Saturday, 30 June 2012

Wordsworthian echoes in Return of the Native

What struck me about the novel wasn't so much the plot as the themes and the possible sources of its characters. Several excellent websites have covered the symbolism of Egdon Heath, but I thought there's extra. Now we know Hardy was acquainted with Romantic poetry - he certainly heard of Keats, who wasn't  a major part of canon at the time. But it is curious that the novel deals a great deal with Darwinism as well as Wordsworth.

Wordsworth's Lucy poems are about a young girl brought up in Nature quite solitary and forlorn, loved by the speaker. Now I doubt she actually existed, but Hardy would have known about the Lucy poems. Eustacia is evidently quite Wordsworthian. Here's from Wordsworth:
She Dwelt among the Untrodden Ways

She dwelt among the untrodden ways
Beside the springs of Dove,
A Maid whom there were none to praise
And very few to love:

A violet by a mossy stone.
Half hidden from the eye!
—Fair as a star, when only one
Is shining in the sky.

She lived unknown, and few could know
When Lucy ceased to be;
But she is in her grave, and, oh,
The difference to me!
Tow Path at Bougival by Camille Pissarro

Like Lucy, Eustacia dwells in nature, in the secluded heath, far away from civilisation and the delights of town. However Lucy seems to be at home with nature while Eustacia detests its dullness, having enjoyed townlife. It's possible Eustacia isn't meant to be a complete Lucy, or this aspect of her could even be an anti-Lucy, to point out that Wordsworth was naïve when it came to nature. But certainly Eustacia embodies many of Lucy's qualities. Like Lucy she lives in a secluded place, and is unpraised and disliked by the villagers. She is beautiful as a goddess, and what is more, she is the only striking beauty there "when only one is shining in the sky," apart from Thomasin, who isn't as beautiful. Hardy even tells the reader you might well wonder what such a queenly woman is doing in Egdon Heath.  Well, Eustacia was certainly known to the villagers as a witchlike creature, so you couldn't say she lived unknown exactly. But no one truly understood this girl while she lived, so you could argue her soul was unknown. In the end she dies.

Now take this from Wordsworth:
A SLUMBER did my spirit seal;
  I had no human fears:
She seem'd a thing that could not feel
  The touch of earthly years.
  
No motion has she now, no force;         5
  She neither hears nor sees;
Roll'd round in earth's diurnal course
  With rocks, and stones, and trees.
As I said she is like an unearthly creature, and died amidst nature, drowning in a pool.

But to come back to Hardy. Eustacia represents a girl living in the midst of nature, but unlike her predecessor she is dissatisfied. Hardy was very much a Darwinist, and he may have thought that a secluded rural life  ill-equipped one for success in the Victorian rat-race. The fact Eustacia dies, and suffered before she died, as she struggles in her life, shows an individual ill at ease with its environment. She died because she was not fit to survive. Unlike her, Thomas survives, being contented living in the heath. She does find it dull and the weather gloomy, but she knows being countrified, she will never be able to live in a city. Thomasin is one who is well-adapted to her environment. And you can find Darwinism in his other novels - Tess of the d'Urbervilles and Jude the Obscure. Eustacia longs for the town and shuns the rest: she is evidently no true heath-dweller. There is also something disturbing in Thomasin's placidity: were she to go to the city she would not be able to adapt, which shows she can be fragile. Clym, too, suffers for his ideals. Originally a diamond-manager in Paris, he decides to teach in Egdon Heath. But his enterprise does not work and he turns blind. In Paris he was dissatisfied and unhappy. So he is yet another on the fringes of survival. He does survive, but as a preacher, not a schoolmaster as he wanted. Fate and his environment has prevented him from educating himself better. One could the the environment is Fate. There's something in that place which seems to curse anyone with education and ideals.
Arthur Hopkins' illustration of Thomasin Yeobright,  from Philip Allingham at the Victorian Web

We are lucky Hardy called Damon Wildeve the Rousseau of Egdon, because Rousseau advocated feeling over reason, which is what he does. He too is unsuccessful, giving up engineering for innkeeping, which is lower down the social scale. Engineering had become a middle-class profession and innkeeping was considered ungentlemanly. He doesn't do well at his new business however. The fact Hardy thought of Rousseau is interesting, because Rousseau believed children should be raised in nature far away from civilisation, and Wordsworth supported Rousseau's ideals. Hardy loved writing about rural life, but being a cynical Victorian, could not agree with the 18th century idea that seclusion in nature develops the individual - at least not the educated individual. It incapacitates them for higher and better things. I think it is not so much Wildeve as Clym who is the Rousseau. Clym is over-idealistic about teaching children who are not suited to his enlightened methods (think of Wittgenstein teaching in a primary school), and he loves nature more than Paris. He worships the heath. And he does worship Eustacia: he sees her as an ideal teacher in his school. It is an ideal of her he sees, as Wordsworth sees Lucy. He thinks she is suited for nature and Rousseauan things, seeing her as a beauty of the heath, like an untamed girl who must be reformed. Though Wordsworth didn't seem to want to reform Lucy. But perhaps I'm contradicting myself, because if you educated natives of the heath that is artificial surely?  But Eustacia follows her feelings rather than reason, and she is quite honest about her feelings, so you can argue she is natural.
Clym Yeobright

As for her goddesslike appearance she is comparable to Lamia or La Belle Dame Sans Merci and she is even compared to the Greek goddesses, a Sphinx and a Pagan, as if Hardy intended her to be great. Quite ironic as her desires are rather shallow. Lamia and LBDSM are not mentioned but their ancestors are probably similar to Hardy's. Lamia is a serpent transformed into a beautiful woman who allures a human man, and when her identity is discovered, turns back into a snake. LBDSM is a fairy who entices a knight in a language he doesn't understand but he thinks it means "I love thee." Clym thinks Eustacia agrees with his ideals, and Eustacia thinks he agrees with hers - leading to much misunderstanding. So she does come to him in false colours. And like Lamia it is she who makes the first move of introduction, in another shape: Lamia as a woman, Eustacia dressed as a boy.

Eustacia Vye
Which leads to another point. Wordsworth is all for natural, Greek myth and Hardy to some extent is for the supernatural, what with Eustacia's witchlike qualities and the way Egdon Heath seems to be almost alive.  Despite being a sceptic Hardy loved incorporating myths into his stories: he even wrote Gothic tales, one called The Withered Arm. Still, the story is full of Darwinism and emphasises good common sense, which sides with nature or should I say natural selection? I don't know what ideal Hardy wanted to bring out, but it is certain he did, or else the story would not be powerful despite its weakness. It also reminds me of one reviewer who said Hardy's ideas are good but the plot rather weak.

I travell’d among unknown men
  In lands beyond the sea;
Nor, England! did I know till then
  What love I bore to thee.
 
’Tis past, that melancholy dream!        45
  Nor will I quit thy shore
A second time, for still I seem
  To love thee more and more.
 
Among thy mountains did I feel
  The joy of my desire;        50
And she I cherish’d turn’d her wheel
  Beside an English fire.
 
Thy mornings show’d, thy nights conceal’d
  The bowers where Lucy play’d;
And thine too is the last green field        55
  That Lucy’s eyes survey’d.
This reminds me of Clym, who having been in Paris, misses England. Eustacia could be his ideal of his own Lucy - a wild rustic English girl.

Thursday, 28 June 2012

My Reading Habits

This meme is from November's Autumn, who got it from A Room of one's Own.


Do you snack while you read? If so, favourite reading snack:
Usually it's potato crisps or shortbread cookies. Something dry.

What is your favourite drink while reading?
Water or something cold.


Do you tend to mark your books as you read, or does the idea of writing in books horrify you?
I never write in my novels. I know it's personal and all that, but I like my books in a tolerable condition :)


How do you keep your place while reading a book? Bookmark? Dog-ears? Laying the book flat open? I used to dog-ear but not now. If I have a bookmark at hand I'll use it, if not I lay it down. Most of the time I take note of the chapter and just look for it again.


Fiction, non-fiction, or both?
I love novels! Well, far more than most non-fiction :P But recently I've found biographies of authors can be just as good if not more absorbing. But don't expect me to read history or economics!

Are you a person who tends to read to the end of a chapter, or can you stop anywhere?
I prefer to end at the chapter, but being busy I stop anywhere.


