gigantea suppresses immutans variegation by interactions with cytokinin and gibberellin signaling pathways.

The immutans (im) variegation mutant of Arabidopsis (Arabidopsis thaliana) is an ideal model to gain insight into factors that control chloroplast biogenesis. im defines the gene for PTOX, a plastoquinol terminal oxidase that participates in the control of thylakoid redox. Here, we report that the im defect can be suppressed during the late stages of plant development by gigantea (gi2), which defines the gene for GI, a central component of the circadian clock that plays a poorly understood role in diverse plant developmental processes. imgi2 mutants are late flowering and display other well-known phenotypes associated with gi2, such as starch accumulation and resistance to oxidative stress. We show that the restoration of chloroplast biogenesis in imgi2 is caused by a development-specific derepression of cytokinin signaling that involves cross talk with signaling pathways mediated by gibberellin (GA) and SPINDLY (SPY), a GA response inhibitor. Suppression of the plastid defect in imgi2 is likely caused by a relaxation of excitation pressures in developing plastids by factors contributed by gi2, including enhanced rates of photosynthesis and increased resistance to oxidative stress. Interestingly, the suppression phenotype of imgi can be mimicked by crossing im with the starch accumulation mutant, starch excess1 (sex1), perhaps because sex1 utilizes pathways similar to gi. We conclude that our studies provide a direct genetic linkage between GI and chloroplast biogenesis, and we construct a model of interactions between signaling pathways mediated by gi, GA, SPY, cytokinins, and sex1 that are required for chloroplast biogenesis.

Alternative oxidases (AOX1a and AOX2) can functionally substitute for plastid terminal oxidase in Arabidopsis chloroplasts.

The immutans (im) variegation mutant of Arabidopsis thaliana is caused by an absence of PTOX, a plastid terminal oxidase bearing similarity to mitochondrial alternative oxidase (AOX). In an activation tagging screen for suppressors of im, we identified one suppression line caused by overexpression of AOX2. AOX2 rescued the im defect by replacing the activity of PTOX in the desaturation steps of carotenogenesis. Similar results were obtained when AOX1a was reengineered to target the plastid. Chloroplast-localized AOX2 formed monomers and dimers, reminiscent of AOX regulation in mitochondria. Both AOX2 and AOX1a were present in higher molecular weight complexes in plastid membranes. The presence of these proteins did not generally affect steady state photosynthesis, aside from causing enhanced nonphotochemical quenching in both lines. Because AOX2 was imported into chloroplasts using its own transpeptide, we propose that AOX2 is able to function in chloroplasts to supplement PTOX activity during early events in chloroplast biogenesis. We conclude that the ability of AOX1a and AOX2 to substitute for PTOX in the correct physiological and developmental contexts is a striking example of the capacity of a mitochondrial protein to replace the function of a chloroplast protein and illustrates the plasticity of the photosynthetic apparatus.

Understanding chloroplast biogenesis using second-site suppressors of immutans and var2.

Chloroplast biogenesis is an essential light-dependent process involving the differentiation of photosynthetically competent chloroplasts from precursors that include undifferentiated proplastids in leaf meristems, as well as etioplasts in dark-grown seedlings. The mechanisms that govern these developmental processes are poorly understood, but entail the coordinated expression of nuclear and plastid genes. This coordination is achieved, in part, by signals generated in response to the metabolic and developmental state of the plastid that regulate the transcription of nuclear genes for photosynthetic proteins (retrograde signaling). Variegation mutants are powerful tools to understand pathways of chloroplast biogenesis, and over the years our lab has focused on immutans (im) and variegated2 (var2), two nuclear gene-induced variegations of Arabidopsis. im and var2 are among the best-characterized chloroplast biogenesis mutants, and they define the genes for plastid terminal oxidase (PTOX) and the AtFtsH2 subunit of the thylakoid FtsH metalloprotease complex, respectively. To gain insight into the function of these proteins, forward and reverse genetic approaches have been used to identify second-site suppressors of im and var2 that replace or bypass the need for PTOX and AtFtsH2 during chloroplast development. In this review, we provide a brief update of im and var2 and the functions of PTOX and AtFtsH2. We then summarize information about second-site suppressors of im and var2 that have been identified to date, and describe how they have provided insight into mechanisms of photosynthesis and pathways of chloroplast development.

