Category Archives: plant science

More Forest Numbers and Tree-Planting Drones

Here’s a new idea under the sun… tree-planting drones.

Drone

Take a minute and think about all of the forest products you use in a day. I’m sitting on one right now as is my laptop. Anyone who’s bought school supplies in the last weeks has also consumed a fair amount. As an avid list-maker, I use my fair share of small pieces of paper for reminders and tasks to be done. Of course, let’s not forget the oxygen. The U.S. Forest Service estimates that forest products account for 4.5% of the total U.S. manufacturing gross domestic product at $190 billion in products annually and employing >900,000 people. There’s little room for doubt, forests are valuable both as they stand and as they fall.

I’ve mentioned before that numbers are important. In this context, numbers about forests can be extremely valuable. New research in Nature this week gives a new estimate of our global tree density. The good news… The earth has nearly an order of magnitude more trees than previously estimated by satellite imagery. The research team puts the total number of trees on the planet at 3.04 trillion. That’s more than 400 trees per person. To you, these numbers are likely just interesting statistics to impress your friends, but to scientists, conservationists and the forestry industry the new estimates of tree density are important for guiding management policies.

The new estimate is based on more than 400,000 ‘ground-sourced measurements of tree density from every continent except Antarctica.’ Translation: A lot of man-hours were spent by humans in a forest collecting tree density data. Of course, the team wasn’t able to count each and every tree, but this is more accurate data than estimating tree numbers from space. Check out the video below for a great data visualization of the world’s forests based on this new research.

If you were paying attention, forest numbers are not static. For various reasons around the globe forests are declining. Current estimates give a net loss of 10 billion trees a year. Some of this loss is due to forest fires, but for decades the forestry industry has become increasingly mechanized to efficiently harvest trees for all of those useful forest products. On the other side of the equation, replanting new trees has not experienced the same industrialization and relies heavily on meticulous man hours and dirty hands. The company BioCarbon Engineering seeks to change this and offers a new scalable model for planting trees using drones. Yes, drones for a noble purpose. The drones are engineered to shoot bullets wrapped in a biodegradable casing and containing soil with pre-germinated seeds into the ground. This could be used for replanting after forest fires or in other areas where forests need help with recovery.

From BioCarbon Engineering

From BioCarbon Engineering

They estimate that they can scale up to 1 billion trees per year. It greatly decreases the amount of human hours involved compared to hand-planting. It’s also more efficient relative to mass seeding because the seeds are pre-germinated, which will eliminate the loss due to bad seeds. The numbers are still not yet in the trees’ favor, but closing the gap with deforestation is a step in the right direction and the Lorax would be proud.

Johnna

*Of course, BioCarbon Engineering could always enter into a relationship with that BIOS Urn company that uses your cremated ashes as the germination medium for tree seeds. Then you and your loved ones could have your ashes with tree seedlings shot almost anywhere to make a stand of trees somewhere in the wilderness rather than just one tree at a time with a single urn. But as I’ve mentioned before, that’s not exactly how photosynthesis and the carbon cycle works.

//The blog has been dark for a record length of time. Only for lack of time and not material. Regularly scheduled posts coming soon.

References and Links:

http://www.nature.com/nature/journal/vaop/ncurrent/full/nature14967.html

http://www.nature.com/news/global-count-reaches-3-trillion-trees-1.18287

For more on the tree-counting research, check out this post over at The Quiet Branches blog.

http://www.biocarbonengineering.com/

http://www.biocarbonengineering.com/blog/deforestation

http://www.biocarbonengineering.com/blog/deforestation-data

http://www.biocarbonengineering.com/blog/what-is-the-social-value-of-a-tree

http://www.msnbc.com/greenhouse/watch/using-drones-to-improve-reforestation-445645891970

http://www.fao.org/docrep/014/am859e/am859e08.pdf

http://www.fs.fed.us/

On becoming a tree…

“As long as you keep getting born, it’s all right to die sometimes”

Orson Scott Card, The Speaker for the Dead.

I’ve written many times about the differences between autotrophs and heterotrophs on this blog. Loyal readers will know I have a hard-line stance that humans fall decidedly into the heterotroph camp. Nevertheless, some art projects blur the lines between person and plant. So I was more than a little intrigued when a link for the Bios Urn came across my Facebook feed.

