Acclimation of Arabidopsis thaliana to the light environment: the role of photoreceptors

The regulation by light of the composition of the photosynthetic apparatus was investigated in photomorphogenic mutants of Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta. Leaf chlorophyll, photosynthesis, photosystem II function, and ribulose-1,5-bisphosphate carboxylase-oxygenase and photosystem II contents were determined for plants grown under high- or low-irradiance growth regimes. Although certain mutant lines had altered chloroplast composition compared to the wild type, all photoreceptor mutants tested were capable of light-dependent changes in chloroplast composition and photosynthetic function, indicating that photoreceptors do not play a central role in the regulation of acclimation at the level of the chloroplast. However, the clear acclimation defect in a det1 signal transduction mutant indicates that photoreceptor-controlled responses either share regulatory components with acclimation, or are important in the expression of components which in turn regulate acclimation. We suggest that the COP/DET/FUS regulatory cluster is a focus for multiple signal transduction pathways, including some of the metabolic signals which form the basis for the acclimatory response. Received: 22 April 1999 / Accepted: 6 June 1999     

Characterization of expressed genes in the SLL2 region of self-compatible Arabidopsis thaliana.

Self-incompatibility in Brassica species is regulated by a set of S-locus genes: SLG, SRK, and SP11/SCR. In the vicinity of the S-locus genes, several expressed genes, SLL2 and SP2/ClpP, etc., were identified in B. campestris. Arabidopsis thaliana is a self-compatible Brassica relative, and its complete genome has been sequenced. From comparison of the genomic sequences between B. campestris and A. thaliana, microsynteny between gene clusters of Arabidopsis and Brassica SLL2 regions was observed, though the S-locus genes, SLG, SRK, and SP11/SCR were not found in the region of Arabidopsis. Almost all genes predicted in this region of Arabidopsis were expressed in both vegetative and reproductive organs, suggesting that the genes in the SLL2 region might not be related to self-incompatibility. Considering the recent speculation that the S-locus genes were translocated as a single unit between Arabidopsis and Brassica, the translocation might have occurred in the region between the SLL2 and SP7 genes.     

Identification of a metabolic bottleneck for cold acclimation in Arabidopsis thaliana

Central carbohydrate metabolism of Arabidopsis thaliana is known to play a crucial role during cold acclimation and the acquisition of freezing tolerance. During cold exposure, many carbohydrates accumulate and a new metabolic homeostasis evolves. In the present study, we analyse the diurnal dynamics of carbohydrate homeostasis before and after cold exposure in three natural accessions showing distinct cold acclimation capacity. Diurnal dynamics of soluble carbohydrates were found to be significantly different in cold-sensitive and cold-tolerant accessions. Although experimentally determined maximum turnover rates for sucrose phosphate synthase in cold-acclimated leaves were higher for cold-tolerant accessions, model simulations of diurnal carbohydrate dynamics revealed similar fluxes. This implied a significantly higher capacity for sucrose synthesis in cold-tolerant than cold-sensitive accessions. Based on this implication resulting from mathematical model simulation, a critical temperature for sucrose synthesis was calculated using the Arrhenius equation and experimentally validated in the cold-sensitive accession C24. At the critical temperature suggested by model simulation, an imbalance in photosynthetic carbon fixation ultimately resulting in oxidative stress was observed. It is therefore concluded that metabolic capacities at least in part determine the ability of accessions of Arabidopsis thaliana to cope with changes in environmental conditions.     