Are you the type of person to throw a book across the room or on the floor if the author irritates you?
No. My wrath is reserved for my pen and table.


If you come across an unfamiliar word, do you stop and look it up right away?
Hardly ever. This leads to misunderstandings, but it's taught me to understand things in context and think for myself.


What are you currently reading?
Tennyson by Christopher Ricks, a biography-cum-analysis of his poems. Also John Clare's biography by Jonathan Bates and Return of the Native by Thomas Hardy.



What is the last book you bought?
John Clare by Jonathan Bate. As the cashier wrapped up my book he looked at it longingly and said "That's a good book." I rather suspect he'd been reading it for free in the store. I actually felt sorry at the moment for depriving him. :(
Also, the day before, Howards End by E.M. Forster.

Do you have a favourite time/place to read?
I like reading in the evening and night at home. Snuggled up in bed especially. It helps me to sleep. Most of the time, nowadays, I go to the bookstore to read in the evening.

Do you prefer series books or stand alones?
I used to love series because you got to know the characters. But generally the best novels are stand alones. Authors who wish to explore certain characters and themes generally put it all in one novel. (Note that much silly fiction comes in trilogies!)



Is there a specific book or author you find yourself recommending over and over?
Villette by Charlotte Brontë, Shirley by the same, Thomas Hardy, Wives and Daughters, Anthony Trollope, Terry Pratchett, George Eliot, KEATS KEATS KEATS. Yes, I'm a Keats fangirl :) And you may have noted almost all of them are Victorian.


How do you organize your books? (by genre, title, author’s last name, etc.)
At home I organise them into classics, Agatha Christie, Harry Potter, Diana Wynne Jones, and Enid Blyton, in separate rows. For the classics, they are arranged by the authors name in alphabetical order. Now I'm at university I tend to put my favourite books on the small shelf, the rest in a large shoebox, not arranged in any particular order. Almost all on the shelf are classics or biographies though :)

The Return of the Native by Thomas Hardy


Return of the Native has perhaps one of the most striking heroines in Thomas Hardy's novels. Not as innocent as Sue Brideshead, or sacrificing as Tess Durbeyfield, Eustacia Vye is nevertheless bewitching - far more witchlike than these two. The novel centres around 4 characters, Thomasin Yeobright, Damon Wildeve, Eustacia Vye and Clym Yeobright. Be warned however that Thomasin is not a typical Hardy heroine, passionate and eager, and we do not hear her thoughts. She is practical, not idealistic, and therefore less struck by tragedy than the rest. It is curious to reflect Hardy liked to  .make his idealistic characters fall into tragic circumstances. He seemed to equate that quality with misfortune.
Thomasin Yeobright has been engaged to Damon Wildeve and have run away to get married in another parish. Her aunt is against Wildeve who hasn't got a steady income. He was an unsuccessful engineer who turned to running an inn for a living. This means that he is better educated than the rest but has fallen. He isn't a steady character either. Unfortunately the license is only valid in another parish so they can't marry. Thomasin runs home and her aunt says now her reputation is ruined she must marry Wildeve. Weeks pass and they don't marry because Thomasin is unsure. She thinks she ought to marry Wildeve since they ran away together and people would gossip, but she does not think him as great as she once did. 
Eustacia Vye portrayed by Catherine Zeta-Jones
In the meantime the other educated person in the district, Eustacia Vye, is dying of boredom in this lonely desolate place, Egdon Heath. She lived in a seaside town called Budmouth in her childhood and misses it, but her family has no money to live in a good way. She lives with her grandfather, a taciturn captain, who seems to be quite a neglectful guardian. They don't mix with the villagers and are called aloof. Eustacia longs for excitement, and her wish is to be loved passionately. Her heroes include Napoleon and powerful figures. Hardy describes her in a way that reminds me of Lamia in Keats. Eustacia is no delicate blonde angel. Her hair is as black as night, her complexion very white, and her features are beautiful in a sensuous way. She is compared to Greek mythology, which seems a bit too much, even for Hardy, but then arguably the novel is meant to reflect certain ancient Greek literary conventions. She looks striking, out of place in Egdon Heath. Even Hardy remarks on it. Eustacia is quite interesting in the beginning, for we get to hear her hopes and wishes, unlike Thomasin's, who is supposed to be the paragon. Although she seems shallow, we can sympathise with her, because she is one of the most intelligent people there and her longings are perfectly justified. 

Some time ago, Wildeve and Eustacia had been lovers (and I mean this in the Victorian sense. Not the modern sense which means you must sleep together, otherwise you're merely dating). Wildeve was quite a charmer, and Eustacia was head in heels in love with him.  They parted later after he deserted her, something which happens again when he takes a long time to marry Thomasin after the elopement. Eustacia had loved him because there was no one better and nothing so interesting as him, and he was the most educated man she knew. I thought it ironic that a love based on shallow grounds should be so passionate rather than flirtatious. But there's Hardy's Darwinism for you. And there's a LOT of Darwinism in this book. But one day while Eustacia lights a bonfire Wildeve sees it and comes to see her. They had used it as a signal in their courtship days. She has heard about his engagement to Thomasin and concludes that since they didn't marry he must still be in love with Eustacia. Which is the wrong picture. But Wildeve is still entranced by her beauty, though he is not always constant to her. The chemistry between them is ill-depicted, as they only seem to discuss their thwarted romance. Hardy is so gloomy. Eustacia urges Wildeve to come to her instead and leave Thomasin but he knows Thomasin is a good girl.
Autumn Leaves by Sir John Everett Millais

In the meantime, an admirer of Thomasin's, Diggory Venn, tries to talk to Eustacia and get her to convince Wildeve to marry Thomasin. Diggory wants Thomasin just to be happy. But Eustacia is too madly in love with Wildeve. Diggory also proposes marriage to Thomasin to protect her reputation but she refuses.

Thomasin's aunt, Mrs Yeobright, tells Wildeve that Thomasin has another suitor and might marry him, hoping this will hurry Wildeve up. It does. When Eustacia hears that Wildeve is jilted, she loses her attraction to him, since someone else doesn't find him so charming. She writes him a letter saying they must cease their understanding.

There's also another reason for this. Mrs Yeobright's son Clym has come home from Paris where he had been working as a jewellery salesman. He is intelligent and better-educated than the rest of the villagers and highly regarded, even though a jewellery salesman may not seem very much to us. In Hardy's former working class roots an ordinary middle-class person with education was considered gentry even though technically they are not. This can be quite confusing to someone accustomed to reading gentry in novels as upper-class or upper-middle class people. Eustacia falls in love with the idea of someone from Paris and arranges to see him. The stable-boy, Charley, who is infatuated with her, is supposed to act a role in a play at the Yeobrights' to celebrate Clym's return. Those day they might invite the whole village, though Eustacia is not included, as she doesn't see the Yeobrights and is standoffish. I think this rather unkind however, because even if Eustacia never visited Mrs Yeobright the latter could have done so to her. Eustacia tells Charley to pretend to be ill and say that Miss Vye's cousin will be playing Charley's part. She takes over his role well, and a few performers suspect her identity.

Eventually she comes to encounter Clym who asks her if she is a woman. She tells him she came because she is bored and depressed and he is curious that a well-spoken lady should dress as a boy, but she will not reveal her identity. I believe she is covered by a hood because he doesn't notice her face.
Victorian schoolroom

Later on, Thomasin and Wildeve marry and move out. Mrs Y is lonely. Clym has ambitions of retiring from the jewelery business to become a schoolmaster in his place. He wants to educate the lowly villagers but Mrs Y says this is foolish, because he will be poor and they cannot be educated to a high level. They may not be interested or have the ability to learn very much. There are so many schoolmasters in that area anyway. But he doesn't listen. He gets to know Eustacia and soon they start walking out together, talking. He hopes she can teach in his future school but she doesn't like the idea and wouldn't be good at it. Eustacia sees him as a man from Paris rather than as an idealist who loves his hometown. While she hates the place he loves it. While she has no inclination for social service he has. She wants to go to Paris some day and hopes to achieve this by marrying him.  And yet we do not feel utterly repugnant towards her. They fall in love, to Mrs Y's disapproval. The chemistry between them is better written than that between her and Wildeve, because it was all sad and thoughtless with Wildeve, and certainly impetuous. Not so with her and Clym. She tells him she would be a bad wife, and not suited to the sort of life he likes. For this I admire her, because she dares to be honest about her weaknesses despite loving Clym, even going to the extent of discouraging their marriage. He says he will go on loving her so they get married and move out. He proposes to read for 6 months to qualify as a schoolmaster in a small cottage and then work. Eustacia hopes he'll change his mind, but anyway goes along with the plan. They seem to be happy initially though she is dissatisfied to be stuck there. 

