seed - plant physiology · plant physiol. vol. 90,1989 relative hsrandnormalizedhsr...

Plant Physiol. (1989) 90, 598-6050032-0889/89/90/0598/08/$01 .00/0

Received for publication August 15, 1988and in revised form January 24, 1989

Heat Shock Response of Germinating Embryos of Wheat1

Effects of Imbibition Time and Seed Vigor

Kenneth W. Helm, Nancy S. Petersen, and Rollin H. Abernethy*Department of Plant, Soil and Insect Sciences, (K.W.H., R.H.A.) and Department of Molecular Biology (N.S.P.),

University of Wyoming, Laramie, Wyoming 82071

ABSTRACT

Seeds frequently face a hostile environment during early ger-mination. In order to determine whether seeds have evolvedunique mechanisms to deal with such environments, a survey ofthe heat shock response in isolated embryos of wheat (Triticumaestivum L.) was undertaken. Embryos simultaneously heatshocked and labeled following several different periods of priorimbibition up to 12 hours synthesized many groups of heat shockproteins (hsps) typical of other plant and animal systems. Also,five developmentally dependent hsps, present only in treatmentsimbibed less than 6 hours prior to heat shock, were detected.These proteins have relative molecular masses of 14, 40, 46, 58,and 60 kilodaltons. One of the developmentally dependent hspsis among the most highly labeled hsps found in eardy imbibedembryos. The possibility that this protein is the Em protein isdiscussed. The hypothesis that the capacity for hsp synthesis isaffected by seed vigor was also tested. The heat shock re-sponses of embryos from two high and two low vigor seed lotswere compared using one- and two-dimensional electrophoresisof labelled protein extracts. The results indicate that both of thelow vigor lots tested had weaker heat shock responses than theirhigh vigor counterparts overall. Not all hsps were relatively lessabundant in low vigor embryos. The developmentally dependenthsps showed little relationship to vigor. Some of the develop-mentally dependent hsps were actually made in greater amounts,relative to other proteins, in the low vigor seed lots. The resultspresented here demonstrate that imbibing embryos are capableof expressing an enhanced heat shock response, and that thisresponse is related to seed vigor.

Upon imbibition, the quiescent seed embryo faces a hostileenvironment. Extremes of temperature and moisture mayconfront the young plant simultaneously or in rapid succes-

sion. The imbibing embryo must survive this environment inorder to germinate. Since the embryo has no developed leafor root system, it is unable to regulate its temperature andwater status through control of transpiration as do matureplants.

In addition to a potentially hostile environment, the em-

bryo must deal with the problem of germination itself. Mem-

' Research was supported in part by National Science Foundationgrant RII-8610680 (R. H. A.) and United States Department ofAgriculture Hatch Funds to the Wyoming Agricultural ExperimentStation. This paper is Wyoming AES number JA- 1582.

branes must go from a disorganized state to the ordered bilayerfound in functional cells. Mitochondria, owing in large meas-ure to the disorganization of their membranes, are inactive inthe quiescent embryo and must be repaired or replaced inorder for germination to occur. Polysomes must be assembled,and new protein and nucleic acid synthesis are initiatedimmediately upon hydration (6). Osborne (18) has demon-strated that the DNA of quiescent rye embryos accumulatesnicks which must also be repaired in order for normal devel-opment of the young plant to proceed.

Seed vigor is a measure of the seed's ability to germinateand establish under less than optimal conditions. Seed lotswhich show high germinability in the laboratory may dem-onstrate poor field emergence. Seed vigor, then, is a manifes-tation of the ability to survive a series of environmentalstresses during germination (1). Deterioration of seed vigormay take place either during maturation on the mother plantor while the seed is in storage. Observed metabolic deficienciesof low vigor seed have been extensively reviewed (1, 19). Thenet effect ofthe lesions is decreased seed vigor and, ultimately,loss of seed viability. In general, the biochemical sites of seeddeterioration can be grouped into the categories of the accu-mulation ofDNA damage, accumulation of membrane dam-age, and loss of protein synthetic ability (1, 3, 5). Indeed,protein synthesis may be required to repair membrane dam-age (5) and has been a subject of interest as a fundamentalcause for loss of vigor.One of the more accurate indicators of seed vigor is the

