Progress 10/01/02 to 09/30/05

Outputs
We have constructed three cDNA libraries from papaya male, hermaphrodite, and female flower buds before meiosis (bud length < 4mm). Protein analysis revealed that cpAP3 shares 60% identity and 75% similarity with the tomato ortholog TDR6, but it shares only 30% identity and 65% similarity with the Arabidopsis AtAP3. Similarly, cpPI shares 70% identity and 85% similarity with the Betula pendula PI ortholog, but only shares 60% identity and 80% similarity with the Arabidopsis AtPI. Within the MADS box region, cpPI both shares greater than 98% amino acid sequence identity (only one amino acid difference) with Antirrhinum PI ortholog GLO and greater than 90% sequence identity with the AtPI. The MADS box region of cpAP3 appears to have more variation from its Arabidopsis counterpart, sharing only 80% sequence identity with AtAP3. The Arabidopsis thaliana SUP (AtSUP) gene was used to screen a papaya BAC library. Positive BAC clones were placed into one of four groups based on the fragment size showing homology to the AtSUP gene. The four fragment sizes were 1.2K, 2.5K, 3.5K and 4.0K. A single BAC clone from each group was sub-cloned into the pPCR-Script Amp vector using Hind III digested fragments. Sequence analysis revealed that the 3.5K positive fragment BAC clone encodes a protein similar to that encoded by the entire coding region of the Arabidopsis thaliana SUP gene. At the protein level, the papaya SUP ortholog, PSUP, shares 43% identity and 52% similarity with Arabidopsis thaliana SUP gene. The 1.2K positive fragment BAC clone shares greatest similarity to an Oryza sativa zinc finger protein. The 2.5K positive fragment BAC clone shares greatest similarity to an Arabidopsis thaliana SUP-like protein. The 4.0K positive fragment BAC clone shares greatest similarity to an Arabidopsis thaliana zinc finger protein. In situ hybridization of PSUP to papaya flower buds proved to be difficult because this gene is known to express at low level for only about 10 hours during flower development in Arabidopsis. However, quantitative PCR analysis revealed that PSUP was highly expressed in female flowers, moderate in hermaphrodite flowers, and low in male flowers. Both cpAP3 and cpPI appear to be expressed in the petals of all three sex types as well as the stamens of male and hermaphrodite flowers. Both genes are expressed with equal intensity in these organs in immature and mature flower buds (data not shown). These results complement the in situ results. In situ hybridization results revealed that both cpAP3 and cpPI mRNA was detected in the floral organ primordia of Sun-Up flowers below 0.5mm in length. This expression of both cpAP3 and cpPI in the flower bud primordia has been observed only in the auxiliary buds in male, female and hermaphrodite flower. For both genes, expression was detected throughout the entire undifferentiated primordia, but not seen in the surrounding vegetative bracts. In flowers ranging from 1.0-3.0mm in length, cpAP3 and cpPI mRNA was also detected at a very high level in the stamen and anther tissue, but not in the petals, sepals or the surrounding vegetative tissues.

Impacts
Malformed fruit is a major problem in papaya production in Hawaii. The malformed fruit is caused by partial transformation of stamens (male parts) to carpels (female parts) of hermaphrodite flowers. The economic consequences are easy to understand since the malformed fruit is unmarketable. The fundamental biological process that causes cat-facing fruit is not clear in papaya, although we know it is controlled by genetic factors that are sensitive to environmental conditions. Two major genes, AP3 and PI, control stamen and carpel development and another major gene, Superman, defining the border of stamen and carpel may play important roles in this process, based on the knowledge obtained in model plant species Arabidopsis on flower development. One of the major genes, AP3, is sensitive to temperature. Three papaya flower cDNA libraries have been constructed to clone these major genes. These two target genes, cpAP3 and cpPI, were cloned and sequenced using degenerate primers designed from orthologs of diverse species. We have also cloned a papaya ortholog of SUPERMAN that serves as an upstream regulator of AP3 and PI. In situ hybridization results suggested that cpAP3 and cpPI were expressed in all three sex types of flowers. However, quantitative real time PCR analyses indicated that papaya SUPERMAN ortholog, PSUP, expressed strongly in female, moderately in hermaphrodite, and weakly in male. Understanding the expression patterns and their regulatory process of these target genes may lead to a solution to reduce malformed fruit in papaya.

Publications

• Ackerman, C.M., Yu, Q., Moore, P.H., Paull, R.E. and Ming, R. 2005. Cloning and characterization of APETALA3 and PISTILLATA orthlogs in papaya. Abstract at the Plant Biology 2005 Conference. Seattle, Washington. July 16 - 20, 2005.