One day Clym's eyesight is ruined, and he cannot become a schoolmaster, so he takes up furze-cutting, to his wife's humiliation. What is worse, to her, is he enjoys it.

After a quarrel between her and Mrs Y, she is furious and refuses to see her again. Clym is upset as he loves his mother. One day Mrs Y visits their house when he is asleep. Eustacia will not answer the door after the first knock. At the same time  Wildeve is in the house as he came to see her. She confides to him her dissatisfaction. The door knocks a second time and opens. Mrs Y sees her son's boots and knows he is at home. She thinks he doesn't want to see her and then leaves. At the sound of the door opening Eustacia thinks Clym has answered it so she doesn't bother. But afterwards she realised he is still sleeping, so he couldn't have seen his mother.

While on the way home Mrs Y is depressed, thinking she is forsaken by her son. She is bitten by an adder and falls unconscious. Clym encounters her and gets the doctor, but she dies. He is remorseful as he had not seen her for two months although she was only 5 miles away. He later finds out the truth about Eustacia's refusal to open the door and gets angry with her. He also discovers Wildeve was in the house with her and thinks they have been having an affair even though they haven't. Eustacia seems to miss her former lover though she doesn't give in to him, I think because she does love Clym. She did not marry him purely for Paris. She married him because she loved him though Paris had a part in it.

Damon Wildeve has inherited 11000 pounds from a dead relative and wants to go on holiday. He asks Eustacia to come with him. She is tempted though she doesn't want to ruin her reputation. Anyway she returns to her grandfather as Clym is angry with her. After he has cooled down he writes a letter to her but she doesn't get to read it, because she has run away - Clym thinks with Wildeve. He runs to look for her, and sees Wildeve. His wife has slipped into the pool and drowned, and in the confusion Wildeve drowns. Clym never gets over her death and becomes a preacher.

Thomasin inherits her late husband's property and brings up their child with great care. The story is supposed to end here, but Hardy's publishers wanted it longer, so he added another part which he resented. In this part Thomasin marries Diggory Venn. Hardy added a note to say that the perceptive reader will decided what the ending is. What ought to have happened was Diggory Venn disappears, wandering alone, and Thomasin remains a widow. It is also truer to character, as Hardy emphasised the fact Venn is an isolated person, not very good with women. Thomasin is also the sort to remain true to her husband, and not remarry even if the husband was rotten. She does not seem to have much passion or sexuality. And given the fact she is presented as pure, it would be expected such a Victorian woman would rather remain a widow, as it was considered commendable then for widows not to remarry.  She also praised her aunt as being noble for not remarrying after the latter's widowhood. So I will not dwell on Hardy's extended ending as he didn't believe in it. He even made Thomasin coquettish and unsteady, which is not the Thomasin we know.

An important question is, did Eustacia intend to elope with Wildeve? She hoped to reunite with Clym but didn't know he had forgiven her. She did signal for Wildeve to meet her, but her thoughts are anguished. She can't bear the idea of being his mistress and asking him for money. She forgot to take money with her and won't ask him. So we do know she did not intend to become his mistress or she would have asked for his money. She was only using him as an escape out of Egdon Heath, and then presumably they would part. Even Wildeve's thoughts are not quite beastly. He plans to leave half his property to his wife and then devote himself chivalrously to Eustacia, which sounds less adulterous than you'd expect. Eustacia has stopped loving him, and sees him only as a gateway to a new world, so her intentions are not adulterous. Many people interpret the last part as adultery, but I don't think so. Eustacia's cries to him are that of a lonely woman wanting to be free rather than a woman in the throes of passion.

Among the main characters, Thomasin is disappointingly drawn because we don't know her thoughts. Hardy could only portray passionate women - indeed many of his heroines have similarities. They could be an Expy as TvTropes would say. Thomasin is unusual in that she is almost sexless, like Sue Bridehead, and is only really attracted to one man. And we never hear Sue's thoughts either. But I commend him for trying another woman, and indeed Thomasin may be fairly realistic. There are many uninteresting people in the world who are not passionate, and some may well be as good and steady as Thomasin. With little to trouble her existence or serenity there is little to write about her. Hardy was best at troubled characters. He could not detail the foibles and subtle relationships in high society, and high society novels tend to be all relationships and little else, no social issues, great passion, etc. Which is being real (like Jane Austen) but it adds more to our emotional understanding rather than intellectual inspiration. But I digress. Hardy is real in the tragic rather than the happy and the ordinary. He will write a good scene of villagers talking in the pub, but real neutral conversation between intelligent people eludes him, unless there is tragedy in it. This is not to say I dislike Hardy. I like and admire Hardy - he brought symbolism the other Victorians didn't, and despite being modern was very much a Romantic. Those who credit the Modernists with consciously doing symbolism would do well to read Hardy.

Damon Wildeve is better-written, because he has more speaking parts, but we only see him as a failed selfish man. Perhaps Hardy intended this, but considering much of the plot is centred on him we see precious little of his thoughts. We do know he goes back to Thomasin, fearing he might lose her, and his passion for Eustacia, but he seems little more than a sensual, dissatisfied lover. Even Eustacia who has these qualities is more sympathetic - but Hardy's heroines are always sympathetic. I think Hardy had a grudge against Wildeve, which is why he is badly written. Being a respectable man he couldn't fully sympathise with Wildeve. But even without hearing his thoughts, I really don't see how he entrances Eustacia. Hardy interestingly refers to him as the Rousseau of Egdon, as Rousseau emphasised feeling and passion rather than reason. Wildeve is unsteady, which is right. But his passion for his two women fluctuates, so how passionate can that be? He is terribly insensitive as well, and is too much of a wastrel to be a Rousseau. Did Hardy try to see a better side of him by comparing him to Rousseau? Perhaps.

We think of this as the passion between Wildeve and Eustacia, but really the principal cast are Eustacia and Clym. In fact the passion is really nothing but shallow fantasies of a secluded girl and Wildeve is not rendered loveable enough to convince you how he charmed two pretty girls. At least, not in his speech. Even Clym sounds more charming and loveable and his conversation is more pleasing. A charmer is good and pleasing speech, for heaven's sake do something with Wildeve's conversation.

Clym in his days of courtship is quite loveable. He accepts Eustacia's self-deprecations, attempting to cheer her up, and says some very sweet things as lovers ought to (unlike Wildeve). His idealism is touching but too unreal for a man accustomed to the jewellery trade in Paris. One would expect such a sensitive's souls ideals would have been shattered by living abroad. Still, these things happen among intelligent men though they are not common. I like the fact someone as good and cheerful and idealistic as him sees past Eustacia's morbidities. Though it is likely he is attracted to the only educated woman in the area. She is the only woman he has loved deeply. One can't help but wonder whether it is her otherworldliness that attracts him. He wishes to reform the villagers into something better: does he see her as an unearthly girl to reform into his plans? She could be a Wordsworthian Lucy or his idea of a Roussean girl, shielded from commerce and industry and close to nature. He loves nature and wants to share it with her. Clym's and Eustacia's expectations, contrary to what they are, are so real as to render the inconsistencies all right.

Eustacia is the most interesting character. Originally she was meant to be more evil, but thankfully Hardy made her a sympathetic anti-heroine which is more real. You may blame her for wanting to go to Paris, for hating her residence, but it is understandable an intelligent educated girl should want more, and hold herself up high. It is not merely pride that makes her so forbidding to the superstitious villagers (they think she is a witch) but her shyness. Why should she have eluded the respectable Yeobrights so long? Pride can't be the full answer: she was probably not at ease with the idea of meeting them. The fact she loved Clym more than Wildeve redeems her, though she loses her faith in her husband.  I do think Clym made her a better person in teaching her to love someone truly for their goodness (to her, not the village) rather than the wild passion in Wildeve. It is more enduring. Her vanity and shallowness is off-putting - does she read nothing useful, or harbour better thoughts? - but having no chemistry or ease is enough to make one a misanthrope. I was angry she was tempted to run away with Wildeve to go to Paris, because that was not only shallow, but unfaithful. But there's no doubt she's the best-drawn character. Her former relationship with Wildeve is not very convincing - I can imagine why she loved him for a while - but why he loved her and had a relationship goes against the fact she is a secluded miss. How would they know each other? And charmers like him don't go for secluded misses who repel most people she comes into contact with. Also, why does she repel everyone? She is pretty and likes gaieties, there surely must be people with similar temperaments. We do know she disliked school during breaktimes, preferring to read. Perhaps she is a misanthrope. But that is inconsistent in liking gaiety and going out to society. People who avoid others do not seek lively society. I feel it is the ideal of going out to society she wants, and were she to confront the reality it would not be likeable to her. Perhaps Hardy did this deliberately. Even so, her school experiences and her stay in Budmouth would surely have warned her, since she didn't have much society there, avoiding it.

