Salt-inducible betaine aldehyde dehydrogenase from sugar beet: cDNA cloning and expression

Members of the Chenopodiaceae, such as sugar beet and spinach, accumulate glycine betaine in response to salinity or drought stress. The last enzyme in the glycine betaine biosynthetic pathway is betaine aldehyde dehydrogenase (BADH). In sugar beet the activity of BADH was found to increase two- to four-fold in both leaves and roots as the NaCl level in the irrigation solution was raised from 0 to 500 mM. This increase in BADH activity was paralleled by an increase in level of translatable BADH mRNA. Several cDNAs encoding BADH were cloned from a gt10 libary representing poly(A)⁺ RNA from salinized leaves of sugar beet plants, by hybridization with a spinach BADH cDNA. Three nearly full-length cDNA clones were confirmed to encode BADH by their nucleotide and deduced amino acid sequence identity to spinach BADH; these clones showed minor nucleotide sequence differences consistent with their being of two different BADH alleles. The clones averaged 1.7 kb and contained an open reading frame predicting a polypeptide of 500 amino acids with 83% identity to spinach BADH. RNA gel blot analysis of total RNA showed that salinization to 500 mM NaCl increased BADH mRNA levels four-fold in leaves and three-fold in the taproot. DNA gel blot analyses indicated the presence of at least two copies of BADH in the haploid sugar beet genome.     

Molecular analysis of a homogentisate phytyltransferase gene from <Emphasis Type="Italic">Lactuca sativa</Emphasis> L.

Tocochromanols, usually known as vitamin E, play a crucial role in human and animal nutrition. The enzyme homogentisate phytyltransferase (HPT) performs the first committed step of the vitamin E biosynthetic pathway. The full-length cDNA encoding HPT was isolated from Lactuca sativa L. by rapid amplification of cDNA ends (RACE). The cDNA, designated as LsHPT, was 1,670 bp long containing an open reading frame (ORF) of 1,185 bp which encoded a protein of 395 amino acids. Sequence analysis indicated that the deduced protein, named as LsHPT, shared high identity with other dicotyledonous HPTs. Real-time fluorescent quantitative PCR (qPCR) analysis revealed that LsHPT was preferentially expressed in mature leaves compared with other tissues. When lettuce plants were subjected to drought and high-light stress treatments, LsHPT expression was markedly increased. Expression of LsHPT in Arabidopsis showed that LsHPT could enhance the α-tocopherol biosynthesis in Arabidopsis. Transient expression of LsHPT via agroinfiltration resulted in 9-fold increase in LsHPT mRNA level and nearly 18-fold enhancement in α-tocopherol content compared with the negative controls.     

Characterization of cDNAs encoding CuZn-superoxide dismutases in Scots pine

A Scots pine (Pinus sylvestris L.) cDNA library was screened with two heterologous cDNA probes (P31 and T10) encoding cytosolic and chloroplastic superoxide dismutases (SOD) from tomato. Several positive clones for cytosolic and chloroplastic superoxide dismutases were isolated, subcloned, mapped and sequenced. One of the cDNA clones (PS3) had a full-length open reading frame of 465 bp corresponding to 154 amino acid residues and showed approximately 85% homology with the amino acid sequences of angiosperm cytosolic SOD counterparts. Another cDNA clone (PST13) was incomplete, but encoded a putative protein with 93% homology to pea and tomato chloroplastic superoxide dismutase. The derived amino acid sequence from both cDNA clones matched the corresponding N-terminal amino acid sequence of the purified mature SOD isozymes. Northern blot hybridizations showed that, cytosolic and chloroplastic CuZn-SOD are expressed at different levels in Scots pine organs. Sequence data and Southern blot hybridization confirm that CuZn-SODs in Scots pine belong to a multigene family. The results are discussed in relation to earlier observations of CuZn-SODs in plants.     

Molecular cloning and characterization of anther-preferential cDNA encoding a putative actin-depolymerizing factor

A cDNA clone, LMP131A, which is preferentially expressed in mature anther was isolated from a lily cDNA library. Northern blot analysis and plaque hybridization expriments showed that the LMP131A mRNA is present at ca. 0.3% of the mRNA in mature pollen and is not detectable in carpel, petal, floral bud, leaf, or root. The clone contains an open reading frame of 139 amino acid residues which shows greater than 40% sequence identity in a 91 amino acid overlap to animal actin-depolymerizing factors (ADF), cofilin and destrin. The sequences at and near the actin-binding site are most conserved. Using the lily clone as a probe, a cDNA clone, BMP1, was isolated from a mature anther library of Brassica napus. The expression pattern of the BMP1 clone was the same as that of the lily clone. The Brassica anther-preferential clone contains an open reading frame which is 79% identical to the lily LMP131A protein. Southern blot analysis showed that there are one or a few copies of the putative ADF genes in B. napus and Arabidopsis thaliana.     

