Plant Development In Arabidopsis Research Articles

Colonization of Solanaceous crops by endophytic and rhizospheric plant growth-promoting bacteria from the native Solanaceae Solanum hindsianum Benth

Plant-associated bacteria are an important tool for sustainable agriculture since the have been shown to play a significant role in the health and productivity of plants through various mechanisms. However, commercial inoculants often fail to produce desired effects due to colonization issues and lack of adaptation to local conditions. In this study, we aimed to evaluate the potential of plant growth-promoting bacteria (PGPB) isolated from Solanum hindsianum, a native plant from the Sonoran Desert, to colonize and promote growth in Solanaceae crops under different irrigation conditions. First, endophytic bacteria were screened for various plant growth-promoting traits, and selected strains were identified as Bacillus, Brevibacillus, Pseudomonas, Acinetobacter, and Siccibacter. Then, endophytic and rhizospheric Pseudomonas isolates, were tested for their tolerance to salinity and osmotic stress and for their ability to induce auxin-responsive gene expression and promote plant development in Arabidopsis. All strains induced strong expression of an auxin reporter gene, while significantly improving root and plant morphology, and showed tolerance to osmotic and salinity stress. In colonization experiments, these strains successfully colonized the root crown and rhizosphere of chili, tomatillo, and tomato plants. However, the endophytic strain showed better colonization ability, especially under low irrigation conditions. Overall, this study demonstrates the potential of native PGPR strains to colonize and promote growth in Solanaceae crops under specific environmental conditions.

DEMETHYLATION REGULATOR 1 regulates DNA demethylation of the nuclear and mitochondrial genomes.

Active DNA demethylation effectively modulates gene expression during plant development and in response to stress. However, little is known about the upstream regulatory factors that regulate DNA demethylation. We determined that the demethylation regulator 1 (demr1) mutant exhibits a distinct DNA methylation profile at selected loci queried by methylation-sensitive polymerase chain reaction and globally based on whole-genome bisulfite sequencing. Notably, the transcript levels of the DNA demethylase gene REPRESSOR OF SILENCING 1 (ROS1) were lower in the demr1 mutant. We established that DEMR1 directly binds to the ROS1 promoter in vivo and in vitro, and the methylation level in the DNA methylation monitoring sequence of ROS1 promoter decreased by 60% in the demr1 mutant. About 40% of the hyper-differentially methylated regions (DMRs) in the demr1 mutant were shared with the ros1-4 mutant. Genetic analysis indicated that DEMR1 acts upstream of ROS1 to positively regulate abscisic acid (ABA) signaling during seed germination and seedling establishment stages. Surprisingly, the loss of DEMR1 function also caused a rise in methylation levels of the mitochondrial genome, impaired mitochondrial structure and an early flowering phenotype. Together, our results show that DEMR1 is a novel regulator of DNA demethylation of both the nuclear and mitochondrial genomes in response to ABA and plant development in Arabidopsis.

Role of HSP90 Family Molecular Chaperone in Regulating Chloroplast Protein Homeostasis and Plant Development in Arabidopsis

Chloroplasts carry out photosynthesis in plants and provide most food sources for living organisms. Either divided or differentiated from proplastids and etioplasts, the biogenesis of thylakoid membranes is the hallmark of chloroplast maturation and functionality, and this process requires a large set of photosynthetic proteins that must be synthesized on cytosolic ribosomes and then imported into the chloroplast stroma. Thylakoid lumen proteins travel further through the stroma and then cross the thylakoid membrane through one of the ancestral translocases. Like the cytosolic environment and the other major organelles, a cohort of molecular chaperones and proteases reside within the chloroplast to ensure proper protein homeostasis. We have identified and confirmed the interaction between the essential plastid HSP90 family heat shock protein HSP90C and a thylakoid lumen located photosystem II subunit PsbO1. To understand the biological significance of the interaction, we adopted transgenic approaches, specific HSP90 inhibitors, biochemical and in organello protein transport assays to study how the HSP90 family protein facilitates the representative lumen proteins transport across the thylakoid membrane. Specifically, we showed that the expression of the molecular chaperone is developmentally regulated, and its expression is highly correlated with some photosynthetic proteins while not with the others. Additionally, we demonstrated that HSP90C physically interacts with the thylakoid SEC translocon subunit, and that its chaperone activity is required for efficient PsbO1 transport. We also investigated the signature sequences of different HSP90 subfamily proteins and provided evidence that the chloroplast located HSP90C contains features specialized for some photosynthetic proteins interaction, and thus the chloroplast maturation and functionality.

