Indian Journ al of Biochemi stry & Biophysics Vol. 37 , December 2000, pp . 453-458

Diurnal regulation of..L-_--'-<---

/' G S J ini vasa-Reddy"t , SlI shm~ Naithani " *, R T lili and P V~Sane Centre For Plant Molecul ar Biology, Nati orITil Botani cal Research Institute, Lucknow 226 00 I, ldia

Accepted 30 May 2000

Li ght regul ates leaf and chl oro pl ast development, together with overall chloroplast gene expression at various levels. Plants respond to diurn al and seasonal changes in li ght by changing expression of photosynthesis genes and metaboli sm. In POPll llis deltoides , a dec iduous tree species, leaf development begins in the month of March and leaf maturati on is attained by summer, whi ch is subsequently foll owed by autumnal senescence and fall. In the present study. di urn al changes in the steady state transcri pt levels of plastid genes were examined in the full y developed leaves during summer season. P!1'r@sults show that steady state level of the psaA/B, fJ ,I'bA, psbEFU and petA transcri pts showed differe nt ial accumulati on during di­urn al cycle in summer. However, there was no signi ficant change in the pigment composition during the day/ni ght cycle. ~ud i es suggest that the diu rnal regu lati on of steady state mR NA accumulation may playa crucial ro le during dai ly ad-justment s in plants life with rapidly changing li ght irradIance and t empe r~ ~ _

Plants adjust to the c hang ing irradi ance and tem­perature during the day and diffe rent seasons by

changes in d i ffe rent photosyntheti c pa ramete rs 1--1.

These adju stments refl ect the changes in inte rna l phys io logical factors and metaboli sm. These phys io­logical processes in hi gher pl ants are characte ri zed by rhythms, whi ch corre late w ith the diurnal (li ght/dark) cycle. Se lec ted bi o logical rhythms can a lso be c lass i­fi ed as circad ian (re lated to the 24 hr cycle of ea rth's rotation). These processes are modulated at va ri ous levels ranging from endogenous flu ctuations of pl as­tid DNA topo log/, transcripti on6

.7

, de novo synthes is of pro te in s~ to post-translati onal modificati ons of

. Q prote ins.

The f irst ev idence that the nuc lear genes coding for thylako id pro te ins are under the control o f the "c ir­cadi an cl ock" was prov ided for the Lhcb gene famil y encoding li ght-harvestin g complex po lypeptides 10.

The finding th at the Lhch mRNA in barley fluctuates during the day has been confinned " and ex tended for

. I I . I J I, ] . I I I . van ous ot le r p ant spec Ies -. ' . n splnac 1, eve s o t mRNAs coding for prote in s tha t be long to the sa me multiprote in complex genera ll y osc ill ate in para ll e l

* Author for corrcspondence at the present address: Dept. or Mo­lecular Biology and Genetics, Co rnell Un iversity, Ithaea- 14850, USA Phone: 1-607-254-4834: Email : sn66 @corncll.eJu Fax : 1-607-255-6249 l' Present add ress: Dept. or Agronomy, Un iversity or Kentuck y, Lexi ngton , KY-40503, USA "These authors cont ributed equa ll y to this stud y

and exhibit maxima that a re spec ific fo r that complex. The mRNAs for PSI prote in s appear pri or to those fo r PSII polypeptides and these, in turn , appear pri or to mRNAs for the three polypeptides constituting the oxygen evo lving complex ' ~. For mRNA that change with hi gh amplitude (e.g., LHC P) osc ill ati ons have a lso been fo und unde r constant conditi ons, indicati ng that a c ircadian osc illator is in vo l ved '~. Transgeni c

tobacco seedlings harboring chimeri c G US gene fu­

sions with 5' -flanking sequences from the spinach genes lhcb , psaF and atpD confirm that the d iffe r­ences in the amplitude as well as the time poin ts of maximum mRNA accumulati on are pe rce ived via cis­regul atory e lements located upstream o f the respec­ti ve ATG codon s ' ~. Minor fluc tuati ons in mRNA lev­

e ls were reported in case of rbcS, rbcL, and psbA and tufA ge l l~s in tomato leaves ' 5. ' 6. Piechull a and Grui ssem" have documented that the express ion of pl astid genes, psaA, psbA and psbB in developing tomato fruit s is pa rti a ll y cont ro ll ed by diurna l rhythms.

