Elizabeth Cordoba

Unravelling the regulatory mechanisms that modulate the MEP pathway in higher plants

The methyl-D-erythritol 4-phosphate pathway is responsible for the biosynthesis of a substantial number of natural compounds of biological and biotechnological importance. In recent years, this pathway has become an obvious target to develop new herbicides and antimicrobial drugs. In addition, the production of a variety of compounds of medical and agricultural interest may be possible through the genetic manipulation of this pathway. To this end, a complete understanding of the molecular mechanisms that regulate this pathway is of tremendous importance. Recent data have accumulated that show some of the multiple mechanisms that regulate the methyl-D-erythritol 4-phosphate pathway in plants. In this review we will describe some of these and discuss their implications. It has been demonstrated that 1-deoxy-D-xylulose-5-phosphate synthase (DXS), the first enzyme of this route, plays a major role in the overall regulation of the pathway. A small gene family codes for this enzyme in most of the plants which have been analysed so far, and the members of these gene families belong to different phylogenetic groups. Each of these genes exhibits a distinct expression pattern, suggesting unique functions. One of the most interesting regulatory mechanisms recently described for this pathway is the post-transcriptional regulation of the level of DXS and DXR proteins. In the case of DXS, this regulation appears conserved among plants, supporting its importance. The evidence accumulated suggests that this regulation might link the activity of this pathway with the plants physiological conditions and the metabolic demand for the final products of this route.

The Arabidopsis ABA-INSENSITIVE (ABI) 4 factor acts as a central transcription activator of the expression of its own gene, and for the induction of ABI5 and SBE2.2 genes during sugar signaling.

The transcription factor ABA INSENSITIVE 4 (ABI4), discovered nearly 10 years ago, plays a central role in a variety of functions in plants, including sugar responses. However, not until very recently has its mechanism of action begun to be elucidated. Modulating gene expression is one of the primary mechanisms of sugar regulation in plants. Nevertheless, the transcription factors involved in regulating sugar responses and their role(s) during the signal transduction cascade remain poorly defined. In this paper we analyzed the participation of ABI4, as it is one of the main transcription factors implicated in glucose signaling during early seedling development. Our studies show that ABI4 is an essential activator of its own expression during development, in ABA signaling and in sugar responses. It is also important for the glucose-mediated expression of the genes ABI5 and SBE2.2. We demonstrate that ABI4 binds directly to the promoter region of all three genes and activates their expression in vivo through at CE1-like element. Previous studies found that ABI4 also functions as a transcriptional repressor of sugar-regulated genes, therefore this transcription factor is a versatile protein with dual functions for modulating gene expression.

An Uncharacterized Apocarotenoid-Derived Signal Generated in ζ-Carotene Desaturase Mutants Regulates Leaf Development and the Expression of Chloroplast and Nuclear Genes in Arabidopsis

A signaling process derived from carotenoids in ζ-carotene desaturase mutants regulates the expression of a variety of chloroplast- and nucleus-encoded genes and dramatically affects early leaf development in Arabidopsis. The signaling molecule involved in this process is an apocarotenoid whose synthesis requires the activity of the carotenoid cleavage dioxygenase CCD4. In addition to acting as photoprotective compounds, carotenoids also serve as precursors in the biosynthesis of several phytohormones and proposed regulatory signals. Here, we report a signaling process derived from carotenoids that regulates early chloroplast and leaf development. Biosynthesis of the signal depends on ζ-carotene desaturase activity encoded by the ζ-CAROTENE DESATURASE (ZDS)/CHLOROPLAST BIOGENESIS5 (CLB5) gene in Arabidopsis thaliana. Unlike other carotenoid-deficient plants, zds/clb5 mutant alleles display profound alterations in leaf morphology and cellular differentiation as well as altered expression of many plastid- and nucleus-encoded genes. The leaf developmental phenotypes and gene expression alterations of zds/clb5/spc1/pde181 plants are rescued by inhibitors or mutations of phytoene desaturase, demonstrating that phytofluene and/or ζ-carotene are substrates for an unidentified signaling molecule. Our work further demonstrates that this signal is an apocarotenoid whose synthesis requires the activity of the carotenoid cleavage dioxygenase CCD4.

