**4. Long-term response: changes in nuclear and chloroplast gene expression**

While short-term responses of the photosynthetic apparatus involve mostly posttranslational mechanisms such as phosphorylation or changes in pH and ion levels, long-term responses are mediated through changes in the expression of specific chloroplast and nuclear genes and their products. Environmental changes such as changes in light quantity and quality lead to changes in the state of the chloroplast which are perceived by the nucleus through a signaling chain referred to as retrograde signaling. The components of this signaling chain are still largely unknown although a few potential retrograde signals have been identified [119]. Among these, tetrapyrroles appear to play a significant role. These compounds are involved in the chlorophyll biosynthetic pathway which needs to be tightly regulated to avoid photo‐ oxidative damage. Mg-protoporphyrin IX (Mg-Proto) was first shown to be involved in the repression of the LHCII genes in retrograde signaling in *Chlamydomonas* [120]. However, such a role for this tetrapyrrole in land plants gave rise to contradictory results and has been questioned [121–123]. In contrast, feeding experiments with Mg-Proto and hemin in *Chlamy‐* *domonas* induce increased expression of the gene of HemA (glutamyl-tRNA reductase) and of the heat shock proteins Hsp70A, Hsp70B and Hsp70E [124–126]. In this alga, both Mg-Proto and hemin are exclusively synthesized in the chloroplast. Genome-wide transcriptional profiling revealed that their exogenous addition to *Chlamydomonas* cells elicits transient changes in the expression of almost 1000 genes [127]. They include only few genes of photo‐ synthetic proteins but several genes of enzymes of the tricarboxylic acid cycle, heme-binding proteins, stress-responsive proteins and proteins involved in protein folding and degradation. Because these tetrapyrroles are not present in the natural environment of the algae, it is likely that these two tetrapyrroles act as secondary messengers for adaptive responses affecting not only organellar proteins but also the entire cell. It is noticeable that these large changes in mRNA levels are not matched by similar changes in protein amount [127].

The synthesis of tetrapyrroles needs to be tightly controlled because some of these chlorophyll or heme precursors are very photodynamic and can cause serious photooxidative damage. In land plants, the conversion of protochlorophyllide (PChlide) to chlorophyllide (Chlide) is light dependent. In the dark, overaccumulation of PChlide is prevented through a negative feedback mediated by the Flu protein which inhibits glutamyl-tRNA reductase at an early step of the tetrapyrrole pathway (**Figure 5**) [128]. Although *Chlamydomonas* is able to synthesize chloro‐ phyll in the dark, it also contains a Flu-like gene called Flp, which gives rise to two transcripts by alternative splicing [129]. The relative levels of the two corresponding Flp proteins correlate with the accumulation of specific porphyrin intermediates some of which have been implicated in a signaling chain from the chloroplast to the nucleus. Moreover, decreased levels of the Flp proteins lead to the accumulation of several porphyrin intermediates and to photobleaching when *Chlamydomonas* cells are transferred from the dark to the light. These Flp proteins therefore appear to act as regulators of chlorophyll synthesis and their expression is controlled by both light and plastid signals.

**Figure 5.** Tetrapyrrole pathway.

Similar findings have been reported for Mca1 and Tca1, two nucleus-encoded proteins that are required for the stability and translation of the *petA* mRNA encoding the Cyt *f* subunit in *C. reinhardtii*. Nitrogen deprivation leads to the proteolytic degradation of these factors and in turn to the loss of the Cyt*b*6*f* complex [109,110]. The response to nitrogen starvation also involves other factors required for the assembly of the Cyt*b*6*f* complex and its hemes [111].

Many soil-dwelling algae like *Chlamydomonas* experience anoxic conditions especially during the night and are able to rapidly acclimate to anaerobiosis by shifting from aerobic to fermen‐ tative metabolism and can thus sustain energy production in the absence of photosynthesis [112–114]. These anaerobic conditions lead to the expression of the oxygen-sensitive hydro‐ genase which catalyzes the production of hydrogen from protons and electrons derived from the photosynthetic electron transport chain. Sulfur deprivation of *Chlamydomonas* cells leads to a significant decline in photosynthetic activity within 24 h although there is no proportional concomitant decline in the levels of the major photosynthetic complexes [115,116]. This decline in electron transport activity is due to the conversion of PSII centers from the QB-reducing to a QB nonreducing center [117]. This system has been used for improving hydrogen production in *Chlamydomonas* cells [118]. These cells as well other microalgal species possess a chloroplast (FeFe)-hydrogenase which acts as an additional sink when the photosynthetic electron transport chain is overreduced under anaerobic conditions. Upon sulfur deprivation, photo‐ synthetic oxygen evolution decreases whereas respiration is maintained resulting in an anaerobic environment in a closed culture system. Although the exact physiological role of algal hydrogenases is not known, they are likely to play a significant role in redox poise,

**3.3. Sulfur deprivation and hydrogen production**

36 Applied Photosynthesis - New Progress

photoprotection and fermentative energy production [114].

