Plant Molecular Biology

Overexpression of StCaM2 in tobacco promotes plant growth and confers increased salinity and drought tolerance by enhancing the photosynthetic efficiency, ROS scavenging, and recovery from membrane injury. Calmodulins (CaMs) are important Ca2+ sensors that interact with effector proteins and drive a network of signal transduction pathways involved in regulating the growth and developmental pattern of plants under stress. Herein, using in silico analysis, we identified 17 CaM isoforms (StCaM) in potato. Expression profiling revealed different temporal and spatial expression patterns of these genes, which were modulated under abiotic stress. Among the identified StCaM genes, StCaM2 was found to have the largest number of abiotic stress responsive promoter elements. In addition, StCaM2 was upregulated in response to some of the selected abiotic stress in potato tissues. Overexpression of StCaM2 in transgenic tobacco plants enhanced their tolerance to salinity and drought stress. Accumulation of reactive oxygen species was remarkably decreased in transgenic lines compared to that in wild type plants. Chlorophyll a fluorescence analysis suggested better performance of photosystem II in transgenic plants under stress compared to that in wild type plants. The increase in salinity stress tolerance in StCaM2-overexpressing plants was also associated with a favorable K+/Na+ ratio. The enhanced tolerance to abiotic stresses correlated with the increase in the activities of anti-oxidative enzymes in transgenic tobacco plants. Overall, our results suggest that StCaM2 can be a novel candidate for conferring salt and drought tolerance in plants.

Soybean flavonoids, a group of important signaling molecules in plant-environment interaction, ubiquitously exist in soybean and are tightly regulated by many genes. Here we reported that GmMYB100, a gene encoding a R2R3 MYB transcription factor, is involved in soybean flavonoid biosynthesis. GmMYB100 is mainly expressed in flowers, leaves and immature embryo, and its level is decreased after pod ripening. Subcellular localization assay indicates that GmMYB100 is a nuclear protein. GmMYB100 has transactivation ability revealed by a yeast functional assay; whereas bioinformatic analysis suggests that GmMYB100 has a negative function in flavonoid biosynthesis. GmMYB100-overexpression represses the transcript levels of flavonoid-related genes in transgenic soybean hairy roots and Arabidopsis, and inhibits isoflavonoid (soybean) and flavonol (Arabidopsis) production in transgenic plants. Furthermore, the transcript levels of six flavonoid-related genes and flavonoid (isoflavonoid and flavone aglycones) accumulation are elevated in the GmMYB100-RNAi transgenic hairy roots. We also demonstrate that GmMYB100 protein depresses the promoter activities of soybean chalcone synthase and chalcone isomerase. These findings indicate that GmMYB100 is a negative regulator in soybean flavonoid biosynthesis pathway.

Amino acid sequences for three members (CMx1, CMx2, and CMx3) of a new subfamily of trypsin/α-amylase inhibitors in wheat have been deduced from the nucleotide sequences of the corresponding cDNAs. A cDNA clone encoding CMx1 was selected from a wheat developing endosperm library using a probe that encoded barley trypsin inhibitor BTI-CMe at low stringency. Sequences corresponding to CMx2 and CMx3 were obtained from cDNA amplified by the polymerase chain reaction. The three CMx sequences contain a premature stop codon after 363 nt, as well as a second stop codon at the same position as in BTI-CMe (nt 439–441). Southern analysis of DNAs from diploid, tetraploid, and hexaploid wheats, as well as from aneuploid lines, indicate that there is a single CMx locus in each of the three genomes of hexaploid wheat, respectively associated with chromosomal arms 4AS, 4BS, and 4DL. These genes are expressed early during endosperm development and not expressed at detectable levels in other tissues. Evolutionary implications are discussed.

Agarose gel electrophoresis of mitochondrial DNA (mtDNA) from the IS1112C male-sterile cytoplasm of Sorghum bicolor (S. bicolor) revealed plasmid-like DNAs additional to the linear N1 and N2 molecules. Mitochondrial plasmids were separated from the principal mitochondrial genome and used in the construction of molecular clones. Clones with EcoRI inserts of 1.7 and 2.3 kb were recovered. Hybridization of these clones to Southern blots of unrestricted and EcoRI-digested IS1112C mitochondrial plasmids indicated the cloned inserts were complete or nearly-complete copies of minicircular DNA molecules. These clones were used to probe Southern blots of mitochondrial genomes from six cytoplasmic male-sterile (CMS) and five male-fertile (MF) lines of S. bicolor, as well as Southern blots of IS1112C chloroplast, kafir chloroplast, IS1112C nuclear, and kafir nuclear genomes. The 2.3 and 1.7 kb plasmids had a very limited distribution among the sorghum entries we examined. We found no evidence for integrated copies of these sequences in any of the mitochondrial, chloroplast, or nuclear genomes probed in this study. However, the 2.3 kb sorghum minicircle did hybridize to the 1.94 kb minicircle from maize mitochondria. Hybridization of the 1.7 and 2.3 kb clones to IS111L2C mitochondrial RNA reveal a transcript of 1.1 kb from the 1.7 kb minicircle and transcripts of 1.9 and 1.4 kb from the 2.3 kb molecule.