Genetic interactions reveal that specific defects of chloroplast translation are associated with the suppression of var2-mediated leaf variegation.

Arabidopsis thaliana L. yellow variegated (var2) mutant is defective in a chloroplast FtsH family metalloprotease, AtFtsH2/VAR2, and displays an intriguing green and white leaf variegation. This unique var2-mediated leaf variegation offers a simple yet powerful tool for dissecting the genetic regulation of chloroplast development. Here, we report the isolation and characterization of a new var2 suppressor gene, SUPPRESSOR OF VARIEGATION8 (SVR8), which encodes a putative chloroplast ribosomal large subunit protein, L24. Mutations in SVR8 suppress var2 leaf variegation at ambient temperature and partially suppress the cold-induced chlorosis phenotype of var2. Loss of SVR8 causes unique chloroplast rRNA processing defects, particularly the 23S-4.5S dicistronic precursor. The recovery of the major abnormal processing site in svr8 23S-4.5S precursor indicate that it does not lie in the same position where SVR8/L24 binds on the ribosome. Surprisingly, we found that the loss of a chloroplast ribosomal small subunit protein, S21, results in aberrant chloroplast rRNA processing but not suppression of var2 variegation. These findings suggest that the disruption of specific aspects of chloroplast translation, rather than a general impairment in chloroplast translation, suppress var2 variegation and the existence of complex genetic interactions in chloroplast development.

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.

Overexpression of a putative Arabidopsis BAHD acyltransferase causes dwarfism that can be rescued by brassinosteroid.

Plant growth and development are ensured through networks of complex regulatory schemes. Genetic approaches have been invaluable in dissecting these regulatory pathways. This study reports the isolation of a semi-dominant dwarf mutant designated abnormal shoot1-1 dominant (abs1-1D) through an Arabidopsis T-DNA activation tagging mutant screen. It was shown that the overexpression of a novel BAHD family acyltransferase gene, ABS1/At4g15400, was the cause of the dwarf phenotype in abs1-1D. Overexpression of ABS1 led to many phenotypic features reminiscent of brassinosteroid (BR) deficient or signalling mutants, and it was shown that exogenously applied BR could effectively rescue the dwarf phenotype of abs1-1D. Furthermore, genetic analyses indicated that abs1-1D interacted, in different ways, with the BR-deficient mutant det2-1, the constitutive BR response mutant bes1-D and the photomorphogenic mutant phyB-1. Moreover, ABS1 expression was activated by BR treatment or in a bes1-D mutant background. Genome-wide transcriptome profiling of abs1-1D revealed clear reprogramming of metabolic pathways, and it was demonstrated that BR biosynthesis genes were activated in abs1-1D and that the flavonoid biosynthesis pathway was repressed in abs1-1D, as well as in det2-1. This work provides new insights into the possible involvement of BAHD acyltransferase in the regulation of plant growth and development, and indicates a possible role of ABS1 in maintaining BR homeostasis.

An Arabidopsis pentatricopeptide repeat protein, SUPPRESSOR OF VARIEGATION7, is required for FtsH-mediated chloroplast biogenesis.

The Arabidopsis (Arabidopsis thaliana) yellow variegated2 (var2) mutant has green- and white-sectored leaves due to loss of VAR2, a subunit of the chloroplast FtsH protease/chaperone complex. Suppressor screens are a valuable tool to gain insight into VAR2 function and the mechanism of var2 variegation. Here, we report the molecular characterization of 004-003, a line in which var2 variegation is suppressed. We found that the suppression phenotype in this line is caused by lack of a chloroplast pentatricopeptide repeat (PPR) protein that we named SUPPRESSOR OF VARIEGATION7 (SVR7). PPR proteins contain tandemly repeated PPR motifs that bind specific RNAs, and they are thought to be central regulators of chloroplast and mitochondrial nucleic acid metabolism in plants. The svr7 mutant has defects in chloroplast ribosomal RNA (rRNA) processing that are different from those in other svr mutants, and these defects are correlated with reductions in the accumulation of some chloroplast proteins, directly or indirectly. We also found that whereas var2 displays a leaf variegation phenotype at 22°C, it has a pronounced chlorosis phenotype at 8°C that is correlated with defects in chloroplast rRNA processing and a drastic reduction in chloroplast protein accumulation. Surprisingly, the cold-induced phenotype of var2 cannot be suppressed by svr7. Our results strengthen the previously established linkage between var2 variegation and chloroplast rRNA processing/chloroplast translation, and they also point toward the possibility that VAR2 mediates different activities in chloroplast biogenesis at normal and chilling temperatures.

