Tight regulation of expression of two Arabidopsis cytosolic Hsp90 genes during embryo development

The spatial and temporal distribution of expression of two cytosolic members of the AtHsp90 gene family was assessed during early development. In stressed transgenic plants bearing the AtHsp90-3 promoter, beta-glucuronidase (GUS) activity was strong in meristematic tissues. Expression was also detected in vascular tissues, leaf veins, siliques, and in pollen sacs. The promoter induced gene expression after heat shock in a time-course dependent manner. AtHsp90-1 promoter activity was low throughout the early stages of embryo development but high just before embryo maturation, with expression most prominent in cotyledons. AtHsp90-3 promoter activity was almost constant and restricted to the root and the cotyledon tips of the embryo. This highly specific spatial distribution of GUS activity changed when the tissues were heat-stressed. Both promoters were also active in unstressed mature pollen grains and during pollen germination. The results shown here indicate that different regulatory and developmental mechanisms control and differentiate the expression of the two cytosolic members of the Arabidopsis AtHsp90 gene family under normal conditions. The developmental and restricted pattern of expression of the AtHsp90-1 and -3 gene promoters in unstressed transgenic plants suggest prominent and distinctive roles of these two genes during different developmental processes.     

Evaluation in tobacco of the organ specificity and strength of therolD promoter,domain A of the 35S promoter and the 35S2 promoter

In order to study the expression in plants of therolD promoter ofAgrobacterium rhizogenes, we have constructed chimaeric genes placing the coding region of thegusA (uidA) marker gene under control of tworolD promoter fragments of different length. Similar results were obtained with both genes. Expression studies were carried out in transformed R₁ progeny plants. In mature transformed tobacco plants, therolD-gus genes were expressed strongly in roots, and to much lower levels in stems and leaves. This pattern of expression was transmitted to progeny, though the ratio of the level of expression in roots relative to that in leaves was much lower in young seedlings. The degree of root specificity inrolD-gus transformants was less than that of a gene constructed with domain A of the CaMV 35S promoter,domA-gus, but the level of root expression was much higher than with the latter gene. However, the level of expression of therolD-gus genes was less than that of agus gene with a 35S promoter with doubled domain B, 35S²-gus. TherolD-gus genes had a distinctive pattern of expression in roots, compared to that of the two other genes, with the strongest GUS activity observed in the root elongation zone and in vascular tissue, and much less in the root apex.     

Seed-specific expression of seven Arabidopsis promoters

Seeds contain storage compounds, from various carbohydrates to proteins and lipids, which are synthesized during seed development. For the purposes of many plant researches or commercial applications, developing promoter systems expressing specifically in seeds or in particular constituents or tissues/compartments of seeds are indispensable. To screen genes dominantly or specifically expressed in seed tissues, we analyzed Arabidopsis ATH1 microarray data open to the public. Thirty-two candidate genes were selected and their expressions in seed tissues were confirmed by RT-PCR. Finally, seven genes were selected for promoter analysis. The promoters of seven genes were cloned into pBI101 vector and transformed into Arabidopsis to assay histochemical β-glucuronidase (GUS) activity. We found that Pro-at3g03230 promoter drove GUS expression in a chalazal endosperm, Pro-at4g27530:GUS expressed in both chalazal endosperm and embryo, Pro-at4g31830 accelerated GUS expression both in radicle and procambium, Pro-at5g10120 and Pro-at5g16460 drove GUS expression uniquely in embryo, Pro-at5g53100:GUS expressed only in endosperm, and Pro-at5g54000 promoted GUS expression in both embryo and inner integument. These promoters can be used for expressing any genes in specific seed tissues for practical application.     

