Eva M. Farré

Starch Synthesis in Potato Tubers Is Regulated by Post-Translational Redox Modification of ADP-Glucose Pyrophosphorylase: A Novel Regulatory Mechanism Linking Starch Synthesis to the Sucrose Supply

Transcriptional and allosteric regulation of ADP-Glc pyrophosphorylase (AGPase) plays a major role in the regulation of starch synthesis. Analysis of the response after detachment of growing potato tubers from the mother plant revealed that this concept requires extension. Starch synthesis was inhibited within 24 h of tuber detachment, even though the catalytic subunit of AGPase (AGPB) and overall AGPase activity remained high, the substrates ATP and Glc-1-P increased, and the glycerate-3-phosphate/inorganic orthophosphate (the allosteric activator and inhibitor, respectively) ratio increased. This inhibition was abolished in transformants in which a bacterial AGPase replaced the potato AGPase. Measurements of the subcellular levels of each metabolite between Suc and starch established AGPase as the only step whose substrates increase and mass action ratio decreases after detachment of wild-type tubers. Separation of extracts on nonreducing SDS gels revealed that AGPB is present as a mixture of monomers and dimers in growing tubers and becomes dimerized completely in detached tubers. Dimerization led to inactivation of the enzyme as a result of a marked decrease of the substrate affinity and sensitivity to allosteric effectors. Dimerization could be reversed and AGPase reactivated in vitro by incubating extracts with DTT. Incubation of tuber slices with DTT or high Suc levels reduced dimerization, increased AGPase activation, and stimulated starch synthesis in vivo. In intact tubers, the Suc content correlated strongly with AGPase activation across a range of treatments, including tuber detachment, aging of the mother plant, heterologous overexpression of Suc phosphorylase, and antisense inhibition of endogenous AGPase activity. Furthermore, activation of AGPase resulted in a stimulation of starch synthesis and decreased levels of glycolytic intermediates.

The ELF4-ELF3-LUX complex links the circadian clock to diurnal control of hypocotyl growth

The circadian clock is required for adaptive responses to daily and seasonal changes in environmental conditions. Light and the circadian clock interact to consolidate the phase of hypocotyl cell elongation to peak at dawn under diurnal cycles in Arabidopsis thaliana. Here we identify a protein complex (called the evening complex)—composed of the proteins encoded by EARLY FLOWERING 3 (ELF3), ELF4 and the transcription-factor-encoding gene LUX ARRHYTHMO (LUX; also known as PHYTOCLOCK 1)—that directly regulates plant growth. ELF3 is both necessary and sufficient to form a complex between ELF4 and LUX, and the complex is diurnally regulated, peaking at dusk. ELF3, ELF4 and LUX are required for the proper expression of the growth-promoting transcription factors encoded by PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and PIF5 (also known as PHYTOCHROME INTERACTING FACTOR 3-LIKE 6) under diurnal conditions. LUX targets the complex to the promoters of PIF4 and PIF5 in vivo. Mutations in PIF4 and/or PIF5 are epistatic to the loss of the ELF4–ELF3–LUX complex, suggesting that regulation of PIF4 and PIF5 is a crucial function of the complex. Therefore, the evening complex underlies the molecular basis for circadian gating of hypocotyl growth in the early evening.

Overlapping and Distinct Roles of PRR7 and PRR9 in the Arabidopsis Circadian Clock

The core mechanism of the circadian oscillators described to date rely on transcriptional negative feedback loops with a delay between the negative and the positive components . In plants, the first suggested regulatory loop involves the transcription factors CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) and the pseudo-response regulator TIMING OF CAB EXPRESSION 1 (TOC1/PRR1). TOC1 is a member of the Arabidopsis circadian-regulated PRR gene family . Analysis of single and double mutants in PRR7 and PRR9 indicates that these morning-expressed genes play a dual role in the circadian clock, being involved in the transmission of light signals to the clock and in the regulation of the central oscillator. Furthermore, CCA1 and LHY had a positive effect on PRR7 and PRR9 expression levels, indicating that they might form part of an additional regulatory feedback loop. We propose that the Arabidopsis circadian oscillator is composed of several interlocking positive and negative feedback loops, a feature of clock regulation that appears broadly conserved between plants, fungi, and animals.

A novel computational model of the circadian clock in Arabidopsis that incorporates PRR7 and PRR9

In plants, as in animals, the core mechanism to retain rhythmic gene expression relies on the interaction of multiple feedback loops. In recent years, molecular genetic techniques have revealed a complex network of clock components in Arabidopsis. To gain insight into the dynamics of these interactions, new components need to be integrated into the mathematical model of the plant clock. Our approach accelerates the iterative process of model identification, to incorporate new components, and to systematically test different proposed structural hypotheses. Recent studies indicate that the pseudo‐response regulators PRR7 and PRR9 play a key role in the core clock of Arabidopsis. We incorporate PRR7 and PRR9 into an existing model involving the transcription factors TIMING OF CAB (TOC1), LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED (CCA1). We propose candidate models based on experimental hypotheses and identify the computational models with the application of an optimization routine. Validation is accomplished through systematic analysis of various mutant phenotypes. We introduce and apply sensitivity analysis as a novel tool for analyzing and distinguishing the characteristics of proposed architectures, which also allows for further validation of the hypothesized structures.

