Expression of a vegetative-storage-protein gene from Arabidopsis is regulated by copper, senescence and ozone

Emerging data suggest that the mechanisms regulating plant copper homeostasis could be implicated in stress and senescence signal transduction pathways. To gain insight into copper-modulated patterns of gene expression, copper-treated Arabidopsis thaliana (L.) Heynh. plants were analysed by mRNA differential display. The experimental conditions were selected using aggregation of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) as a molecular sensor to monitor copper-induced oxidative stress. Two copper-induced messengers encoding a vegetative storage protein (VSP2) were isolated by this technique. Both clones differed in the length of their 3'-untranslated region according to the presence of two polyadenylation signals in this region. VSP2 expression was further studied under natural senescence and various conditions causing oxidative stress, such as ozone exposure, paraquat and H2O2 treatments. The expression of other messengers related to copper homeostasis and detoxification processes was followed in parallel to that of VSP2. Here, we describe specific gene-expression responses to copper treatment, and present arguments connecting copper homeostasis, senescence and antioxidative responses in plants. Our results are consistent with the role of VSPs as temporary nitrogen-storage proteins which accumulate if nutrients are abundant, either in developing organs or in cotyledons and mature leaves subjected to generalized protein mobilization, such as those conditions created under severe oxidative stress.     

The Arabidopsis floral homeotic gene PISTILLATA is regulated by discrete cis-elements responsive to induction and maintenance signals

PISTILLATA is a B-class floral organ identity gene required for the normal development of petals and stamens in Arabidopsis. PISTILLATA expression is induced in the stage 3 flowers (early expression) and is maintained until anthesis (late expression). To explore in more detail the developmentally regulated gene expression of PISTILLATA, we have analyzed the PISTILLATA promoter using uidA (beta)-glucuronidase gene) fusion constructs (PI::GUS) in transgenic Arabidopsis. Promoter deletion analyses suggest that early PISTILLATA expression is mediated by the distal region and that late expression is mediated by the proximal region. Based on the PI::GUS expression patterns in the loss- and gain-of-function alleles of meristem or organ identity genes, we have shown that LEAFY and UNUSUAL FLORAL ORGANS induce PISTILLATA expression in a flower-independent manner via a distal promoter, and that PISTILLATA and APETALA3 maintain PISTILLATA expression (autoregulation) in the later stages of flower development via a proximal promoter. In addition, we have demonstrated that de novo protein synthesis is required for the PISTILLATA autoregulatory circuit.     

A senescence-associated gene of Arabidopsis thaliana is distinctively regulated during natural and artificially induced leaf senescence

We have characterized the structure and expression of a senescence-associated gene (sen1) of Arabidopsis thaliana. The protein-coding region of the gene consists of 5 exons encoding 182 amino acids. The encoded peptide shows noticeable similarity to the bacterial sulfide dehydrogenase and 81% identity to the peptide encoded by the radish din1 gene. The 5-upstream region contains sequence motifs resembling the heat-shock- and ABA-responsive elements and the TCA motif conserved among stress-inducible genes. Examination of the expression patterns of the sen1 gene under various senescing conditions along with measurements of photochemical efficiency and of chlorophyll content revealed that the sen1 gene expression is associated with Arabidopsis leaf senescence. During the normal growth phase, the gene is strongly induced in leaves at 25 days after germination when inflorescence stems are 2–3 cm high, and then the mRNA level is maintained at a comparable level in naturally senescing leaves. In addition, dark-induced senescence of detached leaves or of leaves in planta resulted in a high-level induction of the gene. Expression of the sen1 gene was also strongly induced in leaves subjected to senescence by 0.1 mM abscisic acid or 1 mM ethephon treatment. The induced expression of the gene by dark treatment was not significantly repressed by treatment with 0.1 mM cytokinin or 50 mM CaCl₂ which delayed loss of chlorophyll but not that of photochemical efficiency.     

