Genetic, Hormonal, and Physiological Analysis of...Genetic, Hormonal, and Physiological Analysis of...

Genetic, Hormonal, and Physiological Analysis ofLate Maturity a-Amylase in Wheat1[W][OA]

Jose M. Barrero, Kolumbina Mrva, Mark J. Talbot, Rosemary G. White, Jennifer Taylor,Frank Gubler, and Daryl J. Mares*

Commonwealth Scientific and Industrial Research Organization Plant Industry, Canberra, Australian CapitalTerritory 2601, Australia (J.M.B., M.J.T., R.G.W., J.T., F.G.); and Plant and Pest Science, School of Agriculture,Food, and Wine, University of Adelaide, Waite Campus, Glen Osmond, South Australia 5064, Australia (K.M.,D.J.M.)

Late maturity a-amylase (LMA) is a genetic defect that is commonly found in bread wheat (Triticum aestivum) cultivars and canresult in commercially unacceptably high levels of a-amylase in harvest-ripe grain in the absence of rain or preharvest sprouting.This defect represents a serious problem for wheat farmers, and apart from the circumstantial evidence that gibberellins aresomehow involved in the expression of LMA, the mechanisms or genes underlying LMA are unknown. In this work, we use adoubled haploid population segregating for constitutive LMA to physiologically analyze the appearance of LMA during graindevelopment and to profile the transcriptomic and hormonal changes associated with this phenomenon. Our results show thatLMA is a consequence of a very narrow and transitory peak of expression of genes encoding high-isoelectric point a-amylaseduring grain development and that the LMA phenotype seems to be a partial or incomplete gibberellin response emerging froma strongly altered hormonal environment.

Late maturity a-amylase (LMA), also known as pre-maturity a-amylase, is a genetic defect that is wide-spread in bread wheat (Triticum aestivum) germplasmand can result in unacceptably high levels of a-amylasein harvest-ripe grain in the absence of rain or prehar-vest sprouting (Mares and Mrva, 2008a). The defect isinherited as a recessive trait (Mrva and Mares, 1996a)and is associated with a highly significant quantitativetrait locus (QTL) on the long arm of chromosome 7Band a number of QTLs of lesser significance (Mrva andMares, 2001b; Mares and Mrva, 2008a; Mrva et al., 2008;Emebiri et al., 2010; Tan et al., 2010). In tall genotypessuffering from LMA, the expression of LMA is con-stitutive. In contrast, in the presence of semidwarfing(GA-insensitive) genes such as Reduced Height1 (Rht1)or Rht2, LMA expression is reduced (Mrva and Mares,1996b), highly variable, and appears to be dependenton a cool temperature shock during the middle stagesof grain development (Mrva and Mares, 2001a; Farrelland Kettlewell, 2008; Mares and Mrva, 2008a). In bothcases, LMA is a consequence of the synthesis of high-pI

a-amylase coded by the a-Amylase1 (a-Amy1) genes onwheat chromosomes 6A, 6B, and 6D.

High-pI a-amylase is also synthesized followinggermination, and, together with the inhibitory effectsof the GA insensitivity gene, these factors suggest thatGA is involved in the expression of LMA. However,while there are some points of similarity betweena-amylase production in germination and LMA, thereare a number of differences (Mrva et al., 2006; Maresand Mrva, 2008a). In germinated grain, a-amylase issynthesized by the scutellum and later by the adjacentaleurone, the latter under the influence of GA pro-duced by the embryo. With time, a-amylase synthesisspreads toward the distal end of the grain. In contrast,there is no evidence for the involvement of the embryo,or embryo-derived GA, in LMA, and the enzyme isdistributed approximately equally between proximaland distal halves of the grains. Similarly, whereasgerminating grains can synthesize large amounts ofa-amylase according to an exponential rate curve, thesynthesis of a-amylase in LMA-affected grains rarelyexceeds the amount typical of the early stages of ger-mination. The amount can be sufficient, however, toexceed grain receival standards and result in the down-grading of quality (Mares and Mrva, 2008a).

Apart from the circumstantial evidence that GA issomehow involved in the expression of LMA, the mech-anisms or genes underlying LMA are unknown. GAcontrol of gene expression in mature aleurone has beenextensively studied, and it remains to be determinedwhether similar response pathways are active in LMA-affected grains during grain development. We havedeveloped sets of doubled haploid lines derived from

1 This work was supported by the Australian Grains Research andDevelopment Corporation.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Daryl J. Mares ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a subscrip-

tion.www.plantphysiol.org/cgi/doi/10.1104/pp.112.209502

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the same cross, cv Spica 3 Maringa, expressing con-stitutive LMA (+LMA) or not expressing LMA (–LMA),and we have examined the expression patterns for anumber of genes, including those encoding a-amylases,characteristic of GA-treated aleurone, and enzymes in-volved in the synthesis and catabolism of the plant hor-mone abscisic acid (ABA), over the period of graindevelopment where a-amylase is detected. Second, weused targeted microarrays to identify genes that areup-regulated in +LMA or –LMA genotypes during theinitiation of high-pI a-amylase synthesis. Finally, wecompared the hormonal profile of +LMA and –LMAdeveloping grains. These techniques were used to probea very narrow and transitory peak of expression of genesencoding high-pI a-amylase that is responsible for theLMA phenotype and indicate that the LMA phenotypeseems to be a partial or incomplete GA response emerg-ing from a strongly altered hormonal environment.

RESULTS

Grain Development and the Synthesis of a-Amylase

Doubled haploid genotypes with and without con-stitutive LMA were grown side by side under the sameexperimental conditions. Anthesis to harvest ripeness(12% grain moisture) was completed in 45 d in all ofthe lines examined. Grain moisture declined graduallyfrom around 70% (moisture as a percentage of freshweight) at 12 DPA to around 45% by 35 DPA and thenmore rapidly over the ensuing 10 d (Fig. 1). Maximumgrain dry weight was achieved by 32 to 35 DPA (datanot shown). Grain development as judged by physicalappearance appeared to be similar in +LMA and–LMA genotypes (Fig. 2). Thirty-two DPA grains froma +LMA and a –LMA genotype were imbibed, sec-tioned, and stained, and aleurone cells were observedwith a microscope. About 500 aleurone cells from eachgenotype were examined, but no visual differenceswere observed that could be associated with LMA(Supplemental Fig. S1).

High-pI a-amylase protein remained low or belowthe level of detection until 20 DPA, increased rapidlyin the +LMA genotypes, reaching a maximum at 32 to35 DPA, and then remained at this level through toharvest ripeness (Fig. 3). Samples with elevated high-pI a-amylase protein (measured by ELISA) also hadhigh total a-amylase activity measured either with theAmylazyme assay or with Falling Number (data notshown). Grain moisture content corresponding to thetime of initiation of synthesis of a-amylase was 55% to60%, and grains were just starting to develop a tinge ofyellow.