pho3: a phosphorus-deficient mutant of Arabidopsis thaliana (L.) Heynh

A novel P-deficient mutant of Arabidopsis thaliana, pho3, was isolated by screening for root acid phosphatase (APase) activity in plants grown under low-P conditions. pho3 had 30% less APase activity in roots than the wild type and, in contrast to wild-type plants, root APase activity did not increase in response to growth in low P. However, shoot APase activity was higher in pho3 than in the wild-type plants. In addition, the pho3 mutant had a P-deficient phenotype, even when grown in P-sufficient conditions. The total P content of 11-d-old pho3 plants, grown in agar media with a plentiful supply of P, was about 25% lower than the wild-type level in the shoot, and about 65% lower in the roots. In the rosette leaves of mature soil-grown pho3 plants the total P content was again reduced, to about 50% of wild-type levels. pho3 exhibited a number of characteristics normally associated with low-P stress, including severely reduced growth, increased anthocyanin content (at least 100-fold greater than the wild type in soil-grown plants) and starch accumulation. The results suggest that the mutant is unable to respond to low internal P levels, and may lack a transporter or a signalling component involved in regulating P nutrition. Received: 21 March 2000 / Accepted: 15 August 2000     

Root hair growth in Arabidopsis thaliana is directed by calcium and an endogenous polarity

Tip growth of plant cells has been suggested to be regulated by a tip-focused gradient in cytosolic calcium concentration ([Ca²⁺]_c). However, whether this gradient orients apical growth or follows the driving force for this process remains unknown. Using localized photoactivation of the caged calcium ionophore Br-A23187 we have been able to artificially generate an asymmetrical calcium influx across the root hair tip. This led to a change in the direction of tip growth towards the high point of the new [Ca²⁺]_c gradient. Such reorientation of growth was transient and there was a return to the original direction within 15 min. Root hairs forced to change the direction of their growth by placing a mechanical obstacle in their path stopped, reoriented growth to the side, and grew past the mechanical blockage. However, as soon as the growing tip had cleared the obstacle, growth returned to the original direction. Confocal ratio imaging revealed that a tip-focused [Ca²⁺]_c gradient was always centered at the site of active growth. When the root hair changed direction the gradient also reoriented, and when growth returned to the original direction, so did the [Ca²⁺]_c gradient. This normal direction of apical growth of Arabidopsis thaliana (L.) Heynh. root hairs was found to be at a fixed angle from the root of 85 ± 6.7 degrees. In contrast, Tradescantia virginiana (L.) pollen tubes that were induced to reorient by touch or localized activation of the caged ionophore, did not return to the original growth direction, but continued to elongate in their new orientation. These results suggest that the tip-focused [Ca²⁺]_c gradient is an important factor in localizing growth of the elongating root hair and pollen tube to the apex. However, it is not the primary determinant of the direction of elongation in root hairs, suggesting that other information from the root is acting to continuously reset the growth direction away from the root surface. Received: 22 April 1997 / Accepted: 14 May 1997     

The altered responses of a new mutant long life span 1 to cytokinin in Arabidopsis thaliana

Cytokinins are a class of essential plant hormones regulating plant growth and development.Although the two-component phosphorelay pathway of cytokinin has been well characterized,the intact cytokinin responses regulation picture still needs to be fully depicted.Here we report a new mutant,long life span 1(lls1),which displays dwarf stature,curled leaves,numerous axillary branches and nearly 5-month life span.Exogenous cytokinin could not recover the phenotypes of the mutant.Moreover,mutation in lls1 suppressed the cytokinin-responsive phenotypes,including root and hypocotyl growth inhibition,anthocyanin accumulation,metaxylem promotion in primary root development.The induction of cytokinin-responsive genes,ARR5,AHP5,and CKX3,was also suppressed in lls1.According to quantitative RT-PCR(qRT-PCR) and microarray results,the basal expression of positive factors AHP5,ARR1,and ARR10 were down-regulated,while the negative factors ARR4 and ARR5 were up-regulated.Our results suggested that LLS1 gene might be involved in the regulation of cytokinin signaling.It was mapped to chromosome 4 where no other cytokinin relevant gene has been reported.     

Two amidophosphoribosyltransferase genes of Arabidopsis thaliana expressed in different organs

Amidophosphoribosyltransferase (ATase: EC 2.4.2.14) is a key enzyme in the pathway of purine nucleotide biosynthesis. We have identified several cDNA clones whose amino acid sequences exhibit similarity with the known ATases in a cDNA library of young floral buds of Arabidopsis thaliana. The cDNA clones are derived from two genes homologous with each other. These cDNAs represent the first plant representatives of ATase gene. Structural comparison with ATases of other organisms has revealed that the two genes encode [4Fe-4S] cluster-dependent ATases. Northern blot analysis showed that expression level of the genes is different in three organs; one gene is expressed in flowers and roots, while the other gene is mainly expressed in leaves.     

