José M. Romero

floricaula: A homeotic gene required for flower development in antirrhinum majus

Plants carrying the floricaula (flo) mutation cannot make the transition from inflorescence to floral meristems and have indeterminate shoots in place of flowers. The flo-613 allele carries a Tam3 transposon insertion, which allowed the isolation of the flo locus. The flo gene encodes a putative protein (FLO) containing a proline-rich N-terminus and a highly acidic region. In situ hybridization shows that the flo gene is transiently expressed in the very early stages of flower development. The earliest expression seen is in bract primordia, followed by sepal, petal, and carpel primordia, but no expression is detected in stamen primordia. This pattern of expression has implications for how flo affects phyllotaxis, organ identity, and determinacy. We propose that flo interacts in a sequential manner with other homeotic genes affecting floral organ identity.

ChlamyNET: a Chlamydomonas

BackgroundChlamydomonas reinhardtii is the model organism that serves as a reference for studies in algal genomics and physiology. It is of special interest in the study of the evolution of regulatory pathways from algae to higher plants. Additionally, it has recently gained attention as a potential source for bio-fuel and bio-hydrogen production. The genome of Chlamydomonas is available, facilitating the analysis of its transcriptome by RNA-seq data. This has produced a massive amount of data that remains fragmented making necessary the application of integrative approaches based on molecular systems biology.ResultsWe constructed a gene co-expression network based on RNA-seq data and developed a web-based tool, ChlamyNET, for the exploration of the Chlamydomonas transcriptome. ChlamyNET exhibits a scale-free and small world topology. Applying clustering techniques, we identified nine gene clusters that capture the structure of the transcriptome under the analyzed conditions. One of the most central clusters was shown to be involved in carbon/nitrogen metabolism and signalling, whereas one of the most peripheral clusters was involved in DNA replication and cell cycle regulation. The transcription factors and regulators in the Chlamydomonas genome have been identified in ChlamyNET. The biological processes potentially regulated by them as well as their putative transcription factor binding sites were determined. The putative light regulated transcription factors and regulators in the Chlamydomonas genome were analyzed in order to provide a case study on the use of ChlamyNET. Finally, we used an independent data set to cross-validate the predictive power of ChlamyNET.ConclusionsThe topological properties of ChlamyNET suggest that the Chlamydomonas transcriptome posseses important characteristics related to error tolerance, vulnerability and information propagation. The central part of ChlamyNET constitutes the core of the transcriptome where most authoritative hub genes are located interconnecting key biological processes such as light response with carbon and nitrogen metabolism. Our study reveals that key elements in the regulation of carbon and nitrogen metabolism, light response and cell cycle identified in higher plants were already established in Chlamydomonas. These conserved elements are not only limited to transcription factors, regulators and their targets, but also include the cis-regulatory elements recognized by them.

Chlamydomonas CONSTANS and the evolution of plant photoperiodic signaling.

BACKGROUND The circadian clock controls several important processes in plant development, including the phase transition from vegetative growth to flowering. In Arabidopsis thaliana, the circadian-regulated gene CONSTANS (CO) plays a central role in the photoperiodic control of the floral transition, one of the most conserved flowering responses among distantly related plants. CO is a member of a plant-specific family of transcription factors, and when it arose during the evolution of higher plants is unclear. RESULTS A CO homologous gene present in the genome of the unicellular green alga Chlamydomonas reinhardtii (CrCO) can complement the Arabidopsis co mutation and promote early flowering in wild-type plants when expressed under different promoters. Transcript levels of FLOWERING LOCUS T (FT), the main target of CO, are increased in CrCO transgenic plants in a way similar to those in plants overexpressing CO. In the microalga, expression of CrCO is influenced by day length and the circadian clock, being higher in short photoperiods. Reduction of CrCO expression in Chlamydomonas by RNA interference induces defects in culture growth, whereas algae induced to express high levels of CrCO show alterations in several circadian output processes, such as starch accumulation and the onset of expression of genes that regulate the cell cycle. CONCLUSIONS The effects observed may reflect a conserved role for CrCO in the coordination of processes regulated by photoperiod and the circadian clock. Our data indicate that CO orthologs probably represent ancient regulators of photoperiod-dependent events and that these regulators arose early in the evolutionary lineage that gave rise to flowering plants.