Populus de ltoides (popl a r) is a perennial , dec iduous tree spec ies, which grows well in va ryin g range of agroclimatic conditi ons and has been proved usefu l in agroforestry. In earli e r studies d iurna l and seasonal changes were observed in photosynthet ic characte ris­ti cs of popl ar l eaves~. These c hanges inc lude va rying CO 2 compensation concentrati ons, shift in tempera­ture optima. changes in rates of e lectron transport and in pi gment compos iti o n~. Photosys te m IT, is the pri­mary target which , responds to changes in the envi-

454 INDI AN J. BIOCHEM. BIOPHYS., VOL. 37, DECEMBER 2000

ronment at various levels ranging from light-induced transcription and trans lati on of photosystem II genes 17

to repair and degradation of specific subunits, while photosystem I remains relatively stab le I8

.19

. In the present study, changes in steady state levels of the poplar psaAl8, psbA, psbEFU and petA transcripts were studied during diurnal cyc le in summer. The PsaA and PsaB polypeptides encoded by psaA and psa8 genes, form the heterodimeric core of photo­system I reacti on centre. The psbA gene encodes 0 I protein, which together with 02 protein and cyto­chrome b559 forms the core of photosystem II. The psbE and psbF genes encode a. and ~ subun its of cy­tochrome bm . The psbL and psbJ genes encode PsbL and PsbJ proteins respect ively, also associated with photosystem II, while petA gene encodes cytochrome J, an essential component of cytochrome bd complex. We have prev iously reported differential expression of psbEFU and petC-orJ3 ! operons during leaf de-

I ?O? I . f ve opment- ·- and expressIOn 0 psaAl8, psbA, psbDIC genes during autumnal senescence22. Our present stud ies show diurna l variation in the steady state levels of psaAIB, psbA, psbEFU and petA tran­scripts.

Materials and Methods Plant material

Populus de!toides (S tonevi lle, clone 01 2 1), com­monl y known as poplar or cottonwood tree, is a clo­nally propagated, shall ow rooted, deciduous and fast grow ing tree of family Salicaceae. The present stud­ies were carried on a two-year-old tree, growing in the field of National Botanical Research Inst itute, Lucknow, India.

RNA isolation and northern hybridization

In order to determine diurnal changes tn steady state levels of psaAl8, psbA, psbEFU and petA mRNA, total RNA was iso lated at different time

. f h d . d'b d I' ?0 ?1 pOtnts 0 t e ay tn summer as escn e ear ter- .-. . RNA was separated on a denaturing 1.2% agarose MOPS-formaldehyde ge l and transferred to Zeta­probe membrane (Biorad). The psaAl8 heterologous probe from spinach (a gift from Prof. R. G. Herrmann , Germany) and homologous gene specific probes for psbEFU, petA and 23S rDNA were radi o­labelled and hybridi zed2o.2, . The psbA spec ific 2l-mer oli gonucleotides obtained from Dr. Udo Johanoing­meier, Germany were radiolabelled and hybrid ized22

.

Same blot was used for hybridization with different

probes by stripping the previous probe each time.

Estimation oJphotosynthetic pigments Photosynthetic pigments namely chlorophyll and

carotenoids were extrac ted from the leaves during different time points of the day. Chlorophyll content was estimated as described by Arnon2~ and carote­no ids were calculated according to Duxbury and Yentsch25

.

Results and Discussion Our results show the response of poplar chl oro­

plastic genes to the diurnal cyc le. The steady state levels of psaAl8, psbA, psbEFU and petA transcripts were studied in addi tion to the changes in pigment compos ition at different time points of the day. The pigment composition of the poplar leaves remains more or less stable during the day with minor fluc­tuations (Fig. l).

Diurnal profile oj Photosystem gelle(s) expression In poplar psaAl8, psbA and psbEFU operons give

ri se to single transcripts of 5.8, 1.5 and 1.3 kb respec­tively (Fig. 2A). Previous studies demonstrated that these polycistronic messages are not subj ected to f h .. II ·?O ? ? Th urt er process tng tnto sma er transcnpts- '-- . e 23S rDNA probe was used to confirm equal load ing of RNA in all the lanes since the synthes is of ch loro­plast rRNA is known to reach a steady state level at earl y stage in leaf development and remains more or less stable afterwards2o

.26

. In concurrence with these studies, the 3.4 kb 23S rRNA transcripts were present more or less in similar amounts at all the time po ints of the day (Fig. 2). However, the steady state tran-

2

~ .----- • • • • • --.: 1.5 Vl ~

c!: ~ 0 0 _ 0 0 0 0 0 --eJ

..... Vl .... C ~

E ~

t= ! ~ ! ==t 'Q. 0.5 ~- ~ ~ ~

E 0

5 7 9 II 13 16 19 24

Time ( h r )