On the Role of DGAT1 in Seed Glycerolipid Metabolic Network and Critical Stages of Plant Development in Arabidopsis.

Studies on the model plant Arabidopsis thaliana have uncovered the identities of most enzymatic components involved in seed storage lipid biosynthesis. However, much remains to be learned on how pathway interactions operate in the seed metabolic network. In this study, we dissected seed glycerolipid molecular compositional changes in the Arabidopsis mutant deficient in diacylglycerol acyltransferase 1 (DGAT1). Our results indicate that metabolic adjustments occurred in both phosphatidylcholine synthesis and deacylation in developing seeds. Ultrastructural changes of perturbed oil and protein bodies were also evident in cotyledon parenchyma cells. To unmask the physiological and developmental role associated with DGAT1-mediated neutral lipid biosynthesis, we attempted to combine dgat1 mutation with lpcat2 that harbors a defect in lysophosphatidylcholine acyltransferase 2 (LPCAT2). Disruption in both DGAT1 and LPCAT2 led to an apparent defect in pollen development that manifested as pollen sterility. Collectively, our results highlight a role of DGAT1 in both storage lipid synthesis and plant development.

Functional analysis for domains of maize PPR protein EMP5 in RNA editing and plant development in Arabidopsis

EMP5 and OTP72 are PPR proteins involved in mitochondrial same site RNA editing of rpl16 in maize and Arabidopsis, respectively. Besides rpl16, EMP5 also affects the editing efficiency of some editing sites in other mitochondrial transcripts. Loss of function of EMP5 causes arrested embryogenesis and endosperm development in maize, but no discernable defective phenotype is observed in Arabidopsis otp72 mutant plants. We here report that EMP5 can completely complement Arabidopsis otp72 for Ararpl16-440 editing, and E domain can’t be disrupted for normal function of EMP5. An interesting finding was that overexpression of a set of truncated EMP5s in Col-0 resulted in delayed bolting phenotypes and smaller plants. It seems some truncated EMP5s, such as EMP5-C and EMP5-D, have yet unknown functions, are not responsible for Ararpl16-440 editing any more. The phylogenetic analysis indicated that the functions of most EMP5 orthologs may be conserved, but EMP5 ortholog proteins from Nicotiana and Solanum can’t be found, and the C to U editing of Zmrpl16-458 site was not needed any more in the two species. Therefore, these two primary orthologs maybe already evolve new functions and change to different new proteins during evolutionary history. Overall, our studies revealed that E domain is crucial for mitochondrial RNA editing function of EMP5, and a set of truncated EMP5s still have functions for Arabidopsis normal growth and development, providing new insights into investigating the functions of EMP5 protein.

Two SUMO Proteases SUMO PROTEASE RELATED TO FERTILITY1 and 2 Are Required for Fertility in Arabidopsis.

In plants, the posttranslational modification small ubiquitin-like modifier (SUMO) is involved in regulating several important developmental and cellular processes, including flowering time control and responses to biotic and abiotic stresses. Here, we report two proteases, SUMO PROTEASE RELATED TO FERTILITY1 (SPF1) and SPF2, that regulate male and female gamete and embryo development and remove SUMO from proteins in vitro and in vivo. spf1 mutants exhibit abnormal floral structures and embryo development, while spf2 mutants exhibit largely a wild-type phenotype. However, spf1 spf2 double mutants exhibit severe abnormalities in microgametogenesis, megagametogenesis, and embryo development, suggesting that the two genes are functionally redundant. Mutation of SPF1 and SPF2 genes also results in misexpression of generative- and embryo-specific genes. In vitro, SPF1 and SPF2 process SUMO1 precursors into a mature form, and as expected in vivo, spf1 and spf2 mutants accumulate SUMO conjugates. Using a yeast two-hybrid screen, we identified EMBRYO SAC DEVELOPMENT ARREST9 (EDA9) as an SPF1-interacting protein. In vivo, we demonstrate that EDA9 is sumolyated and that, in spf1 mutants, EDA9-SUMO conjugates increase in abundance, demonstrating that EDA9 is a substrate of SPF1. Together, our results demonstrate that SPF1 and SPF2 are two SUMO proteases important for plant development in Arabidopsis (Arabidopsis thaliana).