Two Arabidopsis ADP-Glucose Pyrophosphorylase Large Subunits (APL1 and APL2) Are Catalytic

ADP-glucose (Glc) pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in starch biosynthesis. Higher plant ADP-Glc PPase is a heterotetramer (α2β2) consisting of two small and two large subunits. There is increasing evidence that suggests that catalytic and regulatory properties of the enzyme from higher plants result from the synergy of both types of subunits. In Arabidopsis (Arabidopsis thaliana), two genes encode small subunits (APS1 and APS2) and four large subunits (APL1–APL4). Here, we show that in Arabidopsis, APL1 and APL2, besides their regulatory role, have catalytic activity. Heterotetramers formed by combinations of a noncatalytic APS1 and the four large subunits showed that APL1 and APL2 exhibited ADP-Glc PPase activity with distinctive sensitivities to the allosteric activator (3-phosphoglycerate). Mutation of the Glc-1-P binding site of Arabidopsis and potato (Solanum tuberosum) isoforms confirmed these observations. To determine the relevance of these activities in planta, a T-DNA mutant of APS1 (aps1) was characterized. aps1 is starchless, lacks ADP-Glc PPase activity, APS1 mRNA, and APS1 protein, and is late flowering in long days. Transgenic lines of the aps1 mutant, expressing an inactivated form of APS1, recovered the wild-type phenotype, indicating that APL1 and APL2 have catalytic activity and may contribute to ADP-Glc synthesis in planta.

Regulatory interaction of photosynthetic nitrate utilization and carbon dioxide fixation in the cyanobacterium Anacystis nidulans

The rate of photosynthetic nitrate utilization in Anacystis nidulans is strongly influenced by the availability of carbon dioxide. This dependence can be relieved by inhibiting the metabolism of the ammonium derived from nitrate reduction. Nitrate uptake seems to be modulated through a sensitive regulatory system integrating the photosynthetic metabolism of carbon and nitrogen, with CO2 fixation products antagonizing the inhibitory effect of ammonium derivatives.

Arabidopsis thaliana Mutants Lacking ADP-Glucose Pyrophosphorylase Accumulate Starch and Wild-type ADP-Glucose Content: Further Evidence for the Occurrence of Important Sources, other than ADP-Glucose Pyrophosphorylase, of ADP-Glucose Linked to Leaf Starch Biosynthesis

It is widely considered that ADP-glucose pyrophosphorylase (AGP) is the sole source of ADP-glucose linked to bacterial glycogen and plant starch biosynthesis. Genetic evidence that bacterial glycogen biosynthesis occurs solely by the AGP pathway has been obtained with glgC⁻ AGP mutants. However, recent studies have shown that (i) these mutants can accumulate high levels of ADP-glucose and glycogen, and (ii) there are sources other than GlgC, of ADP-glucose linked to glycogen biosynthesis. In Arabidopsis, evidence showing that starch biosynthesis occurs solely by the AGP pathway has been obtained with the starchless adg1-1 and aps1 AGP mutants. However, mounting evidence has been compiled previewing the occurrence of more than one important ADP-glucose source in plants. In attempting to solve this 20-year-old controversy, in this work we carried out a judicious characterization of both adg1-1 and aps1. Both mutants accumulated wild-type (WT) ADP-glucose and approximately 2% of WT starch, as further confirmed by confocal fluorescence microscopic observation of iodine-stained leaves and of leaves expressing granule-bound starch synthase fused with GFP. Introduction of the sex1 mutation affecting starch breakdown into adg1-1 and aps1 increased the starch content to 8-10% of the WT starch. Furthermore, aps1 leaves exposed to microbial volatiles for 10 h accumulated approximately 60% of the WT starch. aps1 plants expressing the bacterial ADP-glucose hydrolase EcASPP in the plastid accumulated normal ADP-glucose and reduced starch when compared with aps1 plants, whereas aps1 plants expressing EcASPP in the cytosol showed reduced ADP-glucose and starch. Moreover, aps1 plants expressing bacterial AGP in the plastid accumulated WT starch and ADP-glucose. The overall data show that (i) there occur important source(s), other than AGP, of ADP-glucose linked to starch biosynthesis, and (ii) AGP is a major determinant of starch accumulation but not of intracellular ADP-glucose content in Arabidopsis.

