Reduction of turgor induces rapid changes in leaf translatable RNA

The turgor of pea (Pisum sativum) leaves was reduced by exposing excised pea shoots to a stream of 23°C air for 20 min. Poly(A)⁺ RNA was isolated from control and wilted shoots, translated in vitro and radiolabeled translation products separated by electrophoresis on two-dimensional (isoelectric focusing-sodium dodecyl sulfate) polyacrylamide gels. This analysis showed that the levels of several poly(A)⁺ RNAs increased in wilted plants. Most of the poly(A)⁺ RNAs induced in wilted plants did not accumulate in response to heat shock or exogenously applied ABA even though endogenous ABA levels were found to increase in shoots 30 min after wilting and by 4 h had increased 50-fold (1 versus 0.02 microgram per gram fresh weight). A λgt10 cDNA library was constructed using poly(A)⁺ RNA from wilted shoots which had been incubated for 4 hours. Differential screening of the library identified four clones corresponding to poly(A)⁺ RNAs which are induced in wilted shoots.     

A dehydrin cognate protein from pea (Pisum sativum L.) with an atypical pattern of expression

Dehydrins are a family of proteins characterised by conserved amino acid motifs, and induced in plants by dehydration or treatment with ABA. An antiserum was raised against a synthetic oligopeptide based on the most highly conserved dehydrin amino acid motif, the lysine-rich block (core sequence KIKEK-LPG). This antiserum detected a novel M _r 40 000 polypeptide and enabled isolation of a corresponding cDNA clone, pPsB61 (B61). The deduced amino acid sequence contained two lysine-rich blocks, however the remainder of the sequence differed markedly from other pea dehydrins. Surprisingly, the sequence contained a stretch of serine residues, a characteristic common to dehydrins from many plant species but which is missing in pea dehydrin.The expression patterns of B61 mRNA and polypeptide were distinctively different from those of the pea dehydrins during seed development, germination and in young seedlings exposed to dehydration stress or treated with ABA. In particular, dehydration stress led to slightly reduced levels of B61 RNA, and ABA application to young seedlings had no marked effect on its abundance.The M _r 40 000 polypeptide is thus related to pea dehydrin by the presence of the most highly conserved amino acid sequence motifs, but lacks the characteristic expression pattern of dehydrin. By analogy with heat shock cognate proteins we refer to this protein as a dehydrin cognate.     

A shoot-specific mRNA from pea: nucleotide sequence and regulation as compared to light-induced mRNAs

The regulation of a mRNA encoding a shoot-specific polypeptide from developing pea seedlings was studied and compared to the regulation of mRNAs encoding two major light-induced nuclear-encoded polypeptides, the small subunit of the ribulose 1,5 biphosphate carboxylase (ssRuBPCase) and a polypeptide of the light-harvesting chlorophyll a/b complex (LHCP). By using cDNA clones as probes in Northern blottings of total cellular RNA it was found that both ssRuBPCase and LHCP mRNA could be induced in shoots by white and red light but to lower levels in roots and cotyledons. In contrast, the mRNA for the shoot-specific polypeptide was only found in shoots, and was present approximately two days after the start of germination. The shoot-specific mRNA sequence was predominantly found in stem tissue, irrespective of illumination, both in the young seedlings and adult plants. Only very low amounts could be detected in plumule and leaf. The shoot-specific sequence could also be detected in RNA isolated from developing shoots of another pea cultivar but not in those of other legumes and of cereals. The primary sequence of the complete coding portion and the deduced amino acid sequence of the mRNA encoding the shoot-specific polypeptide was determined. The observed codon usage is non-random and is consistent with data from other high plant genes. Possible polyadenylation signal sequences (AATAAG and AATAAT) were present at 55 and 124 bases 5′ of the poly(A) tail. The polypeptide encoded by the shoot-specific mRNA consists of 196 amino acids with a calculated molecular weight of 21 898. It contains a four times reiterated highly conserved unit of 26 amino acids. The NH₂-terminal end is highly hydrophobic and resembles a signal polypeptide.     

Cloning and characterization of a putative 12-oxophytodienoic acid reductase cDNA induced by osmotic stress in roots of foxtail millet.

Foxtail millet is a gramineous crop with low water requirement. Cloning of osmotic responses-related genes from foxtail millet is a key step for understanding the mechanism of its tolerance to drought. Here we reported the cloning and characterization of a cDNA (SiOPR1) encoding a putative 12-oxophytodienoic acid reductase 1 from foxtail millet by using RACE methods. Sequence analysis showed that SiOPR1 encoded a polypeptide of 374 amino acids with a predicted molecular mass of 41.9 kDa and pI of 5.14. Multiple alignment result showed that OPR1 protein was very conservative among gramineous crops. RNA gel blot analysis results indicated that SiOPR1 was up-regulated by osmotic stress, and its expression was limited in the roots of foxtail millet. However, SiOPR1 expression was not affected by ABA, NaCl and MeJA treatments both in roots and shoots. Therefore, it is suggested that SiOPR1 gene play an important role in response to drought stress.     

