Arginine decarboxylase (ADC) is a key enzyme involved in the synthesis of polyamines, which have been implicated in a wide range of plant responses, including stress. However, regulation of polyamine levels in relation to ADC in response to stress at the molecular level is not well understood. In an attempt to address this question, we first cloned two cDNAs in mustard (Brassica juncea[L.] Czern & Coss var. Indian Mustard), designated MADC2 and MADC3, encoding predicted ADC. MADC2 and MADC3 encode polypeptides of 692 and 680 amino acid residues, respectively. A comparison of deduced amino acid sequence revealed that both were highly homologous to MADC1 (77%), a mustard ADC, and other plant ADCs (63-84%). Northern analysis revealed that ADC transcripts in mustard were generally more abundant in stem and root but were barely detectable in leaf. However, ADC expression in the leaf was up-regulated differentially in response to stress such as chilling, salt and mannitol and to treatments with exogenous polyamines. While chilling induced expression of all three ADC genes, salt predominantly resulted in increased accumulation of MADC3 transcript. Leaves exhibited a similar response to exogenous putrescine, spermidine and spermine, all of which stimulated accumulation of MADC2 and MADC3 transcripts but not MADC1. Furthermore, exogenous putrescine also increased the endogenous levels of spermidine and spermine, while a higher endogenous putrescine and spermidine content was detected in leaf incubated with exogenous spermine. Leaves also responded to chilling, salt and mannitol by increasing the levels of the cellular polyamine content, in which the level of spermine in free and conjugated forms increased most profoundly.

The enzymes glutathione-S-transferases (GSTs, E.C.2.5.1.18) have been associated with detoxification of xenobiotics, limiting oxidative damage and other stress responses in plants. In this study, we report the isolation of a mustard gene, BjGSTF2, homologous to the phi class GSTs and changes in plant growth in vivo and shoot regeneration in vitro were related to GST expression. GST transcripts accumulated differentially in mustard organs, where transcript was most abundant in root. Tissues incubated at high temperature or in the presence of exogenous H2O2, HgCl2, 1-aminocyclopropane-1-carboxylate, salicylic acid and paraquat upregulated GST expression, whereas spermidine was inhibitory. To investigate the in vivo function of GST, transgenic Arabidopsis thalianaplants expressing sense (GST-S6), antisense (GST-A4) and double-stranded BjGSTF2 (GST-DS1) RNAs were generated. GST-S6 was shown to flower two days earlier and was relatively more tolerant to HgCl2 and paraquat, whereas GST-DS1 with least stress tolerance flowered one week later compared to WT and GST-A4. In shoot regeneration response, tissues originated from GST-S6 were highly regenerative, whereas no shoot regeneration was observed in GST-DS1 tissues after 30 days of culture. Results of this study provide the evidence showing that GST plays a role in plant growth and development in vivo and shoot regeneration in vitro.

Role of ethylene in de novo shoot morphogenesis from explants and plant growth of mustard (Brassica juncea cv. India Mustard) in vitro was investigated, by culturing explants or plants in the presence of the ethylene inhibitors aminoethoxyvinylglycine (AVG) and AgNO3. The presence of 20 μM AgNO3 or 5 μM AVG in culture medium containing 5 μM naphthaleneacetic acid and 10 μM benzyladenine were equally effective in promoting shoot regeneration from leaf disc and petiole explants. However, AgNO3 greatly enhanced ethylene production which reached a maximum after 14 days, whereas ethylene levels in the presence of AVG remained low during 3 weeks of culture. The promotive effect of AVG on shoot regeneration was overcome by exogenous application of 25 μM 2‐chloroethylphosphonic acid (CEPA), but AgNO3‐induced regeneration was less affected by CEPA. For whole plant culture, AVG did not affect plant growth, although it decreased ethylene production by 80% and both endogenous levels of 1‐aminocyclopropane‐1‐carboxylate (ACC) synthase and ACC by 70–80%. In contrast, AgNO3 stimulated all 3 parameters of ethylene synthesis. Both AgNO3 and CEPA were inhibitory to plant growth, with more severe inhibition occuring in AgNO3. Leaf discs derived from plants grown with AVG or AgNO3 were highly regenerative on shoot regeneration medium without ethylene inhibitor, but the presence of AgNO3 in the medium was inhibitory to regeneration of those derived from plants grown with AgNO3.

The presence of 1-10 μM aminoethoxyvinylglycine (AVG) or 5-30 μM AgNO3 markedly enhanced shoot regeneration from cotyledon and hypocotyl cultures of eight recalcitrant Brassica campestris and B. juncea genotypes tested. Expiants of B. campestris ssp. chinensis and ssp. parachinensis grown with a high AVG concentration (20 μM), regenerated poorly. All cytokinins tested were equally effective in promoting shoot formation, except that kinetin was inhibitory to shoot regeneration from hypocotyls of B. campestris ssp. pekinensis (cv. Wong Bok). Both AgNO3 and AVG had no effect on percent rooting and number of roots per rooted cutting of Wong Bok, White Sun and Leaf Heading, but AgNO3 was inhibitory to rooting of India Mustard. However, root elongation of all cuttings was markedly inhibited by AVG at concentrations of 5 and 10 μM.