Expression of a-Amylase Genes and GA Response Genesin Mature Aleurone

In order to study which genes encoding a-amylasewere involved in the expression of LMA, specific

primers (Supplemental Table S1) were designed forhigh pI (a-Amy1-1 and a-Amy1-2), low pI (a-Amy2-1),and other known members (a-Amy3-1 and a-Amy4-1)of this family of genes for quantitative reverse tran-scription (qRT)-PCR. As a starting point, the effect ofexogenous GA on the expression of a-Amy genes inmature aleurone from a control wheat variety was in-vestigated. Aleurone layers from mature wheat grainswere harvested and imbibed in control medium or inmedium supplemented with 1 mM GA. After 24 h ofimbibition, RNA was extracted and complementaryDNA (cDNA) was synthesized. The expression of alla-Amy genes was very low in aleurone imbibed incontrol medium, but in the GA treatment, the expres-sion of a-Amy1-1 and a-Amy1-2 was dramatically in-duced (Fig. 4A). a-Amy2-1 was also induced by GA butto a lower extent, while the expression of a-Amy3-1and a-Amy4-1 was not affected (Fig. 4A).

The expression of other known cereal GA responsegenes, including GAMYB, a transcription regulator ofGA signaling in aleurone (Gubler et al., 1995, 1999;Murray et al., 2006), a cell wall-degrading enzyme,(1,3;1,4)-b-glucanase (Mundy and Fincher, 1986), andtwo proteases, triticain-a and triticain-g (Kiyosaki et al.,2009), was also determined in mature aleurone. In com-parison with the expression on control medium, the ex-pression of (1,3;1,4)-b-glucanase was induced 5-fold byGA, triticain-a was induced 14-fold, triticain-g was in-duced 32-fold, and GAMYBwas induced 3-fold (Fig. 4B).

We also studied the effect of GA over several ABAmetabolic wheat genes (NCED1, ABA89OH-1, and

Figure 1. Changes in grain moisture during grain development in+LMA and –LMA genotypes.

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ABA89OH-2; Ji et al., 2011). The ABA biosynthetic geneNCED1 and the ABA catabolic gene ABA89OH-2 werenot significantly affected by GA, although the geneABA89OH-1was induced nearly 4-fold by GA (Fig. 4C).

a-Amy Gene Expression in +LMA and –LMA Genotypesduring Grain Development

The next stage was to determine which a-Amy geneswere involved in LMA and whether the gene expres-sion pattern was similar to the pattern that we found inmature aleurone challenged with GA. The expressionpatterns of the a-Amy genes, therefore, were analyzedduring grain development in +LMA and –LMA geno-types. Based on the a-amylase protein profiling of+LMA and –LMA genotypes (Fig. 3), four develop-mental time points were selected for gene expressionstudies. Aleurone layers were isolated from develop-ing grains at 17 DPA (no a-amylase detected), at 20DPA (just before a-amylase appears in +LMA geno-types), at 23 DPA (as a-amylase increases in +LMA),and at 26 DPA (as a-amylase increases further in+LMA). As an indicator of developmental stage, in thisexperiment, 17, 20, 23, and 26 DPA corresponded tothermal times of 190, 225, 255, and 290 degree daysfrom anthesis (see “Materials and Methods”). At 17DPA, the expression of a-Amy1-1, a-Amy1-2, a-Amy2-1,and a-Amy3-1 was very low, and no differences wereobserved between +LMA and –LMA genotypes. Theexpression of a-Amy4-1 was higher, but no differencesbetween lines were detected (Fig. 5A). At 20 DPA, theexpression profile was almost identical to 17 DPA(Fig. 5B). At 23 DPA, a-Amy1-1 and a-Amy1-2 wereclearly induced in three of the +LMA lines (25, 127,and 52) and remained very low in the other genotypes(Fig. 5C). The expression of a-Amy2-1, a-Amy3-1, anda-Amy4-1 did not change in comparison with the pre-vious time points. At 26 DPA, a-Amy1-1 and a-Amy1-2expression was still high in two of the +LMA lines (127and 52), and a-Amy2-1, a-Amy3-1, and a-Amy4-1 showedthe same expression levels as previously (Fig. 4D). In-terestingly, at 26 DPA in line 52, the expression ofa-Amy1-1 and a-Amy1-2 was much higher than at 23DPA, and in line 25, the expression of these genes at

26 DPA was back to base levels. These results highlightthe transient nature of the expression of a-Amy genesin +LMA genotypes, which only last for around 1 to 4 d.This short a-Amy1-1 and a-Amy1-2 expression windowcould also explain the inability to detect a-Amy1 geneexpression in line 131 despite ELISA data that showedhigh levels of amylase protein in that line. No a-Amy1gene expression was detected in this line at 23 and 26DPA, but it may have been detected if samples had beencollected within the intervals between 20 to 23 DPA.

Expression of GA Response and ABA Metabolic Genes in+LMA and –LMA Genotypes

In the same set of +LMA and –LMA samples, theexpression of four control GA-responsive genes [GAMYB,

Figure 2. Grain appearance for –LMA genotypecv Spica 3 Maringa line 47 (Sp/M#47; top row)and +LMA genotype cv Spica 3 Maringa line 52(Sp/M#52; bottom row) at intervals during de-velopment.

Figure 3. Changes in grain high-pI a-amylase protein content in +LMAand –LMA genotypes during grain development measured by ELISA.Eight technical replicates were performed. Error bars represent SE. OD,Optical density.

Plant Physiol. Vol. 161, 2013 1267

Genes, Hormones, and Physiology of Late Maturity a-Amylase



(1,3;1,4)-b-glucanase, triticain-a, and triticain-g] was alsoanalyzed in order to compare with the response foundin mature aleurone. These genes were clearly inducedby GA in mature aleurone (Fig. 4B); however, no in-crease in their expression in +LMA genotypes duringgrain development was detected. The expression levelsof those GA-responsive genes were very similar be-tween +LMA and –LMA genotypes and did not changebetween 17 and 26 DPA (data not shown). As an ex-ample, their relative expression at 23 DPA is shown inFigure 6A.

Whether the LMA phenotype could be altering theexpression of two important ABA biosynthetic genes(NCED1 and NCED2; Ji et al., 2011) and two ABAcatabolic genes (ABA89OH-1 and ABA89OH-2; Ji et al.,2011) was also investigated. No evidence of differential

expression between +LMA and –LMA genotypes dur-ing grain development was detected at any time point.Again, results from 23 DPA are shown as an example(Fig. 6B).

Microarray Analysis of Gene Expression in Aleurone of+LMA and –LMA Genotypes

A global gene expression analysis using the Agilent44K microarray was used to genetically profile the LMA

Figure 4. GA induction of the a-Amy genes (A), of several GA-responsivegenes (B), and of several ABA metabolic genes (C) in mature aleuronelayers isolated from grains imbibed in control medium or in the presenceof GA. Three biological replicates were performed. Error bars represent SE.

