Analysis of gene sequences indicates that quantity not quality of chloroplast small HSPs improves thermotolerance in C4 and CAM plants.

Chloroplast-localized small heat-shock proteins (Cp-sHSP) protect Photosystem II and thylakoid membranes during heat and other stresses, and Cp-sHSP production levels are related to plant thermotolerance. However, to date, a paucity of Cp-sHSP sequences from C4 or CAM species, or from other extremely heat-tolerant species, has precluded an examination to determine if Cp-sHSP genes or proteins might differ among plants with photosynthetic pathways or between heat-sensitive and heat-tolerant species. To investigate this, we isolated and characterized novel Cp-sHSP genes in four plant species: two moderately heat-tolerant C4 species, Spartina alterniflora (monocot) and Amaranthus retroflexus (eudicot), and two very heat-tolerant CAM species, Agave americana (monocot) and Ferocactus wislizenii (eudicot) (respective genes: SasHSP27.12, ArsHSP26.43, AasHSP26.85 and FwsHSP27.52) by PCR-based genome walking and cDNA RACE. Analysis of these Cp-sHSPs has confirmed the presence of conserved domains common to previously examined species. As expected, the transit peptide was found to be the most variable part of these proteins. Promoter analysis of these genes revealed differences in CAM versus C3 and C4 species that were independent of a general difference between monocots and eudicots observed for the entire protein. Heat-induced gene and protein expression indicated that Cp-sHSP protein levels were correlated with thermotolerance of photosynthetic electron transport, and that in most cases protein and transcript levels were correlated. Thus, available evidence indicates little variation in the amino acid sequence of Cp-sHSP mature proteins between heat-sensitive and -tolerant species, but that variation in Cp-sHSP protein production is related to heat tolerance or photosynthetic pathway (CAM vs. C3 and C4) and is driven by promoter differences. Key message We isolated and characterized four novel Cp-sHSP genes with promoters from wild plants, analysis has shown qualitative and quantitative interspecific variations in Cp-sHSPs of C3, C4, and CAM plant thermotolerance.

A maize line resistant to herbivory constitutively releases (E) -beta-caryophyllene.

Various pests, such as those in the order Lepidoptera, frequently feed on young maize (Zea mays) plants and pose a significant threat to plant development and survival. To manage this problem, maize generates a wide variety of responses to attack by pests, from activation of wound-response pathways to the release of volatile compounds. Mp708, an inbred line resistant to feeding by the larvae of the fall armyworm (Spodoptera frugiperda J.E. Smith Lepidoptera: Noctuidae), has been developed through traditional breeding methods, but its underlying mechanisms of resistance are still not completely understood. Mp708 has been shown to have a moderately high constitutive expression of jasmonic acid (JA) before infestation by fall armyworm. However, Tx601, a genotype susceptible to feeding by fall armyworm, activates JA pathway only in response to feeding, suggesting that Mp708 is "primed" to respond swiftly to an attack. Current research indicates that fall armyworm show a lack of preference to feeding on Mp708, leading to the hypothesis that volatiles constitutively released by the plant may also play an important role in its resistance. Analysis of volatiles released by Mp708 and Tx601 in the presence and absence of fall armyworm larvae identified (E)-beta-caryophyllene, a terpenoid associated with resistance, released constitutively in Mp708. Fall armyworm fed samples of both Mp708 and Tx601 showed high transcript number of tps23, the gene responsible for the synthesis of (E)-beta-caryophyllene. In addition, fall armyworm larvae show a preference for Tx601 whorl tissue over Mp708 tissue, and the dosage of Tx601 whorl with (E)-beta-caryophyllene repels the fall armyworm.

Integration of ethylene and jasmonic acid signaling pathways in the expression of maize defense protein Mir1-CP.

