Peta C. Bonham-Smith

Arabidopsis Ribosomal Proteins RPL23aA and RPL23aB Are Differentially Targeted to the Nucleolus and Are Disparately Required for Normal Development

Protein synthesis is catalyzed by the ribosome, a two-subunit enzyme comprised of four ribosomal RNAs and, in Arabidopsis (Arabidopsis thaliana), 81 ribosomal proteins (r-proteins). Plant r-protein genes exist as families of multiple expressed members, yet only one r-protein from each family is incorporated into any given ribosome, suggesting that many r-protein genes may be functionally redundant or development/tissue/stress specific. Here, we characterized the localization and gene-silencing phenotypes of a large subunit r-protein family, RPL23a, containing two expressed genes (RPL23aA and RPL23aB). Live cell imaging of RPL23aA and RPL23aB in tobacco with a C-terminal fluorescent-protein tag demonstrated that both isoforms accumulated in the nucleolus; however, only RPL23aA was targeted to the nucleolus with an N-terminal fluorescent protein tag, suggesting divergence in targeting efficiency of localization signals. Independent knockdowns of endogenous RPL23aA and RPL23aB transcript levels using RNA interference determined that an RPL23aB knockdown did not alter plant growth or development. Conversely, a knockdown of RPL23aA produced a pleiotropic phenotype characterized by growth retardation, irregular leaf and root morphology, abnormal phyllotaxy and vasculature, and loss of apical dominance. Comparison to other mutants suggests that the phenotype results from reduced ribosome biogenesis, and we postulate a link between biogenesis, microRNA-target degradation, and maintenance of auxin homeostasis. An additional RNA interference construct that coordinately silenced both RPL23aA and RPL23aB demonstrated that this family is essential for viability.

Overexpression of formate dehydrogenase inArabidopsis thaliana resulted in plants tolerant to high concentrations of formate

Summary The potential role played by formate dehydrogenase (FDH) in formate metabolism has been examined by the overexpression of FDH in Arabidopsis thaliana . Three independent transgenic lines were selected and shown to produce elevated amounts of FDH protein with a corresponding elevated FDH activity (2.5-5 fold) over wild-type (WT) plants. Under normal growth conditions, no altered phenotype was observed in these transgenic plants; in growth media supplied with formate, however, significant differences in shoot and root growth, compared to that of WT plants, were observed. WT plants were severely injured if grown in the presence of 16 mmol/L formate, while the transgenic plants were able to grow well. Formate delayed germination of both WT and transgenic seeds at concentrations above 4 mmol/L, but both types of seeds were eventually able to complete more than 95 % germination even at 32 mmol/L formate. Formate markedly inhibited primary root elongation, and its inhibitory action on WT was much stronger than on transgenic plants. Different formate salts affected root elongation similarly, indicating that the formate ion was the major factor inhibiting root growth. Sodium acetate (NaAc), an analogue of formate, also inhibited root elongation, but its action on WT and transgenic plants was the same, indicating that tolerance of transgenic plants to formate toxicity was specific. Transgenic plants showed no significant tolerance to the toxicity of two other one-carbon metabolites, methanol and formaldehyde. A role for FDH in detoxifying formate is proposed.

A rapidAgrobacterium-mediatedArabidopsis thaliana transient assay system

Stable transformation ofArabidopsis thaliana is a lengthy process that involves up to 3 mo of plant growth and seed selection. We have developed a rapid, 3-wk transient assay system to test the functionality ofcis-regulatory regions controlling expression of a reporter gene in plants before undertaking stable transformation. Two-week-oldArabidopsis seedlings were vacuum-infiltrated withAgrobacterium tumefaciens cultures carrying various upstream regulatory regions controllinguidA (β-glucuronidase [GUS]) expression. Seedlings were fixed and stained for GUS activity 3–5 d following infiltration. Regulatory regions tested in this system include the cauliflower mosaic virus (CaMV)35S promoter, the upstream regulatory region of ribosomal protein geneL23A-1, and a temperature-inducible regulatory region (HSP101B) also fromArabidopsis. The percentage of seedlings positive for GUS activity varied depending on the construct used, with the CaMV35S promoter producing the highest number of GUS-positive seedlings. Temperature induction treatments elicited increased GUS expression in seedlings transformed with theHSP101B regulatory region. Regardless of construct, GUS expression levels were higher in seedlings collected 5 d followingAgrobacterium infiltration than those collected 3–4 d postinfiltration.

