The Plant Journal

Cross, Joanna M.; Clancy, Maureen; Shaw, Janine R.; Boehlein, Susan K.; Greene, Thomas W.; Schmidt, Robert R.; Okita, Thomas W.; Curtis Hannah, L.

doi: 10.1111/j.1365-313X.2004.02315.xpmid: 15686515

The heterotetrameric, allosterically regulated enzyme, adenosine‐5′‐diphosphoglucose pyrophosphorylase (AGPase) catalyzes the rate‐limiting step in starch synthesis. Despite vast differences in allosteric properties and a long evolutionary separation, heterotetramers of potato small subunit and maize large subunit have activity comparable to either parent in an Escherichia coli expression system. In contrast, co‐expression of maize small subunit with the potato large subunit produces little activity as judged by in vivo activity stain. To pinpoint the region responsible for differential activity, we expressed chimeric maize/potato small subunits in E. coli. This identified a 55‐amino acid motif of the potato small subunit that is critical for glycogen production when expressed with the potato large subunit. Potato and maize small subunit sequences differ at five amino acids in this motif. Replacement experiments revealed that at least four amino acids of maize origin were required to reduce staining. An AGPase composed of a chimeric potato small subunit containing the 55‐amino acid maize motif with the potato large subunit exhibited substantially less affinity for the substrates, glucose‐1‐phosphate and ATP and an increased Ka for the activator, 3‐phosphoglyceric acid. Placement of the potato motif into the maize small subunit restored glycogen synthesis with the potato large subunit. Hence, a small polymorphic motif within the small subunit influences both catalytic and allosteric properties by modulating subunit interactions.

Nakagawa, Hitoshi; Jiang, Chang‐Jie; Sakakibara, Hitoshi; Kojima, Mikiko; Honda, Ichiro; Ajisaka, Hidetoshi; Nishijima, Takaaki; Koshioka, Masaji; Homma, Tamaki; Mander, Lewis N.; Takatsuji, Hiroshi

doi: 10.1111/j.1365-313X.2004.02316.x

Riddle, Nicole C.; Richards, Eric J.

doi: 10.1111/j.1365-313X.2004.02317.xpmid: 15686517

We investigated the fidelity of epigenetic inheritance in crosses between three accessions of the flowering plant Arabidopsis thaliana (Canary Islands, Cape Verde Islands, and Columbia). Specifically, we examined the cytosine methylation content of the ribosomal RNA genes at the two nucleolus organizer regions (NOR2 and NOR4) in F1 and F2 hybrid individuals derived from reciprocal crosses between the high NOR methylation strain, Columbia, and the two other accessions, both of which have less NOR methylation. In crosses between the Columbia and Cape Verde Islands strains, the cytosine methylation content segregated as an additive Mendelian trait: the high NOR methylation state was tightly associated with the inheritance of the two Columbia‐derived NOR loci. First‐generation hybrid individuals between the Canary Islands and Columbia strains also showed a cytosine methylation content at the NORs intermediate between the parental values, consistent with the epigenetic inheritance of parental methylation patterns. Interestingly, mapping data from F2 individuals derived from a Canary Islands × Columbia cross revealed that NOR2 accounted for nearly all of the NOR methylation variation segregating in the population. NOR4 retains a significant effect on total NOR methylation content only through a complex epistatic interaction with NOR2. Our results indicate that the inheritance of differential cytosine methylation states at NOR loci can be modified by their genetic context, opening up the possibility of genetic dissection of epigenetic inheritance.

Bleuyard, Jean‐Yves; Gallego, Maria E.; Savigny, Florence; White, Charles I.

doi: 10.1111/j.1365-313X.2004.02318.xpmid: 15686518

In addition to the recombinase Rad51, vertebrates have five paralogs of Rad51, all members of the Rad51‐dependent recombination pathway. These paralogs form two complexes (Rad51C/Xrcc3 and Rad51B/C/D/Xrcc2), which play roles in somatic recombination, DNA repair and chromosome stability. However, little is known of their possible involvement in meiosis, due to the inviability of the corresponding knockout mice. We have recently reported that the Arabidopsis homolog of one of these Rad51 paralogs (AtXrcc3) is involved in DNA repair and meiotic recombination and present here Arabidopsis lines carrying mutations in three other Rad51 paralogs (AtRad51B, AtRad51C and AtXrcc2). Disruption of any one of these paralogs confers hypersensitivity to the DNA cross‐linking agent Mitomycin C, but not to γ‐irradiation. Moreover, the atrad51c‐1 mutant is the only one of these to show meiotic defects similar to those of the atxrcc3 mutant, and thus only the Rad51C/Xrcc3 complex is required to achieve meiosis. These results support conservation of functions of the Rad51 paralogs between vertebrates and plants and differing requirements for the Rad51 paralogs in meiosis and DNA repair.