Arabidopsis PLC1 is required for secondary responses to abscisic acid signals

The role of inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) in transducing the abscisic acid (ABA) signal during seed germination and in the stress responses of mature plants is poorly understood. We have considered the contributions of the phospholipase C1 (encoded by AtPLC1) and an Ins(1,4,5)P3 5-phosphatase (encoded by AtIP5PII) to ABA signaling by using a modified version of the glucocorticoid-inducible system to regulate transgene expression. In the presence of the dexamethasone (Dex) inducer, transgenic lines expressing the AtPLC1 antisense and AtIP5PII sense transgenes showed no inhibition of germination and growth by ABA, whereas in the absence of the inducer they were sensitive. In the presence of Dex, these lines accumulated lower Ins(1,4,5)P3 levels upon ABA treatment compared with that of the control transgenic lines. RNA gel blot analysis revealed a decrease in the induction of the ABA-responsive genes RD29a, KIN2, and RD22 but not COR47 in the Dex-induced transgenic plants. In transgenic lines expressing the inducible AtPLC1 sense transgene, an increase in AtPLC1 expression was not sufficient to activate the expression of ABA-responsive genes in vegetative tissues. In vitro experiments demonstrated the induced PLC1 expression when extracts were assayed in the presence of calcium, but no increase in Ins(1,4,5)P3 levels in vivo was detected, suggesting that the PLC1 enzyme was latent. Our results indicate that although an increase in PLC1 activity and increased Ins(1,4,5)P3 levels are necessary for maximal gene induction by ABA, overexpression of AtPLC1 itself is not sufficient to trigger the expression of ABA-responsive genes. We propose that AtPLC1 plays a role in secondary ABA responses.     

Pollen tube growth and guidance is regulated by POP2, an Arabidopsis gene that controls GABA levels

During angiosperm reproduction, pollen grains form a tube that navigates through female tissues to the micropyle, delivering sperm to the egg; the signals that mediate this process are poorly understood. Here, we describe a role for gamma-amino butyric acid (GABA) in pollen tube growth and guidance. In vitro, GABA stimulates pollen tube growth, although vast excesses are inhibitory. The Arabidopsis POP2 gene encodes a transaminase that degrades GABA and contributes to the formation of a gradient leading up to the micropyle. pop2 flowers accumulate GABA, and the growth of many pop2 pollen tubes is arrested, consistent with their in vitro GABA hypersensitivity. Some pop2 tubes continue to grow toward ovules, yet they are misguided, presumably because they target ectopic GABA on the ovule surface. Interestingly, wild-type tubes exhibit normal growth and guidance in pop2 pistils, perhaps by degrading excess GABA and sharpening the gradient leading to the micropyle.     

Phylogenetic and transcriptional analysis of a strictosidine synthase-like gene family in Arabidopsis thaliana reveals involvement in plant defence responses

Protein domains with similarity to plant strictosidine synthase-like (SSL) sequences have been uncovered in the genomes of all multicellular organisms sequenced so far and are known to play a role in animal immune responses. Among several distinct groups of Arabidopsis thaliana SSL sequences, four genes ( AtSSL4–AtSSL7 ) arranged in tandem on chromosome 3 show more similarity to SSL genes from Drosophila melanogaster and Caenorhabditis elegans than to other Arabidopsis SSL genes. To examine whether any of the four AtSSL genes are immune-inducible, we analysed the expression of each of the four AtSSL genes after exposure to microbial pathogens, wounding and plant defence elicitors using real-time quantitative RT-PCR, Northern blot hybridisation and Western blot analysis with antibodies raised against recombinant At SSL proteins. While the AtSSL4 gene was constitutively expressed and not significantly induced by any treatment, the other three AtSSL genes were induced to various degrees by plant defence signalling compounds, such as salicylic acid, methyl jasmonate and ethylene, as well as by wounding and exposure to the plant pathogens Alternaria brassicicola and cucumber mosaic virus . Our data demonstrate that the four SSL-coding genes are regulated individually, suggesting specific roles in basal ( SSL4 ) and inducible ( SSL5-7 ) plant defence mechanisms.     

The Arabidopsis AtEm1 promoter is active in Brassica napus L. and is temporally and spatially regulated.

The promoter of the Arabidopsis thaliana L. AtEm1 gene encoding a late embryogenesis abundant protein was fused to the beta-glucuronidase reporter gene and introduced into Brassica napus. The promoter is highly active in the vascular tissues of embryo and pollen grains and also active in petals, sepals, caulinar leaves, and carpels.     

CPEB1, a histone-modified hypomethylated gene,is regulated by miR-101 and involved in cell senescence in glioma

Epigenetic mechanisms have important roles in carcinogenesis. We certified that the mRNA translation-related gene cytoplasmic polyadenylation element-binding protein 1 (CPEB1) is hypomethylated and overexpressed in glioma cells and tissues. The knockdown of CPEB1 reduced cell senescence by regulating the expression or distribution of p53 in glioma cells. CPEB1 is also regulated directly by the tumor suppressor miR-101, a potential marker of glioma. It is known that the histone methyltransferase enhancer of zeste homolog 2 (EZH2) and embryonic ectoderm development (EED) are direct targets of miR-101. We demonstrated that miR-101 downregulated the expression of CPEB1 through reversing the methylation status of the CPEB1 promoter by regulating the presence on the promoter of the methylation-related histones H3K4me2, H3K27me3, H3K9me3 and H4K20me3. The epigenetic regulation of H3K27me3 on CPEB1 promoter is mediated by EZH2 and EED. EZH2 has a role in the regulation of H3K4me2. Furthermore, the downregulation of CPEB1 induced senescence in a p53-dependent manner.     