accelerated aging test. This test measures a seed's ability towithstand long periods of high temperature and humidity (3).Low vigor seeds show a marked depression in their ability tosurvive and germinate following exposure to such conditions,while high vigor lots demonstrate a high frequency of germi-nation following the test.A brief, sublethal heat shock induces the production of a

number of so called hsps2 in plants (4, 9, 13). Further, such aheat shock has been shown to protect an organism from asecond heat dose that would ordinarily be lethal (15).We hypothesized that the hsr during very early germination

plays an important role in the survival and eventual germi-nation of seeds under stressful conditions. The conditions ofhigh heat and humidity found in the accelerated aging test,

2Abbreviations: hsp, heat shock protein; hs, heat shock; IEF,isoelectric focusing; ANOVA, analysis of variance; hsr, heat shockresponse; Em, early metionine protein.

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WHEAT EMBRYO hsr TO IMBIBITION TIME AND SEED VIGOR

where hsps might be synthesized, led to the formation of thishypothesis. Plants may have evolved extra mechanisms toprotect themselves from the unusual rigors of germination.Low vigor seeds, which do not survive such stresses well,would be expected to have deficient hsr under this hypothesis.Absolute determination that hsps are made during the agingtest is impossible, since the addition of an aqueous mediumcontaining radioactive amino acid would change the waterstatus of the embryo. Such a change might allow artifactualhsp synthesis. Consequently, we have examined the hsrs ofisolated embryos from high and low vigor seed lots of wheatat different times during the first 12 h of imbibition.The wheat germ system is ideal for studying the molecular

events of early germination. Large quantities of embryos canbe isolated in a viable form with ease. Extraction ofDNA andRNA is relatively straightforward, as these embryos are notrich in nucleases. Finally, lysates for in vitro translation maybe prepared from wheat embryos, allowing detailed study oftranslational mechanisms of gene regulation.

MATERIALS AND METHODS

Plant Material

High and low vigor seed lots of Triticum aestivum L. wereobtained from Dr. J. D. Maguire of Washington State Uni-versity, Pullman, WA. Low vigor lots were represented by thecultivars Nugaines 1980 and Wanser 1980. High vigor lotswere Nugaines 1983 and Wanser 1983. Seed vigor ratingswere verified using the accelerated aging test, as described bythe Association of Official Seed Analysts (3), except that theaging treatment was carried out for 24 instead of 48 h. Theaccelerated aging test was done with three replications pertreatment using 100 seeds per replication. From here on,unless noted otherwise, Nugaines 1983 and Wanser 1983 willbe designated as 'Nugaines HV' and 'Wanser HV.' Similarly,Nugaines 1980 and Wanser 1980 will be designated 'NugainesLV' and 'Wanser LV.'

Embryo Isolation

Wheat germ preparations were made according to themethod of Johnston and Stern (12), aliquoted, and stored at-20°C. Undamaged embryos were selected under magnifica-tion from the aliquots just prior to use.

Imbibition, Labeling, and hs

Six embryos from each lot were used for a given treatment.Embryos for all treatments were imbibed for 1.5 or 12 h at21 C, in 20 ,L of an aqueous solution containing 1% sucrose(w/v) and 10 ,Ag/mL of chloramphenicol.

Following imbibition, embryos from each lot were labeledwith [35S] methionine (New England Nuclear, specific activity>800 Ci/mmol) or Tran-35S (ICN, specific activity >800 Ci/mmol) at either 21 or 42°C, for 1.5 h. To label a treatment,embryos were moved from imbibition solution to 15 gL ofthe same solution supplemented with 3 to 6 uCi/AL of label.Labeling was carried out in a water saturated atmosphere tominimize evaporation. Incorporation into protein was meas-

ured according to Mans and Novelli (16). There was noapparent difference in autoradiograms of tissue homogenatesincubated with the two different types of label.