In plants, ethylene and jasmonate control the defense responses to multiple stressors, including insect predation. Among the defense proteins known to be regulated by ethylene is maize insect resistance 1-cysteine protease (Mir1-CP). This protein is constitutively expressed in the insect-resistant maize (Zea mays) genotype Mp708; however, its abundance significantly increases during fall armyworm (Spodoptera frugiperda) herbivory. Within 1 h of herbivory by fall armyworm, Mir1-CP accumulates at the feeding site and continues to increase in abundance until 24 h without any increase in its transcript (mir1) levels. To resolve this discrepancy and elucidate the role of ethylene and jasmonate in the signaling of Mir1-CP expression, the effects of phytohormone biosynthesis and perception inhibitors on Mir1-CP expression were tested. Immunoblot analysis of Mir1-CP accumulation and quantitative reverse-transcriptase polymerase chain reaction examination of mir1 levels in these treated plants demonstrate that Mir1-CP accumulation is regulated by both transcript abundance and protein expression levels. The results also suggest that jasmonate functions upstream of ethylene in the Mir1-CP expression pathway, allowing for both low-level constitutive expression and a two-stage defensive response, an immediate response involving Mir1-CP accumulation and a delayed response inducing mir1 transcript expression.

Degradation of the S. frugiperda peritrophic matrix by an inducible maize cysteine protease.

A unique 33-kDa cysteine protease (Mir1-CP) rapidly accumulates at the feeding site in the whorls of maize (Zea mays L.) lines that are resistant to herbivory by Spodoptera frugiperda and other lepidopteran species. When larvae were reared on resistant plants, larval growth was reduced due to impaired nutrient utilization. Scanning electron microscopy (SEM) indicated that the peritrophic matrix (PM) was damaged when larvae fed on resistant plants or transgenic maize callus expressing Mir1-CP. To directly determine the effects of Mir1-CP on the PM in vitro, dissected PMs were treated with purified, recombinant Mir1-CP and the movement of Blue Dextran 2000 across the PM was measured. Mir1-CP completely permeabilized the PM and the time required to reach full permeability was inversely proportional to the concentration of Mir1-CP. Inclusion of E64, a specific cysteine protease inhibitor prevented the damage. The lumen side of the PM was more vulnerable to Mir1-CP attack than the epithelial side. Mir1-CP damaged the PM at pH values as high as 8.5 and more actively permeabilized the PM than equivalent concentrations of the cysteine proteases papain, bromelain and ficin. The effect of Mir1-CP on the PMs of Helicoverpa zea, Danaus plexippus, Ostrinia nubilalis, Periplaneta americana and Tenebrio molitor also was tested, but the greatest effect was on the S. frugiperda PM. These results demonstrate that the insect-inducible Mir1-CP directly damages the PM in vitro and is critical to insect defense in maize.

Heat-Shock Response in Heat-Tolerant and Nontolerant Variants of Agrostis palustris Huds.

The heat-shock response in heat-tolerant variants (SB) and non-tolerant variants (NSB) of creeping bentgrass (Agrostis palustris Huds.) was investigated. Both variants were derived from callus initiated from a single seed of the cultivar Penncross. SB and NSB synthesized heat-shock proteins (HSPs) of 97, 83, 70, 40, 25, and 18 kD. There were no major differences between SB and NSB in the time or temperature required to induce the heat-shock response. When the HSPs synthesized by SB and NSB were analyzed by two-dimensional gel electrophoresis, it was apparent that SB synthesized two to three additional members of the HSP27 family, which were smaller (25 kD) and more basic than those synthesized by NSB. Analysis of F1 progeny of NSB x SB indicated that 7 of the 20 progeny did not synthesize the additional HSP25 polypeptides. These progeny were significantly less heat tolerant than progeny that did synthesize the additional HSP25 polypeptides. The X2 test of independence (X2 = 22.45, P < 0.001) indicated that heat tolerance and the presence of the additional HSP25 polypeptides are linked traits.

Recovery from Heat Shock in Heat-Tolerant and Nontolerant Variants of Creeping Bentgrass.