Fi g. I- Diurnal changes in the pigment content (ch lorophyll s and carotenoids) of poplar leaves. (Mean values of three independent observations were plotted. (-_-), total ChI; (-D -), ChI a ;

(-t.-) , ChI b; (-.A.-), carotenoidsl

REDDY el at.: DIURNAL REGULATION OF PLASTID GENES 455

(A )

5 7 9 11 13 16 19 24 hr

-------------_._---

E ...... 7_ ....... - .............. 1,,'. 5.8 kb ...... - . - .......... ~ psa/vB ___________ ___________ _--.-1

1.5 kb 1- ....... _ 1 ! psbA

1.3 kb 1- psbE F L J

r 3.4 kl:; I .. 3 • • .7 • 235 rRNA

•••• ~ •• _M __ '''N'''''_· __ .....J

(B) h- 7 9 11 '13 '15 i -. " ) hr v IJ i . i

3.4 kb 235 rRNA

1.3 kb psbE F L J

Fi g. 2-Diurnal Iluctuation in steady statc transcript leve ls of photosystem genes. Changes in stcady statc levels of !1.wA/ B, !lsbA , psbEFU and 235 rRNA transcripts during the 24 hr day/ni gh t cycle in summer season . (A): Diurnal changes in steady state transcript levels of 23S rRNA and psbEFU in autumn . (B): Sizes of the transcripts are shown in kb .

script leve ls of psaAl8 , psbA, and psbEFU showed diurnal flu ctuat ions (Fig. 2A) . The transcript levels of psaAI8 showed a s teep decline at 7 hr as compared to the 5 hr and then agai n showed an inc rease between 9 to II hr. The levels of psaAI8 tran script did not change significantly after II hr except a dip at 19 hr and accumul at ion in ni ght. Si mil arl y, psbA and psbEFU steady state transcript levels were lower at 7 hr in comparison to 5 hr. As shown in Fig. 2A, the levels of photosys tem II transc ripts inc rease around 9 hr before a dip agai n be tween II to 13 hr and re ma ins

more or less stab le afte rwards and tends to accumu­late in the ni ght. The decrease in photosystem II tran­sc ript levels not iced at 7 hr and I I hr were substant ia l and seems to be a characteristi c feature of diurna l express ion in summer. On the contra ry, in autumn transcript level of psbEFU operon re main s more or less constant up to II hr and then showed a steady accumul ation (Fig. 28). The overall levels of psbEFU transcript were found to be s ignifican tl y higher in summer as compared to autumn (Fig. 2). Likewise, the steady state transcript leve ls of pshA,

456 INDI AN J. BIOCHEM. BIOPHYS., VOL. 37, DECEMBER 2000

psbDIC and psaAIB were reported to be low during I . 1 '2 autumna senescence III pop ar - .

In general, steady state transcript levels of psaAIB, psbA and psbEFU exhibit maxima in the night. The diurnal fluctu ati on in transcripts of nuclear encoded photosynthes is genes like Lhcb and cab gene families I b 'dl d' d111 516 H d' lave een WI e y stu Ie "', owever, stu les re-lated to pl as tid encoded genes are limited, Among plas tid genes diurnal flu ctuati ons in steady state tran­script levels have been reported for psbA, psbB, psaA and rbcL from tomato, where low mRNA levels were consistentl y observed in the afternoon foll owed by an increased mRNA accumulation in the eveningl5. The fluctuati on in chl oroplas tic transcript levels in poplar during the day resembles diurnal ex press ion of to­mato pl astid genes documented earlier ls while poplar differs from tomato in the dynamics of steady state mRNA accumulati on during morning hours. The major drop in photosystem I and photosystem II tran­script levels at 7 hr can be correlated to li ght induced degradati on of transcripts accumulated during night , which is then fo ll owed by de novo synthes is of tran­scripts. Light induced increase in the transcripti on of psbA, psbEFU and several other plastid genes have been shown, although the transcripts are present in the etioplas t or in darkl 7, Seasonal vari ati ons in diur­nal express ion pattern of various genes could be due to either phase shift or variati on in the length of the day, Alternati ve ly, seasonal variati on of the diurnal rhythms could be a result of temperature change. It has been reported that leaves primarily respond to the seasonal shift of temperatu re optimum for photosyn­thes is to deal with changing irradi ance and tempera-

4 ture.