Growing Slowly 1 locus encodes a PLS-type PPR protein required for RNA editing and plant development in Arabidopsis.

Most pentatricopeptide repeat (PPR) proteins are involved in organelle post-transcriptional processes, including RNA editing. The PPR proteins include the PLS subfamily, containing characteristic triplets of P, L, and S motifs; however, their editing mechanisms and roles in developmental processes are not fully understood. In this study, we isolated the Arabidopsis thaliana Growing slowly 1 (AtGRS1) gene and showed that it functions in RNA editing and plant development. Arabidopsis null mutants of grs1 exhibit slow growth and sterility. Further analysis showed that cell division activity was reduced dramatically in the roots of grs1 plants. We determined that GRS1 is a nuclear-encoded mitochondria-localized PPR protein, and is a member of the PLS subfamily. GRS1 is responsible for the RNA editing at four specific sites of four mitochondrial mRNAs: nad1-265, nad4L-55, nad6-103, and rps4-377 The first three of these mRNAs encode for the subunits of complex I of the electron transport chain in mitochondria. Thus, the activity of complex I is strongly reduced in grs1 Changes in RPS4 editing in grs1 plants affect mitochondrial ribosome biogenesis. Expression of the alternative respiratory pathway and the abscisic acid response gene ABI5 were up-regulated in grs1 mutant plants Genetic analysis revealed that ABI5 is involved in the short root phenotype of grs1 Taken together, our results indicate that AtGRS1 regulates plant development by controlling RNA editing in Arabidopsis.

Coevolution Pattern and Functional Conservation or Divergence of miR167s and their targets across Diverse Plant Species.

microRNAs (miRNAs), a class of endogenously produced small non-coding RNAs of 20–21 nt length, processed from precursor miRNAs, regulate many developmental processes by negatively regulating the target genes in both animals and plants. The coevolutionary pattern of a miRNA family and their targets underscores its functional conservation or diversification. The miR167 regulates various aspects of plant development in Arabidopsis by targeting ARF6 and ARF8. The evolutionary conservation or divergence of miR167s and their target genes are poorly understood till now. Here we show the evolutionary relationship among 153 MIR167 genes obtained from 33 diverse plant species. We found that out of the 153 of miR167 sequences retrieved from the “miRBase”, 27 have been annotated to be processed from the 3′ end, and have diverged distinctively from the other miR167s produced from 5′ end. Our analysis reveals that gma-miR167h/i and mdm-miR167a are processed from 3′ end and have evolved separately, diverged most resulting in novel targets other than their known ones, and thus led to functional diversification, especially in apple and soybean. We also show that mostly conserved miR167 sequences and their target AUXIN RESPONSE FACTORS (ARFs) have gone through parallel evolution leading to functional diversification among diverse plant species.

RhEXPA4, a rose expansin gene, modulates leaf growth and confers drought and salt tolerance to Arabidopsis