Electrophoresis

One-dimensional SDS-PAGE was done by the method ofLaemmli (14) using the modifications described by Petersenand Mitchell (20). Samples ofequal incorporated radioactivity(>100,000 cpm) were brought to a volume of 20 ,uL withsample buffer (14) and loaded onto each lane of a gel. Acurrent of 13 mA was applied for approximately 16 h. Follow-ing electrophoresis, gels were fixed, stained with CoomassieR-250, and dried. Autoradiography was conducted by expos-ing the dried gels to Kodak SB-5 x-ray film for 7 to 14 d,depending on the amount of radioactivity present. Individualbands on an autoradiogram were quantified with an LKBlaser densitometer.Two-dimensional electrophoresis according to Buzin and

Petersen (8) was done by replacing pH 5-8 ampholines withpH 5-7 ampholines (Bio-Rad). IEF was performed by apply-ing 300 V to 10 tubes, 2.3 x 85 mm, for 21 h (630 V-h/tube).The lower chamber ofthe IEF apparatus was cooled to 18.5°C.The low voltage, as well as the slight cooling, resulted in amore linear pH gradient, with very little smearing in the basicportion of the gels.Two IEF tubes were loaded on to each second dimension

gel. Both tubes of a given pair contained equal incorporatedradioactivity, as described above. A pair of tubes to be loadedon a second dimension consisted of proteins extracted fromeither a high and low vigor control or a high and low vigorheat shock treatment. In this way, gel to gel variability wasminimized and direct comparisons could be made betweenthe heat shock responses ofhigh and low vigor lots. Completedtwo-dimensional gels were treated with Enhance3, dried, andexposed for an amount oftime dependent on the cpm presentin the gel. One-dimensional gels were not treated withEnhance.

Quantification and Normalization of Data

Incorporation of [35S]methionine into protein during hswas quantified as described above and normalized as de-scribed by Smith and Bray (21). For an intravarietal seed lotcomparison at a particular time, the lot displaying the greatestincorporation was set to 100 and the lower figure expressedas a percentage ofthe higher number. Statistical analyses wereperformed on the normalized data.

Quantification of overall hsrs from one-dimensional gels,as well as the comparisons of the relative synthesis of specifichsps from two-dimensional gels were performed using laserdensitometry. Film used for autoradiography (not preflashedprior to exposure), was exposed within the limits of linearity.All densitometry was done using an LKB laser densitometer.Results were analyzed with LKB's Ultroscan software.

3 Mention of trade name or specific supplier is provided for infor-mation only and does not imply endorsement by the University ofWyoming.

599

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Plant Physiol. Vol. 90,1989

Relative hsr and Normalized hsr

For determination of the relative magnitudegeneral, entire lanes of one-dimensional autorzlabeled proteins from heat shocked embryos wThe relative hsr was calculated by dividing tidetected by the densitometer of individual bandtive of hsps 97, 94, 83, 70, 68, 21, 18 and 17 kEo.d. units detected on a given lane. The relative ]

normalized. For each autoradiogram, the large]of a given vigor comparison was set to 100. The 4

was then expressed as a percentage of the largeresulting values are the normalized hsrs. Only hdetectable in all one-dimensional gels, at bothof imbibition were included in the analysis.

Corrected Spot Volumes

The relative synthesis of specific hsps by Iseedlots was determined by scanning selectedtwo-dimensional autoradiograms (boxed in Fig.grated using the LKBs Ultroscan software packalhsp spots (circled in Fig. 3) whose volume di

vigor-dependent were scanned and integrated asvolume of a given hsp spot was expressed as thihsp volume to the sum of the volumes of thespots. This standardization was done in order tofor any minor loading differences between tw4These adjusted values are the corrected spot volt

RESULTS

An autoradiogram demonstrating the hsr durimination in wheat embryos is shown in Figure ]

PIr std.'kD)It

66-

45-

..

a e h

Figure 1. Hsr during early imbibition. Embryo treatrSDS-PAGE, and autoradiography were conducted a:'Materials and Methods." Lanes a-f, embryos were1.5, 3, 6, 9, or 12 h, respectively, then labeled at 2lanes g-l, embryos were imbibed for 0, 1.5, 3, 6,respectively, then labeled at 420C for 1.5 h. Positions;wt standards are given on the left, while positions andof hsps visible in the autoradiogram are given on the r

iment was repeated four times, initially with Nugaines HV(1983) and later with another high vigor cultivar, Nugainesof the hsr in 1986 (shown). The data shows that wheat embryos produce a

idiograms of wide range of hsps at all imbibition times tested, including anrere scanned. hs treatment with no prior imbibition at 2PC. Relative massesIe o.d. units of hsps seen at all time points are 17, 18, 21, 68-70, 83, 94,Is representa- and 97 kD. Three bands, 14 kD and a 40 kD doublet, are) by the total only seen in hs treatments with 3 h or less of prior imbibition.[sredataiwere The 14 kD band appears at low levels in the 2PC controlr relative hsr treatments at the first two time points.

