|Grusak, Michael - Mike|
Submitted to: Journal of Experimental Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/1/1997
Publication Date: N/A
Citation: Interpretive Summary: The phloem transport system in plants is a network of living cells which are involved in the long-distance movement of sugars, mineral ions, and other nutrients between distant parts of the plant. It recently has been shown that this pathway also transports proteins and other large molecules; however, little is known about the exact nature or biological purpose for the movement of these large molecules. Our understanding in this area has been limited because it is difficult to collect the solution (known as phloem sap) within this pathway and because the concentration of proteins in the phloem sap is quite low. In this article, the authors describe the use of a sophisticated technique called matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and how it can be used to characterize and identify phloem sap proteins. Two species were studied, lupin and garden pea, and it was shown that a number of low- molecular-weight proteins were present in the phloem sap of each.Technique previously available were unable to identify phloem sap proteins in this weight range. Low-molecular-weight proteins may be part of a communication network operating in higher plants, especially between shoots and roots. The MALDI-TOF-MS technique will be a useful tool for investigating the physiological significance of these and other transported molecules.
Technical Abstract: The low-molecular-weight (LMW), low-abundance protein composition of lupin and pea phloem exudates was determined using matrix assisted laser desorption/ionization time-of-fight mass spectrometry (MALDI- TOF-MS). Phloem sap was collected from lupin inflorescence stalks and pods (using shallow incisions) or pea seedlings (by placing cut stems in an EDTA solution). Western blot analysis of phloem exudate proteins with either a polyclonal antibody raised against Ricinus communis sieve-tube exudate proteins or pea Rubisco antibody revealed that the collected exudates contained phloem sap, and that contamination with other plant fluids was negligible. Three matrix combinations were tested to assess their ability to facilitate protein ionization. Sinapinic acid in combination with trifluoroacetic acid yielded the cleanest mass spectra, and revealed an array of LMW proteins ranging from 2 to 10 kDa. For pea phloem exudate, the addition of protease inhibitors to the exudate collection solution prevented proteolysis of endogenous proteins; the inhibitors did not interfere with the detection of proteins. The sensitivity of this technique was sufficient to detect changes in LMW phloem peptides throughout plant development in lupin, or to detect differences in the phloem peptide composition of two genotypes of pea. Because only limited sample preparation is required, MALDI-TOF-MS is a useful technique for characterizing complex fluids such as phloem sap.