Seed longevity is crucial for agriculture and plant genetic diversity, but it is limited by cellular damage during storage. Seeds are protected against aging by cellular defenses and by structures such as the seed coat. We have screened an activation-tagging mutant collection of Arabidopsis (Arabidopsis thaliana) and selected four dominant mutants with improved seed longevity (isl1-1D to isl4-1D) under both natural and accelerated aging conditions. In the isl1-1D mutant, characterized in this work, overexpression of the transcription factor ARABIDOPSIS THALIANA HOMEOBOX25 (ATHB25; At5g65410) increases the expression of GIBBERELLIC ACID3-OXIDASE2, encoding a gibberellin (GA) biosynthetic enzyme, and the levels of GA₁ and GA₄ are higher (3.2- and 1.4-fold, respectively) in the mutant than in the wild type. The morphological and seed longevity phenotypes of the athb25-1D mutant were recapitulated in transgenic plants with moderate (4- to 6-fold) overexpression of ATHB25. Simultaneous knockdown of ATHB25, ATHB22, and ATHB31 expression decreases seed longevity, as does loss of ATHB25 and ATHB22 function in a double mutant line. Seeds from wild-type plants treated with GA and from a quintuple DELLA mutant (with constitutive GA signaling) are more tolerant to aging, providing additional evidence for a role of GA in seed longevity. A correlation was observed in several genotypes between seed longevity and mucilage formation at the seed surface, suggesting that GA may act by reinforcing the seed coat. This mechanism was supported by the observation of a maternal effect in reciprocal crosses between the wild type and the athb25-1D mutant.

The stress hormone abscisic acid (ABA) induces expression of defence genes in many organs, modulates ion homeostasis and metabolism in guard cells, and inhibits germination and seedling growth. Concerning the latter effect, several mutants of Arabidopsis thaliana with improved capability for H⁺ efflux (wat1-1D, overexpression of AKT1 and ost2-1D) are less sensitive to inhibition by ABA than the wild type. This suggested that ABA could inhibit H⁺ efflux (H⁺-ATPase) and induce cytosolic acidification as a mechanism of growth inhibition. Measurements to test this hypothesis could not be done in germinating seeds and we used roots as the most convenient system. ABA inhibited the root plasma-membrane H⁺-ATPase measured in vitro (ATP hydrolysis by isolated vesicles) and in vivo (H⁺ efflux from seedling roots). This inhibition involved the core ABA signalling elements: PYR/PYL/RCAR ABA receptors, ABA-inhibited protein phosphatases (HAB1), and ABA-activated protein kinases (SnRK2.2 and SnRK2.3). Electrophysiological measurements in root epidermal cells indicated that ABA, acting through the PYR/PYL/RCAR receptors, induced membrane hyperpolarization (due to K⁺ efflux through the GORK channel) and cytosolic acidification. This acidification was not observed in the wat1-1D mutant. The mechanism of inhibition of the H⁺-ATPase by ABA and its effects on cytosolic pH and membrane potential in roots were different from those in guard cells. ABA did not affect the in vivo phosphorylation level of the known activating site (penultimate threonine) of H⁺-ATPase in roots, and SnRK2.2 phosphorylated in vitro the C-terminal regulatory domain of H⁺-ATPase while the guard-cell kinase SnRK2.6/OST1 did not.

The increased selection pressure of the herbicide glyphosate has played a role in the evolution of glyphosate-resistance in weedy species, an issue that is becoming a threat to global agriculture. The molecular components involved in the cellular toxicity response to this herbicide at the expression level are still unidentified. In this study, we identify the protein kinase GCN2 as a cellular component that fosters the action of glyphosate in the model plant Arabidopsis thaliana. Comparative studies using wild-type and gcn2 knock-out mutant seedlings show that the molecular programme that the plant deploys after the treatment with the herbicide, is compromised in gcn2. Moreover, gcn2 adult plants show a lower inhibition of photosynthesis, and both seedlings and adult gcn2 plants accumulate less shikimic acid than wild-type after treatment with glyphosate. These results points to an unknown GCN2-dependent factor involved in the cascade of events triggered by glyphosate in plants. Data suggest either that the herbicide does not equally reach the target-enzyme in a gcn2 background, or that a decreased flux in the shikimate pathway in a gcn2 plants minimize the impact of enzyme inhibition.

Summary: Intracellular pH must be kept close to neutrality to be compatible with cellular functions, but the mechanisms of pH homeostasis and the responses to intracellular acidification are mostly unknown. In the plantArabidopsis thaliana, we found that intracellular acid stress generated by weak organic acids at normal external pH induces expression of several chaperone genes, includingROF2, which encodes a peptidyl‐prolylcis‐transisomerase of the FK506‐binding protein class. Loss of function ofROF2, and especially double mutation ofROF2and the closely related geneROF1, results in acid sensitivity. Over‐expression ofROF2confers tolerance to intracellular acidification by increasing proton extrusion from cells. The activation of the plasma membrane proton pump (H⁺‐ATPase) is indirect: over‐expression ofROF2activates K⁺uptake, causing depolarization of the plasma membrane, which activates the electrogenic H⁺pump. The depolarization ofROF2over‐expressing plants explains their tolerance to toxic cations such as lithium, norspermidine and hygromycin B, whose uptake is driven by the membrane potential. AsROF2induction and intracellular acidification are common consequences of many stresses, this mechanism of pH homeostasis may be of general importance for stress tolerance.