Arabidopsis mutants of AtABCG22, an ABC transporter gene, increase water transpiration and drought susceptibility

In plants, water vapour is released into the atmosphere through stomata in a process called transpiration. Abscisic acid (ABA) is a key phytohormone that facilitates stomatal closure through its action on guard cells. Recently, ATP-binding cassette (ABC) transporter genes, AtABCG25 and AtABCG40, were shown to be involved in ABA transport and responses. However, the functions of many other AtABCG family genes are still unknown. Here, we identified another ABCG gene (AtABCG22) that is required for stomatal regulation in Arabidopsis. The atabcg22 mutant plants had lower leaf temperatures and increased water loss, implying elevated transpiration through an influence on stomatal regulation. We also found that atabcg22 plants were more suspectible to drought stress than wild-type plants. AtABCG22 was expressed in aerial organs, mainly guard cells, in which the gene expression pattern was consistent with the mutant phenotypes. Using double mutants, we investigated the genetic relationships between the mutations. The atabcg22 mutation further increased the water loss of srk2e/ost1 mutants, which were defective in ABA signalling in guard cells. Also, the atabcg22 mutation enhanced the phenotype of nced3 mutants, which were defective in ABA biosynthesis. Accordingly, the additive roles of AtABCG22 functions in ABA signalling and ABA biosynthesis are discussed.     

Phloem unloading via the apoplastic pathway is essential for shoot distribution of root-synthesized cytokinins

Root-synthesized cytokinins are transported to the shoot and regulate the growth, development, and stress responses of aerial tissues. Previous studies have demonstrated that Arabidopsis (Arabidopsis thaliana) ATP binding cassette (ABC) transporter G family member 14 (AtABCG14) participates in xylem loading of root-synthesized cytokinins. However, the mechanism by which these root-derived cytokinins are distributed in the shoot remains unclear. Here, we revealed that AtABCG14-mediated phloem unloading through the apoplastic pathway is required for the appropriate shoot distribution of root-synthesized cytokinins in Arabidopsis. Wild-type rootstocks grafted to atabcg14 scions successfully restored trans-zeatin xylem loading. However, only low levels of root-synthesized cytokinins and induced shoot signaling were rescued. Reciprocal grafting and tissue-specific genetic complementation demonstrated that AtABCG14 disruption in the shoot considerably increased the retention of root-synthesized cytokinins in the phloem and substantially impaired their distribution in the leaf apoplast. The translocation of root-synthesized cytokinins from the xylem to the phloem and the subsequent unloading from the phloem is required for the shoot distribution and long-distance shootward transport of root-synthesized cytokinins. This study revealed a mechanism by which the phloem regulates systemic signaling of xylem-mediated transport of root-synthesized cytokinins from the root to the shoot.

Phloem unloading via the apoplastic pathway is essential for shoot distribution and long-distance translocation of root-synthesized cytokinins from the root to the shoot through the xylem.     

Arabidopsis mutant of AtABCG26, an ABC transporter gene, is defective in pollen maturation

In plants, pollen is the male gametophyte that is generated from microspores, which are haploid cells produced after meiosis of diploid pollen mother cells in floral anthers. In normal maturation, microspores interact with the tapetum, which consists of one layer of metabolically active cells enclosing the locule in anthers. The tapetum plays several important roles in the maturation of microspores. ATP-binding cassette (ABC) transporters are a highly conserved protein super-family that uses the energy released in ATP hydrolysis to transport substrates. The ABC transporter gene family is more diverse in plants than in animals. Previously, we reported that an Arabidopsis half-size type ABC transporter gene, COF1/AtWBC11/AtABCG11, is involved in lipid transport for the construction of cuticle layers and pollen coats in normal organ formation, as compared to CER5/AtWBC12/AtABCG12. However, physiological functions of most other ABCG members are unknown. Here, we identified another family gene, AtABCG26, which is required for pollen development in Arabidopsis. An AtABCG26 mutant developed very few pollen grains, resulting in a male-sterile phenotype. By investigating microspore and pollen development in this mutant, we observed that there was a slight abnormality in tetrad morphology prior to the formation of haploid microspores. At a later stage, we could not detect exine deposition on the microspore surface. During pollen maturation, many grains in the mutant anthers got aborted, and surviving grains were found to be defective in mitosis. Transmission of the mutant allele through male gametophytes appeared to be normal in genetic transmission analysis, supporting the view that the pollen function was disturbed by sporophytic defects in the AtABCG26 mutant. AtABCG26 can be expected to be involved in the transport of substrates such as sporopollenin monomers from tapetum to microspores, which both are plant-specific structures critical to pollen development.     