The role of peroxiredoxin antioxidant and calmodulin in ABA-primed seeds ofBrassica napusexposed to abiotic stresses during germination

Summary Both peroxiredoxin and calmodulin have been demonstrated previously to be involved in the early stages of seed germination. We believe that the peroxiredoxin antioxidant proteins may play a role in protecting seed from oxidative damages and maintaining seed dormancy, and that calmodulins in regulating metabolic enzymes in the early stages of seed germination. To test this hypothesis two cDNA clones BnPER1 and BnCAM1, encoding peroxiredoxin antioxidant and calmodulin respectively, were isolated from seed of Brassica napus cv. Quest. In dry control seeds, a high level of BnPER1 transcript was present while that of BnCAM was low. Priming with 100 μmol/L ABA, where seeds underwent complete imbibition prior to radicle protrusion then dried back to their original moisture content, reduced the transcript level of BnPER1 and induced the expression of BnCAM1. Coincidentally, ABA-primed seeds exhibited earlier (2-7 days) germination and higher final percent radicle protrusion than non-primed control seeds, under salt (100 mmol/L NaCl) or water stress (20 % PEG 8000) at a low temperature (8 °C). For both ABA-primed and non-primed seeds, the decreased expression of BnPER1 and increased expression of BnCAM1 transcripts was highly correlated to the degree of seed germination. These results suggest that peroxiredoxins are down regulated during seed germination in B. napus, probably due to a reduction in oxidative stresses. On the other hand, elevated calmodulin levels was indicative of increased enzymatic activities required for germination. ABA-priming affected the expression of these genes.

A role for the anaphase promoting complex in hormone regulation.

To increase our knowledge of anaphase promoting complex (APC/C) function during plant development, we characterized an Arabidopsis thaliana T-DNA-insertion line where the T-DNA fell within the 5′ regulatory region of the APC10 gene. The insert disrupted endogenous expression, resulting in overexpression of APC10 mRNA from the T-DNA- internal CaMV 35S promoter, and increased APC10 protein. Overexpression of APC10 produced phenotypes resembling those of known auxin and ethylene mutants, and increased expression of two tested auxin-regulated genes, small auxin up RNA (SAUR) 15 and SAUR24. Taken together, our data suggests that elevated APC10 likely mimics auxin and ethylene sensitive phenotypes, expanding our understanding of proteolytic processes in hormone regulation of plant development.

Transcript profiling demonstrates absence of dosage compensation in Arabidopsis following loss of a single RPL23a paralog

Translation of nucleus-encoded messages in plants is conducted by the cytoplasmic ribosome, an enzyme that is comprised of two RNA/protein subunits. In Arabidopsis thaliana, the 81 different ribosomal proteins (r-proteins) of the cytosolic ribosome belong to gene families with multiple expressed members. Given that ribosomes generally contain only one copy of each r-protein, regulatory mechanisms must exist to ensure their stoichiometric accumulation. These mechanisms must be dynamic, allowing for adjustments to ribosome biogenesis to fulfill biological requirements for protein synthesis during development, and following stress induction of global changes in gene expression. In this study, we investigated whether r-protein paralogs are feedback regulated at the transcript level by obtaining a T-DNA knockout of one member, RPL23aB, from the two-member RPL23a family. Expression of the lone functional paralog in this line, RPL23aA, was compared to the expression of both paralogs in wildtype plants under non-stressed, low temperature-, and high light stresses. RPL23aA expression was not upregulated in RPL23aB knockouts to compensate for paralog-loss, and consequently knockouts showed reduced total abundance of RPL23a transcripts. However, no phenotype developed in RPL23aB knockouts, suggesting that this paralog is dispensable under experimental conditions examined, or that compensation by RPL23aA may occur post-transcriptionally. Patterns of RPL23aA and RPL23aB transcript accumulation in wildtype plants suggest that paralogs respond coordinately to developmental and stress stimuli.

Hairy Canola (Brasssica napus) re-visited: Down-regulating TTG1 in an AtGL3-enhanced hairy leaf background improves growth, leaf trichome coverage, and metabolite gene expression diversity

BackgroundThrough evolution, some plants have developed natural resistance to insects by having hairs (trichomes) on leaves and other tissues. The hairy trait has been neglected in Brassica breeding programs, which mainly focus on disease resistance, yield, and overall crop productivity. In Arabidopsis, a network of three classes of proteins consisting of TTG1 (a WD40 repeat protein), GL3 (a bHLH factor) and GL1 (a MYB transcription factor), activates trichome initiation and patterning. Introduction of a trichome regulatory gene AtGL3 from Arabidopsis into semi-glabrous Brassica napus resulted in hairy canola plants which showed tolerance to flea beetles and diamondback moths; however plant growth was negatively affected. In addition, the role of BnTTG1 transcription in the new germplasm was not understood.ResultsHere, we show that two ultra-hairy lines (K-5-8 and K-6-3) with BnTTG1 knock-down in the hairy AtGL3+ B. napus background showed stable enhancement of trichome coverage, density, and length and restored wild type growth similar to growth of the semi-glabrous Westar plant. In contrast, over-expression of BnTTG1 in the hairy AtGL3+ B. napus background gave consistently glabrous plants of very low fertility and poor stability, with only one glabrous plant (O-3-7) surviving to the T3 generation. Q-PCR trichome gene expression data in leaf samples combining several leaf stages for these lines suggested that BnGL2 controlled B. napus trichome length and out-growth and that strong BnTTG1 transcription together with strong GL3 expression inhibited this process. Weak expression of BnTRY in both glabrous and trichome-bearing leaves of B. napus in the latter Q-PCR experiment suggested that TRY may have functions other than as an inhibitor of trichome initiation in the Brassicas. A role for BnTTG1 in the lateral inhibition of trichome formation in neighbouring cells was also proposed for B. napus. RNA sequencing of first leaves identified a much larger array of genes with altered expression patterns in the K-5-8 line compared to the hairy AtGL3+B. napus background (relative to the Westar control plant). These genes particularly included transcription factors, protein degradation and modification genes, but also included pathways that coded for anthocyanins, flavonols, terpenes, glucosinolates, alkaloids, shikimates, cell wall biosynthesis, and hormones. A 2nd Q-PCR experiment was conducted on redox, cell wall carbohydrate, lignin, and trichome genes using young first leaves, including T4 O-3-7-5 plants that had partially reverted to yield two linked growth and trichome phenotypes. Most of the trichome genes tested showed to be consistant with leaf trichome phenotypes and with RNA sequencing data in three of the lines. Two redox genes showed highest overall expression in K-5-8 leaves and lowest in O-3-7-5 leaves, while one redox gene and three cell wall genes were consistently higher in the two less robust lines compared with the two robust lines.ConclusionThe data support the strong impact of BnTTG1 knockdown (in the presence of strong AtGL3 expression) at restoring growth, enhancing trichome coverage and length, and enhancing expression and diversity of growth, metabolic, and anti-oxidant genes important for stress tolerance and plant health in B. napus. Our data also suggests that the combination of strong (up-regulated) BnTTG1 expression in concert with strong AtGL3 expression is unstable and lethal to the plant.