The promoter of the asi gene directs expression in the maternal tissues of the seed in transgenic barley

The bifunctional alpha-amylase/subtilisin inhibitor (BASI) is an abundant protein in barley seeds, proposed to play multiple and apparently diverse roles in regulation of starch hydrolysis and in seed defence against pathogens. In the Triticeae, the protein has evolved the ability to specifically inhibit the main group of alpha-amylases expressed during germination of barley and encoded by the amyl gene family found only in the Triticeae. The expression of the asi gene that encodes BASI has been reported to be controlled by the hormones abscisic acid (ABA) and gibberellic acid (GA). Despite many studies at the gene and protein level, the function of this gene in the plant remains unclear. In this study, the 5'-flanking region (1033 bp, 1033-asi promoter) and the 3'-flanking region (655 bp) of the asi gene were isolated and characterised. The 1033-asi promoter sequence showed homology to a number of ciselements that play a role in ABA and GA regulated expression of other genes. With a green fluorescent protein gene (gfp) as reporter, the 1033-asi promoter was studied for spatial, temporal and hormonal control of gene expression. The 1033-asi promoter and its deletions direct transient gfp expression in the pericarp and at low levels in mature aleurone cells, and this expression is not regulated by ABA or GA. In transgenic barley plants, the 1033-asi promoter directed tissue-specific expression of the gfp gene in developing grain and germinating grain but not in roots or leaves. In developing grain, expression of gfp was observed specifically in the pericarp, the vascular tissue, the nucellar projection cells and the endosperm transfer cells and the hormones ABA or GA did not regulate this expression. In mature germinating grain gfp expression was observed in the embryo but not in aleurone or starchy endosperm. However, GA induced gfp expression in the aleurone of mature imbibed seeds from which the embryo had been removed. Expression in maternal rather than endosperm tissues of the grain suggests that earlier widespread assumptions that the protein is expressed largely in the endosperm may have been largely based on analysis of mixed grain tissues. This novel pattern of expression suggests that both activities of the protein may be primarily involved in seed defence in the peripheral tissues of the seed.     

桃儿七种子解剖结构及其萌发生长期形态特征

为探明种皮和胚乳是否是限制桃儿七种子萌发的主要因素,利用组织切片和显微技术,对桃儿七种子及其不同萌发期（1、7、14、21、28 d）解剖结构和播种后一定时期内（7~210 d）的植株生长形态进行观察。桃儿七种子由种皮、胚乳和胚构成。种皮包括外种皮和内种皮,外种皮致密规整,由外至内分别为栅状石细胞和表皮层细胞,内种皮由5~6层海绵细胞组成。胚乳占种子体积的绝大部分,包括珠孔胚乳和外胚乳。胚由胚根、胚轴和子叶组成,被致密种皮、多层珠孔胚乳和外胚乳包围。萌发期1~7 d胚根和胚轴开始伸长,7~14 d两片子叶分离,14~21 d胚根突破珠孔胚乳和种皮,21~28 d胚根、胚轴和子叶继续扩张伸长。种子播种210 d后可平均形成3片功能真叶和5条不定根。致密种皮（物理休眠）和多层胚乳（机械休眠）是限制桃儿七种子萌发的两个主要因素。     

The promoter of the geneItr1 from barley confers a different tissue specificity in transgenic tobacco

Tissue-specific expression of the gene coding for trypsin inhibitor BTI-CMe in barley (Itr1) occurs during the first half of endosperm development. In transgenic tobacco, theItr1 promoter drives expression of the β-glucuronidase reporter gene not only in developing endosperm but also in embryo, cotyledons and the meristematic intercotyledonary zone of germinating seedlings. A promoter fragment extending 343 bp upstream of the translation initiation ATG codon was sufficient for full transgene expression, whereas, the proximal 83 bp segment of the promoter was inactive. Possible reasons for the differences in expression patterns are discussed.     

