Differences in wood density and growth of fertilized and nonfertilized loblolly pine associated with a mutant gene, cad-nI

A rare mutant allele (cud-n l ) of the cad gene in loblolly pine (Pinus I I I C ~ I L I L.) causes a deticiency in the production of cinnamyl alcohol dehydrogenase (CAD). Effects associated with this allele were examined by comparing wood density and growth traits of cad-nl heterozygous trees with those of wild-type trees in n 10-year-old openpollinated family trial growing under two levels of fertilization in Scotland County. North Carolina. In all, 200 trees were sampled, with 100 trees for each fertilizer treatment. Wood density measurements were collected from wood cores at breast height using X-ray densitometry. We found that the substitution of a CLI(/-111 f01. il wild-type allclc ('Ccltl) was associated with a significant effect on wood density. The call-111 heterozygotes had a \igniticantly higher wood density (+2.6%) compared with wild-type trees. The higher density was apparently due to the higher percentage of latewood in the heterozygotes. The fertilization effect was highly significanl for both growth and wood density traits. This study indicates that the cad-nl allele could be a valuable gene to tile pulp and paper il~dustry for the purpose of enhancing pulp yields by increasing wood density. R6um6 : Un allkle mutant rare (cad-i l l ) du gkne cad chez le pin i encens (Pi1.1u.s ttrrdtl L.) entraine une tiificience de la production de I'alcool cinnamylique desliydrogenase. Les auteurs ont ktudie les el'fets associis 5 cct allkle en cornparant la densite du bois et les caractkres de croissance des arbres hCtCrozygotes pour cad-1.11 avec les mernes caractkres chez des arbres sauvages. Cette comparaison a CtC effectuCe dans un tcst de descendances rnonoparentales i g i de 10 ans et Ctabli sous deux niveaux de fertilisation dans le cornte de Scotland en Caroline du Nord. En tout. 200 arbres ont CtC ~chantillonnCs i raison de 100 arbres par traitement de fertilisation. Lcs mesures dc densit6 du bois onr CtC obtenues par densitomitrie aux rayons X de carottes de bois prClevCes i hauteur de poiwine. Les auleurs ont obscrvi que la substitution de l'allkle cntl-nl par tin allkle sauvage (Ccitl) ailair un cffet signific:itif sur la densi12 d i ~ bois. LLI densite dl1 bois Ctait significativement plus Clevie (+2,6 5%) chez les lietdrozygotes pour c(irl-t7l co~npar:itiveriient aux arbres sauvages. La plus forte densite Ctait apparemment le rksultat d'un pourcentage P I L L S 2levC de bois linttl chez les IiCterozygotes. L'effct de la fertilisation Ctait Iiautement significatif 5 la fois pour les caractkres de croissance ct la densitt? du bois. Cette etude inclique que I'allkle cclcl-nl poursait comporler une valeur intrins?clue pour i ' i~icl~~strie des pstes er papiers dans le but d'am6liorer le rcndcmcnt en pstc en alignlentant la densite clu boi,. [Traduit par la Rkdaction] Introduction is the only known First-generation carrier of a rare mutant 31lele ( c ad -n l ) o!' the c a d gene. T h e cad-t11 allele confers a reThe southern united states produces 580/0 of the duction i n [he of c i n l l ; l m v ~ ;,lcohol dehydrogenase t imber supply, much of it grown in intensively managed (C.4D: E.C. I . 1 . I . l 95) , which is signil'iuant because CAD plantations o f genetically improved loblolly pine (Pi11i.o cat:~lyzcc the firi~ll s tcp in tlic b iosyn~hcs is of lignin precurtaeda L.) (Wear and Greis 2002). Trees grown from seeds 501-s (>lacK;i> et al. 1995: O'\l;~lIc.!; c.t X I . 1992). derived From sccond-gcncratio~i sced oschayds have cs~i ina tcd gains of 13%-2 1 % in rotation-age woocl volume o i ,e r i ~ n i m I,i?nin is con\,cntiorl;tlIy dcf'incd ;I\ ~i colnplcx hydrophobic seed l o t s ( L i et a l . ,gg9, . One v;llll;lblc nct\vork o!' phcnylpn)p;lnoid units clcl-i~cd li-om thc o s id~ l t i vc ioblolly p i n c selecl,ons nladc by [he C;lrolinu SL;,lc poly~ncl-iz:~tion ot' one 01more ol. rill-cc types o f liydroryuniversity ~~d~~~~~ Cooperative T~~~ ~lnprovclIlcnt Pn,prmll ci r l t i ; i ~ ~ ~ > I ;~lcoliol precursors f Scclcso t.1. C L A. 1 094 ) . '4s S L I C I I , Iignin p l~ iys ;it1 i ~ ~ i p o r r ; ~ ~ i ~ I-olc i l l ~ I L I I ~ L t>iology. cs~~cci ; i l ly Sor Received 28 Scptenlhcr 2004. ,4cceplcd 3 May 2005 Published on the NRC Rc\earcli Web S I I ~ ; ~ t http://clfr.nrc.ca on 19 Augu\t 2005. Q. YU,' S.E. >IcKcand, U. Li, .J.R. Sherrill, and T..J. Muilin. Department of' Forestry. Campus Box 8002. Nol-111 Carolina State University, Raleigh, NC 27695-8002. USA. C.D. Nelson. Southern I~lsrirutc of' Forest Gc~iclics. LSDA Forest Scrvice. Soitthcrn Iicsea~.ch Station. 