Functional characterization of the three genes encoding 1-deoxy-D-xylulose 5-phosphate synthase in maize

The 1-deoxy-D-xylulose 5-phosphate synthase (DXS) enzyme catalyses the first biosynthetic step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. In plants the MEP pathway is involved in the synthesis of the common precursors to the plastidic isoprenoids, isopentenyl diphosphate and dimethylallyl diphosphate, in plastids. DXS is recognized as limiting this pathway and is a potential target for manipulation to increase various isoprenoids such as carotenoids. In Zea mays three dxs genes exist that encode plastid-targeted functional enzymes. Evidence is provided that these genes represent phylogenetically distinctive clades conserved among plants preceding monocot-dicot divergence. There is differential accumulation for each dxs gene transcript, during development and in response to external signals such as light. At the protein level, the analysis demonstrates that in Z. mays, DXS protein is feedback regulated in response to the inhibition of the pathway flow. The results support that the multilevel regulation of DXS activity is conserved in evolution.

ABI4 and its role in chloroplast retrograde communication

The acquisition of plastids is a landmark event in plant evolution. The proper functionality of these organelles depends on strict and continuous communication between the plastids and the nucleus to precisely adjust gene expression in response to the organelle’s requirements. Signals originating from the plastids impact the expression of a variety of nuclear genes, and this retrograde communication is essential to couple the nuclear expression of plastid-localized products with organelle gene expression and, ultimately, functionality. Major advances have been made in this field over the past few years with the characterization of independent retrograde signaling pathways and the identification of some of their components. One such factor is the nuclear transcriptional factor ABI4 (ABA-INSENTIVE 4). ABI4, together with the plastid PPR GUN1 protein, has been proposed to function as a node of convergence for multiple plastid retrograde signaling pathways. ABI4 is conserved among plants and also plays important roles in various critical developmental and metabolic processes. ABI4 is a versatile regulator that positively and negatively modulates the expression of many genes, including other transcriptional factors. However, its mode of action during plastid retrograde signaling is not fully understood. In this review, we describe the current evidence that supports the participation of ABI4 in different retrograde communication pathways. ABI4 is regulated at the transcriptional and post-transcriptional level. A known regulator of ABI4 includes the PTM transcription factor, which moves from the chloroplast to the nucleus. This transcription factor is a candidate for the transmission of retrograde signals between the plastid and ABI4.

Sugar regulation of SUGAR TRANSPORTER PROTEIN 1 (STP1) expression in Arabidopsis thaliana

Highlight text Transcriptional repression of STP1 is rapidly induced by phosphorylatable sugars through an HXK1-independent signalling pathway involving the participation of sugar-responsive cis elements localized in the promoter.

Characterization of Evolutionarily Conserved Motifs Involved in Activity and Regulation of the ABA-INSENSITIVE (ABI) 4 Transcription Factor

In recent years, the transcription factor ABI4 has emerged as an important node of integration for external and internal signals such as nutrient status and hormone signaling that modulates critical transitions during the growth and development of plants. For this reason, understanding the mechanism of action and regulation of this protein represents an important step towards the elucidation of crosstalk mechanisms in plants. However, this understanding has been hindered due to the negligible levels of this protein as a result of multiple posttranscriptional regulations. To better understand the function and regulation of the ABI4 protein in this work, we performed a functional analysis of several evolutionarily conserved motifs. Based on these conserved motifs, we identified ortholog genes of ABI4 in different plant species. The functionality of the putative ortholog from Theobroma cacao was demonstrated in transient expression assays and in complementation studies in plants. The function of the highly conserved motifs was analyzed after their deletion or mutagenesis in the Arabidopsis ABI4 sequence using mesophyll protoplasts. This approach permitted us to immunologically detect the ABI4 protein and identify some of the mechanisms involved in its regulation. We identified sequences required for the nuclear localization (AP2-associated motif) as well as those for transcriptional activation function (LRP motif). Moreover, this approach showed that the protein stability of this transcription factor is controlled through protein degradation and subcellular localization and involves the AP2-associated and the PEST motifs. We demonstrated that the degradation of ABI4 protein through the PEST motif is mediated by the 26S proteasome in response to changes in the sugar levels.

