Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization

Three cDNA clones encoding lipid transfer proteins (LTPs) were isolated by applying the rapid amplification of cDNA ends (RACE) protocol to imbibed seeds and germinating seedlings of Brassica napus. The deduced amino-acid sequences show a great degree of homology and they exhibit the common features shared by all LTPs. Their expression pattern indicates a strong developmental, hormonal, and environmental regulation. They are expressed only in cotyledons and hypocotyls of germinating seedlings and their levels of expression increase upon treatment with cis-abscisic acid and NaCl. Their distribution in the cotyledons of young seedlings is suggestive of a role related to the mobilization of lipid reserves.     

Arabidopsis profilins are functionally similar to yeast profilins: identification of a vascular bundle-specific profilin and a pollen-specific profilin

Four members of the Arabidopsis profilin ( pfn ) multigene family have been cloned, sequenced and analyzed. By RNA gel blot analysis it has been shown that these four genes fall into two groups: one group ( pfn 1 and pfn 2) is expressed in all organs of the plant and the other group ( pfn 3 and pfn 4) in floral tissues only. Based on amino acid sequence alignment Arabidopsis profilins can be divided into the same two groups: PFN1 and PFN2 are 89% identical and PFN3 and PFN4 are 91% identical. Between these two groups they are 71–75% identical. The Arabidopsis profilins bind poly- l -proline and can complement both the Saccharomyces cerevisiae profilin deletion mutant and the Schizosaccharomyces pombe cdc3-124/profilin mutation, showing that the plant profilins are functionally similar to yeast profilins despite the low amino acid sequence homology. Analysis of pfn promoter-GUS fusion genes in transgenic Arabidopsis shows that pfn 2 is specifically expressed in the vascular bundles of roots, hypocotyls, cotyledons, leaves, sepals, petals, stamen filaments and stalks of developing seeds, whereas expression of pfn 4 is restricted to mature and germinating pollen grains.     

A light-independent developmental mechanism potentiates flavonoid gene expression in Arabidopsis seedlings

Throughout the plant kingdom expression of the flavonoid biosynthetic pathway is precisely regulated in response to developmental signals, nutrient status, and environmental stimuli such as light, heat and pathogen attack. Previously we showed that, in developing Arabidopsis seedlings, flavonoid genes are transiently expressed during germination in a light-dependent manner, with maximal mRNA levels occurring in 3-day-old seedlings. Here we describe the relationship between developmental and environmental regulation of flavonoid biosynthesis by examining phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR) mRNA levels in germinating Arabidopsis seedlings as a function of light, developmental stage and temperature. We show that seedlings exhibit a transient potential for induction of these four genes, which is distinct from that observed for chlorophyll a/b-binding protein (CAB). The potential for flavonoid gene induction was similar in seedlings grown in darkness and red light, indicating that induction potential is not linked to cotyledon expansion or the development of photosynthetic capacity. The evidence for metabolic regulation of flavonoid genes during seedling development is discussed.     

Profilin plays a role in cell elongation, cell shape maintenance, and flowering in Arabidopsis

Profilin (PFN) is an ubiquitous, low-M(r), actin-binding protein involved in the organization of the cytoskeleton of eukaryotes including higher plants. PFNs are encoded by a multigene family in Arabidopsis. We have analyzed in vivo functions of Arabidopsis PFN by generating transgenic plants carrying a 35S-PFN-1 or 35S-antisense PFN-1 transgene. Etiolated seedlings underexpressing PFN (PFN-U) displayed an overall dwarf phenotype with short hypocotyls whose lengths were 20% to 25% that of wild type (WT) at low temperatures. Light-grown PFN-U plants were smaller in stature and flowered early. Compared with equivalent cells in WT, most cells in PFN-U hypocotyls and roots were shorter, but more isodiametric, and microscopic observations of etiolated PFN-U hypocotyls revealed a rough epidermal surface. In contrast, light-grown seedlings overexpressing PFN had longer roots and root hair although etiolated seedlings overexpressing PFN were either the same size or slightly longer than WT seedlings. Transgenic seedlings harboring a PFN-1-GUS transgene directed expression in root and root hair and in a ring of cells at the elongating zone of the root tip. As the seedlings matured PFN-1-GUS was mainly expressed in the vascular bundles of cotyledons and leaves. Our results show that Arabidopsis PFNs play a role in cell elongation, cell shape maintenance, polarized growth of root hair, and unexpectedly, in determination of flowering time.     

