How to determine the signal effect of something in certain process?

How to prove the function of chemicals as a signal in physiological process ?  It is a very common question in research of biology,  especial for molecular botany, rather than ecology. However, the logical and careful experiment design is necessary in science study. I like science for it is not expressive skill or  word play.   Today, I read a  traditional article on plant physiology, the same as the name of the published journal.  It is about common reed that I am interested in and have done some works on, so I found it.

 

Zhao L, Zhang F, Guo J, et al. 2004. Nitric Oxide Functions as a Signal in Salt Resistance in the Calluses from Two Ecotypes of Reed. Plant Physiology, 134: 849–857.

 

Calluses from two ecotypes of reed (Phragmites communis Trin.) plant (dune reed [DR] and swamp reed [SR]), which show different sensitivity to salinity, were used to study plant adaptations to salt stress.

Under 200 mm NaCl treatment, the sodium (Na) percentage decreased, but the calcium percentage and the potassium (K) to Na ratio increased in the DR callus, whereas an opposite changing pattern was observed in the SR callus.

Application of sodium nitroprusside (SNP), as a nitric oxide (NO) donor, revealed that NO affected element ratios in both DR and SR calluses in a concentration-dependent manner.

N
-nitro-l-arginine (an NO synthase inhibitor) and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxyde (a specific NO scavenger) counteracted NO effect by increasing the Na percentage, decreasing the calcium percentage and the K to Na ratio.

The increased activity of plasma membrane (PM) H
-ATPase caused by NaCl treatment in the DR callus was reversed by treatment with N
-nitro-l-arginine and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxyde.

Western-blot analysis demonstrated that NO stimulated the expression of PM H
-ATPase in both DR and SR calluses.

These results indicate that NO serves as a signal in inducing salt resistance by increasing the K to Na ratio, which is dependent on the increased PM H
-ATPase activity.

This structure of this paper is very concise. Except the first sentence describing the background and the last sentence drawing a conclusion, every sentence introduced one experiment and its results, providing various evidence for the last conclusion.