Lea mRNAs Research Articles

Abscisic Acid Catabolism Generates Phaseic Acid, a Molecule Able to Activate a Subset of ABA Receptors

Abscisic acid (ABA) catabolic pathways are conventionally thought of as routes for ABA inactivation and, thus, reduction of the well-known ABA responses to cope with stress. However, this point of view was challenged by the finding that some forms of hydroxylated ABA generated by ABA catabolism still maintain biological activity (Zou et al., 1995; Kepka et al., 2011). Hydroxylation at C-8′ position is thought to be the predominant ABA catabolic pathway, and this reaction is catalyzed by the CYP707A family cytochrome P450 monooxygenases (P450s) (Krochko et al., 1998; Kushiro et al., 2004; Saito et al., 2004) (Figure 1).

Isolation of a gene encoding a novel atypical LEA protein from the halophyte Prosopis strombulifera with a sodium salt-specific expression

Salinity is one of the major abiotic stresses that adversely affect the crop productivity and growth of the whole plant. The halophytic Prosopis strombulifera (Fabaceae) shrub is a useful plant model to study the molecular and physiological mechanisms involved in salinity tolerance. In this work, the isolation and characterization of the mRNA for a new atypical LEA protein (PsLEA3) is reported. PsLEA3 has alanine as the most abundant amino acid followed by serine. This characteristic is different from typical LEA proteins which have high glycine content. PsLEA3 shows a negative average of hydropathy and remains localized in the chloroplast. The phylogenetic relationship between related LEA proteins and PsLEA3 showed that this protein belongs to the same group as a LEA protein of Arabidopsis. Northern analysis revealed a differential expression of this atypical LEA mRNA in P. strombulifera plants under NaCl and Na2SO4 treatments. Over-expression in roots of Na2SO4-treated plants from Ψo −1.9 MPa was associated with abscisic acid (ABA) and its conjugate (ABA-glucose ester) accumulation as well as with the root length of these plants, showing an inhibition and toxicity induced by sulphate anion present in the medium.

Seed-specific expression patterns and regulation by ABI3 of an unusual late embryogenesis-abundant gene in sunflower.

We cloned the genomic sequences that correspond to a previously described group 1 late embryogenesis-abundant (Lea) cDNA from sunflower: Ha ds10. The Ha ds10 G1 gene had structural and gene-expression features that depart from those of other group 1 Lea genes. An intron was present at a conserved position but showed a much larger size (1024 bp). Transcription from the Ha ds10 G1 promoter was strictly seed-specific and it originated from at least two close initiation sites. The mRNAs accumulated from stages of embryogenesis that preceded seed desiccation. Ha ds10 G1 mRNA accumulation was moderately induced, by exogenous abscisic acid treatments, in immature seeds but not induced in seedlings. We observed unprecedented changes in Lea mRNA localization associated with seed desiccation: the homogeneous tissue distribution of Ha ds10 G1 mRNAs, which was characteristic of immature embryos, evolved later in embryogenesis to an asymmetric distribution within the cotyledons, with preferential mRNA accumulation in the cells of the palisade parenchyma and provascular bundles. We also showed that, in sunflower embryos, the Ha ds10 G1 promoter could be transiently activated by the Arabidopsis ABI3 transcription factor. We discuss the significance of these results regarding hypotheses of regulation and function of plant genes from the same family.

ABA metabolites induce group 3 LEA mRNA and inhibit germination in wheat

ABA metabolites induce group 3 LEA mRNA and inhibit germination in wheat

Changes in late-embryogenesis-abundant (LEA) messenger RNAs and dehydrins during maturation and premature drying of Ricinus communis L. seeds.

In Ricinus communis L. (castor bean) endosperms, two classes of Late Embryogenesis Abundant (Lea) transcripts were first detected during mid-development (at 30-35 days after pollination, DAP) and peaked at 50 DAP, just prior to the onset of desiccation. Most of the Class I mRNAs declined substantially during desiccation itself; Class II mRNAs remained abundant in the mature dry (60 DAP) seed. Following imbibition, all Lea mRNAs abundant in the mature dry seed declined rapidly (within 5-24 h). Premature drying of developing 35-DAP seeds resulted in the loss of storage-protein mRNAs (Leg B Mat I); following rehydration, mRNAs encoding post-germinative proteins (Germ D91, D30 and D38) increased in the endosperm. The Lea mRNAs present in the developing fresh seed at 35 DAP were preserved, but did not increase in response to premature desiccation; upon rehydration these Lea mRNAs declined within 5 h. During seed development, substantial changes occurred in the synthesis of a subset of LEA proteins referred to as "dehydrins'; in particular, new dehydrin polypeptides were induced between 40 and 60 DAP. Such proteins were not as evident in prematurely dried endosperms. In contrast to the rapid loss of Lea mRNAs during germination, many of the dehydrin proteins abundant in the dried seed persisted following imbibition or rehydration.

