Ripening-related occurrence of phosphoenolpyruvate carboxykinase in tomato fruit

Phosphoenolpyruvate carboxykinase (PEPCK) is present in ripening tomato fruits. A cDNA encoding PEPCK was identified from a PCR-based screen of a cDNA library from ripe tomato fruit. The sequence of the tomato PEPCK cDNA and a cloned portion of the genomic DNA shows that the complete cDNA sequence contains an open reading frame encoding a peptide of 662 amino acid residues in length and predicts a polypeptide with a molecular mass of 73.5 kDa, which corresponds to that detected by western blotting. Only one PEPCK gene was identified in the tomato genome. PEPCK is shown to be present in the pericarp of ripening tomato fruits by activity measurements, western blotting and mRNA analysis. PEPCK abundance and activity both increased during fruit ripening, from an undetectable amount in immature green fruit to a high amount in ripening fruit. PEPCK mRNA, protein and activity were also detected in germinating seeds and, in lower amounts, in roots and stems of tomato. The possible role of PEPCK in the pericarp of tomato fruit during ripening is discussed.     

A role for glutamate decarboxylase during tomato ripening: the characterisation of a cDNA encoding a putative glutamate decarboxylase with a calmodulin-binding site

A tomato fruit cDNA library was differentially screened to identify mRNAs present at higher levels in fruit of the tomato ripening mutant rin (ripening inhibitor). Complete sequencing of a unique clone ERT D1 revealed an open reading frame with homology to several glutamate decarboxylases. The deduced polypeptide sequence has 80% overall amino acid sequence similarity to a Petunia hybrida glutamate decarboxylase (petGAD) which carries a calmodulin-binding site at its carboxyl terminus and ERT D1 appears to have a similar domain. ERT D1 mRNA levels peaked at the first visible sign of fruit colour change during normal tomato ripening and then declined, whereas in fruit of the ripening impaired mutant, rin, accumulation of this mRNA continued until at least 14 days after the onset of ripening. This mRNA was present at much lower levels in other tissues, such as leaves, roots and stem, and was not increased by wounding. Possible roles for GAD, and its product -aminobutyric acid (GABA) in fruit, are discussed.     

cDNA cloning and characterisation of novel ripening-related mRNAs with altered patterns of accumulation in the ripening inhibitor (rin) tomato ripening mutant

A cDNA library produced from mRNA isolated from the pericarp of wild-type tomato fruit (Lycopersicon esculentum Mill. cv Ailsa Craig) at the first visible sign of fruit ripening was differentially screened to identify clones whose homologous mRNAs were present at reduced levels in fruit of the tomato ripening mutant, ripening inhibitor,rin. Five clones were isolated (pERT 1, 10, 13, 14, 15). Accumulation of mRNA homologous to each of these clones increased during the ripening of wild-type fruit and showed reduced accumulation in ripening rin fruit. The levels of three of them (homologous to ERT 1, 13 and 14) were increased by ethylene treatment of the mutant fruit. A further clone, ERT 16 was identified for a mRNA present at a high level in both normal and mutant fruit at early stages of ripening. Database searches revealed no significant homology to the DNA sequence of ERT 14 and 15; however, DNA and derived amino acid sequence of ERT 1 both contain regions of homology with several reported UDP-glucosyl and glucuronosyl transferases (UDPGT) and with a conserved UDPGT motif. A derived amino acid sequence from the ERT 10 cDNA contains a perfect match to a consensus sequence present in a number of dehydrogenases. The ERT 13 DNA sequence has homology with an mRNA present during potato tuberisation. The presence of these mRNAs in tomato fruit is unreported and their role in ripening is unknown. The ERT 16 DNA sequence has homology with a ripening/stress-related cDNA isolated from tomato fruit pericarp.     

Detection of mRNA and hnRNA using a digoxigenin labelled cDNA probe byin situ hybridization on frozen tissue sections

Summary A full-length cDNA clone encoding the constant region of T cell receptor chain was labelled by random priming DNA with digoxigenin-dUTP. The probe was used to estimate the relative amount of the receptor chain mRNA byin situ hybridization on frozen sections from human thymus and lymph nodes. The hybridization was visualized in blue using an anti-digoxigenin antibody conjugated with alkaline phosphatase and a subsequent enzyme-catalysed colour reaction. The distributions of the signal in tissue sections were as expected. Moreover, labelled cells showed hybrids both in the cytoplasm and in the nucleus, and strongly and weakly stained cells were clearly distinguishable. The results indicate that this method ofin situ hybridization should be useful in the detection of specific mRNA in frozen sections.     