A var2 leaf variegation suppressor locus, SUPPRESSOR OF VARIEGATION3, encodes a putative chloroplast translation elongation factor that is important for chloroplast development in the cold.

BACKGROUND: The Arabidopsis var2 mutant displays a unique green and white/yellow leaf variegation phenotype and lacks VAR2, a chloroplast FtsH metalloprotease. We are characterizing second-site var2 genetic suppressors as means to better understand VAR2 function and to study the regulation of chloroplast biogenesis. RESULTS: In this report, we show that the suppression of var2 variegation in suppressor line TAG-11 is due to the disruption of the SUPPRESSOR OF VARIEGATION3 (SVR3) gene, encoding a putative TypA-like translation elongation factor. SVR3 is targeted to the chloroplast and svr3 single mutants have uniformly pale green leaves at 22°C. Consistent with this phenotype, most chloroplast proteins and rRNA species in svr3 have close to normal accumulation profiles, with the notable exception of the Photosystem II reaction center D1 protein, which is present at greatly reduced levels. When svr3 is challenged with chilling temperature (8°C), it develops a pronounced chlorosis that is accompanied by abnormal chloroplast rRNA processing and chloroplast protein accumulation. Double mutant analysis indicates a possible synergistic interaction between svr3 and svr7, which is defective in a chloroplast pentatricopeptide repeat (PPR) protein. CONCLUSIONS: Our findings, on one hand, reinforce the strong genetic link between VAR2 and chloroplast translation, and on the other hand, point to a critical role of SVR3, and possibly some aspects of chloroplast translation, in the response of plants to chilling stress.

Arabidopsis chloroplast FtsH, var2 and suppressors of var2 leaf variegation: a review.

Variegation mutants are ideal model systems to study chloroplast biogenesis. We are interested in variegations whose green and white-sectored leaves arise as a consequence of the action of nuclear recessive genes. In this review, we focus on the Arabidopsis var2 variegation mutant, and discuss recent progress toward understanding the function of VAR2 and the mechanism of var2-mediated variegation. VAR2 is a subunit of the chloroplast FtsH complex, which is involved in turnover of the Photosystem II reaction center D1 protein, as well as in other processes required for the development and maintenance of the photosynthetic apparatus. The cells in green sectors of var2 have normal-appearing chloroplasts whereas cells in the white sectors have abnormal plastids that lack pigments and organized lamellae. To explain the mechanism of var2 variegation, we have proposed a threshold model in which the formation of chloroplasts is due to the presence of activities/processes that are able to compensate for a lack of VAR2. To gain insight into these activities, second-site suppressor screens have been carried out to obtain mutants with non-variegation phenotypes. Cloning and characterization of several var2 suppressor lines have uncovered several mechanisms of variegation suppression, including an unexpected link between var2 variegation and chloroplast translation.

Generation of transgenic maize with enhanced provitamin A content.