It is tempting to ask, as Miss Rigby did on Jane Eyre, why, despite going to a good school, Eustacia never seems to be in contact with her schoolfriends. Friends then were not what they are now: good schoolfriends would regularly write to each other and friendships were treasured greatly. With less mobility each friendship was special. Acquaintances would talk more to each other, and a modern person seeing their conversation would think of them as friends. Though they would not consider each other friends. So between friends things were greater. I won't believe she despised them all, since she loved Budmouth and the girls must have been of a respectable status. I put it down to feeble characterisation, or some clever way of portraying her as unreal and idealistic which may be too contrived. Hardy was never good at writing about the protagonists' side friendships. One gets the idea the characters here are figures rather than living, breathing individuals, meant to promote Hardy's own ideas. Which is not surprising, as he inserts in a lot of references to myths and philosophy, which seems vain but is a boon to the reader because we get to know what Hardy's intentions were. If it hadn't been by Thomas Hardy you wonder whether this would have lasted all those years.

Overall the themes are excellent - better than A Pair of Blue Eyes, and the story is meant to be powerful and it has power. The characters are flimsy, and you wonder how this became known as a masterpiece, but that is due to the passion and tragedy. There's a great deal of Darwinism, a favourite with Hardy (an obsession I should think), and Rousseau and a good deal of Romanticism. If strictly read as an intellectual novel the book beats most. As an overall classic novel it would not win over Jude and Tess. Even the characters in A Pair of Blue Eyes are better-drawn. Return of the Native is a poem in prose form: the materials are haunting, passionate, poetic, and reminiscent of many things in mythology. As it is set some time in the past perhaps that is why it lacks some of the modernity in Hardy's works. But there is little doubt the plot is timeless, and that is why it is a classic.

I'll be writing later on the annotations in the novel. Watch out for them!

Tuesday, 26 June 2012

Fifty Shades Freed, and some comments on silly writing

And so I've completed the entire sadomasochistic trilogy. Phew! To be honest, I read the last one online (someone uploaded a copy) but I checked it at the bookstore and there seems to be some differences. For example, the appendix isn't in the online copy. The bookstore staff are probably fed up I kept on going in to read without buying, but I WILL NOT spend my money on such needless rubbish. Anyway I did buy Villette,  Shirley, Mabinogion and Metamorphoses earlier this year, which is more than what many people my age buy for their own pleasure.

It starts off with the soppy marriage of Christian Grey and Anastasia Steele. He still tries to control her, for example snarling when she goes topless on the beach. Though he has a point. She argues no one else can see her, but actually it seems the security team can because since her attempted assault by her ex-boss, they've been keeping an eye for any potential intruders. A long diatribe on the luxury goods and food they enjoy on holiday. It might as well be an advertisement for some luxury holiday package. I wouldn't be surprised if some of these holiday places offer EL James a lot of money to be their spokesman, since she's popularised luxury living.  I bet you that was her intention, the greedy old cow.

The food descriptions are the only softeners to the blow. Here's a menu of the Fifty Shades series. Some chef decided to capitalise on the book by preparing a meal similar to the one in the book.

Grey acts like a tyrant, over concerned with his wife's security and she is discontented. This book is darker than the previous. Ordinarily that would be a good thing, but darkness is only good for intelligently written books. Stupid authors I feel should confine themselves to comedy and caricature.

There's also the question whether she should change her name. She wants to retain her maiden name, or people will say she got where she is because of her husband, but he wants her to use his name. By the way she has been promoted after the departure of her ex-boss.

In the meantime, her ex-boss Jack Hyde wants to get even. He kidnaps Grey's sister Mia, and calls Anastasia asking for $5 million in ransom. I'm surprised he doesn't ask for more, since her husband is loaded. But she's not allowed to tell anyone about this. So Ana goes to the bank to withdraw from her husband's account. Unfortunately 5 milion being a lot the bank is suspicious so they call Grey, who agrees to let out the money.

The thing is Grey thinks his wife wants to leave him. Ana goes to the meeting spot where she is taken away and Hyde tries to attack her. But she's done the smartest thing she's done so far. She borrowed someone else's phone to take with her, which the kidnappers confiscated. In the bag of money she put her own phone, which can be tracked by Christian.

While the kidnappers are being violent, Grey comes over and the criminals are taken away. One of them was Hyde's junior, who had an affair with him. He had had affairs with all his personal assistants and blackmailed them into helping him and saying he's good. What a horrible man.

Anyway things are resolved about Grey and Ana and they all live happily ever after.  In the epilogue they have 2 children, and there's an appendix after that which details how Grey got his information on Ana. It's written in his point of view, which reminds me of Stephanie Meyer's former intention to publich Twilight from Edward's point of view. Thankfully someone released the draft of Midnight Sun online so SMeyer decided not to publish. Whoever the leaker was, I salute him for saving our brains from more slush and the trees from more cutting.

I have a bone to pick with those silly female authors who keep on using their initials. One would suppose they're ashamed of being women. Not only is this irrelevant in today's day and age, it is a travesty to other women authors. Many intelligent female novelists spell out their first names, and the same for male novelists. Using your initials was more popular in the early 20th century, and it gave a quaint, aloof, intellectual air (possibly because a number of them were used in intelligent books or nonfiction. I think even back then good novelists liked to spell out their first names, except Tolkien and EM Forster). But we can agree that back then initials were a masculine thing and more likely to be intelligent. Like on treatises or something. Even some clever women used their initials eg Edith Nesbit. Not only does it give the first impression you're a man, it is insulting to the old intellectual figures of yore. I can't understand why silly female novelists use 2 initials before their surname. I can only suppose they think (like me) that initials look intellectual, and so adopt it. I won't buy the crap about being discriminated because they're female. No, they know their works are silly and attempt to disguise it by posing as a pseudo-man, because subconsciously they think that masculinity is clever or perceived as clever. (JK Rowling is exempted from this). At least silly male authors use their first name like everyone else and don't act like a hypocrite pretending to be intellectual. I have now come to the conclusion if I see a modern work of fiction with initials it is written by a woman, and not an intelligent work.

Mind you there's some truth that masculine female authors are good. I don't mean tomboyish or lesbian or those who write mainly about men, but adrogynous. The best literature is not exceedingly feminine or masculine, but somewhere in the middle. There are some exceptions (the Brontës and Jane Austen) but even I question the validity of this. They are obviously written by women, but the style of the writing is such that it is not so easy to tell whether a man or woman wrote it. They might even have been written by perceptive men.  Even contemporary male and female literary novelists do not sound too different. But when you get to cheap chick lit and terrible thrillers you can tell the gender quite easily, and they are examples of extreme male and female writing. JK Rowling, who isn't great literature (though a classic) at least doesn't write in too feminine a manner.

Charlotte, whose passions were feminine, was mistaken for a man by some, and though her style was feminine to me, the Victorian standards were different. So much passion and questioning and ideals are often the sort of things we associate with sensitive men.  Apart from the formality of the times, the Victorian man described things with a feminine touch, and idealised friendship. They were also pretty good at writing conversation. Great literature depends on the author's ability to perceive different characters and write them convincingly, which has a wide range of genders, which is why they have to be adrogynous. Mrs Gaskell was excellent in writing both men and women, and so was Jane Austen. Charlotte Brontë is said to have been bad with male characters, and so she was, compared to the great female novelists. But compared to the minor female novelists you see she wasn't that bad at all. Her fault lies in her heroes, who are often over-idealised; give her a minor ordinary male character and he is quite believable. Mr Yorke, some parts of Robert Moore, Mr Helstone and Mr Hall were immediately recognised as real persons in her district. She hardly knew the original men but drew them well. It was when she had some imaginary man in her mind that she had a problem. This fault is common among female novelists (and idealised heroines among male novelists), so we will forgive them because their side characters are so lovable. George Eliot's heroes are not good, but her other male characters are remarkably so, for a woman of her time. Perhaps she wasn't as good as Jane Austen and Mrs Gaskell, but George Eliot was more daring because she probed into the personal lives and thoughts of these men, unlike the other two. If the others had attempted to do the same I'm sure they would have had some problems like the rest of us.