Dependence of nitrate utilization upon active CO2 fixation in Anacystis nidulans: A regulatory aspect of the interaction between photosynthetic carbon and nitrogen metabolism

Specific inhibition of photosynthetic CO2 fixation in Anacystis nidulans cells by D,L-glyceraldehyde resulted in the simultaneous inhibition of nitrate utilization, indicating a dependence of the latter process upon the provision of CO2-fixation products. This dependence was lost in cells treated with L-methionine-D,L-sulfoximine or azaserine, effective inhibitors of ammonium assimilation. In these cells, nitrate uptake could proceed at rates similar to those in control cells even if CO2 fixation was severely inhibited by D,L-glyceraldehyde. The results support the contention that CO2-fixation products participate in the control of nitrate uptake in A. nidulans by preventing the accumulation of certain ammonium derivatives which are negative effectors of nitrate uptake.

Relationship between a 47-kDa cytoplasmic membrane polypeptide and nitrate transport in Anacystis nidulans.

The polypeptide composition of cytoplasmic membranes of the cyanobacterium Anacystis nidulans changes in response to variations in the nitrogen source available to the cells, differing specifically in the amount of a polypeptide of 47-kDa molecular mass. Synthesis of the polypeptide and expression of nitrate transport activity are repressed by ammonium. Transfer of ammonium-grown cells to a medium containing nitrate as the sole nitrogen source results in parallel development of the 47-kDa polypeptide and nitrate transport activity of the cells. These results suggest the involvement of the 47-kDa cytoplasmic membrane polypeptide in nitrate transport by A. nidulans.

Photoperiodic control of carbon distribution during the floral transition in Arabidopsis

The distribution of carbon resources from starch to soluble sugars is crucial to fuel the diverse physiological processes that take place during the floral transition. A multidisciplinary study supports the control of sugar mobilization in Arabidopsis during photoperiodic flowering through regulation of GRANULE BOUND STARCH SYNTHASE expression by the key photoperiodic regulator CONSTANS. Flowering is a crucial process that demands substantial resources. Carbon metabolism must be coordinated with development through a control mechanism that optimizes fitness for any physiological need and growth stage of the plant. However, how sugar allocation is controlled during the floral transition is unknown. Recently, the role of a CONSTANS (CO) ortholog (Cr-CO) in the control of the photoperiod response in the green alga Chlamydomonas reinhardtii and its influence on starch metabolism was demonstrated. In this work, we show that transitory starch accumulation and glycan composition during the floral transition in Arabidopsis thaliana are regulated by photoperiod. Employing a multidisciplinary approach, we demonstrate a role for CO in regulating the level and timing of expression of the GRANULE BOUND STARCH SYNTHASE (GBSS) gene. Furthermore, we provide a detailed characterization of a GBSS mutant involved in transitory starch synthesis and analyze its flowering time phenotype in relation to its altered capacity to synthesize amylose and to modify the plant free sugar content. Photoperiod modification of starch homeostasis by CO may be crucial for increasing the sugar mobilization demanded by the floral transition. This finding contributes to our understanding of the flowering process.

Influence of plant age and growth conditions on nitrate assimilation in roots of Lotus japonicus plants

In roots of Lotus japonicus (Regel) Larsen cv. Gifu, the level of NADH-nitrate reductase (NR) activity and protein, as well as ferredoxin-nitrite reductase activity and nitrate accumulation, were higher in roots of young plants, and decreased in mature plants grown in seed trays. When plants were grown in larger pots, the decrease in NR activity and nitrate accumulation took place at a later stage of growth, suggesting that the cessation of nitrate assimilation in mature plants could be related to both ageing and a lower availability of space for roots. Low NR activity was detected in leaves, whereas nitrate accumulation in this tissue could reach relatively high levels. NR activity and protein, and nitrate accumulation, also decreased in leaves of mature plants, indicating that the diminution in nitrate accumulation and NR in roots from mature plants is not related to a shift of the nitrate assimilation process from roots to leaves. Measurement of the extent of total and active NR according to the phosphorylation/dephosphorylation inactivating mechanism described for spinach leaf NR, indicates that this mechanism is operative on NR from L. japonicus roots, being responsible for a great proportion of inactive NR protein. The amount of inactive NR protein in roots due to the aforementioned phosphorylation mechanism also increased in mature plants.