Progress 10/01/03 to 09/30/04

Outputs
We have constructed two cDNA libraries from papaya hermaphrodite and female flower buds. Ten sets of high macroarray filters were made for each cDNA library. Using the RNA extracted from male flower buds, cDNA was synthesized using the degenerative AP3 primer. These products were run on gel and hybridized to the Arabidopsis AP3 gene. The positive bands were extracted from the gel and cloned into a vector. One clone showed homolgy to this gene, so it was sequenced. On a protein level, blast results showed a 76% similarity and a 61% identity with the Populus AP3 gene, as well as similarity to many other AP3 homologs. This partial AP3 fragment was used to probe the BAC and cDNA libraries to retrieve the full sequence. One clone contained the full cDNA sequence. Probes for the Arabidopsis PI gene were used on a library from young male flowers. Of the clones generated at the protein level, Blast results showed one clone having 83% similarity and 66% identity to the PI homolog of a Betula species, as well as high similarity to many other PI homologs. This partial PI fragment was used to probe the BAC and cDNA libraries to retrieve the full sequence. The three cDNA clones showed strong homolgy to the papaya PI fragment have been cultured and sequenced. Direct sequencing was used to obtain the complete genomic sequence of the papaya PI gene by primer walking. The floral regulatory gene SUPERMAN (SUP). The Arabidopsis thaliana SUP (AtSUP) gene was used to screen a papaya BAC library. Sequence analysis revealed that the 3.5K positive fragment BAC clone encodes a protein similar to that encoded by the coding region of the Arabidopsis thaliana SUP gene. At the protein level, the papaya SUP ortholog, PSUP, shares 43% identity and 52% similarity with AtSUP gene. Preliminary results from in situ hybridization showed that PSUP is expressed in all three sex forms of three papaya genotypes: Kapoho, SunUp and AU9. Very young flower buds of all sexes showed strong expression along the periphery of the flower primordium while expression in the older flower buds is weaker and concentrated in the stamens. In the case of the male flower buds, expression in the stamens is stronger in young buds, but decreases with increasing age of the bud.

Impacts
Understanding the biological process that triggers malformed fruit in papaya is the first step towards solving this problem and improving productivity. Malformed or cat-facing fruit is one of the major problems in papaya production in Hawaii. The malformed fruit is caused by partial transformation of stamens (male parts) to carpels (female parts) of hermaphrodite flowers. The economic consequences are easy to understand since the malformed fruit is unmarketable. The fundamental biological process that causes cat-facing fruit is not clear in papaya. In the last ten months, scientists at UH and HARC have constructed papaya flower cDNA libraries for cloning these major genes. A number of candidate cDNA and BAC clones were identified. Some of the cDNA clones have been partially sequenced, and several matched AP3-like proteins. Three of the four copies of Superman in papaya were cloned and partially sequenced. Identifying the target genes and understanding their expression patterns may lead to a solution to reduce malformed fruit in papaya.

Publications

• Ackerman, C.M., Yu, Q., Moore, P.H., Paull, R.E., Steiger, D.L. and Ming, R. 2004. Cloning and characterization of a papaya Superman ortholog. Abstract at the Plant Biology 2004 Conference. Lake Buena Vista, Florida. July 24-28, 2004.

Progress 10/01/02 to 09/30/03

Outputs
We have constructed two cDNA libraries from papaya hermaphrodite and female flower buds. The titers of hermaphrodite and female cDNA library are 1X1010 pfu/ml and 7.8X109 pfu/ml, respectively. Ten sets of high macroarray filters were made for each cDNA library. We have screened the papaya cDNA and bacterial artificial chromosome (BAC) libraries with AP3 and PI. Although a number of BAC and cDNA clones were identified by AP3 (none by PI), none were confirmed containing the papaya AP3 homologe positive. AP3 and PI belong to MADS box gene family. We used the conserved MADS box regions as probes to screen the cDNA and BAC libraries. Four of the 20 hermaphrodite cDNA clones matched the sequence of AP3 like protein in the GenBank. One of the 15 female cDNA clones matched the sequence of AP3 like protein. The gene Superman defines the border between stamens and carpels of a hermaphrodite flower. We have identified papaya BAC clones containing the Superman genes, and these BAC clones have four different restriction patterns suggesting four different copies of the Superman genes in papaya. We cloned three of the four copies and one appeared to be a pseudo-gene. The second copy contained the full length of the superman gene and shared 85% similarity with the Arabidopsis Superman at the nucleotide level and 55% similarity at the protein level. The third copy has only 150 bp coding sequences in the sub-clone we have sequenced, and it was highly similar to a zinc finger domain of the Superman gene.

Impacts
Understanding the biological process that triggers malformed fruit in papaya is the first step towards solving this problem and improving productivity. Malformed or cat-facing fruit is one of the major problems in papaya production in Hawaii. The malformed fruit is caused by partial transformation of stamens (male parts) to carpels (female parts) of hermaphrodite flowers. The economic consequences are easy to understand since the malformed fruit is unmarketable. The fundamental biological process that causes cat-facing fruit is not clear in papaya. In the last ten months, scientists at UH and HARC have constructed papaya flower cDNA libraries for cloning these major genes. A number of candidate cDNA and BAC clones were identified. Some of the cDNA clones have been partially sequenced, and several matched AP3-like proteins. Three of the four copies of Superman in papaya were cloned and partially sequenced. Identifying the target genes and understanding their expression patterns may lead to a solution to reduce malformed fruit in papaya.

Publications

• No publications reported this period

Progress 10/01/01 to 09/30/02

Outputs
No progress to report. This project was initiated on October 1, 2002.

Impacts
(N/A)

Publications

• No publications reported this period