A brassinosteroid transcriptional network revealed by genome-wide identification of BESI target genes in Arabidopsis thaliana.

Brassinosteroids (BRs) are important regulators for plant growth and development. BRs signal to control the activities of the BES1 and BZR1 family transcription factors. The transcriptional network through which BES1 and BZR regulate large number of target genes is mostly unknown. By combining chromatin immunoprecipitation coupled with Arabidopsis tiling arrays (ChIP-chip) and gene expression studies, we have identified 1609 putative BES1 target genes, 404 of which are regulated by BRs and/or in gain-of-function bes1-D mutant. BES1 targets contribute to BR responses and interactions with other hormonal or light signaling pathways. Computational modeling of gene expression data using Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) reveals that BES1-targeted transcriptional factors form a gene regulatory network (GRN). Mutants of many genes in the network displayed defects in BR responses. Moreover, we found that BES1 functions to inhibit chloroplast development by repressing the expression of GLK1 and GLK2 transcription factors, confirming a hypothesis generated from the GRN. Our results thus provide a global view of BR regulated gene expression and a GRN that guides future studies in understanding BR-regulated plant growth.

Impaired Chloroplast Biogenesis in Immutans, an Arabidopsis Variegation Mutant, Modifies Developmental Programming, Cell Wall Composition and Resistance to Pseudomonas syringae.

The immutans (im) variegation mutation of Arabidopsis has green- and white- sectored leaves due to action of a nuclear recessive gene. IM codes for PTOX, a plastoquinol oxidase in plastid membranes. Previous studies have revealed that the green and white sectors develop into sources (green tissues) and sinks (white tissues) early in leaf development. In this report we focus on white sectors, and show that their transformation into effective sinks involves a sharp reduction in plastid number and size. Despite these reductions, cells in the white sectors have near-normal amounts of plastid RNA and protein, and surprisingly, a marked amplification of chloroplast DNA. The maintenance of protein synthesis capacity in the white sectors might poise plastids for their development into other plastid types. The green and white im sectors have different cell wall compositions: whereas cell walls in the green sectors resemble those in wild type, cell walls in the white sectors have reduced lignin and cellulose microfibrils, as well as alterations in galactomannans and the decoration of xyloglucan. These changes promote susceptibility to the pathogen Pseudomonas syringae. Enhanced susceptibility can also be explained by repressed expression of some, but not all, defense genes. We suggest that differences in morphology, physiology and biochemistry between the green and white sectors is caused by a reprogramming of leaf development that is coordinated, in part, by mechanisms of retrograde (plastid-to-nucleus) signaling, perhaps mediated by ROS. We conclude that variegation mutants offer a novel system to study leaf developmental programming, cell wall metabolism and host-pathogen interactions.

The Mechanism of Variegation in immutans Provides Insight into Chloroplast Biogenesis.

The immutans (im) variegation mutant of Arabidopsis has green and white-sectored leaves due to the absence of fully functional plastid terminal oxidase (PTOX), a plastoquinol oxidase in thylakoid membranes. PTOX appears to be at the nexus of a growing number of biochemical pathways in the plastid, including carotenoid biosynthesis, PSI cyclic electron flow, and chlororespiration. During the early steps of chloroplast biogenesis, PTOX serves as an alternate electron sink and is a prime determinant of the redox poise of the developing photosynthetic apparatus. Whereas a lack of PTOX causes the formation of photooxidized plastids in the white sectors of im, compensating mechanisms allow the green sectors to escape the effects of the mutation. This manuscript provides an update on PTOX, the mechanism of im variegation, and findings about im compensatory mechanisms.

SUPPRESSOR OF VARIEGATION4, a new var2 suppressor locus, encodes a pioneer protein that is required for chloroplast biogenesis.

VAR2 is an integral thylakoid membrane protein and a member of the versatile FtsH class of metalloproteases in prokaryotes and eukaryotes. Recessive mutations in the VAR2 locus give rise to variegated plants (var2) that contain white sectors with abnormal plastids and green sectors with normal-appearing chloroplasts. In a continuing effort to isolate second-site suppressors of var2 variegation, we characterize in this report ems2505, a suppressor strain that has a virescent phenotype due to a missense mutation in At4g28590, the gene for a pioneer protein. We designated this gene SVR4 (for SUPPRESSOR OF VARIEGATION4) and the mutant allele in ems2505 as svr4-1. We demonstrate that SVR4 is located in chloroplasts and that svr4-1 single mutants are normal with respect to chloroplast anatomy and thylakoid membrane protein accumulation. However, they are modestly impaired in several aspects of photochemistry and have enhanced non-photochemical quenching (NPQ) capacity. A T-DNA insertion allele of SVR4, svr4-2, is seedling-lethal due to an early blockage of chloroplast development. We conclude that SVR4 is essential for chloroplast biogenesis, and hypothesize that SVR4 mediates some aspect of thylakoid structure or function that controls NPQ. We propose that in the suppressor strain, photoinhibitory pressure caused by a lack of VAR2 is ameliorated early in chloroplast development by enhanced NPQ capacity caused by reduced SVR4 activity. This would result in an increase in the number of chloroplasts that are able to surmount a threshold necessary to avoid photo-damage and thereby develop into functional chloroplasts.

