DIFFERENTIAL EXPRESSION OF ETHYLENE INDUCED GENES DURING BANANA FRUIT RIPENING Sanjay Mohan Gupta, A.P.Sane and P.Nath Plant Gene Expression Lab, National Botanical Research Institute Rana Pratap Marg Lucknow-226001 India

INTRODUCTION Fruits undergo a complex series of physiological and biochemical changes during final stages of their development and ripening , that include changes in color, flavor, aroma, texture, hardiness etc. These changes make fruit both attractive and edible thus facilitating seed dispersal. Based on the physiological changes that occur during ripening, fruits have been classified as climacteric and non-climacteric. Climacteric fruits such as tomato, avocado, peaches, plums, apple, banana etc. are distinguished from non-climacteric fruits such as strawberry, grapes, citrus fruits etc. by their ability to exhibit high rates of respiration and a burst in ethylene production during onset of ripening. The process of ripening is a highly coordinated and triggered by ethylene in climacteric fruits. Several genes such as those for ethylene biosynthesis, cell wall degradation, pigment biosynthesis, sugar metabolism etc are differentially expressed. The study of the differential expression of these genes not only provides a clue to the mechanism by which fruits undergo process of ripening but also generates possibilities for utilizing these genes for biotechnological uses such as post harvest management. Genes such as ACS, ACO and PG have already been used to control shelf life of climacteric fruits like tomato, melon and avocado and the technology has been patented. However, there is a need to identify newer genes and promoters, which are ripening related and ethylene regulated and modify their expression in order to improve upon quality of the fruit as also to enable tissue specific expression of desired genes. mRNA differential display provides an excellent technique for identification of novel genes.

CO2 and autocatalytic ethylene evolution in Banana upon the onset of ripening (triggered by ethylene treatment) 0.01 0.02 0.03 Ethylene (nmoles.g-1.h-1 0 1 2 3 4 5 6 7 Days Respiratory Climacteric Ethylene biosynthetic activity

Treatments and RNA Isolation Mature green banana fruits were exposed to 100-ppm ethylene in an airtight container for 24 hrs in dark at 22 C in order to induce ripening. Fruits with no ethylene treatment but other conditions similar as above were considered as control. Mature green banana fruits were exposed to 1ppm MCP also for 24 hrs. Three different stages were chosen for RNA isolation from Banana pulp tissue- Pre climacteric - 0 day (untreated) Climacteric - 2 day (ethylene treated) Post Climacteric - 4 day (ethylene treated) 0 day 2day 4 day C2H4 Treated Control

Schematic representation of one-base anchored differential display Differential Display Reverse Transcription-PCR (DDRT-PCR) is powerful tool to study differential gene expression and has been employed recently for studying gene expression changes associated with climacteric fruit ripening and other developmental processes in plants. The advantage of differential display over alternative methods for differential screening includes the ability to compare simultaneously numerous samples for both induction and repression of specific gene expression. Schematic representation of one-base anchored differential display GTTTTTTTTTTT-H GAAAAAAAAAAA-H TAAAAAAAAAAA-H CAAAAAAAAAAA-H 5’-AAGCTTTTTTTTTTTG-3’ dNTPs MMLV-RT I-Reverse transcription 5’AAGCTTGATTGCC-3 (HAP-01) 32P dCTP Taq DNA polymerase II-PCR amplification GATTGCC III-Denaturing polyacrylamide gel (9%) X Y RNA samples _ + DIFFERENTIAL-DISPLAY

Changes in DDRT protocol for standardization Use of 32P ATP for end labeling of anchor primers to get uniform band intensity, instead of using the conventional 32P dCTP in the DDRT-PCR step. Replicates of each DDRT reactions were performed and only those bands which were reproducible and having a size greater then 250 bp chosen for further studies to reduce the number of false positives. Reverse northern blots which involve preparation of Southern blots from the 200 differentially amplified bands and sequential hybridization with 0 day, 2 day and 4 day total RNA probes were not very informative. The most likely reason could be that, these amplified cDNAs may represent a mixture of more than one independent cDNA fragments of similar molecular weight, which could include both differential as well as constitutively expressed genes. The hybridization of the constitutively expressed gene probe to the band on a reverse-northern blot would mask the truly differentially expressed gene making its identification difficult . Changes in DDRT protocol for standardization

Lanes represent days after ethylene treatment. DDRT-PCR was carried out on RNA isolated from three stages of ripening fruit (0 day, 2 day, 4 day) using HAP-01 to HAP-16 series of random primers from Gene-Hunter and three one-base anchored oligo-dT primers [H-(T)11A, H-(T)11G and H-(T)11,C]. Differential display patterns of cDNA from banana pulp tissue treated with 100ppm ethylene Lanes represent days after ethylene treatment. 0 = untreated 2 = 2 day 4 = 4 day. 0 2 4 0 2 4 0 2 4 A G C

Lanes represent days after ethylene treatment. DDRT-PCR was carried out on RNA isolated from three stages of ripening fruit (0 day, 2 day, 4 day) using HAP-01 to HAP-16 series of random primers from Gene-Hunter and three one-base anchored oligo-dT primers [H-(T)11A, H-(T)11G and H-(T)11,C]. Fig: Differential display patterns of cDNA from Banana pulp tissue treated with 100ppm ethylene before elution (a) and after elution (b). Lanes represent days after ethylene treatment. 0 = untreated 2 = 2 day 4 = 4 day. 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4 0 2 4 A G C (a) (b)

Fig: Reamplified cDNA fragments. A total of 200 differentially amplified bands were eluted and reamplified from three stages of banana ripening (0,2&4day). M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 A 622 bp 309 bp 242 bp 404 bp 427 bp 311 bp 500 bp B M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 C M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 D M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 E 404 bp M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 311bp F Fig: Reamplified cDNA fragments. A,C,E: From HAP-1,2,3,4,6 & 8 gel. Marker : pBR322 DNA/ Msp-1 Digest. B,D,F: From HAP-9,10,12,13&14 gel. Marker : ¢174 DNA /Hinf-1 Digest.