BiosUrn

The product offers an alternative to the traditional cemetery as your eternal destination. It’s a special biodegradable urn in which your ashes (or a loved one’s or a pet’s ashes) can be placed along with a tree seed. The whole thing is planted in the ground at a cemetery or other special location. The recycled carbon atoms of your body become the growth medium for a tree that will grow and live on after your death.

biosurn2

It’s beautiful. I get it and as someone who often quotes the Lorax, I’m all for any excuse to plant trees. But honestly, the first thing it made me think of was my all-time favorite book The Speaker for the Dead by Orson Scott Card.* Within the world of this book, humans live on a colony planet that happens to have another species of sentient beings called the Pequeninos. These organisms spend the majority of their life cycle as mammal-like beings, but can pass on into a third life as a tree provided they have been vivisected. It’s pretty gruesome and obviously humans don’t work this way, so cultural misunderstandings ensue.

However, because I’m a stickler for scientific accuracy when it comes to plant science (Hey, someone’s gotta be.), I have one problem with the overly simplistic marketing scheme of the Bios Urn. You don’t actually become the tree. For those of you saying, “I know, I know I won’t be a tree, but in the circle of life my molecules will become part of this tree.” I’ll still be over here at my blog shaking my head, “No, that’s not really how that works.” Here’s a reminder of the photosynthetic equation:

6 CO2 + 6 H2O (+ light) → C6H12O6 + 6 O2

Plants accumulate carbon and mass from atmospheric CO2, not carbon ash or even organic carbon compounds in the soil. The truth is that if you planted your ashes with your tree seed, your carbon would still remain locked in the soil for many years until eventually it is metabolized by microorganisms in the soil and released as CO2 as part of their respiration. This is not a very quick way to turn over your carbon molecules, and your molecules have millennia before they ever become a part of any tree. If you truly wanted your carbon molecules to incorporate into some kind of plant matter, then you need to find a way to carbonate your deceased body. Human carbonated essence could be stored in canisters and then used to supply CO2 to your plant of choice. You could be part of that camellia bush in your front yard, your favorite LSU Oak tree, the General Sherman etc. Scientifically accurate, but good luck marketing that compared to cremation.**

All of this highlights a common misconception about photosynthesis- it’s hard to comprehend how mass can accumulate into living things from air. Our gut tells us mass must come from something more substantial. We memorize the photosynthetic equation in elementary school, but few of us grasp its consequences and really believe it.

“This is how humans are: We question all our beliefs, except for the ones that we really believe in, and those we never think to question.”

Orson Scott Card, The Speaker for the Dead

So, when you die, ask someone to plant a tree or something in your memory. Do it for someone else you’d like to remember, but ashes not really required. Or buy a BiosUrn or put the ashes in a degradable coir starter pot from your local garden center, but you’ll probably want to supplement them with some form of NPK fertilizer. It will help the seed more than your carbon atoms.

Johnna

*It’s a sequel to Ender’s Game for those of you that may not be SciFi freaks.

**Although, in-home soda fountains are a thing now, so if there is a way to carbonate deceased relatives and store them in the handy canisters… well, you get the idea.

Links and References:

https://urnabios.com/

http://en.wikipedia.org/wiki/Speaker_for_the_Dead

The Twelve Days of Christmas Plants

If you’re looking for a holiday related diversion, here’s a linkfest of my posts from last year on the Twelve Days of Christmas plants. Understand the plant science behind the traditions. Use these random facts to quickly change the subject when nosy but well meaning friends and family ask you uncomfortable questions.

The Twelve Days of Christmas Plants

TwelveDays

1. The Christmas Tree

2. Chestnuts

3. Poinsettias

4. Holly

5. Peppermint

6. Mistletoe

7. Grapes

8. Greens and Black-eyed Peas

9. Sugarcane

10. Oranges

11. Pomegranates

12. Boswellia sacra and Commiphora myrrha

 

If you’re craving even more holiday nerdery or you’ve already used my random facts as diversion tactics last year, check out The 2014 Chemistry Advent Calendar over at the Compound Interest blog. Or check out these Yuletide plants gone global you’ve probably never heard of from the John Innes SVC blog.