DIP: a member of the MIP family of membrane proteins that is expressed in mature seeds and dark-grown seedlings of Antirrhinum majus

DiP , a gene from Antirrhinum majus , which encodes a protein with striking homology to other integral membrane proteins, was cloned. The gene was specifically expressed in mature seeds and during seedling germination, particularly in cotyledons of seedlings grown in the dark. The deduced product, called DiP, for dark intrinsic protein, shows strong homology with the MIP family of channel transporters which include; the bovine major intrinsic protein (MIP), the Escherichia coli glycerol facilitator (GlpF), the peribacteroid nodulin-26 (Nod26), and the tonoplast protein from kidney bean (TIP). DiP is most similar to other plant members of this family, and in particular to the tobacco protein TobRB7 which is expressed specifically in roots. However, the expression pattern of diP suggests that its product is functionally more similar to the tonoplast intrinsic protein from kidney bean since it is most highly expressed in the cotyledons of germinating seedlings, before the cells undergo expansion growth and become photosynthetic.     

Hsp101 is necessary for heat tolerance but dispensable for development and germination in the absence of stress

Hsp101 is a molecular chaperone that is required for the development of thermotolerance in plants and other organisms. We report that Arabidopsis thaliana Hsp101 is also regulated during seed development in the absence of stress, in a pattern similar to that seen for LEA proteins and small Hsps; protein accumulates during mid-maturation and is stored in the dry seed. Two new alleles of the locus encoding Hsp101 (HOT1) were isolated from Arabidopsis T-DNA mutant populations. One allele, hot1-3, contains an insertion within the second exon and is null for Hsp101 protein expression. Despite the complete absence of Hsp101 protein, plant growth and development, as well as seed germination, are normal, demonstrating that Hsp101 chaperone activity is not essential in the absence of stress. In thermotolerance assays hot1-3 shows a similar, though somewhat more severe, phenotype to the previously described missense allele hot1-1, revealing that the hot1-1 mutation is also close to null for protein activity. The second new mutant allele, hot1-2, has an insertion in the promoter 101 bp 5' to the putative TATA element. During heat stress the hot1-2 mutant produces normal levels of protein in hypocotyls and 10-day-old seedlings, and it is wild type for thermotolerance at these stages. Thus this mutation has not disrupted the minimal promoter sequence required for heat regulation of Hsp101. The hot1-2 mutant also expresses Hsp101 in seeds, but at a tenfold reduced level, resulting in reduced thermotolerance of germinating seeds and underscoring the importance of Hsp101 to seed stress tolerance.     

The Douglas-fir BiP promoter is functional in Arabidopsis and responds to wounding

A DNA sequence representing the promoter region of the Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) luminal binding protein PmBiP (PmBiPPro1) was isolated using inverse polymerase chain reaction (iPCR). Transient expression analysis of PmBiPPro1 fused to the beta-glucuronidase (GUS) reporter gene demonstrated that this promoter is functional in germinating Douglas-fir embryos. Transgenic Arabidopsis plants containing PmBiPPro1:GUS reporter gene constructs revealed strong staining associated with actively dividing/expanding cells and secretory tissues in developing seedlings. Wounding of cotyledons resulted in an increase in local staining associated with cells surrounding the wound site. Deletion analysis showed that elements necessary for basal-level expression reside within a -261 to +16 bp region, although upstream elements are necessary for higher-level expression in germinating Douglas-fir embryos, developing Arabidopsis seedlings and wounded cotyledons. Correlation of the observed expression pattern with the known function of BiP suggests that pathways controlling expression are highly conserved between angiosperms and gymnosperms.     