Environmental and hormonal regulation of barley late‐embryogenesis‐abundant (Lea) mRNAs is via different signal transduction pathways

ABSTRACTLate‐embryogenesis‐abundant (Lea) genes are induced in immature embryos by ABA and osmotic stress. Using the ABA‐biosynthesis inhibitor norflurazon in combination with ionic and non‐ionic stress, the expression of three distinct groups of Lea transcripts in immature barley embryos was investigated, these groups being embryoand aleurone‐specific B15C mRNAs, embryo‐specific B19 inRNAs, and rab/dehydrin mRNAs, which encode stress‐related proteins that are also expressed in vegetative tissues. The expression of B15C and rab/dehydrins in response to mannitol was not affected by the addition of norflurazon, which decreased the endogenous ABA levels by 89%; this observation is consistent with an ABA‐independent pathway. The expression of B19 was only slightly dependent on ABA. In contrast, induction of the Lea genes by salt (NaCI) required at least 25ümol m−3 endogenous ABA. Together, these results are evidence for different signal transduction pathways linking ionic and non‐ionic osmotic stress to Lea mRNA accumulation in barley embryos.

Simultaneous induction of postabscission and germination mRNAs in cultured dicotyledonous embryos.

Cloned mRNAs identify three programs of gene expression in cotton (Gossypium hirsutum L.) embryos that are associated with the maturation (reserve accumulation) stage, the postabscission stage, which is marked by expression of Late-embryogenesis-abundant (Lea) mRNAs, and germination (broadly defined as including all events through early postgerminative growth). In order to test if the regulation of these programs is the same in other dicotyledonous species, their expression was studied in normal and cultured maturation-stage, postabscission-stage, and mature embryo-stage embryos or seed of oilseed rape (Brassica napus L.), soybean (Glycine max [L.] Merr.), and tobacco (Nicotiana tabacum L.) using cotton and other cDNA probes. During postabscission, Lea mRNAs accumulated in all test species and were induced in earlier maturation-stage embryos by excision and culture on basal medium. Abscisic acid often enhanced this induction in the test species. Germination-specific mRNAs were induced in cultured maturation-stage and postabscission-stage embryos of all test species. These results indicate that the regulation of embryonic and germination programs is similar in all dicotyledons tested. Because excised embryos simultaneously induced postabscission and germination programs, the effects of exogenous growth regulators and other factors on such embryos probably reflect stress responses of germinating mature embryos rather than the identity of endogenous regulators of embryogenesis.

Cotton Lea5 and Lea14 encode atypical late embryogenesis-abundant proteins.

LEA proteins comprise a large group of probable desiccation protectants that in cotton (Gossypium hirsu tum) are developmentally induced during the postabscission stage of embryogenesis (Hughes and Galau, 1989) and are environmentally induced in embryos by desiccation or culture with ABA or high osmoticum (Hughes and Galau, 1991). In other plants, some Lea mRNAs are induced by similar stresses in vegetative tissues (Skriver and Mundy, 1991). Of the many Lea and water stress-related mRNAs that have been sequenced (Skriver and Mundy, 1991), a11 but cotton LealZ, cDNA D34 (Baker et al., 1988), and desiccation-induced Craterostigma cDNA pcC27-45 (Piatkowski et al., 1990) encode proteins that are very hydrophilic (Baker et al., 1988; Skriver and Mundy, 1991). Extensive hydrophilicity is expected of structural and nonstructural proteins that bind or replace water during water stress (Baker et al., 1988; Hughes and Galau, 1989), but other functions might be expected for those that are not remarkably hydrophilic. Of the 18 cloned cotton Lea and LeaA mRNAs (Galau et al., 1986), only Lea5 (cDNA D73) and Leal4 (cDNA D95) are highly induced in mature leaves of water-stressed plants or in water-stressed, detached leaves (D.W. Hughes, G.A. Galau, unpublished observations). We report here that both genes encode proteins with significant hydropathic character and that Leal4 is a homolog of the gene encoding the Craterostigma desiccationinduced cDNA pcC27-45 (Piatkowski et al., 1990). Both alloalleles of Leu5 were recovered, and 3809 nucleotides of Lea5-A clone GD73-14R and 1269 nucleotides of Lea5-D clone GD73-2R were sequenced along with several Lea5 cDNA clones (Table I). Lea5-A and Lea5-D are very similar from at least 240 nucleotides upstream of the transcription start to about 230 nucleotides 3' of the major polyadenylation site, after which they diverge in sequence. The cDNA clones D73 and D96 are transcribed from Lea5-A, and cDNA clone D122 is transcribed from Lea5-D. Clone D73 is the result of an error in excision of the single intron; instead of splicing to Lea5-A nucleotide 2515, as occurs with

