrophosphorylase (glgC), glycogen synthase (glgA), phosphoglucomutase (pgm), and glycogen debranching enzyme (glgX). All six genes are transcribed in the same direction, and analysis with lacZ gene fusions suggests that the first five genes are organized in one operon, although pgm appears to have an additional promoter; glgX is transcribed independently. Surprisingly, the glgA mutant had decreased levels of high-molecular-weight exopolysaccharide after growth on glucose, but levels were normal after growth on galactose. A deletion mutant was constructed in order to generate a nonpolar mutation in glgA. This mutant had a phenotype similar to that of the Tn5 mutant, indicating that the enhanced respiration and symbiotic nitrogen fixation and decreased exopolysaccharide were due to mutation of glgA and not to a polar effect on a downstream gene