Diurnal expression of petA gene encoding apocyto­chromef

The petA gene probe hybridizes to ten mRNA spe­cies of 4.5,3.6,3.3,3.2,2.6,2.5,2.4, 1.7, 1.5 and 1.3 kb in size in poplar (Fig. 3) and show a complex tran­script profil e. The petA transcribes along with psal and ORF3 1 genes, giving ri se to a - 4.5 kb polyc is­troni c transc ript. The 4.5 kb transc ript is subj ected to vari ous post-transcripti onal process ing resulting in smaller mono and oli goc istronic transc ripts. The ORF23 1 (homologue of tobacco ORF229 and pea ORF23 I ) and psal genes are located immediately up­stream of petA gene in poplar (Naithani et al. unpub­li shed results). The pattern for petA gene express ion has been shown to be highl y complex, in tobacco and

5 7 9 11 13 16 19 24 hr ··_·_·_·_· .. ··_·· .. ···· .. ·_·_··_·· .. ··--l kb

I 4.5 j

3.5

3.2

2.4

1.7

pel/!

Fig. 3-Changes in steady state levels of /l elA transcri pts during 24 hr day/n ight cycle. [Northern analys is was carri ed ou t lI sing total RNA iso lated from the leaves at di fferent time po ints of the day in summer. Rad iolabeled gene specific probes were used for hybridi zation].

pea. Out of many mRNA spec ies, two major tran­script spec ies were detected: a 1.8 kb mRNA refl ec t­ing monocistronic petA message and a 5.35 kb mRNA representing a polyc istronic precursor transcript in tobacc027 . The co-transcripti on of petA -ORF231 has been determined in pea, which gives ri se to multiple transcripts (5.5 kb, 4.3 kb, 3.4 kb and 2.7 kb) after post-transcriptional process ing2x .

As opposed to photosystem genes, hi gher leve ls of petA transcript were observed at 5 and 7 hr, however, the steady state levels of petA were fou nd to be low­est during night suggesting de novo synthes is after sunri se. As shown in Fi g. 3, the max ima in steady state levels of petA transcript s at 7 hr was then fo l­lowed by a dip at 9 hI' and subsequent se lec ti ve stabi­li zation of smaller transcri pts. The 2.6, 2.5, 2.4 and 1,7 kb sized transcripts of petA showed max imum fluctu ati on in their levels ex hibiting maxima at 19 hI' (Fig. 3). It remains to be in vestigated whether, the smaller transcripts hybridizing to petA ari se from light induced post-transcriptional process ing of bigger transcripts or due to se lecti ve stabili za tion of smaller transcripts. The post-transcripti onal process ing and selective stabili zation of monocistroni c perA tran­scripts may facilitate differenti al synthes is of gene

REDDY el al.: DI URN AL REGU LATION OF PLASTID G ENES 457

products, encoded within the same operon during changing environmental and developmental condi­tions. The petA gene encodes cytochrome f subunit of cytochrome hJ complex. Interestingly, all pl as tid genes encoding subunits of Cytochrome hJ complex are clustered together with genes encoding compo­nents of different multi subunit complexes and exhibit complex transc ript profil e. Cytochrome h(l complex is composed of six subunits; cytochrome J, cyto­chrome h6, the Rieske-iron-sulphur protein , subunit IV, subunit V and subunit VI. The Rieske-i ron­sul phur protein is encoded by nuclear genome. The genes encoding subunits cytochrome h6 and subunit IV (pe tB-petD) are transcribed as a part of pshB­pshT-pshH-petB-petD polyc istronic message and un­dergo a complex post-transcripti onal process ing gen­erating as many as 14 transc ripts ranging from 5.5 kb to 220 nucleotides in poplar29. The petB-petD dicis­tronic message shows diffe rential stabili zation as compared to pshB-pshT-pshH mono and oligoc is­tronic transcripts29. The petC gene encoding subunit V of cytochrome hri' complex transcribes as ycj7-petC-ORF42 polyc istronic message in mature leaves and as a monoc istronic petC transcript in developing poplar leaves21 . A newly identified subunit VI of the cytochrome h(l is encoded by plastid petN (yc/ 6) gene and co-transcribes with tmC resulting in multi ple transcripts27 . The most abundant spec ies includes two transcripts of - 1.25 and - 0.53 kb . Both transcript spec ies are present at comparable levels in li ght­grown as well as in dark-adapted plants27 . The func­ti onal significance of the clustering of genes encoding subunits of di fferent multi subunit complexes has not been understood but complex process ing events gen­erating multipl e mRNA spec ies and their selec ti ve transcripti on or stabili zati on may fac ilitate des ired stoichiometri c producti on of related subunits.