Developmental and environmental concurrent expression of sunflower dry-seed-stored low-molecular-weight heat-shock protein and Lea mRNAs.

We have cloned and sequenced three different cDNAs from sunflower seed-stored mRNA. Sequence similarities and response to heat-shock identified one of the cDNAs as a low-molecular-weight heat-shock protein (lmw-HSP). The other two clones showed significant sequence similarity to the cotton and carrot late-embryogenesis-abundant (Lea) proteins D-113 and Emb-1, respectively. The three cDNAs showed similar expression patterns during zygotic embryo development, as well as in vegetative tissues of 3-day-old seedlings in response to stress. Maximal accumulation of all three mRNAs was detected in dry seeds and during embryo mid-maturation stage, in the absence of exogenous stress. In seedlings, mRNAs accumulated to lower levels in response to osmotic stress and exogenous abscisic acid (ABA) treatments. A differential time course of response to osmotic stress was observed: lmw-HSP mRNA accumulation was induced earlier than that of Lea mRNAs. The coordinate accumulation of Lea and lmw-HSP transcripts during embryo development and in response to stress and ABA suggests the existence of common regulatory elements for Lea and lmw-HSP genes, and supports the notion that HSPs might have alternative functions in the plant cell.

Sequences of the Cotton Group 2 LEA/RAB/Dehydrin Proteins Encoded by Lea3 cDNAs

The 18 cloned cotton Lea (late embryogenesis-abundant) mRNAs are a diverse group encoding proteins that may be desiccation protectants (5). Lea mRNAs are coordinately induced by ovule abscission in cotton embryos during the postabscission stage before desiccation (5) and in cultured embryos by desiccation, osmotic stress, or exposure to ABA, a plant hormone that may be a transducer of water-related stresses (6). Homologs of the cotton Lea3 cDNA clone D 1I (1) have been identified as rab (responsive to ABA) in rice seedlings exposed to ABA or salt (10) and maize embryos and calli exposed to ABA (9), Dhn (dehydrin) in maize and barley seedlings exposed to water stress (2), pcD27-04 and pcC6-19 in dried leaves and ABA-treated calli of the resurrection plant Craterostigma plantagineum (7), RSLEA2 in dry seeds of radish (8), and TAS14 in tomato seedlings exposed to osmotica or ABA (4). Although the organization and amino acid sequences of these Group 2 LEA/RAB/dehydrin proteins are highly conserved, the carboxy terminus of the cotton D 1I LEA3 protein is singularly divergent. Because the nucleotide sequence of D 11 could encode a more typical terminus if there were frame-shift mutations or sequencing errors (2), we have independently determined the sequence of Dl I and, for comparative purposes, also determined the entire nucleic acid sequence of D147b and part of D71 and D131, three additional Lea3 cDNA clones (Table I). The nucleotide and deduced amino acid sequences of D147b and a comparison with those of Dl I are presented in Figure 1. We note that the published sequence of DIl (1) contains two relatively unimportant errors. First, D 1I contains two additional nucleotides at the 5' end that were not previously reported. Second, D 1 contains a G at D 11 nucleotide 353, not a C as previously reported. The latter cor' Supported by National Institutes of Health grant No. GM29495 to G.A.G. and U.S. Department of Agriculture/Cooperative State Research Service grant No. 91-37100-6615 to T.J.C.