Perhaps I'm harsh. These initialled authors may have a point, because thrillers are generally more intelligent than chick lit, though I wouldn't call them cerebral. (this is true, because romance outsells thrillers.)  I do know thrillers stimulated my mind more than chick lit ever did. Chick lit always put me into a drowsy daze and lowered my IQ. But I'm sure we can all agree female novelists are better at male characters than male novelists at female character (especially good literature).

Speaking of writing styles, I put some of my fiction into Gender Guesser and I came up mostly Weak Male or quite often Weak Female. I thought my writing was feminine, but I do use old-fashioned phrases, which can be mistaken for masculinity. (I attempted to prove this by putting in a Victorian female novelist's work. The results predicted male. When I wrote something modern in a more adrogynous manner I got female I believe which shows you how flawed these algorithms are.) The good news is, after a series of Weak Male results, I am now Weak Female. My writing is now simpler which is the likely cause. It is quite ironic, because those days I couldn't write male characters well, and now my male characters are better. In fact, my lecturer in creative writing was hoping to hear more about my hero, and my male classmates seemed to like him. It was the girls who were puzzled by his character.  On the contrary my heroine fared less well. I refused to idolise my hero: he is flawed (not in the Rochester way) but in the way I hope shy intellectual men can relate to. Of course I cheated: I borrowed a great deal from my biographies - Tennyson and Keats and Darwin. The heroine I fear was borrowed from Charlotte Brontë. I do this because I must write real characters who lived in that era, and studying contemporary persons wouldn't fit the context. I can't empathise with my own era, but Victorian characters come alive to me.

Oh, and some silly author decided to write a porn version of Jane Eyre after the success of Fifty Shades called Jane Eyre Laid Bare, which sends rather disturbing images to my mind. The whole power of Jane Eyre is the unfulfiment of their passion and the impossibility of it. Anyway there are so many porn Jane Eyres in the market (one of the better known is JL Niemann's Rochester). JL Niemann's book is a shame to Charlotte, but the plot is better, if melodramatic and overdone, than most of these so-called sequels.  They do mention art and some intellectual things in the novel which is unusual for romance/erotica. But it will never be a classic.

I can even guess what will come out in a Jane Eyre porn novel. It is likely to deal with the marital relations of Jane and Rochester after marriage. Or, if they are irreverant, it will be a travesty to 19th century manners, and Jane and Rochester have it off before they marry. There will be lurid descriptions of Rochester's bare muscles I can tell you, and how he looks like in tight trousers, as they had in those days. The conversations will deal with their immense desire to remove each other's items of dress. Any intellectual or soul-searching conversation will not exist, and you wonder what was the fuss about the kinship between their souls. Jane will miraculously become beautiful (!!!) and so will Rochester. Everyone will fawn over Jane and she will be popular in the neighbourhood, a travesty to the shy heroine in Charlotte Brontë's original.

And if any reader out here objects to my slant on chick lit, if you don't like my post don't read it. I believe we are all free to express our intellectual opinions. Like if you think George Eliot is dull, fine with me.

Wednesday, 20 June 2012

Light and chloroplast enzyme activity by Peter Schümann



LIGHT and CHLOROPLAST ENZYME ACTIVITY



Peter Schürmann

Université de Neuchâtel, Laboratoire de biologie moléculaire et cellulaire
Rue Emile-Argand 11, CP 158, CH-2009 Neuchatel, Switzerland
peter.schurmann@unine.ch



Introduction 

Light provides the energy needed for the assimilation of carbon by photosynthesis. Light energy absorbed by the photosystems is converted into chemical energy in the form of ATP and NADPH, which serve as co-substrates in the reduction of CO2 to the level of carbohydrates by enzyme reactions of the reductive pentose phosphate cycle or Calvin-Benson cycle. However, this is not the only task light accomplishes in photosynthesis. Light functions also as an essential regulatory element, controlling the activity of enzymes involved in carbon assimilation and related metabolic pathways in chloroplasts. Light activates photosynthetic, anabolic pathways and, at the same time, deactivates catabolic processes, which are required for dark metabolism in the chloroplast. This dual control prevents the operation of futile cycles, which would occur if these opposing reaction pathways were allowed to function simultaneously. In addition, as light is variable in terms of quantity and quality, plants, particularly vascular plants that live at fixed locations, have evolved intricate regulatory networks to adjust their metabolism to changing environmental conditions in order to optimize harnessing light. In this module, I will describe light-dependent mechanisms that regulate the activity of key enzymes in photosynthetic metabolism, and discuss in more detail the ferredoxin/thioredoxin system, which is unique to oxygenic photosynthetic cells. Several recent reviews have addressed different aspects of the regulation of photosynthetic enzymes and provide further detailed information on the topic (Buchanan & Balmer, 2005; Gontero et al., 2006; Mora-Garcia et al., 2006; Schürmann & Buchanan, 2008; Meyer et al., 2009). Instead of citing many original articles I have preferred to refer to reviews wherein the pertinent references can be found. 


Light-Induced Changes in Chloroplasts 

Early experiments with algal cells suggested that light acts as a regulatory signal by activating certain carbon assimilation enzymes, enabling the cells to switch between dark and light metabolism. Different mechanisms have since been found to be engaged in this light dependent regulation, all based on changes of ambient conditions in the chloroplast stroma. Parameters such as pH, concentration of Mg2+and metabolites, and redox potential are subject to light-induced alterations that influence the activity of susceptible enzymes (Buchanan, 1980). 

Coupled to the light-driven electron transport between the photosystems, protons are translocated from the stroma across the thylakoid membrane into the thylakoid lumen (Figure 1). This increases the stromal pH from about pH 7 in the dark, to pH 8 in the light. Such an alkalization by one pH unit affects all stromal enzymes whose activity is strongly pH dependent, and thus serves as a general regulatory factor by providing a favorable pH for activity. 

The transport of protons into the thylakoid lumen is accompanied by a release of Mg2+, the major counter-ion, from the thylakoid membrane into the stroma. This brings about a 1-3 mM increase of the stromal Mg2+ concentration, stimulating the activity of several enzymes that depend on Mg2+ for optimal function. The change in the Mg-ion concentration, therefore, represents another general regulatory element capable of modifying enzyme activities in response to light. 



Figure 1

Figure 1: Light-dependent electron and proton transport in the thylakoid membrane and associated reactions. Electrons released in the oxidation of H2O are transferred in the light through photosystem I (PSI), plastoquinone (PQ), the cytochrome b6f complex, plastocyanin (PC), and photosystem II to ferredoxin (Fdx). Reduced Fdx reduces either thioredoxins (Trx) via ferredoxin:thioredoxin reductase (FTR) or NADP via ferredoxin:NADP reductase (FNR). Protons released into the thylakoid lumen in the oxidation of H2O and by PQ are used for the synthesis of ATP (Figure modified from Meyer et al., 2009).

A third important parameter undergoing light-dependent changes is redox potential. In the light, the stroma becomes much more reducing due to electrons delivered to ferredoxin by non-cyclic electron transport (Figure 1). This redox change is sensed and transmitted to selected enzymes by a redox cascade known as the ferredoxin/thioredoxin system. It is composed of three proteins, ferredoxin (Fdx), ferredoxin:thioredoxin reductase (FTR), and thioredoxin (Trx), and causes reversible structural changes in the target proteins that modify their catalytic activity. This regulatory system is more specific than pH and Mg2+ ion concentration changes, since it entails specific protein-protein interactions within the redox cascade, and with selected target enzymes. Consequently, only those proteins that have developed a redox sensitive "receiver" structure are affected. 


Components of the Ferredoxin/Thioredoxin System 

All components of the regulatory cascade are small, soluble stromal proteins containing either an iron-sulfur cluster, a redox-active disulfide bridge or both. They have been extensively studied, and their structures determined (Dai et al., 2000). 