The scutellar vascular bundle-specific promoter of the wheat HD-Zip IV transcription factor shows similar spatial and temporal activity in transgenic wheat, barley and rice

An HD‐Zip IV gene from wheat, TaGL9, was isolated using a Y1H screen of a cDNA library prepared from developing wheat grain. TaGL9 has an amino acid sequence distinct from other reported members of the HD‐Zip IV family. The 3′ untranslated region of TaGL9 was used as a probe to isolate a genomic clone of the TaGL9 homologue from a BAC library prepared from Triticum durum L. cv. Langdon. The full‐length gene containing a 3‐kb‐long promoter region was designated TdGL9H1. Spatial and temporal activity of TdGL9H1 was examined using promoter‐GUS fusion constructs in transgenic wheat, barley and rice plants. Whole‐mount and histochemical GUS staining patterns revealed grain‐specific expression of TdGL9H1. GUS expression was initially observed between 3 and 8 days after pollination (DAP) in embryos at the globular stage and adjacent to the embryo fraction of the endosperm. Expression was strongest in the outer cell layer of the embryo. In developed wheat and barley embryos, strong activity of the promoter was only detected in the main vascular bundle of the scutellum, which is known to be responsible for the uptake of nutrients from the endosperm during germination and the endosperm‐dependent phase of seedling development. Furthermore, this pattern of GUS staining was observed in dry seeds several weeks after harvesting but quickly disappeared during imbibition. The promoter of this gene could be a useful tool for engineering of early seedling vigour and protecting the endosperm to embryo axis pathway from pathogens during grain desiccation and storage.     

Expression and maintenance of embryogenic potential is enhanced through constitutive expression of AGAMOUS-Like 15

The MADS domain protein AGL15 (AGAMOUS-Like 15) has been found to preferentially accumulate in angiosperm tissues derived from double fertilization (i.e. the embryo, suspensor, and endosperm) and in apomictic, somatic, and microspore embryos. Localization to the nuclei supports a role in gene regulation during this phase of the life cycle. To test whether AGL15 is involved in the promotion and maintenance of embryo identity, the embryogenic potential of transgenic plants that constitutively express AGL15 was assessed. Expression of AGL15 was found to enhance production of secondary embryos from cultured zygotic embryos, and constitutive expression led to long-term maintenance of development in this mode. Ectopic accumulation of AGL15 also promoted somatic embryo formation after germination from the shoot apical meristem of seedlings in culture. These results indicate that AGL15 is involved in support of development in an embryonic mode.     

Molecular mechanisms and hormonal regulation underpinning morphological dormancy: a case study using Apium graveolens (Apiaceae)

Underdeveloped (small) embryos embedded in abundant endosperm tissue, and thus having morphological dormancy (MD) or morphophysiological dormancy (MPD), are considered to be the ancestral state in seed dormancy evolution. This trait is retained in the Apiaceae family, which provides excellent model systems for investigating the underpinning mechanisms. We investigated Apium graveolens (celery) MD by combined innovative imaging and embryo growth assays with the quantification of hormone metabolism, as well as the analysis of hormone and cell-wall related gene expression. The integrated experimental results demonstrated that embryo growth occurred inside imbibed celery fruits in association with endosperm degradation, and that a critical embryo size was required for radicle emergence. The regulation of these processes depends on gene expression leading to gibberellin and indole-3-acetic acid (IAA) production by the embryo and on crosstalk between the fruit compartments. ABA degradation associated with distinct spatiotemporal patterns in ABA sensitivity control embryo growth, endosperm breakdown and radicle emergence. This complex interaction between gibberellins, IAA and ABA metabolism, and changes in the tissue-specific sensitivities to these hormones is distinct from non-MD seeds. We conclude that the embryo growth to reach the critical size and the associated endosperm breakdown inside MD fruits constitute a unique germination programme.     