一种鉴定金花茶人工杂交F_1代杂种苗的简易方法

本试验选用了两个以金花茶作父本的人工杂交组合F_1代实生苗,经根尖体细胞染色体观察鉴定四倍体杂种。一、云南野山茶(Camellia pitardii var.yunnanica六倍体)×金花茶(C.Chrysantha二倍体)。结果为:所获56株子叶和下胚轴为红色的F_1代杂种苗中,有55株(98.2％)是真正的四倍体杂种(X~2=0.0128,D.f.=1,0.95>p>0.90);所获50株子叶和下胚轴为黄白色的杂种苗中,有13株(26％)为真正四倍体杂种(X~2=27.38,D.f.=13,0.02>p>0.01)。二、云南山茶花(C.reticulata六倍体)×金花茶。结果为:在78株子叶和下胚轴为红色的杂交苗中,有77株(98.7％)为真正四倍体杂种(X~2=0.0128,D.f.=1,0.95>p>0.90);而在78株子叶及下胚轴为黄白色的杂种苗中,只有21株(26.9％)是真正四倍体杂种(X~2=41.65,D.f.=24,0.02>p>0.01) 在多数杂种实生苗中的这种红色素,是因种子直感现象而发生的父本金花茶的一种遗传性状。金花茶的另一特征——多子叶现象(3枚以上),则在一些F_1代杂种苗或杂种种子中表现得不明显。因此,利用F_1代杂种苗子叶和下胚轴所具有的红色特征,在杂种种子萌发期用来鉴别以金花茶为亲本的F_1代杂种的真伪,是一种简便、快速的、有发展前途的方法。     

Changes in leucyl-tRNAs and aminoacyl tRNA synthetases in developing and aging soybean cotyledons

Leucine specific tRNA of soybean cotyledons was frationated into six peaks (1–6). The relative amounts of Leu-tRNA 5 and 6 are lower in developing cotyledons than in germinating cotyledons. Leu-tRNA synthetase from developing cotyledons is less active in aminoacylating Leu-tRNA 5 and 6 compared to enzyme from 5-day-old germinating cotyledons. Leu-tRNA synthetase from cotyledons of germinating seedlings and developing cotyledons can be fractionated into three peaks (1–3). Peak 1 in the developing cotyledon is about 36% less than peak 1 from 5-day-old germinating cotyledons. Peaks 2 and 3 from developing cotyledons are about 10 and 18% higher than from germinating cotyledons, respectively. Peak 1 from developing cotyledons acylates all six species of Leu-tRNA in contrast with peak 1 from germinating cotyledons, which essentially acylates only Leu-tRNA 5 and 6. The specificity of peaks 2 and 3 towards Leu-tRNA 1–4 is identical in both the organs.     

Adenosylhomocysteinase and adenosine nucleosidase activities in Lupinus luteus cotyledons during seed formation and germination

The activities of adenosylhomocysteinase (EC 3.3.1.1) and adenosine nucleosidase (EC 3.2.2.7) were assayed in extracts from yellow lupin (Lupinus luteus L.) cotyledons at different stages of seed formation and seedling development. Adenosylhomocysteinase activity was demonstrated in all the cotyledon extracts examined. Its lowest level was found in the dry seeds and the highest, in 4-day-old seedling cotyledons. Extracts from the cotyledons of maturating seeds, dry seeds, and seedlings up to the second day of growth exhibited no adenosine nucleosidase activity. Adenosine nucleosidase activity appeared in the cotyledons of 2-day-old seedlings and its highest level was reached in 4-to 5-day-old seedlings. There is no inhibitor of adenosine nucleosidase in the maturating and dry yellow lupin seeds. No activator of a possible zymogen form of adenosine nucleosidase from maturating or dry seeds occurs in the growing seedlings.