Differential gene expression analysis provides new insights into the molecular basis of iron deficiency stress response in the citrus rootstock Poncirus trifoliata (L.) Raf.

Iron chlorosis is one of the major abiotic stresses affecting fruit trees and other crops in calcareous soils and leads to a reduction in growth and yield. Usual remediation strategies consist of amending iron to soil, which is an expensive practice, or using tolerant cultivars, which are difficult to develop when not available. To understand the mechanisms underlying the associated physiopathy better, and thus develop new strategies to overcome the problems resulting from iron deficiency, the differential gene expression induced by iron deficiency in the susceptible citrus rootstock Poncirus trifoliata (L.) Raf. have been examined. The genes identified are putatively involved in cell wall modification, in determining photosynthesis rate and chlorophyll content, and reducing oxidative stress. Additional studies on cell wall morphology, photosynthesis, and chlorophyll content, as well as peroxidase and catalase activities, support their possible functions in the response to iron deficiency in a susceptible genotype, and the results are discussed.

Development of a citrus genome-wide EST collection and cDNA microarray as resources for genomic studies.

A functional genomics project has been initiated to approach the molecular characterization of the main biological and agronomical traits of citrus. As a key part of this project, a citrus EST collection has been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in the citrus genome. Functional annotation of unigenes which have Arabidopsis orthologues (68% of all unigenes) revealed gene representation in every major functional category, suggesting that a genome-wide EST collection was obtained. A Citrus clementina Hort. ex Tan. cv. Clemenules genomic library, that will contribute to further characterization of relevant genes, has also been constructed. To initiate the analysis of citrus transcriptome, we have developed a cDNA microarray containing 12,672 probes corresponding to 6875 putative unigenes of the collection. Technical characterization of the microarray showed high intra- and inter-array reproducibility, as well as a good range of sensitivity. We have also validated gene expression data achieved with this microarray through an independent technique such as RNA gel blot analysis.

Genetics of the DST-mediated mRNA decay pathway using a transgene-based selection.

mRNA sequences that control abundance, localization and translation initiation have been identified, yet the factors that recognize these sequences are largely unknown. In this report, a transgene-based strategy designed to isolate mutants of Arabidopsis thaliana that fail to recognize these sequences is described. In this strategy, a selectable gene and a screenable marker gene are put under the control of the sequence element being analysed and mutants are selected with altered abundance of the corresponding marker RNAs. The selection of mutants deficient in recognition of the DST (downstream) mRNA degradation signal is used as a test-case to illustrate some of the technical aspects that have facilitated success. Using this strategy, we report the isolation of a new mutant, dst3, deficient in the DST-mediated mRNA decay pathway. The targeted genetic strategy described circumvents certain technical limitations of biochemical approaches. Hence, it provides a means to investigate a variety of other mechanisms responsible for post-transcriptional regulation.

New molecular phenotypes in the dst mutants of Arabidopsis revealed by DNA microarray analysis.

In this study, DNA microarray analysis was used to expand our understanding of the dst1 mutant of Arabidopsis. The dst (downstream) mutants were isolated originally as specifically increasing the steady state level and the half-life of DST-containing transcripts. As such, txhey offer a unique opportunity to study rapid sequence-specific mRNA decay pathways in eukaryotes. These mutants show a threefold to fourfold increase in mRNA abundance for two transgenes and an endogenous gene, all containing DST elements, when examined by RNA gel blot analysis; however, they show no visible aberrant phenotype. Here, we use DNA microarrays to identify genes with altered expression levels in dst1 compared with the parental plants. In addition to verifying the increase in the transgene mRNA levels, which were used to isolate these mutants, we were able to identify new genes with altered mRNA abundance in dst1. RNA gel blot analysis confirmed the microarray data for all genes tested and also was used to catalog the first molecular differences in gene expression between the dst1 and dst2 mutants. These differences revealed previously unknown molecular phenotypes for the dst mutants that will be helpful in future analyses. Cluster analysis of genes altered in dst1 revealed new coexpression patterns that prompt new hypotheses regarding the nature of the dst1 mutation and a possible role of the DST-mediated mRNA decay pathway in plants.

Mutants of Arabidopsis defective in a sequence-specific mRNA degradation pathway.