Novel mode of hormone induction of tandem tomato invertase genes in floral tissues

The genomic organization of two extracellular invertase genes from tomato (Lin5 and Lin7), which are linked in a direct tandem repeat, and their tissue-specific and hormone-inducible expression are shown. Transient expression analysis ofLin5 promoter sequences fused to the -glucuronidase (GUS) reporter gene (uidA) demonstrates a specific expression of Lin5during tomato fruit development. A Lin5 promoter fragment was fused to the truncated nos promoter to analyse hormone induction via GUS reporter gene activity in transiently transformed tobacco leaves. A specific up-regulation of GUS activity conferred by this Lin5 promoter fragment in response to gibberellic acid (GA), auxin and abscisic acid (ABA) treatment was observed, indicating a critical role of the regulation of Lin5 by phytohormones in tomato flower and fruit development. In situ hybridization analysis of Lin7 shows a high tissue-specific expression in tapetum and pollen. These results support an important role for Lin5 and Lin7 extracellular invertases in the development of reproductive organs in tomato and contribute to unravel the underlying regulatory mechanisms.     

An mRNA that specifically accumulates in maize roots delineates a novel subset of developing cortical cells

A near full-length cDNA clone (pZRP3) corresponding to an mRNA that accumulates specifically in roots of maize was isolated. The ZRP3 mRNA is ca. 600 nucleotides in length. The amino acid sequence of the predicted polypeptide is rich in leucine (16%), proline (11%), and cysteine (8.5%). The zrp3 gene appears to be expressed exclusively in roots, whereas other ZRP3-related genes are expressed in additional organs of the maize plant. In situ hybridization shows that ZRP3 mRNA accumulation is largely confined to the cells of the cortical ground meristem. Furthermore, accumulation of this mRNA occurs within a distinct subset of cortical cells, the inner three to four cell layers.Journal paper number J-14572 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa Project Number 2997.     

A dioxygenase gene (Ids2) expressed under iron deficiency conditions in the roots of Hordeum vulgare

A zapII cDNA library was constructed from mRNA isolated from Fe-deficient barley roots and screened with cDNA probes made from mRNA of Fe-deficient and Fe-sufficient (control) barley roots. Seven clones were selected. Among them a clone having the putative full-length mRNA of dioxygenase as judged by northern hybridization was selected and named Ids2 (iron deficiency-specific clone 2). Using a cDNA fragment as probe, two clones from the genomic library (EMBL-III) were isolated and one was sequenced. The predicted amino acid sequence of Ids2 resembled that of 2-oxoglutarate-dependent dioxygenase. Ids2 is expressed in the Fe-deficient barley roots but is not in the leaves. The expression is repressed by the availability of Fe. Ids2 was also strongly expressed under Mn deficiency and weakly under Zn deficiency or excess NaCl (0.5%). The upstream 5-flanking region of Ids2 has a root-specific cis element of the CaMV 35S promoter and a nodule-specific element of leghemoglobin, a metal regulatory element (MRE) and several Cu regulatory elements (UAS) of yeast metallothionein (CUP1).     

Characterization of a cDNA encoding a proline-rich 14 kDa protein in developing cortical cells of the roots of bean (Phaseolus vulgaris) seedlings

A cDNA clone, corresponding to mRNAs preferentially expressed in the roots of bean (Phaseolus vulgaris L.) seedlings, was isolated. This clone contains a 381 bp open reading frame encoding a polypeptide of 13.5 kDa, designated PVR5 (Phaseolus vulgaris root 5). The amino acid sequence of this clone is rich in proline (13.5%) and leucine (12.7%) and shares significant amino acid sequence homology with root-specific and proline-rich proteins from monocots (maize and rice), and proline-rich proteins from dicots (carrot, oilseed rape, and Madagascar periwinkle). The precise biological roles of these polypeptides are unknown. PVR5 mRNA accumulation is developmentally regulated within the root, with high levels at the root apex and declining levels at distances further from the root tip. In situ hybridization shows that PVR5 mRNA specifically accumulates in the cortical ground meristem in which maximal cell division occurs. Southern blot analysis suggests that genomic DNA corresponding to PVR5 cDNA is encoded by a single gene or a small gene family.     

A histidine decarboxylase-like mRNA is involved in tomato fruit ripening

DNA sequencing of a tomato ripening-related cDNA, TOM 92, revealed an open reading frame with homology to several pyridoxal 5-phosphate histidine decarboxylases, containing the conserved amino acid residues known to bind pyridoxal phosphate and -fluoromethylhistidine, an inhibitor of enzyme activity. TOM 92 mRNA accumulated during early fruit ripening and then declined. Fruit of the ripeningimpaired tomato mutant, ripening inhibitor (rin), did not accumulate TOM 92 mRNA, and its accumulation was not restored by treatment of fruit with ethylene. The TOM 92 mRNA was not detected in tomato leaves and unripe fruit.     

