Keting Chen

Dynamics of the antioxidant system during seed osmopriming, post-priming germination, and seedling establishment in Spinach (Spinacia oleracea)

Osmopriming is a pre-sowing treatment that improves seed germination performance and stress tolerance. To understand osmopriming physiology, and its association with post-priming stress tolerance, we investigated the antioxidant system dynamics during three stages: during osmopriming, post-priming germination, and seedling establishment. Spinach seeds (Spinacia oleracea L. cv. Bloomsdale) were primed with -0.6 MPa PEG at 15°C for 8 d, and dried at room temperature for 2 d. Unprimed and primed germinating seeds/seedlings were subjected to a chilling and desiccation stresses. Seed/seedling samples were collected for antioxidant assays and germination performance and stress tolerance were evaluated. Our data indicate that: (1) during osmopriming the transition of seeds from dry to germinating state represses the antioxidant pathways (residing in dry seeds) that involve CAT and SOD enzymes but stimulates another pathway (only detectable in imbibed seeds) involving APX; (2) a renewal of antioxidant system, possibly required by seedling establishment, occurs after roughly 5 d of germination; (3) osmopriming strengthens the antioxidant system and increases seed germination potential, resulting in an increased stress tolerance in germinating seeds. Osmopriming-mediated promotive effect on stress tolerance, however, may diminish in relatively older (e.g. ~5-week) seedlings.

Selection of Reference Genes for Normalizing Gene Expression During Seed Priming and Germination Using qPCR in Zea mays and Spinacia oleracea

Quantitative real-time RT-PCR (qPCR) has been widely used to investigate gene expression during seed germination, a process involving seed transition from dry/physiologically inactive to hydrated/active state. This transition may result in altered expression of many housekeeping genes (HKGs), conventionally used as internal controls, thereby posing a challenge about selection of HKGs in such scenarios. The objectives of this study included identifying valid reference genes for seed priming and germination studies, both of which involve the transition of seed hydration status, and assessing whether or not findings derived from the “seed model” used in this study would also be applicable to other plant species. Eight commonly used HKGs were evaluated in maize seeds during hydropriming and germination. Using Bestkeeper, geNorm, and NormFinder, we provided a rank of stability for these HKGs. Actdf, UBQ, βtub, 18S, Act, and GAPDH were adjudged as valid internal controls by geNorm and NormFinder. Under the second objective, we conducted a case study with spinach seeds collected during osmopriming and germination. Our results indicate that the conclusions derived from maize were applicable to spinach as well, in that 18S exhibited greater expression stability than GAPDH in osmoprimed and germinated seeds; this held true even under stress conditions. While both of these genes were rejected by BestKeeper, we found that 18S exhibited stable expression when “dry” and “hydrated” seeds were analyzed as separate data sets. Although this approach precludes the comparison between “hydrated” and “dry” seeds, it still provides effective comparison among samples of same hydration status.

Aquaporin expression during seed osmopriming and post-priming germination in spinach

Aquaporins (AQPs) are proteinaceous channels known to regulate transmembrane water transport, and therefore may be important component of imbibition during osmopriming and germination. To explore the association between AQPs and osmopriming-led enhanced germination performance, we studied the expression patterns of four spinach (Spinacia oleracea) AQP coding genes (SoPIP1;1, SoPIP1;2, SoPIP2;1, and SoδTIP) during osmopriming and subsequent germination under optimal conditions, chilling and drought. All these genes were up-regulated within 2–4 d of priming (phase II-imbibition). We hypothesize such up-regulation to facilitate the pressure potential-driven cell expansion and increase germination potential of primed seeds. Our data during post-priming germination suggest that SoPIP1;1 and SoδTIP were more closely associated with enhanced germination performance. In general, all AQPs were downregulated under chilling and drought. However, under chilling, SoPIP2;1 was expressed at relatively higher level in primed seeds that also exhibited greater chilling tolerance, while SoPIP1;2 and SoδTIP exhibited opposite pattern. Similarly, SoPIP1;1, SoPIP2;1, and SoδTIP exhibited higher expression in primed seeds that also had greater drought tolerance.