The first protein in the chain, receiving electrons from photosystem 1 (Figure 1), is ferredoxin. Plant-type Fdxs are 11 kDa acidic proteins that contain a single [2Fe-2S] cluster with a low redox potential of approximately -470 mV at pH 7.9, the pH of the stroma upon illumination. Fdx can carry one electron and deliver it to FTR in a non-covalent interaction between the two proteins. It is the first soluble electron carrier in the chloroplast, and distributes electrons to a number of enzymes in addition to FTR. The most important is Fdx:NADP reductase, the flavoprotein that produces NADPH needed for the reductive step in the Calvin-Benson cycle. Enzymes involved in N- and S-metabolism, i.e., nitrite reductase, glutamate synthase, and sulfite reductase, also obtain electrons from Fdx (Hase et al., 2006). 

Ferredoxin:thioredoxin reductase, exclusively found in oxygenic photosynthetic cells, is the central enzyme of the regulatory cascade. It is an ß-heterodimer composed of a variable (7-13 kDa) and a catalytic (13 kDa) subunit. Size and primary structure of the variable subunit vary significantly from different species, whereas the catalytic subunit is highly conserved, including the arrangement of 6 Cys in two Cys-Pro-Cys, and one Cys-His-Cys motifs. This latter subunit contains the essential elements for catalysis, a [4Fe-4S] cluster and an adjacent redox-active disulfide. FTR is a flat, concave, disk-like molecule, well adapted to its function that necessitates simultaneous interaction with electron donor and acceptor (Figure 2). 


Figure 2

Figure 2. Crystal structure of the FTR heterodimer seen from front (A) and from the side (B). The variable subunit (green) is a heart-shaped, open ß-barrel structure, and the catalytic subunit (blue) an entirely -helical structure sitting on top of the variable subunit. The catalytically essential elements are located in the center of the disk, where the protein is only about 10 Å thick (adapted from Dai et al., 2000).

The [4Fe-4S] cluster is close to the surface that carries some positive charges on one side. The redox-active disulfide is on the opposite side whose surface is more hydrophobic, thus permitting interaction with different types of Trxs. Both FTR surfaces are highly conserved, allowing simultaneous docking of negatively charged Fdx on the cluster side and Trx on the disulfide side (Figure 3) (Dai et al., 2007; Xu et al., 2009). 



Figure 3

Figure 3. Crystal structure of the ternary complex Fdx-FTR-Trxf. Whereas the interaction between Fdx (blue) and FTR (beige, brown) is noncovalent, Trx f (green) is covalently linked through a disulfide bond (colored in yellow) between the active sites of the two proteins (Figure taken from Dai et al., 2007). This complex represents the transient one-electron-reduced reaction intermediate, which has been stabilized by using an active-site mutant of Trx f (Glauser et al., 2004).

The third member of the regulatory cascade is thioredoxin. Trxs are ubiquitous, low-molecular weight protein disulfide reductases with a large number of biological functions (Arner & Holmgren, 2000). They catalyze thiol-disulfide exchange reactions based on reversible oxidation of two cysteine thiol groups to a disulfide, accompanied by the transfer of two protons. Plants contain a large number of Trxs varying in primary structure, function and location (Meyer et al., 2008). Four different types are found in chloroplasts, Trx fmx and y, which all share the canonical active-site sequence -Trp-Cys-Gly-Pro-Cys-. However, only Trxs f and m are involved in enzyme regulation; the other two appear to function in detoxification (removal of reactive oxygen species, ROS). Trx f, originally described as a specific activator protein for chloroplast fructose-1,6-bisphosphatase, is of eukaryotic origin and present only in this group of organisms. Discovered as an activator protein for chloroplast NADP-dependent malate dehydrogenase in C3 and C4 plants Trx m is found in oxygenic prokaryotes, algae and land plants. All four chloroplast Trxs are reduced by FTR in the light, and reoxidized by O2 or other oxidants in the dark. The chloroplast Trxs have the typical basic structure observed in all known Trxs (Figure 4). 


Figure 4

Figure 4. Crystal structures of spinach Trx f and Trx m. The two Trxs have the same overall 3-D structure (cartoon representations to the left), with a central five-stranded ß-sheet, surrounded by four -helices, as found in prokaryotic and human Trxs. Significant differences present in their primary sequences are reflected in the distribution of amino acids and charges on their surfaces, as seen in the corresponding representations to the right. Residue properties: blue is charged positive; red is charged negative; pale cyan is polar; white is hydrophobic/aromatic and proline; yellow is cysteine; and A is the active site. (Figure courtesy of Guido Capitani and Martin Schaerer.)

The surface properties are probably responsible for specificity in the protein-protein interactions. Several targets are very selective, and are activated efficiently only by Trx f, whereas others are also activated by Trx m. Since experiments exploring Trx specificity have not always been performed under the same conditions or with proteins from the same species, certain results differ. In general, results favor Trx f as the primary activator protein. So far, a target specific for Trx m has not been identified. 


The Mechanism of Reduction of Target Enzymes by Thioredoxin 

On its way from the thylakoid membranes to target enzymes, the light signal is transformed from an electron signal to a dithiol signal that can be perceived by proteins possessing a regulatory disulfide bridge. This transformation is performed by FTR through a novel mechanism involving a [4Fe-4S] cluster, and an associated disulfide bridge (Walters et al., 2005, 2009). The proposed mechanism accommodates defined features of the participants: (i) the reduction of a disulfide bond requires two electrons; (ii) Fdx is a one-electron donor; and (iii) FTR has a single Fdx docking site. Hence, the two-step mechanism proposed involves two consecutive electron transfers from Fdx to FTR, with the concomitant formation of a transient one-electron-reduced intermediate stabilized by the [4Fe-4S] cluster (Figure 5). 


Figure 5

Figure 5. Mechanism of Trx reduction by FTR. The first electron, delivered by Fdx and transmitted through the [4Fe-4S] cluster, cleaves the active-site disulfide, and produces a surface-exposed thiolate and a cysteine-based thiyl radical (1→2). This radical becomes stabilized by covalent attachment of the cysteine to the closest Fe atom of the cluster, thereby forming an oxidized (3+), five-coordinate cluster. The surface exposed thiolate then acts as a nucleophile, and attacks the disulfide bond of a Trx, forming the one-electron-reduced heterodisulfide intermediate (2→3). This reaction anchors a Trx molecule to a docking area designed to fit the protein. The Fdx docking area on the opposite side of FTR becomes free for a second interaction with reduced Fdx, which transfers a second electron to the [4Fe-4S] cluster. This transfer results in the reduction of the cluster-ligated Cys, and reestablishes the original 2+oxidation state of the cluster (3→4). The newly reduced internal Cys attacks and cleaves the heterodisulfide linkage between FTR and Trx, thus releasing reduced Trx. In a last step (4→1), the closing of the active-site disulfide completes the reaction cycle.

The reduced Trx, in turn, reduces the regulatory disulfide of the target protein by a thiol-disulfide exchange reaction. Such reactions with Trx also proceed via the formation of a transient mixed disulfide between Trx and the target protein, shown in red in the scheme below. 


Scheme 1



Target Proteins of the Ferredoxin/Thioredoxin System 

Most well studied light-regulated enzymes are members of the Calvin-Benson cycle. This cyclic pathway of CO2 incorporation can be divided into three phases: carboxylation, reduction, and regeneration (Figure 6). 



Figure 6

Figure 6. Calvin-Benson cycle and associated light-regulated enzymes. (Modified from Ruelland & Miginiac-Maslow 1999)

In the dark, light control mechanisms switch off the carboxylation and reduction phases, and disconnect the regeneration phase by turning off three enzymes catalyzing irreversible reactions, two positioned early in the regeneration phase (fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase), and a third at the end (phosphoribulokinase). Thus, the energy-demanding reactions of the cycle are turned off, while enzymes catalyzing reversible reactions of the regeneration phase that are needed for carbohydrate transformations in dark metabolism remain active. Further, in contrast to members of the Calvin-Benson cycle, the first enzyme of the oxidative pentose phosphate pathway, glucose 6-phosphate dehydrogenase, which provides reducing equivalents for dark metabolism, responds to light in an opposing manner, i.e., oxidative activation in the dark and reductive deactivation in the light. 