Two Tomato Expansin Genes Show Divergent Expression and Localization in Embryos during Seed Development and Germination

Expansins are plant proteins that can induce extension of isolated cell walls and are proposed to mediate cell expansion. Three expansin genes were expressed in germinating tomato (Lycopersicon esculentum Mill.) seeds, one of which (LeEXP4) was expressed specifically in the endosperm cap tissue enclosing the radicle tip. The other two genes (LeEXP8 and LeEXP10) were expressed in the embryo and are further characterized here. LeEXP8 mRNA was not detected in developing or mature seeds but accumulated specifically in the radicle cortex during and after germination. In contrast, LeEXP10 mRNA was abundant at an early stage of seed development corresponding to the period of rapid embryo expansion; it then decreased during seed maturation and increased again during germination. When gibberellin-deficient (gib-1) mutant seeds were imbibed in water, LeEXP8 mRNA was not detected, but a low level of LeEXP10 mRNA was present. Expression of both genes increased when gib-1 seeds were imbibed in gibberellin. Abscisic acid did not prevent the initial expression of LeEXP8 and LeEXP10, but mRNA abundance of both genes subsequently decreased during extended incubation. The initial increase in LeEXP8, but not LeEXP10, mRNA accumulation was blocked by low water potential, but LeEXP10 mRNA amounts fell after longer incubation. When seeds were transferred from abscisic acid or low water potential solutions to water, abundance of both LeEXP8 and LeEXP10 mRNAs increased in association with germination. The tissue localization and expression patterns of both LeEXP8 and LeEXP10 suggest developmentally specific roles during embryo and seedling growth.     

Vacuolar H+-ATPase Is Expressed in Response to Gibberellin during Tomato Seed Germination

Completion of germination (radicle emergence) by gibberellin (GA)-deficient (gib-1) mutant tomato (Lycopersicon esculentum Mill.) seeds is dependent upon exogenous GA, because weakening of the endosperm tissue enclosing the radicle tip requires GA. To investigate genes that may be involved in endosperm weakening or embryo growth, differential cDNA display was used to identify mRNAs differentially expressed in gib-1 seeds imbibed in the presence or absence of GA₄₊₇. Among these was a GA-responsive mRNA encoding the 16-kD hydrophobic subunit c of the V₀ membrane sector of vacuolar H⁺-translocating ATPases (V-ATPase), which we termed LVA-P1. LVA-P1 mRNA expression in gib-1 seeds was dependent on GA and was particularly abundant in the micropylar region prior to radicle emergence. Both GA dependence and tissue localization of LVA-P1 mRNA expression were confirmed directly in individual gib-1 seeds using tissue printing. LVA-P1 mRNA was also expressed in wild-type seeds during development and germination, independent of exogenous GA. Specific antisera detected protein subunits A and B of the cytoplasmic V₁ sector of the V-ATPase holoenzyme complex in gib-1 seeds only in the presence of GA, and expression was localized to the micropylar region. The results suggest that V-ATPase plays a role in GA-regulated germination of tomato seeds.     

Molecular cloning, characterization and expression of cDNA encoding translationally controlled tumor protein (TCTP) from Jatropha curcas L

A cDNA encoding translationally controlled tumor protein (TCTP) of Jatropha curcas L., JcTCTP, was isolated from an endosperm cDNA library. JcTCTP consisted of a 5?? untranslated region (UTR) of 526 bp, a 3?? UTR of 377 bp and an open reading frame (ORF) of 507 bp, encoding a protein of 168 amino acid residues, which contained two signature sequences of TCTP family. Its deduced amino acid sequence was similar to the other known plants TCTPs in a range of 77.4?C92.3%. Expression of JcTCTP was the highest in the stem, endosperm at embryo formation stage and embryo of J. curcas tissues, and the lowest in the endosperm at seminal leaf embryo stage and flower, demonstrating a pattern of temporal and spatial specific expression.     