Characterization of tomato endo-β-1,4-glucanase Cel1 protein in fruit during ripening and after fungal infection

The tomato (Lycopersicon esculentum Mill.) endo--1,4-glucanase (EGase) Cel1 protein was characterized in fruit using specific antibodies. Two polypeptides ranging between 51 and 52 kDa were detected in the pericarp, and polypeptides ranging between 49 and 51 kDa were detected in locules. The polypeptides recognized by Cel1 antiserum in fruit are within the size range predicted for Cel1 protein and could be derived from heterogeneous glycosylation. Cel1 protein accumulation was examined throughout fruit ripening. Cel1 protein appears in the pericarp at the stage in which many ripening-related changes start, and remains present throughout fruit ripening. In locules, Cel1 protein is already present at the onset of fruit ripening and remains constant during fruit ripening. This pattern of expression supports a possible role for this EGase in the softening of pericarp tissue and in the liquefaction of locules that takes place during ripening. The accumulation of Cel1 protein was also analyzed after fungal infection. Cel1 protein and mRNA levels are down-regulated in pericarp after Botrytis cinerea infection but are not affected in locular tissue. The same behavior was observed when fruits were infected with Penicillium expansum, another fungal pathogen. Cel1 protein and mRNA levels do not respond to wounding. These results support the idea that the tomato Cel1 EGase responds to pathogen infection and supports a relationship between EGases, plant defense responses and fruit ripening.This revised version was published online in August 2004 with corrections to Fig. 1 and Fig. 5.     

Avocado cellulase: nucleotide sequence of a putative full-length cDNA clone and evidence for a small gene family

A cDNA library was prepared from ripe avocado fruit (Persea americana Mill. cv. Hass) and screened for clones hybridizing to a 600 bp cDNA clone (pAV5) coding for avocado fruit cellulase. This screening led to the isolation of a clone (pAV363) containing a 2021 nucleotide transcribed sequence and an approximately 150 nucleotide poly(A) tail. Hybridization of pAV363 to a northern blot shows that the length of the homologous message is approximately 2.2 kb. The nucleotide sequence of this putative full-length mRNA clone contains an open reading frame of 1482 nucleotides which codes for a polypeptide of 54.1 kD. The deduced amino acid composition compares favorably with the amino acid composition of native avocado cellulase determined by amino acid analysis. Southern blot analysis of Hind III and Eco RI endonuclease digested genomic DNA indicates a small family of cellulase genes.     

Isolation and characterisation of cDNA clones for tomato polygalacturonase and other ripening-related proteins

Summary Gene expression during the ripening of tomato fruit was investigated by cDNA cloning and hybrid-select translation. A cDNA library was prepared from poly(A)-containing mRNA from ripe tomato fruit and sreened by differential hybridization. 146 ripening-related cDNA clones were found. Eleven groups and eight unique clones have been identified so far. The sizes of the cloned cDNA inserts were determined and type-members for seven groups were used in hybrid selection experiments. Six of the seven clones encode translation products corresponding to six ripening related polypeptides detected previously by in vitro translation of total cytoplasmic RNA (14). One cDNA group codes for a M_r 48 000 protein that was identified as polygalacturonase on the basis of immunoprecipitation with specific antiserum raised against tomato polygalacturonase. re]19840918 rv]19850613 ac]19850618     

Changes in ripening-related processes in tomato conditioned by the alc mutant

Summary The alc mutation affects the ripening and storability of tomato fruit. The alteration of fruit color in alc lines is due to a reduction in total pigment and a reduction in lycopene relative to total carotinoids. Polygalacturonase (PG) activity is reduced to less than 5% of normal, and the isozymes PG2a and PG2b are absent in alc fruit. The level of anti-PG precipitable proteins is also reduced to less than 5% of normal. Total polyA + mRNA is not significantly reduced in ripening alc fruit, but hybridization of polyA + mRNA to different ripening-related cDNA clones showed that specific mRNAs are present at reduced levels in the mutant. Specific mRNA levels were reduced to 10%–80% of normal levels, depending on the cDNA clone used as the probe. PG mRNA was present at 5%–10% of the normal level.All effects of alc on fruit ripening are relived in the line Alcobaca-red, which arose spontaneously from the original alc line, Alcobaca. The Alcobaca-red trait segregates as a single dominant trait at or very near the alc locus, and it is probably the result of a reverse mutation at the alc locus.The chromosomal locations of regions homologous to 5 ripening-related cDNA probes were determined. Regions homologous to 4 of these probes map to chromosomes other than chromosome 10, indicating that the effects of alc are transactive. A cDNA clone for PG was homologous to only one chromosomal region. This region is located on chromosome 10, which is also the chromosome on which alc and nor are located.     

