The Cytosolic Ca2+ Concentration Gradient of Sinapis alba Root Hairs as Revealed by Ca2+-Selective Microelectrode Tests and Fura-Dextran Ratio Imaging

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Abstract
Using Ca2+-selective microelectrodes and fura 2-dextran ratio imaging, the cytosolic free [Ca2+] was measured in Sinapis alba root hair cells. Both methods yielded comparable results, i.e. values between 158 to 251 nM for the basal [Ca2+] of the cells and an elevated [Ca2+] of 446 to 707 nM in the tip region. The zone of elevated [Ca2+] reaches 40 to 60 [mu]m into the cell and is congruent with the region of inwardly directed Ca2+ net currents measured with an external Ca2+- selective vibrating electrode. The channel-blocker La3+ eliminates these currents, stops growth, and almost completely eliminates the cytosolic [Ca2+] gradient without affecting the basal level of the ion. Growth is also inhibited by pressure-injected dibromo-1,2-bis(o-aminophenoxy)ethane-N,N,N[prime],N[prime]-tetraacetic acid, which causes a decrease in the [Ca2+] in the tip in a concentration-dependent manner. Indole-3-acetic acid, used as a model stimulus, decreases cytosolic free [Ca2+] by 0.2 to 0.3 pCa units in the tip, but only by about 0.1 pCa unit in the shank. Nongrowing root hairs may or may not display a [Ca2+] gradient, but still reversibly respond to external stimuli such as La3+, Ca2+, or indole-3-acetic acid with changes in cytosolic free [Ca2+]. During short time periods, dicyclohexylcarbodiimide inhibition of the plasma membrane H+-ATPase, which stops growth, does not abolish the [Ca2+] gradient, nor does it change significantly the basal [Ca2+] level. We conclude that the cytosolic [Ca2+] gradient and an elevated [Ca2+] in the tip, as in other tip-growing cells, is essential for tip growth in root hairs; however, its presence does not indicate growth under all circumstances. We argue that with respect to Ca2+, tip growth regulation and responses to external signals may not interfere with each other. Finally, we suggest that the combination of the methods applied adds considerably to our understanding of the role of cytosolic free [Ca2+] in signal transduction and cellular growth.