GENE EXPRESSION IN PECAN SOMATIC EMBRYOS

Repetitive somatic embryogenic lines of pecan (Carya illinoensis) were obtained and subcultured on basal WPM, following a one week induction of zygotic embryo tissue on modified WPM with 6 mg/L NAA. Gene expression of somatic embryos has been studied and compared with that occurring in zygotic embryos. Somatic embryos simultaneously expressed mRNA classes that are specific to each of the zygotic embryo cotyledon (Cot), maturation (Mat), and post abscission stages (Late embryogenesis, Lea). Somatic embryos exhibiting such multiple, nonregulated gene expression patterns have a low germination rate. Treatments found to enhance embryo germination (cold and desiccation) may be effective in part, by modifying gene expression patterns. Some of the Cot and Mat mRNA classes decreased following such treatments, while Lea mRNAs were not effected. Cold and desiccation treatments appear to coordinate gene expression in pecan somatic embryos, which might be associated with embryo germination.

Common amino acid sequence domains among the LEA proteins of higher plants

LEA proteins are late embryogenesis abundant in the seeds of many higher plants and are probably universal in occurrence in plant seeds. LEA mRNAs and proteins can be induced to appear at other stages in the plant's life by desiccation stress and/or treatment with the plant hormone abscisic acid (ABA). A role in protecting plant structures during water loss is likely for these proteins, with ABA functioning in the stress transduction process. Presented here are conserved tracts of amino acid sequence among LEA proteins from several species that may represent domains functionally important in desiccation protection. Curiously, an 11 amino acid sequence motif is found tandemly repeated in a group of LEA proteins of vastly different sizes. Analysis of this motif suggests that it exists as an amphiphilic α helix which may serve as the basis for higher order structure.

Temporally modular gene expression during cotyledon development.

The regulation of cotton embryogenesis has been addressed by measuring the abundance of 47 mRNAs in cotyledons from the late cotyledon stage through early germination. There are at least 11 distinct classes of coordinately expressed mRNAs. Their expression patterns appear to result from unique combinations of five temporal abundance components. These are associated with the cotyledon stage, the endogenous concentration of free abscisic acid, maturation (reserve accumulation), ovule abscission, and germination. This modularity suggests that only a few global regulatory factors orchestrate gene expression with many genes responding to several of them. Significant expression associated only with postabscission or free abscisic acid is restricted to that of the Lea mRNAs earlier suggested to be a component of the embryo's preparation for desiccation.

Accumulation kinetics of cotton late embryogenesis-abundant mRNAs and storage protein mRNAs: Coordinate regulation during embryogenesis and the role of abscisic acid

The accumulation of total RNA transcripts of 18 late embryo-abundant ( Lea) gene families, each encoding two closely related Lea mRNAs, was measured in cotyledon total RNA during embryogenesis and germination of Gossypium hirsutum L. by RNA dot hybridization. Transcript abundance of the three storage protein families was also followed. The Lea mRNAs belong to only two related groups of commonly regulated mRNAs. The transcript level of each of the 6 members of Class I has two transient maxima during early maturation and a maximum level at 3 days prior to desiccation. The transcript level of each of the 12 members of Class II increases abruptly in late maturation with a maximum concentration at 3 days before desiccation (Class IIA) or at desiccation (Class IIB). Several patterns of early accumulation also exist within Class II, some overlapping with those of storage protein or Lea Class I mRNAs. The concentrations of Lea mRNAs increase at least 10- to 1700-fold during embryogenesis and decline 15- to 220-fold during the first day of germination. Earlier studies indicated that most Lea mRNAs, but no storage protein mRNAs, are induced in excised embryos exposed to abscisic acid (ABA). Free (+)ABA was measured during embryogenesis using a monoclonal antibody-based ELISA. The ABA concentration shows maxima correlating with the maxima in early Class I Lea mRNA abundance. However, ABA declines during the accumulation of Class I and Class II Lea mRNAs in late embryogenesis. Consequently, ABA could be an endogenous regulator of Class I Lea mRNAs during early maturation but is not the primary regulator of Lea mRNAs during late maturation. In contrast, cotyledon water potential exhibits a decrease that correlates with the late induction of Lea mRNAs. These findings are consistent with the hypothesis that low water potential is the endogenous regulator of some ABA-inducible mRNAs. The cessation of vascular flow to the embryo may also be involved in the disappearance of storage protein mRNAs and the late induction of Lea mRNAs.