 

Johnna

The Dangerous Double Life of a Distinctive Diazotroph

For many people around the world, tonight is one of the most anticipated nights of the year. In that spirit, here’s some new science, not under the sun, but under the moonlight and starlight. New research from Wegener, Nagarajan and Pakrasi* describe something new about the Photosystem II D1 protein, an unexpected source that photosynthesis researchers had long written off as conquered or fully explained.

Why would we care about photosynthesis at night anyway? It all comes down to the distinctive lifestyle of the cyanobacterium Cyanothece, which belongs to an interesting class called unicellular diazotrophs (word of the day). Like all cyanobacteria, they perform oxygenic photosynthesis but they have the bonus biochemical ability of nitrogen-fixation.

Nitrogen-fixers are capable of pulling nitrogen gas (N2) out of the air and turning it into ammonia which the cells can use as a nitrogen source for important molecules like protein and DNA. Other microbes can perform nitrogen-fixation, and you may remember them as the root nodule symbionts of legumes. If you’re not a nitrogen-fixer, you have to rely on ammonia or other nitrate compounds present in the soil, sea or your diet (depending on what kind of organism you are) to make the required nitrogenous biomolecules. Since the Earth’s atmosphere is 78% N2, you don’t have to be great at math to figure out that acquiring the ability to tap into the atmospheric nitrogen source advances your species in the game of life a considerable number of squares.

What does this have to do with photosynthesis? Why don’t more plants and cyanobacteria get in on this nitrogen-fixation biochemistry? There’s a critical fatal flaw in the photosynthesis/nitrogen-fixation biochemical tango. Nitrogenase, the enzyme responsible for the nitrogen-fixation reaction is hopelessly and irreversibly killed by oxygen. Oxygen destroys the protein enzyme that’s already been made and tells the genome not to even bother wasting energy trying to make it new. If you’ve been paying attention to the blog, oxygenic photosynthesizers like cyanobacteria and plants make oxygen via their PSII enzyme every time light shines on them. So for nonphotosynthetic soil bacteria and other microbes, it’s not a big deal to avoid oxygen and go anaerobic to fix nitrogen. Cyanobacteria, however, must lead dangerous double lives to combine these two opposing biochemical processes.

Anabeana spherica via Wikimedia Commons The more round cell in the center of the string is the nitrogen-fixing heterocyst.

Some diazotrophic cyanobacteria solve this problem by separating these two processes in space. Species like Anabeana can go through a special type of development where the cells divide themselves, but incompletely to form a connected network like beads on a string. Every tenth cell (heterocyst) gets an extra layer of cell wall, destroys its photosynthetic machinery, and commits itself to a life of nitrogen-fixation. These cells share their fixed nitrogen compounds with their neighbors, which in turn share their fixed-carbon from photosynthesis. This is a fascinating process still hasn’t given up all of its secrets and remains an active area of research.

Cyanothece 51142 by Michelle Liberton, Pakrasi lab

Cyanothece 51142 by Michelle Liberton, Pakrasi lab

Cyanothece, on the other hand, is a more ruggedly individualistic species that manages to perform both oxygenic photosynthesis and nitrogen-fixation within a single cell. It accomplishes this through a carefully controlled circadian rhythm. During the day, oxygenic photosynthesis occurs. During the night, nitrogen-fixation occurs. It sounds so simple, yet this elegance is far from easy. This is a true circadian process, in that, while some cues are definitely derived from light signals, the cells also have a strong internal clock that keeps the time of day and controls what biochemical machinery is active. Thus, near the hour of dawn and dusk, the cells are actively preparing for the metabolism to come. Also, if you were to train a culture of these cells under a certain day/night cycle for several days then switch them to continuous light, they would still maintain their circadian biochemical cycle for several days after the switch.

One issue that has puzzled researchers for some time is that the photosynthetic machinery, like PSII, is actively shut down at night. It’s not just that it’s dark, so there won’t be any photosynthetic oxygen production. Apparently, that’s not good enough. The cells actively shut it down. When researchers take culture samples of these cells during their dark period and shine light on them to check photosynthetic capability, there is no signal. Sure, there’s probably little chance any significant photosynthesis is occurring at night, but Cyanothece isn’t taking any chances. For these cells, no moonlight, floodlights from passing ships, comets, or even the blessed Eastern Star is going to interfere with their nocturnal nitrogen fixation.