Nitrate does not compete with abscisic acid as a substrate of AtNPF4.6/NRT1.2/AIT1 in Arabidopsis

We identified a member of the Arabidopsis NRT1/PTR FAMILY (NPF), AtNPF4.6, as an abscisic acid (ABA) transporter, AIT1. AtNPF4.6 was originally characterized as a low-affinity nitrate transporter NRT1.2. We hypothesized that the competition between nitrate and ABA as substrates for AtNPF4.6 might be involved in the interactions between nitrate and ABA signaling. However, the ABA transport activity of AtNPF4.6 was not inhibited by an excess amount of nitrate. In addition, the npf4.6 mutant was less sensitive to ABA than the wild type during germination irrespective of nitrate concentrations in the media. Furthermore, nitrate promoted germination of both wild type and npf4.6 in the presence of ABA. These results do not support the idea of a physiological linkage between nitrate and ABA signals through AtNPF4.6.     

The Arabidopsis A4 subfamily of lectin receptor kinases negatively regulates abscisic acid response in seed germination

Abscisic acid (ABA) is an important plant hormone for a wide array of growth and developmental processes and stress responses, but the mechanism of ABA signal perception on the plasma membrane remains to be dissected. A previous GeneChip analysis revealed that a member of the A4 subfamily of lectin receptor kinases (LecRKs) of Arabidopsis (Arabidopsis thaliana), At5g01540 (designated LecRKA4.1), is up-regulated in response to a low dose of ABA in the rop10-1 background. Here, we present functional evidence to support its role in ABA response. LecRKA4.1 is expressed in seeds and leaves but not in roots, and the protein is localized to the plasma membrane. A T-DNA knockout mutant, lecrka4.1-1, slightly enhanced ABA inhibition of seed germination. Interestingly, LecRKA4.1 is adjacent to two other members of the A4 subfamily of LecRK genes, At5g01550 (LecRKA4.2) and At5g01560 (LecRKA4.3). We found that loss-of-function mutants of LecRKA4.2 and LecRKA4.3 exhibited similarly weak enhancement of ABA response in seed germination inhibition. Furthermore, LecRKA4.2 suppression by RNA interference in lecrka4.1-1 showed stronger ABA inhibition of seed germination than lecrka4.1-1, while the response to gibberellic acid was not affected in lecrka4.1-1 and lecrka4.1-1; LecRKA4.2 (RNAi) lines. Expression studies, together with network-based analysis, suggest that LecRKA4.1 and LecRKA4.2 regulate some of the ABA-responsive genes. Taken together, our results demonstrate that the A4 subfamily of LecRKs has a redundant function in the negative regulation of ABA response in seed germination.     

Nitric oxide suppresses the inhibitory effect of abscisic acid on seed germination by S-nitrosylation of SnRK2 proteins

Nitric oxide (NO) plays important roles in plant development, and biotic and abiotic stress responses. In a recent study, we showed that endogenous NO negatively regulates abscisic acid (ABA) signaling in guard cells by inhibiting sucrose nonfermenting 1 (SNF1)-related protein kinase 2.6 (SnRK2.6)/open stomata 1(OST1) through S-nitrosylation. Application of NO breaks seed dormancy and alleviates the inhibitory effect of ABA on seed germination and early seedling growth, but it is unclear how NO functions at the stages of seed germination and early seedling development. Here, we show that like SnRK2.6, SnRK2.2 can be inactivated by S-nitrosoglutathione (GSNO) treatment through S-nitrosylation. SnRK2.2 and the closely related SnRK2.3 are known to play redundant roles in ABA inhibition of seed germination in Arabidopsis. We found that treatment with the NO donor SNP phenocopies the snrk2.2snrk2.3 double mutant in conferring ABA insensitivity at the stages of seed germination and early seedling growth. Our results suggest that NO negatively regulates ABA signaling in germination and early seedling growth through S-nitrosylation of SnRK2.2 and SnRK2.3.     