Vitamin A deficiency (VAD) affects over 250 million people worldwide and is one of the most prevalent nutritional deficiencies in developing countries, resulting in significant socio-economic losses. Provitamin A carotenoids such as beta-carotene, are derived from plant foods and are a major source of vitamin A for the majority of the world's population. Several years of intense research has resulted in the production of 'Golden Rice 2' which contains sufficiently high levels of provitamin A carotenoids to combat VAD. In this report, the focus is on the generation of transgenic maize with enhanced provitamin A content in their kernels. Overexpression of the bacterial genes crtB (for phytoene synthase) and crtI (for the four desaturation steps of the carotenoid pathway catalysed by phytoene desaturase and zeta-carotene desaturase in plants), under the control of a 'super gamma-zein promoter' for endosperm-specific expression, resulted in an increase of total carotenoids of up to 34-fold with a preferential accumulation of beta-carotene in the maize endosperm. The levels attained approach those estimated to have a significant impact on the nutritional status of target populations in developing countries. The high beta-carotene trait was found to be reproducible over at least four generations. Gene expression analyses suggest that increased accumulation of beta-carotene is due to an up-regulation of the endogenous lycopene beta-cylase. These experiments set the stage for the design of transgenic approaches to generate provitamin A-rich maize that will help alleviate VAD.

PTOX Mediates Novel Pathways of Electron Transport in Etioplasts of Arabidopsis.

The immutans (im) variegation mutant of Arabidopsis defines the gene for PTOX (plastid terminal oxidase), a versatile plastoquinol oxidase in chloroplast membranes. In this report we used im to gain insight into the function of PTOX in etioplasts of dark-grown seedlings. We discovered that PTOX helps control the redox state of the plastoquinone (PQ) pool in these organelles, and that it plays an essential role in etioplast metabolism by participating in the desaturation reactions of carotenogenesis and in one or more redox pathways mediated by PGR5 (PROTON GRADIENT REGULATION 5) and NDH (NAD(P)H dehydrogenase), both of which are central players in cyclic electron transport. We propose that these elements couple PTOX with electron flow from NAD(P)H to oxygen, and by analogy to chlororespiration (in chloroplasts) and chromorespiration (in chromoplasts), we suggest that they define a respiratory process in etioplasts that we have termed "etiorespiration". We further show that the redox state of the PQ pool in etioplasts might control chlorophyll biosynthesis, perhaps by participating in mechanisms of retrograde (plastid-to-nucleus) signaling that coordinate biosynthetic and photoprotective activities required to poise the etioplast for light development. We conclude that PTOX is an important component of metabolism and redox sensing in etioplasts.

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.

Genetic modification of low phytic acid 1-1 maize to enhance iron content and bioavailability.

High phytate content in staple food crops is a major barrier to successful iron biofortification. We have exploited the low phytic acid 1-1 (lpa1-1) mutant of maize to generate transgenic plants with up-to 70 µg/g seed iron through the endosperm-specific overexpression of soybean ferritin, resulting in more than 2-fold improvement in iron bioavailability. The levels of bioavailable seed iron achieved in this study greatly exceed any achieved thus far and closely approach values estimated to have a nutritional impact on target populations. Gene expression studies reveal a large induction of the YS1 transporter in leaves and severe repression of an iron acquisition gene DMAS1 in roots, suggesting significant alterations in the iron homeostatic mechanisms in transgenic lpa1-1. Furthermore, preliminary tests show that the high-iron lpa1-1 seeds have higher germination rates and seedling vigor when compared to those of the nontransgenic seeds, which may help improve their value to plant breeders.

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.

Mutations in SUPPRESSOR OF VARIEGATION1, a factor required for normal chloroplast translation, suppress var2-mediated leaf variegation in Arabidopsis.

The Arabidopsis thaliana yellow variegated2 (var2) mutant is variegated due to lack of a chloroplast FtsH-like metalloprotease (FtsH2/VAR2). We have generated suppressors of var2 variegation to gain insight into factors and pathways that interact with VAR2 during chloroplast biogenesis. Here, we describe two such suppressors. Suppression of variegation in the first line, TAG-FN, was caused by disruption of the nuclear gene (SUPPRESSOR OF VARIEGATION1 [SVR1]) for a chloroplast-localized homolog of pseudouridine (Psi) synthase, which isomerizes uridine to Psi in noncoding RNAs. svr1 single mutants were epistatic to var2, and they displayed a phenotypic syndrome that included defects in chloroplast rRNA processing, reduced chloroplast translation, reduced chloroplast protein accumulation, and elevated chloroplast mRNA levels. In the second line (TAG-IE), suppression of variegation was caused by a lesion in SVR2, the gene for the ClpR1 subunit of the chloroplast ClpP/R protease. Like svr1, svr2 was epistatic to var2, and clpR1 mutants had a phenotype that resembled svr1. We propose that an impairment of chloroplast translation in TAG-FN and TAG-IE decreased the demand for VAR2 activity during chloroplast biogenesis and that this resulted in the suppression of var2 variegation. Consistent with this hypothesis, var2 variegation was repressed by chemical inhibitors of chloroplast translation. In planta mutagenesis revealed that SVR1 not only played a role in uridine isomerization but that its physical presence was necessary for proper chloroplast rRNA processing. Our data indicate that defects in chloroplast rRNA processing are a common, but not universal, molecular phenotype associated with suppression of var2 variegation.

