Carole L. Bassett
Levels of the large subunit (LSU) of rubisco (ribulose 1,5 bisphosphate carboxylase/oxygenase) were measured in vegetative and floral organs of `Violet' Japanese morning glory (Ipomoea [Pharbitis] nil Roth). Identification of the LSU in polypeptides separated by two-dimensional gel electrophoresis allowed estimation of the relative abundance of this polypeptide in the organs examined. Further quantitation was achieved by immunoblotting protein extracts either alone or in combination with various amounts of extracts from other organs. The amount of LSU decreased in the order leaves > cotyledons > sepals > corollas > androecium, gynoecium. The relative abundance of LSU in sepals (74%) compared to photosynthetically competent organs [leaves (100%) and cotyledons (81%)] suggests that sepals may be photosynthetically competent in supporting development of the other floral organs.
D. Michael Glenn and Carole Bassett
Plant ash content has been highly correlated with plant water use efficiency (WUE) and Δ13C (Δ) in field crops and grassland species and proposed as a selection criteria for WUE. δ18O (δ) has also been correlated with transpiration in herbaceous plants. The objectives of the study were to 1) evaluate the relationship of shoot ash (ASH) with Δ and δ in ‘Empire’ apple over a 3-year period; 2) determine if yearly variation significantly affected the relationship of ASH with Δ and δ; and 3) evaluate the value of the relationship between ASH content with Δ and δ for a population of Malus sieversii. ‘Empire’ leaf area index (LAI) was negatively correlated with ASH content and positively correlated with Δ. Δ was negatively correlated with ‘Empire’ ASH. There were no yearly effects at a site. Within the Malus sieversii accessions, there was a grouping based on ASH that could be identified and this outlier group also had the lowest δ of the accessions. There were no correlations of leaf area, number, length, width, or stomata number with δ, Δ, or ASH for the Malus sieversii accessions. Rather than a substitute for Δ measurement in assessing WUE, ash content analysis adds an additional dimension to understanding the dynamics of WUE in apple. This work has identified a unique population of Malus meriting further study.
Michael Wisniewski, Carole Bassett and Lawrence V. Gusta
Carole L. Bassett and Timothy S. Artlip
During the past several years, we have been interested in genes and gene-products involved in various aspects of ripening and maturation in peach (Prunus persica) fruit. The ethylene biosynthetic and signal transduction pathways are of particular interest due to the role of this hormone in such processes. Recently, we isolated a cDNA encoding a homologue of the ethylene receptor ETR1 from a near fully ripe (20–60N) peach fruit cDNA library. This cDNA clone, PpETR1, is nearly 2300 bp in length, with a 5' untranslated region of 268 bp, a 3' untranslated region of 150 bp, and an ORF of 1881 bp, encoding a protein of 70 kDa. The cDNA is most closely related to an ETR1 homologue from apple (Malus domestica), i.e., 95% identity at the amino acid level, but shows considerable similarity to Arabidopsis thaliana ETR1, as well. A comparison of the similarity among cloned ETR1 genes from a range of plant species will be presented.
Ann M. Callahan and Carole L. Bassett
NADP-dependent Malic Enzyme (NADP-ME, EC 184.108.40.206) catalyzes the decarboxylation of malate, resulting in the release of CO2. In C3 plants the enzyme does not contribute CO2 directly to photosynthesis. Rather, it is associated with the supplemental synthesis of glycolytic and Krebs Cycle intermediates, although it may also be involved in regulating intracellular pH. NADP-ME activity increases during ripening of several fruits e.g. tomato and apple, usually in association with increased respiration of the developing fruit. We examined expression of NADP-ME during ripening in peach using a cDNA probe derived from F. trinervia (C4 dicot). The probe hybridized to a single RNA species of the predicted size and was low in abundance as expected for a C3 NADP-ME. As fruit matured, the RNA levels increased to a maximum around 133-140 days after bloom (fully ripe). NADP-ME RNA was not detectable from leaves isolated at the same time.