Two different techniques were used to isolate potential cDNAs for acyl-CoA: 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPA-AT) enzymes from Limnanthes douglasii. Both heterologous screening with the maize pMAT1 clone and in vivo complementation of the Escherchia coli mutant JC201 which is deficient in LPA-AT activity, were carried out. Clones identified by these procedures were different. Homology searches demonstrated that the clone isolated by heterologous probing, pLAT1, encodes a protein which is most similar to the maize (open reading frame in pMAT1) and yeast SLC1 proteins, which are putative LPA-AT sequences. This L. douglasii sequence shows much lower homology to the E. coli LPA-AT protein PlsC, which is the only LPA-AT sequence confirmed by over-expression studies. The clone isolated by complementation, pLAT2, encodes a protein with homology to both SLC1 and PlsC. It was not possible to over-express the complementing protein encoded by pLAT2 but further experimentation on membranes from complemented JC201 demonstrated that they possess a substrate specificity distinctly different from PlsC and similar to Limnanthes sp. microsome specificity. This data strongly supports the contention that pLAT2 is an LPA-AT clone. Northern blot analysis revealed different expression patterns for the two genes in pLAT1 and pLAT2. Transcription of the gene encoding the insert of pLAT2 occurred almost exclusively in developing seed tissue, whilst the cDNA of pLAT1 hybridised to poly(A)+ mRNA from seed, stem and leaf, demonstrating more widespread expression throughout the plant. Southern blot analysis indicated that the cDNA of pLAT2 was transcribed from a single-copy gene while that for pLAT1 was a member of a small gene family.

Relatively few indica rice full-length cDNAs were available to aid in the annotation of rice genes. The data presented here described the sequencing and analysis of 10,096 full-length cDNAs from Oryza sativa subspecies indica Guangluai 4. Of them, 9,029 matched rice genomic sequences in publicly-available databases, and 1,200 were identified as new rice genes. Comparison with the knowledge-based Oryza Molecular Biological Encyclopedia japonica cDNA collection indicated that 3,316 (41.6%) of the 7,965 indica-japonica cDNA pairs showed no distinct variations at protein level (2,117 indica-japonica cDNA pairs showed fully identical and 1,199 indica-japonica cDNA pairs showed no frame shift). Moreover, 3,645 (45.8%) of the indica-japonica pairs showed substantial differences at the protein level due to single nucleotide polymorphisms (SNPs), insertions or deletions, and sequence-segment variations between indica and japonica subspecies. Further experimental verifications using PCR screening and quantitative reverse transcriptional PCR revealed unique transcripts for indica subspecies. Comparative analysis also showed that most of rice genes were evolved under purifying selection. These variations might distinguish the phenotypic changes of the two cultivated rice subspecies indica and japonica. Analysis of these cDNAs extends known rice genes and identifies new ones in rice.

ELISA was used to determine PR la protein and TMV accumulation in local necrotic lesions produced on salicylic acid and water sprayed Nicotiana tabacum cv Xanthi-nc leaves. The amount of PR la protein produced is the result of an interaction between the salicylic acid treatment and lesion growth. The implication of these observations for experiments investigating the relationship between PR proteins and resistance are discussed. The distribution of TMV and PR la protein in and around single local necrotic lesions up to 14 days after inoculation was measured by ELISA. The highest concentration of TMV was in the centre of the lesion and decreased rapidly with distance from the centre. In contrast there was very little PR la protein in the centre of the lesion, the largest amounts were just outside the centre, and the concentration then decreased with distance from the centre. This is the distribution that might be expected for a substance closely associated with the restriction of virus spread.

Brassinosteroids (BRs) are steroid hormones that are essential for plant growth and development. To gain insight into potential sites of BR synthesis, we studied promoter activities of the two Arabidopsis BR C-6 oxidase genes (CYP85A1 and CYP85A2) in transgenic plants carrying promoter fusions with the GUS, GFP or LUC reporter genes. BR-dependent feedback regulation of the GUS reporter constructs indicated that their expression corresponded to those of the native genes. Both the CYP85A1 and CYP85A2 promoters showed maximum activity during the first week following germination, particularly in the vascular tissues. Compared to CYP85A2, CYP85A1 expression was weaker and confined to the early stages of seedling development. Stronger CYP85A2 promoter activity was evident in both juvenile and adult plants. Comparison of the 5′-UTR and TATA box sequences of CYP85A1 and CYP85A2 revealed high homology, indicating a relatively recent gene duplication. We also found that transgenic Arabidopsis plants harbouring the tomato DWARF promoter-GUS fusion had similarities in the expression pattern to the Arabidopsis genes suggesting common transcriptional regulation of CYP85 genes in the two species.