Identification and Comparative Analysis of CBS Domain-Containing Proteins in Soybean (Glycine max) and the Primary Function of GmCBS21 in Enhanced Tolerance to Low Nitrogen Stress.

Nitrogen is an important macronutrient required for plant growth, and is a limiting factor for crop productivity. Improving the nitrogen use efficiency (NUE) is therefore crucial. At present, the NUE mechanism is unclear and information on the genes associated with NUE in soybeans is lacking. cystathionine beta synthase (CBS) domain-containing proteins (CDCPs) may be implicated in abiotic stress tolerance in plants. We identified and classified a CBS domain-containing protein superfamily in soybean. A candidate gene for NUE, GmCBS21, was identified. GmCBS21 gene characteristics, the temporal expression pattern of the GmCBS21 gene, and the phenotype of GmCBS21 overexpression in transgenic Arabidopsis thaliana under low nitrogen stress were analyzed. The phenotypes suggested that the transgenic Arabidopsis thaliana seedlings performed better under the nitrogen-deficient condition. GmCBS21-overexpressing transgenic plants exhibit higher low nitrogen stress tolerance than WT plants, and this suggests its role in low nitrogen stress tolerance in plants. We conclude that GmCBS21 may serve as an excellent candidate for breeding crops with enhanced NUE and better yield.

Diagnostico de homocistinuria y deficiencia de Adenilosuccinato Liasa a través de técnicas químicas, bioquímicas y moleculares / Diagnose of Homocistinuria and deficiency of Adenilosuccinato Liasa through technical chemical, biochemical and molecular

Entre los errores innatos del metabolismo (EIM) que son defectos bioquímicos de origen genético se encuentra: la homocistinuria y la deficiencia de adenilosuccinato liasa (EC 4.3.2.2) (ADSL), la primera es frecuentemente producida por deficiencia de la cistationina ß sintasa (EC 4.2.1.22) (CßS) enzima que interviene en el catabolismo del aminoácido esencial metionina, la segunda es un defecto en el metabolismo de las purinas. Las manifestaciones clínicas de estas deficiencias son: para la CßS se comprometen sistemas del organismo como el ocular, músculo esquelético, vascular y sistema nervioso central; en el caso de ADSL, se presenta retardo mental, convulsiones, rasgos autistas, movimientos involuntarios, espasmo e hipoplasia cerebral. En este trabajo se escribe la secuencia utilizada en el diagnóstico de la homocistinuria y de la ADSL, a partir del uso de métodos químicos, bioquímicos y moleculares. Se estudiaron pacientes con sospecha clínica de estar afectados por un EIM; se emplearon las pruebas químicas como el nitroprusiato de sodio y de plata; separación de aminoácidos en plasma y orina por cromatografía de capa fina, técnicas bioquímicas para cuantificar la enzima CßS y técnicas moleculares para identificar la mutación que produce la homocistinuria. En el caso de la ADSL el diagnóstico se realizó por medio del test de Bratton Marshall con el cual se identifica la presencia de metabolitos de las purinas; luego por cromatografía de alta resolución (HPLC) para separar, identifica y cuantifica las succinilpurinas. Para el caso de la homocistinuria los resultados de nitroprusiato de sodio y de plata fueron positivos, la cuantificación enzimática mostró deficiencia de CßS y se llegó a identificar la presencia de una mutación. En el caso de la deficiencia de ADSL el test de Bratton Marshall fue positivo y la cuantificaron de metabolitos de las purinas se encontró aumentada.

Homocystinuria. Evidence for three distinct classes of cystathionine beta-synthase mutants in cultured fibroblasts.

We have compared in vivo pyridoxine responsiveness with in vitro cystathionine beta-synthase activity in extracts of confluent fibroblasts from 14 synthase-deficient patients. Enzyme activity was measured with and without addition of its cofactor, pyridoxal-5'-phosphate, using a radioisotopic assay which detects as little as 0.25% of control activity. Six of seven lines from responsive patients had measurable activity without the added cofactor (0.6-15% of mean control). Two of these lines showed a five- and sevenfold stimulation of cystathionine beta-synthase activity with added pyridoxal-5'-phosphate; in the other four, the cofactor addition increased activity only modestly, as in controls. Two of seven lines from nonresponsive patients had measurable activity (each 3% of mean control) which increased two- and fivefold with the added cofactor. Cystathionine beta-synthase activity was undetectable in one line from a responsive patient and in five lines from nonresponsive ones. To characterize control and mutant synthase further, dissociation constants for pyridoxal-5'-phosphate were estimated and thermostability (54 degrees C) was studied in two control and five mutant lines. In one mutant, both parameters were normal; in the others, the affinity for the cofactor was reduced 3-to 11-fold and thermostability was much impaired. We conclude that at least three general classes of cystathionine beta-synthase mutants exist: those with no residual activity; those with reduced activity and normal affinity for pyridoxal-5' phosphate; and those with reduced activity and a reduced affinity for the cofactor. Pyridoxine responsiveness in vivo cannot be correlated simply with the presence or absence of residual synthase activity in vitro or with stimulation of in vitro enzyme activity by cofactor.