The Arabidopsis dual-affinity nitrate transporter gene AtNRT1.1 (CHL1) is regulated by auxin in both shoots and roots

The AtNRT1.1 (CHL1) gene of Arabidopsis encodes a dual-affinity nitrate transporter and contributes to both low and high affinity nitrate uptake. Localization studies have shown that CHL1 expression is preferentially targeted to nascent organs and growing regions of roots and shoots in Arabidopsis. In roots, CHL1 expression is concentrated in the tips of primary and lateral roots and is activated during lateral root initiation. In shoots, strong CHL1 expression is found in young leaves and developing flower buds. These findings suggest that CHL1 expression might be regulated by a growth signal such as the phytohormone auxin. To test this, auxin regulation of CHL1 was examined. Using transgenic Arabidopsis plants containing CHL1::GUS/GFP DNA constructs, it was found that treatment with exogenous auxin or introduction of the auxin overproducing mutations (yucca and rooty) resulted in a strong increase in CHL1::GUS/GFP signals in roots and leaves. When mature roots were treated with auxin to induce lateral root formation, CHL1::GFP signals were dramatically enhanced in dividing pericycle cells and throughout primordia development. RNA blot analysis showed that CHL1 mRNA levels in whole seedlings increase within 30 min of auxin treatment. The distribution of CHL1 expression in Arabidopsis roots and shoots was found to be similar to that of DR5::GUS, a synthetic, auxin-responsive gene. These results indicate that auxin acts as an important signal regulating CHL1 expression and contributes to the targeting of CHL1 expression to nascent organs and root tips in Arabidopsis.     

The unusual Arabidopsis extensin gene atExt1 is expressed throughout plant development and is induced by a variety of biotic and abiotic stresses

We detail the expression of the Arabidopsis thaliana (L.) Heynh. atExt1 extensin gene. atExt1 is normally expressed in roots and inflorescences, and is induced by wounding, exogenously supplied salicylic acid, methyl jasmonate, auxins and brassinosteroids. Northern assays and histochemical analysis of transgenics expressing an atExt1:: gus fusion show that this gene is also induced by the brassica pathogen Xanthomonas campestris pv. campestris and that this induction is restricted to tissues close to the site of infection. Expression at regions of abscission and senescence also implicates atExt1 in these important developmental processes.     

The Arabidopsis glucosyltransferase UGT76B1 conjugates isoleucic acid and modulates plant defense and senescence

Plants coordinate and tightly regulate pathogen defense by the mostly antagonistic salicylate (SA)- and jasmonate (JA)-mediated signaling pathways. Here, we show that the previously uncharacterized glucosyltransferase UGT76B1 is a novel player in this SA-JA signaling crosstalk. UGT76B1 was selected as the top stress-induced isoform among all 122 members of the Arabidopsis thaliana UGT family. Loss of UGT76B1 function leads to enhanced resistance to the biotrophic pathogen Pseudomonas syringae and accelerated senescence but increased susceptibility toward necrotrophic Alternaria brassicicola. This is accompanied by constitutively elevated SA levels and SA-related marker gene expression, whereas JA-dependent markers are repressed. Conversely, UGT76B1 overexpression has the opposite effect. Thus, UGT76B1 attenuates SA-dependent plant defense in the absence of infection, promotes the JA response, and delays senescence. The ugt76b1 phenotypes were SA dependent, whereas UGT76B1 overexpression indicated that this gene possibly also has a direct effect on the JA pathway. Nontargeted metabolomic analysis of UGT76B1 knockout and overexpression lines using ultra-high-resolution mass spectrometry and activity assays with the recombinant enzyme led to the ab initio identification of isoleucic acid (2-hydroxy-3-methyl-pentanoic acid) as a substrate of UGT76B1. Exogenously applied isoleucic acid increased resistance against P. syringae infection. These findings indicate a novel link between amino acid-related molecules and plant defense that is mediated by small-molecule glucosylation.