‘Senescence‐associated vacuoles’ are involved in the degradation of chloroplast proteins in tobacco leaves

Abstract

SummaryMassive 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.

The Endosomal Protein CHARGED MULTIVESICULAR BODY PROTEIN1 Regulates the Autophagic Turnover of Plastids in Arabidopsis

“…Many membrane-bound carriers that deliver plastid contents to the vacuole for degradation during leaf senescence have been characterized. The RECA-GFP bodies seen in chmp1 cells morphologically resemble Rubiscocontaining bodies (RCBs) and senescence-associated vacuoles previously documented to arise during leaf senescence and proposed to help transport stromal contents like Rubisco and RECA-GFP to the vacuole for breakdown (Chiba et al, 2003;Martínez et al, 2008), but appear distinct from the much smaller ATI1-PS bodies (50 to 100 nm) (Michaeli et al, 2014). Our results indicate that not only stromal but also envelope proteins such as MEX1-YFP become incorporated into these bodies.…”

“…Through these receptors, autophagosomes selectively engulf peroxisomes (pexophagy), mitochondria (mitophagy), endoplasmic reticulum (reticulophagy), RNAs (RNautophagy), ribosomes (ribophagy), and other cellular components. Chloroplast dismantling during senescence also involves the delivery of chloroplastic constituents to vacuoles for degradation (Chiba et al, 2003;Martínez et al, 2008;Ono et al, 2013). ATG8-decorated bodies containing Rubisco and other stromal proteins accumulate in the vacuolar lumen of wild-type Arabidopsis plants but not in autophagy mutants Wada et al, 2009).…”

Ultrastructure of autophagy in plant cells

“…Otegui et al 110 and Martínez et al 111 found bodies other than RCBs in senescent cells containing chloroplasts. It was suggested that these bodies, called senescence-activated vacuoles (SAVs), are different from RCBs, since they show chlorophyll autofluorescence, which is not observed in RCBs.…”

“…101 The SAVs contain Rubisco as well as SAG12, a specific senescence-associated cysteine protease. 110 Martínez et al 111 showed intense proteolytic activity in SAVs. Protease inhibitors delay the decrease in Rubisco levels in isolated SAVs held at 30 °C.…”

Expression of Sweet Potato Senescence-Associated Cysteine Proteases Affect Seed and Silique Development and Stress Tolerance in Transgenic Arabidopsis

“…Substrate proteins must be transported and sequestered into these lytic vacuoles before degradation. Therefore, senescence-associated vacuoles are lytic vacuoles and involved in the degradation of imported chloroplast proteins in tobacco leaves (Martı´nez et al, 2008). The molecular mechanisms for vacuolar protein degradation and nutrient recycling pathway in senescent leaves are generally not clear.…”