Heterologous expression of yeast Hxt2 in Arabidopsis thaliana alters sugar uptake, carbon metabolism and gene expression leading to glucose tolerance of germinating seedlings

The hexose transporter 2 gene (Hxt2) from Saccharomyces cerevisiae was expressed in Arabidopsis thaliana under control of the 35S promoter. Several independent transgenic lines were selected after confirming single gene insertion by southern blot analysis in the T4 generation. Northern blots revealed the presence of heterologous transcript. Radiolabeling experiments revealed an increased rate of incorporation of the non-metabolizable analog 3-O-methyl-[U-14C]-glucose. This confirmed that the yeast Hxt2 transporter was functional in Arabidopsis. No phenotypic changes at the vegetative and reproductive stages could be detected in the transgenic lines when compared to wild type plants. Shortly after germination some differences in development and glucose signaling were observed. Transgenic seedlings cultivated in liquid medium or on solid agar plates were able to grow with 3% glucose (producing bigger plants and longer roots), while development of wild type plants was delayed under those conditions. Metabolite analysis revealed that the Hxt2 transgenic lines had higher rates of sugar utilization. Transcriptional profiling showed that particular genes were significantly up- or down-regulated. Some transcription factors like At1g27000 were repressed, while others, such as At3g58780, were induced. The mRNA from classical sugar signaling genes such as STP1, Hxk1, and ApL3 behaved similarly in transgenic lines and wild type lines. Results suggest that the Hxt2 transgene altered some developmental processes related to the perception of high carbon availability after the germination stage. We conclude that the developmental arrest of wild type plants at 3% glucose not only depends on Hxk1 as the only sugar sensor but might also be influenced by the route of hexose transport across the plasma membrane.

Understanding the Mechanisms that Modulate the MEP Pathway in Higher Plants

The methyl-d-erythritol 4-phosphate (MEP) pathway is responsible for the biosynthesis of an impressive number of natural compounds of biological and biotechnological importance. In recent years, this pathway has become a clear target to develop new herbicides and antimicrobial drugs. Additionally, the production of a variety of compounds of medical and agricultural interest may be possible through genetic manipulation of this pathway. To this end, a full understanding of the molecular mechanisms that regulate this pathway is of tremendous importance. Our work has shown that the MEP pathway is subjected to multiple levels of regulation, some of them being conserved among different plant species. In this chapter, we describe some of these regulatory mechanisms.

Shedding light on the methylerythritol phosphate (MEP)-pathway: long hypocotyl 5 (HY5)/phytochrome-interacting factors (PIFs) transcription factors modulating key limiting steps

The plastidial methylerythritol phosphate (MEP) pathway is an essential route for plants as the source of precursors for all plastidial isoprenoids, many of which are of medical and biotechnological importance. The MEP pathway is highly sensitive to environmental cues as many of these compounds are linked to photosynthesis and growth and light is one of the main regulatory factors. However, the mechanisms coordinating the MEP pathway with light cues are not fully understood. Here we demonstrate that by a differential direct transcriptional modulation, via the key-master integrators of light signal transduction HY5 and PIFs which target the genes that encode the rate-controlling DXS1, DXR and HDR enzymes, light imposes a direct, rapid and potentially multi-faceted response that leads to unique protein dynamics of this pathway, resulting in a significant difference in the protein levels. For DXS1, PIF1/HY5 act as a direct activation/suppression module. In contrast, DXR accumulation in response to light results from HY5 induction with minor contribution of de-repression by PIF1. Finally, HDR transcription increases in the light exclusively by suppression of the PIFs repression. This is an example of how light signaling components can differentially multi-target the initial steps of a pathway whose products branch downstream to all chloroplastic isoprenoids. These findings demonstrate the diversity and flexibility of light signaling components that optimize key biochemical pathways essential for plant growth.