**expression**

**4. Long-term response: changes in nuclear and chloroplast gene**

While short-term responses of the photosynthetic apparatus involve mostly posttranslational mechanisms such as phosphorylation or changes in pH and ion levels, long-term responses are mediated through changes in the expression of specific chloroplast and nuclear genes and their products. Environmental changes such as changes in light quantity and quality lead to changes in the state of the chloroplast which are perceived by the nucleus through a signaling chain referred to as retrograde signaling. The components of this signaling chain are still largely unknown although a few potential retrograde signals have been identified [119]. Among these, tetrapyrroles appear to play a significant role. These compounds are involved in the chlorophyll biosynthetic pathway which needs to be tightly regulated to avoid photo‐ oxidative damage. Mg-protoporphyrin IX (Mg-Proto) was first shown to be involved in the repression of the LHCII genes in retrograde signaling in *Chlamydomonas* [120]. However, such a role for this tetrapyrrole in land plants gave rise to contradictory results and has been questioned [121–123]. In contrast, feeding experiments with Mg-Proto and hemin in *Chlamy‐*

The heme and chlorophyll biosynthetic pathways branch at protoporphyrin IX (Prot IX). GTR, glutamine tRNA reductase, is subjected to feedback inhibition by heme and FLU. In most land plants, conversion of PChlide (protochlorophyllide) to Chlide is light-dependent (L, in *Chlamydomonas* this conversion also occurs in the dark, *D*). Through its negative feedback on GTR, FLU prevents overaccumulation of PChlide in the dark. The steps affected by the *gun* and *hy* mutations which affect retrograde signaling are indicated. GSA, glutamate 1-semial‐ dehyde; ALA, 5-aminolevulinic acid; Chl, chlorophyll; Fc, ferrochelatase, Hmox1, heme oxygenase; BV, biliverdin; Pcy, bilin reductase; PCB, phytocyanobilin, CHLH, CHLI and CHLD are subunits of Mg-chelatase. Reproduced from Ref. 126 with permission.

Additional evidence for the involvement of tetrapyrroles in retrograde signaling comes from the identification of a functional bilin biosynthesis pathway in *Chlamydomonas*[130,131]. In this pathway, protoporphyrin IX is converted to protoheme and Mg-Proto by Fe- and Mgchelatase, respectively. While heme is used as prosthetic group for many hemoproteins, a portion of heme is converted to biliverdin IXa by heme oxygenase (Hmox1) and in the next step by a ferredoxin-dependent phytochromobilin synthase (PcyA) to phytochromobilin which serves as chromophore of phytochromes (**Figure 5**). However, *Chlamydomonas* as well as other chlorophytes do not produce phytochromes, raising questions on the role of this pathway in these algae. Some clues came from the analysis of a mutant of *Chlamydomonas* deficient in Hmox1 whose phototrophic growth is compromised and in which the increase of chlorophyll upon a dark-to-light transition no longer occurs [131]. Comparative transcriptomic studies of wild-type and *hmox1* cells revealed a set of nuclear genes that are up-regulated by bilins and that comprise several oxygen-dependent redox enzymes such as mono-and dioxygenases, proteins with redox cofactors and enzymes of oxidative amino acid metabolism. These results raise the possibility that bilins operate within a retrograde signaling pathway that evolved in chlorophytes for the detoxification of ROS generated during sudden dark to light shifts. It remains to be seen whether bilins assume additional roles in chlorophytes besides ensuring smooth daily transitions from dark to light with minimal photooxidative damage.

A further striking example of the action of tetrapyrroles as mediators for plastid-to-nucleuscommunication is the identification of a tetrapyrrole-regulated ubiquitin ligase for cell cycle coordination from organelle to nuclear DNA replication in the red alga *Cyanidioschyzon merolae* [132–134].

Redox changes within the photosynthetic electron transport chain occur upon changes in light quality and quantity, CO2 levels, nutrient availability and elevated temperature. As a result of unequal excitation of PSI and PSII or of insufficient electron acceptor capacity on the PSI acceptor side, the redox state of the plastoquinone pool is altered. In this case, chloroplast gene expression is affected in land plants [135] although the evidence is less convincing in algae. However, in these organisms, there is unambiguous evidence that nuclear gene expression is affected [136]. A possible candidate for sensing the redox state of the plastoquinone pool is the chloroplast protein kinase Stt7/STN7 which is known to be activated when plastoquinol occupies the Qo site of the Cyt*b*6*f* complex [21,22]. During experiments in which plants were shifted from light preferentially absorbed by PSI to light preferentially absorbed by PSII, the expression levels of 937 genes changed significantly in *Arabidopsis* [137]. 800 of these changes were dependent on STN7, indicating that most of these genes are under redox control.

In all situations in which the redox poise of the plastoquinone pool is affected, the relative sizes of the PSII and PSI antenna sizes play an important role. Several factors involved in antenna size were identified through a genetic screen in *Chlamydomonas*[138]. One of these factors, Tla1 functions as a regulator of chlorophyll content and antenna size and is localized in the chloroplast envelope. In the *tla1* mutant, thylakoid membranes were disorganized, appressed grana membranes were lost and accumulation of the PSII core proteins was reduced [138]. The second identified factor Tla2 corresponds to FtsY required for insertion of proteins into thylakoid membranes [139] and the third, Tla3 corresponds to SRP43, a component of chlor‐ oplast SRP, known to be essential for the integration of LHCII proteins into the thylakoid membrane [140].

Another protein regulating antenna size in *Chlamydomonas* is Nab1, a cytoplasmic repressor of translation of specific Lcbm isoforms [141]. By binding selectively to the mRNAs of these proteins with Lhcm6 mRNA as its principal target, it sequesters the RNA in translationally silent nucleoprotein complexes. The activity of Nab1 is regulated through a cysteine-based redox control and also by arginine methylation [141,142]. This protein apparently senses the increased or decreased demand for LHCII protein synthesis through changes in the cytosolic redox state although the underlying molecular mechanisms are still unknown.