Menges, Margit; De Jager, Sarah M.; Gruissem, Wilhelm; Murray, James A.H.

doi: 10.1111/j.1365-313X.2004.02319.xpmid: 15686519

Arabidopsis has over 80 genes encoding conserved and plant‐specific core cell cycle regulators, but in most cases neither their timing of expression in the cell cycle is known nor whether they represent redundant and/or tissue‐specific functions. Here we identify novel cell cycle regulators, including new cyclin‐dependent kinases related to the mammalian galactosyltransferase‐associated protein kinase p58, and new classes of cyclin‐like and CDK‐like proteins showing strong tissue specificity of expression. We analyse expression of all cell cycle regulators in synchronized Arabidopsis cell cultures using multiple approaches including Affymetrix microarrays, massively parallel signature sequencing and real‐time reverse transcriptase polymerase chain reaction, and in plant material using the results of over 320 microarray experiments. These global analyses reveal that most core cell cycle regulators are expressed across almost all tissues and more than 85% are expressed at detectable levels in the cell suspension culture, allowing us to present a unified model of transcriptional regulation of the plant cell cycle. Characteristic patterns of D‐cyclin expression in early and late G1 phase, either limited to the re‐entry cycle or continuously oscillating, suggest that several CYCD genes with strong oscillatory regulation in late G1 may play the role of cyclin E in plants. Alone amongst the six groups of A and B type cyclins, members of CYCA3 peak in S‐phase suggest it is a major component of S‐phase kinases, whereas others show a peak in G2/M. 82 genes share this G2/M regulatory pattern, about half being new candidate mitotic genes of previously unknown function.

Tillemans, Vinciane; Dispa, Laurence; Remacle, Claire; Collinge, Mélanie; Motte, Patrick

doi: 10.1111/j.1365-313X.2004.02321.xpmid: 15686520

Serine/arginine‐rich (SR) proteins constitute an important class of splicing regulators in higher eukaryotes that share a modular structure consisting of one or two N‐terminal RNA recognition motif (RRM) domains and a C‐terminal RS‐rich domain. Herein, we have investigated the in vivo functional distribution of Arabidopsis SR factors. Agrobacterium‐mediated transient transformation revealed nuclear speckled distribution and the overall colocalization of fluorescent protein (FP)‐tagged SR factors in both tobacco and Arabidopsis cells. Their overall colocalization in larger nucleoplasmic domains was further observed after transcriptional and phosphorylation/dephosphorylation inhibition, indicating a close functional association between SR factors, independent of their phosphorylation state. Furthermore, we demonstrated in vivo the conserved role of the RS and RRM domains in the efficient targeting of Arabidopsis SR proteins to nuclear speckles by using a series of structural domain‐deleted mutants of atRSp31 and atRSZp22. We suggest additional roles of RS domain such as the shuttling of atRSZp22 between nucleoplasm and nucleolus through its phosphorylation level. The coexpression of deletion mutants with wild‐type SR proteins revealed potential complex associations between them. Fluorescence recovery after photobleaching demonstrated similar dynamic properties of SR factors in both tobacco transiently expressing cells and Arabidopsis transgenics. Cell cycle phase‐dependent organization of FP‐tagged SR proteins was observed in living tobacco BY‐2 cells. We showed that atRSp31 is degraded at metaphase by fluorescence quantification. SR proteins also localized within small foci at anaphase. These results demonstrate interesting related features as well as potentially important differences between plant and animal SR proteins.

Zhu, Cong; Perry, Sharyn E.

doi: 10.1111/j.1365-313X.2004.02320.xpmid: 15686521

AGL15 is an Arabidopsis thaliana MADS‐domain regulatory factor that not only preferentially accumulates during embryogenesis but is also expressed at lower levels after the completion of germination. To better understand the control of expression of AGL15, a series of 5′ and internal deletions within the regulatory regions of AGL15 was generated. Regions important for the level of expression, including a region involved in expression in response to auxin, were identified. Additionally, AGL15 expression was found to respond to AGL15 accumulation amounts and to altered forms of AGL15. This feedback loop is at least in part due to direct regulation, as assessed by in vivo and in vitro binding of AGL15 to its own regulatory regions and by site‐directed mutagenesis studies.