Figure 5. Analysis of a-Amy gene expression in aleurone isolated from+LMA and –LMA genotypes at intervals during grain development.Three replicates were performed. Error bars represent SE.


Barrero et al.



phenotype. We did not find probes matching thewheat a-Amy genes in the microarray, but we wantedto find other genes associated with LMA. Gene ex-pression in three +LMA lines (25, 127, and 52) and inthree –LMA lines (41, 109, and 47) was studied at 20DPA (before any a-amylase protein is detectable; Fig. 3)and at 23 DPA (when a-amylase protein is first ob-served). For the microarray analysis, each of the threedoubled haploid lines in the +LMA or the –LMA groupwas treated as a biological replicate. First, probes with62-fold change between +LMA and –LMA groupsand P , 0.01 were considered. Using those cutoff cri-teria, only 37 probes were identified as differentiallyexpressed at 20 and/or 23 DPA. Then, and thinkingthat only a very reduced proportion of aleurone cells inan LMA variety express LMA (about 2%; Mrva et al.,2006), we decided to use less stringent criteria to avoidthe effects of possible gene expression dilution. So probeswith 61.5-fold change between +LMA and –LMAgroups and P , 0.01 were considered. Eighty-threeprobes satisfied the cutoff criteria at 20 and/or 23DPA: 56 of them were up-regulated in the +LMAgenotypes (Table I) and 27 were up-regulated in the–LMA genotypes (Table II). Three probes from Table Iand three probes from Table II were selected forvalidation by qRT-PCR, and the results obtained werevery similar to those found by the microarray analysis(Supplemental Fig. S2). The normalized expressionvalues for all the probes in all samples are given inSupplemental Table S2.Out of the 56 probes up-regulated in the +LMA

genotypes (Table I), 50 were found at 20 DPA, 36 at

23 DPA, and 30 at both developmental points. Seven-teen of those probes are annotated as “unknown,” andthe majority of the rest show very little similarity withknown proteins. The probe A_99_P239876 showed thebigger fold change at 20 DPA (5.58-fold) and showssimilarity with a member of the Translational initiationfactor2 family. This probe had a 2.70-fold change at23 DPA. Several other probes had a similar expressionpattern, showing a bigger fold change at 20 DPA thanat 23 DPA, but few of these are annotated. The probesA_99_P286731 (Programmed cell death protein2) andA_99_P244556 (NucleaseI) followed that trend and arewell annotated. Another group of probes displayed abigger fold change at 23 DPA than at 20 DPA. Fromthis group, three different probes were annotated asWali proteins (for wheat aluminum-induced proteins;Snowden et al., 1995). Probes A_99_P484557 andA_99_P216921 (Wali6) were up-regulated 2.30- and1.64-fold at 20 DPA and 5.66- and 3.55-fold at 23 DPA,respectively. The probe A_99_P462667 (Wali3) was up-regulated 1.82-fold at 20 DPA and 4.59-fold at 23 DPA.Five other probes were annotated as Wali5 proteins,and they have similar fold change at both 20 and23 DPA. The ABA-inducible protein WRAB1 (probeA_99_P218621), the Pathogenesis-related protein1.1precursor (A_99_245726), a Xyloglucan endotrans-glycosylase (A_99_P265761), and a 1,4-b-D-mannanendohydrolase (A_99_P470812) also displayed higherfold change at 23 DPA than at 20 DPA.

On the other hand, out of the 27 probes up-regulatedin the –LMA genotypes (Table II), 25 were found at20 DPA, 19 at 23 DPA, and 17 at both developmentalpoints. Again, many probes were poorly annotated,and eight of them were classified as unknown. Theprobe A_99_P142933 was found to be strongly up-regulated at 20 DPA (44.60-fold change) and also at23 DPA (10.02-fold change). This probe matched awheat cDNA with no similarity to known sequences.The second most highly up-regulated probe by LMA(A_99_P224251) encodes a wheat ribosomal proteinand is up-regulated 5.02- and 4.51-fold at 20 and 23DPA, respectively. The third probe (A_99_P153962) inthis group does not shown homology to known genes.The fourth probe (A_99_P230121) encoded a g-thioninprotein and was up-regulated 3.08- and 2.48-fold at 20and 23 DPA, respectively. The fifth probe (A_99_P407792)showed 76% similarity with the rice brassinosteroidbiosynthetic gene Diminuto and was up-regulated 2.88-and 2.15-fold at 20 and 23 DPA, respectively. From therest of the list, the probe A_99_P498137 matched amanganese superoxide dismutase and was 1.52-foldup-regulated at 23 DPA in –LMA lines.

Expression of LMA-Altered Genes in GA-TreatedMature Aleurone

To test if some of the genes found to be alteredby LMA were also induced or repressed in maturealeurone after the GA treatment, primers were

Figure 6. Expression of GA-responsive (A) and ABA metabolic (B)genes in developing aleurone isolated from +LMA and –LMA genotypesat 23 DPA. Three replicates were performed. Error bars represent SE.







Table I. Probes up-regulated in +LMA lines

Fifty-six probes were significantly overexpressed in the +LMA lines more than 1.5-fold (highlighted in boldface) at 20 and/or at 23 DPA.

Probe Name Probe Identifier

Fold Change

P Description20

DAA

23

DAA

A_99_P239876 TA64425_4565 5.58 2.70 3.4E-10 Os05g0592600 protein; rice ssp. japonica, partial (15%) [TC317164];Initiation factor2 family protein

A_99_P490267 TC334665 5.34 3.15 9.0E-09 UnknownA_99_P433057 TC299231 4.01 2.38 2.1E-08 UnknownA_99_P241121 TA64772_4565 3.52 2.26 2.3E-03 UnknownA_99_P241126 CK207885 3.21 2.04 7.0E-03 Wheat Functional Genomics of Abiotic Stress (FGAS) Project: Library 5 Gate 7

wheat cDNA, mRNA sequence [CK207885]A_99_P229111 TA61320_4565 3.05 2.49 4.9E-08 Predicted protein; Ostreococcus lucimarinus, partial (6%) [TC289607]A_99_P230131 TA61561_4565 3.02 2.33 2.4E-03 UnknownA_99_P217041 TA57043_4565 2.48 6.17 5.0E-04 Wheat cDNA, clone: SET3_D18, cv Chinese SpringA_99_P231166 TA61862_4565 2.37 1.82 2.7E-06 UnknownA_99_P229201 TA61342_4565 2.33 1.99 2.5E-08 Predicted protein; Chlamydomonas reinhardtii, partial (3%) [TC285456]A_99_P484557 TA57006_4565 2.30 5.66 1.3E-04 Wali6 protein; wheat, partial (85%) [TC331873]A_99_P383537 TA106400_4565 2.27 1.81 8.1E-08 Polysaccharide deacetylase precursor; Pseudomonas putida, partial (5%)

