Elina Welchen

Differential expression of the arabidopsis cytochrome c genes Cytc-1 and Cytc-2. evidence for the involvement of TCP-domain protein-binding elements in anther- and meristem-specific expression of the Cytc-1 gene

The promoters of the Arabidopsis (Arabidopsis thaliana) cytochrome c genes, Cytc-1 and Cytc-2, were analyzed using plants transformed with fusions to the β-glucuronidase coding sequence. Histochemical staining of plants indicated that the Cytc-1 promoter directs preferential expression in root and shoot meristems and in anthers. In turn, plants transformed with the Cytc-2 promoter fusions showed preferential expression in vascular tissues of cotyledons, leaves, roots, and hypocotyls, and also in anthers. Quantitative measurements in extracts prepared from different organs suggested that expression of Cytc-1 is higher in flowers, while that of Cytc-2 is higher in leaves. The analysis of a set of deletions and site-directed mutants of the Cytc-1 promoter indicated that a segment located between −147 and −156 from the translation start site is required for expression and that site II elements (TGGGCC/T) located in this region, coupled with a downstream internal telomeric repeat (AAACCCTAA), are responsible for the expression pattern of this gene. Proteins present in cauliflower nuclear extracts, as well as a recombinant protein from the TCP-domain family, were able to specifically bind to the region required for expression. We propose that expression of the Cytc-1 gene is linked to cell proliferation through the elements described above. The fact that closely located site II motifs are present in similar locations in several genes encoding proteins involved in cytochrome c-dependent respiration suggests that these elements may be the target of factors that coordinate the expression of nuclear genes encoding components of this part of the mitochondrial respiratory chain.

Overrepresentation of Elements Recognized by TCP-Domain Transcription Factors in the Upstream Regions of Nuclear Genes Encoding Components of the Mitochondrial Oxidative Phosphorylation Machinery

We have observed that a cis-acting regulatory element, known as site II, is overrepresented in the promoters of nuclear genes encoding components of the oxidative phosphorylation (OxPhos) machinery from both Arabidopsis ( Arabidopsis thaliana ) and rice ( Oryza sativa ). Site II elements have been

TCP transcription factors: architectures of plant form

Abstract After its initial definition in 1999, the TCP family of transcription factors has become the focus of a multiplicity of studies related with plant development at the cellular, organ, and tissue levels. Evidence has accumulated indicating that TCP transcription factors are the main regulators of plant form and architecture and constitute a tool through which evolution shapes plant diversity. The TCP transcription factors act in a multiplicity of pathways related with cell proliferation and hormone responses. In recent years, the molecular pathways of TCP protein action and biochemical studies on their mode of interaction with DNA have begun to shed light on their mechanism of action. However, the available information is fragmented and a unifying view of TCP protein action is lacking, as well as detailed structural studies of the TCP-DNA complex. Also important, the possible role of TCP proteins as integrators of plant developmental responses to the environment has deserved little attention. In this review, we summarize the current knowledge about the structure and functions of TCP transcription factors and analyze future perspectives for the study of the role of these proteins and their use to modify plant development.

Coordination of plant mitochondrial biogenesis: keeping pace with cellular requirements

Plant mitochondria are complex organelles that carry out numerous metabolic processes related with the generation of energy for cellular functions and the synthesis and degradation of several compounds. Mitochondria are semiautonomous and dynamic organelles changing in shape, number, and composition depending on tissue or developmental stage. The biogenesis of functional mitochondria requires the coordination of genes present both in the nucleus and the organelle. In addition, due to their central role, all processes held inside mitochondria must be finely coordinated with those in other organelles according to cellular demands. Coordination is achieved by transcriptional control of nuclear genes encoding mitochondrial proteins by specific transcription factors that recognize conserved elements in their promoter regions. In turn, the expression of most of these transcription factors is linked to developmental and environmental cues, according to the availability of nutrients, light–dark cycles, and warning signals generated in response to stress conditions. Among the signals impacting in the expression of nuclear genes, retrograde signals that originate inside mitochondria help to adjust mitochondrial biogenesis to organelle demands. Adding more complexity, several nuclear encoded proteins are dual localized to mitochondria and either chloroplasts or the nucleus. Dual targeting might establish a crosstalk between the nucleus and cell organelles to ensure a fine coordination of cellular activities. In this article, we discuss how the different levels of coordination of mitochondrial biogenesis interconnect to optimize the function of the organelle according to both internal and external demands.