Shutting down photosynthesis is no small feat. Again, if you’ve been paying attention to the blog, you will note that I’ve ranted on numerous occasions about how complex PSII is and how difficult it is to assemble  and reassemble from its individual parts. As far as energetics are concerned, it would be a losing battle for Cyanothece to completely disassemble its PSII at dusk only to resynthesize it again the next morning. I don’t care how hard-up it is for a nitrogen source. As it turns out, Cyanothece and other unicellular diazotrophic cyanobacteria solve this problem in a more graceful way, for which Wegener et al have provided new experimental evidence.

Cyanothece, like all other oxygenic photosynthesizers, contains multiple copies of the psbA gene encoding the D1 protein, which is a core subunit of PSII and critical for function. Since the advent of the genomic era, the genomes of numerous photosynthetic organisms have been sequenced revealing these multiple psbA genes, most of which have little or no protein sequence differences. These subtle changes have been linked to cells’ ability to fine-tune photosynthesis under certain conditions. However, Cyanothece and its cousins have a psbA gene copy that is particularly different- psbA4. If one were to take all of the site-directed mutants that photosynthesis researchers made to sort out PSII function over the last few decades since molecular biology techniques were available and threw them altogether into a single mutant gene, you would get psbA4. If PSII contained the D1 protein encoded by psbA4, there would be no C-terminal processing and no active site for water-splitting and oxygen production.

Why would any self-respecting photosynthetic organism retain such an utterly non-functional PSII subunit? The answer is because it does serve a critical function in the daily cycle of Cyanothece, just not during the day. The D1 protein encoded by the psbA4 gene, called a sentinel D1 protein (sD1), is made only for the night, when it does the only thing it still can do- fit right in the center of the PSII complex and prevent PSII function of any kind. There’s no chance of any oxygen production while sentinel D1 is on duty, but the cells can maintain most of the other PSII components in a stable complex (meaning they don’t have to be degraded and made fresh every morning). Then, Cyanothece can turn photosynthesis back on in the morning by replacing a single protein in its PSII complexes. The report by Wegener et al gives the first biochemical characterization of PSII complexes containing a sentinel D1 protein, providing evidence that this strategy works for controlling PSII oxygen production.

sentinel

Performing oxygenic photosynthesis and nitrogen-fixation within the same cell is biochemically problematic, but the sentinel D1 protein appears to be the secret weapon for this dangerous double life. Through this strategy oxygenic photosynthesis and nitrogen fixation are “Always together, eternally apart.”** Many questions remain regarding the regulation of the transition from functional D1 to sentinel D1 at dusk and the reverse replacement at dawn. There are still outstanding questions regarding the D1 turnover event for any PSII in any photosynthetic organism, so it’s not at all clear what parts of this process Cyanothece uses or whether new factors are involved. Obviously, there must also be some sophisticated regulation to prevent the synthesis of sentinel D1 during the day.

While this work focuses on an organism you’ve never heard of, it has some interesting biotech implications for the future. As I mentioned, being able to both fix CO2 through photosynthesis and N2 through nitrogenase puts you at a significant metabolic advantage. And no, I don’t mean you. Remember, we’ve been through this- people are not photosynthetic. It would be great if one day we could engineer certain crop plants to combine these traits. Legumes already have this trick covered through symbiosis, but many other staple crops require tons of nitrogen fertilizer. If researchers could unlock the secrets of Cyanothece’s double life and translate it to plants, then it may be possible to engineer versions of crop plants that don’t require so much nitrogen fertilizer. Thus, one night in the future, engineered crop plants may be fixing nitrogen thanks to the addition of a variant gene they already had.

Johnna

*I know I’ve posted frequently about Pakrasi lab (my thesis lab) research, but this topic was just developing as I was leaving the lab and it’s too cool to pass up.

**Bonus points to you if you get the movie reference.

References and Links:

http://www.jbc.org/content/early/2014/12/18/jbc.M114.604124.full.pdf+html?sid=8f38f7c6-96c1-4085-bbb0-efc6ca425624

A Green Deal for Black Friday

It’s Black Friday. The blog may have been dark for the last couple of weeks while I’ve been busy with my day job, but today I have a special deal for all my readers. I’ve been writing about the holiday plants we use in our celebrations for the past year and now you can have all of those posts to yourself in convenient downloadable PDF format*. All this plant science for the low, low price of absolutely free! How’s that for a green Black Friday Deal?

