Summary: Massive degradation of photosynthetic proteins is the hallmark of leaf senescence; however the mechanism involved in chloroplast protein breakdown is not completely understood. As small ‘senescence‐associated vacuoles’ (SAVs) with intense proteolytic activity accumulate in senescing leaves of soybean and Arabidopsis, the main goal of this work was to determine whether SAVs are involved in the degradation of chloroplastic components. SAVs with protease activity were readily detected through confocal microscopy of naturally senescing leaves of tobacco (Nicotiana tabacum L.). In detached leaves incubated in darkness, acceleration of the chloroplast degradation rate by ethylene treatment correlated with a twofold increase in the number of SAVs per cell, compared to untreated leaves. In a tobacco line expressing GFP targeted to plastids, GFP was re‐located to SAVs in senescing leaves. SAVs were isolated by sucrose density gradient centrifugation. Isolated SAVs contained chloroplast‐targeted GFP and the chloroplast stromal proteins Rubisco (ribulose‐1,5‐bisphosphate carboxylase/oxygenase) and glutamine synthetase, but lacked the thylakoid proteins D1 and light‐harvesting complex II of the photosystem II reaction center and photosystem II antenna, respectively. In SAVs incubated at 30°C, there was a steady decrease in Rubisco levels, which was completely abolished by addition of protease inhibitors. These results indicate that SAVs are involved in degradation of the soluble photosynthetic proteins of the chloroplast stroma during senescence of leaves.

Breakdown of leaf proteins, particularly chloroplast proteins, is a massive process in senescing leaves. In spite of its importance in internal N recycling, the mechanism(s) and the enzymes involved are largely unknown. Senescence-associated vacuoles (SAVs) are small, acidic vacuoles with high cysteine peptidase activity. Chloroplast-targeted proteins re-localize to SAVs during senescence, suggesting that SAVs might be involved in chloroplast protein degradation. SAVs were undetectable in mature, non-senescent tobacco leaves. Their abundance, visualized either with the acidotropic marker Lysotracker Red or by green fluorescent protein (GFP) fluorescence in a line expressing the senescence-associated cysteine protease SAG12 fused to GFP, increased during senescence induction in darkness, and peaked after 2–4 d, when chloroplast dismantling was most intense. Increased abundance of SAVs correlated with higher levels of SAG12 mRNA. Activity labelling with a biotinylated derivative of the cysteine protease inhibitor E-64 was used to detect active cysteine proteases. The two apparently most abundant cysteine proteases of senescing leaves, of 40kDa and 33kDa were detected in isolated SAVs. Rubisco degradation in isolated SAVs was completely blocked by E-64. Treatment of leaf disks with E-64 in vivo substantially reduced degradation of Rubisco and leaf proteins. Overall, these results indicate that SAVs contain most of the cysteine protease activity of senescing cells, and that SAV cysteine proteases are at least partly responsible for the degradation of stromal proteins of the chloroplast.

Cellular proteins are extensively degraded during leaf senescence, and this correlates with an up-regulation of protease gene expression, particularly cysteine proteases. The objectives of this work were (i) to detect cysteine proteases associated with senescence of wheat leaves under different conditions and (ii) to find out their subcellular location. Activity labelling of cysteine proteases with the biotinylated inhibitor DCG-04 detected five bands at 27, 36, 39, 42, and 46 kDa in leaves of wheat senescing under continuous darkness. In-gel activity assays showed that these proteases are only active in an acid milieu (pH 4), and their activity increased several-fold in senescing leaves. Fractionation experiments showed that the senescence-associated cysteine proteases of 36, 39, 42, and 46 kDa localize to a vacuolar-enriched fraction. The vacuolar cysteine proteases of 36, 39, and 42 kDa increased in activity in attached flag leaves senescing naturally during post-anthesis, and in attached leaves of plants subjected to a period of water deficit. Thus, the activity of these vacuolar cysteine proteases is associated with developmental (post-anthesis) senescence and with senescence induced by stress factors (i.e. protracted darkness or drought). This suggests that vacuoles are involved in senescence-associated cellular degradation, and that different senescence-inducing factors may converge on a single degradation pathway.

Leaf senescence is characterized by massive degradation of chloroplast proteins, yet the protease(s) involved is(are) not completely known. Increased expression and/or activities of serine, cysteine, aspartic, and metalloproteases were detected in senescing leaves, but these studies have not provided information on the identities of the proteases responsible for chloroplast protein breakdown. Silencing some senescence-associated proteases has delayed progression of senescence symptoms, yet it is still unclear if these proteases are directly involved in chloroplast protein breakdown. At least four cellular pathways involved in the traffic of chloroplast proteins for degradation outside the chloroplast have been described (i.e., “Rubisco-containing bodies,” “senescence-associated vacuoles,” “ATI1-plastid associated bodies,” and “CV-containing vesicles”), which differ in their dependence on the autophagic machinery, and the identity of the proteins transported and/or degraded. Finding out the proteases involved in, for example, the degradation of Rubisco, may require piling up mutations in several senescence-associated proteases. Alternatively, targeting a proteinaceous protein inhibitor to chloroplasts may allow the inhibitor to reach “Rubisco-containing bodies,” “senescence-associated vacuoles,” “ATI1-plastid associated bodies,” and “CV-containing vesicles” in essentially the way as chloroplast-targeted fluorescent proteins re-localize to these vesicular structures. This might help to reduce proteolytic activity, thereby reducing or slowing down plastid protein degradation during senescence.

Exposure to abiotic stresses accelerates leaf senescence in most crop plant species, thereby reducing photosynthesis and other assimilatory processes. In some cases, genotypes with delayed leaf senescence (i.e. ‘stay-green’) show stress resistance, particularly in cases of water deficit, and this has led to the proposal that senescence delay improves crop performance under some abiotic stresses. In this review, we summarize the evidence for increased resistance to abiotic stress, mostly water deficit, in genotypes with delayed senescence, and specifically focus on the physiological mechanisms and agronomic conditions under which the stay-green trait may ameliorate grain yield under stress.