By PCR using a RAPD primer (5'-GCCTGCCTCACG-3'), we detected a DNA fragment which was preferentially amplified from mRNAs prepared from the salt-stressed leaves. We subsequently isolated the full length cDNA clone from a cDNA library prepared from the leaves of salt-stressed barley. The clone was 1283 bp in length and contained an open reading frame predicted to encode a polypeptide of 290 amino acids with the estimated molecular mass of 32.4 kDa. The protein has a sequence which is identical, except for one residue, to the N-terminal region of barley nuclease, expressed at the stage of seed germination (Fig. 21.1).3 Therefore, we concluded that the clone is the gene equivalent to barley nuclease (Nuclease I) and designated this gene as Bnuc1. Bnuc1 protein has a putative signal sequence of twenty-three amino acids at its N-terminus, but no other organelle targeting sequence. This may imply that the protein is secreted to the extracellular space.

The transcript of Bnuc1 gene in non-stressed leaves was difficult to detect by Northern blot analysis. However, the expression level was increased markedly under salt stress. It has been reported that barley nuclease is produced in the aleurone layer of seeds and is secreted from the layer in response to GA.4 These

Table 21.1. Summary of the clones

isolated by differential





RNA helicase




yeast SSF




observations suggest that barley nuclease degrades the nucleic acids stored in the endosperm to provide the embryo with nucle-otides or phosphates for heterotrophic growth. The Bnuc1 gene was not expressed in germinating seeds and the expression did not respond to GA. These result suggest that the Bnuc1 gene does not encode the previously reported barley nuclease, but encodes another member of the family. Actually, in Southern blot analysis using Bnuc1 cDNA as a probe, several bands were detected, suggesting that nuclease genes constitute a gene family on the barley genome.

It has been considered that nuclease I enzymes have two major roles, alteration of gene expression and remobilization of nutri-ents.5 Since Bnuc1 protein has a signal sequence at its N-terminus (Fig. 21.1), different from any nuclear-targeting sequence, Bnuc1 protein does not seem to be a nuclease associated with chromatin.6,7 So we postulate that Bnuc1 protein plays a role in remobilization of nutrients, such as nucleotides and phosphates, for the benefit of young fresh organs under salt stress. Accumulation of salt ions during salt stress is harmful to leaves and causes premature senescence. The aged leaves certainly yellowed first in this situation. Previously, it was reported that sodium and chloride ions accumulate preferentially in older leaves in barley8 and rice9,10 during salt stress, while glycinebetaine, one of the compatible solutes and osmoprotectants, accumu lates preferentially in the younger leaves in barley.8 These observations suggest that, in order to survive under high salinity conditions, barley plants have a preference for growth of the fresh tissues which have more sufficient metabolic and physiological activity, and limit the damage by salt to the older tissues which no longer have optimal cellular functions. To examine the spatial expression of Bnucl gene, we followed the kinetics of its transcript accumulation in individual leaves during salt treatment. Bnucl transcript started accumulating in the older leaves within 24 h. By contrast, the signal was hardly detectable in young leaves. Nuclease activity corresponding to the estimated molecular mass of Bnuc1 protein actually increased more in the older leaves under salt stress.

Gan and Amasinoll hypothesized that premature leaf senescence caused by salt stress has an obvious adaptive value, allowing the plant to complete its life cycle even under stressful conditions. They further claimed that plants have evolved mechanisms by which leaf senescence can be induced by limited water and nutrient availability to reallocate nutrients to reproductive organs and to eliminate water consumption by older, less productive leaves. Quite recently, it was also reported that nuclease activity with approximately the same molecular mass as the one we have found increases during leaf aging in barley.12 These observations suggest that during adaptation to salt stress, a nutrient recycling system similar to that occurring during natural leaf senescence is induced, and the barley nuclease I, Bnuc1, likely functions in the system.

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