A Chloroplast-Targeted Heat Shock Protein 70 (HSP70) Contributes to the Photoprotection and Repair of Photosystem II during and after Photoinhibition

Abstract

Dark-grown Chlamydomonas reinhardtii cultures that were illuminated at low fluence rates before exposure to high-light conditions exhibited a faster rate of recovery from photoinhibition than did dark-grown cells that were directly exposed to photoinhibitory conditions. This pretreatment has been shown to induce the expression of several nuclear heat shock protein 70 (HSP70) genes, including HSP70B, encoding a chloroplast-localized chaperone. To investigate a possible role of plastidic HSP70B in photoprotection and repair of photosystem II, which is the major target of photoinhibition, we have constructed strains overexpressing or underexpressing HSP70B. The effect of light stress on photosystem II in nuclear transformants harboring HSP70B in the sense or antisense orientation was monitored by measuring variable fluorescence, flash-induced charge separation, and relative amounts of various photosystem II polypeptides. Underexpression of HSP70B caused an increased light sensitivity of photosystem II, whereas overexpression of HSP70B had a protective effect. Furthermore, the reactivation of photosystem II after photoinhibition was enhanced in the HSP70B-overexpressing strain when compared with the wild type, both in the presence or absence of synthesis of chloroplast-encoded proteins. Therefore, HSP70B may participate in vivo both in the molecular protection of the photosystem II reaction centers during photoinhibition and in the process of photosystem II repair.

Analysis of the mutational effects of theCOP/DET/FUSloci on genome expression profiles reveals their overlapping yet not identical roles in regulatingArabidopsisseedling development

“…Some of the light stress-induced marker genes, e.g. late embryogenesis abundant protein (Dunaeva and Adamska, 2001), metallothionein (Dunaeva and Adamska, 2001), HSP70 (Schroda et al, 1999), glutathione reductase (Karpinski et al, 1997) and NAD(P)H dehydrogenases (Endo et al, 1999), were also induced in dark-grown cop1 or det1 null mutants. Third, we found that the genes encoding chlorophyll antennae and thylakoid membrane proteins began to be less induced in weak cop1 mutants, repressed in light-grown strong cop1 or weak det1 mutants, and severely repressed in cop1 or det1 null mutants (Fig.…”

Two Glycerol-3-Phosphate Dehydrogenases from Chlamydomonas Have Distinct Roles in Lipid Metabolism

“…The PCR products were cloned into pGEM-T Easy vector for propagation and sequencing (GATC Biotech). Each cDNA was ligated into the NheI and EcoRI sites of pCB740 (Schroda et al, 1999), so that GPD2 and GPD3 were under the control of the HSP70A-RBCS2 tandem promoter, and selection was performed in the absence of Arg. Successful generation of the pCB740-GPD2 and pCB740-GPD3 plasmids was confirmed by sequencing.…”

Ferritin is required for rapid remodeling of the photosynthetic apparatus and minimizes photo‐oxidative stress in response to iron availability in Chlamydomonas reinhardtii

“…The 5¢ fragment was then inserted after the stop codon of the ferritin coding region in the reverse orientation. The RNAi cassette was excised by digestion with BsrGI (followed by Klenow treatment) and SpeIT, and ligated into the pCB740 vector (Schroda et al, 1999) digested with NheI and EcoRV.…”

“…Two constructs were designed. A classical RNAi construct, using the expression vector pCB740 (Schroda et al, 1999) with 440 bp of the 5¢ coding sequence of ferritin in the antisense orientation, was generated. The second RNAi construct contained a cassette consisting of 410 bp of the coding region of ferritin in antisense orientation separated by a 310-bp long spacer and was inserted in the RNAi vector designed by Rohr et al (2004).…”