Drought and high salinity are major environmental conditions limiting plant growth and development. Expansin is a cell-wall-loosening protein known to disrupt hydrogen bonds between xyloglucan and cellulose microfibrils. The expression of expansin increases in plants under various abiotic stresses, and plays an important role in adaptation to these stresses. We aimed to investigate the role of the RhEXPA4, a rose expansin gene, in response to abiotic stresses through its overexpression analysis in Arabidopsis. In transgenic Arabidopsis harboring the Pro RhEXPA4 ::GUS construct, RhEXPA4 promoter activity was induced by abscisic acid (ABA), drought and salt, particularly in zones of active growth. Transgenic lines with higher RhEXPA4 level developed compact phenotypes with shorter stems, curly leaves and compact inflorescences, while the lines with relatively lower RhEXPA4 expression showed normal phenotypes, similar to the wild type (WT). The germination percentage of transgenic Arabidopsis seeds was higher than that of WT seeds under salt stress and ABA treatments. Transgenic plants showed enhanced tolerance to drought and salt stresses: they displayed higher survival rates after drought, and exhibited more lateral roots and higher content of leaf chlorophyll a under salt stress. Moreover, high-level RhEXPA4 overexpressors have multiple modifications in leaf blade epidermal structure, such as smaller, compact cells, fewer stomata and midvein vascular patterning in leaves, which provides them with more tolerance to abiotic stresses compared to mild overexpressors and the WT. Collectively, our results suggest that RhEXPA4, a cell-wall-loosening protein, confers tolerance to abiotic stresses through modifying cell expansion and plant development in Arabidopsis.

RhNAC2 and RhEXPA4 Are Involved in the Regulation of Dehydration Tolerance during the Expansion of Rose Petals

Dehydration inhibits petal expansion resulting in abnormal flower opening and results in quality loss during the marketing of cut flowers. We constructed a suppression subtractive hybridization library from rose (Rosa hybrida) flowers containing 3,513 unique expressed sequence tags and analyzed their expression profiles during cycles of dehydration. We found that 54 genes were up-regulated by the first dehydration, restored or even down-regulated by rehydration, and once again up-regulated by the second dehydration. Among them, we identified a putative NAC family transcription factor (RhNAC2). With transactivation activity of its carboxyl-terminal domain in yeast (Saccharomyces cerevisiae) cell and Arabidopsis (Arabidopsis thaliana) protoplast, RhNAC2 belongs to the NAC transcription factor clade related to plant development in Arabidopsis. A putative expansin gene named RhEXPA4 was also dramatically up-regulated by dehydration. Silencing RhNAC2 or RhEXPA4 in rose petals by virus-induced gene silencing significantly decreased the recovery of intact petals and petal discs during rehydration. Overexpression of RhNAC2 or RhEXPA4 in Arabidopsis conferred strong drought tolerance in the transgenic plants. RhEXPA4 expression was repressed in RhNAC2-silenced rose petals, and the amino-terminal binding domain of RhNAC2 bound to the RhEXPA4 promoter. Twenty cell wall-related genes, including seven expansin family members, were up-regulated in Arabidopsis plants overexpressing RhNAC2. These data indicate that RhNAC2 and RhEXPA4 are involved in the regulation of dehydration tolerance during the expansion of rose petals and that RhEXPA4 expression may be regulated by RhNAC2.

The translation elongation factor eEF-1Bβ1 is involved in cell wall biosynthesis and plant development in Arabidopsis thaliana.

The eukaryotic translation elongation factor eEF-1Bβ1 (EF1Bβ) is a guanine nucleotide exchange factor that plays an important role in translation elongation. In this study, we show that the EF1Bβ protein is localized in the plasma membrane and cytoplasm, and that the transcripts should be expressed in most tissue types in seedlings. Sectioning of the inflorescence stem revealed that EF1Bβ predominantly localizes to the xylem vessels and in the interfascicular cambium. EF1Bβ gene silencing in efβ caused a dwarf phenotype with 38% and 20% reduction in total lignin and crystalline cellulose, respectively. This loss-of-function mutant also had a lower S/G lignin monomer ratio relative to wild type plants, but no changes were detected in a gain-of-function mutant transformed with the EF1Bβ gene. Histochemical analysis showed a reduced vascular apparatus, including smaller xylem vessels in the inflorescence stem of the loss-of-function mutant. Over-expression of EF1Bβ in an eli1 mutant background restored a WT phenotype and abolished ectopic lignin deposition as well as cell expansion defects in the mutant. Taken together, these data strongly suggest a role for EF1Bβ in plant development and cell wall formation in Arabidopsis.