Reamplified cDNA fragments. A total of 200 differentially amplified bands were eluted and reamplified from three stages of banana ripening (0,2&4day). M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 A 622 bp 309 bp 242 bp 404 bp 427 bp 311 bp 500 bp B M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Reamplified cDNA fragments. A: HAP-01 gel Marker : pBR322 DNA/ Msp-1 Digest. B: HAP-02 gel Marker : ¢174 DNA /Hinf-1 Digest. Total Number of differential bands obtained 200 Number of Bands tested on northern blot 85 Number of positives obtained 27 Up-regulated genes 21 Down-regulated genes 6

Expression of twenty one up regulated genes in Banana fruit tissue treated with 100 ppm ethylene. 0 = untreated , 2 = 2day ethylene treated , 4 = 4day ethylene treated. H10-3 H1-18 H1-4 H8-19 H10-1 H9-5 H9-8 H3-5 H4-11 H8-20 H8-21 A B H3-15 H9-3 H9-6 H1-26 H1-35 H9-2 H12-8 H13-2 H14-4 H4-1 0 2 4 0 2 4 A: Ethidium bromide stained gel shows equal RNA loading. B: Northern blot hyb. with 23s rRNA probe.

Expression of Six down regulated gene in banana fruit tissue treated with 100 ppm ethylene. 0 = untreated , 2 = 2day ethylene treated , 4 = 4day ethylene treated A: Ethidium bromide stained gel shows equal RNA loading. B: Northern blot hyb. with 23s rRNA probe. A H2-10 0 2 4 H6-1 B H6-10 H12-5 H6- 9 H6- 19 0 2 4

Seq. identity & %homology Sequence homology of 27 clones novel 0.80 133 Up H8-21 CF542265 0.90 153 H4-11 CF542264 1.1 223 H1-26 175 H8-20 CF542225 1.2 191 H8-19 CF542224 1.45 291 H3-15 CF519299 98%, -1,3 glucanase M.acuminata 1.36 271 H3-5 CF542263 0.75 257 Down H6-10 CF542223 205 H2-10 CF519300 91%, pBANUD75 1.4 370 H6-9 CF519306 86% At2g 43780 A. thaliana 0.8 266 H6-19 Accession No. Seq. identity & %homology Transcriptsize (Kb) cDNA (bp) C2H4 reguln. Clone Sequence homology of 27 clones CF569225 CF569226

84%, Isoflavone reductase Seq. identity & %homology novel 1.0 151 Up H9-6 CF542262 1.5 167 H9-5 1.6 149 H9-3 2.0 177 H9-2- 1.7 135 H9-8 CF542266 1.2 246 H1-35 CF519305 84%, Isoflavone reductase P.communis 1.42 266 H4-1 1.3 104 H1-18 105 H1-4 CF519303 99%, Pectate lyase M.acuminata 184 H10-3 CF519302 164 H10-1 Accession No. Seq. identity & %homology Transcriptsize (Kb) cDNA (bp) C2H4 reguln. Clone CF569228 CF569229 CF569227 CF569222 CF569224 CF569223

Seq. identity & %homology * Out of 27clones , 8 gave known identity. CF519301 Up H 13-2 Accession No. Seq. identity & %homology Transcriptsize (Kb) cDNA (bp) C2H4 reguln. Clone H6-1 H12-5 H12-8 H14-4 Down 248 169 114 113 124 1.7 1.3 1.8 1.4 0.65 82%, pSbaNS5 protein S.bicolor 89%, pBANED141 M.acuminata CF519304 novel CF542267 CF542268 CF569230

Tissue specific & developmental. Fig: Characterization of some ethylene up regulated genes on different northern blots. R S Ps L Fuop Fop S1 S2 S3 S4 IAA/ ABA treated Tissue specific & developmental. H3-5 H3-15 H8-19 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 C 0 1 2 3 4 5 6 7 Ethylene treated MCP + Ethylene treated

CONCLUSION FUTURE PLAN DDRT-PCR of RNA isolated from various stages of ripening banana resulted in the identification of putative 27 differentially expressed cDNA fragments. Out of these, 21 were found to be up regulated and 6 were down regulated. Out of 27 clones, 11 clones were sequenced. Three clones show homology to known genes viz., -1,3 glucanase, pBAN UD75 and At2g43780, while the others do not show any homology to known genes. Expression of H3-5, H3-15 and H8-19 genes are ethylene induced, inhibited by MCP and are totally fruit specific. FUTURE PLAN Sequence analysis of the remaining ethylene regulated and ripening related genes in banana fruit. Attempts are being made to get full length cDNA and promoter sequence of differentially expressed cDNA fragments through 5’RACE and Genome-Walking methods using reverse primers. Time-course study of expression during ripening and tissue-specificity of the remaining genes by northern analysis. Study of the promoter sequence of some important ethylene regulated and ripening related genes.

ACKNOWLEDGEMENTS CSIR is acknowledged for the JRF and SRF fellowship to SMG. Department of Biotechnology, India, is acknowledged for funding the project.