Abscisic acid accumulation in the roots of nutrient-limited plants: its impact on the differential growth of roots and shoots

We describe the involvement of abscisic acid (ABA) in the control of differential growth of roots and shoots of nutrient limited durum wheat plants. A ten-fold dilution of the optimal concentration of nutrient solution inhibited shoot growth, while root growth remained unchanged, resulting in a decreased shoot/root ratio. Addition of fluridone (inhibitor of ABA synthesis) prevented growth allocation in favour of the roots. This suggests the involvement of ABA in the redirecting of growth in favour of roots under limited nutrient supply. The ABA content was greater in shoots and growing apical root parts of starved plants than in nutrient sufficient plants. Accumulation of ABA in shoots of nutrient deficient plants was linked to a decrease in leaf turgor. Increased flow of ABA in the phloem apparently contributed to the accumulation of ABA in the apical part of the roots. Thus, partitioning of growth between roots and shoots of wheat plants limited in mineral nutrients appears to be modulated by accumulation of ABA in roots. This ABA may originate in the shoots, where its synthesis is stimulated by the loss of leaf turgor.     

Characterisation of a pea hsp70 gene which is both developmentally and stress-regulated

A pea pod cDNA library was screened for sequences specific to lignifying tissue. A cDNA clone (pLP19) encoding the C-terminal region of a hsp70 heat shock protein hybridised only to pod mRNA from pea lines where pod lignification occurred. Expression of pLP19 was induced by heat shock in leaves, stems and roots of pea and chickpea plants. Four different poly(A) addition sites were observed in cDNAs derived from the same gene as pLP19. This gene was fully sequenced; unlike most hsp70 genes, it contains no introns. The 5-flanking sequence contains heat shock elements and other potential regulatory sequences.     

Construction and application of EST library from Setaria italica in response to dehydration stress

Foxtail millet is a gramineous crop with low water requirement. Despite its high water use efficiency, less attention has been paid to the molecular genetics of foxtail millet. This article reports the construction of subtracted cDNA libraries from foxtail millet seedlings under dehydration stress and the expression profile analysis of 1947 UniESTs from the subtracted cDNA libraries by a cDNA microarray. The results showed that 95 and 57 ESTs were upregulated by dehydration stress, respectively, in roots and shoots of seedlings and that 10 and 27 ESTs were downregulated, respectively, in roots and shoots. The expression profile analysis showed that genes induced in foxtail millet roots were different from those in shoots during dehydration stress and that the early response to dehydration stress in foxtail millet roots was the activation of the glycolysis metabolism. Moreover, protein degradation pathway may also play a pivotal role in drought-tolerant responses of foxtail millet. Finally, Northern blot analysis validated well the cDNA microarray data.     

Isolation and characterization of a cDNA clone encoding the major hsp70 of the pea chloroplastic stroma

The 70-kD heat shock proteins (hsp70s) are a group of ubiquitous, highly conserved molecular chaperones that have been implicated in a variety of processes, ranging from DNA replication to protein folding and transport. To learn more about the evolution and possible functions of higher plant chloroplastic hsp70s, we isolated a cDNA clone encoding the major stromal hsp70 of pea chloroplasts, which we term CSS1 (Chloroplastic Stress Seventy). This cDNA clone encodes a 75,490-D protein that is very closely related to an hsp70 from the cyanobacterium, Synechocystis. CSS1 is nuclear encoded and synthesized as a higher molecular mass precursor with a chloroplastic transit peptide approximately 65 amino acids long. CSS1 mRNA was detected in RNA samples from leaves and roots of pea (Pisum sativum) plants grown at 18°C but increased 9- and 6-fold, respectively, after brief exposure of the plants to elevated temperature. We discuss the possible role(s) of CSS1 in chloroplastic protein transport and other processes.     

Molecular cloning of pea mRNAs encoding a shoot-specific polypeptide and light-induced polypeptides

Summary The molecular cloning of cDNA corresponds to pea seedling mRNA sequences encoding a shoot-specific polypeptide, the small subunit of the ribulose 1,5 biphosphate carboxylase and a component of the light-harvesting chlorophyll a/b complex is described. cDNA prepared from polysomal poly(A)RNA of light-grown shoots was enriched for shoot-specific and light-induced sequences by heterologous liquid hybridization with mercurated polysomal poly(A)RNA of dark-grown roots, followed by sulfhydryl chromatography. Cloned shoot-specific sequences were identified by 2D electrophoretic analysis of hybrid release translation products. The cloned shoot-specific sequence corresponded to a mRNA of 850 nt present both in light-and dark-grown shoots, and produced anin vitro translation product of M_r27 500 and isoelectric point of 4.7.     