r value. The A comparison of the results of the accelerated aging test,s thatlue.rTe incorporation of [35S]methionine, and the ability of the four

sps that were seed lots examined to synthesize a number of different hspsL.5 andl12 h is summarized in Table I. All seed lots were of high viability,

as indicated by greater than 90% germination in a standardgermination test at 2PC.

Vigor ratings, based on the accelerated aging test, are de-fined as the percentage of seeds able to germinate following

HV and LV the accelerated aging treatment divided by the percentage ofhsp spots on seeds able to germinate at 2PC. A lower numerical rating is3) and inte- indicative of lower seed vigor.Two non- Total protein synthesis during hs was estimated determiningenot appear the incorporation of [35S]methionine into protein (TCA) pre-

controls. The cipitable counts). The average total incorporation of label pere ratio of the embryo, average normalized incorporation, and the results oftwo control one way ANOVA tests, performed on the normalized data,compensate are given in Table I. All of the ANOVA tests indicated that

o IEF tubes. LV seed lots incorporated significantly less label than did theumes. HV lots, with the exception of Wanser, following 12 h of

imbibition. Diminished capacity for protein synthesis in lowvigor seed lots has been observed previously (1).For both Nugaines and Wanser, the 1980 seed lots displayed

ing early ger- less vigor than did the 1983 lots (hence the designations 'HV'1. The exper- and 'LV'). The average relative hsr was reduced in the low

vigor lots when the embryos were heat shocked following 90Mr hsp min of 2PC imbibition (Fig. 2). The hsrs, based on the one-Nkm dimensional gel information, of the high and low vigor lots-94_g7 were nearly the same following 12 h of imbibition. ANOVA

* -83 tests performed on the normalized hsrs (defined in "Materials*_ -83

-68-70 and Methods") are different at the 0.01 level of significanceat the earlier time ofimbibition. Following 12 h ofimbibition,

*- the differences between the high and low vigor lots were lesspronounced. Results of one way ANOVA tests are summa-

1-40 rized in Table I. Representative fluorograms of two-dimen-1 sional gels of protein extracts from heat shocked embryos of

the four seed lots are shown in Figure 3, A (Nugaines) and B(Wanser). The two-dimensional gels show four groups of hsps

L|-21 expressed only during early germination, as well as numerousI w -17-18 differences in the hsrs of the high and low vigor lots. In

- 14 general, the differences in the hsr between high and low vigorseed lots may be summarized as follows: (a) the develop-mentally dependent hsps were either unchanged or showed

nent, labeling, greater relative synthesis in the low vigor lots (b) the 94 to 97s described in kD group of proteins showed variable results, (c) the 68 to 70

10° for 1.5 h kD group showed unchanged or greater synthesis in the low9, and 12 h, vigor lots after 1.5 h of imbibition and lower relative synthesisand Mrs of mol in the low vigor lots after 12 h of imbibition, and (d) all ofcalculated Mrs the other groups of hsps showed lower synthesis in the lowight. vigor lots. The corrected volumes of the spots representing

600 HELM ET AL.

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Table I. Viability, Vigor, and Protein Synthesis Characteristics of Wheat SeedlotsVigor ratings, germination after 5 d at 200C, mean cpm of incorporation, normalized values of

incorporation during hs, and normalized heat shock responses (Nhsr) of the four wheat seed lotsNugaines HV, Nugaines LV, Wanser HV, and Wanser LV were determined as described in "Materialsand Methods." The label incorporation values were obtained from five different replications. Values inparentheses represent the averages of these replications, and are given in cpm x 1 03/embryo. TheNhsrs shown represent averages computed from four different labelings, with a single one-dimensionalgel per labeling per treatment (sometimes, protein extracts from different varieties were run on separategels). One way analyses of variance were performed between high and low vigor lots, within a variety,for both imbibition times on the normalized incorporation and Nhsr data. High vigor response wasdifferent from the low vigor response at the 0.01 level of significance (**) and at the 0.05 level ofsignificance (*) for [35S]methionine incorporation or hs data.