Differential transcript accumulation and subcellular localization of Arabidopsis ribosomal proteins

Arabidopsis cytoplasmic ribosomes are an assembly of four rRNAs and 81 ribosomal proteins (RPs). With only a single molecule of each RP incorporated into any given ribosome, an adequate level of each RP in the nucleolus is a prerequisite for efficient ribosome biogenesis. Using Genevestigator (microarray data analysis tool), we have studied transcript levels of 192 of the 254 Arabidopsis RP genes, as well as the sub-cellular localization of each of five two-member RP families, to identify the extent to which these two processes contribute to the nucleolar pool of RPs available for ribosome biogenesis. While transcript levels from different RP genes show up to a 300-fold difference across the RP population, this difference is drastically reduced to ∼8-fold when considering RP gene families. Under various stimuli, while the transcript level for most RP genes remains unchanged some show a significantly increased or decreased level. Subcellular localization studies in tobacco not only showed differential targeting of RPs to the cytoplasm, nucleus and nucleolus, but also differential nucleolar import rates. This degree of variation in gene regulation and subcellular localization of RPs hints at the possibility of extra-ribosomal functions for some RP isoforms.

Evolutionary divergence of ribosomal protein paralogs in Arabidopsis

The majority of proteins in plants are synthesized by the cytoplasmic ribosome. In Arabidopsis, this massive (~3.2 MDa), two-subunit (40S small subunit [SSU] and 60S large subunit [LSU]) enzyme is comprised of 81 ribosomal proteins (RPs; 33 SSU, 48 LSU) that assemble around a catalytic core of 4 ribosomal RNAs (rRNA; 1 SSU, 3 LSU).1,2 None of the Arabidopsis RPs are encoded by single genes, but rather derive from families of 2–7 members that encode nearly-identical isoforms, are independently regulated, dispersed throughout the genome, and largely all transcriptionally active.2 To gain an understanding of why so many plant RP paralogs exist, we recently analyzed function and localization of the two-member Arabidopsis RPL23a family (RPL23aA and RPL23aB).3 RPL23aB was found to be unnecessary for normal development, while a relatively small decrease in transcript levels of RPL23aA resulted in development of a severe phenotype. Isoforms exhibited differences in nucleolar-targeting, which may result from disparity in putative nuclear/nucleolar localization signals (NLS/NoLSs). We postulate a role for ribosome biogenesis in the primary regulation of auxin homeostasis and plant development, and discuss properties of high efficiency NoLSs. Addendum to: Degenhardt RF, Bonham-Smith PC. Arabidopsis ribosomal proteins RPL23aA and -B are differentially targeted to the nucleolus and are disparately required for normal development. Plant Physiol 2008; In press.

Brassica Villosa a Potential Tool to Improve the Insect or DiseaseResistance of Brassica Crop Species

Outward growths on plant epidermal cells are known as trichomes or hair cells and they act as a barrier for plant predators. Brassica napus (canola) is the major oil seed crop in the world but lacks trichomes on them but a wild relative of canola, Brassica villosa has dense trichomes on them. Transcriptomic study of B. villosa indicated differential expression of trichome, photosynthesis light reactions, major carbohydrates, cellulose, lipid and amino acid metabolism, sulfur assimilation, metal handling/binding, hormones, biotic stress, redox, RNA regulation/ transcription, post-translational modification, signalling, cell vesicle transport, development, secondary metabolism and miscellaneous genes. And bio-chemical results from B. villosa trichomes confirmed accumulation of metals and a unique alkaloid-like compound in them. These results from B. villosa opened the doors for using this species as a potential tool for the improvement of insect or disease resistance in Brassica crop species.