Our study shows vari ati ons in steady state mRN A level of photosystem I (psaAlB) and photosystem II (pshA and pshEFU) genes. The polyc istronic psaAIB, pshA and pshEFU operons transcribed as single polyc istronic transcript were not subjected to post­transcripti onal process ing leading to smaller tran­script spec ies in poplar. The petA gene encoding apo­cytochrome / subunit of cytochrome hri' complex shows a complex transcript profil e as a result of post­transcripti onal process ing of polyc istronic transcript. The rati o of bigger polyc istronic message and the processed mono or oli goc istronic mRNA spec ies varies during the diurnal cyc le, suggesting li ght-

induced selective accumulati on of monoc istronic perA transcript. The steady state mRNA levels at any given time point, represent a dynamic equilibrium between interrelated processes ; such as transcripti on, post­transcriptional process ing, RNA stability and degra­dation. The regulati on of gene express ion at vari ous levels in changing environmental and climatic condi ­ti ons is crucial for the surviva l of the plant. Diffe ren­ti al accumulation of steady state transcript levels may be an important step of the complex regulatory proc­esses to achieve required ex press ion of photosynthe­S IS genes.

Acknowledgement We thank Dr. Udo Johanningmeier, Germany fo r

pshA spec ific oligonucleotides, Prof. R G Herrmann for the spinach psaAIB probes. We also th ank De­partment of Biotechnology, Government of India and Council of Scientific and Industri al Research, Gov­ernment of India for fin ancial support.

References I Powels S B & Critch ley C (1980) Plalll Physiol 65 , 11 8 1-

11 87 2 Azcon-Bieto J. Farq uhar G D & Cabal lero A ( 198 1) PlallIa

152,497-504 3 Wong S-C. Cowan I R & Farquhar G D ( 1985) Plalll PhY.I'iol

78,826-829 4 Singh M, C haturved i R & Sane P V ( 1996) PhOlos\'Illhelica

32, 11 -2 1 5 Salvador M L, Kl ein U & Bogorad L ( 1998) Mol Cell Bioi

18, 7235-7242 6 Mill ar A J , Short S R & Chua q -I ( 1992) Plan I Cell 4,

1075- 1087 7 G iuli ano G, Ho lTman N E, Ko K, Sco lnik P & Cashmore A

R ( 1988) EMBO J 7,3635-3642 8 Carte r P J, Nimmo H G, Few~on C A & W il kin s M B (1991)

EMBO J 10,2063-2068 9 Kim M , C hristopher D A & Mu ll et J E ( 1999) Plalll Phrsiol

I 19, 663-670 10 Kloppstech. K ( 1985) Planla 165 , 502-506 II Piechulla B & G rui ssem W ( 1987) EMBO J 6 , 3593-3599 12 Nagy F, Kay S A & C hua N- H ( 1988) Gelles Devel 2, 376-

382 13 Mill ar A J & Kay S A ( 199 1) Plall l Cell 3, 54 1-550 14 Oelmull er R, Sch neide rbauer A. Herrmann R G &

Kl ooppsteck K ( 1995) Mol Gell Gell el, 246, 478-484 15 Pi echull a B ( 1988) Planl Mol Bioi I I , 345-353 16 Piechull a B ( 1989) Plalll Mo l Bioi 12,3 17-327 17 Kl ein R R & Mulle t J E ( 1987) J Bioi Gelll 262, 434 1-4348 18 Ishikawa Y, Nakatani E, Henmi T , Feljani A, Harada Y,

Tamura N & Yamamoto Y (1999) Biochilll Bioflhys Acta 14 13, 147- 158

19 Ortega J M , Ro ncel M & Loshada M ( 1999) FEBS Letl458 , 87-92

458 INDIAN J. BIOCHEM. BIOPHYS., VOL. 37, DECEMBER 2000

20 Naithani S, Trivedi P K, Tuli R & Sane P V ( 1997) J Celie! 76, 61-72

21 Naithnni S, Trived i P K & Sane P V (1997) Biochelll Mol BioI III! 43 , 433-442

22 Reddy M S S, Trivedi P K, Tuli R & Sane P V (1997) Bio­chelll Mol Bioi 111 1 43 , 677-684

23 Reddy M S S, Trivedi P K, Tuli R & Sane P V (1997) J Celi e! 77, 77-83

24 Arnon D I ( 1949) Plall! Physiol24 , 1-15

25 Duxbury A C & Yentsch C S (1956) MOllograph J Marill e Research 15 ,92-1 0 I

26 Callow J A, Callow M E & Wool house H W (1972) Cell Differ I, 79-90

27 Willey D L & Gray J C ( 1990) Plall! Mol Bioi, 15 ,347-356 28 Hager M, Biehler K, 1I1erhaus J, Rur S & Bock R ( 1999)

£MBO J 18, 5834-5842 29 Dixit R, Trivedi P K, Nath P & Sane P V ( 1999) Cllrr Celie!

36, 165-172