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Salinity (NaCl) stress impairs plant growth and inflicts severe crop losses. In roots, increasing extracellular NaCl causes Ca2+ influx to elevate cytosolic free Ca2+ ([Ca2+]cyt) as a second messenger for adaptive signaling. Amplification of the signal involves plasma membrane reduced nicotinamide adenine dinucleotide phosphate oxidase activation, with the resultant reactive oxygen species triggering Ca2+ influx. The genetic identities of the Ca2+-permeable channels involved in generating the [Ca2+]cyt signal are unknown. Potential candidates in the model plant Arabidopsis (Arabidopsis thaliana) include annexin1 (AtANN1). Here, luminescent detection of [Ca2+]cyt showed that AtANN1 responds to high extracellular NaCl by mediating reactive oxygen species-activated Ca2+ influx across the plasma membrane of root epidermal protoplasts. Electrophysiological analysis revealed that root epidermal plasma membrane Ca2+ influx currents activated by NaCl are absent from the Atann1 loss-of-function mutant. Both adaptive signaling and salt-responsive production of secondary roots are impaired in the loss-of-function mutant, thus identifying AtANN1 as a key component of root cell adaptation to salinity.

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NADPH oxidase activity is involved in plant adaptation and development. The reactive oxygen species sourced by NADPH oxidase activity may contribute to wall strength and protoplast volume adjustment. Root hair bulge apices of the NADPH oxidase mutant rhd2/Atrbohc were more robust than the kjk cellulose synthase mutant, but burst more readily than the wild type (WT). Root epidermal wall appeared impaired in rhd2/Atrbohc, as revealed by the number of protoplasts released by wall-degrading enzymes. Root hair bulges of rhd2/Atrbohc burst more than the WT when challenged in situ with hypo-osmotic low ionic strength medium. Inhibition of NADPH oxidase activity with diphenylene iodonium caused WT to phenocopy the rhd2/Atrbohc bursting in response to hypo-osmotic shock. This implicates RHD2/AtRBOHC in softening the cell wall to permit protoplast expansion. Overall, the results point to a role for RHD2/AtRBOHC in contributing to wall strength.

Damage can be signalled by extracellular ATP (eATP) using plasma membrane (PM) receptors to effect cytosolic free calcium ion ([Ca 2+ ] cyt ) increase as a second messenger. The downstream PM Ca 2+ channels remain enigmatic. Here, the Arabidopsis thaliana Ca 2+ channel subunit CYCLIC NUCLEOTIDE-GATED CHANNEL2 (CNGC2) was identified as a critical component linking eATP receptors to downstream [Ca 2+ ] cyt signalling in roots.Extracellular ATP-induced changes in single epidermal cell PM voltage and conductance were measured electrophysiologically, changes in root [Ca 2+ ] cyt were measured with aequorin, and root transcriptional changes were determined by quantitative real-time PCR. Two cngc2 loss-offunction mutants were used: cngc2-3 and defence not death1 (which expresses cytosolic aequorin).Extracellular ATP-induced transient depolarization of Arabidopsis root elongation zone epidermal PM voltage was Ca 2+ dependent, requiring CNGC2 but not CNGC4 (its channel cosubunit in immunity signalling). Activation of PM Ca 2+ influx currents also required CNGC2. The eATP-induced [Ca 2+ ] cyt increase and transcriptional response in cngc2 roots were significantly impaired.CYCLIC NUCLEOTIDE-GATED CHANNEL2 is required for eATP-induced epidermal Ca 2+ influx, causing depolarization leading to [Ca 2+ ] cyt increase and damage-related transcriptional response.