Rolf E. Christoffersen

Isolation and characterization of a cellulase gene family member expressed during avocado fruit ripening.

SummaryWe present in this paper the structural analysis of two members of a small cellulase gene family, designated cel1 and cel2, from avocado. These genes were isolated by screening a λ EMBL3 genomic library with a ripening-induced cellulase cDNA. Restriction endonuclease and Southern blot analyses showed that the cel1 gene is highly homologous to the cellulase cDNA and thus represents a ripening-related cellulase gene. The other cellulase gene, cel2, is closely related to cel1, but is divergent at its 5′ end. The nucleotide sequence of a 5 kb region encompassing the cel1 gene was determined. Four previously characterized cellulase cDNAs from ripe fruit are identical to the eight exons of the cel1 gene. RNase protection and primer extension analyses were used to define the transcription start site of cel1 and to quantitate cel1 transcripts in ripening fruit. The cel1 mRNA was present at a low level in unripe fruit and increased 37-fold during ripening. Partial DNA sequence analysis of cel2 and comparison to the cel1 sequence revealed a high degree of similarity both at the DNA and deduced amino acid sequence levels. No characterized cellulase cDNAs derived from ripe fruit represent cel2 transcripts. These data suggest that the cel1 gene is responsible for a major portion, if not all, of the cellulase transcripts in ripe fruit. The DNA sequence of 1.4 kb of 5′ flanking DNA of the cel1 gene was compared to the upstream sequence of other ethylene-regulated genes. Several interesting upstream sequence motifs were identified and are discussed.

Nucleotide sequence of a ripening-related cDNA from avocado fruit.

Fruit ripening is a developmental process involving a series of coordinated biochemical and physiological changes, leading to a soft edible fruit [2]. A number of specific mRNAs increase with ripening of mature avocado fruit [3]. Among these are the messages for cellulase [3], a cytochrome P-450 oxidase [ 1 ], and a small number of other genes, as yet unidentified [4, 10]. In order to understand the function and regulation of ripening-related genes, we have analyzed the

Cytochrome P-450-catalysed monoterpenoid oxidation in catmint (Nepeta racemosa) and avocado (Persea americana); evidence for related enzymes with different activities

A cytochrome P-450 present in ripening avocado (Persea americana) fruit mesocarp (CYP71A1) had previously been shown to metabolize the monoterpenoids nerol and geraniol (Hallahan et al. (1992) Plant Physiol. 98, 1290-1297). Using DNA encoding CYP71A1 as a hybridization probe, we have shown by Southern analysis that a related gene is present in the catmint, Nepeta racemosa. RNA blot analysis, together with Western analysis of catmint leaf polypeptides using avocado cyt P-450 antiserum, showed that a closely related gene is expressed in catmint leaves. Cytochrome P-450 in catmint microsomes catalysed the specific hydroxylation of nerol and geraniol at C-10, whereas avocado CYP71A1, in either avocado microsomes or heterologously expressed in yeast, catalysed 2,3- or 6,7-epoxidation of these substrates. These results suggest that orthologous genes of the CYP71 family are expressed in these two plant species, but catalyse dissimilar reactions with monoterpenoid substrates.

Ripening-related polygalacturonase cDNA from avocado.

During the ripening of avocado (Persea americana) fruit, extensive cell wall degradation leads to a dramatic softening of the mesocarp tissue. Two enzymes have been suggested to play a critica1 role in this ripening-associated cell wall degradation in many fruits, including avocado, viz. cellulase (endo-~-1,4-glucanase) and endo-PG (Awad and Young, 1979; Fischer and Bennett, 1991). Although the avocado cellulase cDNA (Tucker et al., 1987) and gene (Cass et al., 1990) have been cloned and sequenced, the avocado PG has not previously been characterized at the molecular level. The ripe avocado fruit is known to contain numerous electrophoretic variants of the PG protein (Kanellis et al., 1991), but whether these represent different gene products or posttranslational modifications of one or more primary polypeptides is unknown. In tomato fruit, the various PG isoforms that accumulate during ripening (PG1, PG2A, and PG2B) are derived from a single gene (Fischer and Bennett, 1991). Using heterologous hybridization with a tomato cDNA probe, we have identified a putative avocado PG cDNA (pAVOpg) from a library generated with RNA from ripe avocado fruit (Table I). The cDNA contains an insert of 1725 bp that detects a 1900-nucleotide mRNA on hybridization to ripe fruit RNA and no detectable signal in mRNA from unripe fruit. This pattem of expression during ripening in avocado parallels that previously reported for PG enzyme activity (Awad and Young, 1979). Sequence analysis of the pAVOpg cDNA revealed an open reading frame of 453 amino acids, which is similar in size to that reported for avocado PG protein (Kanellis et al., 1991). This putative avocado PG sequence shows close similarity to PG sequences derived from both tomato and com, 52 and 36% identity, respectively. In addition, it contains an octapeptide motif that is conserved in PG sequences derived from plant, fungal, and bacterial species (Bairoch, 1992). This octapeptide is contained within a span of 14 residues (TCGPGHGISIGSLG) that is identical among the known plant PG sequences and thus should allow characterization of other plant PG genes using degenerate oligonucleotide primers in polymerase chain reaction experiments.

Avocado fruit protoplasts : a cellular model system for ripening studies

SummaryMesocarp protoplasts were isolated from mature avocado fruits (Persea americana cv. Hass) at varying stages of propylene-induced ripening. Qualitative changes in the pattern of radiolabel incorporation into polypeptides were observed in cells derived from fruit at the different stages. Many of these differences correlate with those observed during radiolabeling of polypeptides from fresh tissue slices prepared from unripe and ripe fruit. Protoplasts isolated from fruit treated with propylene for one day or more were shown to synthesize cellulase (endo-ß-1,4-glucanase) antigen, similar to the intact propylene-treated fruit. These results suggest that the isolated protoplasts retain at least some biochemical characteristics of the parent tissue. The cells may also be used in transient gene expression assays. Protoplasts isolated from preclimacteric and climacteric fruit were equally competent in expressing a chimeric test gene, composed of the CaMV 35S RNA promoter fused to the bacterial chloramphenicol acetyltransferase gene, which was introduced by electroporation.

Enhancing Undergraduate Success in Biology Through the Biomentors Program

Abstract Many undergraduates who wish to pursue degrees in science, particularly students from underrepresented groups, drop out of science majors before realizing their goal. This study examines the effectiveness of a mentoring program — called Biomentors — aimed at promoting success in biology courses for undergraduates beginning their coursework toward a bachelors degree in the biological sciences. Students enrolled in the Biomentors program met twice a week in a small group with an advanced biology major under the supervision of a faculty member to explore effective learning strategies for success in an introductory-level biology course they were taking. Students who participated in the Biomentors program scored significantly higher (based on total points earned) than other students enrolled in the course across two cohorts (d = 0.36 in the fall quarter of 2014; d = 0.34 in the winter quarter of 2015). The biomentors group significantly outscored the control group even when the effects of gender, parent income level, parent education level, total SAT score, and cumulative GPA were statistically controlled using a stepwise regression. Overall, the results encourage further investigation of the effectiveness of peer-mentoring programs that emphasize domain-specific learning strategies for college students beginning as science majors.