ATP-synthase and NADP-malate dehydrogenase, two enzymes associated with CO2 assimilation, are also light-controlled. Whereas ATP-synthase provides the ATP needed for the Calvin-Benson cycle, NADP-malate dehydrogenase of C3 plants participates in a shuttle system by exporting surplus reducing equivalents from the chloroplast to the cytoplasm, thereby maintaining a favorable redox equilibrium in the plastid. In certain C4 plants, NADP-malate dehydrogenase is a member of a CO2 concentrating mechanism and is, therefore, directly involved in CO2 assimilation. Enzymes involved in lipid biosynthesis, starch and nitrogen metabolism have also been found to be regulated by the Fdx/Trx system in chloroplasts (Schürmann & Buchanan, 2008). In the past decade, proteomic results have greatly expanded the list of putative targets in chloroplasts and other parts of the plant (Buchanan & Balmer, 2005). While a number of these candidates have been shown to be authentic targets, most still await confirmation. 

All enzymes targeted by Trx have at least one regulatory disulfide bridge, which is absent in cytoplasmic counterparts. The regulatory Cys are not embedded in a conserved consensus motif, and are separated by a variable number of residues, sometimes located on a loop structure absent from non-light-regulated isoforms. The mechanisms by which the enzyme activities are light-modulated are as diverse as the regulatory disulfide motifs. These observations suggest that light regulation has evolved individually for each target enzyme. 


Target Enzymes in the Calvin-Benson Cycle 

Phosphoribulokinase (PRK) is the last enzyme of the regeneration phase and is unique to the Calvin-Benson cycle. In presence of Mg2+PRK catalyzes the ATP-dependent phosphorylation of ribulose 5-phosphate to ribulose-1,5-bisphosphate, the acceptor molecule for the carboxylation reaction. This homodimeric protein of ~80 kDa contains a redox-sensitive disulfide bridge formed between two Cys separated by ~40 residues, which are proposed to be located on a loop involved in binding ATP (Harrison et al., 1998) close to the N-terminus of each subunit. Neither of the two Cys is directly involved in binding ATP, but formation of the disulfide in the oxidized enzyme perturbs the ATP binding site, and inactivates the enzyme. PRK appears to be the only known Trx-dependent enzyme with a regulatory Cys as part of its active site. 

Moreover, in the dark PRK is further deactivated by forming aggregates with glyceraldehyde-3-phosphate dehydrogenase and the small chloroplast protein CP12. In the light, the enzyme is released and activated most efficiently by reduced Trx f (see below) (Marri et al., 2009). 

Ribulose-1,5-bisphosphate-carboxylase/oxygenase (RuBisCo), the sole enzyme of the carboxylation phase and also unique to the Calvin-Benson cycle, catalyzes the addition of CO2 and H2O to ribulose-1,5-bisphosphate to yield two molecules of 3-phosphoglycerate. In a side reaction, RuBisCo uses O2 instead of CO2 to produce one molecule each of 3-phosphoglycerate and 2-phosphoglycolate. This oxygenase activity drains carbon from the cycle that is partly recovered by photorespiration in peroxisomes and mitochondria. Oxygenation proceeds more slowly than carboxylation, and its magnitude depends on the relative amounts of O2 and CO2 in the environment. RuBisCo has long been known to be activated by light that is due in part to increasing Mg2+ concentration and alkalization of the stroma. In certain plant species light activates additionally via the Fdx/Trx system, however, not directly the enzyme, but via RuBisCo activase, an enzyme that in most plants consists of two isoforms. Following reductive activation by Trx f, the activase dissociates and, in an ATP requiring reaction, removes a wide variety of inhibitory sugar phosphates from the active site of RuBisCo, thereby restoring full enzyme activity. The larger of the two isoforms contains in a C-terminal extension (CTE) two Cys separated by 18 residues that form a redox-active disulfide. Through reduction of this disulfide, the sensitivity of the activase to ADP inhibition is greatly diminished, and its activity is increased. The redox changes in the larger isoform, also alter the activity of the smaller isoform presumably through cooperative interactions (Figure 7). 


Figure 7

Figure 7. Model for activation of RuBisCo activase. Experimental results suggest that the disulfide carrying the CTE is located near the ATP binding site and selectively interferes with ATP (but not ADP) binding and ATP hydrolysis. Reduction of the disulfide by Trx f is accompanied by a conformational change, probably rendering the reduced extension more flexible. This change removes the CTE blocking the ATP binding site, a mechanism reminiscent of the situation observed for glyceraldehyde-3-phosphate dehydrogenase and NADP-dependent malate dehydrogenase (see below).

Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) catalyzes the freely reversible reduction of 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate in the presence of NAD(P)H in the sole reductive step of the Calvin-Benson cycle. This enzyme, which is specific to photosynthetic tissue, exhibits dual cofactor specificity toward pyridine nucleotides with a kinetic preference for NADP(H). This activity is regulated by metabolites, reduction via Trxs, and reversible aggregate formation. The NADH-dependent activity of the enzyme, needed for chloroplast dark metabolism, is constitutive and insensitive to any kind of regulation. 

GAPDH is found in two isoforms with different regulatory properties, a minor homotetrameric A4-isoform, known as the non-regulatory GAPDH, and a major heterotetrameric A2B2-isoform, modulated by Trx and metabolites. The two types of subunits of the A2B2-GAPDH are quite similar. The major difference is the presence of a flexible CTE of about 30 residues in the B-subunit containing two invariant Cys, and a large number of negatively charged residues. The CTE is highly homologous to the C-terminus of CP12, which participates in the aggregation of non-regulatory GAPDH with PRK. 

Redox-dependent association-dissociation appears to be the main mechanism responsible for the light induced activity changes in GAPDH. In the dark both isoforms are found as large aggregates with strongly inhibited NADPH-dependent activity. Upon reduction by Trx f these aggregates dissociate and GAPDH regains full NADPH-linked activity (Figures 8 and 9). 



Figure 8

Figure 8. Schematic representation of association-dissociation of A2B2-GAPDH. In the A2B2-isoform the redox-sensitive disulfide is formed by the Cys in the CTE. In the oxidized enzyme, the CTE is positioned in a cleft between subunits, restricting access to the coenzyme binding site and allowing formation of higher oligomers that maintain the enzyme in a kinetically inhibited conformation. Reduction by Trx f dissociates the aggregates, and releases the CTE giving access to the coenzyme binding site and reestablishing full NADPH-dependent activity (Sparla et al., 2002).


Figure 9

Figure 9. Schematic representation of association-dissociation of A4-GAPDH, CP-12 and PRK. In the homotetrameric A4-isoform, which has no CTE, the small protein, CP12, with its four Cys mimics the CTE. When oxidized, the Cys form two disulfides, one in the N-terminal, and one in the C-terminal region of CP12, which then acts as a linker forming noncovalent complexes between GAPDH and PRK. These complexes aggregate to higher molecular structures keeping both enzymes inactive in the dark. In the light, Trx f reduces the disulfide bonds on CP12 and PRK, releasing both enzymes in active form (Marri et al., 2008, 2009).

Fructose-1,6-bisphosphatase (FBPase) hydrolyzes fructose-1,6-bisphosphate to fructose-6-phosphate and inorganic phosphate. The reaction has a high negative free energy, making it irreversible, and an important point of regulation in the beginning of the regeneration phase. FBPase is a homotetramer of 160 kDa, very similar in primary structure to its cytoplasmic counterpart, however, containing an insert in the middle of the primary sequence of each subunit. This insert forms a flexible loop and contains three cysteines, two of them separated by ~18 residues, which form the regulatory disulfide (Figure 10). 



Figure 10

Figure 10. Schematic representation of the activation mechanisms of FBPase. In the oxidized enzyme the disulfide bond locks the 20 Å distant active site, through the movement of two ß-strands, in an inactive configuration that does not permit binding of essential Mg2+ ions. Reduction by Trx f only loosens the loop structure and moves through the ß-strands a glutamate back into the active site making it catalytically competent to bind Mg2+ and substrate (Chiadmi et al., 1999; Dai et al., 2000).

In essence, light reduction of the regulatory disulfide has an allosteric effect on the distant active site by greatly decreasing the Mg2+requirement for efficient catalysis (Balmer et al., 2001). This, in concert with the alkalization and the increase of available Mg2+ in the stroma, greatly boosts FBPase activity. 