Recovery from the heat-shock response was tested in heat-tolerant (selected bentgrass [SB]) and nontolerant (nonselected bentgrass [NSB]) variants of creeping bentgrass (Agrostis palustris Huds.) SB increased incorporation of radioactive amino acids into protein 2 h earlier than NSB when leaf blades were incubated at the recovery temperature following heat shock. Electrophoresis indicated that heat-shock protein (HSP) synthesis decreased and normal protein synthesis increased at 4 h in SB and at 6 to 8 h in NSB. Increased synthesis of normal proteins was not due to increased abundance of normal mRNAs, which were equivalent in SB and NSB at 4 h. But at 4 h, more of the normal mRNA population was associated with polysomes in SB than in NSB. Synthesis of HSP70 and HSP18 decreased earlier in SB than in NSB. The decreased synthesis of these HSPs appeared to be correlated with decreased mRNA abundance. But at 4 h, some of the HSP18 mRNA may have been associated with heat-shock granules in SB. Synthesis of HSP25 continued through the 8-h recovery in both variants. Although the abundance of HSP25 was equivalent in SB and NSB during heat shock and recovery, more HSP25 mRNA was associated with polysomes in SB than in NSB.

Rapid qualitative protease microassay (RPM).

A rapid qualitative protease microassay (RPM) was developed as an alternative to conventional assays of cysteine protease activity in HPLC fractions. Using this technique protease activity in samples could be visually determined within 5 min. The method was sensitive to 3.3x10(-7) U/mL of papain and detected cysteine protease activity in dilute HPLC fractions with activity of 5.4x10(-5) U/mL. Because the method monitors the decolorization of Coomassie Brilliant Blue stained substrate, it can be modified to detect other classes of proteases.

Influence of whorl region from resistant and susceptible corn genotypes on fall armyworm (Lepidoptera: Noctuidae) growth and development.

The effect of diets prepared from whorl tissue of resistant and susceptible corn genotypes, Zea mays L., on the larval growth, development, and physiology of fall armyworm, Spodoptera frugiperda (J. E. Smith), was analyzed. Larvae reared on an optimized artificial diet had a higher growth rate and developed faster than those reared on lyophilized whorl tissue from resistant and susceptible genotypes. Larvae reared on the resistant material were smaller and had a longer developmental period. Larvae reared on yellow-green and green whorl sections from resistant plants were significantly smaller than those reared on the same sections of susceptible plants. There was no significant difference in weight when larvae were reared on the yellow whorl regions from either resistant or susceptible lines. Physiological indices were determined for larvae fed resistant and susceptible lyophilized and fresh whorl material. Larvae fed resistant lyophilized material had significantly lower growth rate (GW) and efficiency of conversion of ingested food to body substance (ECI) than those reared on artificial diet or susceptible material. However, there were no significant differences in consumption index (CI), approximate digestibility (AD) and efficiency of conversion of digested food to body substance (ECD) between larvae reared on lyophilized tissue from resistant and susceptible genotypes. Larvae reared on fresh yellow-green whorl sections from resistant plants had significantly lower GW, ECI, and ECD than those reared on susceptible material. In contrast, no significant differences in any of the estimated food consumption and utilization indices were observed between larvae reared on fresh yellow whorl sections from resistant or susceptible plants. These results suggest that some components of whorls from resistant plants, especially the yellow-green region, inhibit food utilization in fall armyworm larvae.

Storage Protein Synthesis during Oat (Avena sativa L.) Seed Development.

Oat (Avena sativa L.) seeds harvested at 2-day intervals from anthesis to maturity were tested for their ability to incorporate [(35)S]sulfate into protein. Incorporation of [(35)S]sulfate into TCA-insoluble material began 2 to 4 days postanthesis (DPA), reached a peak 14 to 16 DPA, and was barely detectable by 24 DPA. Incorporation of label into globulin was parallel to total protein accumulation, and averaged about 85% of the total protein synthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total protein extracted from developing seeds indicated that some polypeptides coinciding with the alpha and beta globulin subunits were present 2 to 4 DPA, but the full complement of globulin polypeptides was not present until 10 DPA. Immunoprecipitation of in vivo labeled seed extracts showed that globulin polypeptides and the 59 kilodalton precursor were present at early stages of development (4 DPA). Quantitation of dot blot analysis, using an oat globulin cDNA clone as a probe, indicated that one species of oat globulin mRNA was most abundant 15 DPA, which is during the peak time of storage protein synthesis.