Lack of cytochrome c in Arabidopsis decreases stability of Complex IV and modifies redox metabolism without affecting Complexes I and III

We studied the role of cytochrome c (CYTc), which mediates electron transfer between Complexes III and IV, in cellular events related with mitochondrial respiration, plant development and redox homeostasis. We analyzed single and double homozygous mutants in both CYTc-encoding genes from Arabidopsis: CYTC-1 and CYTC-2. While individual mutants were similar to wild-type, knock-out of both genes produced an arrest of embryo development, showing that CYTc function is essential at early stages of plant development. Mutants in which CYTc levels were extremely reduced respective to wild-type had smaller rosettes with a pronounced decrease in parenchymatic cell size and an overall delay in development. Mitochondria from these mutants had lower respiration rates and a relative increase in alternative respiration. Furthermore, the decrease in CYTc severely affected the activity and the amount of Complex IV, without affecting Complexes I and III. Reactive oxygen species levels were reduced in these mutants, which showed induction of genes encoding antioxidant enzymes. Ascorbic acid levels were not affected, suggesting that a small amount of CYTc is enough to support its normal synthesis. We postulate that, in addition to its role as an electron carrier between Complexes III and IV, CYTc influences Complex IV levels in plants, probably reflecting a role of this protein in Complex IV stability. This double function of CYTc most likely explains why it is essential for plant survival.

Nuclear and mitochondrial genes encoding cytochrome c oxidase subunits respond differently to the same metabolic factors

Abstract We have identified in the Arabidopsis thaliana Heyhn. nuclear genome a gene encoding COX6a, a homologue of subunit 6a of the mitochondrial cytochrome c oxidase (COX, E.C. 1.9.3.1) from animals and fungi. The expression of this gene and of the previously identified nuclear and mitochondrial genes, respectively, that encode subunit 6b (COX6b) and subunit 2 of the same enzyme was analyzed by northern blot. The analysis indicated that incubation of plants in solutions containing metabolizable sugars produced an increase in transcript levels for the nuclear genes but not for the mitochondrial one. The effect of carbohydrates showed the same time-dependence for COX6a and COX6b. Incubation of plants in solutions with different nitrogen sources also produced changes in expression. The behavior of the nuclear genes was very similar to that previously observed for genes encoding cytochrome c and cytochrome oxidase subunit 5b, suggesting the operation of a common regulatory mechanism for components of the mitochondrial respiratory chain encoded in the nucleus, but not for their counterparts encoded in the mitochondria. Regulation of the expression of mitochondrial genes, if any, may operate at a different level.

Plants contain two SCO proteins that are differentially involved in cytochrome c oxidase function and copper and redox homeostasis

Two Arabidopsis thaliana genes (HCC1 and HCC2), resulting from a duplication that took place before the emergence of flowering plants, encode proteins with homology to the SCO proteins involved in copper insertion during cytochrome c oxidase (COX) assembly in other organisms. Heterozygote HCC1 mutant plants produce 25% abnormal seeds with defective embryos arrested at the heart or torpedo stage. These embryos lack COX activity, suggesting that the requirement of HCC1 during the early stages of plant development is related with its COX assembly function. Homozygote HCC2 mutant plants develop normally and do not show changes in COX2 levels. These plants display increased sensitivity of root growth to increased copper and a higher expression of miR398 and other genes that respond to copper limitation, in spite of the fact that they have a higher copper content than the wild type. HCC2 mutant plants also show increased expression of stress-responsive genes. The results suggest that HCC1 is the protein involved in COX biogenesis and that HCC2, that lacks the cysteines and histidine putatively involved in copper binding, functions in copper sensing and redox homeostasis. In addition, plants that overexpress HCC1 have an altered response of root elongation to changes in copper in the growth medium and increased expression of two low-copper-responsive genes, suggesting that HCC1 may also have a role in copper homeostasis.