PDS activity acts as a rheostat of retrograde signaling during early chloroplast biogenesis.

Chloroplasts are crucial for the process of photosynthesis, as well as for developmental and environmental sensing. One of the important mechanisms of sensing is retrograde (plastid-to-nucleus) signaling, whereby the state of the chloroplast is signaled to the nucleus, resulting in alterations in gene expression for chloroplast proteins, usually at the transcriptional level. Retrograde signaling was early studied in carotenoid-deficient plants that contain, upon exposure to high light, photooxidized plastids that arise because of an inability to quench ROS produced during the light reactions of photosynthesis. Phytoene desaturase (PDS) is required for one of the early steps of the carotenogenic pathway, and impaired PDS activity during early chloroplast biogenesis results in a highly reduced plastoquinone pool (high excitation pressure), accumulation of the colorless C(40) intermediate, phytoene, and white photooxidized plastids. Here, we discuss results from global transcript profiling of white leaf tissues of Arabidopsis that are blocked at the PDS step in three different ways--two by mutation (immutans & pds3) and one by inhibitor treatment (norflurazon). We show that the molecular phenotypes of the three tissues bear many similarities, but that there are also significant tissue-specific differences. We propose that PDS acts as a rheostat of excitation pressure-mediated retrograde signaling during chloroplast development, and speculate that whether the rheostat is set high (as in pds3 and NF-treated seedlings), intermediate (as in im) or low (as in WT) is a crucial determinant of the suite of genes that is expressed during chloroplast biogenesis.

Chloroplast photooxidation-induced transcriptome reprogramming in Arabidopsis immutans white leaf sectors.

Arabidopsis (Arabidopsis thaliana) immutans (im) has green and white sectoring due to the action of a nuclear recessive gene, IMMUTANS. The green sectors contain normal-appearing chloroplasts, whereas the white sectors contain abnormal chloroplasts that lack colored carotenoids due to a defect in phytoene desaturase activity. Previous biochemical and molecular characterizations of the green leaf sectors revealed alterations suggestive of a source-sink relationship between the green and white sectors of im. In this study, we use an Affymetrix ATH1 oligoarray to further explore the nature of sink metabolism in im white tissues. We show that lack of colored carotenoids in the im white tissues elicits a differential response from a large number of genes involved in various cellular processes and stress responses. Gene expression patterns correlate with the repression of photosynthesis and photosynthesis-related processes in im white tissues, with an induction of Suc catabolism and transport, and with mitochondrial electron transport and fermentation. These results suggest that energy is derived via aerobic and anaerobic metabolism of imported sugar in im white tissues for growth and development. We also show that oxidative stress responses are largely induced in im white tissues; however, im green sectors develop additional energy-dissipating mechanisms that perhaps allow for the formation of green sectors. Furthermore, a comparison of the transcriptomes of im white and norflurazon-treated white leaf tissues reveals global as well as tissue-specific responses to photooxidation. We conclude that the differences in the mechanism of phytoene desaturase inhibition play an important role in differentiating these two white tissues.

Variegation mutants and mechanisms of chloroplast biogenesis.

Variegated plants typically have green- and white-sectored leaves. Cells in the green sectors contain normal-appearing chloroplasts, whereas cells in the white sectors lack pigments and appear to be blocked at various stages of chloroplast biogenesis. Variegations can be caused by mutations in nuclear, chloroplast or mitochondrial genes. In some plants, the green and white sectors have different genotypes, but in others they have the same (mutant) genotype. One advantage of variegations is that they provide a means of studying genes for proteins that are important for chloroplast development, but for which mutant analysis is difficult, either because mutations in a gene of interest are lethal or because they do not show a readily distinguishable phenotype. This paper focuses on Arabidopsis variegations, for which the most information is available at the molecular level. Perhaps the most interesting of these are variegations caused by defective nuclear gene products in which the cells of the mutant have a uniform genotype. Two questions are of paramount interest: (1) What is the gene product and how does it function in chloroplast biogenesis? (2) What is the mechanism of variegation and why do green sectors arise in plants with a uniform (mutant) genotype? Two paradigms of variegation mechanism are described: immutans (im) and variegated2 (var2). Both mechanisms emphasize compensating activities and the notion of plastid autonomy, but redundant gene products are proposed to play a role in var2, but not in im. It is hypothesized that threshold levels of certain activities are necessary for normal chloroplast development.