Isolation of a choline monooxygenase cDNA clone from Amaranthus tricolor and its expressions under stress conditions

INTRODUCTIONAmaranth is a C4 dicotyledonous mesophytecrop plant. A. tricofor is a major variety for veg-etable and ornamental crops, and is widely culti-vated in the wor1d. Osmoprotectant glycine betaine(GB) was detected in Amaranthaceae, A. HyPochon-driacus L[2] and A. Caudatus L[3, 4]. GB iswidespread and an effective osmoprotectant in manyplants[3]. We studied the photosynthetic adaptationmechanism of A. trico1or under salt stress due to ac-cumulation of GB[5].GB is synthesized …     

Differential accumulation of mRNAs in drought-tolerant and susceptible common bean cultivars in response to water deficit

The physiological response to drought was measured in two common bean varieties with contrastive susceptibility to drought stress. A subtractive cDNA library was constructed from the two cultivars, Phaseolus vulgaris'Pinto Villa' (tolerant) and 'Carioca' (susceptible). 18 cDNAs displayed protein-coding genes associated with drought, cold and oxidative stress, signal transduction, plant defense, chloroplast function and unknown function. A cDNA coding for an aquaporin (AQP) was selected for further analyses. The open reading frames (ORFs) of AQPs from 'Pinto Villa' and 'Carioca' were compared and despite their similarity, accumulated differentially in the plant organs, as demonstrated by Northern blot and in situ hybridization. A phylogenetic analysis of the deduced amino acid sequence with other AQPs suggested a tonoplast-located protein. Under drought conditions, the levels of AQP mRNA from the susceptible cultivar decreased to undetectable levels; by contrast, 'Pinto Villa' mRNA was present and restricted the phloem tissue. This would allow 'Pinto Villa' to maintain vascular tissue functions under drought stress.     

Hypoxically inducible barley alanine aminotransferase: cDNA cloning and expression analysis

A 1.75 kb cDNA containing the entire coding sequence of the hypoxically inducible alanine aminotransferase (AlaAT) from barley roots was isolated and sequenced. This clone has an open reading frame of 1446 bp, and a deduced amino acid sequence of 482 residues, giving an estimated protein molecular mass of 52 885 Da. RNA blot analysis of barley root tissue showed a 4-fold increase of a single AlaAT-2 mRNA band after 12–24 hours of hypoxic stress, followed by a decrease in message levels after 48 h of hypoxic conditions. AlaAT-2 protein concentration increased in a similar pattern to AlaAT activity in root tissue, to almost 6-fold the aerobic level after 96 h of hypoxic stress. AlaAT-2 activity increased more than 2-fold in roots of Panicum miliaceum exposed to hypoxia, and is the same isoform as the light inducible AlaAT in P. miliaceum leaves. The unique expression patterns of AlaAT-2 in root and leaf tissue upon exposure to different environmental stimuli is also discussed.     

Isolation and characterization of a cDNA-clone coding for potato type A phytochrome

We have isolated and sequenced a cDNA clone encoding the apoprotein of a potato phytochrome. Based on the deduced amino acid sequence, which shows 78% amino acid identity to the Arabidopsis phyA and 50% identity to the Arabidopsis phyB open reading frame, we have classified this cDNA clone as potato phyA phytochrome. The amino acid immediately preceding cysteine 323, which is the homologue of oat cystein 321, to which the chromophore has been shown to be attached, is a tyrosine residue. This contrasts with six other type A phytochrome sequences from both monocots and dicots that encode serine in this position. As already observed in three other cDNAs isolated from dicot species, the potato phyA clone encodes a short open reading frame (13 amino acids) preceding the phyA open reading frame (1123 amino acids), supporting the idea that this type of leader sequence might be involved in the regulated expression of the phytochrome apoprotein. Southern blot analysis revealed a single phyA gene as well as other related phytochrome sequences in the potato genome. phyA mRNA levels varied in different organs and were modulated by white light; in seedlings and sprouts, highest levels of mRNA were detected in the etiolated stage. Upon illumination with white light, mRNA levels decreased to the amount found in leaves of re-etiolated plants. Lowest expression was observed in leaves of plants grown in the light, in tubers irrespective of light treatment, and in roots of plants grown in the dark. In roots of plants grown in the light, elevated levels of phyA mRNA were detected. Using a monoclonal antibody generated against pea phytochrome as an immunochemical probe, the protein was only detectable in protein extracts from etiolated seedlings and sprouts.     