A segment containing a G-box and an ACGT motif confers differential expression characteristics and responses to the Arabidopsis Cytc-2 gene, encoding an isoform of cytochrome c

Sequences required for the expression of Cytc-2 (At4g10040), one of two cytochrome c genes from Arabidopsis thaliana, were characterized using plants transformed with deleted and mutagenized promoter fragments fused to gus. These studies indicated that a region containing a G-box and an ACGT motif is essential for expression. Mutation of the ACGT motif causes a complete loss of expression, while mutation of the G-box causes decreased expression in aerial parts and abolishes expression in roots and induction by environmental factors. Upstream located site II elements are required for maximal expression, mainly in reproductive tissues, and maximal induction by different factors. One-hybrid screenings allowed the identification of transcription factors from the bZIP and bHLH families that interact mainly with the G-box. Four of these factors were able to bind to the Cytc-2 promoter in vitro and in transactivation assays in Arabidopsis. Analysis of available microarray data indicated that the bZIP transcription factors share expression characteristics with the Cytc-2 gene, suggesting that they act as mediators of its response to tissue-specific, environmental, and metabolic conditions. Site II elements interact with a TCP family protein and may co-ordinate the expression of the Cytc-2 gene with that of other respiratory chain components. A model is proposed for the evolution of the Cytc-2 gene through the incorporation of a segment containing a G-box and an ACGT motif into an ancestral gene that contained site II elements. This may have reduced the importance of site II elements for basal expression and conferred new responses to environmental factors.

Plant mitochondria under pathogen attack: A sigh of relief or a last breath?

Plants constitute excellent sources for pathogen nutrition and survival. To fight against pathogen attack, higher plants have developed a sophisticated immune system responsible for pathogen recognition and activation of downstream defense responses. After pathogen perception, mitochondria play an important role in the defense strategy of the plant cell, integrating and amplifying diverse signals such as salicylic acid, nitric oxide, reactive oxygen species (ROS) or pathogen elicitors. Signals perceived by mitochondria usually impact on their normal function, destabilizing the organelle, generating changes in respiration, membrane potential and ROS production. At this stage, mitochondria produce several signals influencing the redox state of the cell and promoting changes in the expression of nuclear genes by mitochondrial retrograde regulation. At more advanced stages, they promote programmed cell death in order to avoid pathogen propagation to the whole plant. Recent evidence indicates that plants and pathogens have evolved mechanisms to modulate the immune response by acting on mitochondrial functions. In this review, we summarize knowledge about the involvement of mitochondria in different aspects of the response of plants to pathogen attack.

Mitochondria and copper homeostasis in plants.

Copper (Cu) and other transition metals are essential for living organisms but also toxic when present in excess. To cope with this apparent paradox, organisms have developed sophisticated mechanisms to acquire, transport and store these metals. Particularly, plant mitochondria require Cu for the assembly and function of cytochrome c oxidase (COX), the terminal enzyme of the respiratory chain. COX assembly is a complex process that requires the action of multiple factors, many of them involved in the delivery and insertion of Cu into the enzyme. In this review, we summarize what is known about the processes involved in Cu delivery to mitochondria and how these processes impact in Cu homeostasis at the cellular level. We also discuss evidence indicating that metallochaperones involved in COX assembly play additional roles in signaling pathways related to changes in Cu and redox homeostasis and the response of plants to stress. We propose that cysteine-rich proteins present in the mitochondrial intermembrane space are excellent candidates as sensors of these changes and transducers of signals originated in the organelle to the rest of the cell.