Carole L. Bassett and D. Michael Glenn
Reflective films are used in orchard management to improve fruit coloration. Numerous physiological studies on the effects of application of these films have been conducted, including variation of angles of light incidence and reflection, spectral determination of reflected light, and effects on photosynthesis and pigment development. At present there have been no studies on the effects of these treatments on gene expression, particularly with regard to genetic factors controlling light perception and genes targeted by specific regulators. We sampled a fully developed leaf from apple (Malus ×domestica) branches of mature trees in 2 consecutive years. The grass between the tree rows was left untreated, sprayed with a formulation of a reflective particle film (RPF), or covered with a single layer of an aluminized plastic film (APF). Leaves at the same branch position were sampled between 1000 and 1200 hr on sunny days. Nine different gene transcripts associated with light perception, sorbitol transport, and actin (control) were measured by semiquantitative reverse transcription–polymerase chain reaction and five by quantitative reverse transcription–polymerase chain reaction. Transcript abundance of most genes was unaffected by treatment or slightly enhanced by RPF. These results are consistent with earlier observations indicating that application of this film stimulated carbon partitioning to the fruit likely through the reduced red/far-red ratio of reflected light. In contrast, the level of several gene transcripts, including MdPif3, MdPhyB, and MdSot3, was reduced by application of the APF. Only one gene, MdElip1A/B, was significantly and dramatically elevated by APF.
Carole Bassett, Ann Callahan and Linda Dunn
A gene (Lhcb2*Pp1) encoding a type II chlorophyll a/b-binding protein (LHCB) was isolated from peach [Prunus persica (L.) Batsch]. The gene was sequenced and compared to a variety of other genes encoding LHCB polypeptides associated with photosystem II. Similarity at the nucleotide and amino acid level was highest between Lhcb2*Pp1 and other type II genes and was lowest with type I and III genes from other species. Expression of Lhcb2*Pp1 was followed by determining abundance of transcripts in developing leaves of field grown trees in the absence of flowering. Expression was monitored at three times during the first phase of the growing season and was shown to be highest in leaves which were at or near full expansion at each sampling time. These results are consistent with the function of this gene and indicate that it can serve as a marker of photosynthetic maturity under field conditions.
Carole L. Bassett and Ann M. Callahan
Leaf expansion in peach (Prunus persica) cv Loring was monitored by measuring the increase in blade length during the spring and early summer of 1994, a season in which no flowers were observed on the sampled trees. Expansion was correlated with time after vegetative budbreak and with leaf position on growing apical shoots. In preliminary studies, information from these measurements was used to identify the relative maturity of leaves during the growing season in order to define sampling times that would represent “old,” “mature,” and “young” leaves. Leaves in these categories were sampled and pooled on two different dates, and total RNA was isolated from each sample. The RNAs were examined by Northern blot analysis using a 32P-labeled cDNA clone encoding a peach cab (chlorophyll a/b binding protein) gene. Estimates of abundance based on the intensities of RNA bands hybridizing to the probe indicated that RNAs representing the cab gene family were most abundant in “mature” leaves. Further examination of abundance in pooled, individual leaves representing positions 1 through 19 (numbered acropetally) revealed a substantial decline in abundance in leaves from positions 1 through 5, which were already showing signs of senescence. These results are consistent with enhanced expression of the cab gene in the most photosynthetically active leaves.
Carole Bassett, Mike Nickerson, Tim Artlip and Michael Wisniewski
To identify components of a possible signal transduction pathway associated with woody plant cold acclimation, a cDNA library prepared from peach bark collected in December was screened using a small DNA fragment from a maize receptor-like protein kinase (ZMPK1) as probe. Six isolates were obtained and partially sequenced to confirm protein kinase identity. One isolate (PPPKB5) contained a relatively large insert (about 1.9 kbp) and was selected for further analysis. Both strands of PPPKB5 were sequenced and the derived amino acid sequence was compared with a variety of known plant protein kinases, leading to the tentative identification of this clone as encoding a partial cDNA for a serine/threonine class protein kinase. PPPKB5 has a classic protein kinase catalytic domain with all the features required for phosphorylation activity and with greater similarity to the known plant receptor-like protein kinases (RLKs) than other classes of these enzymes. In addition, it contains a potential transmembrane region separating the catalytic portion of the molecule from the receptor region. The receptor region has greatest identity with the class of RLKs known as the S-locus type based on conservation of a stretch of 10 cysteines on the amino-terminal side of the transmembrane region. PPPKB5 shares greater amino acid identity with ZMPK1 (50%) than with the Brassica, rice or Arabidopsis S-locus RLKs (34%–40%). Furthermore, PCR analysis of `Loring' suggests that, like maize ZMPK1, the genomic sequence encoding PPPKB5 contains no introns, in contrast to the genomic sequences of Brassica and Arabidopsis which contain six.