A simple technique for the preparation and storage of sucrose gradients.

A method for preparing multiple sucrose gradients by quickly freezing layers of sucrose has been developed. These gradients may be stored in the freezer indefinitely, and thawed from 8 to 24 h at 4 degrees C before use. The middle region of the resulting sucrose gradients was linear. Thawing time and centrifugation had little effect on the shape of the gradient. The method is applicable for both small- and large-volume centrifuge tubes. Gradients prepared in the same batch were nearly identical.

Quantitation of oat globulin by radioimmunoassay.

A radioimmunoassay was used to determine the globulin content of developing oat (Avena sativa L. var Coker 6622) seeds. The globulin content increased from 0.32 to 2.37 milligrams per seed from 2 to 21 days post anthesis. The amount of globulin did not increase from 21 to 30 days post anthesis. When the total protein of seeds harvested 21, 24, and 30 days post anthesis was measured by micro-Kjeldahl analysis, it was determined to be 3.15 milligrams per seed for each sampling date. When the amount of globulin per seed was compared to this value, the relative proportion of globulin to total seed protein was approximately 75%.

Biochemical characterization of rice glutelin.

The two major subunits of rice glutelin, the acidic (alpha) and basic (beta) polypeptides were purified by chromatofocusing and cation exchange chromatography, respectively. The molecular weight range of the alpha polypeptides was 28.5 to 30.8 kilodaltons and the molecular weight range of the beta polypeptides was 20.6 to 21.6 kilodaltons. Electrofocusing in polyacrylamide gels showed that the isoelectric points of the alpha and beta polypeptides were 6.5 to 7.5 and 9.4 to 10.3, respectively. At least 12 polypeptides of the alpha-group and nine polypeptides of the beta-group could be separated by electrofocusing. The amino acid compositions of whole glutelin, and the purified alpha and beta subunits were analyzed. The alpha subunit contained more glutamic acid/glutamine, serine, and glycine, and less alanine, lysine, aspartic acid/asparagine, and isoleucine than the beta subunit. A comparison of the amino acid composition of rice glutelin subunits with those of the 11S proteins from eight other plant species indicated that there is more similarity between the beta subunits than the alpha subunits of several diverse plant species.

Transcription in Isolated Wheat Nuclei: I. ISOLATION OF NUCLEI AND ELIMINATION OF ENDOGENOUS RIBONUCLEASE ACTIVITY.

Nuclei isolated from embryos of wheat (var. Yamhill) incorporated [(3)H]UTP into a trichloroacetic acid-insoluble product linearly for 60 minutes. When the RNA synthesized in vitro was analyzed on a sucrose gradient, the amount of RNA in the 4S region increased with longer incubation times. These data and the absence of higher molecular weight RNA of specific size classes in our work (and previously published reports) suggested that nuclear fractions from plant tissue contained active nucleases. This was confirmed when wheat nuclei were mixed with [(3)H]yeast RNA (4, 18, 26S). All of the radioactive yeast RNA was degraded within 30 minutes to species sedimenting between 4 and 10S. The inclusion of high salt (125 millimolar (NH(4))(2)SO(4), 100 millimolar KCl), EGTA, and exogenous RNA or DNA reduced but did not eliminate endogenous RNase activity. Wheat embryo nuclei were further purified by centrifugation on a gradient of a polyvinylpyrrolidone-coated colloidal silica suspension (Percoll). These nuclei were ellipsoidal, free of cytoplasmic material, and lacked endogenous nuclease activity when assayed with [(3)H]yeast RNA. Sucrose gradients were not as effective as Percoll gradients in purifying nuclei free of RNase activity. The Percoll method of isolating nuclei and the RNase assay reported here will be useful in isolating plant nuclei that are capable of synthesizing distinct RNA species in vitro.