Methylation of gibberellins by Arabidopsis GAMT1 and GAMT2.

Arabidopsis thaliana GAMT1 and GAMT2 encode enzymes that catalyze formation of the methyl esters of gibberellins (GAs). Ectopic expression of GAMT1 or GAMT2 in Arabidopsis, tobacco (Nicotiana tabacum), and petunia (Petunia hybrida) resulted in plants with GA deficiency and typical GA deficiency phenotypes, such as dwarfism and reduced fertility. GAMT1 and GAMT2 are both expressed mainly in whole siliques (including seeds), with peak transcript levels from the middle until the end of silique development. Within whole siliques, GAMT2 was previously shown to be expressed mostly in developing seeds, and we show here that GAMT1 expression is also localized mostly to seed, suggesting a role in seed development. Siliques of null single GAMT1 and GAMT2 mutants accumulated high levels of various GAs, with particularly high levels of GA(1) in the double mutant. Methylated GAs were not detected in wild-type siliques, suggesting that methylation of GAs by GAMT1 and GAMT2 serves to deactivate GAs and initiate their degradation as the seeds mature. Seeds of homozygous GAMT1 and GAMT2 null mutants showed reduced inhibition of germination, compared with the wild type, when placed on plates containing the GA biosynthesis inhibitor ancymidol, with the double mutant showing the least inhibition. These results suggest that the mature mutant seeds contained higher levels of active GAs than wild-type seeds.

Pathways of intracellular communication: tetrapyrroles and plastid-to-nucleus signaling.

Retrograde plastid-to-nucleus signaling plays a central role in coordinating nuclear and plastid gene expression. The gun (genomes uncoupled) mutants of Arabidopsis have been used to demonstrate that Mg-protoporphyrin (Mg-Proto) acts as a plastid signal to repress the transcription of nuclear photosynthesis genes (1). It is unclear how Mg-Proto triggers repression, but several components of this pathway have been recently identified. These include the products of GUN4 and GUN5. GUN5 is the ChlH subunit of Mg-chelatase, which produces Mg-Proto, and GUN4 is a regulator of ChlH activity (2). GUN4 might also play a role in photoprotection and in the trafficking of Mg-Proto.

A proteomic analysis of maize chloroplast biogenesis.

Proteomics studies to explore global patterns of protein expression in plant and green algal systems have proliferated within the past few years. Although most of these studies have involved mapping of the proteomes of various organs, tissues, cells, or organelles, comparative proteomics experiments have also led to the identification of proteins that change in abundance in various developmental or physiological contexts. Despite the growing use of proteomics in plant studies, questions of reproducibility have not generally been addressed, nor have quantitative methods been widely used, for example, to identify protein expression classes. In this report, we use the de-etiolation ("greening") of maize (Zea mays) chloroplasts as a model system to explore these questions, and we outline a reproducible protocol to identify changes in the plastid proteome that occur during the greening process using techniques of two-dimensional gel electrophoresis and mass spectrometry. We also evaluate hierarchical and nonhierarchical statistical methods to analyze the patterns of expression of 526 "high-quality," unique spots on the two-dimensional gels. We conclude that Adaptive Resonance Theory 2-a nonhierarchical, neural clustering technique that has not been previously applied to gene expression data-is a powerful technique for discriminating protein expression classes during greening. Our experiments provide a foundation for the use of proteomics in the design of experiments to address fundamental questions in plant physiology and molecular biology.