Baunsgaard, Lone; Lütken, Henrik; Mikkelsen, René; Glaring, Mikkel A.; Pham, Tam T.; Blennow, Andreas

doi: 10.1111/j.1365-313X.2004.02322.xpmid: 15686522

An Arabidopsis thaliana gene encoding a homologue of the potato α‐glucan, water dikinase GWD, previously known as R1, was identified by screening the Arabidopsis genome and named AtGWD3. The AtGWD3 cDNA was isolated, heterologously expressed and the protein was purified to apparent homogeneity to determine the enzymatic function. In contrast to the potato GWD protein, the AtGWD3 primarily catalysed phosphorylation at the C‐3 position of the glucose unit of preferably pre‐phosphorylated amylopectin substrate with long side chains. An Arabidopsis mutant, termed Atgwd3, with downregulated expression of the AtGWD3 gene was analysed. In Atgwd3 the amount of leaf starch was constantly higher than wild type during the diurnal cycle. Compared with wild‐type leaf starch, the level of C‐3 phosphorylation of the glucosyl moiety of starch in this mutant was reduced. Taken together, these data indicate that the C‐3 linked phospho‐ester in starch plays a so far unnoticed specific role in the degradation of transitory starch.

Ivashikina, Natalya; Hedrich, Rainer

doi: 10.1111/j.1365-313X.2004.02324.xpmid: 15686523

Non‐selective slow vacuolar (SV) channels mediate uptake of K+ and Na+ into vacuolar compartment. Under salt stress plant cells accumulate Na+ in the vacuole and release vacuolar K+ into the cytoplasm. It is, however, unclear how plants mediate transport of K+ from the vacuole without concomitant efflux of toxic Na+. Here we show by patch‐clamp studies on isolated Arabidopsis thaliana cell culture vacuoles that SV channels do not mediate Na+ release from the vacuole as luminal Na+ blocks this channel. Gating of the SV channel is dependent on the K+ gradient across the vacuolar membrane. Under symmetrical K+ concentrations on both sides of the vacuolar membrane, SV channels mediate potassium uptake. When cytoplasmic K+ decreases, SV channels allow K+ release from the vacuole. In contrast to potassium, Na+ can be taken up by SV channels, but not released even in the presence of a 150‐fold gradient (lumen to cytoplasm). Accumulation of Na+ in the vacuole shifts the activation potential of SV channels to more positive voltages and prevents gradient‐driven efflux of K+. Similar to sodium, under physiological conditions, vacuolar Ca2+ is not released from vacuoles via SV channels. We suggest that a major Arabidopsis SV channel is equipped with a positively charged intrinsic gate located at the luminal side, which prevents release of Na+ and Ca2+, but permits efflux of K+. This property of the SV channel guarantees that K+ can shuttle across the vacuolar membrane while maintaining Na+ and Ca2+ stored in this organelle.

Evans, Nicola H.; McAinsh, Martin R.; Hetherington, Alistair M.; Knight, Marc R.

doi: 10.1111/j.1365-313X.2004.02325.xpmid: 15686524

Ozone is responsible for more crop losses than any other air pollutant. The changes in gene expression, which occur in plants in response to ozone, have been well characterized, yet little is known about how ozone is perceived or the signal transduction steps that follow. The earliest characterized response to ozone is an elevation in cytosolic‐free calcium, which takes place within seconds of exposure. In this study, the calcium response to ozone was investigated in Arabidopsis thaliana seedlings using a variety of fumigation protocols. Ozone elicited distinct calcium responses in the aerial tissue and roots of seedlings. The calcium response in the cotyledons and leaves was biphasic and sensitive to the rate at which the ozone concentration increased. The response in the root was monophasic and insensitive to the rate of increase in ozone concentration. Experiments utilizing inhibitors of antioxidant metabolism demonstrated that the magnitude of the first peak in calcium in the aerial tissues was dependent upon the redox status of the plant. Seedlings were shown to be able to distinguish between ozone and hydrogen peroxide, producing a calcium signal in response to one of these reactive oxygen species (ROS) when they had become refractory to the other. Pre‐treatment with ozone altered the calcium response to hydrogen peroxide and vice versa, indicating that the calcium response to a given ROS may reflect the stress history of the plant. These data suggest ROS signalling is more sophisticated than previously realized and raise questions over current models of ozone perception.