The rice EP3 and OsFBK1 E3 ligases alter plant architecture and flower development, and affect transcript accumulation of microRNA pathway genes and their targets.

ERECTA PANICLE 3 (EP3) and ORYZA SATIVA F-BOX KELCH 1 (OsFBK1) proteins share 57% and 54% sequence identity with the Arabidopsis F-box protein HAWAIIAN SKIRT (HWS). Previously we showed that EP3 is a functional orthologue of HWS. Here we demonstrate that OsFBK1 is another functional orthologue of HWS and show the complexity of interaction between EP3 and OsFBK1 genes at different developmental stages of the plant. qRT-PCR expression analyses and studies of EP3-GFP and OsFBK1-RFP promoter reporter lines demonstrate that although EP3 and OsFBK1 expression can be detected in the same tissues some cells exclusively express EP3 or OsFBK1 whilst others co-express both genes. Loss, reduction or gain-of-function lines for EP3 and OsFBK1, show that EP3 and OsFBK1 affect plant architecture, organ size, floral organ number and size, floral morphology, pollen viability, grain size and weight. We have identified the putative orthologue genes of the rice microRNA pathway for ORYZA SATIVA DAWDLE (OsDDL) and ORYZA SATIVA SERRATE (OsSE), and demonstrated that EP3 and OsFBK1 affect their transcript levels as well as those of CROWN ROOT DEFECT 1/ORYZA SATIVA Exportin-5 HASTY (CRD1/OsHST), ORYZA SATIVA DICER-LIKE 1 (OsDCL) and ORYZA SATIVA WEAVY LEAF1 (OsWAF1). We show that EP3 affects OsPri-MIR164, OsNAM1 and OsNAC1 transcript levels. OsNAC1 transcripts are modified by OsFBK1, suggesting two independent regulatory pathways, one via EP3 and OsMIR164 and the other via OsFBK1. Our data propose that EP3 and OsFBK1 conjointly play similar roles in rice to how HWS does in Arabidopsis.

Decreased photosynthesis in the erect panicle 3 (ep3) mutant of rice is associated with reduced stomatal conductance and attenuated guard cell development.

The ERECT PANICLE 3 gene of rice encodes a peptide that exhibits more than 50% sequence identity with the Arabidopsis F-box protein HAWAIIAN SKIRT (HWS). Ectopic expression of the Os02g15950 coding sequence, driven by the HWS (At3g61950) promoter, rescued the hws-1 flower phenotype in Arabidopsis confirming that EP3 is a functional orthologue of HWS. In addition to displaying an erect inflorescence phenotype, loss-of-function mutants of Os02g15950 exhibited a decrease in leaf photosynthetic capacity and stomatal conductance. Analysis of a range of physiological and anatomical features related to leaf photosynthesis revealed no alteration in Rubisco content and no notable changes in mesophyll size or arrangement. However, both ep3 mutant plants and transgenic lines that have a T-DNA insertion within the Os02g15950 (EP3) gene exhibit smaller stomatal guard cells compared with their wild-type controls. This anatomical characteristic may account for the observed decrease in leaf photosynthesis and provides evidence that EP3 plays a role in regulating stomatal guard cell development.

Divide and shape: an endosymbiont in action.

The endosymbiotic evolution of the plastid within the host cell required development of a mechanism for efficient division of the plastid. Whilst a model for the mechanism of chloroplast division has been constructed, little is known of how other types of plastids divide, especially the proplastid, the progenitor of all plastid types in the cell. It has become clear that plastid shape is highly heterogeneous and dynamic, especially stromules. This article considers how such variation in morphology might be controlled and how such plastids might divide efficiently.

Plastids unleashed: their development and their integration in plant development.

Derived by endosymbiosis from ancestral cyanobacteria, chloroplasts integrated seamlessly into the biology of their host cell. That integration involved a massive transfer of genes to the cell's nucleus, with the modification of pre-existing processes, like plastid division and the operation of the plastid genetic machinery and the emergence of new ones, like the import of proteins translated in the cytoplasm. The uncovering in molecular detail of several of these processes reveals a merger of mechanisms of symbiont and host origin. Chloroplasts acquired roles as part of the biology of land plants by differentiating into a variety of interconvertible plastid forms according to the cell type. How these conversions take place, or how new problems, like the regulation of the plastid population size in cells, have been solved, is barely starting to be understood. Like the whole plant and as a result of the requirements and dangers associated with photosynthetic activity, chloroplasts in particular are under the control of environmental cues. Far from being passive targets of cellular processes, plastids are sources of signals of plastid-nuclear communication, which regulate activities for their own biogenesis. Plastids are also sources of developmental signals, in whose absence tissue architecture or cell differentiation are aberrant, in a cell-autonomous fashion. Over evolutionary time, plastids also contributed many genes for activities that are no longer directly associated with them (like light perception or hormone function). The overall picture is one in which plastids are at both the receiving and the acting ends in plant development, in both ontogenic and evolutionary terms.

