With the completion of the Arabidopsis genome sequencing project, the next major challenge is the large-scale determination of gene function. As a model organism for agricultural biotechnology, Arabidopsis presents the opportunity to provide key insights into the way that gene function can affect commercial crop production. In an attempt to aid in the rapid discovery of gene function, we have established a high throughput phenotypic analysis process based on a series of defined growth stages that serve both as developmental landmarks and as triggers for the collection of morphological data. The data collection process has been divided into two complementary platforms to ensure the capture of detailed data describing Arabidopsis growth and development over the entire life of the plant. The first platform characterizes early seedling growth on vertical plates for a period of 2 weeks. The second platform consists of an extensive set of measurements from plants grown on soil for a period of ∼2 months. When combined with parallel processes for metabolic and gene expression profiling, these platforms constitute a core technology in the high throughput determination of gene function. We present here analyses of the development of wild-type Columbia (Col-0) plants and selected mutants to illustrate a framework methodology that can be used to identify and interpret phenotypic differences in plants resulting from genetic variation and/or environmental stress.

With the completion of the Arabidopsis genome sequencing project, the next major challenge is the large-scale determination of gene function. As a model organism for agricultural biotechnology, Arabidopsis presents the opportunity to provide key insights into the way that gene function can affect commercial crop production. In an attempt to aid in the rapid discovery of gene function, we have established a high throughput phenotypic analysis process based on a series of defined growth stages that serve both as developmental landmarks and as triggers for the collection of morphological data. The data collection process has been divided into two complementary platforms to ensure the capture of detailed data describing Arabidopsis growth and development over the entire life of the plant. The first platform characterizes early seedling growth on vertical plates for a period of 2 weeks. The second platform consists of an extensive set of measurements from plants grown on soil for a period of ∼2 months. When combined with parallel processes for metabolic and gene expression profiling, these platforms constitute a core technology in the high throughput determination of gene function. We present here analyses of the development of wild-type Columbia (Col-0) plants and selected mutants to illustrate a framework methodology that can be used to identify and interpret phenotypic differences in plants resulting from genetic variation and/or environmental stress.

It has been shown that 1-aminocyclopropane-1-carboxylic acid (ACC) is the immediate precursor of ethylene, which is derived from C-2 and C-3 of ACC. When [1- ¹⁴ C]ACC was administered to etiolated mungbean ( Vigna radiata ) hypocotyls, ≈16% of the ACC was converted to ethylene and about 10% of the radioactivity was converted to [ ¹⁴ C]asparagine in 7 hr. In etiolated epicotyls of common vetch ( Vicia sativa ), after 7 hr about 14% of the ACC was converted to ethylene and 16% of the radioactivity was converted to β-cyanoalanine plus γ-glutamyl-β-cyanoalanine. Itis known that in most plants cyanide is metabolized to asparagine via the intermediate β-cyanoalanine, whereas in a fewplants such as V. sativa , β-cyanoalanine is converted to the conjugate γ-glutamyl-β-cyanoalanine. We confirmed that [ ¹⁴ C]cyanide was metabolized into [ ¹⁴ C]asparagine in mungbean and into [ ¹⁴ C]cyanoalanine plus its conjugate in V. sativa . Moreover, after feeding plant tissue with [1- ¹⁴ C]ACC, [ ¹⁴ C]asparagine isolated from mungbean and β-[ ¹⁴ C]cyanoalanine from V. sativa were labeled in the C-4 position, as would be expected if these two compounds were derived from [ ¹⁴ C]cyanide. When the conversion of ACC to ethylene in V. sativa tissue was inhibited by high temperature (41°C), the conversion of [1- ¹⁴ C]ACC to β-[ ¹⁴ C]cyanoalanine and γ-glutamyl-β-[ ¹⁴ C]cyanoalanine was similarly inhibited. When [ carboxyl - ¹⁴ C]ACC was administered to mungbean and V. sativa , ¹⁴ CO ₂ was recovered in an amount equivalent to the amount of ethylene produced. These data indicate that in the conversion of ACC to ethylene the carboxyl group yields CO ₂ , and C-1 is released as HCN.

The mechanisms involved in the posttranslational targeting of membrane proteins are not well understood. The light-harvesting chlorophyll proteins (LHCP) of the thylakoid membrane are a large family of hydrophobic proteins that are targeted in this manner. They are synthesized in the cytoplasm, translocated across the chloroplast envelope membranes into the stroma, bound by a stromal factor to form a soluble intermediate, “transit complex”, and then integrated into the thylakoid membrane by a GTP dependent reaction. Signal recognition particle (SRP), a cytoplasmic ribonucleoprotein, is known to mediate the GTP dependent cotranslational targeting of proteins to the endoplasmic reticulum. We show that chloroplasts contain an SRP consisting of, cpSRP54, a homologue of SRP54 and a previously undescribed 43-kDa polypeptide (cpSRP43) instead of an RNA. We demonstrate that both subunits of cpSRP are required for the formation of the transit complex with LHCP. Furthermore, cpSRP54, cpSRP43, and LHCP are sufficient to form a complex that appears to be identical to authentic transit complex. We also show that the complex formed between LHCP and cpSRP, together with an additional soluble factor(s) are required for the proper integration of LHCP into the thylakoid membrane. It appears that the expanded role of cpSRP in posttranslational targeting of LHCP has arisen through the evolution of the 43-kDa protein.

Diurnal oscillations in steady‐state mRNA levels and transcription rates were measured for seven transcripts (five of which encode chloroplast‐localized proteins) in tomato seedlings: photosystem I and photosystem II chlorophyll a/b binding proteins (CAB/I and CAB/II), small subunit of RuBisCO (RBCS), actin, subunit II of the photosystem I reaction center (PSAD), subunit I of the photosystem II oxygen‐evolving enzyme (OEE1), and a biotin‐binding protein of unknown function. CAB/II mRNA levels were found to oscillate greater than 20‐fold, showing a peak at noon, while only marginal diurnal oscillations are seen in RBCS transcripts. The oscillations are at least partially controlled at the transcriptional level. Transcription rates of both CAB/II and RBCS, measured by nuclear run‐on experiments, were found to oscillate, with a peak around 8 a.m. Transcription rates of the ‘biotin’ clone also oscillated, with a peak around noon. Transfer of plants to constant darkness or constant light conditions alters the amplitude of the transcriptional oscillation, but does not abolish it, suggesting that it is at least partially controlled by a circadian clock. The oscillations are still visible after three days in complete darkness, and have a period very close to 24 h. The oscillator phase can be reset by out‐of‐phase light treatment.