[TC302730]A_99_P306396 TA83585_4565 2.13 1.58 5.8E-07 TolA protein; Vibrionales sp. bacterium, partial (13%) [TC285028]A_99_P488392 TC333744 2.09 1.63 6.2E-08 Possible Ribosomal L29e protein family; Synechococcus sp., partial (12%)

[TC333744]A_99_P217741 TA57367_4565 2.06 1.51 6.5E-03 UnknownA_99_P461432 TC319251 2.04 1.57 3.1E-03 Chromosome chr18 scaffold_1, whole-genome shotgun sequence; grape

(Vitis vinifera), partial (29%) [TC319251]A_99_P289376 TA78618_4565 2.03 1.31 7.3E-03 UnknownA_99_P277691 TA75212_4565 2.00 1.55 2.1E-08 UnknownA_99_P244466 TA65608_4565 1.98 1.38 6.4E-07 Wheat cDNA, clone: WT011_H23, cv Chinese SpringA_99_P534342 TC353270 1.94 1.40 3.4E-09 UnknownA_99_P429037 TC296078 1.92 1.50 2.9E-05 PE-PGRS family protein; Mycobacterium tuberculosis, partial (4%)

[TC296078]A_99_P557882 TC361978 1.90 1.43 4.6E-04 Monosaccharide transporter6; rice ssp. japonica, partial (8%) [TC361978]A_99_P457092 DN829647 1.82 1.51 1.1E-03 cDNA library; wheat cDNA, mRNA sequence [DN829647]A_99_P462667 TC319988 1.82 4.59 6.5E-05 Wali3 protein; wheat, partial (81%) [TC319988]A_99_P293411 TA79766_4565 1.79 1.27 6.9E-03 Fasciclin-like protein FLA1; wheat, partial (10%) [TC335892]A_99_P236421 TA63445_4565 1.79 1.80 2.6E-03 Wali5 protein; wheat, complete [TC306694]A_99_P236391 TA63438_4565 1.76 1.50 2.9E-03 UnknownA_99_P543232 TA63453_4565 1.76 1.63 3.4E-03 Wali5 protein; wheat, partial (34%) [TC356640]A_99_P236411 TA63442_4565 1.75 1.52 1.8E-03 Wali5 protein; wheat, partial (34%) [TC356640]A_99_P236486 TA63458_4565 1.75 1.61 1.8E-03 UnknownA_99_P419937 TC289170 1.74 1.94 7.9E-04 Os04g0639200 protein; rice ssp. japonica, partial (38%) [TC289170]A_99_P519152 CK161960 1.71 1.49 3.9E-03 Wheat FGAS: Library 4 Gate 8 wheat cDNA, mRNA sequence [CK161960]A_99_P286731 TA77840_4565 1.70 1.21 7.8E-04 Programmed cell death protein2, C-terminal domain-containing protein,

expressed; rice ssp. japonica, partial (32%) [TC295481]A_99_P421652 TC290507 1.69 1.33 2.3E-07 Predicted protein; Monosiga brevicollis, partial (3%) [TC290507]A_99_P251481 TA67572_4565 1.68 1.49 6.8E-03 Chromosome chr18 scaffold_1, whole-genome shotgun sequence; grape,

partial (63%) [TC337280]A_99_P278691 TA75509_4565 1.67 1.16 4.8E-03 Os09g0552400 protein; rice ssp. japonica, partial (18%) [TC332503].

RmlC-like cupin family proteinA_99_P495327 TC336890 1.67 1.12 6.2E-03 Ribonucleotide reductase, barrel domain; Burkholderia thailandensis,

partial (3%) [TC336890]A_99_P442867 TC306694 1.66 1.54 5.9E-03 Wali5 protein; wheat, complete [TC306694]A_99_P216921 TA57006_4565 1.64 3.55 9.1E-05 Wali6 protein; wheat, partial (80%) [TC332871]A_99_P236461 TA63453_4565 1.64 1.55 3.7E-03 UnknownA_99_P236451 TA63451_4565 1.63 1.55 5.4E-03 Wali5 protein; wheat, complete [TC319425]A_99_P124250 CN012056 1.63 1.20 2.3E-03 Wheat Fusarium graminearum-infected spike cDNA library, mRNA

sequence [CN012056]A_99_P244556 TA65629_4565 1.61 1.28 3.3E-03 Nuclease I; barley, partial (77%) [TC288687]A_99_P236491 TA63459_4565 1.61 1.62 7.3E-03 UnknownA_99_P543167 CK161960 1.60 1.41 4.7E-03 Wheat FGAS: Library 4 Gate 8 wheat cDNA, mRNA sequence [CK161960]A_99_P415582 TC285524 1.57 1.32 3.5E-06 UnknownA_99_P329196 TA90299_4565 1.57 1.48 4.1E-03 Unknown

(Table continues on following page.)


Barrero et al.



developed for three putative Wali genes (A_99_P462667,A_99_P484557, and A_99_P236421) up-regulated in+LMA genotypes during grain development and forthree probes (A_99_P142933, A_99_P224251, andA_99_P407792) that were up-regulated in –LMA

genotypes (the differential expression of all those geneswas validated by qRT-PCR; Supplemental Fig. S2).Expression of those genes was studied by qRT-PCR inmature aleurone challenged with GA (Fig. 7). The ex-pression of two out of three genes up-regulated in

Table I. (Continued from previous page.)


Fold Change

P Description20

DAA

23

DAA

A_99_P255741 TA68758_4565 1.55 21.02 6.7E-03 Cupin family protein, expressed; rice ssp. japonica, partial (4%) [TC337126]A_99_P394452 TA109072_4565 1.51 1.33 4.7E-03 UnknownA_99_P236396 CK161960 1.51 1.42 4.8E-03 Wheat FGAS: Library 4 Gate 8 wheat cDNA, mRNA sequence [CK161960]A_99_P218621 TA57596_4565 1.48 1.65 2.9E-03 ABA-inducible protein WRAB1; wheat, complete [TC288919]A_99_P245726 TA65929_4565 1.35 2.37 5.3E-04 Pathogenesis-related protein1.1 precursor; wheat, complete [TC285151]A_99_P265761 TA71620_4565 1.28 1.80 1.9E-03 Xyloglucan endotransglycosylase; Musa acuminata, partial (21%) [TC329070]A_99_P213376 TA55414_4565 1.24 1.76 5.2E-03 UnknownA_99_P469557 DN829463 1.13 1.53 1.1E-03 cDNA library wheat cDNA, mRNA sequence [DN829463]A_99_P470812 TA86664_4565 21.01 1.55 2.0E-03 1,4-b-D-Mannan endohydrolase; barley var distichum, partial (33%)

[TC324673]

Table II. Probes up-regulated in –LMA lines

Twenty-seven probes were overexpressed in the –LMA lines more than 1.5-fold (highlighted in boldface) at 20 and/or at 23 DPA.


