ABSCISIC ACID INSENSITIVE 4 coordinates eoplast formation to ensure acquisition of seed longevity during maturation in Medicago truncatula.

Seed longevity, the capacity to remain alive during dry storage, is pivotal to germination performance and is essential for preserving genetic diversity. It is acquired during late maturation concomitantly with seed degreening and the de-differentiation of chloroplasts into colorless, non-photosynthetic plastids, called eoplasts. As chlorophyll retention leads to poor seed performance upon sowing, these processes are important for seed vigor. However, how these processes are regulated and connected to the acquisition of seed longevity remains poorly understood. Here, we show that such a role is at least provided by ABSCISIC ACID INSENSITIVE 4 (ABI4) in the legume Medicago truncatula. Mature seeds of Mtabi4 mutants contained more chlorophyll than wild-type seeds and exhibited a 75% reduction in longevity and reduced dormancy. MtABI4 was necessary to stimulate eoplast formation, as evidenced by the significant delay in the dismantlement of photosystem II during the maturation of mutant seeds. Mtabi4 seeds also exhibited transcriptional deregulation of genes associated with retrograde signaling and transcriptional control of plastid-encoded genes. Longevity was restored when Mtabi4 seeds developed in darkness, suggesting that the shutdown of photosynthesis during maturation, rather than chlorophyll degradation per se, is a requisite for the acquisition of longevity. Indeed, the shelf life of stay green mutant seeds that retained chlorophyll was not affected. Thus, ABI4 plays a role in coordinating the dismantlement of chloroplasts during seed development to avoid damage that compromises the acquisition of seed longevity. Analysis of Mtabi4 Mtabi5 double mutants showed synergistic effects on chlorophyll retention and longevity, suggesting that they act via parallel pathways.

The seed-specific heat shock factor A9 regulates the depth of dormancy in Medicago truncatula seeds via ABA signalling.

During the later stages of seed maturation, two key adaptive traits are acquired that contribute to seed lifespan and dispersal, longevity and dormancy. The seed-specific heat shock transcription factor A9 is an important hub gene in the transcriptional network of late seed maturation. Here, we demonstrate that HSFA9 plays a role in thermotolerance rather than in ex situ seed conservation. Storage of hsfa9 seeds of Medicago truncatula and Arabidopsis had comparable lifespan at moderate storage relative humidity (RH), whereas at high RH, hsfa9 seeds lost their viability much faster than wild type seeds. Furthermore, we show that in M. truncatula, Mthsfa9 seeds acquired more dormancy during late maturation than wild type. Transient expression of MtHSFA9 in hairy roots and transcriptome analysis of Mthsfa9 Tnt1 insertion mutants identified a deregulation of genes involved in ABA biosynthesis, catabolism and signalling. Consistent with these results, Mthsfa9 seeds exhibited increased ABA levels and higher sensitivity to ABA. These data suggest that in legumes, HSFA9 acts as a negative regulator of the depth of seed dormancy during seed development via the modulation of hormonal balance.

Molecular and environmental factors regulating seed longevity.

Seed longevity is a central pivot of the preservation of biodiversity, being of main importance to face the challenges linked to global climate change and population growth. This complex, quantitative seed quality trait is acquired on the mother plant during the second part of seed development. Understanding what factors contribute to lifespan is one of the oldest and most challenging questions in plant biology. One of these challenges is to recognize that longevity depends on the storage conditions that are experimentally used because they determine the type and rate of deleterious conditions that lead to cell death and loss of viability. In this review, we will briefly review the different storage methods that accelerate the deteriorative reactions during storage and argue that a minimum amount of information is necessary to interpret the longevity data. Next, we will give an update on recent discoveries on the hormonal factors regulating longevity, both from the ABA signaling pathway but also other hormonal pathways. In addition, we will review the effect of both maternal and abiotic factors that influence longevity. In the last section of this review, we discuss the problems in unraveling cause-effect relationship between the time of death during storage and deteriorative reactions leading to seed ageing. We focus on the three major types of cellular damage, namely membrane permeability, lipid peroxidation and RNA integrity for which germination data on seed stored in dedicated seed banks for long period times are now available.

ABI5 Is a Regulator of Seed Maturation and Longevity in Legumes.

The preservation of our genetic resources and production of high-quality seeds depends on their ability to remain viable and vigorous during storage. In a quantitative trait locus analysis on seed longevity in Medicago truncatula, we identified the bZIP transcription factor ABSCISIC ACID INSENSITIVE5 (ABI5). Characterization of Mt-abi5 insertion mutant seeds revealed that both the acquisition of longevity and dormancy were severely impaired. Using transcriptomes of developing Mt-abi5 seeds, we created a gene coexpression network and revealed ABI5 as a regulator of gene modules with functions related to raffinose family oligosaccharide (RFO) metabolism, late embryogenesis abundant (LEA) proteins, and photosynthesis-associated nuclear genes (PhANGs). Lower RFO contents in Mt-abi5 seeds were linked to the regulation of SEED IMBIBITION PROTEIN1 Proteomic analysis confirmed that a set of LEA polypeptides was reduced in mature Mt-abi5 seeds, whereas the absence of repression of PhANG in mature Mt-abi5 seeds was accompanied by chlorophyll and carotenoid retention. This resulted in a stress response in Mt-abi5 seeds, evident from an increase in α-tocopherol and upregulation of genes related to programmed cell death and protein folding. Characterization of abi5 mutants in a second legume species, pea (Pisum sativum), confirmed a role for ABI5 in the regulation of longevity, seed degreening, and RFO accumulation, identifying ABI5 as a prominent regulator of late seed maturation in legumes.

An emerging picture of the seed desiccome: confirmed regulators and newcomers identified using transcriptome comparison.

Desiccation tolerance (DT) is the capacity to withstand total loss of cellular water. It is acquired during seed filling and lost just after germination. However, in many species, a germinated seed can regain DT under adverse conditions such as osmotic stress. The genes, proteins and metabolites that are required to establish this DT is referred to as the desiccome. It includes both a range of protective mechanisms and underlying regulatory pathways that remain poorly understood. As a first step toward the identification of the seed desiccome of Medicago truncatula, using updated microarrays we characterized the overlapping transcriptomes associated with acquisition of DT in developing seeds and the re-establishment of DT in germinated seeds using a polyethylene glycol treatment (-1.7 MPa). The resulting list contained 740 and 2829 transcripts whose levels, respectively, increased and decreased with DT. Fourty-eight transcription factors (TF) were identified including MtABI3, MtABI5 and many genes regulating flowering transition and cell identity. A promoter enrichment analysis revealed a strong over-representation of ABRE elements together with light-responsive cis-acting elements. In Mtabi5 Tnt1 insertion mutants, DT could no longer be re-established by an osmotic stress. Transcriptome analysis on Mtabi5 radicles during osmotic stress revealed that 13 and 15% of the up-regulated and down-regulated genes, respectively, are mis-regulated in the mutants and might be putative downstream targets of MtABI5 implicated in the re-establishment of DT. Likewise, transcriptome comparisons of the desiccation sensitive Mtabi3 mutants and hairy roots ectopically expressing MtABI3 revealed that 35 and 23% of the up-regulated and down-regulated genes are acting downstream of MtABI3. Our data suggest that ABI3 and ABI5 have complementary roles in DT. Whether DT evolved by co-opting existing pathways regulating flowering and cellular phase transition and cell identity is discussed.