One of the ways a cell can rapidly and tightly regulate gene expression is to target specific mRNAs for rapid decay. A number of mRNA instability sequences that mediate rapid mRNA decay have been identified, particularly from multicellular eukaryotes, but pinpointing the cellular components that play critical roles in sequence-specific decay in vivo has been more difficult. In contrast, general pathways of mRNA degradation in yeast have been well established through the analysis of mutants affecting the general mRNA decay machinery. Strategies to isolate mutants in sequence-specific mRNA decay pathways, although extremely limited so far, have the potential to be just as powerful. In the study reported here, a selection in transgenic plants allowed the isolation of rare mutants of Arabidopsis thaliana that elevate the abundance of mRNAs that contain the plant mRNA instability sequence called DST (downstream element). This instability sequence is highly conserved in unstable small auxin up RNA (SAUR) transcripts. Genetic analysis of two dst mutants isolated via this selection showed that they are incompletely dominant and represent two independent loci. In addition to affecting DST-containing transgene mRNAs, mutations at both loci increased the abundance of the endogenous DST-containing SAUR-AC1 mRNA, but not controls lacking DST sequences. That these phenotypes are caused by deficiencies in DST-mediated mRNA decay was supported by mRNA stability measurements in transgenic plants. Isolation of the dst mutants provides a means to study sequence-specific mRNA degradation in vivo and establishes a method to isolate similar mutants from other organisms.

Identification of BFN1, a bifunctional nuclease induced during leaf and stem senescence in Arabidopsis.

Nuclease I enzymes are responsible for the degradation of RNA and single-stranded DNA during several plant growth and developmental processes, including senescence. However, in the case of senescence the corresponding genes have not been reported. We describe the identification and characterization of BFN1 of Arabidopsis, and demonstrate that it is a senescence-associated nuclease I gene. BFN1 nuclease shows high similarity to the sequence of a barley nuclease induced during germination and a zinnia (Zinnia elegans) nuclease induced during xylogenesis. In transgenic plants overexpressing the BFN1 cDNA, a nuclease activity of about 38 kD was detected on both RNase and DNase activity gels. Levels of BFN1 mRNA were extremely low or undetectable in roots, leaves, and stems. In contrast, relatively high BFN1 mRNA levels were detected in flowers and during leaf and stem senescence. BFN1 nuclease activity was also induced during leaf and stem senescence. The strong response of the BFN1 gene to senescence indicated that it would be an excellent tool with which to study the mechanisms of senescence induction, as well as the role of the BFN1 enzyme in senescence using reverse genetic approaches in Arabidopsis.

Arginase, Arginine Decarboxylase, Ornithine Decarboxylase, and Polyamines in Tomato Ovaries (Changes in Unpollinated Ovaries and Parthenocarpic Fruits Induced by Auxin or Gibberellin).

Arginase (EC 3.5.3.1) activity has been found in the ovaries and Young fruits of tomato (Lycopersicon esculentum Mill. cv Rutgers).Changes in arginase, arginine decarboxylase (EC 4.1.1.19), and ornithine decarboxylase activity (EC 4.1.1.17) and levels of free and conjugated putrescine, spermidine, and spermine were determined in unpollinated ovaries and in parthenocarpic fruits during the early stages of development induced by 2,4-dichlorophenoxyacetic acid (2,4-D) or gibberellic acid (GA3). Levels of arginase, free spermine, and conjugates of the three polyamines were constant in unpollinated ovaries and characteristic of a presenescent step. A marked decrease in arginase activity, free spermine, and polyamine conjugates was associated with the initiation of fruit growth due to cell division, and when cell expansion was initiated, the absence of arginase indicated a redirection of nitrogen metabolism to the synthesis of arginine. A transient increase in arginine decarboxylase and ornithine decarboxylase was also observed in 2,4-D-induced fruits. In general, 2,4-D treatments produced faster changes than GA3, and without treatment, unpollinated ovaries developed only slightly and senescence was hardly visible. Sensitivity to 2,4-D and GA3 treatment remained for at least 2 weeks postanthesis.

N4-Hexanoylspermidine, a New Polyamine-Related Compound That Accumulates during Ovary and Petal Senescence in Pea.