Characterization of a cDNA encoding a novel plant poly(A) polymerase

We have isolated cDNA clones encoding a novel factor (PAP-I) that is a component of a multi-subunit poly(A) polymerase from pea seedlings. The encoded protein, when isolated from appropriately engineered Escherichia coli, was active as a poly(A) polymerase, either with an associated RNA binding cofactor (PAP-III) or with free poly(A) as an RNA substrate. The latter observation indicates that PAP-I is in fact a poly(A) polymerase. PAP-I bore a striking resemblance to an as yet uncharacterized cyanobacterial protein. This observation suggested a possible chloroplast localization for PAP-I. This hypothesis was tested and found to be substantiated; immunoblot analysis identified PAP-I in chloroplast but not nuclear extracts. Our results suggest that PAP-I is a component of the machinery that adds poly(A) to chloroplast RNAs.     

The sequence of chick alpha-actinin reveals homologies to spectrin and calmodulin

We have sequenced a cDNA, isolated from a chick embryo fibroblast lambda gt11 library, that encodes all 887 amino acids of alpha-actinin. Sequence from 10 different peptides from chick smooth muscle alpha-actinin was found to match that derived from the cDNA. The deduced protein sequence can be divided into three distinct domains: (a) the N-terminal 240 amino acid contains a highly conserved region (compared with Dictyostelium alpha-actinin) which probably represents the actin-binding domain, (b) amino acids 270-740 contain four repeats of a spectrin-like sequence, and (c) the C-terminal sequence contains two EF-hand Ca2+-binding sites. Each of these sites is defective in at least one oxygen-containing Ca2+-chelating amino acid side chain, suggesting that they are nonfunctional. Southern blots suggest that the alpha-actinin cDNA described here hybridizes to only one gene in chicken. Northern blots reveal only one size class of mRNA in fibroblasts and smooth muscle, but no hybridizing species could be detected in skeletal muscle poly(A+) RNA. The results are consistent with the view that smooth and skeletal muscle alpha-actinins are encoded by separate genes, which are considerably divergent.     

两个小麦14-3-3基因的特征、亚细胞定位及其对非生物胁迫的响应(英文)

为了探讨14-3-3基因在小麦逆境胁迫应答中的调控作用,利用RACE技术克隆了两个包含完整编码框的14-3-3基因(命名为Ta14R1和Ta14R2),其中Ta14R1 cDNA长999 bp,编码262个氨基酸,而Ta14R2 cDNA长897 bp,编码261个氨基酸。Ta14R1/Ta14R2-GFP融合载体瞬时表达结果显示,Ta14R1和Ta14R2蛋白均定位于细胞质和细胞膜,但不在叶绿体中。荧光定量PCR分析表明,Ta14R1和Ta14R2均在萌发1 d的胚芽鞘中表达量最高;在高温、低温、模拟干旱和ABA处理下,两个基因在小麦的根和叶中都受胁迫诱导而且显著上调表达,推测这两个14-3-3基因通过依赖ABA的非生物胁迫响应途径发挥作用,可能参与了小麦中高温、低温和干旱胁迫的耐受调节过程。     

Nucleotide sequences of two pea cDNA clones encoding the small subunit of ribulose 1,5-bisphosphate carboxylase and the major chlorophyll a/b-binding thylakoid polypeptide

Two major chloroplast proteins are encoded by nuclear genes and synthesized on free cytoplasmic ribosomes: the small subunit of ribulose 1,5-bisphosphate carboxylase and the apoprotein components of the chlorophyll a/b light harvesting complex. We have recently reported the isolation of two cDNA clones from pea which encode both the small subunit of ribulose 1,5-bisphosphate carboxylase (pSS15) and the polypeptide 15 (pAB96), the major chlorophyll a/b binding protein (Broglie, R., Bellemare, G., Bartlett, S., Chua, N.-H., and Cashmore, A. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 7304-7308). To further characterize these clones, we determined their nucleotide sequence. Clone pSS15 contains a 691-base pair cDNA insert which encodes the entire 123 amino acids of the mature small subunit protein. In addition, this clone also encodes 33 amino acids of the NH2-terminal transit peptide extension and 148 nucleotides of the 3' noncoding region preceding the poly(A)tail. A second cDNA clone (pAB96) contains an 833-nucleotide insert which encodes most of polypeptide 15. The DNA sequence of this cloned cDNA was used to deduce the previously undetermined amino acid sequence of this integral thylakoid membrane protein. The nucleotide sequence of the cDNA clone, pSS15, should provide information concerning the role of the transit sequence in the transport of cytoplasmically synthesized chloroplast proteins. Similarly, the deduced amino acid sequence of polypeptide 15 will provide information for predicting its orientation in thylakoid membranes as well as its role in binding chlorophyll.     

Isolation and expression analysis of two rice genes encoding the major intrinsic protein

We isolated two rice cDNAs (rMip1 and rTip1) which are homologous to the genes encoding the major intrinsic protein (Mip) (soybean nod-26 and Arabidopsis -Tip), respectively. Expression of rTip1 in shoots and roots of rice seedlings was enhanced by water stress, salt stress and exogenous ABA. rMip1 was expressed only in shoots. Although mRNA level of rMip1 in shoots was induced to a small extent by exogenous ABA, it did not show any increase under water or salt stress over the course of 12 h. On the basis of the differential expression patterns and evolutional distinctions, it is suggested that the possible channel proteins encoded by rMip1 and rTip1 genes may function in different transport systems.