Transcription in Isolated Wheat Nuclei: II. CHARACTERIZATION OF RNA SYNTHESIZED IN VITRO.

Nuclei free of RNase activity were isolated from 3-hour-imbibed wheat (var. Yamhill) embryos by centrifugation through a discontinuous gradient of Percoll. The maximum rate of RNA synthesis observed in these nuclei was approximately 5 picomoles [(3)H]UTP per milligram DNA per minute. Two monovalent cation optima were found when measured in the presence of 15 millimolar MgCl(2) or 2 millimolar MnCl(2); 15 and 75 millimolar (NH(4))(2)SO(4). At the high monovalent cation optimum, the rate of RNA synthesis was linear for the first 10 to 15 minutes of incubation and still increasing after 3 hours. RNA synthesized in vitro (30-minute pulse followed by a 30-minute chase) showed distinct 18 and 26S RNA peaks comprising 13 and 17% of the total radioactivity, respectively. The over-all pattern of RNA synthesized in vitro was similar to the in vivo pattern. Approximately 40 to 50% of the RNA synthesized was inhibited by alpha-amanitin at 4 micrograms per milliliter. The newly synthesized 6 to 10S RNA appeared to be selectively inhibited by alpha-amanitin.

Cell-free Synthesis of Globulin by Developing Oat (Avena sativa L.) Seeds.

The primary storage protein synthesized during oat (Avena sativa L.) groat development is a globulin. Polysomes were isolated from oat groats 12 days after anthesis. These polysomes directed the incorporation of radioactive amino acids into protein in a cell-free protein synthesis system containing wheat germ supernatant. The Mg(2+) optimum was 4 mm, the pH optimum was 6-8, and the amount of amino acid incorporation depended on polysome concentration. Incorporation of amino acids was linear for about 10 min and approached a maximum after 20 min. Using the initiation inhibitor, T-2 toxin, it was determined that about 36% of the amino acid incorporation was due to the initiation of new polypeptide chains. The in vitro product co-electrophoresed with authentic oat groat globulin on polyacrylamide-sodium dodecyl sulfate (SDS) gels. The cyanogen bromide peptides of the in vitro product partially corresponded with those from authentic globulin when electrophoresed on polyacrylamide-SDS gels. These data suggest that the in vitro product is primarily oat globulin. The polysome population was separated into membrane-bound and free polysomes. Membrane-bound polysomes synthesized about twice the amount of protein as did free polysomes. Products synthesized in vitro on both types of polysomes were essentially the same.

Isolation and characterization of oat globulin messenger RNA.

When polyadenylated RNA, isolated from membrane-bound polysomes extracted from developing oat (Avena sativa L.) seeds, was translated in vitro in the rabbit reticulocyte system, two polypeptides of about 58 and 60 kilodaltons were immunoprecipitated by anti-oat globulin antibody. No electrophoretic bands corresponding to the 40 and 20 kilodalton polypeptides of oat globulin were present. However, when in vivo labeled extracts were immunoprecipitated with anti-oat globulin antibody, three groups of polypeptides (60, 40, and 20 kilodaltons) were present. It therefore seems probable that the two large polypeptides (58 and 60 kilodaltons) were precursors of the 40 and 20 kilodalton polypeptides. When the polyadenylated RNA coding for these polypeptides was size fractionated on a sucrose density gradient, it sedimented near the 18S region of the gradient. Translation of the RNA from the gradient fractions and immunoprecipitation of translation products indicated that the template for the 58 to 60 kilodalton ;putative' precursors of oat globulin was probably the RNA which was approximately 18S in size.

Heterologous expression of maize (Zea mays L.) Mir1 cysteine proteinase in eukaryotic and prokaryotic expression systems.