CIRCADIAN CLOCK-ASSOCIATED 1 and LATE ELONGATED HYPOCOTYL regulate expression of the C-REPEAT BINDING FACTOR (CBF) pathway in Arabidopsis

The C-REPEAT BINDING FACTOR (CBF) cold-response pathway has a prominent role in cold acclimation, the process whereby certain plants increase tolerance to freezing in response to low nonfreezing temperatures. In Arabidopsis, the CBF pathway is characterized by rapid induction of the C-REPEAT BINDING FACTOR 1 (CBF1), CBF2, and CBF3 genes, which encode transcriptional activators, followed by induction of the CBF-targeted genes known as the “CBF regulon.” Expression of the CBF regulon results in an increase in freezing tolerance. Previous studies established that CBF1, CBF2, and CBF3 are subject to circadian regulation and that their cold induction is gated by the circadian clock. Here we present the results of genetic analysis and ChIP experiments indicating that both these forms of regulation involve direct positive action of two transcription factors that are core components of the clock, i.e., CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). In plants carrying the cca1-11/lhy-21 double mutation, cold induction of CBF1, CBF2, and CBF3 was greatly impaired, and circadian regulation of CBF1 and CBF3 was essentially eliminated; circadian regulation of CBF2 continued, although with significantly reduced amplitude. Circadian regulation and cold induction of three CBF regulon genes, i.e., COLD-REGULATED GENE15A (COR15A), COR47, and COR78, also were greatly diminished in plants carrying the cca1-11/lhy-21 double mutation. Furthermore, the cca1-11/lhy-21 double mutation resulted in impaired freezing tolerance in both nonacclimated and cold-acclimated plants. These results indicate that CCA1/LHY-mediated output from the circadian clock contributes to plant cold tolerance through regulation of the CBF cold-response pathway.

Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga Nannochloropsis oceanica CCMP1779

Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica–specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus.

PRR3 Is a Vascular Regulator of TOC1 Stability in the Arabidopsis Circadian Clock

The pseudoresponse regulators (PRRs) participate in the progression of the circadian clock in Arabidopsis thaliana. The founding member of the family, TIMING OF CAB EXPRESSION1 (TOC1), is an essential component of the transcriptional network that constitutes the core mechanism of the circadian oscillator. Recent data suggest a role in circadian regulation for all five members of the PRR family; however, the molecular function of TOC1 or any other PRRs remains unknown. In this work, we present evidence for the involvement of PRR3 in the regulation of TOC1 protein stability. PRR3 was temporally coexpressed with TOC1 under different photoperiods, yet its tissue expression was only partially overlapping with that of TOC1, as PRR3 appeared restricted to the vasculature. Decreased expression of PRR3 resulted in reduced levels of TOC1 protein, while overexpression of PRR3 caused an increase in the levels of TOC1, all without affecting the amount of TOC1 transcript. PRR3 was able to bind to TOC1 in yeast and in plants and to perturb TOC1 interaction with ZEITLUPE (ZTL), which targets TOC1 for proteasome-dependent degradation. Together, our results indicate that PRR3 might function to modulate TOC1 stability by hindering ZTL-dependent TOC1 degradation, suggesting the existence of local regulators of clock activity and adding to the growing importance of posttranslational regulation in the design of circadian timing mechanisms in plants.

The interactions between the circadian clock and primary metabolism.

Primary metabolism in plants is tightly regulated by environmental factors such as light and nutrient availability at multiple levels. The circadian clock is a self-sustained endogenous oscillator that enables organisms to predict daily and seasonal changes. The regulation of primary metabolism by the circadian clock has been proposed to explain the importance of circadian rhythms in plant growth and survival. Recent transcriptomic and metabolomic analyses indicate a wide spread circadian regulation of different metabolic processes. We review evidence of circadian regulation of pathways in primary metabolism, discuss the challenges faced for discerning the mechanisms regulating circadian metabolic oscillations and present recent evidence of regulation of the circadian clock by metabolites.

The regulation of plant growth by the circadian clock

Circadian regulated changes in growth rates have been observed in numerous plants as well as in unicellular and multicellular algae. The circadian clock regulates a multitude of factors that affect growth in plants, such as water and carbon availability and light and hormone signalling pathways. The combination of high-resolution growth rate analyses with mutant and biochemical analysis is helping us elucidate the time-dependent interactions between these factors and discover the molecular mechanisms involved. At the molecular level, growth in plants is modulated through a complex regulatory network, in which the circadian clock acts at multiple levels.

Acceleration of potato tuber sprouting by the expression of a bacterial pyrophosphatase

Potato is a globally important crop. Unfortunately, potato farming is plagued with problems associated with the sprouting behavior of seed tubers. The data presented here demonstrate that using transgenic technology can influence this behavior. Transgenic tubers cytosolically expressing an inorganic pyrophosphatase gene derived from Escherichia coli under the control of the tuber-specific patatin promoter display significantly accelerated sprouting. The period of presprouting dormancy for transgenic tubers planted immediately after harvest is reduced by six to seven weeks when compared to wild-type tubers. This study demonstrates a method with which to regulate dormancy, an important aspect of potato crop management.