Isaac John

Isolation and analysis of cDNAs encoding tomato cysteine proteases expressed during leaf senescence.

Several cDNAs for mRNAs that change in abundance during tomato leaf senescence were isolated. In this paper we report molecular cloning and expression analysis of two cysteine proteases. SENU2 is identical to the cDNA C14 which encodes a cysteine protease previously shown to be expressed in response to extremes of temperature in tomato fruit [43]. SENU3 cDNA clone was 1.2 kb in length and hybridized to a transcript of 1.4 kb which suggested that the clone was not full-length. The missing 5′ end was isolated using rapid amplification of cDNA ends (RACE). Southern blot analysis of tomato genomic DNA indicates that SENU3 is encoded by a single or low copy gene. SENU3 was also shown to have significant homology with known cysteine proteases. These two senescence-associated cysteine proteases are also expressed during other developmental processes, including seed germination, consistent with a role in protein turnover. SENU2 and SENU3 mRNAs were detectable in young fully expanded leaves and increased in abundance with leaf age, reaching a maximum during the later stages of visible leaf senescence. Such a pattern of expression suggests that the onset of leaf senescence is a gradual event. Analysis of senescence in transgenic plants deficient in ethylene biosynthesis, in which leaf senescence is delayed, indicated that enhanced accumulation of SENU2 and SENU3 mRNA was similarly delayed but not prevented.

Whole Plant Senescence

Publisher Summary This chapter discusses causes of monocarpic senescence and senescence in polycarpic plants and clones. Whole plant senescence is viewed primarily in terms of leaf senescence and it is measured mainly through chloroplastic parameters such as chlorophyll loss and decreased photosynthesis. In monocarpic plants where senescence and death closely follow reproductive development, the senescence (monocarpic senescence) is often controlled by the developing reproductive structures. In these cases, removal of the reproductive structures or prevention of their development usually prolongs the life of the plant. Monocarpic plants cease their vegetative growth fairly abruptly early in their reproductive phase. Conversely, the perennial polycarpic pattern requires continued vegetative growth. This prominent shift (diversion) in growth-related allocation of resources in monocarpy seems to be part of a reproductive strategy that optimizes reproductive output for the plants. This diversion/withdrawal is often quite prominent that leads to monocarpic senescence.

Cloning and characterization of tomato leaf senescence-related cDNAs

Senescence-related cDNA clones designated SENU1, 4, 5 (senescence up-regulated) and SEND32, 33, 34, 35 and 36 (senescence down-regulated) isolated from a tomato leaf cDNA library [9] were characterized. Southern analysis showed that SEND32 is encoded by a single-copy gene while SEND33, 34, 35, 36 and SENU1 and SENU5 are members of small gene families. DNA and protein database searches revealed that SEND32, SEND35, SENU1 and SENU5 are novel cDNAs of unknown function. SEND33 encodes ferredoxin, SEND34 encodes a photosystem II 10 kDa polypeptide and SEND36 encodes catalase. The SENU4 sequence is identical to the P6 tomato protein previously reported to be pathogenesis-related [46]. The mRNA levels of SENU1, 4 and 5 increased during leaf senescence and SENU1 and SENU5 were also expressed at high levels during leaf development and in other plant organs. The SENU4 mRNA was associated more specifically with leaf senescence, although low expression was also detected in green fruit. The mRNAs for all SEND clones decreased during tomato leaf development and senescence and all except SEND32 were expressed at low levels in other plant organs. The accumulation of mRNA homologous to SENU4 and the decrease in abundance of SEND32 provide good molecular markers for leaf senescence.