Characterization of tt15, a novel transparent testa mutant of Arabidopsis thaliana (L.) Heynh.

The Arabidopsis thaliana seed coat typically has a brown color due to the accumulation of flavonoid pigments in the testa. Mutants of A. thaliana with defects in pigment biosynthesis often produce seeds that are olive brown or even yellow in appearence, and the responsible genetic loci are referred to as TRANSPARENT TESTA (TT). Large-scale screening for mutants affected in seed development and complementation analysis of a candidate mutant line with all published A. thalianatt mutants identified a new tt locus designated tt15. The tt15 mutation maps to the lower part of chromosome 1. Mutant plants produced pale greenish-brown seeds whose dormancy was slightly reduced. The phenotype was consistent with the maternal origin of the testa. Analysis of pigment accumulation and the study of expression patterns of genes involved in flavonoid biosynthesis in tt15 plants and seeds indicated a seed-specific phenotype. Most notable was a reduction of the cyanidin and quercetin content of tt15 seeds. Received: 2 October 1998 / Accepted: 10 October 1998     

Brassinosteroids, microtubules and cell elongation in Arabidopsis thaliana. II. Effects of brassinosteroids on microtubules and cell elongation in the bul1 mutant

In order to elucidate the involvement of brassinosteroids in the cell elongation process leading to normal plant morphology, indirect immunofluorescence and molecular techniques were use to study the expression of tubulin genes in the bul1-1 dwarf mutant of Arabidopsis thaliana (L.) Heynh., the characteristics of which are reported in this issue (M. Catterou et al., 2001). Microtubules were studied specifically in the regions of the mutant plant where the elongation zone is suppressed (hypocotyls and petioles), making the reduction in cell elongation evident. Indirect immunofluorescence of α-tubulin revealed that very few microtubules were present in mutant cells, resulting in the total lack of the parallel microtubule organization that is typical of elongating cells in the wild type. After brassinosteroid treatment, microtubules reorganized and became correctly oriented, suggesting the involvement of brassinosteroids in microtubule organization. Molecular analyses showed that the microtubule reorganization observed in brassinosteroid-treated bul1-1 plants did not result either from an activation of tubulin gene expression, or from an increase in tubulin content, suggesting that a brassinosteroid-responsive pathway exists which allows microtubule nucleation/organization and cell elongation without activation of tubulin gene expression. Received: 28 April 2000 / Accepted: 6 October 2000     

Genetic control of male fertility in Arabidopsis thaliana: structural analyses of postmeiotic developmental mutants

Seven new male-sterile mutants (ms7–ms13) of Arabidopsis thaliana (L.) Heynh. (ecotype columbia) are described that show a postmeiotic defect of microspore development. In ms9 mutants, microspores recently released from the tetrad appear irregular in shape and are often without exines. The earliest evidence of abnormality in ms12 mutants is degeneration of microspores that lack normal exine sculpturing, suggesting that the MS12 product is important in the formation of pollen exine. Teratomes (abnormally enlarged microsporocytes) are also occasionally present and each has a poorly developed exine. In ms7 mutant plants, the tapetal cytoplasm disintegrates at the late vacuolate microspore stage, apparently causing the degeneration of microspores and pollen grains. With ms8 mutants, the exine of the microspores appears similar to that of the wild type. However, intine development appears impaired and pollen grains rupture prior to maturity. In ms11 mutants, the first detectable abnormality appears at the mid to late vacuolate stage. The absence of fluorescence in the microspores and tapetal cells after staining with 4′,6-diamidino-2-phenylindole (DAPI) and the occasional presence of teratomes indicate degradation of DNA. Viable pollen from ms10 mutant plants is dehisced from anthers but appears to have surface abnormalities affecting interaction with the stigma. Pollen only germinates in high-humidity conditions or during in-vitro germination experiments. Mutant plants also have bright-green stems, suggesting that ms10 belongs to the eceriferum (cer) class of mutants. However, ms10 and cer6 are non-allelic. The ms13 mutant has a similar phenotype to ms10, suggesting is also an eceriferum mutation. Each of these seven mutants had a greater number of flowers than congenic male-fertile plants. The non-allelic nature of these mutants and their different developmental end-points indicate that seven different genes important for the later stages of pollen development have been identified. Received: 14 August 1997 / Accepted: 7 October 1997     

