Kazuko Yamaguchi-Shinozaki

The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression

The complete nucleotide sequence (155 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA has been determined. It contains two copies of an identical 25 339 bp inverted repeat, which are separated by a 86 684 bp and a 18 482 bp single‐copy region. The genes for 4 different rRNAs, 30 different tRNAs, 39 different proteins and 11 other predicted protein coding genes have been located. Among them, 15 genes contain introns. Blot hybridization revealed that all rRNA and tRNA genes and 27 protein genes so far analysed are transcribed in the chloroplast and that primary transcripts of the split genes hitherto examined are spliced. Five sequences coding for proteins homologous to components of the respiratory‐chain NADH dehydrogenase from human mitochondria have been found. The 30 tRNAs predicted from their genes are sufficient to read all codons if the ‘two out of three’ and ‘U:N wobble’ mechanisms operate in the chloroplast. Two sequences which autonomously replicate in yeast have also been mapped. The sequence and expression analyses indicate both prokaryotic and eukaryotic features of the chloroplast genes.

Six chloroplast genes (ndhA-F) homologous to human mitochondrial genes encoding components of the respiratory chain NADH dehydrogenase are actively expressed: determination of the splice sites in ndhA and ndhB pre-mRNAs.

SummarySequences (ndhA-F) homologous to human mitochondrial genes for components of the respiratory chain NADH dehydrogenase have been found in the chloroplast DnA of tobacco. The ndhA, D, E and F sequences corresponding to the mitochondrial URF1, 4, 4L and 5 are located in the small single copy region, the ndhB sequence corresponding to URF2 in the inverted repeat and the ndhC sequence corresponding to URF3 in the large single copy region of the chloroplast DNA. Northern blot hybridization revealed that all six ndh sequences are actively expressed in the chloroplasts. The ndhA and ndhB sequences contain single introns and the splice sites of their pre-mRNAs were determined by reverse transcription analysis. These findings suggest that potential components of an NADH dehydrogenase are synthesized in the chloroplasts.

The complete nucleotide sequence of the tobacco chloroplast genome

The c o m p l e t e n u c i e o t i d e sequence [155 ,844 bp) o f t o b a c c o ( N i c o t i a n a tabecum v a r . B r i g h t y e l l o w 4) c h l o r o p l a s t DNA [ S h i n o z a k i e t e l . 1986) is p r e s e n t e d . The c i r c u l a r DNA [see F ig . 1) i8 i nea r zed by c u t t i n g a t the j u n c t i o n JLA between IR A and LSC JLA is d e s i g n a t e d ze ro and numbered p r o c e e d i n g t owa rds LSC The DNA s t r a n d which codes f o r the l a r g e s u b u n i t of r i b u l o s e l , % b 8phospha te c a r b o x y l a s e is d e s i g n a t e d as A s t r a n d and the comp lemen ta ry s t r a n d as B s t r a n d . The B s t r a n d i8 shown he re . 6enes are boxed . The n o m e n c l a t u r e f o r genes f o l l o w s the p r o p o s a l s of H a l l i c k and B o t t o m l e y [1983, see Tab le 1) . A s t e r i s k s i n d i c a t e s p l i t genes and [C) deno tes genes l o c a t e d on the comp lemen ta ry s t r a n d ( t h e A s t r a n d ) .

Processing of precursor tRNAs in a chloroplast lysate: Processing of the 5′-end involves endonucleolytic cleavage by an RNase P-like enzyme and precedes 3′-end maturation

An in vitro tRNA processing system using either a spinach or tobacco chloroplast lysate has been constructed. Monomeric tRNAPhe precursors were prepared using an SP6 transcription vector system. tRNA precursors were processed to mature tRNA molecules by incubation in an S30 fraction of a chloroplast lysate. Both 5′ leader and 3′ extension sequences were processed endonucleolytically. Processing of the 5′ leader was demonstrated to precede 3′‐end maturation. RNase P‐like endoribonuclease is likely to function in the 5′‐end processing.

Molecular Mechanisms of Plant Responses and Tolerance of Drought and Cold Stress

Plants respond and adapt to a variety of environmental stresses including drought, cold and high salinity to survive in severe stress conditions. These stresses induce various physiological and biochemical responses in plants. Moreover, a variety of genes have been described that respond to these stresses at transcriptional level (Shinozaki and Yamaguchi-Shinozaki, 1997, 2000). Their gene products are thought to function in stress tolerance and response. Many stress-inducible genes have been used to improve stress tolerance of plants by gene transfer. It is important to analyze functions of stress-inducible genes not only for further understanding of molecular mechanisms of stress tolerance and response of higher plants but also for improvement of stress tolerance of crops by gene manipulation.

Water Stress-Induced Genes inArabidopsis thaliana

Plants respond to conditions of severe environmental changes or stresses, such as dehydration, low temperature, high-salinity with a number of physiological and developmental changes. We have isolated more than thirty genes that respond to dehydration stresses at transcriptional level. In the present study, we will report the expression and function of differentArabidopsis genes involved in the kinase cascade and in the proline biosynthesis. MAP kinases play key roles in the signalling pathway activated by growth factors and in stress response in animals. The mRNA levels of three protein kinases, ATMEKK1 (MAP kinase kinase kinase), ATMPK3 (MAP kinase) and ATPK19 (ribosomal S6 kinase), increased markedly and simultaneously in response to touch, cold and salinity stresses. These results suggest that MAP kinase cascades, which are thought to respond to a variety of extracellular signals, are regulated not only at the post-translational level but also at the transcriptional level in plants. It is also suggested that MAP kinase cascades in plants may function in transducing signals under environmental stresses. Proline is thought to be a compatible osmolyte like glycine betaine, which plays a role in counteracting the effects of osmotic stress in plants. We have isolated two cDNAs fromArabidopsis (cAtPSCS andcAtPSCR) encoding P5CS (Δ1-pyrroline-5-carboxylate synthetase) and P5CR (Δ1-pyrroline-5-carboxylate reductase) which play key roles in the biosynthesis of proline in plants under water stress. Northern blot analysis revealed that theAtP5CS gene was induced by dehydration, high salt and treatment with ABA. Moreover, a simultaneous accumulation of proline was observed as a result of these treatments ofArabidopsis plants. By contrast, theAtPSCR gene was not induced to a significant extent by dehydration or high-salt stress. These observations suggest that theAtP5CS gene plays a principal role in the biosynthesis of proline inArabidopsis under osmotic stress.