Nitrate responses of Arabidopsis cho1 mutants

Abstract

We reported a loss-of-function of an Arabidopsis double AP2 transcription factor CHOTTO1 (CHO1) gene results in the altered responses to high concentrations of nitrate (∼50 mM). Nitrate up to 10 mM promotes growth of the wildtype seedling, but inhibits it under higher concentrations. The cho1 seedlings responded to nitrate up to 10 mM similarly to the wildtype, but the inhibitory effect of excess nitrate is less prominent in the mutants. This phenotype is restricted to the cotyledons, and growth of the hypocotyl and roots of the cho1 mutants is inhibited by excess nitrate. The cho1 mutations caused the upregulation of two nitrate transporter genes, AtNRT1.4 and At1g52190. Altered nitrate distribution and storage may explain the phenotypes of the cho1 mutants.

CHO1 is a Growth Repressor Downstream of ABI4

Abscisic acid (ABA) is a plant hormone that regulates germination and seedling growth.¹ The cho1 mutants were isolated by their ability to germinate and grow in the presence of (−)-R-ABA, an ABA analog.² The majority of (−)-R-ABA-insensitive mutants were allelic to the abi4 mutants, whereas the collection of (+)-S-ABA-insensitive mutants included abi3, abi4 and abi5 in a similar proportion (data not shown). This suggests that (−)-R-ABA action requires the ABI4 prominently. Four (−)-R-ABA-insensitive mutations fell into two new loci, designated as chotto1 (cho1) and chotto2 (cho2). Map-based cloning revealed that CHO1 is identical to the AINTEGUMENTALIKE5 (AIL5) that encodes a double AP2 transcription factor.³ AIL5 was reported as a homologue of AINTEGUMENTA (ANT) that plays a key role in the development of ovules and flowers and that is thought to function in the regulation of stem cell identity and mitotic activity.^4,5 Overexpression of AIL5/CHO1 causes an increase in the size of flowers, as is the case of the ANT overexpressor.⁴ Moreover, AIL5/CHO1 is shown to bind in vitro to the ANT-binding sequence,⁶ suggestive of its role as a growth regulator.

Genetic analysis indicated that the cho1 mutations did not remarkably affect the phenotypes of abi4 mutants, but enhanced the abi5 phenotypes.³ In addition, cho1 mutants displayed glucose-insensitive growth in a similar manner to the abi4 mutants, whereas abi5 mutants are sensitive to glucose.³ CHO1 is expressed in both developing and imbibed seeds and its expression is induced most prominently after imbibition of dry seeds. This induction is dependent on ABI4, because the abi4 mutants failed to induce CHO1.³ Collectively, we concluded that CHO1 is a growth repressor that acts downstream of ABI4.

Nitrate Response of cho1 Mutants

The phenotypes of the cho1 mutants resembled those of the abi4 mutants, except for its response to excess supply of nitrate. Nitrate (1–10 mM) plays a positive role in seedling growth: it promotes the expansion and greening of cotyledons as well as root growth. However, supply of excess nitrate inhibits the expansion of cotyledons, elongation of hypocotyl and root growth. On 50 mM KNO₃, germinated wildtype cotyledons were green, but they became bleached after a prolonged period of incubation. This contrasts to the growth inhibition by excess glucose, which blocks greening of cotyledons. Excess supply of NH₄NO₃, but not of NH₄Cl, KCl or tryptone, showed the similar effect. This suggests that this inhibition is cased by nitrate itself. It is also notable that excess supply of KNO₃ inhibited growth when applied alone (or with buffer), but Murashige & Skoog medium (that includes 18.8 mM KNO₃ + 20.6 mM NH₄NO₃) did not cause such inhibition. This suggests that other nutrients and minerals complemented the inhibitory effect of nitrate.

Altered nitrate responses of the cho1 mutants can be seen only when excess nitrate is supplied. The cho1 seedlings were comparable to the wildtype on agarose plates supplemented with less than 10 mM KNO₃. The growth promotion by nitrate in cho1 seedlings looks similar to the wildtype: it can be visible above 1 mM and is optimal around 10 mM. However, the cho1 seedlings grown on 50 mM KNO₃ are still able to expand the cotyledons and to maintain chlorophylls after a prolonged period of incubation. In contrast to the “healthy looking” cotyledons, hypocotyl elongation and root growth of the cho1 seedlings were inhibited similarly to the wildtype. The effective concentration range of nitrate on the cho1 phenotype is different from that on the nitrate induction of an ABA catabolism gene in which moderate concentrations (10 mM or less) of KNO₃ decrease the ABA content.⁷

The CHO1 expression was high in 1-day-imbibed seeds, and was reduced thereafter. The β-glucuronidase (GUS) reporter driven by a 2.2 kb CHO1 promoter (pCHO1::GUS) showed its expression was low and seen in the vasculature of roots on the control medium (Fig. 1A). On the other hand, pCHO1::GUS seedlings on excess nitrate showed the intense staining at the veins of the cotyledons, the junction between hypocotyl and root, and root tips (Fig. 1B). Also, a weak staining was observed over the cotyledons, which was not seen in the pCHO1::GUS seedlings grown on the control medium (Fig. 1A and B).

Figure 1.

Responses to excess nitrate. Expression patterns of pCHO1:GUS in 3-day-old seedlings grown on agarose media (A) or on agarose media supplemented with 40 mM KNO3 (B). (C) Relative expression levels of AtNRT1.4 and At1g52190 in the wildtype (WT) and cho1-3 (cho1) mutant. Microarray expression data on dry seeds and 2-day seedlings were obtained from Yamagishi et al. (2009).³ Exp. 1 and Exp. 2 indicate two independent experiments.

Nitrate plays a role as both nutrient and signal.⁸ A low concentration of nitrate (<1 µM) can induce expression of a large number of genes.⁹ The growth inhibition by excess nitrate on wildtype seedlings, which is associated with the cho1 phenotype, requires ∼50 mM. The simplest explanation is that this inhibition is due to the imbalance of nitrate metabolism or its transport. Microarray expression data on wildtype and cho1 mutant suggests this may be the case. Plant possesses two types of structurally distinct nitrate transporters, AtNRT1 and AtNRT2 families.¹⁰ The Arabidopsis genome contains 53 AtNRT1 genes that compose a superfamily of nitrate/peptide transporters and 7 AtNRT2 genes that encode high-affinity nitrate transporters.^11,12 Microarray data showed that two AtNRT1-type nitrate transporter genes, AtNRT1;4 and At1g52190 were upregulated in the cho1 seedlings (Fig. 1C). Other AtNRT1 genes and AtNRT2 genes show no remarkable changes in expression patterns in the mutant relative to the wildtype. AtNRT1;4 is expressed in the petiole and mid vein of the shoots and is responsible for the distribution and storage of nitrate.¹³ It is notable that both AtNRT1;4 and At1g52190 genes are abundantly expressed in shoots, which is correlated with the shoot-specific phenotypes of the cho1 mutants. The upregulation of these genes may explain the altered nitrate responses of the cho1 mutants. Nitrate can be moved, stored, metabolized and trigger the biological processes. Chasing the function of nitrate in plants will provide us a multidimensional picture of the plant life.

Abbreviations

ABA

abscisic acid

ABI

abscisic acid-insensitive

Addendum to: Yamagishi K, Tatematsu K, Yano R, Preston J, Kitamura S, Takahashi H, et al. CHOTTO1, a double AP2 domain protein of Arabidopsis thaliana, regulates germination and seedling growth under excess supply of glucose and nitrate. Plant Cell Physiol. 2009;50:330–340. doi: 10.1093/pcp/pcn201.