L,L-Diaminopimelate Aminotransferase from Chlamydomonas reinhardtii: A Target for Algaecide Development

In some bacterial species and photosynthetic cohorts, including algae, the enzyme l,l-diaminopimelate aminotransferase (DapL) (E.C. 2.6.1.83) is involved in the anabolism of the essential amino acid L-lysine. DapL catalyzes the conversion of tetrahydrodipicolinate (THDPA) to l,l-diaminopimelate (l,l-DAP), in one step bypassing the DapD, DapC and DapE enzymatic reactions present in the acyl DAP pathways. Here we present an in vivo and in vitro characterization of the DapL ortholog from the alga Chlamydomonas reinhardtii (Cr-DapL). The in vivo analysis illustrated that the enzyme is able to functionally complement the E. coli dap auxotrophs and was essential for plant development in Arabidopsis. In vitro, the enzyme was able to inter-convert THDPA and l,l-DAP, showing strong substrate specificity. Cr-DapL was dimeric in both solution and when crystallized. The structure of Cr-DapL was solved in its apo form, showing an overall architecture of a α/β protein with each monomer in the dimer adopting a pyridoxal phosphate-dependent transferase-like fold in a V-shaped conformation. The active site comprises residues from both monomers in the dimer and shows some rearrangement when compared to the apo-DapL structure from Arabidopsis. Since animals do not possess the enzymatic machinery necessary for the de novo synthesis of the amino acid l-lysine, enzymes involved in this pathway are attractive targets for the development of antibiotics, herbicides and algaecides.

Redox regulation of auxin signaling and plant development in Arabidopsis

Thioredoxin (NTR/TRX) and glutathione (GSH/GRX) are the two major systems which play a key role in the maintenance of cellular redox homeostasis. They are essential for plant development, cell division or the response to environmental stresses. In a recent article,1 we studied the interplay between the NADP-linked thioredoxin and glutathione systems in auxin signaling genetically, by associating TRX reductase (ntra ntrb) and glutathione biosynthesis (cad2) mutations. We show that these two thiol reduction pathways interfere with developmental processes. This occurs through modulation of auxin activity as shown by genetic analyses of loss of function mutations in a triple ntra ntrb cad2 mutant. The triple mutant develops almost normally at the rosette stage but fails to generate lateral organs from the inflorescence meristem, producing almost naked stems that are reminiscent of mutants affected in PAT (polar auxin transport) or biosynthesis. The triple mutant exhibits other defects in processes regulated by auxin, including a loss of apical dominance, vasculature defects and reduced secondary root production. Furthermore, it has lower auxin (IAA) levels and decreased capacity for PAT, suggesting that the NTR and glutathione pathways influence inflorescence meristem development through regulation of auxin transport and metabolism.

A nuclear-encoded mitochondrial gene AtCIB22 is essential for plant development in Arabidopsis

Complex I (the NADH:ubiquinone oxidoreductase) of the mitochondrial respiratory chain is a complicated, multi-subunit, membrane-bound assembly and contains more than 40 different proteins in higher plants. In this paper, we characterize the Arabidopsis homologue (designated as AtCIB22) of the B22 subunit of eukaryotic mitochondrial Complex I. AtCIB22 is a single-copy gene and is highly conserved throughout eukaryotes. AtCIB22 protein is located in mitochondria and the AtCIB22 gene is widely expressed in different tissues. Mutant Arabidopsis plants with a disrupted AtCIB22 gene display pleiotropic phenotypes including shorter roots, smaller plants and delayed flowering. Stress analysis indicates that the AtCIB22 mutants' seed germination and early seedling growth are severely inhibited by sucrose deprivation stress but more tolerant to ethanol stress. Molecular analysis reveals that in moderate knockdown AtCIB22 mutants, genes including cell redox proteins and stress related proteins are significantly up-regulated, and that in severe knockdown AtCIB22 mutants, the alternative respiratory pathways including NDA1, NDB2, AOX1a and AtPUMP1 are remarkably elevated. These data demonstrate that AtCIB22 is essential for plant development and mitochondrial electron transport chains in Arabidopsis. Our findings also enhance our understanding about the physiological role of Complex I in plants.