1.5 h Imbibition 12 h Imbibition

Seed Lot Vigor StandardSeetRating Germination incorration Normalized Nhsr Incorporation Nomaized Nhsrincorporation incorporation

cpm x 10-3 cpm x 10-3Nugaines HV 100 93 (1300) 1 00** 1 00** (1601) 1 00** 99Nugaines LV 49 100 (487) 44 81 (697) 44 91

Wanser HV 100 97 (1434) 1 00** 1 00** (1327) 86 100*Wanser LV 76 99 (568) 42 84 (851) 64 92

Mr stdNkO

93-

45-

31-E411llllllllllll-2,4-t8~~~~~~~~~~~~~~~-222

a b c d e f g h ij k I m n o p

Figure 2. Comparison of the hsrs of high and low vigor embryosfrom Nugaines and Wanser wheat. Embryo treatment, labeling, SDS-PAGE, and autoradiography were done as described in "Materialsand Methods." Each set of four lanes has the seed lots NugainesHV, Nugaines LV, Wanser HV, and Wanser LV in that order. Lanesa-d, embryos imbibed 1.5 h then labeled at 21 0C for 1.5 h, lanes e-h, embryos imbibed 1.5 h then labeled at 420C for 1.5 h; lanes i-I,

embryos imbibed 12 h then labeled at 210C for 1.5 h, lanes m-p,embryos imbibed for 12 h, then labeled at 420C for 1.5 h. Positionsand Mrs of mol wt standards are given on the left, while positionsand calculated Mrs of hsps included in the calculation of normalizedhsrs are given on the right.

hsps seen in Figure 3, determined densitometrically, are givenin Table II. There are no dramatic, qualitative differences inthe hsrs between the two varieties.

DISCUSSION

This study established that a germinating wheat embryo isable to mount a complete hsr from the very earliest time ofimbibition. In fact, wheat embryos are able to synthesize a

complete set of hsps when heat shocked simultaneously withthe initiation of imbibition. Many of the hsps found in other

plant and animal systems are also present in heat shockedwheat embryo protein extracts (Fig. 1). Groups of proteins ofsimilar mass to hsps 97, 94, 83, 18, and 17 have been reportedin other plant systems including maize (9) and soybean (13,15). The profile of hsps that we have observed after 12 h ofgermination closely resembles that reported by Mansfield andKey (17) in 3 d old wheat seedlings. The ubiquitous hsp 68to 70 is nearly universal in its occurrence and it is no surprisethat is also seen in heat shocked wheat embryos.

In both Nugaines and Wanser, particularly at the earliertime, we observed the synthesis of members ofthe hsp 97 and94 kD group, as well as proteins belonging to the 68 to 70 kDgroup at 2lC. It is not certain whether these are actual hspsor so called hs cognates (1 1). Similar developmental regulationof hsps in the absence of heat stress has been seen in a hostofother organisms (7). To date, the functions ofthese proteinsin development, if any, remains unknown.An exciting result of this study is the discovery of several

hsps of 60, 58, 46, 40, and 14 kD that are expressed very earlyin germination. They are detectable only at early imbibitiontimes (Fig. 3, A and B). The 14 kD protein appears to bemade at low levels at ambient temperature through 3 h ofimbibition prior to labeling. It is possible that these proteinshave evolved specifically to meet particular demands of earlygermination. We have previously shown that wheat embryosare unusually tolerant of high temperatures during early ger-mination (2). This tolerance diminishes after 6 to 8 h ofimbibition, coincident with the loss of ability to make theseproteins. Alternatively, the developmentally dependent hspsmay be required for protection ofthe axis from stress induceddamage during embryogenesis, and what we observe is rem-nant expression during germination.