Fold Change

P Description20

DAA

23

DAA

A_99_P142933 CJ929649 44.60 10.02 3.6E-09 Y. Ogihara unpublished cDNA library; wheat cDNA clone whchan3h03 59,mRNA sequence [CJ929649]

A_99_P224251 TA59797_4565 5.02 4.51 8.5E-03 Wheat ribosomal protein L17 mRNA, complete coding sequenceA_99_P153962 CJ926746 3.84 2.48 2.5E-10 Y. Ogihara unpublished cDNA library, wheat cDNA clone whchan30p15 59,

mRNA sequence [CJ926746]A_99_P230121 TA61559_4565 3.08 2.48 2.7E-03 g-Thionin; barley, partial (98%) [TC336410]A_99_P407792 TA63249_4565 2.88 2.15 1.0E-09 Os10g0397400 protein; rice ssp. japonica, partial (76%) [TC278422]; cell

elongation protein DIMINUTOA_99_P448897 TC310916 2.65 1.80 5.1E-07 UnknownA_99_P287416 TA78047_4565 2.56 1.76 5.6E-08 Os03g0848200 protein; rice ssp. japonica, partial (40%) [TC320092]; phytaseA_99_P434382 TA60205_4565 2.31 1.72 9.8E-03 40S ribosomal protein S15a-1; Arabidopsis (Arabidopsis thaliana), complete

[TC300266]A_99_P443517 TC307152 2.21 1.82 2.5E-03 UnknownA_99_P208196 TA53518_4565 2.18 1.35 7.6E-04 UnknownA_99_P363836 TA101312_4565 2.17 1.82 4.1E-03 CUP-SHAPED COTYLEDON3; Petunia hybrida, partial (26%) [TC307683]A_99_P210321 TA54232_4565 2.14 1.32 7.8E-03 Ferredoxin-NADP(H) oxidoreductase; wheat, partial (10%) [TC336047]A_99_P215456 TA56293_4565 2.14 1.55 1.1E-03 UnknownA_99_P215306 TA56235_4565 2.05 1.44 5.6E-03 Probable conjugal transfer protein; Caulobacter sp., partial (4%) [TC315249]A_99_P215471 TA56315_4565 1.97 1.59 6.1E-04 UnknownA_99_P427842 TC295283 1.91 1.65 9.5E-09 Wheat cDNA, clone: SET4_C16, cv Chinese SpringA_99_P198871 TA50571_4565 1.87 1.59 2.4E-03 UnknownA_99_P201276 TA51341_4565 1.75 1.17 8.0E-03 UnknownA_99_P260276 TA70074_4565 1.74 1.58 7.0E-04 Os05g0595300 protein; rice ssp. japonica, partial (21%) [TC350416]; CCT

domain-containing proteinA_99_P104810 CK159501 1.73 1.87 4.3E-03 Wheat FGAS: TaLt5 wheat cDNA, mRNA sequence [CK159501]A_99_P225791 TA60205_4565 1.71 1.44 5.6E-03 40S ribosomal protein S15a-1; Arabidopsis, partial (77%) [TC354179]A_99_P260256 TA70070_4565 1.69 1.58 7.2E-04 Os05g0595300 protein; rice ssp. japonica, partial (19%) [TC307377]; CCT

domain-containing proteinA_99_P462517 TA103921_4565 1.59 1.23 3.3E-03 Calcineurin B-like protein; rice ssp. japonica, partial (11%) [TC319899]A_99_P251506 TA67578_4565 1.54 1.31 6.6E-03 UnknownA_99_P260606 TA70166_4565 1.52 1.09 5.0E-04 Type IV pilus assembly PilZ; Acidothermus cellulolyticus, partial (9%)

[TC302599]A_99_P498137 TA52414_4565 1.39 1.52 4.0E-03 Manganese superoxide dismutase; wheat, partial (68%) [TC338095]A_99_P269046 TA72621_4565 1.38 1.50 1.6E-04 CTV.22; Poncirus trifoliata, partial (15%) [TC299309]






+LMA was shown to be actually repressed by GA inmature aleurone, and the other one was not detected inmature aleurone (Fig. 7A). The expression of the threegenes up-regulated by –LMA was either not signifi-cantly affected by GA or was not detected in maturealeurone (Fig. 7B). This result shows a different geneexpression signature in LMA-affected developing al-eurone and in mature aleurone treated with GA.

Hormone Profiling of LMA

Deembryonated developing grains (endosperm,testa, and pericarp) from three +LMA and three –LMAgenotypes at 23 DPA were isolated for hormone quan-tification. Several ABA-related compounds (neo-phaseicacid, 79-hydroxy-ABA, phaseic acid, dihydrophaseicacid, and cis- and trans-ABA), auxins (indole-3-aceticacid [IAA], indole-3-butyric acid, IAA-Asp, IAA-Glu,IAA-Ala, and IAA-Leu), cytokinins (cis- and trans-zeatin,cis- and trans-zeatin riboside, cis- and trans-zeatin-O-glucoside, dihydrozeatin, dihydrozeatin riboside,isopentenyladenine, and isopentenyladenosine riboside),and GAs (GA1, GA3, GA4, GA7, GA8, GA9, GA19, GA20,GA24, GA29, GA34, GA44, GA51, and GA53) were assayed,and their endogenous levels and full names are givenin Supplemental Table S3. Compounds that weredetected and quantifiable are presented in Figure 8.

Major changes in some hormones between the twosets of genotypes tested were observed. First, the ABAcontent was about 2-fold higher in +LMA (about 200ng g21 dry weight) than in –LMA samples (about 100ng g21 dry weight; Fig. 8A). In addition, several GAswere more abundant in +LMA than in –LMA samples;in particular, GA19 was about 20-fold more abundant(Fig. 8B). By comparison, auxin (IAA) was clearly moreabundant in –LMA than in +LMA (20,000 versus 5,000ng g21 dry weight; Fig. 8C), while cytokinins were notsignificantly altered (Fig. 8D).

These results show a deeply altered hormone profilein developing +LMA grains and suggest that, in LMAgenotypes, the mechanisms controlling hormone ho-meostasis are altered. As an indirect indicator of theratio of GA to ABA (which is critical in many plantprocesses, including germination), it is worth notingthat the ratio of GA19 to ABA appeared to be about10 times higher in +LMA than in –LMA developinggrains.

DISCUSSION

Grain Development and Expression of LMA

Even when LMA-affected grains are visually andanatomically identical to LMA-free grains, we haveshown by detailed analysis that LMA represents asignificant deviation from the normal pattern of eventsin grain development in wheat. Why and how thechanges in hormone levels and gene expression aremainly buffered so grains appear anatomically similaris intriguing. LMA has not been found so far in Aegi-lops tauschii (DD genome) or Triticum monococcum(AmAm genome; D.J. Mares and K. Mrva, unpublisheddata). On the other hand, relatively high frequencies ofLMA in durum wheat (Triticum turgidum durum;AABB genome), in synthetic hexaploid wheat (T. tur-gidum durum 3 A. tauschii), and of course in breadwheat (AABBDD) have been reported (Mares andMrva, 2008b). That suggests that LMA seems to be asyndrome introduced during wheat domestication andbreeding rather than a common phenomenon found inother crop cereals or undomesticated wheat ancestors.