Here is the link to download:

Holiday Plants: An Autotrophic Almanac

 

Johnna

*Sorry, the Youtube video links don’t work any more, but the rest of the links are still hot.

Binding P’s and Q’s, Minding T’s and I’s

When it comes to nailing down the location of P and Q in plant Photosystem II, you have to be careful how you cross your t’s and dot your i’s.

A longstanding question in the area of Photosystem II research involves the complement of proteins on the lumenal side of the enzyme in plants vs. cyanobacteria. This region of the complex serves to split water into molecular oxygen- a seemingly conserved reaction in all oxygenic photosynthetic organisms. However, it has long been known that the extrinsic proteins associated with the lumenal face of the complex differ significantly between plants and cyanobacteria.

Differences in the lumenal extrinsic proteins, plants vs. cyanobacteria

Differences in the lumenal extrinsic proteins, plants vs. cyanobacteria

Studies from various disciplines in recent years (structural, mutants, proteomic) have layered on more questions. Biochemical work on plants indicates there are three extrinsic proteins- PsbO, PsbP and PsbQ. Biochemical analysis and structural studies of cyanobacteria have shown that their PSII complexes contain PsbO, PsbU and PsbV. Other proteomic analysis and mutant studies have shown that cyanobacteria also contain homologues of PsbP and PsbQ (aka CyanoP and CyanoQ, respectively). Sure, this accumulation of data sounds like alphabet soup to those outside of our field, but it also leaves photosynthesis researchers wondering how appropriate it is to use structural information from cyanobacteria to infer anything about PSII structure in plants.

For all the knowledge we’ve gained, we’ve been comparing green apples to blue-green oranges. New research from the Bricker lab has used chemical crosslinking and mass spectrometry to gain more information on the organization of extrinsic proteins in plant PSII- directly from plant material. I’ve written about these techniques before in another paper addressing a similar problem from the cyanobacterial extrinsic protein vantage point. In a publication available electronically this week, Mummadisetti et al provide new information on the arrangement of the PsbP and PsbQ proteins in higher plant PSII. This work goes beyond what either of the individual protein crystal structures (not in the context of the PSII complex) could tell us. Distance constraints from the crosslinking data were used to guide modeling studies to fill in gaps in the solution structure of PsbP as well as identify interaction sites with PSII membrane components and another extrinsic protein, PsbQ. Altogether this gives us a more complete picture of an important enzyme.

In addition to the satisfaction that comes with publishing this great work in a top journal, the authors have also been featured in a research highlight by the university. Since I like to take things a step further on this blog, this post will feature the behind the scenes story of how this project came to be a publication worthy of a press release- especially with regard to how the story is told with imagery.

As is typical of any press release, an image is used to capture the essence of the research. In the featured image, we see Manju Mummadisetti without lab coat and gloves, holding her PSII membranes proudly aloft the pristine ice bucket next to a carefully positioned bag of spinach leaves that have yet to meet their demise in the Waring blender. I get it. In photosynthesis research labs we always have a tube or bubbling flask of something green-ish that easily fits the stereotype non-scientists have of what scientists must do all day long. Every. Day.

Credit: Louisiana State University

Credit: Louisiana State University

Long time readers of this blog will realize that in this image Manju is breaking the first rule of biochemistry. Fast. And. Cold. No self-respecting biochemist would gaze longingly at their biochemical sample that wasn’t on ice or in the cold room. Moreover, photosynthesis researchers opt for darkness or dim light for their preparations in order to keep activity low and avoid damage. I can say unequivocally that Manju knows and obeys all of the rules of biochemistry. This sample was only for practice or for show. A more true picture would look like this.

What real research looks like

What real research looks like

Notice the lab coat and gloves. Samples are kept in the ice bucket. For all you know, there’s nothing in there at all green or otherwise.*

Manju spent considerably more time gazing at data on her computer screen than she ever did her green samples. In the picture below, she is analyzing mass spec data and evaluating the validity of calls made by the software. p > 0.005 need not apply for her results, but anything better is painstakingly recorded on a brown paper towel.

005

But really, Manju’s research is more appropriately captured in other images like this one.