Arabidopsis ROP9 and ROP10 GTPases differentially regulate auxin and ABA responses

Auxin and abscisic acid (ABA) are major plant hormones that act together to modulate numerous aspects of plant growth and development, including seed germination, primary root elongation, and lateral root formation. In this study, we analyzed the loss-of-function mutants of two closely related ROP (Rho of plants) GTPases, ROP9 and ROP10, and found that these ROP GTPases differentially regulate the auxin and ABA responses. rop9 and rop10 mutations enhanced the ABA-induced suppression of seed germination, primary root growth, and lateral root formation and the expression of ABA-responsive genes, whereas rop9 but not rop10 suppressed auxin-induced root phenotypes and auxin-responsive gene expression. These results suggest that both ROP9 and ROP10 function as negative regulators of ABA signaling, and that ROP9, but not ROP10, functions as a positive regulator of auxin signaling. Previously, ROPinteractive CRIB motif-containing protein 1 (RIC1) was reported to participate in auxin and ABA responses, and to have a similar effect as ROP9 and ROP10 on gene expression, root development, and seed germination. Because RIC proteins mediate ROP GTPase signaling, our results suggest that ROP9 and ROP10 GTPases function upstream of RIC1 in auxin- and ABA-regulated root development and seed germination.     

MOTHER OF FT AND TFL1 regulates seed germination and fertility relevant to the brassinosteroid signaling pathway

Brassinosteroids (BRs) are a family of plant steroid hormones that play diverse roles in many aspects of plant growth and development. For example, BRs promote seed germination by counteracting the inhibitory effect of ABA and regulate plant reproductive development, thus affecting seed yield. We have recently reported that MOTHER OF FT AND TFL1 (MFT) regulates seed germination through a negative feedback loop modulating ABA signaling in Arabidopsis. Here, we show that MFT function is also relevant to the BR signaling pathway. In mft loss-of-function mutants, the application of BR could not fully antagonize the inhibitory effect of exogenous ABA on seed germination, suggesting that BR promotes seed germination against ABA partly through MFT. In addition, mft enhances the low-fertility phenotype of det2 in which BR biosynthesis is blocked. This phenotype, together with the observation that MFT is expressed in gametophytes and developing seeds, suggests that MFT and BR play redundant roles in regulating fertility. Therefore, these results suggest that MFT affects seed germination and fertility relevant to the BR signaling pathway.Key words: Arabidopsis, brassinosteroid, abscisic acid, fertility, seed germinationPlant hormones exert profound effects on many fundamental processes during plant growth and development. With respect to seed development and germination, it has long been known that abscisic acid (ABA) and gibberellin (GA) are two major types of phytohormones that play antagonistic roles in regulating these events. Not until recently, another group of phytohormones, namely brassinosteroids (BRs), has also been found to counteract the inhibitory effect of ABA on seed germination.^1,2 In addition, BR has been suggested to act in parallel with GA to promote cell elongation and germination.^1,3,4BRs are a class of polyhydroxysteroids that are found in a wide variety of plant species.⁵ They can be detected in almost every plant tissue, with the highest abundance in the pollen and seeds.⁶ The most active component in the family of BRs is 24-epibrassinolide (BL), which is capable of activating BR signaling.⁶ In Arabidopsis, when the early steps of BR biosynthesis are blocked, the resulting defects include reduced male fertility under normal growth conditions^7,8 and decreased germination percentage in the presence of exogenous ABA.¹ Thus, BR plays an indispensible role in the control of seed development and also contributes to the regulation of seed germination.We have previously reported that MOTHER OF FT AND TFL1 (MFT) responds to both ABA and GA signals to regulate seed germination.⁹ Here we show that MFT functions in regulating seed germination and fertility, which is also relevant to the BR signaling pathway. Thus, MFT seems to function specifically in seeds in response to various phytohormones.