The targeting and accumulation of ectopically expressed oleosin in non-seed tissues of Arabidopsis thaliana

 Full-length and N-terminal deletions of a sunflower (Helianthus annuus L.) oleosin protein were expressed ectopically in transgenic Arabidopsis thaliana (L.) Heynh. Immunological detection of the sunflower protein revealed that it accumulated in a range of non-oil-storing tissues, including leaves, roots and petals. This accumulation was shown to result from deposition in the microsomal membrane fraction. Expression in oil-storing tissues (such as seeds) of oleosin N-terminal deletions revealed impaired transfer from the endoplasmic reticulum to the oil body. In non-oil-storing tissues, accumulation in the microsomal membrane fraction was progressively reduced by N-terminal deletion. These data confirm the role of the endomembrane system in the targeting of the oleosin and its intimate relationship with oil-body biogenesis. Received: 26 August 1999 / Accepted: 4 October 1999     

Expression of CKS1At in Arabidopsis thaliana indicates a role for the protein in both the mitotic and the endoreduplication cycle

Although endoreduplication is common in plants, little is known about the mechanisms regulating this process. Here, we report the patterns of endoreduplication at the cellular level in the shoot apex of Arabidopsis thaliana L. Heynh. plants grown under short-day conditions. We show that polyploidy is developmentally established in the pith, maturing leaves, and stipules. To investigate the role of the cell cycle genes CDC2aAt, CDC2bAt, CYCB1;1, and CKS1At in the process of endoreduplication, in-situ hybridizations were performed on the vegetative shoot apices. Expression of CDC2aAt, CDC2bAt, and CYCB1;1 was restricted to mitotically dividing cells. In contrast, CKS1At expression was present in both mitotic and endoreduplicating tissues. Our data indicate that CDC2aAt, CDC2bAt, and CYCB1;1 only operate during mitotic divisions, whereas CKS1At may play a role in both the mitotic and endoreduplication cycle. Received: 11 May 1998 / Accepted: 29 September 1998     

Impact of soluble sugar concentrations on the acquisition of freezing tolerance in accessions of Arabidopsis thaliana with contrasting cold adaptation – evidence for a role of raffinose in cold acclimation

In the present study the cold acclimation potential of two accessions of Arabidopsis thaliana was investigated. Significant variation was found for basic tolerance as well as the capacity to acclimate to freezing temperatures. During cold acclimation, levels of soluble sugars increased in both genotypes, but raffinose accumulation discriminated the more tolerant accession Col‐0 from C24. Concentrations of other compatible solutes such as proline and glutamine were also higher in cold‐acclimated Col‐0 than C24 plants. Changes of invertase activity during cold exposure corresponded to changes in sucrose and fructose, but not glucose concentrations and were consistent with an initial chilling response and a later decline in hexose metabolization. When vacuolar invertase was suppressed by siRNA expression, reduced sucrolytic activity resulted in elevated leaf sucrose concentration, whereas the fructose content was strongly reduced. This led to elevated freezing tolerance in the cold‐tolerant genotype Col‐0, but not in C24. The most pronounced metabolic changes in invertase‐inhibited Col‐0 plants occurred for proline and glutamine concentrations, indicating indirect metabolic effects of altered sugar concentrations.     