Typically, low-pI (a-Amy2) a-amylase synthesizedin the pericarp of grains shortly after anthesis disap-pears rapidly as the grain matures, and there is nofurther synthesis of any a-amylase unless rain falls onthe ripe crop and induces sprouting (Mares and Gale,1990). The results reported here not only confirm thesynthesis of high-pI a-amylase in +LMA genotypesduring grain development but place this brief periodof enzyme synthesis precisely in relation to changes ingrain moisture, dry weight, and appearance. The pe-riod in grain development when LMA is expressedappears to coincide with other important changeswithin the developing grain, notably the decline in GAand rise in ABA as well as the onset of grain dehy-dration commencing with the seed coat. The aleuroneof developing grain is capable of producing a-amylaseif challenged with exogenous GA, but only if the grainis first dried artificially (King, 1976; Armstrong et al.,1982; Jiang et al., 1996). Gale and Lenton (1987) failedto detect a peak of new GA synthesis preceding LMAexpression. An alternative explanation is that LMAmay represent a loss of function of a gene or genes thatnormally inhibit the capability of the aleurone of de-veloping grains to respond to GA and that for a shortperiod there is sufficient endogenous GA to triggera-Amy1 gene expression. Consistent with this proposal,

Figure 7. Expression in GA-treated mature aleurone of some LMA-affected genes. The expression of three LMA up-regulated genes (A)and three LMA down-regulated genes (B) was studied. Three biologicalreplicates were performed. Error bars represent SE. nd, Not detected.


Barrero et al.




previous reports for wheat, barley (Hordeum vulgare),and rice (Oryza sativa) indicate that the synthesis ofGA precedes that of ABA during grain developmentbut that the phases of synthesis overlap (King, 1976;Radley, 1979; Mounla et al., 1980; Yang et al., 2003;Eradatmand et al., 2011).

Genetic Characterization of the a-Amylase Response toGA in Mature Aleurone

It is well established that in the mature cereal aleu-rone, GA triggers a series of events that are charac-terized by the synthesis and secretion of severalhydrolytic enzymes (a-amylase, glucanases, proteases,etc.) driven by the GAMYB transcription factor (Murrayet al., 2006) and that lead to the degradation of theendosperm and finally to programmed cell death (PCD)in the aleurone (Fath et al., 2000). In order to compareat the genetic level the typical a-amylase production inthe mature aleurone with the abnormal productionduring LMA in developing aleurone, the genetic re-sponse to added GA in mature aleurone was firstcharacterized. The results for GA-treated mature al-eurone confirmed that the genes encoding high-pIa-amylase (a-Amy1-1 and a-Amy1-2) are strongly in-duced after the GA treatment, whereas genes encodingother a-amylases are lowly expressed (a-Amy2-1 anda-Amy3-1) or not induced (a-Amy4-1). In addition tothe a-Amy1 genes, genes encoding the wheat GAMYB

transcription factor, a glucanase [(1,3;1,4)-b-glucanase],and two proteases (triticain-a and triticain-g) were alsoclearly induced by the treatment in mature aleurone,as expected. There was also evidence of a cross-talkresponse between GA and ABA, as we found the up-regulation of the ABA catabolic gene ABA89OH-1 inthe GA-treated aleurone, which possibly could lead toa decrease in ABA concentration.

Genetic Characterization of the a-Amylase Responseduring LMA

Abnormal production of a-amylase in +LMA geno-types has been well characterized at the enzymatic andcellular levels. Previous studies have shown that high-pI a-amylase is produced in small pockets of cellsrandomly distributed across the aleurone layer duringa particular developmental window (Mrva et al., 2006).We failed to find any anatomical differences betweenaleurone cells from a constitutive +LMA genotype anda –LMA genotype, although this may have been dueto a failure to section affected cells previously shownto be sparsely distributed in the aleurone (Mrva et al.,2006). However, what our anatomical studies show isthat aleurone cells from +LMA and –LMA genotypesare at a similar developmental stage when LMA appears.

The results reported here suggest that the geneticsignature of LMA expression is similar to that of GA-treated mature aleurone only in the expression of the

Figure 8. Hormonal analysis of LMAand non-LMA grains. ABA, GAs, auxins,and cytokinins were quantified in deem-bryonated half-grains isolated at 23 DPA.Two technical replicates per sample wereassayed. For details, see SupplementalTable S3.







a-Amy1 genes. Expression of these genes coincidedwith the appearance of high-pI a-amylase protein at23 and 26 DPA in +LMA but not in –LMA genotypes.Unlike GA-treated aleurone, no expression of otherGA-regulated genes like GAMYB, glucanase, or pro-tease genes was detected. Lazarus et al. (1985) reportedthat the pattern of a-Amy1 mRNA accumulation inwheat aleurone resembled that of nonamylase genesregulated by GA. In this context, the LMA phenotypeappears to be a partial or incomplete GA response.Baulcombe and Buffard (1983) noted, however, thata-AmymRNA was by far the most abundant species intissue incubated with GA. It is possible that the overalllevel of gene expression in LMA-affected aleuronetissue is so much reduced compared with GA-treatedmature aleurone that transcripts of other GA-regulatedgenes are below the level of detection.

These results confirm that high-pI a-amylases arethe enzymes involved in the LMA phenomenon. Aninteresting observation is that the induction of thea-Amy1 genes in the +LMA samples appeared to betransitory in nature over a very narrow time frame.The induction of the a-Amy1 genes only persisted forbetween 1 and 4 d. This is consistent with an earlystudy by Lazarus et al. (1985) that reported that mRNAaccumulation commenced 6 to 12 h after the start ofincubation with GA, reaching a peak at 24 h, except fora-AmymRNA, which declined after 48 h. The transientexpression is also consistent with observations thathigh-pI a-amylase protein increases rapidly during themiddle stages of grain development in LMA geno-types but then plateaus at a level that is relatively lowcompared with GA-challenged aleurone or germina-tion. Nevertheless, there can still be unacceptably highlevels of activity at harvest ripeness, causing low fall-ing numbers in LMA genotypes (Mares and Mrva,2008a).