Pattern Recognition Receptors Require N-Glycosylation to Mediate Plant Immunity

N-Glycans attached to the ectodomains of plasma membrane pattern recognition receptors constitute likely initial contact sites between plant cells and invading pathogens. To assess the role of N-glycans in receptor-mediated immune responses, we investigated the functionality of Arabidopsis receptor kinases EFR and FLS2, sensing bacterial translation elongation factor Tu (elf18) and flagellin (flg22), respectively, in N-glycosylation mutants. As revealed by binding and responses to elf18 or flg22, both receptors tolerated immature N-glycans induced by mutations in various Golgi modification steps. EFR was specifically impaired by loss-of-function mutations in STT3A, a subunit of the endoplasmic reticulum resident oligosaccharyltransferase complex. FLS2 tolerated mild underglycosylation occurring in stt3a but was sensitive to severe underglycosylation induced by tunicamycin treatment. EFR accumulation was significantly reduced when synthesized without N-glycans but to lesser extent when underglycosylated in stt3a or mutated in single amino acid positions. Interestingly, EFR(N143Q) lacking a single conserved N-glycosylation site from the EFR ectodomain accumulated to reduced levels and lost the ability to bind its ligand and to mediate elf18-elicited oxidative burst. However, EFR-YFP protein localization and peptide:N-glycosidase F digestion assays support that both EFR produced in stt3a and EFR(N143Q) in wild type cells correctly targeted to the plasma membrane via the Golgi apparatus. These results indicate that a single N-glycan plays a critical role for receptor abundance and ligand recognition during plant-pathogen interactions at the cell surface.

SIZ1 Controls Cell Growth and Plant Development in Arabidopsis Through Salicylic Acid

The post-translational conjugation of small ubiquitin-related modifiers (SUMOs) to other proteins is involved in regulation of many processes in eukaryotic development; although its role in plant development is beginning to be dissected. Previously, we demonstrated that the siz1 mutant, which is impaired in SUMO E3 ligase, showed a dwarf-like shoot phenotype with accumulation of salicylic acid (SA), and the expression of nahG, a bacterial salicylate hydroxylase that catabolizes SA, in siz1 reduced the SA level and suppressed dwarfism. Herein, we provide evidence that the SIZ1 gene controls cell division and elongation through regulation of the SA level. Mature siz1-2 and siz1-3 plants exhibited a dwarf-like shoot phenotype that is attributable to decreased leaf cell volume and number relative to the wild type. Cell division and expansion defects caused by siz1 were also suppressed by the expression of nahG. Expression of XTH8 and XTH31, encoding xyloglucan endotransglycosylase/hydrolase, which are thought to facilitate leaf cell expansion, was down-regulated in siz1 leaves. However, reduced XTH8 and XTH31 expression in siz1 plants was restored in nahG siz1-2 plants. These results indicate that SIZ1 regulates cell growth and plant development with regulation of SA accumulation. Also, XTH8 and XTH31 genes may be responsible for reduced leaf cell expansion.

Overexpression of a gibberellin inactivation gene alters seed development, KNOX gene expression, and plant development in Arabidopsis.

We have examined the role of gibberellins (GAs) in plant development by expression of the pea GA 2-oxidase2 (PsGA2ox2) cDNA, which encodes a GA inactivating enzyme, under the control of the MEDEA (MEA) promoter. Expression of MEA:PsGA2ox2 in Arabidopsis caused seed abortion, demonstrating that active GAs in the endosperm are essential for normal seed development. MEA:PsGA2ox2 plants had reduced ovule number per ovary and exhibited defects in phyllotaxy and leaf morphology which were partly suppressed by GA treatment. The leaf architecture and phyllotaxy defects of MEA:PsGA2ox2 plants were also restored by sly1-d which reduces DELLA protein stability to increase GA response. MEA:PsGA2ox2 seedlings had increased expression of the KNOTTED1-like homeobox (KNOX) genes, BP, KNAT2 and KNAT6, which are known to control plant architecture. The expression of KNOX genes is also altered in wild-type plants treated with GA. These results support the conclusion that GAs can suppress the effects of elevated KNOX gene expression, and raise the possibility that localized changes in GA levels caused by PsGA2ox2 alter the expression of KNOX genes to modify plant architecture.