It is possible that the 14 kD hsp that we observed may beidentical to the so-called Em protein, first described by Thomp-son and Lane (22). Three reasons suggest that this is the case.The Mr of hsp 14 is similar to that reported for Em (25). Theisoelectric point of Em, according to the results of Thompson

601




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Figure 3. Two-dimensional analysis of the heat shock responses in embryos of Nugaines (A) and Wanser (B) wheat. Embryo treatment, labeling,two-dimensional electrophoresis, and fluorography were conducted as described in 'Materials and Methods." Two-dimensional electrophoresiswas performed on three separate labelings, although only the autoradiograms shown were scanned with the densitometer. Two-dimensionalgels of 21 0C profiles were performed for all seed lots. As there were no distinguishable differences between 21 0C controls of high and low vigorembryos, the autoradiograms whose exposures most closely match the autoradiogeams of heat shocked embryos are shown here. Seed lotdates, imbibition times, temperatures, and calculated Mrs of hsps are given in the figures. Panels are shown with the IEF dimension horizontally,with the acid side on the right, and the SDS-PAGE dimension vertically. Effective pi range in this system is about pH 4.0 to 7.3. Hsps are

delineated by boxes. Control spots used in the densitometer analysis are identified by circles.

and Lane (22), is similar to that ofhsp 14. The hsp is expressedonly during early germination, in a manner much like thatreported for Em by Williamson and Quatrano (24). Finally,the abundant synthesis of hsp 14 is very reminiscent of theEm protein. The evidence for the identity of hsp 14 we present

here is in no way conclusive. However, we are actively inves-tigating the possibility that hsp 14 is the Em protein at thistime.Another major finding of this research is the reduced rela-

tive synthesis of a number of hsps in low vigor embryos. Other

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investigators have shown that low vigor embryos synthesizeless protein (1) and transcribe less mRNA under stressfulconditions (21) than their high vigor counterparts. Our dataagree with these findings (Table I), in that overall proteinsynthesis is reduced in low vigor seed lots under stressfulconditions. The electrophoretic analyses of the synthesizedproteins further show that the reduction is in part due to adimunition of hsp synthesis. Not only do low vigor embryoshave reduced capacity to make proteins in general, they are

unable to respond efficiently to an applied stress. This effectis apparent in the embryos heat shocked after 1.5 or 12 h ofimbibition at 2 IC. Figures 2 and 3 show that after both timesof imbibition, the reduction of hsp synthesis in low vigor

embryos is most pronounced in the 17 to 18 kD group, thetwo 21 kD proteins, and hsp 83. Synthesis of hsp 25 is nearlyundetectable in low vigor embryos. These specific decreasedresponses to a stimulus (heat shock) point to lesions in theprotein synthetic system, in addition to the generalized decayof DNA demonstrated by Osborne (18). It is possible thateither the low vigor embryos' ability to sense stress is impairedor that specific factors required for gene expression are missingin such embryos. Loss of the ability to sense heat might be aresult of deterioration of hs sensing proteins such as the hsactivator protein in Drosophila (26). Deterioration of anyother component of the embryos' transcriptional systemmight also result in a decreased ability to make a specific

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Table II. Densitometric Comparison of Synthesis of hsps Delineated in Figure 3, A and BVolumes of spots were determined densitometncally and normalized using encircled spots, as

described in "Materials and Methods." Dashed lines indicate that a given protein was not visible in aparticular autoradiogram. Meaningful comparisons can only be made for high/low vigor pairs of thesame variety and prior imbibition time, as each such comparison represents a single two-dimensionalseparation.

Corrected Spot Volumes with Imbibition Time Prior to Labeling:

1.5h 12hMl pi

Nugaines Wanser Nugaines Wanser

HV LV HV LV HV LV HV LV

kD

97 6.4 2.28 1.25 1.07 2.00 1.80 0.77 0.18 0.1394 6.3-6.5 3.57 1.74 3.38 4.00 1.57 0.24 0.70 0.1083 6.9 3.66 1.38 1.34 1.07 1.65 0.45 3.93 2.0570-68 5.3-5.6 14.32 15.45 11.87 15.31 4.71 2.57 5.78 3.5360-58 6.0 3.59 4.01 3.88 3.82 -46 5.6 0.91 0.62 0.45 0.37 -40 3.5 0.05 2.07 0.90 1.2925 5.2 0.81 0.06 0.52 0.16 0.55 0.00 0.46 0.3021 5.8 0.36 0.20 0.23 0.27 0.48 0.10 0.24 0.0721 6.1 0.20 0.13 0.13 0.15 0.56 0.11 0.54 0.0617-18 3.8 0.34 0.85 0.54 0.22 0.53 0.0917-18 4.6 1.40 0.54 0.84 0.4617-18 5.6 9.83 5.38 6.20 5.68 9.73 4.40 4.69 2.3517-18 5.9 10.13 6.27 8.65 7.57 9.33 4.51 8.49 3.8117-18 6.2 0.05 0.00 0.05 0.03 0.23 0.00 0.13 0.0217-18 6.8 0.85 0.13 1.39 0.94 0.84 0.16 2.01 0.6114 5.5 4.88 9.09 2.72 2.0 -

protein. Finally, decay of translation initiation or elongationfactors might prevent the translation of hsp messages. Vigorrelated deficiencies of the initiation factor eIF-2 have beenpreviously observed (10).There are several hsps that show greater relative synthesis

in low vigor embryos. With one exception, all are develop-mentally dependent hsps found only during early imbibition.The 14 and 40 kD proteins, after 90 min of imbibition, arethe main examples. The 70 kD group is also made in largerproportion in the low vigor treatments, after 1.5 h of imbibi-tion. Two other sets of proteins, 58 to 60 kD and 45 kD, aremade in roughly equal proportions in high and low vigorembryos. Perhaps these proteins are being made in responseto some other heat induced damage which is more profoundin the low vigor embryos. Another possibility is that the abilityto synthesize these proteins is protected from degradativeprocesses that affect the expression of other genes. Thus,synthesis of such proteins is preserved, while the capacity forsynthesis of "unprotected" gene products decays along withthe vigor of the seed. The Em protein, which may be hsp 14,can be thought of as having such protection in that its mRNAis stored in the dry and early imbibed embryo. Translation isthe only process that has to occur in order for the protein tobe synthesized (24). Vierling and Sun (23) have observedmessages encoding hsp 70, as well as a variety of the low molwt hsps in quiescent axes of pea. We have detected mRNAfor hsp 70 in dry wheat embryos (our unpublished data).Perhaps the early germinating embryo relies at least in parton stored messages to synthesize hsps. The presence of stored

hsp mRNA might explain some of the variable deficienciesin the hsrs of low vigor embryos. After the messages decay,the embryo would rely entirely on newly transcribed RNAfor its hsr.An important point ofthe preceding discussion is that vigor

related differences in the hsr cannot be easily categorized intoa single, overall decline in the response. The relative synthesisof a number of hsps is either unchanged or greater in the lowvigor lots. The synthesis of other hsps, such as hsps 83, 25,and some of the 17 to 18 group is considerably decreased inthe low vigor lots. The data in Figure 3 and Table II showthat the decreased hsrs in low vigor embryos are due toreductions in the relative synthesis of some, but not all hsps.Therefore, the moderate decline in relative hsrs seen in lowvigor lots is due to fairly large decreases in the synthesis ofspecific hsps. The decreased hsrs observed in the low vigorlots are due to specific lesions in the gene expression apparatiof low vigor embryos, and do not represent a general inabilityto respond to stress.

If the germinating embryo relies on a full set of hsps forprotection against heat induced damage, then marked declinesin the synthesis ofa small number these proteins would makethe seedling more susceptible to stress. Such embryos wouldbe, by definition, low vigor.The results presented here support the idea that the germi-

nating embryo relies on its ability to produce a full set of hspsto survive heat stress. Low vigor embryos are deficient in theirability to synthesize a number of these proteins efficiently.Lowered seed vigor is a composite of many biochemical

604 HELM ET AL.




lesions (1). The reduced hsr that we have observed is probablya manifestation of the numerous initial effects of loss of seedvigor. Later, this reduction in hsr could become a part of thecomposite of biochemical lesions, leading to a further decayofvigor by making the seeds more vulnerable to stress induceddamage. In this model, decreased hsrs are both a result, anda cause of lowered seed vigor.

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