Global Gene Expression Analysis of LMA

The transcriptome comparison between developingaleurone from +LMA and –LMA genotypes shows alimited number of abnormally expressed genes. Oneexplanation for that is the apparent simplicity of thealeurone tissue, formed by just a single layer of iden-tical cell types, which may have a reduced number ofgenes being expressed. Another possible explanation isthat only about 2% of the aleurone cells in the +LMAgenotypes undergo LMA (Mrva et al., 2006), so geneexpression changes associated with LMA may be di-luted. Of the 56 probes up-regulated in LMA-pronealeurone (Table I), three have been previously relatedto PCD (Initiation factor2 family protein, Programmedcell death protein2, and NucleaseI; Young and Gallie,2000), consistent with the involvement of GA in LMA,as GA response leads to PCD in mature aleurone (Fathet al., 2000). Similarly, another two genes up-regulatedduring LMA have been reported to be induced by GAin rice and barley (Pathogenesis-related protein precursor

and Xyloglucan endotransglycosylase; Yang et al.,2004; Chen and An, 2006). However, we have foundseveral genes related to stress (Wali3, Wali5, and Wali6;Snowden et al., 1995) and ABA (ABA-inducible proteinWRAB1; Tsuda et al., 2000) that are also up-regulatedin the LMA samples and that suggest higher levelsof ABA. The Wali genes affected by LMA belong to aparticular group showing similarity to the Bowman-Birk protease inhibitors (Snowden et al., 1995), whichare very abundant in seeds and have several putativefunctions, including the regulation of exogenous andendogenous seed proteases (Black et al., 2006). Thisclass of Wali genes was not induced by GA in maturealeurone but was actually repressed, thus indicatingclear discrepancies between the LMA phenomenonand the GA effects in mature aleurone.

Twenty-seven probes were up-regulated in the al-eurone of –LMA genotypes (Table II). Of these, themost striking fold-change expression difference wasrecorded for the probe A_99_P142933, possibly sug-gesting a principal role for the gene in repressing LMAduring grain development. However, the lack of sim-ilarity between that sequence and any known genemakes it difficult to speculate about its function. In-terestingly, this gene was not induced by GA inmature aleurone. A gene encoding a g-thionin was up-regulated at both 20 and 23 DPA, and these proteinshave been shown to inhibit insect a-amylase (Pelegriniand Franco, 2005). Another probe up-regulated at both20 and 23 DPA shows great similarity with the ricebrassinosteroid biosynthetic gene Diminuto (Hong et al.,2005) and could suggest a deficiency in brassinoste-roids in the LMA-prone aleurone. Interestingly, a barleyhomolog of this gene was found to be down-regulatedby GA in mature barley aleurone (Chen and An, 2006),but we could not detect its expression in mature wheataleurone. A probe with similarity to manganese su-peroxide dismutase is also up-regulated in the non-LMA aleurone, and this protein is an antioxidant thathas been shown to retard aleurone PCA (Fath et al.,2000).

In summary, microarray results show a very limitednumber of genes affected by LMA. The expression ofsome of them could be explained by an increase in GAsignaling in the developing aleurone; however, severalothers seem to be more related to stress signals andpossibly driven by ABA. This is in marked contrastwith mature aleurone, where GA and ABA act in op-position and are not found together (Fath et al., 2000).

Hormonal Changes Associated with LMA

Several lines of evidence, such as the reduction ofLMA in GA-insensitive phenotypes and the micro-array analysis, suggest that hormones are involved inthe development of LMA. We have proved that bydetailed hormone measurements. We found that ABAconcentration was 2-fold higher in the samples withLMA, consistent with the microarray expression data,


Barrero et al.



in which stress- and ABA-related genes were up-regulated in +LMA aleurone. Several other ABA-relatedmetabolites were assayed, but their concentrationswere similar in all samples. In relation to GAs, 14metabolites were assayed, but only GA19, GA44,GA29, GA8, and GA24 were detected and quantifiable(Supplemental Table S3). All but one (GA24) of thequantified GAs belong to the early 13-hydroxylationbiosynthetic pathway (Sponsel and Hedden, 2004),which suggests that this is the major biosynthetic routein wheat grains. Some other GA compounds have beenfound in wheat grains (Gale and Lenton, 1987), butdue to the lack of standards, they were not analyzed.Neither the active form GA1 nor GA4, the active formin the nonhydroxylation pathway, was detected, but20- and 6-fold increases in the precursors GA19 andGA44, respectively were found. Importantly, the pres-ence of GA8, which is an inactive form derived fromGA1, was detected in +LMA samples but not in –LMAgenotypes. These changes provide strong evidence foractive GA and GA flux in the +LMA samples but notin the LMA-free genotypes. In addition, large differ-ences in the levels of auxins (IAA) between +LMA and–LMA were observed. Auxins levels were 4 timeshigher in –LMA samples, in apparent contradiction ofprevious studies that reported a positive effect of auxinon the production of GA (Singh and Paleg, 1986).Whether the changes in concentration of these hor-

mones are cause or effect of the expression of LMAremains unclear. However, the large differences inhormone content are more likely to have originatedfrom differences existing in the whole tissue and notjust from the small number of aleurone cells that pro-duce LMA. This scenario envisages a deeply alteredhormonal environment across the entire aleurone of+LMA genotypes during and prior to the expression ofLMA. Under these conditions, some of the aleuronecells could escape their normal regulation and triggera partial GA response, producing very transientlya-Amy expression. This model would fit very well withthe increased concentration of GAs and ABA observedin +LMA genotypes: ABA could suppress almost to-tally the GA effect, but in some cells, small changes inhormones and/or sensitivity could generate abnormalgene expression. Whether changes in GAs and ABAconcentrations occur simultaneously or if one precedesthe other is still to be investigated. At the gene ex-pression level, changes in genes related to the hor-mones GA and ABA have been identified. On the basisof the microarray analysis, genes related to GA wouldappear to be altered earlier (20 DPA) than the genesrelated to ABA (at 23 DPA), suggesting that changes inGAs would appear before changes in ABA. In additionto the changes in endogenous hormones reported inthis study, earlier studies have also highlighted thepossibility that LMA expression may also be associ-ated with increased GA sensitivity in LMA-prone linescompared with non-LMA lines (Mares and Mrva, 2008a;Kondhare et al., 2012). A detailed hormonal profilingand analysis of GA signaling components during grain

development is required to resolve these criticalquestions.

MATERIALS AND METHODS

Plant Material

Sets of tall genotypes of bread wheat (Triticum aestivum) with and withoutLMA were selected from a doubled haploid population, cv Spica rht (LMA)/cv Maringa Rht1 (non-LMA), based on a consistent phenotype over severalseasons and alleles at the LMA 7B QTL (Mrva and Mares, 2001b). Doubledhaploid lines 41, 47, 84, and 109, which have a non-LMA phenotype, werecompared with lines 25, 52, 127, and 131, which express constitutive LMA. Tallgenotypes were selected to avoid the confounding effects of the semidwarfing,GA-insensitive gene Rht1 on LMA expression (Mares and Mrva, 2008a). Plantswere grown side by side in pots in a glasshouse, and spikes were tagged atanthesis. Spikes (10 per sampling time) were sampled at 12, 17, 20, 23, 26, 29,32, 35, 40, and 45 DPA for determination of grain moisture, grain dry weight,grain appearance, high-pI a-amylase abundance, and isolation of aleuronetissue for preparation of mRNA. Between anthesis and grain maturity, themean minimum and maximum temperatures were 16°C (range, 12.8°C–20.5°C) and 25.3°C (range, 21.4°C–31.4°C), respectively. Thermal time calculationswere based on a summation of physiologically effective temperatures post-anthesis (thermal time after anthesis = [average daily temperature – basaltemperature for postanthesis development] 3 number of days after anthesis,where basal temperature for postanthesis development is 9.5°C; Angus et al.,1981; Slafer and Savin, 1991).

