Dismantling of Arabidopsis thaliana mesophyll cell chloroplasts during natural leaf senescence

Abstract

One of the earliest events in the process of leaf senescence is dismantling of chloroplasts. Mesophyll cell chloroplasts from rosette leaves were studied in Arabidopsis thaliana undergoing natural senescence. The number of chloroplasts decreased by only 17% in fully yellow leaves, and chloroplasts were found to undergo progressive photosynthetic and ultrastructural changes as senescence proceeded. In ultrastructural studies, an intact tonoplast could not be visualized, thus, a 35S-GFP::δ-TIP line with a GFP-labeled tonoplast was used to demonstrate that chloroplasts remain outside of the tonoplast even at late stages of senescence. Chloroplast DNA was measured by real-time PCR at four different chloroplast loci, and a fourfold decrease in chloroplast DNA per chloroplast was noted in yellow senescent leaves when compared to green leaves from plants of the same age. Although chloroplast DNA did decrease, the chloroplast/nuclear gene copy ratio was still 31:1 in yellow leaves. Interestingly, mRNA levels for the four loci differed: psbA and ndhB mRNAs remained abundant late into senescence, while rpoC1 and rbcL mRNAs decreased in parallel to chloroplast DNA. Together, these data demonstrate that, during senescence, chloroplasts remain outside of the vacuole as distinct organelles while the thylakoid membranes are dismantled internally. As thylakoids were dismantled, Rubisco large subunit, Lhcb1, and chloroplast DNA levels declined, but variable levels of mRNA persisted.

Chloroplast DNA in Mature and Senescing Leaves: A Reappraisal

“…It appeared that the ptDNA decline during senescence was correlated with an ;60% reduction of plastid numbers per cell (Supplemental Figures 1A, 2, and 3) and not caused primarily by DNA degradation in organelles as reported by Tymms et al (1983). In Arabidopsis, tobacco, and maize, a significant decrease of ptDNA was found only very late, in tissue neighboring necrotic leaf sectors ( Figures 1A, 1C, and 2F; compare with real-time qPCR of Rowan et al, 2009;Evans, et al, 2010).…”

“…First, undetectability of stainable DNA, even if the plastids were not treated with DNase during the isolation procedure, is not a valid criterion per se to postulate the absence of DNA or to assess nature and impact of changes of in-gel DNA structures remaining after lysis of embedded chloroplasts Rowan et al, 2004Rowan et al, , 2009Oldenburg et al, 2006;Shaver et al, 2006). Besides possible technical problems with insufficient dye penetration (Selldé n and Leech, 1981;Evans et al, 2010), leaf tissue, especially if more mature, is known to contain endogenous nucleases that can be sufficiently active to destroy accessible DNA in organelle preparations (Selldé n and Leech, 1981). This can affect not only nuclear DNA and DNA of broken chloroplasts, but also DNA of morphologically intact plastids Sugar beet (A), tobacco (B), and maize (C).…”

“…Second, ptDNA in senescing and necrotic tissue appears to be more difficult to visualize by DAPI. The failure of Bendich and coworkers to observe DNA staining in most chloroplasts of mature and senescent mesophyll cells in situ, in addition to possible dye penetration problems (Selldé n and Leech, 1981;Evans et al, 2010), could also be caused by ploidy reduction of nucleoids with leaf ageing (Figures 2C and 2J; Supplemental Data Sets 1, 2, and 4, panels 42,52 to 55,61,63,100,103,133,and 134,and Supplemental Methods) and DNA dispersion with progressing senescence ( Figures 2C, 2E, and 2H; Supplemental Data Set 1, panels 61 and 65 to 80). Although DAPI is capable of tracing relative DNA quantities down to a single plastid chromosome (James and Jope, 1978), this sensitivity may only be reached when the DNA is in a sufficiently condensed state.…”

“…However, since contradictory data were reported for the same species that were grown under comparable, if not identical, conditions (Rowan et al, 2004(Rowan et al, , 2009Li et al, 2006;Oldenburg et al, 2006;Shaver et al, 2006;Zoschke et al, 2007;Evans et al, 2010;Udy et al, 2012), it is apparent that some of them must reflect methodological insufficiencies of the experimental approaches employed.…”

Ultrastructure of autophagy in plant cells

“…It has been demonstrated, rather, that chloroplasts remain in the cytoplasm even until late stages of degradation and that many thylakoid membranes become dismantled internally. [102][103][104] Chlorophyll degradation inside chloroplasts apparently takes place initially by dephytylation performed by chlorophyllase, followed by removal of the magnesium ion, and cleavage of the porphyrin ring. An intermediate degradation molecule is transported from the chloroplast to the vacuole, where final degradation takes place.…”