Sedoheptulose-1,7-bisphosphatase (SBPase) is the third of the three enzymes unique to the Calvin-Benson cycle. SBPase catalyzes the hydrolysis of sedoheptulose-1,7-bisphosphate, another irreversible step early in the regeneration phase of the cycle. Activity of the enzyme strictly depends on reduction by Trx f and is further regulated by stromal pH and Mg2+ level as well as by substrate and the products of the reaction (Schürmann & Buchanan 2008). A homodimer of 70 kDa, SBPase contains a regulatory disulfide bridge in the N-terminal region with the two Cys separated by four residues. The active site is located in the C-terminal part of the primary sequence. Based on homology modeling the disulfide is situated in a loop, which might function similarly as observed for FBPase, i.e., by acting on nearby ß-strands that modify the conformation of the distant catalytic site (Chiadmi et al., 1999). 


Additional Chloroplast Target Enzymes 

ATP-synthase, the chloroplast coupling factor CFOCF1, is not a member of the Calvin-Benson cycle, but provides ATP needed for the kinase reactions of the cycle. This enzyme is a membrane protein complex composed of the integral membrane portion CFO and the hydrophilic CF1consisting of five subunits. It uses the proton-motive force across the thylakoid membrane to synthesize ATP from ADP and Pi. ATP synthase is a latent enzyme, and its activity is regulated in vivo by the transmembrane electrochemical proton gradient inducing conformational rearrangements (Ort & Oxborough, 1992). The potential gradient thus acts as a driving force for phosphorylation, and as an activator for the reversible conversion of the complex to catalytic competence. 

In addition to this electrochemical activation, ATP synthase is subject to redox regulation by the Fdx/Trx system. The structural element allowing for thiol modulation is comprised of two Cys separated by five residues in the CF1 -subunit, forming a disulfide bond in the oxidized enzyme. Reduction of CF1 is very rapid, even in weak light, probably due to the positive redox potential of the disulfide, thus permitting a higher rate of ATP formation at limiting electrochemical potential. Trx-linked regulation also allows the enzyme to be switched off in the dark to avoid wasteful ATP hydrolysis (Samra et al., 2006; Wu et al., 2007). This is in line with the observation that in organisms such as cyanobacteria, CF1 lacks the regulatory disulfide segment seen in the chloroplast counterpart. In these organisms, CF1 is required for both photo- and oxidative-phosphorylation and, therefore, should not be turned off in the dark. 

Glucose-6-phosphate-dehydrogenase catalyzes the first committed step of the oxidative pentose phosphate pathway, the oxidation of glucose 6-phosphate to 6-phosphogluconate with concomitant reduction of NADP. The chloroplast isoform is strictly light regulated by the Fdx/Trx system, but in contrast to enzymes of the Calvin-Benson cycle, is deactivated by reduction in the light, and activated by oxidation in the dark. This response is essential in providing for differential control of the two opposing carbon pathways in the chloroplast, minimizing the simultaneous occurrence of carbohydrate synthesis (via the Calvin-Benson cycle) and degradation (via the oxidative pentose phosphate pathway). In land plants the two Cys engaged in the regulatory disulfide bridge are found in a nine residue motif, located in the N-terminal half of the protein near the NADP-binding domain and active site. Based on homology modeling, the regulatory disulfide motif is part of an exposed loop, accessible to Trx. Whereas Trx m has been thought to be the unique regulatory protein, recent results show that reduced Trx f is equally efficient in deactivating the enzyme (Nee et al., 2009). 

NADP-dependent malate dehydrogenase (NADP-MDH) is a chloroplast enzyme present in green algae and different types of land plants. The enzyme catalyzes the reduction of oxaloacetate to malate using NADPH as reductant in a reaction that strictly requires light-activation via the Fdx/Trx system. In C3 plants, NADP-MDH functions in a shuttle mechanism exporting surplus reducing equivalents in the form of malate from chloroplasts to the cytosol, thereby helping to maintain a favorable redox equilibrium in the stroma. In C4 plants of the NADP-malic enzyme type (maize, sugarcane or sorghum), NADP-MDH functions as a member of a carbon trapping and transport system of mesophyll cells. It reduces oxaloacetate, the first CO2 fixation product that has been imported in the chloroplast after formation from phosphoenolpyruvate and CO2 in the cytosol. The newly formed malate is then transported into chloroplasts of the bundle-sheath cells where it is decarboxylated by malic enzyme, thereby releasing CO2 and generating NADPH. The CO2 is fixed by RuBisCo, and the NADPH is used in the Calvin-Benson cycle, together with ATP generated by photophosphorylation. 

A homodimer of 85 kDa, chloroplast NADP-MDH differs from its NAD-dependent cytosolic homolog by the presence of Cys-containing N- and C-terminal extensions. In the oxidized enzyme, these Cys form two disulfide bridges, one within the N-terminal extension, and one between a Cys on the C-terminal extension and a Cys in the core of the protein. Reduction of either disulfide has a differential effect on enzyme activity, but only a reduction of both disulfides provides maximal activity. This can be understood on the basis of the molecular structure (Figure 11). 


Figure 11

Figure 11. Schematic representation of the activation of MDH. The highly flexible N-terminal extension is located at the interface between subunits, where it makes a number of mainly hydrophobic contacts with both the catalytic domain of one subunit, and the coenzyme-binding domain of the other. The reduction of the disulfide is thought to relax this rigid structure, and free the catalytic domain to adopt a productive conformation. The C-terminal extension of the oxidized enzyme is held against the core structure by the disulfide bond, and shields the entrance to the active site by occupying the position of the natural substrate oxaloacetate. Negative charges at the tip of the extension, a penultimate glutamate and the C-terminal carboxylate, are instrumental in this inhibitory action by mimicking the dicarboxylic substrate. The negative charges can also interact with bound NADP+ and, in this way, inhibit the enzyme. Trx reduction of the C-terminal disulfide destabilizes the extension rendering it very mobile thereby permitting access to the active site (Miginiac-Maslow & Lancelin, 2002).

Trxs f and m are both capable of activating NADP-MDH, but as Trx f is more efficient under certain in vitro conditions, it may be the primary activator (Schürmann & Jacquot 2000; Schürmann & Buchanan, 2001). 

Redox Potentials and Target Enzyme Activation 

The redox potential difference between Fdx and Trxs indicates that Trx reduction is thermodynamically very favorable (Table 1). 


Table 1


A comparison of the redox potentials of the target enzymes further suggests an order in the action of Trxs on the targets upon illumination. The group of targets with the most positive redox potentials will be reduced very rapidly, thereby turning off the oxidative pentose phosphate pathway, and preparing acceptor molecules for carboxylation. This rapid-response group includes G6PDH, the aggregates of PRK and non-regulatory GAPDH linked by CP12 and ATP-synthase. Then the activation of RuBisCo via RuBisCo activase starts carboxylation, and, finally, the activation of the two phosphatases and GAPDH (regulatory form) enables regeneration of the CO2 acceptor. Depending on the electron pressure, different redox equilibria can be reached, resulting in defined degrees of activation. These equilibria could be further modified by effector molecules that allow fine-tuning of the enzymes. 

The redox potential of NADP-MDH is more negative than that of the Trxs, thus requiring an excess of reduced Trx for activation. This requirement is consistent with the role of the enzyme in C3 plants, where it transports excess reducing equivalents from the chloroplast to the cytosol (see Figure 6). 


Concluding Remarks 

Experiments with algal cells and chloroplasts initiated in the 1960s provided evidence that light not only supplies energy for CO2assimilation, but also is perceived as a regulatory signal that modifies the activity of chloroplast enzymes (Buchanan et al., 2002; Bassham, 1971). This function enables chloroplasts to switch from dark to light metabolism and to adjust enzyme activities in response to changing light conditions. Research in subsequent decades has established the elements involved in transmitting the light signal to enzymes, leading to the discovery of Trxs in plants and to their involvement in enzyme regulation, work that has given birth to the field of redox biology. The redox-based mechanism by which this occurs in oxygenic photosynthesis has become known as the Fdx/Trx system of enzyme regulation. Research in recent years has added structural and mechanistic details, which have provided a better understanding of the mechanism of signal transfer via this mechanism. Future studies should explore the Trx-linked enzymes that have been identified in proteomic studies, and further elucidate details of the changes that proteins undergo when regulated by redox.