Grain Moisture and Grain Dry Weight

Duplicate samples of 20 grains were removed from the central part of thespikes, using only grains from the first and second florets. They were imme-diately weighed, dried at 100°C for 2 d, and weighed again. Grain moisturecontent was calculated as percentage fresh weight from the difference.

Determination of High-pI a-Amylase Content

For the extraction of a-amylase, eight replicates each of five deembryonatedgrains per sampling time were crushed, mixed with 1 mL of 0.85 M NaClcontaining 0.018 M CaCl2 on a vortex mixer, incubated at 37°C overnight, andthen centrifuged for 10 min at 14,000 rpm in a microfuge. An aliquot of 100 mLwas used in the ELISA determination of high-pI a-amylase protein. High-pIa-amylase protein was assayed in a 96-well plate format using a modificationof the sandwich ELISA reported by Verity et al. (1999). Plates were coatedwith a rabbit anti-wheat a-amylase polyclonal antibody, blocked with bovineserum albumin, incubated with extracts of grain, washed, and then incubatedwith a mouse anti-barley high-pI a-amylase monoclonal antibody. The plateswere again washed and incubated with horseradish peroxidase-labeled don-key anti-mouse antibody (Sigma) before adding color developer, 3,39,5,59-tetramethylbenzidine substrate (Elisa Systems), and then recording opticaldensity at 595 nm in a microplate reader. Color change was measured witha microplate spectrophotometer, and a-amylase protein in extracts wasexpressed as the mean of the optical density of eight microplate wells minusthe optical density for a non-LMA control, a bulk sample of Sunco grainpreviously shown to be ELISA negative, samples of which were included onall ELISA plates. The polyclonal and monoclonal antibodies are currentlymaintained by the South Australian Research and Development Institute andmade available under a research-only agreement between the Grains Researchand Development Corporation of Australia and Bayer AG, the holder of thepatent covering the use of these antibodies for the determination of a-amylasein wheat. a-Amylase activity in grain extracts was determined using theAmylazyme dye-labeled substrate assay (Megazyme International).

Isolation of Aleurone Tissue

Based on preliminary experiments, it was anticipated that high-pI a-amylasewould be synthesized in +LMA genotypes beginning at 20 to 23 DPA. Conse-quently, grain from samples collected at 17, 20, 23, and 26 DPA was used for theisolation of grain coat plus aleurone, according to the method described byMrvaet al. (2006). Fifty grains per sample were deembryonated, the crease tissue was






removed, and the aleurone was cleaned of starchy endosperm with a scalpel.The tissue was immediately frozen in liquid nitrogen and stored at –80°C untilrequired for RNA isolation.

Mature aleurone layers were isolated from grains of cv Spica as describedpreviously (Chrispeels and Varner, 1967), except that the embryoless half-grains were imbibed only for 24 h. The isolated layers were incubated inflasks containing 2 mL of 10 mM CaCl2, 150 mg mL21 cefotaxime, 50 units mL21

nystatin, and either no hormone (control) or 1 mM GA3 at 25°C for 24 h. Thelayers were snap frozen in liquid nitrogen and stored at 280°C until requiredfor RNA isolation.

Gene Expression Analysis

Samples used in the microarray experiment were dissected from +LMA and–LMA developing aleurone layers. RNA was prepared using the hexadecyl-trimethylammonium method described by Chang et al. (1993). Twenty aleu-rone layers were ground in liquid nitrogen, and the powder was added to5 mL of hot extraction buffer. Following purification, 25 mg was further pu-rified on a Qiagen RNeasy column (Qiagen). The RNA quality was checked onformaldehyde gel (1.2% agarose). Probe synthesis, labeling, and hybridizationto the Wheat Gene Expression 44K chip, which contains 43,663 unique probestargeted to 40,606 unique ESTs (Agilent Technologies), were carried out at theAustralian Genome Research Facilities. Microarray analyses were performedon three independent +LMA doubled haploid lines and three independent–LMA doubled haploid lines, which were treated as biological replicates.

Expression data were analyzed in R. Raw data were corrected for back-ground (BGMedianSignal) and normalized between samples using quantilenormalization. Fold change values and statistically significant differential ex-pression were calculated using the Limma (Linear Models for Microarray Data;Smyth, 2005) package from Bioconductor (www.bioconductor.org). Differen-tially expressed genes with P, 0.01 and fold change thresholds of absolute 1.5were used in final comparisons. The normalized expression of all the probes inall the samples was extracted and is shown in Supplemental Table S1.

For real-time PCR, RNA from developing or mature aleurone was isolatedusing the same protocol as described in the microarray section. A total of 2 mgof total RNA was then used to synthesize cDNA using SuperScript III (Invi-trogen Life Sciences) following the supplier’s recommendations in 20-mL re-actions. RNA extractions were performed on three biological replicates of 15embryos isolated from hydrated or dry grains. cDNA was diluted 50-fold, and10 mL was used in 20-mL PCRs with Platinum Taq (Invitrogen Life Sciences)and SYBR Green (Invitrogen). Reactions were run on a Rotor-gene 3000A real-time PCR machine (Corbett Research), and data were analyzed with Rotor-gene software using the comparative quantification tool. The expression ofTaActin1 (Ji et al., 2011) was used as an internal control to normalize geneexpression. Three biological replicates were performed for each experiment.Primer sequences are given in Supplemental Table S1.

Hormone Analysis

Twenty deembryonated 23-DPA grains from +LMA (lines 25, 52, and 131)and –LMA (lines 41, 47, and 84) genotypes were frozen in liquid nitrogen andlyophilized. Hormone profiling was carried out at the Plant BiotechnologyInstitute of the National Research Council of Canada (http://www.nrc-cnrc.gc.ca/eng/facilities/pbi/plant-hormone.html) following the methods de-scribed by Chiwocha et al. (2003) and Zaharia et al. (2005) by ultra-performanceliquid chromatography-electrospray ionization-tandem mass spectrometry.Two technical replicates were analyzed.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Microsections of aleurone cell layers from2LMAand 1LMA developing grains.

Supplemental Figure S2. Validation of selected microarray probes byqRT-PCR.

Supplemental Table S1. Primers used for qRT-PCR.

Supplemental Table S2. Normalized microarray expression in all samples.

Supplemental Table S3. Hormonal analysis of 23 DPA1LMA and 2LMAdeveloping grains.

ACKNOWLEDGMENTS

We thank Trijntje Hughes, Kerrie Ramm, and Hai-Yunn Law for theirexpert and invaluable technical assistance. We also thank Drs. Peter Chandlerand Jean-Philippe Ral for critical reading of the manuscript.

Received October 21, 2012; accepted January 14, 2013; published January 15,2013.

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