The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression

We have isolated and determined DNA sequence for the 5-flanking regions of three Arabidopsis thaliana polyubiquitin genes, UBQ3, UBQ10, and UBQ11. Comparison to cDNA sequences revealed the presence of an intron in the 5-untranslated region at the same position immediately upstream of the initiator methionine codon in each of the three genes. An intron at this position is also present in two sunflower and two maize polyubiquitin genes. An intron is also found in the 5-untranslated regions of several animal polyubiquitin genes, although the exact intron position is not conserved among them, and none are in the same position as those in the higher plant polyubiquitin genes. Chimeric genes containing the 5-flanking regions of UBQ3, UBQ10, and UBQ11 in front of the coding regions for the reporter enzyme Escherichia coli -glucuronidase (GUS) were constructed. When introduced transiently into Arabidopsis leaves via microprojectile bombardment, all resulted in readily detectable levels of GUS activity that were quantitatively similar. The introns of UBQ3 and UBQ10 in the corresponding promoter fragments were removed by replacement with flanking cDNA sequences and chimeric genes constructed. These constructs resulted in 2.5- to 3-fold lower levels of marker enzyme activity after transient introduction into Arabidopsis leaves. The UBQ10 promoter without the 5 intron placed upstream of firefly luciferase (LUX) resulted in an average of 3-fold lower LUX activity than from an equivalent construct with the UBQ10 intron. A UBQ3 promoter cassette was constructed for the constitutive expression of open reading frames in dicot plants and it produced readily detectable levels of GUS activity in transient assays.     

Harpin induces mitogen-activated protein kinase activity during defence responses in Arabidopsis thaliana suspension cultures

Elicitation of Arabidopsis thaliana (L.) Heynh. suspension cultures with the bacterial protein harpin (from Pseudomonas syringae pv. syringae) induced the activation of two kinases of 39 and 44 kDa, as demonstrated by in-gel kinase assays using myelin basic protein (MBP) as a substrate. Both these kinases appeared to be tyrosine-phosphorylated upon activation, as demonstrated by treatment with tyrosine phosphatase and immunoprecipitation using an anti-phosphotyrosine monoclonal antibody. An inhibitor of mammalian mitogen-activated protein kinase (MAPK) activation, PD98059, inhibited harpin-induced MBPK activation, but did not inhibit the activity of these kinases. PD98059 also inhibited harpin-induced programmed cell death and defence gene expression, suggesting the involvement of harpin-induced MAPKs in defence responses in Arabidopsis thaliana. Received: 23 February 1999 / Accepted: 22 July 1999     

Chlorosis caused by two recessively interacting genes reveals a role of RNA helicase in hybrid breakdown in Arabidopsis thaliana

Hybrids often differ in fitness from their parents. They may be superior, translating into hybrid vigour or heterosis, but they may also be markedly inferior, because of hybrid weakness or incompatibility. The underlying genetic causes for the latter can often be traced back to genes that evolve rapidly because of sexual or host–pathogen conflicts. Hybrid weakness may manifest itself only in later generations, in a phenomenon called hybrid breakdown. We have characterized a case of hybrid breakdown among two Arabidopsis thaliana accessions, Shahdara (Sha, Tajikistan) and Lövvik‐5 (Lov‐5, Northern Sweden). In addition to chlorosis, a fraction of the F₂ plants have defects in leaf and embryo development, and reduced photosynthetic efficiency. Hybrid chlorosis is due to two major‐effect loci, of which one, originating from Lov‐5, appears to encode an RNA helicase (AtRH18). To examine the role of the chlorosis allele in the Lövvik area, in addition to eight accessions collected in 2009, we collected another 240 accessions from 15 collections sites, including Lövvik, from Northern Sweden in 2015. Genotyping revealed that Lövvik collection site is separated from the rest. Crosses between 109 accessions from this area and Sha revealed 85 cases of hybrid chlorosis, indicating that the chlorosis‐causing allele is common in this area. These results suggest that hybrid breakdown alleles not only occur at rapidly evolving loci, but also at genes that code for conserved processes.