A previously unknown polyamine conjugate that accumulates in senescing ovaries of pea (Pisum sativum L.) was shown by mass spectrometry, nuclear magnetic resonance, and chemical synthesis to be N4-hexanoylspermidine (hexanoyl-spd) This structure was indicated by analysis of the dansylated polyamine using fast atom bombardment mass spectrometry, following purification by high-performance liquid chromatography. Furthermore, acid hydrolysis of the compound yielded spermidine and hexanoic acid. 1H-nuclear magnetic resonance suggested that spermidine was substituted at N4 in the conjugate. Hexanoyl-spd was synthesized, and its didansyl derivative was shown to have an identical mass spectrum and high-performance liquid chromatography retention time as the derivatized natural compound. Further confirmation of its structure was obtained by comparison of the synthetic and natural polyamines as trifluoroacetyl derivatives using gas chromatography-mass spectrometry. This new polyamine conjugate is present in pea ovaries at low levels at anthesis and its concentration remains low in developing seeded fruit or in parthenocarpic fruit that have been induced by application of growth regulators to emasculated flowers or by topping the plant. Conjugate levels are also low in parthenocarpic fruit induced naturally in the slender (la crys) mutant. However, levels of hexanoyl-spd increase progressively in senescing petals and ovaries, beginning at anthesis or 2 d later, respectively.

Expression of arginine decarboxylase is induced during early fruit development and in young tissues of Pisum sativum (L.).

A cDNA coding for arginine decarboxylase (ADC, EC 4.1.1.19) has been isolated from a cDNA library of parthenocarpic young fruits of Pisum sativum (L.). The deduced aminoacid sequence is 74%, 46% and 35% identical to ADCs from tomato, oat and Escherichia coli, respectively. When the pea ADC cDNA was put under the control of the galactose inducible yeast promoter CYC1-GAL10 and introduced into Saccharomyces cerevisiae, it conferred galactose-regulated expression of the ADC activity. The ADC activity expressed in S. cerevisiae was inhibited 99% by alpha-DL-difluoromethylarginine (DFMA), a specific inhibitor of ADC activity. No activity was detected in the untransformed S. cerevisiae, nor when it was transformed with an antisense ADC construct. This provides direct evidence that the ADC cDNA from pea encoded a functional, specific ADC activity and that S. cerevisiae is able to process correctly the protein. In the pea plant, gene expression of the ADC is high in young developing tissues like shoot tips, young leaflets and flower buds. Fully expanded leaflets and roots have much lower, but still detectable, levels of the ADC transcript. In the ovary and fruit, they are developmentally regulated, showing high levels of expression during the early stages of fruit growth, which in pea is mainly due to cell expansion. The observed changes in the steady-state levels of ADC mRNA alone, however, cannot account for the differences in ADC activity suggesting that other regulatory mechanisms must be acting.

Arginine Decarboxylase and Putrescine Oxidase in Ovaries of Pisum sativum L. (Changes during Ovary Senescence and Early Stages of Fruit Development).

Enzymatic activities involved in putrescine metabolism in ovaries of Pisum sativum L. during ovary senescence and fruit set were investigated. Accumulation of putrescine was observed during incubation of extracts from gibberellic acid-treated unpollinated ovaries (young developing fruits) but not in extracts from untreated ovaries (senescent ovaries). Extracts from pea ovaries showed arginine decarboxylase (ADC) activity, but ornithine decarboxylase and arginase activity were not detected. ADC activity decreased in presenescent ovaries and increased markedly after induction of fruit set with gibberellic acid. Increases in ADC activity were also observed with application of other plant growth substances (benzy-ladenine and 2,4-dichlorophenoxyacetic acid), after pollination, and in the slender (la crys) pea mutant. By contrast, putrescine oxidase activity increased in presenescent ovaries but did not increase during early fruit development. All of these results suggest that ADC and putrescine oxidase are involved in the control of putrescine metabolism. Ovary senescence is characterized by the absence of putrescine biosynthesis enzymes and increased levels of putrescine oxidase and fruit development by an increase in ADC and a constant level of putrescine oxidase.

Correlation between Ornithine Decarboxylase and Putrescine in Tomato Plants Infected by Citrus Exocortis Viroid or Treated with Ethephon.

We have investigated the arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) activities and the levels of conjugated polyamines to explain the decrease of free putrescine level caused by citrus exocortis viroid (CEVd) and ethephon treatment in tomato (Lycopersicon esculentum Mill. cv Rutgers) plants (J.M. Belles, J. Carbonell, V. Conejero [1991] Plant Physiol 96: 1053-1059). This decrease correlates with a decrease in ODC activity in CEVd-infected or ethephon-treated plants; ADC activity was not altered. CEVd infection had no effect on polyamine conjugates, and ethephon produced a decrease in putrescine conjugates. Interference with ethylene action by silver ions prevented the decrease in ODC activity and in free and conjugated putrescine. It is suggested that changes in putrescine level after CEVd infection and ethephon treatment are regulated via ODC activity and that conjugation is not involved.