It's harder than you think

It’s harder than you think

It may not make for click bait, but carefully filling out FedEx shipping forms is an essential part of her research. After Manju prepares her samples, they must be shipped overnight on dry ice (Fast. And. Cold.) to collaborators in Cincinnati for the mass spectroscopy analysis. These shipments are carefully planned so that someone is available to receive them and perform the analysis. This isn’t always easy over the summer when it is necessary to coordinate the travel schedules of half a dozen researchers in two different labs.

On one such occasion, forms and labels were not filled out appropriately. Not because Manju forgot to dot and i or cross a t, but because she did. Packages with dry ice require a special hazard label that must include all address information for both the sender and recipient in a very small area. We never knew how critical it was to avoid crossing that dotted line of the diamond.**

Notice how the last 'ti' in Manju's name infringes into the label

Notice how the last ‘ti’ in Manju’s name crosses over the dotted line and into the hazard label. Such a distraction could interfere with proper handling of this hazardous substance.

IMG_0038 (2)

Really FedEx? Her name is 20 characters long! Plus, the all caps just makes it seem like they’re yelling. It’s that serious.

A diligent FedEx employee at the Baton Rouge office rejected the shipment and sent it back to the lab at LSU the next day. I know rules are rules, but really this is on the verge of Gas Station Manager Syndrome. The samples were fine and promptly placed back in the freezer. A flurry of NSFW text messages about the situation were exchanged among people still on vacation. A new shipment date was coordinated and new forms were completed. It went away without a hitch and was promptly turned into data.

A new lesson was learned that day- Thou shalt not write within the dotted line of the hazard label. It’s an extension of a long-standing research rule- Obey arbitrary formatting and paperwork requests from people that control what you need.

 

Johnna

*Full disclosure- there was NO sample in that ice bucket. No one in the lab had any membranes prepped that day and real biochemists don’t pull good samples out of the freezer for a photo op.

**It’s like crossing the streams in Ghostbusters apparently. It would be bad.

References and Links:

http://www.pnas.org/content/early/2014/10/24/1415165111.short

http://www.lsu.edu/ur/ocur/lsunews/MediaCenter/News/2014/10/item73226.html

Plant Skulls

snapgdragon seed pod skull dragons skullIf you didn’t know this was a plant science blog, you might think these were macbre trophies of some ancient tribe. This isn’t so much an appropriate plant costume for Halloween as it is an interesting confluence of floral anatomy and human propensity for recognizing facial forms.

Snapdragons or Antirrhinum sp. are a colorful staple of summer gardens. We’re more used to seeing them look like this image with tall inflorescences boasting clusters of ruffled flowers in a variety of color schemes.

Antirrhinum majus Credit: Michael Apel via Wikimedia Commons

What’s responsible for this spooky transition from delicate flowers to haunting faces? It all comes down to the snapdragon’s flower structure and its bilateral symmetry. The skulls are really the seed pods of the plant after the flowers have been pollinated and the petals have withered away. Dissecting the flowers in their prime shows the ovary at the base of the bloom. The pollen-containing stamens and the style emerge from the orifices in the ovary. These structures leave behind gaping holes that look like a mouth and eye sockets.

Snapdragon flower anatomy

Snapdragon flower anatomy

The striking resemblance of these seed pods to human skulls has led to their association with supernatural powers. They were purported to help women stay young and beautiful as well as protect humans of all ages from witchcraft and evil spirits. I don’t have any scientific evidence of that, but if you’re looking for new ideas for botanical Halloween decorations that go beyond cucurbits and mums, dried snapdragon stems with seed pods make a wicked wreath.

Johnna

Links and References:

http://en.wikipedia.org/wiki/Antirrhinum_majus

http://en.wikipedia.org/wiki/Antirrhinum

http://www.missouribotanicalgarden.org/PlantFinder/PlantFinderDetails.aspx?kempercode=a561

http://www.kuriositas.com/2013/07/the-dragons-skull-macabre-appearance-of.html

http://www.waitwow.com/snapdragons-make-cute-flowers-die-turn-skulls/

http://gardenofeaden.blogspot.com/2014/02/the-dragons-skull-seed-pod.html

http://weirdworldwonders.com/the-dragons-skull-snapdragon-seed-pods/

https://s10.lite.msu.edu/res/msu/botonl/b_online/library/snapdragon/flower.html

http://www.flowersplans.com/snapdragon-flower-dry/

http://www.gardenguides.com/126335-history-snapdragons.html