27332 bli\sissil,pi 67, Saucier. MS 39574-0-3-13, U S A . mcch~inical suppol-t, w:ircs tr;lnhl>orr, ;11ic1 p; l~hogcn I C S I S ~ ~ I I C C fxincc cr : I ] . 1080). 111 Lrccs. Iligh lc\,cl\ 01 ' ligrlin ; ~ s c contained irl wood iind account l'or l5?i-70'3 01' tile dry \vciglit o!' wood (\Vlicttcn and Scdcl-ol'l' 1905 1. In thc pulp and paper induxtl-y. lignin mils1 be I-cniovccl by C I I C I I I ~ C : ~ ~ rrc;iLliicnts 1h;ll arc c o \ ~ I y to the Inill ;itid thc ctlvisonlncnt (P)c:ui ;lritl Eri ksson I OY4 ). llrosc~iy 1.1-01ii t l ~ c C . L L ~ I I 1 Ii)i~ncic~:ire ~vidcly p1:inlcd t h r o ~ ~ g h out [he \oi~thr_.~-n L11iilcd St;\ics i l l ol)cra~ion:~l allcl cs j~cs imcl i 1:iI 1~I;111t~~tions. Brccc l i~~g wol-1; using this sclcctcd genotypc psoduccs ~ h r c c types 01' trccs with scspcct lo tlic ctrtl locus. Can. I . For. Kcs. 35: 1773-1730 t 2M)S) (101: 10. I I .JO/X05I 0 3 6 ) 2005 NK(.I C;~nacl ; t Can. J . For. Res. Vol. 35, 2005 With self-pollination, partially CAD-deficient heterozygotes (Cadlcad-nl), wild-type homozygotes (Cucll Cad), and totally CAD-deficient homozygotes (call-nllcccd-nl) are produced (MacKay ct al. 1997; Ralph et al. 1997). Only lieterozygotcs 'and wild-type homozygotes are produced with cross-pollination. Wu et al. (1999) reported that heterozygous cad-nl trees produced 14% more debarked wood volume at age 4 years. compared with wild-type trees. In addition, Ditntnel et 21. (200 1) 1-epol-ted that homozygous cad-rrl trees had poor growth and low pulp yields (due at least in part to their inbred nature), compared with other genotypes, although they produced wood that was more easily delignified. Previous studies of cad-nl heterozygotes yielded inconsistent results on pulping and bleaching. In one study, haf t cooks of 4and 6-year-old heterozygous trees resulted in kappa numbers (i.e., lignin contents) that were significantly lower than wild-type trees. Additionally, significantly less energy was required (15%-25% lower H-Sactor) to pulp to a given kappa number than for wild-type trees, and the pulp of the lieterozygotes was brighter and stronger (Diinmel ct al. 2001). Conversely, Dimme1 et al. (2002) found no apparent differences in ease of delignification or pulp yield between heterozygous and wild-type trees that were 14 years old. The effects of the cad-nl allele in loblolly pine are caused by a frame shift mutation in the coding region of the call gene (Gill et al. 2003) that causes a reduction of cad mRNA and CAD enzyme activity (Stasolla et al. 2003). Heterozygous and homozygous cad-nI trees have, respectively, only 50% and 1% of the normal (i.e., homozygous wild type) levels of CAD expression (MacKay et al. 1997). There are also differences in the chemical composition of lig~iin between ccrcl-nl hetcrozygous and wild-typc trees, rcsi~lting in LL large difference in the extractability of lignin and potential benefits to the pulp and paper industry (MacKay et al. 1999; Lapierre et al. 2000). To date, no studies have focused on the effect of thc c ~ ~ t l r z l allele on physical properties of wood, although wood density (LC., spccilic gravity) of a few cud-ILI hcterozygous trees was measured in one study (Dimmel et al. 2002). Wood density is an important trait in loblolly pine, because it is highly correlated with wood strength. wood stiffness, and pulp yield (Haygreen and Bowyer 1996; Fuust et al. 1999). For example, a change of 0.02 in wood spec~fic gravity is eqirivalerit to a ch~uige of 23 kg dry rnas\/m3 (Zobel and Jet1 1995). In this paper, wc report o n tlic cfl'ccti :~ssociatcd with this Inutant allele hy co~nparing \ilooci dcllsity ancl growth traits ot c.citl-/r 1 hctcro~ygotcs with tlio\c ol' wildtype trees. Thcsc ~nc:~surc~i~cnts were made In a 10-year-old opcn-pollinated trtmily trial $1-owing undcr two Ic~cl5 of fcrt~lization. Thc >peciHc objccti\cs of thc study WCI-c ( I ) to analyze the quantitrttivc effccts on growth and wood dctisity associated with the mutant cud-111 allelc and (2) to examine the magnitude of (ud genotype by nutrient ~ntcsactions o n these lrai ts. Materials and methods Plant nlaterials The licld test 1s loci~tcd ill Scoil:lnd County. Nostli C'trolina. adjacent to the USDA Forest Scsvicc North Casollnr~ Starc Univcrsity Sou tlic:~st Trcc Kc\casch :lnd Ectuc:~t~on Sitc (SETRES). The test was established in November and December of 1993 with con taincr-grown seedlings from 10 open-pollin~lted ktmilies of loblolly pine. The soil is very infertile and solnewhat cxccssivcly cirrtined. The fertilizcd and itnfcrtilized (i.e.. control) plots. each consisting of 100 trees, were replicated over 10 rrtndomized complete blocks. The trces were plxnted at a 1.5 m x 2.1 In spacing, with a 12-m buffer al.ound each treatment plot to minimize the intluencc of adjaccnt fcrt~lizcr trcatmcnts. Fertilizer was applied annually to maintain an optimiun supply of macroand micronutrients its deter~iiined by foliiuanalysis to sti~nitlate rapid growth in fertilized plots. Through the first 10 growing seasons, the major nutrient additions (kglha) have been 817 N, 83 P, 57 K, 9 Ca, 52 Mg, and 179 S as well as micronutrient additions (kglha) of 1.7 B, 2.0 Cu. 5.0 Fe. 5.0 Mn. and 2.0 Zn. Growth measurements Height was measured annually through age 10 years (except for years 7 and 9), and DBH (diameter at breast height) was lncasured annually starting in year 3. Thirty-three trees (20 and 13 from control and fertilized treatments, respectivcly) wcrc randomly sclected for destl-uctivc sampling at age 10 to generate an equalion to cstinialc totill inside-bark volume ( y 111') I'rom total height (H. ni). and DBH ( D , mni). Once felled, each tree was bucked at heights of 0, 1.1. and 2.4 m, and ciery 1.2 m thereafter, Inside and outside bark diameters were measured for each stem section. Inside bark diameters were used in Smalian's log volume equation to estimate the inside-bark voliune of each bolt (Ave~y and Burkhat-t 1994). The equation is V = L x (B + b)/2, where V is the total section volume; L is the lensth of bolt: B is the basal arca of the bolt's large end; and h i j the basal area of the bolt's s~iiall clid. Thc stem section \olumes wesc summed to product a whole trce, inside-bark volume. Thc whole tree volume was then used to devclop it PI-ediction equation. V = Bo + B , (DBI-I'H). a \ huggcilccl 13) Spul-I( IYSli. The cxp1:lnatosy i~nriablcc fcst~lizcr (XI), X,X~>'H, rind (D'N)' isere addcd to the ccluation based on their significiuice (p I 0.05), lo product the thllo\vtng cqurition: i\I~cre XI = 1 fatthe fcstilized trc;ttmcnt. and .Yl = O for thc ~ o ~ i t r o l . Tlic riatio ti on cxpla~necl by [ h i \ model (R' = 0.98) l i ~ ~ l i l y ~ign~l.ic:~tik (1) < 0 0001 1. \Voocl densit. measurements 'rhc seed p:u-cnt used iu this study is :i sclectcd sccondgctics;ttion dcsccnd:int of the ( . ( ~ ( ! I I / f'ouncics t1i:lt is known to bc ;I ( . c I ~ I ~ I hctc~-ozygot~. In .plt~gi~st 7003. a rot~tl of' 200 heal thy tl-ccs wcl-c s;~ndo~nly sclectcd from fi\je blocks (3-0 trees per rsc:~tlncnt per blockj. ;\ 12-~iim tot-c \V;IS s:~~iiplctl fl-otn each trce at bsci~st liciglit 1'0s woocl qi1:11ity analyses. Corcs wcrc sectioned longitcrdinally to psoducc n strip approxitiialcly 2 Inm thick. The saniples wesc conditioned to :i uniform moistusc cotitcnt 01' 8% bcl'osc they wcrc scanned. Wood clctisity was rnc:~si~rcct itsitlg X S L I ~ dcr1sitomctr.y. Each \tsip \vas sc;lnnccl I'ro~u pit11 to tllc 17;~rk on ;L QklS Trcc Ri ug i l r~ :~ l~zc t " ' (moclel Qss-0 l \c. C>i~in[c.k h4casurc1iicnt Systems. Illc.. Ktioxvillc, 'I'cnncssec). 'I'llc last growth sing was exclilcled bccausc ol' missing I:~tcivood on cores collected in