An mRNA Putatively Coding for an O-Methyltransferase Accumulates Preferentially in Maize Roots and Is Located Predominantly in the Region of the Endodermis

ZRP4, a 1.4-kb mRNA that preferentially accumulates in roots of young Zea mays L. plants, was identified by isolation of the corresponding cDNA clone. Genomic Southern analysis indicates that the zrp4 gene is represented once in the corn genome. The deduced ZRP4 polypeptide of 39,558 D is rich in leucine, serine, and alanine. Comparison of the deduced ZRP4 polypeptide sequence to polypeptide sequences of previously cloned plant and animal genes indicates that ZRP4 may be an O-methyltransferase. The ZRP4 mRNA preferentially accumulates in young roots and can be detected only at low levels in leaf, stem, and other shoot organs. ZRP4 mRNA accumulation is developmentally regulated within the root, with very low levels of accumulation in the meristematic region, higher levels in the regions of cell elongation, highest levels in the region of cell maturation, and low levels in the mature regions of the root. ZRP4 mRNA is predominantly located in the endodermis, with lower levels in the exodermis. An intriguing possibility is that the ZRP4 mRNA may code for an O-methyltransferase involved in suberin biosynthesis.

Characterization of two cDNA clones for mRNAs expressed during ripening of melon (Cucumis melo L.) fruits

In vitro translation of mRNAs and polyacrylamide gel electrophoresis of proteins from melons revealed that several mRNAs increased in amount during ripening, indicating the existence of other ripening genes in addition to those cloned previously. To identify ripening-related genes we have screened a ripe melon cDNA library and isolated two novel cDNA clones (MEL2 and MEL7) encoding unidentified proteins. Southern analysis revealed that MEL2 and MEL7 are encoded by low-copy-number genes. The MEL2 cDNA clone is near full-length, corresponds to a 1600 nucleotide mRNA that accumulates during ripening and encodes a predicted protein rich in hydrophobic amino acids. The MEL7 cDNA clone is full-length, corresponds to a mRNA of 0.7 kb which accumulates during early ripening stages and is also present at low levels in other organs of the melon plant. The MEL7 predicted polypeptide is 17 kDa and shows significant homology with the major latex protein from opium-poppy. Wounding and ethylene treatment of unripe melon fruits 20 days after anthesis showed that MEL2 and MEL7 mRNAs are only induced by ethylene.

Isolation and characterisation of a melon cDNA clone encoding phytoene synthase.

A cDNA clone (MEL5), encoding a protein homologous to phytoene synthase (PSY), has been isolated from a climacteric melon fruit cDNA library, using the tomato cDNA clone TOM5 [34] as a heterologous probe. MEL5 hybridised to a transcript of 1.65 kb which suggested that the 1.36 kb clone, isolated originally, was not full-length. The missing 5′ end was isolated by a reverse transcriptase-polymerase chain reaction (RT-PCR)-based method. This enabled the full sequence of the protein to be deduced and the cleavage site of the transit peptide for chromoplast import to be predicted. Northern analysis of RNA extracted from fruit samples of different ripening stages as well as from roots, leaves and flower petals was used to examine the expression pattern of the corresponding mRNA. The transcript corresponding to MEL5 is present at low quantities in unripe (green) fruit, reaches its highest levels when the fruit turns from green to orange and persists at lower levels during later ripening stages. A similar transcript was also detected in flower petals and in trace amounts in leaves and roots. Genomic Southern analysis indicates that the clone is homologous to a low-copy-number gene family. Sequence analysis showed a high degree of conservation among plant PSYs.

zrp2: a novel maize gene whose mRNA accumulates in the root cortex and mature stems

A near full-length cDNA clone (pZRP2) was isolated from a cDNA library constructed from maize root mRNAs. The predicted polypeptide has a calculated molecular mass of 66 975 Da, is largely hydrophilic, and contains 26 repeats of a motif the consensus sequence of which is RKATTSYG[S][D/E][D/E][D/E][D/E][P]. The function of the putative protein remains to be elucidated. The ZRP2 mRNA accumulates to the highest levels in young roots, and is also present in mature roots and stems of maize. Further analysis of young roots indicates that the lowest level of ZRP2 mRNA is near the root tip, with relatively high levels throughout the remainder of the root. In situ hybridization reveals that ZRP2 mRNA accumulates predominantely in the cortical parenchyma cells of the root. In vitro nuclear run-on transcription experiments indicate a dramatically higher level of zrp2 gene transcription in 3-day old roots than in 5-day old leaves. A zrp2 genomic clone, which includes the transcribed region and 4.7 kb of upstream sequence, was isolated and characterized.