Genetic Evidence for the Role of Isopentenyl Diphosphate Isomerases in the Mevalonate Pathway and Plant Development in Arabidopsis

Isopentenyl/dimethylallyl diphosphate isomerase (IPI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are the universal C(5) units of isoprenoids. In plants, IPP and DMAPP are synthesized via the cytosolic mevalonate (MVA) and plastidic methylerythritol phosphate (MEP) pathways, respectively. However, the role of IPI in each pathway and in plant development is unknown due to a lack of genetic studies using IPI-defective mutants. Here, we show that the atipi1atipi2 double mutant, which is defective in two Arabidopsis IPI isozymes, exhibits dwarfism and male sterility under long-day conditions and decreased pigmentation under continuous light, whereas the atipi1 and atipi2 single mutants are phenotypically normal. We also show that the sterol and ubiquinone levels in the double mutant are <50% of those in wild-type plants, and that the male-sterile phenotype is chemically complemented by squalene, a sterol precursor. In vivo isotope labeling experiments using the atipi1atipi2 double mutant revealed a decrease in the incorporation of MVA (in its lactone form) into sterols, with no decrease in the incorporation of MEP pathway intermediates into tocopherol. These results demonstrate a critical role for IPI in isoprenoid biosynthesis via the MVA pathway, and they imply that IPI is essential for the maintenance of appropriate levels of IPP and DMAPP in different subcellular compartments in plants.

AKRP and EMB506 are two ankyrin repeat proteins essential for plastid differentiation and plant development in Arabidopsis

EMB506 is a chloroplast protein essential for embryo development, the function of which is unknown. A two-hybrid interaction screen was performed to provide insight into the role of EMB506. A single interacting partner, AKRP, was identified among a cDNA library from immature siliques. The AKR gene (Zhang et al., 1992, Plant Cell 4, 1575-1588) encodes a protein containing five ankyrin repeats, very similar to EMB506. Protein truncation series demonstrated that both proteins interact through their ankyrin domains. Using reverse genetics, we showed that loss of akr function resulted in an embryo-defective (emb) phenotype indistinguishable from the emb506 phenotype. Transient expression of the signal peptide of AKRP fused to green fluorescent protein demonstrated the chloroplast localization of AKRP. The ABI3 promoter was used to express AKR in a seed-specific manner in order to analyse the post-embryonic effect of AKR loss of function in akr/akr seedlings. Homozygous fertile and viable akr/akr plants were obtained. These plants exhibited mild to severe defects in chloroplast and leaf cellular organization. We conclude that EMB506 and AKRP are involved in crucial and tightly controlled events in plastid differentiation linked to cell differentiation, morphogenesis and organogenesis during the plant life cycle.

Arabidopsis myosin XI mutant is defective in organelle movement and polar auxin transport.

Myosins are eukaryotic molecular motors moving along actin filaments. Only a small set of myosin classes is present in plants, in which myosins have been found to play a role in cytoplasmic streaming and chloroplast movement. Whereas most studies have been done on green algae, more recent data suggest a role of higher plant myosin at the postcytokinetic cell wall. Here we characterize a loss-of-function mutation for a myosin of plant-specific class XI and demonstrate myosin functions during plant development in Arabidopsis. T-DNA insertion in MYA2 caused pleiotropic effects, including flower sterility and dwarf growth. Elongation of epidermal cells, such as in hypocotyls and anther filaments, was reduced by up to 50% of normal length. This effect on anther filaments is responsible for flower sterility. In the meristems of root tips, it was evident that cell division was delayed and that cell plates were mislocated. Like zwichel, a kinesin-related mutation causing two-branched trichomes, the mya2 knockout causes branching defects, but here the trichomes remained unbranched. Growth was also impaired in pollen tubes and root hairs, cells that are highly dependent on vesicle transport. A failure in vesicle flow could be directly confirmed, because cytoplasmic streaming of vesicles and, more so, of large endoplasmic reticulum-based organelles was slowed. The defect in vesicle trafficking was accompanied by failures in basipetal auxin transport, measured in stem segments of inflorescences. This result strongly suggests a causal link between auxin-dependent processes and the distribution of vesicles and membrane-bound molecules by plant myosin.