Reduced gravitropism in inflorescence stems and hypocotyls, but not roots, of Arabidopsis mutants with large plastids.

The sites of gravity perception are columella cells in roots and endodermal cells in hypocotyls and inflorescence stems. Since plastids are likely to play a role in graviperception, we investigated gravitropism in plastid mutants of Arabidopsis. Previous studies have shown that the arc6 and arc12 (accumulation and replication of chloroplasts) mutants have an average of two large plastids per leaf mesophyll cell. In this study, we found that these arc mutants have altered plastid morphology throughout the entire plant body, including the cells involved in gravity perception. There were no major differences in total starch content per cell in endodermal and columella cells of the wild-type (WT) compared to arc6 and arc12 as assayed by iodine staining. Thus, the total mass of plastids per cell in arc6 and arc12 is similar to their respective WT strains. Results from time course of curvature studies demonstrated that the plastid mutation affected gravitropism only of inflorescence stems and hypocotyls, but not roots. Thus, roots appear to have different mechanisms of gravitropism compared to stems and hypocotyls. Time course of curvature studies with light-grown seedlings were performed in the presence of latrunculin B (Lat-B), an actin-depolymerizing drug. Lat-B promoted gravitropic curvature in hypocotyls of both the WT and arc6 but had little or no effect on gravitropism in roots of both strains. These results suggest that F-actin is not required for hypocotyl gravitropism.

Division and dynamic morphology of plastids.

Plastid division is fundamental to the biology of plant cells. Division by binary fission entails the coordinated assembly and constriction of four concentric rings, two internal and two external to the organelle. The internal FtsZ ring and external dynamin-like ARC5/DRP5B ring are connected across the two envelopes by the membrane proteins ARC6, PARC6, PDV1, and PDV2. Assembly-stimulated GTPase activity drives constriction of the FtsZ and ARC5/DRP5B rings, which together with the plastid-dividing rings pull and squeeze the envelope membranes until the two daughter plastids are formed, with the final separation requiring additional proteins. The positioning of the division machinery is controlled by the chloroplast Min system, which confines FtsZ-ring formation to the plastid midpoint. The dynamic morphology of plastids, especially nongreen plastids, is also considered here, particularly in relation to the production of stromules and plastid-derived vesicles and their possible roles in cellular communication and plastid functionality.

The suffulta mutation in tomato reveals a novel method of plastid replication during fruit ripening.

Mutant alleles at the suffulta locus of tomato dramatically affect the pattern of plastid division throughout the plant, resulting in few, greatly enlarged chloroplasts in leaf and stem cells. suffulta plants are compromised in growth and have distinctly pale stems. The green developing tomato fruit are generally paler compared with the wild type, but ripe red fruit are much more similar in colour and pigment content. By using plastid-targeted green fluorescent protein, the underlying plastid phenotypes in the ripening suffulta fruit reveal that enlarged chlorophyll-containing chloroplasts degenerate and give rise to a wild type-like population of chromoplasts in ripe fruit by a process of plastid budding and fragmentation, resulting in a heterogeneous population of plastid-derived structures which eventually become chromoplasts. In stomatal guard cells, plastid-derived structures lacking chlorophyll, but containing GFP, are also observed, especially in guard cells which completely lack normal chloroplasts. How this novel 'replication' process in suffulta relates to conventional plastid division and stromule formation is discussed.

Mutations in the RETICULATA gene dramatically alter internal architecture but have little effect on overall organ shape in Arabidopsis leaves.

A number of mutants have been described in Arabidopsis, whose leaf vascular network can be clearly distinguished as a green reticulation on a paler lamina. One of these reticulate mutants was named reticulata (re) by Rédei in 1964 and has been used for years as a classical genetic marker for linkage analysis. Seven recessive alleles of the RE gene were studied, at least four of which seem to be null. Contrary to many other leaf mutants studied in Arabidopsis, very little pleiotropy was observed in the external morphology of the re mutants, whose only aberration obvious at first sight is the reticulation exhibited by cotyledons and leaves. The re alleles caused a marked reduction in the density of mesophyll cells in interveinal regions of the leaf, which does not result from perturbed plastid development in specific cells, but rather from a dramatic change in internal leaf architecture. Loss of function of the RE gene seems to specifically perturb mesophyll cell division in the early stages of leaf organogenesis. The leaves of re mutants were nearly normal in shape in spite of their extremely reduced mesophyll cell density, suggesting that the epidermis plays a major role in regulating leaf shape in Arabidopsis. The RE gene was positionally cloned and found to be expressed in all the major organs studied. RE encodes a protein of unknown function and is identical to the LCD1 gene, which was identified based on the increased sensitivity to ozone caused by its mutant allele lcd1-1. Double mutant analyses suggest that RE acts in a developmental pathway that involves CUE1 but does not include DOV1.

A comparison of the Thlaspi caerulescens and Thlaspi arvense shoot transcriptomes.

