Phytoglobins in the nuclei, cytoplasm and chloroplasts modulate nitric oxide signaling and interact with abscisic acid

Abstract

Summary Symbiotic hemoglobins provide O2 to N2‐fixing bacteria within legume nodules, but the functions of non‐symbiotic hemoglobins or phytoglobins (Glbs) are much less defined. Immunolabeling combined with confocal microscopy of the Glbs tagged at the C‐terminus with green fluorescent protein was used to determine their subcellular localizations in Arabidopsis and Lotus japonicus. Recombinant proteins were used to examine nitric oxide (NO) scavenging in vitro and transgenic plants to show S‐nitrosylation and other in vivo interactions with NO and abscisic acid (ABA) responses. We found that Glbs occur in the nuclei, chloroplasts and amyloplasts of both model plants, and also in the cytoplasm of Arabidopsis cells. The proteins show similar NO dioxygenase activities in vitro, are nitrosylated in Cys residues in vivo, and scavenge NO in the stomatal cells. The Cys/Ser mutation does not affect NO dioxygenase activity, and S‐nitrosylation does not significantly consume NO. We demonstrate an interaction between Glbs and ABA on several grounds: Glb1 and Glb2 scavenge NO produced in stomatal guard cells following ABA supply; plants overexpressing Glb1 show higher constitutive expression of the ABA responsive genes Responsive to ABA (RAB18), Responsive to Dehydration (RD29A) and Highly ABA‐Induced 2 (HAI2), and are more tolerant to dehydration; and ABA strongly upregulates class 1 Glbs. We conclude that Glbs modulate NO and interact with ABA in crucial physiological processes such as the plant's response to dessication.

Plant hemoglobins: a journey from unicellular green algae to vascular plants

“…Physiological work using transgenic plants with altered concentrations of Glbs has shown that these proteins play an important role in plant development and morphogenesis, and largely supports the theory that they do so by modulating NO concentrations (Hill, ; Hebelstrup et al , ). Glbs may act through NO modulation to affect the signaling pathways of hormones including auxins (Elhiti et al , ), cytokinins (Ross et al , ; Wang et al , ), salicylate (Mur et al , ), jasmonates (Mur et al , ; Mira et al , ), ethylene (Mur et al , ; Kapoor et al , ; Hartman et al , ) and abscisic acid (ABA; Kapoor et al , ; Rubio et al , ). Two exemplary studies, concerning the response of Arabidopsis to biotic and abiotic stress, will illustrate the triple crosstalk between Glbs, hormones and NO.…”

“…There is solid support for the NO scavenging reaction of Glbs in vivo . In Arabidopsis the reaction can be carried out by both Atha1 and Atha2 (Hebelstrup & Jensen, ; Kuruthukulangarakoola et al , ) and has been observed in stomatal cells, where a crosstalk between Glbs, NO and ABA may occur (Rubio et al , ). Recent work with Arabidopsis and barley has shown that NO can be ‘fixed’ into nitrogen metabolism in plants overexpressing, respectively, Atha1 or Atha2 (Kuruthukulangarakoola et al , ) and Hvul1‐1 (Zhang et al , ).…”

“…Other studies conclude that Glbs are preferentially localized to the nuclei and cytoplasm. This is the case in alfalfa ( Medicago sativa ) suspension cells (Seregélyes et al , ), Arabidopsis roots and leaves (Hebelstrup et al , ), and L. japonicus roots, leaves and nodules (Sainz et al , ; Rubio et al , ). Glbs are absent from mitochondria, but there are indications that Glbs occur in chloroplasts.…”

“…Also, Bvul1‐1 contains a chloroplast transit peptide (Leiva‐Eriksson et al , ). Very recent work using quantitative immunogold labeling of overexpressing and silenced/knockout lines of Arabidopsis, combined with confocal microscopy of Glbs tagged with green fluorescent protein, has shown the presence of several Glbs in the chloroplasts and amyloplasts (Rubio et al , ). These Glbs might play a role associated with photosynthesis and/or NO homeostasis because chloroplasts are a source of NO within the cell (Jasid et al , ).…”

Recommendations on terminology and experimental best practice associated with plant nitric oxide research

“…Leghemoglobins (Lb) and symbiotic hemoglobins (sym‐Hb) are present in either legume species or actinorhizal/nonlegume symbiotic nodules, respectively. The presence of hemoglobin‐like proteins in organs not associated with symbiotic interactions led to the term nonsymbiotic hemoglobins (Hill, ; Rubio et al ., ). These nonsymbiotic hemoglobins are thought to function as key scavengers of NO under various environmental and stress conditions (Hill, ).…”