Effects of gibberellins, darkness and osmotica on endosperm rupture and class I β-1,3-glucanase induction in tobacco seed germination

The “Havana 425” cultivar of Nicotiana tabacum L. is photodormant. Gibberellins (e.g. 10^?5 M GA₄ or GA₇) can substitute for light in releasing dormancy. Measurements of β-1,3-glucanase activity, mRNA accumulation and the activity of the class I β-1,3-glucanase B promoter indicated that class I β-1,3-glucanases are induced by GA₄ in the dark in association with germination. As in the light, this induction occurred prior to endosperm rupture and was localized exclusively in the micropylar region of the endosperm where the radicle will penetrate. Abscisic acid (ABA, 10^?5 M) did not appreciably affect GA-induced release of photodormancy or seed-coat rupture, but it delayed endosperm rupture and inhibited the rate of class I β-1,3-glucanase accumulation. Seeds imbibed in the light in the presence of osmotica, e.g. 0.04 M polyethylene glycol 6000, showed delayed seed-coat and endosperm rupture, delayed onset of β-1,3-glucanase induction, and decreased rates of β-1,3-glucanase accumulation. These delays were shortened by GA₄ treatment. Our results suggest that GAs and ABA act at two distinct sites during germination and that expansive growth of the embryo acts in two ways by triggering β-1,3-glucanase induction and by providing force for endosperm penetration. This provides further support for our working hypothesis that class I β-1,3-glucanases promote endosperm weakening and facilitate radicle penetration.     

Studies on the early development of zygotic and synergid embryo and endosperm in polyembryonic rice ApIII

A morphological, cytological and embryological investigation was conducted on the early development of embryo and endosperm in polyembryonic rice (Oryza sativa L.) ApIII. We found that the percentage of single-, twin- and triple-seedlings from mature caryopses was 82.4–85.6%, 11.3–14.6% and 2.0–3.8%, respectively. From twin-seedlings one of the plants and in the triple-seedlings usually two seedlings were very weak having a mortality of about 50–60% and over 95%, respectively, and most of the dead seedlings were haploid. In addition, among 674 young caryopses the percentage of single-, double- and triple-embryo sacs was 77.60%, 18.10% and 4.30%, respectively. The development of both embryo and endosperm was observed in the embryo sacs of 653 young caryopses, while embryo development occurred in absence of endosperm in the embryo sacs of 21 ones. Furthermore, after pollen tube penetration, frequently one, rarely two, additional embryos arise via apogamy from the typical specialized synergid of normal egg apparatus with a collapsed cap-neck region that had a damaged filiform apparatus at the micropylar end. Finally, the formation of a strikingly degenerated endosperm cell region is a specialized functional structure that serves for the active transport of metabolites from endosperm to the developing embryo. Taken together, these results suggest that synergid apogamy seems to be the unique reason of polyembryony, poor growth and high mortality of many seedlings may be due to haploidy, and high genetic stability of polyembryony occurs in rice ApIII.     

Locus- and Site-Specific DNA Methylation of 19 kDa Zein Genes in Maize

An interesting question in maize development is why only a single zein gene is highly expressed in each of the 19-kDa zein gene clusters (A and B types), z1A2-1 and z1B4, in the immature endosperm. For instance, epigenetic marks could provide a structural difference. Therefore, we investigated the DNA methylation of the arrays of gene copies in both promoter and gene body regions of leaf (non-expressing tissue as a control), normal endosperm, and cultured endosperm. Although we could show that expressed genes have much lower methylation levels in promoter regions than silent ones in both leaf and normal endosperm, there was surprisingly also a difference in the pattern of the z1A and z1B gene clusters. The expression of z1B gene is suppressed by increased DNA methylation and activated with reduced DNA methylation, whereas z1A gene expression is not. DNA methylation in gene coding regions is higher in leaf than in endosperm, whereas no significant difference is observed in gene bodies between expressed and non-expressed gene copies. A median CHG methylation (25–30%) appears to be optimal for gene expression. Moreover, tissue-cultured endosperm can reset the DNA methylation pattern and tissue-specific gene expression. These results reveal that DNA methylation changes of the 19-kDa zein genes is subject to plant development and tissue culture treatment, but varies in different chromosomal locations, indicating that DNA methylation changes do not apply to gene expression in a uniform fashion. Because tissue culture is used to produce transgenic plants, these studies provide new insights into variation of gene expression of integrated sequences.