Whole-genome transcriptome profiling is revealing how biological systems are regulated at the transcriptional level. This study reports the development of a robust method to profile and compare the transcriptomes of two nonmodel plant species, Thlaspi caerulescens, a zinc (Zn) hyperaccumulator, and Thlaspi arvense, a nonhyperaccumulator, using Affymetrix Arabidopsis thaliana ATH1-121501 GeneChip arrays (Affymetrix, Santa Clara, CA, USA). Transcript abundance was quantified in the shoots of agar- and compost-grown plants of both species. Analyses were optimized using a genomic DNA (gDNA)-based probe-selection strategy based on the hybridization efficiency of Thlaspi gDNA with corresponding A. thaliana probes. In silico alignments of GeneChip probes with Thlaspi gene sequences, and quantitative real-time PCR, confirmed the validity of this approach. Approximately 5000 genes were differentially expressed in the shoots of T. caerulescens compared with T. arvense, including genes involved in Zn transport and compartmentalization. Future functional analyses of genes identified as differentially expressed in the shoots of these closely related species will improve our understanding of the molecular mechanisms of Zn hyperaccumulation.

Plastid and stromule morphogenesis in tomato.

By using green fluorescent protein targeted to the plastid organelle in tomato (Lycopersicon esculentum Mill.), the morphology of plastids and their associated stromules in epidermal cells and trichomes from stems and petioles and in the chromoplasts of pericarp cells in the tomato fruit has been revealed. A novel characteristic of tomato stromules is the presence of extensive bead-like structures along the stromules that are often observed as free vesicles, distinct from and apparently unconnected to the plastid body. Interconnections between the red pigmented chromoplast bodies are common in fruit pericarp cells suggesting that chromoplasts could form a complex network in this cell type. The potential implications for carotenoid biosynthesis in tomato fruit and for vesicles originating from beaded stromules as a secretory mechanism for plastids in glandular trichomes of tomato is discussed.

Stromule formation is dependent upon plastid size, plastid differentiation status and the density of plastids within the cell.

Stromules are motile extensions of the plastid envelope membrane, whose roles are not fully understood. They are present on all plastid types but are more common and extensive on non-green plastids that are sparsely distributed within the cell. During tomato fruit ripening, chloroplasts in the mesocarp tissue differentiate into chromoplasts and undergo major shifts in morphology. In order to understand what factors regulate stromule formation, we analysed stromule biogenesis in tobacco hypocotyls and in two distinct plastid populations in tomato mesocarp. We show that increases in stromule length and frequency are correlated with chromoplast differentiation, but only in one plastid population where the plastids are larger and less numerous. We used tobacco hypocotyls to confirm that stromule length increases as plastids become further apart, suggesting that stromules optimize the plastid-cytoplasm contact area. Furthermore, we demonstrate that ectopic chloroplast components decrease stromule formation on tomato fruit chromoplasts, whereas preventing chloroplast development leads to increased numbers of stromules. Inhibition of fruit ripening has a dramatic impact on plastid and stromule morphology, underlining that plastid differentiation status, and not cell type, is a significant factor in determining the extent of plastid stromules. By modifying the plastid surface area, we propose that stromules enhance the specific metabolic activities of plastids.

ARC6 is a J-domain plastid division protein and an evolutionary descendant of the cyanobacterial cell division protein Ftn2.

Replication of chloroplasts is essential for achieving and maintaining optimal plastid numbers in plant cells. The plastid division machinery contains components of both endosymbiotic and host cell origin, but little is known about the regulation and molecular mechanisms that govern the division process. The Arabidopsis mutant arc6 is defective in plastid division, and its leaf mesophyll cells contain only one or two grossly enlarged chloroplasts. We show here that arc6 chloroplasts also exhibit abnormal localization of the key plastid division proteins FtsZ1 and FtsZ2. Whereas in wild-type plants, the FtsZ proteins assemble into a ring at the plastid division site, chloroplasts in the arc6 mutant contain numerous short, disorganized FtsZ filament fragments. We identified the mutation in arc6 and show that the ARC6 gene encodes a chloroplast-targeted DnaJ-like protein localized to the plastid envelope membrane. An ARC6-green fluorescent protein fusion protein was localized to a ring at the center of the chloroplasts and rescued the chloroplast division defect in the arc6 mutant. The ARC6 gene product is related closely to Ftn2, a prokaryotic cell division protein unique to cyanobacteria. Based on the FtsZ filament morphology observed in the arc6 mutant and in plants that overexpress ARC6, we hypothesize that ARC6 functions in the assembly and/or stabilization of the plastid-dividing FtsZ ring. We also analyzed FtsZ localization patterns in transgenic plants in which plastid division was blocked by altered expression of the division site-determining factor AtMinD. Our results indicate that MinD and ARC6 act in opposite directions: ARC6 promotes and MinD inhibits FtsZ filament formation in the chloroplast.