1. 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
India

2. 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.

3. 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
Days
Respiratory Climacteric
Ethylene biosynthetic activity

4. 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 day (ethylene treated)
Post Climacteric - 4 day (ethylene treated)
0 day day day
C2H4 Treated
Control

5. 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

6. 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

7. 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 day = 4 day. A G C

8. 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 day = 4 day. A G C
(a)
(b)

9. 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 A
622 bp
309 bp
242 bp
404 bp
427 bp
311 bp
500 bp B M C M D M M E
404 bp M
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.

10. Reamplified cDNA fragments.
A total of 200 differentially amplified bands were eluted and reamplified from three stages of banana ripening (0,2&4day). M A
622 bp
309 bp
242 bp
404 bp
427 bp
311 bp
500 bp B M
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

11. 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
A: Ethidium bromide stained gel shows equal RNA loading.
B: Northern blot hyb. with 23s rRNA probe.

12. 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
H6-1 B
H6-10
H12-5
H6- 9
H6- 19

13. 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

14. 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

15. 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

16. 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 C
Ethylene treated
MCP + Ethylene treated

17. 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.

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