Several heterologous expression systems were tested for their ability to express a unique maize cysteine proteinase Mir1. A baculovirus-based expression system using Trichoplusia ni larvae as host resulted in the expression of Mir1 that was correctly processed and exhibited proteinase activity. Expression in Escherichia coli resulted in accumulation of Mir1, but it had limited solubility and enzymatic activity. Large quantities of Mir1 were produced when Pichia pastoris was used as the host, but the enzyme was insoluble and inactive.

Association of a 33-kilodalton cysteine proteinase found in corn callus with the inhibition of fall armyworm larval growth.

Protein patterns of callus from corn (Zea mays L.) inbreds that are either resistant or susceptible to fall armyworm (Spodoptera frugiperda [J.E. Smith]) were analyzed by two-dimensional electrophoresis. Fall armyworm larvae reared on callus initiated from resistant inbreds were significantly smaller than those reared on callus of susceptible inbreds. A 33-kD protein found in callus from the resistant inbreds Mp704 and Mp708 was absent in callus from the susceptible inbreds Tx601 and Ab24E. However, a 36-kD protein found in Ab24E callus immunoreacted with polyclonal antibody raised against the 33-kD protein. When Mp704 nonfriable callus changed to friable, larval growth was not inhibited and the 33-kD protein was absent. There was a significant negative correlation between the concentration of the 33-kD protein in the callus and the weight of the larvae feeding on the callus in the F2 progeny of Mp704 x Tx601. The N-terminal amino acid sequence of the 33-kD protein suggested that it was cysteine proteinase. Purification of the 33- (Mp708) and 36-kD (Ab24E) proteins indicated that they were both cysteine proteinases. The 33-kD cysteine proteinase had 7-fold higher specific activity than the 36-kD enzyme.

Characterization of three distinct cDNA clones encoding cysteine proteinases from maize (Zea mays L.) callus.

In previous work, a 33 kDa cysteine proteinase was found in callus initiated from maize (Zea mays L.) resistant to fall armyworm feeding. A callus cDNA library from the maize inbred Mp708 was screened with oligonucleotides derived from the N-terminal amino acid sequence of the 33 kDa proteinase and several cDNA clones were isolated and sequenced. A cDNA clone encoding the 33 kDa cysteine proteinase, mir1, was identified. Two additional clones, mir2 and mir3, encoding putative cysteine proteinases were also identified. mir2 and mir3 are distinct from mir1 and each other, but show a high degree of homology. All of the mir cDNA clones map to distinct sites on the maize genome. Amino acid sequences encoded by the mir clones are similar to other known cysteine proteinases and are most closely related to the oryzain-alpha and -beta precursors. The ERFNIN motif and a 12 amino acid conserved sequence are present in the propeptide region of the putative proteinases encoded by mir clones. mir2 and mir3 appear to have C-terminal extensions. The phylogenetic tree of nucleotide sequences of mir1, mir2, mir3 and other representative cysteine proteinases from protozoa, plants and animals was constructed.

A unique 33-kD cysteine proteinase accumulates in response to larval feeding in maize genotypes resistant to fall armyworm and other Lepidoptera.

Plants respond to insect feeding with a number of defense mechanisms. Using maize genotypes derived from Antiquan germ plasm that are resistant to Lepidoptera, we have demonstrated that a unique 33-kD cysteine proteinase accumulates in the whorl in response to larval feeding. The abundance of the proteinase increased dramatically at the site of larval feeding after 1 hr of infestation and continued to accumulate for as long as 7 days. The 33-kD cysteine proteinase was most abundant in the yellow-green portion of the whorl-the normal site of larval feeding and the tissue that has the greatest inhibitory effect on larval growth in bioassays. The proteinase was expressed in response to wounding and was found in senescent leaves. It may be a marker of programmed cell death. The gene coding for the proteinase, mir1, has been transformed into Black Mexican Sweet callus. When larvae were reared on callus expressing the proteinase, their growth was inhibited approximately 60 to 80%. The expression of a cysteine proteinase, instead of a cysteine proteinase inhibitor, may be a novel insect defense mechanism in plants.