Understanding the cellular mechanism of recovery from freeze–thaw injury in spinach: possible role of aquaporins, heat shock proteins, dehydrin and antioxidant system

Recovery from reversible freeze-thaw injury in plants is a critical component of ultimate frost survival. However, little is known about this aspect at the cellular level. To explore possible cellular mechanism(s) for post-thaw recovery (REC), we used Spinacia oleracea L. cv. Bloomsdale leaves to first determine the reversible freeze-thaw injury point. Freeze (-4.5°C)-thaw-injured tissues (32% injury vs <3% in unfrozen control) fully recovered during post-thaw, as assessed by an ion leakage-based method. Our data indicate that photosystem II efficiency (Fv/Fm) was compromised in injured tissues but recovered during post-thaw. Similarly, the reactive oxygen species (O2 (•-) and H2 O2 ) accumulated in injured tissues but dissipated during recovery, paralleled by the repression and restoration, respectively, of activities of antioxidant enzymes, superoxide dismutase (SOD) (EC. 1.14.1.1), and catalase (CAT) (EC.1.11.1.6) and ascorbate peroxidase (APX) (EC.1.11.1.11). Restoration of CAT and APX activities during recovery was slower than SOD, concomitant with a slower depletion of H2 O2 compared to O2 (•-) . A hypothesis was also tested that the REC is accompanied by changes in the expression of water channels [aquaporines (AQPs)] likely needed for re-absorption of thawed extracellular water. Indeed, the expression of two spinach AQPs, SoPIP2;1 and SoδTIP, was downregulated in injured tissues and restored during recovery. Additionally, a notion that molecular chaperones [heat shock protein of 70 kDa (HSP70s)] and putative membrane stabilizers [dehydrins (DHNs)] are recruited during recovery to restore cellular homeostasis was also tested. We noted that, after an initial repression in injured tissues, the expression of three HSP70s (cytosolic, endoplasmic reticulum and mitochondrial) and a spinach DHN (CAP85) was significantly restored during the REC.

Is expression of aquaporins (plasma membrane intrinsic protein 2s, PIP2s) associated with thermonasty (leaf-curling) in Rhododendron?

It is postulated that leaf thermonasty (leaf curling) in rhododendrons under sub-freezing temperatures is caused by water redistribution due to extracellular freezing. We hypothesize that aquaporins (AQPs), the transmembrane water-channels, may be involved in regulating water redistribution and thus leaf curling. Our experimental system includes two Rhododendron species with contrasting leaf curling behavior whereby it was observed in R. catawbiense but not in R. ponticum. We compared leaf movements and the expression of two AQPs, i.e. R. catawbiense/ponticum plasma-membrane intrinsic protein 2 (Rc/RpPIP2;1 and Rc/RpPIP2;2), in the two species under freezing-rewarming and dehydration-rehydration cycles. To determine the relationship between extracellular freezing and leaf-curling, we monitored leaf-curling in R. catawbiense with or without controlled ice-nucleation. Our data indicate that extracellular freezing may be required for leaf curling. Moreover, in both species, PIP2s were up-regulated at temperatures that fell in ice-nucleation temperature range. Such up-regulation could be associated with the bulk-water efflux caused by extracellular freezing. When leaves were frozen beyond the ice-nucleation temperature range, PIP2s were continuously down-regulated in R. catawbiense along with the progressive leaf curling, as also observed for RcPIP2;2 in dehydrated leaves; as leaves uncurled during re-warming/rehydration, RcPIP2 expression was restored. On the other hand, R. ponticum, a non-curling species, exhibited substantial up-regulation of RpPIP2s during freezing/dehydration. Taken together, our data suggest that RcPIP2 down-regulation was associated with leaf curling. Moreover, the contrasting PIP2 expression patterns combined with leaf behavior of R. catawbiense and R. ponticum under these two cycles may reflect different strategies employed by these two species to tolerate/resist cellular dehydration.