An elicitor-induced cDNA from aerial yam (Dioscorea bulbifera L.) encodes a pathogenesis-related type 4 protein

We established Dioscorea bulbifera (aerial yam) cell suspension cultures to study the expression of defense-related genes upon elicitation with the yam pathogenic ascomycete Colletotrichum gloeosporioides. The induction of phenylalanine-ammonia lyase (PAL) mRNA, coding for a key enzyme involved in phytoalexin biosynthesis, was observed upon elicitation. Using RT-PCR, we isolated an elicited cDNA clone with an open reading frame of 453 nucleotides which showed high homology to cDNA sequences of pathogenesis-related proteins belonging to the PRP-4 group. Yam PRP-4 expression is increased in elicited cell cultures as well as in elicited leaves, and is encoded by a small multigene family. This is the first example for the cloning of a cDNA that might be involved in defense reactions of yam plants. Received: December 1997 / Revision received: June 1998 / Accepted: November 1998     

Molecular cloning of an alanine aminotransferase from NAD-malic enzyme type C4 plant Panicum miliaceum

We have determined the nucleotide sequence of a cDNA encoding AlaAT-2, which is believed to function in the C4-pathway of Panicum miliaceum. An open reading frame (1446 bp) encodes a protein of 482 amino acid residues. The deduced amino acid sequence of AlaAT-2 shows 44.2 and 44.8% homology with the amino acid sequences of AlaATs from rat and human livers, respectively. Northern blot analysis showed that the gene encoding AlaAT-2 in mesophyll and bundle sheath cells was the same and transcribed similarly in the cells. The level of translatable mRNA for AlaAT-2 increased dramatically during greening.     

Molecular characterization of a cDNA clone encoding glutamine synthetase from a gymnosperm,Pinus sylvestris

A full-length cDNA clone (pGSP114) encoding glutamine synthetase was isolated from a gt11 library of the gymnosperm Pinus sylvestris. Nucleotide sequence analysis showed that pGSP114 contains an open reading frame encoding a protein of 357 amino acid residues with a calculated molecular mass of 39.5 kDa. The derived amino acid sequence was more homologous to cytosolic (GS1) (78–82%) than to chloroplastic (GS2) (71–75%) glutamine synthetase in angiosperms. The lack of N-terminal presequence and C-terminal extension which define the primary structure of GS2, also supports that the isolated cDNA encodes cytosolic GS. Southern blot analysis of genomic DNA from P. sylvestris and P. pinaster suggests that GS may be encoded by a small gene family in pine. GS mRNA was more abundant in cotyledons and stems than in roots of both Scots and maritime pines. Western blot analysis in P. sylvestris seedlings showed that only one GS polypeptide, similar in size to GS1 in P. pinaster, could be detected in several different tissues. Our results suggest that cytosolic GS is mainly responsible for glutamine biosynthesis in pine seedlings.This paper is dedicated to the memory of Dr. Jesús S. Olavarría.     

Molecular cloning and characterization of 4-hydroxyphenylpyruvate dioxygenase gene from Lactuca sativa

Vitamin E has been found to be associated with an important antioxidant property in mammals and plants. In photosynthetic organisms, the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD; E.C. 1.13.11.27) plays an important role in the vitamin E biosynthetic pathway. The full-length cDNA encoding HPPD was isolated from Lactuca sativa L. by rapid amplification of cDNA ends (RACE). The cDNA, designated as LsHPPD, was 1743 base pairs (bp) long containing an open reading frame (ORF) of 1338 bp encoding a protein of 446 amino acids. Sequence analysis indicated that LsHPPD shared high identity with HPPD from Medicago truncatula L. Real-time fluorescent quantitative PCR (qPCR) analysis revealed that LsHPPD was preferentially expressed in mature leaves compared with other tissues and that the LsHPPD expression was sensitive to high light and drought stress treatments. Transient expression of LsHPPD via agroinfiltration resulted in 12-fold increase in LsHPPD mRNA expression level and 4-fold enhancement in α-tocopherol content compared with the negative control. A decrease in chlorophyll content and inhibition of photosystem II were observed during stress treatments and agroinfiltration.     

Molecular characterization of the soybean L-asparaginase gene induced by low temperature stress

L-asparaginase (EC 3.5.1.1) catalyzes the hydrolysis of the amide group of L-asparagine, releasing aspartate and NH4+. We isolated a low temperature-inducible cDNA sequence encoding L-asparaginase from soybean leaves. The full-length L-asparaginase cDNA, designated GmASP1, contains an open reading frame of 1,258 bp coding for a protein of 326 amino acids. Genomic DNA blotting and fluorescence in situ hybridization showed that the soybean genome has two copies of GmASP1. GmASP1 mRNA was induced by low temperature, ABA and NaCl, but not by heat shock or drought stress. E. coli cells expressing recombinant GmASP1 had 3-fold increased L-asparaginase activity. A possible function of L-asparaginase in the early response to low temperature stress is discussed.