Coordinate accumulation of homeologous transcripts of seven cotton Lea gene families during embryogenesis and germination

One of two related patterns of total transcript accumulation are seen during embryogenesis for 18 cotton Lea (Late embryogenesis-abundant) gene families in the allotetraploid cotton Gossypium hirsutum L. cv Coker 201. Coordinate accumulation in each class is complex, suggesting that Lea mRNA abundance is regulated by several events. Each of the Lea gene families probably contains two active homeologous genes (alloalleles), one in each of cotton's two subgenomes. It is of interest whether both transcripts of a Lea family are regulated the same or whether the complexity of total transcript accumulation is due to different regulation of the two transcripts. The two polypeptides encoded by the homeologous transcripts of 7 Lea families can be distinguished on two-dimensional gels. The majority of Lea transcripts in total RNAs is shown to be functional in vitro throughout development; thus in vitro translation should faithfully measure their relative abundance. The ratio of the two transcripts of each was followed during embryo maturation when Lea transcript concentration increases an average of 70-fold and during the first 12 hr of germination when Lea transcripts decline in concentration an average of 50-fold. For 6 of the Lea families, the relative level of the two transcripts is invariant throughout this period. The two constituent transcripts of the seventh, Lea 9 , change 5-fold in relative concentration during late maturation and 3-fold during germination. Both transcripts still follow the same temporal pattern of accumulation; only their rates of change are somewhat different. These Lea families are a random sample of the 18 described. If multiple events affect the mRNA abundance of each Lea family, such events then each affect their individual transcripts in a similar way.

Translation efficiency of Lea mRNAs in cotton embryos: minor changes during embryogenesis and germination

In earlier studies, only two major patterns of transcript accumulation were seen for 18 late embryogenesis abundant (Lea) gene families in cotton (Gossypium hirsutum L.) during embryogenesis and early germination. Each of these gene families probably comprises two active alloalleles. The two polypeptides encoded by seven of the Lea families can be distinguished, and analysis of their translation in vitro indicated that regulation of the homeologous transcript abundance was similar in each.In the present study, two-dimensional gel electrophoresis of polypeptides synthesized in excised embryos was employed to determine if LEA polypeptide synthesis is regulated at the translational level. The relative in vivo synthesis rate of each of the two polypeptides of 7 Lea families was compared with the relative concentration of their transcripts measured earlier by in vitro translation. For 4 families, the relative translational efficiencies of the homeologous mRNAs do not change during embryogenesis. However, there are changes of 1.5-3-fold in the other 3 families. The translation efficiencies of all transcripts of 9 of the Lea family mRNAs in vivo can be calculated from the fraction of total protein radioactivity incorporated in each LEA polypeptide family and the fractional abundance of Lea family transcripts measured by RNA dot hybridization. Lea mRNAs are found to be translated at near average rates throughout embryogenesis and early germination. These observations suggest that regulation of Lea gene expression at the translational level is minor.

Abscisic acid induction of cloned cotton late embryogenesis-abundant (Lea) mRNAs

Earlier studies found that cotton (Gossypium hirsutum L.) cotyledons contain several mRNAs which are more abundant during late embryogenesis than in mid-embryogenesis or early germination. They are here termed 'Late embryogenesis-abundant' mRNAs, encoded by Lea loci. Complementary DNA clones for 18 such mRNA sequences, defined at a hybridization criterion of Tm-15°C, were identified in a mature embryo cDNA library by differential cDNA hybridization. At a lower hybridization criterion, some sequence homology was found within several of these cloned Lea mRNA sequences. Each Lea mRNA sequence comprises 0.04-1.3% of mature embryo poly(A)(+) mRNA, a level ten-fold to several hundred-fold higher than in young embryo or 24 h seedling poly(A)(+) mRNA. Of 18 Lea mRNA sequences examined in cultured young embryos, the level of at least 13 are specifically increased by exogenous abscisic acid (ABA), several to a level near that in normal mature embryos. However, the abundance of several of the sequences does not appear to be significantly modulated by ABA. The LEA polypeptides encoded by 10 Lea mRNA sequences were identified by hybrid-arrested translation. They include most of the late embryogenesis-abundant, ABA-inducible, polypeptides previously identified. Preliminary results suggest that many of the individual Lea mRNA sequences are transcribed from 1-3 genes in each of cotton's two subgenomes.