Tomato extensin and extensin-like cDNAs: structure and expression in response to wounding

Two tomato cDNA libraries were synthesized from poly(A)⁺ RNAs isolated from unwounded and wounded tomato stems. These cDNA libraries were packaged in gt10 and screened by in situ plaque hybridization with a tomato extensin gene clone (pTom 5.10). Several cDNA clones were identified and isolated from both libraries in this manner and subjected to restriction enzyme digestion, Southern gel blot hybridization, RNA gel blot hybridization, and DNA sequence analyses. From these analyses, the various cDNA clones were found to fall into one of five distinct classes (classes I–V). Class I clones hybridized to a 4.0 kb mRNA which accumulated markedly after wounding and encoded an extensin characterized largely by Ser-(Pro)₄-Ser-Pro-Ser-(Pro)₄-(Tyr)₃-Lys repeats. Class II clones hybridized to a 2.6 kb mRNA which showed no accumulation following wounding and encoded an extensin containing Ser-(Pro)₄-Ser-Pro-Ser-(Pro)₄-Thr-(Tyr)_1–3-Ser repeats. Class III clones hybridized to a 0.6 kb mRNA which greatly accumulated in response to wounding and encoded a glycine-rich protein (GRP) with (Gly)_2–6-Tyr-Pro and(Gly)_2–6-Arg repeats. Class IV clones contained both class I and class III DNA sequences and consequently hybridized to both the 4.0 kb and the 0.6 kb wound-accumulating mRNAs; these clones encoded a portion of a GRP sequence on one DNA strand and encoded a portion of an extensin sequence on the other DNA strand. Class V clones hybridized to a 2.3 kb mRNA which decreased following wounding and encoded a GRP sequence characterized by (Gly)_2–5-Arg repeats.     

cDNA cloning and developmental expression of pea nodulin genes

A cDNA library prepared from pea nodule poly(A)⁺ RNA was screened by differential hybridization with cDNA probes synthesized from root and nodule RNA respectively. From the cDNA clones that hybridized exclusively with the nodule probe five clones, designated pPsNod 6, 10, 11, 13 and 14 and each containing unique sequences, were further characterized together with one leghemoglobin and one root-specific cDNA clone. In vitro translation of RNA selected by the pPsNod clones showed that the corresponding genes encode nodulins with molecular weights ranging from 5 800 to 19 000. During pea root nodule development expression of the five PsNod genes starts more or less concomitantly with the onset of nitrogen fixing activity in the nodules and the time course of appearance and accumulation of the nodulin mRNAs is similar to that of leghemoglobin mRNA. In ineffective pea root nodules expression of the PsNod genes is induced but the final accumulation levels of the mRNAs are markedly reduced to various degrees. The expression of another nodulin gene, designated ENOD2, was followed using a heterologous soybean cDNA clone as probe. In pea root nodules the ENOD2 gene is expressed at least five days before the PsNod and leghemoglobin genes, and in contrast to the PsNod mRNAs the concentration of the ENOD2 mRNA is the same in wild type and fix ^- nodules. The results described suggest that in root nodules several regulatory mechanisms exist which determine the final nodulin mRNA amounts accumulating in the root nodule.     

Molecular cloning of tomato pectin methylesterase gene and its expression in rutgers, ripening inhibitor, nonripening, and never ripe tomato fruits

We have purified pectin methylesterase (PME; EC 3.1.11) from mature green (MG) tomato (Lycopersicon esculentum Mill. cv Rutgers) pericarp to an apparent homogeneity, raised antibodies to the purified protein, and isolated a PME cDNA clone from a λgtll expression library constructed from MG pericarp poly(A)⁺ RNA. Based on DNA sequencing, the PME cDNA clone isolated in the present study is different from that cloned earlier from cv Ailsa Craig (J Ray et al. [1989] Eur J Biochem 174:119-124). PME antibodies and the cDNA clone are used to determine changes in PME gene expression in developing fruits from normally ripening cv Rutgers and ripening-impaired mutants ripening inhibitor (rin), nonripening (nor), and never ripe (Nr). In Rutgers, PME mRNA is first detected in 15-day-old fruit, reaches a steady-state maximum between 30-day-old fruit and MG stage, and declines thereafter. PME activity is first detectable at day 10 and gradually increases until the turning stage. The increase in PME activity parallels an increase in PME protein; however, the levels of PME protein continue to increase beyond the turning stage while PME activity begins to decline. Patterns of PME gene expression in nor and Nr fruits are similar to the normally ripening cv Rutgers. However, the rin mutation has a considerable effect on PME gene expression in tomato fruits. PME RNA is not detectable in rin fruits older than 45 days and PME activity and protein begin showing a decline at the same time. Even though PME activity levels comparable to 25-day-old fruit were found in root tissue of normal plants, PME protein and mRNA are not detected in vegetative tissues using PME antibodies and cDNA as probes. Our data suggest that PME expression in tomato pericarp is highly regulated during fruit development and that mRNA synthesis and stability, protein stability, and delayed protein synthesis influence the level of PME activity in developing fruits.